Journal Of The Egyptian Public Health Association

Methicillin-Resistant Staphylococcus aureus nasal carriage among primary school-aged children from Jordan: Prevalence, antibiotic resistance and molecular

characteristics--Manuscript Draft--

Manuscript Number: JEPHA-D-14-00006R1

Full Title: Methicillin-Resistant Staphylococcus aureus nasal carriage among primary school-aged children from Jordan: Prevalence, antibiotic resistance and molecularcharacteristics

Article Type: Original Article

Keywords: Community-associated methicillin resistant Staphylococcus aureus, Prevalence, riskfactors for MRSA, school-age children, Jordan.

Corresponding Author: Hamed Alzoubi, Ph.DMutah UniversityKarak, JORDAN

Corresponding Author SecondaryInformation:

Corresponding Author's Institution: Mutah University

Corresponding Author's SecondaryInstitution:

First Author: Hamed Alzoubi, Ph.D

First Author Secondary Information:

Order of Authors: Hamed Alzoubi, Ph.D

Amin Aqel, Ph.D

Sameeh Al-Sarayreh, Ph.D

Enas Al-Zayadneh, Consultant pediatrician

Order of Authors Secondary Information:

Manuscript Region of Origin: JORDAN

Abstract: Background and aim: Colonization with Methicillin-Resistant Staphylococcus aureus(MRSA) increases the risk of subsequent infections with an increased mortality andmorbidity. Children were suggested to be a major asymptomatic reservoir forcommunity associated (CA) - MRSA with an ability to quickly spread the MRSA withincommunity. Therefore, the availability of epidemiological and antibiotic susceptibilitydata of CA-MRSA will be useful for the infection control and management policies. Thisstudy aimed to assess the nasal carriage and antibiotic susceptibility of MRSA inprimary school-aged children from Jordan.Subjects and Methods: A total of 210 nasal swabs were collected from 6-11 years oldchildren. Isolated MRSA and its SCCmec typing, Spa type and PVL toxin wereidentified following culture, biochemical and polymerase chain reaction. Antibiogramwas determined by the disc diffusion method.Results: The prevalence of CA-MRSA was 7.1%. Allergic rhinitis and recent antibioticexposure were the only significant risk factors for MRSA nasal carriage amongchildren. Resistance to erythromycin, trimethoprim-sulfamethoxazole and tetracyclinewas 33.4%, 20% and 13.4% respectively. All isolates were susceptible to theremaining non beta-lactam antibiotics used in this study, in particular, linezolid andmupirocin. All MRSA isolates were SCCmec type IV and PVL toxin negative and themajority were Spa type t223.Conclusion and recommendations: This is the first study to assess the MRSAprevalence among 6-11 years old children in Jordan. The prevalence in communitychildren is within the range compared to other studies in other countries. Theantibiogram, SCCmec and Spa types of the isolated MRSA are much similar to what

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was found previously in Jordan. However, all isolates were PVL toxin negative. Thestudy recommends increasing the public awareness of MRSA and the properantibiotics dispensing. Future studies to follow up on the changing epidemiology of theCA-MRSA in Jordan are also recommended.

Response to Reviewers: reviewer 1:grammatical corrections were done as requestedreviewer 2:1-reference for fusidic acid susceptibility added2-a paragraph was added in methods on MRSA detection using cefoxitin3- all other grammatical and formatting corrections were done

all above are included in this revised MS.Kind regards

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1

Methicillin-Resistant Staphylococcus aureus nasal carriage among primary school-aged children from Jordan: Prevalence, antibiotic resistance and

molecular characteristics

Hamed M. Alzoubi1 *

, Amin A. Aqel1, Sameeh A. Al-Sarayreh

2,

Enas Al-Zayadneh3

1Microbiology and Immunology Department,

2Biochemistry and Molecular Biology

Department, 3Department of pediatrics, Faculty of Medicine, Mu'tah University, Al-

Karak, Jordan.

