fnbB Genes Research Articles

Effect of Salicylic Acid on the gene expression of FnbA and FnbB genes in Staphylococcus hominis.

Staphylococcus hominis is an opportunistic pathogen that expresses surface proteins, which are adhesive proteins that play a major role in biofilm formation. Biofilm is a protective layer that provides S. hominis bacteria with greater antibiotic resistance and promotes its adherence to biomedical surfaces, facilitating its entry into the bloodstream. This research aimed to investigate the activity of Salicylic Acid (SA) and its effect on the gene expression of biofilm genes (FnbA and FnbB genes). A total of 150 blood specimens were collected from patients. The specimens were cultured in broth media of the BacT/ALERT® system and subcultured on blood and chocolate agar. Bacteria were detected using the VITEK2 system. FnbA and FnbB genes were detected using PCR. The broth microdilution method performed the minimum inhibitory concentration (MIC) of Salicylic acid (SA) on S. hominis isolates with both genes. Detection of the gene expression levels of FnbA and FnbB genes was assessed using Real-Time PCR(RT-PCR). The results showed that out of the 150 specimens collected, 35 were S. hominis. The detection of S. hominis bacteria was performed by PCR amplification of two genes FnbA and FnbB and showed 100% and 17.14% of isolates were positive for genes FnbA and FnbB, respectively. The expression of FnbA and FnbB genes was decreased in samples treated with SA compared with untreated ones. In conclusion, there is a significant impact of SA on the prevention of biofilm formation of S. hominis through the suppression of gene expression, specifically FnbA and FnbB. This could enhance susceptibility to antimicrobial treatments. However, more research is required to determine whether SA leads to the selection of resistant bacteria.

Virulence Determinants of Buffalo Mastitis Originated Streptococcus agalactiae Isolates

Streptococci as a cause of mastitis have become the major concern to the dairy industry worldwide due to huge economic losses. Streptococcus agalactiae is a major contagious mastitis pathogen and continues to be a major cause of mastitis in dairy cattle and buffaloes. The aim of the study was to investigate the virulence determinants of S. agalactiae strains isolated from buffalo milk. Within the scope of the study, 24 S. agalactiae isolates from buffalo mastitis were examined. Biofilm production of isolates was investigated phenotypically by CRA method. It was observed that 18 (75%) isolates were positive for biofilm production. The presence of hylB, fnbB, scpB and spb1 virulence genes in S. agalactiae isolates were investigated by PCR. It was determined that 19 (79.17%) of the isolates were positive for scpB and 6 (25%) for fnbB virulence genes. None of the isolates were found to contain hylB and spb1 virulence genes. The antibiotic resistance profiles of the isolates among kanamycin, ampicillin, enrofloxacin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole antibiotic discs were determined by Kirby Bauer Disc Diffusion Method. Resistance were evaluated as % 41.7, % 45.9, % 25, % 12.5, % 20.9, and % 33.3, respectively. RAPD-PCR patterns of all isolates were determined using the ERIC-2 primer. The dendrograms of the RAPD patterns were plotted with the UPGMA method. It was determined that the isolates showed similarity between 59-95%. In conclusion, the research confirms the prevalence of various virulence genes in S. agalactiae isolated from buffalo mastitis. Further studies are therefore necessary to determine the molecular epidemiology and variability of S. agalactiae isolated from buffaloes, with the aim of improving mastitis control programs with regard to S. agalactiae.

High clonal diversity of Staphylococcus aureus isolates from children's playgrounds in Hungary.

Staphylococcus aureus is one of the most important human pathogenic bacteria and environmental surfaces play an important role in the spread of the bacterium. Presence of S. aureus on children's playgrounds and on toys was described in international studies, however, little is known about the prevalence and characteristics of S. aureus at playgrounds in Europe. In this study, 355 samples were collected from playgrounds from 16 cities in Hungary. Antibiotic susceptibility of the isolates was tested for nine antibiotics. Presence of virulence factors was detected by PCR. Clonal diversity of the isolates was tested by PFGE and MLST. The overall prevalence of S. aureus was 2.81% (10/355) and no MRSA isolates were found. Presence of spa (10), fnbA (10), fnbB (5), icaA (8), cna (7), sea (2), hla (10), hlb (2) and hlg (6) virulence genes were detected. The isolates had diverse PFGE pulsotypes. With MLST, we have detected isolates belonging to ST8 (CC8), ST22 (CC22), ST944 and ST182 (CC182), ST398 (CC398), ST6609 (CC45), ST3029 and ST2816. We have identified a new sequence type, ST6609 of CC45. S. aureus isolates are present on Hungarian playgrounds, especially on plastic surfaces. The isolates were clonally diverse and showed resistance to commonly used antibiotics. These data reinforce the importance of the outdoor environment in the spread for S. aureus in the community.

