Evaluation of the Staph-Zym system with staphylococci isolated from bovine intramammary infections

A total of 148 staphylococci isolated from bovine intramammary infections were used to evaluate the Staph-Zym system (ROSCO, Taastrup, Denmark). The overall accuracy of the system was 91.9%. The system correctly identified all strains of Staphylococcus aureus, Staphylococcus simulans, and Staphylococcus xylosus and 95% of Staphylococcus intermedius strains. Of 33 Staphylococcus hyicus strains, 31 (93.9%) were classified correctly by the Staph-Zym system, as well as 8 (80%) of 10 Staphylococcus chromogenes strains. All 11 Staphylococcus epidermidis strains and the 1 Staphylococcus haemolyticus strain included in the study were identified, but the Staph-Zym system had difficulty distinguishing strains of Staphylococcus warneri and Staphylococcus hominis from other species in the S. epidermidis group. The Staph-Zym system correctly identified all six S. xylosus strains and two of three Staphyloccus sciuri strains. The Staph-Zym system was considered an acceptable alternative to conventional methods for identification of bovine mammary gland isolates.     

Collagen binding, elastase production, and slime production associated with coagulase-negative staphylococci isolated from bovine intramammary infections.

Collagen binding, elastase production, slime production, and associated somatic cell counts were determined with 160 strains of coagulase-negative staphylococci isolated from bovine intramammary infections. Mean binding values for type I and II collagen with Staphylococcus epidermidis, S. chromogenes, and S. hyicus strains were 5.8, 6.6, and 7.4 and 4.3, 4.2, and 4.9%, respectively. Eleven of 28 (39.3%) S. epidermidis, 1 of 38 (2.6%) S. chromogenes, and 1 of 94 (1.1%) S. hyicus strains were elastase positive. Slime production was noted with 12 (42.9%) S. epidermidis, 1 (2.6%) S. chromogenes, and 11 (11.7%) S. hyicus strains. No differences in somatic cell counts were observed with type I or type II collagen binding, elastase production, or slime production with S. epidermidis or S. chromogenes. However, somatic cell counts associated with S. hyicus strains with collagen type I binding affinities of greater than 5 and type II binding affinities of greater than 3 were 320.7 x 10(3) compared with 163.9 x 10(3) for strains with lower binding affinities.     

Synergistic hemolysis associated with coagulase-negative staphylococci isolated from bovine mammary glands.

A total of 353 coagulase-negative staphylococcus (CNS) isolates from infected bovine mammary glands were tested for cytolysin production by using the synergistic hemolysin assay (SHA). Overall, 34.6% of CNS isolates were SHA positive. Human-associated, coagulase-negative staphylococcal species contained the greatest number of SHA-positive strains. Milk leukocyte levels expressed as somatic cell counts (SCC) were elevated with SHA-positive Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus warneri strains. Elevated SCC levels were associated with strains of Staphylococcus hyicus and Staphylococcus chromogenes. However, no difference in SCC levels was observed between SHA-positive and SHA-negative strains. Results indicated that the SHA was a sensitive test for the detection of cytolysin-producing CNS.     

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.     

Staphylococci express a receptor for human transferrin: identification of a 42-kilodalton cell wall transferrin-binding protein.

