Detection of superantigenic toxin genes in Staphylococcus aureus strains from subclinical bovine mastitis

The aim of this study was to determine the presence of genes encoding enterotoxins (sea-sej) and toxic shock syndrome toxin-1 (tst) of Staphylococcus aureus strains (n = 130) isolated from subclinical bovine mastitis in Turkey by polymerase chain reaction. Sixty-one (46.9%) isolates were found to contain one or more toxin genes. The most frequently found enterotoxin genes were seg (16.2%) and sei (16.2%), followed by sec (15.4%), sed (10.8%), and sej (10.8%), respectively. The tst gene was detected in seven (5.4%) isolates. None of S. aureus strains harbored sea, seb, see, and seh genes. Since these toxins are recognized agents of staphylococcal food poisoning, it must be considered that the consuming raw milk and raw milk products would pose public health risk as high prevalence of toxigenic S. aureus was found in this study.     

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.     

原料乳和临床乳房炎金黄色葡萄球菌毒力基因检测及药敏分析

对临床乳房炎（57株）和原料乳（44株）金黄色葡萄球菌菌株,用PCR方法检测mecA基因、PVL基因、ETs基因、SEs基因和TSST-1基因;采用CLSI指导说明执行琼脂稀释法药敏性试验。结果显示原料乳菌株中,84.09%携带有毒素基因,其中PVL的检出率为84.09%,肠毒素的检出率为52.27%,主要流行的肠毒素基因为sea（56.82%）,均未检测到携带mecA、ETs、TSST-1、sei和sej基因的菌株;同时得到10种毒素基因型,其主要流行的毒素基因型为PVL＋sea（29.55%）和PVL（27.27%）。临床菌株中,78.95%携带有毒素基因,其中PVL的检出率为28.07%,肠毒素的检出率为77.19%,主要流行的肠毒素基因为sea（47.37%）,没有检测到携带ETs、TSST-1和seh基因菌株;同时得到25种毒素基因型,其主要流行的毒素基因型为sea（19.30%）,其次是seb（7.02%）,sea＋sed＋sej（3.51%）和PVL＋sea＋seb＋sec＋seg＋sei（3.51%）。6株（10.53%）携带有mecA基因菌株均含有较多毒素基因。原料乳分离株对甲氧苄啶和头孢西丁的耐药率较高,分别为100%和86.36%,其次对氯霉素、红霉素、苯唑西林、头孢哌酮和庆大霉素的耐药率分别为11.36%、4.55%、2.27%、2.27%和6.82%,所有原料乳菌株均对环丙沙星敏感,同时得到8种耐药谱,多重耐药率达22%;临床乳房炎菌株对红霉素和甲氧苄啶的耐药率较高,分别为100%和71.93%,其次对氯霉素、庆大霉素、环丙沙星、头孢西丁和苯唑西林的耐药率分别为28.07%、26.07%、24.56%、19.30%和7.02%,临床乳房炎菌株对头孢哌酮和四环素的敏感率为100%,同时得到13种耐药谱,多重耐药率达77.19%。所有原料乳和临床乳房炎菌株均对万古霉素和阿米卡星敏感。临床乳房炎菌株携带的毒素基因和多重耐药率比原料奶菌株高,同时在临床乳房炎乳中检测到MRSA菌株,提示我们应加强乳及其乳制品的管理,并对奶牛乳房炎加以重视。     

奶牛乳房炎病例中金黄色葡萄球菌毒素基因的检测

金黄色葡萄球菌是引起奶牛乳房炎的重要病原菌之一，其不仅给奶业生产造成严重损失，而且携带毒素基因的金黄色葡萄球菌在适当环境温度、pH、介质下可产生毒素，影响乳产品质量安全，危害人体健康。金黄色葡萄球菌常产生的、临床意义较大的毒素为肠毒素（SEs）、剥脱毒素（ETs）和中毒休克毒素-1（TSST-1）三类。国外有较多奶牛乳房炎金黄色葡萄球菌毒素基因流行情况的调查报道，国内少有这方面的研究。因此，本文就我国重要奶产地呼和浩特地区奶牛乳房炎金黄色葡萄球菌的sea、seb、sec、sed、see、eta、etb、tst等8种常见毒素基因的流行情况进行调查研究，以期为食源性疾病的监控和分子检测提供科学依据。     

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.     

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.     

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.     

Resistance situation and enterotoxin production capacity of Staphylococcus aureus strains from bovine mastitis milk samples]

In total, 63 S. aureus strains from mastitis milk samples of different animals in 57 farms were isolated. In 14 (22%) of the S. aureus strains resistancies against one or several of the examined antibiotics could be observed whereby six resistance patterns were found. 14.3% of the strains were penicillin resistant. 34 (54%) of the 63 S. aureus produced enterotoxins (SE). Three strains formed SEA, 21 SEC, three SED and seven strains 2 SE, SEAC, SEAD or SEBD.     

