Virulence Factors Of Staphylococci Research Articles

Detection of Some Virulence Factors in Staphylococci Isolated from Mastitic Cows and Ewes

Abstract About 150 million families around the world are engaged in milk production. However, inflammation of the mammary gland (mastitis) remains a major problem in dairy ruminants that affects the quality of milk worldwide. The aim of this study was the examination of udder health with detection of contagious and environmental pathogens causing mastitis in 960 and 940 dairy cows and ewes, respectively. The presence of selected virulence factors such as: the formation of haemolysis, gelatinase, biofilm, hydrolyse DNA, and resistance to antibiotics with detection of methicillin resistance gene (mecA), were determined in selected virulence factors associated with isolated staphylococci. These isolated staphylococci with selected virulence factors can have untoward effects on the severity of mastitis. The results of our study indicated that, in addition to the major udder pathogens (S. aureus, S. uberis, and S. agalactiae) causing mastitis, non-aureus staphylococci (NAS), is a major risk to dairy cows and ewes. NAS, such as S. chromogenes, S. warneri, and S. xylosus isolated from infected animals with clinical and chronic mastitis, had the highest representation of virulence factors in comparison to less virulent strains. In addition, the isolates of S. aureus and NAS demonstrated 77.0 % and 44.2 % resistance to one or more antimicrobial classes from mastitic milk samples obtained from dairy cows and ewes, respectively. Due to the high resistance to β-lactamantibiotics in two isolates of S. aureus and two species of NAS isolated from cows’ mastitic milk samples, the presence of a methicillin-resistant gene mecA poses serious complications for the treatment and a serious health risk to milk consumers.

Antimicrobial resistance and virulence factors in staphylococci isolated from canine otitis externa

Antimicrobial resistance and virulence factors in staphylococci isolated from canine otitis externa

Dairy Cows' Udder Pathogens and Occurrence of Virulence Factors in Staphylococci.

Simple SummaryDairy farmers and those involved in primary milk production face the challenge of maintaining the health of their animals. Although the level of technological support during milking has increased and appropriate veterinary measures are available, mastitis remains a major health problem in dairy cows as well as a major threat to dairy farm profitability. The results of this study indicate that, in addition to the major udder pathogens (S. aureus, S. uberis and S. agalactiae) causing dairy mastitis, non-aureus staphylococci (NAS) are a major risk to dairy cows during early lactation. NAS, such as S. chromogenes, S. warneri and S. xylosus, which were isolated from animals with clinical mastitis characterized by mild, moderate, or severe symptoms and persistent infections, had the highest representation of virulence factors (production of hemolysis, gelatinase and biofilm; the ability to hydrolyze DNA; resistance to antibiotics) in comparison to less virulent strains. In addition, isolates S. aureus and NAS showed resistance to one or more antimicrobials in 77.2%, and in 16 (11.7%) isolates, multi-drug resistance to three or more antimicrobial classes was confirmed. Due to the high resistance to β-lactam-antibiotics in two isolates of S. aureus and two species of NAS, the presence of a methicillin-resistant gene, mecA, was confirmed, which potentially poses serious complications for the treatment of dairy cows and a serious health risk to milk consumers.This study investigated 960 Slovak and Czech spotted cattle from four different conventional (non-organic) dairy herds located in Eastern Slovakia and Czechia during early lactation (14–100 days after calving). Dairy cows were examined clinically; milk from fore-stripping of each udder quarter was subjected to sensory examination and assessed by the California mastitis test (CMT), and laboratory analyses of bacterial pathogens in milk, including virulence factors, were conducted. Positive CMT scores (1–3) for one or more quarters were detected in 271 (28.2%) of the examined animals. Out of 230 infected milk samples, representing 24.0% of all dairy cows, staphylococci (59.1% of positive findings) were the most commonly isolated organisms, followed by E. coli (11.3%), streptococci Str. uberis (9.1%) and Str. agalactiae (3.4%), and enterococci (6.1%). From 136 isolates of S. aureus (38 isolates) and non-aureus staphylococci (NAS; 98 isolates), virulence factors and their resistance to 14 antimicrobials were detected using the disk diffusion method, with PCR detection of the methicillin resistance gene, mecA. An increased incidence of clinical and chronic forms of mastitis has been reported in mastitic cows in which staphylococci, especially S. aureus and NAS (S. chromogenes, S. warneri, and S. xylosus), have been detected and compared to other isolated udder pathogens. From those species, S. aureus and isolates of NAS mentioned above showed multiple virulence factors that are more likely to hydrolyze DNA, hemolysis, produce gelatinase and biofilm, and have multi-drug resistance as compared to other less virulent staphylococci. Generally, the isolated staphylococci showed 77.2% resistance to one or more antimicrobials, in particular to aminoglycosides, β-lactams, macrolides, or cephalosporins. Isolates that showed the ability to form a biofilm were more resistant to more than one antimicrobial than isolates without biofilm production. Multi-drug resistance to three or more antimicrobial classes was recorded in 16 isolates (11.7%), and the presence of the mecA gene was also confirmed in two isolates of S. aureus and two species of NAS.

