Methicillin-resistant Staphylococcus Epidermidis Isolates Research Articles

Methicillin Resistant Staphylococci Isolated from Goats and Their Farm Environments in Saudi Arabia Genotypically Linked to Known Human Clinical Isolates: a Pilot Study.

ABSTRACTWe conducted a pilot whole genome sequencing (WGS) study to characterize the genotypes of nine methicillin resistant staphylococci (MRS) isolates recovered from goats and their farm environments in Eastern Province, Saudi Arabia, between November 2019 to August 2020. Seven out of nine isolates were methicillin resistant Staphylococcus aureus (MRSA), and two were methicillin resistant Staphylococcus epidermidis (MRSE). All MRSA isolates possessed genotypes previously identified to infect humans, including isolates harboring ST6-SCCmec IV-t304 (n = 4), ST5-SCCmec VI- t688 (n = 2) and ST5-SCCmec V-t311 (n = 1). 2 MRSA isolates possessed plasmids that were genetically similar to those identified in S. aureus isolates recovered from humans and poultry. In contrast, plasmids found in three MRSA isolates and one MRSE isolate were genetically similar to those recovered from humans. All MRSA isolates harbored the host innate modulate genes sak and scn previously associated with human infections. The genotypes of MRSE isolates were determined as ST35, a well-known zoonotic sequence type and ST153, which has been associated with humans. However, the MRSE isolates were untypeable due to extra ccr complexes identified in their SCCmec elements. Moreover, we identified in ST153 isolate SCCmec element also harbored the Arginine Catabolic Mobile Element (ACME) IV. All MRS isolates were phenotypically resistant to trimethoprim-sulfamethoxazole, an antibiotic for the decolonization of MRS. Three isolates carried antibiotic resistance genes in their SCCmec elements that were not previously described, including those encoding fusidic acid resistance (fusC) and trimethoprim resistance (dfrC) incorporated in the MRSA SCCmec VI.IMPORTANCE Our findings demonstrate a possible cross-transmission of methicillin resistant staphylococci between goats and their local environments and between goats and humans. Due to ever increasing resistance to multiple antibiotics, the burden of MRS has a significant impact on livestock farming, public health, and the economy worldwide. This study highlights that implementing a holistic approach to whole genome sequencing surveillance in livestock and farm environments would aid our understanding of the transmission of methicillin resistant staphylococci and, most importantly, allow us to implement appropriate infection control and hygiene practices.

Detection and characterization of methicillin-resistant and susceptible coagulase-negative staphylococci in milk from cows with clinical mastitis in Tunisia.

ObjectivesThis study investigated prevalence of methicillin-resistant (MR) and methicillin-susceptible (MS) coagulase-negative staphylococci (CNS) and the implicated mechanisms of resistance and virulence in milk of mastitis cows. In addition, the presence of SCCmec type was analyzed in MR Staphylococcus epidermidis (MRSE). ResultsThree hundred milk samples from cows with clinical mastitis were obtained from 30 dairy farms in different regions of Tunisia. Sixty-eight of the 300 tested samples contained CNS strains. Various CNS species were identified, with Staphylococcus xylosus being the most frequently found (40%) followed by Staphylococcus warneri (12%). The mecA gene was present in 14 of 20 MR-CNS isolates. All of them were lacking the mecC gene. The SCCmecIVa was identified in four MRSE isolates. Most of CNS isolates showed penicillin resistance (70.6%) and 58.3% of them carried the blaZ gene. MR-CNS isolates (n = 20) showed resistance to erythromycin, tetracycline and trimethoprim-sulfametoxazole harboring different resistance genes such us erm(B), erm(T), erm(C), mph(C) or msr(A), tet(K) and dfr(A). However, a lower percentage of resistance was observed among 48 MS-CNS isolates: erythromycin (8.3%), tetracycline (6.2%), streptomycin (6.2%), clindamycin (6.2%), and trimethoprim-sulfametoxazole (2%). The Inu(B) gene was detected in one Staphylococcus xylosus strain that showed clindamycin resistance. The virulence gene tsst-1 was observed in one MR-CNS strain. DiscussionCoagulase-negative staphylococci containing a diversity of antimicrobial resistance genes are frequently detected in milk of mastitis cows. This fact emphasizes the importance of identifying CNS when an intramammary infection is present because of the potential risk of lateral transfer of resistant genes among staphylococcal species and other pathogenic bacteria.

