Abstract
For a long time, Staphylococcus epidermidis, as a member of the coagulase-negative staphylococci, was considered as part of the physiological skin flora of the human being with no pathogenic significance. Today, we know that S. epidermidis is one of the most prevalent causes for implant-associated and nosocomial infections. We performed pheno- and genotypic analysis (ica, IS256, SCCmec types, agr groups) of biofilm formation in 200 isolates. Fifty percent were genetically ica-positive and produced biofilm. Among all studied isolates, agr II and III and SCCmec type I were the most prevalent, whereas within the selected multi-resistant isolates (29%), agr I and III and SCCmec type II dominated. SCCmec type I and mecA-negative S. epidermidis isolates were associated with agr II. The majority of the blood culture and biopsy isolates were assigned to agr III and SCCmec type I, whereas agr II was predominantly detected in mecA-negative S. epidermidis isolated from catheter and implant materials. MLST analysis revealed the major clonal lineages of ST2, ST5, ST10, and ST242 (total 13 STs). ST2 isolates from blood cultures were icaA/D-positive and harbored SCCmec types II and III and IS256, whereas the icaA/D- and IS256-positive ST23 isolates were assigned to SCCmec types I and IV.
Keywords: S. epidermidis, biofilm, ica, antibiotic resistance, agr, SCCmec, IS256
Introduction
Among the coagulase-negative staphylococci, Staphylococcus epidermidis strains have long been regarded as apathogenic, but their important role as pathogens and their increasing incidence have been recognized and studied in the recent years [1–3]. During the past two decades, S. epidermidis has emerged as one of the major pathogens in nosocomial infections. Infections caused by this organism generally occur in newborns, elderly individuals, immunocompromised patients, and especially in patients with indwelling catheters and other implanted devices. Its major pathogenicity lies in its ability to form biofilm on polymeric surfaces [4, 5]. As catheters and implanted devices are increasingly used in medical practice, infection due to S. epidermidis is now regarded as among the top five microbial causes of nosocomial infections [6].
Biofilm formation has long been suspected of making a major contribution to S. epidermidis infections after implantation of medical devices, in particular catheters [7, 8]. The use of biofilm formation by S. epidermidis strains as a marker of pathogenicity has led to conflicting results because of difficulties in the standardization of measuring biofilm production [7, 8] and phase variation in the expression of biofilm [9, 10]. S. epidermidis is believed to produce biofilm in two stages: rapid initial attachment of the bacteria to polymer surfaces via a surface-associated autolysin encoded by the gene atlE [11], followed by cell proliferation and the production of polysaccharide intercellular adhesion encoded by the ica operon, including four genes: icaA, icaD, icaB, and icaC [12]. The ica operon was shown to be more prevalent in clinical S. epidermidis isolates obtained from catheter-related infections and presumed sepsis than in isolates from normal skin flora of healthy control groups [9, 13]. That means pathogenicity appears to be a complex phenomenon, which could be accounted for not only by large genetic differences but also by small variations in gene contents. This prompted us to perform a detailed comparative pheno- and genotypic analysis of S. epidermidis isolates from different sampling sites, complementing the previous approaches used to differentiate pathogenic strains from the non-pathogenic ones [14].
Other research groups have shown that staphylococcal biofilm formation is a highly variable factor, which is influenced by both regulatory processes and genetic mechanisms, such as phase variations, mutations, and chromosomal rearrangements [15–18]. The observation that some of these genetic processes are mediated by the action of insertion sequence (IS) elements prompted us to investigate the distribution of common staphylococcal IS elements among S. epidermidis strains of clinical and commensal origin. Previous investigations demonstrated that IS256 can be involved in phase variation of biofilm formation in S. epidermidis. Biofilm formation is mainly mediated by the expression of the icaADBC operon, which encodes enzymes for the production of the polysaccharide intercellular adhesin PIA [10]. To investigate a possible association between the ica genes, biofilm formation, and IS256, all strains were tested for the presence of the ica genes and biofilm formation as described previously [9]. Staphylococcus accessory gene regulator (agr) locus influences the expression of many virulence genes in staphylococci. Three allelic groups of agr, which generally inhibit the regulatory activity of each other, are described within the S. epidermidis [19]. The gene structure and sequences of the S. epidermidis agr system are very similar to those of S. aureus, and the locus may play a comparable role. The agr system, in both S. aureus and S. epidermidis, is approximately 3.5 kb in size with 68% similarity and comprises the genes agrA, agrB, agrC, and agrD, which are co-transcribed (RNAII) [19]. In this study, we focused on the agr group specificity and SCCmec types of the respective strains, besides the biofilm production, the antibiotic resistance profile, and the presence of IS256 and ica operon. SCCmec is a mobile genetic element that carries the mecA gene, which is responsible for methicillin resistance in staphylococci. We studied the correlation between the antibiotic resistance, including resistance to macrolides and lincosamides, IS256 presence, biofilm formation, and agr group specificity as well as the SCCmec types in nosocomial S. epidermidis isolates.
Materials and methods
Bacterial isolates, identification, and antibiotic susceptibility testing
In this study, a total of 200 S. epidermidis isolates (blood culture, catheter tips, drain catheters, implants, and biopsies) from hospitalized patients at the University Clinic Magdeburg, Germany, were analyzed.
The identification and susceptibility testing of the S. epidermidis isolates (penicillin, oxacillin, trimethoprim/sulfamethoxazole, erythromycin, clindamycin, gentamicin, ciprofloxacin, levofloxacin, tetracycline, vancomycin, teicoplanin, and linezolid) were performed by the automated VITEK 2® system (bioMérieux, Marcy-l’Etoile, France) [20]. The results were interpreted in accordance to Clinical Laboratory Standards Institute (CLSI) [21] guidelines.