Abstract:

Background and aim: Colonization with Methicillin-Resistant Staphylococcus

aureus (MRSA) increases the risk of subsequent infections with an increased

mortality and morbidity. Children were suggested to be a major asymptomatic

reservoir for community associated (CA) - MRSA with an ability to quickly spread

the MRSA within community. Therefore, the availability of epidemiological and

antibiotic susceptibility data of CA-MRSA will be useful for the infection control and

management policies. This study aimed to assess the nasal carriage and antibiotic

susceptibility of MRSA in primary school-aged children from Jordan.

Subjects and Methods: A total of 210 nasal swabs were collected from 6-11 years

old children. Isolated MRSA and its SCCmec typing, Spa type and PVL toxin were

identified following culture, biochemical and polymerase chain reaction. Antibiogram

was determined by the disc diffusion method.

Results: The prevalence of CA-MRSA was 7.1%. Allergic rhinitis and recent

antibiotic exposure were the only significant risk factors for MRSA nasal carriage

among children. Resistance to erythromycin, trimethoprim-sulfamethoxazole and

tetracycline was 33.4%, 20% and 13.4% respectively. All isolates were susceptible to

the remaining non beta-lactam antibiotics used in this study, in particular, linezolid

and mupirocin. All MRSA isolates were SCCmec type IV and PVL toxin negative and

the majority were Spa type t223.

Conclusion and recommendations: This is the first study to assess the MRSA

prevalence among 6-11 years old children in Jordan. The prevalence in community

children is within the range compared to other studies in other countries. The

antibiogram, SCCmec and Spa types of the isolated MRSA are much similar to what

was found previously in Jordan. However, all isolates were PVL toxin negative. The

Final reviewed manuscript JEPHA-D-14-00006 Alzoubi et al Jordan

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study recommends increasing the public awareness of MRSA and the proper

antibiotics dispensing. Future studies to follow up on the changing epidemiology of

the CA-MRSA in Jordan are also recommended.

Keywords:

Community-associated methicillin resistant Staphylococcus aureus, Prevalence, risk

factors for MRSA, school-age children, Jordan.

Corresponding author:

Hamed Mahmoud Alzoubi MD, PhD Medical Microbiology.

Chairman of the Microbiology and Immunology Department,

Faculty of Medicine, Mu'tah University,

P.O Box 7, Alkarak, Jordan.

Tel.: 00962-799238884; Fax: 00962-32397180

Email: dr_alzoubi@yahoo.com

INTRODUCTION

Methicillin resistant staphylococcus aureus (MRSA) was first recognized in

early 1960s soon after introduction of methicillin.(1)

Since then, it has been confined

to health care settings. However, since 1990s, the number of MRSA infections in

populations that lack exposure to health care settings has increased. This has led to the

recognition of community associated MRSA (CA-MRSA) strains.(2-4)

CA-MRSA strains are mainly distinguished from healthcare associated MRSA

(HA-MRSA) using molecular techniques.(4)

The SCCmec in CA-MRSA belongs to

type IV or V and is smaller than that of HA-MRSA SSCmec which belongs to type I,

II or III. Additionally CA-MRSA strains frequently carry the gene for PVL (Panton

Valentine Leukociden) toxin which is rarely found in HA-MRSA.(4, 5)

CA-MRSA infections can affect healthy young patients leading to serious

community infections with increased mortality and morbidity such as skin and soft

tissue infections, necrotizing pneumonia and sepsis.(3, 5)

Colonization with MRSA has

been suggested to increase the risk of developing subsequent infections.(6)

Therefore,

such colonization represents a threat to the carrier and community since infections

caused by MRSA can be difficult and more expensive to treat compared to those

caused by Methicillin sensitive staphylococcus aureus (MSSA) strains.(7)

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The risk factors for carriage of CA-MRSA in the community are not fully

defined.(8)

However, it was found that the carriage rate of CA-MRSA among children

is higher than in adults.(9)

Additionally, adults living with young children are at

increased risk of MRSA colonization.(10)

Therefore, it was suggested that children

could be a major asymptomatic reservoir for CA-MRSA with an ability to quickly

spread MRSA within other different community populations.(8, 11, 12)

As a result,

studying the nasal carriage of MRSA is a necessary sentinel approach to assess the

MRSA burden and its risk factors in a population.

