research article fecal colonization with extended-spectrum...
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Research ArticleFecal Colonization with Extended-Spectrum Beta-Lactamase andAmpC-Producing Escherichia coli
Mohamed H Al-Agamy12 Taghrid S El Mahdy3 and Atef M Shibl14
1Department of Pharmaceutics Microbiology Division College of Pharmacy King Saud University PO Box 2457Riyadh 11451 Saudi Arabia2Department of Microbiology and Immunology Faculty of Pharmacy Al-Azhar University Cairo Egypt3Microbiology and Immunology Department Faculty of Pharmacy Helwan University Cairo Egypt4College of Medicine Alfaisal University Riyadh Saudi Arabia
Correspondence should be addressed to Mohamed H Al-Agamy elagamy71yahoocom
Received 18 December 2015 Accepted 10 May 2016
Academic Editor Wejdene Mansour
Copyright copy 2016 Mohamed H Al-Agamy et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited
Background Extended-spectrum120573-lactamases (ES120573Ls) andAmpC 120573-lactamases cause120573-lactam resistance in Escherichia coli Fecalcolonization by ES120573L- andor AmpC-positive E coli is a source of nosocomial infectionsMethods In order to investigate inpatientfecal colonization by ES120573Ls and AmpC antibiotic sensitivity tests were conducted andminimum inhibitory concentrations (MICs)were determined using the disk diffusion method and E-test respectively Characterization of ES120573L and AmpC was performedusing E-test strips and a set of PCRs and DNA sequence analyses were used to characterize the ES120573L and AmpC genes ResultsThe whole collection of E coli isolates (119899 = 50) was sensitive to imipenem tigecycline colistin and fosfomycin while 26 of theisolates showed reduced susceptibility to ceftazidime (MIC ge 4 120583gmL) ES120573L was phenotypically identified in 26 (1350) of caseswhile AmpC activity was detected in two ES120573L-producing E coli isolates All ES120573L-producing E coli were positive for the CTX-M gene eleven isolates carried 119887119897119886CTX-M-15 and two isolates carried 119887119897119886CTX-M-14 gene Two CTX-M-positive E coli isolates carried119887119897119886CMY-2 Conclusions The alimentary tract is a significant reservoir for ES120573L- andor AmpC-producing E coli which may lead tonosocomial infection
1 Introduction
A remarkable increase in fecal colonization rates with ex-tended-spectrum beta-lactamase- (ES120573L-) AmpC-plasmidmediated- andor carbapenemases-producing Enterobac-teriaceae has been reported in many regions worldwide[1 2] Infections caused by Enterobacteriaceae which areresistant to 120573-lactams are coupled with the inappropriateuse of antibiotics andor a prolonged period of hospitaladmission The rising use of carbapenems for empiricaltreatment of nosocomial infections has led to fast global dis-semination of carbapenemase-positive enterobacterial strains[3] ES120573Ls arise through point mutations in TEM-1TEM-2 and SHV-1 However over the last three decades non-TEM and non-SHV ES120573Ls strains have been detected pri-marily CTX-M Enterobacteriaceae that produce CTX-Menzymes have shown rapid and concerning dissemination
and have been documented as the most prevalent etio-logical infectious agents [4] ES120573L confers resistance topenicillins cephalosporins and monobactam (aztreonam)but they are susceptible to cephamycins (cefoxitin andcefotetan) and carbapenems (imipenem meropenem anddoripenem) and are typically reserved by inhibitors ofAmbler class A 120573-lactamase (clavulanic acid tazobactamor sulbactam) Most ES120573Ls can hydrolyze fourth-generationcephalosporins While AmpC 120573-lactamases confer resistanceto penicillins third-generation cephalosporins monobac-tam and cephamycins they are sensitive to carbapenem andare not inhibited by 120573-lactamase inhibitors however they areinhibited by cloxacillin [5ndash7]
Numerous studies in Saudi Arabia have focused on theidentification of ES120573L-producing strains from clinical speci-mens [4] but there are few reports on the fecal colonizationof ES120573L-producing isolates in Saudi Arabia Therefore in
Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 3704150 7 pageshttpdxdoiorg10115520163704150
2 BioMed Research International
the present study we determine the incidence of ES120573L-andor AmpC cephalosporinase-producing Escherichia coliisolates in human fecal flora and investigated the genesencoding the corresponding enzymes
2 Materials and Methods
21 Bacterial Identification Fifty different E coli isolates wereisolated from 50 stool samples of different inpatients carriersunder nonoutbreak conditions at a hospital in Riyadh SaudiArabia from April 2014 to June 2014 Briefly fresh stoolspecimens were aseptically collected and transported to themicrobiology laboratory Stool samples were suspended insterile phosphate-buffered saline pH 7 A 100120583L volume wasdirectly inoculated onto blood agar andEosinMethyleneBlueagar (Oxoid Microbiology Products Hampshire UK) After48 h incubation at 37∘C the isolated organisms were identi-fied by conventional procedures and automated identificationsystems with the API20E identification kit (bioMerieuxMarcy lrsquoEtoile France)These isolates were preserved in brainheart infusion broth containing 20 glycerol at minus70∘C
22 Phenotypic Detection of ES120573L The isolates showingreduced susceptibility to ceftazidime (CAZ) cefotaxime(CTX) or aztreonam (ATM) (minimum inhibitory concen-tration (MIC) ge 1 120583gmL or zone diameter le 22mm) wereselected for screening of ES120573L production (Clinical and Lab-oratory Standards Institute (CLSI) 2014) E-test ES120573L stripswere used in accordance with the manufacturerrsquos instruc-tions to evaluate ES120573L production The CAZceftazidime+ clavulanate- (CAZCAL-) ES120573L E-test strip was used todetect ES120573L production The test is considered positive ifthe ratio of MIC of CAZCAL is ge8 To inhibit AmpC120573-lactamase the CAZCAL-ES120573L E-test was carried outon cloxacillin Mueller-Hinton agar and the results wereinterpreted in a similar manner
23 Phenotypic Detection of AmpC The isolates showingreduced susceptibility to cefoxitin (FOX) or cefotetan (CTT)(zone diameter of 18 or 16mm resp) were selected forscreening of AmpC enzyme production [9] The phenotypicdetection test consists of a strip containing CTT on one endand CTT-cloxacillin (CTTCXT) on the other end Ratios oftheMICs of CTTCXT ge 8 are considered to indicate positiveAmpC 120573-lactamase production
24 Susceptibility Testing MICs for the isolates showing aphenotype of producing ES120573L and AmpC activities weredetermined by using E-test strips (bioMerieux MarcylrsquoEtoile France) Interpretation was based on the Clinical andLaboratory Standards Institute (CLSI) criteria [9]Escherichiacoli ATCC 25922 strains were used as reference strainsThe following antibiotics were tested piperacillin (PIP)piperacillintazobactam (TZP) CAZ CAZCAL CTXcefepime (FEP) ATM FOX CTT CTTCXT imipenem(IMI) gentamicin (GM) amikacin (AK) ciprofloxacin (CI)colistin (COL) tigecycline (TGC) and fosfomycin (FOS)
25 Screening for the Presence of 120573-Lactamase Genes Theisolate was cultured in 2mL of Tryptic Soy Broth (DifcoFranklin Lakes NJ USA) A 200120583L volume of overnightculture was heated at 99∘C in a heat block for 10minThe obtained DNA was used in polymerase chain reaction(PCR) assays on a Techne FlexigeneThermal Cycler (TechneDuxford Cambridge UK) Positive and negative controlswere included in all PCR assays All PCR products wereanalyzed on 08 agarose gels (incorporated with 05mgLethidium bromide) and then visualized under UV light(Pharmacia LKB Biotechnology AB Gothenburg Sweden)and photographed using a documentation system (CE DP-CF-011C European Union)
The PCR primers used are listed in Table 1 The primerswere used to search for class A 120573-lactamase genes (119887119897119886TEM119887119897119886SHV 119887119897119886OXA-1 and 119887119897119886CTX-M families) and class C 120573-lactamase genes (119887119897119886CMY 119887119897119886MOX 119887119897119886FOX 119887119897119886DHA 119887119897119886ACC119887119897119886ACT 119887119897119886MIR 119887119897119886EBC 119887119897119886CIT and 119887119897119886BIL) PCR assays wereconducted as previously described [8]
26 Sequencing of 120573-Lactamase Genes Purification of PCRamplicons was performed using a PCR purification kit(Qiagen Hilden Germany) PCR products of bla genes weresequenced on both strands using PCR primers to determinetheir molecular types DNA sequences were analyzed usingthe ABI 3100 Genetic Analyzer (Applied Biosystems FosterCity CA USA) according to the manufacturerrsquos recommen-dations
3 Results
31 Bacterial Identification Fifty fecal E coli samples wereisolated randomly from hospitalized patients in RiyadhSaudi Arabia The patients were treated for noninfectiousdiseases under nonoutbreak conditions Escherichia coli iso-lates were identified manually and according to the API20Eidentification kit (bioMerieux Marcy lrsquoEtoile France)
32 Characterization of 119887119897119886ES120573L and 119887119897119886AmpC Thirteen of the50 E coli isolates which showed reduced susceptibility toCAZ CTX or ATM (MIC ge 1 120583gmL or inhibition zonele 22mm) were selected for screening of ES120573L and AmpCenzyme production using CAZCAL-ES120573L and CTTCXT-AmpC E-test strips Thirteen isolates were positive for ES120573Land two ES120573L-positive isolates producedAmpC120573-lactamase
PCRwas used to detect 119887119897119886ES120573L genes andAmpC plasmid-mediated genes in ES120573L- and AmpC-positive E coli isolates(119899 = 13) The results of PCR and DNA sequencing of blagenes are shown in Table 2
Eleven of 13 ES120573L-producing E coli isolates were found tocontain CTX-M-15 while two isolates harbored 119887119897119886CTX-M-14CMY-2-positive isolates (119899 = 2) were concomitant withCTX-M-15 All ES120573L-producing E coli isolates (119899 = 13)were positive for 119887119897119886TEM-1 while eight (615) isolates carried119887119897119886OXA-1 In contrast three (23) isolates were found tocontain 119887119897119886SHV-1
BioMed Research International 3
Table1Prim
ersu
sedfora
mplificatio
nof
thetested120573-la
ctam
aseg
enes
(Dallenn
eetal2010
[8])
PCRtype
Target
Prim
erSequ
ence
ofprim
ers(51015840-31015840
)Amplified
prod
ucts
(bp)
Multip
lexIT
EMSHV
and
OXA-
1-like
TEM
MultiT
SO-T
for
CATT
TCCG
TGTC
GCC
CTTA
TTC
800
MultiT
SO-T
rev
CGTT
CATC
CATA
GTT
GCC
TGAC
SHV
MultiT
SO-S
for
AGCC
GCT
TGAG
CAAAT
TAAAC
713
MultiT
SO-S
rev
ATCC
CGCA
GAT
AAAT
CACC
AC
OXA-
1MultiT
SO-O
for
GGCA
CCAG
ATTC
AAC
TTTC
AAG
564
MultiT
SO-O
rev
GAC
CCCA
AGTT
TCCT
GTA
AGTG
Multip
lexIICT
X-M
grou
p1grou
p2andgrou
p9
CTX-
Mgrou
p1
MultiC
TXMGp1
for
TTAG
GAART
GTG
CCGCT
GYA
688
MultiC
TXMGp1-2
rev
CGAT
ATCG
TTGGTG
GTR
CCAT
CTX-
Mgrou
p2
MultiC
TXMGp2
for
CGTT
AAC
GGCA
CGAT
GAC
404
MultiC
TXMGp1-2
rev
CGAT
ATCG
TTGGTG
GTR
CCAT
CTX-
Mgrou
p9
MultiC
TXMGp9
for
TCAAG
CCTG
CCGAT
CTGGT
561
MultiC
TXMGp9
rev
TGAT
TCTC
GCC
GCT
GAAG
CTX-
Mgrou
p825
CTX-
Mgrou
p825
CTX-
Mg825for
AAC
RCRC
AGAC
GCT
CTAC
326
CTX-
Mg825rev
TCGAG
CCGGAASG
TGTY
AT
Multip
lexIIIA
CCFOX
MOX
DHAC
ITand
EBC
ACC-
1and
ACC-
2MultiC
aseA
CCfor
CACC
TCCA
GCG
ACTT
GTT
AC346
MultiC
aseA
CCrev
GTT
AGCC
AGCA
TCAC
GAT
CC
FOX-
1toFO
X-5
MultiC
aseFOX
for
CTAC
AGTG
CGGGTG
GTT
T162
MultiC
aseFOX
rev
CTAT
TTGCG
GCC
AGGTG
AMOX-
1MOX-
2CM
Y-1CM
Y-8to
CMY-11
andCM
Y-19
MultiC
aseM
OX
for
GCA
ACAAC
GAC
AAT
CCAT
CCT
895
MultiC
aseM
OX
rev
GGGAT
AGGCG
TAAC
TCTC
CCAA
DHA-
1and
DHA-
2MultiC
aseD
HA
for
TGAT
GGCA
CAGCA
GGAT
ATTC
997
MultiC
aseD
HA
rev
GCT
TTGAC
TCTT
TCGGTA
TTCG
LAT-1toLA
T-3BIL-1CM
Y-2to
CMY-7
CMY-12
toCM
Y-18and
CMY-21
toCM
Y-23
MultiC
aseC
ITfor
CGAAG
AGGCA
ATGAC
CAGAC
538
MultiC
aseC
ITrev
ACGGAC
AGGGTT
AGGAT
AGY
ACT-1and
MIR-1
MultiC
aseEBC
for
CGGTA
AAG
CCGAT
GTT
GCG
683
4 BioMed Research International
Table2Antim
icrobialsusceptib
ilityprofi
leso
fES120573
L-andAmpC
-produ
cing
Ecoliiso
latesfrom
fecalsam
ples
andassociated
resistancep
atterns
Isolates
number
119864-te
stMIC
(mgL)
Resistanceg
enes
PIP
TZP
CTX
CAZ
CAZCA
LFE
PAT
MFO
XCT
TCT
TCX
TIM
IGM
AK
CICO
LTG
CFO
SEC
1gt256lt1gt256
32322
48
0032
025
lt05lt05
006
05
21lt0016
025
0016
TEM-1+C
TX-M
-15+
OXA-
1EC
2gt256lt1gt256
161605
416
0065
025
lt05lt05
012
153
025lt0016
025lt0016
TEM-1+C
TX-M
-15+
+SHV-1
EC3
gt256
64gt256gt256
gt324
12gt256
0125
025
lt05lt05
025
643
4lt0016
0125lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
4gt256
8gt256
32320064
816
0032
006
lt05lt05
006
124
4lt0016
0125lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
5gt256
32gt256
161605
412
0125
0125lt05lt05
003
83
6lt0016
025lt0016
TEM-1+C
TX-M
-15+
SHV-1
EC6
gt256
8gt256gt256
gt324
192gt256
025
025
lt05lt05
006
192
48gt32lt0016
025lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
7gt256
8gt256gt256
gt324
128
192
025
025
lt05lt05
012
128
16gt32lt0016
025lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
8gt256lt1gt256gt256
gt324
96128
025
025
lt05lt05
006
9605
025lt0016
025
0125
TEM-1+C
TX-M
-14+
OXA-
1EC
9gt256
16gt256
44006
43
30032
0032lt05lt05
006
82
025lt0016
038lt0016
TEM-1+C
TX-M
-15
EC10
gt256
4gt256
660125
46
0032
0032lt05lt05
0125
161
05lt0016
025lt0016
TEM-1+C
TX-M
-15+
SHV-1
EC11
gt256lt1gt256
48gt321
824
0032
0032lt05lt05
025
232
1lt0016
025lt0016
TEM-1+C
TX-M
-14+
OXA-
1EC
12gt256gt256gt256gt256gt32gt4
48gt256
6432
3232
025gt256
192
075lt0016
025
0016
TEM-1+C
TX-M
-15+
CMY-2+
OXA-
1EC
13gt256
128gt256gt256gt32gt4
64gt256
6448
gt32gt32
025
128
4gt32lt0016
038
0016
TEM-1+C
TX-M
-15+
CMY-2
PIP
piperacillin
TZP
piperacillintazobactam
CAZ
cefta
zidime
CAZCA
Lcefta
zidimecefta
zidime+cla
vulanicacidC
TXc
efotaxim
eFE
Pcefepime
ATMa
ztreon
amF
OX
cefoxitin
CTT
cefotetan
CTTCX
Tcefotetancefotetan+clo
xacillin
IMIim
ipenem
GMgentamicinA
Kam
ikacinC
Iciprofl
oxacinC
OLcolistin
TGC
tigecyclin
eFO
Sfosfo
mycin
BioMed Research International 5
