Staphylococcus aureus exosecretions and bovine mastitis

The role of Staphylococcus aureus and coagulase-negative staphylococcal exosecretions in bovine udder infection was tested by monitoring the cows' response to in vivo inoculation of bacterial exosecretions into udder quarters. Twenty Israeli-Holstein dairy cows were included in the study; two or three of the udder quarters of each cow were intracisternally inoculated with 0.04-0.05 mg/quarter (total proteins) of the various sterile bacterial exosecretions in a sterile pyrogen-free saline. Each udder was inoculated with two or three different bacterial exosecretions or placebo (Columbia Broth). Cows were monitored for 96 h post-inoculation for rectal temperature, heart and respiratory rates, alimentary tract activity (rumen contraction), udder temperature, pain, oedema and udder size. Milk samples were examined bacteriologically and for somatic cell count, N-acetyl-D-glucosaminidase (NAGase) activity and somatic cell differentiation. No enterotoxins (beta-G) or toxic shock syndrome toxin-1 were detected in response to any of the bacteria tested. Control quarters or those inoculated with Columbia Broth, showed similar NAGase and somatic cell count values throughout the experiment. Twelve of the 18 strains tested, induced inflammation in the inoculated quarters while six did not. Of the 12 strains causing local inflammation, only six were found significantly different from the control and were considered as high response (group 1). The other six that caused a local inflammation did not differ significantly from the control, and were considered to be moderate response (group 2). The six S. aureus isolates that did not cause an inflammatory response were considered to have low response (group 3). In all quarters inoculated with S. aureus bacterial exosecretions belonging to groups 1 and 2, the polymorphonuclear cells and macrophages were proportionally increased while CD4+ and CD8+ T-lymphocyte populations decreased. One-dimensional NuPAGE (7%) Tris-acetate gel electrophoresisof the bacterial exosecretions revealed four different bands appearing between 36 and 31 kDa, marked from top to bottom as A, B, C and D. An association was found between the combinations of expressed bands and the cow responses: the majority of the cases could be linked to the expression of bands B and C.     

The use of liposomally-entrapped gentamicin in the treatment of bovine Staphylococcus aureus mastitis.

The effect of incorporation of gentamicin in liposomes on intracellular killing of Staphylococcus aureus was studied in vitro in cultured bovine mammary macrophages, and in experimental bovine mastitis. Liposomes were prepared by reverse-phase evaporation and ranged in size from 0.1 to 1.0 micron in diameter (mean 0.51 micron), with an encapsulation efficiency of gentamicin of 27.4%. Liposomes were taken up by in vitro cultured macrophages but intracellular killing of S. aureus over 12 h was not significantly enhanced when treatment with liposomally-entrapped gentamicin was compared to free gentamicin. Treatment of experimentally-induced S. aureus mastitis in five lactating Holstein cows (20 quarters) failed to show significant differences in bacterial counts when treatment with liposomally-entrapped gentamicin was compared to treatment with free gentamicin or blank liposomes plus free gentamicin. Gentamicin concentrations exceeded the in vitro determined minimum inhibitory concentration for 48 h when quarters were treated with 50 mg gentamicin on two occasions 24 h apart.     

Genotyping of Staphylococcus aureus isolated from bovine mastitis.