The aim of this study is to assess the nasal carriage of MRSA among primary

school-aged children in Al-Karak province, Jordan. It also aims to describe the risk

factors for colonization, antibiotic resistance and molecular characteristics of CA-

MRSA. This may serve as a guide for infection control and management policies of

CA-MRSA infections.

SUBJECTS AND METHODS

Study design and data collection:

This cross-sectional study was carried out from November, 2012 to May, 2013, at Al-

Karak province in Jordan. Approval for the study was obtained from the scientific and

the ethics committees at the faculty of medicine, University of Mutah, Jordan. Parents

gave written informed consent.

The sample:

The sample size for this study was calculated using Kish's formula for cross-sectional

studies,(13)

and at a prevalence of community MRSA of approximately 13% in Jordan

and a neighboring country.(14, 15)

Based on that, the minimum sample size required for

this study was 174 samples. However, 210 samples were collected from 210 children

aged 6-11 years attending the University-Model school at Al-Karak, Jordan. A

questionnaire was filled in with information on illnesses, having a family member

who is a healthcare worker, antibiotic consumption and hospital admission over the

last 3 months. Hospital admission was an exclusion criterion in this study.

Bacterial detection, antimicrobial susceptibility testing and molecular

characterization of MRSA isolates:

This was carried out as previously described.(16)

Briefly, nasal swabs were inoculated

on Mannitol Salt Agar (MSA, BBL Microbiology System, Becton Dickinson

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Company, MD, U.S.A) and incubated at 35 ± 1°C and examined for growth after 24-

48 hours. Single distinctive morphotype of a mannitol-positive colony was selected

from an MSA plate, subcultured to a nutrient agar (BBL Microbiology Systems,

Becton Dickinson, Company, MD, U.S.A.), and incubated overnight at 37Co in a

humidified incubator. Colonies growing on Nutrient agar were identified as S. aureus

by Gram’s staining, anaerobic utilization of glucose and mannitol, catalase production

and tube coagulase test. Identifying the MRSA isolates was then carried out using

30µg/ml cefoxitin disc in Mueller-Hinton agar.

Antimicrobial susceptibility to fusidic acid, erythromycin, mupirocin, gentamicin,

linezolid, teicoplanin, ciprofloxacin, trimethoprim-sulfamethoxazole, tetracyclin,

rifampicin, cefoxitin was performed by disk diffusion, according to the European

Committee on Antimicrobial Susceptibility Testing for fusidic acid, and according to

the Clinical Laboratory Standards Institute (CLSI) guidelines for the remaining

antibiotics.(17, 18)

S. aureus ATCC 25923 was used as control strain. Discs were

purchased from Oxoid, Hampshire, England.

Methicillin resistance genes (mecA and mecC) plus the Panton-Valentine Leucocidin

gene (Luk-PV) were detected by multiplex PCR as previously published,(19)

isolates Spa-typing was carried out as described by Harmsen et al.,(20)

and

the SCCmec typing was performed according to Milheirico et al.(21)

Statistical analysis

The statistical analysis was conducted using STATA10 (Stata Corp. 2007. Stata

Statistical Software: Release 10. College Station, TX: StataCorp LP, USA) to

evaluate the significance of potential risk factors. A P value of <0.05 was considered

as significant.

RESULTS

Study population included 109 males and 101 females. A total of 15 MRSA

were isolated from 7 males and 8 females. The prevalence of MRSA nasal carriage is

therefore 7.1% (15/210). The distribution of the isolated MRSA according to different

variables and statistical significance is shown in table 1. The only significant variables

for MRSA nasal carriage are receiving antibiotics over the last 3 months (P = 0.037)

and suffering of allergic rhinitis (P = 0.022).

