Molecular epidemiology of nasal isolates of methicillin-resistant Staphylococcus aureus from Jordan

Journal of Infection and Public Health (2015) 8, 90—97

Molecular epidemiology of nasal isolates of methicillin-resistant Staphylococcus aureus from Jordan Amin A. Aqel a,∗, Hamed M. Alzoubi a, Anna Vickers b, Bruno Pichon b, Angela M. Kearns b a

Microbiology and Immunology Department, Faculty of Medicine, Mu’tah University, Alkarak 61710, Jordan b Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Public Health England, 61 Colindale Avenue, London NW9 5EQ, UK Received 5 March 2014 ; received in revised form 9 April 2014; accepted 24 May 2014

KEYWORDS MRSA; Healthcare workers; Healthy individuals; SCCmecIVa

Summary Asymptomatic carriage of methicillin-resistant Staphylococcus aureus (MRSA) can predispose the host to a wide range of infections. To inform public health strategies, this study sought to determine the prevalence and the phenotypic and genotypic characteristics of MRSA from nasal swabs of health care workers (HCWs) and other healthy individuals in Jordan. Overall, 716 nasal swabs were collected from 297 HCWs, 141 adults and 278 children in the community. MRSA was recovered from 56 (7.8%) nasal swabs, which represented carriage rates of 10.1%, 4.3% and 7.2% among HCWs, adults and children, respectively. The MRSA isolates were resistant to oxacillin (100%), erythromycin (42.8%), tetracycline (37.5%), clindamycin (5.3%), fucidin (5.3%), and ciprofloxacin (3.5%). A total of 17 different spa types belonging to eight different clonal complexes (CCs) were identified. All isolates were mecA positive, and mecC-MRSA was not detected. Analysis of the staphylococcal cassette chromosome mec (SCCmec) elements revealed that the majority (54; 96.4%) of the samples harbored the smaller type IV and V elements (the most common were SCCmec IVa or IVc, and there were two each of the IVg and V elements), and two were nontypable. The genes for Panton-Valentine leukocidin (luk-PV) were detected in 5.4% of the study isolates. A tst-positive, CC22-MRSA-SCCmecIVa clone (spa type t223) was identified as the dominant MRSA lineage among the nasal carriage isolates from both HCWs and other individuals (adults and children) in the community.

∗ Corresponding author at: Mu’tah University, Faculty of Medicine, Microbiology Department, P.O. Box 7, Mu’tah 61710, Alkarak, Jordan. Tel.: +962 3 238 6287x3208; fax: +962 3 239 7180. E-mail addresses: [email protected], [email protected] (A.A. Aqel).

http://dx.doi.org/10.1016/j.jiph.2014.05.007 1876-0341/© 2014 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Molecular epidemiology of nasal isolates of MRSA from Jordan

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These findings provide important information for public health personnel for the formulation of effective infection prevention and control strategies. Studies to further our understanding of the distribution, pathogenicity, transmissibility and fitness of this lineage would be prudent. © 2014 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Introduction Methicillin-resistant Staphylococcus aureus (MRSA) has long been recognized as a major nosocomial pathogen that is associated with severe morbidity and mortality [1]. Worldwide, MRSA infections and outbreaks have become an increasing problem not only in healthcare but also in community settings [2]. Studies from various countries have sought to establish the nasal carriage rate of MRSA in the community. The figures vary widely across the different locations and populations that have been surveyed. Published data show the nasal carriage rates of MRSA in Europe and the USA range from <0.1% to 2.5%, and rates up to 10.5% have been reported in Africa [3—8]. In countries in the Middle East (including Saudi Arabia, Kuwait, Lebanon and Palestine), 0—13.2% MRSA nasal carriage rates have been observed [9—14]. Studies in Jordan suggest high MRSA carriage rates of 7.5—19% among healthy individuals [15,16]. An additional concern is that a 3-year study (2003—2005) reported an MRSA rate of 56% among invasive isolates of S. aureus from multiple medical centers in Jordan [17]. Another study from Jordan [18] indicated that of the 232 S. aureus isolates recovered from diverse clinical samples (abscesses, wounds, skin infections, blood and nasal swabs), 62% were MRSA. An understanding of the molecular epidemiology of MRSA is crucial for the formulation and implementation of appropriate infection prevention and control measures [19,20]. Despite the high MRSA rates, few reports have been published on the molecular epidemiology of MRSA in Jordan [16,21,22]. To our knowledge, a detailed analysis of the nasal strains of MRSA that occur in healthcare and community settings in Jordan has not been published. The objectives of the current study were to establish the prevalence and phenotypic and genotypic characteristics of MRSA from nasal swabs from HCWs and other healthy individuals (adults and children) in southern Jordan.

