Antimicrobial resistance and toxin gene profiles of Staphylococcus aureus strains from Holstein milk

Isolation of Staphylococcus aureus (Staph. aureus) from Holstein milk samples with mastitis and nonmastitis was conducted to estimate its prevalence, antimicrobial resistance and toxin genes. A total of 353 milk samples were collected from three Chinese Holstein herds. Fifty‐three Staph. aureus isolates collected from 29 Staph. aureus‐positive samples were characterized via antimicrobial susceptibility, toxin genes and Pulsed‐field Gel Electrophoresis (PFGE) profiles. The prevalence of Staph. aureus was 4·0–9·5% in mastitic and 7·3–11·5% in nonmastitic samples in the analysed herds. Approximately 61·0% of Staph. aureus strains isolated from mastitis cows were resistant to ≥10 antimicrobials compared with 0% of isolates with nonmastitis. The most frequently observed super antigenic toxin gene was pvl (41·5%) followed by seh + pvl (13·2%). We did not find mecA‐positive methicillin‐resistant Staph. aureus (MRSA) strains, while mecA‐negative MRSA strains were identified in the three herds. PFGE results suggested potential transmission of Staph. aureus strains in different farms. These results open new insights into Staph. aureus transmission and antimicrobial resistance of Holstein dairy cows and into developing strategies for udder health improvement of dairy cattle.     

Molecular population and virulence factor analysis of Staphylococcus aureus from bovine intramammary infection

Staphylococcus aureus isolates from cows in Ireland (n = 102) and the USA (n = 42) were characterized by RAPD-PCR and analysed for the production of a number of putative virulence factors. Of these strains 63 representative isolates were screened for the corresponding virulence factor genes by PCR or Southern hybridization or both. The isolates were divided into 12 distinct clonal types on the basis of their RAPD fingerprint profiles. Of the isolates, 107 (74.3%) tested positive for clumping factor in a slide agglutination test, all 24 RAPD type 7 isolates being negative for clumping factor. PCR analysis of region R, a repeat region of the clfA gene, revealed eight region-R sizes. There was a strong association between RAPD type and the clfA region-R genotype among Irish isolates. Of the RAPD type 7 isolates, 21 (87.5%) coproduced toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin C (SEC). Over 90% of isolates demonstrated haemolytic activity on sheep or rabbit red blood cells and all isolates harboured the gamma-haemolysin (hlg) locus. Of the Irish isolates, all those of RAPD type 7 were sensitive to penicillin G, whereas 86% of RAPD types 4 and 5 strains were resistant. Furthermore, RAPD types 5 and 7 were more likely to be associated with clinical mastitis whereas RAPD type 4 isolates were more often associated with a latent infection. The current study identifies some of the putative virulence factors produced by the predominant clonal types of bovine Staph. aureus that may be considered as components of a vaccine.     

The survival of Staphylococcus aureus in raw milk with different cell count

The antibacterial activity of the individual udder quarter milk sample cannot be evaluated by the number of somatic cells. Milk with the highest number of cells is not always the most bactericidal. However, when the data were obtained from a material large enough for statistical calculations, negative and highly significant correlation coefficient (-0.465, P less than or equal to 0.01) between number of somatic cells in milk and number of bacteria surviving in it, and negative and significant correlation coefficient (-0.203, P 0.05) between number of somatic cells in milk and number of bacteria surviving in whey, were found. Results of the analysis of variance demonstrated highly significant differences as regards number of cells and bacteria surviving in milk and in whey between cows, quarters and stimulation time. Nevertheless, among ten investigated cows, three cows with high cell count and high antibacterial activity in milk and whey, highly reacting to stimulant, were found.     