Biofilm production on Congo Red Agar (CRA)
Biofilm formation by S. epidermidis was screened according to the Freeman et al. [22] method for Staphylococcus isolates, which requires the use of a specially prepared solid medium brain heart infusion (BHI) broth supplemented with 5% sucrose and Congo red. The medium was composed of BHI (37 g/l), sucrose (50 g/l), agar (10 g/l), and Congo red stain (0.8 g/l). Congo red was prepared as a concentrated aqueous solution and autoclaved at 121 °C for 15 min separately from other medium constituents and was then added when the agar had cooled to 55 °C. Plates were inoculated and incubated aerobically for 24–48 h at 37 °C. Positive result was indicated by black colonies with a dry crystalline consistency.
DNA extraction
Chromosomal DNA was isolated from overnight cultures grown on blood agar at 37 °C. Genomic DNA was extracted by using the Qiagen® DNA extraction kit according to the manufacturer’s suggestions (Qiagen, Hilden, Germany) with the modification that 20 µl of lysostaphin (Sigma; 1 mg/ml) and 20 µl lysozyme (Qiagen; 100 mg/ml) were added at the cell lysis step. The concentration of the DNA was assessed spectrophotometrically [20].
PCR method for amplification of ica operon
The presence of the entire ica operon in biofilm-producing strains and in non-producing S. epidermidis was checked by amplification of the gene loci, encompassing a region of the icaADBC locus, as described [23].
PCR method for detection of IS256 insertion element
PCR detection of the insertion element IS256 was performed as described before [24].
agr group-specific multiplex PCR
For analysis of the agr group specificity, PCR was carried with specific primers to amplify different agr groups according to the modified protocol of Li et al. [25]. The A primers were selected from conserved sequences to amplify a 1022-bp fragment of agr that is common to agr groups I, II, and III. The B primers were selected from the hypervariable region common to agr groups II and III. The PCR result for agr group I was negative but that of groups II and III was positive. With the C primers, the typing was specific for agr group II, whereas agr groups I and III were negative.
Determination of SCCmec types
SCCmec types I to IV were determined by a multiplex PCR with specific primers and subsequent visualization of the amplified DNA fragment patterns by agarose gel electrophoresis as described by Oliveira et al. [26] and Kozitskaya et al. [27].
Multilocus sequence typing (MLST)
All isolates were analyzed by three MLST protocols, as previously described by Wisplinghoff et al. [28]. The sequences of both strands of all PCR products were resolved with an ABI 3700 automated sequencer (PE Applied Biosystems) with BigDye fluorescent terminators and the primers used in the initial PCR amplification. The alleles at each locus were distinguished by using sequence software (available from http://www.mlst.net). Allelic profiles, consisting of the allele numbers at each of the seven loci, were assigned to sequence types (STs).
Results
Two hundred Staphylococcus epidermidis isolates from hospitalized patients at the University Clinic of Magdeburg were collected and analyzed. Approximately one third was recovered from blood culture, whereas the other specimens were catheter tips, implant materials, and biopsies/punctures. The catheter tips were sub-grouped into central venous catheters, drain catheters, and other catheter types. Specimens like punctures, abscesses, and intra-surgical materials were grouped into biopsies/punctures. Few isolates were recovered from implants, total joint arthroplasty, pacemaker, and intrauterine devices.
Antibiotic resistance of S. epidermidis
Over 91% of the isolates were penicillin (PEN)-resistant and 76% oxacillin (OXA)-resistant. High resistance rates were detected against erythromycin (ERY, 70.5%), clindamycin (CLI, 67.5%), and ciprofloxacin (CIP, 57%). Resistance toward gentamicin (GEN) was observed in 46% of the isolates and toward trimethoprim-sulfamethoxazole (SXT) in 25% of the isolates. All isolates were susceptible toward vancomycin, teicoplanin, and linezolid. One hundred and fifty-two isolates were PEN- and OXA-resistant, whereas the antibiotic resistance profile PEN–OXA–CIP was observed in 103 isolates. Additional GEN resistance was detected in 71 of the isolates. PEN–OXA–CIP–GEN–ERY resistance profile shared 62 (31%) isolates.
Multi-resistant profile (PEN–OXA–CIP–GEN–ERY–CLI) shared 41 (29.5%) isolates, and broader antibiotic resistance profile (PEN–OXA–CIP–GEN–ERY–CLI–SXT) was detected in 15.5% of the S. epidermidis isolates. Only 4% of the S. epidermidis isolates were susceptible to all tested antibiotics.
Biofilm formation
Thirty-two percent of the S. epidermidis isolates produced black colonies on the Congo red agar (CRA) and were therefore classified as biofilm producers. S. epidermidis isolates, which were gained from implants, produced biofilms most predominantly (46.2%). One third of the isolates from drain catheters and biopsies produced biofilm phenotypically. Seventy-five percent of the S. epidermidis isolates from blood culture and central venous catheter tips produced no biofilm phenotypically.
Prevalence of ica genes
By use of PCR, the icaA/D genes were detected in a duplex amplification assay. Sixty-one S. epidermidis isolates were icaA/D-positive and produced biofilm phenotypically. Three icaA/D-negative S. epidermidis isolates somehow produced biofilm phenotypically. In total, 136 isolates were phenotypic biofilm-negative by use of Congo red agar. Sixty-three of these were icaA/D-positive and 73 icaA/D-negative (Table 1).
Table 1.
Analysis of pheno- and genotypic biofilm production
| Biofilm |
|||
|---|---|---|---|
| icaA/D | Positive | Negative | Total |
| Positive | 61 | 63 | 124 |
| Negative | 3 | 73 | 76 |
| Total | 64 | 136 | 200 |
Of the 124 icaA/D-positive isolates, only 49% were able to produce biofilm in vitro. In 76 S. epidermidis isolates, the ica genes were not detectable. On Congo agar, approximately 96% of the icaA/D-negative isolates did not produce biofilm in vitro.
