Species distribution and antibiotic sensitivity pattern of coagulase-negative Staphylococci other than Staphylococcus epidermidis isolated from various clinical specimens

This study was undertaken to determine the species distribution and antibiotic resistance patterns of coagulase-negative Staphylococci (CoNS) other than Staphylococcus epidermidis. A total of 142 CoNS (except S. epidermidis) strains were isolated from a variety of clinical specimens in neutropenic patients at the Bone Marrow Transplant Centre of Tunisia between 2002 and 2004. All CoNS isolates were further identified by Api ID32 STAPH and ITS-PCR and antibiotic sensitivity was performed by disc diffusion method. Staphylococcus haemolyticus was the commonest species (38%) followed by Staphylococcus hominis (36%). All isolates were sensitive to vancomycin and 8 (6%) strains showed a reduced sensitivity to teicoplanin. Resistance to penicillin G and methicillin was 84 and 60%, respectively. Methicillin-resistant CoNS strains were determined to be more resistant to antibiotics than methicillin-susceptible CoNS strains. The mecA gene was detected by PCR in 65% (92/142) CoNS isolates. Out of 92 mecA-positive isolates, 90 were phenotypically methicillin-resistant and two were methicillin-susceptible. Phylogenetic analysis, carried out to study the evolution of mecA genes between different Staphylococcal species, revealed a high homology for such genes among Staphylococci.


INTRODUCTION
Cancer patients are particularly susceptible to nosocomial infections because of their compromised immune system (Ashour et al., 2007).Over the two decades, neutropenic patients have been infected with changing spectrum of bacterial organisms (Zinner et al., 1999) and antimicrobial-resistant gram-positive strains are becoming increasingly frequent in these patients.CoNS have emerged as a major cause of infection in immuno-deficient compromised patients, especially in those with indwelling foreign bodies (von Eiff et al., 2001).Although Staphylococcus epidermidis causes most CoNS *Corresponding author.E-mail: onsboucham@yahoo.fr or onsbouchami@gmail.comTel: 00 216 71 577 413/00 216 96 653 590.Fax: 00 216 71 565 623 infections, many other species have been identified in association with human infections (Zinner et al., 1999).They have become a serious problem due to associated methicillin resistance, leading to significant limitations in therapeutic options (Abbassi et al., 2008).CoNS have historically been more resistant to antimicrobials, including the β-lactam antibiotics, than Staphylococcus aureus and some hospitals reveal rates of oxacillin resistance in CoNS approaching 90% (John et al., 2007;Martins et al., 2007).In reports from different parts of Europe, the oxacillin resistance in CoNS varies between 70 and 80% (Agvald-Öhman et al., 2004;Sader et al., 2004) and similar high rates of resistance are also reported from the United States, Canada and Latin America (Diekema et al., 2001;Vincent et al., 2000).In addition, cross resistance to non-β-lactam agents has been a recurrent theme over the past 40 years in the CoNS.Correct identification of CoNS species has become important in clinical laboratories, since several species have been recognized as potential pathogens, especially in a nosocomial setting (Layer et al., 2006).Hence, convenient, reliable and inexpensive identification methods are needed to identify most of the CoNS and discriminate between the species, commonly implicated in the majority of infections.
Methicillin-resistance is mediated by the mecA gene, which is carried by a mobile genomic element designated Staphylococcal cassette chromosome mec (SCCmec) (IWG-SCC, 2009).Several recent reports suggest that in CoNS, mecA gene is highly conserved (Ito et al., 2001;Rahimi et al., 2009) and have shown that this gene has been actively transmitted from one Staphylococcal species to another (Suzuki et al., 1992).CoNS were suggested to be active players in the horizontal transfer of mecA gene.Previous studies have shown that a mecA homologue ubiquitous in Staphylococcus sciuri may have been the evolutionary precursor of the structural gene of PBP2a (Couto et al., 1996).
Thus, the present study was undertaken with the primary aim of studying species distribution and antimicrobial resistance patterns of CoNS other than S. epidermidis isolated from a variety of clinical specimens in neutropenic patients at the Bone Marrow Transplant Centre of Tunisia with particular reference to phenotypic and genotypic expression of methicillin resistance.Also, we aimed to access the phylogeny and the evolution of mecA genes.