33 Antimicrobial Resistance Pattern of ES120573L and AmpCEnzyme-Positive Isolates The MICs of 17 antimicrobialagents were determined for E coli fecal isolates The resultsof the susceptibility pattern for ES120573L and AmpC enzyme-producing E coli isolates are illustrated in Table 2
4 Discussion
The human and animal alimentary tracts are vital reservoirsfor ES120573L- carbapenemases- and AmpC enzyme-producingEnterobacteriaceae Patient-to-patient transmission of resis-tant microorganisms may occur in hospitals [10 11] Theoveruse of antibiotics has recently been associated with theemergence of resistant intestinal bacteria particularly ES120573L-carbapenemases- and AmpC enzyme-producing Enterobac-teriaceae Numerous studies have demonstrated that expo-sure to 120573-lactam antibiotics is a risk factor for the selection ofmultidrug-resistant E coli [12] Therefore the current studyexamined the antimicrobial resistance patterns of fecal Ecoli isolates as well as the molecular basis for their 120573-lactamresistance mechanisms using phenotypic and genotypicmethods The present study included 50 patients admitted toa hospital in Riyadh Saudi Arabia from April 2014 to June2014 These patients were treated for noninfectious diseasesunder nonoutbreak conditions Fifty fecal stool specimenscollected from the 50 patients were cultured on blood agarand EMB agar as described in Materials and Methods Fiftysuspected E coli isolates were selected and the isolates wereidentified by conventional procedures and using the API20Eidentification kit All isolates were identified as E coli
Production of 120573-lactamases is the main mechanism of120573-lactam resistance in Gram-negative bacteria including Ecoli [6 7] Several 120573-lactamases (ES120573Ls AmpC enzymes andcarbapenemases) have been previously reported in fecal Ecoli isolates [13ndash15]
In the present study 100 of 50 E coli isolates were foundto be sensitive to imipenem and 13 (26) of 50 isolates wereresistant or showed reduced susceptibility to CAZ CTX-M or ATM The carbapenem susceptibility results indicatedthat our isolates did not harbor carbapenemase while 26(1350) of the E coli isolates harbored ES120573L andor AmpC 120573-lactamase Two of 13 isolates exhibited reduced susceptibilityto cephamycins (FOX and CTT)
Phenotypic screening for the presence of different typesof 120573-lactamases was conducted using E coli isolatesThirteenE coli isolates were selected for screening of ES120573L andAmpC 120573-lactamase production using CAZCAL-ES120573L andCTTCXT-AmpC E-test strips were used to detect ES120573Lproduction Using phenotypic detection methods all isolateswere found to produce ES120573L while two isolates phenotyp-ically produced AmpC enzyme A battery of PCR assayswas conducted to detect 119887119897119886ES120573L genes and AmpC plasmid-mediated genes in the 13 E coli isolates Therefore class Aand class C 120573-lactamase genes were tested The PCR-purifiedproduct was subjected to DNA sequencing to identify thegene variants The results of molecular characterization ofbla genes are shown in Table 2 PCR amplification andDNA sequencing analyses of the PCR products showed
that all isolates possessed a CTX-M-type ES120573L and that119887119897119886CTX-M-15 was present in 1 isolate while two isolates con-tained 119887119897119886CTX-M-14 Other CTX-M families were not detectedThe gene encoding CMY-2 enzyme was detected in two Ecoli isolates CMY-2-positive isolates are concomitant withCTX-M-15 All E coli isolates (119899 = 13) were positivefor 119887119897119886TEM-1 while 615 and 23 of the isolates contained119887119897119886OXA-1 and 119887119897119886SHV-1 respectivelyThe increase in expressionof the AmpC 120573-lactamases may mask the recognition ofES120573Ls [16] Therefore in the present study the genotypicmethods revealed that all 13 strains were ES120573L CTX-M-positive while phenotypic methods showed that 11 strainswere ES120573L-positive and two strains were AmpC enzyme-positive AmpC-producing strains producingCTX-M-15mayact as a dormant reservoir for ES120573Ls
Numerous studies have documented a remarkableincrease in intestinal colonization rates with ES120573L- andAmpC enzyme-producing Enterobacteriaceae in manycountries [2 10 11 14ndash17]The prevalence of ES120573L-producingE coli fecal isolates varies widely from country to countryfrom region to region and at different time periods Ahigh incidence of fecal carriage rate of ES120573L-producing Ecoli has been observed in Asia Africa and South America[13 14 18ndash21] while a significantly lower prevalence ofES120573L-producing E coli fecal isolates was reported in mostEuropean countries [22 23] In Argentina the rate of fecalcarriage of Enterobacteriaceae-resistant strains to third-generation cephalosporins was 268 [20] Villar et al [20]reported that 2022 and 67 of fecal strains were colonizedby ES120573L- and AmpC enzyme-producing Enterobacteriaceae[20] In Egypt Al-Agamy et al reported that 226 and322 of hospitalized patients were colonized by ES120573L-and AmpC-positive E coli respectively The 119887119897119886CTX-M-likegene was the predominant ES120573L gene detected in 714of ES120573L-producing E coli isolates [21] In a recent studyin Egypt Bassyouni et al reported that 21 and 3 ofpatients were colonized by ES120573L- and AmpC-producing Ecoli respectively They also found that 119887119897119886SHV gene was thepredominant ES120573L gene detected in 818 of the resistant Ecoli isolates [13] In Korea 203 of fecal Enterobacteriaceaemembers were ES120573Ls [19] In India the prevalence ofES120573L-positive E coli isolates was 19 in healthy volunteersfrom the community [14] In Libya 134 and 67 of E coliisolates were ES120573Ls- and AmpC-positive respectively [18]In a previous study in Saudi Arabia 177 of strains werefound to be ES120573L-positive [24] A high (261) prevalencewas detected in inpatients followed by outpatients (154)and the lowest prevalence rate (131) was detected in healthyindividuals [24] In the present study the prevalence of ES120573L-producing E coli was 26 Despite differences in the dateand region of isolation the prevalence of fecal carriage rateof ES120573L in the present study (26) was in agreement withthe prevalence (261) reported by Kader et al In contrastthe prevalence rate of ES120573L-producing Enterobacteriaceaewas 29 among healthy Swedish children Escherichia colicontaining CTX-M 120573-lactamase predominated and onlyone E coli isolate harbored genes encoding for CMY [22]The carriage rate of ES120573L- and AmpC enzyme-producing Ecoli was 357 and 238 among 84 Danish army recruits
6 BioMed Research International
respectively 119887119897119886CTX-M-14 gene was the predominant ES120573Lgene detected in three (100) ES120573L-producing E coliisolates while 119887119897119886CMY-2 was detected in two AmpC enzyme-producing E coli isolates [23] In Spain the prevalence ofES120573L and AmpC enzyme carriers was 506 and 059respectively [25] 119887119897119886CTX-M genes were the ES120573L dominatinggenes (9615) and CTX-M-14 was the most prevalent gene(50) followed by CTX-M-15 (40) CMY-2 was the mostprevalent gene (8125) followed by DHA-1 (1875) [25]
MICs were determined for ES120573L- and AmpC enzyme-producing E coli (119899 = 13) isolates The results of MIC areshown in Table 2
In conclusion a high incidence of carriage of ES120573L-positive E coli fecal isolates among hospitalized patients inRiyadh was detected reaching 26 with 119887119897119886CTX-M-15 (846)being the most predominant gene The emergence of fecalcarriage of CMY-2-producing E coli among hospitalizedpatients has been reported to be 4These outcomes empha-size the importance of the intestinal tract as a reservoir forES120573L- and AmpC enzyme-producing E coli which may leadto nosocomial infection The admission of colonized fecalcarriers of ES120573L- and AmpC-positive E coli to the medicalsetting increases the possibility of other patients acquiringinfection in the same hospital Our results emphasize thenecessity for continuous surveillance in hospitals to detectthe ES120573L- AmpC enzyme- and carbapenemase-producingstrains andmultidrug strains as well applying effective strate-gies for antimicrobial therapy and infection control measuresto decrease the abuse and misuse of antimicrobial agentsagainst resistant strains and to prevent their spread
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
The authors extend their appreciation to the Deanship ofScientific Research at King Saud University for funding thework through the research group Project no RGP-038
References
[1] A Bhargava K Hayakawa E Silverman et al ldquoRisk factors forcolonization due to carbapenem-resistant enterobacteriaceaeamong patients exposed to long-term acute care and acute carefacilitiesrdquo Infection Control and Hospital Epidemiology vol 35no 4 pp 398ndash405 2014
[2] P-L Woerther C Burdet E Chachaty and A AndremontldquoTrends in human fecal carriage of extended-spectrum 120573-lactamases in the community toward the globalization of CTX-Mrdquo Clinical Microbiology Reviews vol 26 no 4 pp 744ndash7582013
[3] N Karah B Haldorsen N O Hermansen et al ldquoEmergenceof OXA-carbapenemase- and 16S rRNA methylase-producinginternational clones of Acinetobacter baumannii in NorwayrdquoJournal of Medical Microbiology vol 60 no 4 pp 515ndash521 2011
[4] S Yezli A M Shibl and Z A Memish ldquoThemolecular basis of120573-lactamase production in Gram-negative bacteria from Saudi
Arabiardquo Journal of Medical Microbiology vol 64 no 2 pp 127ndash136 2015
[5] M Guzman-Blanco J A Labarca M V Villegas and EGotuzzo ldquoExtended spectrum 120573-lactamase producers amongnosocomial Enterobacteriaceae in Latin Americardquo BrazilianJournal of Infectious Diseases vol 18 no 4 pp 421ndash433 2014
[6] Y Pfeifer A Cullik andWWitte ldquoResistance to cephalosporinsand carbapenems in Gram-negative bacterial pathogensrdquo Inter-national Journal ofMedicalMicrobiology vol 300 no 6 pp 371ndash379 2010
[7] R Canton and T M Coque ldquoThe CTX-M beta-lactamasepandemicrdquo Current Opinion in Microbiology vol 9 no 5 pp466ndash475 2006
[8] C Dallenne A da Costa D Decre C Favier and G ArletldquoDevelopment of a set of multiplex PCR assays for the detectionof genes encoding important 120573-lactamases in Enterobacteri-aceaerdquo Journal of Antimicrobial Chemotherapy vol 65 no 3 pp490ndash495 2010
[9] Clinical and Laboratory Standards Institute (CLSI) ldquoPer-formance standards for antimicrobial susceptibility testingTwenty-fourth informational supplement performance stan-dards for antimicrobial susceptibility testingrdquoDocumentM100-24 Clinical and Laboratory Standards Institute (CLSI) WaynePa USA 2014
[10] J A Severin E S Lestari W Kloezen et al ldquoFaecal carriageof extended-spectrum 120573-lactamase-producing Enterobacteri-aceae among humans in Java Indonesia in 2001-2002rdquo TropicalMedicine and International Health vol 17 no 4 pp 455ndash4612012
[11] B Li J-Y Sun Q-Z Liu L-Z Han X-H Huang and Y-X Ni ldquoHigh prevalence of CTX-M 120573-lactamases in faecalEscherichia coli strains from healthy humans in Fuzhou ChinardquoScandinavian Journal of Infectious Diseases vol 43 no 3 pp170ndash174 2011
[12] G V Sanchez R N Master J A Karlowsky and J M BordonldquoIn vitro antimicrobial resistance of urinary Escherichia coliisolates among US outpatients from 2000 to 2010rdquo Antimicro-bial Agents andChemotherapy vol 56 no 4 pp 2181ndash2183 2012
[13] R H Bassyouni S N Gaber and A AWegdan ldquoFecal carriageof extended-spectrum 120573-lactamase- and AmpC- producingEscherichia coli among healthcare workersrdquo Journal of Infectionin Developing Countries vol 9 no 3 pp 304ndash308 2015
[14] D Mathai V A Kumar B Paul et al ldquoFecal carriage ratesof extended-spectrum 120573-lactamase-producing Escherichia coliamong antibiotic naive healthy human volunteersrdquo MicrobialDrug Resistance vol 21 no 1 pp 59ndash64 2015
[15] S B Joslashrgensen Oslash Samuelsen A Sundsfjord et al ldquoHighprevalence of faecal carriage of ESBL-producing Enterobacteri-aceae in Norwegian patients with gastroenteritisrdquo ScandinavianJournal of Infectious Diseases vol 46 no 6 pp 462ndash465 2014
[16] N O Yilmaz N Agus E Bozcal O Oner and A UzelldquoDetection of plasmid-mediated AmpC 120573-lactamase in E coliand K pneumoniaerdquo Indian Journal of Medical Microbiologyvol 31 no 1 pp 53ndash59 2013
[17] T M H Bui I Hirai S Ueda et al ldquoCarriage of Escherichiacoli producing CTX-M-type extended-spectrum 120573-lactamasein healthy Vietnamese individualsrdquo Antimicrobial Agents andChemotherapy vol 59 no 10 pp 6611ndash6614 2015
[18] S F Ahmed M M M Ali Z K Mohamed T A Moussa andJ D Klena ldquoFecal carriage of extended-spectrum 120573-lactamasesand AmpC-producing Escherichia coli in a Libyan communityrdquo
BioMed Research International 7
Annals of Clinical Microbiology and Antimicrobials vol 13article 22 2014
[19] Y J Ko H-W Moon M Hur C-M Park S E Cho andY-M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[20] H E Villar M N Baserni and M B Jugo ldquoFaecal carriage ofESBL-producing enterobacteriaceae and carbapenem-resistantGram-negative bacilli in community settingsrdquo Journal of Infec-tion in Developing Countries vol 7 no 8 pp 630ndash634 2013
[21] M H Al-Agamy M S Ali M M Salem and T R MohamedldquoFaecal colonization by extended-spectrum beta-lactamase-producing Escherichia coli from hospitalized patientsrdquo NewEgyptian Journal of Microbiology vol 19 pp 285ndash314 2008
[22] J Kaarme Y Molin B Olsen and A Melhus ldquoPrevalence ofextended-spectrum beta-lactamase-producing Enterobacteri-aceae in healthy Swedish preschool childrenrdquo Acta Paediatricavol 102 no 6 pp 655ndash660 2013
[23] A M Hammerum C H Lester L Jakobsen and L J PorsboldquoFaecal carriage of extended-spectrum 120573-lactamase-producingand AmpC 120573-lactamase-producing bacteria among Danisharmy recruitsrdquo Clinical Microbiology and Infection vol 17 no4 pp 566ndash568 2011