In vitro activity of oritavancin in combination with rifampin or gentamicin against prosthetic joint infection-associated methicillin-resistant Staphylococcus epidermidis biofilms

This study evaluated the in vitro activity of oritavancin in combination with rifampin or gentamicin against methicillin-resistant Staphylococcus epidermidis (MRSE) biofilms. Oritavancin, rifampin, and gentamicin were tested against 20 MRSE isolates recovered from prosthetic joint infection (PJI). Time-kill studies were used to evaluate the activities of the three antimicrobial agents individually vs. combinations of oritavancin with rifampin or gentamicin against MRSE biofilms formed on Teflon coupons. At 24 h, the combination of oritavancin and rifampin resulted in a significant (P≤0.001) reduction in biofilm density compared with either antimicrobial alone for 85% (17/20) of isolates. Oritavancin combined with gentamicin showed a significant (P≤0.001) reduction in biofilm density compared with either antimicrobial alone against 55% (11/20) of isolates at 24 h. Synergy (defined as a ≥2 log10 cfu/cm2 decrease at 24 h for the antimicrobial combination compared with the most active single antimicrobial) was observed against 65% (13/20) of the isolates for oritavancin in combination with rifampin and 35% (7/20) of the isolates for oritavancin in combination with gentamicin. Oritavancin in combination with rifampin or gentamicin demonstrated bactericidal activity (defined as a ≥3 log10 cfu/cm2 reduction at 24 h from the starting biofilm bacterial density) for 85% (17/20) and 80% (16/20) isolates, respectively. Our study suggests that oritavancin and rifampin combination therapy may be an option for antimicrobial management of PJIs caused by MRSE.

Methicillin-Resistant Staphylococcus epidermidis in Iran: A Systematic Review and Meta-Analysis

Objective: Methicillin-resistant Staphylococcus epidermidis (MRSE) remains one of the most prevalent drug-resistant bacteria causing health care infections. Limited data are available about how the frequency of MRSE changed in Iran over the past years. The current study aimed at determining the frequency of MRSE in different cities of Iran. Methods: Databases including Web of Sciences, Scopus, Embase, Medline, and Iranian databases were searched to find studies addressing the frequency of MRSE in Iran published from Mar 2006 to Jan 2016. The data were analyzed using comprehensive meta-analysis version 2.2 (Biostat). Of the 139 records identified in the databases, 15 studies met the inclusion criteria. Results: The analyses showed that the frequency of MRSE infections was 73.9 95% confidence interval (95% CI) 61.4-83.4 among culture-positive cases of S. epidermidis in different parts of Iran. The frequency of MRSE was higher in the studies conducted from 2011 to 2015, based on further stratified analyses. Conclusions: The regular surveillance on antimicrobial susceptibility pattern and formulation of definite antibiotic policy may control high rate of MRSE associated infections in Iran. Moreover, rapid and reliable diagnosis of MRSE isolates and regular screening of the personnel and surfaces of hospitals in terms of MRSE are indispensable. © 2018, Archives of Clinical Infectious Diseases.

Diversity and antimicrobial susceptibility of cultivable commensal bacteria from 18 to 22 year-old healthy people′s nose and skin

