Characterization of resistance to selected antibiotics and Panton-Valentine leukocidin-positive Staphylococcus aureus in a healthy student population at a Malaysian University

Abstract

Introduction

This study aims to assess the antimicrobial susceptibility profiles of Staphylococcus aureus strains isolated from university students and to determine the prevalence of constitutive and inducible clindamycin resistance, the latter being able to cause therapeutic failure due to false in vitro clindamycin susceptibility.

Methods

S. aureus strains were isolated from the nasal swabs of 200 health sciences students of a Malaysian university. Twelve classes of antibiotics were used to evaluate the antimicrobial susceptibility profiles with the macrolide-lincosamide-streptogramin B (MLS_B) phenotype for inducible clindamycin resistance determined by the double-diffusion test (D-test). Carriage of resistance and virulence genes was performed by PCR on S. aureus isolates that were methicillin resistant, erythromycin resistant and/or positive for the leukocidin gene, pvl (n=15).

Results

Forty-nine isolates were viable and identified as S. aureus with four of the isolates characterized as methicillin-resistant S. aureus (MRSA; 2.0%). All isolates were susceptible to the antibiotics tested except for penicillin (resistance rate of 49%), erythromycin (16%), oxacillin (8%), cefoxitin (8%) and clindamycin (4%). Of the eight erythromycin-resistant isolates, iMLS_B was identified in five isolates (three of which were also MRSA). The majority of the erythromycin-resistant isolates harbored the msrA gene (four iMLS_B) with the remaining iMLS_B isolate harboring the ermC gene.

Conclusion

The presence of MRSA isolates which are also iMLS_B in healthy individuals suggests that nasal carriage may play a role as a potential reservoir for the transmission of these pathogens.

Introduction

Staphylococcus aureus is a Gram positive and coagulase producing bacterium usually considered as a normal flora or commensal of the skin and nasal cavities of humans and various animal species.¹ Previous studies have shown that 20-50% of the human population are often colonized with S. aureus.^2,3 Methicillin-resistant S. aureus (MRSA) is one of the major risk pathogens associated with evolution of antimicrobial resistance. Additionally, antibiotics from the macrolide-lincosamide-streptogramin B (MLS_B) family are usually drugs of choice for the treatment of staphylococcal infections. However, resistance to erythromycin (a macrolide) is usually associated with resistance to clindamycin (a lincosamide) and to type B streptogramin, and this cross-resistance is normally mediated by the erythromycin ribosomal methylase (erm) genes.⁴ Three MLS_B phenotypes are typically identified in staphylococci: a constitutive resistant (cMLS_B) phenotype, an inducible clindamycin resistant (iMLS_B) phenotype, and a clindamycin-susceptible, macrolide-streptogramin B-resistant (MS) phenotype. It is difficult to detect the iMLS_B phenotype in routine laboratory testing as they appear clindamycin-susceptible and erythromycin resistant in vitro. This false susceptibility to clindamycin in strains with the iMLS_B phenotype may lead to therapeutic failure.⁴

PVL is a bi-component pore-forming toxin with the ability to destroy and lyse leukocytes.⁵ The presence and emergence of pvl-positive MRSA has been previously described in clinical isolates or hospital-associated MRSA (HA-MRSA).⁶ A key feature of community-acquired (CA) MRSA is the presence of SCCmec types IV and V, as well as (in some cases) their co-existence with the pvl gene.⁷ Recently, MRSA infection is no longer confined to patients with known risk factors or exposed to healthcare settings. Emergence of CA-MRSA has been documented from several regions across the globe and imposes important clinical implications especially among patients without traditional risk factors.⁸

Previous studies reported that pvl-positive MSSA strains have been associated with outbreaks of skin and soft tissue infections (SSTI) among communities as well as from nosocomial infection. The pvl prevalence in MSSA infections varies between countries and is reported with higher prevalence in African countries.⁹ Interestingly, there is evidence showing that pvl-positive S. aureus strains, particularly MSSA, may emerge and become pvl-positive MRSA by acquiring the SCCmec elements comprising the mecA gene through horizontal gene transfer, thus conferring the methicillin-resistance capability.¹⁰

Thus, the presence and emergence of MSSA-pvl-positive isolates among healthy nasal carriers should not be neglected. Unfortunately, reports on the prevalence of pvl among S. aureus isolated from healthy individuals in the community in Malaysia are lacking. Here, we described the antimicrobial susceptibility profiles and carriage of resistance and virulence determinants in a collection of S. aureus strains isolated from healthy students in a major tertiary university in Malaysia. Interestingly we observed the occurrence of S. aureus isolates that showed inducible MLS_B phenotype, were pvl-positive and harbored several other virulence genes. Selected isolates were characterized by SCCmec typing, staphylococcal protein A (spa) typing and accessory regulator (agr) typing for comprehending their genetic background.

