Molecular Characterization and Toxicity Confirmation of LukM/F′-PV Producing Staphylococcus aureus Isolated from Bovine Mastitis Samples in Mysore, India

The widespread status of subclinical condition of bovine mastitis is often associated with the production of leukotoxin M/F′-PV producing Staphylococcus aureus. The present study aims for the profiling of such leukotoxin producers through conventional and molecular methods in parallel to their leukotoxicity. The incidence of this particular pathogen was assessed in mastitis infected Holstein–Friesian cattle, where eight isolates of staphylococci were found to be present in 20 % of collected samples. Being intermediately resistant to vancomycin, they showed characteristic double zone hemolysis on 7 % sheep blood agar and typical type II reaction for coagulase test indicating the pathogenic attributes. Further with RAPD-PCR and 16S rDNA-RFLP, epidemiological specificity and genotypic relatedness of isolates to S. aureus was confirmed. Subsequently, the presence of leukotoxin (lukM) gene in native isolates was detected by leukotoxin gene specific PCR. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay evaluated for secreted leukotoxin in cell free supernatant was estimated to be 223 toxic units which had an LD₅₀ cytotoxic activity on bovine neutrophil. Thus, the data acquired during study can be of prime diagnostic method for timely and accurate analysis of subclinical mastitis samples which goes undetected at consumer level.     

Survey of potential factors involved in the low frequency of CP5 and CP8 expression in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from mastitis of dairy cattle from Argentina,Chile, and Uruguay

Staphylococcus aureus produces capsular polysaccharides (CPs) both in vivo and under defined culture conditions being serotypes 5 and 8 the most prevalent. S. aureus isolates that fail to produce CP5 or CP8 are defined as non-typeable (NT). Loss of capsule expression, however, may lead to S. aureus persistence in a chronically infected host. The prevalence of NT strains of S. aureus isolated from bovine mastitis varies according to the geographic origin of the strain. The aims of this work were to detect phenotypically and genotypically the capsular profile of 144 S. aureus isolated from bovine mastitis in Argentina, Chile, and Uruguay and explore the factors that are considered to be associated with capsule expression as presence of IS257, IScap, and agr typing of non-related collection. The detection of the IS257, IScap, cap genes, and agr typing was performed using PCR. The detection and quantification of capsular polysaccharide production were performed by ELISA assays. We found that 96% of the S. aureus isolates investigated carried cap5(8) genes but over 75% of strains do not express capsule in the three countries studied. However, only 6 isolates from Argentina carried the IScap element that totally suppressed the expression of the capsule, suggesting that other factors could influence on CP expression. Moreover, the agrI/NT association was statistically significant suggesting that this profile is a phenomenon observed not only in other parts of the world but also in our region.     

Isolation,Biochemical and Molecular Identification,and In-Vitro Antimicrobial Resistance Patterns of Bacteria Isolated from Bubaline Subclinical Mastitis in South India

Buffaloes are the second largest source of milk. Mastitis is a major impediment for milk production, but not much information is available about bubaline mastitis, especially subclinical mastitis. The aim of this study was to (a) investigate the application of various tests for the diagnosis of bubaline subclinical mastitis, (b) identify the major bacteria associated with it, and (c) evaluate the antibiotic resistance pattern of the bacteria. To this end, 190 quarter milk samples were collected from 57 domesticated dairy buffaloes from organized (64 samples) and unorganized (126 samples) sectors. Of these, 48.4%, 40.0%, 45.8%, 61.1%, and 61.6% were positive for subclinical mastitis by somatic cell count, electrical conductivity, California mastitis test, bromothymol blue test, and N-acetyl glucosaminidase test, respectively. As compared to the gold standard of somatic cell count, California mastitis test performed the best. However, a combination of the two methods was found to be the best option. Microbiological evaluation, both by biochemical methods as well as by monoplex and multiplex polymerase chain reaction, revealed that coagulase-negative staphylococci were the most predominant (64.8%) bacteria, followed by streptococci (18.1%), Escherichia coli (9.8%) and Staphylococcus aureus (7.3%). Most of the pathogens were resistant to multiple antibiotics, especially to β-lactam antibiotics. We propose that California mastitis test be combined with somatic cell count for diagnosis of subclinical mastitis in domestic dairy buffaloes. Further, our results reveal high resistance of the associated bacteria to the β-lactam class of antibiotics, and a possible major role of coagulase-negative staphylococci in causing the disease in India.     

