Abstract
Aim:
To determine the genetic makeup of methicillin-sensitive/methicillin-resistant Staphylococcus aureus (MSSA/MRSA) from nasal colonization and environmental contamination in dental clinics.
Materials and Methods:
Nasal swabs from students and health care workers and environmental swabs were obtained at two academic dental clinics in the United Arab Emirates. The StaphyType DNA microarray-based assay was used for molecular characterization.
Results:
Forty-eight S. aureus isolates were identified phenotypically (nasal: n = 43; environmental: n = 5), but 6 of these were assigned to S. argenteus by genotyping. These were CC(argenteus)2596, CC(arg)2250-MSSA, CC(arg)2250-MSSA-(Panton Valentine leukocidin [PVL]+) (n = 2), and CC(arg)2198-MSSA (n = 2). MRSA nasal colonization rate was 5.4% (n/N = 8/146) with the following strain affiliations: CC5-MRSA-[IV+fus+ccrAB], “Maltese Clone”; CC6-MRSA-IV, “WA MRSA-51”; CC22-MRSA-IV (PVL+/tst+); CC22-MRSA-[IV+fus+ccrAA/(C)]; and two each of CC5-MRSA-[VI+fus] and CC97-MRSA-[V/VT+fus]. The SCC-borne fusidic acid resistance (fusC) gene was detected in MRSA (n = 5) and MSSA (n = 1). Some MSSA strains, CC1-MSSA-[fus+ccrAB1] and ST1278-MSSA-[ccrA1], harbored recombinase genes. A CC30-MSSA harbored ACME locus/arc-genes, while ST1278-MSSA-[ccrA1] had an ACME-III element. Enterotoxin genes were commonly carried, but tst-1 gene was found in only CC22, CC30, and CC34 strains, while pvl genes were identified in CC(arg)2250 and CC22-MRSA-IV. Of the 51 noncoagulase staphylococci (CoNS) identified, 18 were mecA positive.
Conclusion:
The findings demonstrate the first report of rare strains (ST1278 MSSA, CC(arg)2198, CC(arg)2596, and PVL+CC(arg)2250) in our region. Detection of MSSA with recombinase genes and ACME loci alongside mecA-positive CoNS is of clinical significance as this could provide a milieu for acquisition and transfer of SCC-elements, either with different ACME types, with fusC or the mecA gene resulting in conversion of MSSA into MRSA.
Introduction
Staphylococcus spp. are common human colonizers with Staphylococcus aureus and Staphylococcus epidermidis being predominant species. 1 Persistent colonization of the anterior nares with S. aureus occurs in 20–30% of the population, and this nasal carriage plays a key role in the epidemiology and pathogenesis of diseases.1,2 Indeed, nasal carriage of highly transmissible clones has been linked with the accelerated spread of community acquired methicillin-resistant S. aureus (CA-MRSA). 3
Methicillin-resistant S. aureus (MRSA) was first described in 1961 and is now established as an important cause of nosocomial infections accounting worldwide for significant patient morbidity and mortality. 4 Both hospital acquired MRSA (HA-MRSA) and CA-MRSA strains contribute to the high burden of infection. MRSA nasal carriage rates of 5–50% among health care workers (HCWs) have been reported with higher rates in settings of close clinical contact. 5 In the Arabian Gulf region, available data indicate that there is a significant rate of MRSA colonization among HCWs, including medical students.6,7 Research on the population structure and genotyping of S. aureus isolates in the hospital setting is changing our understanding of the diversity of S. aureus isolates present.8–11
HCWs serve as a reservoir of infection/vector for dissemination among patients through contaminated hands and hospital equipment. The threat posed by MRSA colonization in the dental care setting is as grave as those in the hospital setting especially as the nature of care provided by dental health care workers (DHCWs) necessitates close contact with patients. MRSA colonization among dental students, particularly those in the clinical years, has been reported.12–14 Findings from another study have indicated that MRSA carriage rates among DHCWs and dental students are lower in comparison to nondental HCWs. 15
Furthermore, nosocomial transmission of MRSA through the surfaces of the dental operatory has been reported. 16 However, in contrast to the magnitude of research in the hospital setting, data on S. aureus colonization and environmental contamination in dentistry remain limited. In addition, there is very little information on the population structure of S. aureus isolates from the dental setting in terms of identification of strain assignments and genetic characterization. 14 In the Arabian Gulf region, there are no reports on the genotypic characterization of methicillin-sensitive Staphylococcus aureus (MSSA)/MRSA circulating in dental settings. To address this paucity of data, this study was carried out to characterize the molecular structure of S. aureus associated with nasal colonization and environmental contamination in two academic dental units.