Methods

Bacterial culture and growth maintenance

A total of 49 S. aureus isolates, previously collected from the anterior nares of 200 healthy undergraduate students from the Health Sciences program, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia in 2012 (n=30) and 2013 (n=19) were viable for further analysis in this study. The isolates had been kept in 15% glycerol stock at -20°C and revived on mannitol salt phenol red agar (MSA, Oxoid, Lenexa, KS, USA) and incubated at 37 °C for 24 hours. Presumptive S. aureus colonies with yellowish colonies were further subcultured onto nutrient agar supplemented with 3.5% NaCl prior to further phenotypic identification. Confirmation of presumptive S. aureus strains was performed by Gram staining, determination of the presence of coagulase and catalase activity as well as genotypic identification by using nuc-PCR.¹¹ All nuc-positive S. aureus were further characterized as MRSA by PCR detection of mecA gene as described previously.¹¹ Positive controls were used for genotypic identification including MRSA ATCC 33591, MSSA ATCC 22932, IMR S5-pvl-positive and an in-house MSSA KT/312045 isolate that was previously characterized as pvl-positive.¹²

Antibiotic susceptibility test

Kirby-Bauer disc diffusion was used to identify the susceptibility profiles of the S. aureus isolates using the panel of antibiotics as suggested previously by Magiorakos et al.¹³ (Table 1) and interpreted using the breakpoints set by the Clinical and Laboratory Standards Institute (CLSI, 2016)¹⁴ and British Society for Antimicrobial Chemotherapy (BSAC).¹⁵ Determination of the MLS_B phenotype for the erythromycin resistant isolates was performed using the double-diffusion test (D-test) based on the CLSI (2016) guidelines.

Table 1. List of antibiotics used and susceptibility pattern of 49 isolates of S. aureus.

Antibiotic agents		Resistant n (%)	Susceptible n (%)
PEN	Penicillin	49 (100%)	0 (0%)
FOX	Cefoxitin	4 (8%)	45 (92%)
OX	Oxacillin	4 (8%)	45 (92%)
E	Erythromycin	8 (16%)	41 (84)
DA	Clindamycin	2 (4%)	47 (96%)
DO	Doxycycline	0 (0%)	49 (100%)
TE	Tetracycline	0 (0%)	49 (100%)
CN	Gentamicin	0 (0%)	49 (100%)
SXT	Trimethoprim/ sulfamethoxazole	0 (0%)	49 (100%)
CIP	Ciprofloxacin	0 (0%)	49 (100%)
MXF	Moxifloxacin	0 (0%)	49 (100%)
TEC	Teicoplanin	0 (0%)	49 (100%)
VA	Vancomycin	0 (0%)	49 (100%)
C	Chloramphenicol	0 (0%)	49 (100%)
QD	Quinupristin/ dalfopristin	0 (0%)	49 (100%)
LZD	Linezolid	0 (0%)	49 (100%)
*TGC	Tigecycline	0 (0%)	49 (100%)
*FD	Fusidic acid	0 (0%)	49 (100%)
*FOS	Fosfomycin	0 (0%)	49 (100%)
MUP	Mupirocin	0 (0%)	49 (100%)
Total		49 isolates

All antimicrobial susceptibility breakpoint interpretation was performed according to CLSI 2016 except for *Breakpoint guideline BSAC (2014).

Molecular detection of genes encoding virulence and antimicrobial resistance

The presence of several resistance and virulence genes in the S. aureus isolates was determined by PCR. The following genes conferring resistance to the respective antibiotics were screened: blaZ (penicillin), mupA, ileS (mupirocin), aac(6’)-aph(2”) (gentamicin), ermA, ermB, ermC, msrA (erythromycin), vanA, vanB (vancomycin), cfr (linezolid), tetA, tetB (tetracycline), and fusB, fusC, fusD (fusidic acid).^16,17 Additionally PCR was also performed to detect 17 different virulence genes for enterotoxins (sea, seb, sec, sed, see, seg, sei, sej), exfoliative-toxins (eta, etb, etd), cytotoxin (lukS/lukF), and adhesins (hla, hlg, ica, efb, sasX).^6,16,18 Amplification products were separated by electrophoresis in a 1.5% (w/v) agarose gel (Promega, USA) in a 1X tris-borate-EDTA (TBE) running buffer (Bioline, London, UK). Following staining in a 1 µg/mL GelRed solution (10 mg/mL) (Biothium, Fremont, CA, USA), the PCR products were visualized and documented using an Alpha Imager gel documentation system (Thermo Fisher Scientific, Waltham, MA, USA). A 100 bp molecular weight marker (Bioline Hyperladder) was electrophoresed alongside samples to enable the size of the PCR products to be compared and determined.

