Characterization of coagulase-negative staphylococci from urinary tract specimens.

Species of coagulase-negative staphylococci isolated from urine specimens submitted from both inpatients and outpatients to the clinical microbiology laboratory of a teaching hospital were identified with a biotyping system, with species then correlated by clinical features and antimicrobial susceptibility. Of 145 isolates, 102 (70%) were Staphylococcus epidermidis, 24 (17%) were Staphylococcus saprophyticus, 7 (4.7%) were Staphylococcus haemolyticus, 4 (2.8%) were Staphylococcus hominis, 3 (2.1%) were Staphylococcus simulans, and 5 (3.4%) were other species. Features characterizing persons with bacteriuria with S. saprophyticus compared with bacteriuria with any other species included female sex (95% versus 52%), young age (median age, 22 years versus 61 years), ambulatory status (hospital outpatients, 86% versus 23%), and absence of indwelling catheters (4.5% versus 49%). All other coagulase-negative staphylococci were isolated in a setting suggesting nosocomial acquisition, were more frequently resistant to common antimicrobial agents (42% multiply resistant versus 4.2% of S. saprophyticus), and were not distinguished by clinical features. Novobiocin susceptibility, with a sensitivity of 100% and specificity of 96%, provided a simple and reliable test for differentiation of S. saprophyticus from other coagulase-negative staphylococci and should be routinely used for urinary tract specimens in the clinical laboratory.     

Clinical comparison of the AutoMicrobic system gram-positive identification card, API Staph-Ident, and conventional methods in the identification of coagulase-negative Staphylococcus spp.

In an effort to rapidly identify coagulase-negative staphylococci (CNS), a clinical comparison was conducted with the AutoMicrobic system Gram-Positive Identification Card (GPI) (Vitek Systems, Inc.), the API Staph-Ident (Analytab Products), and the conventional methods of W. E. Kloos and K. H. Schleifer (W. E. Kloos and K. H. Schleifer, J. Clin. Microbiol. 1:82-88, 1975). CNS isolates tested included 157 from blood and 33 from urine in pure culture at greater than 10(5) CFU/ml. S. epidermidis accounted for 79.6 and 60.6% of the isolates from blood and urine, respectively. S. saprophyticus was the next most frequent urine isolate (27.4%). Other CNS species were isolated from blood and urine specimens with frequencies of less than 5%. Overall, the GPI correctly identified 158 (83.2%) of the 190 CNS, whereas the Staph-Ident identified 124 (65.3%) without further testing. This resulted in the GPI and Staph-Ident correctly identifying 95.9 and 74.5% of the S. epidermidis and 100 and 33% of the S. saprophyticus, respectively. The GPI misidentified 8 (47%) of the S. hominis and S. warneri isolates as S. saprophyticus, indicating the need for novobiocin testing. These data suggest that the GPI is a more definitive method for the rapid identification of S. epidermidis than the Staph-Ident and that both systems require additional testing to identify S. saprophyticus.     

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.     

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.     

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.     

Is resistance to novobiocin a reliable test for confirmation of the identification of Staphylococcus saprophyticus?

Staphylococcus saprophyticus, a coagulase-negative staphylococcus (CNS), causes acute urinary tract infection predominantly in young women (15-30 years). In the clinical microbiology laboratory identification and differentiation of S. saprophyticus from other CNS usually depends solely upon the demonstration of resistance to the antimicrobial agent novobiocin. Phenotypic characteristics of 36 novobiocin-resistant CNS isolated from the urine of patients with acute urinary tract infections were further analysed and the homogeneity of the isolates assessed. The organisms were speciated by the API STAPH identification system. Twenty-one isolates were S. saprophyticus (p greater than or equal to 97%), and there was one strain each of S. epidermidis, S. hominis and S. simulans (p greater than or equal to 97%). Of the remainder, three isolates were unidentifiable and a further nine had the characteristics associated with more than one species of CNS. Additional tests, including carbohydrate fermentation, antibiotic sensitivity and fluorogenic substrate utilisation, were performed on all isolates. Computer analysis of the results confirmed that testing for resistance to novobiocin selects a heterogeneous group of CNS composed of several different species.     

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.     

Recognition of Staphylococcus saprophyticus in urine cultures by screening colonies for production of phosphatase.