Characterization of Staphylococcus aureus Isolates from Buffalo, Bovine, Ovine, and Caprine Milk Samples Collected in Rio de Janeiro State, Brazil

Eighty-four staphylococcal isolates were obtained from milk samples from cows, sheep, goats, and buffalo with subclinical mastitis and from colonization samples from ostriches. The animals were hosted in 18 small dairy herds and an ostrich breeding located in 10 municipalities of the state of Rio de Janeiro, Brazil. Thirty isolates were identified as Staphylococcus aureus by biochemical and molecular techniques and were comparatively characterized by phenotypic and genotypic methods. The molecular characterization by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) revealed five clonal types (PFGE A, spa type t359, sequence type 747 [ST747]; PFGE B, spa type t1180, ST750; PFGE C, spa type t605, ST126; PFGE D, spa type t127, ST751; and PFGE F, spa type t002, ST5). None of the isolates harbored the Panton-Valentine leukocidin or exfoliative toxin D gene. The detection of major clone A (in 63% of the isolates) in different herds, among all animal species studied, and in infection and colonization samples evidenced its geographical spread among Rio de Janeiro State and no host preference among the animal species. Comparison with S. aureus from a human origin suggested that all but one clone found in the present study might be animal specific.     

Xenorhabdus antibiotics: a comparative analysis and potential utility for controlling mastitis caused by bacteria

Aims: The role of antibiotics produced by bacterial symbionts of entomopathogenic nematodes is to suppress growth of microbes in the soil environment. These antibiotics are active against Gram‐positive and Gram‐negative bacteria, and were tested against mastitis isolates from dairy cows. Methods and Results: Two bioassays were adapted for Xenorhabdus antibiotics; an overlay method on agar plates, and serially diluted, cell‐free, Xenorhabdus cultures. The antimicrobial activities of the liquid cultures of 13 strains from five Xenorhabdus species were further evaluated. Antimicrobial activities of the type strains of X. nematophila, X. budapestensis and X. szentirmaii were tested on mastitis isolates of Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae with both bioassays. A previously reported antibiotic from X. nematophila, nematophin, was synthesized in three steps from tryptamine and 4‐methyl‐2‐oxovaleric acid sodium salt. Conclusions: The antibiotics of all three Xenorhabdus strains were powerful in either bioassay, but the sensitivity of the isolates differed from each other. While Kl. pneumoniae was the least susceptible, Staph. aureus had the highest sensitivity to each Xenorhabdus strain. Xenorhabdus szentirmaii and X. budapestensis were more potent antibiotic producers than X. nematophila, and raceme nematophin was ineffective against all mastitis isolates. Significance and Impact of the Study: These results indicate that Xenorhabdus antibiotics are effective against mastitis isolates and should be further evaluated for their potential in mastitis control or prevention.     

Detection of the intercellular adhesion loci (ica) in clinicalStaphylococcus aureus strains responsible for hospital acquired auricular infection

In clinicalStaphylococcus aureus strains, the presence of theica genes, biofilm formation and susceptibility to antibiotics are considered important factors of virulence. In this study, 35 strains ofS. aureus, isolated from auricular infection, were investigated for slime production using Congo red agar (CRA) method, antibiotic susceptibility, presence ofmecA gene, and presence oficaA andicaD gene. The results show that 60% of strains weremecA positive when tested by PCR although 25.7% of strains were oxacillin resistant when tested with ATB STAPH. Qualitative slime production ofS. aureus using CRA revealed that 74.3% ofS. aureus were slime producers. All the strains carried theica gene.     

Contribution of Lactococcus lactis Reducing Properties to the Downregulation of a Major Virulence Regulator in Staphylococcus aureus,the agr System

Staphylococcus aureus is a major cause of food poisoning outbreaks associated with dairy products, because of the ingestion of preformed enterotoxins. The biocontrol of S. aureus using lactic acid bacteria (LAB) offers a promising opportunity to fight this pathogen while respecting the product ecosystem. We had previously established the ability of Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, to downregulate a major staphylococcal virulence regulator, the accessory gene regulator (agr) system, and, as a consequence, agr-controlled enterotoxins. In the present paper, we have shown that the oxygen-independent reducing properties of L. lactis contribute to agr downregulation. Neutralizing lactococcal reduction by adding potassium ferricyanide or maintaining the oxygen pressure constant at 50% released agr downregulation in the presence of L. lactis. This downregulation still occurred in an S. aureus srrA mutant, indicating that the staphylococcal respiratory response regulator SrrAB was not the only component in the signaling pathway. Therefore, this study clearly demonstrates the ability of L. lactis reducing properties to interfere with the expression of S. aureus virulence, thus highlighting this general property of LAB as a lever to control the virulence expression of this major pathogen in a food context and beyond.     