Virulence Factors and Prevalence of Udder´s Pathogens in Dairy Cows during the Peripartal Period

Data presented in this study described the prevalence of udder pathogens and presence of virulence factors in staphylococci isolated from mastitis in dairy cows. The practical part of study was realized in five different dairy herds of Slovak spotted cattle breed located in Eastern Slovakia with conventional (non-organic) farming. At the beginning of lactation (during first two months after calving) were comprehensively investigated 960 cows from monitored herds. The comprehensive examination included clinical examination, sensory analysis of milk from fore stripping of each udder quarter, followed by assessment of the California mastitis test (CMT) and laboratory analyses of bacteria pathogens. Screening the health status of the mammary gland revealed that 314 cows (32.7%) had positive CMT score (1-3) for one or more quarters. Out of 230 infected milk samples, representing 24.0% of all dairy cows examined, were the most commonly isolated staphylococci (59.1% of positive findings), followed by E. coli (11.3%), streptococci Str. uberis (9.1%), Str. agalactiae (3.4%), and enterococci (6.1%). From 136 isolates of coagulase negative stafylococci (98 isolates) and S. aureus (38 isolates) were detected some virulence factors such as production of hemolysins (lysines ß and δ), gelatinase, biofilm, and hydrolyze of DNA. Isolated S. aureus, S. chromogenes and S. warneri had the most numerous representation of detected virulence factors, as demonstrated by the increased incidence of clinical forms of mastitis compared to less virulent strains.

Diversity of staphylococcal species in food producing animals in Spain, with detection of PVL-positive MRSA ST8 (USA300)

This work aimed to determine the prevalence, diversity, antibiotic-resistance phenotype/genotype and virulence factors in staphylococci of farm-animals. Nasal samples of 117 farm-animals (calve: 72; lamb: 37; goat: 8) were collected from one slaughterhouse in La Rioja/Spain and cultured for staphylococci and methicillin-resistant Staphylococcus (MRS) recovery. Identification was performed by MALDI-TOF. Antimicrobial resistance phenotype/genotype was determined by susceptibility testing and specific PCRs. Molecular typing (spa-typing, multilocus-sequence-typing, agr-typing, SCCmec), and detection of 12 virulence genes and human Immune-evasive-cluster (IEC) genes were performed by PCR/sequencing in S. aureus. Two marker genes of arginine catabolic mobile element (ACME) were determined by PCR (USA300-MRSA detection). Staphylococci were identified in 50%, 54% and 21% of goat, lamb and calve samples, respectively. Among the 13 S. aureus isolates recovered, 11 were susceptible to all antimicrobials tested, and two were multidrug-resistant-MRSA [beta-lactams (blaZ, mecA), macrolides [(msr(A)/msr(B)] and fluoroquinolones]. The MSSA harboured either tst or enterotoxin genes, while the MRSA harboured the lukF/lukS-PV genes. Five sequence-types were detected. The two MRSA strains (from lamb and goat) were typed as t5173/ST8/agr-I/SCCmec-IVa/ACME-positive, corresponding to USA300 clone, and were IEC-B-positive. Among the 47 coagulase-negative staphylococci (CoNS), six species were identified, predominating S. simulans (n = 25) and S. sciuri (n = 11). Fifty-three percent of CoNS showed resistance to at least one antimicrobial agent (six multidrug-resistant strains), and the following resistance phenotypes/genotypes were detected: streptomycin [27.6%; ant(6)-Ia, str], tetracycline [23.4%; tet(M), tet(L), tet(K)], clindamycin [19.1%; lnu(A), vgaA], erythromycin [10.6%; erm(C), msr(A)/msr(B)], chloramphenicol (8.5%; fexA), tobramycin (6.4%), penicillin-cefoxitin (4.3%; blaZ, mecA), and SXT (2.1%). The detection of the MRSA-USA300 lineage in food animals is worrisome and should be further monitored.

