Abstract

Objectives: To observe the prevalence of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and Panton-Valentine leukocidin (PVL)-positive S. aureus, this study was carried out in sheep and sheep keepers in Van, Türkiye.

Materials and methods: S. aureus strains were recovered from 200 privates nose swab samples from sheep and sheep keepers between July 2019 and June 2020. Methicillin resistance was determined by the oxacillin and cefoxitin disc diffusion method, the minimum inhibitory concentration (MIC) of oxacillin, and mecA gene detection. VRSA resistance was determined by the disc diffusion method, the MIC of vancomycin, and screening for the vanA and vanB genes. The PVL gene was also detected in MRSA strains.

Results: Fifteen of the 44 isolated strains of S. aureus were MRSA (2 of them were VRSA) and 29 were methicillin-sensitive S. aureus. All MRSA isolates were highly resistant to oxacillin (MIC 256 mg/mL). When compared with polymerase chain reaction (PCR), the sensitivity and specificity of the oxacillin disc diffusion method were 93.33% and 100% respectively; for the cefoxitin disc diffusion method and MIC of oxacillin both the sensitivity and specificity were 100%. Four (26.67%) MRSA isolates were positive for PVL genes which were also mecA positive. The MRSA strains were highly resistant to ciprofloxacin (93.33%), ceftriaxone (86.63%), azithromycin (73.33%), gentamycin (73.33%), and amoxiclav (66.67%). All (100%) MRSA strains were sensitive to linezolid and 86.67% were sensitive to vancomycin. The VRSA strains had an MIC 256 mg/mL for vancomycin and were positive for the vanB gene but negative for the vanA gene.

Keywords: The results of this study provide insight into the high proportion of MRSA and presence of VRSA in Van, Türkiye.

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Introductı on

Infections caused by Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), are emerging as a major public health problem in hospital and community settings, causinga wide range of diseases. The emergence and spread of both health care and community-associated MRSA has made infection control intervention and treatment challenging [1].

MRSA has evolved after acquiring the mecA gene that encodes the penicillin-binding protein 2a which confers resistance to methicillin and other b-lactam antibiotics [2].The mecA is a highly conserved gene found only in methicillin-resistant strains with no allelic equivalent in methicillin-susceptible Staphylococci,making it a useful marker for b-lactam resistance [3]. Detection of the mecA gene is the gold standard for identifying MRSA[4] but this testis not available in many clinical laboratories in developing countries and is relatively expensive. Different methods have been developed for the detection of MRSA including minimum inhibitory concentrations (MIC), the oxacillin screening agar method, and disc diffusion testing. The major problem in routine screening is that MRSA strains are heterogeneous in the expression of resistance to b-lactam agents [5].

S. aureus produces numerous virulence factors, including Panton-Valentine Leukocidin (PVL), which is a pore forming cytotoxin more often identified in community-associated MRSA strains than hospital-associated strains [6]. PVL-positive Staphylococcal infection typically presents as a life-threatening infection of the skin and soft tissues, bone, or joints, although it can also lead to necrotizing pneumonia, a severe and often fatal condition involving primarily young, healthy patients [3,6].

In 1996, VISA (vancomycin-intermediate S. aureus)was first reported in Japan [7], and then in 2002 vancomycin-resistant S. aureus (VRSA) was first reported in the USA [8]. Subsequent isolation of VRSA from different countries has confirmed that the emergence of these strains is a global issue [9-12].

The aim of this study was to determine the prevalence of MRSA and VRSA in nose swabs of sheep and sheep keepers using antimicrobial susceptibility profiles and to detect the PVL and mecA genes in MRSA as well as the vanA and vanB genes in VRSA.

Materı als And Methods

Samples

Two hundred privates swab samples were collected from sheep and sheep keepers admitted to Van Yüzüncü Yıl University, Faculty of Pharmacy, Pharmaceutical Microbiology Laboratory in Van, Türkiye over a 12-month period from July 2019 to June 2020.

Inclusion criteria

Nose swabs collected from sheep and sheep keepers admitted to irrespective of age, sex, and antibiotic intake. Nose swaps samples received in the Department of Pharmaceutical Microbiology for culture and sensitivity irrespective of age, sex, and history of antibiotic intake were also included.

Ethical issues

The human part of the study was carried out due to Approval of Clinical Researches Ethics Committee of Van Yüzüncü Yıl University Medical Faculty, Dated on 16th February 2018 and Decision Number: 2018/19. Animal part of the study was performed due to the Approval of Animal Experiments Local Ethics Committee of Van Yüzüncü Yıl University (decision number: 2018/01).