Histopathological and parasitological study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum

Background

A nationwide survey on the microbial etiology of cases of subclinical mastitis in dairy cows was carried out on dairy farms in Sweden. The aim was to investigate the microbial panorama and the occurrence of antimicrobial resistance. Moreover, differences between newly infected cows and chronically infected cows were investigated.

Methods

In total, 583 quarter milk samples were collected from 583 dairy cows at 226 dairy farms from February 2008 to February 2009. The quarter milk samples were bacteriological investigated and scored using the California Mastitis Test. Staphylococci were tested for betalactamase production and presence of resistance was evaluated in all specific udder pathogens. Differences between newly infected cows and chronically infected cows were statistically investigated using logistic regression analysis.

Results

The most common isolates of 590 bacteriological diagnoses were Staphylococcus (S) aureus (19%) and coagulase-negative staphylococci (CNS; 16%) followed by Streptococcus (Str) dysgalactiae (9%), Str. uberis (8%), Escherichia (E.) coli (2.9%), and Streptococcus spp. (1.9%). Samples with no growth or contamination constituted 22% and 18% of the diagnoses, respectively. The distribution of the most commonly isolated bacteria considering only bacteriological positive samples were: S. aureus - 31%, CNS - 27%, Str. dysgalactiae - 15%, Str. uberis - 14%, E. coli - 4.8%, and Streptococcus spp. - 3.1%. There was an increased risk of finding S. aureus, Str. uberis or Str. dysgalactiae in milk samples from chronically infected cows compared to findings in milk samples from newly infected cows. Four percent of the S. aureus isolates and 35% of the CNS isolates were resistant to penicillin G. Overall, resistance to other antimicrobials than penicillin G was uncommon.

Conclusions

Staphylococcus aureus and CNS were the most frequently isolated pathogens and resistance to antimicrobials was rare.     

Extensive Genomic Diversity among Bovine-Adapted Staphylococcus aureus: Evidence for a Genomic Rearrangement within CC97

Staphylococcus aureus is an important pathogen associated with both human and veterinary disease and is a common cause of bovine mastitis. Genomic heterogeneity exists between S. aureus strains and has been implicated in the adaptation of specific strains to colonise particular mammalian hosts. Knowledge of the factors required for host specificity and virulence is important for understanding the pathogenesis and management of S. aureus mastitis. In this study, a panel of mastitis-associated S. aureus isolates (n = 126) was tested for resistance to antibiotics commonly used to treat mastitis. Over half of the isolates (52%) demonstrated resistance to penicillin and ampicillin but all were susceptible to the other antibiotics tested. S. aureus isolates were further examined for their clonal diversity by Multi-Locus Sequence Typing (MLST). In total, 18 different sequence types (STs) were identified and eBURST analysis demonstrated that the majority of isolates grouped into clonal complexes CC97, CC151 or sequence type (ST) 136. Analysis of the role of recombination events in determining S. aureus population structure determined that ST diversification through nucleotide substitutions were more likely to be due to recombination compared to point mutation, with regions of the genome possibly acting as recombination hotspots. DNA microarray analysis revealed a large number of differences amongst S. aureus STs in their variable genome content, including genes associated with capsule and biofilm formation and adhesion factors. Finally, evidence for a genomic arrangement was observed within isolates from CC97 with the ST71-like subgroup showing evidence of an IS431 insertion element having replaced approximately 30 kb of DNA including the ica operon and histidine biosynthesis genes, resulting in histidine auxotrophy. This genomic rearrangement may be responsible for the diversification of ST71 into an emerging bovine adapted subgroup.     

Application of optical coherence tomography enhances reproducibility of arthroscopic evaluation of equine joints

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.     