[24] A A Kader A Kumar and K A Kamath ldquoFecal car-riage of extended-spectrum120573-lactamase-producingEscherichiacoli and Klebsiella pneumoniae in patients and asymptomatichealthy individualsrdquo Infection Control and Hospital Epidemiol-ogy vol 28 no 9 pp 1114ndash1116 2007
[25] A Garrido C Seral M J Gude et al ldquoCharacterization ofplasmid-mediated 120573-lactamases in fecal colonizing patients inthe hospital and community setting in Spainrdquo Microbial DrugResistance vol 20 no 4 pp 301ndash304 2014
Submit your manuscripts athttpwwwhindawicom
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International Journal of
Microbiology
2 BioMed Research International
the present study we determine the incidence of ES120573L-andor AmpC cephalosporinase-producing Escherichia coliisolates in human fecal flora and investigated the genesencoding the corresponding enzymes
2 Materials and Methods
21 Bacterial Identification Fifty different E coli isolates wereisolated from 50 stool samples of different inpatients carriersunder nonoutbreak conditions at a hospital in Riyadh SaudiArabia from April 2014 to June 2014 Briefly fresh stoolspecimens were aseptically collected and transported to themicrobiology laboratory Stool samples were suspended insterile phosphate-buffered saline pH 7 A 100120583L volume wasdirectly inoculated onto blood agar andEosinMethyleneBlueagar (Oxoid Microbiology Products Hampshire UK) After48 h incubation at 37∘C the isolated organisms were identi-fied by conventional procedures and automated identificationsystems with the API20E identification kit (bioMerieuxMarcy lrsquoEtoile France)These isolates were preserved in brainheart infusion broth containing 20 glycerol at minus70∘C
22 Phenotypic Detection of ES120573L The isolates showingreduced susceptibility to ceftazidime (CAZ) cefotaxime(CTX) or aztreonam (ATM) (minimum inhibitory concen-tration (MIC) ge 1 120583gmL or zone diameter le 22mm) wereselected for screening of ES120573L production (Clinical and Lab-oratory Standards Institute (CLSI) 2014) E-test ES120573L stripswere used in accordance with the manufacturerrsquos instruc-tions to evaluate ES120573L production The CAZceftazidime+ clavulanate- (CAZCAL-) ES120573L E-test strip was used todetect ES120573L production The test is considered positive ifthe ratio of MIC of CAZCAL is ge8 To inhibit AmpC120573-lactamase the CAZCAL-ES120573L E-test was carried outon cloxacillin Mueller-Hinton agar and the results wereinterpreted in a similar manner
23 Phenotypic Detection of AmpC The isolates showingreduced susceptibility to cefoxitin (FOX) or cefotetan (CTT)(zone diameter of 18 or 16mm resp) were selected forscreening of AmpC enzyme production [9] The phenotypicdetection test consists of a strip containing CTT on one endand CTT-cloxacillin (CTTCXT) on the other end Ratios oftheMICs of CTTCXT ge 8 are considered to indicate positiveAmpC 120573-lactamase production
24 Susceptibility Testing MICs for the isolates showing aphenotype of producing ES120573L and AmpC activities weredetermined by using E-test strips (bioMerieux MarcylrsquoEtoile France) Interpretation was based on the Clinical andLaboratory Standards Institute (CLSI) criteria [9]Escherichiacoli ATCC 25922 strains were used as reference strainsThe following antibiotics were tested piperacillin (PIP)piperacillintazobactam (TZP) CAZ CAZCAL CTXcefepime (FEP) ATM FOX CTT CTTCXT imipenem(IMI) gentamicin (GM) amikacin (AK) ciprofloxacin (CI)colistin (COL) tigecycline (TGC) and fosfomycin (FOS)
25 Screening for the Presence of 120573-Lactamase Genes Theisolate was cultured in 2mL of Tryptic Soy Broth (DifcoFranklin Lakes NJ USA) A 200120583L volume of overnightculture was heated at 99∘C in a heat block for 10minThe obtained DNA was used in polymerase chain reaction(PCR) assays on a Techne FlexigeneThermal Cycler (TechneDuxford Cambridge UK) Positive and negative controlswere included in all PCR assays All PCR products wereanalyzed on 08 agarose gels (incorporated with 05mgLethidium bromide) and then visualized under UV light(Pharmacia LKB Biotechnology AB Gothenburg Sweden)and photographed using a documentation system (CE DP-CF-011C European Union)
The PCR primers used are listed in Table 1 The primerswere used to search for class A 120573-lactamase genes (119887119897119886TEM119887119897119886SHV 119887119897119886OXA-1 and 119887119897119886CTX-M families) and class C 120573-lactamase genes (119887119897119886CMY 119887119897119886MOX 119887119897119886FOX 119887119897119886DHA 119887119897119886ACC119887119897119886ACT 119887119897119886MIR 119887119897119886EBC 119887119897119886CIT and 119887119897119886BIL) PCR assays wereconducted as previously described [8]
26 Sequencing of 120573-Lactamase Genes Purification of PCRamplicons was performed using a PCR purification kit(Qiagen Hilden Germany) PCR products of bla genes weresequenced on both strands using PCR primers to determinetheir molecular types DNA sequences were analyzed usingthe ABI 3100 Genetic Analyzer (Applied Biosystems FosterCity CA USA) according to the manufacturerrsquos recommen-dations
3 Results
31 Bacterial Identification Fifty fecal E coli samples wereisolated randomly from hospitalized patients in RiyadhSaudi Arabia The patients were treated for noninfectiousdiseases under nonoutbreak conditions Escherichia coli iso-lates were identified manually and according to the API20Eidentification kit (bioMerieux Marcy lrsquoEtoile France)
32 Characterization of 119887119897119886ES120573L and 119887119897119886AmpC Thirteen of the50 E coli isolates which showed reduced susceptibility toCAZ CTX or ATM (MIC ge 1 120583gmL or inhibition zonele 22mm) were selected for screening of ES120573L and AmpCenzyme production using CAZCAL-ES120573L and CTTCXT-AmpC E-test strips Thirteen isolates were positive for ES120573Land two ES120573L-positive isolates producedAmpC120573-lactamase
PCRwas used to detect 119887119897119886ES120573L genes andAmpC plasmid-mediated genes in ES120573L- and AmpC-positive E coli isolates(119899 = 13) The results of PCR and DNA sequencing of blagenes are shown in Table 2
Eleven of 13 ES120573L-producing E coli isolates were found tocontain CTX-M-15 while two isolates harbored 119887119897119886CTX-M-14CMY-2-positive isolates (119899 = 2) were concomitant withCTX-M-15 All ES120573L-producing E coli isolates (119899 = 13)were positive for 119887119897119886TEM-1 while eight (615) isolates carried119887119897119886OXA-1 In contrast three (23) isolates were found tocontain 119887119897119886SHV-1
BioMed Research International 3
Table1Prim
ersu
sedfora
mplificatio
nof
thetested120573-la
ctam
aseg
enes
(Dallenn
eetal2010
[8])
PCRtype
Target
Prim
erSequ
ence
ofprim
ers(51015840-31015840
)Amplified
prod
ucts
(bp)
Multip
lexIT
EMSHV
and
OXA-
1-like
TEM
MultiT
SO-T
for
CATT
TCCG
TGTC
GCC
CTTA
TTC
800
MultiT
SO-T
rev
CGTT
CATC
CATA
GTT
GCC
TGAC
SHV
MultiT
SO-S
for
AGCC
GCT
TGAG
CAAAT
TAAAC
713
MultiT
SO-S
rev
ATCC
CGCA
GAT
AAAT
CACC
AC
OXA-
1MultiT
SO-O
for
GGCA
CCAG
ATTC
AAC
TTTC
AAG
564
MultiT
SO-O
rev
GAC
CCCA
AGTT
TCCT
GTA
AGTG
Multip
lexIICT
X-M
grou
p1grou
p2andgrou
p9
CTX-
Mgrou
p1
MultiC
TXMGp1
for
TTAG
GAART
GTG
CCGCT
GYA
688
MultiC
TXMGp1-2
rev
CGAT
ATCG
TTGGTG
GTR
CCAT
CTX-
Mgrou
p2
MultiC
TXMGp2
for
CGTT
AAC
GGCA
CGAT
GAC
404
MultiC
TXMGp1-2
rev
CGAT
ATCG
TTGGTG
GTR
CCAT
CTX-
Mgrou
p9
MultiC
TXMGp9
for
TCAAG
CCTG
CCGAT
CTGGT
561
MultiC
TXMGp9
rev
TGAT
TCTC
GCC
GCT
GAAG
CTX-
Mgrou
p825
CTX-
Mgrou
p825
CTX-
Mg825for
AAC
RCRC
AGAC
GCT
CTAC
326
CTX-
Mg825rev
TCGAG
CCGGAASG
TGTY
AT
Multip
lexIIIA
CCFOX
MOX
DHAC
ITand
EBC
ACC-
1and
ACC-
2MultiC
aseA
CCfor
CACC
TCCA
GCG
ACTT
GTT
AC346
MultiC
aseA
CCrev
GTT
AGCC
AGCA
TCAC
GAT
CC
FOX-
1toFO
X-5
MultiC
aseFOX
for
CTAC
AGTG
CGGGTG
GTT
T162
MultiC
aseFOX
rev
CTAT
TTGCG
GCC
AGGTG
AMOX-
1MOX-
2CM
Y-1CM
Y-8to
CMY-11
andCM
Y-19
MultiC
aseM
OX
for
GCA
ACAAC
GAC
AAT
CCAT
CCT
895
MultiC
aseM
OX
rev
GGGAT
AGGCG
TAAC
TCTC
CCAA
DHA-
1and
DHA-
2MultiC
aseD
HA
for
TGAT
GGCA
CAGCA
GGAT
ATTC
997
MultiC
aseD
HA
rev
GCT
TTGAC
TCTT
TCGGTA
TTCG
LAT-1toLA
T-3BIL-1CM
Y-2to
CMY-7
CMY-12
toCM
Y-18and
CMY-21
toCM
Y-23
MultiC
aseC
ITfor
CGAAG
AGGCA
ATGAC
CAGAC
538
MultiC
aseC
ITrev
ACGGAC
AGGGTT
AGGAT
AGY
ACT-1and
MIR-1
MultiC
aseEBC
for
CGGTA
AAG
CCGAT
GTT
GCG
683
4 BioMed Research International
Table2Antim
icrobialsusceptib
ilityprofi
leso
fES120573
L-andAmpC
-produ
cing
Ecoliiso
latesfrom
fecalsam
ples
andassociated
resistancep
atterns
Isolates
number
119864-te
stMIC
(mgL)
Resistanceg
enes
PIP
TZP
CTX
CAZ
CAZCA
LFE
PAT
MFO
XCT
TCT
TCX
TIM
IGM
AK
CICO
LTG
CFO
SEC
1gt256lt1gt256
32322
48
0032
025
lt05lt05
006
05
21lt0016
025
0016
TEM-1+C
TX-M
-15+
OXA-
1EC
2gt256lt1gt256
161605
416
0065
025
lt05lt05
012
153
025lt0016
025lt0016
TEM-1+C
TX-M
-15+
+SHV-1
EC3
gt256
64gt256gt256
gt324
12gt256
0125
025
lt05lt05
025
643
4lt0016
0125lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
4gt256
8gt256
32320064
816
0032
006
lt05lt05
006
124
4lt0016
0125lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
5gt256
32gt256
161605
412
0125
0125lt05lt05
003
83
6lt0016
025lt0016
TEM-1+C
TX-M
-15+
SHV-1
EC6
gt256
8gt256gt256
gt324
192gt256
025
025
lt05lt05
006
192
48gt32lt0016
025lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
7gt256
8gt256gt256
gt324
128
192
025
025
lt05lt05
012
128
16gt32lt0016
025lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
8gt256lt1gt256gt256
gt324
96128
025
025
lt05lt05
006
9605
025lt0016
025
0125
TEM-1+C
TX-M
-14+
OXA-
1EC
9gt256
16gt256
44006
43
30032
0032lt05lt05
006
82
025lt0016
038lt0016
TEM-1+C
TX-M
-15
EC10
gt256
4gt256
660125
46
0032
0032lt05lt05
0125
161
05lt0016
025lt0016
TEM-1+C
TX-M
-15+
SHV-1
EC11
gt256lt1gt256
48gt321
824
0032
0032lt05lt05
025
232
1lt0016
025lt0016
TEM-1+C
TX-M
-14+
OXA-
1EC
12gt256gt256gt256gt256gt32gt4
48gt256
6432
3232
025gt256
192
075lt0016
025
0016
TEM-1+C
TX-M
-15+
CMY-2+
OXA-
1EC
13gt256
128gt256gt256gt32gt4
64gt256
6448
gt32gt32
025
128
4gt32lt0016
038
0016
TEM-1+C
TX-M
-15+
CMY-2
PIP
piperacillin
TZP
piperacillintazobactam
CAZ
cefta
zidime
CAZCA
Lcefta
zidimecefta
zidime+cla
vulanicacidC
TXc
efotaxim
eFE
Pcefepime
ATMa
ztreon
amF
OX
cefoxitin
CTT
cefotetan
CTTCX
Tcefotetancefotetan+clo
xacillin
IMIim
ipenem
GMgentamicinA
Kam
ikacinC
Iciprofl
oxacinC
OLcolistin
TGC
tigecyclin
eFO
Sfosfo
mycin
BioMed Research International 5
33 Antimicrobial Resistance Pattern of ES120573L and AmpCEnzyme-Positive Isolates The MICs of 17 antimicrobialagents were determined for E coli fecal isolates The resultsof the susceptibility pattern for ES120573L and AmpC enzyme-producing E coli isolates are illustrated in Table 2
4 Discussion
The human and animal alimentary tracts are vital reservoirsfor ES120573L- carbapenemases- and AmpC enzyme-producingEnterobacteriaceae Patient-to-patient transmission of resis-tant microorganisms may occur in hospitals [10 11] Theoveruse of antibiotics has recently been associated with theemergence of resistant intestinal bacteria particularly ES120573L-carbapenemases- and AmpC enzyme-producing Enterobac-teriaceae Numerous studies have demonstrated that expo-sure to 120573-lactam antibiotics is a risk factor for the selection ofmultidrug-resistant E coli [12] Therefore the current studyexamined the antimicrobial resistance patterns of fecal Ecoli isolates as well as the molecular basis for their 120573-lactamresistance mechanisms using phenotypic and genotypicmethods The present study included 50 patients admitted toa hospital in Riyadh Saudi Arabia from April 2014 to June2014 These patients were treated for noninfectious diseasesunder nonoutbreak conditions Fifty fecal stool specimenscollected from the 50 patients were cultured on blood agarand EMB agar as described in Materials and Methods Fiftysuspected E coli isolates were selected and the isolates wereidentified by conventional procedures and using the API20Eidentification kit All isolates were identified as E coli
Production of 120573-lactamases is the main mechanism of120573-lactam resistance in Gram-negative bacteria including Ecoli [6 7] Several 120573-lactamases (ES120573Ls AmpC enzymes andcarbapenemases) have been previously reported in fecal Ecoli isolates [13ndash15]
In the present study 100 of 50 E coli isolates were foundto be sensitive to imipenem and 13 (26) of 50 isolates wereresistant or showed reduced susceptibility to CAZ CTX-M or ATM The carbapenem susceptibility results indicatedthat our isolates did not harbor carbapenemase while 26(1350) of the E coli isolates harbored ES120573L andor AmpC 120573-lactamase Two of 13 isolates exhibited reduced susceptibilityto cephamycins (FOX and CTT)
Phenotypic screening for the presence of different typesof 120573-lactamases was conducted using E coli isolatesThirteenE coli isolates were selected for screening of ES120573L andAmpC 120573-lactamase production using CAZCAL-ES120573L andCTTCXT-AmpC E-test strips were used to detect ES120573Lproduction Using phenotypic detection methods all isolateswere found to produce ES120573L while two isolates phenotyp-ically produced AmpC enzyme A battery of PCR assayswas conducted to detect 119887119897119886ES120573L genes and AmpC plasmid-mediated genes in the 13 E coli isolates Therefore class Aand class C 120573-lactamase genes were tested The PCR-purifiedproduct was subjected to DNA sequencing to identify thegene variants The results of molecular characterization ofbla genes are shown in Table 2 PCR amplification andDNA sequencing analyses of the PCR products showed
that all isolates possessed a CTX-M-type ES120573L and that119887119897119886CTX-M-15 was present in 1 isolate while two isolates con-tained 119887119897119886CTX-M-14 Other CTX-M families were not detectedThe gene encoding CMY-2 enzyme was detected in two Ecoli isolates CMY-2-positive isolates are concomitant withCTX-M-15 All E coli isolates (119899 = 13) were positivefor 119887119897119886TEM-1 while 615 and 23 of the isolates contained119887119897119886OXA-1 and 119887119897119886SHV-1 respectivelyThe increase in expressionof the AmpC 120573-lactamases may mask the recognition ofES120573Ls [16] Therefore in the present study the genotypicmethods revealed that all 13 strains were ES120573L CTX-M-positive while phenotypic methods showed that 11 strainswere ES120573L-positive and two strains were AmpC enzyme-positive AmpC-producing strains producingCTX-M-15mayact as a dormant reservoir for ES120573Ls