Insertion element IS256
The PCR analysis revealed that 105 of the total 200 S. epidermidis isolates (52.5%) were positive for the insertion element IS256. When the multi-resistance was considered within the total 200 isolates, the positivity for IS256 was 95.1%. In phenotypic biofilm-negative and icaA/D-positive isolates, the insertion element IS256 was detected in 63.5%.
agr group specificity
The accessory gene regulator (agr) system occurs in commensals and pathogenic S. epidermidis isolates and regulates the production of virulence factor genes. Multiplex PCR analysis was performed to group the isolates. Thirty-nine (19.5%) S. epidermidis isolates were grouped in agr I and 66 (33%) in agr II. Another 70 (35%) isolates were categorized in agr III. The remaining 25 (12.5%) of the isolates were agr-negative.
Among 41 S. epidermidis isolates with multi-resistance (PEN–OXA–CIP–GEN–CLI–ERY) and icaA/D positivity, agr groups I and III were the most prevalent, whereas agr group II was infrequent, and nine multi-resistant isolates (22%) were agr-negative.
SCCmec typing
SCCmec type I was most prevalent and was detected in 79 (39.5%) of the isolates. SCCmec type II was carried by 25 (12.5%) S. epidermidis isolates and SCCmec type III by 14 (7%) isolates. SCCmec type IV was detected in 10 (5%) isolates. Twenty-five isolates (12.5%) were mecA-positive but were not SCCmec-typeable. Forty-seven S. epidermidis isolates (23.5%) were negative for mecA. Among these, 42 isolates were susceptible toward oxacillin.
In 39% of the multi-resistant isolates (PEN–OXA–CIP–GEN–CLI–ERY), SCCmec type II was detected as the most frequent type. SCCmec type I was detectable in 19.5% and SCCmec type III in 12.2% of the multi-resistant isolates. SCCmec type IV was infrequent in among the unselected isolates of our study population, but increased in amount within the multi-resistant isolates up to 14.6%. Approximately 10% of the multi-resistant S. epidermidis isolates were non-typeable, and another 5% were mecA-negative.
Correlation between antibiotic resistance profile, SCCmec types and agr
SCCmec type II isolates were significantly resistant to all antibiotics, excluding tetracycline, vancomycin, teicoplanin, and linezolid. SCCmec type III isolates indicated less resistance toward clindamycin (CLI) and erythromycin (ERY) but were mostly resistant toward ciprofloxacin (CIP). S. epidermidis isolates which carried SCCmec type IV were mostly resistant toward erythromycin (ERY), clindamycin (CLI), and ciprofloxacin (CIP), but demonstrated less resistance rates against tetracycline (TET), gentamicin (GEN), and sulfamethoxazole-trimethoprim (SXT).
Approximately all agr II isolates carried either SCCmec type I or were mecA-negative and non-typeable, whereas agr group I isolates were associated with the different SCCmec types and those which were non-typeable. The agr III isolates showed similar correlation. The infrequent agr-negative S. epidermidis isolates did not associate with a particular SCCmec type as well.
Correlation between agr group specificity and SCCmec type among the multi-resistant S. epidermidis isolates
SCCmec type II and the agr groups I and III were most predominantly detected in multi-resistant isolates. Also, a significant correlation between agr group I and SCCmec type II was observed. Furthermore, in multi-resistant isolates, SCCmec type III was exclusively associated with agr group III. Between agr group III, agr-negative isolates, and SCCmec type II, a slight correlation was observed, with no significant preference for SCCmec type II.
SCCmec type and agr group specificity in dependency of the clinical samples
In S. epidermidis, isolates from central venous catheters, drains, and implants agr group II were most prevalent, whereas agr group III dominated in the isolates from blood cultures and biopsies (Table 2).
Table 2.
Distribution of agr groups in the different specimens (*CV, central venous)
| agr Group | Blood culture (%) | CV* catheter (%) | Drain catheters (%) | Biopsies (%) | Implants (%) |
|---|---|---|---|---|---|
| I | 23.4 | 21.6 | 14.3 | 18.2 | 7.7 |
| II | 21.9 | 41.2 | 46.4 | 22.7 | 61.5 |
| III | 48.4 | 23.5 | 32.1 | 36.4 | 15.4 |
| Negative | 6.3 | 13.7 | 7.2 | 22.7 | 15.4 |
In S. epidermidis, isolates from blood cultures, catheters, and biopsies SCCmec I were the frequent type, whereas 50% of the isolates from implants were mecA-negative and non-typeable (Table 3).
Table 3.
Distribution of SCCmec types in different specimens (*CV, central venous)
| SCCmec type | Blood culture (%) | CV* catheter (%) | Drain catheters (%) | Biopsies (%) | Implants (%) |
|---|---|---|---|---|---|
| I | 29.7 | 49.0 | 50.0 | 40.9 | 23.1 |
| II | 10.9 | 13.8 | 21.4 | 9.1 | 7.7 |
| III | 15.6 | 3.9 | 0.0 | 4.5 | 0.0 |
| IV | 1.6 | 7.8 | 7.2 | 4.5 | 7.7 |
| Non-typeable | 21.9 | 7.8 | 0.0 | 13.7 | 7.7 |
| Negative | 20.3 | 17.7 | 21.4 | 27.3 | 53.8 |
Multilocus sequence typing (MLST) of selected S. epidermidis isolates
Due to economic reasons, 40 S. epidermidis isolates were selected for MLST analysis, 14 were isolated from biopsies, catheter tips, implants, and drain catheter and were resistant against penicillin, oxacillin, gentamicin, and ciprofloxacin. Additional 26 S. epidermidis isolates were from blood cultures due to clinical significance. One isolate was not assignable to a particular sequence type. The results of the MLST analysis are depicted in Table 4.
Table 4.