Characterization of Colonizing Staphylococcus aureus Isolated from Surgical Wards' Patients in a Nigerian University Hospital

In contrast to developed countries, only limited data on the prevalence, resistance and clonal structure of Staphylococcus aureus are available for African countries. Since S. aureus carriage is a risk factor for postoperative wound infection, patients who had been hospitalized in surgical wards in a Nigerian University Teaching Hospital were screened for S. aureus carriage. All S. aureus isolates were genotyped (spa, agr) and assigned to multilocus sequence types (MLST). Species affiliation, methicillin-resistance, and the possession of pyrogenic toxin superantigens (PTSAg), exfoliative toxins (ETs) and Panton-Valentine Leukocidin (PVL) were analyzed. Of 192 patients screened, the S. aureus carrier rate was 31.8 % (n = 61). Of these isolates, 7 (11.5%) were methicillin-resistant (MRSA). The isolates comprised 24 spa types. The most frequent spa types were t064, t084, t311, and t1931, while the most prevalent MLST clonal complexes were CC5 and CC15. The most frequent PTSAg genes detected were seg/sei (41.0%) followed by seb (29.5%), sea (19.7%), seh (14.7%) and sec (11.5). The difference between the possession of classical and newly described PTSAg genes was not significant (63.9% versus 59.0% respectively; P = 0.602). PVL encoding genes were found in 39.3% isolates. All MRSA isolates were PVL negative, SCCmec types I and VI in MLST CC 5 and CC 30, respectively. Typing of the accessory gene regulator (agr) showed the following distribution: agr group 1 (n = 20), group II (n = 17), group III (n = 14) and group IV (n = 10). Compared to European data, enterotoxin gene seb and PVL-encoding genes were more prevalent in Nigerian methicillin-susceptible S. aureus isolates, which may therefore act as potential reservoir for PVL and PTSAg genes.     

Bacterial Competition for Human Nasal Cavity Colonization: Role of Staphylococcal agr Alleles

We examined the bacterial aerobic nasal flora of 216 healthy volunteers to identify potential competitive interactions among different species, with special emphasis on the influence of staphylococcal agr alleles. The Staphylococcus aureus colonization rate correlated negatively with the rate of colonization by Corynebacterium spp. and non-aureus staphylococci, especially S. epidermidis, suggesting that both Corynebacterium spp. and S. epidermidis antagonize S. aureus colonization. Most of the S. aureus and S. epidermidis isolates were agr typed by a PCR method. Only one S. aureus agr (agr_Sa) allele was detected in each carrier. Multiple logistic regression of the two most prevalent agr_Sa alleles (agr-1_Sa and agr-2_Sa) and the three S. epidermidis agr (agr_Se) alleles showed a specific influence of the agr system. The results of this model did not support conclusions drawn from previous in vitro agr-specific cross-inhibition experiments. Our findings suggest that the agr alleles, which are strongly linked to the bacterial genetic background, may simply be associated with common biological properties—including mediators of bacterial interference—in the strains that bear them.     

Prevalence of antibiotic resistant mastitis pathogens in dairy cows in Egypt and potential biological control agents produced from plant endophytic actinobacteria

Dairy production is threatened by antibiotic resistant pathogens worldwide, and alternative solutions to treat mastitis are not available. The prevalence of antibiotic resistant strains is not well known in less developed countries. The prevalence of pathogenic bacteria and their resistance to 21 commercial antibiotics were studied in milk samples taken from 122 dairy cows suffering from the symptoms of mastitis in Egypt. The bacterial species were identified with molecular methods, and antibiotic resistance was studied with disc diffusion method. The prevalence of Streptococcus aureus, Escherichia coli and Pseudomonas aeruginosa were 30%, 17% and 3.5%, respectively. Most (90%) of the S. aureus strains showed resistance to penicillin whereas only 10% of the strains were resistant to oxacillin. Nearly half (40%) of E. coli strains showed resistance to streptomycin. Six P. aeruginosa strains showed resistance to several antibiotics, including ceftriaxone, enrofloxacin and levofloxacin. This points out that despite P. aeruginosa was not common, it should be followed up carefully. Potential biocontrol agents against antibiotic resistant mastitis bacteria were searched among 30 endophytic actinobacterial strains derived from wild medicinal plants. Three plants, namely Mentha longifolia, Malva parviflora and Pulicaria undulata were chosen for a more detailed study; their endophytic actinobacteria were used to prepare metabolic extracts. The crude metabolites of the actinobacteria were extracted with ethyl acetate. All metabolic extracts inhibited the growth of S. aureus, methicillin-resistant Staphylococcus aureus (MRSA), E. coli and P. aeruginosa in vitro. The 16S rRNA sequence analysis revealed that the most efficient actinobacterial strains were two Micromonospora sp. and one Actinobacteria bacterium. We conclude that the combination of the metabolites of several endophytic actinobacteria derived from several medicinal plants would be the most efficient against pathogens. Different metabolite cocktails should be studied further in order to develop novel biocontrol agents to treat antibiotic resistant mastitis bacteria in dairy cows.     