Isolation of S. aureus

S. aureus was identified by observing opaque colonies on blood agar media, Gram's staining, positive catalase and coagulase tests, and mannitol fermentation on mannitol salt agar [13].

Antimicrobial susceptibility testing

Standard disc diffusion techniques as recommended by the Clinical Laboratory Standards Institute (CLSI) were performed for susceptibility testing of oxacillin, cefoxitin, gentamycin, cotrimoxazole, ciprofloxacin, ceftriaxone, amoxiclav, vancomycin, and linezolid (oxoid, UK) for all S. aureus [14].

Detection of MRSA and VRSA by the disc diffusion technique

Screening for methicillin-resistance was determined using the Kirby-Bauer disc diffusion method with 1 mg oxacillin and 30 mg cefoxitin discs. VRSA was detected by a 30 mg vancomycin disc. Three to fivewere emulsifiedinto3 mLofsterile normal saline. The turbidity of the suspension was compared with the 0.5 McFarland turbidity standard and the suspension was incubated on Muellere Hinton agar plates at 37C for 24 hours. An inhibition zone diameter of 10 mm around the oxacillin disc was considered resistance; 11e12 mm indicated intermediate, and 13 mm was considered sensitive. For the cefoxitin disc, an inhibition zone diameter of 21 mmwasconsidered resistance and 22 mmwas considered sensitive [14].

Detection of MRSA by the MIC of oxacillin and VRSA by MIC of vancomycin

The MIC of oxacillin by the agar dilution method was determined according to CLSI guidelines. An MIC of oxacillin 4 mg/mL was considered MRSA and 2 mg/mL was considered methicillinsensitive S. aureus (MSSA) [14]. For preparation of the oxacillin stock solution, a 500-mg base of oxacillin was added to 50 mL distilled water to a concentration of 10 mg/mL. For each plate, 50 mL Muellere Hinton medium was prepared and impregnated with 10 mL, 20 mL, 40 mL, 80 mL,160 mL, 320 mL, 640 mL, or 1280 mLof the oxacillin stock solution to achieve concentrations of 2 mg/mL, 4 mg/mL, 8 mg/mL, 16 mg/mL, 32 mg/mL, 64 mg/mL, 128 mg/mL, or 256 mg/mL per plate, respectively.

For detection of VRSA, an MIC of vancomycin 16 mg/mL was considered resistant, 2 mg/mL was sensitive, and 4e8 mg/mL was VISA [14]. A vial of a 500 mg base of commercially available vancomycin injection was added to 50 mL distilled water to a concentration of 10 mg/mL. For each plate, 50 mL sterile MuellereHinton agar was prepared and impregnated with 10 mL, 20mL, 40mL,80mL,160mL,320mL, 640mL, or1280mLofvancomycin stock solution to achieve a concentration of 2 mg/mL, 4 mg/mL, 8 mg/ mL, 16 mg/mL, 32 mg/mL, 64 mg/mL, 128 mg/mL, or 256 mg/mL per plate, respectively.

DNA extraction

DNA was extracted using the boiling method. Bacterial colonies were suspended in 300 mL of distilled water and boiled for 10 minutes in a heat block, then placed on ice for 5 minutes. After centrifugation at 13,000 g at 4⸰C for 5 minutes, the supernatant was placed in a microtube and kept at 4⸰C until used as a DNA template [15].

Amplification of mecA and PVL genes

The mecA gene was amplified as described previously [16], using primers mecA-F: 50-AAAATCGATGGTAAAGGTTGGC-30 and mecA-R: 50-AGTTCTGCAGTACCGGATTTTGC-30. The DNA of the S.aureus ATCC 43300 and ATCC 25923 strains was used as positive and negative controls, respectively, for this polymerase chain reaction (PCR) assay of mecA. The PCR assay was performed in a total volume of 25 mL containing 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.25 mM each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP), and 1 U of Taq DNA polymerase (Promega Corporation, USA). DNA amplification was carried out using the following thermal cycling profile: initial denaturation at 95⸰C for 10 minutes, 35 cycles of amplification (denaturation at 95⸰C for 45 seconds, annealing at 55⸰C for 45 seconds, and extension at 72⸰C for 1 minute), and a final extension at 72⸰C for 10 minutes in a thermal cycler (Mastercycler gradient, Eppendorf AG, Germany). PCR products were analyzed on 1.5% agarose gel with 0.53 Tris-borate EDTA buffer. A 100-bp DNA ladder (Promega Corporation) was used as the molecular size marker. The gels were stained with 1% ethidium bromide and visualized under UV light [16].

