world journal of pharmaceutical research aziz et al. sjif

www.wjpr.net Vol 5, Issue 5, 2016.

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Aziz et al. World Journal of Pharmaceutical Research

GENETIC ELEMENTS RESPONSIBLE FOR EXTREME DRUG

RESISTANCE (XDR) IN KLEBSIELLA PNUMONIAE VAR PNUMONIAE

ISOLATED FROM CLINICAL SAMPLES OF IRAQI PATIENTS

Rawa Abdul Redha Aziz1*

and Sawsan Sajid Al-Jubori2

1Dept. of Biology, Collage of Science, Kufa University, Najaf, Iraq.

2Dept. of Biology, Collage of Science, Al-Mustansiriyah University, Baghdad, Iraq.

ABSTRACT

Twenty four isolates primarily diagnosed as Klebsiella pneumoniae

were isolated from different samples from patients submitted to

Baghdad hospitals\Iraq. Bacterial diagnosis was performed using

complementary Vitek system. A house keeping gene(primers were

designed for the 16s and 23s rRNA region) was used for genotypic

diagnosis with amplified size 639bp.The results of antimicrobial

susceptibility testing (disc diffusion method) revealed that most

isolates displayed extremely high drug resistance (XDR) patterns. The

highest resistance percentage was towards Ampicillin (95.83%)

followed by Ceftazidime (87.50%) compared with other used antibiotic

groups. Results also reveal that 16\24 (66.67%) isolates were resistant

to Colistin sulphate, while only 2\24 (8.33%) isolates were resistant to Tigecycline. The MIC

test for polymyxin B revealed that 22/24 (91.67%) isolates were resistant to ˃32µg/ml of this

antibiotics. The results of modified Hodge test and rapid ESβLs production test revealed

positivity of 100%(24/24) and 58.33% (14/24), respectively. About genotypic screening test

for antibiotic resistance genes, the results revealed that the highest percentage were towards

aminoglycoside modifying enzyme group type aac(6)-Ib followed by aac(3)-I (100% and

75%, respectively) as compared with other genes in the same group. For efflux pump gene

about 88% of the isolates were harbored mexX gene and only 29% harbored mexY. Also

16.67% of the isolates harbor the metalo β-lactamase gene blaIMP-1 while none of the

isolates were positive to blaVIM gene. The ESBLs genes bla SHV, bla AMP-C, and bla CMY

revealed 58.33%, zero%, and 33.33% among K. pneumonia local isolates tested in this study.

The highest percentage was towards rmtF (66.67%) followed by rmtB (54.17%) as compared

World Journal of Pharmaceutical Research SJIF Impact Factor 6.805

Volume 5, Issue 5, 01-23. Research Article ISSN 2277– 7105

*Corresponding Author

Dr. Rawa Abdul Redha

Aziz

Dept. of Biology, Collage of

Science, Kufa University,

Najaf, Iraq.

Article Received on

26 Feb 2016,

Revised on 15 March 2016,

Accepted on 08 April 2016

DOI: 10.20959/wjpr20165-6020

http://www.wjpr.net/


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with other gene in the methylation group. Some of the isolates encode the quinolones

resistance genes ParC2 and GyrA (54.17% and 75%, respectively). For oxacillinace genes

(OXA58, OXA51, OXA24, and OXA23), only OXA 23 was detected (8.33%) among K.

pneumonia isolates. As a conclusion, most isolates harbor more than one resistant

mechanisms which might considered a sign for emergency pan drug resistant not only XDR

pattern.

KEYWORDS: XDR Klebsiella pneumonia, ESBLs, Hodege test, Different resistance

mechanism.

1. INTRODUCTION

K. pneumonia is opportunistic pathogen can be carried asymptomatically in the intestinal trac,

skin, nose and throat of healthy individuals but can also cause a range of infections in

hospitalized patients, most commonly pneumonia, wound, soft tissue, or urinary tract

infections.[1]

Increasing reports of infections due to multidrug resistance (MDR) and

extremely drug resistance XDR fermentative Enterobacteriaceae such as Klebsiella spp.

might become the worst health problems and issues.[1,2]

It is pointed out that MDR pathogens

could be resistant towards 3-5 antibiotic classes such as antipseudomonal cephalosporins,

antipseudomonal carbapenems, β-lactam–β-lactamase inhibitor, fluoroquinolones, and

aminoglycosides, while XDR pathogens are susceptibility to only 2 or less antibiotic

classes.[3]

However, PDR (pan drug resistance) pathogens have no options for treatment as

they diminished susceptibility to all classes.[3]