Objective To investigate the diversity and antimicrobial susceptibility of cultivable commesal bacteria from 18 to 22 year-old healthy people′s nose and skin. Methods From June to August 2017, 18 to 22 year-old healthy people (n=210) were swabbed on skin and nose and cultured with blood plates at Renji Hospital, School of Medicine, Shanghai Jiao Tong University; Species determination was performed using matrix assisted laser desorption Lonization-time of flight-mass spectrometry(MALDI-TOF-MS); Susceptibility testing was performed on the major species by the disc diffusion method; Genomic characteristics of methicillin resistant Staphylococcus epidermidis(MRSE) were determined by pulsed field gel electrophoresis (PFGE) method; SCCmec typing was tested by polymerase chain reaction (PCR); Statistical analysis was performed using SPSS software and association statistics were tested using Chi-Square tests. Results In total, 25 genera were identified of cultivable bacteria from 210 healthy people′s nose (1497 isolates) and skin (941 isolates). Staphylococcus isolates from nose and skin accounted for 82.03% and 80.23%respectively.Eleven species were identified of all Staphylococcus isolates and coagulase-negative Staphylococci(CoNS)from nose and skin accounted for 90.72% and 99.21% respectively.Furthermore, a variety of other species and differences between men and women were observed. Susceptibility testing was done on 631 Staphylococcus isolates, which were sensitive to most antibiotics but show high prevalence of resistance towards penicillin (76.55%), erythromycin (41.20%), clindamycin (10.77%) and trimethoprim/sulfamethoxazole(10.14%). Methicillin Resistant Staphylococcus (MRS) (45 isolates) showed higher prevalence of resistance towards penicillin (χ2=12.17, P<0.001), erythromycin (χ2=10.80, P=0.001), levofloxacin (χ2=20.24, P<0.001) and trimethoprim/sulfamethoxazole (χ2=58.57, P<0.001) compared to methicillin sensitive Staphylococcus (MSS)(586 isolates). Moreover, multidrug resistance (MDR) was observed in 42.16% of 631 isolates and MRS showed a significantly higher proportion than MSS(100.00% vs 37.71%, χ2=66.49, P<0.001). PFGE generated 23 groups out of 33 MRSE isolates. SCCmec genotyping of MRSE showed the most prevalent type was SCCmecⅣ (66.67%). Conclusions Obvious genus and species diversity and genetic diversity were observed on cultivable bacteria from 18 to 22 year-old healthy people′s nose and skin, and CoNS was in the majority. Staphylococcus isolates from healthy people′s nose and skin were sensitive to most antibiotics, but show high prevalence of resistance towards penicillin, erythromycin, clindamycin and trimethoprim/sulfamethoxazole. In addition, MDR was serious especially in MRS. Commensal bacteria may act as reservoir for resistance genes facilitating bacteria infection. (Chin J Lab Med, 2018, 41: 196-202) Key words: Commensal bacteria; Genus and species diversity; Staphylococcus; Antimicrobial resistance; Pulsed field gel electrophoresis

Role of Insertion Sequence Element IS256 as a Virulence Marker and Its Association with Biofilm Formation among Methicillin-Resistant Staphylococcus epidermidis from Hospital and Community Settings in Chennai, South India

The objective of this study was to detect the association of biofilm formation with IS256 among clinical and carrier isolates of methicillin-resistant Staphylococcus epidermidis (MRSE). A total of 71 MRSE isolates were included in this study. Phenotypic detection of biofilm formation was done by Congo red agar method. Detection of genes associated with biofilm formation (icaAD, aap and atlE) and insertion sequence IS256 was done by polymerase chain reaction. Of the 71 MRSE isolates,19/40 (47.5%) clinical isolates from hospital settings and 11/31 (35.5%) carrier isolates from community settings respectively were found to be positive for all the three genes tested, namely, icaAD+, aap+ and atlE+ genes. Nearly 80% of clinical isolates were found to harbour IS256, whereas only 13% of community isolates harboured IS256.