The PVL gene was amplified as described previously [6,15]. Primers lukSF- forward (50- ATCATTAGGTAAAATGTCTGGACATGATCCA-30) and lukSF- reverse (50- GCATCAASTGTATTGGATAGCAAAAGC-30) directed against the PVL S and F precursor genes (lukS/F-PV) were used. DNA amplification was carried out using the following thermal cycling profile: initial denaturation at 95⸰C for 10 minutes, 33 cycles of amplification (denaturation at 95⸰C for 30 seconds, annealing at 55⸰C for 45 seconds, and extension at 72⸰C for 1 minute), and a final extension at 72⸰C for 10 minutes in a thermal cycler (Mastercycler gradient, Eppendorf AG). The PCR products were analyzed on 1.5% agarose gel with 0.53 Tris-borate EDTA buffer. A100-bp DNA ladder (Promega Corporation)was used as the molecular size marker. The gels were stained with 1% ethidium bromide and visualized under UV light [15]. The DNA of the S. aureus ATCC 25923strain was used as the negative control for the PVL gene assay.

Amplification of the vanA and vanB genes

The VRSA isolates were amplified using the primers for vanA (vanA F 5’-ATGAATAGAATAAAAGTTGCAATA-3’ and R 5’-CCCCTTTAACGCTAATACGATCAA-3’) and vanB (vanB F5’- GTGACAAACCGGAGGCGAGGA-3’ and R 5’- CCGCCATCCTCCTGCAAAAAA-3’) [11,17]. The PCR assay was performed in a total volume of 25 µL containing 10mM Tris-HCl, 50mM KCl, 1.5mM MgCl2, 0.25mM each of deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP), and 1U of Taq DNA polymerase (Promega Corporation). DNA amplification was carried out using the following thermal cycling profile: initial denaturation at 95⸰C for 10 minutes, 33 cycles of amplification (denaturation at 95⸰C for 45 seconds, annealing at 54⸰C for 45 seconds, and extension at 72⸰C for 1 minute), and a final extension at 72⸰C for 10 minutes in a thermal cycler (Mastercycler gradient, Eppendorf AG). The PCR products were analyzed on 1.5% agarose gel with 0.53 Tris-borate-EDTA buffer. A 100-bp DNA ladder (Promega Corporation) was used as the molecular size marker. The gels were stained with 1% ethidium bromide and visualized under UV light [11].

Results

Fifteen of the 44 isolated strains of S. aureus (34.09%) were MRSA as identified by the cefoxitin disc diffusion method and the MIC of oxacillin, and all of them were mecA positive on PCR. Fourteen (31.82%) strains were resistant to methicillin based on the oxacillin disc diffusion method. Four (26.67%) of the 15 mecA positive strains were alsopositive for the PVL gene. MRSA colonization was highest in patients aged between 40 years and 55 years with male and female distributions of 73.33% and 26.67%, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value of the three phenotypic methods in comparison with the molecular method. The cefoxitin disc diffusion method was superior to the oxacillin disc diffusion method for detection of MRSA and was comparable to the MIC of oxacillin. All the isolates were susceptible to linezolid. MRSA strains showed higher resistance than the other strains to all antimicrobials except for vancomycin. All (100%) of the PVL positive strains were sensitive to amoxiclav, vancomycin, and linezolid, three (75%) were sensitive to azithromycin, two (50%) were resistant to ceftriaxone and gentamycin, and three (75%) were resistant to ciprofloxacin.

The MIC for vancomycin of one of the two VRSA strains was >256mg/mL and the other was 256mg/mL. Both were positive for the vanB gene and neither was positive for the vana gene. Both VRSA strains were sensitive to linezolid, one was sensitive to gentamycin and the other was sensitive to azithromycin. No VISA was observed.

Dıscussıon

Infections caused by drug resistant S. aureus are increasing World wide due to the emergence of MRSA and VRSA. The prompt and accurate detection of MRSA and VRSA isolates could be useful for determining the appropriate treatment strategy and subsequent effective management and control of corresponding infections.