Virulence Factors in Staphylococci Isolated From Nasal Cavities of Footballers

AimThis study aimed to investigate the rate of Panton-Valentine Leukocidin producing Staphylococcus aureus and methicillin (mecA) and slime (icaA/icaD) genes in staphylococcal strains isolated from nasal cavities of footballers. Materials and MethodsNasal swab samples were taken from each footballers and a healthy control group for the isolation of staphylococcal strains. The polymerase chain reaction technique was used to determine Panton-Valentine Leukocidin, mecA and icaA/icaD genes in staphylococcal isolates. ResultsAmong 91 S. aureus strains, the presence of mecA gene was detected as 9.9%. This ratio was 17.9% (27 of 151) among the coagulase-negative staphylococci. A significant difference was found between coagulase-negative staphylococci and S. aureus isolates regarding the presence of mecA gene (P < 0.001). As for the genes of the slime, icaA/icaD genes were detected in 198 of 242 (81.8%) strains. The occurrence of slime genes was 91.2% and 89.4% among the S. aureus coagulase and negative staphylococci, respectively (P > 0.05). There was a statistically significant difference between the frequency of the mecA and slime genes when compared with the healthy control group and the football players (P < 0.01). Of 91 isolates, 22 were found to be methicillin resistant by the oxacillin disc diffusion method, whereas the remaining (220) were methicillin susceptible. Methicillin resistance was detected as 14.9% by the polymerase chain reaction method, whereas it was found as 9.1% by phenotypic methods. ConclusionsEarly and accurate diagnosis of virulent staphylococcal strains is crucial because the virulent coagulase-negative and coagulase-positive staphylococcal strains in the nasal floras of footballers may be major potential sources of superficial and deep tissue infections.

Virulence factors and ability of staphylococci from bovine milk and the cowshed environment to biofilm formation

The aim of this study was to examine virulence factors and the ability of S. aureus and CNS species isolated from milk of cows with mastitis to form biofilm, and to compare them with virulence factors of staphylococci from milk of cows without mastitis and cowshed environment. Most of S. aureus strains from cows with mastitis showed haemolytic activity (93.9%), among them 72.7% and 21.2% produced alpha- and beta-haemolysin, respectively. S. aureus from cows with mastitis symptoms produced proteases (above 48%) and esterase (42.4%). The highly significant relationship between the number of S. xylosus strains producing haemolysins (62%) and the origin of these strains from milk of cows with mastitis was observed. The ability to produce proteases was significantly associated with S. sciuri from milk of cows with mastitis. The ability of biofilm formation by staphylococcal strains from milk of cows with mastitis was greater than in strains from milk of cows without mastitis and the difference was significant (p < or = 0.05). The highest percentage of strains from milk of cows with mastitis were weak biofilm formers (48.6%), while 40% and 11.4% of strains were moderate and strong biofilm producers, respectively. S. xylosus showed the highest ability to form biofilm, while the lowest ability to form biofilm was observed in S. aureus and S. epidermidis. In conclusion, production of exotoxins and enzymes, and ability of biofilm formation shown by many CNS isolated from milk of cows with mastitis symptoms indicates that these features are important in pathogenesis of this disease.

