Investigation of SCCmec types I-IV in Clinical Isolates of Methicillin-Resistant Coagulase-Negative Staphylococci in Ahvaz, Southwest Iran.

Abstract Today methicillin resistant coagulase-negative staphylococci (MR-CoNS) are important in terms of causing significant nosocomial infections. Besides, MR-CoNS are confirmed as the reservoir of SCCmec elements that carry mecA (methicillin-resistant) gene. Hence, the present study was designed to evaluate the susceptibility pattern, prevalence and diversity of SCCmec types I, II, III, and IV in MR-CoNS strains. In this cross-sectional study, 44 clinical isolates of MR-CoNS were identified using the cefoxitin disc method and further confirmation by polymerase chain reaction (PCR) amplification of the mecA gene. Antimicrobial susceptibility of isolates was investigated by disc diffusion. The identification of CoNS was done by amplification and sequencing of the tuf gene. Multiplex PCR method was done for the determination of SCCmec types. In the present study, the Staphylococcus epidermidis and Staphylococcus haemolyticus were the most predominant isolates with a prevalence of 45.4%. The highest resistance rates were observed against erythromycin (84.1%) and clindamycin (75%). Multiplex PCR revealed the SCCmec type I as the predominant type in the present study. Our study showed that there was no significant relationship between the presence of different types of SCCmec elements and resistance to antibiotics. The present study highlighted a frequent prevalence of MR-CoNS harboring SCCmec type genes in Ahvaz, southwest of Iran. Thus, the molecular typing and periodical monitoring of their drug resistance pattern should be considered in national stewardship programs to designing useful antibiotic prescription strategies.


Introduction
Coagulase-negative staphylococci (CoNS) are gram-positive catalase-positive bacteria, with no ability to clot blood plasma. More than 40 species of CoNS are known as normal flora of the human skin and mucous membranes [1]. In recent years, CoNS have been established as important agents of nosocomial and community-acquired infections especially in patients with medical devices and immunocompromized patients. Staphylococcus epidermidis and Staphylococcus haemolyticus are identified as the most common species isolated from clinical samples [2].  [14] tuf-R CCATTTCAGTACCTTCTGGTAA mecA-F TCCAGATTACAACTTCACCAGG mecA 162 [15] mecA-R CCACTTCATATCTTGTAACG 1272F1 GCCACTCATAACATATGGAA IS1272 415 [16] 1272R1 CATCCGAGTGAAACCCAAA β ATTGCCTTGATAATAGCCYTCT ccrA2-B 937 [16] α3 TAAAGGCATCAATGCACAAACACT IS1272 CGTCTATTACAAGATGTTAAGGATAAT ccrC 518 [16] ccrCR CCTTTATAGACTGGATTATTCAAAATAT (MDR) isolates (resistant to three or more of antimicrobials) were estimated according to previously described definitions [13]. Staphylococcus aureus ATCC 29213 and ATCC 33591 were used as control strains.

Detection of inducible clindamycin resistance
All MR-CoNS isolates that showed resistance to erythromycin and susceptibility to clindamycin in AST were evaluated for inducible clindamycin resistance by D-zone test. The D-zone test was performed by placing a 15 μg erythromycin and 2 μg clindamycin discs at 15-25 mm edge-to-edge distance on Mueller-Hinton agar plates (Merck, Darmstadt, Germany) inoculated by tested isolates. The plates were then incubated at 35 + − 1 • C for 18 + − 2 h. The isolates that were showed flattening of the clindamycin inhibition zone adjacent to the erythromycin disc were considered as D-zone test positive (inducible clindamycin resistance), and those with a circular inhibition zone were classified as D-zone test negative [12].

DNA extraction
DNA extraction was done by the boiling method [14]. The isolates were cultured on nutrient agar (Merck, Darmstadt, Germany) incubated at 37 • C for 24 h. The bacterial suspension was prepared in microtubes containing 500 μl TE (10 mM Tris/HCl, 1 mM EDTA, pH 8.0). The suspension was heated at 95 • C for 5 min and then centrifuged at 14,000 rpm for 10 min at 4 • C. The supernatant was used as a template for polymerase chain reaction (PCR). Assessment of DNA concentration and quality were performed by measuring the absorbance of A260 and A280 nm with the spectrophotometer and agarose gel electrophoresis, respectively.