The present study was designed to comparatively investigate 25 Staphylococcus aureus strains isolated from bovine subclinical mastitis. The S. aureus strains, obtained from six different farms at five locations in one region of Germany, were characterized by phenotypic and genotypic methods. The S. aureus could be identified and further characterized by their cultural, biochemical and hemolytic properties. To analyze the epidemiological relationship the isolates were subjected to DNA fingerprinting by macrorestriction analysis of their chromosomal DNA, by PCR amplification of the gene encoding the 16S-23S rRNA intergenic spacer, by PCR amplification of the gene encoding the IgG binding region and the X region of protein A and by amplifying, and subsequent, digestion of the gene encoding staphylococcal coagulase. The macrorestriction analysis revealed five DNA restriction patterns with DNA patterns I, III and IV occurring in three, four, and three different farms, respectively. In addition, clones with different DNA patterns could be found within one herd. The PCR products for the spacer DNA, the spa gene encoding the X region of protein A and the coa gene encoding coagulase corresponded mostly to the pattern observed by DNA fingerprinting. Amplification of the gene encoding the IgG binding region revealed sizes of 620 bp for 20 of the isolates and 280 bp for four isolates indicating, for the latter, a deletion of segments in this region. These findings show, that single, widely distributed clones seemed to be responsible for cases of bovine subclinical mastitis found in one region of Germany.     

奶牛乳腺炎金黄色葡萄球菌超抗原的检测

用PCR方法对分离到的临床型乳腺炎金黄色葡萄球菌124株.隐性乳腺炎金黄色葡萄球菌213株,进行超抗原毒素sea,seb,sec,sed,see和tst基因的榆测.结果表明:奶牛临床型乳腺炎金黄色葡萄球菌肠毒素基因和tst基因的阳性率为27.42%,隐性乳腺炎金黄色匍萄球菌肠毒素基因的阳性率为1.41%,奶牛乳腺炎金黄色葡萄球菌产生毒素的类型以SEA,TSST-1和SEC为主,对于肠毒素基因和tst基因PCR阳件的金黄色葡萄球菌同时用ELISA和RPLA两种方法进行检测,3种检测方法结果基本一致.     

Characteristics of Staphylococcus aureus from subclinical bovine mastitis in Brazil

A total of 127 Staphylococcus aureus strains isolated from milk samples of cows with subclinical bovine mastitis was examined for biotype, phage pattern, in-vitro antibiotic susceptibilities and ability to produce enterotoxins. The majority of the strains showed features consistent with bovine rather than human origin. All strains were sensitive to the antibiotics tested, except penicillin and streptomycin. Enterotoxigenicity was observed in 6 (4.7%) strains and only enterotoxins A and C were produced.     

Characterization of Staphylococcus aureus strains isolated from bovine mastitis in Japan

Fifty-three strains of Staphylococcus aureus isolated from cows affected with mastitis from 21 prefectures in Japan were characterized by phenotypic and genotypic methods. Thirty-three (62.3%) strains showed biotype K-beta+CV:A, coagulase type VI, and sensitivity to bovine phages of group III or IV. These 33 strains could be subdivided into two groups on the basis of the production of staphylococcal enterotoxins (SEs) and on toxic shock syndrome toxin-1 (TSST-1). By pulsed-field gel electrophoresis, the 16 SEC- and TSST-1-producing strains showed similar patterns that differed by only a few fragments, suggesting that they were genetically closely related. Fifteen of 17 non SEC-producing strains which did not produce any other SEs and TSST-1 were genetically different from the SEC-producing strains and showed genetic diversity.     

Antimicrobial drug susceptibility of Staphylococcus aureus isolated from bovine mastitis

Isolates of Staphylococcus aureus (106) from bovine mastitis were tested for susceptibility to antimicrobial agents. beta-lactamase was produced by 69.8 per cent of isolates and 7.5 per cent were resistant to streptomycin (minimum inhibitory concentration more than 32 micrograms/ml). Resistance to other agents was rare. Intrinsic resistance or tolerance to beta-lactam antibiotics was not found.     