MLST analysis of the S. epidermidis isolates (n = 39) recovered from the different specimen types
| Sequence type (ST) |
|||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Specimen | ST2 | ST5 | ST6 | ST10 | ST21 | ST23 | ST32 | ST43 | ST48 | ST59 | ST130 | ST135 | ST242 |
| Blood culture | 6 | 4 | 1 | 2 | 2 | 1 | 1 | – | 2 | 3 | 2 | – | 1 |
| CV catheter | – | – | – | – | – | 1 | – | 1 | – | – | – | 1 | 2 |
| Drain catheter | – | – | – | 1 | – | – | – | – | – | – | – | 1 | 2 |
| Implants | – | – | – | – | – | 1 | – | 1 | – | – | – | – | – |
| Biopsies | – | – | – | 1 | – | – | – | – | – | – | 1 | 1 | – |
| Total | 6 | 4 | 1 | 4 | 2 | 3 | 1 | 2 | 2 | 3 | 3 | 3 | 5 |
In total, 13 different STs were revealed. Approximately 47% of the isolates were assigned to the sequence types ST2, ST5, ST10, and ST242. Among these, ST2 occurred in 15% (n = 6 of 40), followed by ST242 (12.5%) and ST5 and ST10 with each 10%. The blood culture isolates were frequently typed as ST2; only a few were assigned to ST5 and ST59. S. epidermidis isolates from catheter tips were frequently assigned to ST242.
Occurrence of IS256, icaA/D, mecA and agr in sequence typed S. epidermidis isolates
A number of the S. epidermidis isolates carried the insertion element IS256 and the icaA/D genes. Correlation between SCCmec type I and sequence types ST5 and ST10 was observed. All ST2 and ST242 isolates were positive for icaA/D genes and the insertion element IS256 but were assigned to different SCCmec types. Table 5 summarizes the results for each sequence type.
Table 5.
Sequence types (frequent STs highlighted in bold) of S. epidermidis isolates and their genetic determinants (agr, icaA/D, SCCmec)
|
icaA/D |
IS256 |
SCCmec |
agr |
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ST (n) | pos | neg | pos | neg | I | II | III | IV | NT | neg | I | II | III | neg |
| 2 (6) | 6 | – | 5 | 1 | – | 3 | 3 | – | – | – | 2 | – | 3 | 1 |
| 5 (4) | 1 | 3 | 1 | 3 | 3 | – | – | – | 1 | – | – | 3 | 1 | – |
| 6 (1) | 1 | – | 1 | – | 1 | – | – | – | – | – | – | 1 | – | – |
| 10 (4) | 1 | 3 | 1 | 3 | 4 | – | – | – | – | – | 2 | – | 2 | – |
| 21 (2) | – | 2 | – | 2 | – | – | 1 | – | – | 1 | 1 | 1 | – | – |
| 23 (3) | 3 | – | 3 | – | 1 | – | – | 1 | 1 | – | – | 1 | 2 | – |
| 32 (1) | – | 1 | – | 1 | – | – | – | – | – | 1 | – | 1 | – | – |
| 43 (2) | 2 | – | 2 | – | – | 1 | 1 | – | – | – | – | – | 2 | – |
| 48 (2) | 2 | – | – | 2 | – | – | – | – | 2 | – | – | – | 2 | – |
| 59 (3) | 2 | 1 | 1 | 2 | – | – | – | – | 2 | 1 | – | 1 | 2 | – |
| 130 (3) | 1 | 2 | 1 | 2 | 1 | – | – | – | 1 | 1 | 1 | – | 2 | – |
| 135 (3) | 3 | – | 3 | – | 1 | 1 | 1 | – | – | – | – | – | 3 | – |
| 242 (5) | 5 | – | 4 | 1 | 1 | 2 | 1 | 1 | – | – | 1 | – | 4 | – |
| n, number of isolates; neg, negative; pos, positive; NT, non-typeable | ||||||||||||||
Discussion
Approximately 91% of the S. epidermidis isolates were penicillin-resistant and 76% oxacillin-resistant. Our population was slightly more resistant compared to the study of Kresken et al. [29]. Their data revealed that 60% were oxacillin-resistant, and only 87% were penicillin-resistant. Our isolates were more resistant toward the other antibiotics as well: erythromycin (70.5% versus 55.3%), ciprofloxacin (57% versus 46.7%), and clindamycin (67.5% versus 34.8%).
Biofilm formation and insertion element IS256
Sixty-two percent of our isolates were icaA/D-positive, much more than what was determined in a study by Diemond-Hernández et al. [30] for S. epidermidis. Approximately 32% of our isolates were able to produce biofilm phenotypically. Thus, only 50% of these were ica-positive and produced biofilm on Congo red agar. Chaieb et al. [31] determined higher proportion of icaA/D-positive isolates (72.7%), which phenotypically produced biofilm. Probably, other factors besides the ica genes may have an influence in biofilm formation [14]. Other factors, e.g., environment, nutrition subinhibitory concentrations of diverse antibiotics (tetracycline, gentamicin, clindamycin, teicoplanin), and stress (temperature, osmolarity), might have a significant role in biofilm formation and/or repression [17].
In our population, a quarter of the isolates from blood cultures and catheter tips produced biofilm. However, more isolates from implants (46.2%) produced biofilm. The comparison between the different specimens is not equivalent, since 64 isolates were from blood cultures and only 13 isolates were recovered from implants. The isolates in our study and elsewhere, which produced biofilm, were less susceptible toward a number of antibiotics [32–36]. A possible reason for the higher antibiotic resistance may be the reduced growth of the bacteria within the biofilm or the insufficient uptake of antibiotics [37]. Also, Saginur et al. [38] determined a broader antibiotic resistance in biofilm-positive S. epidermidis and recommended combinations of antibiotics with rifampicin for sufficient therapy. Three of our isolates were biofilm producers although ica genes were not detected. Chaieb et al. [31] described ica-positive S. epidermidis isolates which did not produce biofilm and S. epidermidis which was ica-negative but was somehow able to produce biofilm. Similar results were reported by Kogan et al. [39]. In our study, two of the three biofilm producers were IS256-positive. Possible IS256 insertion within the ica gene complex might disrupt the PCR detection of ica genes. Ziebuhr et al. [9, 10] described a phase variation process. Upon activation of this operon, a polysaccharide intercellular adhesin (PIA) is synthesized which supports bacterial cell-to-cell contacts and triggers the production of thick, multilayered biofilms. PIA synthesis undergoes a phase variation process. The phase variation and transposition of IS256 into the ica gene complex might be the reason for the icaA/D-positive isolates which did not phenotypically produce biofilm since 63.5% of the isolates were IS256-positive. Thus, naturally occurring insertion sequence element is actively involved in the modulation of expression of a Staphylococcus virulence factor, e.g., biofilm formation.