Molecular typing of isolates

Four MRSA isolates were further characterized for SCCmec typing using a multiplex PCR method which generated a clear and easily discriminated pattern for the 5 main SCCmec types (I to V) with specific primers as described by Boye et al.¹⁹ The isolates that showed unrelated patterns were classified as non-typeable (NT). Meanwhile, the same four MRSA with eleven MSSA isolates with either pvl-positive or MLS_B phenotypes were further characterized for spa and agr group typing. The polymorphic X-region of the spa gene was amplified and submitted to 1^st Base Laboratory, Malaysia, for sequencing services and assembled using CLC Workbench Version 5.0 (CLCbio, Boston, MA, USA) prior to being imported and analyzed with DNAgear.²⁰ Spa types were determined using the spa database website (http://spaserver.ridom.de). Meanwhile typing of the accessory gene regulator (agr) group was also performed as previously described using a multiplex PCR primer set, comprising a common forward primer (Pan) and reverse primers (agr1, agr2, agr3 and agr4) that were specific to each agr group.²¹

Statistical analysis

The frequencies of S. aureus isolates as well as susceptibility towards antibiotics were counted and the resistance percentages were calculated by 95% confidence interval. The correlation between isolates and both resistance and virulence genes was determined by Chi-square and Fisher’s exact test. Statistical significance was identified as p<0.05. All statistical analysis was performed by using JMP 10.1 (SAS Institute, Cary, NC, USA).

Ethical approval

Informed written and signed consents were obtained from recruited volunteers. The study and method of obtaining consent were approved by the University Research Ethics Committee of the Universiti Putra Malaysia (JKEUPM) Reff: UPM/FPSK/100-9/2-JKEUP(JSB(U)_Dec (12)07).

Results

Bacterial characterization and antibiotics susceptibility testing

All forty-nine presumptive S. aureus isolates that produced yellow colonies were confirmed as S. aureus genotypically by nuc-PCR identification. The percentage of S. aureus carriers among healthy undergraduate students was (24.5%). Four out of 49 (8%) isolates were positive for the mecA gene and thus categorized as MRSA, while the remaining 45 isolates (95%) were MSSA. Antimicrobial susceptibility testing indicated that all isolates were resistant to penicillin whereas resistance rates to cefoxitin and oxacillin were much lower, at 8% each (or 4/49 isolates).

All isolates were susceptible to vancomycin, teicoplanin, linezolid, ciprofloxacin, norfloxacin, mupirocin, fusidic acid, rifampicin, tigecycline, doxycycline, tetracycline, gentamycin, trimethoprim/sulfamethoxazole, moxifloxacin, chloramphenicol, quinupristin/dalfopristin and fosfomycin (Table 1). Five (11%) out of 45 MSSA isolates were erythromycin resistant followed by clindamycin at 4% (2/45 isolates). Among five erythromycin-resistant MSSA isolates, iMLS_B was observed in two isolates, cMLS_B also in two isolates, and MS in the remaining isolate (Table 2). Two (4%) MSSA isolates were multidrug-resistant (MDR) with resistance towards 3 antimicrobial classes (penicillin, erythromycin and clindamycin). Meanwhile for MRSA, three (75%) out of four isolates were erythromycin resistant with iMLS_B phenotypes, and there were no MDR isolates detected.

Table 2. Details of phenotypic, virulence and resistance determinants of 15 selected isolates of S. aureus.

