Species-specific sensitivity of coagulase-negative Staphylococci to single antibiotics and their combinations

The activity of beta-lactam antibiotics (oxacillin, cloxacillin, cephalotin), vancomycin, gentamicin and rifampicin applied in vitro individually and in combination against 37 nosocomial methicillin-resistant strains of coagulase-negative staphylococci (CNS) was assessed to demonstrate the heterogeneity of this group of bacteria and estimate the chance of the efficacy of such therapy. The strains belonged to four species: Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus cohnii, Staphylococcus hominis. They originated from a hospital environment and from the skin of medical staff of the intensive care unit of a paediatric ward at a university hospital. All strains were methicillin-resistant, according to CLSI standards, but individual strains differed in MIC(ox) values. Susceptibility to other tested antibiotics was also characteristic for the species. The increased susceptibility to antibiotics in combinations, tested by calculating the fractional inhibitory concentration (FIC) index, concerned 26 out of 37 investigated strains and it was a feature of a particular species. Combinations of vancomycin and cephalotin against S. epidermidis and oxacillin with vancomycin were significant, as well as cephalotin and rifampicin in growth inhibition of multiresistant S. haemolyticus strains.     

The amino acid sequences and activities of synergistic hemolysins from Staphylococcus cohnii

Staphylococcus cohnii ssp. cohnii and S. cohnii ssp. urealyticus are a coagulase-negative staphylococci considered for a long time as unable to cause infections. This situation changed recently and pathogenic strains of these bacteria were isolated from hospital environments, patients and medical staff. Most of the isolated strains were resistant to many antibiotics. The present work describes isolation and characterization of several synergistic peptide hemolysins produced by these bacteria and acting as virulence factors responsible for hemolytic and cytotoxic activities. Amino acid sequences of respective hemolysins from S. cohnii ssp. cohnii (named as H1C, H2C and H3C) and S. cohnii ssp. urealyticus (H1U, H2U and H3U) were identical. Peptides H1 and H3 possessed significant amino acid homology to three synergistic hemolysins secreted by Staphylococcus lugdunensis and to putative antibacterial peptide produced by Staphylococcus saprophyticus ssp. saprophyticus. On the other hand, hemolysin H2 had a unique sequence. All isolated peptides lysed red cells from different mammalian species and exerted a cytotoxic effect on human fibroblasts.     

Coagulase-Negative,Novobiocin-Resistant Staphylococci on the Skin of Animals and Man,on Meat and in Milk

The occurrence of coagulase-negative, novobiocin-resistant staphylococci, i.e. Staphylococcus cohnii, Staphylococcus saprophyticus, Staphylococcus sciuri and Staphylococcus xylosus, on the skin of animals and man has been studied. On cultures from cats, cows, dogs, guinea pigs, mice, rabbits and sheep studied, such organisms were predominant among the coagulase-negative staphylococci. From the skin of the hands of 21 of 38 persons whose professions brought them into contact with animals, e.g. inséminât ors, slaughterhouse workers and veterinarians, coagulase-negative, novobiocin-resistant staphylococci were isolated. This finding contrasted with that regarding 50 persons lacking such contacts, of whom only 1 harboured such bacteria. S. saprophyticus was isolated only from those slaughterers presenting with wounds on their hands. Coagulase-negative, novobiocin-resistant staphylococci were also isolated from every second specimen collected from the surface of meat at a slaughterhouse. No difference in the culture results could be demonstrated from specimens collected before and after cutting-up of the carcasses. Of 26 strains of coagulase-negative, DNase-negative staphylococci isolated from milk with pathological CMT, all but 5 were novobiocin-resistant. Fifteen were classified as S. xylosus, 4 as S. sciuri and 1 as S. cohnii. Of another 15 DNase-positive strains, 3 were resistant to novobiocin. Finally, clinical infections with coagulase-negative, novobiocin-resistant staphylococci in man, e.g. urinary tract infections caused by S. saprophyticus, are considered in relation to possible contagious reservoirs and modes of spread.     