Material and methods Study design and sample collection The sample size of the current study was calculated using Kish’s formula for cross-sectional studies [23] based on MRSA prevalences of 5.8%, 7.5% and 13% [15,21,24] in HCWs, adults and children, respectively, in Jordan. The minimum sample sizes required were 84 samples from HCWs, 107 samples from adults and 174 samples from children. The randomly collected samples examined in this study (297, 141 and 278 from HCWs, adults and children, respectively) exceeded these figures. Between May 2011 and April 2012, non-duplicate nasal swabs were collected from the study population. Written informed consent and a questionnaire about general information and specific risk factors for MRSA carriage were obtained from each participant or, in the cases of the children, their parents or guardians. The ethics and scientific committee of the faculty of Medicine, Mu’tah University approved this study. All HCWs (medical doctors, nurses, technicians, domestic and administrative staff) were screened while working in the nursery, maternity, pediatric, medical, surgery and intensive care wards of Alkarak governmental hospital in Jordan. No MRSA outbreaks were apparent during the sampling period. All other adults and children were randomly selected from Alkarak province (population of 249,100 inhabitants in 2012). Nasal swabs were immersed in nutrient broth (Oxoid, Cambridge, UK) containing 5% NaCl and incubated for 3 h at 37 ◦ C. Each broth was subcultured on mannitol salt agar (Oxoid, Cambridge, UK) and incubated at 35 ◦ C for 24—72 h. Presumptive identifications of S. aureus were based on colony morphology, Gram staining, and positive catalase and slide agglutination reactions (bioMérieux, Marcy l’Etoile, France). The MRSA isolates were identified by disk diffusion using

92 1 ␮g oxacillin and 30 ␮g cefoxitin discs in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines [25]. Presumptive MRSA samples were then forwarded to the Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit (Public Health England, Colindale, London) for further characterization.

Antibiotic susceptibility testing The minimum inhibitory concentrations (MICs) of antibiotics were determined by agar dilution (British Society for Antimicrobial Chemotherapy method, BSAC [26]). The following antimicrobials were tested: vancomycin, mupirocin, gentamicin, rifampicin, daptomycin, teicoplanin, linezolid, erythromycin, tetracycline, clindamycin, fucidin, and ciprofloxacin.

PCR-based detection of virulence and resistance genes All study isolates were examined with real-time multiplex PCR [27] to detect the thermonuclease gene (nuc), methicillin resistance genes (mecA and mecC), and the Panton-Valentine leukocidin gene (luk-PV). A total of 29 representative isolates (including at least one of each spa type) were subjected to multiplex PCR [28] to detect the genes encoding the following: enterotoxins A-E and G-J (sea-see and seg-sej), toxic shock syndrome toxin-1 (tst), and exfoliative toxins A, B and D (eta, etb and etd). The characterizations of the staphylococcal cassette chromosome mec (SCCmec) elements were performed on all isolates according to the protocol ¸o [29]. described by Milheiric

Spa typing Spa typing was performed as previously described [30]. Multi-locus sequence type clonal complex (MLST-CC) assignments were inferred based on spa typing data and by reference to the spa server (http://spa.ridom.de/mlst.shtml) and MLST database (http://saureus.mlst.net).

