Molecular analysis of Pseudomonas aeruginosa: epidemiological investigation of mastitis outbreaks in Irish dairy herds.

Pseudomonas aeruginosa is a pathogen in both humans and animals. This bacterium, most often associated with respiratory infections in cystic fibrosis patients, was found to be the causative agent in bovine mastitis outbreaks among 11 Irish dairy herds. Epidemiological findings suggested that the infection was spread to all herds by teat wipes that had been contaminated with this organism. Two molecular-typing strategies were used in an attempt to determine the genomic relationship(s), if any, of the P. aeruginosa strains isolated from the various herds and to verify whether the same strain was responsible for each outbreak. Thirty-six isolates from the mastitis outbreaks were tested and compared to fourteen clinical isolates from Cork University Hospital. With one exception, all outbreak-linked strains produced identical patterns when ribotyped with ClaI and PvuII enzymes. Eight of the clinical isolates gave the same ClaI ribotype pattern as the mastitis-causing strains. However, PvuII proved more discriminatory, with only the outbreak isolates producing identical patterns. Similar results were obtained with RW3A-primed DNA amplification fingerprinting, with all outbreak isolates except one displaying the same fingerprint array. The clinical strains produced several fingerprint patterns, all of which were different from those of the mastitis-causing isolates. Fine-resolution DNA fingerprinting with a fluorescence-labelled RW3A primer also identified a number of low-molecular-weight polymorphisms that would have remained undetected by conventional methods. These data support the view that the same P. aeruginosa strain was responsible for the mastitis outbreaks in all 11 herds.     

Molecular Analysis of Pseudomonas aeruginosa: Epidemiological Investigation of Mastitis Outbreaks in Irish Dairy Herds

Pseudomonas aeruginosa is a pathogen in both humans and animals. This bacterium, most often associated with respiratory infections in cystic fibrosis patients, was found to be the causative agent in bovine mastitis outbreaks among 11 Irish dairy herds. Epidemiological findings suggested that the infection was spread to all herds by teat wipes that had been contaminated with this organism. Two molecular-typing strategies were used in an attempt to determine the genomic relationship(s), if any, of the P. aeruginosa strains isolated from the various herds and to verify whether the same strain was responsible for each outbreak. Thirty-six isolates from the mastitis outbreaks were tested and compared to fourteen clinical isolates from Cork University Hospital. With one exception, all outbreak-linked strains produced identical patterns when ribotyped with ClaI and PvuII enzymes. Eight of the clinical isolates gave the same ClaI ribotype pattern as the mastitis-causing strains. However, PvuII proved more discriminatory, with only the outbreak isolates producing identical patterns. Similar results were obtained with RW3A-primed DNA amplification fingerprinting, with all outbreak isolates except one displaying the same fingerprint array. The clinical strains produced several fingerprint patterns, all of which were different from those of the mastitis-causing isolates. Fine-resolution DNA fingerprinting with a fluorescence-labelled RW3A primer also identified a number of low-molecular-weight polymorphisms that would have remained undetected by conventional methods. These data support the view that the same P. aeruginosa strain was responsible for the mastitis outbreaks in all 11 herds.     

Genotyping of Staphylococcus aureus isolated from humans, bovine subclinical mastitis and food samples in Argentina

The aim of the present study was to characterize genotypically 45 Staphylococcus aureus strains isolated from humans, bovine subclinical mastitis and food samples in Argentina by rep-PCR and PCR amplification of virulence genes. Resistances to various antibiotics could be observed for the human S. aureus, less pronounced for the bovine strains, but not for the eight S. aureus isolated from food samples. The strains could be classified genotypically by rep-PCR and by amplification of the genes encoding protein A, coagulase, clumping factor, the collagen adhesin domains A and B, capsular polysaccharide 5 and 8, the accessory gene regulator agr classes I to III, and the S. aureus gene regulator sae. rep-PCR analyses and the different gene patterns revealed that the strains could be divided into seven groups mostly matching with the origin of the isolates. The present study describes genotypic variations of S. aureus strains isolated from different origins in Argentina. The study provides a valuable insight into molecular specificities of this important pathogen.     