About 52.5% of the S. epidermidis isolates were IS256-positive, but 95% of the multi-resistant isolates were IS256-positive. Supporting our data, Gu et al. [24] determined similar results and concluded that IS256 is a good marker gene to differentiate between invasive and non-invasive isolates. We observed the correlation between the IS256 and the icaA/D genes. In 79% of the IS256-positive isolates, the icaA/D genes were detectable. Same results were reported by Kozitskaya et al. [14]. Moreover, 47% of the IS256- and ica-positive S. epidermidis isolates shared a multi-resistant profile (PEN–OXA–CIP–GEN–ERY–CLI), and 66% were resistant toward PEN–OXA–GEN. Also, Kozitskaya et al. [14] described the correlation between IS256 and the resistance toward oxacillin and gentamicin. The insertion element IS256 more frequently occurs in multi-resistant and ica-positive isolates.
agr group specificity and antibiotic resistance
In contrast to S. aureus, in S. epidermidis, three agr groups have been described [25, 40]. In our population, agr II and III were most prevalent with 33% and 35%, respectively. Twenty percent of the isolates were assigned to agr I, and 25 isolates were agr-negative. Li et al. [25] detected two thirds of their clinical isolates agr I, whereas agr II and III were prevalent in 19.3% and 12.5% of the isolates from Chinese population. The difference in the distribution may be caused by geographical and environmental factors.
In multi-resistant isolates, the distribution of the agr groups shifted in a manner that agr I and III dominated with each 34.1%, whereas agr II was detectable in 9.8% of the multi-resistant isolates. Twenty-two percent among these were agr-negative. The fact that agr I was relatively higher in multi-resistant isolates than in the general population could be due to the clinical relevance of such isolates and may emphasize the pathogenic potential. This finding was supported by Li et al. [25]. Among the multi-resistant group, agr-negative isolates could be mutants. These agr mutants may reflect high primary adhesion during the first phase of biofilm formation and therefore benefit during colonization [41].
Vuong and co-workers described S. epidermidis isolates, which were permanently agr-negative [42]. The agr-negative isolates were observed more dominantly in implant-associated infections and were more successful in colonization of surfaces [42].
Besides the agr system, there is another quorum-sensing system luxS in S. epidermidis. Similar to agr, the luxS system has an inhibitory influence during biofilm formation [43] and probably plays a particular role in the regulation of biofilm production in agr-negative isolates.
SCCmec typing
SCCmec type I was most prevalent in the study isolates (39.5%), whereas 23.5% were not SCCmec typeable. Mombach Pinheiro Machado et al. [44] studied 129 methicillin-resistant coagulase-negative staphylococci isolated from blood cultures and determined SCCmec type I as most prevalent in S. epidermidis; similar to our study, SCCmec IV was infrequent, and very few isolates were non-typeable. The difference in distribution of the SCCmec between our study and that of Mombach Pinheiro Machado et al. [44] in Brazil was the number of SCCmec types II and III. They had no SCCmec type II in their population. In our population, the proportion was 12.5% for this type. Possible rationale for this difference may be the variant therapy, the divergent local antibiotic resistance, and the patient population with their environment. Miragaia et al. [45] studied exclusively methicillin-resistant S. epidermidis, and SCCmec IV was the most prevalent type, followed by type III. A number of isolates were also non-typeable. Wisplinghoff et al. [28] determined a different distribution of SCCmec types in methicillin-resistant S. epidermidis isolates as well. They detected SCCmec IV as the most dominant type followed by types II and III, rarely SCCmec type I.
The different distribution of the SCCmec types in S. epidermidis isolates shown in the diverse studies indicates that the genetic element SCCmec is easily being transferred between the different staphylococci [28, 45–48].
SCCmec type II isolates were mostly resistant toward all antibiotics, excluding tetracycline, glycopeptides, and linezolid, whereas SCCmec type IV isolates in comparison to other types showed more resistance toward tetracycline and trimethoprim-sulfamethoxazole. The SCCmec non-typeable isolates shared the resistance profile against clindamycin and erythromycin, in contrast to SCCmec type III isolates with low resistance rates toward both antibiotics. The results for SCCmec types I and III were supported by those of Mombach Pinheiro Machado et al. [44], although they detected for both types higher resistance rates against gentamicin and trimethoprim-sulfamethoxazole and for SCCmec type I lower resistance rates toward clindamycin and erythromycin. For SCCmec type III isolates, Mombach Pinheiro Machado et al. [44] described much higher resistance rates toward clindamycin and erythromycin. The divergent rates may be reflected by the geographical location of the study population, administration, and consumption of different antibiotics.
Multilocus sequence typing
Within our 40 S. epidermidis isolates, ST2 (15%), ST242 (12.5%), ST5, and ST10 (each 10%) dominated. Kozitskaya et al. [27] sequenced S. epidermidis isolates which were collected from German, Irish, Norwegian, and North American population. ST27 was the dominant sequence type they identified, followed by ST2 and sporadically ST5. Both ST2 and ST5 were mostly mecA-negative and were sporadically ica gene-positive. Miragaia et al. [45] studied S. epidermidis from patients and healthy controls in 17 countries, including Denmark, Island, Portugal, and Bulgaria. They identified 74 different sequence types with ST2 as the most prevalent type. ST2 was assigned to SCCmec types II, III, IV, and non-typeable. Li et al. [49] studied 80 S. epidermidis isolates from patients in Shanghai and determined 16 different sequence types. Also, in their study, ST2 was most prevalent. ST2 was ica-positive, phenotypically produced biofilm, and was a carrier of IS256 and SCCmec type III.