S. aureus isolate	Year	Phenotypic resistance profiles	MLSB phenotype	AMR genes	Virulence genes	spa type	SCCmec type	agr type
MRSA
1.	2012	PEN, FOX, OX	-	mecA,blaZ	efb, ica, seg, hla, hlg	t186	III	IV
2.	2012	PEN, FOX, OX, E	iMLSB	mecA, blaZ, msrA	efb, ica, sec, sei, hla, hlg	t701	I	III
3.	2012	PEN. FOX, OX, E	iMLSB	mecA, blaZ, msrA	pvl, efb, ica, sec, sei, hla, hlg	t145	I	III
4.	2013	PEN, FOX, OX, E	iMLSB	mecA, blaZ, msrA	efb, ica, seg, hla, hlg	NT	I	III
MSSA
1.	2012	PEN	-	blaZ	pvl, efb, ica, sec, sei, hla, hlg	t548	-	IV
2.	2012	PEN	-	blaZ	pvl, efb, ica, see, seg, hla, hlg	t159	-	III
3.	2012	PEN	-	blaZ	pvl, efb, ica, sea, sei, hla, hlg	t14331	-	I
4.	2012	PEN	-	blaZ	pvl, efb, ica, sec, sei, hla, hlg	t336	-	I
5.	2012	PEN	-	blaZ	pvl, efb, ica, see, seg, hla, hlg	t548	-	I
6.	2012	PEN	-	blaZ	pvl, efb, ica, see, hla, hlg	t026	-	III
7.	2012	PEN, E	iMLSB	blaZ, msrA	pvl, efb, ica, hla, hlg	t6290	-	I
8.	2013	PEN, E, DA	cMLSB	blaZ, msrA	efb, ica, hla, hlg	NT	-	III
9.	2013	PEN, E	MS	blaZ	efb, ica, hla, hlg	t1381	-	III
10.	2013	PEN, E, DA	cMLSB	blaZ, msrA	efb, ica,hla, hlg	t14503	-	III
11.	2013	PEN, E	iMLSB	blaZ, ermC	efb, ica, hla, hlg	NT	-	I

AMR – antimicrobial resistance gene; cMLSB – constitutive macrolide-lincosamide-streptogramin B resistant; DA – clindamycin; E – erythromycin; FOX – cefoxitin; iMLSB – inducible macrolide-lincosamide-streptogramin B resistant; MS – macrolide-streptogramin B-resistant; NT – non-typeable; OX – oxacillin; PEN – penicillin.

Molecular typing

In this study, four methicillin-resistant S. aureus and 11 MSSA isolates with either erythromycin-resistant MLS_B phenotypes (iMLS_B or cMLS_B) or pvl-positive, or both MLS_B and pvl-positive, were selected. As shown in Table 2, the four MRSA isolates showed only two SCCmec types, whereas three of the MRSA isolates harbored SCCmec I (75%) and the remaining one had SCCmec III (25%). Furthermore, a total of 11 different spa types were identified in both MSSA and MRSA isolates, where only two MSSA isolates were of the same spa type (t548) albeit from different agr types (type I and IV). Nevertheless, three isolates (two MSSA and one MRSA) were non-typeable. Meanwhile, three agr genotypes were observed where the most frequent agr type was agr III (54%), followed by agr I (33%) and agr IV (13%) (Table 2). No agr type II was observed in whole isolates. The results also indicated that three of the MRSA isolates that were typed with SCCmec I were of agr type III while the remaining MRSA isolate which contained SCCmec III was of agr type IV. Nevertheless, agr types I and III were predominant among MSSA isolates (33% each) and one MSSA isolate was of agr type IV.

Prevalence of antimicrobial resistance and virulence determinants

The 15 isolates subjected earlier to molecular typing were further screened for resistance and virulence determinants using PCR as indicated in Table 3. All isolates were positive for the blaZ gene, which was not surprising as all the isolates were penicillin resistant, there were no significant differences between MRSA and MSSA (p=1).

Table 3. Antimicrobial resistance and virulence gene distribution among MRSA and MSSA isolates.

