Expression and lytic efficacy assessment of the Staphylococcus aureus phage SA4 lysin gene

Treatment of bovine mastitis caused by Staphylococcus (S.) aureus is becoming very difficult due to the emergence of multidrug-resistant strains. Hence, the search for novel therapeutic alternatives has become of great importance. Consequently, bacteriophages and their endolysins have been identified as potential therapeutic alternatives to antibiotic therapy against S. aureus. In the present study, the gene encoding lysin (LysSA4) in S. aureus phage SA4 was cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clone revealed a single 802-bp open reading frame encoding a partial protein with a calculated mass of 30 kDa. Results of this analysis also indicated that the LysSA4 sequence shared a high homology with endolysin of the GH15 phage and other reported phages. The LysSA4 gene of the SA4 phage was subsequently expressed in Escherichia coli. Recombinant LysSA4 induced the lysis of host bacteria in a spot inoculation test, indicating that the protein was expressed and functionally active. Furthermore, recombinant lysin was found to have lytic activity, albeit a low level, against mastitogenic Staphylococcus isolates of bovine origin. Data from the current study can be used to develop therapeutic tools for treating diseases caused by drug-resistant S. aureus strains.     

PCR-based detection of genes encoding virulence determinants in Staphylococcus aureus from bovine subclinical mastitis cases

The present study was carried out to genotypically characterize Staphylococcus aureus (S. aureus) isolated from bovine mastitis cases. A total of 37 strains of S. aureus were isolated during processing of 552 milk samples from 140 cows. The S. aureus strains were characterized phenotypically, and were further characterized genotypically by polymerase chain reaction using oligonucleotide primers that amplified genes encoding coagulase (coa), clumping factor (clfA), thermonuclease (nuc), enterotoxin A (entA), and the gene segments encoding the immunoglobulin G binding region and the X region of protein A gene spa. All of the isolates yielded an amplicon with a size of approximately 1,042 bp of the clfA gene. The amplification of the polymorphic spa gene segment encoding the immunoglobulin G binding region was observed in 34 isolates and X-region binding was detected in 26 isolates. Amplification of the coa gene yielded three different products in 20, 10, and 7 isolates. The amplification of the thermonuclease gene, nuc, was observed in 36 out of 37 isolates. All of the samples were negative for the entA gene. The phenotypic and genotypic findings of the present strategies might provide an understanding of the distribution of the prevalent S. aureus clones among bovine mastitis isolates, and might aid in the development of steps to control S. aureus infections in dairy herds.     

兔金黄色葡萄球菌的分离鉴定

本研究从福建省某养殖场病兔分离到3株革兰氏阳性球菌,并对其进行了16S rRNA扩增、生化反应、药敏试验等检测,结果显示该病原菌为金黄色葡萄球菌。     

Difference in virulence between Staphylococcus aureus isolates causing gangrenous mastitis versus subclinical mastitis in a dairy sheep flock

Staphylococcus aureus mastitis in dairy sheep ranges from subclinical mastitis to lethal gangrenous mastitis. Neither the S. aureus virulence factors nor the host-factors or the epidemiological events contributing to the different outcomes are known. In a field study in a dairy sheep farm over 21 months, 16 natural isolates of S. aureus were collected from six subclinical mastitis cases, one lethal gangrenous mastitis case, nasal carriage from eight ewes and one isolate from ambient air in the milking room. A genomic comparison of two strains, one responsible for subclinical mastitis and one for lethal gangrenous mastitis, was performed using multi-strain DNA microarrays. Multiple typing techniques (pulsed-field-gel-electrophoresis, multiple-locus variable-number, single-nucleotide polymorphisms, randomly amplified polymorphic DNA, spa typing and sas typing) were used to characterise the remaining isolates and to follow the persistence of the gangrenous isolate in ewes’ nares. Our results showed that the two strains were genetically closely related and they shared 3 615 identical predicted open reading frames. However, the gangrenous mastitis isolate carried variant versions of several genes (sdrD, clfA-B, sasA, sasB, sasD, sasI and splE) and was missing fibrinogen binding protein B (fnbB) and a prophage. The typing results showed that this gangrenous strain emerged after the initial subclinical mastitis screening, but then persisted in the flock in the nares of four ewes. Although we cannot dismiss the role of host susceptibility in the clinical events in this flock, our data support the hypothesis that S. aureus populations had evolved in the sheep flock and that S. aureus genetic variations could have contributed to enhanced virulence.     