Efficacy of Staphylococcus aureus vaccines for bovine mastitis: a systematic review

Bovine mastitis is the main cause of economic loss in milk production worldwide and Staphylococcus aureus is the agent most frequently associated with the disease. The aim of this systematic review was to assess the efficacy of vaccines for bovine mastitis caused by S. aureus and suggest the immunotherapeutic protocols that have achieved the best and/or most promising results. An electronic search was made of the PubMed and Web of Science databases in November 2009. Only studies that tested vaccines in vivo in cows were included. The experimental design, methodological quality, type of vaccine and results of the studies were analyzed. Twenty-four papers were selected for this review. In general, bacterin-toxoid vaccines, vaccines of DNA-recombinant protein and recombinant protein alone were investigated in the studies selected. This systematic review suggests that vaccines that employ new technologies (DNA and/or recombinant protein vaccines) and some long-standing bacterins have achieved good results, which supports their use in the prevention and control of bovine mastitis caused by S. aureus. However, methodological differences and in some cases, a lack of more severe scientific criteria (such as double blind protocols) hinder the assessment of the effectiveness of these vaccines.     

Absence of encapsulation in strains of Staphylococcus aureus isolated from bovine mastitis

Thirty of 104 strains of Staphylococcus aureus isolated from clinical cases of bovine mastitis in England grew as diffuse colonies in serum soft agar (SSA), 45 grew as mixed diffuse and compact colonies and 29 yielded compact colonies only. The compact strains grew as diffuse colonies in SSA after one passage in the mammary gland of mice. However, none of the strains had an unstained halo when examined by the India ink technique and there was a 99.99 per cent reduction in the viable numbers of the bacteria in 30 representative strains 24 hours after inoculation into the peritoneal cavity of mice. By contrast the truly encapsulated strain M had an unstained halo by the India ink technique and resisted phagocytic killing in the peritoneal cavity. It is concluded that these strains from cases of mastitis are not encapsulated and that growth as diffuse colonies in SSA is not a reliable test of encapsulation.     

Molecular typing of enterotoxigenic Staphylococcus aureus isolated from bovine mastitis in Korea

One hundred and sixty-six Staphylococcus aureus isolates from mastitic milk samples from different cows on 26 farms were investigated for staphylococcal enterotoxins(SEs) and toxic shock syndrome toxin-1(TSST-1) by polymerase chain reaction(PCR) and reverse passive latex agglutination assay(RPLA). SEs and the TSST-1 gene were detected in thirty-seven isolates based on a multiplex PCR; SEA was detected in 32 isolates, SEB in 3 isolates, SEC in 1 isolate, and SEA and the TSST-1 gene in 1 isolate. Of the 37 enterotoxigenic isolates, thirty-three isolates were enterotoxigenic according to RPLA, where 29 isolates produced SEA, 3 isolates produced SEB, and 1 isolate produced SEC. The enterotoxin-producing S. aureus isolates were further characterized by pulsed-field gel electrophoresis(PFGE). A macrorestriction analysis revealed 11 PFGE patterns. Among the 33 enterotoxigenic S. aureus isolates, 45.4% exhibited the same PFGE pattern I. Accordingly, although the enterotoxin-producing S. aureus isolates from bovine mastitis were genetically diverse, 1 common genotype prevailed on the farms, indicating that PFGE pattern I isolates may be the most disseminated in Korea.     

Isolation of L-form variants after antibiotic treatment in Staphylococcus aureus bovine mastitis

An unstable L-form of Staphylococcus aureus was identified in milk samples from 3 quarters of 2 cows after treatment with cloxacillin. Milk samples incubated on standard 5% blood agar plates were culture-negative for 7 to 30 days after treatment, but S aureus was reisolated in 80% of 66 samples by additional culturing on enriched L-form media when incubated in 10% CO2 at 37 C. The organism was identified at various phases of reversion of L-form agar and was confirmed on transfer to blood agar plates.     

