spa typing and enterotoxin gene profile of Staphylococcus aureus isolated from bovine raw milk in Korea

Staphylococcus aureus is a major etiological pathogen of bovine mastitis, which triggers significant economic losses in dairy herds worldwide. In this study, S. aureus strains isolated from the milk of cows suffering from mastitis in Korea were investigated by spa typing and staphylococcal enterotoxin (SE) gene profiling. Forty-four S. aureus strains were isolated from 26 farms in five provinces. All isolates grouped into five clusters and two singletons based on 14 spa types. Cluster 1 and 2 isolates comprised 38.6% and 36.4% of total isolates, respectively, which were distributed in more than four provinces. SE and SE-like toxin genes were detected in 34 (77.3%) isolates and the most frequently detected SE gene profile was seg, sei, selm, seln, and selo genes (16 isolates, 36.3%), which was comparable to one of the genomic islands, Type I νSaβ. This is a first report of spa types and the prevalence of the recently described SE and SE-like toxin genes among S. aureus isolates from bovine raw milk in Korea. Two predominant spa groups were distributed widely and recently described SE and SE-like toxin genes were detected frequently.     

PCR-based detection of genes encoding virulence determinants in Staphylococcus aureus from bovine subclinical mastitis cases

The present study was carried out to genotypically characterize Staphylococcus aureus (S. aureus) isolated from bovine mastitis cases. A total of 37 strains of S. aureus were isolated during processing of 552 milk samples from 140 cows. The S. aureus strains were characterized phenotypically, and were further characterized genotypically by polymerase chain reaction using oligonucleotide primers that amplified genes encoding coagulase (coa), clumping factor (clfA), thermonuclease (nuc), enterotoxin A (entA), and the gene segments encoding the immunoglobulin G binding region and the X region of protein A gene spa. All of the isolates yielded an amplicon with a size of approximately 1,042 bp of the clfA gene. The amplification of the polymorphic spa gene segment encoding the immunoglobulin G binding region was observed in 34 isolates and X-region binding was detected in 26 isolates. Amplification of the coa gene yielded three different products in 20, 10, and 7 isolates. The amplification of the thermonuclease gene, nuc, was observed in 36 out of 37 isolates. All of the samples were negative for the entA gene. The phenotypic and genotypic findings of the present strategies might provide an understanding of the distribution of the prevalent S. aureus clones among bovine mastitis isolates, and might aid in the development of steps to control S. aureus infections in dairy herds.     

In vitro susceptibility of Staphylococcus aureus strains isolated from cows with subclinical mastitis to different antimicrobial agents

Sensitivity to commercial teat dips (nonoxinol-9 iodine complex and chlorhexidine digluconate) of 56 Staphylococcus (S.) aureus strains isolated from quarter milk samples of various German dairy herds treated with different teat dipping schemes was investigated in this study. The minimum inhibitory concentration was determined using a broth macrodilution method according to the German Veterinary Association guidelines. The main objective of the current study was to induce in vitro resistance induction of S. aureus to chemical disinfectants. Ten different strains were repeatedly passed ten times in growth media with sub-lethal concentrations of disinfectants. Nine strains showed a significant reduction in susceptibility to the nonoxinol-9 iodine complex but only one strain developed resistance to chlorhexidine digluconate. Stability of the acquired resistance was observed in all S. aureus strains adapted to the nonoxinol-9 iodine complex and chlorhexidine digluconate. In contrast, simultaneous resistance to different antibiotics was not observed in any of the ten investigated S. aureus strains. However, the isolates exhibited a high degree of resistance to penicillin G. Based on these results, resistance of S. aureus to chemical disinfectants may be more likely to develop if the chemicals are used at concentrations lower than that required for an optimal biocidal effect.     

Difference in virulence between Staphylococcus aureus isolates causing gangrenous mastitis versus subclinical mastitis in a dairy sheep flock