Results Of the 716 nasal swabs collected, 56 (7.8%) yielded MRSA (Table 1). The MRSA nasal carriage rates of the HCWs, other adults and children were 10.1%, 4.3% and 7.2%, respectively. Antibiotic exposure and occupation were significant risk factors for MRSA nasal carriage among the healthy adults and HCWs,

A.A. Aqel et al. respectively (Table 1). All MRSA isolates were mecA positive, and mecC-MRSA were not detected. The molecular characterization data and antibiotic susceptibility profiles are summarized in Table 2. The majority of MRSA isolates harbored SCCmec type IV; 42 (75%) encoded SCCmecIVa, 10 (17.8%) encoded SCCmecIVc, one harbored SCCmecIVg and one harbored SCCmecV. Two isolates were SCCmec non-typable. A total of 17 different spa types of varying toxin gene compositions that belonged to eight diverse CCs were identified (i.e., CC1, CC5, CC15, CC22, CC30, CC45, CC80 and CC97). Overall, spa types t223 (n = 22, 39%) and t386 (n = 8, 14%), which belong to CC22 and CC1, respectively, were predominant. Resistance to the following antimicrobials was noted: oxacillin (100%), erythromycin (42.8%), tetracycline (37.5%), clindamycin (5.3%), fucidin (5.3%), and ciprofloxacin (3.5%). All MRSA isolates were susceptible to vancomycin, mupirocin, gentamicin, rifampicin, daptomycin, teicoplanin and linezolid. Three MRSA isolates (5.4%) were luk-PV positive; two of these were from adults (spa types t044 and t803, which belong to MLST-CC 80 and 15, respectively), and one was from a HCW (spa type t044, also belonging to MLST-CC 80).

Discussion Previous studies have provided some insight into the molecular epidemiology of MRSA in the Middle East, but differing results have been reported according to the cohorts that have been investigated. For example, in a recent study conducted by Sabri [31], the most prevalent MRSA among clinical isolates from hospitalized patients in three Arabian countries (Jordan, Palestine and Iraq) was the pandemic HA-MRSA lineage ST239-SCCmecIII (spa type t932), and Khalil [22] reported that the most common MRSA recovered from the nasal and stool samples of children in Jordan was the ST80-IV PVL-positive CA-MRSA clone. In contrast, Al-Bakri reported that SCCmecIV-MRSA spa types t9519, spa type t223 and spa type t044 were identified among 76%, 14.7% and 5.9%, respectively, of all MRSA isolates from adults in Jordan [16]. In the present study, the collective data revealed that the overall prevalence of the nasal carriage of MRSA was 7.8% (ranging from 4.3% in adults to 10.1% in HCWs) and highlight a previously unrecognized genetic heterogeneity. The recovered 56 MRSA isolates were belonged to eight diverse MLST-CCs and harbored different SCCmec sub-types; these findings suggest that these strains had multiple

Molecular epidemiology of nasal isolates of MRSA from Jordan Table 1

Characteristics of MRSA positive individuals versus negatives by study group.

Study group

Demographic data

HCWs (n = 297)

Gender Male Female Age (years) 20—24 25—50 Occupation Nurse Othersb

Healthy adults (n = 141)

Healthy children (n = 278)

a b ‡

93

Gender Male Female Age (years) 20—24 25—50 Antibiotic exposure Yes No Gender Male Female Age (years) 6—11 12—18

Total

MRSA-positive

Percentage (%)

125 172

11 19

8.8 11

35 262

1 29

2.8 11

P valuea 0.52

0.13 0.002‡ 95 202

17 13

17.8 6.4

60 81

2 4

3.3 4.9

115 26

4 2

3.4 7.6

19 122

5 1

26.3 0.8

136 142

13 7

9.5 4.9

168 110

15 5

8.9 4.5

0.64

0.33 <0.0001‡

0.13

0.16

P values for each category refer to comparison of MRSA positives versus negatives. Medical doctors, nurses, technicians, domestic and administrative staff. Significant P value < 0.05.

origins. The majority of the MRSA isolates (54; 96.4%) harbored the smaller SCCmec elements of SCCmecIV or V (2 were non-typable). The following two major clones were identified: CC22-IVa (spa types t223, t309 and t6397), and CC1-IVa (spa types t386 and t693). These clones were variously resistant to erythromycin, tetracycline, clindamycin and/or ciprofloxacin. Interestingly, both lineages are among the most common MRSA isolates that are identified in screening samples from children in Jordan [22]. Data from Jordan [16] and neighboring countries (i.e., namely Palestine, Lebanon and Saudi Arabia) also revealed that SCCmecIV is commonly identified in nasal and clinical MRSA isolates [14,31—33]. In the study published recently by Al-Bakri, spa type t223 was found to be the second most common type in Jordanian adults [16]. Moreover, the ST22-MRSA-IVa clone (spa t223) is dominant in Gaza Strip and accounts for 64% of the MRSA in healthy children and their parents [12]. Importantly, although this clone belongs to the same MLST-CC as the pandemic healthcareassociated EMRSA-15 strain, it differs in SCCmec type (IVa versus IVh) and tst status, which is