Investigation of biofilm formation ability, antimicrobial resistance and the staphylococcal cassette chromosome mec patterns of methicillin resistant Staphylococcus epidermidis with different sequence types isolated from children

This study investigated the molecular characterizations of 80 methicillin resistant Staphylococcus epidermidis (MRSE) collected during 2012–2013 in Tehran Children's Medical Center, Iran. About 90% of MRSE isolates were multi-drug resistant (MDR) and the highest resistance was observed to cotrimoxazole and they were quite sensitive to quinupristin-dalfopristin and linezolid. Though vanA gene was not detected, the majority of isolates showed intermediate resistance to vancomycin (MIC90 16 μg/ml). Resistance to mupirocin was observed in 18 isolates. Staphylococcal cassette chromosome mec (SCCmec) types V, III, IV and II were detected in 23.75%, 7.5%, 6.25% and 5% of isolates respectively, in some of which the additional parts of mec or ccr complexes were observed. In 57.5% MRSE isolates SCCmec types were not classified. 41.2% of MRSE isolates were carrying intercellular adhesion (ica) operon and 40% had strong or intermediate biofilm. The types of arginine catabolic mobile element (ACME) were limited to type I and II. Nine sequence types (STs) were seen in mupirocin resistant MRSE isolates. The common STs were ST2, ST5 and ST22 with 27.7% (5/18), 22.2% (4/18) and 16.6% (3/18) frequencies, respectively. ST23, ST54 and ST179 plus three novels STs 580, 581,588 were also observed. The majority of STs, 83.3% (15/18) belonged to clonal complex 2 (CC2). The spread of antibiotic resistance and virulence factors among MRSE species is an alarming sign in Children's Hospitals. The combination of these two issues leads to increase the chance of successfully establishing of common STs in hospital environments, and promotes the device-related infections and bacteremia.

Construction of mutant strains of methicillin resistant Staphylococcus epidermidis with psm-mec gene deletion

Objective To construct mutant strains of methicillin resistant Staphylococcus epidermidis (MRSE) with psm-mec gene deletion and to investigate the function of psm-mec gene. Methods The drug sensitivity test and DNA sequence analysis were performed to screen out the tetracycline and chloramphenicol sensitive clinical strains of MRSE, whose upstream and downstream sequences of psm-mec gene were identical to those of the Staphylococcus epidermidis reference strain RP62A. The recombinant plasmid pBT2-Δpsm-mec was constructed by using the fusion PCR and a temperature sensitive shuttle plasmid. After being identified, the plasmid was transformed into the Staphylococcus aureus RN4220 strain by electroporation, and then transformed into the selected clinical isolates of MRSE. The mutant strains of MRSE with psm-mec deletion were screened out and identified after homologous recombination. The differences in biofilm formation between the mutant and wild-type strains were analyzed for further elucidation the relationships between the psm-mec gene and biofilm formation in MRSE strains. Results Three clinical MRSE isolates for the construction of mutant strains with psm-mec gene deletion were screened out and identified by using drug sensitivity test and sequence alignment analysis. The mutants constructed via homogenous recombination were screened out and identified. Compared with the corresponding wild-type strains, the three mutants with psm-mec gene deletion showed significantly decreased ability of biofilm formation, demonstrating that the psm-mec genes strains induced the biofilm formation of MRSE. Conclusion The Δpsm-mec mutant strains were successfully constructed. The psm-mec gene played an important role in the biofilm formation of Staphylococcus epidermdis. Key words: Staphylococcus epidermidis; psm-mec; Homologous recombination; Mutant; Biofilm formation

Characterization of Some Genotypic and Phenotypic Traits of Biofilm Producing Clinical Isolates of Methicillin Resistant Staphylococcus Epidermidis

Staphylococcus epidermidis is a major commensal bacterium. Various strains of methicillin resistant S. epidermidis are capable of forming biofilms and it is found to be associated with many hospital-acquired infections. Bacterial biofilms, which are micro-colonies encased in extracellular polysaccharide material, mediated by gene products of the icaADBC operon, are the sources of many bacterial infections which is so difficult to respond to routine treatments. In this research, we investigated the biofilm forming capacity of a 100 methicillin resistant staphylococcus epidermidis isolates, isolated from different clinical specimens delivered to the Diagnostic Microbiology Laboratories and Surveillance Laboratory, Faculty of Medicine, Alexandria University, in relation to the icaADBC gene cluster. Also, the minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) assays were used to evaluate the antibiotic sensitivity patterns of these MRSE isolates in their planktonic and biofilm phases to Vancomycin and Linezolid. The results showed that only 27 isolates (27%) produced detectable biofilm, and icaADBC gene was detected in only 5 of these isolates. Moreover, there was no statistical association between the presence of the gene and the biofilm status. All 27 biofilm producing isolates were susceptible to both Vancomycin and Linezolid in their planktonic state, but the MBEC values of Vancomycin were higher than those of Linezolid in almost all strains, with an agreement between both MBEC values in 15/27 (55.5%) of isolates and disagreement in 12/27 (44.5%) of isolates, and this was statistically significant (p<0.05). In conclusion, this study indicates that the presence of icaADBC gene is not always associated with in-vitro formation of biofilm. Although Vancomycin and Linezolid continued to be effective for planktonic MRSE infection, their sub-MIC concentrations can induce biofilm formation.