Numerous studies have documented a remarkableincrease in intestinal colonization rates with ES120573L- andAmpC enzyme-producing Enterobacteriaceae in manycountries [2 10 11 14ndash17]The prevalence of ES120573L-producingE coli fecal isolates varies widely from country to countryfrom region to region and at different time periods Ahigh incidence of fecal carriage rate of ES120573L-producing Ecoli has been observed in Asia Africa and South America[13 14 18ndash21] while a significantly lower prevalence ofES120573L-producing E coli fecal isolates was reported in mostEuropean countries [22 23] In Argentina the rate of fecalcarriage of Enterobacteriaceae-resistant strains to third-generation cephalosporins was 268 [20] Villar et al [20]reported that 2022 and 67 of fecal strains were colonizedby ES120573L- and AmpC enzyme-producing Enterobacteriaceae[20] In Egypt Al-Agamy et al reported that 226 and322 of hospitalized patients were colonized by ES120573L-and AmpC-positive E coli respectively The 119887119897119886CTX-M-likegene was the predominant ES120573L gene detected in 714of ES120573L-producing E coli isolates [21] In a recent studyin Egypt Bassyouni et al reported that 21 and 3 ofpatients were colonized by ES120573L- and AmpC-producing Ecoli respectively They also found that 119887119897119886SHV gene was thepredominant ES120573L gene detected in 818 of the resistant Ecoli isolates [13] In Korea 203 of fecal Enterobacteriaceaemembers were ES120573Ls [19] In India the prevalence ofES120573L-positive E coli isolates was 19 in healthy volunteersfrom the community [14] In Libya 134 and 67 of E coliisolates were ES120573Ls- and AmpC-positive respectively [18]In a previous study in Saudi Arabia 177 of strains werefound to be ES120573L-positive [24] A high (261) prevalencewas detected in inpatients followed by outpatients (154)and the lowest prevalence rate (131) was detected in healthyindividuals [24] In the present study the prevalence of ES120573L-producing E coli was 26 Despite differences in the dateand region of isolation the prevalence of fecal carriage rateof ES120573L in the present study (26) was in agreement withthe prevalence (261) reported by Kader et al In contrastthe prevalence rate of ES120573L-producing Enterobacteriaceaewas 29 among healthy Swedish children Escherichia colicontaining CTX-M 120573-lactamase predominated and onlyone E coli isolate harbored genes encoding for CMY [22]The carriage rate of ES120573L- and AmpC enzyme-producing Ecoli was 357 and 238 among 84 Danish army recruits
6 BioMed Research International
respectively 119887119897119886CTX-M-14 gene was the predominant ES120573Lgene detected in three (100) ES120573L-producing E coliisolates while 119887119897119886CMY-2 was detected in two AmpC enzyme-producing E coli isolates [23] In Spain the prevalence ofES120573L and AmpC enzyme carriers was 506 and 059respectively [25] 119887119897119886CTX-M genes were the ES120573L dominatinggenes (9615) and CTX-M-14 was the most prevalent gene(50) followed by CTX-M-15 (40) CMY-2 was the mostprevalent gene (8125) followed by DHA-1 (1875) [25]
MICs were determined for ES120573L- and AmpC enzyme-producing E coli (119899 = 13) isolates The results of MIC areshown in Table 2
In conclusion a high incidence of carriage of ES120573L-positive E coli fecal isolates among hospitalized patients inRiyadh was detected reaching 26 with 119887119897119886CTX-M-15 (846)being the most predominant gene The emergence of fecalcarriage of CMY-2-producing E coli among hospitalizedpatients has been reported to be 4These outcomes empha-size the importance of the intestinal tract as a reservoir forES120573L- and AmpC enzyme-producing E coli which may leadto nosocomial infection The admission of colonized fecalcarriers of ES120573L- and AmpC-positive E coli to the medicalsetting increases the possibility of other patients acquiringinfection in the same hospital Our results emphasize thenecessity for continuous surveillance in hospitals to detectthe ES120573L- AmpC enzyme- and carbapenemase-producingstrains andmultidrug strains as well applying effective strate-gies for antimicrobial therapy and infection control measuresto decrease the abuse and misuse of antimicrobial agentsagainst resistant strains and to prevent their spread
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
The authors extend their appreciation to the Deanship ofScientific Research at King Saud University for funding thework through the research group Project no RGP-038
References
[1] A Bhargava K Hayakawa E Silverman et al ldquoRisk factors forcolonization due to carbapenem-resistant enterobacteriaceaeamong patients exposed to long-term acute care and acute carefacilitiesrdquo Infection Control and Hospital Epidemiology vol 35no 4 pp 398ndash405 2014
[2] P-L Woerther C Burdet E Chachaty and A AndremontldquoTrends in human fecal carriage of extended-spectrum 120573-lactamases in the community toward the globalization of CTX-Mrdquo Clinical Microbiology Reviews vol 26 no 4 pp 744ndash7582013
[3] N Karah B Haldorsen N O Hermansen et al ldquoEmergenceof OXA-carbapenemase- and 16S rRNA methylase-producinginternational clones of Acinetobacter baumannii in NorwayrdquoJournal of Medical Microbiology vol 60 no 4 pp 515ndash521 2011
[4] S Yezli A M Shibl and Z A Memish ldquoThemolecular basis of120573-lactamase production in Gram-negative bacteria from Saudi
Arabiardquo Journal of Medical Microbiology vol 64 no 2 pp 127ndash136 2015
[5] M Guzman-Blanco J A Labarca M V Villegas and EGotuzzo ldquoExtended spectrum 120573-lactamase producers amongnosocomial Enterobacteriaceae in Latin Americardquo BrazilianJournal of Infectious Diseases vol 18 no 4 pp 421ndash433 2014
[6] Y Pfeifer A Cullik andWWitte ldquoResistance to cephalosporinsand carbapenems in Gram-negative bacterial pathogensrdquo Inter-national Journal ofMedicalMicrobiology vol 300 no 6 pp 371ndash379 2010
[7] R Canton and T M Coque ldquoThe CTX-M beta-lactamasepandemicrdquo Current Opinion in Microbiology vol 9 no 5 pp466ndash475 2006
[8] C Dallenne A da Costa D Decre C Favier and G ArletldquoDevelopment of a set of multiplex PCR assays for the detectionof genes encoding important 120573-lactamases in Enterobacteri-aceaerdquo Journal of Antimicrobial Chemotherapy vol 65 no 3 pp490ndash495 2010
[9] Clinical and Laboratory Standards Institute (CLSI) ldquoPer-formance standards for antimicrobial susceptibility testingTwenty-fourth informational supplement performance stan-dards for antimicrobial susceptibility testingrdquoDocumentM100-24 Clinical and Laboratory Standards Institute (CLSI) WaynePa USA 2014
[10] J A Severin E S Lestari W Kloezen et al ldquoFaecal carriageof extended-spectrum 120573-lactamase-producing Enterobacteri-aceae among humans in Java Indonesia in 2001-2002rdquo TropicalMedicine and International Health vol 17 no 4 pp 455ndash4612012
[11] B Li J-Y Sun Q-Z Liu L-Z Han X-H Huang and Y-X Ni ldquoHigh prevalence of CTX-M 120573-lactamases in faecalEscherichia coli strains from healthy humans in Fuzhou ChinardquoScandinavian Journal of Infectious Diseases vol 43 no 3 pp170ndash174 2011
[12] G V Sanchez R N Master J A Karlowsky and J M BordonldquoIn vitro antimicrobial resistance of urinary Escherichia coliisolates among US outpatients from 2000 to 2010rdquo Antimicro-bial Agents andChemotherapy vol 56 no 4 pp 2181ndash2183 2012
[13] R H Bassyouni S N Gaber and A AWegdan ldquoFecal carriageof extended-spectrum 120573-lactamase- and AmpC- producingEscherichia coli among healthcare workersrdquo Journal of Infectionin Developing Countries vol 9 no 3 pp 304ndash308 2015
[14] D Mathai V A Kumar B Paul et al ldquoFecal carriage ratesof extended-spectrum 120573-lactamase-producing Escherichia coliamong antibiotic naive healthy human volunteersrdquo MicrobialDrug Resistance vol 21 no 1 pp 59ndash64 2015
[15] S B Joslashrgensen Oslash Samuelsen A Sundsfjord et al ldquoHighprevalence of faecal carriage of ESBL-producing Enterobacteri-aceae in Norwegian patients with gastroenteritisrdquo ScandinavianJournal of Infectious Diseases vol 46 no 6 pp 462ndash465 2014
[16] N O Yilmaz N Agus E Bozcal O Oner and A UzelldquoDetection of plasmid-mediated AmpC 120573-lactamase in E coliand K pneumoniaerdquo Indian Journal of Medical Microbiologyvol 31 no 1 pp 53ndash59 2013
[17] T M H Bui I Hirai S Ueda et al ldquoCarriage of Escherichiacoli producing CTX-M-type extended-spectrum 120573-lactamasein healthy Vietnamese individualsrdquo Antimicrobial Agents andChemotherapy vol 59 no 10 pp 6611ndash6614 2015
[18] S F Ahmed M M M Ali Z K Mohamed T A Moussa andJ D Klena ldquoFecal carriage of extended-spectrum 120573-lactamasesand AmpC-producing Escherichia coli in a Libyan communityrdquo
BioMed Research International 7
Annals of Clinical Microbiology and Antimicrobials vol 13article 22 2014
[19] Y J Ko H-W Moon M Hur C-M Park S E Cho andY-M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[20] H E Villar M N Baserni and M B Jugo ldquoFaecal carriage ofESBL-producing enterobacteriaceae and carbapenem-resistantGram-negative bacilli in community settingsrdquo Journal of Infec-tion in Developing Countries vol 7 no 8 pp 630ndash634 2013
[21] M H Al-Agamy M S Ali M M Salem and T R MohamedldquoFaecal colonization by extended-spectrum beta-lactamase-producing Escherichia coli from hospitalized patientsrdquo NewEgyptian Journal of Microbiology vol 19 pp 285ndash314 2008
[22] J Kaarme Y Molin B Olsen and A Melhus ldquoPrevalence ofextended-spectrum beta-lactamase-producing Enterobacteri-aceae in healthy Swedish preschool childrenrdquo Acta Paediatricavol 102 no 6 pp 655ndash660 2013
[23] A M Hammerum C H Lester L Jakobsen and L J PorsboldquoFaecal carriage of extended-spectrum 120573-lactamase-producingand AmpC 120573-lactamase-producing bacteria among Danisharmy recruitsrdquo Clinical Microbiology and Infection vol 17 no4 pp 566ndash568 2011
[24] A A Kader A Kumar and K A Kamath ldquoFecal car-riage of extended-spectrum120573-lactamase-producingEscherichiacoli and Klebsiella pneumoniae in patients and asymptomatichealthy individualsrdquo Infection Control and Hospital Epidemiol-ogy vol 28 no 9 pp 1114ndash1116 2007
[25] A Garrido C Seral M J Gude et al ldquoCharacterization ofplasmid-mediated 120573-lactamases in fecal colonizing patients inthe hospital and community setting in Spainrdquo Microbial DrugResistance vol 20 no 4 pp 301ndash304 2014
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 3
Table1Prim
ersu
sedfora
mplificatio
nof
thetested120573-la
ctam
aseg
enes
(Dallenn
eetal2010
[8])
PCRtype
Target
Prim
erSequ
ence
ofprim
ers(51015840-31015840
)Amplified
prod
ucts
(bp)
Multip
lexIT
EMSHV
and
OXA-
1-like
TEM
MultiT
SO-T
for
CATT
TCCG
TGTC
GCC
CTTA
TTC
800
MultiT
SO-T
rev
CGTT
CATC
CATA
GTT
GCC
TGAC
SHV
MultiT
SO-S
for
AGCC
GCT
TGAG
CAAAT
TAAAC
713
MultiT
SO-S
rev
ATCC
CGCA
GAT
AAAT
CACC
AC
OXA-
1MultiT
SO-O
for
GGCA
CCAG
ATTC
AAC
TTTC
AAG
564
MultiT
SO-O
rev
GAC
CCCA
AGTT
TCCT
GTA
AGTG
Multip
lexIICT
X-M
grou
p1grou
p2andgrou
p9
CTX-
Mgrou
p1
MultiC
TXMGp1
for
TTAG
GAART
GTG
CCGCT
GYA
688
MultiC
TXMGp1-2
rev
CGAT
ATCG
TTGGTG
GTR
CCAT
CTX-
Mgrou
p2
MultiC
TXMGp2
for
CGTT
AAC
GGCA
CGAT
GAC
404
MultiC
TXMGp1-2
rev
CGAT
ATCG
TTGGTG
GTR
CCAT
CTX-
Mgrou
p9
MultiC
TXMGp9
for
TCAAG
CCTG
CCGAT
CTGGT
561
MultiC
TXMGp9
rev
TGAT
TCTC
GCC
GCT
GAAG
CTX-
Mgrou
p825
CTX-
Mgrou
p825
CTX-
Mg825for
AAC
RCRC
AGAC
GCT
CTAC
326
CTX-
Mg825rev
TCGAG
CCGGAASG
TGTY
AT
Multip
lexIIIA
CCFOX
MOX
DHAC
ITand
EBC
ACC-
1and
ACC-
2MultiC
aseA
CCfor
CACC
TCCA
GCG
ACTT
GTT
AC346
MultiC
aseA
CCrev
GTT
AGCC
AGCA
TCAC
GAT
CC
FOX-
1toFO
X-5
MultiC
aseFOX
for
CTAC
AGTG
CGGGTG
GTT
T162
MultiC
aseFOX
rev
CTAT
TTGCG
GCC
AGGTG
AMOX-
1MOX-
2CM
Y-1CM
Y-8to
CMY-11
andCM
Y-19
MultiC
aseM
OX
for
GCA
ACAAC
GAC
AAT
CCAT
CCT
895
MultiC
aseM
OX
rev
GGGAT
AGGCG
TAAC
TCTC
CCAA
DHA-
1and
DHA-
2MultiC
aseD
HA
for
TGAT
GGCA
CAGCA
GGAT
ATTC
997
MultiC
aseD
HA
rev
GCT
TTGAC
TCTT
TCGGTA
TTCG
LAT-1toLA
T-3BIL-1CM
Y-2to
CMY-7
CMY-12
toCM
Y-18and
CMY-21
toCM
Y-23
MultiC
aseC
ITfor
CGAAG
AGGCA
ATGAC
CAGAC
538
MultiC
aseC
ITrev
ACGGAC
AGGGTT
AGGAT
AGY
ACT-1and
MIR-1
MultiC
aseEBC
for
CGGTA
AAG
CCGAT
GTT
GCG
683
4 BioMed Research International
Table2Antim
icrobialsusceptib
ilityprofi
leso
fES120573
L-andAmpC
-produ
cing
Ecoliiso
latesfrom
fecalsam
ples
andassociated
resistancep
atterns
Isolates
number
119864-te
stMIC
(mgL)
Resistanceg
enes
PIP
TZP
CTX
CAZ
CAZCA
LFE
PAT
MFO
XCT
TCT
TCX
TIM
IGM
AK
CICO
LTG
CFO
SEC
1gt256lt1gt256
32322
48
0032
025
lt05lt05
006
05
21lt0016
025
0016
TEM-1+C
TX-M
-15+
OXA-
1EC
2gt256lt1gt256
161605
416
0065
025
lt05lt05
012
153
025lt0016
025lt0016
TEM-1+C
TX-M
-15+
+SHV-1
EC3
gt256
64gt256gt256
gt324
12gt256
0125
025
lt05lt05
025
643
4lt0016
0125lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
4gt256
8gt256
32320064
816
0032
006
lt05lt05
006
124
4lt0016
0125lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
5gt256
32gt256
161605
412
0125
0125lt05lt05
003
83
6lt0016
025lt0016
TEM-1+C
TX-M
-15+
SHV-1
EC6
gt256
8gt256gt256
gt324
192gt256
025
025
lt05lt05
006
192
48gt32lt0016
025lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
7gt256
8gt256gt256
gt324
128
192
025
025
lt05lt05
012
128
16gt32lt0016
025lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
8gt256lt1gt256gt256
gt324
96128
025
025
lt05lt05
006
9605
025lt0016
025
0125
TEM-1+C
TX-M
-14+
OXA-
1EC
9gt256
16gt256
44006
43
30032
0032lt05lt05
006
82
025lt0016
038lt0016
TEM-1+C
TX-M
-15
EC10
gt256
4gt256
660125
46
0032
0032lt05lt05
0125
161
05lt0016
025lt0016
TEM-1+C
TX-M
-15+
SHV-1
EC11
gt256lt1gt256
48gt321
824
0032
0032lt05lt05
025
232
1lt0016
025lt0016
TEM-1+C
TX-M
-14+
OXA-
1EC
12gt256gt256gt256gt256gt32gt4
48gt256
6432
3232