Materials and Methods
Study site and population
The study was carried out from April to November 2018 at the Hamdan Bin Mohammed College of Dental Medicine (HBMCDM-MBRU/Dubai Dental Clinic, Dubai and College of Dentistry, Ajman University (CoD-AU), Ajman). Ethical approval was obtained from the ethics committee at each study site. Signed consent was obtained from participants. Dental students in clinical clerkship (years 4 and 5) at CoD-AU and full-time DHCWs at both facilities were eligible to participate. Eligible DHCWs included dentists, dental assistants, and dental hygienists.
Sample collection
Nasal swabs were collected from the anterior nares using sterile premoistened cotton wool tipped swabs. The moistened swab was introduced into the anterior nares one at a time and rotated gently but firmly for about four to five times. The swabs were transported to the laboratory in Amies transport media. At the time of sample collection, a questionnaire was completed by each participant to obtain demographic data and risk factors for MRSA data, especially with regard to previous hospitalizations, previous surgery, antibiotic use during the preceding 90 days, chronic skin conditions, history of care for known MRSA patients, or contact with companion/farm animals.
Environmental swabs were obtained during clinic hours at the same visit when sampling of participants was done. Samples were collected from a total of 10 randomly selected dental treatment rooms at the facilities. Five to 10 surfaces (e.g., dental chair seat and arm, the adjoining sink, and counter rest) within the immediate area of the dental operatory unit in each treatment room were swabbed using cotton wool tipped swabs. The swabs from each dental treatment room were pooled and processed as a single sample.
Bacterial isolation
The swabs were processed in the laboratory by vortexing them individually in brain heart infusion broth (BHIB) containing 6.5% NaCl for 2 minutes, followed by incubation of broth at 35–37°C for 24–48 hours. Subculture was done from turbid BHIB tubes onto mannitol-salt agar and MRSA chromogenic agar. Bacterial identification was carried out using conventional methods, that is, VITEK2 (BioMerieux, Marcy L'Etoile, France) and MALDI-TOF (Bruker Daltonics, Bremen, Germany). MRSA and MSSA isolates were stored at −80°C pending molecular characterization.
Molecular characterization
Molecular characterization was carried out using the StaphyType DNA microarray (Abbott [Alere Technologies GmbH], Jena, Germany) based assay on all isolates identified phenotypically as S. aureus. Target genes and primer and probe sequences, as well as the detailed protocol, have been published previously.8,17 DNA microarray assays were carried out in accordance to manufacturer's instructions as previously described.8,10,17 Selected isolates were tested using a second array for further characterization of their SCC elements as previously described. 18
Results
Demographic information
Of the 146 participants, 50 (34.2%) were clinical clerkship students and 96 (65.8%) were DHCWs. The age range was 19–52 years (mean ± standard deviation [SD]: 27.9 ± 7.9 years). Duration of work experience among DHCWs ranged from 1 to 20 years (mean ± SD: 4.9 ± 4.6 years). None of the participants had cared for a known MRSA patient in the preceding 6 months, and usage of antibiotics and surgical history in this time period was reported by 23.3% (n = 34) and 5.5% (n = 8) of participants, respectively. Twenty-seven percent (n = 39) participated in team sports, and 10.3% (n = 15) had companion animals at home.
Nasal carriage of staphylococcal species and environmental contamination
Among the 146 participants, 89 (60.9%) were colonized with staphylococcal species. Six individuals had a combination of two staphylococcal species. Table 1 shows the distribution of staphylococcal isolates identified from nasal swabs. The most common staphylococcal species was S. aureus (n = 38/89; 42.6%) followed by S. epidermidis (n/N = 19/89; 21.3%). Among the coagulase-negative staphylococci, the presence of the mecA gene was found in S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus lugdunensis (Table 1). Nasal colonization with MRSA was detected in 8 (5.4%) participants, including five DHCWs and three students. None of the eight individuals with MRSA nasal colonization had identifiable risk factors. From the environmental sampling, five S. aureus isolates were identified (MSSA: n = 4; MRSA: n = 1).
Distribution of Staphylococcal Isolates Identified from Nasal Swabs
Three people had a combination of S. epidermidis mecA + S. warneri; One person each was found to have a combination of S. epidermidis + S. warneri; S. epidermidis + S. haemolyticus mecA; and S. warneri + S. capitis.
These were identified as S. aureus phenotypically but confirmed as S. argenteus on genotyping.