MDR Klebsiella pneumonia remains

susceptible only to more agents such as the polymyxins, and it convert to PDR if they resist

this antibiotic.[3]

Usually Klebsiella pneumonia has the ability to resist wide range of

antibiotics via different mechanisms including production of Extended spectrum β-

lactamases (ESBLs)and carbapenemases, production of aminoglycoside-modifying enzymes,

over expression of efflux pumps, reduction in porin channels, and target-site alteration

including changing in penicillin binding proteins and topoisomerases.[4]

K. pneumoniae is

resistant to penicillins extended-spectrum β-lactamase, and expanded-spectrum

cephalosporins through the production of β-lactamases that are encoded mainly by the bla

SHV, bla TEM, and amp C genes. β-lactams such as imipenem and meropenem, which are

highly resistant to hydrolysis by TEM, SHV, and AmpC β -lactamases remain effective

antibiotic options.[4]

Klebsiella pneumonia contain many plasmids that differ in numbers and

molecular weight, carrying different types of genes including those encoding extended-



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spectrum β-lactamases (ESBLs), AmpC β-lactamases, inhibitor resistant TEM β-lactamases

and metallo β-lactamase enzymes. These enzymes confer resistance to various antimicrobial

agents including the third and fourth generation cephalosporins, cephamycins, monobactam

βlactamase, β-lactamase/inhibitor combinations and carbapenems.[5]

Due to the dearth of

antimicrobial effect with novel mechanisms of action, very few options remain to treat the

infections caused by XDR pathogens.[6]

Klebsiella pneumonia has emerged as a common

cause of serious epidemic and nosocomial infections in hospitals, resulting in high morbidity

and mortality.[3,6]

ESBL-producing K. pneumoniae is resistance to other antibiotics, including

fluoroquinolones, aminoglycosides, trimethoprim, and sulfamethoxazoles.[7]

It is also

reported that clinical outcome in bacteraemic infections caused by ESBL-producing K.

pneumoniae appears to be worse than that of patients with non-ESBL-producing isolates.[8]

This prevalence of ESBLs has limited the available therapeutic options and necessitated the

increased use of carbapenems against Klebsiella pneumoniae infections. In response to use of

carbapenems, carbapenem resistance has been emerged.[9]

Modified Hodge test (MHT) has

been widely used as primary screening test for carbapenemase activity.[10]

In our study, we

focus on the detection of Iraqi extensively drug-resistant (XDR) among the isolates and

studying their resistance pattern towards different antimicrobial groups by PCR.

2. MATERIAL AND METHODS

2.1. Collection and diagnosis of Bacterial isolates Twenty four isolates of Klebsiella were

obtained from patients admitted to several teaching hospitals in Baghdad during a period

between March 2015 to September 2015. They were collected from the midstream urine from

patients with urinary tract infections (5 isolates),from bacteraemia (7 isolates), wound swabs

from Burn unit (4 isolates), 2 isolates were collected form sputum , ear, and pus (for each ),

finally 1 isolate collected from both of wounds infections and stool. Isolates were re-

diagnosed by Vitek 2 compact automated system (Biomeriux, USA), and the probability was

99% Klebsiella pneumonia ssp pneumonia.

2.2 Genotyping detection for isolates Specific primers were designed for the 16srRNA and

23s rRNA region (housekeeping gene, amplified size was 639 bp) using Geneious Software/

primer 3. The sequence for the forward primer was 3′ TGTACACACCGCCCGTC-5′ and for

the reverse was R-3′GGTACTTAGATGTTTCAGTTC-5′. Total DNA was extracted using

Genomic DNA Extraction Kit (Wizards, Promega, USA) following manufacture’s protocol

and was used as a template for the PCR process. The mixture of PCR composed from 12.5 of



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GoTaq®Green Master Mix (2x), 5 µl template DNA, 1.5 µl primers (for each) final

concentration 0.6pmol/µl and nuclease free water up to 25 µl (4.5 µl). PCR products were

stored at -20 ºC, then nucleotides sequence was carried out at NICEM Company, USA.

Results of sequencing were analyzed using genious soft ware 7.01R compareid with the

NCBI standered strain.

