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Journal of Applied Microbiology Dec 2011To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus,...
AIM
To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli in milk.
METHODS AND RESULTS
A two-tube mPCR assay was developed. The accuracy of the mPCR was evaluated using 56 standard reference strains and 705 strains comprising of E. coli (n = 99), staphylococci (n = 522) and streptococci (n = 84). The threshold of detection of the mPCR assay was 10 fg of genomic DNA and <10(3) CFU ml(-1). A comparative evaluation of mPCR with culture method using 115 milk samples from subclinical mastitis showed mPCR to be more efficacious. Subsequently, the mPCR showed successful detection of target bacteria, when applied directly for the assessment of 36 bulk milk samples.
CONCLUSION
The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time.
SIGNIFICANCE AND IMPACT OF THE STUDY
The assay will be useful for the detection of mastitis, testing bacteriological safety of milk and for species level differentiation. The assay will be of value in the dairy sector for diagnosis and research. The early and accurate identification of pathogens will enable timely interventions for the treatment and control of bovine mastitis.
Topics: Animals; Bacteria; Cattle; DNA, Bacterial; Female; Food Contamination; Limit of Detection; Mastitis, Bovine; Milk; Multiplex Polymerase Chain Reaction; Sensitivity and Specificity; Species Specificity
PubMed: 21972842
DOI: 10.1111/j.1365-2672.2011.05169.x -
Frontiers in Microbiology 2019is a commensal colonizer of both humans and animals, but also an opportunistic pathogen responsible for a multitude of diseases. In recent years, colonization of pigs...
is a commensal colonizer of both humans and animals, but also an opportunistic pathogen responsible for a multitude of diseases. In recent years, colonization of pigs by methicillin resistant has become a problem with increasing numbers of humans being infected by livestock strains. In colonization and virulence factor expression is controlled by the quorum sensing system, which responds to and is activated by self-generated, autoinducing peptides (AIPs). AIPs are also produced by coagulase negative staphylococci (CoNS) commonly found as commensals in both humans and animals, and interestingly, some of these inhibit activity. Here, we have addressed if cross-communication occurs between and CoNS strains isolated from pig nares, and if so, how properties such as host factor binding and biofilm formation are affected. From 25 pig nasal swabs we obtained 54 staphylococcal CoNS isolates belonging to 8 different species. Of these, none were able to induce as monitored by reporter gene fusions to regulated genes but a number of -inhibiting species were identified including , , , , and . After establishing that the inhibitory activity was mediated via AgrC, the receptor of AIPs, we synthesized selective AIPs to explore their effect on adhesion of to fibronectin, a host factor involved in colonization. Here, we found that the CoNS AIPs did not affect adhesion of except for strain 8325-4. When individual CoNS strains were co-cultured together with we observed variable degrees of biofilm formation which did not correlate with interactions. Our results show that multiple CoNS species can be isolated from pig nares and that the majority of these produce AIPs that inhibit . Further they show that the consequences of the interactions between CoNS and are complex and highly strain dependent.
PubMed: 31611856
DOI: 10.3389/fmicb.2019.02212 -
Microbiological Research 2009The aim of this study is to determine antibiotic resistance patterns and slime production characteristics of coagulase-negative Staphylococci (CoNS) caused nosocomial...
The aim of this study is to determine antibiotic resistance patterns and slime production characteristics of coagulase-negative Staphylococci (CoNS) caused nosocomial bacteremia. A total of 200 CoNS strains were isolated from blood samples of patients with true bacteremia who were hospitalized in intensive care units and in other departments of Istanbul University Cerrahpasa Medical Hospital between 1999 and 2006. Among 200 CoNS isolates, Staphylococcus epidermidis was the most prevalent species (87) followed by Staphylococcus haemolyticus (23), Staphylococcus hominis (19), Staphylococcus lugdunensis (18), Staphylococcus capitis (15), Staphylococcus xylosus (10), Staphylococcus warneri (8), Staphylococcus saprophyticus (5), Staphylococcus lentus (5), Staphylococcus simulans (4), Staphylococcus chromogenes (3), Staphylococcus cohnii (1), Staphylococcus schleiferi (1), and Staphylococcus auricularis (1). Resistance to methicillin was detected in 67.5% of CoNS isolates. Methicillin-resistant CoNS strains were determined to be more resistant to antibiotics than methicillin-susceptible CoNS strains. Resistance rates of methicillin-resistant and methicillin-susceptible CoNS strains to the antibacterial agents, respectively, were as follows: gentamicin 90% and 17%, erythromycin 80% and 37%, clindamycin 72% and 18%, trimethoprim-sulfamethoxazole 68% and 38%, ciprofloxacin 67% and 23%, tetracycline 60% and 45%, chloramphenicol 56% and 13% and fusidic acid 25% and 15%. None of the strains were resistant to vancomycin and teicoplanin. Slime production was detected in 86 of 200 CoNS strains. Resistance to methicillin was found in 81% of slime-positive and in 57% of slime-negative strains. Our results indicated that there is a high level of resistance to widely used agents in causative methicillin-resistant CoNS strains. However fusidic acid has the smallest resistance ratio, with the exception of glycopeptides. Additionally, most S. epidermidis strains were slime-positive, with statistically significant (p<0.001) association between methicillin resistance and slime production.