Influence of papain in biofilm formed by methicillin-resistant Staphylococcus epidermidis and methicillin-resistant Staphylococcus haemolyticus isolates

Methicillin-resistant Staphylococcus epidermidis (MRSE) and methicillin-resistant Staphylococcus haemolyticus (MRSHa) are important coagulase-negative staphylococci. They are often isolated from bacteremia in humans mainly due to their ability to form biofilm on the surfaces of medical devices. Papain is a complex mixture of proteolytic enzymes and peroxidases extracted from the latex of Carica papaya and it is recognized by accelerating the healing process of wounds. This study aimed to evaluate the ability of the MRSE and MRSHa isolates to produce biofilms. Besides this, the ability of papain to inhibit the formation of biofilms or to disrupt the ones already formed by those bacteria was analyzed. Thirty MRSHa and 30 MRSE were isolated from bacteremia and used in this study. It was observed that papain has ability to reduce biofilms formed by MRSE (p &lt; 0.06) and by MRSHa (p = 0.0005). In addition, papain was able to disrupt mature biofilms made by MRSE (p = 0.014). No antibacterial activity of papain was observed for any isolates of MRSE and MRSHa tested. Papain has been demonstrated as a potential product for reducing biofilm.

Complete sequence of a multiresistance plasmid from a methicillin-resistant Staphylococcus epidermidis ST5 isolated in a small animal clinic

