Characteristics of the staphylococci isolated from the udder of cows with mastitis

A total of 175 strains of Staphylococcus aureus and 67 strains of Staphylococcus epidermidis were studied, isolated from 486 samples of milk secretion taken aceptically from the individual quarters of the udder of cows affected with subclinical and purulent (clinical) mastitis. The staphylococci were referred to as the causative agent of mastitis in case they were the only microflora in the seedings of the investigated material. Tests were applied as given in Fig. 1 to characterize the strains. It was found that mastitis in cows could be due to both plasma coagulating staphylococci (Staphylococcus aureus) and coagulase-negative Staphylococcus epidermidis organisms. The two Staphylococcus species were isolated from cows with clinical and subclinical mastitis. The division between pathogenic and nonpathogenic Staphylococcus strains by the plasma-coagulating symptom proved impossible, and this made it necessary to use other tests for pathogenicity. It became evident that the thing Staph. aureus and Staph. epidermidis had in common when isolated from cows with mastitis was the production of a gold-like pigment and delta hemolysin. Similarly to Staph. aureus isolated animals, the bovine Staph. epidermidis organisms did not possess fibrinolysin and rarely produced hemolysin. The isolated organisms belonging to the coagulase-positive staphylococci corresponded by their basic properties to Staphylococcus aureus var. bovis as described in the literature. The cultures of Staphylococcus epidermidis isolated under similar conditions showed in a considerable per cent of the cases somewhat different behaviour.     

Antibiotic sensitivity of various Staphylococcus species isolated from newborn infants with purulent septic infections

Sensitivity of 120 staphylococcal strains belonging to different species isolated from newborn infants with pyoseptic infections was studied with respect to 13 antibiotics. Methicillin, cephaloridin, rifampicin, ristomycin, gentamicin and novobiocin were mainly highly active against the isolates, while some of the strains were resistant. The coagulase negative species were as a whole characterized by higher resistance levels with respect to some antibiotics, as compared to Staph. aureus. The predominant part of the coagulase negative strains with exceedingly high resistance levels did not belong to Staph. epidermidis. The sensitivity of staphylococci depended on the source of their isolation. At large the strains isolated from purulent excretions of localized foci were characterized by lower levels of resistance to a number of antibiotics (benzylpenicillin, tetracycline, erythromycin, methicillin and others) as compared to the strains isolated from the blood.     

Incidence of nasal carriers of Staphylococcus aureus in and outside hospital environment and antibiotic sensitivity of isolated staphylococcus strains

This study was carried out on 100 nasal swabs collected from medical personnel (nurses and doctors) and patients inside hospital environment and also from 50 individuals outside hospital. The swabs were inoculated on different culture media for isolation of /staphylococci which were further identified as S. aureus either by classic bacteriologic methods or by one of rapid screening test of S. aureus. The isolated strains were tested for antibiotic sensitivity to some of B-Lactam antibiotics and to other antibiotics. The results showed that significantly higher percentage of coagulase + ve Staph. were isolated from newborn nursery (90%), operating theatre (71.4%) and hemodialysis unit (60%) than those isolated from intensive care unit, cancer chemotherapy, surgery, chest, internal medicine departments (25%, 26.6%, 31.2%, 33.3%, 50%) respectively. It also showed significant difference in isolation rate between persons at the hospital (patients, doctors and nurses) 44% and controls (normal population) 26%. Most isolates of coagulase + ve Staph. were resistant to penicillin G (93.2%), Streptomycin (77.3%), tetracycline (61.4%) and sensitive to cefamandole (95.4%). All coagulase+ve Staph. isolates were resistant to sulphonamide and methicillin and all sensitive to vancomycin.     

Staphylococci outside the hospital. Staphylococcus aureus in sheep

Biochemical properties were studied in Staph. aureus strains obtained from the anterior nares of healthy sheep and from the udders of ewes suffering from purulent mastitis. Of the total number of 84 isolated staphylococcal strains 75 (89.3%) were classified as the C biotype. These undoubtedly sheep-adapted staphylococci produced pigment and beta hemolysin, they were growing on crystal violet agar as the negative type in violet colonies lacking both fibrinolysin and alpha hemolysin. All of them coagulated human plasma within one hour after inoculation. In bovine plasma 27 strains (36%) formed the coagulum within 3 hours, 16 (21.3%) within 24 hours, and the remaining 32 strains (42.7%) only within 72 hours. Mannitol was fermented after five days only by 33 cultures (44%). The staphylococci were sensitive to the applied antibiotics without exception. All these sheep-adapted staphylococci had analogous biochemical features to the earlier discussed staphylococcal strains obtained by the authors from the nasal cavities of cattle. Next two strains were denoted as deficit variants of the C biotype because of their lack of pigment. Of quite a different character were 3 strains (3.6%) of the A biotype and one strain identified as the E biotype. The former were presumably transferred to sheep from man while the latter from a dog. The remaining 3 strains could not be subdivided according to the classificatory criteria used here.     

