Time course of biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates

Biofilm formation is considered a selective advantage for staphylococci mastitis isolates, facilitating bacterial persistence in the udder. It requires attachment to mammary epithelium, proliferation and accumulation of cells in multilayers and enclosing in a polymeric matrix, being regulated by several loci. As biofilm formation can proceed through different pathways and time ranges, its detection may differ according to the time of observation. This study aimed at evaluating the time course evolution of biofilm production in Staphylococcus aureus (n = 26) and Staphylococcus epidermidis (n = 29) mastitis isolates by Fluorescent In Situ Hybridisation. Biofilm-forming ability increased with incubation time for both species: for S. aureus, 34.6%, 69.2% and 80.8% of the isolates were able to produce biofilm at 24, 48 and 72 h, respectively. For S. epidermidis, 44.8%, 62.1% and 75.9% of the isolates were biofilm-positive at 24, 48 and 72 h, respectively. No significant difference was found between species at each time point (Friedman's test, p > 0.05). For S. aureus, although a significant difference was found between 24 and 48 h (Wilcoxon matched paired test, p < 0.05), no significant difference was found between 24 and 48 h (p > 0.05). For S. epidermidis, significant differences were found between each time point (p < 0.05). Bacterial biofilms may impair eradication of chronic mastitis, rendering antibiotherapy less effective. Detection of biofilm-forming ability in mastitis isolates may provide useful information for the establishment of a more adequate therapeutic regimen, in view of the antimicrobial concentrations required for bacterial control. However, it is essential that biofilm formation time course is taken into consideration.     

Biofilm-forming ability profiling of Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates

Biofilm-forming ability has been increasingly recognized as an important virulence factor in Staphylococci, facilitating their persistence in the host, evading its defences and allowing bacterial survival at high antimicrobial concentrations. Staphylococcus aureus remains a major pathogen of chronic mastitis, but in the last years Staphylococcus epidermidis has emerged as a relevant mastitis pathogen. The present work aimed at the evaluation of the biofilm-forming ability of Staphylococci field isolates from bovine subclinical mastitis and at the development of a fluorescent in situ hybridisation (FISH) protocol that would allow the direct observation of biofilm formation in milk samples. The analysis of phenotypic expression in Congo Red Agar (CRA) and by FISH, showed that 37.5% of the S. aureus isolates produced biofilm, while by optical density measurement only 18.75% isolates revealed this phenotype. The results showed a fair agreement according to the kappa coefficient test (kappa = 0.259). Regarding S. epidermidis mastitis isolates, 37.5% revealed the ability to produce biofilm, but only four isolates were positive by all methods. This agreement was moderate (kappa = 0.467). The application of FISH to artificially contaminated milk samples allowed the direct observation of biofilm production by 37.5% isolates, showing total agreement with the CRA results. This method better mimics the in vivo conditions, especially in terms of the presence of calcium and iron, which in high concentrations, respectively, are known to inhibit or induce biofilm production.     

Changes in blood and milk lymphocyte sub-populations during acute and chronic phases of Staphylococcus aureus induced bovine mastitis

Staphylococcus aureus (S. aureus) often causes long-lasting chronic sub-clinical udder infections in dairy cows. To investigate if this can be due to a negative impact of S. aureus on lymphocytes important for the immune defence, alterations in proportions and expression intensity of CD4+, CD8+, WC1+, B and IL-2R+ lymphocytes was studied in blood and milk, as S. aureus mastitis developed from acute clinical to chronic sub-clinical form. Six healthy dairy cows were inoculated with S. aureus in one udder quarter per cow, and one quarter per cow acted as an uninfected control. Blood samples, and milk samples from infected and non-infected quarters were collected before infection and for five weeks after infection. All infected quarters developed acute clinical mastitis, of which five turned into chronic sub-clinical mastitis. In infected quarters, the proportions of all lymphocyte sub-sets, except WC1+ cells, differed in acute phase compared to pre-infection, while the dominant finding in the chronic phase was increased expression intensities per cell. An impact on blood lymphocytes and milk lymphocytes in non-infected quarters also occurred, mainly during the chronic phase. The most prominent finding was the increased proportion and expression of B-lymphocytes in blood, infected and non-infected quarters during chronic sub-clinical mastitis. As S. aureus can invade and survive intracellularly, a preferential stimulation of B-cells, suggesting development of a humoral response, may not be sufficient to eliminate intracellular bacteria, which could explain the persistence of the infection.     

