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Year : 2020  |  Volume : 38  |  Issue : 2  |  Page : 162--168

Genotypic and phenotypic characterisation of clinical isolates of methicillin-resistant Staphylococcus aureus in two different geographical locations of Iran

Shiva Ahmadishoar1, Nadia Kazemi Pour1, Javid Sadeghi2, Mohammad Reza Nahaei2, Babak Kheirkhah1,  
1 Department of Microbiology, Kerman Branch, Islamic Azad University, Kerman, Iran
2 Department of Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

Correspondence Address:
Dr. Nadia Kazemi Pour
Department of Microbiology, Kerman Branch, Islamic Azad University, Kerman
Iran

Abstract

Background: Methicillin-resistant Staphylococcus aureus (MRSA) has become more prevalent all over the world and it is important to determine MRSA prevalence and typing in different regions. The present study was carried out to determine the prevalence and frequency of circulating molecular types of MRSA isolates as well as their antibiotics susceptibility in Tabriz and Kerman cities of Iran. Materials and Methods: A total of 230 S. aureus isolates were collected from Tabriz (n=125) and Kerman (n=105) during January to December 2018. MRSA isolates were identified by PCR amplification of nuc and mec A genes. Antibiotic susceptibility of MRSA isolates were determined by Kirby-Bauer disk diffusion method. Multiplex PCR was exploited to detect various types of SCCmec. Results: The MRSA prevalence was 51/125 (40.8%) in Tabriz and 60/105 (57.1%) in Kerman. Overall, 36/51 (70.58%) and 15/51 (29.41%) isolates and 37/60 (61.66%) and 23/60 (38.34%) isolates were isolated from inpatients and outpatients in Tabriz and Kerman, respectively. Almost all of the isolates were resistant to penicillin and all of them were sensitive to linezolid. Thirty five (68.2%) and 34(56.6%) of MRSA isolates in Tabriz and Kerman were determined as MDR, respectively. SCCmec typing showed that the frequent SCCmec type in both Tabriz and Kerman cities was SCCmec III (56.86% and 55%, respectively). Conclusion: The high prevalence of MRSA makes it necessary to revisit the antibiotics administration by physicians. Indeed, periodic evaluation of antibacterial susceptibility patterns of the MRSA strains is required for efficient treatment of MRSA infections.

How to cite this article:
Ahmadishoar S, Pour NK, Sadeghi J, Nahaei MR, Kheirkhah B. Genotypic and phenotypic characterisation of clinical isolates of methicillin-resistant Staphylococcus aureus in two different geographical locations of Iran.Indian J Med Microbiol 2020;38:162-168

How to cite this URL:
Ahmadishoar S, Pour NK, Sadeghi J, Nahaei MR, Kheirkhah B. Genotypic and phenotypic characterisation of clinical isolates of methicillin-resistant Staphylococcus aureus in two different geographical locations of Iran. Indian J Med Microbiol [serial online] 2020 [cited 2020 Dec 4 ];38:162-168
Available from: https://www.ijmm.org/text.asp?2020/38/2/162/293900

Full Text



 Introduction



Staphylococcus aureus (S. aureus) is an opportunistic bacterium, which is the most frequent cause of hospital and community-acquired infections.[1] The bacterium is responsible for a wide range of infections, including endocarditis, folliculitis, food toxicity, chronic osteomyelitis, pneumonia, septic arthritis, skin and deep tissue infections, etc.[2]

The frequency of methicillin-resistant S. aureus (MRSA) isolates is increasing,[3],[4] and this issue can lead to severe therapeutic dilemmas and exacerbate the control of infections in hospitals.[5] MRSA, which was first reported in the 1960s,[6] has become endemic in hospitals and health-care settings worldwide. The term methicillin-resistance is a classic term that implies resistance to all beta-lactam antibiotics, except for recently introduced anti-MRSA cephalosporins, such as ceftobiprole. The mec A gene, which encodes for a modified penicillin-binding protein, PBP2a, with decreased beta-lactams affinity,[7] is responsible for methicillin-resistance among bacteria, including MRSA. The gene is located on a mobile genetic element defined as staphylococcal cassette chromosome mec (SCCmec).[8] Till now, 13 types of SCCmec elements have been characterised (I-XIII) that each type has its own specific characteristics. Among them, Types I, II and III of SCCmec are the most prevalent types of SCCmec among hospital-acquired MRSA (HA-MRSA), whereas Types IV and V are the most seen types found in the community-acquired MRSA (CA-MRSA) strains.[9] For efficient epidemiological surveillance and control of health-care-associated MRSA infections, it is of great importance to determine the antibiotics susceptibility pattern as well as the molecular type of the bacteria. There are many methods for molecular typing of MRSA isolates, such as spa and SCCmec typing, pulsed-field gel electrophoresis and multilocus sequence typing.[10] The objective of this study was to identify the patterns of antibacterial susceptibility and SCCmec typing in MRSA clinical isolates from the hospital and community-acquired infections in Tabriz and Kerman cities, located in northwest and southeast of Iran, respectively.