The first nosocomial outbreak of methicillin-resistant Staphylococcus aureus in horses in Sweden

Background

Methicillin-resistant Staphylococcus aureus (MRSA) in animals is a rare finding in Sweden. In horses, MRSA was first detected in a screening survey in 2007. In 2008, six clinical cases occurred in an equine hospital, indicating an outbreak.

Method

All MRSA isolates detected, 11 spa-type t011 and one t064 (n = 12), in infected horses (n = 10) and screening of horses (n = 2) in Sweden from December 2007 to March 2010 were retrospectively analysed with pulsed-field gel electrophoresis (PFGE) using Cfr9I and ApaI restriction enzymes, to study relationship between the isolates. Medical records of infected horses and outbreak investigation notes were scrutinised to monitor the clinical outcome and other aspects of the outbreak.

Results

Eight of the 10 infected horses were linked to one equine hospital and two to another hospital in the same region. The six horses infected with MRSA in 2008 underwent surgery during the period 22 May-7 July in one of the hospitals. Four more infections linked to the two hospitals were notified between 2009 and March 2010.Nine of the 11 spa-type t011 isolates had identical Cfr9I and ApaI PFGE pattern. All six infected horses from 2008 presented with this MRSA. Two t011 isolates differed in one and two bands, respectively, in PFGE.Nine horses suffered from surgical site infections (SSI). No antimicrobials were used following the MRSA diagnosis and the infections cleared. The time from surgery to MRSA diagnosis differed greatly between the horses (range 15-52 days).

Conclusions

Association in time and space of six horses infected with an identical MRSA strain of spa-type t011 confirmed an outbreak. Two isolates found in 2009 and 2010 in the outbreak hospital were closely related to the outbreak strain, indicating one circulating strain. Both spa-type t011 and t064 have been reported in horses in Europe prior to these findings. The observation that the infections cleared although antimicrobials were not used is encouraging for future prudent use of antimicrobials. The time from surgery to bacteriological diagnosis was not acceptable in most cases, as contagious spread was a risk. Sampling when symptoms of infection are noticed and accurate analysis are thus important.     

Genotypic characterization of Staphylococcus aureus isolated from bovines, humans, and food in Indonesia

The present study determined the genetic relationships between 41 Staphyloccocus (S.) aureus isolates from bovines, humans, and food using a single enzyme amplified fragment length polymorphism (AFLP) technique. We evaluated the prevalence of staphylococcal enterotoxin (SE) genes and other virulence gene determinants by PCR. The identification of S. aureus was based on culturing and biochemical tests, and by amplifying a specific section of the 23S rRNA gene. PCR amplification of the SE genes (sea, seb, sec, see, seg, seh, and sei) singly or in combination was observed. Most isolates of bovine origin harbored hla (84%) and cap5 (74%), while most isolates from humans harbored hla (73%), cap8 (91%), and fnbA (100%). Strains from food sources were positive for hla (100%), cap5 (100%), and cap8 (64%) unlike isolates from humans or bovines. A single enzyme AFLP analysis revealed a correlation between AFLP clusters of some strains and the source of the isolates The genotypic results of the present study might help to better understand the distribution of prevalent S. aureus clones among humans, bovines, and food and will help control S. aureus infections in Indonesia.     

Risk factors for methicillin-resistant Staphylococcus aureus (MRSA) infection in dogs and cats: a case-control study

Risk factors for methicillin-resistant Staphylococcus aureus (MRSA) infection in dogs and cats were investigated in an unmatched case-control study. A total of 197 animals from 150 veterinary practices across the United Kingdom was enrolled, including 105 MRSA cases and 92 controls with methicillin-susceptible S. aureus (MSSA) infection. The association of owners and veterinarian staff with the human healthcare sector (HCS) and animal-related characteristics such as signalment, antimicrobial and immunosuppressive therapy, and surgery were evaluated as putative risk factors using logistic regression. We found that significant risk factors for MRSA infection were the number of antimicrobial courses (p = 0.005), number of days admitted to veterinary clinics (p = 0.003) and having received surgical implants (p = 0.001). In addition, the odds of contact with humans which had been ill and admitted to hospital (p = 0.062) were higher in MRSA infected pets than in MSSA controls. The risk factors identified in this study highlight the need to increase vigilance towards identification of companion animal groups at risk and to advocate responsible and judicious use of antimicrobials in small animal practice.     

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.     