Protein expression by Streptococcus uberis in co-culture with bovine mammary epithelial cells

Three strains of Streptococcus uberis isolated from dairy cows with mastitis were co-cultured with a bovine mammary epithelial cell line (MAC-T) in Dulbecco's modified Eagle's medium without fetal bovine serum. Protein profiles from culture supernatants and bacterial pellets among different treatments were compared by electrophoresis. There were proteins induced or having increased expression in both supernatant and surface-associated samples from S. uberis co-cultured with MAC-T cells. Some of these proteins were recognized by antibodies in serum obtained from a cow infected by S. uberis . In supernatant samples, there were two distinct protein bands at 35 and 36.8 kDa for all three strains of S. uberis co-cultured with MAC-T cells. These two bands were absent when bacterial protein synthesis was inhibited by chloramphenicol. This study clearly indicates that bacterial protein expression was regulated in response to co-culture with mammary epithelial cells.     

Coagulase gene polymorphism of Staphylococcus aureus isolated from goat mastitis in Brazilian dairy herds

AIMS: To investigate the coagulase gene polymorphism of Staphylococcus aureus isolated from mastitic goat's milk. METHODS AND RESULTS: A typing procedure based on coagulase gene polymorphism was used to discriminate S. aureus isolated from goat mastitis. Thirty-six strains collected from goats belonging to herds from northern Ceara State and Serrana region of Rio de Janeiro State were analysed. Based on restriction fragment length polymorphism of 3' end coagulase gene, the goat strains were grouped into 11 types. In northern Ceara herds, the predominant type was found in 60% of the strains, while in the Serrana region herds the two most common accounting for 62.5% of the strains. CONCLUSIONS: The analysis of the coa gene proved to be useful for typing S. aureus from goat mastitis. Although the results showed that goats from the studied regions were infected by S. aureus strains harbouring more than one coagulase genotype, only one or two types predominated. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of the prevalent strains within a herd or region is a necessity. The important virulence factors could be identified in such strains and this information can then be used as a specific base to develop S. aureus mastitis control measures.     

Characterization of PauB, a Novel Broad-Spectrum Plasminogen Activator from Streptococcus uberis

A bovine plasminogen activator of atypical molecular mass ( approximately 45 kDa) from Streptococcus uberis strain SK880 had been identified previously (L. B. Johnsen, K. Poulsen, M. Kilian, and T. E. Petersen. Infect. Immun. 67:1072-1078, 1999). The strain was isolated from a clinical case of bovine mastitis. The isolate was found not to secrete PauA, a bovine plasminogen activator expressed by the majority of S. uberis strains. Analysis of the locus normally occupied by pauA revealed an absence of the pauA open reading frame. However, an alternative open reading frame was identified within the same locus. Sequence analysis of the putative gene suggested limited but significant homology to other plasminogen activators. A candidate signal peptide sequence and cleavage site were also identified. Expression cloning of DNA encoding the predicted mature protein (lacking signal peptide) confirmed that the open reading frame encoded a plasminogen activator of the expected size, which we have named PauB. Both native and recombinant forms of PauB displayed an unexpectedly broad specificity profile for bovine, ovine, equine, caprine, porcine, rabbit, and human plasminogen. Clinical and nonclinical field isolates from nine United Kingdom sites were screened for the pauB gene and none were identified as carrying it. Similarly, clinical isolates from 20 Danish herds were all found to encode PauA and not PauB. Therefore, PauB represents a novel but rare bacterial plasminogen activator which displays very broad specificity.     