suggestive of different epidemiologies and evolutionary pathways [34]. In accordance with previous reports, enterotoxin genes were commonly detected in the study isolates [35,36]. In contrast, the prevalence of the PVL genes was low. Only three MRSA isolates (5.4%) encoded the PVL genes; two (t044) of these belonged to the so-called European clone of CAMRSA and were recovered from adults (one of whom was a HCW). These finding parallel those of Al-Bakri [16] who found only two PVL-positive MRSA isolates, both of which belonged to spa type t044. These results also mirror those of Biber [12] who found that ST80-IV was the dominant PVL-MRSA strain in healthy children and their parents throughout the Gaza Strip. Conversely, Khalil [22] observed that 34% of the MRSA from nasal and stool samples from children up to one year of age who attended Jordan University Hospital in Amman were PVL-positive. Interestingly, tst positive ST22-MRSA-IVa spa t223 was found to be the predominant nonmultiresistant lineage in the nasal swabs from 500 healthy preschool children in Palermo, Italy [37]. The same MRSA clone was prevalent in the neonatal

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Table 2 Sources of MRSA (#)

Phenotypic and genotypic characteristics of MRSA isolates. Spa type (#)

Spa repeat succession

MLST-CC

SCCmec type

Toxin gene profile (no. isolates tested)

Resistant toa

Children (20)

t012 (1) t021 (2) t214 (3) t223 (9) t253 (1) t386 (2) t6397 (1) t790 (1)

15-12-16-02-16-02-25-17-24-24 15-12-16-02-16-02-25-17-24 26-23-17-34-17-20-17-12-17-16-16 26-23-13-23-05-17-25-17-25-16-28 15-12-16-02-16-02-25-17-24-24-24-24 07-23-13 26-13-23-05-17-25-17-25-16-28 26-23-13-23-31-29-17-25-17-25-16-28

30 30 5 22 30 1 22 22

IVc IVg, IVa IVa IVa IVc IVa IVa IVa

seg, sei, tst seg, sei (n = 1) seg, sei (n = 1) seg, sei, tst (n = 2) seg, sei, tst seg, seh, sei (n = 1) seg, sei, tst sec, seg, sei,

S R S [R] S R R R

S S S [R] S R R R

S S S S S S S S

S S S S S S S S

Fusidic acid S S S S S S S S

Adults (6)

t044 (1) t1234 (1) t1651 (2) t223 (1) t803 (1)

07-23-12-34-34-33-34 07-23-12-12-34-34-34-33-34 08-16-02-43-34-16-02-17-16 26-23-13-23-05-17-25-17-25-16-28 07-23-02-12-23

80 97 30 22 15

IVc NT IVc IVa NT

seh, etd, luk-PV — sec, tst (n = 1) ND etd, luk-PV

S S S S S

R R S R S

S S S S S

S S S S S

R S S S S

HCWs (30)

t018 (2) t012 (1) t021 (2) t044 (1) t10683 (2) t223 (12) t309 (2) t370 (1) t386 (6) t693 (1)

15-12-16-02-16-02-25-17-24-24-24 15-12-16-02-16-02-25-17-24-24 15-12-16-02-16-02-25-17-24 07-23-12-34-34-33-34 35-17-34-17-20-17-12-17-16-16 26-23-13-23-05-17-25-17-25-16-28 26-23-05-17-25-17-25-16-28 09-34-17-34-16-34 07-23-13 7

30 30 30 80 5 22 22 45 1 1

IVc IVc IVa, V IVc IVa IVa IVa IVc IVa IVa

seg, sei, tst (n = 2) tst seg, sei (n = 1) seh, etd, luk-PV [sed], seg, sei (n = 2) seg, sei, tst seg, sei, tst seg, sei seh (n = 2) seh