Identification of CoNS species by tuf gene
Confirmation of MR-CoNS species was done by amplification and partial sequencing of the tuf gene using primers described previously in Table 1 [15]. The S. epidermidis ATCC 49134 was used as positive control in the amplification reaction. The obtained sequences of tuf gene for each isolate were aligned separately using MEGA 5 (Molecular Evolutionary Genetics Analysis) software and compared with all existing sequences of CoNS annotated in GenBank database (http://www.ncbi.nlm.nih.gov/BLAST). Verified isolates were stored in trypticase soy broth (Merck, Darmstadt, Germany) containing 20% (v/v) glycerol at −70 • C for further analysis.

Detection of the mecA gene and SCCmec typing
Detection of the mecA gene was performed by polymerase chain reaction (PCR), using primers described previously (Table 1) [16]. SCCmec typing of isolates was determined by multiplex PCR described by Boye et al [17].

2b. Distributed of MR-CoNS isolates from hospital wards
Hospital wards Women's ward ATCC 29247 strains (for mecA gene), S. aureus NCTC 10442 (for Type I), S. aureus N315 (for Type II), S. aureus 85/2082 (for Type III), S. aureus JCSC 4744 (for Type IV) and the sterile deionized water were included with each PCR run as positive and negative controls, respectively.

Statistical analysis
The analysis was performed by using SPSS™ software, version 22.0 (IBM Corporation, Armonk, NY, U.S.A.). The results are presented as descriptive statistics in terms of relative frequency. Values are expressed as the percentages of the group (categorical variables). Chi-square or Fisher's exact tests were used to determine the significance of differences. A difference was considered statistically significant if the P-value was < 0.05.

Multi-drug resistance profiles
According to the AST, 34 (77%) of 44 MR-CoNS isolates that were resistant to at least three different classes of antimicrobial agents considered as MDR, with 20 different patterns ( Table 4). The pattern 1   (rifampin-gentamycin-erythromycin-clindamycin-ciprofloxacin) with a frequency rate of 18.8% was the most prevalent resistance profile (Table 4).

PCR amplification of the mecA gene and SCCmec typing
The presence of mecA was confirmed in 44 MR-CoNS isolates by PCR. According to our results 27 isolates showed single type, including type I (n = 20, 45.5%), type IV (n = 4, 9.1%), type III (n = 2, 4.5%) and type II (n = 1, 2.3%). Six isolates had two types, III+ I (n = 5, 11.4%) and IV+ III (n = 1, 2.3%). Eleven (25%) isolates showed no band for SCCmec I-IV (Table 5). The 11 MR-CoNS isolates that lacking SCCmec I-IV were tested by single-plex PCR for the four loci, but no SCCmec types were detected in these isolates. Fifty-five percent of SCCmec type I positive MR-CoNS isolates (11/20) belonged to S. haemolyticus species. The assessment of association between presence of different SCCmec types and antibiotic resistance in 44 MR-CoNS isolates showed that there was no significant relationship (P > 0.05) ( Table 6).