Effect of US signal value on blocking of a CS-US association

To evaluate whether clinical Klebsiella isolates differ from nonclinical strains with respect to bacteriocin susceptibility patterns, a total of 452 Klebsiella pneumoniae and K. oxytoca strains isolated from different sources were examined. Bacteriocin typing was done by a modification of the scrape-and-point method, using a set of eight producer strains. 96% of the strains were typable. Forty-one different bacteriocin susceptibility patterns were observed. While two thirds of the K. oxytoca isolates belonged to only three different bacteriocin types, the K. pneumoniae strains showed a more heterogeneous distribution of patterns. No differences in pattern distribution were observed between isolates from clinical, fecal, or environmental sources. Certain bacteriocins showed a very broad spectrum of activity; e.g. 93% of all isolates were susceptible to bacteriocin type 3. The results suggest that nonclinical Klebsiella strains do not show other bacteriocin susceptibility types than clinical isolates do.     

In vitro activities of the oxazolidinone antibiotics U-100592 and U-100766 against Staphylococcus aureus and coagulase-negative Staphylococcus species

U-100592 and U-100766 are closely related antibiotics of the oxazolidinone class. Their in vitro activities were determined against 100 isolates of Staphylococcus aureus and 100 isolates of coagulase-negative Staphylococcus species by broth and agar dilution test methods. The MICs of both compounds by either test method at which 50 and 90% of isolates are inhibited were 2 and 4 micrograms/ml, respectively, for S. aureus and 1 to 2 micrograms/ml for coagulase-negative staphylococci. Time-kill assay with selected strains indicated a primarily bacteriostatic effect against staphylococci.     

Production and properties of a staphylococcin genetically controlled by the staphylococcal plasmid for exfoliative toxin synthesis

Previous data from this laboratory showed that certain phage group 2 staphylococci contain a large 56S virulence plasmid containing genes that code for both exfoliative toxin (ET) and a specific staphylococcin. Optimal cultural conditions for bacteriocin production were similar to those found for ET production. The bacteriocin is an extracellular product produced in small quantities that can be neither extracted from cell pellets with 1 M NaCl nor induced with mitomycin C. The staphylococcin is active against a wide variety of gram-positive organisms and also against group 2 staphylococcal strains that have been cured of the plasmid carrying the staphylococcin marker. The bacteriocin is not inactivated by oxidation, mechanical agitation, or boiling for 15 min. It is sensitive to the action of trypsin and Pronase but not lysostaphin and is stable within a pH range of 4 to 9. It has an isoelectric point of approximately 7.7. Removal of the ampholytes and glycerol from electrofocused staphylococcin preparations resulted in total loss of bacteriocin activity.     

Sensitivity to antibiotics in coagulase-negative staphylococci isolated from patients with central venous catheters

We examined 25 coagulase-negative staphylococci isolated from children, of whom 17 with leukaemia and 8 with terminal renal failure. Strain identification performed by api Staph system revealed the presence of S. epidermidis in 21 children, S. hominis in 3 patients and S. haemolyticus in 1 patient. By diffusion method we examined the activity of penicillin, methicillin, cephalexin, cephtriaxon, lincomycin, erythromycin, vancomycin, co-trimoxasol, gentamicin, amikacin, chloramphenicol, rifampicin and fusidic acid. MICs of seven antibiotics were obtained by agar dilution method. MIC50 and MIC90 were as follows: /ml Methicillin 3.13 mg/ml and 50 mg/ml, lincomycin 100 mg/ml and 100 mg/ml, gentamicin 25 mg/ml and 100 mg/ml, chloramphenicol 6.25 mg/ml and 50 mg/ml, amikacin 1.56 mg/ml and 100 mg/ml, rifampicin 0.09 mg/ml and 12.5 mg/ml, fusidic acid 6.25 mg/ml and 12.5 mg/ml, vancomycin 1.56 mg/ml and 3.13 mg/ml. These data show that the examined strains are highly resistant to numerous antibiotics. Thirty six percent of all strains were resistant to methicillin, 88% to lincomycin, 60% to gentamicin, 52% to chloramphenicol, 24% to amikacin, 52% to rifampicin and 56% to fuscidic acid. All the examined strains were sensitive to vancomycin.     