S S S S [R] [R] R R R R

S S S R S [R] R S [R] R

S S S S S S R S [R] S

S S S S S S S S [R] S

S S S R S S R S S S

Erythro-mycin

Tetracy-cline

Clinda-mycin

Ciproflo-xacin

MLST-CC, multilocus sequence type clonal-complex; SCCmec, staphylococcal cassette chromosome mec; luk-PV, Panton-Valentine leukocidin genes; tst, toxic shock syndrome toxin-1 gene; etd, exfoliative toxin D gene; sec, sed, seg, she and sei, genes for enterotoxin genes C, D, G, H and I. All isolates tested negative for enterotoxin genes A, B, E, J and exfoliative toxin genes A and B. NT, non-typable; ND, not done; results in brackets [] were variable; —, negative for all toxin genes tested; R, resistant; S, susceptible. a All isolates were susceptible to vancomycin, mupirocin, gentamicin, rifampicin, daptomycin, teicoplanin and linezolid.

A.A. Aqel et al.

Molecular epidemiology of nasal isolates of MRSA from Jordan intensive care unit (NICU) and had a colonization rate of 88.8% among infants in Palermo, Italy [38]. Given these findings, it seems likely that this lineage is more widely distributed than currently appreciated. Further studies are necessary to assess the risk factors for harboring and disseminating this clone across a range of community and hospital settings. In our study population, risk factors, such as antibiotic exposure and occupation, were associated with increased MRSA nasal carriage. Uncontrolled antibiotic consumption has been suggested to facilitate and increase nasal MRSA colonization [39]. Similarly, among HCWs, the nursing occupation was significantly associated with MRSA nasal colonization, which is most likely attributable to patient contact. The MRSA isolates recovered in this study were susceptible to a broad range of agents, and none were multi-resistant. The most commonly encountered resistance was to erythromycin (42.1%), followed by tetracycline (36.8%), which likely reflects the widespread use of these antibiotics in Jordan. MRSA isolates harboring the larger SCCmec types (I, II, and III), which are often associated with HA-MRSA strains, were absent in our study. Although MRSA isolates with the larger SCCmec elements have remained prevalent in the healthcare environment, those with smaller SCCmec types are considered to be ‘‘fitter’’ and have proven to be successful both in community and healthcare settings. The infiltration of MRSA isolates with ‘‘community-like’’ phenotypes/genotypes into hospitals may result in strains such as those identified herein becoming established as nosocomial pathogens in addition to the extant HA-MRSA. Indeed, such infiltration has previously been documented in some countries; for example, the PVL-positive USA300 clone of CAMRSA dominates in some hospitals in North America and is responsible for approximately 70% of hospital acquired infections [40]. Similarly, ST59-IV has become an important nosocomial pathogen in hospitals in Taiwan [41]. Given these findings, the possibility of clones with smaller SCCmec types, such as those identified in the present study, emerging as successful pathogens in healthcare and community environments in Jordan and elsewhere is a cause for increasing concern. This study highlights the value of molecular typing in the monitoring of trends in the emergence, spread and epidemiology of MRSA strains nationally and internationally. In view of the high MRSA rates associated with carriage and infection in healthcare and community settings in Jordan, these results support the need for MRSA screening policies to identify carriers and subsequent referral to local

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infection prevention and control teams for decolonization, as has previously been recommended [42]. Actively managed antibiotic policies and stewardship in combination with surveillance are required to prevent both further increases in MRSA rates and the emergence of multidrug-resistant MRSA strains. Further studies are required to compare the epidemiologies of MRSA clones across the Middle East with those of other geographical regions across the world. Studies to further our understanding of the distribution, pathogenicity, transmissibility and fitness of the tst-positive ST22-MRSA-IVa lineage would also be prudent.

Funding No funding sources.

Competing interests None declared.

Ethical approval Not required.

Acknowledgments The visit to PHE (formerly the Health Protection Agency [HPA]), London was supported by the Daniel Turnberg UK/Middle East Travel Fellowship Scheme. We appreciate the assistance of the staff of the Staphylococcus Reference Service (PHE). Many thanks to Mr. Ahmad Khazar and the MRSA study group in the faculty of Medicine, Mu’tah University for sample collection and primary processing. This work was supported by grant number 65/14/120 from the Deanship of Academic Research, Mu’tah University, Jordan.

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