Identification of bap and icaA genes involved in biofilm formation in coagulase negative staphylococci isolated from feline conjunctiva

Bap and icaA genes are commonly known to be involved in the biofilm formation. The prevalence of bap and icaA genes and biofilm formation was determined in conjunctival isolates of coagulase negative staphylococci (CNS) collected from cats. The study was conducted on 90 archival CNS isolates collected from feline conjunctiva obtained from clinically healthy cats and cats with ocular problems. Biofilm formation was examined using the microtiter plate (MTP) method. The prevalence of icaA and bap genes was determined using polymerase chain reaction (PCR). Genetic profiles of the bap-positive isolates were examined using the modified random amplified polymorphic DNA (RAPD) method. Of the 90 CNS isolates investigated, 58.9 % (53/90) were confirmed to form biofilms on a polystyrene plate after 24 h, and the intensity of the biofilm production varied strongly between positive strains. Among the biofilm-producing isolates, 24.5 % (13/53) carried the icaA gene and 3.8 % (2/53) carried the bap gene. Among the isolates that did not produce biofilms, the icaA gene and bap gene were detected in 8.1 % (3/37) and 2.7 % (1/37) of isolates, respectively. This is the first report demonstrating that CNS isolated from feline conjunctiva can potentially be a bap gene reservoir. Preliminary comparison of the genetic profiles of three bap-positive isolates collected from cats showed that each of the isolates has a different genetic background with a high similarity with the human strain of S. epidermidis.     

The occurrence and significance of plasma coagulase negative staphylococci from the genital tract of horses

Classification based on biochemical characteristics of 389 strains of plasma-coagulase-negative (plc-) staphylococci isolated from the genital tract of mares and stallions resulted in the following distribution of species: St. sciuri 130 (33.4%), St. equorum 42 (10.8%), St. xylosus 16 (4.1%), St. epidermidis 35 (9.0%), St. simulans 24 (6.2%), St. haemolyticus 33 (8.5%), St. warneri 18 (4.6%), St. lentus 12 (3.1%), St. hyicus 11 (2.8%). Strains of St. cohnii, St. capitis, St. gallinarum, St. saprophyticus and St. hominis have only been found sporadically (a. 1%). 48 (12.3%) strains could not be classified. With regard to species distribution of isolates from stallions and mares. 63.7% of the isolates from stallions belonged to St. sciuri and 9.3% to St. lentus, whereas in isolates of mares these species numbered only 24.9% and 0.4%, respectively. On the other hand the species St. equorum (14.9% vs. 6.8%), St. epidermidis (14.5% vs. 1.7%), St. haemolyticus (14.0% vs. 1.7%), St. warneri (7.7% vs. 0.8%) and St. xylosus (5.9% vs. 2.5%) predominated in mares. St. simulans was found occurring equally in mares and stallions (7.7% vs. 5.9%). Comparing the staphylococcal species of healthy mares and of mares which have not become pregnant after copulation no indication was found for a significant role of certain plc- staphylococci in infertility. All of the 389 isolates were tested for production of protein A, i.e. Fc-fragment binding receptors, using a microenzyme-assay with peroxidase-labelled rabbit immunoglobulin G. With this method cell-bound or extracellular Fc-receptors could not be detected in anyone of the plc- staphylococcal strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of coagulase negative staphylococci from cases of subclinical mastitis in dairy cattle in Kampala,Uganda

Background

Coagulase negative staphylococci (CNS) are the most common pathogens leading to subclinical mastitis (SCM) in dairy cattle in Uganda. Coagulase negative staphylococci can vary between bacterial species in how they cause disease. The aim of the study was to characterize CNS, from cows with SCM in Uganda, at the species level.

Findings

Quarter milk samples (n = 166) were collected from 78 animals with SCM. Bacteriological analyses were carried out at Makerere University, Kampala, Uganda and at the National Veterinary Institute (SVA), Uppsala, Sweden. The most common pathogens found in milk samples from cows with SCM were CNS (31.7%). Two species of CNS were found, S. epidermidis (85%) and S. haemolyticus (15%). Of the CNS isolates, 16/20 (80%) were positive for β-lactamase production (β+).

Conclusions

In milk samples from cows with SCM caused by CNS, S. epidermidis was most prevalent, followed by S. haemolyticus.     

Production of staphylococcal enterotoxins and TSST-1 by coagulase negative staphylococci isolated from ruminant mastitis.

The production of staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 by 40 coagulase negative staphylococci isolated from sheep, goat and cow mastitis was studied. Both ELISA double sandwich and Western blot were used to detect the production of these toxins. Only two strains of S. xylosus were enterotoxigenic, producing SEC. TSST-1 was seen to be produced by 5 strains of S. xylosus, 1 S. sciuri and 2 S. epidermidis. Results obtained by ELISA and by Western blot agreed in all cases except in one strain of S. epidermidis which was only positive using ELISA.     