Sir, In a previous study, we identified a methicillin-resistant Staphylococcus epidermidis (MRSE) clone being present in nine different samples obtained from two cats hospitalized in a small animal clinic, an employee and the clinic environment. Members of this MRSE clone exhibited SCCmec type IV, dru type dt10a, multilocus sequence type ST5 and very similar or indistinguishable SmaI macrorestriction patterns. These MRSE isolates were resistant to aminoglycosides/ aminocyclitols, b-lactams, fluoroquinolones, lincosamides, macrolides, chloramphenicol/florfenicol, pleuromutilins, sulphonamides, tetracyclines and trimethoprim, and shared the same plasmid profile including one plasmid of 28 kb in addition to several smaller plasmids. The aim of this study was to investigate this 28 kbplasmidfor itsstructureandthepresenceofresistancegenes. Plasmids from all nine isolates were transferred into Staphylococcus aureus RN4220 by protoplast transformation with selection on regeneration plates supplemented with tiamulin (10 mg/L), trimethoprim (30 mg/L), tetracycline (20 mg/L), erythromycin (10 mg/L) or florfenicol (20 mg/L). The resistance phenotype of the transformants was determined by broth microand macrodilution according to documents VET01-A4 and VET01-S2 (formerly M31-A3) of the CLSI. The antimicrobial agents tested are listed in Table S1 (available as Supplementary data at JAC Online). Plasmid DNA from one representative transformant, namely that from the feline MRSE isolate 47, was cut separately with HindIII and BglII. The resulting fragments were cloned into pBluescript II SK+ and sequenced starting with the M13 universal and reverse primers. Sequence determination was completed by primer walking on both strands. Sequence analysis was performed with the basic local alignment tools BLASTN and BLASTP (http://www.ncbi. nlm.nih.gov/BLAST), the ORF Finder program (http://www.ncbi. nlm.nih.gov/gorf/gorf.html) and the IS finder program (https:// www-is.biotoul.fr//) (all URLs last accessed in July 2013). From each of the nine isolates, the 28 kb plasmid was successfully transferred into S. aureus RN4220 after selection on plates supplemented with tetracycline, tiamulin or trimethoprim. All the transformed plasmids showed indistinguishable restriction patterns with BglII, EcoRI and HindIII. All nine transformants exhibited increased MICs compared with the empty recipient S. aureus RN4220 (Table S1) for clindamycin, tobramycin, neomycin, minocycline, tetracycline, tiamulin, trimethoprim/sulfamethoxazole and trimethoprim. Sequence analysis of one representative plasmid, designated pSWS47, showed that it had a total size of 28743 bp and revealed 27 open reading frames, 9 of which were truncated (Figure 1). The mosaic structure of plasmid pSWS47 strongly suggested that this plasmid has been developed from parts of plasmids and transposons previously described in staphylococci, but also other Gram-positive bacteria, including pUR4128 from a methicillinresistant S. aureus (MRSA), pSERP from human MRSE, pERGB from human MRSA, pPECL-3 and pPECL-4 from Pediococcus claussenii, pMD5057 from Lactobacillus plantarum and pSAP016A from S. epidermidis (Figure 1). Similarity to segments of the aforementioned plasmids was often terminated by insertion sequences, such as IS431, IS257 or IS1310. This might point towards the ability of insertion sequences to mediate the integration of small plasmids into larger plasmids. During such interplasmid recombination processes, insertion sequences may also be truncated, as seen in the present case. Moreover, closer inspection of the 8 bp sequences usually found immediately upand downstream of complete IS431 and IS257 revealed that none of these 8 bp sequences represented direct repeats (Figure 1). This observation suggested that multiple recombination processes, in which these insertion sequences had been involved, might have taken place. This scenario may also be supported by the detection of two segments of the same repU gene disrupted by IS257 and another three intact plasmid replication genes, a plasmid recombination gene, a relaxase gene and two plasmid mobilization genes, originating at least in part from different ancestral plasmids (Figure 1). Plasmid pSWS47 carried five functionally active resistance genes, accounting for the resistance phenotype observed: vga(A) (clindamycin and tiamulin resistance), dfrK (trimethoprim resistance), aadD (neomycin and tobramycin resistance), tet(L) and tet(M) (tetracycline and tetracycline/minocycline resistance) (Figure 1). When comparing plasmid pSWS47 with other genetic elements carrying resistance genes, three interesting observations were made. First, plasmid pSWS47 also harboured an inactive blaZ b-lactamase gene. Sequence analysis revealed an additional adenine residue at position 21227, which resulted in a frameshift mutationintheblaZgene.Second,thestartcodonforthereadingframeof the 20 amino acid peptide in the tet(L) translational attenuator was changed from ATG to AGG in pSWS47. However, MIC testing (32 mg/ L) of the4079 bpHindIIIclonethatcarried the tet(L)geneand its regulatory region confirmed that the tet(L) gene is functionally active despite this mutation. This observation is in agreement with thedetection of functionally active tet(L) genes in Pasteurella multocida and Mannheimia spp. that lacked the entire translational attenuator. Third, a segment that corresponded to the plasmid pMD5057 from

Characterization of a cfr-positive methicillin-resistant Staphylococcus epidermidis strain of the lineage ST22 implicated in a life-threatening human infection

The cfr gene was identified in 3 methicillin-resistant Staphylococcus epidermidis (MRSE) isolates of lineage ST22 implicated in a fatal human infection. MRSE isolates displayed an indistinguishable pulsed-field gel electrophoresis profile and SCCmec type III, and showed a multiresistance phenotype. The presence of cfr, fexA, aac(6′)-aph(2″), and dfrS1 genes was confirmed by polymerase chain reaction and sequencing. A mutation in 23S rRNA gene (C2534T) and amino acid changes and/or insertions in L3 and L4 proteins were detected. The cfr and fexA genes were located in a conjugative plasmid of approximately 45 kb and in the chromosome.