2.3. Antimicrobial sensitivity tests: Resistance tested for the isolates towards thirty types of

different antibiotics including β- Lactams, aminoglycoside, tetracyclines and quinolones

groups besides other groups. Susceptibility was determined based on the interpretative

criteria recommended by the Clinical and Laboratory Standards Institute (CLSI).[11]

The

antibiotics were: from β- Lactams, Ampicillin (25mcg), Piperacillin(100µg), Cefixime (5µg),

Cefazolin(30µg), Cephalothin(30µg), Cefotaxime(30µg), Ceftriaxone(30µg),

Ceftazidime(30µg), Cefepime(30µg), Amoxicillin/clavulanic acid (20/10µg), Ticarcillin-

clavulanic acid(75/10µg), Aztreonam(30µg), Imipenem(10µg), Meropenem (10µg) and

Doripenem(10µg). In the Aminoglycoside group: Kanamycin(30 µg), Gentamycin (10µg),

Tobramycin(10µg), Netilmicin(30µg) and Amikacin(10µg). The quinolones group include

Naldixic acid(30µg), Levofloxacin(5µg), Gatifloxacin(5µg), Ofloxacin(5µg). Other

antibiotics include sulpha group: Trimethoprim (5µg) and Trimethoprime-

Sulphamethoxazole (1.25/23.75µg), and Tetracyclines: Tetracycline(30µg) and

Doxycycline(30µg). Tigecycline(15µg) and colistin sulphate(25µg) were also tested.[12, 13]

Polymyxin B was also used to achieve the minimum inhibitory concentration (break point

2µg/ml).[14]

E. coli HB101 used as the negative control strain .Phylogenic analysis for

antibiotic susceptibility pattern was done using Tamura –Nei genetic destine model and

UPGMA tree build method in order to analyze antibiogram similarity.

2.4: Indirect three dimensional agar diffusion method to detect ESBLs The detection of

ESBLs production was performed using modified indirect three dimensional method in which

Muller Hinton Agar (MHA) plates were seeded with a lawn of a standard strain E.coliHB101

adjusted to MacFarland (0.5) standard. A slit was performed inside the MHA plate using a

sharp scalpel emerged with the test isolate. The following discs were placed 2mm away

towards the performed line. They were amoxicillin/clavulanic acid(20/10µg), ceftazidime(30

µg), ceftriaxone(30 µg), and cefexime(5µg). The plate was incubated at 37ºC for16-18 hours.

The distortion of inhibition zone around the antibiotic indicates ESBLs production.[15]



5


2.5: Modified Hodge test (MHT) Modified Hodge test (MHT) was performed according

to[10]

for detection of carbapenemase production. Briefly, 5 ml of brain heart infusion broth

culture for Escherichia coli HB101 equal to 0.5 McFarled was prepared then 100μl from.

1:10 dilution was streaked as lawn on to a Mueller Hinton agar plate. Imipenem susceptibility

disk(10μg/disc) was placed in the center of the test area. The test isolate was streaked in a

straight line from the edge of the disk to the edge of the plate. The plate was incubated

overnight at 37º C for 24 hours. After 24 hours, MHT positive test (carbapenemase producer)

showed a clover leaf-like indentation of the Escherichia coli 25922 growing along the test

organism growth streak within the disk diffusion zone. MHT negative test represent by

normal growth for the standard strain or without the formation of clover leaf-like shape.

2.6: Detection of antibiotic resistant genes using PCR amplification technique All the

twenty four isolates were subjected to molecular screening study using PCR amplification

technique to detect XDR profile. Different primers (table 2) were used. PCR mixture was

composed from 5μl template DNA which was extracted using Genomic DNA Extraction Kit

(Wizards, Promega, USA), 12.5μl of GoTaq®Green Master Mix, 1.5μl from forward and

reverse primers (final concentration 0.6pmol\ μl).The volume was completed to 25μl with

nuclease free water. PCR was run under the following conditions: primary denaturation step

95°Cfor 5 min; 30 -40 repeated cycles (according to gene listed in table 2)of 94°C for 30sec ,

43- 62 °C annealing temperature (table-2) for 60 sec and 72°C for 1 min then final extension

step at 72 °C for 6 min. PCR products were electrophoresed in 1% agarose gel and visualized

under UV ligh.[16]



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Table 2: Primers used for detection antibiotic resistant genes.

Ref. Tm Size product Sequences (5'_3')

R

Sequences (5'_3')

F Primer

This study 59 639 R-GGTACTTAGATGTTTCAGTTC F-TGTACACACCGCCCGTC 16s RNA& 23s RNA

Lianes et.al.,[17]

62 326 R- GAT CTG CTC GAC GCG GGT

CAG CG

F- TGA AGG CGG CCC TGG ACA TCA

GC mexX

Xavier et.al.,[18]

59 250 R-AGCGGGATCGACCAGCTTTC F- CCGCTACAACGGCTATCCCT MexY

Xavier et.al.,[18]

43 227 R- GGCATACGGGAAGAAGT F-AGCCCGCATGGATTTGA aac(3)-I

Ndegwa et.al.,[19]