Molecular characterization using DNA microarrays
Forty-eight isolates from nasal (n = 43) and environmental (n = 5) sources, which were identified phenotypically as S. aureus, were genotyped. We found upon genotyping that six of these (five nasal and one environmental) were S. argenteus (Table 2). The genotyped isolates were distributed into 19 clonal complexes (CC), 4 of them being assigned to S. argenteus (these CCs are labelled “arg” henceforth). The predominant CC were CC22 and CC30 (n = 7 each), five each for CC5 and CC15. There were three each for CC97, CC361, and CC(arg)2250, while two isolates each belonged to CC398, CC(arg)2198, and ST1278. Other CC occurring as single isolates were CC1, CC6, CC7, CC8, CC10, CC34, CC188, CC2885, and CC(arg)2596. The five environmental isolates belonged to CC22-MSSA, CC30-MSSA, CC2885-MSSA, CC(arg)2250-MSSA (PVL+), and CC361-MRSA-[V/VT+fus]. Table 2 shows the CC and strain assignments of all genotyped isolates.
Molecular Characterization of DNA Microarray Genotyped Isolates
Positive in one isolate.
Positive in two isolates.
Positive in three isolates.
Positive in all isolates except one.
agr, accessory gene regulator; ccr, cassette chromosome recombinase gene; blaZ, beta-lactamase; blaI, beta lactamase repressor (inhibitor); blaR, beta-lactamase regulatory protein; sea, enterotoxin A; seb, enterotoxin B; sec, enterotoxin C; sed, enterotoxin D; sek, enterotoxin K; seq, enterotoxin Q; egc cluster, enterotoxins g,i,m,n,o,u; sak, staphylokinase; scn, staphylococcal complement inhibitor; chp, chemotaxis-inhibiting protein (CHIPS); hla, hemolysin alpha; hlb, hemolysin beta; cna, collagen-binding adhesin; fnbA, fibronectin-binding protein A; fnbB, fibronectin-binding protein B; mecA, alternate penicillin binding protein 2, defining MRSA; Q6GD50 (fusC), hypothetical protein associated with fusidic acid resistance; Q9XB68-dcs, hypothetical protein from SCCmec elements; ermC, rRNA adenine N-6-methyl-transferases causing erythromycin/clindamycin resistance; dfrA, dihydrofolate reductase; tetM/K, tetracycline resistance markers; fexA, chloramphenicol/florfenicol exporter; linA, lincosamide nucleotidyl transferase; msrA, energy-dependent efflux of erythromycin; mpbBM, probable lysylphosphatidyl glycerol synthetase; aadD, aminoglycoside adenyl-transferase, tobramycin resistance; aphA3, 3′5′-aminoglycoside phosphotransferase, neo-/kanamycin resistance; sat, streptothricin-acetyl-transferase; far1, fusidic acid resistance.
env, environmental; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensitive Staphylococcus aureus; NA, not applicable; PVL, Panton Valentine leukocidin.
Virulence genes
One isolate (CC30-MSSA) harbored the ACME locus/arc-genes. The two ST1278 were further characterized using a second array because of the presence of ccrA1 and were shown to harbor an ACME-III element, that is, opp genes. The toxic shock syndrome toxin 1 gene (tst1) was present only in isolates belonging to CC22 (n = 4), CC30 (n = 2), and CC34 (n = 1), and the latter also carried the enterotoxin H gene. Only three isolates were found to be positive for the Panton Valentine leukocidin (pvl) genes (Table 2). These comprised two CC(arg)2250 and one CC22-MRSA-IV (PVL+/tst+). While all S. aureus isolates harbored combinations of the protease genes (splA, splB, splE, sspA, sspB, sspP), these were not detectable in the S. argenteus isolates.
Although all isolates harbored the clumping factor genes (clfA, clfB), the intercellular adhesion protein genes (icaA, icaC, icaD) were present only in S. aureus, while in S. argenteus only icaA was detectable. In addition, none of the S. argenteus yielded signals for the cell surface elastin binding protein gene (ebpS), which was present in all S. aureus isolates. With regard to other toxigenic virulence genes, none of the isolates harbored the exfoliative toxin genes (etA, etB, etD) or the epidermal cell differentiation inhibitor genes (edinA, edinB, edinC). Majority of the isolates harbored different combinations of the enterotoxin genes (Table 2). In contrast to the S. aureus isolates, none of the S. argenteus yielded signals for hemolysin alpha and beta genes (hla, hlb). Table 2 shows the details of virulence genes found in the isolates.