025gt256
192
075lt0016
025
0016
TEM-1+C
TX-M
-15+
CMY-2+
OXA-
1EC
13gt256
128gt256gt256gt32gt4
64gt256
6448
gt32gt32
025
128
4gt32lt0016
038
0016
TEM-1+C
TX-M
-15+
CMY-2
PIP
piperacillin
TZP
piperacillintazobactam
CAZ
cefta
zidime
CAZCA
Lcefta
zidimecefta
zidime+cla
vulanicacidC
TXc
efotaxim
eFE
Pcefepime
ATMa
ztreon
amF
OX
cefoxitin
CTT
cefotetan
CTTCX
Tcefotetancefotetan+clo
xacillin
IMIim
ipenem
GMgentamicinA
Kam
ikacinC
Iciprofl
oxacinC
OLcolistin
TGC
tigecyclin
eFO
Sfosfo
mycin
BioMed Research International 5
33 Antimicrobial Resistance Pattern of ES120573L and AmpCEnzyme-Positive Isolates The MICs of 17 antimicrobialagents were determined for E coli fecal isolates The resultsof the susceptibility pattern for ES120573L and AmpC enzyme-producing E coli isolates are illustrated in Table 2
4 Discussion
The human and animal alimentary tracts are vital reservoirsfor ES120573L- carbapenemases- and AmpC enzyme-producingEnterobacteriaceae Patient-to-patient transmission of resis-tant microorganisms may occur in hospitals [10 11] Theoveruse of antibiotics has recently been associated with theemergence of resistant intestinal bacteria particularly ES120573L-carbapenemases- and AmpC enzyme-producing Enterobac-teriaceae Numerous studies have demonstrated that expo-sure to 120573-lactam antibiotics is a risk factor for the selection ofmultidrug-resistant E coli [12] Therefore the current studyexamined the antimicrobial resistance patterns of fecal Ecoli isolates as well as the molecular basis for their 120573-lactamresistance mechanisms using phenotypic and genotypicmethods The present study included 50 patients admitted toa hospital in Riyadh Saudi Arabia from April 2014 to June2014 These patients were treated for noninfectious diseasesunder nonoutbreak conditions Fifty fecal stool specimenscollected from the 50 patients were cultured on blood agarand EMB agar as described in Materials and Methods Fiftysuspected E coli isolates were selected and the isolates wereidentified by conventional procedures and using the API20Eidentification kit All isolates were identified as E coli
Production of 120573-lactamases is the main mechanism of120573-lactam resistance in Gram-negative bacteria including Ecoli [6 7] Several 120573-lactamases (ES120573Ls AmpC enzymes andcarbapenemases) have been previously reported in fecal Ecoli isolates [13ndash15]
In the present study 100 of 50 E coli isolates were foundto be sensitive to imipenem and 13 (26) of 50 isolates wereresistant or showed reduced susceptibility to CAZ CTX-M or ATM The carbapenem susceptibility results indicatedthat our isolates did not harbor carbapenemase while 26(1350) of the E coli isolates harbored ES120573L andor AmpC 120573-lactamase Two of 13 isolates exhibited reduced susceptibilityto cephamycins (FOX and CTT)
Phenotypic screening for the presence of different typesof 120573-lactamases was conducted using E coli isolatesThirteenE coli isolates were selected for screening of ES120573L andAmpC 120573-lactamase production using CAZCAL-ES120573L andCTTCXT-AmpC E-test strips were used to detect ES120573Lproduction Using phenotypic detection methods all isolateswere found to produce ES120573L while two isolates phenotyp-ically produced AmpC enzyme A battery of PCR assayswas conducted to detect 119887119897119886ES120573L genes and AmpC plasmid-mediated genes in the 13 E coli isolates Therefore class Aand class C 120573-lactamase genes were tested The PCR-purifiedproduct was subjected to DNA sequencing to identify thegene variants The results of molecular characterization ofbla genes are shown in Table 2 PCR amplification andDNA sequencing analyses of the PCR products showed
that all isolates possessed a CTX-M-type ES120573L and that119887119897119886CTX-M-15 was present in 1 isolate while two isolates con-tained 119887119897119886CTX-M-14 Other CTX-M families were not detectedThe gene encoding CMY-2 enzyme was detected in two Ecoli isolates CMY-2-positive isolates are concomitant withCTX-M-15 All E coli isolates (119899 = 13) were positivefor 119887119897119886TEM-1 while 615 and 23 of the isolates contained119887119897119886OXA-1 and 119887119897119886SHV-1 respectivelyThe increase in expressionof the AmpC 120573-lactamases may mask the recognition ofES120573Ls [16] Therefore in the present study the genotypicmethods revealed that all 13 strains were ES120573L CTX-M-positive while phenotypic methods showed that 11 strainswere ES120573L-positive and two strains were AmpC enzyme-positive AmpC-producing strains producingCTX-M-15mayact as a dormant reservoir for ES120573Ls
Numerous studies have documented a remarkableincrease in intestinal colonization rates with ES120573L- andAmpC enzyme-producing Enterobacteriaceae in manycountries [2 10 11 14ndash17]The prevalence of ES120573L-producingE coli fecal isolates varies widely from country to countryfrom region to region and at different time periods Ahigh incidence of fecal carriage rate of ES120573L-producing Ecoli has been observed in Asia Africa and South America[13 14 18ndash21] while a significantly lower prevalence ofES120573L-producing E coli fecal isolates was reported in mostEuropean countries [22 23] In Argentina the rate of fecalcarriage of Enterobacteriaceae-resistant strains to third-generation cephalosporins was 268 [20] Villar et al [20]reported that 2022 and 67 of fecal strains were colonizedby ES120573L- and AmpC enzyme-producing Enterobacteriaceae[20] In Egypt Al-Agamy et al reported that 226 and322 of hospitalized patients were colonized by ES120573L-and AmpC-positive E coli respectively The 119887119897119886CTX-M-likegene was the predominant ES120573L gene detected in 714of ES120573L-producing E coli isolates [21] In a recent studyin Egypt Bassyouni et al reported that 21 and 3 ofpatients were colonized by ES120573L- and AmpC-producing Ecoli respectively They also found that 119887119897119886SHV gene was thepredominant ES120573L gene detected in 818 of the resistant Ecoli isolates [13] In Korea 203 of fecal Enterobacteriaceaemembers were ES120573Ls [19] In India the prevalence ofES120573L-positive E coli isolates was 19 in healthy volunteersfrom the community [14] In Libya 134 and 67 of E coliisolates were ES120573Ls- and AmpC-positive respectively [18]In a previous study in Saudi Arabia 177 of strains werefound to be ES120573L-positive [24] A high (261) prevalencewas detected in inpatients followed by outpatients (154)and the lowest prevalence rate (131) was detected in healthyindividuals [24] In the present study the prevalence of ES120573L-producing E coli was 26 Despite differences in the dateand region of isolation the prevalence of fecal carriage rateof ES120573L in the present study (26) was in agreement withthe prevalence (261) reported by Kader et al In contrastthe prevalence rate of ES120573L-producing Enterobacteriaceaewas 29 among healthy Swedish children Escherichia colicontaining CTX-M 120573-lactamase predominated and onlyone E coli isolate harbored genes encoding for CMY [22]The carriage rate of ES120573L- and AmpC enzyme-producing Ecoli was 357 and 238 among 84 Danish army recruits
6 BioMed Research International
respectively 119887119897119886CTX-M-14 gene was the predominant ES120573Lgene detected in three (100) ES120573L-producing E coliisolates while 119887119897119886CMY-2 was detected in two AmpC enzyme-producing E coli isolates [23] In Spain the prevalence ofES120573L and AmpC enzyme carriers was 506 and 059respectively [25] 119887119897119886CTX-M genes were the ES120573L dominatinggenes (9615) and CTX-M-14 was the most prevalent gene(50) followed by CTX-M-15 (40) CMY-2 was the mostprevalent gene (8125) followed by DHA-1 (1875) [25]
MICs were determined for ES120573L- and AmpC enzyme-producing E coli (119899 = 13) isolates The results of MIC areshown in Table 2
In conclusion a high incidence of carriage of ES120573L-positive E coli fecal isolates among hospitalized patients inRiyadh was detected reaching 26 with 119887119897119886CTX-M-15 (846)being the most predominant gene The emergence of fecalcarriage of CMY-2-producing E coli among hospitalizedpatients has been reported to be 4These outcomes empha-size the importance of the intestinal tract as a reservoir forES120573L- and AmpC enzyme-producing E coli which may leadto nosocomial infection The admission of colonized fecalcarriers of ES120573L- and AmpC-positive E coli to the medicalsetting increases the possibility of other patients acquiringinfection in the same hospital Our results emphasize thenecessity for continuous surveillance in hospitals to detectthe ES120573L- AmpC enzyme- and carbapenemase-producingstrains andmultidrug strains as well applying effective strate-gies for antimicrobial therapy and infection control measuresto decrease the abuse and misuse of antimicrobial agentsagainst resistant strains and to prevent their spread
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
The authors extend their appreciation to the Deanship ofScientific Research at King Saud University for funding thework through the research group Project no RGP-038
References
[1] A Bhargava K Hayakawa E Silverman et al ldquoRisk factors forcolonization due to carbapenem-resistant enterobacteriaceaeamong patients exposed to long-term acute care and acute carefacilitiesrdquo Infection Control and Hospital Epidemiology vol 35no 4 pp 398ndash405 2014
[2] P-L Woerther C Burdet E Chachaty and A AndremontldquoTrends in human fecal carriage of extended-spectrum 120573-lactamases in the community toward the globalization of CTX-Mrdquo Clinical Microbiology Reviews vol 26 no 4 pp 744ndash7582013
[3] N Karah B Haldorsen N O Hermansen et al ldquoEmergenceof OXA-carbapenemase- and 16S rRNA methylase-producinginternational clones of Acinetobacter baumannii in NorwayrdquoJournal of Medical Microbiology vol 60 no 4 pp 515ndash521 2011
[4] S Yezli A M Shibl and Z A Memish ldquoThemolecular basis of120573-lactamase production in Gram-negative bacteria from Saudi
Arabiardquo Journal of Medical Microbiology vol 64 no 2 pp 127ndash136 2015
[5] M Guzman-Blanco J A Labarca M V Villegas and EGotuzzo ldquoExtended spectrum 120573-lactamase producers amongnosocomial Enterobacteriaceae in Latin Americardquo BrazilianJournal of Infectious Diseases vol 18 no 4 pp 421ndash433 2014
[6] Y Pfeifer A Cullik andWWitte ldquoResistance to cephalosporinsand carbapenems in Gram-negative bacterial pathogensrdquo Inter-national Journal ofMedicalMicrobiology vol 300 no 6 pp 371ndash379 2010
[7] R Canton and T M Coque ldquoThe CTX-M beta-lactamasepandemicrdquo Current Opinion in Microbiology vol 9 no 5 pp466ndash475 2006
[8] C Dallenne A da Costa D Decre C Favier and G ArletldquoDevelopment of a set of multiplex PCR assays for the detectionof genes encoding important 120573-lactamases in Enterobacteri-aceaerdquo Journal of Antimicrobial Chemotherapy vol 65 no 3 pp490ndash495 2010
[9] Clinical and Laboratory Standards Institute (CLSI) ldquoPer-formance standards for antimicrobial susceptibility testingTwenty-fourth informational supplement performance stan-dards for antimicrobial susceptibility testingrdquoDocumentM100-24 Clinical and Laboratory Standards Institute (CLSI) WaynePa USA 2014
[10] J A Severin E S Lestari W Kloezen et al ldquoFaecal carriageof extended-spectrum 120573-lactamase-producing Enterobacteri-aceae among humans in Java Indonesia in 2001-2002rdquo TropicalMedicine and International Health vol 17 no 4 pp 455ndash4612012
[11] B Li J-Y Sun Q-Z Liu L-Z Han X-H Huang and Y-X Ni ldquoHigh prevalence of CTX-M 120573-lactamases in faecalEscherichia coli strains from healthy humans in Fuzhou ChinardquoScandinavian Journal of Infectious Diseases vol 43 no 3 pp170ndash174 2011
[12] G V Sanchez R N Master J A Karlowsky and J M BordonldquoIn vitro antimicrobial resistance of urinary Escherichia coliisolates among US outpatients from 2000 to 2010rdquo Antimicro-bial Agents andChemotherapy vol 56 no 4 pp 2181ndash2183 2012
[13] R H Bassyouni S N Gaber and A AWegdan ldquoFecal carriageof extended-spectrum 120573-lactamase- and AmpC- producingEscherichia coli among healthcare workersrdquo Journal of Infectionin Developing Countries vol 9 no 3 pp 304ndash308 2015
[14] D Mathai V A Kumar B Paul et al ldquoFecal carriage ratesof extended-spectrum 120573-lactamase-producing Escherichia coliamong antibiotic naive healthy human volunteersrdquo MicrobialDrug Resistance vol 21 no 1 pp 59ndash64 2015
[15] S B Joslashrgensen Oslash Samuelsen A Sundsfjord et al ldquoHighprevalence of faecal carriage of ESBL-producing Enterobacteri-aceae in Norwegian patients with gastroenteritisrdquo ScandinavianJournal of Infectious Diseases vol 46 no 6 pp 462ndash465 2014
[16] N O Yilmaz N Agus E Bozcal O Oner and A UzelldquoDetection of plasmid-mediated AmpC 120573-lactamase in E coliand K pneumoniaerdquo Indian Journal of Medical Microbiologyvol 31 no 1 pp 53ndash59 2013
[17] T M H Bui I Hirai S Ueda et al ldquoCarriage of Escherichiacoli producing CTX-M-type extended-spectrum 120573-lactamasein healthy Vietnamese individualsrdquo Antimicrobial Agents andChemotherapy vol 59 no 10 pp 6611ndash6614 2015
[18] S F Ahmed M M M Ali Z K Mohamed T A Moussa andJ D Klena ldquoFecal carriage of extended-spectrum 120573-lactamasesand AmpC-producing Escherichia coli in a Libyan communityrdquo
BioMed Research International 7
Annals of Clinical Microbiology and Antimicrobials vol 13article 22 2014
[19] Y J Ko H-W Moon M Hur C-M Park S E Cho andY-M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[20] H E Villar M N Baserni and M B Jugo ldquoFaecal carriage ofESBL-producing enterobacteriaceae and carbapenem-resistantGram-negative bacilli in community settingsrdquo Journal of Infec-tion in Developing Countries vol 7 no 8 pp 630ndash634 2013
[21] M H Al-Agamy M S Ali M M Salem and T R MohamedldquoFaecal colonization by extended-spectrum beta-lactamase-producing Escherichia coli from hospitalized patientsrdquo NewEgyptian Journal of Microbiology vol 19 pp 285ndash314 2008