Staphylococcus aureus and the coagulase-negative staphylococci are commonly responsible for peritonitis in renal patients undergoing continuous ambulatory peritoneal dialysis. To simulate growth conditions in vivo, staphylococci isolated from peritoneal infections were cultured in used human peritoneal dialysate (HPD). Immunoblotting experiments using cell wall preparations from these staphylococci revealed the presence of the host iron-binding glycoprotein transferrin bound to S. aureus, S. epidermidis, S. capitis, S. haemolyticus, and S. hominis but not to S. warneri or S. saprophyticus. Similar results were obtained by incubating broth-grown staphylococci with human transferrin, although, in contrast to S. aureus, the coagulase-negative staphylococci bound more transferrin after growth in iron-restricted broth. To determine whether the staphylococci express a saturable specific receptor for human transferrin, the interaction of human 125I-transferrin with the staphylococci was examined. Both S. aureus and S. epidermidis bound the radiolabelled iron-saturated ligand in a time- and concentration-dependent manner. From competition binding assays, the affinity (Kd) and number of receptors were estimated for S. epidermidis (Kd, 0.27 microM; 4,200 receptors per cell) and S. aureus (Kd, 0.28 microM; 4,200 receptors per cell). S. epidermidis but not S. aureus receptor activity was partially iron regulated. Human apotransferrin and iron-saturated transferrin and rabbit and rat transferrins competed equally well for the staphylococcal receptor. Bovine and porcine transferrins and ovotransferrin as well as human and bovine lactoferrins were much less effective at competing with human transferrin. Treatment of whole staphylococci with protease abolished transferrin binding, indicating the involvement of cell surface protein. Western blots (immunoblots) of cell wall preparations probed with human transferrin revealed the presence of a 42-kDa transferrin-binding protein common to both S. aureus and S. epidermidis. On Western strip blots, the binding of human transferrin to this protein was blocked by labelled human transferrin but not by albumin, immunoglobulin G, or bovine transferrin or ovotransferrin. To assess the conservation of the 42-kDa transferrin-binding protein, cell wall proteins of S. epidermidis, S. haemolyticus, S. capitis, S. hominis, S. warneri, and S. saprophyticus were Western blotted and probed with human transferrin. Only S. warneri and S. saprophyticus lacked the 42-kDa wall protein, consistent with their inability to bind transferrin. These data show that the staphylococci express a specific receptor for human transferrin based at least in part on a common 42-kDa cell wall protein.     

Interaction of coagulase-negative Staphylococcus species with bovine mammary epithelial cells.

Three coagulase-negative Staphylococcus species (CNS) (Staphylococcus epidermidis, Staphylococcus xylosus and Staphylococcus hyicus), from the milk of cows with mastitis, were used to evaluate adherence to and internalization by bovine mammary epithelial cells, and to investigate involvement of host cell signal transduction and host cell cytoskeleton rearrangement on internalization of CNS. S. xylosus showed highest adherence and internalization values of the species evaluated. Host cell cytoskeleton polymerization and protein kinase (PK) phosphorylation were required for internalization of CNS. Both protein kinase C (PKC) and tyrosine kinase (TPK) pathways were involved, but internalization of S. xylosus occurred preferentially through epidermal growth factor TPK activity. S. epidermidis and S. hyicus seemed to exploit other TPK pathways. Results of this study showed that S. xylosus, S. hyicus and S. epidermidis were able to adhere and internalize bovine mammary cells in a process that appeared to be receptor(s) mediated and exploited host signal transduction and cytoskeleton to induce an uptake signal.     

Binding of fibronectin to Staphylococcus strains.

Fibronectin, a major protein component of plasma and loose connective tissue has previously been shown to bind to several strains of Staphylococcus aureus. We examined a large number of strains of different species of Staphylococcus with respect to their ability to bind fibronectin. The relative numbers of strains defined as fibronectin-binders among the different species were as follows: S. aureus (22 of 23), S. haemolyticus (5 of 5), S. warneri (8 of 11), S. hyicus (5 of 6), S. hominis (13 of 17), S. saprophyticus (11 of 20), S. epidermidis (4 of 7), and S. simulans (8 of 10). Only three species showed a predominance of nonbinders over binders: S. capitis (4 of 14), S. xylosus (0 of 4), and S. cohnii (3 of 11). These data indicate that staphylococcal species isolated from soft tissue infections frequently have the ability to bind fibronectin and suggest that the ability to bind to this protein may contribute to the virulence of coagulase-positive and coagulase-negative staphylococci.     

Effect of milk on fibronectin and collagen type I binding to Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis.

Tryptic soy broth (TSB)-grown cells of Staphylococcus aureus isolated from acute and chronic bovine mastitis bound mainly 125I-fibronectin (Fn) [corrected], whereas strains of nine species of coagulase-negative staphylococci showed a predominant interaction with 125I-collagen (Cn) [corrected] type I. A particle agglutination assay (PAA) was used to examine the interaction of coagulase-negative staphylococci with 125I-Fn and 125I-Cn immobilized on latex. All 368 coagulase-negative staphylococci demonstrated high 125I-Cn and moderate to low 125I-Fn interactions in the PAA. Cn-PAA reactivity was high among strains of Staphylococcus xylosus (84.2%), Staphylococcus simulans (77.8%), Staphylococcus epidermidis (76.7%), and Staphylococcus hyicus (74.3%), whereas all six Staphylococcus capitis strains clumped Cn-PAA reagent. Incubating TSB-grown cells in 10% skim milk for 1 h decreased the 125I-Fn- and 125I-Cn-binding affinity in most of the S. aureus and coagulase-negative staphylococci, while growth in 10% skim milk for 18 h resulted in more than 90% decrease or complete loss of interaction with these proteins. Decreased 125I-Fn binding in the presence of milk was correlated with protease production but not with 125I-Cn binding.     