Antimicrobial resistance genes
Gene	MRSA n=4	MSSA n=11	Total n=15	OR	*95%CI	p-value
mecA	4	0	4 (27%)	-	- -	<0.05
blaZ	4	9	13 (87%)	-	- -	1
ermA	0	0	0	-	- -	-
ermB	0	0	0	-	- -	-
ermC	0	1	1 (6%)	-	- -	1
msrA	3	3	6 (40%)	8	0.58-110	0.24
aac(6′)-aph	0	0	0	-	-	-
cfr	0	0	0	-	-	-
fusB	0	0	0	-	-	-
fusC	0	0	0	-	-	-
fusD	0	0	0	-	-	-
ileS	0	0	0	-	-	-
tetA	0	0	0	-	-	-
tetB	0	0	0	-	-	-
vanA	0	0	0	-	-	-
vanB	0	0	0	-	-	-
Virulence genes
Gene	MRSA n=4	MSSA n=11	Total n=15 (%)	OR	*95%CI	p-value
pvl	1	7	8 (53)	0.58	0.04-7.66	1
sea	0	1	1 (7)	0	- -	1
seb	0	0	0	-	-	-
sec	2	2	4 (27)	4.5	0.37-54	0.52
sed	0	0	0	-	-	-
see	0	3	3 (20)	0	- -	0.52
seg	2	2	4 (27)	4.5	0.37-54	0.52
sei	2	3	5 (33%)	2.7	0.25-28	0.56
sej	0	0	0	-	-	-
efb	4	11	15 (100)	-	- -	1
ica	4	11	15 (100)	-	- -	1
hla	4	11	15 (100)	-	- -	1
hlg	4	11	15 (100)	-	- -	1
eta	0	0	0	-	-	-
etb	0	0	0	-	-	-
etd	0	0	0	-	-	-
sasX	0	0	0	-	-	-

95%CI – 95% confidence interval; OR – odds ratio.

For the eight erythromycin-resistant isolates, most of them (6/8) harbored the msrA gene (four iMLS_B and two cMLS_B). Only one erythromycin-resistant isolate, an iMLS_B MSSA, was positive for the ermC gene. Intriguingly, the single erythromycin-resistant MSSA strain that displayed the MS phenotype did not show any positive results for the macrolide-resistance genes that were screened by PCR in this study (i.e., ermA, ermB, ermC and msrA) (Table 2). All 15 isolates screened were negative for the other antibiotic resistance genes, which correlated well with their susceptibility phenotypes: vancomycin (vanA and vanB genes), mupirocin (mupA, ileS), gentamicin (aac(6’)-aph(2”)), linezolid (cfr), tetracycline (tetA, tetB), and fusidic acid (fusB, fusC, fusD). There were no significant differences between MRSA and MSSA for blaZ (p=1.000), ermC (p=1.000) and msrA (p=0.235).

Eight (16%) S. aureus isolates harbored the cytotoxin pvl gene, of which only one was MRSA and the other seven were MSSA isolates. All 15 isolates were positive for four (efb, ica, hla and hlg) out of six adhesion genes as shown in Table 2. Additionally, for enterotoxin genes, ten (67%) out of 15 isolates harbored at least one or two staphylococcal enterotoxin (SE) genes. Most of the SE genes were sei (33% or 5/15 isolates) followed by sec (27% or 4/15 isolates), seg (27% or 4/15 isolates), see (20% or 3/15 isolates) and sea (7% or 1/15 isolates). Among the four MRSA, two isolates harbored only seg whereas the other two isolates harbored both the sec and sei genes. Meanwhile, one MSSA isolate (ND19) was positive for only the see gene, while five other MSSA isolates were positive for a combination of either sea+sei (ND11), sec+sei (ND6 and ND13) or see+seg (ND10 and ND15) genes. Furthermore, all MSSA isolates were negative for all SEs genes. Nevertheless, no adhesion (sasX), exfoliative-toxin (eta, etb and etd) and enterotoxin (tsst) genes were detected among all 15 isolates tested.

Discussion

S. aureus is a major pathogen with a high virulence that causes both HA and CA infections globally, including skin and soft tissue infection with varying severity, from mild to life-threatening infections such as sepsis and bacteremia.¹⁸ This study showed that the MRSA proportion was only 8%, which is similar to a recent study at a similar setting in nasal isolates.² The proportion of MRSA carriage in our collection was in concordance with other worldwide studies investigating the prevalence of MRSA carriage that varied within 0.34 to 12% rates in medical students and healthcare centres.^2,3,22

Among the four MRSA strains, three isolates were detected as SCCmec I and one isolate was identified as SCCmec III. Although the number of MRSA in this study is low, the predominant occurrence of SCCmec I from this study is consistent with a previous study in a similar setting by Mat Azis et al., indicating the persistency of MRSA with SCCmec I.² Similarly, another previous study reported a high prevalence of SCCmec I (68%) among all MRSA isolates from nares of healthcare workers in Nepal.²³ Additionally, spa typing showed a diverse genetic background of the isolates in this collection. Of the 11 different spa types identified, spa type t548 was the predominant clone involving two MSSA isolates with different agr types and pvl positive character. The associated sequence type (ST) for the spa type t548 was ST-5 and ST-97. Similarly, other clinical MRSA isolates positive for the pvl gene also shared the same spa type, t548.²⁴ Furthermore, one of our pvl-positive MRSA isolates with spa type t701 (SCCmec I) was also reported as the most frequently identified MRSA in Iran, except for the SCCmec III, which is different from our isolates.²⁵ Meanwhile another study in Malaysia, reported the same spa type, t701, among nasal S. aureus isolates from persistent carriers.² On the other hand, three non-typeable (NT) spa strains were detected in this study, either due to spa mutations or due to absent spa. However, NT spa strains may still have the potential to induce invasive disease.²⁶