Resistant S. aureus Isolates Capable of Producing Biofilm from the Milk of Dairy Cows with Subclinical Mastitis in Slovakia

Staphylococcus spp. is the most common cause of mastitis, with a significantly low cure rate. Bacterial characteristics like adhesion and biofilm formation, as well as extracellular factors, can affect the pathogenesis of staphylococcal mastitis. The study’s objectives were to confirm S. aureus, assess their antibiotic resistance, identify methicillin resistance genes, verify biofilm formation, and detect biofilm-associated genes from bovine mastitis samples using multiplex PCR (mPCR). From 215 milk samples, six were confirmed as S. aureus. Most isolates were sensitive to all measured antibiotics. One isolate was identified as an inducible form of MLSB resistance (macrolides, lincosamides, and streptogramin B resistance), while the other two isolates were resistant to penicillins and carboxypenicillins. In S. aureus cultures used for methicillin resistance genotypic analysis by PCR, the mecA and mecC genes were not found. Biofilm formation phenotypes were determined in four strains. An mPCR analysis revealed that all strains of S. aureus carried icaABCD, agrA, srtA, fnbA, clfA, and clfB genes. Only in one isolate was the fnbB gene detected; the bap gene was not detected in any of the isolates. This emphasizes the importance of using appropriate treatment and continuous monitoring of S. aureus to prevent the spread of antibiotic-resistant strains in dairy cow farms.

Pathogenic potential of meat-borne coagulase negative staphylococci strains from slaughterhouse to fork.

This study aimed to determine the prevalence of coagulase-negative staphylococci (CoNS) in meat processing lines for their pathogenic potential associated with biofilm formation, staphylococcal toxin genes, and antibiotic resistance in obtained isolates. Out of 270 samples, 56 isolates were identified as staphylococcal with their species level, and their antimicrobial resistance profiles were also determined with the BD Phoenix™ system. Among these, CoNS were found in 32 isolates, including S. epidermidis (22%), S. warneri (22%), S. cohnii (9%), S. schleiferi (9%), S. capitis (6%), S. haemolyticus (6%), S. lugdunensis (6%), S. chromogenes (6%), S. kloosii (3%), S. sciuri (3%), S. lentus (3%), and S. caprae (3%). Biofilm formation was observed in 78.1% of CoNS isolates, with 56% being strong biofilm producers; and the frequency of the icaA, fnbA, and fnbB genes were 43.7% and 34.3%, and 9.3% in isolates, respectively. Twenty-five (78.1%) of these strains were resistant to at least one antimicrobial agent, 20 (80%) of which exhibited multidrug resistance (MDR). Regarding genotypic analyses, 15.6%, 22.2%, 87.5%, and 9% of isolates, were positive for blaZ, ermC, tetK, and aacA-aphD, respectively. In 8 (25%) of all isolates had one or more staphylococcal toxin genes: the sed gene was the most frequent (12.5%), followed by eta (9.3%), tst-1 (6.25%), and sea (3.1%). In conclusion, this study highlights meat; and meat products might be reservoirs for the biofilm-producing MDR-CoNS, which harbored several toxin genes. Hence, it should not be ignored that CoNS may be related to foodborne outbreaks.

Characterization of biofilm formation in uropathogenic Staphylococcus aureus and their association with antibiotic resistance