Identification of coagulase-negative staphylococci with the API staph system.

A kit for the identification of staphylococci based on the biochemical criteria proposed by Kloos and Schleifer (W.E. Kloos and K.H. Schleifer, J. Clin. Microbiol., 1:82-88, 1975) is now available commercially. The system was used to identify 100 strains of coagulase-negative staphylococci isolated from various body sites as the primary etiological agent of clinical infection. The increasing importance of staphylococci and their resistance to antibiotics provided the rationale for such an investigation. Over 90% of the Staphylococcus isolates were easily identified as to their species on the basis of their reaction profile to 19 biochemical tests included in the kit. The remainder, which showed minor variations, could also be assigned to the various species. Identification of the isolates was as follows: S. epidermidis, 54; S. haemolyticus, 5; S. simulans, 2; S. hominis, 1; S. capitis, 4; S. cohnii, 2; S. warneri, 2; S. xylosus, 8; and S. saprophyticus, 22. Antibiotic sensitivity patterns were determined for each of the isolates. Novobiocin resistance was detected in strains of S. saprophyticus and S. xylosus, a property hitherto recognized in Micrococcus sp. type 3 causing bacteriuria in young women. Resistance to penicillin was widespread among strains of several species, whereas resistance to tetracycline was mainly confined to strains of S. epidermidis. General resistance to sulfamethoxazole and nalidixic acid was found among all strains, with almost uniform sensitivity to the other drugs tested.     

Correlation of oxacillin MIC with mecA gene carriage in coagulase-negative staphylococci

The National Committee for Clinical Laboratory Standards has recently changed the oxacillin breakpoint from >/=4 mg/liter to >/=0. 5 mg/liter to detect methicillin-resistant coagulase-negative staphylococci (CoNS) because the previous breakpoint lacked sensitivity. To determine the correlation between the new oxacillin breakpoint and the presence of the mecA gene, 493 CoNS of 11 species were tested. The presence of the mecA gene was determined by PCR, and oxacillin susceptibility was determined by the agar dilution method with Mueller-Hinton agar containing 2% NaCl and oxacillin (0. 125 to 4.0 mg/liter). The new breakpoint correctly classified all CoNS strains with mecA as methicillin resistant and strains of Staphylococcus epidermidis, S. haemolyticus, and S. hominis without mecA as methicillin susceptible. The breakpoint of >/=0.5 mg/liter was not specific for S. cohnii, S. lugdunensis, S. saprophyticus, S. warneri, and S. xylosus, in that it categorized 70 of 74 strains of these species without mecA (94.6%) as methicillin resistant. The results of this study indicate that the new oxacillin breakpoint accurately identifies strains of CoNS with mecA but is not specific for strains of certain species of CoNS without mecA.     

Widespread distribution of disinfectant resistance genes among staphylococci of bovine and caprine origin in Norway

We demonstrate here a widespread distribution of genes mediating efflux-based resistance to quaternary ammonium compounds (QACs) in staphylococci from unpasteurized milk from 127 dairy cattle herds and 70 dairy goat herds. QAC resistance genes were identified in 21% of the cattle herds (qacA/B, smr, qacG, and qacJ) and in 10% of the goat herds (qacA/B and smr). Further examination of 42 QAC-resistant bovine and caprine isolates revealed the following genes: qacA/B (12 isolates) was present in four different species of coagulase-negative staphylococci (CoNS), smr (27 isolates) was detected in eight different CoNS species and in Staphylococcus aureus on a previously reported plasmid (pNVH99), qacG (two isolates) was detected on two plasmids (pST94-like) in Staphylococcus cohnii and Staphylococcus warneri, and qacJ (two isolates) was found in Staphylococcus hominis and Staphylococcus delphini on a plasmid (pNVH01) previously found in equine staphylococci. Isolation of indistinguishable pulsed-field gel electrophoresis (PFGE) CoNS types from tank milk and mammary quarter milk samples in a dairy cattle herd suggested that these QAC-resistant staphylococci were of intramammary origin. Indistinguishable or closely related PFGE types of bovine QAC-resistant CoNS were observed in different herds. One particular bovine S. warneri PFGE type was isolated repeatedly from samples collected during a 30-month period in a herd, showing long-term persistence. In conclusion, it seems that the widespread distribution of staphylococci carrying QAC resistance genes in Norwegian dairy cattle and goat herds is the result of both the intra- and interspecies spread of QAC resistance plasmids and the clonal spread of QAC-resistant strains.     