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。     

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.     

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.     

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.     

Genetic diversity and antimicrobial susceptibility profiles among mastitis-causing Staphylococcus aureus isolated from bovine milk samples

OBJECTIVE: To determine whether particular antimicrobial susceptibility profiles of bovine mastitis-causing Staphylococcus aureus isolates were associated with specific S aureus genotypes. SAMPLE POPULATION:357 S aureus isolates recovered from milk samples submitted for diagnostic bacteriologic testing from 24 dairy herds. PROCEDURES: Antimicrobial susceptibility of S aureus isolates was assessed by determining minimum inhibitory concentrations (MICs) to 14 antimicrobial agents. After digestion of S aureus genomic DNA by SmaI, electrophoretic patterns were obtained via pulsed-field gel electrophoresis (PFGE) and used to classify isolates into types. Gels were analyzed, and data were used to prepare dendrograms. RESULTS: 308 of 357 (86%) S aureus isolates were susceptible to all antimicrobials evaluated. Forty-nine S aureus isolates were resistant to 1 or more antimicrobials; of these isolates, 37 were resistant only to penicillin, 9 were resistant to penicillin and erythromycin, 2 were resistant to tetracycline, and 1 was resistant to erythromycin. Isolates were assigned to 7 PFGE types. An association was found between PFGE type and antimicrobial susceptibility profile. Organisms with resistance to at least one of the tested antimicrobial agents were identified in only 4 of the 7 types of S aureus. CONCLUSIONS AND CLINICAL RELEVANCE: Antimicrobial resistance was uncommon among the mastitis-causing S aureus isolates identified in the milk samples. A limited number of genotypes were associated with mastitis in these herds. Antimicrobial resistance phenotypes were associated with particular S aureus PFGE types; this association may have implications for future treatment and control of S aureus-associated mastitis in cattle.     

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.     

Population diversity of Staphylococcus aureus isolated from bovine mastitis in Brazilian dairy herds

Population diversity was evaluated in strains of Staphylococcus aureus isolated from bovine mastitis in Brazilian dairy herds by PCR-RFLP and sequencing of the 3'-terminal portion of the coagulase gene, and the susceptibility of strains to antimicrobials. The results showed great diversity in S. aureus population studied and the existence of predominant clones that account for most infections. No associations between the predominant types observed in the PCR-RFLP and the forms of presentation of the mastitis or to any of the different patterns of antimicrobial resistance were observed.     

新疆部分地区奶牛乳房炎金黄色葡萄球菌荚膜多糖分型的研究

为调查新疆奶牛乳房炎金黄色葡萄球菌主要流行血清型,本研究采用玻板凝集和双重PCR方法对不同地区奶牛乳房炎乳样中分离的117株金黄色葡萄球菌进行血清分型.结果表明荚膜多糖5型占10.26％(12/117),8型占13.68％(16/117),336型占64.96％(76/117),未分型菌株占11.11 ％(13/117).该研究为新疆奶牛乳房炎金黄色葡萄球菌疫苗菌株的筛选提供了依据.     

Characterization of enterotoxigenic Staphylococcus aureus strains isolated from bovine mastitis in north-east Switzerland

Thirty-four strains of enterotoxin-producing Staphylococcus aureus obtained from milk samples of 34 dairy cows suffering from mastitis from 34 different farms in north-east Switzerland were identified and further characterized by pheno- and genotypic methods. This included the identification of staphylococcal enterotoxin (SE) types, an antibiotic resistance testing, the appraisal of hemolysis, the egg yolk reaction, the detection of the clumping factor and protein A by means of a latex agglutination, the PCR amplification of a S. aureus specific part of the gene encoding the 16S-23S rRNA "intergenic spacer" region and a species specific part of the 23S rRNA-gene, the PCR amplification of the clumping factor (clfA) gene, the X region and the IgG-binding region of the protein A (spa) gene, the coagulase (coa) gene and additionally a macrorestriction analysis of the chromosomal DNA. Within the 26 cultures which formed a single SE, there were 23 SEC- and three SED-formers. Eight cultures were SEAD formers. It was remarkable that 22 SEC formers were also positive for TSST-1. Eighteen of the 23 SEC-formers could be classified as being of the same phenotype. Most of the cultures of one enterotoxin type also showed a great uniformity in the size and number of repeats of the X region as well as in the size of the IgG-binding region of protein A gene and in the size of the coagulase gene. Macrorestriction analysis revealed 11 PFGE patterns. These were in part only different from each other in a few fragments and thus displayed close clonal relations. The results of the present investigation show that a broad distribution of identical or closely related enterotoxin-producing S. aureus clones seem to contribute to the bovine mastitis problem in north-east Switzerland.