Serotyping of Dutch Staphylococcus aureus strains from carriage and infection

International epidemiological studies have shown that clinical isolates of Staphylococcus aureus are usually capsulated with either type 5 or 8 capsular polysaccharides (CPs). Because all noncapsulated strains were found to be cross-reactive with polysaccharide 336 (336PS) antibodies, the noncapsulated strains were denoted as type 336PS. The capsular types of 162 Dutch methicillin-susceptible S. aureus strains derived from individuals living in the Rotterdam area were determined. The serotype distribution was 28.4% serotype 5, 53.7% type 8, and 17.9% type 336PS. Serotyping was in agreement with genotyping by amplified fragment length polymorphism (AFLP) and multi locus sequence typing (MLST). Among 49 nasal carriage isolates from healthy children 24.5% belonged to serotype 5, 67.3% were type 8 and 8.2% were type 336PS. For 28 adult patients on chronic ambulatory peritoneal dialysis (CAPD) the serotype incidences among carriage isolates obtained from the nose, catheter exit-site, and abdominal skin were 45.1%, 41.2% and 13.7%, respectively. Among S. aureus strains deriving from blood cultures, the serotype incidences were 17.7% serotype 5, 53.2% type 8, and 29.0% type 336PS. Apparently, type 336PS strains are more prevalent (P=0.017) among bacteraemia isolates as compared with the nasal carriage isolates obtained from healthy children and CAPD patients. In conclusion, all Dutch S. aureus isolates belonged to types 5, 8, or 336PS, which is in agreement with data from other countries. Thus, addition of the 336PS conjugate to a type 5- and type 8-CP protein conjugate vaccine would significantly extend the vaccine coverage.     

牛乳源金黄色葡萄球菌EsxA蛋白的表达及免疫原性分析

为分析牛乳源金黄色葡萄球菌(Staphylococcus aureus)EsxA蛋白的免疫原性,构建EsxA-p ET-28a重组表达质粒,重组质粒经诱导表达后进行SDS-PAGE和Western blotting鉴定。用纯化后重组EsxA蛋白免疫小鼠,用间接ELISA检测免疫小鼠血清中的IgG、IgG1和IgG2a水平;免疫小鼠经S.aureus菌株攻击后,检测小鼠肝、脾、肾组织荷菌数和免疫保护率,观察S.aureus菌株攻击后小鼠肝、脾、肾病理组织学变化。结果表明,成功诱导表达了EsxA重组蛋白,该重组蛋白免疫小鼠后血清抗体效价可达1∶900,与对照相比,重组蛋白免疫后可减少小鼠肝、脾、肾组织的荷菌数,减轻这些脏器的病理损伤,对免疫小鼠保护率达75%。上述结果表明,该重组Esx A蛋白具有良好的免疫原性。     

A Staphylococcus aureus mouse keratitis topical infection model: cytokine balance in different strains of mice

Staphylococcus is a leading cause of the potentially blinding disease microbial keratitis. Even with the use of antibiotic therapy, the host inflammatory response continues to damage the cornea, which may lead to blindness. Manipulation of the host response may help improve patient outcome from this devastating disease. We aim to understand the contribution of the host response to Staphylococcus aureus infection. A S. aureus keratitis mouse model was developed in both C57BL/6 and BALB/c mice using two different strains of S. aureus (8325-4 and Staph 38). Twenty-four hours postinfection, mice were killed and eyes were harvested for enumeration of bacteria, polymorphonuclear leucocytes, chemokines and cytokines. The laboratory strain 8325-4 was not as virulent as the clinical isolate Staph 38. In vitro data showed a 250-fold increase in invasion of human corneal epithelial cells by Staph 38 compared to 8325-4. BALB/c mice were susceptible to S. aureus infection whereas C57BL/6 mice were resistant. The resistant C57BL/6 mice were polarized towards a Th2 response, which may be protective for these mice. IL-4, IL-10 and IL-6 were elevated significantly in C57BL/6 mice infected with Staph 38 (P < 0.05). Macrophage inflammatory peptide (MIP)-2 was also significantly elevated in C57BL/6 mice (P < 0.001). The susceptible BALB/c mice had a muted cytokine response, which suggests that S. aureus might be 'walled off' during infection and might avoid host defences. IL-4, IL-10 and IL-6 cytokines may be protective during Gram-positive corneal infection and therefore may be useful for adjunct therapies in the treatment of this disease.     

Protection induced in mice vaccinated with recombinant collagen-binding protein (CnBP) and alpha-toxoid against intramammary infection with Staphylococcus aureus

Mice vaccinated with a combination of two Staphylococcus aureus antigens consisting of a recombinant collagen-binding protein (CnBP) and alpha-toxoid (alpha-toxoid) were significantly protected from intramammary challenge infection with S. aureus. The average number of bacteria recovered from the glands of mice vaccinated with the combination of CnBP/alpha-toxoid was significantly lower compared to the average number of bacteria recovered from the glands of mice vaccinated with only CnBP or alpha-toxoid or controls (P< or =0.01). Histopathological examination of mammary glands of mice vaccinated with CnBP together with alpha-toxoid showed no pathological changes, whereas glands of mice vaccinated with CnBP or alpha-toxoid alone developed severe mastitis and showed both focal and disseminated necrosis.     