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The results also show that 33% (5/15) and 67% (10/15) of the 15 isolates were

distributed among age groups 6-<9 and 9-<12 year respectively. However, age was

not found to be statistically significant (P = 0.25) for MRSA nasal carriage in the

current study. Similarly, gender (P = 0.67) and having a family member working as a

healthcare worker (P = 0.12) were not statistically significant for MRSA nasal

carriage among study population.

Table 1: Potential risk factors and P values of the 210 analyzed samples obtained

from the school age children, Al Kark, Jordan

Variable Age (years) Gender Antibiotics

last 3

months

Allergic

rhinitis

HCW-

family

member

6-<9 9-<12 M F Y N Y N Y N

MRSA

Positive

5 10 7 8 9 6 7 8 3 12

MRSA

Negative

95 100 102 93 65 130 41 154 16 179

Total

(Number)

100 110 109 101 74 136 48 162 19 191

P value 0.25 0.67 0.037* 0.022* 0.12

*Significant P value < 0.05

Antibiotic susceptibility pattern is shown in Table 2. None of the 15 MRSA

isolates (0%) isolates was susceptible to cefoxitin. Whereas 66.6%, 80% and 86.6% of

the isolates were susceptible to erythromycin, trimethoprim-sulfamethoxazole and

tetracycline respectively. All isolates were susceptible to the remaining antibiotics

used in this study as shown in table 2.

The MecA gene was detected in all MRSA isolates, whereas none of them

were positive for the mecC and Panton-Valentine Leucocidin gene (Luk-PV). Nine

isolates were of t223 Spa type, followed by t214, t386 and t012 which were detected

in 3, 2 and 1 isolates respectively. The IVa-SCCmec lineage was detected in 14

MRSA isolates and IVc- SCCmec was detected in one isolate.

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Table 2: Antibiotic susceptibility pattern of MRSA isolates from primary school

students, Al Kark, Jordan

Pathogen

/ number

Antibiotic susceptibility (number, %)

MRSA

/15

E Mup G LZD Teic Cip SXT Tet Rd F Cef

(10)

66.6

(15)

100

(15)

100

(15)

100

(15)

100

(15)

100

(12)

80

(13)

86.6

(15)

100

(15)

100

0

0

Abbreviations: E: Erythromycin, , Mup: Mupirocin, G: Gentamicin, LZD: Linezolid,

Teic: Teicoplanin , Cip: Ciprofloxacin, SXT: Trimethoprim-sulfamethoxazole, Tet:

Tetracyclin, Rd: Rifampicin, F: Fusidic acid, Cef: Cefoxitin.

DISCUSSION

MRSA infections are globally emerging as an escalating infection in the

community and can no longer be considered as a healthcare associated infection only.

Therefore, hospital based infection control measures alone are not enough to fight the

increasing MRSA infections in the community.(6)

CA-MRSA have been shown to

infiltrate hospital settings.(22)

Epidemiological and antibiotic susceptibility data for

CA-MRSA can be of paramount importance for practitioners to guide their infection

control and management policies.

In the current study, the prevalence of CA-MRSA nasal carriage among

children aged 6-11 years was 7.1%. This is nearly similar to the 7.4% prevalence rate

recently found among healthy Jordanian adult volunteers.(23)

However, in a study

carried out by Shehabi et al,(14)

the prevalence of MRSA among Jordanian infants was

13.2%, which is significantly higher than what was found in the current study. In

another study among children aged 1.8-5.5 years in Gaza, the prevalence of CA-

MRSA was 12 %.(15)

Such higher rate of prevalence might be explained by factors

such as difference in study population and exposure to different potential risk factors.

Another important factor can be the difference in the age of the studied population.

Our study is the first in Jordan to study the primary school-age children, whereas the

previously mentioned studies (14, 15)

focused on infants and adults. Therefore, the

results of the current study covers an important age span which should, together with

the previous studies, provide a more comprehensive picture about CA-MRSA

prevalence among different ages. Additionally, this study should form a start to follow

up on the changing epidemiology of the CA-MRSA in Jordan and, probably, the

region.