56 482 R- CTC GAA TGC CTG GCG TGT TT F- TTG CGA TGC TCT ATG AGT GGC

TA aac(6)-Ib-

Ndegwa et.al.,[19]

57 364 RGGAACAAGACCCGTTCAATTCAA

TTCATCAAGTTT

FGACGACGACAAGGATATGGAATTG

CCCAATATTATT ant(4’)IIb-

Haldorsen et.al.,[20]

55 800 R-CAC GCG GGG AAA CGC GAG AA F-TAT CTC GGC GGC GGT CGA GT aph(3′)VI

Vaziri et.al.,[21]

59 200 R-TTCAGTTCCGTTTCCCAGCGG F-AAGATCCACTATCGCCAGCAG blaSHV

Vaziri et.al.,[21]

58 1150 R-GTTGGGGTAGTTGCGATTGG F-ATGCAACAACGACAATCCATC blaAmpC

Oliveira et.al.,[22]

50 1014 R-TGGAACGAAGGCTACGTA F-GACAGCCTCTTTCTCCACA blaCMY

Ramazanzadeh et. al.,[23]

55 500 R-AACCAGTTTTGCCTTACCAT F-CTACGCCAGCAGAGTCTTTG IMP-1

Mendes et.al.,[24]

57 382 R-AATGCGCAGCACCAGGATAG F-GTTTGGTCGCATATCGCAAC VIM

Haldorsen et.al.,[20]

58 635 R-TTG CTT CCA TGC CCT TGC C F-CTA GCG TCC ATC CTT TCC TC RmtA

Doi et.al.,[25]

59 584 R-CTC AAA CTC GGC GGG CAA GC F-CCC AAA CAG ACC GTA GAG GC RmtB

Mendes et.al.,[24]

53 711 R-ATC CCA ACA TCT CTC CCA CT F-CGA AGA AGT AAC AGC CAA AG RmtC

Tijet at.al., [26]

52 500 R-CGATGCGACGATCCATTC F-TCAAAAAGGAAAAGGACGTG RmtD

Lee et.al.,[27]

51 453 R-GGCAGGAGCTTCATCAGAA F-GCGATACAGAAAACCGAAGG RmtF

Morovat et.al., [28]

53 353 R5'-GGATTGCACTTCATCTTGG-3' F 5'-TAATGCTTTGATCGGCCTTG-3' OXA 51


53 599 R 5'-CCCCTTGCGCTCTACATAC-3 F 5'-AAGTATTGGGGCTTGTGCTG-3 OXA58


53 501 R 5'-ATTTCTGACCGCATTTCCAT-3 F 5'-GATCGGATTGGAGAACCAGA-3 OXA23


53 246 R 5'-AGTTGAGCGAAAAGGGGATT-3 F 5'-GGTTAGTTGGCCCCCTTAAA-3' OXA24

This study 57 886 R-TTCCGGGAACTCGGTTAACG F-GGCATTTATTCGTCACCGCC gyr A1

This study 57.1 314 R-TGATTTCACCTGAGGACGGC F-GTTACCGTATGCGAGCGGTA Par C2



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3. RESULTS AND DISCUSSION

Klebsiella pneumoniae is a leading cause of hospital-acquired (HA) infections and serious

community-acquired (CA) infections ,including pyogenic liver abscess, pneumonia, and

meningitis.[3]

In the current study , all the 24 isolates were diagnosed depending on a

housekeeping gene 16S rRNA gene and 23SrRNA and gave a positive result (figure 1) .The

amplified DNA segment (figure 2) as compareid with the standered strain Klebsiella

pneumoniae subsp.pneumoniae KPNIH29: CP009863 (NCBI) was exactely 639 bp start

from 18,101 bp to 17,467 bp in the genomic map and passing throug 3 important

housekeeping genes (red area) the 16s rRNA, tRNA-Glu and 23s rRNA. Pariwise identity

was 91% which represent the percentage of residues that are identical in the alignment. Some

differences were recognized between the local isolate and the recorded NCBI strain as clear

with gaps in the upper green identity line. The reason of choosing 16S rRNA gene to identify

Klebsiella pneumonia isolates in this study is that the widespread use of this gene sequence

for bacterial identification and taxonomy of many non cultured bacteria, and the fact that it is

an essential part of the description of a novel organism.[28]

Figure(1): Agarose gel electrophoresis (1% agarose, 5 V/cm for 90min) for 16SrRNA

gene of Klebsiella pneumonia isolates .lane M : 100bp DNA ladder, lanes 1,2,3,4,5,6, and

7 are positive results with 639bp amplicon of Klebsiella pneumonia blood 1,2, 3, 4, 5, 6,7.