[22] J Kaarme Y Molin B Olsen and A Melhus ldquoPrevalence ofextended-spectrum beta-lactamase-producing Enterobacteri-aceae in healthy Swedish preschool childrenrdquo Acta Paediatricavol 102 no 6 pp 655ndash660 2013
[23] A M Hammerum C H Lester L Jakobsen and L J PorsboldquoFaecal carriage of extended-spectrum 120573-lactamase-producingand AmpC 120573-lactamase-producing bacteria among Danisharmy recruitsrdquo Clinical Microbiology and Infection vol 17 no4 pp 566ndash568 2011
[24] A A Kader A Kumar and K A Kamath ldquoFecal car-riage of extended-spectrum120573-lactamase-producingEscherichiacoli and Klebsiella pneumoniae in patients and asymptomatichealthy individualsrdquo Infection Control and Hospital Epidemiol-ogy vol 28 no 9 pp 1114ndash1116 2007
[25] A Garrido C Seral M J Gude et al ldquoCharacterization ofplasmid-mediated 120573-lactamases in fecal colonizing patients inthe hospital and community setting in Spainrdquo Microbial DrugResistance vol 20 no 4 pp 301ndash304 2014
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
4 BioMed Research International
Table2Antim
icrobialsusceptib
ilityprofi
leso
fES120573
L-andAmpC
-produ
cing
Ecoliiso
latesfrom
fecalsam
ples
andassociated
resistancep
atterns
Isolates
number
119864-te
stMIC
(mgL)
Resistanceg
enes
PIP
TZP
CTX
CAZ
CAZCA
LFE
PAT
MFO
XCT
TCT
TCX
TIM
IGM
AK
CICO
LTG
CFO
SEC
1gt256lt1gt256
32322
48
0032
025
lt05lt05
006
05
21lt0016
025
0016
TEM-1+C
TX-M
-15+
OXA-
1EC
2gt256lt1gt256
161605
416
0065
025
lt05lt05
012
153
025lt0016
025lt0016
TEM-1+C
TX-M
-15+
+SHV-1
EC3
gt256
64gt256gt256
gt324
12gt256
0125
025
lt05lt05
025
643
4lt0016
0125lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
4gt256
8gt256
32320064
816
0032
006
lt05lt05
006
124
4lt0016
0125lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
5gt256
32gt256
161605
412
0125
0125lt05lt05
003
83
6lt0016
025lt0016
TEM-1+C
TX-M
-15+
SHV-1
EC6
gt256
8gt256gt256
gt324
192gt256
025
025
lt05lt05
006
192
48gt32lt0016
025lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
7gt256
8gt256gt256
gt324
128
192
025
025
lt05lt05
012
128
16gt32lt0016
025lt0016
TEM-1+C
TX-M
-15+
OXA-
1EC
8gt256lt1gt256gt256
gt324
96128
025
025
lt05lt05
006
9605
025lt0016
025
0125
TEM-1+C
TX-M
-14+
OXA-
1EC
9gt256
16gt256
44006
43
30032
0032lt05lt05
006
82
025lt0016
038lt0016
TEM-1+C
TX-M
-15
EC10
gt256
4gt256
660125
46
0032
0032lt05lt05
0125
161
05lt0016
025lt0016
TEM-1+C
TX-M
-15+
SHV-1
EC11
gt256lt1gt256
48gt321
824
0032
0032lt05lt05
025
232
1lt0016
025lt0016
TEM-1+C
TX-M
-14+
OXA-
1EC
12gt256gt256gt256gt256gt32gt4
48gt256
6432
3232
025gt256
192
075lt0016
025
0016
TEM-1+C
TX-M
-15+
CMY-2+
OXA-
1EC
13gt256
128gt256gt256gt32gt4
64gt256
6448
gt32gt32
025
128
4gt32lt0016
038
0016
TEM-1+C
TX-M
-15+
CMY-2
PIP
piperacillin
TZP
piperacillintazobactam
CAZ
cefta
zidime
CAZCA
Lcefta
zidimecefta
zidime+cla
vulanicacidC
TXc
efotaxim
eFE
Pcefepime
ATMa
ztreon
amF
OX
cefoxitin
CTT
cefotetan
CTTCX
Tcefotetancefotetan+clo
xacillin
IMIim
ipenem
GMgentamicinA
Kam
ikacinC
Iciprofl
oxacinC
OLcolistin
TGC
tigecyclin
eFO
Sfosfo
mycin
BioMed Research International 5
33 Antimicrobial Resistance Pattern of ES120573L and AmpCEnzyme-Positive Isolates The MICs of 17 antimicrobialagents were determined for E coli fecal isolates The resultsof the susceptibility pattern for ES120573L and AmpC enzyme-producing E coli isolates are illustrated in Table 2
4 Discussion
The human and animal alimentary tracts are vital reservoirsfor ES120573L- carbapenemases- and AmpC enzyme-producingEnterobacteriaceae Patient-to-patient transmission of resis-tant microorganisms may occur in hospitals [10 11] Theoveruse of antibiotics has recently been associated with theemergence of resistant intestinal bacteria particularly ES120573L-carbapenemases- and AmpC enzyme-producing Enterobac-teriaceae Numerous studies have demonstrated that expo-sure to 120573-lactam antibiotics is a risk factor for the selection ofmultidrug-resistant E coli [12] Therefore the current studyexamined the antimicrobial resistance patterns of fecal Ecoli isolates as well as the molecular basis for their 120573-lactamresistance mechanisms using phenotypic and genotypicmethods The present study included 50 patients admitted toa hospital in Riyadh Saudi Arabia from April 2014 to June2014 These patients were treated for noninfectious diseasesunder nonoutbreak conditions Fifty fecal stool specimenscollected from the 50 patients were cultured on blood agarand EMB agar as described in Materials and Methods Fiftysuspected E coli isolates were selected and the isolates wereidentified by conventional procedures and using the API20Eidentification kit All isolates were identified as E coli
Production of 120573-lactamases is the main mechanism of120573-lactam resistance in Gram-negative bacteria including Ecoli [6 7] Several 120573-lactamases (ES120573Ls AmpC enzymes andcarbapenemases) have been previously reported in fecal Ecoli isolates [13ndash15]
In the present study 100 of 50 E coli isolates were foundto be sensitive to imipenem and 13 (26) of 50 isolates wereresistant or showed reduced susceptibility to CAZ CTX-M or ATM The carbapenem susceptibility results indicatedthat our isolates did not harbor carbapenemase while 26(1350) of the E coli isolates harbored ES120573L andor AmpC 120573-lactamase Two of 13 isolates exhibited reduced susceptibilityto cephamycins (FOX and CTT)
Phenotypic screening for the presence of different typesof 120573-lactamases was conducted using E coli isolatesThirteenE coli isolates were selected for screening of ES120573L andAmpC 120573-lactamase production using CAZCAL-ES120573L andCTTCXT-AmpC E-test strips were used to detect ES120573Lproduction Using phenotypic detection methods all isolateswere found to produce ES120573L while two isolates phenotyp-ically produced AmpC enzyme A battery of PCR assayswas conducted to detect 119887119897119886ES120573L genes and AmpC plasmid-mediated genes in the 13 E coli isolates Therefore class Aand class C 120573-lactamase genes were tested The PCR-purifiedproduct was subjected to DNA sequencing to identify thegene variants The results of molecular characterization ofbla genes are shown in Table 2 PCR amplification andDNA sequencing analyses of the PCR products showed
that all isolates possessed a CTX-M-type ES120573L and that119887119897119886CTX-M-15 was present in 1 isolate while two isolates con-tained 119887119897119886CTX-M-14 Other CTX-M families were not detectedThe gene encoding CMY-2 enzyme was detected in two Ecoli isolates CMY-2-positive isolates are concomitant withCTX-M-15 All E coli isolates (119899 = 13) were positivefor 119887119897119886TEM-1 while 615 and 23 of the isolates contained119887119897119886OXA-1 and 119887119897119886SHV-1 respectivelyThe increase in expressionof the AmpC 120573-lactamases may mask the recognition ofES120573Ls [16] Therefore in the present study the genotypicmethods revealed that all 13 strains were ES120573L CTX-M-positive while phenotypic methods showed that 11 strainswere ES120573L-positive and two strains were AmpC enzyme-positive AmpC-producing strains producingCTX-M-15mayact as a dormant reservoir for ES120573Ls
Numerous studies have documented a remarkableincrease in intestinal colonization rates with ES120573L- andAmpC enzyme-producing Enterobacteriaceae in manycountries [2 10 11 14ndash17]The prevalence of ES120573L-producingE coli fecal isolates varies widely from country to countryfrom region to region and at different time periods Ahigh incidence of fecal carriage rate of ES120573L-producing Ecoli has been observed in Asia Africa and South America[13 14 18ndash21] while a significantly lower prevalence ofES120573L-producing E coli fecal isolates was reported in mostEuropean countries [22 23] In Argentina the rate of fecalcarriage of Enterobacteriaceae-resistant strains to third-generation cephalosporins was 268 [20] Villar et al [20]reported that 2022 and 67 of fecal strains were colonizedby ES120573L- and AmpC enzyme-producing Enterobacteriaceae[20] In Egypt Al-Agamy et al reported that 226 and322 of hospitalized patients were colonized by ES120573L-and AmpC-positive E coli respectively The 119887119897119886CTX-M-likegene was the predominant ES120573L gene detected in 714of ES120573L-producing E coli isolates [21] In a recent studyin Egypt Bassyouni et al reported that 21 and 3 ofpatients were colonized by ES120573L- and AmpC-producing Ecoli respectively They also found that 119887119897119886SHV gene was thepredominant ES120573L gene detected in 818 of the resistant Ecoli isolates [13] In Korea 203 of fecal Enterobacteriaceaemembers were ES120573Ls [19] In India the prevalence ofES120573L-positive E coli isolates was 19 in healthy volunteersfrom the community [14] In Libya 134 and 67 of E coliisolates were ES120573Ls- and AmpC-positive respectively [18]In a previous study in Saudi Arabia 177 of strains werefound to be ES120573L-positive [24] A high (261) prevalencewas detected in inpatients followed by outpatients (154)and the lowest prevalence rate (131) was detected in healthyindividuals [24] In the present study the prevalence of ES120573L-producing E coli was 26 Despite differences in the dateand region of isolation the prevalence of fecal carriage rateof ES120573L in the present study (26) was in agreement withthe prevalence (261) reported by Kader et al In contrastthe prevalence rate of ES120573L-producing Enterobacteriaceaewas 29 among healthy Swedish children Escherichia colicontaining CTX-M 120573-lactamase predominated and onlyone E coli isolate harbored genes encoding for CMY [22]The carriage rate of ES120573L- and AmpC enzyme-producing Ecoli was 357 and 238 among 84 Danish army recruits
6 BioMed Research International
respectively 119887119897119886CTX-M-14 gene was the predominant ES120573Lgene detected in three (100) ES120573L-producing E coliisolates while 119887119897119886CMY-2 was detected in two AmpC enzyme-producing E coli isolates [23] In Spain the prevalence ofES120573L and AmpC enzyme carriers was 506 and 059respectively [25] 119887119897119886CTX-M genes were the ES120573L dominatinggenes (9615) and CTX-M-14 was the most prevalent gene(50) followed by CTX-M-15 (40) CMY-2 was the mostprevalent gene (8125) followed by DHA-1 (1875) [25]
MICs were determined for ES120573L- and AmpC enzyme-producing E coli (119899 = 13) isolates The results of MIC areshown in Table 2
In conclusion a high incidence of carriage of ES120573L-positive E coli fecal isolates among hospitalized patients inRiyadh was detected reaching 26 with 119887119897119886CTX-M-15 (846)being the most predominant gene The emergence of fecalcarriage of CMY-2-producing E coli among hospitalizedpatients has been reported to be 4These outcomes empha-size the importance of the intestinal tract as a reservoir forES120573L- and AmpC enzyme-producing E coli which may leadto nosocomial infection The admission of colonized fecalcarriers of ES120573L- and AmpC-positive E coli to the medicalsetting increases the possibility of other patients acquiringinfection in the same hospital Our results emphasize thenecessity for continuous surveillance in hospitals to detectthe ES120573L- AmpC enzyme- and carbapenemase-producingstrains andmultidrug strains as well applying effective strate-gies for antimicrobial therapy and infection control measuresto decrease the abuse and misuse of antimicrobial agentsagainst resistant strains and to prevent their spread
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
The authors extend their appreciation to the Deanship ofScientific Research at King Saud University for funding thework through the research group Project no RGP-038
References
[1] A Bhargava K Hayakawa E Silverman et al ldquoRisk factors forcolonization due to carbapenem-resistant enterobacteriaceaeamong patients exposed to long-term acute care and acute carefacilitiesrdquo Infection Control and Hospital Epidemiology vol 35no 4 pp 398ndash405 2014
[2] P-L Woerther C Burdet E Chachaty and A AndremontldquoTrends in human fecal carriage of extended-spectrum 120573-lactamases in the community toward the globalization of CTX-Mrdquo Clinical Microbiology Reviews vol 26 no 4 pp 744ndash7582013
[3] N Karah B Haldorsen N O Hermansen et al ldquoEmergenceof OXA-carbapenemase- and 16S rRNA methylase-producinginternational clones of Acinetobacter baumannii in NorwayrdquoJournal of Medical Microbiology vol 60 no 4 pp 515ndash521 2011
[4] S Yezli A M Shibl and Z A Memish ldquoThemolecular basis of120573-lactamase production in Gram-negative bacteria from Saudi
Arabiardquo Journal of Medical Microbiology vol 64 no 2 pp 127ndash136 2015
[5] M Guzman-Blanco J A Labarca M V Villegas and EGotuzzo ldquoExtended spectrum 120573-lactamase producers amongnosocomial Enterobacteriaceae in Latin Americardquo BrazilianJournal of Infectious Diseases vol 18 no 4 pp 421ndash433 2014
[6] Y Pfeifer A Cullik andWWitte ldquoResistance to cephalosporinsand carbapenems in Gram-negative bacterial pathogensrdquo Inter-national Journal ofMedicalMicrobiology vol 300 no 6 pp 371ndash379 2010
[7] R Canton and T M Coque ldquoThe CTX-M beta-lactamasepandemicrdquo Current Opinion in Microbiology vol 9 no 5 pp466ndash475 2006
[8] C Dallenne A da Costa D Decre C Favier and G ArletldquoDevelopment of a set of multiplex PCR assays for the detectionof genes encoding important 120573-lactamases in Enterobacteri-aceaerdquo Journal of Antimicrobial Chemotherapy vol 65 no 3 pp490ndash495 2010
[9] Clinical and Laboratory Standards Institute (CLSI) ldquoPer-formance standards for antimicrobial susceptibility testingTwenty-fourth informational supplement performance stan-dards for antimicrobial susceptibility testingrdquoDocumentM100-24 Clinical and Laboratory Standards Institute (CLSI) WaynePa USA 2014
[10] J A Severin E S Lestari W Kloezen et al ldquoFaecal carriageof extended-spectrum 120573-lactamase-producing Enterobacteri-aceae among humans in Java Indonesia in 2001-2002rdquo TropicalMedicine and International Health vol 17 no 4 pp 455ndash4612012