Identification of coagulase-negative staphylococci from farm animals

The species identity of 661 strains of coagulase-negative staphylococci isolated from the skin and nares of cattle, pigs, poultry, goats and sheep was determined. They belonged either to the novobiocin-sensitive species Staphylococcus hyicus, Staph. simulans, Staph. epidermidis, Staph. haemolyticus and Staph. warneri or to the novobiocin-resistant species Staph. sciuri, Staph. lentus, Staph. xylosus, Staph. cohnii. Staph. saprophyticus and Staph. gallinarum ; twenty-one strains remained unidentified. The staphylococcal flora of the farm animals studied differed markedly from that associated with man; several species which do not occur in man were isolated and novobiocin-resistant strains, which occur infrequently in man, were present in large numbers in animals. Two simplified schemes for the identification of staphylococci from farm animals and man are presented.     

Identification of coagulase-negative staphylococci from farm animals

The species identify of 661 strains of coagulase-negative staphylococci isolated from the skin and nares of cattle, pigs, poultry, goats and sheep was determined. They belonged either to the novobiocin-sensitive species Staphylococcus hyicus, Staph. simulans, Staph. epidermidis, Staph. haemolyticus and Staph. warneri or to the novobiocin-resistant species Staph. sciuri, Staph. lentus, Staph. xylosus, Staph. cohnii, Staph. saprophyticus and Staph. gallinarum; twenty-one strains remained unidentified. The staphylococcal flora of the farm animals studied differed markedly from that associated with man; several species which do not occur in man were isolated and novobiocin-resistant strains, which occur infrequently in man, were present in large numbers in animals. Two simplified schemes for the identification of staphylococci from farm animals and man are presented.     

Antimicrobial susceptibility of staphylococci isolated from otitis externa in dogs

Samples were obtained from 65 unmedicated adult dogs, processed for isolation of Staphylococcus species and tested for susceptibility to penicillin G, gentamicin, oxacillin, tetracycline, trimethoprim-sulphamethoxazole, streptomycin, ampicillin and rifampin. Forty-four isolates were obtained, which represents 67.7% of samples. Coagulase-negative species were most commonly found, and the most frequently isolated staphylococcus species were Staph. epidermidis and Staph. aureus. Other species, such as Staph. simulans, Staph. haemolyticus, Staph. saprophyticus and Staph. intermedius were also isolated. Resistance to antibiotics was frequently observed, with 90.9% of the isolates showing resistance to at least one drug. The most active antimicrobial agents against staphylococci isolated from otitis externa of dogs were rifampin and oxacillin. Multidrug resistance was a common finding, and one strain of Staph. haemolyticus species, was resistant to all tested antimicrobial agents. Resistance to three or more different drugs was a common finding, observed in 16 strains (36.4%) of both coagulase-positive and coagulase-negative staphylococci. This study highlights the emergence of cases of otitis externa determined by coagulase-negative staphylococcus strains and once more emphasizes the need for bacterial culture with species identification and susceptibility testing of swab specimens from the ear canal in order to choose appropriate antimicrobial agents.     

Epidemiological investigation of an animal house based upon phage-typing and biotyping of Staphylococcus epidermidis.

The value of biotyping and phage-typing coagulase-negative staphylococci in the epidemiological investigation of a laboratory animal house was clearly demonstrated. In the animal rooms in which conventional bacteriological methods revealed equal bacterial contamination between a conventional unit and one housing specified-pathogen-free rodents, biotyping identified Staphylococcus cohnii as the only species in the latter, compared to S. warneri, S. hominis, S. saprophyticus. S. xylosus abd S. epidermidis as well as S. cohnii in the conventional unit. Similarly, phagetyping revealed 2 phage types in the specified-pathogen-free compared to 7 in the conventional unit. Thus biotyping and phage-typing provided evidence for the existence of a barrier between these units that had presented similar gross bacteriological findings.     

