A study of coagulase-negative staphylococci isolated from clinically significant infections at an Australian teaching hospital

Some 151 isolates of coagulase-negative staphylococci isolated from patients at an Australian teaching hospital were characterized by biochemical analysis, antibiotic sensitivity patterns and slime production. S. epidermidis was the predominant species (64%) isolated from clinically significant infections, and all S. epidermidis isolates from true bacteremias produced slime. Forty-nine per cent were resistant to methicillin and 61% to gentamicin. S. haemolyticus isolates from clinically significant infections also showed antibiotic resistance and 80% were resistant to more than five antibiotics. The importance of coagulase-negative staphylococci as pathogens in this large teaching hospital was confirmed.     

Staphylococcal peritonitis in patients on continuous peritoneal dialysis.

During 1984, 35 patients undergoing continuous peritoneal dialysis experienced 77 cases of peritonitis with 55 cases (71.4%) related to staphylococci. Coagulase-negative staphylococci were isolated in 41 cases, while Staphylococcus aureus was found in 14. A coexisting tunnel infection was more often associated with S. aureus (7/14) than with coagulase-negative staphylococci (2/41) (P less than 0.01). Likewise, eradication of the infection necessitated catheter removal more frequently with S. aureus (5/14) than with coagulase-negative staphylococci (2/41) (P less than 0.01). Of the 41 coagulase-negative staphylococci, 35 were characterized as to species, adherence, and production of two exopolysaccharides. Staphylococcus epidermidis was the most frequent coagulase-negative species (29/35). Peritonitis cases caused by coagulase-negative staphylococci that lacked adherence and exopolysaccharides were more frequently associated with complications (4/6) than were those organisms with either or both properties of adherence or exopolysaccharide production (5/29). There were no appreciable differences in antibiotic susceptibilities. Staphylococcal peritonitis remains a significant cause of morbidity in continuous peritoneal dialysis patients. The incidence of complications was not directly linked to staphylococcal properties of adherence or exopolysaccharide production.     

Species identification and antibiotic susceptibilities of coagulase-negative staphylococci isolated from clinical specimens

Identification of potentially significant coagulase-negative staphylococci isolated from clinical specimens was performed along with antibiotic susceptibility determinations, S. epidermidis accounted for 75% of these isolates, with S. haemolyticus and S. hominis being the second and third most frequently encountered species, respectively. Although there were many instances of single blood culture isolations of questionable significance, all three species were also found in multiple blood cultures from individual patients, indicating the ability to cause significant bacteremia. The most common source for most species was blood, except for S. saprophyticus and S. simulans, which were found more frequently in urine. Of urinary tract isolates, however, S. epidermidis was more common than S. saprophyticus. Antibiotic susceptibility profiles demonstrated that S. haemolyticus and S. epidermidis were frequently multiply antibiotic resistant. S. haemolyticus had a higher percentage of isolates that were oxacillin, cephalothin, aminoglycoside, erythromycin, and clindamycin resistant than did S. epidermidis. We found that species identification could be of benefit for both epidemiological as well as patient care purposes, and that this additional information is readily available, using convenient and rapid new methods.     

Studies on clinical isolates of coagulase-negative staphylococci resistant to methicillin. Evidence of cross-resistance between methicillin and cephalothin

The in vitro susceptibility to cephalothin and cefuroxime of 195 isolates of methicillin-resistant coagulase-negative staphylococci was determined by the agar-diffusion test, using 7.5% NaCl-supplemented agar. The distribution of the inhibition zone diameters for isolates of S. epidermidis (S. biotype 1) as well as for S. haemolyticus (S. biotype 4) was trimodal. While 4% of the isolates were found susceptible to cefuroxime, 39% of the S. epidermidis/S. hominis (S. biotype 1) isolates and 34% of the S. haemolyticus (S. biotype 4) isolates were found susceptible to cephalothin by this method. Eight of these isolates (six S. epidermidis, two S. haemolyticus) were selected for susceptibility testing by the tube-dilution method, together with four isolates (three S. haemolyticus, one S. epidermidis) found resistant to cephalothin by the agar-diffusion test. The first-mentioned isolates were all found susceptible to cephalothin with MICs less than or equal to 2 micrograms/l, while the last-named all were resistant with MICs greater than or equal to 16 micrograms/ml. Population analyses revealed sub-populations of highly resistant bacteria in all methicillin-resistant isolates of S. epidermidis (S. biotype 1), as well as in all isolates of S. haemolyticus (S. biotype 4). We thus concluded that methicillin-resistance in isolates of coagulase-negative staphylococci implies resistance to cephalosporins and that the difference between S. epidermidis and S. haemolyticus as regards cephalosporin-susceptibility is quantitative and not qualitative. Eighty-nine per cent of the 195 methicillin-resistant isolates in this study were resistant to penicillin and at least one more antibiotic. We therefore think that resistance to penicillin and one or more non-beta-lactam antibiotics strongly suggests methicillin-resistance and that such isolates should be further tested on hypertonic media.     