Prevalence of Clonal Complexes and Virulence Genes among Commensal and Invasive Staphylococcus aureus Isolates in Sweden

Staphylococcus aureus encodes a remarkable number of virulence factors which may contribute to its pathogenicity and ability to cause invasive disease. The main objective of this study was to evaluate the association between S. aureus invasiveness and bacterial genotype, in terms of the presence of virulence genes and affiliation to clonal complexes. Also, the significance of different virulence genes, mainly adhesins, for the development of infective endocarditis was investigated.DNA microarray technology was used to analyze 134 S. aureus isolates, all methicillin-susceptible, derived from three groups of clinically well-characterized patients: nasal carriers (n=46), bacteremia (n=55), and bacteremia with infective endocarditis (n=33).Invasive isolates were dominant in four of the major clonal complexes: 5, 8, 15, and 25. Of the 170 virulence genes examined, those encoding accessory gene regulator group II (agr II), capsule polysaccharide serotype 5 (cap5), and adhesins such as S. aureus surface protein G (sasG) and fibronectin-binding protein B (fnbB) were found to be associated with invasive disease. The same was shown for the leukocidin genes lukD/lukE, as well as the genes encoding serine protease A and B (splA/splB), staphylococcal complement inhibitor (scn) and the staphylococcal exotoxin-like protein (setC or selX). In addition, there was a trend of higher prevalence of certain genes or gene clusters (sasG, agr II, cap5) among isolates causing infective endocarditis compared to other invasive isolates. In most cases, the presence of virulence genes was linked to clonal complex affiliation.In conclusion, certain S. aureus clonal lineages harboring specific sets of virulence genes seem to be more successful in causing invasive disease.     

Investigation of the lytic ability of South African bacteriophages specific for Staphylococcus aureus,associated with bovine mastitis

Bovine mastitis is an infectious disease of the mammary glands of dairy cattle primarily causaled by the bacterium, Staphylococcus aureus subsp. aureus Rosenbach1884. Traditional control of this organism was through the use of antibiotics. However, S. aureus is developing resistance towards these chemotherapeutic agents faster than they are being developed. Bacteriophages can serve as an alternative control measure for the disease. This study investigated the prevalence of phages and S. aureus within the South African dairy environment, as well as infectivity of phage isolates against antibiotic-resistant S. aureus. The four S. aureus strains used in the study displayed resistance to representative antibiotics from both the β-lactamases and non-β-lactamases, macrolides, aminoglycosides and glycopeptides. Susceptibility was only noted towards the tetracycline antibiotics. Twenty-eight phages were isolated and screened against four strains of S. aureus. Only six phages showed biocontrol potential based on their wide host range, high titres and common growth requirements. Morphological and preliminary genomic analysis was carried out on the three best performing phages. At an optimal titre of between 6.2 × 10⁷ and 2.9 × 10⁸ pfu.ml^?1, the phages were able to reduce live bacterial cell counts between 64% and 95%. In addition, these six phages showed further infectivity towards S. aureus strains that were isolated from different milk-producing regions during a farm survey. The phages isolated in this study show reasonable potential for in vivo applications.     

Characterisation of Australian MRSA strains ST75- and ST883-MRSA-IV and analysis of their accessory gene regulator locus

Background

Community-acquired methicillin-resistant Staphylococcus aureus have become a major problem in Australia. These strains have now been isolated throughout Australia including remote Indigenous communities that have had minimal exposure to healthcare facilities. Some of these strains, belonging to sequence types ST75 and ST883, have previously been reported to harbour highly divergent alleles of the housekeeping genes used in multilocus sequence typing.