We found that the prevalence of MRSA was 34.09%, which correlates with previous reports from other regions of Bangladesh, Pakistan, and India [18-21]. However, some studies have reported much higher rates of 64-88% [17,22]. The exact cause of the relatively low prevalence of MRSA in the present study in comparison toot her countries is not known but it is possible that the prevalence of MRSA is still low in Türkiye. Moreover, the samples in the present study were taken from nose swaps of sheep and sheep keepers. The prevalence could have been higher if the samples had been taken from patients in the burn unit or casualty ward. The prevalence of MRSA varies significantly in different regions,which suggests a need for periodic evaluation of MRSA [23]. The increasing prevalence of MRSA is a threat to management of cases allover the world, including Türkiye.

As described in other studies [24,25], our MRSA isolates were resistant to most commonly used antibiotics. All MRSA isolates were sensitive to linezolid and 86.67% were sensitive to vancomycin, similar to other studies. This suggests that these drugs could be suitable treatment options [19,20,26]. As these drug sare expensive and some times not available in the local Pharmaceutical market, treatment options in Türkiye are limited for infections caused by MRSA.

In this study, MRSA strains had an MIC ofoxacillin 256 mg/mL. Some authors from Iran reported that most MRSA strains are highly resistant to oxacillin(MIC 256 mg/mL),similar to our study[17]. The higher MIC of oxacillin in the present study might be due to the fact that common antibiotics are sold over the counter in Banglades hand anybody can buy them without a doctor's advice. This might cause development of resistance to antibiotics.

Some authors from Bangladesh reported a PVL positive S.aureus rate of 15.25%, but no PVL was detected in MRSA strains [27]. However, in this study, the PVL gene was detected in four(26.67%) of the MRSA isolates which were hospital acquired MRSA. In the present study, however, MSSA strains were not screened for the PVL gene. Therefore, a surveillance mechanisms hould be set up to detect PVL genes in both MRSA and MSSA in Bangladesh. Usually PVL positive strain sare found in community acquired MRSA butthe PVL gene is not restricted to community acquired strains [28]. Some authors from India reported that 68-85% of MRSA strains were positive for the PVL gene, which is much higher than in the present study[21,29].The higher percentages in those studies might be due to misuse of antibiotics causing selective pressure for development of resistant strains along with the PVL virulence factor in those countries. The presence of PVL is associated with increased virulence of certain strains of S.aureus and is the cause of necrotic lesions involving the skin and mucosa which are very difficult to manage, such as necrotic hemorrhagic pneumonia. Therefore, early detection of the PVL gene in S.aureus may be important to manage cases and to assess the outcome after treatment.

In the present study, both the sensitivity and specificity of the cefoxitin disc diffusion method and the MIC of oxacillin were 100% when compared with PCR and these results are similar to those in other studies [30-32]. The sensitivity and specificity of the oxacillin disc diffusion method were 93.33% and 100% respectively in the present study, which correlates with another study [33]. We showed the cefoxitin disc diffusion method correlated better with the presence of the mecA gene. Cefoxitin is a potent inducer of the mecA regulatory system and is being widely used as a marker for mecA gene-mediated methicillin resistance [4]. Some reports showed almost no discrepancy between cefoxitin susceptibility testing and PCR results for the mecA gene [23,34]. The oxacillin disc diffusion method is used to identify MRSA in most laboratoriesin Türkiye. However, false negative as well as false positive results may be obtained using this method. This problem can be overcome by using cefoxitin discs instead of oxacillin discs to detect MRSA.

VRSA had not been detected previously in Bangladesh butthe present study Show edits emergence, which is a problem in treatment. Both VRSA isolates in the present study were resistant to several antimicrobials, but not linezolid, which is inaccordance with other reports[10]. In the present study, one VRSA strain had an MIC of vancomycin of 256mg/mL and the other was >256mg/mL, whereas VRSA isolated in other countries had MICs 32-1024mg/mL [9-11,35]. In the present study, however, VRSA strains were not tested for MICs>256mg/mL. Most VRSA isolated Worldwide carries the vana gene. However, in our study, neither strain was positive for the vanA gene but both strains were positive for the vanB gene. Some authors from India, Cairo, and Iran have reported vanB gene positive VRSA [12,36,37]. The prevalence of the vanA gene among VRSA isolates has been reported in some studies worldwide. The reason behind the absence of vanA gene in the present study might be differences in the geographical distribution of van genes in VRSA. The low number of isolated VRSA strains might also be the reason. In the present study, no VISA was identified.

Funding

This project was supported by Van Yüzüncü Yıl University Scientific Research Project Center. The project code is defined as TSA-2018-6938 (TAP).

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