Topics: Bacteremia; Coagulase; Cross Infection; Drug Resistance, Bacterial; Hospital Units; Humans; Staphylococcal Infections; Staphylococcus; Turkey
PubMed: 17475456
DOI: 10.1016/j.micres.2007.03.004 -
Frontiers in Veterinary Science 2021An understanding of the spatio-temporal distribution of several groups of mastitis pathogens can help to inform programs for the successful control and management of...
An understanding of the spatio-temporal distribution of several groups of mastitis pathogens can help to inform programs for the successful control and management of mastitis. However, in the absence of an active surveillance program such information is not readily available. In this retrospective study we analyzed passive surveillance data from a diagnostic laboratory with an aim to describe the spatio-temporal trend of major mastitis pathogens between 2008 and 2017 in Ontario dairy cattle. Data for all milk culture samples submitted to the Animal Health Laboratory (AHL) at the University of Guelph between 2008 and 2017 was accessed. Descriptive analyses were conducted to identify the major pathogens and Chi-square goodness-of-fit tests were used to compare between multiple proportions. Likewise, univariable logistic regression analysis was performed to determine if there was a change in the probability of isolating the major mastitis pathogens depending on geography or time. Seasonality was assessed by calculating the seasonal relative risk (RR). Of a total of 85,979 milk samples examined, more than half of the samples (61.07%) showed no growth and the proportion of samples that showed no growth almost halved during the study period. Of the samples (36.21%, = 31,133) that showed any growth, the major bacterial pathogens were (15.60%), Non-aureus Staphylococci (NAS) (5.04%), spp. (2.96%), and (2.00%). Of the NAS, the major species reported were (69.02%), (14.45%), (12.99%), and (2.13%). A temporal change in the prevalence of contagious pathogens like and spp. was observed with an increasing odds of 1.06 and 1.62, respectively. Likewise, except for , the prevalence of all the major environmental mastitis pathogens increased during the study period. The isolation of most of the pathogens peaked in summer, except for , and which peaked in spring months. Interestingly, a regional pattern of isolation of some bacterial pathogens within Ontario was also observed. This study showed a marked spatio-temporal change in the prevalence of major mastitis pathogens and suggests that a regional and seasonal approach to mastitis control could be of value.
PubMed: 34805334
DOI: 10.3389/fvets.2021.742696 -
JAAD Case Reports Nov 2016
PubMed: 27957522
DOI: 10.1016/j.jdcr.2016.08.015 -
Antimicrobial Agents and Chemotherapy May 2004LasA protease is a staphylolytic endopeptidase secreted by Pseudomonas aeruginosa. We have examined the effectiveness of LasA protease in the treatment of staphylococcal...
LasA protease is a staphylolytic endopeptidase secreted by Pseudomonas aeruginosa. We have examined the effectiveness of LasA protease in the treatment of staphylococcal keratitis caused by methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates in a rabbit model. Keratitis was induced by intrastromal injection of the bacteria. The eyes were treated topically, and the efficacy of LasA protease was compared to those of lysostaphin (a staphylolytic protease secreted by Staphylococcus simulans) and vancomycin. When treatment was initiated early (4 h) after infection, practically all of the MSSA- and MRSA-infected corneas were sterilized by LasA protease, and its efficacy in eradicating the bacteria was comparable to those of lysostaphin and vancomycin. By contrast, most of the control corneas were heavily infected, with median values of 4.5 x 10(6) (MSSA) and 5 x 10(5) (MRSA) CFU/cornea (P < 0.001). When treatment was initiated late (10 h) after infection, LasA protease reduced the numbers of CFU in both MSSA- and MRSA-infected corneas by 3 to 4 orders of magnitude compared to the numbers of CFU for the controls (median values, 1,380 and 30 CFU/cornea, respectively, for the treated animals compared to 1.2 x 10(6) and 5 x 10(5) CFU/cornea for the respective controls [P = 0.001]), and it was more effective than vancomycin in eradicating MRSA cells (P = 0.02). In both the early- and the late-treatment protocols, the clinical scores for eyes treated with LasA protease were significantly lower than those for the eyes of the corresponding controls and comparable to those for the lysostaphin- and vancomycin-treated eyes. We conclude that LasA protease is effective in the treatment of experimental S. aureus keratitis in rabbits and may have potential for the treatment of disease in humans.
Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Colony Count, Microbial; Cornea; Humans; Keratitis; Lysostaphin; Metalloendopeptidases; Methicillin; Methicillin Resistance; Microbial Sensitivity Tests; Penicillins; Pseudomonas aeruginosa; Rabbits; Staphylococcal Infections; Staphylococcus aureus; Vancomycin
PubMed: 15105121
DOI: 10.1128/AAC.48.5.1681-1687.2004 -
Infection and Immunity Jul 1981An encapsulated strain of Staphylococcus simulans (strain 76) isolated from a clinical case of bovine mastitis was demonstrated by the India ink technique, using both a...
An encapsulated strain of Staphylococcus simulans (strain 76) isolated from a clinical case of bovine mastitis was demonstrated by the India ink technique, using both a light microscope and an electron microscope. The strain failed to kill mice after intraperitoneal inoculation but resisted ingestion by peritoneal phagocytic cells. The behavior of the strain helps to delineate the antiphagocytic role of the capsule.
Topics: Animals; Cattle; Coagulase; Mastitis, Bovine; Mice; Peritoneal Cavity; Phagocytosis; Staphylococcus
PubMed: 7263067
DOI: 10.1128/iai.33.1.304-308.1981 -
Journal of Dairy Science Jul 2017The effect of non-aureus staphylococci (NAS) in bovine mammary health is controversial. Overall, NAS intramammary infections (IMI) increase somatic cell count (SCC),...
The effect of non-aureus staphylococci (NAS) in bovine mammary health is controversial. Overall, NAS intramammary infections (IMI) increase somatic cell count (SCC), with an effect categorized as mild, mostly causing subclinical or mild to moderate clinical mastitis. However, based on recent studies, specific NAS may affect the udder more severely. Some of these apparent discrepancies could be attributed to the large number of species that compose the NAS group. The objectives of this study were to determine (1) the SCC of quarters infected by individual NAS species compared with NAS as a group, culture-negative, and major pathogen-infected quarters; (2) the distribution of NAS species isolated from quarters with low SCC (<200,000 cells/mL) and high SCC (≥200,000 cells/mL), and clinical mastitis; and (3) the prevalence of NAS species across quarters with low and high SCC. A total of 5,507 NAS isolates, 3,561 from low SCC quarters, 1,873 from high SCC quarters, and 73 from clinical mastitis cases, were obtained from the National Cohort of Dairy Farms of the Canadian Bovine Mastitis Research Network. Of quarters with low SCC, high SCC, or clinical mastitis, 7.6, 18.5, and 4.3% were NAS positive, respectively. The effect of NAS IMI on SCC was estimated using mixed-effect linear regression; prevalence of NAS IMI was estimated using Bayesian analyses. Mean SCC of NAS-positive quarters was 70,000 cells/mL, which was higher than culture-negative quarters (32,000 cells/mL) and lower than major pathogen-positive quarters (129,000 to 183,000 cells/mL). Compared with other NAS species, SCC was highest in quarters positive for Staphylococcus capitis, Staphylococcus gallinarum, Staphylococcus hyicus, Staphylococcus agnetis, or Staphylococcus simulans. In NAS-positive quarters, Staphylococcus xylosus (12.6%), Staphylococcus cohnii (3.1%), and Staphylococcus equorum (0.6%) were more frequently isolated from quarters with low SCC than other NAS species, whereas Staphylococcus sciuri (14%) was most frequently isolated from clinical mastitis cases. Finally, in NAS-positive quarters, Staphylococcus chromogenes, S. simulans, Staphylococcus epidermidis, and Staphylococcus haemolyticus were isolated with similar frequency from among low SCC and high SCC quarters and clinical mastitis cases. Staphylococcus chromogenes, S. simulans, S. xylosus, S. haemolyticus, S. epidermidis, S. agnetis, Staphylococcus arlettae, S. capitis, S. gallinarum, S. sciuri, and Staphylococcus warneri were more prevalent in high than in low SCC quarters. Because the NAS are a large, heterogeneous group, considering them as a single group rather than at the species, or even subspecies level, has undoubtedly contributed to apparent discrepancies among studies as to their distribution and importance in IMI and mastitis.
