Nasal carriage of sequence type 22 MRSA and livestock-associated ST398 clones in Tangier, Morocco

Introduction: This study aimed to provide data of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage as well as to determine the genetic lineages of this circulating MRSA in the Tangier community. Methodology: Between 2012 and 2013 two subpopulations consisting of randomly chosen healthy volunteers and outpatients in 11 healthcare facilities were screened. The antibiotic resistance phenotype was determined by disk diffusion. Toxin Panton-Valentin Leukocidin (PVL), toxic shock syndrome toxin-1 gene (tst), and mecA were detected by polymerase chain reaction (PCR). Nasal swabs were obtained from persons with no identified risk factors for MRSA acquisition. MRSA molecular typing was performed by pulsed-field gel electrophoresis (PFGE), staphylococcal chromosomal cassette mec, and Staphylococcus protein A (spa) typing. Results: A total of400 subjects (33.3%) were nasally colonized with S. aureus, and 17 (1.4%) were nasal carriers of MRSA. The analysis did not identify age, gender, and the two subpopulations as predictors for MRSA colonization. MRSA were more likely to harbor the tst gene (p < 0.05). This work highlighted a low prevalence of nasal MRSA carriage, with 52.94% belonging to sequence type (ST) ST22. The remaining isolates were distributed as singletons (ST8, ST1, and ST398), whereas approximately one-third of MRSA was not identified, including three novel spa-types (t13247, t13248, and t13249). Conclusions: Although we highlighted the current clones present in the Tangier community, they are limited in space and time. Therefore, further studies would be required to obtain a comprehensive picture of the dissemination of MRSA in the community, hospital, and livestock.


Introduction
Staphylococcus aureus is an important bacterial pathogen that causes infections ranging from mild infections of skin and soft tissue to serious invasive diseases [1]. Pathogenicity of S. aureus is largely due to its ability to express a broad variety of virulenceassociated factors and to colonize humans as well as animals [2]. S. aureus nasal carriage is a key source of clinical S. aureus infection, though other body sites may also be clinically important [3]. Because methicillinresistant S. aureus (MRSA) was first identified among hospitalized patients and is a major problem in healthcare facilities, it has been referred to as healthcare-associated (HA) MRSA) for almost three decades [2]. Since the 1990s, MRSA infections attributed to community-acquired MRSA (CA-MRSA) have been increasingly recognized in the community [4]. Traditionally, CA-MRSA strains colonize or cause infections in persons with no prior history of healthcareassociated risk factors and seem to be more susceptible to antimicrobial drugs. These strains cause distinct clinical syndromes, have diverse genetic backgrounds, carry smaller SCCmec elements, most commonly SCCmec type IV or type V, and often harbor the Panton-Valentine leukocidin (PVL) [5]. However, the distinction between HA-MRSA and CA-MRSA remains problematic due to blurred boundaries between them and lack of consistently stable markers [5,6]. Since 2003, MRSA strains from livestock (for example, pigs) or animal products, termed livestock-associated MRSA (LA-MRSA) have emerged worldwide, which reveals the truly zoonotic potential of S. aureus/MRSA [7]. Recently, LA-MRSA, notably the sequence type (ST) 398 or related STs clustered in clonal complex (CC) 398, have been found in the community and have spread into hospitals [8].
To date, few studies documenting CA-MRSA in Tangier have been conducted. Therefore, we sought to investigate the prevalence of nasal carriage, the virulence, and the antimicrobial resistance of S. aureus, and to assess the current molecular epidemiology of MRSA in the community setting of Tangier.

Study design and patient recruitment
The present study was conducted over a four-month period, from 10 September 2012 to 16 January 2013. A total of 11 healthcare facilities, the Emergency Department of Tangier's CHR (Centre Hospitalier Régional) and 10 outpatient care centers situated in different regions of the city representing various socioeconomic statuses, were selected. The emergency department of CHR and the 10 outpatient care centers act as major referral health facilities in Tangier, and serve a population of one million people. The study involved outpatients and healthy volunteers attending those settings at the time of the study. The healthy population consisted of patients attending for contraception and prenuptial check-ups, children coming for vaccinations, and the individuals accompanying them. The current study was approved by the local ethics committee in 2012.
After obtaining consent from patients older than 18 years of age, or from younger patients' parents, 1,200 persons were screened for S. aureus carriage. Exclusion criteria for the study population were individuals who were non-residents of Tangier, those who had been hospitalized within the past year, had a device or prosthesis placement, dialysis, close contact with a healthcare worker (family member), factors beyond immunosuppression (diabetes mellitus, malignancy, and current chemotherapy), as well as healthy volunteers who had taken antibiotics in the past two months. Information regarding patient demographics, clinical presentation, residence, and household contacts was collected through standardized questionnaires and medical records.
The standardized questionnaires used in this study include the following data: age, gender, residence area, work status and income, working in a healthcare environment or having a close contact with a healthcare worker (family member), antimicrobial drug therapy history, relationship among various people screened, animal contact, previous institutional admission in the last 12 months (hospital, clinics, day-care attendance), medical procedure in the last 12 months (surgery, dialysis, catheterization, drainage), history of travel, type and length of infection, and history of skin infection (e.g., abscess, furuncles).