Phenolphthalein diphosphate was incorporated into a primary blood agar medium for use in performing quantitative urine cultures. Phosphatase-negative staphylococci, such as Staphylococcus saprophyticus, were differentiated from phosphatase-positive species, such as Staphylococcus epidermidis, by spot testing colonies on filter paper saturated with 1 N NaOH. Phosphatase-positive colonies turned pink within seconds, and phosphatase-negative colonies showed no color. None of 55 S. saprophyticus isolates showed production of phosphatase on this medium. Of 193 consecutive coagulase-negative staphylococci isolated from the urine of 190 adolescent female patients, 84% were phosphatase positive, non-S. saprophyticus species; 16% were phosphatase-negative and indicated S. saprophyticus (22), Staphylococcus haemolyticus (4), Staphylococcus simulans (2), Staphylococcus warneri (1), and Staphylococcus hominis (1). Phosphatase activity was variable in the other flora encountered in the urine cultures. Mixtures of phosphatase-positive and -negative organisms did not cause false-positive reactions.     

Frequency of isolation of Staphylococcus lugdunensis in consecutive urine cultures and relationship to urinary tract infection

Recent reports associate Staphylococcus lugdunensis with severe infection in humans. The frequency of this microorganism in urine cultures is unknown. Five hundred isolates of coagulase-negative staphylococci (CoNS) were recovered from 4,652 consecutive urine specimens submitted for culture to the Mayo Clinic Microbiology Laboratory. Thirty-one (6%) of 500 isolates of CoNS were identified as S. lugdunensis. In no case was S. lugdunensis isolated in pure culture; 29 (94%) of 31 S. lugdunensis isolates were part of mixed nonpathogenic flora. Medical records were reviewed for 30 of the 31 patients from whom these 31 isolates were isolated. Twenty-one (70%) of the 30 evaluable patients were not treated with antibiotics; the remaining 9 (30%) of 30 patients were treated with antibiotics that may be effective against S. lugdunensis. S. lugdunensis may be an unrecognized yet infrequent cause of urinary tract infection.     

Evaluation of RapiDEC Staph for identification of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus.

RapiDEC Staph is a test for presumptive identification of the principal human staphylococcal species, Staphylococcus aureus, S. epidermidis, and S. saprophyticus. The test includes control and test cupules for fluorogenic detection of coagulase and chromogenic substrates for alkaline phosphatase and beta-galactosidase. These tests identify S. aureus, S. epidermidis, and S. saprophyticus, respectively. Positive results with both chromogenic substrates provide a presumptive identification of S. xylosus or S. intermedius (S. xylosus-S. intermedius). Test cupules are inoculated with an organism suspension, and reactions are read after a 2-h incubation. RapiDEC-Staph was evaluated with 303 clinical and stock staphylococcal strains. Identifications were compared with those obtained by the tube coagulase test, a latex slide coagulase test (StaphAUREX), another commercial identification system (Staph-TRAC), and additional conventional tests. RapiDEC-Staph correctly identified 100% of 130 S. aureus strains, 70.3% of 74 S. epidermidis strains, and 81.3% of 32 S. saprophyticus strains. Four of five S. xylosus isolates were called S. xylosus-S. intermedius. Unidentified S. epidermidis and S. saprophyticus strains were called "Staphylococcus spp." Among the 62 other coagulase-negative staphylococci, 4 were misidentified as S. epidermidis and 7 were misidentified as S. saprophyticus. While the sensitivity and specificity of the fluorogenic coagulase test for S. aureus were 100%, failure to detect alkaline phosphatase activity in several S. epidermidis isolates resulted in fewer correct identifications by the RapiDEC-Staph test for this species.     

Comparative performances of six agglutination kits assessed by using typical and atypical strains of Staphylococcus aureus.

Six commercial agglutination tests designed for the identification of Staphylococcus aureus were compared by using a strain collection which included 512 staphylococci representing 33 species (318 isolates of Staphylococcus aureus [including 144 oxacillin resistant], 46 S. epidermidis isolates, 15 S. haemolyticus isolates, 12 S. saprophyticus isolates, 29 S. schleiferi isolates, 30 S. lugdunensis isolates, and 62 other coagulase-negative staphylococci). This group also included a proportion of strains with unusual phenotypes (e.g., 19 coagulase-negative S. aureus isolates, 26 clumping factor-negative S. aureus isolates, and 4 S. aureus isolates each with a double deficiency). The overall sensitivity for identification of typical and atypical S. aureus was high with the Staphaurex Plus test (Murex Biotech) (99.7%), the Pastorex Staph Plus test (Sanofi Diagnostics Pasteur) (99.7%), and the Slidex Staph Plus test (bioMérieux) (100%). The overall rate of specificity was affected by the unusual inclusion in this study of a high proportion of non-S. aureus species, such as S. lugdunensis and S. schleiferi, which express a clumping factor and therefore produce a positive result with the agglutination tests.     