 Materials and Methods



Isolation and data collection

A total of 230 S. aureus isolates were obtained from seven hospitals in Tabriz (n = 125) and Kerman (n = 105) during 11 months of the study period. The patients from whom isolates were collected were 2–67 years old. All of the clinical specimens were taken to the laboratory within 2 h of collection. The strains were confirmed through biochemical tests, such as the ability to grow on mannitol salt agar, catalase, tube coagulase and DNase production. Furthermore, they were grown on sheep blood agar to observe the haemolysis properties. MRSA isolates were determined on Mueller–Hinton agar plates by cefoxitin disc (30 μg) in accordance with the Clinical and Laboratory Standards Institute (CLSI) guideline.[11] MRSA isolates were stored in TSB broth (Merck, Germany) containing 30% glycerol at −70°C for further investigations.

Antimicrobial susceptibility testing

Antibiotic susceptibility test of the 111 MRSA isolates was determined by Kirby-Bauer disk diffusion method compliance with the CLSI-2019 guidelines, with the following antibiotics: oxacillin (OX 1 μg), ciprofloxacin (CIP 5 μg), penicillin (PG 10 u), clindamycin (CD 2 μg), erythromycin (E 15 μg), linezolid (LZD 30 μg), cefoxitin (FOX 10 μg), gentamicin (GM 10 μg), rifampin (RA 5 μg), and trimethoprim-sulphamethoxazole (SXT 2.5 μg). Multidrug resistance (MDR), in addition to beta-lactams, has been defined as resistance to 3 or more specific classes of antibiotics.[12] All antibiotic disks used in this analysis were provided by Mast, UK. In conjunction with each test run, S. aureus ATCC 25923 and ATCC 29213 strains were used as control strains. To detect inducible clindamycin resistance, D-test was exploited according to the CLSI guidelines. For this aim, following the preparation of the isolates' suspension (equivalence to 0.5 MacFarland turbidity), it was inoculated into MHA plates. A clindamycin disk (2 μg) and an erythromycin disk (15 μg) were placed on MHA plates (15 mm edge to edge). Following incubation at 35°C, isolates with resistance to both clindamycin and erythromycin were considered to be constitutive resistance phenotype (cMLSB). Isolates showing resistance to erythromycin and susceptible to clindamycin with a D-shaped zone around the clindamycin disk and flattened from the side of erythromycin disk were considered as inducible resistance phenotype (iMLSB). S. aureus ATCC 25923 was used as the control strain.

Genomic DNA extraction

For molecular identification and typing, DNA extraction was performed by DNA Mini Kit (Favorgene, Taiwan), according to the manufacturer's instructions. Lysostaphin (SigmaeAldrich, USA) was used for cell wall lysis with a final concentration of 15 μg/ml. UV spectrophotometer was used to determine the concentration of DNA.

Molecular detection of methicillin-resistantStaphylococcus aureus using polymerase chain reaction amplification of nuc and mecA genes

All of the isolates were identified as MRSA by polymerase chain reaction (PCR) amplification of nuc and mec A genes using primers listed in [Table 1]. PCR reactions were prepared in 25 μl solutions containing 2.5 μl of PCR buffer (×10), 2 μl of DNA template, 1.5 μl of MgCl2 (50 mM), 0.5 μl of dNTPs (10 mM), 0.5 μl of each primer, 0.25 μl of Taq DNA polymerase (5u/μl) and 14.25 μl of distilled water. Following 30 cycles of denaturation at 93°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s, the amplified fragments were electrophoresed using agarose gel electrophoresis and visualised by ethidium bromide staining.{Table 1}

Staphylococcal cassette chromosome mec typing by multiplex polymerase chain reaction

Various types of SCCmec were detected with multiplex PCR. The primer sequences are shown in [Table 1]. The final volume of each PCR reaction was 25 μl, consisting of 4 μl of DNA template, 0.5 μl of dNTPs (10 mM), 0.75 μl of MgCl2 (50 mM), 2.5 μl of PCR buffer (×10), 0.5 μM of βF, α3R, ccrCR and ccrCF primers, 0.3 μM of 1272F, 1272R, 5Rmec A and 5R431 primers [3.2 μl in total], 13.8 μl of distilled water and 0.25 μl of Taq DNA polymerase (5u/μl). DNA was amplified using an automated thermal cycler (Eppendorf, Germany), and the conditions of multiplex PCR were as follows: initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 60 s and final extension at 72°C for 10 min. The types of SCCmec were identified by comparison of isolated banding patterns with control stains of MRSA that were provided by Dr. Javid Sadeghi (Tabriz University of Medical Science, Iran).