Daptomycin for methicillin-resistant Staphylococcus epidermidis native-valve endocarditis: a case report

Coagulase-negative staphylococci (CoNS) have been increasing in importance as a cause of native valve endocarditis (NVE). Most cases of NVE caused by CoNS are attributable to Staphylococcus epidermidis. NVE caused by CoNS acquired in a nosocomial setting may differ from cases acquired in the community in several ways. It may be associated with hemodialysis, the presence of a long-term indwelling central catheter or pacemaker, or a recent invasive procedure; nosocomial cases may have a higher rate of methicillin resistance among CoNS isolates, and so be more likely to be treated with vancomycin. Unfortunately, NVE caused by methicillin-resistant CoNS has been associated with significantly higher rates of persistent bacteremia and in-hospital mortality than methicillin-susceptible isolates. The poor outcomes in these cases point to the need for alternative therapies with potent activity against methicillin-resistant CoNS. In our medical center, a 76-year-old man presented with native-valve endocarditis and positive blood cultures for methicillin-resistant Staphylococcus epidermidis (MRSE). During each of three 6-week courses of treatment with vancomycin, blood cultures were negative, but they once again became positive for MRSE when vancomycin was discontinued. The minimum inhibitory concentration of the MRSE isolates for vancomycin remained stable at 2 μg/mL. Eventually, treatment with daptomycin was initiated (500 mg [7 mg/kg]) 3 times/week for 6 weeks. Over the following year, no positive cultures for MRSE were detected.

Commensal isolates of methicillin-resistant Staphylococcus epidermidis are also well equipped to produce biofilm on polystyrene surfaces

To study biofilm production and to detect icaAD, atlE and aap genes in 137 isolates of methicillin-resistant Staphylococcus epidermidis (MRSE) obtained from healthy individuals from the community (35 isolates), from hospitalized patients at the Antônio Pedro University Hospital (25 isolates) and from individuals from a home-care system (HCS; 77 isolates). Biofilm production was determined in vitro using polystyrene inert surfaces. icaAD, atlE and aap genes were detected using PCR. Hybridization experiments were also carried out to confirm some PCR results. Antimicrobial susceptibility testing was carried out using the NCCLS methods. Although many of the commensal MRSE isolates produced biofilms, the percentage of biofilm producers was significantly higher (P = 0.0107) among hospital isolates (76%) than among isolates from the community (60%) and from the HCS (57%). An association was observed between multiresistance and biofilm production for isolates obtained from healthy individuals from the community and from household contacts from the HCS (P < 0.0001). The concomitant presence of the ica operon and atlE and aap genes was associated with the strong biofilm-producer phenotype (P < 0.0001). Because many of the commensal MRSE isolates obtained from nares produced biofilms and carried icaAD, aap and atlE genes, biofilms or such genetic elements should not be used as markers for clinical significance. The biofilm environment seems to increase genetic exchanges and hence may contribute to multiresistance phenotypes.

Genetic Diversity among Methicillin-ResistantStaphylococcus epidermidis(MRSE)

We selected 106 methicillin-resistant Staphylococcus epidermidis (MRSE) and 22 methicillin-susceptible S. epidermidis (MSSE) hospital isolates--each with a different PFGE pattern--for more detailed documentation of genetic diversity. The 106 MRSE isolates showed extensive variation in the SmaI DNA fragments hybridizing with the DNA probe for mecA, the molecular size of which varied from as low as 20 kb up to over 500 kb. Parallel variation was also observed in the size of DNA fragments hybridizing with the chromosomal genes orfX and gyrA, and this was also observed in MSSE isolates. In contrast, SmaI fragments associated with the housekeeping genes murE and aroE, both located distantly from orfX, showed little size variation. Typing for the mec complex and ccr identified 10 different SCCmec structures and a large number of strains (21 isolates) that were non-typeable. The majority of strains studied (36%) carried a SCCmec type IV-like structure, including strains with non-related PFGE profiles. On the other hand, closely related strains often carried different types of SCCmec. The findings indicate that the acquisition and/or loss of mobile genetic elements, including various structural types of SCCmec, may occur frequently in the vicinity of the orfX gene on the S. epidermidis chromosome.