Sequential analysis of staphylococcal colonization of body surfaces of patients undergoing vascular surgery.

Slime-producing coagulase-negative staphylococci are pathogens in vascular surgery by virtue of their ability to adhere to and persist on prosthetic graft material. Inguinal and abdominal skin sites were cultured in 41 patients upon hospitalization, and slime production and antimicrobial susceptibility were assessed in all recovered staphylococcal isolates. Twenty-one patients eventually underwent lower-extremity revascularization. In the operative population, cultures were also obtained on the day of surgery and fifth postoperative day. All 21 patients received perioperative cefazolin. Of 327 coagulase-negative staphylococci recovered, Staphylococcus epidermidis (47%), S. haemolyticus (21%), and S. hominis (10%) were the predominant isolates. Slime-producing coagulase-negative staphylococci were recovered from 17 of 21 patients at admission but only from 8 of 21 patients on day 5 postoperation (P less than 0.05). S. epidermidis isolates demonstrated increasing multiple resistance from admission to 5 days postoperation to methicillin, gentamicin, clindamycin, erythromycin, and trimethoprim-sulfamethoxazole (P less than 0.05). All coagulase-negative staphylococcal isolates were susceptible to ciprofloxacin and vancomycin. Slime-producing capability was not associated with increased methicillin resistance for the recovered isolates. The data demonstrate that patients enter the hospital colonized with slime-producing strains of coagulase-negative staphylococci and that during hospitalization the staphylococcal skin burden shifts from a predominately susceptible to a resistant microbial population, which may enhance the importance of slime production as a risk factor in lower-extremity revascularization.     

Nationwide German multicenter study on prevalence of antibiotic resistance in staphylococcal bloodstream isolates and comparative in vitro activities of quinupristin-dalfopristin

Antibiotic-resistant gram-positive bacteria have become an increasing problem in the last two decades. In order to evaluate the prevalence of antibiotic resistance in staphylococcal bloodstream isolates in Germany, 2,042 staphylococci collected in 21 tertiary-care hospitals were investigated during a 3-year period (March 1996 to March 1999). Altogether, 1,448 S. aureus isolates and 594 coagulase-negative staphylococci (CoNS) that comprised 13 different species were included. Furthermore, the antistaphylococcal activities of quinupristin-dalfopristin were compared with those of eight other compounds by the broth microdilution method. The rates of oxacillin resistance in Staphylococcus aureus, S. epidermidis, S. haemolyticus, and other CoNS were 13.5, 69, 90, and 34%, respectively. In oxacillin-resistant strains high rates of resistance (up to 100%) to erythromycin, clindamycin, ciprofloxacin, and gentamicin were also observed. However, no strain appeared to be resistant to vancomycin or quinupristin-dalfopristin. The streptogramin combination exhibited excellent in vitro activity against all staphylococcal species tested, regardless of the patterns of resistance to other drug classes. In terms of MICs at which 90% of the isolates are inhibited, quinupristin-dalfopristin was 2 times more active against S. aureus isolates, 4 to 16 times more active against S. haemolyticus, and 8 to 32 times more active against S. epidermidis than vancomycin or teicoplanin.     

Comparative virulence of human isolates of coagulase-negative staphylococci tested in an infant mouse weight retardation model.