Staphylococcus aureus mastitis in dairy sheep ranges from subclinical mastitis to lethal gangrenous mastitis. Neither the S. aureus virulence factors nor the host-factors or the epidemiological events contributing to the different outcomes are known. In a field study in a dairy sheep farm over 21 months, 16 natural isolates of S. aureus were collected from six subclinical mastitis cases, one lethal gangrenous mastitis case, nasal carriage from eight ewes and one isolate from ambient air in the milking room. A genomic comparison of two strains, one responsible for subclinical mastitis and one for lethal gangrenous mastitis, was performed using multi-strain DNA microarrays. Multiple typing techniques (pulsed-field-gel-electrophoresis, multiple-locus variable-number, single-nucleotide polymorphisms, randomly amplified polymorphic DNA, spa typing and sas typing) were used to characterise the remaining isolates and to follow the persistence of the gangrenous isolate in ewes’ nares. Our results showed that the two strains were genetically closely related and they shared 3 615 identical predicted open reading frames. However, the gangrenous mastitis isolate carried variant versions of several genes (sdrD, clfA-B, sasA, sasB, sasD, sasI and splE) and was missing fibrinogen binding protein B (fnbB) and a prophage. The typing results showed that this gangrenous strain emerged after the initial subclinical mastitis screening, but then persisted in the flock in the nares of four ewes. Although we cannot dismiss the role of host susceptibility in the clinical events in this flock, our data support the hypothesis that S. aureus populations had evolved in the sheep flock and that S. aureus genetic variations could have contributed to enhanced virulence.     

Expression and lytic efficacy assessment of the Staphylococcus aureus phage SA4 lysin gene

Treatment of bovine mastitis caused by Staphylococcus (S.) aureus is becoming very difficult due to the emergence of multidrug-resistant strains. Hence, the search for novel therapeutic alternatives has become of great importance. Consequently, bacteriophages and their endolysins have been identified as potential therapeutic alternatives to antibiotic therapy against S. aureus. In the present study, the gene encoding lysin (LysSA4) in S. aureus phage SA4 was cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clone revealed a single 802-bp open reading frame encoding a partial protein with a calculated mass of 30 kDa. Results of this analysis also indicated that the LysSA4 sequence shared a high homology with endolysin of the GH15 phage and other reported phages. The LysSA4 gene of the SA4 phage was subsequently expressed in Escherichia coli. Recombinant LysSA4 induced the lysis of host bacteria in a spot inoculation test, indicating that the protein was expressed and functionally active. Furthermore, recombinant lysin was found to have lytic activity, albeit a low level, against mastitogenic Staphylococcus isolates of bovine origin. Data from the current study can be used to develop therapeutic tools for treating diseases caused by drug-resistant S. aureus strains.     

Efficacy of specific egg yolk immunoglobulin (IgY) to bovine mastitis caused by Staphylococcus aureus

The objective of this study was to estimate the efficacy of specific egg yolk immunoglobulin (IgY) to bovine mastitis caused by Staphylococcus aureus. Eighteen lactating cows with clinical mastitis and 18 lactating cows with experimental mastitis (1 quarter per cow) were randomly assigned to three treatments: IgY (20mg/ml) infusion, penicillin (100mg/ml) infusion and no infusion. Treatments for clinical mastitis and experimental mastitis were performed by a 6-day course of intramammary infusion with a dosage of 10ml at an interval of 12h. Milk samples were collected at morning milking time for testing color, clot, somatic cell counts (SCC) and bacterial count. For most of the cows treated with IgY and penicillin, the milk color and clot recovered to normal form during the therapy course. The milk SCCs and bacterial counts of treated cows decreased compared to those of untreated cows (p<0.05). The cure rates by IgY for experimental and clinical mastitis were 83.3% and 50%, respectively, and those by penicillin were 66.7% and 33.3%, respectively. These results showed the potential of specific IgY to be an alternative therapy for mastitis caused by S. aureus.     

Associations between bacterial genotype and outcome of bovine clinical Staphylococcus aureus mastitis

Background

Staphylococcus aureus is an important cause of clinical mastitis in dairy cows worldwide. The cure rate after antimicrobial treatment of clinical S. aureus mastitis is very variable due to both cow and bacterial factors. Studies have shown that bacterial genotype might affect short-term bacteriological and clinical cure, but the long-term outcome has been less studied. The objectives of this study were to investigate associations between bacterial genotype and long-term outcome of veterinary-treated clinical mastitis (VTCM) caused by S. aureus during a follow-up period of 120 days and to study genotype variation among Swedish S. aureus isolates. S. aureus isolates from cases of VTCM were genotyped by pulsed-field gel electrophoresis. Long-term outcome measurements used were somatic cell count (SCC), additional diagnoses of VTCM, milk yield and culling. Isolates were classified into clusters (>80% similarity) and pulsotypes (100% similarity). Clusters and pulsotypes were grouped according to occurrence. Multivariable mixed-effect linear regression models including cow and bacterial factors with possible influence on SCC or milk yield were used to calculate differences in SCC or milk yield between groups. Additional outcome measures were calculated using a test of proportions.