Related clones containing SCCmec type IV predominate among clinically significant Staphylococcus epidermidis isolates.

SCCmec is a mobile genetic element that carries the gene (mecA) mediating methicillin resistance in staphylococci. For Staphylococcus aureus, four SCCmec types have been described, one (type IV) of which has been associated with newly identified community-acquired methicillin-resistant S. aureus. However, the distribution of SCCmec types among S. epidermidis is not known. SCCmec typing of a collection of 44 methicillin-resistant Staphylococcus epidermidis (MRSE) isolates recovered between 1973 and 1983 from the blood of patients with prosthetic valve endocarditis (PVE) was performed by PCR amplification of key genetic elements (mecA, mecI, IS1272, and ccrAB). Of the 44 isolates, 1 (2%) harbored SCCmec type I, 15 (34%) harbored type II, 12 (28%) harbored type III, and 16 (36%) harbored type IV. The complete nucleotide sequence of SCCmec type IV was determined for 16 isolates and found to be identical in size (24 kb) and 98% homologous to DNA sequences published for S. aureus. Type IV SCCmec was also common (5 of 10 isolates) among a geographically dispersed collection of 10 recent (1998 to 2001) S. epidermidis bloodstream isolates. Multilocus sequence typing (MLST) (using the same seven genes presently employed for S. aureus MLST) of these MRSE isolates and of 10 additional recent geographically dispersed methicillin-susceptible isolates demonstrated that all 16 PVE isolates and 2 of 5 recent isolates harboring type IV SCCmec were in three related clonal groups. All three MSSE PVE isolates recovered from patients between 1976 and 1979 were in the same clonal groups as type IV SCCmec MRSE isolates. These data support the hypothesis of intra- and interspecies transfer of type IV SCCmec and suggest that there are clonal associations in S. epidermidis that correlate with SCCmec type.

Source and route of methicillin-resistant Staphylococcus epidermidis transmitted to the surgical wound during cardio-thoracic surgery. Possibility of preventing wound contamination by use of special scrub suits

The objective of this study was to trace the source and route of transmission of methicillin-resistantStaphylococcus epidermidis (MRSE) in the surgical wound during cardio-thoracic surgery, and to investigate the possibility of reducing wound contamination by wearing special scrub suits. In total 65 elective operations for coronary artery bypass grafting (CABG) with or without concomitant valve replacement were investigated. All staff present in the operating room wore conventional scrub suits during 33 operations and special scrub suits during 32 operations. Samples were taken from the hands of the scrubbed team after surgical scrub but before putting on sterile gowns and gloves, and from patients' skin (incisional area of sternum and vein harvesting area of legs) after preoperative skin preparation with chlorhexidine gluconate. Air samples were taken during operations. Samples were also taken from the wound just before closure. Total counts of bacteria on sternal skin and from the wound (cfu/cm2) were calculated as well as total counts of bacteria in the air (cfu/m3). Strains of MRSE recovered from the different sampling sites were compared by pulsed field gel electrophoresis (PFGE). It was found that wearing special scrub suits did not reduce the number of air-samples where MRSE was found compared with conventiona<$>l scrub suits. The risk factor most strongly associated with MRSE in the wound at the end of the operation was preoperative carriage of MRSE on sternal skin; RR 2.42 [95% CI 1.43–4.10], P= 0.021. By use of PFGE, it was possible to identify the probable source for four MRSE isolates recovered from the wound. In three cases the source was the patients own skin. Finding MRSE in air-samples, or on the hands of the scrubbed team, were not risk factors for the recovery of MRSE in the wound at the end of operation. In conclusion, with a total bacterial air count around 20cfu/m3and a low proportion of MRSE, the reduction of total air counts by use of tightly woven special scrub suits did not reduce air counts of MRSE or wound contamination with MRSE. The patients' sternal skin was the main source for wound contamination with MRSE