[11] B Li J-Y Sun Q-Z Liu L-Z Han X-H Huang and Y-X Ni ldquoHigh prevalence of CTX-M 120573-lactamases in faecalEscherichia coli strains from healthy humans in Fuzhou ChinardquoScandinavian Journal of Infectious Diseases vol 43 no 3 pp170ndash174 2011
[12] G V Sanchez R N Master J A Karlowsky and J M BordonldquoIn vitro antimicrobial resistance of urinary Escherichia coliisolates among US outpatients from 2000 to 2010rdquo Antimicro-bial Agents andChemotherapy vol 56 no 4 pp 2181ndash2183 2012
[13] R H Bassyouni S N Gaber and A AWegdan ldquoFecal carriageof extended-spectrum 120573-lactamase- and AmpC- producingEscherichia coli among healthcare workersrdquo Journal of Infectionin Developing Countries vol 9 no 3 pp 304ndash308 2015
[14] D Mathai V A Kumar B Paul et al ldquoFecal carriage ratesof extended-spectrum 120573-lactamase-producing Escherichia coliamong antibiotic naive healthy human volunteersrdquo MicrobialDrug Resistance vol 21 no 1 pp 59ndash64 2015
[15] S B Joslashrgensen Oslash Samuelsen A Sundsfjord et al ldquoHighprevalence of faecal carriage of ESBL-producing Enterobacteri-aceae in Norwegian patients with gastroenteritisrdquo ScandinavianJournal of Infectious Diseases vol 46 no 6 pp 462ndash465 2014
[16] N O Yilmaz N Agus E Bozcal O Oner and A UzelldquoDetection of plasmid-mediated AmpC 120573-lactamase in E coliand K pneumoniaerdquo Indian Journal of Medical Microbiologyvol 31 no 1 pp 53ndash59 2013
[17] T M H Bui I Hirai S Ueda et al ldquoCarriage of Escherichiacoli producing CTX-M-type extended-spectrum 120573-lactamasein healthy Vietnamese individualsrdquo Antimicrobial Agents andChemotherapy vol 59 no 10 pp 6611ndash6614 2015
[18] S F Ahmed M M M Ali Z K Mohamed T A Moussa andJ D Klena ldquoFecal carriage of extended-spectrum 120573-lactamasesand AmpC-producing Escherichia coli in a Libyan communityrdquo
BioMed Research International 7
Annals of Clinical Microbiology and Antimicrobials vol 13article 22 2014
[19] Y J Ko H-W Moon M Hur C-M Park S E Cho andY-M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[20] H E Villar M N Baserni and M B Jugo ldquoFaecal carriage ofESBL-producing enterobacteriaceae and carbapenem-resistantGram-negative bacilli in community settingsrdquo Journal of Infec-tion in Developing Countries vol 7 no 8 pp 630ndash634 2013
[21] M H Al-Agamy M S Ali M M Salem and T R MohamedldquoFaecal colonization by extended-spectrum beta-lactamase-producing Escherichia coli from hospitalized patientsrdquo NewEgyptian Journal of Microbiology vol 19 pp 285ndash314 2008
[22] J Kaarme Y Molin B Olsen and A Melhus ldquoPrevalence ofextended-spectrum beta-lactamase-producing Enterobacteri-aceae in healthy Swedish preschool childrenrdquo Acta Paediatricavol 102 no 6 pp 655ndash660 2013
[23] A M Hammerum C H Lester L Jakobsen and L J PorsboldquoFaecal carriage of extended-spectrum 120573-lactamase-producingand AmpC 120573-lactamase-producing bacteria among Danisharmy recruitsrdquo Clinical Microbiology and Infection vol 17 no4 pp 566ndash568 2011
[24] A A Kader A Kumar and K A Kamath ldquoFecal car-riage of extended-spectrum120573-lactamase-producingEscherichiacoli and Klebsiella pneumoniae in patients and asymptomatichealthy individualsrdquo Infection Control and Hospital Epidemiol-ogy vol 28 no 9 pp 1114ndash1116 2007
[25] A Garrido C Seral M J Gude et al ldquoCharacterization ofplasmid-mediated 120573-lactamases in fecal colonizing patients inthe hospital and community setting in Spainrdquo Microbial DrugResistance vol 20 no 4 pp 301ndash304 2014
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 5
33 Antimicrobial Resistance Pattern of ES120573L and AmpCEnzyme-Positive Isolates The MICs of 17 antimicrobialagents were determined for E coli fecal isolates The resultsof the susceptibility pattern for ES120573L and AmpC enzyme-producing E coli isolates are illustrated in Table 2
4 Discussion
The human and animal alimentary tracts are vital reservoirsfor ES120573L- carbapenemases- and AmpC enzyme-producingEnterobacteriaceae Patient-to-patient transmission of resis-tant microorganisms may occur in hospitals [10 11] Theoveruse of antibiotics has recently been associated with theemergence of resistant intestinal bacteria particularly ES120573L-carbapenemases- and AmpC enzyme-producing Enterobac-teriaceae Numerous studies have demonstrated that expo-sure to 120573-lactam antibiotics is a risk factor for the selection ofmultidrug-resistant E coli [12] Therefore the current studyexamined the antimicrobial resistance patterns of fecal Ecoli isolates as well as the molecular basis for their 120573-lactamresistance mechanisms using phenotypic and genotypicmethods The present study included 50 patients admitted toa hospital in Riyadh Saudi Arabia from April 2014 to June2014 These patients were treated for noninfectious diseasesunder nonoutbreak conditions Fifty fecal stool specimenscollected from the 50 patients were cultured on blood agarand EMB agar as described in Materials and Methods Fiftysuspected E coli isolates were selected and the isolates wereidentified by conventional procedures and using the API20Eidentification kit All isolates were identified as E coli
Production of 120573-lactamases is the main mechanism of120573-lactam resistance in Gram-negative bacteria including Ecoli [6 7] Several 120573-lactamases (ES120573Ls AmpC enzymes andcarbapenemases) have been previously reported in fecal Ecoli isolates [13ndash15]
In the present study 100 of 50 E coli isolates were foundto be sensitive to imipenem and 13 (26) of 50 isolates wereresistant or showed reduced susceptibility to CAZ CTX-M or ATM The carbapenem susceptibility results indicatedthat our isolates did not harbor carbapenemase while 26(1350) of the E coli isolates harbored ES120573L andor AmpC 120573-lactamase Two of 13 isolates exhibited reduced susceptibilityto cephamycins (FOX and CTT)
Phenotypic screening for the presence of different typesof 120573-lactamases was conducted using E coli isolatesThirteenE coli isolates were selected for screening of ES120573L andAmpC 120573-lactamase production using CAZCAL-ES120573L andCTTCXT-AmpC E-test strips were used to detect ES120573Lproduction Using phenotypic detection methods all isolateswere found to produce ES120573L while two isolates phenotyp-ically produced AmpC enzyme A battery of PCR assayswas conducted to detect 119887119897119886ES120573L genes and AmpC plasmid-mediated genes in the 13 E coli isolates Therefore class Aand class C 120573-lactamase genes were tested The PCR-purifiedproduct was subjected to DNA sequencing to identify thegene variants The results of molecular characterization ofbla genes are shown in Table 2 PCR amplification andDNA sequencing analyses of the PCR products showed
that all isolates possessed a CTX-M-type ES120573L and that119887119897119886CTX-M-15 was present in 1 isolate while two isolates con-tained 119887119897119886CTX-M-14 Other CTX-M families were not detectedThe gene encoding CMY-2 enzyme was detected in two Ecoli isolates CMY-2-positive isolates are concomitant withCTX-M-15 All E coli isolates (119899 = 13) were positivefor 119887119897119886TEM-1 while 615 and 23 of the isolates contained119887119897119886OXA-1 and 119887119897119886SHV-1 respectivelyThe increase in expressionof the AmpC 120573-lactamases may mask the recognition ofES120573Ls [16] Therefore in the present study the genotypicmethods revealed that all 13 strains were ES120573L CTX-M-positive while phenotypic methods showed that 11 strainswere ES120573L-positive and two strains were AmpC enzyme-positive AmpC-producing strains producingCTX-M-15mayact as a dormant reservoir for ES120573Ls
Numerous studies have documented a remarkableincrease in intestinal colonization rates with ES120573L- andAmpC enzyme-producing Enterobacteriaceae in manycountries [2 10 11 14ndash17]The prevalence of ES120573L-producingE coli fecal isolates varies widely from country to countryfrom region to region and at different time periods Ahigh incidence of fecal carriage rate of ES120573L-producing Ecoli has been observed in Asia Africa and South America[13 14 18ndash21] while a significantly lower prevalence ofES120573L-producing E coli fecal isolates was reported in mostEuropean countries [22 23] In Argentina the rate of fecalcarriage of Enterobacteriaceae-resistant strains to third-generation cephalosporins was 268 [20] Villar et al [20]reported that 2022 and 67 of fecal strains were colonizedby ES120573L- and AmpC enzyme-producing Enterobacteriaceae[20] In Egypt Al-Agamy et al reported that 226 and322 of hospitalized patients were colonized by ES120573L-and AmpC-positive E coli respectively The 119887119897119886CTX-M-likegene was the predominant ES120573L gene detected in 714of ES120573L-producing E coli isolates [21] In a recent studyin Egypt Bassyouni et al reported that 21 and 3 ofpatients were colonized by ES120573L- and AmpC-producing Ecoli respectively They also found that 119887119897119886SHV gene was thepredominant ES120573L gene detected in 818 of the resistant Ecoli isolates [13] In Korea 203 of fecal Enterobacteriaceaemembers were ES120573Ls [19] In India the prevalence ofES120573L-positive E coli isolates was 19 in healthy volunteersfrom the community [14] In Libya 134 and 67 of E coliisolates were ES120573Ls- and AmpC-positive respectively [18]In a previous study in Saudi Arabia 177 of strains werefound to be ES120573L-positive [24] A high (261) prevalencewas detected in inpatients followed by outpatients (154)and the lowest prevalence rate (131) was detected in healthyindividuals [24] In the present study the prevalence of ES120573L-producing E coli was 26 Despite differences in the dateand region of isolation the prevalence of fecal carriage rateof ES120573L in the present study (26) was in agreement withthe prevalence (261) reported by Kader et al In contrastthe prevalence rate of ES120573L-producing Enterobacteriaceaewas 29 among healthy Swedish children Escherichia colicontaining CTX-M 120573-lactamase predominated and onlyone E coli isolate harbored genes encoding for CMY [22]The carriage rate of ES120573L- and AmpC enzyme-producing Ecoli was 357 and 238 among 84 Danish army recruits
6 BioMed Research International
respectively 119887119897119886CTX-M-14 gene was the predominant ES120573Lgene detected in three (100) ES120573L-producing E coliisolates while 119887119897119886CMY-2 was detected in two AmpC enzyme-producing E coli isolates [23] In Spain the prevalence ofES120573L and AmpC enzyme carriers was 506 and 059respectively [25] 119887119897119886CTX-M genes were the ES120573L dominatinggenes (9615) and CTX-M-14 was the most prevalent gene(50) followed by CTX-M-15 (40) CMY-2 was the mostprevalent gene (8125) followed by DHA-1 (1875) [25]
MICs were determined for ES120573L- and AmpC enzyme-producing E coli (119899 = 13) isolates The results of MIC areshown in Table 2
In conclusion a high incidence of carriage of ES120573L-positive E coli fecal isolates among hospitalized patients inRiyadh was detected reaching 26 with 119887119897119886CTX-M-15 (846)being the most predominant gene The emergence of fecalcarriage of CMY-2-producing E coli among hospitalizedpatients has been reported to be 4These outcomes empha-size the importance of the intestinal tract as a reservoir forES120573L- and AmpC enzyme-producing E coli which may leadto nosocomial infection The admission of colonized fecalcarriers of ES120573L- and AmpC-positive E coli to the medicalsetting increases the possibility of other patients acquiringinfection in the same hospital Our results emphasize thenecessity for continuous surveillance in hospitals to detectthe ES120573L- AmpC enzyme- and carbapenemase-producingstrains andmultidrug strains as well applying effective strate-gies for antimicrobial therapy and infection control measuresto decrease the abuse and misuse of antimicrobial agentsagainst resistant strains and to prevent their spread
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
The authors extend their appreciation to the Deanship ofScientific Research at King Saud University for funding thework through the research group Project no RGP-038
References
[1] A Bhargava K Hayakawa E Silverman et al ldquoRisk factors forcolonization due to carbapenem-resistant enterobacteriaceaeamong patients exposed to long-term acute care and acute carefacilitiesrdquo Infection Control and Hospital Epidemiology vol 35no 4 pp 398ndash405 2014
[2] P-L Woerther C Burdet E Chachaty and A AndremontldquoTrends in human fecal carriage of extended-spectrum 120573-lactamases in the community toward the globalization of CTX-Mrdquo Clinical Microbiology Reviews vol 26 no 4 pp 744ndash7582013
[3] N Karah B Haldorsen N O Hermansen et al ldquoEmergenceof OXA-carbapenemase- and 16S rRNA methylase-producinginternational clones of Acinetobacter baumannii in NorwayrdquoJournal of Medical Microbiology vol 60 no 4 pp 515ndash521 2011
[4] S Yezli A M Shibl and Z A Memish ldquoThemolecular basis of120573-lactamase production in Gram-negative bacteria from Saudi
Arabiardquo Journal of Medical Microbiology vol 64 no 2 pp 127ndash136 2015
[5] M Guzman-Blanco J A Labarca M V Villegas and EGotuzzo ldquoExtended spectrum 120573-lactamase producers amongnosocomial Enterobacteriaceae in Latin Americardquo BrazilianJournal of Infectious Diseases vol 18 no 4 pp 421ndash433 2014
[6] Y Pfeifer A Cullik andWWitte ldquoResistance to cephalosporinsand carbapenems in Gram-negative bacterial pathogensrdquo Inter-national Journal ofMedicalMicrobiology vol 300 no 6 pp 371ndash379 2010
[7] R Canton and T M Coque ldquoThe CTX-M beta-lactamasepandemicrdquo Current Opinion in Microbiology vol 9 no 5 pp466ndash475 2006
[8] C Dallenne A da Costa D Decre C Favier and G ArletldquoDevelopment of a set of multiplex PCR assays for the detectionof genes encoding important 120573-lactamases in Enterobacteri-aceaerdquo Journal of Antimicrobial Chemotherapy vol 65 no 3 pp490ndash495 2010