华北地区牛源无乳链球菌的分离鉴定及生物学特性

【目的】了解华北地区牛源无乳链球菌的生物学特性。【方法】在2012到2015年间从内蒙古自治区、河北、北京等地隐性乳房炎557份奶牛乳样中分离、收集无乳链球菌。采用纸片扩散法和PCR的方法对这些菌株分别进行耐药谱测定、荚膜分子分型、表面蛋白基因及毒力因子的检测。【结果】无乳链球菌的分离率为5.03%,其药物敏感性与其他地区无明显差别。分离到的28株无乳链球菌均属于荚膜Ia型,且毒力基因基本相同并且其表面蛋白均属于未定型。【结论】华北不同地区的无乳链球菌有相似的药物敏感性和毒力基因。为奶牛乳房炎无乳链球菌疫苗的研制及药物防治提供理论依据。     

牛源多杀性巴氏杆菌的分离与初步鉴定

【目的】本研究旨在对引起犊牛呼吸道综合征的多杀性巴氏杆菌进行分离鉴定,分析其亲缘关系和毒力基因的分布情况。【方法】收集2017年8月至2018年4月疑似患有犊牛呼吸道综合征的病牛鼻拭子进行细菌分离培养,对菌落形态和染色疑似巴氏杆菌的菌株进行16S rRNA测序和血清型鉴定,选择巴氏杆菌7类23种毒力基因,筛查临床分离株的毒力基因的分布。【结果】从8个省份的237份病料中分离出31株多杀性巴氏杆菌,分离率为13.1%。16S rRNA测序分析表明31株A型多杀性巴氏杆菌属于同一亚群,其序列同源性与中国分离株HB01以及国外分离株USDA-ARS-USMARC-60712、USDA-ARS-USMARC-60214、ATCC 43137以及36950亲缘关系较近。对分离出的31株A型多杀性巴氏杆菌的7类共23种毒力基因鉴定,结果显示31株多杀性巴氏杆菌所携带的毒力因子大多分布在17–19个,且集中度较高。【结论】A型多杀性巴氏杆菌为犊牛呼吸道综合症的主要流行血清型,通过对多杀性巴氏杆菌的临床分离株进化树和毒力基因分析,内蒙古、黑龙江、新疆、山西以及河北的7株分离株进化来源于同一分支,且均缺失毒力基因tadD和hgbA及携带毒力基因hsf-1,提示着其亲缘关系可能与其携带的特定毒力基因存在一定相关性。该研究为犊牛呼吸道综合征的病原学调查和多杀性巴氏杆菌流行病学调查提供了参考数据。     

Genetic variation among Staphylococcus aureus strains from Norwegian bulk milk

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex.     

Characterization of Staphylococcus aureus strains involved in human and bovine mastitis

Staphylococcus aureus is one of the main etiological agents of mastitis in different mammalian species. At present, it is unknown whether strains isolated from human mastitis cases share phenotypic properties and genetic background with those obtained from animal mastitis cases. Therefore, the objective of this study was to characterize S. aureus strains isolated from women with lactational mastitis and to compare them with the strains responsible for bovine mastitis and noninfectious strains. All the strains were genotyped by both pulsed field gel electrophoresis and multilocus sequence typing and submitted to a characterization scheme that included diverse assays related to pathogenic potential and antibiotic resistance. Apart from siderophore production, no significant association was observed between the strains from bovine and human mastitis. Statistical differences between human- and bovine-mastitis-associated strains were detected for some traits and virulence determinants, such as the presence of prophages and cna and hlb genes, which were more frequently found within the bovine group. On the contrary, resistance to penicillin was significantly higher among strains isolated from human lactational mastitis, probably related to the common presence of the blaZ gene. A high genetic diversity was found among the strains involved in mastitis in breastfeeding women.     