High diversity of coagulase negative staphylococci species in wild boars,with low antimicrobial resistance rates but detection of relevant resistance genes

This work was focused to determine the prevalence and the species diversity of coagulase-negative staphylococci (CoNS) in wild boars, and to study their antimicrobial resistance phenotype and genotype. Nasal samples of 371 wild boars from six Spanish regions were collected for CoNS recovery. The identification was performed by MALDI-TOF mass-spectrometry. Antimicrobial susceptibility for eight antimicrobial agents was studied by disc-diffusion method and the presence of 31 antimicrobial resistance genes by PCR.CoNS were detected in nasal samples of 136/371 animals tested (36.6%), and 161 isolates were obtained (1–3/animal); a high diversity of species was found (n = 17), with predominance of S. sciuri (n = 64), S. xylosus (n = 21) and S. chromogenes (n = 17). Among CoNS isolates, 22.4% showed resistance to at least one antimicrobial tested. Tetracycline-resistance phenotype was the most frequently detected (10.5%), generally mediated by tet(K) gene [associated or not with tet(L)]. Other relevant resistance genes were identified including unusual ones [mecA, erm(B), erm(F), mphC, erm(43), msr(A)/msr(B), lnu(A), dfrG, fexA, and cat_pC221].This is the first study in which CoNS isolates from wild boars are analysed. The knowledge of antimicrobial phenotype and genotype of CoNS in natural ecosystems is highly important since these staphylococcal species can act as vectors of relevant antimicrobial resistance mechanisms.     

Antimicrobial resistance of Staphylococcus aureus and coagulase negative staphylococci isolated from mastitis milk samples from sheep and goats

Staphylococcus aureus and coagulase negative staphylococci (CNS) were isolated from ovine and caprine mastitis milk samples originating from more than 40 Swiss farms. CNS dominated as causal microorganisms of mastitis in small ruminants. By restriction fragment length polymorphism (RFLP) analysis of the groEL gene and sequencing of 16S rDNA, various CNS species were identified, albeit certain of them predominated. For susceptibility testing of mastitis pathogens to selected antibiotics, minimal inhibitory concentrations were determined. Of the 67 S. aureus and 208 CNS strains, 31.3 % and 8.2 % were resistant to penicillin, 29.9 % and 1.0 % to ampicillin, 1.5 % and 10.6 % to erythromycin, and 3.0 % and 7.7 % to tetracycline, respectively. Moreover, 10 CNS strains (4.8 %) were resistant to oxacillin and one CNS strain to sulfamethoxazole/trimethoprim. The results obtained describe for the first time the resistance situation of mastitis pathogens from sheep and goats in Switzerland. However, accompanying and preventing measures are also of importance in mastitis control of small ruminants.     

Identification and antimicrobial susceptibility of coagulase positive staphylococci isolated from healthy dogs and dogs suffering from otitis externa

A total of 67 strains of coagulase positive staphylococci isolated from healthy dogs and dogs suffering from otitis externa were studied. Twenty-two isolates were from healthy dogs (five from hound dogs and 17 from companion dogs) and 45 from dogs suffering otitis externa (14 from hound dogs and 31 from companion dogs). Presumptive identification was attempted using the following tests: production of acetoin, anaerobic utilization of mannitol, acid production from mannitol, presence of beta-galactosidase, and growth on P agar supplemented with different concentrations of acriflavine. Susceptibility of staphylococci to 16 antibiotics was determined. Most effective antibiotics were imipenem, amoxycillin/clavulanic acid, ciprofloxacin, tobramycin, gentamicin and marbofloxacin. Penicillin, ampicillin and polymyxin B showed the lowest activity. There were no significant differences in antimicrobial susceptibility among isolates from healthy dogs and dogs suffering from otitis externa.     

Phage typing coagulase-positive staphylococci from rooks and gulls

Phage typing was performed on 86 strains of Staphylococcus aureus and 25 strains of Staphylococcus intermedius from rooks and gulls with human, bovine, chicken and canine phages. Eighty per cent of the S aureus strains and 64 per cent of the S intermedius strains were typable. The S aureus biotype D strains of rook origin were specifically lysed at routine test dilution (RTD) by chicken phages from groups I or I + IV, by human phages belonging to groups I and M, and partly by canine phage 58. The other rook and gull S aureus strains did not show characteristic phage patterns. The S intermedius strains isolated from both species of birds could be typed only with canine phages and this correlated with their classification into biotypes. All the biotype 1 strains tested but only two of 12 biotype 2 strains were lysed with canine phages at RTD.     