Establishment of canine and feline cells expressing canine signaling lymphocyte activation molecule for canine distemper virus study

Antimicrobial therapy is a useful tool to control bovine mastitis caused by Staphylococcus aureus, as consequence an increase in staphylococci resistant cases has been registered. Alternative strategies are desirable and bacteriocins represent attractive control agents to prevent bovine mastitis. The aim of this work was to evaluate the activity of five bacteriocins synthesized by Bacillus thuringiensis against S. aureus isolates associated to bovine mastitis. Fifty S. aureus isolates were recovered from milk composite samples of 26 Holstein lactating cows from one herd during September 2007 to February 2008 in México and susceptibility of those isolates to 12 antibiotics and 5 bacteriocins from B. thuringiensis was evaluated. S. aureus isolates were mainly resistant to penicillin (92%), dicloxacillin (86%), ampicillin (74%) and erythromycin (74%); whereas susceptibility to gentamicin, trimethoprim and tetracycline was detected at, respectively, 92%, 88%, and 72%. All S. aureus isolates showed susceptibility to the five bacteriocins synthesized by B. thuringiensis, mainly to morricin 269 and kurstacin 287 followed by kenyacin 404, entomocin 420 and tolworthcin 524. Our results showed that S. aureus isolates had differences in the antimicrobial resistance patterns and were susceptible to bacteriocins produced by B. thuringiensis, which could be useful as an alternative method to control bovine mastitis.     

奶牛乳房炎金黄色葡萄球菌荚膜多糖主要血清型的鉴定

为了解内蒙古地区奶牛乳房炎金黄色葡萄球菌荚膜多糖的主要血清型,试验从内蒙古地区9个牧场共采集236份奶牛乳房炎患牛的奶样,采用常规微生物方法、生物化学反应方法、动物试验和PCR方法对金黄色葡萄球菌荚膜多糖的血清型进行鉴定。结果表明:经常规微生物学检验分离得到162株葡萄球菌,其中金黄色葡萄球菌(S.aureus)124株、表皮葡萄球菌(S.epidermidis)29株、腐生葡萄球菌(S.saparophytics)9株。动物试验得到116株致病性S.aureus。PCR方法鉴定出荚膜多糖5型S.aureus23株,占致病性S.aureus的19.83%;荚膜多糖8型S.aureus61株,占致病性S.aureus的52.59%;荚膜多糖5型和8型S.aureus占致病性S.aureus的72.41%。说明5型和8型S.aureus是内蒙古地区奶牛乳房炎金黄色葡萄球菌荚膜多糖的主要血清型。     

Prevalence of coagulase-negative staphylococci in bovine mastitis in Zimbabwe

This study was carried out to determine the prevalence of coagulase-negative staphylococci in clinical and subclinical mastitis in commercial and small-scale farms in Zimbabwe. Thirty five quarter milk samples from clinical mastitis cases and 371 quarter milk samples from cows with subclinical mastitis were cultured for bacterial pathogens. The most frequent pathogens isolated in clinical mastitis were the enteric bacteria (31.4%), followed by coagulase negative staphylococci (22.9%) and then Staphylococcus aureus (17.1%), whereas in subclinical mastitis S. aureus (34.2%) and coagulase-negative staphylococci were (33.2%) the most common. Bacillus species were only isolated in milk samples from subclinical mastitis. Coagulase-negative staphylococci were observed in mixed infections with other bacteria in only 2.2 of the 406 milk samples from clinical and subclinical mastitis where they were isolated together with Bacillus species in 6 of the 9 mixed infection cases. About 95% of the milk samples from which 131 coagulase-negative staphylococci were isolated had correspondingly high somatic cell counts. The coagulase-negative staphylococci isolated most frequently were S. chromogenes (7.9%), S. epidermidis (7.4%) and S. hominis (5.9%). They were all associated with high somatic cell counts. All the coagulase-negative staphylococci isolates were susceptible to cloxacillin and erythromycin, and more than 90% of the isolates were susceptible to neomycin, penicillin and streptomycin. The highest resistance was to tetracycline (17.6%), followed by lincomycin (13.7%). About 8% of the isolates were resistant to both penicillin and streptomycin.     