Thirty-five oxacillin-resistant S. epidermidis isolates from Brazil were studied by Iorio et al. [50]. ST2 (45.7%) was the predominant sequence type, followed by ST23 (25.7%). All ST2 isolates were positive for the ica genes and carried SCCmec type III or were non-typeable (four isolates). ST23 isolates were also ica-positive and were mostly SCCmec type IV carriers.
In our study, ST2 was also the most dominant type and icaA/D-positive. About 83.33% of these isolates were carriers of IS256 and equally of SCCmec types II and III. Therefore, our data support the data of Li et al. [49] and Iorio et al. [50]. Our ST23 isolates, similar to those of Iorio et al. [50], were exclusively ica-positive. However, Iorio et al. [50] assigned the ST23 isolates to SCCmec type IV, whereas our ST23 isolates were grouped into SCCmec I and IV. Differences were also observed between the studies concerning ST5 and ST10. In our study, both sequence types were carriers of SCCmec I and were up to 75% positive for icaA/D but IS256-negative. In contrast, Li et al. [49] detected ST5 as a carrier of SCCmec V and ST10 exclusively as SCCmec IV carrier and, in most cases, IS256-positive. Consistent with our data, they detected low rate of icaA/D-positive isolates typed as ST5 and ST10.
An explanation for the divergent results may be the study population; the above-stated groups compared isolates from patients and healthy individuals, whereas our study population and that of Li et al. [49] included only hospitalized patients.
In our study, most of the ST2 isolates were acquired from blood cultures, followed by ST5 and ST59 isolates. ST242 isolates were recovered from catheter tips. Otherwise, there was no significant correlation between the identified STs and the sampling type or location.
Acknowledgements
We thank the Federal State Saxony-Anhalt and the University of Magdeburg, Faculty of Medicine, for funding the staphylococci project (Leistungsorientierte Mittelvergabe, LOM) and the former director Prof. Wolfgang König for supporting the project idea. We express our sincere gratitude to Mrs. Elisabeth Mertens for technical assistance, the patients, clinicians, and the study participants.
Contributor Information
A. Mertens, 1Medical Microbiology, Otto-von-Guericke-University Clinic, Magdeburg, Germany.
B. Ghebremedhin, 2Medical Microbiology Helios Wuppertal, Faculty of Medicine, University of Witten/ Herdecke, Witten, Email: beniam.ghebremedhin@med.ovgu.de, Germany.
References
- 1.Kloos WE, Bannerman TL. Update on clinical significance of coagulase-negative staphylococci. Clinical microbiology reviews. 1994 Jan 1;7(1) doi: 10.1128/cmr.7.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.von Eiff C, Peters G, Heilmann C. Pathogenesis of infections due to coagulase-negative staphylococci. The Lancet infectious diseases. 2002 Nov 1;2(11) doi: 10.1016/s1473-3099(02)00438-3. [DOI] [PubMed] [Google Scholar]
- 3.Bannerman TL. Staphylococcus, Micrococcus, and other catalase-positive cocci that grow aerobically. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH , editors. Manual of Clinical Microbiology. Washington, D.C.: American Society Microbiology; 2003. pp. 384–404. [Google Scholar]
- 4.Mack D. Molecular mechanisms of Staphylococcus epidermidis biofilm formation. The Journal of hospital infection. 1999 Dec 1;43 Suppl doi: 10.1016/s0195-6701(99)90074-9. [DOI] [PubMed] [Google Scholar]
- 5.von Eiff C, Heilmann C, Peters G. New aspects in the molecular basis of polymer-associated infections due to staphylococci. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology. 1999 Dec 1;18(12) doi: 10.1007/s100960050417. [DOI] [PubMed] [Google Scholar]
- 6.Thylefors JD, Harbarth S, Pittet D. Increasing bacteremia due to coagulase-negative staphylococci: fiction or reality? Infection control and hospital epidemiology : the official journal of the Society of Hospital Epidemiologists of America. 1998 Aug 1;19(8) doi: 10.1086/647878. [DOI] [PubMed] [Google Scholar]
- 7.Christensen GD, Baldassarri L, Simpson WA. Colonization of medical devices by coagulase-negative staphylococci. In: Bisno AL, Waldvogel FA, editors. Infections associated with indwelling medical devices. 2. Washington, D.C.: American Society for Microbiology; pp. 45–78. [Google Scholar]
- 8.Ammendolia MG, Di Rosa R, Montanaro L, Arciola CR, Baldassarri L. Slime production and expression of the slime-associated antigen by staphylococcal clinical isolates. Journal of clinical microbiology. 1999 Oct 1;37(10) doi: 10.1128/jcm.37.10.3235-3238.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Ziebuhr W, Heilmann C, Götz F, Meyer P, Wilms K, Straube E, Hacker J. Detection of the intercellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates. Infection and immunity. 1997 Mar 1;65(3) doi: 10.1128/iai.65.3.890-896.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Ziebuhr W, Krimmer V, Rachid S, Lössner I, Götz F, Hacker J. A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256. Molecular microbiology. 1999 Apr 1;32(2) doi: 10.1046/j.1365-2958.1999.01353.x. [DOI] [PubMed] [Google Scholar]
- 11.Heilmann C, Hussain M, Peters G, Götz F. Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface. Molecular microbiology. 1997 Jun 1;24(5) doi: 10.1046/j.1365-2958.1997.4101774.x. [DOI] [PubMed] [Google Scholar]
- 12.Heilmann C, Schweitzer O, Gerke C, Vanittanakom N, Mack D, Götz F. Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis. Molecular microbiology. 1996 Jun 1;20(5) doi: 10.1111/j.1365-2958.1996.tb02548.x. [DOI] [PubMed] [Google Scholar]