Susceptibility of staphylococci to natural and synthetic iron chelators

A total of 237 strains of staphylococci belonging to 27 species were tested for susceptibility to natural and synthetic iron-chelators. Coagulase-negative staphylococci were much more susceptible than coagulase-positive ones: 82 out of 148 coagulase-negative strains were susceptible to desferrioxamine B, rhodotorulic acid and ovotransferrin and 7 out of 89 coagulase-positive strains. A correlation between the susceptibility to iron-chelators species affiliation and the origin of strain was not found.     

Cross protection between a strain of Staphylococcus epidermidis and eight other species of coagulase-negative staphylococci

Passive protective activity of rabbit antiserum prepared by a representative capsular type II strain of Staphylococcus epidermidis in mice was absorbed out with homologous capsular type strains of S. simulans, S. cohnii, S. xylosus, S. hominis, S. capitis, S. hyicus, S. haemolyticus, and S. saprophyticus in addition to the homologous strain. The minimum amount of the strains required for absorption differed greatly, depending upon the strain. No absorption of the activity was shown with a strain of capsular type I and III of S. epidermidis, S. simulans, and S. cohnii, and a strain of capsular type III of S. hominis. These results suggest a possible capsular type specificity in the cross protection between strains of S. epidermidis and other species of coagulase-negative staphylococci.     

Plasmids of<Emphasis Type="Italic">Staphylococcus cohnii</Emphasis> isolated from the intensive-care unit

Numerous isolates of both subspecies of Staphylococcus cohnii were found in the environment of the intensive-care unit of a pediatric hospital. These isolates carried in their cells many plasmids, up to fourteen, of a wide range of sizes (< 2 to > 56 kb). Striking was the occurrence of large plasmids not very common in staphylococci. These were present in > 80% of S. cohnii isolates. Fifty-two different plasmid profiles were found in 79 investigated isolates belonging to S. cohnii ssp. cohnii and S. cohnii ssp. urealyticus. Isolates similar in plasmid profiles were grouped in antibiotic-resistance clusters established for 9 antibiotics (gentamicin, ciprofloxacin, clindamycin, erythromycin, tetracycline, chloramphenicol, mupirocin, trimethoprim-sulfamethoxazole, vancomycin) using the method of unweighted pair group mathematical averages (UPGMA). Many isolates were multiresistant to antibiotics and produced bacteriocins.     

Antimicrobial sensivity and ability to slime production of koagulaze-negative staphylococci

The aim of this study was to assess the ability of slime production ofcoagulase-negative staphylococci (CONS) and evaluate the susceptibility of bacteria to antibiotics. Strains were isolated from clinical specimens obtained from hospitalized patients. The most frequently isolated species were S. epidermidis (51%), S. hominis (18%), S. haemolyticus (13%). The result of this study shows that 61% of S.epidermidis produce slime on CRA (Congo red agar), whereas none of the tested S. haemolyticus strains has this ability. All examined strains were susceptible to vancomycin, linezolid and quinupristin/ dalfopristin. The majority of strains were susceptible to minocycline, fusid acid, nitrofurantoin and rifampicin. Sixty six percent of isolates were determined as methicillin-resistant coagulase-negative staphylococci.     

In vitro activity of fosfomycin against 'problem' gram-positive cocci.

Fosfomycin was active in vitro against 54 of 60 'problem' Gram-positive cocci (20 methicillin-resistant Staphylococcus aureus, 20 coagulase-negative staphylococci and 20 enterococci). Its activity was significantly greater under anaerobic conditions, especially against coagulase-negative staphylococci. Mutants resistant to fosfomycin were readily demonstrated, but their growth was prevented by rifampicin or ciprofloxacin. The combinations rifampicin+fosfomycin and ciprofloxacin+fosfomycin showed MIC synergy. It is concluded that fosfomycin in an appropriate combination would be a valuable addition to the small and dwindling range of antibiotics active against problem Gram-positive cocci.     