Lanes ,10 ,11 and 12 are positive results with 639bp amplicon of Klebsiella pneumonia

Urine 1, 2, and 3; lane 8 and 9 show negative results.



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Figure 2: The housekeeping genes used for diagnosis localKlebsiella isolates.

Antibiotics are widely used in clinical settings whether for treating simple cases or life-

threatening infections caused by microorganisms. Now a days, many antibiotic resistant

gram negative bacteria come up and have made global concern such as Klebsiella

pneumonia.[29]

The broad use of antibiotics had created a strong selective pressure, which

consistently had resulted in the survival and spread of resistance that has evolved with the

increased number, volume and diversity of antimicrobial applications. Among the 24 isolates,

rate of resistance (figure3)for penicillin group the rates were 95.83% for ampicillin, 79.17%

for piperacillin, 58.33% for ticarcillin-clavulante, 70.83% for amoxicillin/clavulanic acid.

While for cephalosporins ,the percentage of resistance were 87.50% for ceftazidime, 83.33%

for cefotaxime, 79.17% for ceftriaxone, 79.17% for cefepime, 75.00% for cefazolin, 66.60%

for cephalothin, 58.33% for cefixime. As for Monobactam the rat was 62.50% for aztreonam.

Resistance towards aminoglycoside group were38%, 20.83%, 4.17% and 29.17% for

kanamycin, gentamycin, amikacin, and tobramycin, respectively, but none of the isolates

were able to resist netilmicin. Tetracyclines was also tested and the results revealed that

62.50% of the isolates showed resistance for tetracycline and the rate decreased to 58.33%

for doxycycline. In addition, flouroquinolones sensitivity test showed that 20.83% of the

isolates resisted each of gatifloxacin, florofloxacin, and levofloxacin, while nalidixic acid

resistance was 66.67%. Finaly rate of resistance toward thrimethoprim and trimethoprime-

sulfamethoxazole were 70.83% and 70.80%, respectively. Similar work was done by Radha

et.al where antibiotic sensitivity test of 25 K. pneumoniae isolates ranged between

intermediate and resistant according to the National committee for clinical laboratory

standard (NCCLS).[29]

Moreover, the results of the current study revealed that there was no



9


resistant isolates towards netlimicin, meropenem, and doripenem, however, 29.17% of

Klebsiella pneumoniae isolates were resistant to imipenem.It has been revealed that, ESBL

producers showed resistance pattern towards imipenem due to the production of

carbapenemase, so these bacteria cannot be eliminated by carbapenems that are the usual

drug of choice against them.[30]

It is reported that 62 (50%) K. pneumonia isolates resistant

to imipenem.[31]

It is important to reveal that 16(66.67%) K. pneumonia isolates (K. pne.b4,

b5, b6, b7, U2, U3, U4, U5, S2, W1, ES2, Bu1, Bu2, Bu3, Bu4, and P1) were resistant to

colistin sulphate, while only two(8.33%) isolates (K. pneumonia b5 and W1) were resistant

to a tigecycline. It was pointed out that tigecycline, a broad-spectrum glycylcycline, is

tigecycline is considered as therapeutic option for MDR gram positive and gram negative

bacteria, and it was found to be effective against Klebsiella infections.[31]

However, Rhee

found that their local Klebsiella pneumoniae collected from patients with diabetes mellitus

and chronic renal failure on He modialysis in South Korea displayed intermediate pattern to

tigecyline, so he concluded that the isolates could be defined as XDR.[32]

Galani et. al.

showed that colistin sulphate was less active against Klebsiella pneumonia tested in their

study[13]

although colistin sulphate (polymyxin E) has been considered an effective

therapeutic option for such XDR pathogen.[33]

Finally, minimum inhibitory concentration

test(MIC) of Polymyxin revealed that 91.67% (22/24) isolates were resistant at 32µg/ml

(break point ≥2µg/ml), leading to conclude that K. pneumoniae isolates are PDR pathogens.

This conclusion is supported in literature, for example Falagas and his team found that the

mortality rate was 41.7% among hospitalized patients at a tertiary-care center when tested 23

Klebsiella pneumoniae isolates. They conclude that PDR gram-negative bacterial infections

are associated with considerable mortality, although not as high as expected given the fact

that the isolates were resistant to all tested antibiotics, including polymyxins.[14]

It was clear

that each isolate showed its ability to resist more than one antibiotic group. As illustrated in

figure(4) each isolates of K. pne. B5, ES2, and Bu2were able to resist 23-24 antibiotics

related to different groups followed by K. pne. W1andP1 that were resistant to 21 antibiotics

as compared with other local isolates tested.