- 13.Barbier-Frebourg N, Lefebvre S, Baert S, Lemeland J-F. PCR-Based assay for discrimination between invasive and contaminating Staphylococcus epidermidis strains. Journal of clinical microbiology. 2000 Feb 1;38(2) doi: 10.1128/jcm.38.2.877-880.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Kozitskaya S, Cho SH, Dietrich K, Marre R, Naber K, Ziebuhr W. The bacterial insertion sequence element IS256 occurs preferentially in nosocomial Staphylococcus epidermidis isolates: association with biofilm formation and resistance to aminoglycosides. Infection and immunity. 2004 Feb 1;72(2) doi: 10.1128/IAI.72.2.1210-1215.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Conlon KM, Humphreys H, O’Gara JP. icaR encodes a transcriptional repressor involved in environmental regulation of ica operon expression and biofilm formation in Staphylococcus epidermidis. Journal of bacteriology. 2002 Aug 1;184(16) doi: 10.1128/JB.184.16.4400-4408.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Fitzpatrick F, Humphreys H, Smyth E, Kennedy CA, O’Gara JP. Environmental regulation of biofilm formation in intensive care unit isolates of Staphylococcus epidermidis. The Journal of hospital infection. 2002 Nov 1;52(3) doi: 10.1053/jhin.2002.1309. [DOI] [PubMed] [Google Scholar]
- 17.Rachid S, Ohlsen K, Witte W, Hacker J, Ziebuhr W. Effect of subinhibitory antibiotic concentrations on polysaccharide intercellular adhesin expression in biofilm-forming Staphylococcus epidermidis. Antimicrobial agents and chemotherapy. 2000 Dec 1;44(12) doi: 10.1128/aac.44.12.3357-3363.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Ziebuhr W, Dietrich K, Trautmann M, Wilhelm M. Chromosomal rearrangements affecting biofilm production and antibiotic resistance in a Staphylococcus epidermidis strain causing shunt-associated ventriculitis. International journal of medical microbiology : IJMM. 2000 Mar 1;290(1) doi: 10.1016/S1438-4221(00)80115-0. [DOI] [PubMed] [Google Scholar]
- 19.Otto M, Echner H, Voelter W, Götz F. Pheromone cross-inhibition between Staphylococcus aureus and Staphylococcus epidermidis. Infection and immunity. 2001 Mar 1;69(3) doi: 10.1128/IAI.69.3.1957-1960.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Ghebremedhin B, Olugbosi MO, Raji AM, Layer F, Bakare RA, König B, König W. Emergence of a community-associated methicillin-resistant Staphylococcus aureus strain with a unique resistance profile in Southwest Nigeria. Journal of clinical microbiology. 2009 Sep 1;47(9) doi: 10.1128/JCM.00648-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.CLSI . Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard. Pennsylvania, USA: Clinical and Laboratory Standards Institute; 2009. [Google Scholar]
- 22.Freeman DJ, Falkiner FR, Keane CT. New method for detecting slime production by coagulase negative staphylococci. Journal of clinical pathology. 1989 Aug 1;42(8) doi: 10.1136/jcp.42.8.872. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Arciola CR, Baldassarri L, Montanaro L. Presence of icaA and icaD genes and slime production in a collection of staphylococcal strains from catheter-associated infections. Journal of clinical microbiology. 2001 Jun 1;39(6) doi: 10.1128/JCM.39.6.2151-2156.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Gu J, Li H, Li M, Vuong C, Otto M, Wen Y, Gao Q. Bacterial insertion sequence IS256 as a potential molecular marker to discriminate invasive strains from commensal strains of Staphylococcus epidermidis. The Journal of hospital infection. 2005 Dec 1;61(4) doi: 10.1016/j.jhin.2005.04.017. [DOI] [PubMed] [Google Scholar]
- 25.Li M, Guan M, Jiang XF, Yuan FY, Xu M, Zhang WZ, Lu Y. Genetic polymorphism of the accessory gene regulator (agr) locus in Staphylococcus epidermidis and its association with pathogenicity. Journal of medical microbiology. 2004 Jun 1;53(Pt 6) doi: 10.1099/jmm.0.05406-0. [DOI] [PubMed] [Google Scholar]
- 26.Oliveira DC, de Lencastre H. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrobial agents and chemotherapy. 2002 Jul 1;46(7) doi: 10.1128/AAC.46.7.2155-2161.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Kozitskaya S, Olson ME, Fey PD, Witte W, Ohlsen K, Ziebuhr W. Clonal analysis of Staphylococcus epidermidis isolates carrying or lacking biofilm-mediating genes by multilocus sequence typing. Journal of clinical microbiology. 2005 Sep 1;43(9) doi: 10.1128/JCM.43.9.4751-4757.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Wisplinghoff H, Rosato AE, Enright MC, Noto M, Craig W, Archer GL. Related clones containing SCCmec type IV predominate among clinically significant Staphylococcus epidermidis isolates. Antimicrobial agents and chemotherapy. 2003 Nov 1;47(11) doi: 10.1128/AAC.47.11.3574-3579.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Kresken M, Hafner D, Witte W, Reinert RR. Resistenzentwicklung bei Staphlokokken und anderen grampositiven Erregern gegenüber Chemotherapeutika im mitteleuropäischen Raum. Chemoth J . 1999;8:136–144. [Google Scholar]
- 30.Diemond-Hernández B, Solórzano-Santos F, Leaños-Miranda B, Peregrino-Bejarano L, Miranda-Novales G. Production of icaADBC-encoded polysaccharide intercellular adhesin and therapeutic failure in pediatric patients with Staphylococcal device-related infections. BMC infectious diseases. 2010 Mar 15;10 doi: 10.1186/1471-2334-10-68. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Chaieb K, Mahdouani K, Bakhrouf A. Detection of icaA and icaD loci by polymerase chain reaction and biofilm formation by Staphylococcus epidermidis isolated from dialysate and needles in a dialysis unit. The Journal of hospital infection. 2005 Nov 1;61(3) doi: 10.1016/j.jhin.2005.05.014. [DOI] [PubMed] [Google Scholar]