奶牛乳房炎金黄色葡萄球菌荚膜多糖研究概况

金黄色葡萄球菌是引起奶牛乳房炎的一种主要致病菌,其中94%～100%的金黄色葡萄球菌都含有荚膜多糖.荚膜多糖共分11个血清型(CP1～CP11),而5型和8型占临床分离株的70%～80%,为优势血清型;由于荚膜多糖的抗吞噬作用和黏附作用,决定了金黄色葡萄球菌的致病性和免疫原性.因此,基于荚膜多糖的疫苗可能是更有效的抗奶...     

牛源耐甲氧西林金黄色葡萄球菌的检测

本研究旨在了解甘肃地区奶牛乳房炎金黄色葡萄球菌的耐药性和耐甲氧西林金黄色葡萄球菌的感染情况,为奶牛乳房炎的防制提供理论依据。采用KB纸片扩散法,检测17株金黄色葡萄球菌对8种不同抗菌药物的敏感性;再用琼脂稀释法检测了苯唑西林、万古霉素对金黄色葡萄球菌的最小抑菌浓度(MICs);头孢西丁纸片扩散法和PCR扩增特异性mecA耐药基因对所有受试菌株进行全面的耐甲氧西林金黄色葡萄球菌检测。结果表明,菌株对青霉素、磺胺异恶唑具有较强抗性,而对环丙沙星、头孢唑啉、万古霉素和苯唑西林全敏感;头孢西丁纸片扩散法未能检测出表型为MRSA的阳性菌株,而PCR方法却检测出8株mecA基因阳性菌株,且这些菌株的苯唑西林MIC均小于2 μg/mL。菌株的耐药情况较严重,对甲氧西林敏感而携带mecA基因的菌株高频存在于被调查地区的奶牛场中。     

Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome

Based on our clinical experience on bovine mastitis, we hypothesized that subtypes of Staphylococcus aureus (S. aureus) exist which differ in their contagious and pathogenic properties. In order to investigate this hypothesis, we analyzed strains of S. aureus isolated from spontaneous intramammary infection (IMI) with their virulence gene patterns and genotypes obtained by PCR amplification of the 16S-23S rRNA intergenic spacer (RS-PCR). The genotypes were then associated with epidemiological and clinical data including 26 herds. The results demonstrated a high association between genotypes and virulence gene patterns as well as between epidemiological and pathogenic properties of S. aureus. In particular, genotype B was related to high contagiosity and increased pathogenicity whereas the other types (C, OG) were found with infection of single cows. Because of the high clinical relevance, our results indicate the need to subtype the IMI-associated strains of S. aureus in the future.     

CNS species and antimicrobial resistance in clinical and subclinical bovine mastitis

Coagulase-negative staphylococci (CNS) are often associated with bovine mastitis. Knowledge about the relative importance of specific CNS species in different types of mastitis, and differences in antimicrobial resistance among CNS species is, however, scarce. Therefore, the aims of this study were to compare prevalence and antimicrobial susceptibility of CNS species in clinical and subclinical mastitis using material from two national surveys. Overall, Staphylococcus chromogenes and Staphylococcus epidermidis were the most common CNS species found followed by Staphylococcus simulans and Staphylococcus haemolyticus. S. epidermidis was significantly more prevalent in subclinical than in clinical mastitis, and a similar trend was observed for Staphylococcus saprophyticus, while Staphylococcus hyicus was significantly more common in clinical mastitis. The prevalence of β-lactamase producing isolates varied markedly between CNS species, and was significantly higher in S. epidermidis and S. haemolyticus (～ 40%), than in S. simulans and S. chromogenes where none or a few of the isolates produced β-lactamase. Resistance to more than one antimicrobial substance occurred in 9% and 7% of the clinical and subclinical isolates, respectively. In conclusion, the distribution of CNS species differed between clinical and subclinical mastitis indicating inter-species variation of pathogenicity and epidemiology. Overall, the prevalence of antimicrobial resistance was low, but some variation between CNS species was observed.     