Results

The isolates (n = 185) were divided into 18 clusters and 29 pulsotypes. Two pulsotypes were classified as common, and were found in 64% of the cases of VTCM. Remaining isolates were classified as less common or rare pulsotypes. The distribution was similar at cluster level. Outcome was calculated from follow-up data on 111 cows. Significantly lower SCC during the follow-up period was found in cows infected with common clusters compared to in cows infected with less common/rare clusters. The proportion of cows with SCC <200 000 cells/ml during the whole follow-up period was significantly higher in the group common clusters than in the group less common/rare clusters. Bacterial genotype did not influence the other outcome parameters.

Conclusions

In Sweden, two S. aureus pulsotypes, identified in about 64% of clinical S. aureus cases, were widespread. Cows infected with the common genotypes had significantly lower SCC during 120 days after treatment compared to cows infected with less common or rare genotypes.     

金黄色葡萄球菌融合肠毒素的构建及高效表达

以食物中毒性金黄色葡萄球菌毒素为研究对象,利用PCR方法分别扩增出金黄色葡萄球菌肠毒素SEB1、SEB2、SED1、SED2、SEE基因片段。将克隆得到的5个毒素基因片段采用柔性的Linker序列(Gly4Ser)3进行串联(SEB1-SEB2-SED1-SED2-SEE),通过重叠延伸PCR法扩增出了融合毒素T,目的基因全长1 835 bp,测序结果同源性达99%。将融合基因克隆到pET-28a( )原核表达载体,转化大肠杆菌后成功表达了融合蛋白,蛋白主要以包涵体形式存在,占菌体总蛋白的29.6%。     

金黄色葡萄球菌及其肠毒素等指标在发酵牛肉加工过程中变化规律的研究

作者对自然状态发酵牛肉、接种金黄色葡萄球菌的发酵牛肉和接种发酵剂(植物乳杆菌、戊糖片球菌)与金黄色葡萄球菌的发酵牛肉中金黄色葡萄球菌及其肠毒素等指标在加工过程中的变化规律做了研究。研究结果表明,自然发酵牛肉中没有金黄色葡萄球菌及其肠毒素;接种发酵剂和金黄色葡萄球菌的发酵牛肉中金黄色葡萄球菌先是上升然后快速下降,且金黄色葡萄球菌肠毒素为阴性;接种金黄色葡萄球菌的发酵牛肉中金黄色葡萄球菌及其肠毒素都显著高于接种发酵剂的发酵牛肉(P＜0.05)。由试验结果可知,发酵剂植物乳杆菌和戊糖片球菌对金黄色葡萄球菌有很强的抑制作用。     

Genotypic characterization of Staphylococcus aureus isolated from bovines, humans, and food in Indonesia

The present study determined the genetic relationships between 41 Staphyloccocus (S.) aureus isolates from bovines, humans, and food using a single enzyme amplified fragment length polymorphism (AFLP) technique. We evaluated the prevalence of staphylococcal enterotoxin (SE) genes and other virulence gene determinants by PCR. The identification of S. aureus was based on culturing and biochemical tests, and by amplifying a specific section of the 23S rRNA gene. PCR amplification of the SE genes (sea, seb, sec, see, seg, seh, and sei) singly or in combination was observed. Most isolates of bovine origin harbored hla (84%) and cap5 (74%), while most isolates from humans harbored hla (73%), cap8 (91%), and fnbA (100%). Strains from food sources were positive for hla (100%), cap5 (100%), and cap8 (64%) unlike isolates from humans or bovines. A single enzyme AFLP analysis revealed a correlation between AFLP clusters of some strains and the source of the isolates The genotypic results of the present study might help to better understand the distribution of prevalent S. aureus clones among humans, bovines, and food and will help control S. aureus infections in Indonesia.     

Genetic basis of penicillin resistance of S. aureus isolated in bovine mastitis

Background

The blaZ gene encoding penicillin resistance can be located either chromosomally or on plasmids. The aim of this study was to investigate the genetic relationships and to determine the location of the blaZ gene in S. aureus isolated in bovine mastitis in Finland and Sweden.

Methods

Seventy-eight β-lactamase positive S. aureus isolates from bovine mastitis (34 from Finland and 44 from Sweden) were included in the study. The localization of blaZ gene was determined by Southern blotting. The blaZ genes of the isolates were sequenced and the sequences were translated to beta-lactamase proteins and further grouped as different protein signatures. The isolates and, as control, 33 Swedish and 36 Finnish beta-lactamase negative isolates were typed with pulsed-field gel electrophoresis (PFGE).