Growth of coagulase-negative staphylococci on colistin-nalidixic acid agar and susceptibility to polymyxins.

Colistin-nalidixic acid agar, although recently recommended as a replacement for blood agar for primary plating of urine specimens ( Fung et al., J. Clin. Microbiol. 16:632-636, 1982), has also been reported to suppress the growth of some strains of staphylococci that are susceptible to colistin (polymyxin E). The susceptibility of 11 species of staphylococci to polymyxins was determined, and the ability of these species to grow on colistin-nalidixic acid agar was examined. Although the MICs for most of the strains tested were 8 micrograms/ml or less, only a few coagulase-negative staphylococci grew on or were inhibited by colistin-nalidixic acid agar. This descrepancy was explained by the antagonistic effects that medium components, such as physiological concentrations of magnesium and calcium and 5% sheep blood, had on the activity of polymyxin. Colistin-nalidixic acid agar is still recommended for routine urine processing; however, the poor growth of 13% of the Staphylococcus saprophyticus strains tested suggests that blood agar should be included in the primary plating battery of urine specimens obtained from female outpatients.     

Colonization of the female genital tract with Staphylococcus saprophyticus.

The prevalence of colonization by Staphylococcus saprophyticus of the urogenital tracts of 276 women from an outpatient gynecology practice was determined by using selective and enrichment culture techniques. Nineteen subjects (6.9%) were found to be colonized by S. saprophyticus. The rectum was the most frequent site of colonization and was responsible for 40% of the isolates; this was followed in decreasing order by the urethra, urine, and cervix. Women colonized by S. saprophyticus were more likely to have experienced a urinary tract infection in the previous 12 months (P = 0.058; odds ratio, 2.844; 95% confidence interval, 1.054 to 7.671). Patients colonized by S. saprophyticus tended to have had their menstrual periods more recently (P = 0.066), experienced sexual intercourse more recently (P = 0.168), and had a recent or concurrent diagnosis of vaginal candidiasis (P = 0.111; odds ratio, 2.393; 95% confidence interval, 0.877 to 6.528). A seasonal variation in colonization was observed, with colonization most likely occurring during the summer and fall. Follow-up for an average of 6.75 months failed to document any colonized woman progressing to symptomatic urinary tract infection. In addition, 21 women colonized by non-S. saprophyticus, novobiocin-resistant, coagulase-negative staphylococci were identified and characterized.     

Clinical Evaluation of a Novel Chromogenic Agar Dipslide for Diagnosis of Urinary Tract Infections

 The performance of a novel dipslide (DipStreak; Novamed, Israel) consisting of chromogenic agar (Uriselect 3; Sanofi Pasteur, France) and blood agar media was evaluated prospectively and compared to that of conventional urine culture for the diagnosis of urinary tract infection. A total of 1070 clean-catch urine specimens obtained from 251 hospitalized patients and 819 outpatients were processed. The overall performance of the DipStreak was as follows: sensitivity, 95.7%; specificity, 99.2%; agreement, 89.8%; accuracy, 98%; positive predictive value, 98.5%; and negative predictive value, 97.7%. A total of 270 urine specimens were positive by both DipStreak and conventional culture. The chromogenic agar allowed rapid identification of organisms in 211 (78.1%) cultures, while isolates in the other 59 (21.9%) cultures remained unidentified. The results indicate that the DipStreak device coupled with the Uriselect 3 agar represents a convenient and accurate method for inoculation of urine specimens, quantitation of bacteria, diagnosis of significant bacteriuria, and presumptive identification of isolates.     

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.     

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.     