Antimicrobial Resistance and Other Related Virulence Factors in Staphylococcus Spp isolated from Food, Environmental and Humans in Italy

We investigated the presence of antibiotic-resistances and others important virulence factors in coagulase negative and positive staphylococci, isolated from several sources (food, environmental and clinical). Thirty-four staphylococci were isolated and identified (28 coagulase-positive were identified as Staphylococcus aureus, 6 remaining Coagulase Negative Staphylococci were identified as 3 S. epidermidis, 2 S. warneri, 1 S. hycus). All the strains were analyzed both with phenotypic and genotypic assays in order to evaluate the virulence factors and the susceptibility against antibiotics and to correlate them to the sources of isolation. Results showed that isn't a direct correlation between features researched and isolation, and the different virulence factors are as widespread as antibiotic-resistance. The isolation from food of a strain of Staphylococcus aureus with a high methicillin resistance was very interesting. 15 of the 34 strains were found to possess multi-drug resistance and in 7 cases with Minimum Inhibitory Concentration(MIC) really very high; also in this case there is no distinction between the coagulase-positive and coagulase-negative staphylococci and their origin of isolation.

Lipase, protease, and biofilm as the major virulence factors in staphylococci isolated from acne lesions

Staphylococci involve infections in association with a number of bacterial virulence factors. Extracellular enzymes play an important role in staphylococcal pathogenesis. In addition, biofilm is known to be associated with their virulence. In this study, 149 staphylococcal isolates from acne lesions were investigated for their virulence factors including lipase, protease, and biofilm formation. Coagulase-negative staphylococci were demonstrated to present lipase and protease activities more often than coagulase-positive staphylococci. A microtiter plate method (quantitative method) and a Congo red agar method (qualitative method) were comparatively employed to assess biofilm formation. In addition, biofilm forming ability was commonly detected in a coagulase-negative group (97.7%, microtiter plate method and 84.7%, Congo red agar method) more frequently than in coagulase-positive organisms (68.8%, microtiter plate method and 62.5%, Congo red agar method). This study clearly confirms an important role for biofilm in coagulasenegative staphylococci which is of serious concern as a considerable infectious agent in patients with acnes and implanted medical devices. The Congo red agar method proved to be an easy method to quickly detect biofilm producers. Sensitivity of the Congo red agar method was 85.54% and 68.18% and accuracy was 84.7% and 62.5% in coagulase-negative and coagulase-positive staphylococci, respectively, while specificity was 50% in both groups. The results clearly demonstrated that a higher percentage of coagulasenegative staphylococci isolated from acne lesions exhibited lipase and protease activities, as well as biofilm formation, than coagulase-positive staphylococci.

Genotypes, Antibiotic Resistance, and Virulence Factors of Staphylococci from Ready-to-Eat Food

Sixty-seven staphylococcal isolates belonging to 12 species were obtained from 70 ready-to-eat food products. Staphylococcus aureus (n=25), and Staphylococcus epidermidis (n=13) were dominant. Susceptibility to penicillin, oxacillin, tetracycline, clindamycin, gentamicin, erythromycin, ciprofloxacin, and vancomycin was determined. All investigated S. aureus isolates were resistant to at least one antibiotic, and fifteen isolates were resistant to four and more antibiotics. Thirty-eight coagulase-negative staphylococci (CNS) isolates were resistant to at least one antibiotic, and seventeen to four and more antibiotics. Fifteen CNS isolates were mecA positive, and grew in the presence of 6 μg/mL oxacillin. All S. aureus isolates were mecA-negative. Arginine catabolic mobile element (ACME) was found in seven S. epidermidis isolates. Five S. epidermidis isolates harbored ica operon, ACME and were able to form biofilm. Three of them also possessed IS256 element and were mecA-positive. The expression of icaA gene was comparable in five ica-positive S. epidermidis isolates. One of six mecA positive S. epidermidis isolates was classified as sequence type (ST)155, one as ST110, and two as ST88. Two methicillin-resistant Staphylococcus epidermis (MRSE) belonged to new STs, that is, ST362, and ST363. Enterotoxin genes were found in 92% of S. aureus isolates. No enterotoxin gene was detected in analyzed CNS population. We show that ready-to-eat products are an important source of antibiotic-resistant CNS and potentially virulent strains of S. epidermidis, including genotypes undistinguishable from hospital-adapted clones.