Human infections caused by coagulase-negative staphylococci have steadily increased in numbers and severity. Causes may be the use of artificial prostheses, immunocompromising chemotherapy and radiation therapy, and sophisticated surgical techniques, to name a few. Although the infectivity of coagulase-negative staphylococci as a group has been well documented for humans, attempts to study the pathogenesis of infections caused by individual species of coagulase-negative staphylococci have been hampered by the lack of an animal model that is not refractory to infection by these organisms. In the study reported here, a 2-day-old-mouse weight retardation test was used to assay the virulence of 60 clinical and reference strains of coagulase-negative staphylococci. These strains represented eight species of coagulase-negative staphylococci. The most virulent strains were demonstrated to be of the species Staphylococcus haemolyticus, S. saprophyticus, and S. epidermidis. The data further suggest that production of slime is a marker of virulence in S. epidermidis and that intraspecies differences in virulence occur.     

Problems with the laboratory identification and evaluation of coagulase-negative staphylococci

During a 105 day period in mid 1984, 796 isolates of coagulase-negative staphylococci were recovered from routine specimens handled in the Microbiology Laboratory, Dunedin Hospital. Of these isolates 504 (63.3%) were from wounds, 170 (21.4%) from urines, 58 (7.3%) from intravascular catheter tips, 44 (5.5%) from blood cultures and 20 (2.5%) from sputa. Presumptive identification of 315 consecutive isolates revealed 175 (55.6% of total) strains of Staphylococcus epidermidis, 44 (14.0%) S. capitis, 36 (11.4%) S. haemolyticus/hominis, 29 (9.2%) S. warneri, 19 (6.0%) S. simulans and 12 (3.8%) members of the S. saprophyticus group. Using laboratory generated criteria, 44.8%, 25.9% and 4.5% of coagulase-negative isolates from catheter tips, urines and blood cultures respectively, were deemed pathologically potentially significant. Although more common, S. epidermidis did not appear to be significantly more virulent than other members of the epidermidis species group or S. simulans; 67% of the S. saprophyticus group isolates from urine were considered pathologically significant. Antibiograms on the 796 coagulase-negative isolates revealed 63.2% resistant to penicillin, 22.6% to methicillin, 34.8% to cephradine, 27.5% to gentamicin, and 14.4% to erythromycin; multiple resistance was common. Methicillin resistance was a feature of S. saprophyticus group strains. With isolates from catheter tips and blood cultures, a significantly higher percentage of those regarded as significant were gentamicin resistant. Apart from penicillin, antibiotic resistance was not so marked in strains of coagulase-positive staphylococci recovered over the same period.     

Species identification of coagulase-negative staphylococcal isolates from blood cultures.

Coagulase-negative staphylococci generally are not fully identified, are called Staphylococcus epidermidis, and are considered contaminants when isolated from blood cultures. In a cancer hospital during 6 months, 46 patients had multiple blood cultures (mean, 3.1) which yielded coagulase-negative staphylococci. Species identification of these showed that 10 of the 46 (22%) were not S. epidermidis. Similarly, 96 coagulase-negative staphylococci isolated from only one of multiple blood cultures from patients and thought to be skin contaminants were identified. Of 96 of the staphylococci, 14 (16%) of the latter group were not S. epidermidis. Species found included S. haemolyticus, S. hominis, S. warneri, S. simulans, and S. xylosus. Eight isolates of these species were methicillin resistant, and all eight were mannitol fermenters. The results suggest that these species invasively infect cancer patients with the same frequency at which the species colonize. No one species was identified as being more pathogenic than the others. Routine species identification of coagulase-negative staphylococci from blood cultures of cancer patients contributed little to management except to occasionally distinguish multiple-episode culture contamination by different species from sustained bacteremia with the same species.     

Species identification and susceptibility to 17 antibiotics of coagulase-negative staphylococci isolated from clinical specimens.

A total of 299 isolates of gram-positive, catalase-positive, coagulase-negative cocci were isolated from a variety of specimens collected from patients at a large university hospital, and 281 (94%) were identified as staphylococci by established methods. Using the scheme of Kloos and Schleifer, we determined the species of the coagulase-negative staphylococci. Staphylococcus epidermidis was the cause of all bacteremias and the most commonly isolated species from bone, joint, and wound infections. Staphylococcus haemolyticus was the second most common isolate from wound infections, and Staphylococcus saprophyticus was the most commonly isolated species from urinary tract infections. Antibiograms to 17 antimicrobial agents were performed by a microdilution technique, and the results revealed that S. epidermidis was resistant to a water spectrum of antimicrobial agents than the other species of staphylococci were.     