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Figure 3: the percentage of different antibiotic groups against Klebsiella pneumonia.

Tetracycline (T), Meropenem (MEM), Cefepime (FEP), piperacillin (PRL), Aztreonam

(ATM), Amikacin (AK), Ticarcillin +Clavulanic acid (TIM), Trimethoprim I, Levofloxacin

(LEV), Naldixic acid (NA), Imipenem (IPM), Gentamicin (CN), Cephalothin(KF),

Ceftazidime (CAZ), Ampicillin (AP), Kanamycin (K), Gatifloxacin (GAT), Cefazolin (CZ),

Cefixime(CFM), Tobramycin(TOB), Ofloxacin (OFX), Trimethoprim +Sluphomethoxazole

(SXT), Doripenem (DOR), Doxycycline (DXT), Cefotaxime (CTX), Ceftriaxone (CRO),

Amoxicillin+Clavulanic acid (AMC), netilmicin (NET).

Figure 4: illustrate number of resisted antibiotics related to different by K. pneumonia



11


Figure 5, dendrogram illustrating phylogenic analysis of Antibiotic sensitivity test toward 30

different antibiotics using Global alignment \Tamura-Nei (UPGMA) in Geneious software .

The tree was with 47 nodes and 24 Tips. The data is separated into 2 groups (A and B). The

phylogenic tree of the current study reflects a high diversity between isolates. It is clear that

cluster A2 shows K.pne U4 having different antibiogram pattern compared with other

isolates in the same group. Similarities in antibiogram pattern on the other hand were

appeared in cluster B1b2, precisely K. pne. B5 and K. pne. Bu2since they did not have

differences and the coefficient was (0). Also, similar phenotypic pattern was noticed between

K.pneb1 and b3(B1a cluster) making them segregated within the same clone (red color). The

isolate K.pne .p2 at (0.259) bosses the most differ pattern hence it segregate alone as with the

stander strain.

Figure 5: The phylogenic tree for antibiogram phenotype(47 nodes and 24 Tips) for

local K. pneumonia isolates tested.

Modified Hodge test were performed since this test has been widely used fo rscreening of

carbapenemase activity.[34]

As it is known that carbapenemases (KPC) are β-lactamase

enzymes that inactivate the carbapenems (ertapenem, imipenem andmeropenem), and these

are responsible for many epidemicsin Greece, India, and USA, especially when

carbapenemase producers spread in patients with identical risk factors (patients receiving

broad-spectrum anti-biotherapy, patients in intensive care units, immune compromised

patients, transplant patients, surgical patients.[35]

Therefore, early identification of

carbapenemase producers in clinical infections is mandatory to prevent development of those



12


hospital-based outbreaks.[36]

All the isolates gave a positive result (100%). Figure( 6 )shows

some of the positive tested isolates presented in leaf –shape.

Figure 6: positive results of Modified Hodage test for Klebsiella pneumonia. Isolates no.

1,3,5,13, and 20 showed positive results (leaf shape), while no 16 showed negative

results.

Moreover, it is revealed that K. pneumoniaeis identified to be the mostcommon entero

bacterial species for spreading ESBL genes in health care facilities during the past 30

years.[36, 37]

Resistance to fluoroquinolones, co-trimoxazole, and trimethoprim is frequently

observed among ESBLs producers.[6]

Therefore, results of screening for extended spectrum β-

lactmases via the modified indirect method in this study showed14 isolates (58.33%) were

positive in that 11 isolates (45.83%) were resistant to Amoxicillin/clavulanic acid, 9 (37.5%)

isolates were resistant to Ceftriaxone, 8 (33.33%) to Ceftazidime, 4 (16.67%) were resistant

to Cefixime. Similar work has been done by Dhara et. al., however, their results demonstrated

81.48%ESBLs prevalence among (54 isolates) and the susceptibility pattern of ESBL positive

isolates showed all 2nd

generation cephalosporins, 3rd

generation cephalosporins

andaztreonam were resistant.[38]

Also it has been reported that 148 (61%) of K. pneumoniae

isolates from different areas in Iran were ESBLs producing.[39]

It has been reported that the increasing clinical incidence of antibiotic-resistant bacteriais a

major global health care issue. It has been grounded on data that characterization ofantibiotic

resistance determinants at the genomic level plays a critical role in understanding, and

potentially controlling, the spread of MDR or XDR pathogens. Of particular concern is the

spread of resistant Klebsiella pneumoniae which can harbor both extended-spectrum β-



13


lactamases (ESBL)and carbapenemases capable of hydrolyzing newer carbapenem drugs.