奶牛乳房炎金黄色葡萄球菌的分离鉴定及药敏试验

为深入了解内蒙古自治区、河北省、黑龙江省、上海市和山东省奶牛体内金黄色葡萄球菌的耐药情况,从5个省市自治区共收集247份患乳房炎奶牛乳样,进行金黄色葡萄球菌的分离。通过染色镜检、PCR和微量肉汤稀释法鉴定金黄色葡萄球菌并检测其耐药性。结果表明金黄色葡萄球菌分离率12.15%。所分离得到金黄色葡萄球菌对氨曲南的耐药率最高,达到了100%;其次为青霉素(80%)、氧氟沙星(43.33%)、头孢西丁(43.33%)和美罗培南(43.33%),耐药率均高于40%。耐药率低于10%的为万古霉素(全部敏感)、苯唑西林(全部敏感)、利奈唑胺(全部敏感)、阿莫西林-克拉维酸(全部敏感)、氯霉素(3.33%)、复方新诺明(3.33%)、利福平(6.67%)和环丙沙星(6.67%)。检测结果为奶牛乳房炎的防控和临床用药提供了参考依据。     

多重PCR快速检测奶牛乳房炎3种主要病原体

奶牛乳房炎是引起奶牛业经济损失的一种重要疫病,目前还没有快速、特异检测奶牛乳房炎主要致病原的方法。本试验根据金黄色葡萄球菌、无乳链球菌、大肠杆菌各自保守的16S或23S rRNA基因序列,合成了3对特异性引物,建立了三重PCR检测方法。特异性试验表明,该方法对所有参与测试的金黄色葡萄球菌、无乳链球菌和大肠杆菌都能扩增出各自的阳性条带,而对所有参与测试的对照菌株则不能扩增出任何条带。敏感性试验表明该方法能检测到4个菌的金黄色葡萄球菌、无乳链球菌和2个菌的大肠杆菌。对送检的乳房炎奶样36份直接进行PCR检测,金黄色葡萄球菌阳性7份,无乳链球菌阳性2份,大肠杆菌阳性6份。     

Efficacy of specific egg yolk immunoglobulin (IgY) to bovine mastitis caused by Staphylococcus aureus

The objective of this study was to estimate the efficacy of specific egg yolk immunoglobulin (IgY) to bovine mastitis caused by Staphylococcus aureus. Eighteen lactating cows with clinical mastitis and 18 lactating cows with experimental mastitis (1 quarter per cow) were randomly assigned to three treatments: IgY (20mg/ml) infusion, penicillin (100mg/ml) infusion and no infusion. Treatments for clinical mastitis and experimental mastitis were performed by a 6-day course of intramammary infusion with a dosage of 10ml at an interval of 12h. Milk samples were collected at morning milking time for testing color, clot, somatic cell counts (SCC) and bacterial count. For most of the cows treated with IgY and penicillin, the milk color and clot recovered to normal form during the therapy course. The milk SCCs and bacterial counts of treated cows decreased compared to those of untreated cows (p<0.05). The cure rates by IgY for experimental and clinical mastitis were 83.3% and 50%, respectively, and those by penicillin were 66.7% and 33.3%, respectively. These results showed the potential of specific IgY to be an alternative therapy for mastitis caused by S. aureus.     

Antimicrobial susceptibility of coagulase-negative Staphylococcus species isolated from bovine milk