Isolation and identification of nasal S. aureus isolates
Samples were collected from both anterior nares by rotating a sterile swab (cotton-tipped polyester swab), which was inoculated into enrichment broth, brainheart infusion broth (BHI) (Oxoid, Basingstoke, UK), and incubated overnight at 37°C. A loopful of broth was sub-cultured on Mannitol agar (Oxoid, Basingstoke, UK). Suspicious colonies were identified as S. aureus by colony morphology and catalase and DNase tests. Presumptive S. aureus were confirmed by PCR for 16S rRNA and nuc genes, as previously described [9]. S. aureus ATCC29213 was used as the positive control.

Exotoxin gene detection
All the S. aureus isolates were tested for the presence of PVL genes and the tst gene by PCR [11]. MW2 (PVL-positive) and FRI913 (tst-positive) isolates were used as positive controls for PCR.

MRSA molecular typing
SCCmec types were determined by multiplex PCR using previously published primers that identified mec and complex genes [12]. Pulsed-field gel electrophoresis (PFGE) was performed by SmaI. Restriction digestion and patterns were normalized and compared using the Dice coefficient and unweighted pair group method with arithmetic mean (UPGMA) clustering method with Bio Numerics software version 2.5 (Applied Maths, Sint-Martens-Latem, Belgium). S. aureus subspecies NCTC 8325 was used as a control strain, and PFGE EMRSA-15 type L1a (CQ2001-SA05-E) was included for comparison. Strains showing the same PFGE pattern were grouped as a pulsotype and assigned alphabetically (A, B, C, etc.) as previously described [13,14]. The polymorphic X region of the spa gene was amplified using primers spa-1113f and spa-1514r [15]. Sequences were analyzed using Ridom Staph Type software version 1.4 (Ridom GmbH) and were synchronized with the global spa type database via the Staph Type server (www.spaserver.ridom.de). Clustering analysis into spa clonal complexes (spa-CCs) was carried out using the clustering algorithm based upon repeat pattern (BURP) with default settings. S. aureus isolates having fewer than five repeat units were excluded from the clustering analysis [16].

Statistical analyses
A univariate analysis was used to assess predictors for MRSA carriage among outpatients and healthy volunteers. Comparisons between proportions were drawn with Fisher's exact test and the Chi-squared test. Odds ratio (OR) and 95% confidence intervals (CI) were also calculated. Differences showing a p value < 0.05 were considered significant. Calculations were performed using SPSS version 20.0 software (IBM, Armonk, USA).

Antimicrobial susceptibility profiles
As Table 2 illustrates, MRSA isolates were susceptible or, alternatively, resistant to a limited number of antibiotics. None was resistant to  1 The differences between MSSA and MRSA in healthy volunteers and between MSSA and MRSA in outpatients; 2 The ratio of risk of being MRSA in the presence of different characteristics; 3 Overall comparison between healthy volunteers and outpatients; statistical significance at p < 0.05; 4 The ratio of risk of being outpatients in the presence of different characteristics. ciprofloxacin, pristinamycin, rifampicin, and fusidic acid. MRSA strains were resistant when compared to methicillin-sensitive S. aureus (MSSA); the difference was statistically significant regarding only tetracycline and co-trimoxazole (p < 0.0001).

Virulence factor genes
Of the isolates detected, 46 (11.5%) were PVL positive, and 62 (15.5%) were tst positive. The distribution of toxin genes in different groups is illustrated in Table 1. A total of12 isolates harbored both PVL and tst genes: 4 strains were from outpatients, and 8 belonged to healthy volunteers.