Characteristics of Korean-Saanen goat milk caseins and somatic cell counts in comparison with Holstein cow milk counterparts

Characteristics of α- and β-casein fractions in the milk of Korean-Saanen goats were compared with those of Holstein cow milk using capillary electrophoresis (CE) analysis. The αs₁-CN content of the Saanen goat milk samples varied from 2.4% to 9.3% of total proteins. Total αs-CN content of the goat milk varied from 10.1% to 17.0%. Total β-CN content containing β₁-CN and the β₂-CN varied from 49.6% to 61.0% of total proteins. Average αs-CN to β-CN ratio of the Saanen goat milk from different farms was 0.24 ± 0.04, ranging from 0.17 to 0.33. The αs-CN (αs₁-CN + αs₀-CN) to β-CN (β_A1-CN + β_A2-CN) ratio of Holstein cow milk was 0.81, which was much higher than that of Korean-Saanen goat milk. The goat milk samples having more than 1.5 million cells/ml somatic cell counts (SCC) contained higher αs-CNs (P < 0.01) and lower β-CNs (P < 0.05) contents than milks with <1.5 million SCC. This resulted in a higher αs-CN to β-CN ratio (P < 0.01) in the milk with >1.5 million SCC.     

Characterization of hemolytic activity of Staphylococcus aureus strains isolated from bovine mastitic milk

Beta (beta) and delta (delta)-hemolysin of Staphylococcus aureus strains were cultured in vitro in milk lactoserum (whey) prepared from both healthy and mastitis bovine milk. Production of beta- and delta-hemolysins were detected in 12 out of 50 strains studied. The association between N-acetyl-beta-D-glucosaminidase (NAGase) activity, plasmin activity (PL) and trypsin inhibitory capacity (TIC), known as inflammatory indicators for mastitis, and hemolytic activity were also studied. Mastitic milk decreased directly the lytic effect of both beta-and delta-hemolysins of S. aureus on hemolytical blood agar plates. S. aureus in healthy milk samples produced more beta-hemolysin (3 times) and delta-hemolysin (2 times) when compared to S. aureus supernatants in milk from infected quarters. Furthermore, beta- and delta-hemolysis correlated negatively with TIC and NAGase and PL activities. Addition of reduced glutathione (GSH) or beta-mercaptoethanol into the artificial medium enhanced hemolysins activity.     

Serum and milk iron levels during sheep intramammary infection caused by coagulase-negative staphylococci

The concentration of iron (Fe) in the milk and serum of sheep was determined before and during experimental intramammary infection (IMI) by coagulase-negative staphylococci (C-NS). Fe concentration of normal milk and serum samples was 0.24 Μg/mL and 1.56 Μg/mL respectively. Presence of C-NS in the mammary gland resulted in a significant increase in milk-iron concentration (p < 0.001) and a decrease in the serum-iron concentration. Serum-iron concentration was significantly decreased (p = 0.04) one d after the intramammary introduction of C-NS and 29 d later (p = 0.03).     

Changes in antibiotic sensitivity in strains of Staphylococcus aureus, 1952-78.

Two hundred strains of Staphylococcus aureus isolated from outpatients with infections of the skin and subcutaneous tissues were tested for sensitivity to penicillin, erythromycin, tetracycline, sodium fusidate, methicillin, clindamycin, chloramphenicol, and gentamicin. One hundred and sixty-three (81.5%) of the strains were resistant to penicillin and 16 (8%) resistant to tetracycline. Incidence of resistance to other antibiotics was low. No strain was resistant to chloramphenicol, gentamicin, or methicillin. When compared with results of earlier studies, there was an increase in the incidence of resistance to penicillin and tetracycline, but no appreciable increase in resistance to other antibiotics.     