Results

In 26 out of 34 Finnish isolates (76.5%) and in 25 out of 44 Swedish isolates (56.8%) the blaZ gene was localized on a plasmid. Six different protein signatures were found. One signature was found only in four Swedish isolates, but all other signatures were found both in Finnish and Swedish isolates. The PFGE results revealed a diversity of S. aureus clones. The protein signatures were not clearly associated with certain pulsotypes.

Conclusions

The plasmid location of the blaZ gene was not statistically significantly more common in Finland than in Sweden, and hence does not explain the higher proportion of penicillin-resistant isolates of S. aureus causing bovine mastitis in Finland compared to Sweden.     

Bovine-associated MRSA ST398 in The Netherlands

During routinely screening (50.000 milk samples on an annual basis) 14 MRSA ST398 strains were identified in the period of January 2008 to September 2008 in 14 different dairy herds located in the provinces Overijssel and Gelderland, The Netherlands. Molecular analysis was performed by Cfr9I PFGE, ST398-specific diagnostic PCR, spa typing, SCCmec typing and Panton-Valentine Leukocidin (PVL) gene PCR. The molecular analyses of 14 MRSA (one MRSA strain per herd) strains revealed that all strains belong to ST398 with 3 closely related spa types (t011, t108 and t889, all commonly found in pigs) and carry 2 different SCCmec types, IVa and V. All MRSA strains were resistant to two or more classes of antibiotics and also PVL negative. The majority of farms (n = 9, 64%) harboured combined livestock with both cows and pigs present. Our study contributes to the growing evidence that MRSA ST398 is transmitted among various animal species and can be considered as an etiological agent of mastitis in dairy cows.     

金黄色葡萄球菌烈性噬菌体的分离鉴定和最佳保存方法研究

基于细菌rRNA区域序列的PCR技术,通过设计特异性引物从奶样中快速、准确地鉴定奶牛乳房炎主要致病菌金黄色葡萄球菌。采用金黄色葡萄球菌与挤奶前从奶牛乳房上洗刷下来的污水经过3次富集的方法,通过双层琼脂平板法,分离纯化得到了强裂解性的奶牛乳房炎主要致病菌金黄色葡萄球菌的噬菌体。本试验分离的是可收缩尾的噬菌体,其头部为二十面体,约50 nm×62.5 nm,尾部长约187.5 nm,尾宽约37.5 nm,有尾鞘、基板等结构。纯化后在双层琼脂平板上形成的噬菌斑大而整齐,边缘光滑,透明,直径约2 mm,具有明显的强裂解性噬菌体特性,通过测定,该噬菌体的最佳感染复数为0.01,效价大于10⁹ PFU/mL。对噬菌体的不同保存方法进行比较,结果表明,将噬菌体置于-80 ℃加入7% DMSO冷藏是较好的保存方法。     

Determination of staphylococcal exotoxins, SCCmec types, and genetic relatedness of Staphylococcus intermedius group isolates from veterinary staff, companion animals, and hospital environments in Korea

The Staphylococcus (S.) intermedius group (SIG) has been a main research subject in recent years. S. pseudintermedius causes pyoderma and otitis in companion animals as well as foodborne diseases. To prevent SIG-associated infection and disease outbreaks, identification of both staphylococcal exotoxins and staphylococcal cassette chromosome mec (SCCmec) types among SIG isolates may be helpful. In this study, it was found that a single isolate (one out of 178 SIG isolates examined) harbored the canine enterotoxin SEC gene. However, the S. intermedius exfoliative toxin gene was found in 166 SIG isolates although the S. aureus-derived exfoliative toxin genes, such as eta, etb and etd, were not detected. SCCmec typing resulted in classifying one isolate as SCCmec type IV, 41 isolates as type V (including three S. intermedius isolates), and 10 isolates as non-classifiable. Genetic relatedness of all S. pseudintermedius isolates recovered from veterinary staff, companion animals, and hospital environments was determined by pulsed-field gel electrophoresis. Strains having the same band patterns were detected in S. pseudintermedius isolates collected at 13 and 18 months, suggesting possible colonization and/or expansion of a specific S. pseudintermedius strain in a veterinary hospital.     