奶牛乳房炎金黄色葡萄球菌基因多态性分型试验

利用随机引物AP-7,建立引物随机多态性扩增(RAPD)体系对71株引起内蒙古和贵州地区奶牛乳房炎的金黄色葡萄球菌分离株进行基因分型研究。结果表明,71株金黄色葡萄球菌均得到清晰的RAPD指纹图谱,扩增产物为2～9条带,产物大小为240～4 500bp。菌株共分为6个基因型,其中Ⅰ型17株(占23.9%)、Ⅱ型3株(占4.2%)、Ⅲ型33株(占46.5%)、Ⅳ型15株(占21.1%)、Ⅴ型2株(占2.8%)、Ⅵ型2株(占2.8%)。Ⅰ型为内蒙古地区的流行优势菌群,Ⅲ型为贵州地区的流行优势菌群。两地区各基因型菌株比例有明显差异,这可能与奶牛养殖业水平和环境差异有关。     

Milk whey induction of agglutination in ovine and bovine mastitis Staphylococcus aureus

A total of 59 mastitis staphylococcic strains were tested for growth agglutination upon supplementation of growth media with ovine and bovine milk whey and mammary secretions from dry cows. Differences were observed when comparing bacterial species or origins (ovine vs. bovine) of bacteria and whey. All of the ovine and bovine S. aureus strains tested, but only 4 among 22 other ovine mastitis staphylococcic strains, showed growth agglutination in Todd Hewitt broth (THB) supplemented with greater than or equal to 30% (v/v) ovine milk whey. None of the strains agglutinated during growth in regular THB medium. Ovine whey had an agglutination induction capacity higher than bovine whey (P less than 0.005), concerning the number of responsive ovine and bovine S. aureus strains. There were no differences between whey samples from different ewes with regard to their capacity to induce agglutination. Ovine S. aureus strains were more responsive than bovine strains of this bacterial species, concerning the number of responsive strains (P less than 0.001) to bovine whey (greater than or equal to 30% in THB), the proportion of responsive strains at low (10%) ovine whey concentration (P less than 0.001), and the strength of reaction (precipitation timing and clump size). Secretions from dry cows systematically induced agglutination in all of the bovine and ovine S. aureus strains tested.     

Chloramphenicol resistance plasmids in Staphylococcus aureus isolated from bovine subclinical mastitis.

Chloramphenicol resistance (CmR) could be detected in 11 of 217 Staphylococcus aureus isolates from bovine subclinical mastitis. All isolates were assigned to biotypes A or C. The CmR-determinants were found to be located exclusively on small plasmids of approximately 4.6 kb as revealed by protoplast transformation. The 11 CmR-plasmids could be differentiated on the basis of restriction endonuclease analyses. The restriction maps of these CmR-plasmids identified two separate groups. One group demonstrated homology to the plasmid pC 221, the other to the plasmid pC 223. Both prototype plasmids, pC 221 and pC 223, had been isolated from S. aureus of human origin.     

Extended antimicrobial susceptibility assay for Staphylococcus aureus isolates from bovine mastitis growing in biofilms

Staphylococcus aureus is one of the most prevalent causes of bovine mastitis. The antimicrobial treatment of this disease is currently based on antimicrobial susceptibility tests according to CLSI standards. However, various studies have shown that there is a discrepancy between the results of this standard susceptibility test and the actual cure rate of the applied antimicrobial treatment. Increasing evidence suggests that biofilm formation by S. aureus is associated with this problem. The currently available antimicrobial susceptibility assays for bacteria growing in biofilms, are not considered reliable enough for routine application. Therefore, the objective of this study was to further develop a susceptibility test for bacteria growing in biofilm, suitable for routine testing of the antimicrobial susceptibility of S. aureus. With the expansion of the available MBEC assay to an extended biofilm susceptibility test, that comprises 2 and 4 consecutive days of antimicrobial challenge, the antimicrobial susceptibility for S. aureus growing in biofilm was further analysed. The results showed clear differences between strains and various antimicrobial agents with respect to the effect of longer duration of the antimicrobial challenge on the eradication of S. aureus growing in biofilm. The extended biofilm susceptibility test also indicates that each bacterial strain requires a specific duration of antimicrobial therapy, which cannot be derived from a standard susceptibility test or from a 24-h biofilm susceptibility test.     