The predominant agr genotype in this collection of isolates among MRSA and MSSA was agr type III (54%), the majority of this agr were pvl-positive, which is in agreement with other reports indicating the predominance of the same agr type among S. aureus.²¹ Most of our agr type I MSSA isolates were positive for the pvl gene and this is in contrast with a previous study conducted by Afroz et al., indicating that the presence of pvl among MSSA was clustered within agr type II and III.²⁷ Our results also revealed that agr type IV only existed in two isolates (13%) of MRSA and MSSA strains. Likewise, a previous study showed that this type of agr IV is an uncommon feature in S aureus isolates.²⁸

Our isolates collection showed the presence of pvl-positive isolates (16%), where one out of eight were MRSA, while the remaining were harbored in MSSA. Our findings showed a higher proportion of pvl-positive isolates compared with previous studies in Malaysia that reported 5.5% pvl-positive MSSA among pigs and pigs handler samples, conducted by Neela et al.²⁹ Neela et al. also stated that the prevalence of pvl-positive isolates in Malaysian carriage displayed an increasing pattern, from 5.5% to 16%, indicating that the community-pvl is able to cause MSSA infections worldwide. Community-associated S. aureus surveillance must not be underestimated, since a high prevalence of pvl-positive MSSA has been reported in our nearest neighboring countries such as Indonesia and Thailand, meanwhile a low prevalence of pvl among MRSA was reported in Indonesia.^8,30

Moreover, in this collection of isolates, the classical SEs (sea - see) as well as seg, sei and sej were screened in MRSA and MSSA. Four MRSA isolates harbored at least one or two SEs, while six MSSA harbored two SEs and only one isolate carried a single SE. The presence of SEs among nasal isolates become a public health concern as these genes are often associated with foodborne poisoning, toxic shock syndrome and various other toxin-mediated diseases.¹⁶ In this present study, the highest rate of SE was sei with 33%, which differed from other studies that reported that the sea gene was common among S. aureus isolates from nasal samples, while another report found a high prevalence of the sei gene from invasive strains of both MRSA and MSSA.³¹ The occurrence of the sea gene in this collection was low at 7% compared with other studies previously reported elsewhere.^18,32 Additionally, our results also showed that eight (53%) (4 MRSA and 4 MSSA) isolates were positive for either sec or seg. Previous reports also showed the presence of sec among the clinical MRSA isolates from Terengganu referral Hospital, east coast of Malaysia, involving 2 MRSA and 3 MSSA isolates.¹⁸ Although both seg and sei genes are located in the same enterotoxin gene cluster (egc) operon, all of our isolates did not harbor both genes simultaneously, which is similar to data reported by other researchers.³¹ Likewise, none of the eta, etb and etd genes were detected among all tested isolates; similar results were also reported elsewhere.³³

The major limitation of this study was that the bacterial sample collection was not constructed in a systematic consecutive manner, and some volunteers did not allow the use of further details on demographic as well as retrospective data examination. Therefore, this study does not represent a prevalence study but it shows the characterization of the 49 isolates with a further analysis on 15 isolates that carried antimicrobial resistance and virulence genes of interest only. Nevertheless, as inclusion of isolates is random and no bias can be reasonably expected, the proportion may reflect some epidemiological perspective to a certain extent in providing insight into the distribution of antimicrobial susceptibility occurrences with both resistance as well as virulence genes determinants in MRSA and MSSA. Thus, data obtained here can be used for monitoring the presence of virulence and resistance determinant genes in S. aureus isolated from nasal carriers.

Conclusions

Interestingly, the presence of pvl-positive MSSA carriage and MLS_B phenotypes in our study suggests that nasal carriage may play an important role as potential reservoir and transmission of these pathogens in posing disease threat regardless of methicillin resistance. Therefore, although the risk of human infection with both MRSA and MSSA from nasal carriage is often considered rare, its potential risk to the public health should not be undermined.