Frequently associated with ESBL-producing K. pneumonia isresistance to other antibiotics,

including fluoroquinolones, aminoglycosides, trimethoprim, and sulfamethoxazoles.[40, 30]

Therefore, many genes related to different antibiotic resistance mechanisms were tested.

Figure 7 represents 1:aac(6)-Ib, 2:aac(3)-I, 3: ant(4’)IIb-, and 4:aph(3′)VI, genes related to

aminoglycoside modifying enzymes.5:mexX and 6:mexY, the efflux pump genes. 7: blaIMP-

1 represent MBL gene 8: multiplex oxacillinase genes (OXA 58, OXA 51, OXA 24, and

OXA23). 9: 16s ribosomal methylation enzymes such as rmtA, 10: rmtD, 11: rmtF,

12:multiplex of rmtB and the ESBLs encoding gene bla SHV. 13: bla CMY. Finally,

14:ParC2 and 15: GyrA1, quinolone resistance genes.

1 2

3 4

5 6



14


7 8

9 10

11 12

13 14



15


15

Figure 7: Agarose gel electrophoreses(1% agarose, 50 V/cm for 90 min) for some

amplified virulence genes: 1) aac(6)-Ib (482 bp), 2) acc(3)-I (227bp), 3)

ant(4’)IIb(364bp), 4) aph(3)-VI (800bp), 5) MexX (326bp), 6) MexY (250bp), 7) bla IMP-1

(500bp), 8)multiplex OXA (58, 51, 24, and 23) (599, 353, 246, and 501bp), 9)rmtA(635bp),

10) rmtD (500 bp), 10) rmtF (453bp), 12)multiplex rmtB (584 bp) and bla SHV (200bp),

13)blaCMY (1014bp), 14)Par C2 (314bp), and 15) Gyr A1 (886 bp).

Results revealed that all the isolates harbored more than one resistant gene .The most

dominant gene among aminoglycoside modifying enzymes tested was aac(6)-Ib(100%),

followed by aac(3)-I(75%), ant(4’)Iib- (45.83%) and the amikacin resistance geneaph(3)-VI

also called (aphA6 ) (8.33%) (figure 8). It has been known that aminoglycoside are

important options for the treatment of infection caused by carbapenem- resistant K.

pneumonia strains. The inactivation by enzymatic modification is the most prevalent

mechanisms of resistance specially class aph(3)-VI and ant(4’)Iib- confer high- level

resistance towards amikacinand are infrequently encountered. Althoughaac (6)Ib is common

among Enterobacteriaceae, the clinical relevance is largely unknown.[41]

Aac(6)-Ib, the most

prevalent aminoglycoside modifying enzyme that confers resistance to tobramycin,

kanamycin, and amikacin was first identified in K. 15arboring isolates in 1986.[42]

Bremmer

reported that 20(70%), aac(6)Ib positive K. pneumonia isolates were resistant to amikacin,

however, majority showed regrowth supporting the idea that aac(6)-Ib confer intermediated

amikacin resistant.[41]

The percentage of Efflux pump genes were 88% and 29.17% for mexX and mexY,

respectively (figure 8). mexX and mexY (RND; resistance-nodulation-division) are located on

operon within genomic chromosome of Klebsiella pneumonia.[43]

There are increasing

evidence that the role of efflux pumps in antibiotic resistance in bacteria is significant.[43]

Despite high- level resistance may not occur as a result of MDR efflux pumps alone, the



16


association of over- expression of these genes amongst highly resistant clinical isolates

cannot be ignored.[44]

The percentages of resistance for16s ribosomal methylation enzymes were rmtA (20.83%),

rmtB (54.17%), rmtC (zero%), rmtD(33.3%), and rmtF (66.67%) (figure 8). Those enzymes

confer high level resistance to most clinically useful aminoglycosides by inhibiting their

access to the site of action.[45]

As can be seen here, the most dominant gene among local

isolates was rmtF that confers high-level resistance to most clinically relevant

aminoglycosides. This resistance determinant appears to be spreading quickly among

enterobacteria associated with other emerging resistance mechanisms, including NDM-1

carbapenemase.[27]

Morover, rmtB has been detected in various species belonging to the

family Enterobacteriaceae, including K. pneumonia, however, only 0.3% presence of rmtB

among K. pneumonia isolates in Taiwan screened by amikacin and confirmed by PCR.