Frequency of the isolation of staphylococci from domestic animals and strain identification

Staphylococci occur in donkeys more frequently than in other animals, and only from donkeys coagulase-negative staphylococci, characteristic of humans (S. hominis, S. capitis, S. cohnii), were isolated. Least frequently staphylococcal carrier state was registered in cats; in these animals only coagulase-negative strains were found to occur. From 30 donkeys coagulase-positive staphylococci belonging to 47 S. aureus strains were isolated. These strains differed from known ecological variants in their biological properties, thus suggesting the existence of S. aureus ecovar specific for donkeys. These strains did not coagulate human, bovine and ovine plasma, but coagulated rabbit plasma in 100% of cases and donkey plasma only in 53% of cases; at the same time they relatively often produced delta hemolysin, rarely phosphatase and hyaluronidase and never fibrinolysin. These strains were typed by KPC phages, mainly 116 and 117.     

Automated systems in the identification and determination of methicillin resistance among coagulase negative staphylococci

Coagulase-negative staphylococci (CoNS) are an important cause of nosocomial bacteremia, specially in patients with indwelling devices or those submitted to invasive medical procedures. The identification of species and the accurate and rapid detection of methicillin resistance are directly dependent on the quality of the identification and susceptibility tests used, either manual or automated. The objective of this study was to evaluate the accuracy of two automated systems--MicroScan and Vitek--in the identification of CoNS species and determination of susceptibility to methicillin, considering as gold standard the biochemical tests and the characterization of the mecA gene by polymerase chain reaction, respectively. MicroScan presented better results in the identification of CoNS species (accuracy of 96.8 vs 78.8%, respectively); isolates from the following species had no precise identification: Staphylococcus haemolyticus, S. simulans, and S. capitis. Both systems were similar in the characterization of methicillin resistance. The higher discrepancies for gene mec detection were observed among species other than S. epidermidis (S. hominis, S. saprophyticus, S. sciuri, S. haemolyticus, S. warneri, S. cohnii), and those with borderline MICs.     

Standardization of salt aggregation test for reproducible determination of cell-surface hydrophobicity with special reference to Staphylococcus species

The laboratory conditions for reproducible routine determination of staphylococcal cell-surface hydrophobicity by the salt aggregation test were standardized. Fresh bacterial suspensions standardized to 5 times 10⁹ cfu/ml gave the most reproducible results with both Staphylococcus aureus and coagulase-negative staphylococci. For relatively hydrophobic strains a 5-min reading time was necessary to detect bacterial aggregation in ammonium sulphate solutions ranging from 0.1 M to 1.5 M, pH 6.8. A × 10 hand lens facilitated reading aggregations. Overnight storage of bacterial suspensions at 20C reduced cell-surface hydrophobicity of all species, while storage at 4C reduced the hydrophobic nature of Staph. aureus strains. The hydrophobicity of coagulase-negative staphylococci rarely changed at 4C. A 10-fold dilution of fresh, standardized bacterial suspensions made it impossible to detect bacterial aggregation in ammonium sulphate solutions even with a hand lens. Under standardized conditions three types of staphylococcal cell aggregations were observed. The first looked like the slide agglutination for O antigens of Enterobacteriaceae, the second resembled H-agglutination, while the third had a filamentous appearance. These patterns indicated that more than one component might contribute to cell-surface hydrophobicity of both Staph. aureus and coagulase-negative staphylococci, or the same component might have different position on the cell surface.     

Standardization of salt aggregation test for reproducible determination of cell-surface hydrophobicity with special reference to Staphylococcus species

The laboratory conditions for reproducible routine determination of staphylococcal cell-surface hydrophobicity by the salt aggregation test were standardized. Fresh bacterial suspensions standardized to 5 x 10(9) cfu/ml gave the most reproducible results with both Staphylococcus aureus and coagulase-negative staphylococci. For relatively hydrophobic strains a 5-min reading time was necessary to detect bacterial aggregation in ammonium sulphate solutions ranging from 0.1 M to 1.5 M, pH 6.8. A x 10 hand lens facilitated reading aggregations. Overnight storage of bacterial suspensions at 20 degrees C reduced cell-surface hydrophobicity of all species, while storage at 4 degrees C reduced the hydrophobic nature of Staph. aureus strains. The hydrophobicity of coagulase-negative staphylococci rarely changed at 4 degrees C. A 10-fold dilution of fresh, standardized bacterial suspensions made it impossible to detect bacterial aggregation in ammonium sulphate solutions even with a hand lens. Under standardized conditions three types of staphylococcal cell aggregations were observed. The first looked like the slide agglutination for O antigens of Enterobacteriaceae, the second resembled H-agglutination, while the third had a filamentous appearance. These patterns indicated that more than one component might contribute to cell-surface hydrophobicity of both Staph. aureus and coagulase-negative staphylococci, or the same component might have different position on the cell surface.     