Effects of intra-mammary bacterial infection with coagulase negative staphylococci and stage of lactation on shedding of epithelial cells and infiltration of leukocytes into milk: comparison among cows, goats and sheep

The effects of mammary gland bacterial infection and stage of lactation on leukocyte infiltration into the mammary gland were compared among cows, goats and sheep. Animals were at two stages of lactation: mid or late. In mid-lactation animals, bacterial-free glands and coagulase negative Staphylococcus (CNS)-infected glands were compared. In late lactation only uninfected glands were studied. Of mid-lactation bacteria-free animals, goats had the highest number of leukocytes and % polymorphonuclears (PMNs), whereas sheep had the lowest and leukocytes number in cows were intermediate between sheep and goats. Based on %PMN, two cell clusters were found in sheep, which overlapped with the parallel cell clusters of cows and goats, but with a slightly higher number of leukocytes in each cell cluster. At late lactation, goats had higher values for %PMN and leukocyte numbers in comparison to cows, which had a similar cellular profile to sheep. The cellular immune response to CNS infection was similar for the three animal species, although the number of cells was different, while the basal cell level at mid-lactation and especially at the end of lactation was species specific.     

Evaluation of latex agglutination tests for establishing coagulase status of staphylococci from milk.

A total number of 640 staphylococci isolated from cows' milk were tested by latex agglutination and coagulase tests. About 50% of coagulase positive and 5% of coagulase negative staphylococci were positive to the latex agglutination tests. Latex agglutination tests were found to be not satisfactory for determining the coagulase status of staphylococci isolated from cows' milk.     

Characterization of staphylococci associated with clinical and subclinical bovine mastitis

Attempts were made to identify 900 species of staphylococci or micrococci recovered from samples of bovine milk examined for mastitis pathogens. The presence and identity of haemolysins was recorded together with results of disc diffusion antibiotic sensitivity tests. The occurrence of clinical mastitis was also noted and somatic cell counts (SCC) were performed on milk samples which were normal in appearance. Eight hundred and thirty-one coagulase positive staphylococci were obtained, of which 810 were S. aureus and 21 were S. intermedius. Of 65 coagulase negative staphylococci the species of 19 could not be determined by the identification systems used. The remainder were identified as S. hyicus sub sp. hyicus (1), S. hyicus sub sp. chromogenes (19), S. haemolyticus (17), S. hominis (3), S. epidermidis (4), S. capitis (1) and either S. hominis or S. warneri (1). Four other isolates could not clearly be assigned to the genus Staphylococcus or Micrococcus and were designated irregular strains. No micrococci were identified. The presence of alpha, beta, or delta haemolysins occurring singly or in various combinations was identified in 98.3% of coagulase positive staphylococci and in 60% of coagulase negative staphylococci. Epsilon haemolysin was detected in 47.6% of the coagulase negative staphylococci and in 9.5% of S. intermedius. All staphylococci were sensitive to tetracycline (30 microg), novobiocin (1.6 microg), nafcillin (30 microg), methicillin (10 microg) and cephalothin (30 microg) and variable numbers of each species were sensitive to penicillin (2 iu) and streptomycin (10 microg). One non-identified species of coagulase negative staphylococcus was sensitive to erythromycin (0.4 microg) the remaining staphylococci were resistant. Each of the four irregular strains was sensitive to erythromycin and novobiocin. Clinical mastitis was associated with 30.6% of coagulase positive staphylococci, 15.3% of coagulase negative staphylococci, and two of the four irregular strains (50%). Subclinical mastitis as determined by SCC of 500 x 10(3) or greater was associated with 92.7% of coagulase positive and 37.5% of coagulase negative staphylococci.     

Phage typing of staphylococci isolated from dairy cows in New Zealand

Four red deer calves (Cervus elaphus) died with severe nephritis apparently associated with infection by Leptospira interrogans serovar pomona. The sera of 12 in-contact red deer calves were examined for leptospiral agglutinins and nine showed titres to pomona consistent with recent infection. Two also showed titres of 1:100 to serovar hardjo. The urine of five of these in-contact calves was examined periodically over a period of nine months. All were initially leptospiruric, four being infected with pomona and one with hardjo. In four animals leptospiruria could only be detected for up to six months, but one animal infected with pomona was leptospiruric for at least eight months.

The apparent source of infection was from infected cattle, and it is suggested that deer are unlikely to act as maintenance hosts for serovar pomona.     

Identification and characterization of staphylococci isolated from cats

Staphylococcus simulans was found to be the most common staphylococcal species present on the healthy skin and in the nares of cats. S. aureus was the most frequent Staphylococcus species in lesions. Most of the S. aureus strains showed the characteristics of the human ecovar. Other strains isolated formed a heterogenic collection in which not less than 13 species were represented. One of these, S. epidermidis, was almost exclusively associated with lesions. It was concluded that coagulase-positive staphylococci are not endogenous to cats. Staphylococci from other sources, most frequently from man, are often associated with lesions in these animals.