Identification of clinical staphylococcal isolates from humans by internal transcribed spacer PCR

The emergence of coagulase-negative staphylococci not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was used to identify a collection of 617 clinical staphylococcal isolates. The amplicons were resolved in high-resolution agarose gels and visually compared with the patterns obtained for the control strains of 29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%) were identified by ITS-PCR and included 11 species: 302 isolates of Staphylococcus epidermidis, 157 of S. haemolyticus, 79 of S. aureus, 21 of S. hominis, 14 of S. saprophyticus, 8 of S. warneri, 6 of S. simulans, 2 of S. lugdunensis, and 1 each of S. caprae, S. carnosus, and S. cohnii. All species analyzed had unique ITS-PCR patterns, although some were very similar, namely, the group S. saprophyticus, S. cohnii, S. gallinarum, S. xylosus, S. lentus, S. equorum, and S. chromogenes, the pair S. schleiferi and S. vitulus, and the pair S. piscifermentans and S. carnosus. Four species, S. aureus, S. caprae, S. haemolyticus, and S. lugdunensis, showed polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a valuable alternative for the identification of staphylococci, offering, within the same response time and at lower cost, higher reliability than the currently available commercial systems.     

Synergistic hemolysis exhibited by species of staphylococci.

The synergistic hemolysis reactions of 61 reference strains and 189 clinical isolates representing 17 species of staphylococci were examined on plates of Trypticase soy blood agar (BBL Microbiology Systems, Cockeysville, Md.). Some or all of the strains of Staphylococcus aureus, S. epidermidis, S. capitis, S. cohnii, S. haemolyticus, S. hyicus, S. simulans, S. warneri, and S. xylosus produced a delta-hemolysin that gave synergistic, complete hemolysis of washed human, sheep, and ox blood cells in an area of beta-lysin activity from strains of S. aureus and S. intermedius. Strains of the same nine species were positive with a commercial beta-lysin paper disk designed for presumptive identification of group B streptococci; most of these strains also gave synergistic, complete hemolysis with exotoxin from a strain of Corynebacterium pseudotuberculosis. None of the strains of S. auricularis, S. carnosus, S. caseolyticus, S. hominis, S. intermedius, S. saprophyticus, S. sciuri, or S. lentus were positive by any of these tests for synergistic hemolysis. These results indicate that a synergistic hemolysis test could prove very useful for differentiating these species; they also suggest that one role of some of these organisms in human infections could be that of a synergist. Further studies of synergism may clarify the clinical significance of these results.     

Comparison of the MicroScan system with the API Staph-Ident system for species identification of coagulase-negative staphylococci.

To evaluate the accuracy of the MicroScan System (American Hospital Supply Corp., Sacramento, Calif.) for identification of coagulase-negative staphylococci, we tested 175 clinical isolates of coagulase-negative staphylococci. The results obtained by the MicroScan system were compared with those of the API Staph-Ident system (Analytab Products, Plainview, N.Y.). Forty-three discrepancies between the two systems were resolved by the conventional method of Kloos and Schleifer (W.E. Kloos and K.H. Schleifer, J. Clin. Microbiol. 1:82-88, 1975). The MicroScan and the Staph-Ident systems correctly identified 146 (86.4%) and 154 (88%) of 175 strains, respectively. The API system failed to identify phosphatase-negative Staphylococcus epidermidis. The MicroScan system demonstrated the greatest accuracy in the identification of S. epidermidis and S. saprophyticus, whereas lesser accuracy was achieved with S. hominis, S. warneri, and S. sciuri.     

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.     

Evaluation of the Staph-Ident and STAPHase systems for identification of staphylococci from bovine intramammary infections.

The Staph-Ident and STAPHase systems (Analytab Products, Plainview, N.Y.) were compared with conventional methods for identification of staphylococci isolated from bovine intramammary infections. Adjunct testing by colony morphology, pigmentation, and biochemical tests was conducted to resolve discrepant identifications. The initial accuracies of the conventional scheme and Staph-Ident were 92.1 and 89.2%, respectively. Staphylococcus hyicus subsp. chromogenes could not be identified by means of the Staph-Ident test, but the addition of pigment production as a key character permitted identification of most strains. The final accuracy of the Staph-Ident was 94.3%. The STAPHase system was as accurate as the conventional tube coagulase method. The Staph-Ident and STAPHase systems are acceptable alternatives to conventional methods for identification of staphylococcal species isolated from bovine intramammary infections.     

