Epidemiological and Inducible Resistance in Coagulase Negative Staphylococci

Background and Objectives: Coagulase negative staphylococci (CNS) are potential pathogens with the increased use of implants in hospitals. Macrolide, lincosamide and streptogramin B (MLSB) are used in the treatment of staphylococcal infections. The aim of this study was to molecular detection of inducible clindamycin resistance and genetic pattern in CNS isolates and their transmission between hospitals. Materials and Methods: 110 CNS strains, isolated from hospitalized patients in the intensive care unit and infectious wards of Besat and Toohid hospitals, Sanandaj. Methicillin resistance was done by agar screen test and the resistance inducible Clindamycin by the D-Test. Multiplex PCR was performed, using primers specific for erm (A, B, C, and TR) genes. Diversity of strains was determined by ERIC–PCR technique based on the similarities between DNA fingerprints by using Jaccards coefficient in the SAHN program of the NTSYS-pc software. Results: Of the 110 isolates, 64(58.2%) were methicillin -resistant CNS (MRCNS), 48(43.6%) were resistant to erythromycin (ERCNS). Out of 48 Erythromycin-resistant strains 5 (10.4%) were iMLSB phenotypes that 4 isolates showed genes erm by Multiplex PCR. The ERIC–PCR profiles allowed typing of the 110 isolates into 90 ERIC-types which were grouped into fourteen main clusters (C1–C14). Conclusion: The results of this study also showed that most of CNS isolated produced different genomic fingerprint patterns, therefore, source of infection is differen t.


Introduction
Coagulase negative staphylococci (CNS), now considered to be as significant potential pathogens with the increased use of implants in hospitals (Azuka, & Idahosa, 2013). However, with upraise use modern medicine that show dependence to medical device materials to production biofilm also CNS identified mainly to cause nosocomial infections (Azuka, & Idahosa, 2013;Dryden, 2010;Caseya, Lambertb, & Elliotta;, Most of diseases or infections assumed by CNS are significant as a result of hospitalization (Dryden, 2010). CNS have historically been more resistant to antimicrobials including the -lactam antibiotics, than S. aureus (Longauerova, 2006).

Materials and Methods
Collection and culture of isolates. Totally 110 Coagulase negative staphylococci isolated from hospitalized patients in the intensive care unit and infectious wards of Besat and Toohid hospitals in Sanandaj. In this study 23 samples were collected in Besat hospital, and 87 samples were from Toohid hospital. Sampling was performed with sterile swab from the throat (29.1%) and nose (70.9%). The isolates were sub cultured on to blood agar and were incubated at 37 0 C overnight before they were used so were identified by using usual microbiological methods include colonial morphology, Gram stain, negative mannitol test and a test tube coagulase reaction, susceptibility to bacitracin and novobiocin (Pal, Sharma, Sharma, & Vyas, 2010;Moosavian, & Darban, 2010;Moosavian & Wadowsky, 2010). All the isolates were suspended in 20% glycerol stock and kept at -70 o C for long-term storage (Varges, Penna, Martins, Martins, & Lilenbaum, 2009).

Methicillin resistance.
To identify strains of methicillin-resistant Coagulase negative staphylococci used specific medium and added Mueller Hinton agar medium, 4% NaCl and 6 mg/L oxacillin. Coagulase negative staphylococci strains were cultured and incubated at temperatures of 35°C for 24 hours. The growth more than one colony was showed resistance to methicillin (Franklin, Matthew, Karen, Michael, George, Dwigh, & et al, 2010).
Detection of inducible resistance erm gene expression. Inducible Clindamycin resistance was performed according to CLSI guidelines by using D -Test method. A 0.5 McFarland equivalent suspension of organisms was inoculated onto a Mueller -Hinton agar (MHA) plate, the ER (15 μg) disk was placed 15-26 mm (edge to edge) apart from CL (2 μg) disk on MHA. Plates were surveyed after 16-18 hours of incubation at 35 °C ( Figure  1) (Franklin et al, 2010;Schreckenberger, Ilendo, & Ristow, 2004). DNA extraction. Genomic DNA extraction of Coagulase negative staphylococci strains were performed by use of Cinna Pure kit (Cinagen, Iran).
Multiplex PCR for gene erm. The specific primers for erm genes were obtained are shown in Table 1 (Matthew, Sullivan, Cai, Kong, Zeng, & Gilbert, 2006).
Computer-Assisted ERIC-PCR DNA Fingerprint Analysis. Data matrix was formed based on presence or absence of bands and analyzed using the NTSYS-pc software (version 2.02 K, Applied Biostatistics, Inc, NY, USA). Dendrograms of dissimilarity were produced for every case. The similarity between the strains was determined on the basis of the Jaccard similarity. The dendrogram was produced on the basis of the averaged similarity of the matrix with the use of the algorithm of the Unweighted Pair-Group Method (UPGMA) in the SAHN program of the NTSYS-pc software. This method has been used to show relations between similar groups (Gadepalli, Dhawan, Mohanty, Kapil, Das, & Chaudhry, 2006).