Biofilm production, a marker of pathogenic potential of colonizing and commensal staphylococci

Biofilm is one of the known virulence factors of staphylococci, a human and animal pathogen and commensal. Some of the strains become invasive under favorable conditions while others do not cause disease. Early detection and management of potentially pathogenic staphylococci is the essential step to prevent device-associated infections. There is also a need to evaluate one simple method for the detection of potential pathogens. Hence this study was planned to study the difference in potential of commensal, colonizing and invasive strains of staphylococci to produce biofilm. We used one qualitative (Congo red agar) and one quantitative (microtiter plate) method for detection of biofilm production and evaluated the sensitivity and specificity of Congo red agar method by using microtiter plate method as a gold standard. We consecutively enrolled staphylococcal strains isolated from peripheral intravenous device (IVD), venous blood, site of IVD insertion and nasal mucosa of patients admitted to pediatric ward with peripheral intravenous devices in place for more than 48 h. Total 100 invasive, 50 colonizing and 50 commensal isolates were studied. Of 100 invasive isolates 74% (74/100) were biofilm positive while only 68% (34/50) colonizing and 32% (16/50) commensal isolates were biofilm positive. The difference in biofilm production by commensal, colonizing and invasive strains was statistically significant ( p < 0.0001). Sensitivity and specificity of Congo red agar test for detection of biofilm producers were 90.63% and 90.79% for Staphylococcus aureus and 75.86% and 96.88% respectively for coagulase negative staphylococci. CRA is a method that could be used to determine whether an isolate has the potential for biofilm production or not.

Antibiotic‐induced SOS response promotes horizontal dissemination of pathogenicity island‐encoded virulence factors in staphylococci

Although mobile genetic elements have a crucial role in spreading pathogenicity-determining genes among bacterial populations, environmental and genetic factors involved in the horizontal transfer of these genes are largely unknown. Here we show that SaPIbov1, a Staphylococcus aureus pathogenicity island that belongs to the growing family of these elements that are found in many strains, is induced to excise and replicate after SOS induction of at least three different temperate phages, 80alpha, phi11 and phi147, and is then packaged into phage-like particles and transferred at high frequency. SOS induction by commonly used fluoroquinolone antibiotics, such as ciprofloxacin, also results in replication and high-frequency transfer of this element, as well as of SaPI1, the prototypical island of S. aureus, suggesting that such antibiotics may have the unintended consequence of promoting the spread of bacterial virulence factors. Although the strains containing these prophages do not normally contain SaPIs, we have found that RF122-1, the original SaPIbov1-containing clinical isolate, contains a putative second pathogenicity island that is replicated after SOS induction, by antibiotic treatment, of the prophage(s) present in the strain. Although SaPIbov1 is not induced to replicate after SOS induction in this strain, it is transferred by the antibiotic-activated phages. We conclude that SOS induction by therapeutic agents can promote the spread of staphylococcal virulence genes.

Infective endocarditis: new concepts in pathogenesis

Gram-positive cocci account for the large majority of cases of infective endocarditis. Pathogenesis investigations of endocarditis have therefore focused on purported virulence factors in staphylococci, viridans group streptococci and enterococci. In addition to novel molecular techniques that have been adapted for use in the examination of gram-positive cocci, animal models of experimental endocarditis have been employed to support or discount the role of specific bacterial components in production of infective endocarditis. This review details recent work that addresses endocarditis pathogenesis and highlights pertinent findings from these investigations.

Structure of the pheromone peptide of the Staphylococcus epidermidis agr system

The agr quorum-sensing system is responsible for the regulation of several virulence factors in staphylococci, with an extracellular pheromone peptide as signalling molecule. By monitoring the biological activity of synthetic peptides, it could be demonstrated that the pheromone of the agr system in Staphylococcus epidermidis is an octapeptide containing a thiolester linkage between the central cysteine and the C-terminal carboxyl group. The peptide was active at nanomolar concentrations. The N-terminus of the peptide pheromone, which is encoded as part of a protein precursor, proved to be crucial for biological activity.