Detection of this resistance mechanism may pose a challenge in clinical laboratories.[24]

The multiplex oxacillinase genes (OXA 58, OXA 51, OXA 24, and OXA23) showed positive

results for only OXA 23 (8.33%) (figure 7). It is reported that OXA-23 confers high-level

carbapenem resistance.[45]

The emergence of carbapenem-resistant K. pneumoniae strains will

have a serious impact on remaining therapeutic options. β-Lactamase producing strains are

clinically significant, as they are difficult to treat.[46]

Moreover, different types of ESBLs and MBLs arethe major enzymes that are increasing in

pathogenic microorganisms worldwide.[30]

The rate of MBL genes in this study was 16.67%

for blaIMP-1 and zero % for blaVIM (figure 7). Also the results showed ESBLs encoding

genes bla AMP-C for which no positive results (0.00%), however, bla SHV and bla CMY

showed positivity (58.33% and 33.33%, respectively). Ulyashova et. al., revealed in their

work that K. pneumonia isolates did not expressed blaVIM.[47]

It has been revealed by

Hammond et al.,that 13/21 clinical isolates tested in their study expressed bla SHV which is

prevalent in gram- negative bacteria, and it can hydrolyze penicillin and cephalosporines but

not extended spectrum antibiotics oxyimino cephalosporine and monobactams.[47]

Reports of

broad spectrum β-lactamase types such as SHV, mediated by plasmids, have proliferated with

the increased identification of K. pneumonia strains and with the use of cephalosporin

during recent years.[48]

Acquired MBL genes are located on integron that reside on mobile

genetic elements such as plasmids or transposons, thus, enabling widespread

dissemination.[45]

According to research done in Mexican hospital, 31 strains of Klebsiella



17


pneumonia isolated from septicemic pediatric pataints could harbor bla SHV gene and

considered as MDR.[48]

Some of these multidrug- resistant isolates produce extended-

spectrum β- lactamases (ESBLs) that are able to hydrolyze expanded- spectrum

cephalosporins (e.g., ceftriaxone, cefotaxime, and ceftazidime), aztreonam, and related

oxyimino-β- lactams. The substitution of one or more amino acids in bla SHV gene has

altered the configuration of the active site.[48]

ParC2 andGyrA1 (quinolone resistance genes) exhibited54.17% and 75% , respectively, and

this is the first time in Iraq to document the existence of quinolons resistant genes among

Iraqi Klebsiella isolates. Flouroquinolones have been frequently prescribed as empirical

therapy against most hospital and community infections due to increased appearance of

multiple drug resistant gram-negative pathogens and to the disease severity. In countries with

extensive clinical use of quinolones, fluoroquinolone resistance has been a problem in

clinical medicine for its limiting of available agents in the treatment of many types of

infection.[49]

Klebsiella pneumonia isolates showed mutation in either gyrA or ParC or both

genes in different codons resulting in resistance towards quinolones, and these multiple

alterations of gyrA and parC have been associated with ciprofloxacin resistance.[50]

It has

been shown that 77 of 120 test strain K. pneumonia harbor gyrA and par C genes and could

then resist quinolones.[51]

Twenty-two K. pneumonia isolates in other study were resistant to

gatifloxacin, trovafloxacin, moxifloxacin, levofloxacin, and ciprofloxacin, whereas only 7

were clinafloxacin resistant. This resistant mechanism related to alteration in gyrA and par C

genes, too.[52]

Results obtained in this study support data in literature which revealed that K. pneumonia

clinical isolates have been shown to manifest all three broad mechanisms of drug resistance

in gram-negative bacteria, which are the acquisition of novel antibiotic catalytic genes,

mutations of antibiotic targets and membrane proteins, and differential expression of specific

genes such as those for efflux pumps which mediate drug effects.[40]

In this study, K.

pneumonia Sputum 2 and Stool 1 isolates noticed to have 13 genes each out of the 22

antibiotic resistant genes tested compared with other isolates that have range of 3-12 genes

(Figure 9).

In conclusion, antibiotic resistance among various species of bacteria is a global public health

problem.Molecular analysis has been performed to quantify the hypothesis of these isolates

being extremely drug resistant(XDR); therefore, it could be concluded from the results



18


explained above that most of the 24 K. pneumonia local Iraqi isolates covered not only

extremely drug resistance (XDR) pattern but also being pan drug resistant (PDR) pathogens.

A sign of emergency of un treatable isolates

Figure 8: Prevalence and percentage of Antibiotics resistance genes within K.

pneumonia local Iraqi isolates.

Figure 9: The number of resisted genes within twenty four Klebsiella pneumonia isolates

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