IntroductionThe increase in antibiotic resistance and biofilm formation in Staphylococcus aureus is a serious public health problem, complicating the treatment of infections caused by these bacteria, including urinary tract infections (UTIs). This research aimed to assess the correlation between antibiotic resistance and the capacity for biofilm formation in S. aureus strains isolated from patients with UTIs. MethodsThe antibiotic susceptibility testing was conducted using disc diffusion and broth micro-dilution method. The blaZ and mecA genes were investigated in all penicillin and cefoxitin-resistant S. aureus strains, using PCR. Biofilm formation was investigated using the Congo red agar and microtitre plate methods. All S. aureus strains were screened for genes encoding adhesion and biofilm formation (spa, fnb AB, clfA, bap) and intercellular adhesin (icaADB and icaR). ResultsAmong 20 strains of S. aureus, 55% were found to be multidrug-resistant (MDR) and methicillin-resistant (MRSA). All strains were biofilm producers, including 55% moderate biofilm producers and 35% strong biofilm producers. A significant correlation (p-value <0.01) was observed between MDR and non-MDR strains in terms of biofilm production. All strains were sensitive to linezolid, and 20% exhibited the constitutive clindamycin resistance phenotype. The spa, fnbB, and clfA genes were present in all strains followed by the bap (95%) and fnbA (90%). The icaA (90%) and icaB (60%) genes were most detected. The coexistence of icaA, icaADB, and icaAD genes was significantly associated with MRSA strains. In contrast, the icaR gene was significantly detectable in the methicillin-susceptible group of strains. ConclusionThis study uncovered a significant association between the formation of biofilms by uropathogenic S. aureus and antibiotic resistance, highlighting a noteworthy concern for public health.

S. aureus biofilm metabolic activity correlates positively with patients’ eosinophil frequencies and disease severity in chronic rhinosinusitis

Chronic rhinosinusitis (CRS) is a persistent inflammation of the sinus mucosa. Recalcitrant CRS patients are unresponsive to medical and surgical interventions and often present with nasal polyps, tissue eosinophilia, and Staphylococcus aureus dominant mucosal biofilms. However, S. aureus sinonasal mucosal colonisation occurs in the absence of inflammation, questioning the role of S. aureus in CRS pathogenesis. Here, we aimed to investigate the relationship between S. aureus biofilm metabolic activity and virulence genes, innate immune cells, and disease severity in CRS. Biospecimens, including sinonasal tissue and nasal swabs, and clinical datasets, including disease severity scores, were obtained from CRS patients and non-CRS controls. S. aureus isolates were grown into biofilms in vitro, characterised, and sequenced. The patients’ innate immune response was evaluated using flow cytometry. S. aureus was isolated in 6/19 (31.58%) controls and 23/53 (43.40%) CRS patients of 72 recruited patients. We found increased S. aureus biofilm metabolic activity in relation to increased eosinophil cell frequencies and disease severity in recalcitrant CRS cases. Mast cell frequencies were higher in tissue samples of patients carrying S. aureus harbouring lukF.PV, sea, and fnbB genes. Patients with S. aureus harbouring lukF.PV and sdrE genes had more severe disease. This offers insights into the pathophysiology of CRS and could lead to the development of more targeted therapies.

Sponge-derived fatty acids inhibit biofilm formation of MRSA and MSSA by down-regulating biofilm-related genes specific to each pathogen.

A promising approach for the development of next-generation antimicrobials is to shift their target from causing bacterial death to inhibiting virulence. Marine sponges are an excellent potential source of bioactive anti-virulence molecules (AVM). We screened fractions prepared from 26 samples of Irish coastal sponges for anti-biofilm activity against clinically relevant pathogens. Fifteen fractions from eight sponge species inhibited biofilm of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), and/or Listeria monocytogenes without causing growth inhibition. Gas chromatograph/mass spectroscopy analyses of Mycale contarenii fractions revealed the presence of myristic acid and oleic acid. These fatty acids repressed transcription of the fibronectin-binding protein fnbA and fnbB genes and the polysaccharide intercellular adhesin icaADBC operon, which are required for MRSA and MSSA biofilm formation, respectively. This study illustrates the potential of AVM from Irish coastal sponges to specifically target bacterial virulence phenotypes, in this case, repression of biofilm formation via decreased transcription of biofilm-associated genes in MSSA and MRSA.

Increased biofilm formation by Staphylococcus aureus clinical isolates on surfaces covered with plasma proteins.

Introduction. Biofilm formation is a major virulence factor associated with Staphylococcus aureus infections. However, the influence of plasma proteins on biofilm formation of clinical isolates in vitro remains unclear.Hypotheses. We hypothesized that coating surfaces with plasma proteins might induce biofilm formation by S. aureus of different clonal lineages.Aim. To evaluate biofilm production by clinical S. aureus isolates of different clonal lineages isolated in Rio de Janeiro hospitals and investigated the presence of biofilm-associated genes.Methodology. This study assessed biofilm production of 60 S. aureus isolates in polystyrene microtitre plates with and without fibrinogen or fibronectin. The biochemical composition of the biofilm matrices was determined and the biofilm formation on fibrinogen-coated surfaces was also evaluated by confocal laser scanning microscopy. The presence of biofilm-related genes was detected by PCR, and the typing and functionality of agr operon was also evaluated.Results. Most of the isolates (45 %) were weak biofilm producers or non-producers. However, most of them presented a significant increase in biofilm production on plates covered with plasma proteins. There was no significant difference in biofilm formation between methicillin-resistant and -susceptible S. aureus isolates, or between different clonal lineages, except for ST30-IV (weak producers) and ST239-III (strong producers). The fnbB gene was associated with higher biofilm production.Conclusion. An increase in biofilm production in the presence of plasma proteins highlights the importance of investigating biofilm formation by S. aureus clinical isolates under different conditions since this virulence factor contributes to persistent infections and increased resistance to antimicrobials.