Lecithinase reaction of Staphylococcus aureus strains of different origin on Baird-Parker medium

J.E.S. MATOS, R.J. HARMON AND B.E. LANGLOIS. 1995. Staphylococcus aureus produces one or more enzymes with lipolytic activity, but differences between strains have been reported (Owens and John 1975; O'Toole 1987; Rollof et al. 1987). The biological and biochemical properties of these enzymes have been investigated and results were recently reviewed (Kötting et al. 1984).
Baird-Parker medium (Baird-Parker 1962) is a selective medium commonly used for the isolation of Staph. aureus. The presence of egg yolk in this medium permits the detection of two reactions due to lipolytic activity of staphylococci: (1) Lecithinase reaction, a zone of precipitate in the medium surrounding the colonies; and (2) Lipase reaction or 'pearly layer', an iridescent film in and immediately surrounding colonies, visible by reflected light (iridescent sheen or 'oil in water').
In this study, human and bovine strains, previously biotyped according to the scheme of Devriese et al. (1984), were compared for production of a zone of precipitation, lecithinase reaction, on Baird-Parker medium.
Bovine and human strains of Staph. aureus were compared for production of the egg yolk reaction (lecithinase reaction) on Baird-Parker medium and the results were related to their biotypes and site of origin of the sample. Human strains and strains biotyped as human biotypes had higher percentage of positive results than bovine isolates and/or biotypes. However, all strains isolated from body sites of heifers produced a positive reaction regardless of the biotype.     

Detection of early pregnancy-specific proteins in Holstein milk

Bovine pregnancy is commonly diagnosed by rectal palpation or ultrasonography and changes in progesterone concentration. To determine a simpler and less expensive diagnostic method, we sought to identify early pregnancy-specific proteins in bovine milk by comparing samples collected from pregnant and non-pregnant Holstein cattle. Of the 600-700 protein spots visible on 2-DE gel images, 39 were differentially expressed in milk from pregnant and non-pregnant cattle. Antibodies generated against synthetic peptides of milk whey proteins expressed specifically during pregnancy were used to confirm protein expression patterns. Western blot analysis showed that the levels of expression of lactoferrin (lactotransferrin) and alpha1G T-type calcium channel subunit (alpha-1G) were higher in samples from pregnant than non-pregnant cattle. These findings suggest that assays for pregnancy-specific milk proteins may be used to diagnose pregnancy in cattle.     

Antibiotic resistance of Staphylococcus aureus strains isolated in two different Warsaw hospitals]

S. aureus strains isolated in the same period from different specimens obtained from patients of two different hospitals were compared. The significant differences were observed in the frequency of resistance determinants between strains of these hospitals. The most important was the difference in the prevalence of MRSA. In the first hospital the percentage of MRSA was 40% whereas in the second one only 20%. The resistance to the other antibiotics was also compared, and independently from the compared group: MRSA, MSSA or all, the prevalence of resistance determinants was higher in the first hospital than in the second. Although the frequencies of MRSA in both investigated hospitals were relatively high comparing to the other European countries and in the first hospital even alarming, isolated MRSA strains are less resistant to other antibiotics than MRSA in other countries.     

Mechanism of compact-colony formation by strains of Staphylococcus aureus in serum soft agar.

Compact-colony forming active substance (CCFAS), the material responsible for the compact colonies of Staphylococcus aureus observed in serum soft agar, was found to be an alkaline-stable, associated polysaccharide containing galactose, N-acetylglucosamine, ribitol, phosphorus and a small quantity of alanine. This substance, when extracted from strains unable to produce protein A clumping factor, was able to absorb the serum-reacting factor whereas a teichoic acid preparation of one strain could not. The formation of CCFAS was unaffected by the age of the cells, whereas when staphylococci were cultured at alkaline pH, young cells produced more clumping factor than old ones. Both fibrinogen and its degradation products were capable of inducing compact colonies in a strain of S. aureus. The ability of human sera to interact in compact-colony formation was independent of the immunoglobin content. Thus neither protein A, clumping factor, nor teichoic acid participate in the CCFAS reaction.     

Persistence of Staphylococcus aureus L-form during experimental lung infection in rats

The course of pulmonary infection in rats infected by intranasal inoculation with a Staphylococcus aureus stable protoplast L-form was studied. Blood and bronchoalveolar samples were taken on days 3, 7, 14 and 30 after challenge and were investigated by microbiological, electron-microscopic, cytochemical and cytometric methods. The electron microscopic data and isolation of L-form cultures from bronchoalveolar samples at all experimental times demonstrated the ability of S. aureus L-form cells to internalize, replicate and persist in the lungs of infected rats to the end of the observation period, in contrast to the S. aureus parental form. It was found that persisting L-form evoked ineffectual phagocytose by alveolar macrophages and low but long-lasting inflammatory reaction in rats. The experimental model of pulmonary infection with S. aureus L-form suggests that the cell-wall-deficient bacterial forms may be involved in the pathogenesis of chronic and latent lung infections.