Bacterial strains and identification
Organisms from clinical samples were cultured as per the routine procedures.A total of 142 consecutive non-repeat clinically significant CoNS strains belonging to different species, sampled between 2002 and 2004 from neutropenic patients hospitalized at the Bone Marrow Transplant Centre of Tunisia, were analyzed.Bacterial strains were recovered from different pathological specimens and were initially identified by conventional tests including: colony morphology (size and pigment), Gram staining, catalase test, coagulase tests, DNase tests and manitol fermenting.One CoNS isolate of each colony-morphology type, from each site and sampling occasion, was stored in glycerin-containing broth at -20°C until further analysis.
The identification at species level was carried out by Api ID32 STAPH (bioMérieux, Marcy l'Etoile, France) in accordance with the manufacturer's instructions.All isolates were confirmed for species identification by ITS-PCR, according to previously described methodology (Couto et al., 2001).

Susceptibility testing
Antibiotic susceptibility was determined using the Kirby Bauer disc diffusion method according to the recommendation of the French Society of Microbiology «Comité de l'Antibiogramme de la Société Française de Microbiologie» (CA-SFM) (http://www.sfm.asso.fr) on MH agar (Difco).Antimicrobial drugs tested included penicillin G Bouchami et al. 1299 (6

DNA extraction
Chromosomal DNA was extracted from each CoNS strain by the small-scale phenol extraction method (Depardieu et al., 2004) with modifications: Cells from 1.5 ml of Staphylococci from an overnight shaken culture in brain heart infusion broth were harvested (15,000 × g, 5 min); suspended in 150 μl of a solution containing 10 mM Tris (pH 8.0), 1 mM EDTA, and lysostaphin (2 mg/ml; Sigma); and incubated at 37°C for 30 min.The resulting protoplasts were lysed with 3µl of proteinase K (200 µg/ml) and sodium dodecyl sulfate (10%) for 30 min at 55°C, and after two phenol-chloroform extractions, total DNA was recovered in the supernatant after centrifugation (15,000 × g, 5 min).

Detection of the mecA gene by PCR
In addition to the phenotypic determination of methicillin resistance, a simplex PCR assay which permits the detection of the mecA gene was performed as previously described (Frebourg et al., 2002) with modifications.The PCR primers for mecA were mecA-F, 5'-GGCTATCGTGTCACAATCGTT-3' and mecA-R, 5'-TCACCTTGTCCGTAACCTGA-3' containing a ClaI restriction site.The reaction mixture (50 µl total) containing 2 µl extracted DNA, 10 mM Tris/HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 µM each dNTP, 1.25 U Taq DNA polymerase and 0.5 µM each primer was used for PCR to amplify the mecA gene in an automated thermal cycler (Promega).DNA amplification thermal cycling profile was as follows: initial denaturation at 94°C for 2 min, followed by 30 cycles of amplification (94°C for 1 min, 55°C for 1 min and 72°C for 2 min), ending with the 72°C for 5 min.The amplified product of 683 bp was detected by ethidium bromide staining following 1.5% agarose gel electrophoresis.The positive and negative control strains used in mecA detection were S. epidermidis RP62A and S. aureus ATCC25923, respectively.

Restriction analysis by PCR-RFLP
In order to confirm the presence of mecA gene, a PCR-RFLP was performed according to the manufacturer's recommendations (Promega, Madison, Wisconsin, USA).Fifteen (15) µl of each mecA amplified product were mixed and digested with 10U of ClaI restriction enzyme (Promega, USA), and incubated at 37°C for 3 h.The sizes of the restriction fragments were documented by electrophoresis, ehtidium bromide, UV transillumination and photography.

Statistical analysis
Proportions were compared using the Chi-square test.The Chisquared test was used to assess the statistical significance for a confidence level of 95% (α = 0.05).