Topics: Animals; Bayes Theorem; Canada; Cattle; Cell Count; Female; Mammary Glands, Animal; Mastitis, Bovine; Milk; Staphylococcal Infections; Staphylococcus
PubMed: 28456402
DOI: 10.3168/jds.2016-12479 -
Journal of Dairy Science Dec 1991A recombinant mucolytic protein, lysostaphin, was evaluated as a potential intramammary therapeutic for Staphylococcus aureus mastitis in dairy cattle. Lysostaphin, a... (Comparative Study)
Comparative Study
A recombinant mucolytic protein, lysostaphin, was evaluated as a potential intramammary therapeutic for Staphylococcus aureus mastitis in dairy cattle. Lysostaphin, a product of Staphylococcus simulans, enzymatically degrades the cell wall of Staphylococcus aureus and is bactericidal. Thirty Holstein-Freisian dairy cattle in their first lactation were infected with Staphylococcus aureus (Newbould 305, ATCC 29740) in all quarters. Infections were established and monitored for somatic cell counts and Staphylococcus aureus colony-forming units 3 wk prior to subsequent treatment. Infected animals were injected through the teat canal with a single dose of recombinant lysostaphin (dose response 1 to 500 mg) or after three successive p.m. milkings with 100 mg of recombinant lysostaphin in 60 ml of sterile phosphate-buffered saline. Animals were considered cured if the milk remained free of Staphylococcus aureus for a total of 28 milkings after last treatment. Kinetic analysis of immunologically active recombinant lysostaphin demonstrated that a minimum bactericidal concentration was maintained in the milk for up to 36 to 48 h after a single infusion of 100 mg of recombinant lysostaphin. The cure rate of quarters receiving recombinant lysostaphin (100 mg in sterile phosphate-buffered saline, administered over three consecutive p.m. milkings) was 20% compared with 29% for sodium cephapirin in saline and 57% for a commercial antibiotic formulation, respectively. An improved formulation of recombinant lysostaphin may prove to be an effective alternative to antibiotic therapy for bovine mastitis.
Topics: Animals; Cattle; Cell Wall; Cephapirin; Female; Lysostaphin; Mammary Glands, Animal; Mastitis, Bovine; Milk; Penicillin G Procaine; Recombinant Proteins; Staphylococcal Infections; Staphylococcus aureus
PubMed: 1787188
DOI: 10.3168/jds.S0022-0302(91)78612-8 -
Antimicrobial Agents and Chemotherapy Jun 2013Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pyogenes (group A streptococcus [GrAS]) cause serious and sometimes fatal human diseases. They are...
Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pyogenes (group A streptococcus [GrAS]) cause serious and sometimes fatal human diseases. They are among the many Gram-positive pathogens for which resistance to leading antibiotics has emerged. As a result, alternative therapies need to be developed to combat these pathogens. We have identified a novel bacteriophage lysin (PlySs2), derived from a Streptococcus suis phage, with broad lytic activity against MRSA, vancomycin-intermediate S. aureus (VISA), Streptococcus suis, Listeria, Staphylococcus simulans, Staphylococcus epidermidis, Streptococcus equi, Streptococcus agalactiae (group B streptococcus [GBS]), S. pyogenes, Streptococcus sanguinis, group G streptococci (GGS), group E streptococci (GES), and Streptococcus pneumoniae. PlySs2 has an N-terminal cysteine-histidine aminopeptidase (CHAP) catalytic domain and a C-terminal SH3b binding domain. It is stable at 50 °C for 30 min, 37 °C for >24 h, 4°C for 15 days, and -80 °C for >7 months; it maintained full activity after 10 freeze-thaw cycles. PlySs2 at 128 μg/ml in vitro reduced MRSA and S. pyogenes growth by 5 logs and 3 logs within 1 h, respectively, and exhibited a MIC of 16 μg/ml for MRSA. A single, 2-mg dose of PlySs2 protected 92% (22/24) of the mice in a bacteremia model of mixed MRSA and S. pyogenes infection. Serially increasing exposure of MRSA and S. pyogenes to PlySs2 or mupirocin resulted in no observed resistance to PlySs2 and resistance to mupirocin. To date, no other lysin has shown such notable broad lytic activity, stability, and efficacy against multiple, leading, human bacterial pathogens; as such, PlySs2 has all the characteristics to be an effective therapeutic.
Topics: Animals; Anti-Bacterial Agents; Bacteremia; Coinfection; Disease Models, Animal; Enzyme Therapy; Enzymes; Humans; Methicillin-Resistant Staphylococcus aureus; Mice; Microbial Sensitivity Tests; Staphylococcal Infections; Streptococcal Infections; Streptococcus Phages; Streptococcus pyogenes; Treatment Outcome
PubMed: 23571534
DOI: 10.1128/AAC.02526-12