Sensitive test for screening for Staphylococcus aureus in bovine mastitis by broth cultivation and PCR

An assay was developed and evaluated for screening for Staphylococcus aureus in milk samples from cases of bovine mastitis by overnight cultivation in a broth containing 7.5 per cent sodium chloride, followed by pcr to amplify the nuc gene. The assay could detect concentrations of S aureus as low as 1 colony-forming unit/ml milk. Among 106 milk samples collected from individual quarters of lactating cows in one dairy herd and from a bulk tank, S aureus was detected in nine samples by the pcr assay but in only three samples by conventional microbiological culture.     

Phenotypic and genotypic characterization of bovine mastitis isolates of Staphylococcus aureus for biofilm formation

Staphylococcus aureus is one of the most common pathogens responsible for contagious mastitis in ruminants. The ability of S. aureus to form biofilm in vivo is considered to be a major virulence factor influencing its pathogenesis in mastitis. The objectives of the study were to examine in vitro slime production, biofilm formation, and the presence of the ica gene locus and icaA and icaD genes in S. aureus isolates from bovine mastitis. Thirty-two of the 35 isolates tested produced slime on Congo red agar, whereas only 24 of the isolates were found to produce biofilm in vitro. However, all the 35 isolates possessed the ica locus, icaA and icaD genes. This study indicates a high prevalence of the ica genes among S. aureus mastitis isolates, and their presence is not always associated with in vitro formation of slime or biofilm. A combination of phenotypic and genotypic tests is recommended for investigating biofilm formation in S. aureus.     

Comparison of methods for the determination of antimicrobial resistance in Staphylococcus aureus from bovine mastitis

The results of three standard methods (broth dilution, agar dilution, disk diffusion) and an experimental modification of the microdilution method for determination of resistance to ampicillin, cephalotin, cloxacillin, neomycin, novobiocin, penicillin and streptomycin were compared using 151 Staphylococcus aureus isolates obtained from cases of mastitis. The accuracy of the dilution methods was compared by determination of minimum inhibition concentrations (MIC, MIC50, MIC90 and modal MIC) and by assessment of the agreement within the tolerance of +/-1 dilution step in 2-fold dilution series. The results of the dilution methods were further compared with those of the reference disk diffusion method and the strains were classified as sensitive or resistant using the interpretation criteria for human strains. The comparisons indicated that MIC characteristics and the final classification as sensitive or resistant were method-dependent. Resistance to aminoglycoside antibiotics was observed more often when using broth dilution methods, especially when the broth was supplemented with lactose.     

奶牛乳房炎金黄色葡萄球菌脉冲场凝胶电泳分型研究

为探讨新疆北疆奶牛乳房炎致病菌的流行规律,本研究采用Sma Ⅰ酶切,脉冲场凝胶电泳(PFGE)分型的方法对43株分离自新疆6个规模奶牛场隐性乳房炎奶样的金黄色葡萄球菌进行了分子分型比较研究.结果表明,所有菌株都能被PFGE法分型,43株金黄色葡萄球菌可分为A、B、C、D和E 5个基因型.A型株(22株,51.2%)有13个亚型,相似度在81.8%～96.8%之间,在5个奶牛场均分离到,是主要的流行株;B型(25.6%)、C型(14.0%)、D型(7.0%)各型别内菌株间的相似度为100%,E型仅有1株.不同地区主要流行株有差别:乌鲁木齐主要流行A型菌株,昌吉以A型和B型菌株为主,而奎屯主要流行C型和B型.有2个奶牛场流行株只有1个基因型,也有2种基因型(3个牛场)或3种基因型(1个牛场)同时存在,但以1种为优势株.这些结果提示,不同地区主要流行株有差别,多数奶牛场以1种流行株感染为主,不同牛场可能流行相同的菌株,在较大地域范围内某些流行株具有侵染优势.