RESULTS
Based on our identification methods, we isolated a total of 142 coagulase-negative Staphylococcal isolates from different clinical specimens collected during the study period (2002)(2003)(2004)  Antimicrobial susceptibility testing revealed that all CoNS isolated from various clinical sources were sensitive to vancomycin, while 60% were resistant to methicillin.Methicillin-resistant strains showed MIC ≥256 µg/ml in 55% of cases.Sensitive strains showed MIC ranged from 0.016 to 0.25 µg/ml.Penicillin resistance was frequent (84%).Isolates showed also high rates of resistance to erythromycin (69%), cotrimoxazole (53%), gentamicin (50%), ofloxacine (47%), tetracycline (39%) and to fusidic acid (38%).Lower resistance rates were detected for rifampin (15%), fosfomycin (10%), chloramphenicol (6%) and pristinamycin (2%) Reduced susceptibility was observed for teicoplanin (6%).Resistance to antibiotics was seen more in the methicillin-resistant isolates compared with those that were methicillin sensitive.We also found that S. haemolyticus and S. hominis were the most resistant among the CoNS species other than Staphylococcus epidermidis studied (Table 2).The resistance to antibiotics in the methicillin resistant (MRCoNS) compared with those that were methicillin sensitive (MSCoNS) are displayed in Figure 1.All (100%) of the MRCoNS and MSCoNS were penicillin resistant.A coexisting resistance to a different antibiotic was significantly (p<0.0001)higher in MRCoNS compared with MSCoNS in the present study, with the exception of pristinamycin and fosfomycin.
Of the 90 methicillin-resistant strains isolated in this study, 66 strains (73%) were found to be multidrug resistant, as shown in Table 2. Sequencing of mecA gene was performed for six MRCoNS and the sequence alignment was carried out using ClustalW 1.8.BLAST search at the GenBank database with the mecA sequences for other species of CoNS displayed that CoNS isolates from our study were clearly closely related to GU301101 S. hominis, 007168 S. haemolyticus, GU370073 S. cohnii isolates and X52592 S. epidermidis (Ito et al., 2001;Takeuchi et al., 2005;Zong et al., 2010) in the database, with a nucleotide sequence identity of 100%.More interesting, a similar homology (100%) was found between mecA from the six CoNS isolates from our collection and the mecA from S. aureus (X52593 S. aureus) (Ryffel et al., 1990) (Figure 2).Likewise, all the examined isolates were related to Y13096 S. sciuri (Ito et al., 2001) with 85% identity.Phylogenic construction was carried out using MEGA 4.0 based on the nucleotide sequence of the mecA genes for all the tested isolates.

DISCUSSION
Despite the introduction of antimicrobial therapy and the recent improvements of medical services, CoNS are recognized as a major cause of nosocomial infections, especially in neutropenic patients.In our centre, the epidemiology of S. epidermidis has been extensively studied, but little known about that of the other CoNS (Abbassi et al., 2008;Bouchami et al., 2007).At present, resistance of Staphylococcus to methicillin is a problem of global proportions.This has underlined the need for species identification which is important in monitoring the reservoir and distribution of CoNS involved in infections and determining the etiological agent (Huebner et al., 1999).In this study, phenotypic and genotypic characteristics were used in the identification of the CoNS other than S. epidermidis isolates.Because of the similarity of the biochemical traits of the CoNS species, the correct identification of these species is not easy (Couto et al., 2001;Shittu et al., 2006).Molecular methods are highly desirable to permit a more precise 0.5 0.4 0.3 0.2 0.1 0.0 and full identification at species and subspecies level and must be used as valuable alternatives to commercial systems for identification of CoNS.S. haemolyticus (38%) and S. hominis (36%) were the commonest species which is consistent with the reports of its isolation from clinical samples documented in various published studies (Cuevas et al., 2004;Secchi et al., 2008).However, the species of CoNS isolated in this study were slightly different from those isolated by Tan et al. (2006).It suggests that the distribution of CoNS species is variable and may differ from one country to another (Mohan et al., 2002).
The highest percentage of CoNS species was collected from catheter (31%) and blood (29%) samples mainly recovered from the haematological unit (30%).CoNS are the microorganisms most commonly isolated from blood and catheter in neutropenic patients (von Eiff et al., 2001).Several studies have found corresponding figures for the species distribution among clinical samples of CoNS (Singh et al., 2008).Infection with S. epidermidis, and less commonly with S. haemolyticus and S. hominis, usually involves implantation of medical devices (Akpaka et al., 2006).
Results of antibiotic susceptibility testing showed multidrug resistance and variability in sensitivity and resistance patterns, similar to the study of Mohan et al. (2002) and Pathak et al. (1994).In our study, maximum resistance was observed towards penicillin (84%) followed by erythromycin (69%), oxacillin (60%), cotrimoxazole (53%), gentamicin (50%), ofloxacine (47%), tetracycline (39%) and to fusidic acid (38%).These resistance rates were similar or even higher compared to previous reports for clinical isolates (Mohan et al., 2002).All isolates were sensitive to vancomycin, which is included in empirical therapy for neutropenic patients, 6% S. haemolyticus isolates showed a reduced susceptibility to teicoplanin.Reduced susceptibility to teicoplanin is observed in about 30% of S. haemolyticus and more rarely in S. epidermidis (Achour et al., 2008).It was widely accepted that S. haemolyticus is uniquely predisposed among CoNS to develop glycopeptides resistance as this was the first CoNS species in which vancomycin and teicoplanin resistance was identified (Schwalbe et al., 1987).It is noteworthy that 73% of the MRCoNS isolates were resistant to more than four antibiotics which confirm the large spread of multidrugresistant CoNS isolated from clinical samples as previously reported (Santos et al., 2000;Diekema et al., 2001;Koksal et al., 2007).The heavy use of several antibiotics in certain hospital facilities may select for multiple-resistant commensal organisms including MRCoNS.S. haemolyticus and S. hominis were slightly more resistant to the antibiotic agents other than βlactams than were the other CoNS species other than S. epidermidis studied.This correlated well with the study conducted by Minto et al. (1999) who reported a greater resistance in S. haemolyticus and S. hominis than in Bouchami et al. 1303 S. epidermidis.Interestingly, the high oxacillin resistance rate in our study (60%) was associated with a high resistance level (MIC ≥256 µg/ml in 55% of cases) similarly to what was observed in other studies (Cuevas et al., 2004;Perez et al., 2008) where methicillin resistance rates were high and increased even progressively.In addition, a coexisting resistance to a different antibiotic was significantly higher in methicillinresistant CoNS compared with methicillin-sensitive CoNS.
More than half (65%) of the isolates were mecApositive with highest percentages in S. haemolyticus and S. hominis (47 and 43%, respectively).This wide distribution of the mecA gene in isolates belonging to nine CoNS species has been also previously demonstrated (Secchi et al., 2008).Such a wide distribution of the mecA gene seems to be explained by the following two hypotheses (i) the mecA gene was carried by a common ancestor cell of both S. aureus and CoNS species and the gene has been inherited by all Staphylococcal species of the present day (ii) the mecA gene has been actively transmitted from one Staphylococcal species to another (Suzuki et al., 1992).
In the present study, there were two mecA-positive methicillin-sensitive (MICs = 0.064-0.125µg/ml) isolates.This trend has been observed in CoNS and S. aureus isolates.The discordance of PCR and disc diffusion method could be caused by the heteroresistant nature of the Staphylococci or by absence of mecA gene expression on phenotype level (Martineau et al., 2000).
Comparative analysis of nucleotide sequences of mecA genes showed that CoNS from our study shared 100% sequence identity with GU301101 S. hominis, 007168 S. haemolyticus and GU370073 S. cohnii isolates, X52592 S. epidermidis and X52593 S. aureus, and 85% identity with Y13096 S. sciuri.Altogether, the results suggest that the mecA gene was very well conserved among Staphylococcal species.Similar results were reported that mecA gene is highly conserved among Staphylococcal species (Petinaki et al., 2001;Ito et al., 2001;Rahimi et al., 2009).The mecA gene is considered to have originated in some coagulase-negative Staphylococcus species (Wu et al., 1996) and was then transferred into S. aureus to generate methicillin-resistant S. aureus MRSA (Suzuki et al., 1993;Musser et al., 1992).In human medicine, it has been demonstrated that horizontal transfer from a primitive Staphylococci species, S. sciuri, into an S. aureus chromosome may be occurred (Wu et al., 1996).It is likely that the SCCmec serves as the carrier of the mecA gene moving across Staphylococcal species (Ito et al., 2001;Noto et al., 2006).