A simple and inexpensive method for the identification of Staphylococcus epidermidis and Staphylococcus hominis

Eight hundred and ninety-two strains of Staphylococcus species were identified by means of desferrioxamine susceptibility and fermentation results of three carbohydrates, with the API Staph system (bioMérieux, France) as reference method. No identification could be obtained for 34 strains with API Staph. Of the remaining 858 strains, identical identification was obtained with 842 (98.1%). All 707 strains identified as Staphylococcus epidermidis or Staphylococcus hominis by the API Staph system were found to be desferrioxamine susceptible, and all but 5 (3.3%) of 151 strains identified as other staphylococcal species were found to be resistant, yielding an identification correlation of 99.4% for desferrioxamine. The five additional strains which were susceptible to desferrioxamine were identified as Staphylococcus capitis (2 strains), Staphylococcus lugdunensis (2 strains), and Staphylococcus warneri (1 strain) by API Staph, and as Staphylococcus epidermidis (1 strain), Staphylococcus hominis (3 strains), and one other staphylococcal species by the experimental system.     

Molecular analysis of rifampin-resistant Mycobacterium tuberculosis isolated from Korea by polymerase chain reaction-single strand conformation polymorphism sequence analysis

The production of and sensitivity to bacteriocin-like activity among 44 strains of black-pigmented anaerobes isolated from periodontal sites were evaluated by both an overlay and an agar diffusion method. The species studied were Porphyromonas gingivalis, Prevotella intermedia and the closely related species Pr. nigrescens. Pr. intermedia strains (90%) produced bacteriocin-like activity against Pr. nigrescens and all Pr. nigrescens were active against Pr. intermedia. Both species showed a high degree of activity against P. gingivalis, whereas only one P. gingivalis strain produced bacteriocin-like activity against either of the other two species. Both Pr. nigrescens and Pr. intermedia showed some activity (40% and 20%, respectively) against other strains of the same species. Such bacteriocin production might be expected to influence the distribution of these black-pigmented species in vivo. Of 224 periodontal sites sampled, only 2.6% yielded mixed cultures of black-pigmented species and of these only one strain, a P. gingivalis isolate, produced bacteriocin-like activity against any of the other strains isolated from these sites. These data support the concept that local production of bacteriocin-like activity in vivo may contribute to the selection of the black-pigmented bacterial profile in subgingival sites.     

Accuracy of methods using somatic cell count and N-acetyl-beta-D-glucosaminidase activity in milk to assess the bacteriological cure of bovine clinical mastitis

This study examined the capability of milk somatic cell count (SCC) and NAGase activity to discriminate between quarters that had been cured versus those that had not been cured at 4 wk after antimicrobial therapy for clinical mastitis. The distribution of microorganisms that were isolated before therapy from 630 quarters with mastitis was as follows: 225 strains of Staphylococcus aureus, 96 strains of coagulase-negative staphylococci, 152 strains of streptococci (Streptococcus dysgalactiae and Streptococcus uberis), and 157 strains of coliform bacteria. Bacteriological cure rates were 35% for mastitis caused by Staph. aureus, 75% for mastitis caused by coagulase-negative staphylococci, 66% for mastitis caused by streptococci, and 72% for mastitis caused by coliforms. Diagnostic accuracy of milk SCC and NAGase and their interquarter ratios for predicting bacteriological status of the control samples was assessed by calculating sensitivity, specificity, and accuracy and by means of receiver operating characteristic analysis. The efficiency of milk SCC and NAGase for predicting bacteriological cure was greatest for cows that had been infected with Staph. aureus. The main problem in detecting coagulase-negative staphylococci was low sensitivity, and the main problem in detecting streptococci and coliforms was low specificity. Receiver operating characteristic analysis is not completely suitable for the detection of mastitis because reference method bacteriology and indirect tests can never fully agree. To assess the recovery of cows from mastitis caused by Staph. aureus, bacteriology should be supplemented with an examination of milk SCC or NAGase activity at threshold values such as those presented here.     

Routes of pathogenic staphylococcal contamination of slaughter poultry

The contamination of birds with pathogenic staphylococci was followed up during their slaughter handling and trimming at two poultry-dressing houses. It was found that water cooling and evisceration were mostly contributing to contamination. Taking part in the dissemination of Staphylococcus infection were also the female workers on the slaughter belt, especially those that had wounds on their hands. Studied were the properties of a total of 881 strains of staphylococci, 43.35 per cent of them being defined as Staph. aureus, and 56.64 per cent--as Staph. edidermidis.     