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。     

In vitro susceptibility of Staphylococcus aureus strains isolated from cows with subclinical mastitis to different antimicrobial agents

Sensitivity to commercial teat dips (nonoxinol-9 iodine complex and chlorhexidine digluconate) of 56 Staphylococcus (S.) aureus strains isolated from quarter milk samples of various German dairy herds treated with different teat dipping schemes was investigated in this study. The minimum inhibitory concentration was determined using a broth macrodilution method according to the German Veterinary Association guidelines. The main objective of the current study was to induce in vitro resistance induction of S. aureus to chemical disinfectants. Ten different strains were repeatedly passed ten times in growth media with sub-lethal concentrations of disinfectants. Nine strains showed a significant reduction in susceptibility to the nonoxinol-9 iodine complex but only one strain developed resistance to chlorhexidine digluconate. Stability of the acquired resistance was observed in all S. aureus strains adapted to the nonoxinol-9 iodine complex and chlorhexidine digluconate. In contrast, simultaneous resistance to different antibiotics was not observed in any of the ten investigated S. aureus strains. However, the isolates exhibited a high degree of resistance to penicillin G. Based on these results, resistance of S. aureus to chemical disinfectants may be more likely to develop if the chemicals are used at concentrations lower than that required for an optimal biocidal effect.     

Reservoirs of Staphylococcus aureus in meat sheep and dairy cattle

The objective of the study was to investigate reservoirs and transmission of S. aureus in ewes and lambs in 3 meat sheep flocks. Repeated sampling of milk, teat skin, nasal- and vaginal mucous membranes was performed and samples were analysed for S. aureus. For comparison, samples were also collected from cows and young heifers in 3 dairy cattle herds. Selected isolates were compared by pulsed-field gel electrophoresis (PFGE). S. aureus was detected in 8 (1.5%) of 520 milk samples from ewes and in 38 (6.4%) of 588 milk samples from cows. From body site swabs, S. aureus was found in 394 (32.6%) of 1208 samples from sheep and in 67 (16.0%) of 420 samples from cattle. The proportion of S. aureus-positive nasal swabs from ewes and cows were 56.7% and 13.9%, respectively. From lambs, 58.2% of the nasal swabs were S. aureus-positive. In each flock, one S. aureus pulsotype predominated. Identical S. aureus pulsotypes were found in milk and from body sites. Paired S. aureus isolates from the nasal cavity of (i) ewes and their lambs, (ii) twins and (iii) from repeated swabs of individual ewes were compared by PFGE, and in the majority of cases the two isolates were identical. The results contribute new knowledge indicating frequent transmission of S. aureus between the dam and her lambs and within animals in a flock. In contrast to cattle, S. aureus is frequently present in the nose of sheep which may represent the primary reservoir of S. aureus in sheep flocks.     

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.     

Genetic basis of penicillin resistance of S. aureus isolated in bovine mastitis

Background

The blaZ gene encoding penicillin resistance can be located either chromosomally or on plasmids. The aim of this study was to investigate the genetic relationships and to determine the location of the blaZ gene in S. aureus isolated in bovine mastitis in Finland and Sweden.

Methods

Seventy-eight β-lactamase positive S. aureus isolates from bovine mastitis (34 from Finland and 44 from Sweden) were included in the study. The localization of blaZ gene was determined by Southern blotting. The blaZ genes of the isolates were sequenced and the sequences were translated to beta-lactamase proteins and further grouped as different protein signatures. The isolates and, as control, 33 Swedish and 36 Finnish beta-lactamase negative isolates were typed with pulsed-field gel electrophoresis (PFGE).

Results

In 26 out of 34 Finnish isolates (76.5%) and in 25 out of 44 Swedish isolates (56.8%) the blaZ gene was localized on a plasmid. Six different protein signatures were found. One signature was found only in four Swedish isolates, but all other signatures were found both in Finnish and Swedish isolates. The PFGE results revealed a diversity of S. aureus clones. The protein signatures were not clearly associated with certain pulsotypes.

Conclusions

The plasmid location of the blaZ gene was not statistically significantly more common in Finland than in Sweden, and hence does not explain the higher proportion of penicillin-resistant isolates of S. aureus causing bovine mastitis in Finland compared to Sweden.     

Associations between bacterial genotype and outcome of bovine clinical Staphylococcus aureus mastitis

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.     

