Phage types and antimicrobial resistance among Danish bovine Staphylococcus aureus isolates since the 1950s

A total of 292 bovine Staphylococcus aureus isolates obtained from the 1950s (86 isolates), 1992 (107 isolates), and 2000 (99 isolates) were examined for antimicrobial susceptibility and phage typing. The same types of S. aureus (80, 52, 3A, 3A/3C, 42E, 77) were found among the isolates from all three time periods, representing 43.3% of the typeable isolates. This indicates that the Danish S. aureus population related to bovine mastitis has remained relatively unchanged over the last 50 years. The occurrence of antimicrobial resistance has remained low in Denmark in comparison to other countries in Europe.     

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.     

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.     

禽源金黄色葡萄球菌耐药性监测

从临床样品中分离鉴定得到183株金黄色葡萄球菌,采用微量肉汤稀释法监测其对24种抗菌药物的耐药性。结果表明,183株金黄色葡萄球菌对24种抗菌药物均呈现不同程度的耐药性,对常用的青霉素类、磺胺类药物及甲氧苄啶严重耐药,耐药率高达81%以上;其中检测出MRSA42株,检出率为22.95%。94.00%的金黄色葡萄球菌菌株呈现多重耐药,耐药谱型主要分布在3～8耐之间,以7耐菌株所占比例最大(15.30%),共有119种耐药谱型。     

Molecular types and antibiotic resistance of Staphylococcus aureus isolates from bovine mastitis in a single herd in China

The molecular diversity, antibiotic resistance patterns and presence of resistance genes were determined in Staphylococcus aureus isolates from cases of bovine mastitis in a dairy cattle herd in China. Multiple locus variable number tandem repeat analysis was used for molecular typing. Resistance was determined through minimum inhibitory concentrations and resistance genes were detected by PCR. There was low molecular diversity; one predominant strain (type I) accounted for the majority of cases of S. aureus mastitis in the herd and this strain had a high frequency of resistance to penicillin and tetracycline. The most prevalent resistance genes were blaZ, ermC and tetM.     

Methicillin resistance genes and in vitro biofilm formation among Staphylococcus aureus isolates from bovine mastitis in India

IntroductionBiofilms, an assemblage of microbial cells irreversibly associated with a surface and enclosed in a matrix of polysaccharide material pose serious health challenges, resulting in high economic losses. The emergence of methicillin-resistant S. aureus (MRSA) infections and ability to form biofilms in dairy animals is of emerging concern for livestock and public health owing to their association with serious infections. The present study was undertaken to examine the presence of methicillin resistance genes among the biofilm forming Staphylococcus aureus strains isolated from cases of acute and subacute bovine mastitis. A total of 150 mastitic milk samples referred to Veterinary Clinical Complex, Shuhama (Aulesteng) SKUAST-K were screened in present study. The methicillin resistant Staphylococcus aureus isolates were also screened for in vitro biofilm forming ability.ResultsA total of 80 (53.33%) S. aureus isolates were recovered from cases of bovine mastitis of which 20 (25%) were methicillin (mecA) gene positive. Of the 20 mecA positive isolates, 20% were positive for SCCmec I, 35% for SCCmec IV and 45% for SCCmec V subtypes. In vitro antibiotic sensitivity testing of MRSA revealed complete resistance towards methicillin and other pencillin group of antibiotics.ConclusionA significant correlation was observed between in vitro biofilm formation and presence of methicillin resistance gene in S aureus isolates recovered from acute and subacute mastitis. The Staphylococcus aureus isolates positive for methicillin resistance gene (mecA) were either strong or moderate biofilm formers.     

Comparison between diffusion and dilution methods for testing antimicrobial susceptibility of Staphylococcus aureus cultures derived from bovine mastitis

One hundred Staphylococcus aureus strains from bovine mastitis were tested for susceptibility to eight antimicrobials by the paper disc diffusion method (Kirby-Bauer) and a commercially prepared dilution system. Except in the case of penicillin and possibly ampicillin, comparison of the two methods showed excellent correlation. Agreement was perfect with methicillin, chloramphenicol, gentamicin, and kanamycin.     

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.     

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.     

The use of a new phage set for typing poultry strains of Staphylococcus aureus obtained from seven countries

1. Eighty per cent of poultry strains of Staphylococcus aureus tested from French, Belgian, English, West German, Japanese and Argentinian sources were typable using a set of typing phages isolated in this laboratory. Strains from Bulgaria, however, with few exceptions were not typable with this phage set. 2. Strains isolated from lesions generally resembled those from apparently healthy poultry. 3. The existence of two distinct Staph. aureus biotypes on poultry was confirmed by isolates from six of the countries; one of these biotypes closely resembled Staph. aureus variety gallinae as described by Witte et al. (1977).     

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.     

Minimum inhibitory concentrations of 20 antimicrobial agents against Staphylococcus aureus isolated from bovine intramammary infections in Japan

Minimum inhibitory concentrations (MICs) of 20 antimicrobial agents were determined against 51 isolates of Staphylococcus aureus from bovine intramammary infections. Fourteen (27.4%) isolates were resistant to benzylpenicillin, but none of the isolates was resistant to cloxacillin, nafcillin, or cephems. Among aminoglycosides, gentamicin was the most active, with an MIC50 of 0.2 microg/ml, followed by kanamycin, with an MIC50 of 0.78 microg/ml. Five isolates (9.8%) were resistant to dihydrostreptomycin, three isolates (5.9%) to kanamycin and two isolates (3.9%) to gentamicin. Resistance to erythromycin was observed in two isolates (3.9%). Tylosin was less active than erythromycin, with MIC50s of 1.56 microg/ml versus 0.39 microg/ml, but none of the isolates was resistant to this antibiotic. Oxytetracycline MICs were situated in the range of 0.39-1.56 microg/ml for 48 susceptible isolates. Although 19 (37.3%) isolates were resistant to one or more antimicrobial agents, a single resistance pattern was most frequent: benzylpenicillin (12 isolates), dihydrostreptomycin (two isolates) and kanamycin (one isolate). There were no isolates resistant to antimicrobial agents such as methicillin, lincomycin, clindamycin, chloramphenicol, florfenicol and virginiamycin, which have not been approved for use in cattle husbandry in Japan.     

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。     

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 resistance and resistance genes in Staphylococcus aureus strains from rabbits

Fifty-six Staphylococcus aureus isolates recovered between 1998 and 2003 from 31 rabbit farms with and without problems of chronic staphylococcosis, were screened for resistance to enrofloxacin, erythromycin, gentamicin, lincomycin, neomycin, penicillin and tetracyclines using the agar dilution test. For penicillin, a disk diffusion test was also performed. The detection of tetP(B), tet(K), tet(L), tet(M), tet(O), tet(T), tet(W), erm(A), erm(B), erm(C) and mec(A) genes was done via a PCR assay. Four isolates showed resistance to erythromycin and lincomycin. These isolates were positive for the erm(C) gene in the PCR. Eleven strains were resistant to tetracyclines and all harboured the tet(K) gene. In the agar dilution test, five isolates showed resistance to penicillin, whereas in the disk diffusion test 12 isolates showed resistance. None of these 12 resistant isolates carried the mec(A) gene. Only one strain showed resistance to gentamicin, and all strains were susceptible to enrofloxacin and neomycin. This study demonstrates that resistance to antimicrobial agents in S. aureus isolates originating from rabbits is relatively rare compared to resistance in S. aureus isolates originating from other animals and humans.     

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.