Coagulase-negative Staphylococcus (CNS) isolates (n=168) obtained from milk from heifers and dairy cows were screened for minimum inhibitory concentration (MIC) to antimicrobials used commonly for mastitis therapy. Of the 10 CNS species included in the study, the predominant species were Staphylococcus chromogenes (n=61), Staphylococcus epidermidis (n=37), Staphylococcus hyicus (n=37), and Staphylococcus simulans (n=16). The majority of CNS was susceptible to ampicillin, oxacillin, cephalothin, and ceftiofur. Erythromycin and pirlimycin were also very effective in vitro inhibitors of CNS. The only exception was observed with S. epidermidis. Of 37 S. epidermidis evaluated, 13 (35%) exhibited efflux-based resistance to erythromycin (> or =16 microg/ml) encoded by msrA and one isolate carried ermC encoding ribosomal methylase-based resistance to both erythromycin (> or =64 microg/ml) and pirlimycin (> or =64 microg/ml). A total of 17 S. epidermidis, 11 S. chromogenes, and one S. hyicus exhibited phenotypic resistance to ampicillin (> or =0.5 microg/ml). Constitutive beta-lactamase production was observed in all ampicillin resistant isolates except 4 S. epidermidis that exhibited inducible beta-lactamase production. Induced beta-lactamase production was also observed in 13 S. epidermidis that were phenotypically susceptible to the entire MIC panel. All isolates that produced beta-lactamase either constitutively or by induction carried blaZ. S. epidermidis (n=12, 32%) that were resistant to methicillin (oxacillin > or =0.5 microg/ml) carried low affinity penicillin-binding protein encoded by mecA. Most multi-drug resistant (MDR) S. epidermidis (> or =2 resistance genes) were resistant to ampicillin, erythromycin and methicillin. All except one MDR S. epidermidis had icaAB, which encodes for polysaccharide intercellular adhesion. Based on pulsed field gel electrophoresis, MDR S. epidermidis were closely related genotypically, and were isolated from different cows on the same farm suggesting clonal dissemination. Bovine S. epidermidis share antimicrobial resistance patterns and virulence determinants of strains observed in human infections. Studying CNS at the species level can provide valuable information about species-specific differences that can be vital data for effective mastitis therapy and management.     

Phenotypic and genotypic characterization of bovine mastitis isolates of Staphylococcus aureus for biofilm formation

Staphylococcus aureus is one of the most common pathogens responsible for contagious mastitis in ruminants. The ability of S. aureus to form biofilm in vivo is considered to be a major virulence factor influencing its pathogenesis in mastitis. The objectives of the study were to examine in vitro slime production, biofilm formation, and the presence of the ica gene locus and icaA and icaD genes in S. aureus isolates from bovine mastitis. Thirty-two of the 35 isolates tested produced slime on Congo red agar, whereas only 24 of the isolates were found to produce biofilm in vitro. However, all the 35 isolates possessed the ica locus, icaA and icaD genes. This study indicates a high prevalence of the ica genes among S. aureus mastitis isolates, and their presence is not always associated with in vitro formation of slime or biofilm. A combination of phenotypic and genotypic tests is recommended for investigating biofilm formation in S. aureus.     

金黄色葡萄球菌α-溶血素基因的克隆与序列分析

参考Gary S Gray和Michael Kehce在《传染与免疫》杂志上发表的关于金黄色葡萄球菌α溶血素蛋白的基因序列,设计合成1对引物,从分离自牛体的野生株金黄色葡萄球菌中提取细菌总DNA,对α溶血素蛋白基因进行PCR扩增,产物经琼脂糖凝胶电泳分析,呈现1条约960 bp的条带,回收纯化后,将其克隆至pMD18 T质粒载体中,进行核苷酸序列分析,然后与报道的α溶血素蛋白基因进行比较。结果表明:所鉴定的野生株金黄色葡萄球菌的α溶血素蛋白基因序列与报道的核苷酸的同源性高达99.99%以上,证实为金黄色葡萄球菌Wood 46株α溶血素蛋白基因。     

胸腺嘧啶依赖型金黄色葡萄球菌小菌落突变株的分离鉴定

本试验对由患慢性奶牛乳房炎奶样中分离出的1株疑似金黄色葡萄球菌小菌落突变株(SCVs)进行形态观察、金黄色葡萄球菌相关保守基因片段(nuc、nucA、16S rDNA) 多重PCR扩增鉴定、药敏试验、生理生化特性研究及补偿试验。结果显示分离出1株金黄色葡萄球菌SCVs,该菌含有金黄色葡萄球菌菌种特异性基因nuc和nucA;与金黄色葡萄球菌质控菌株ATCC 25923的抑菌圈大小明显不同;菌落形态主要表现为菌落细小、生长缓慢、溶血能力下降;凝固酶活性下降;耐盐能力降低;革兰氏染色为革兰氏阳性球菌,呈葡萄状排列;补偿试验鉴定该金黄色葡萄球菌SCVs为胸腺嘧啶依赖型。结果表明成功分离鉴定出1株胸腺嘧啶依赖型金黄色葡萄球菌SCVs,为由金黄色葡萄球菌SCVs引起的奶牛慢性乳房炎的预防和控制及其致病机制的研究奠定前期基础。     