Phosphatase-novobiocin-mannose-inhibition test (PNMI-test) for routine identification of the coagulase-negative staphylococcal urinary tract pathogens S. epidermidis and S. saprophyticus

A modified Kloos/Schleifer-scheme proved to be useful in identifying coagulase-negative staphylococci isolated from urine. S. epidermidis (44.2%) and S. saprophyticus (21.5%) were the most frequent species. Analysis of patients confirmed both species as urinary pathogens. Using an abbreviated scheme of 6 characteristics, S. saprophyticus was mis-classified in 19.5% of cases. A Phosphatase-Novobiocin-Mannose-Inhibition Test (PNMI-Test) together with a high NaCl concentration (10%) in combination with a coagulase test seems to be an acceptable compromise for routine identification of the three most important staphylococcal urinary tract pathogens, S. aureus, S. epidermidis, and S. saprophyticus. The technical and financial expenditure can be reduced considerably, because an extended identification has to be applied only to strains which cannot be identified by the PNMI-Test.     

Slime production by bovine milk Staphylococcus aureus and identification of coagulase-negative staphylococcal isolates.

Staphylococcus aureus isolates from bovine milk were assessed for capsule or slime production. When pure S. aureus cultures in milk were inoculated directly into serum-soft agar constituted with a modified staphylococcus 110 medium, 100% of the isolates grew with diffuse colony morphology. Diffuse colony morphology was rapidly lost on subculture and was more rapidly lost in brain heart infusion-serum-soft agar. No evidence was seen for encapsulation in India ink preparations or by the clumping factor test. It was concluded that freshly isolated S. aureus strains produce slime, not true capsules. During examination of the 84 milk samples that grew staphylococci in addition to S. aureus (27.4%), a significant number of coagulase-negative staphylococcal species were encountered and identified by conventional tests as S. simulans (41.7%), S. xylosus (11.9%), S. epidermidis (3.6%), S. saprophyticus (3.6%), S. hyicus (2.9%), S. cohnii (1.2%), S. haemolyticus (1.2%), and S. warneri (1.2%). Five isolates (6.0%) were not identified. Attempts were also made to identify the isolates by the API Staph-Ident system, which gave an overall accuracy of 45.2%. The susceptibilities of the isolates to a variety of antibiotics were determined, and they appeared to be less resistant than human clinical isolates.     

Occurrence of Staphylococcus lugdunensis in consecutive clinical cultures and relationship of isolation to infection.

Consecutive record review over a 63-month period revealed 229 Staphylococcus lugdunensis isolates, or 10.1% of the staphylococcal species that were not Staphylococcus aureus or Staphylococcus epidermidis. A total of 155 S. lugdunensis specimens were isolated from sites over the entire bodies of the 143 patients studied. The most common clinical diagnoses were skin and skin structure infections (55.4%) and blood and vascular catheter infections (17.4%). For 40% of the reviewed specimens, S. lugdunensis was the sole agent isolated, and for 60% of specimens, S. lugdunensis was isolated as part of mixed flora. In only 15.4% of clinically reviewed specimens was S. lugdunensis clearly a culture contaminant or colonizing organism. The pattern of human infection identified in this study emphasizes the predominance of skin and soft tissue S. lugdunensis infections over deep serious infections such as endocarditis, peritonitis, infected hip prosthesis, and osteomyelitis and vascular-associated infections. S. lugdunensis should be included along with S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus saprophyticus as a coagulase-negative species of Staphylococcus pathogenic for humans.     

Clinical significance of coagulase-negative staphylococci.

Although coagulase-negative staphylococci (C-NS) have been implicated in certain human infections, they are generally regarded as contaminants, and their clinical significance is questioned. To assess their role as pathogens, we studied 205 isolates of C-NS from wounds and body fluids (blood, urine, pleural and peritoneal fluids, etc.). Patient's charts were reviewed, and, by using strict criteria, a determination was made regarding the clinical significance of these isolates. The organisms were then identified to determine whether certain species of C-NS were associated with specific infections. S epidermidis sensu stricto accounted for 81% of the C-NS isolated. The frequencies of other species were: S. haemolyticus (6%), S. hominis (5%), S. capitis (4%), S. warneri (3%), and others (1%). Only two isolates were novobiocin resistant; neither was identified as S. saprophyticus. By using our criteria, 22% of the C-NS were considered to be clinically significant, and the majority of these (93%) was S. epidermidis. The most common source of the clinically relevant C-NS isolates was wounds. These data suggest that identification of C-NS species other than S. epidermidis may be of limited value in predicting clinical significance.