Aureocin A70 production is disseminated amongst genetically unrelated Staphylococcus aureus involved in bovine mastitis

Aims: The main aim of this study was to analyse the genetic relationship amongst 46 Staphylococcus aureus Bac⁺ strains isolated in Brazil from 12 geographically distant dairy herds, including 34 isolates that produce the antimicrobial peptide aureocin A70. Methods and Results: The comparison of 46 Staph. aureus Bac⁺ strains was performed by pulsed‐field gel electrophoresis (PFGE). Thirteen different pulsotypes were identified, and the subtype A₁ was the most prevalent one. Nine strains belong to pulsotype F, the second most prevalent and mostly confined to a single herd. The PFGE patterns of the 34 Staph. aureus aureocin A70‐producers, isolated in Brazil, were also compared with those of strains isolated from bovine mastitis cases in Argentina and revealed that these strains are not genetically related. Conclusions: Although a previous study has suggested that a prevalent pulsotype of aureocin A70‐producer Staph. aureus involved in bovine mastitis is disseminated in Argentina, this does not occur in Brazil. Additionally, it was possible to demonstrate that closely related staphylococcal strains can produce distinct staphylococcins. Significance and Impact of the Study: This study corroborates the hypothesis of horizontal gene transfer of aureocin A70 genes amongst distinct staphylococcal strains involved in bovine mastitis, giving them a selective advantage when colonizing the mammary glands.     

Production of enterotoxins and toxic shock syndrome toxin by Staphylococcus aureus isolated from bovine mastitis in Brazil

The production of toxic shock syndrome toxin 1 (TSST-1) and enterotoxins (SE) A, B, C and D by bovine mastitis isolates of Staphylococcus aureus was evaluated by immunodiffusion using the Optimum-Sensitivity Plate method. S. aureus strains were isolated from bovine mastitis in 23 dairy herds in the state of Minas Gerais, Brazil, during 1994-9. Of 127 isolates, 83 (65.04%) produced one or several toxins, and among them production of SE was found in 54 (43.0%) isolates, of which 1138 (29.09%) secreted enterotoxin identified as type D. TSST-1 was found in 5829 (45.723.0%) isolates.     

Genes coding for virulence determinants of Campylobacter jejuni in human clinical and cattle isolates from Alberta, Canada, and their potential role in colonization of poultry

Forty nine Campylobacter jejuni isolates from cattle feces collected from Alberta feedlots and 50 clinical C. jejuni isolates from people in Alberta were tested for the presence of 14 genes encoding putative virulence factors by PCR. These included genes implicated in adherence and colonization (flaC, cadF, docC, racR, jlpA, peb1, and dnaJ), invasion (virB11, ciaB, pldA, and iamA) and protection against harsh conditions (htrA, cbrA, and sodB). The genes examined were widely distributed in both the cattle fecal isolates and the human isolates. Of the isolates tested, 67% contained all of the genes except virB11. The cadF gene was found in 100% of the isolates tested. The presence or absence of virulence-associated genes was not associated with the ability of the organism to colonize birds. All of the C. jejuni isolates used to challenge birds were able to colonize the animals regardless of virulence gene profile. While some diversity in the profile of the occurrence of virulence-associated genes in C. jejuni exists, the distribution of these putative virulence-associated genes isolates from feedlot cattle feces and humans in Alberta was similar. In addition it was not possible to predict the ability of the selected isolates to colonize young chicks based on the presence of these genes coding for virulence determinants.     

Genomic and proteomic characterization of Staphylococcus aureus mastitis isolates of bovine origin

Staphylococcus aureus colonizes and infects humans as well as animals. In the present study, 17 S. aureus strains isolated from cows suffering from mastitis were characterized. The well-established multilocus sequence typing (MLST) technique and a diagnostic microarray covering 185 S. aureus virulence and resistance genes were used for genetic and epidemiological analyses. Virulence gene expression studies were performed by analyzing the extracellular protein pattern of each isolate on 2-D gels. By this way, a pronounced heterogeneity of the extracellular proteome between the bovine isolates has been observed which was attributed to genome plasticity and variation of gene expression. Merely 12 proteins were expressed in at least 80% of the isolates, i.e. Atl, Aur, GlpQ, Hla, LtaS, Nuc, PdhB, SAB0846, SAB2176, SAB0566, SspA, and SspB forming the core exoproteome. Fifteen extracellular proteins were highly variably expressed and only present in less than 20% of the isolates. This includes the serine proteases SplB, C, and F, and the superantigens SEC-bov, SEL and TSST-1. Compared to human isolates we identified at least six proteins with significantly different expression frequencies. While SAB0846 was expressed more frequently in bovine isolates, LytM, EbpS, Spa, Geh, and LukL1 were seen less frequently in these isolates.     