- 32.Evans RC, Holmes CJ. Effect of vancomycin hydrochloride on Staphylococcus epidermidis biofilm associated with silicone elastomer. Antimicrobial agents and chemotherapy. 1987 Jun 1;31(6) doi: 10.1128/aac.31.6.889. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Nichols WW, Dorrington SM, Slack MP, Walmsley HL. Inhibition of tobramycin diffusion by binding to alginate. Antimicrobial agents and chemotherapy. 1988 Apr 1;32(4) doi: 10.1128/aac.32.4.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Gristina AG, Jennings RA, Naylor PT, Myrvik QN, Webb LX. Comparative in vitro antibiotic resistance of surface-colonizing coagulase-negative staphylococci. Antimicrobial agents and chemotherapy. 1989 Jun 1;33(6) doi: 10.1128/aac.33.6.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Vuong C, Otto M. Staphylococcus epidermidis infections. Microbes and infection / Institut Pasteur. 2002 Apr 1;4(4) doi: 10.1016/s1286-4579(02)01563-0. [DOI] [PubMed] [Google Scholar]
- 36.Vuong C, Kocianova S, Voyich JM, Yao Y, Fischer ER, DeLeo FR, Otto M. A crucial role for exopolysaccharide modification in bacterial biofilm formation, immune evasion, and virulence. The Journal of biological chemistry. 2004 Dec 24;279(52) doi: 10.1074/jbc.M411374200. [DOI] [PubMed] [Google Scholar]
- 37.Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clinical microbiology reviews. 2002 Apr 1;15(2) doi: 10.1128/CMR.15.2.167-193.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Saginur R, StDenis M, Ferris W, Aaron SD, Chan F, Lee C, Ramotar K. Multiple combination bactericidal testing of staphylococcal biofilms from implant-associated infections. Antimicrobial agents and chemotherapy. 2006 Jan 1;50(1) doi: 10.1128/AAC.50.1.55-61.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Kogan G, Sadovskaya I, Chaignon P, Chokr A, Jabbouri S. Biofilms of clinical strains of Staphylococcus that do not contain polysaccharide intercellular adhesin. FEMS microbiology letters. 2006 Feb 1;255(1) doi: 10.1111/j.1574-6968.2005.00043.x. [DOI] [PubMed] [Google Scholar]
- 40.Dufour P, Jarraud S, Vandenesch F, Greenland T, Novick RP, Bes M, Etienne J, Lina G. High genetic variability of the agr locus in Staphylococcus species. Journal of bacteriology. 2002 Feb 1;184(4) doi: 10.1128/jb.184.4.1180-1186.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Vuong C, Gerke C, Somerville GA, Fischer ER, Otto M. Quorum-sensing control of biofilm factors in Staphylococcus epidermidis. The Journal of infectious diseases. 2003 Sep 1;188(5) doi: 10.1086/377239. [DOI] [PubMed] [Google Scholar]
- 42.Vuong C, Kocianova S, Yao Y, Carmody AB, Otto M. Increased colonization of indwelling medical devices by quorum-sensing mutants of Staphylococcus epidermidis in vivo. The Journal of infectious diseases. 2004 Oct 15;190(8) doi: 10.1086/424487. [DOI] [PubMed] [Google Scholar]
- 43.Xu L, Li H, Vuong C, Vadyvaloo V, Wang J, Yao Y, Otto M, Gao Q. Role of the luxS quorum-sensing system in biofilm formation and virulence of Staphylococcus epidermidis. Infection and immunity. 2006 Jan 1;74(1) doi: 10.1128/IAI.74.1.488-496.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Mombach Pinheiro Machado AB, Reiter KC, Paiva RM, Barth AL. Distribution of staphylococcal cassette chromosome mec (SCCmec) types I, II, III and IV in coagulase-negative staphylococci from patients attending a tertiary hospital in southern Brazil. Journal of medical microbiology. 2007 Oct 1;56(Pt 10) doi: 10.1099/jmm.0.47294-0. [DOI] [PubMed] [Google Scholar]
- 45.Miragaia M, Thomas JC, Couto I, Enright MC, de Lencastre H. Inferring a population structure for Staphylococcus epidermidis from multilocus sequence typing data. Journal of bacteriology. 2007 Mar 1;189(6) doi: 10.1128/JB.01484-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Hanssen AM, Kjeldsen G, Sollid JU. Local variants of Staphylococcal cassette chromosome mec in sporadic methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative Staphylococci: evidence of horizontal gene transfer? Antimicrobial agents and chemotherapy. 2004 Jan 1;48(1) doi: 10.1128/AAC.48.1.285-296.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Miragaia M, Couto I, de Lencastre H. Genetic diversity among methicillin-resistant Staphylococcus epidermidis (MRSE). Microbial drug resistance (Larchmont, N.Y.) 2005 Summer;11(2) doi: 10.1089/mdr.2005.11.83. [DOI] [PubMed] [Google Scholar]
- 48.Miragaia M, de Lencastre H, Perdreau-Remington F, Chambers HF, Higashi J, Sullam PM, Lin J, Wong KI, King KA, Otto M, Sensabaugh GF, Diep BA. Genetic diversity of arginine catabolic mobile element in Staphylococcus epidermidis. PloS one. 2009 Nov 6;4(11) doi: 10.1371/journal.pone.0007722. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Li M, Wang X, Gao Q, Lu Y. Molecular characterization of Staphylococcus epidermidis strains isolated from a teaching hospital in Shanghai, China. Journal of medical microbiology. 2009 Apr 1;58(Pt 4) doi: 10.1099/jmm.0.007567-0. [DOI] [PubMed] [Google Scholar]
- 50.Iorio NL, Caboclo RF, Azevedo MB, Barcellos AG, Neves FP, Domingues RM, dos Santos KR. Characteristics related to antimicrobial resistance and biofilm formation of widespread methicillin-resistant Staphylococcus epidermidis ST2 and ST23 lineages in Rio de Janeiro hospitals, Brazil. Diagnostic microbiology and infectious disease. 2012 Jan 1;72(1) doi: 10.1016/j.diagmicrobio.2011.09.017. [DOI] [PubMed] [Google Scholar]