Methodology/Principal Findings

ST75-MRSA-IV and ST883-MRSA-IV isolates were characterised in detail. Morphological features as well as 16S sequences were identical to other S. aureus strains. Although a partial rnpB gene sequence was not identical to previously known S. aureus sequences, it was found to be more closely related to S. aureus than to other staphylococci. Isolates also were screened using diagnostic DNA microarrays. These isolates yielded hybridisation results atypical for S. aureus. Primer directed amplification assays failed to detect species markers (femA, katA, sbi, spa). However, arbitrarily primed amplification indicated the presence of unknown alleles of these genes. Isolates could not be assigned to capsule types 1, 5 or 8. The allelic group of the accessory gene regulator (agr) locus was not determinable. Sequencing of a region of agrB, agrC and agrD (approximately 2,100 bp) revealed a divergent sequence. However, this sequence is more related to S. aureus agr alleles I and IV than to agr sequences from other Staphylococcus species. The predicted auto-inducing peptide (AIP) sequence of ST75 was identical to that of agr group I, while the predicted AIP sequence of ST883 was identical to agr group IV.

Conclusions/Significance

The genetic properties of ST75/ST883-MRSA may be due to a series of evolutionary events in ancient insulated S. aureus strains including a convergent evolution leading to agr group I- or IV-like AIP sequences and a recent acquisition of SCCmec IV elements.     

Prevalence of bovine subclinical mastitis and isolation of its major causes in Bishoftu Town,Ethiopia

Objective

A cross-sectional study was conducted from November 2015 to March 2016 to estimate the prevalence, to assess the risk factors and to isolate the major etiological agent of subclinical mastitis in Bishoftu town. The study was conducted on 262 cross breed lactating cows selected from 12 intensively managed dairy farms. California mastitis test (CMT) and bacteriological culture methods were used as diagnostic tools.

Result

From 262 cows examined, 105 (40.1%) and from 1048 quarters examined, 170 (16.1%) were positive for sub-clinical mastitis using CMT. All CMT positive samples were cultured for etiological agent identification. From 170 samples cultured, 153 were positive for known subclinical mastitis pathogens. The dominant bacteria isolated were Staphylococcus species from these Staphylococcus aureus (44.9%) was the major one followed by Streptococcus spp. (25.3%) and other gram negative enteric bacteria, Escherichia coli (8.8%). Age, body condition score, milk yield, and number of parity were considered as potential risk factors; among these, age and number of parity have statistically significance association with the occurrence of subclinical mastitis (P < 0.05) both in the CMT and the bacteriological tests.

     

Antimicrobial susceptibility and invasive ability of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from mastitis from dairy backyard systems

Fifteen (15) backyard farms were investigated to determine the antimicrobial susceptibility and invasion ability of S. aureus isolates from cows with subclinical mastitis in México. A total of 106 cows were sampled and 31 S. aureus isolates were recovered. S. aureus isolates were resistant to penicillin class antibiotics and susceptible to gentamicin and cetyltrimethylammonium bromide. STA9 and STA13 isolates were resistant to erythromycin (MIC > 25 microg/ml) and lincomycin (STA13, MIC > 25 microg/ml; STA9, MIC > 100 microg/ml). STA9 isolate harbors the erm(B) and msr(A) genes, whereas STA13 isolate harbors the erm(C) gene. STA9 and STA13 isolates contains the lnu(A) gene. Only 5 isolates (STA11, STA13, STA14, STA15 and STA21) were able to internalize in bovine mammary epithelial cells. These results indicate that S. aureus isolates from dairy backyard farms showed differences in the antimicrobial susceptibility patterns and invasion ability in bovine mammary epithelial cells. This kind of evaluations should be performed in different dairy regions, since resistance patterns and isolate diversity vary on a per-region basis.     

Isolation and characterization of methicillin-resistantStaphylococcus aureus from livestock and poultry meat

Meat samples from sheep, bovine, camel and poultry were collected from Amman area and were processed and tested for the presence of methicillin (oxacillin) resistantStaphylococcus aureus (MRSA). Identity ofS. aureus was ensured by Gram-staining and a battery of biochemical tests. From 1260 meat samples, 157S. aureus positive isolates were identified. Of the 157 isolates, 30 were resistant to methicillin levels greater than 2 μg/ml and only 15 weremecA-positive MRSA originating mainly from sheep and chicken. Subjecting themecA-positive MRSA to antibiotic susceptibility testing revealed that all isolates were resistant to β-lactam antibiotics (ampicillin, penicillin, and oxacillin) and were sensitive to vancomycin, trimethoprim, chloramphenicol and cephalothin. Randomamplified polymorphic DNA (RAPD) analysis ofmecA-positive animal isolates generated six different patterns. Comparing these results with results of isolates of human origin of our laboratory there is some molecular epidemiological relatedness between both and could be a possible source of infections through consuming contaminated meat products, direct contact or meat processing.