Dissimilarity of ccrAB gene sequences between methicillin-resistant Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus among bovine isolates in Korea

The sequences of the ccrAB genes from bovine-, canine- and chicken-originating methicillin-resistant Staphylococcus (S.) epidermidis (MRSE) and bovine methicillin-resistant Staphylococcus (S.) aureus (MRSA) were compared to investigate the frequency of intra-species horizontal transfer of the staphylococcal cassette chromosome mec (SCCmec) complex. Nineteen MRSE strains were isolated from bovine milk, chickens, and dogs, and their genetic characteristics were investigated by multilocus sequence typing and SCCmec typing. Among the animal MRSE strains, the most frequent SCCmec type was type IV, which consisted of the type B mec complex and ccrAB type 2. The ccrA2 and ccrB2 genes were sequenced from the bovine, chicken and canine MRSE strains and compared with those of the bovine MRSA strains. The sequences generally clustered as MRSA and MRSE groups, regardless of the animal source. Additionally, no bovine MRSE sequence was associated with the bovine MRSA groups. Although most of the bovine MRSE and MRSA isolates possessed SCCmec type IV sequences, our results suggest that the intra-species gene transfer of the SCCmec complex between bovine S. aureus and bovine S. epidermidis strains is not a frequent event.     

我国部分地区奶牛乳房炎病例中金黄色葡萄球菌血清型及毒力调查

为了解我国奶牛乳房炎病例中金黄色葡萄球菌(S.aureus)流行血清型和不同血清型菌株的毒力情况,本研究采用玻板凝集和双重PCR方法分别对北京、山西、内蒙古、山东、浙江和新疆奶牛乳房炎乳样中分离的191株S.aureus进行血清分型并从各血清型中随机选取100株菌以小白鼠进行毒力测定.结果表明:这些地区牛源S.aureus中,336PS型为流行血清型,占60.2％(115/191),CP8型占19.4％(37/191),CP5型占11.5％(22/191),未分型菌株占8.9％(17/191).毒力测定显示:336PS、CP8和CP5型菌株中强毒力菌株分别占65.1％、62.9％及61.9％,未分型菌株中强毒力菌株为22.2％.该调查为我国奶牛乳房炎S.aureus疫苗菌株的筛选提供了依据.     

Relationship of bovine NOS2 gene polymorphisms to the risk of bovine tuberculosis in Holstein cattle

Many studies suggest significant genetic variation in the resistance of cattle and humans to infection withMycobacterium bovis, the causative agent of zoonotic tuberculosis. The inducible nitricoxide synthase (iNOS which is encoded by the NOS2 gene) plays a key role in the immunologicalcontrol of a broad spectrum of infectious agents. This study aimed to investigate the influence of geneticvariations in the promoter of the NOS2 gene on bovine tuberculosis (bTB) susceptibility. Inthis study, the NOS2 genes of 74 bTB-infected Holstein cows and 90 healthy controls weregenotyped using PCR followed by nucleotide sequencing. Polymorphisms at rs207692718, rs109279434, rs209895548,rs385993919, rs433717754, rs383366213, rs466730386, rs715225976, rs525673647, rs720757654 and g.19958101T>Gin the promoter region of the NOS2 gene were detected. The g.19958101T>G SNP produced twodifferent conformation patterns (TT and TG) and the TG genotype was over-represented in the bTB group (20.27%)compared with the control group (2.22%). The TG genotype frequency of the g.19958101T>G variant wassignificantly higher in bTB cattle than in healthy controls (OR, 11.19; 95% CI, 2.47–50.73;P=0.0002). The G allele of the g.19958101T>G polymorphism was more frequent in bTB groupwhen compared to control group (10.14% versus 1.11%). Furthermore, the G allele was a risk factor for bTBsusceptibility (OR, 10.04; 95% CI, 2.26–44.65; P=0.0002). In conclusion, the g.19958101T>Gpolymorphism of the NOS2 gene may contribute to the susceptibility of Holstein cattle tobTB.     

牛源金黄色葡萄球菌凝固酶基因的分型

为了了解我国不同地区奶牛场金黄色葡萄球菌基因型的分布情况,采用凝固酶基因（Coa）扩增的分型方法,对在奶牛乳房炎奶样中所分离到的26株金黄色葡萄球菌进行了分型。结果显示,26株金黄色葡萄球菌共分为5个基因型,黑龙江省分离株以PCR 3型为主（10/16）。发现在同一牛群中的分离株可以存在相同的基因型,也可以存在不同的基因型;而不同牛群中的分离株也可属于同一基因型。这种结果提示,在某个或某些牛群中存在某一金黄色葡萄球菌的流行型,但也可能存在多个金黄色葡萄球菌的克隆。