Conclusion
This study showed variability in species distributions and in the antibiotic susceptibility pattern of CoNS other than S. epidermidis.The determination of species of CoNS could help in determining the contribution of each species to antibiotic resistance in the hospital and help in designing effective surveillance and control strategies.In addition to the disk susceptibility tests which are widely available, PCR can ensure results to properly guide antimicrobial therapy.The demonstration that the mecA gene is highly conserved in Staphylococci species may aid in the understanding and management of methicillinresistant among Staphylococci.

Figure 2 .
Figure 2. Phylogenetic tree for comparison of mecA gene sequences from CoNS isolates from our study (underlined) with sequences available on GenBank database.The tree was constructed by the Neighbour-Joining (NJ) method using MEGA version 4.0 (Source: Tamura et al., 2007).

Table 1 .
Species distribution of CoNS isolated from a variety of clinical sources.
from 142 neutropenic patients in the Bone Marrow Transplant Center of Tunisia.The species distribution of the isolates is shown in Table1.S.
S. lugdunensis (2%), S. capitis (1.5%) and S. saprophyticus (1.5%).The wards with the highest number of isolates included the hematological unit with 67 (47%) isolates and the graft unit with 42 (30%) isolates.The remaining 33 (23%) CoNS isolates were from outpatients.Patients from the graft (transplant) unit, haematological unit and outpatients were neutropenic or were receiving immunosuppressant drugs.Importantly, hospitalized patients had a central vein catheter.As illustrated in Table1, the largest number of isolates was from catheter (31%) and blood cultures (29%).Only 6.5% were isolated from graft tissue and 6% from Pus.The remainder of CoNS (28%) was isolated from other sources such as cerebrospinal fluid, parenteral alimentation, nose, respiratory tract, throat, urine, genital and stool cultures.

Table 2 .
Antibiotic resistance profile of the two species most frequently isolated and the other CoNS.