Genomic Comparative Study of Bovine Mastitis Escherichia coli

Escherichia coli, one of the main causative agents of bovine mastitis, is responsible for significant losses on dairy farms. In order to better understand the pathogenicity of E. coli mastitis, an accurate characterization of E. coli strains isolated from mastitis cases is required. By using phylogenetic analyses and whole genome comparison of 5 currently available mastitis E. coli genome sequences, we searched for genotypic traits specific for mastitis isolates. Our data confirm that there is a bias in the distribution of mastitis isolates in the different phylogenetic groups of the E. coli species, with the majority of strains belonging to phylogenetic groups A and B1. An interesting feature is that clustering of strains based on their accessory genome is very similar to that obtained using the core genome. This finding illustrates the fact that phenotypic properties of strains from different phylogroups are likely to be different. As a consequence, it is possible that different strategies could be used by mastitis isolates of different phylogroups to trigger mastitis. Our results indicate that mastitis E. coli isolates analyzed in this study carry very few of the virulence genes described in other pathogenic E. coli strains. A more detailed analysis of the presence/absence of genes involved in LPS synthesis, iron acquisition and type 6 secretion systems did not uncover specific properties of mastitis isolates. Altogether, these results indicate that mastitis E. coli isolates are rather characterized by a lack of bona fide currently described virulence genes.     

Virulence properties of uropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> isolated from children with urinary tract infection in Korea

Urinary tract infections (UTIs) are one of the most common types of bacterial infection in humans in various parts of the world and are caused mainly by uropathogenic Escherichia coli (UPEC). A total of 58 UPEC isolates from urine were characterized by serotyping and pulsed-field gel electrophoresis (PFGE). The majority of the UPEC strains belonged to serogroups O2 and O6. The UPEC strains were grouped under different pulsotypes and majority of them belonged to serogroups O2 and O6. Among the 14 virulence factors considered, 13 were present in various serogroups. The virulence genes fimH and sfa were present in all the isolates while none of the isolates carried lt-1. The strains exhibited 36 different virulence patterns, of which 11, referred to as UP (UPEC pattern) 1 to UP 11 were most common. Antibiotic resistance profiling of the UPEC isolates revealed that the serogroups O2 and O6 contain the highest number of resistant strains. The data from the current study depicting the distribution of UPEC strains among various serogroups and pulsotypes, and the occurrence of virulence genes and antibiotics resistance offer useful information on the epidemiological features of UPEC in Korea for the enhanced surveillance of potential emergence of UPEC.     

Incubation of Streptococcus uberis with extracellular matrix proteins enhances adherence to and internalization into bovine mammary epithelial cells

Two strains of Streptococcus uberis (UT 888 and UT 366) isolated from cows with clinical mastitis were co-cultured with bovine mammary epithelial cells (MAC-T) with and without laminin, fibrinogen, fibronectin or collagen. Incubation of S. uberis with extracellular matrix proteins (ECMPs) increased adherence to and internalization into MAC-T cells. Both strains of S. uberis exhibited greater adherence when co-cultured in the presence of collagen than with any other ECMP. However, adherence was always higher when strains were co-cultured with ECMP than in medium alone. S. uberis UT 888 adhered better to MAC-T cells than S. uberis UT 366. The influence of ECMPs on bacterial internalization into MAC-T cells was similar to adherence, however, differences among ECMPs were less noticeable. S. uberis UT 888 had a higher internalization index than S. uberis UT 366. It is possible that ECMPs induce or up-regulate proteins that selectively adhere to ECMPs which could serve as a bridge between the eukaryotic cell and the bacterial pathogen that leads to internalization of the ECMP-bound pathogen into the mammary epithelial cell.     

Antimicrobial susceptibility and genetic relatedness of bovine Stenotrophomonas maltophilia isolates from a mastitis outbreak

Aims: To evaluate the antimicrobial susceptibility and genetic relatedness of 11 Stenotrophomonas maltophilia isolates from an outbreak of bovine clinical mastitis in one herd and two isolates from two separate mastitis cases in two other herds. Methods and Results: Thirteen S. maltophilia isolates were obtained from milk samples from 11 cows from three dairy herds in Japan during 2008. We tested their susceptibility to 14 antimicrobials by broth microdilution and identified their genotypes by enterobacterial repetitive intergenic consensus 2 (ERIC2)‐PCR. Every cow had acute mild mastitis (slightly watery foremilk with flakes) without systemic symptoms and all resolved within 3–5 weeks of diagnosis. Eleven of the 13 isolates derived from nine cows in one herd over a 7‐month period exhibited a closely related ERIC2 type (A). The remaining two isolates derived from two cows from two other herds exhibited two distinct ERIC2 types (B and C). Most of the 13 isolates exhibited susceptibility to trimethoprim‐sulfamethoxazole, chloramphenicol, minocycline and levofloxacin; however, they were resistant to four β‐lactams, kanamycin, gentamicin and oxytetracycline. They were intermediate to enrofloxacin. Conclusions: Eleven closely related S. maltophilia isolates were involved in a herd outbreak of mastitis to some extent. Bovine S. maltophilia isolates exhibited resistance to many classes of antimicrobials. Significance and impact of study: This is a rare report of a herd outbreak of bovine mastitis involving closely related S. maltophilia isolates.     

Riboprint and virulence gene patterns for Bacillus cereus and related species

A total of 72 Bacillus cereus strains and 5 Bacillus thuringiensis strains were analyzed for their EcoRI ribogroup by ribotyping and for the presence or absence of seven virulence-associated genes. From these 77 strains, 42 distinctive ribogroup were identified using EcoRI, but the two species could not be discriminated by their EcoRI ribogroup. The 77 strains were also examined by PCR for the presence of seven virulence-associated genes, cerAB, pi-plc, entFM, bceT, hblA, hblC, and hblD. All five Bacillus thuringiensis strains were positive for these genes. Although differences in the patterns of virulence genes were observed among the different B. cereus strains, within any given ribogroup the patterns of the seven virulence genes was the same. Pulsed-field gel electrophoresis (PFGE) analysis in combination with available chromosomal maps for a selected group of B. cereus strains revealed significant differences in their chromosome size and the placement of virulence genes. Evidence for significant rearrangements within the B. cereus chromosome suggests the mechanism through which the pattern of virulence-associated genes varies. The results suggest linkage between ribogroups and virulence gene patterns as well as no apparent containment of the latter within any particular species boundary.     

A novel IS-like element frequently inserted in a putative virulence regulator in bovine mastitis isolates of Streptococcus dysgalactiae

Streptococcus dysgalactiae, a Lancefield group C streptococcus, is commonly isolated from bovine mastitis. We recently identified a putative regulon in two S. dysgalactiae strains, 8215 and Epi9, consisting of two consecutive genes, dmg and dem, coding for a possible regulatory protein and an M-like protein with fibrinogen- and IgG-binding-properties, respectively. During these studies a short sequence homologous to an IS element was found to be inserted in the dmg gene of strain 8215. The present investigation describes the complete sequence of this IS-like element, named ISSdy1, which consists of 1218 bp and contains two ORFs, flanked by imperfect repeats. The nucleotide sequence of the IS-like element shows 82% identity to the previously reported sequence of IS199 from Streptococcus mutans V403. The deduced amino acid sequences of the ORFs also revealed high homology to transposases from IS elements in Enterococcus faecium, Escherichia coli, and Shigella dysenteriae, all belonging to the IS3 family. We studied the distribution of ISSdy1 in 57 S. dysgalactiae isolates using PCR analysis with specific primers derived from the IS element. Ninety-eight percent of the isolates contained the ISSdy1 element. Surprisingly, in the majority of studied strains a copy of the IS-like element was found to be inserted in the dmg gene, a putative virulence regulator.