Significance of urinary isolates of coagulase-negative Micrococcaceae.

Of 16,347 urine cultures submitted to the hospital laboratory, 68 (0.4%) specimens from 50 patients yielded greater than 10(4) coagulase-negative staphylococci/ml in pure culture. A total of 62 of 63 organisms available for study were staphylococci: 45 Staphylococcus epidermidis (predominantly subgroup 1), 15 Staphylococcus saprophyticus (subgroup 3), and 2 Staphylococcus aureus. Twenty-one patients had "probable" urine infections. Eight patients had two or more positive urine cultures, and all isolates from the same patients were identical (by morphology, antibiotic susceptibility, and hemolytic pattern). Nine (75%) of the 12 isolates of S. saprophyticus, which were novobiocin resistant and nonhemolytic on the synergistic hemolysis test, were from patients with probable urinary infection. Eight were young women with acute symptoms and pyuria. Differences in the glucose and mannitol fermentation tests with different media may lead to difficulties in identification. Novobiocin resistance cannot be relied upon to differentiate isolates of S. saprophyticus from S. epidermidis.     

Species-specific and ubiquitous DNA-based assays for rapid identification of Staphylococcus epidermidis.

Staphylococcus epidermidis is an aerobic gram-positive coccus that is now recognized among the coagulase-negative staphylococci as an etiological agent with an important range of pathogenicity in humans. Several diagnostic kits based on biochemical or immunological reactions can efficiently identify Staphylococcus aureus. However, these tests are often unreliable for the identification of coagulase-negative staphylococcal species including S. epidermidis. Since DNA-based assays for the species-specific identification of S. epidermidis remain unavailable, we have developed such tests in order to improve the accuracy and the rapidity of tests for the diagnosis of S. epidermidis infections. On the basis of the results of hybridization assays with clones randomly selected from an S. epidermidis genomic library, we identified a chromosomal DNA fragment which is specific and 100% ubiquitous for the identification of S. epidermidis. This 705-bp fragment was sequenced and used to design PCR amplification primers. PCR assays with the selected primers were also highly specific and ubiquitous for the identification from bacterial cultures of clinical isolates of S. epidermidis from a variety of anatomic sites. While three strains of S. capitis were misidentified as S. epidermidis with the API Staph-Ident system and 2.5% of the S. epidermidis identifications were inconclusive with the MicroScan Autoscan-4 system, the PCR assay was highly specific and allowed for the correct identification of all 79 S. epidermidis strains tested. The PCR assays developed are simple and can be performed in about 1 h. The DNA-based tests provide novel diagnostic tools for improving the diagnosis of S. epidermidis infections.     

Use of trehalose-mannitol-phosphatase agar to differentiate Staphylococcus epidermidis and Staphylococcus saprophyticus from other coagulase-negative staphylococci.

Using a plate medium containing trehalose, mannitol, and phenolphthalein diphosphate (TMPA), we differentiated significant clinical isolates of Staphylococcus epidermidis by their lack of acid production in 18 h from other coagulase-negative staphylococci, with our results having a sensitivity (R. S. Galen and S. R. Gambino, Beyond Normality: The Predictive Value and Efficiency of Medical Diagnoses) of 100%, a specificity of 89.9%, and a positive predictive value of 94.8%. With a Taxo A bacitracin disk, which differentiates Staphylococcus species from Micrococcus species, no zone of inhibition was seen for 96% of all staphylococcal strains, with 5 of 26 strains of Staphylococcus saprophyticus exhibiting zone diameters up to 10 mm. By using resistance to a 5-microgram novobiocin disk, we differentiated S. saprophyticus, with our results having a sensitivity of 100%, a specificity of 97.1%, and a positive predictive value of 83.9% on TMPA. These two species represented 77.8% of coagulase-negative staphylococci isolated. Reference strains fo Staphylococcus and Micrococcus species were differentiated by TMPA. The cost of TMPA was compared with that of another method. TMPA was found to offer an inexpensive, sensitive method for rapidly differentiating coagulase-negative Staphylococcus isolates.     