Antibiotic resistance genes
All MRSA and majority of MSSA isolates (except CC7-MSSA, CC10-MSSA, CC(arg)2198-MSSA, and CC(arg)2250-MSSA [PVL+]) harbored the beta lactamase operon (blaZ, blaI, blaR). Phenotypic antibiotic susceptibility profile was in keeping with detection of antibiotic resistance genes. The SCC-borne fusidic acid resistance gene (fusC) was found only in the environmental MRSA and five of the eight nasal MRSA, which were phenotypically resistant to fusidic acid.
The following antibiotic resistance genes were absent from all isolates: erythromycin/clindamycin resistance gene (ermB), macrolide efflux protein A mefA, acetyl-transferase inactivating streptogramin A gene (vatA), chloramphenicol acetyltransferase gene (cat), mupirocin resistance protein gene (mupR), quaternary ammonium compound resistance genes (qacA, qacC), vancomycin resistance genes (vanA, vanB), and teicoplanin resistance gene (vanZ) (Table 2). Details on antibiotic resistance genes found in the isolates are shown in Table 2.
Discussion
The findings from this study indicate a high carriage of staphylococcal species as nasal colonizers among DHCWs and students. In addition, we found that 5.4% of participants had MRSA nasal colonization, which is higher than 3.1% found among dental students in Seoul, South Korea, 12 but considerably lower than 21–26% reported from other studies in the dental setting.13,14 In the Arabian Gulf region, MRSA rates among medical students and HCWs in a nondental tertiary care facility have varied with some reports showing levels similar to those found in this study and others demonstrating significantly higher carriage rates.6,7,19 The high prevalence of coagulase negative staphylococci nasal colonizers harboring the mecA gene along with co-colonization of S. aureus is of concern as these isolates could potentially serve as a reservoir for SCCmec elements and the mecA gene in the dental setting.
DNA microarray-based analysis of the S. aureus isolates revealed a wide clonal diversity, which is in accordance with previous reports from the Arabian Gulf region.10,19–21 S. aureus exhibits a clonal population structure with some clones predominating in geographical regions. Indeed, the predominant CC identified in this study were also similar to those reported as nasal colonizers among medical students and HCWs, as well as from infections in patients in Saudi Arabia.9,10,19–22
The MRSA strains identified in this study especially CC5 and CC22 MRSA are representative of common strains circulating in the region. The CC5-MRSA-[IV+fus+ccrAB] “Maltese Clone” is prevalent in health care facilities and is associated with clinical infections.9,10,22 CC5-MRSA-VI was first described in Portugal and has been reported in other centers in Europe, Latin America, and United States.23,24 However, the CC5-MRSA-[VI+fus] identified in this study, as well as those reported as nasal colonizer among HCWs in a tertiary care facility in Saudi Arabia, 19 uniformly differs from the Portuguese strain (HDE288) 24 in the absence of dcs.
For CC22 MRSA, six distinguishable strains based on SCCmec IV subtypes and virulence factors have been reported from this region. 11 Furthermore, recent identification of a novel CC22-MRSA variant in Saudi Arabia is suggestive of the expanding repertoire of CC22-MRSA. 25 The CC22-MRSA-IV (PVL+/tst+) reported in this study appears to be an emerging clone in the Arabian Gulf region, as well as beyond.22,25–27 Although CC97-MRSA-IV and CC361-MRSA-V have previously been reported in United Arab Emirates, 23 the identification of CC97-MRSA-[V/VT+fus] and CC361-MRSA-[V/VT+fus] in this study represents the first description of these strains in the United Arab Emirates. Indeed, CC361-MRSA-[V/VT+fus] appears to be emerging in the region as it was recently identified in Kuwait and Saudi Arabia.22,25
With the exception of CC22-MRSA-IV (PVL+/tst+), all other MRSA strains carried the fusC gene as additional payload on the SCCmec element. The high occurrence of fusC carriage in MRSA strains from the Arabian Gulf region has been reported and is thought to be linked with fusidic acid misuse/overuse.11,22,25,28 In addition, a SCCfus element was identified in a MSSA strain, the CC1-MSSA-[fus+ccrAB1] strain, which had also been reported as a nasal colonizer among HCWs in Saudi Arabia. 19 Locally in the dental setting, fusidic acid misuse tends to occur in the context of treatment of angular cheilitis. These findings highlight the importance of promoting judicious antibiotic use through adequate antibiotic stewardship programs and implementation of antibiotic prescription-only policy to prevent over the counter availability.