抑制奶牛乳房炎源金黄色葡萄球菌的乳酸菌的筛选

为获得抑制奶牛乳房炎源金黄色葡萄球菌(S. aureus)的乳酸菌(LAB),本研究从新疆巴音布鲁克牧区鲜牛乳和哈萨克族乳制品奶疙瘩样品中分离培养LAB,通过传统的分离鉴定与16S rDNA基因序列测序相结合的方法鉴定LAB种类,同时以临床奶牛乳房炎源金黄色葡萄球菌S. aureus N2为指示菌,采用双层琼脂扩散法检测分离株的抑菌能力。通过测定生长曲线确定分离株的生长稳定期,进而利用硫酸铵沉淀法透析提取稳定期内分离株的细菌素,并检测其细菌素抑菌效价。结果显示:从样品中筛选获得5株能够抑制指示菌生长的LAB,分别为希氏乳杆菌、干酪乳杆菌、粪肠球菌、戊糖片球菌和乳酸乳球菌亚种。生长规律曲线表明20 h^30 h为5株LAB的稳定期,此期培养液pH值维持在3.8~4.5。从培养20 h的5株LAB上清液中提取到了细菌素,经检测其具有抑菌活性,抑菌效价分别为457 IU/mL、1 023 IU/mL、676 IU/mL、1 862 IU/mL和1 023 IU/mL。本研究结果表明5株LAB通过在生长稳定期内维持较低酸性环境(pH<4.5),代谢产生细菌素对乳房炎源S. aureus发挥抑制生长作用。本研究为S. aureus性奶牛乳房炎的生物防治提供了实验依据。     

奶牛乳房炎金黄色葡萄球菌8种毒力基因的PCR检测

为研究奶牛乳房炎金黄色葡萄球菌(Staphylococcus aureus,SA)主要毒力因子的分布情况,本试验利用PCR对70株临床型乳房炎、55株隐性乳房炎金黄色葡萄球菌的8种主要毒力基因进行检测。结果显示,nuc、ClfA、TSST-1、PVL、Hla、Hlb、FnBPA和FnBPB 8种毒力基因在临床型乳房炎SA菌株中的检出率分别为:100.0%(70株)、100.0%(70株)、0(0株)、5.7%(4株)、100.0%(70株)、11.4%(8株)、97.1%(68株)和100.0%(70株);在隐性乳房炎SA菌株中的检出率分别为:100.0%(55株)、100.0%(55株)、0(0株)、70.9%(39株)、98.2%(54株)、9.1%(5株)、100.0%(55株)和100.0%(55株)。结果表明,nuc、ClfA、Hla、Hlb和FnBPA 5种毒力基因是引起奶牛乳房炎的SA的最主要毒力基因;PVL基因可能是引起隐性乳房炎的重要致病基因。     

A retrospective study of the aetiology and temporal distribution of bovine clinical mastitis in smallholder dairy herds in the Dar es Salaam region of Tanzania

A 31-year record-based retrospective study was carried out to determine the aetiology and temporal distribution of bovine clinical mastitis in smallholder dairy herds in the Dar es Salaam region of Tanzania over the period November 1971-December 2002. Laboratory information on 1964 quarter samples from 1365 cows in 281 smallholder dairy herds were retrieved, compiled and studied. Eighty-eight percent of the quarter samples were culture-positive and the predominant mastitis pathogens isolated were Staphylococcus aureus (25.7%), Streptococcus agalactiae (15.4%), Klebsiella pneumoniae (14.3%) and Escherichia coli (14.1%). Other isolates included Pseudomonas aeruginosa (7.5%), Streptococcus dysgalactiae (5.2%) and Streptococcus uberis (4.2%). Contagious mastitis pathogens were isolated from 45.6% of the culture-positive samples, whereas environmental and miscellaneous pathogens were isolated from 48.2% and 5.7%, respectively. Thirty percent of the miscellaneous mastitis pathogens were Candida species. The results demonstrate a steady increase in clinical Candida albicans mastitis. The prevalence of Candida albicans has increased from 1% in 1971 to 17.0% in November 2002. Conversely, despite some fluctuations, the prevalence of Staphylococcus aureus, Streptococcus agalactiae, E. coli and K. pneumoniae remain above 10%. The possible risk factors for these observations are discussed.