Antimicrobial resistance in coagulase-negative staphylococci

Patterns of resistance to antimicrobial agents were studied in 193 strains of coagulase-negative staphylococci isolated from hospital patients. Strains isolated from patients with malignant disease were significantly more often resistant to sulphonamide, trimethoprim, gentamicin and methicillin than were strains from other sources. Susceptibility to various beta-lactam antibiotics and aminoglycosides was investigated in members of the two most frequent species: Staphylococcus epidermidis and S. haemolyticus. S. haemolyticus strains were not only more often resistant to methicillin than S. epidermidis strains (respectively 81% and 17%) but they were more highly resistant (mean MICs respectively 85 and 19 mg/L). Methicillin-resistant S. haemolyticus strains were highly resistant to nine other beta-lactam antibiotics, whereas methicillin-resistant S. epidermidis strains showed both lower levels and a narrower spectrum of cross-resistance. Resistance to methicillin in members of both species was "heterogeneous", i.e., only a minority of cells in a culture showed significant resistance. Almost all gentamicin-resistant strains were sensitive to netilmicin and amikacin; rifampicin, vancomycin and teicoplanin were also highly active in vitro.     

Composition and density of microflora in the subungual space of the hand.

There were significant quantitative differences in the composition and density of microflora in different areas of the hands of 26 adult volunteers. The subungual spaces had an average log10 CFU of 5.39, compared with a range from 2.55 to 3.53 for other hand sites. In quantitative cultures from five subungual spaces in 26 subjects, coagulase-negative staphylococci were the dominant organisms, with Staphylococcus epidermidis, S. haemolyticus and S. hominis being the most frequently isolated species. Other bacteria recovered from subungual spaces included gram-negative bacilli in 42.3% of subjects, with Pseudomonas species composing 31.3% of this group, and coryneforms in 42.3% of subjects, with multiply resistant JK group coryneforms making up 12.5%. Yeasts were isolated from 69.0% of subjects sampled, with 51.3% of the yeasts identified as Candida parapsilosis. The subungual coagulase-negative staphylococci were susceptible to most antibiotics, with resistance to penicillin, ampicillin, and erythromycin detected in 23 to 38% of isolates.     

Isolation of methicillin-resistant coagulase-negative staphylococci from chickens.

Methicillin-resistant coagulase-negative staphylococci were isolated from the nares and skin of 1- to 8-week-old healthy chickens in three flocks from a farm. Isolation of methicillin-resistant coagulase-negative staphylococci was positive for 72 (25.7%) of the 280 chickens tested, with the frequency varying from 2.2 to 100% according to flock. A total of 45 appropriate isolates were selected and subjected to identification. Of the 45 methicillin-resistant coagulase-negative staphylococcal isolates selected, 37 were identified as Staphylococcus sciuri, 5 were identified as Staphylococcus epidermidis, and 3 were identified as Staphylococcus saprophyticus. The distribution of the species was different among the flocks. Comparative analysis of the SmaI-digested chromosomal DNA by pulsed-field gel electrophoresis revealed that the isolates could have originated from a single clone of each of S. sciuri and S. saprophyticus and three clones of S. epidermidis. By two methods based on the PCR technique, the mecA gene was detected in all five representative isolates of each methicillin-resistant coagulase-negative staphylococcal clone. The nucleotide sequence of a PCR fragment obtained from an isolate of S. sciuri was completely identical to the corresponding region of mecA genes reported in human methicillin-resistant Staphylococcus aureus isolates and Staphylococcus epidermidis isolates. The representative methicillin-resistant coagulase-negative staphylococcal isolates were resistant to many beta-lactam antibiotics, and some isolates were also resistant to macrolide and aminoglycoside antibiotics. This is the first evidence of the existence of methicillin-resistant coagulase-negative staphylococci from animals possessing the mecA gene.     