金黄色葡萄球菌及其肠毒素等指标在发酵牛肉加工过程中变化规律的研究

作者对自然状态发酵牛肉、接种金黄色葡萄球菌的发酵牛肉和接种发酵剂(植物乳杆菌、戊糖片球菌)与金黄色葡萄球菌的发酵牛肉中金黄色葡萄球菌及其肠毒素等指标在加工过程中的变化规律做了研究。研究结果表明,自然发酵牛肉中没有金黄色葡萄球菌及其肠毒素;接种发酵剂和金黄色葡萄球菌的发酵牛肉中金黄色葡萄球菌先是上升然后快速下降,且金黄色葡萄球菌肠毒素为阴性;接种金黄色葡萄球菌的发酵牛肉中金黄色葡萄球菌及其肠毒素都显著高于接种发酵剂的发酵牛肉(P＜0.05)。由试验结果可知,发酵剂植物乳杆菌和戊糖片球菌对金黄色葡萄球菌有很强的抑制作用。     

Phenotypic and genotypic characterization of Staphylococcus aureus isolated from raw camel milk samples

In the present study 320 milk samples collected from 160 apparently healthy camels of three different locations in Sudan were investigated for the presence of Staphylococcus aureus resulting in the isolation of this bacterial pathogen from 28 milk samples from 24 camels. Twenty-five S. aureus were identified phenotypically and by PCR mediated amplification of species-specific genes or gene segments. Investigation of the S. aureus for toxinogenic potential revealed that three S. aureus strains were positive for the enterotoxin encoding gene sec and the genes seg, sei, sem, sen and seo, representing the egc gene cluster. In addition all 25 S. aureus were positive for the superantigen-like encoding gene ssl7 (set1). Partial sequencing of gene sec of the three S. aureus strains yielded an almost complete sequence identity to the sequence of the sec variant sec2. However, all three sec2 genes of the present study showed a deletion of one base causing a frame shift and a corresponding earlier stop codon.According to the present results, the raw camel milk collected from three locations in Sudan seems to be, at least at this stage, of minor importance as vector causing staphylococcal food poisoning.     

亚抑菌浓度穿心莲提取物对减缓金黄色葡萄球菌α-溶血素介导的A549细胞损伤作用的影响

金黄色葡萄球菌是一种广泛存在的重要致病菌,可引起人类和动物许多严重的感染。另外,α-溶血素是金黄色葡萄球菌最重要的毒力因子之一。本研究通过乙醇回流提取方法,获得中草药穿心莲提取物,然后通过最低抑制浓度(MIC)值测定、溶血活性测定、Western blotting、RT-PCR、LDH及Live/dead等方法研究分析穿心莲提取物对金黄色葡萄球菌α-溶血素分泌的影响及对A549细胞的保护作用。试验结果表明,穿心莲提取物对金黄色葡萄球菌的MIC值为2048 μg/mL,且呈剂量依赖性地抑制金黄色葡萄球菌α-溶血素分泌,并能有效减缓金黄色葡萄球菌菌液上清对A549细胞的损伤。提示,穿心莲提取物是一种潜在的抗金黄色葡萄球菌感染的药物。     

Effect of intramammary infusion of bacterial lipopolysaccharide on experimentally induced Staphylococcus aureus intramammary infection

Mastitis due to Staphylococcus aureus is a significant problem in the dairy industry and is refractory to antibiotic treatment and/or vaccine prevention. Relative to other mastitis-causing pathogens, S. aureus elicits a diminutive host inflammatory response during intramammary infection. To determine whether induction of a heightened inflammatory response could influence outcome of infection, the highly pro-inflammatory molecule bacterial lipopolysaccharide (LPS) was infused into udder quarters experimentally infected with S. aureus. Relative to S. aureus-infected udder quarters receiving saline, quarters infused with LPS demonstrated a heightened inflammatory response as demonstrated by the induction of TNF-alpha and higher milk somatic cell counts and albumin levels. Although there was no overall effect on bacterial clearance, a trend toward reduced bacterial numbers during the immediate pro-inflammatory response following LPS infusion was observed suggesting that this novel approach to treating S. aureus intramammary infection may warrant further investigation.     

构树叶提取物对金黄色葡萄球菌的抑制作用及机理研究

为了研究构树叶提取物的抑菌作用及其机理,以金黄色葡萄球菌为供试菌,采用牛津杯法测定构树叶提取物的抑菌效果,通过测定供试菌生长曲线、细胞膜渗透性和蛋白质合成来研究构树叶提取物的抑菌机理。结果表明,构树叶水提取物和75%乙醇提取物抑菌圈直径分别为17.5和18.0 mm,最小抑菌浓度均为6.8 mg/mL,最小杀菌浓度均为12.50 mg/mL。构树提取物能显著抑制金黄色葡萄球菌蛋白质合成,从而抑制其生长,但对其细胞膜的通透性影响不大。因此,构树叶提取物通过抑制金黄色葡萄球菌蛋白质合成发挥其抑菌效果。