Discussion
In accordance with several studies, this research shows that nearly one-third of the screened population is colonized by S. aureus [2]. MRSA colonization rate (1.4%) appears also to be relatively close to that observed in the global European population [17], yet this rate remains low compared to MRSA carriage (3.3%) in a neighboring Algerian community [18]. The distribution of virulence genes among the two subpopulations was not statistically significant (Table  1), and this statistical insignificance concerned even the presence of the two virulence genes (OR = 2.56; 95% CI = 0.760-8.866; p > 0.05). However, it differs between MSSA and MRSA; tst was found to be significantly associated with MRSA both in healthy volunteers and outpatients (Table 1), whereas no MRSA was PVL positive. This could be partially explained by the fact that the MRSA tst positive isolates belong to the major MRSA clonal sequence type ST22 found (a biased notion).
Furthermore, in sharp contrast to previous studies in North Africa, Europe, the Middle East, and recent sporadic reports in sub-Saharan Africa, the predominant CA-MRSA isolates ST80, which are mainly PVL positive [18,19], were not identified in the current study. In addition, the ST1 and ST8 found differ from USA 400 and USA 300, respectively, in being PVL negative [20].
Distinct ST22 have been found worldwide in hospitals as well as communities; the most common is the epidemic HA-MRSA EMRSA-15 [20,21]. It has been recently reported in healthy individuals (without associated healthcare risk factors) in Italy and the Middle East [22][23][24]. The origin of ST22-MRSA-IV strains circulating in the Tangier community could be HA-EMRSA-15 types, which are disseminated from hospitals to community environments, as several studies have indicated [6,25]. This hypothesis might find its justification in the fact that the discharged patients continue their wound dressing elsewhere in the outpatient care centers. Nevertheless, the susceptibility pattern of our clone (Table 2) does not match that of HA-EMRSA-15, whose isolates are uniformly resistant to ciprofloxacin and susceptible to co-trimoxazole and minocycline [26]. ST22-MRSA-IV strains could be a variant of UK-EMRSA-15 originating from ST22-MSSA spa t223 by means of local evolution, as suggested by Biber et al. [23], knowing that the ST22-MRSA-IV strains observed in the present study showed traits similar to the predominant MRSA clone in the Gaza Strip [23] and to the one described in healthy Italian preschool children [22]. This clone mainly lacks exotoxin PVL, exhibits spa t223, and less often carries exotoxin gene tst. Nonetheless, the two hypotheses cannot be definitively confirmed since there is no study determining MRSA clones circulating in Tangier's hospital settings or any genetic information regarding the MSSA strain. Importantly, two strains (numbers 4 and 7) colonizing a healthy mother and her infant shared identical PFGE and spa types (Table 3). It should be noted that this was the only case in our study relating to household contact carriage. This finding may suggest that this kind of transmission of S. aureus could easily occur as previously described [27,28].
The relevant finding of the present study concerns the MRSA-ST398-t011 strain, which was isolated from an outpatient without any history of livestock or pet exposure. Elhani et al. [29] isolated one MRSA-ST398-t899 from the nasal sample of a farmer in Tunisia. According to the author, this was the first report of ST398 in humans in Africa. Although colonization of LA-MRSA in African countries seems to be scarce, prudence is needed and further investigations must be undertaken to understand the origin and the dissemination of this clone within our population.
Although our study provides the baseline in the epidemiology of MRSA colonization in Tangier, it has limitations. Firstly, it was limited in space and time. Secondly, surveying only nares and screening participants who are intermittently colonized may result in the underestimation of MRSA prevalence. And finally, five (29.41%) of the MRSA isolates could not be clustered.

Conclusions
We found a relatively low prevalence of nasal MRSA carriage within persons attending 11 healthcare facilities situated in different regions of the city and representing various socio-economic statuses. The molecular study highlights, for the first time, the current clones present in the Tangier community with a high prevalence of a non-multiresistant, tst-positive, PVLnegative t223 ST22-MRSA-IV clone and the presence of one ST 398 clone, and an unclassified third MRSA clone, which require further attention. The number of MRSA isolates available for our investigation was limited and might not reflect the whole epidemiology and MRSA strain types circulating in Tangier. Therefore, to obtain a comprehensive picture of CA-MRSA dissemination, other populations need to be addressed and continuous efforts to understand the epidemiology of this microorganism are necessary, not only in the community but also in the hospital and livestock.