[9] Clinical and Laboratory Standards Institute (CLSI) ldquoPer-formance standards for antimicrobial susceptibility testingTwenty-fourth informational supplement performance stan-dards for antimicrobial susceptibility testingrdquoDocumentM100-24 Clinical and Laboratory Standards Institute (CLSI) WaynePa USA 2014
[10] J A Severin E S Lestari W Kloezen et al ldquoFaecal carriageof extended-spectrum 120573-lactamase-producing Enterobacteri-aceae among humans in Java Indonesia in 2001-2002rdquo TropicalMedicine and International Health vol 17 no 4 pp 455ndash4612012
[11] B Li J-Y Sun Q-Z Liu L-Z Han X-H Huang and Y-X Ni ldquoHigh prevalence of CTX-M 120573-lactamases in faecalEscherichia coli strains from healthy humans in Fuzhou ChinardquoScandinavian Journal of Infectious Diseases vol 43 no 3 pp170ndash174 2011
[12] G V Sanchez R N Master J A Karlowsky and J M BordonldquoIn vitro antimicrobial resistance of urinary Escherichia coliisolates among US outpatients from 2000 to 2010rdquo Antimicro-bial Agents andChemotherapy vol 56 no 4 pp 2181ndash2183 2012
[13] R H Bassyouni S N Gaber and A AWegdan ldquoFecal carriageof extended-spectrum 120573-lactamase- and AmpC- producingEscherichia coli among healthcare workersrdquo Journal of Infectionin Developing Countries vol 9 no 3 pp 304ndash308 2015
[14] D Mathai V A Kumar B Paul et al ldquoFecal carriage ratesof extended-spectrum 120573-lactamase-producing Escherichia coliamong antibiotic naive healthy human volunteersrdquo MicrobialDrug Resistance vol 21 no 1 pp 59ndash64 2015
[15] S B Joslashrgensen Oslash Samuelsen A Sundsfjord et al ldquoHighprevalence of faecal carriage of ESBL-producing Enterobacteri-aceae in Norwegian patients with gastroenteritisrdquo ScandinavianJournal of Infectious Diseases vol 46 no 6 pp 462ndash465 2014
[16] N O Yilmaz N Agus E Bozcal O Oner and A UzelldquoDetection of plasmid-mediated AmpC 120573-lactamase in E coliand K pneumoniaerdquo Indian Journal of Medical Microbiologyvol 31 no 1 pp 53ndash59 2013
[17] T M H Bui I Hirai S Ueda et al ldquoCarriage of Escherichiacoli producing CTX-M-type extended-spectrum 120573-lactamasein healthy Vietnamese individualsrdquo Antimicrobial Agents andChemotherapy vol 59 no 10 pp 6611ndash6614 2015
[18] S F Ahmed M M M Ali Z K Mohamed T A Moussa andJ D Klena ldquoFecal carriage of extended-spectrum 120573-lactamasesand AmpC-producing Escherichia coli in a Libyan communityrdquo
BioMed Research International 7
Annals of Clinical Microbiology and Antimicrobials vol 13article 22 2014
[19] Y J Ko H-W Moon M Hur C-M Park S E Cho andY-M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[20] H E Villar M N Baserni and M B Jugo ldquoFaecal carriage ofESBL-producing enterobacteriaceae and carbapenem-resistantGram-negative bacilli in community settingsrdquo Journal of Infec-tion in Developing Countries vol 7 no 8 pp 630ndash634 2013
[21] M H Al-Agamy M S Ali M M Salem and T R MohamedldquoFaecal colonization by extended-spectrum beta-lactamase-producing Escherichia coli from hospitalized patientsrdquo NewEgyptian Journal of Microbiology vol 19 pp 285ndash314 2008
[22] J Kaarme Y Molin B Olsen and A Melhus ldquoPrevalence ofextended-spectrum beta-lactamase-producing Enterobacteri-aceae in healthy Swedish preschool childrenrdquo Acta Paediatricavol 102 no 6 pp 655ndash660 2013
[23] A M Hammerum C H Lester L Jakobsen and L J PorsboldquoFaecal carriage of extended-spectrum 120573-lactamase-producingand AmpC 120573-lactamase-producing bacteria among Danisharmy recruitsrdquo Clinical Microbiology and Infection vol 17 no4 pp 566ndash568 2011
[24] A A Kader A Kumar and K A Kamath ldquoFecal car-riage of extended-spectrum120573-lactamase-producingEscherichiacoli and Klebsiella pneumoniae in patients and asymptomatichealthy individualsrdquo Infection Control and Hospital Epidemiol-ogy vol 28 no 9 pp 1114ndash1116 2007
[25] A Garrido C Seral M J Gude et al ldquoCharacterization ofplasmid-mediated 120573-lactamases in fecal colonizing patients inthe hospital and community setting in Spainrdquo Microbial DrugResistance vol 20 no 4 pp 301ndash304 2014
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
6 BioMed Research International
respectively 119887119897119886CTX-M-14 gene was the predominant ES120573Lgene detected in three (100) ES120573L-producing E coliisolates while 119887119897119886CMY-2 was detected in two AmpC enzyme-producing E coli isolates [23] In Spain the prevalence ofES120573L and AmpC enzyme carriers was 506 and 059respectively [25] 119887119897119886CTX-M genes were the ES120573L dominatinggenes (9615) and CTX-M-14 was the most prevalent gene(50) followed by CTX-M-15 (40) CMY-2 was the mostprevalent gene (8125) followed by DHA-1 (1875) [25]
MICs were determined for ES120573L- and AmpC enzyme-producing E coli (119899 = 13) isolates The results of MIC areshown in Table 2
In conclusion a high incidence of carriage of ES120573L-positive E coli fecal isolates among hospitalized patients inRiyadh was detected reaching 26 with 119887119897119886CTX-M-15 (846)being the most predominant gene The emergence of fecalcarriage of CMY-2-producing E coli among hospitalizedpatients has been reported to be 4These outcomes empha-size the importance of the intestinal tract as a reservoir forES120573L- and AmpC enzyme-producing E coli which may leadto nosocomial infection The admission of colonized fecalcarriers of ES120573L- and AmpC-positive E coli to the medicalsetting increases the possibility of other patients acquiringinfection in the same hospital Our results emphasize thenecessity for continuous surveillance in hospitals to detectthe ES120573L- AmpC enzyme- and carbapenemase-producingstrains andmultidrug strains as well applying effective strate-gies for antimicrobial therapy and infection control measuresto decrease the abuse and misuse of antimicrobial agentsagainst resistant strains and to prevent their spread
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
The authors extend their appreciation to the Deanship ofScientific Research at King Saud University for funding thework through the research group Project no RGP-038
References
[1] A Bhargava K Hayakawa E Silverman et al ldquoRisk factors forcolonization due to carbapenem-resistant enterobacteriaceaeamong patients exposed to long-term acute care and acute carefacilitiesrdquo Infection Control and Hospital Epidemiology vol 35no 4 pp 398ndash405 2014
[2] P-L Woerther C Burdet E Chachaty and A AndremontldquoTrends in human fecal carriage of extended-spectrum 120573-lactamases in the community toward the globalization of CTX-Mrdquo Clinical Microbiology Reviews vol 26 no 4 pp 744ndash7582013
[3] N Karah B Haldorsen N O Hermansen et al ldquoEmergenceof OXA-carbapenemase- and 16S rRNA methylase-producinginternational clones of Acinetobacter baumannii in NorwayrdquoJournal of Medical Microbiology vol 60 no 4 pp 515ndash521 2011
[4] S Yezli A M Shibl and Z A Memish ldquoThemolecular basis of120573-lactamase production in Gram-negative bacteria from Saudi
Arabiardquo Journal of Medical Microbiology vol 64 no 2 pp 127ndash136 2015
[5] M Guzman-Blanco J A Labarca M V Villegas and EGotuzzo ldquoExtended spectrum 120573-lactamase producers amongnosocomial Enterobacteriaceae in Latin Americardquo BrazilianJournal of Infectious Diseases vol 18 no 4 pp 421ndash433 2014
[6] Y Pfeifer A Cullik andWWitte ldquoResistance to cephalosporinsand carbapenems in Gram-negative bacterial pathogensrdquo Inter-national Journal ofMedicalMicrobiology vol 300 no 6 pp 371ndash379 2010
[7] R Canton and T M Coque ldquoThe CTX-M beta-lactamasepandemicrdquo Current Opinion in Microbiology vol 9 no 5 pp466ndash475 2006
[8] C Dallenne A da Costa D Decre C Favier and G ArletldquoDevelopment of a set of multiplex PCR assays for the detectionof genes encoding important 120573-lactamases in Enterobacteri-aceaerdquo Journal of Antimicrobial Chemotherapy vol 65 no 3 pp490ndash495 2010
[9] Clinical and Laboratory Standards Institute (CLSI) ldquoPer-formance standards for antimicrobial susceptibility testingTwenty-fourth informational supplement performance stan-dards for antimicrobial susceptibility testingrdquoDocumentM100-24 Clinical and Laboratory Standards Institute (CLSI) WaynePa USA 2014
[10] J A Severin E S Lestari W Kloezen et al ldquoFaecal carriageof extended-spectrum 120573-lactamase-producing Enterobacteri-aceae among humans in Java Indonesia in 2001-2002rdquo TropicalMedicine and International Health vol 17 no 4 pp 455ndash4612012
[11] B Li J-Y Sun Q-Z Liu L-Z Han X-H Huang and Y-X Ni ldquoHigh prevalence of CTX-M 120573-lactamases in faecalEscherichia coli strains from healthy humans in Fuzhou ChinardquoScandinavian Journal of Infectious Diseases vol 43 no 3 pp170ndash174 2011
[12] G V Sanchez R N Master J A Karlowsky and J M BordonldquoIn vitro antimicrobial resistance of urinary Escherichia coliisolates among US outpatients from 2000 to 2010rdquo Antimicro-bial Agents andChemotherapy vol 56 no 4 pp 2181ndash2183 2012
[13] R H Bassyouni S N Gaber and A AWegdan ldquoFecal carriageof extended-spectrum 120573-lactamase- and AmpC- producingEscherichia coli among healthcare workersrdquo Journal of Infectionin Developing Countries vol 9 no 3 pp 304ndash308 2015
[14] D Mathai V A Kumar B Paul et al ldquoFecal carriage ratesof extended-spectrum 120573-lactamase-producing Escherichia coliamong antibiotic naive healthy human volunteersrdquo MicrobialDrug Resistance vol 21 no 1 pp 59ndash64 2015
[15] S B Joslashrgensen Oslash Samuelsen A Sundsfjord et al ldquoHighprevalence of faecal carriage of ESBL-producing Enterobacteri-aceae in Norwegian patients with gastroenteritisrdquo ScandinavianJournal of Infectious Diseases vol 46 no 6 pp 462ndash465 2014
[16] N O Yilmaz N Agus E Bozcal O Oner and A UzelldquoDetection of plasmid-mediated AmpC 120573-lactamase in E coliand K pneumoniaerdquo Indian Journal of Medical Microbiologyvol 31 no 1 pp 53ndash59 2013
[17] T M H Bui I Hirai S Ueda et al ldquoCarriage of Escherichiacoli producing CTX-M-type extended-spectrum 120573-lactamasein healthy Vietnamese individualsrdquo Antimicrobial Agents andChemotherapy vol 59 no 10 pp 6611ndash6614 2015
[18] S F Ahmed M M M Ali Z K Mohamed T A Moussa andJ D Klena ldquoFecal carriage of extended-spectrum 120573-lactamasesand AmpC-producing Escherichia coli in a Libyan communityrdquo
BioMed Research International 7
Annals of Clinical Microbiology and Antimicrobials vol 13article 22 2014
[19] Y J Ko H-W Moon M Hur C-M Park S E Cho andY-M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[20] H E Villar M N Baserni and M B Jugo ldquoFaecal carriage ofESBL-producing enterobacteriaceae and carbapenem-resistantGram-negative bacilli in community settingsrdquo Journal of Infec-tion in Developing Countries vol 7 no 8 pp 630ndash634 2013
[21] M H Al-Agamy M S Ali M M Salem and T R MohamedldquoFaecal colonization by extended-spectrum beta-lactamase-producing Escherichia coli from hospitalized patientsrdquo NewEgyptian Journal of Microbiology vol 19 pp 285ndash314 2008
[22] J Kaarme Y Molin B Olsen and A Melhus ldquoPrevalence ofextended-spectrum beta-lactamase-producing Enterobacteri-aceae in healthy Swedish preschool childrenrdquo Acta Paediatricavol 102 no 6 pp 655ndash660 2013
[23] A M Hammerum C H Lester L Jakobsen and L J PorsboldquoFaecal carriage of extended-spectrum 120573-lactamase-producingand AmpC 120573-lactamase-producing bacteria among Danisharmy recruitsrdquo Clinical Microbiology and Infection vol 17 no4 pp 566ndash568 2011
[24] A A Kader A Kumar and K A Kamath ldquoFecal car-riage of extended-spectrum120573-lactamase-producingEscherichiacoli and Klebsiella pneumoniae in patients and asymptomatichealthy individualsrdquo Infection Control and Hospital Epidemiol-ogy vol 28 no 9 pp 1114ndash1116 2007
[25] A Garrido C Seral M J Gude et al ldquoCharacterization ofplasmid-mediated 120573-lactamases in fecal colonizing patients inthe hospital and community setting in Spainrdquo Microbial DrugResistance vol 20 no 4 pp 301ndash304 2014
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 7
Annals of Clinical Microbiology and Antimicrobials vol 13article 22 2014
[19] Y J Ko H-W Moon M Hur C-M Park S E Cho andY-M Yun ldquoFecal carriage of extended-spectrum 120573-lactamase-producing Enterobacteriaceae in Korean community and hos-pital settingsrdquo Infection vol 41 no 1 pp 9ndash13 2013
[20] H E Villar M N Baserni and M B Jugo ldquoFaecal carriage ofESBL-producing enterobacteriaceae and carbapenem-resistantGram-negative bacilli in community settingsrdquo Journal of Infec-tion in Developing Countries vol 7 no 8 pp 630ndash634 2013
[21] M H Al-Agamy M S Ali M M Salem and T R MohamedldquoFaecal colonization by extended-spectrum beta-lactamase-producing Escherichia coli from hospitalized patientsrdquo NewEgyptian Journal of Microbiology vol 19 pp 285ndash314 2008
[22] J Kaarme Y Molin B Olsen and A Melhus ldquoPrevalence ofextended-spectrum beta-lactamase-producing Enterobacteri-aceae in healthy Swedish preschool childrenrdquo Acta Paediatricavol 102 no 6 pp 655ndash660 2013
[23] A M Hammerum C H Lester L Jakobsen and L J PorsboldquoFaecal carriage of extended-spectrum 120573-lactamase-producingand AmpC 120573-lactamase-producing bacteria among Danisharmy recruitsrdquo Clinical Microbiology and Infection vol 17 no4 pp 566ndash568 2011
[24] A A Kader A Kumar and K A Kamath ldquoFecal car-riage of extended-spectrum120573-lactamase-producingEscherichiacoli and Klebsiella pneumoniae in patients and asymptomatichealthy individualsrdquo Infection Control and Hospital Epidemiol-ogy vol 28 no 9 pp 1114ndash1116 2007
[25] A Garrido C Seral M J Gude et al ldquoCharacterization ofplasmid-mediated 120573-lactamases in fecal colonizing patients inthe hospital and community setting in Spainrdquo Microbial DrugResistance vol 20 no 4 pp 301ndash304 2014
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology