Identification of staphylococci from bovine mastitis and an examination of their susceptibility to antibiotics and beta-lactamase production

Strains of Staphylococcus species isolated from bovine mastitic milk at 66 dairy farms in Japan during the period from November 1988 to May 1989 were identified, and examined for their drug susceptibility and beta-lactamase production in order to clarify an epidemiological aspect of bovine mastitis caused by staphylococci. The results of bacteriological identification showed that the most predominant species was S. xylosus. Other major species isolated were S. aureus, S. sciuri and S. hyicus. Thirty-eight (71.7%) isolates of S. xylosus, 21 (45.7%) of S. aureus and 5 (71.4%) of S. epidermidis were positive for beta-lactamase production. Most of the beta-lactamase-producers of S. aureus were classified as high producers, although all of the beta-lactamase-positive S. xylosus isolates remained to be low producers. All isolates of S. aureus were sensitive to methicillin and cloxacillin at 6.25 micrograms/ml and 1.56 micrograms/ml, respectively, and none of methicillin-resistant S. aureus were detected. Isolates of other species were considered to be susceptible to 6 beta-lactams, in contrast to human isolates, but antibacterial activities of penicillin G and ampicillin were affected more strongly by beta-lactamase than those of methicillin, cloxacillin, cefazolin and cefoperazone.     

In vitro antibiotic sensitivity test of Staphylococcus aureus isolated from mastitic milk

The antibiotic susceptibility of 2297 strains of Staphylococci aureus isolated from mastitic milk has been tested. The percentage of strains of S. aureus "resistant" to penicillin was 16.5%. Approximately 97% of these strains were susceptible both to tetracyclines and sulfonamides. The frequency of "resistant" strains is in good agreement with the results from a comparable survey done ten years ago. In this investigation, however, the strains of S. aureus showed great variation in their sensitivity to penicillin according to their geographical origin. Thus, the percentage "resistant" strains within counties varied from a minimum of 9.26 to a maximum of 53.05.     

New findings on the biotyping of Staphylococcus aureus isolated during milk processing]

A detailed analysis of biotypes of Staphylococcus aureus, as related to their origin and enterotoxigenicity, was performed, using 432 strains isolated from bulk milk, milking machines, quarter milk samples collected from mastitic cows, and cowherds and milkers. All strains coagulated rabbit blood plasma and produced thermonuclease (Tab. I). Human strains differed from bovine ones mostly in the production of alpha-haemolysin (94%) and fibrinolysin (66%). Biotypes C1 (35%) and C2 (38%) dominated clearly among the strains isolated from quarter milk samples. The findings of 13% of biotype A and 8% of biotype D suggest that other sources of udder infections than mastitic cows were involved. Almost 19% of human strains and two strains isolated from quarter milk samples were identified as the recently defined type G. The production of enterotoxins (Tab. III) of was associated mostly with strains of human origin (69%) and with biotypes G (35%) and A (31%). Three enterotoxigenic strains belonged to the biotype B and one strain was not classifiable.     

Prevalence of coagulase gene polymorphism in Staphylococcus aureus isolates causing bovine mastitis.

This study was conducted to investigate polymorphism of the coagulase gene of Staphylococcus aureus causing bovine mastitis. One hundred eighty-seven strains of S. aureus were isolated from bovine mastitic milk samples obtained from 187 different Danish dairy farms. The isolates were characterised for restriction fragment length polymorphism (RFLP) of the coagulase gene. A variable region of the coagulase gene was amplified using the polymerase chain reaction (PCR) followed by AluI restriction enzyme digestion. A total of 15 different RFLP patterns were observed. The predominant pattern was found in 35% of the isolates. The ease of analysing coagulase gene polymorphisms among a large number of strains, and the multiple distinct polymorphic patterns generated, supports the use of this technique in epidemiological investigations of bovine mastitis. The predominating variants may have predelection for causing intramammary infections.     

Efficacy of targeted 5-day combined parenteral and intramammary treatment of clinical mastitis caused by penicillin-susceptible or penicillin-resistant Staphylococcus aureus

Combined parenteral and intramammary treatment of mastitis caused by Staphylococcus aureus was compared to parenteral treatment only. Cows with clinical mastitis (166 mastitic quarters) caused by S. aureus treated by veterinarians of the Ambulatory Clinic of the Faculty of Veterinary Medicine during routine farm calls were included. Treatment was based on in vitro susceptibility testing of the bacterial isolate. Procaine penicillin G (86 cases due to beta-lactamase negative strains) or amoxycillin-clavulanic acid (24 cases due to beta-lactamase positive strains) was administered parenterally and intramammarily for 5 days. Efficacy of treatments was assessed 2 and 4 weeks later by physical examination, bacteriological culture, determination of CMT, somatic cell count and NAGase activity in milk. Quarters with growth of S. aureus in at least one post-treatment sample were classified as non-cured. As controls we used 41 clinical mastitis cases caused by penicillin-susceptible S. aureus isolates treated with procaine penicillin G parenterally for 5 days and 15 cases due to penicillin-resistant isolates treated with spiramycin parenterally for 5 days from the same practice area. Bacteriological cure rate after the combination treatment was 75.6% for quarters infected with penicillin-susceptible S. aureus isolates, and 29.2% for quarters infected with penicillin-resistant isolates. Cure rate for quarters treated only parenterally with procaine penicillin G was 56.1% and that for quarters treated with spiramycin 33.3%. The difference in cure rates between mastitis due to penicillin-susceptible and penicillin-resistant S. aureus was highly significant. Combined treatment was superior over systemic treatment only in the beta-lactamase negative group.     

Immunobiology of bovine mammary gland: apoptosis of somatic cells in milk during naturally occurring mastitis

Mastitis remains a major cause of economic losses in dairy herds. It is believed, that the enhancement of natural defense mechanisms in mammary gland may be helpful in the treatment of bovine mastitis. Our study was designed to assess the apoptosis of leukocytes isolated from bovine milk during subclinical staphylococcal mastitis. Milk samples were collected from cows naturally infected with one pathogen--Staphylococcus aureus and from animals with mastitis caused by several pathogens, including S. aureus. It has been determined that the rate of apoptosis was lower in mastitic milk, as compared with control samples, although varied significantly between groups. High percentage of apoptotic cells was detected in milk with high counts of pathogenic bacteria. In all groups the rate of apoptosis was dependent on the bacterial load, although various correlations were identified. Thus, it is postulated, that the rate of apoptosis of somatic cells in mastitic milk is related to the etiology of infection and is determined by the bacterial load.     

Molecular typing of Staphylococcus aureus based on PCR-RFLP of coa gene and RAPD analysis

The aim of this study was molecular identification of S. aureus strains isolated from mastitic milk samples and establishing the genetic relationship between strains isolated from cows belonging to the same herd. In all 43 isolated strains the gap gene (930 bp) was amplified, which enabled their affiliation to the Staphylococcus genus to be established. PCR-RFLP with AluI endonuclease of the gap gene as well as nuc (450 bp) and coa (1130 bp) gene amplification allowed precise S. aureus species identification. One hundred percent of the genetic relationship between strains was established via RAPD-PCR and coa-typing.     

Biochemical and serologic characterization of Klebsiella strains from bovine mastitis and the environment of the dairy cow

Klebsiella strains isolated from the cow and its environment were biochemically and serologically characterized and evaluated for their susceptibility to normal bovine serum. Thirty-one different biotypes of Klebsiella were identified among 288 cattle and environmental strains. Of these, 56.2% were indole-positive, a greater percentage than expected for Klebsiella. Biotypes 1/1/1 and 5/1/1, most frequently isolated and constituting about 37% of the total isolates, would be considered K pneumoniae by standard biochemical typing procedures. Of 65 cattle and environmental strains studied serologically, 11 serotypes, 14 biotypes, and 29 bioserotypes were identified, indicating the diversity of Klebsiella strains present in the herd. When strains from mastitic milk (n = 19) and the environment (n = 22) were compared, no bioserotype distinction or grouping that related to isolation source was obvious. The predominant bioserotype from both sources was 5/1/1-K35 (21.0% and 22.7% of the strains from mastitic milk and the environment, respectively). The growth inhibition by bovine serum of strains isolated from mastitic milk, the environment, and udder skin was similar. However, strains isolated from the mouth and rectum of the cow were significantly (P less than 0.05) more inhibited by serum.     

Induction of anti-phagocytic surface properties of Staphylococcus aureus from bovine mastitis by growth in milk whey

The respiratory burst activity of bovine polymorphonuclear (PMN) cells in response to milk whey- and TSB-grown S. aureus strains isolated from bovine mastitis was studied in whole blood chemiluminescence (CL) and in a CL system with purified bovine neutrophils. In both cases milk whey-grown S. aureus strains elicited significantly less CL than homologous strains grown in TSB. Ingestion of milk whey-grown S. aureus strains by bovine neutrophils was also considerably lower than that of the corresponding homologous organisms grown in TSB. Binding of complement factor C3 to serum-opsonized milk whey-grown S. aureus strains was lower compared with TSB-grown homologous organisms. Moreover, 5 of 6 S. aureus strains grown in milk whey were significantly more resistant to in vivo clearance from the peritoneal cavity of mice compared with homologous bacteria grown in TSB. S. aureus strains grown in TSB exhibited hydrophobic surface properties, whereas homologous strains grown in milk whey were hydrophilic.     

The expression of capsule in serum-soft agar by Staphylococcus aureus isolated from bovine mastitis

Strains of Staphylococcus aureus were isolated from bovine mastitis, subcultured and maintained in the laboratory for up to 3 years. Encapsulation was assessed by production of a diffuse colony in serum-soft agar. Eight (4%) of 200 strains were encapsulated. Three rapid passages of the remaining 192 strains through either brain-heart infusion broth containing 30% serum or modified 110 medium retrieved the capsule in 75%, but this was rapidly lost after subculture on blood agar. The stimulation of capsule production was studied in 18 of these strains by addition of various components to the passaging medium. Heat-labile factors in serum, milk and mastitic milk enhanced capsule production while bovine serum albumin, an extract of polymorphonuclear leucocytes, NaCl and immunoglobulins had minimal effect. The results indicate that encapsulation is common in bovine staphylococci and while it is lost on subculture, may be retrieved under appropriate conditions.     

甘肃地区牛源金黄色葡萄球菌分子鉴定及RAPD分型

本研究目的是分离鉴定引起甘肃地区奶牛乳房炎的金黄色葡萄球菌,掌握其基因型情况。利用16S、23SrRNA保守序列PCR扩增对乳房炎奶样中的金黄色葡萄球菌进行鉴定,并进行RAPD基因分型。结果表明,310份奶样中共分离出金黄色葡萄球菌100株,RAPD结果显示这100株金黄色葡萄球菌均可得到清晰的RAPD指纹图谱,扩增产物在2～7条带之间,具有多种带型组成。通过聚类分析100株菌产生11个基因型,其中Ⅰ型4株,Ⅱ型4株,Ⅲ型10株,Ⅳ型13株,Ⅴ型7株,Ⅵ型24株,Ⅶ型16株,Ⅷ型6株,Ⅸ型4株,Ⅹ型10株,Ⅺ型2株。Ⅵ型为该地区的流行优势菌群,不同牛场各基因型菌株分布有明显差异。本研究说明牛场的环境与养殖条件对病原菌流行传播有明显的影响,这一结果对地区性奶牛乳房炎的防治提供了可靠的理论依据。     

Molecular typing of Staphylococcus aureus isolated from bovine mastitic milk on the basis of toxin genes and coagulase gene polymorphisms

A total of 270 strains of Staphylococcus aureus, isolated from mastitic milk, were investigated by the polymerase chain reaction for the presence of genes encoding enterotoxins (sea to sej) and a toxic shock syndrome toxin (tst). One hundred eighty three (67.8%) bovine isolates possessed either one or more toxin genes and the most common pattern that coexisted in S. aureus was tst, sec, seg, and sei. Coagulase genotyping revealed 15 patterns, and 161 of the 270 isolates (59.6%) belonged to the coagulase genotype B1. Further, these 161 isolates possessed at least two enterotoxin genes. However, the role of these toxins in udder pathogenicity remains unclear. Moreover, the predominant isolate possessed the enterotoxin genes supporting the theory that superantigenic toxins are important for the udder pathogenesis.     

Leukotoxin family genes in Staphylococcus aureus isolated from domestic animals and prevalence of lukM-lukF-PV genes by bacteriophages in bovine isolates

Leukotoxin family genes in Staphylococcus aureus isolated from domestic animals were examined by polymerase chain reaction. LukS and lukF genes were detected in all 48 avian and 72 porcine isolates of S. aureus. LukE and lukD genes, located in a putative staphylococcal pathogenicity island (Sapln3/Saplm3), were recognized in 44 (91.7%) of 48 avian isolates, but these genes were not detected in porcine isolates. In 297 bovine isolates collected from mastitic cow's milk and bulk milk from dairy farms in two regions, lukM and lukF-PV(P83) genes in addition to lukS-lukF and lukE-lukD genes were detected in 100 (62.5%) of the 160 isolates from Ishikawa and in118 (86.1%) of the 137 isolates from Hokkaido. When the lysogeny of S. aureus bovine isolates was examined by treatment with mitomycin C, clearing of the culture due to cell lysis was observed in 34 (91.9%) of 37 lukM-lukF-PV(P83) genes--positive isolates. In addition, we isolated a novel lukM-lukF-PV(P83)-carrying (designated phiLukM), and revealed that the lukM-lukF-PV(P83) genes were located very close to an amidase gene on the temperate phage genomes. These results suggest horizontal transmission of lukM-lukF-PV(P83) genes by temperate bacteriophages in S. aureus of bovine origin.     

Comparison of phenotypic and genotypic detection of penicillin G resistance of Staphylococcus aureus isolated from bovine intramammary infection

Resistance of Staphylococcus aureus to penicillin G is common among isolates from bovine mastitis. We determined phenotypic resistance to penicillin G for 151 S. aureus isolates derived from dairy cows with intramammary infection by two methods. The methods were determination of minimum inhibitory concentration (MIC) by a standard agar dilution technique and direct testing of beta-lactamase production using a chromogenic cephalosporin, nitrocefin. The results from these tests were compared with the presence of the beta-lactamase (blaZ) gene in the isolates, which was detected by polymerase chain reaction (PCR). Testing beta-lactamase production with nitrocefin was more predictive for the presence of the blaZ gene than the agar dilution method and the results of the former agreed highly with the presence of the blaZ gene in the isolates. In contrast, the resistance breakpoint generally used in the agar dilution method may be too high for prediction of penicillin resistance in S. aureus isolates with borderline MICs. Using this method, 40% of the isolates possessing the blaZ gene were classified as susceptible; however, majority of these isolates produced beta-lactamase when tested with nitrocefin.     

Identification of a bovine mastitis Escherichia coli subset

Eleven Escherichia coli isolates from clinical bovine mastitis cases (mastitic strains) and 11 from the cowshed environment (environmental strains) were compared, to determine if the former were a subset of the latter. The mastitic and environmental strains could not be distinguished according to O antigen and antibiotic sensitivity. All mastitic isolates showed significantly (P<0.0001) faster growth in milk and faster lactose fermentation than most (approximately 64%) environmental strains, but growth rates in nutrient broth did not differ. The rates of lactose fermentation and growth in milk were positively correlated. Adhesion and phagocytosis of mastitic strains by bovine PMN were significantly (P<0.0001) lower than those of environmental strains, and correlated negatively with growth in milk and lactose fermentation. The average percentages of killing by bovine leukocytes in the two sources were not statistically different. All mastitic strains were serum sensitive, whereas most ( approximately 72%) environmental ones were resistant. Finally, pulse-field gel electrophoresis revealed two main pulse type clusters, sharing a similarity coefficient of 79%. Cluster 1 comprised only environmental strains, whereas cluster 2 comprised mostly mastitic strains and only three environmental ones. Four mastitic strains shared a similarity coefficient of less than 74% with the other strains and were not included in the clusters. Our results suggest that clinical bovine mastitis E. coli isolates may form a subset of the general environmental E. coli population; they seem better able to multiply in the udder medium and to evade the host cellular innate immune response, and are genetically distinct from most environmental strains.     

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.     

贵州地区牛源金黄色葡萄球菌分离株16S rRNA分子鉴定及RAPD分型研究

利用16S rRNA保守序列对引起贵州地区奶牛乳房炎的金黄色葡萄球菌进行分离鉴定,建立随机多态性扩增体系对金黄色葡萄球菌分离株进行基因分型研究。结果表明,69份奶样中共分离出金黄色葡萄球菌37株,DNA随机多态性扩增（random amplified polymorphic DNA,RAPD）结果显示这37株金黄色葡萄球菌扩增产物在1～7条带之间,产物大小为400～4500 bp。菌株可分为7个基因型,其中Ⅰ型7株（18.9%）、Ⅱ型2株（5.4%）、Ⅲ型2株（5.4%）、Ⅳ型1株（2.7%）、Ⅴ型19株（51.4%）、Ⅵ型1株（2.7%）、Ⅶ型5株（13.5%）,Ⅴ型为该地区的流行优势菌群。地理和气候环境对病原菌流行传播的影响是病原菌基因型分布差异的重要原因。     

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。     

Validation of DNAse reaction for identification of Staphylococcus aureus strains in routine mastitis diagnosis

In routine mastitis diagnostic S. aureus strains are often identified using DNAse reaction. However, this enzyme activity is not limited only to S. aureus strains. Furthermore, the strength of the DNAse reaction between different strains varies strongly. These factors lead to the fact that the results are often not comparable between laboratories. The aim of these investigations was to validate the DNAse reaction for routine identification of S. aureus in mastitis diagnostic and to set a critical limit for interpretation of DNAse reaction. The results of 189 strains isolated from bovine mastitis milk samples show that colonies with beta delta or delta-haemolysis and DNAse reactions of > or = 2 mm can reliably be identified (sensitivity 91.2%, specificity 96.9%) as S. aureus.     

Relevance of sensitivity testings (MIC) of S. aureus to predict the antibacterial action in milk.

Bacterial susceptibility testings were carried out in parallel Iso-sensitest broth (ISB) and bovine milk cultures using 16 antibacterials and 4 sensitive strains of mastitic isolates of Staphylococcus aureus. Bacterial activities were analyzed by continuous turbidity monitoring (broth cultures), continuous fluorometric monitoring of the resazurin-reducing redox activity, and by analyzing the triphenyltetrazolium (TTC)-reducing capacity at the end of the incubation period. To obtain an equipotent bacteria-suppressing activity, milk cultures required in general several times more antibiotic than the respective ISB cultures. Antibacterial activities of sulfadoxine-trimethoprim, vancomycin, novobiocin, macrolides, aminoglycosides and oxytetracycline were most effectively suppressed by milk. Aminoglycosides suffered additionally from reduction of oxygen in the incubation environment. The beta-lactams (penicillin G, oxacillin, cephalothin, ceftiofur, ampicillin, ampicillin-clavulanic acid), gentamicin and enrofloxacin showed extremely variable sensitivity results depending on the S. aureus/milk combination.