Methicillin-resistant Staphylococcus aureus (MRSA) is considered as one of the most important multiple drug resistant (MDR) pathogens in intensive care units (ICUs) [1, 2]. Over the past decades, the incidence of MRSA isolates has increased dramatically worldwide [2]. Vancomycin is a drug of choice for the treatment of MDR S. aureus infections [3]. Resistance to vancomycin can be linked to the presence of the vanA gene or thickening of the bacterial cell wall [4]. Vancomycin resistant S. aureus (VRSA) was first reported in the USA in 2002 [5]. By the end of 2015, several VRSA strains had been reported in different countries, which made treatment more complicated [5, 6]. The first VRSA isolate from Iran was reported in Tehran in 2008 [7]. Herein, the authors describe the emergence of the first VRSA isolates from two hospitalized patients in Kerman, southeastern Iran.

Materials and methods

From February 2015 to May 2016, 205 S. aureus isolates were collected from patients admitted to three university hospitals affiliated with the Kerman University of Medical science in Kerman, Iran. These isolates were obtained from various samples such as urine, blood, cerebrospinal fluid, wound and respiratory tract. All isolates were identified as S. aureus by positive Gram staining, as well as positive tests for catalase, coagulase, DNAse and fermentation of mannitol [8]. Polymerase chain reaction (PCR)-mediated amplification of the nuc gene was performed to confirm these isolates genotypically [9].

The antibiotic susceptibility profile of isolates was determined by the disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) recommendations [10]. The following antibiotic disks were employed: gentamicin (10 µg), amikacin (30 µg), erythromycin (15 µg), clindamycin (2 µg), tetracycline (30 µg), ciprofloxacin (5 µg), trimethoprim/sulfamethoxazole (1.25/23.75 µg) and linezolid (30 µg). Screening for MRSA and VRSA isolates was carried out by detection of resistance to a cefoxitin disk (30 µg) and growing on Brain Hearth Infusion agar (BHI; Difco, BD, NJ, USA) containing 6 µg/ml vancomycin (Sigma-Aldrich, MO, USA), respectively. Also, the minimum inhibitory concentration (MIC) of vancomycin was determined using the broth microdilution method. S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were used as standard strains in the antimicrobial susceptibility tests.

The total genomic DNA of VRSA strains was extracted by Exgene™ Clinic SV (GeneALL, Seoul, Korea) according to manufacturer’s guidelines. The oligonucleotide primers used for amplification of the mecA, vanA, vanB, ermA, ermB, ermC, mrsA/B and pvl genes are listed in Table 1. The PCR amplifications for the above genes were carried out as described previously [11–13]. Finally, SCCmec and spa typing was performed as described previously [14, 15].

Table 1 List of primers used in this study
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Results

In this study, 100 (48.78%) of the 205 isolates were determined as MRSA by phenotyping methods. Two MRSA isolates were identified as VRSA and both isolates were vanA positive (Fig. 1). These isolates were from two women with pneumonia, hospitalized in the same ICU. It is not known exactly how long these women were hospitalized before specimen collection, but it is clear that the specimens had been collected at least 4 days after hospitalization. One of the isolates had been obtained in February 2015 from the bronchial aspirate of a 76-year-old woman with a history of diabetes mellitus and haemodialysis, who was not treated and died. According to our information, this patient had been admitted to the ICU on arrival. The other isolate had been obtained in April 2015 from the bronchial aspirate of a 66-year-old woman with pneumonia. This patient had been transferred from the urology unit (no further data available). Both VRSA isolates were resistant to gentamicin, amikacin, erythromycin, clindamycin, tetracycline, ciprofloxacin, trimethoprim/sulfamethoxazole and cefoxitin, but they were susceptible to linezolid. Genetic characteristics of and clinical information pertaining to these isolates are shown in Table 2.

Fig. 1
figure 1

Agarose gel electrophoresis of PCR-amplified vanA gene. N negative control, P positive control, 1 and 2 positive strains, M size marker (100 bp)

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Table 2 Genetic characteristics and clinical information of two VRSA strains
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Discussion

During the past decade, VRSA strains have been reported from different countries such as the USA, Portugal and India [3, 5, 6]. This study is the first report of the emergence of VRSA in the southeast of Iran. In contrast to previously reported VRSA isolates that were susceptible to gentamicin and other antibiotics [2–8], the isolates presented here showed a MDR phenotype. Although MRSA strains with SCCmec III and spa t030 have been reported from different countries, such as China, Germany, Denmark, Sweden and even other regions in Iran, none of them showed vancomycin resistance [16–19]. VRSA strains with spa type t292 (SCCmec IV) and t019 (SCCmec IV) have been detected in Brazil and the USA, respectively [5, 20]. In 2012, one VRSA with SCCmec III, spa t037 and pvl negative was reported by Azimian et al, in the northeast of Iran [8]. In another study in Iran, one VRSA isolate harbouring vanA (MIC: 64 µg/ml) was reported by Aligholi, et al. [7]. Therefore, it seems that the incidence of VRSA in Iran is increasing. In the present report, both VRSA isolates belonged to SCCmec III and spa type t030, with a different presence of erm and mrsA/B genes. These findings confirm that these isolates have acquired new resistance genes during persistence in ICU and hospitals. Also, SCCmec III is found predominantly in healthcare-associated MRSA isolates and is transferred by person-to-person spread in the hospital. Since no VRSA strain was observed in the authors’ subsequent epidemiological studies, it seems these isolates have been not transmitted from patient to other patient, or to healthcare workers.

Conclusion

Since a high rate of MRSA isolates has been reported in Iran, finding VRSA isolates is a serious threat for Iranian hospital settings. Therefore, proper infection-control policies, appropriate antimicrobial agents management and improved awareness of healthcare personnel are needed to prevent the emergence and transmission of VRSA isolates in Iran. Due to the importance of VRSA emergence, these cases were reported to the infection control committee of the affected hospital. As no further VRSA was detected during the next year of specimen collection in this study (until May 2016), it can be concluded that the more strict preventive measures taken to control dissemination of resistant strains in the hospital setting were effective.