The MSSA and MRSA strains we identified were found to harbor similar types of virulence and antibiotic resistance genes. The absence of exfoliative toxins and the low prevalence of the pvl and tst-1 genes, as well as the widespread carriage of scn, chp, and sak virulence genes, are similar to MSSA isolates found in HCWs.19,20 However, in contrast to previous reports, the CC15-MSSA in this study carried the largest repertoire of antibiotic resistance and virulence genes. Although the implication of this is unknown, the concern remains that conversion of these CC15-MSSA strains into CC15-MRSA strains with an expanded repertoire of resistance and virulence could occur.
In addition to the presence of highly prevalent S. aureus clonal groups, our findings also identified the relatively rare ST1278-MSSA, as well as the emerging S. argenteus. ST1278-MSSA is related to the CC1 lineage and it has been identified in small numbers from human nasal colonization samples in Palestine, Israel, and France, as well as animals in Algeria.29–32 It is represented by publicly available sequence data of three isolates (SAMEA1523264, SAMEA2384354, SAMEA2710431).
ST1278 differs—based on our hybridization profiles, as well as on these sequences—from canonical CC1 in capsule type (five rather than eight), MLST profile (arcC-18 rather than −1), and lmrP allele. All other markers are identical to canonical CC1. Since some of the markers covered by the array (such as seh and cna) yielded the same results for CC1 and ST1278, and since they are localized between arcC and capsule gene/lmrP, two separate genomic replacements could be assumed. In addition, the two ST1278-MSSA strains harbored an ACME-III locus (opp genes) accompanied by ccrA1. We postulate that presence of SCC- or mecA-positive coagulase negative staphylococci in our dental setting as shown in this study could provide the milieu for acquisition of SCC elements, either with different ACME types or with the mecA gene by MSSA strains possibly resulting in conversion into MRSA.
S. argenteus is a recently named staphylococcus species, which had previously been misidentified as S. aureus. 33 Although S. argenteus is catalase and coagulase positive like S. aureus, phenotypically it differs from S. aureus as it lacks pigmentation of colonies due to the absence of the carotenoid pigment staphyloxanthin. S. argenteus has been shown to be of clinical relevance.33–38 Previously identified S. argenteus from our region belonged to CC(arg)225010,25 and was indeed associated with clinical infection. The findings in this study highlight an emergence of S. argenteus from nasal colonization and environmental contamination. It also marks the first description of CC(arg)2198 and CC(arg)2596 in our region.
Widespread occurrence of S. argenteus in clinical infections has been demonstrated in studies from South East Asia.34,38–40 There is significant population movement to and from the United Arab Emirates, which is driven by tourism and the expatriate work force, and we hypothesize that this could explain the introduction of S. argenteus strains into our setting. S. argenteus strains normally do not carry PVL with the notable exception being two CC2250 isolates from clinical infections in the French Indian Ocean Islands, one nasal colonizer in Myanmar, and most significantly, several cases from Thailand.34,36,39 The identification of two CC(arg)2250-MSSA (PVL+) in this study also buttresses the travel link hypothesis. Furthermore, the rare S. aureus CC2885 identified in this study was recently discovered in Laos. 38 Its detection, as also discussed for S. argenteus, suggests an epidemiological link to Laos/South East Asia, either by expatriate patients or health care personnel. The finding of CC(arg)2250-MSSA (PVL+) and ST2885-MSSA as environmental contaminants reinforces the need for implementation of adequate disinfection protocol to limit their dissemination.
Conclusions
The findings demonstrate similarities between the prevalence and characteristics of MRSA strains present in the dental setting and those reported from other health care facilities. The identification of MSSA strains harboring recombinase genes and the presence of rare S. argenteus strains highlight the need for continued surveillance and infection control practices. Regular screening of health care providers in the dental setting and prevention of fusidic acid misuse are recommended.
Ethical Approval
Obtained from both the ethics committees from both study sites.
Mohammed Bin Rashid University of Medicine and Health Sciences, Institutional Review Board—Reference No. MBRU-IRB-2017-00.
Ajman University, Research Ethics Committee—Reference No. F-H-17-12-01.
Footnotes
Disclosure Statement
E.M., A.R., D.G., R.E., and S.M. were employees of Alere Technologies GmbH/Abbott, Jena, Germany at the time the study was carried out. This did not influence the study design and they have no competing interests to declare. None of the other authors has any financial or other relationships that may constitute a conflict of interest.
Funding Information
This study was supported by Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, United Arab Emirates (College of Medicine, MBRU Internal Research Grant No. MBRU-CM-RG2017-03). The funding body did not influence or play any role in the study design, collection, analysis and interpretation of data, writing of the report, and decision to submit the article for publication.