Hippocampal age differences reoccur after modification of stimulus configurations in rabbit jaw movement conditioning

A bacteriocin-like activity produced by Enterococcus faecium BC25, isolated from the the rumen of a cow, was partially purified and characterized. The active substance was prepared by ammonium sulfate and chloroform/methanol precipitation of culture supernatant. The bacteriocin was a protein of molecular mass 17.5 kDa. Activity was inactivated by trypsin and proteinase K. The bacteriocin BC25 inhibited growth of amylolytic ruminal strains of Streptococcus bovis, including S. bovis AO 24/85. Results showed that bacteriocine substance BC25 has a bacteriostatic effect when present in concentrations exceeding 125 AU/ml. Agar overlays and batch culture growth experiments proved that E. faecium BC25 was producing bacteriocin that inhibited the growth of S. bovis.     

Staphylococcal strains involved in bovine mastitis are inhibited by Staphylococcus aureus antimicrobial peptides

The inhibitory activity of five bacteriocin (Bac)-producer strains of Staphylococcus aureus was tested against bacteria pathogenic for cattle. Sixty-five epidemiologically unrelated strains of Staph. aureus involved in bovine mastitis were used as indicators in an agar diffusion test. Bacteriocins produced by four strains could inhibit only a limited number of test organisms. However, all 65 indicator strains proved to be susceptible to the combined action of both bacteriocins encoded by pRJ9, a Bac plasmid found in strain A53. Therefore, the bacteriocins produced by this strain may represent new antimicrobial peptides with potential applications in the prevention and treatment of bovine mastitis.     

Molecular markers of hemostatic activation and inflammation following major injury: effect of therapy with IFN-gamma

The aim of this study was to develop a simple, reliable, and inexpensive in-house system for routine species identification of staphylococci in clinical practice. The system combines 15 key tests (including carbohydrate fermentation) performed in micro-well strips and antimicrobial disk diffusion susceptibility tests performed on standardised paper disk method antibiotic sensitivity medium agar. Twenty-eight staphylococcal reference strains belonging to 18 different species were correctly identified using this in-house system. A total of 291 clinical staphylococci isolates were evaluated with the in-house system and a conventional identification scheme. The in-house system identified 281 (96.6%) of these 291 isolates. Eleven different species were recognised. The five species most frequently identified were Staphylococcus epidermidis (48.6%), Staphylococcus aureus (27.8%), Staphylococcus haemolyticus (8.2%), Staphylococcus hominis (5.7%), and Staphylococcus warneri (5.3%). There was an agreement of 86.3% between the species identification obtained with the in-house system and the conventional identification scheme. All coagulase-negative isolates initially identified as species other than Staphylococcus epidermidis as well as indistinctly identified isolates were also evaluated with a commercial identification system. The agreement between species identification obtained with the in-house system and the commercial system for 101 identified isolates was 73%. Several isolates that were difficult to distinguish with the conventional scheme and/or the commercial system were identified with the aid of the antimicrobial susceptibility test included in the in-house system. The described test scheme should be of value for identification of clinically significant staphylococci species.     

Evaluation of Biolog for identification of members of the family Micrococcaceae

The Biolog Identification System (Biolog, Inc., Hayward, Calif.) was challenged at two separate laboratories with 113 coded isolates, including 33 type strains of staphylococci, 5 strains of Micrococcus spp., and 1 strain of Stomatococcus mucilaginosus. Test parameters between the sites were controlled as much as possible. Discrepancies were arbitrated by using conventional biochemicals. Overall accuracies (correct to the species level) upon initial testing were 47.7 and 59.3%, respectively, at the two laboratories. After repeat testing of isolates generating "no identification" responses or errors, the overall accuracies increased to 69.0 and 74.3% at the two sites, respectively, revealing no significant difference in the final results at the two laboratories (78 of 113 versus 84 of 113; P > 0.05). Error rates were 7.1% at one site and 9.7% at the other. The Biolog is not yet accurate enough to serve as a primary method for identifying staphylococci.     

Bacteriocins of Streptococcus bovis

Forty-seven strains of Streptococcus bovis were tested for bacteriocin production. Fourteen were found to produce bacteriocins, while all 47 were sensitive to at least one of these bacteriocins. The bacteriocins, on the basis of their host range on S. bovis strains, formed six groups. A representative of each group was selected and characterized by temperature stability, sensitivity to trypsin and lipase, sedimentation by centrifugation, ability to pass through dialysis tubing, host range on other bacterial species, and conditions for production in liquid media. A correlation between mannitol fermentation and bacteriocin production was noted.     