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Variable prevalence rates of 8.8%, 10.8%, and 4% among primary school-age

children were previously found in different countries.(24-26)

Therefore, the prevalence

of 7.1% found in our study is still within the range of what was found in some other

countries.

Factors such age, sex and having members of the family working in healthcare

setting were not found to be statistically significant for MRSA carriage in this study.

On the other hand, recent antibiotic exposure and having allergic rhinitis significantly

increased the risk of MRSA nasal carriage.

Antibiotic exposure was previously suggested to be associated with CA-

MRSA carriage among children.(27)

Antibiotic abuse and misuse is believed to be

common in the community where this study was carried out. This is may be due to

absence of restriction on antibiotic prescriptions and the proper antibiotics

prescription guidelines. However, this aspect is still hypothetical and it needs further

investigations. The presence of allergic rhinitis was found to be a significant factor for

CA-MRSA carriage in the studied children. This is in agreement with the recently

published study by Cevik et al. (28)

which revealed that MRSA nasal carriage was

significantly higher among allergic rhinitis patients compared to control healthy

people. The carriage of MRSA among allergic rhinitis patients should be considered

as a factor that may increase the co-morbidities and complications therefore, proper

diagnosis and antibiotic selection is necessary to manage community infections such

as skin and soft tissue infection in this group of patients.

Resistance to erythromycin, trimethoprim-sulfamethoxazole and tetracycline

was 33.4%, 20% and 13.4% respectively. The resistance to these antibiotics was

nearly similar to some of the previous studies in Jordan.(14, 16)

No resistance for the

remaining antibiotics that were used in this study, in particular to linezolid which was

also not much different from what was found by some other studies in Jordan.(16, 23)

Such narrow antibiotic resistance profile is consistent with the characteristic of the

CA-MRSA.(29)

All of the isolated MRSA in the current study belonged to the IV-

SCCmec lineage which is consistent with MRSA of community origin that usually

harbor type IV or V,(4)

and is nearly similar to what was found previously in the

region.(14, 15, 23)

The Spa type t223 was predominant among all MRSA isolates and

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represented 60% of all detected Spa types. This type was found as the second

commonest type by Al-Bakri et al. (23)

, where it represented 14.7% preceded by the

Spa type t9519 which was detected in 76% in the same study. The significance of Spa

types is still debatable with some suggestions of an association between the Spa type

and CA-MRSA invasiveness. However, such suggestions have not been confirmed

experimentally and further research is needed to assess that.(4, 30)

Unexpectedly, the

PVL toxin was not detected in all CA-MRSA isolates of this study. The PVL-positive

MRSA isolates in studies carried out recently in Jordan by Shehabi et al. (14)

and Al-

Bakri et al. (23)

were 28% and 5% respectively. Rare occurrence of PVL toxins in the

CA-MRSA was previously reported.(31)

However, the absence of PVL toxin in our

isolates is reported for the first time in Jordan and the neighboring countries according

to our best knowledge and literature search. A proposed hypothesis for such finding is

that PVL genes might be less common in isolates causing asymptomatic colonization

compared to those causing clinically apparent infection.(4)

However, further

investigations might be needed in the future.

CONCLUSION AND RECOMMENDATIONS: This study showed a prevalence

rate of MRSA nasal carriage among primary school-aged children in Jordan

comparable to those in other countries. The antibiotic susceptibility pattern is not

alarming and is approximately consistent with CA-MRSA. All MRSA isolates

belonged to SCCmec type IV and 60% were Spa type t223. The absence of PVL toxin

in all MRSA isolates is a finding that worth further studying. Further studies are

required to sample children for MRSA nasal carriage in the future to follow up on the

epidemiology pattern and antibiotic susceptibility.

Acknowledgment:

We would like to acknowledge Mu’tah University / Jordan for the financial support of

this work. We would also like to acknowledge Ahmad Khazar Zayed Makableh,

medical student, at the faculty of medicine Mutah University, for his help in the

practical part of this study.

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