 Results



Sample collection

In this cross-sectional study, 111 MRSA isolates were collected from seven medical centers (four in Northwest [Tabriz] and three in the Southeast [Kerman]) during January to December 2018.

The isolates were recovered from wound infections (n = 74, 32.17%), blood cultures (n = 44, 19.13%), urinary tract infections (n = 52, 22.6%), skin abscesses (n = 22, 9.56%), cerebrospinal fluid (n = 5, 2.17%), body fluids (n = 22, 9.56%) and other specimens (n = 11, 4.78%) which are shown in [Table 2]. Fifty-one out of 125 (40.8%) S. aureus isolates of Tabriz and 60 out of 105 (57.1%) of Kerman isolates, were identified as MRSA.{Table 2}

Antimicrobial susceptibility testing

The antimicrobial susceptibility test results are shown in [Table 3]. Methicillin resistance was identified by disk diffusion using cefoxitin (30 μg). Furthermore, it was found that (96.7% and 99%) of isolates in Tabriz and (96.66% and 99%) of isolates in Kerman were resistant to cefoxitin and penicillin, respectively, but all of them were susceptible to linezolid. Our findings showed that (9.8% and 78.43%) of MRSA isolates in Tabriz and (11.6% and 46.6%) of MRSA isolates in Kerman were iMLSB and cMLSB, respectively.{Table 3}

Molecular detection of methicillin-resistant Staphylococcus aureus

As it was explained in the Materials and Methods section, mecA and nuc genes were used to molecularly detect MRSA. [Figure 1] shows the presence of mec A gene in some isolates. Thirty-five (68.2%) and 34 (56.6%) of methicillin-resistant S. aureus isolates in Tabriz and Kerman were recorded as MDR, respectively.{Figure 1}

Staphylococcal cassette chromosome mec typing

The majority of MRSA isolates carried SCCmec III. Twenty-nine isolates (56.86%) and 33 isolates (55%) were recorded positive for this type in Tabriz and Kerman, respectively. Other frequent SCCmec type in Tabriz were SCCmec IV (11 isolates, 21.56%), SCCmec I (6 isolates, 11.76%), SCCmec V (3 isolates, 5.88%), SCCmec II (2 isolates, 3.92%) and in Kerman were SCCmec V (13 isolates, 21.66%), SCCmec I (11 isolates, 18.34%), SCCmec IV (3 isolates, 5%) which are shown in [Table 4] and [Figure 2].{Table 4}{Figure 2}

 Discussion



The widespread emergence of MDR bacteria, including MRSA, results in higher antibiotic therapy costs and restricts therapeutic options.[13],[14] In this research, we investigated the resistance of S. aureus isolates to different antibiotics, which were collected from healthcare and community-acquired infections in southeast and northwest of Iran. The prevalence of MRSA isolates in various regions of Iran was reported at 20%–90%.[15],[16],[17],[18] In our study, the rate of MRSA isolates was 40.8% in Tabriz and 57.1% in Kerman, lower than the other previous reports from other cities of Iran, such as Ahvaz (87.2%) and Tehran (74%).[15],[19] The discrepancy among the MRSA outbreak in Iran supports the use of various infection control measures and inappropriate use of antibiotics in hospitals and community. Compatible with the other researches,[20],[21],[22] we have found that the most effective antibiotics for MRSA isolates are vancomycin and linezolid.

The antibiotic susceptibility tests demonstrated high resistance of MRSA isolates to beta-lactam antibiotics such as penicillin (99% in Tabriz and 97.8% in Kerman), followed ciprofloxacin 84.3% in Tabriz and 56.66% in Kerman, which was consistent with the findings of previous studies in Iran,[16],[18] Italy [23] and Scotland.[24] This can be demonstrated by the widespread use of beta-lactam antibiotics in hospitals to the treatment of various infections. Although several investigators have been reported with a low resistance to ciprofloxacin in MRSA isolates,[25],[26] was consistent with the study conducted in Italy and Scotland [24] in the current survey, high resistance to ciprofloxacin was seen due to differences in antibiotic consumption, the extent of efflux pumps expression and different rates of mutation in gyrase.