Gel electrophoretic analysis of penicillin-binding proteins of coagulase negatibve staphylococci

We analyzed gel electrophoretic banding patterns of penicillin-binding proteins (PBPs) of 16 type strains of coagulase-negative staphylococci (CNS). S. epidermidis, S. haemolyticus, S. saprophyticus, S. hominis, S. xylosus, S. simulans, S. warneri, S. capitis, S. saccharolyticus, S. auricularis, S. caseolyticus, S. gallinarum, S. hycus subsp. hycus, S. cohnii, S. caprae, and S. sciuri subsp. sciuri. The PBP profile of each CNS species was found to be unique and was clearly distinguishable from those of the rest of the species. Together with the previous work of other researchers, this study substantiates the applicability of the PBP profile analysis to the identification of clinical CNS strains.     

Platelet aggregation induced by strains of various species of coagulase-negative staphylococci

Major species of coagulase-negative staphylococci (CNS) were tested for their ability to induce platelet aggregation in rabbit platelet-rich plasma (PRP). Among 11 species of CNS tested, a majority of the strains of 10 species of CNS (S. epidermidis, S. simulans, S. capitis, S. hyicus, S. sciuri, S. cohnii, S. xylosus, S. hominis, S. haemolyticus, S. warneri) caused induction of the platelet aggregation and serotonin release, while S. saprophyticus did not show such activity. The addition of aspirin (10 mM) or quinacrine (1 mM) to PRP resulted in no remarkable effect on the platelet aggregation induced by these strains and it was shown that the platelet aggregation did not require arachidonate pathways. Complement system components were shown to be one of the plasma factors required for platelet aggregation by ten strains of each species of CNS. The bacterial substance participating in the platelet aggregation by ten species of CNS tested was indicated to be heat-stable and trypsin-resistant, while the activity of a strain of S. epidermidis was susceptible to trypsin.     

Prevalence and antimicrobial susceptibility of staphylococci isolated from saliva of clinically normal cats

Samples were collected from 150 adult cats, processed for isolation of Staphylococcus species and tested for susceptibility to penicillin G, gentamicin, oxacillin, enrofloxin and tetracycline. Methicillin resistance was also determined. One hundred and four isolates were obtained (69.3% of samples). Coagulase-negative species were most common, and the most frequently isolated (33 samples) species was Staph. felis. Other coagulase-negative species, such as Staph. haemolyticus, Staph. simulans, Staph. epidermidis and Staph. saprophyticus were also isolated. Coagulase-positive staphylococci were obtained from 30 cats, and the only species recovered in this group was Staph. intermedius. Resistance to antibiotics was frequently observed, with 68.2% of the isolates showing resistance to at least one drug. Resistance to Penicillin G was observed in 68 of the 104 isolates (65.4%), 23 samples were resistant to oxacillin (22.1%), 33 to tetracycline (31.7%) and 24 to enrofloxin (23.1%). Gentamicin was the most active antimicrobial agent. The role of these microorganisms in the saliva of cats is discussed.     

Rapid detection of methicillin resistance in teicoplanin-resistant coagulase-negative staphylococci by a penicillin-binding protein 2' latex agglutination method

Thirty-five clinical isolates of coagulase-negative staphylococci with decreased glycopeptide sensitivity were examined by a penicillin-binding protein (PBP2′) latex agglutination (LA) test and were compared to the detection of the mecA gene by PCR, and oxacillin susceptibility determined minimum inhibitory concentrations. The latex test demonstrated high sensitivity and specificity for detecting methicillin resistance in coagulase-negative staphylococci after PBP2′ induction with oxacillin.