Sensitivity of 858 strains of staphylococci to 27 antibiotics

Susceptibility to 27 antimicrobial agents of 858 strains of staphylococci was determined. Tested strains belonged to the following species: S. epidermidis, S. saprophyticus, S. haemolyticus, S. hominis, S. simulans, S. warneri, S. cohnii, S. xylosus and S. intermedius. The antibiotics were: penicillin G, amoxycillin, augmentin, oxacillin, streptomycin, kanamycin, tobramycin, dibekacin, amikacin, gentamicin, sisomycin, netilmicin, doxycycline, minocycline, chloramphenicol, erythromycin, josamycin, clindamycin, pristinamycin, rifampin, fusidic acid, fosfomycin, trimethoprim, sulfamethoxazole, cotrimoxazole, and vancomycin. The ATB system was used, with the criteria for categorization recommended by the Antibiotic Sensitivity Testing Committee. Penicillin-resistance, that was found in all species, was high for hospital-acquired strains (67 to 75%) but also for some other strains (32% for S. simulans). Oxacillin-resistance varied across species (0% for the least prevalent hospital strains, 6% for S. epidermidis and 28% for S. haemolyticus). All strains were susceptible to vancomycin. For some drugs, resistance was a characteristic of the species: resistance to fosfomycin was often found for S. saprophyticus, S. haemolyticus, S. warneri, S. cohnii, and S. capitis; resistance to trimethoprim was common for S. simulans, and S. haemolyticus. S. haemolyticus was the most resistant species, a fact that justifies routine identification of this pathogen in clinical specimens.     

Characteristics of coagulase-negative staphylococci that help differentiate these species and other members of the family Micrococcaceae.

One hundred reference strains and 1,240 clinical isolates representing 26 species of the family Micrococcaceae were used to evaluate the potential of tests for synergistic hemolysis, adherence to glass, pyroglutamyl-beta-naphthylamide hydrolysis, and susceptibility to a set of five antimicrobial agents for differentiating these species and strains within the species. Sixty-eight percent of the clinical isolates exhibited synergistic hemolysis; 69% of the clinical staphylococci but none of the micrococci or stomatococci were adherence positive, and 92% of the strong positive adherence reactions were produced by strains of Staphylococcus epidermidis. Strains from 15 of the species were pyroglutamyl-beta-naphthylamide positive, but this test separated Staphylococcus xylosus from other novobiocin-resistant staphylococci and Staphylococcus intermedius from other coagulase-positive species. A polymyxin B disk helped differentiate S. epidermidis from most other coagulase-negative staphylococci, and a bacitracin disk (10 U) helped differentiate Staphylococcus haemolyticus from most other novobiocin-susceptible staphylococci. All strains that were susceptible to furazolidone and resistant to Taxo A disks (bacitracin, 0.04 U; BBL Microbiology Systems, Cockeysville, Md.) were staphylococci. We observed a 91% correlation between species identification obtained with the Staph-Ident system (Analytab Products, Plainview, N.Y.) and conventional methods; but the micrococci and stomatococci were incorrectly identified as staphylococci with Staph-Ident, and several isolates of S. epidermidis were misidentified as Staphylococcus hominis because they were alkaline phosphatase negative. Both these problems can be prevented by adding the simple tests we describe to those already recommended when the Staph-Ident system is used to identify isolates of gram-positive, catalase-positive cocci.     

Enterotoxin and toxic shock syndrome toxin-one production by staphylococci isolated from mastitis in sheep.

From 160 staphylococci isolated from ovine mastitis, 125 were identified as coagulase-positive staphylococci (CPS) and 35 as coagulase-negative staphylococci (CNS). Of these, 108 (87.8%) S. aureus produced at least one of the staphylococcal enterotoxins (SE) described. However, no CNS was found to be enterotoxigenic. Enterotoxin C (SEC) was the type most frequently produced. TSST-1 was shown to be produced by 91 (74.0%) of S. aureus, almost invariably in combination with SEC. Three CNS strains were also found to produce TSST-1 (two strains of S. xylosus and one strain of S. epidermidis).