Discussion
MR-CoNS are one of the most important causative agents of human infections in which the MDR property is more visible than the MS-CoNS [18]. Owing to the paucity of information about the frequency of SCCmec types among   [19,20,21,22]. The rate of methicillin resistance in our study was 48.8% that was close to Namvar et al. [19] and Hajiahmadi et al. [23] results with 47.2% and 50%, respectively. In our study, the most common MR-CoNS isolates belonged to both of S. epidermidis and S. haemolyticus (45.45%) which was consistent with previous reports from Iran and other countries [3,23,24,25]. One of the species that so far has not been reported from other regions of Iran, but was isolated in Ahvaz city, was S. petrasii. Staphylococcus haemolyticus was the predominant isolate from urine specimens in our study (31.8%). Our results confirmed the results of previous studies in which the most common species isolated from urine was S. haemolyticus [3,26]. In the present study resistance to eight antibiotics was considered. The highest resistance was observed to erythromycin (84.1%). Nahaei et al. closest to our findings showed the high rate of resistance to erythromycin among MR-CoNS (84.8%) [21].
In the present investigation, the susceptibility patterns of MR-CoNS were at variance with some other studies from Iran. For example in Hajiahmadi et al. [23] and Havaei et al. [27] studies the highest resistance was observed to trimethoprim-sulfamethoxazole (56%) and ciprofloxacin (65%) respectively. This could be because of different protocols and panels of antibiotics used in various hospitals. Although in our study the lowest resistance was observed to linezolid (22.7%), this rate of resistance was high in comparison with many studies in Iran and other countries [23,28,29,30]. According to multinational and multicenter surveillance studies, more than 99% of coagulase-negative staphylococci and S. aureus clinical strains are sensitive to linezolid (31) [31]. The common mechanisms for linezolid resistance are mutations in the linezolid 23S rRNA binding site and acquisition of a plasmid-borne ribosomal methyltransferase gene called the cfr (chloramphenicol/florfenicol resistance) gene. Later is more worrisome because of its rapid spread and possibility of its transfer to more pathogenic organisms, such as S. aureus [8]. The cfr enzyme causes resistance to several other antibiotic classes besides oxazolidinones, such as phenicols, lincosamides, pleuromutilins and streptogramin compounds [32]. In different countries such as Italy, Spain, USA, Mexico, and China, the emergence of cfr in nosocomial staphylococci has been reported [32,33]. Additionally, the possibility of cfr gene transmission from veterinary isolates, such as S. sciuri, S. warneri, S. aureus, S. hyicus and Enterococci has been reported [34]. Because the linezolid isn't used routinely as a common antibiotic in our region, it is difficult to accept this percentage of resistance. Therefore, for a better understanding of high resistance reason to linezolid and some other antibiotics, more studies about the existence of cfr gene among CoNS can be helpful. Moreover, the result of inducible resistance to clindamycin assessed by D-test was 4.5%. There is little information about the rate of inducible resistance to clindamycin among MR-CoNS in Iran. Aghazadeh et al. [29] and Abdollahi et al. [35] closest to our findings showed the rate of inducible clindamycin resistance among MR-CoNS 6% and 5% respectively. According to Emaneini et al. [36] results, 14.1% of MR-CoNS had inducible resistance to clindamycin.
In the present study, SCCmec typing permitted the differentiation of types I-IV among the MR-CoNS strains. The SCCmec is a mobile genetic element widely distributed among MR-CoNS species that varies depending on the host species, various environments and geographical locations [16]. In the present study, the most abundant SCCmec type among MR-CoNS was type I (45.5%), whereas in other studies in Iran SCCmec type IV, III and V were more frequent [20,23,28]. It isn't easy to compare our results with other results in Iran because in many studies the diversity of SCCmec type has been evaluated only among S. epidermidis [19,23,28]. Additionally, the high percentage of SCCmec type V among S. epidermidis in some studies can be associated with the presence of a large number of commensal samples, among which a higher prevalence rate of SCCmec type V was proven [37]. Different countries have reported varied SCCmec types in MR-CoNS. For example, SCCmec type I has been reported to be the most predominant in India, whereas SCCmec types II and III have been found to be the most common in Nigeria and China respectively [25,38]. In our study, six isolates had two types: III+ I (n = 5, 11.4%) and IV+ III (n = 1, 2.3%). This was not surprising as the co-existence of two SCCmec elements appears to be common in MR-CoNS [25,39,40]. In our study, carriage of SCCmec elements was not associated with resistance to tested antibiotics. In a study by Garza-Gonzalez et al. [24], a significant correlation between the presence of the SCCmec type III and resistance to meropenem (P<0.05) was reported. Machado et al. [40] showed that isolates with SCCmec type III were more resistant to non-β-lactam antimicrobials than isolates with other SCCmec types, although the increase in resistance was statistically significant only for clindamycin (P = 0.021), rifampicin (P = 0.010) and levofloxacin (P = 0.005).
To date, more than ten SCCmec types have been reported in CoNS [25]. A limitation of our study was that we only tried to search for the most common SCCmec types in Iran. Our PCR targeted only SCCmec types I-IV, but 11 isolates in this study were negative for these four types indicating that the MR-CONS isolates in our region have SCCmec types belonging to the other established types or have novel types. Generally, performing diverse typing methods can afford better and more valuable epidemiological data about the clonal diversity of the isolates that can be advantageous for outbreak researches [41,42]. Another limitation of the present study was the lack of determination of the minimum inhibitory concentrations of tested antibiotics.

Conclusion
Our study was the first study for the determination of frequency and diversity of SCCmec types in MR-CoNS isolates in Ahvaz, Iran. The present study highlighted a frequent prevalence of multidrug-resistant CoNS harboring SCCmec type genes. The SCCmec type I was the most common in Ahvaz city, southwest of Iran. Since CoNS are important causes of nosocomial and community-acquired infections especially in patients with medical devices, molecular typing and periodical monitoring of their drug resistance pattern should be considered in national stewardship programs to designing useful antibiotic prescription policies and hospital infection control strategies.