Gentamicin is one of the few heat-stable antibiotics used to treat S. aureus infections in combination with other antibiotics. In our study, the rate of resistance to gentamicin was (84.3%) in Tabriz and (60%) in Kerman, consistent with the study of Goudarzi et al. in Tehran,[22] but higher than 10% stated by Havaei et al. in Isfahan.[25]

The principle mechanism of resistance to aminoglycoside is antibiotic inactivation with plasmid or transposon-mediated aminoglycoside modification of enzymes. Erythromycin is commonly used in the diagnosis of staphylococcal infections as a protein synthesis inhibitor. The resistance rates for clindamycin and erythromycin in this study were 88.2%, 86.2% in Tabriz and 43.2%, 68.33% in Kerman, respectively. The incidence of MRSA resistance to erythromycin examined by Mahdiyoun et al. in Sari and Tehran [27] was 84.4%. In a research performed by Eksi et al., in 2011,[28] the rates of resistance to clindamycin and erythromycin in Turkey were reported to be 39.6% and 53.4%, respectively. The rate of resistance to clindamycin in the current study in Kerman was lower than Mahdiyoun et al.'s study in Iran (67.4%),[27] while it was greater than Eski's investigation in Turkey.[28] The clindamycin resistance can be structural or induced in S. aureus strains.[29] Mutations cause structural resistance, leading to treatment failure.[30] The MLSB resistance pattern varies between geographical regions.[31] Our results revealed that (9.8% and 78.43%) of MRSA isolates in Tabriz and (11.6% and 46.6%) of MRSA isolates in Kerman were iMLSB and cMLSB, respectively. The results of this study were consistent with the results of a study by Seifi et al.[32] and Mansouri and Sadeghi [33] in Iran.

As noted in the literature, the use of glycopeptide antibiotics for the treatment of MRSA bacteraemia has increased over the last decade.[34] In this study, linezolid had a similar in vitro susceptibility to vancomycin in the treatment of infections with MRSA.

As an alternative drug to treat MRSA infections, trimethoprim-sulfamethoxazole has recently been considered. It was previously documented that the rate of resistance to cotrimoxazole in Iran ranges between 19.3% and 69%;[13] however, in our study, 28 isolates (54.9%) in Tabriz and 17 isolates (28.33%) in Kerman were found to be resistant to cotrimoxazole.

The frequency of resistance to rifampicin was 39.21% in Tabriz and 40% in Kerman in this study, which is higher than Switzerland's recorded rate (2%).[35]

The MDR-MRSA prevalence varies with different geographical areas. In this study, the rate of MDR isolates was 68.2% in Tabriz and 56.6% in Kerman that is lower than that reported from Taiwan (95.5%).[36]

Most reports have shown that SCCmec Type III between HA-MRSA isolates is the most common type of SCCmec. Goudarzi et al. reported in 2017 that the SCCmec Type III was the most prevalent type of MRSA isolates obtained from burn patients.[37] SCCmec Type III was the most common type of SCCmec between HA-MRSA isolates in other studies conducted in Iran by Montazeri et al., Japoni-Nejad et al. and Shekarabi et al.,[4],[17],[38] In similar studies, SCCmec Type III was the most frequent type of MRSA isolates from different samples in Spain, Malaysia and Brazil.[39],[40],[41]

In this study, SCCmec Type III was the most common type of MRSA between HA-MRSA and CA-MRSA isolates. On the basis of the literature, Types I, II and III are the most common types of SCCmec in hospital-acquired infections (HA-MRSA), while SCCmec Types IV and V in community-acquired MRSA (CA-MRSA).[42] Our results confirmed that the most frequent types in the community were SCCmec Types III and IV in Tabriz and SCCmec III in Kerman. These results demonstrated that HA-MRSA was spread from hospital to community and CA-MRSA was spread from community to hospital settings. Infection control activities, therefore, play a key role in preventing the spread of CA-MRSA infections to hospital patients and vice versa. Since SCCmec IV is commonly located on small mobile elements, it is generally possible to transfer horizontal genes between S. aureus, the CA-MRSA isolates carrying SCCmec IV were transferred from community to the health-care workers.[1],[7],[43] In the present study, all of CA-MRSA isolates carrying SCCmec Type IV in Tabriz were MDR and majority of MRSA isolates with SCCmec Type III (24 (68.75%) and 30 (88.23%) in Tabriz and Kerman) had MDR phenotype and these isolates were resistant to ciprofloxacin, cotrimoxazole, gentamicin, erythromycin, clindamycin. Therefore, the identification of S. aureus carriage between staffs and patients helps to reduce the transmission of MRSA and infections in health-care settings.

According to our study, the frequency of MRSA isolates in both Tabriz and Kerman is high. We suggest using new infection control policies to reduce the spread of MRSA isolates in hospitals. Many MRSA isolates implicated in HA-MRSA derived from community-associated infections and vice versa. Therefore, the appearance of MRSA isolates is a troubling issue that can lead to therapeutic limitations.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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