Development of the quantitative micro-test for slime production by coagulase-negative staphylococci

The macro-test for slime production by coagulase-negative staphylococci was adapted to a spectrophotometric micro-test assay to obtain more objective and quantitative information on slime production. A total of 135 isolates of coagulase-negative staphylococci (70 macro-test-positive and 65 macro-test-negative) were tested by both methods. The quantitative micro-test optical density readings were (mean ±SD) 1.176 ± 0.294 and 0.130 ± 0.095 for the macro-test-positive and -negative groups, respectively. The micro-test was reproducible and demonstrated quantitative differences in slime production among the different species of coagulase-negative staphylococci. Aside fromStaphylococcus epidermidis, the majority of the coagulase-negative staphylococci had very low optical density readings, indicating little or no slime production under the conditions employed in this assay. This test allows one to study the relative production of slime by different strains and species of coagulase-negative staphylococci, and may be useful in studying the effects of different conditions, such as antibiotic exposure, on slime production.     

Detection of methicillin-resistant staphylococci by using the polymerase chain reaction.

A polymerase chain reaction (PCR)-based test was developed for the detection of mecA in staphylococci. To facilitate this process, a rapid cell lysis procedure was established for the release of DNA from staphylococcal strains. Primers based on the DNA sequence of the mecA gene from Staphylococcus aureus were used in PCRs to screen for the presence of this gene in a total of 98 staphylococcal isolates. Fifty-one isolates were mecA positive (17 S. aureus strains and 34 coagulase-negative staphylococci including S. epidermidis, S. haemolyticus, and S. simulans). Results obtained with PCRs were generally consistent with those of standard microbiological assays. PCRs designed to detect the femA gene (factor essential for methicillin resistance) revealed the presence of the gene in all S. aureus strains examined regardless of the susceptibility profiles of the strains to methicillin. In contrast, femA could not be detected in coagulase-negative staphylococci by PCR with the same primers. Low-stringency hybridization suggested the presence of a gene structurally related to femA in S. epidermidis and other coagulase-negative staphylococci examined.     

Rapid identification of fibronectin, vitronectin, laminin, and collagen cell surface binding proteins on coagulase-negative staphylococci by particle agglutination assays.

Seventeen strains of ten different species of coagulase-negative staphylococci were shown to interact with collagen, laminin, fibronectin, and vitronectin immobilized on latex beads. Different species of coagulase-negative staphylococci have different capacities to agglutinate proteins. Cells of 18 strains of Staphylococcus haemolyticus reacted more strongly than did cells of 18 Staphylococcus epidermidis strains with proteincoated latex beads, although no significant difference in cell surface hydrophobicity or charge could be shown. The cell surface receptors of S. haemolyticus were more heat and protease resistant than were Staphylococcus aureus receptors. Strains of Staphylococcus saprophyticus isolated from urinary tract infections showed a high capacity to adhere to laminin. The ability to agglutinate fibronectin and collagen was common among coagulase-negative staphylococci isolated from other infections; 55% (31 of 56) and 63% (35 of 56) agglutinated fibronectin and/or collagen. S. haemolyticus and S. epidermidis bound to both N-terminal (29-kDa) and C-terminal (120-kDa) fragments of fibronectin.     

Application of gas-liquid chromatographic analysis of cellular fatty acids for species identification and typing of coagulase-negative staphylococci.

Gas-liquid chromatography (GLC) of bacterial cellular fatty acids was used to analyze 264 isolates of coagulase-negative staphylococci, of which 178 were Staphylococcus epidermidis. The presence and amounts of individual fatty acids were determined to generate fatty acid profiles for each of the seven coagulase-negative species tested. The fatty acid profiles were then analyzed by computerized correlation and cluster analysis to calculate mean correlation values between isolates belonging to the same or different species, as well as to establish cluster analysis dendrograms. These data ultimately allowed the clustering of individual samples into species-specific clusters. Species identification by the GLC clustering was highly consistent with species identification by biochemical assays; the results were similar in 92.4% of the cases. The GLC profile correlation analysis was further used to analyze multiple blood isolates from 60 patients in order to determine the usefulness of this methodology in establishing identity, as well as differences, between consecutive patient isolates. The correlation between those multiple S. epidermidis isolates determined to be identical by standard techniques (such as the antibiogram, biotype, and plasmid profile) was significantly (P less than 0.001) higher than that between random isolates of the same species. The correlation coefficient was greater than 97 for 40 (97.6%) of the 41 patients with multiple identical blood isolates, compared with less than 95 in all 19 (100.0%) patients with multiple nonidentical isolates. The successful use of the computerized GLC analysis in this study demonstrated its appropriate application for species identification and typing of coagulase-negative staphylococci.