Development of a PCR-restriction fragment length polymorphism assay using the nucleotide sequence of the Helicobacter hepaticus urease structural genes ureAB

Infection with Helicobacter hepaticus causes chronic active hepatitis in certain strains of mice and is associated with hepatocellular carcinoma in A/JCr mice. Like the gastric helicobacters, H. pylori and H. mustelae, H. hepaticus possesses a high level of urease activity. However, the H. hepaticus urease structural gene sequences have not been previously determined, and the role of the urease enzyme in colonization and in pathogenesis is not known. PCR was used to amplify a portion of the urease structural genes from H. hepaticus genomic DNA. Amplified DNA fragments were cloned, and the nucleotide sequence was determined. The deduced amino acid sequence of the partial H. hepaticus ureA gene product was found to exhibit 60% identity and 75% similarity to the predicted H. pylori UreA. The deduced amino acid sequence of a partial H. hepaticus ureB gene product exhibited 75% identity and 87% similarity to the predicted H. pylori UreB. Diversity among H. hepaticus isolates was evaluated by means of a restriction fragment length polymorphism (RFLP) assay. The 1.6-kb fragments within the ureAB open reading frames, amplified from 11 independent isolates, were digested with the restriction endonuclease HhaI. Three distinct RFLP patterns were observed. Identical RFLP profiles were noted in sequential isolates of one strain of H. hepaticus during an 18 month in vivo colonization study, suggesting that the urease genes of H. hepaticus are stable. The urease genes among H. hepaticus strains were also well conserved, showing 98.8 to 99% nucleotide sequence identity among three isolates analyzed. These findings indicate that H. hepaticus has urease structural genes which are homologous to those of the gastric Helicobacter species and that these gene sequences can be used in a PCR and RFLP assay for diagnosis of this important murine pathogen.     

Detection of bacteriocins of lactic acid bacteria isolated from foods and comparison with pediocin and nisin

A total of 663,533 colonies from 72 dairy and meat sources showed a detection rate of 0.2% for bacteriocin producers using direct plating techniques. A further 83,000 colonies from 40 fish and vegetable sources showed a detection rate of 3.4% for bacteriocin producers using selective enrichment procedures. A collection of seven purified isolates showing a different host spectrum of bacteriocin activity and with the ability to produce bacteriocins in broth culture were compared with nisin and pediocin (with respect to their inhibitory activity, determined by the critical dilution method), against various indicator bacteria in agar and broth. The sensitivity of Listeria species to various bacteriocins was influenced by the agar and broth test systems used. A Lactobacillus curvatus strain was found to be the most suitable indicator for quantitating antimicrobial effects of all the bacteriocins investigated in both agar and broth test systems. The bacteriocin-producing isolates were characterized by biochemical reactions and DNA restriction enzyme profiles and taxonomic identification revealed species of Lactobacillus, Carnobacterium and Lactococcus assigned on the basis of 16S rDNA sequences.     

Phylogeny of species in the family Neisseriaceae isolated from human dental plaque and description of Kingella oralis sp. nov [corrected

Fourteen human periodontal isolates recovered from a purported Eikenella corrodens-selective medium containing 1 microgram of clindamycin per ml displayed biochemical traits which differed from those described for E. corrodens. These organisms were gram-negative rods which corroded agar. The isolates were oxidase positive and urease, indole, and esculin negative. They differed from E. corrodens in catalase, nitrate reduction, lysine decarboxylase, and ornithine decarboxylase activities. One isolate, strain UB-294, was presumptively identified as Kingella denitrificans. A second isolate, strain UB-204, differed from E. corrodens by being catalase positive and nitrate reduction negative. Twelve isolates, including strain UB-38T (T = type strain), were phenotypically similar to Kingella kingae except that they did not produce acid from maltose and were not beta-hemolytic. Essentially complete (1,480-base) 16S rRNA sequences were determined for strains UB-38T, UB-204, and UB-294 and the type strains of Neisseria animalis, Neisseria canis, Neisseria denitrificans, Neisseria elongata, Neisseria flavescens, Neisseria macaca, and Neisseria polysaccharea. These sequences were compared with the previously published sequences of six other species belonging to the family Neisseriaceae. On the basis of the results of the comparative sequence analysis, UB-294 was confirmed as a K. denitrificans strain, UB-204 was identified as a member of a new species which may belong in the genus Eikenella, and UB-38T was identified as a member of a new species of the genus Kingella, for which we propose the name Kingella oralis [corrected]. Since strain UB-204 was the only representative of a new species, it was not named.(ABSTRACT TRUNCATED AT 250 WORDS)