Results
The total 110 Coagulase negative staphylococci isolates was collected 23(20.9%) specimens from Beast hospital and 87(79.1%) specimens from Toohid hospital which are shown in Table 2. The antimicrobial susceptibility of 110 Coagulase negative staphylococci isolates was determined in vitro (Table 3). Of the 110 isolates, 64(58.2%) methicillin -resistant CNS (MRCNS) and 46(41.8%) methicillin-sensitive CNS (MSCNS), a total of 110 CNS isolates were found 48(43.6%) to be resistant to erythromycin (ERCNS) and were tested for inducible resistance by double disk approximation test (D-Test). Out of 48 Erythromycin-resistant strains 5 (10.4%) were iMLS B phenotypes 4 (80%) iMLS B phenotypes were observed to be methicillin-resistant.   Genetic studies conducted on the erythromycin-resistant isolates, searching for genes erm, Among the iMLS B phenotypes 5 isolates, The erm(A) gene was detected in 80% (4/5) of the these isolates, erm(B) in 80% (4/5), erm(C) in 80% (4/5), and erm(TR) in 20% (1/5) isolates.  (Figure 3), the clustering results of ERIC-PCR, tested strains were classified in 14 groups. The similarity coefficient between 27 to 50 percent. Maximum strain was in the thirteenth group (Figure 4), a dendrogram that included all profiles was constructed on the basis of the levels of similarity. The thirteenth group with the largest number was formed by 13 CNS strains isolated from Toohid hospital. The eight group with the lowest number contained 2 strains isolated from Toohid hospital. (Table 4) The agarose gel electrophoresis of the fragments amplified by REP-PCR is shown in figure 3. The profiles generated with primer ERIC contained several bands
So the different resistance to antimicrobials was found within isolates collected from Besat and Toohid hospitals that these results may indicate differences in Therapeutic measures among these hospitals, particularly, differences in the type drugs and frequency of their use, and so exposure to Patients antimicrobials.
Rep-PCR determination the genetic diversity and also transmission trace of Coagulase negative staphylococci clinical isolates bacterial in the community and hospitals. This is the first study carried out in hospitals of Sanandaj which describes the genetic relationships among CNS isolates hospitalized patients in the intensive care unit and infectious ward of Besat and Toohid hospitals by rep-PCR. As shown in previous reports, rep-PCR was proven to be a highly discriminate and rapid screening method to classify a large number of isolates into clusters (Moosavian, & Darban, 2010;Moosavian, & Wadowsky, 2010;Reinoso, Bettera, Odieno, & Bogni, 2007;Ross, Merz, Farkosh, Carroll, 2005) Most of the strains (11.8 %) were grouped in cluster 13. Genetic heterogeneity among CNS isolates was observed within hospitals. In totally 16 groups obtained with similarity coefficient 100 percent (same profiles within hospitals). Fourteen groups were found in Toohid hospital (strains 63-64; 5,100; 70-71; 72-73; 90-91; 107-108; 105-106; 98-99; 15-17; 49-50; 10-11; 39-40; 102-104; 109-110), two predominant profiles were identified in Besat hospital (strains 1-4; 80-81). Although There wasn't any same isolates between of two hospitals, isolates obtained from the different wards of hospitals: strains 10, 11 that belonged to intensive care unit and infectious wards hospital Toohid and 1-3, 4 belonged to intensive care unit and infectious wards hospital Besat showed similar genotype which indicate clonal transmission of CNS in wards of hospitals. Among of them only one sample was shown erm genes by multiplex PCR that it belonged to infectious ward hospital Toohid it could cause frequent uses of this drugs that it lead to resistance, especially resistance induction, and consequent failure to be treated with clindamycin.

Conclusion
To conclude, this study showed the genetic relationship in isolates CNS in two hospitals. Most of the isolates showed unique pattern which indicate that the rate of transmission resistant strains are very low in Sanandaj hospitals. The remaining patterns showed similarity in most characteristics which was indicated of similar origin of dissemination.