Characterization of Biofilm Formation Ability, Virulence Factors and Antibiotic Resistance Pattern of Staphylococcus aureus Isolates from Subclinical Bovine Mastitis

Background Staphylococcus aureus is the common mastitis pathogen. This pathogen has the tendency to biofilm forming, and happens antibiotic resistance. ObjectivesThe aim of this study is characterization of biofilm formation ability, virulence factors and antibiotic resistance pattern of S. aureus isolates from subclinical mastitis.MethodsThe lactating dairy cows were screened for subclinical mastitis. The isolates were identified by phenotypic method and presence of the nuc gene. The biofilm forming & quantification was characterized by using a colorimetric assay. The S. aureus biofilm gene targeted by using PCR detection. The antimicrobial susceptibility of isolates was performed by using DAD method. The lowest antimicrobial agent preventing visible growth was construed by MIC50. Antibiotic susceptibility and MBECs for bacteria embedded in biofilms were determined by XTT method.ResultThe results highest resistance rate was seen against Penicillin, and the Ceftiofur and Ciprofloxacin had the lowest resistance rate. The MIC50 of Ceftiofur was founded 2 μg/ml. The results showed the 100% of the isolates were biofilm producers, and most of them formed strong biofilms. The frequency of icaAD, fnbAB and bap as virulence genes encoding biofilm was identified. The icaD and fnbB genes had the highest and lowest frequencies, respectively. The MBEC results for bacterial biofilm showed resistant to Ceftiofur in biofilm state; however, these strains were susceptible to this agent in the planktonic state.ConclusionsBiofilm formation is a significant virulence factor that, were detected at a high rate and antibiotic-resistant, and responsible for subclinical bovine mastitis that does not respond to routine treatments. Based on this results, we should look for more appropriate treatment strategies to prevent the formation or dispersion of biofilms.

EXPLORING THE STAPHYLOCOCCUS AUREUS IN PATIENTS INFECTED OF THE TERTIARY-CARE UNIVERSITY HOSPITAL: RESULTS OF THE RETROSPECTIVE COHORT STUDY

Objective: To establish a baseline of knowledge regarding about inappropriate therapy, virulence and resistance in a cohort of patients infected with S. aureus. Methods: Retrospective cohort study in tertiary-care university hospital was employed to evaluate the risk factors and the impact of inappropriate therapy among patients with Staphylococcus aureus infections, resistance and virulence. To assess the presence of the genes was performed PCR. Results: Patients with MRSA were older and hospitalized 17 days longer than those with MSSA infection, which were in ICU with a bloodstream infection. 50.0% received inadequate antibiotic therapy and we found virulence factors associated with MRSA (mecA, LukS, fnbB and clfA genes). Conclusion: These data show that surveillance studies related to Staphylococcus aureus infections remain essential to identify resistance and inform policy on resistance.

Reductive evolution of virulence repertoire to drive the divergence between community- and hospital-associated methicillin-resistant Staphylococcus aureus of the ST1 lineage

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) of the ST1-SCCmecIV lineage has been associated with community-acquired (CA) infections in North America and Australia. In Brazil, multi-drug resistant ST1-SCCmecIV MRSA has emerged in hospital-associated (HA) diseases in Rio de Janeiro. To understand these epidemiological differences, genomic and phylogenetic analyses were performed. In addition, virulence assays were done for representative CA – and HA-MRSA strains. Despite the conservation of the virulence repertoire, some genes were missing in Brazilian ST1-SCCmecIV including lukSF-PV, fnbB, and several superantigen-encoded genes. Additionally, CA-MRSA lost the splDE while HA-MRSA strains conserved the complete operon. Most of these variable genes were located in mobile genetic elements (MGE). However, conservation and maintenance of MGEs were often observed despite the absence of their associated virulence markers. A Bayesian phylogenetic tree revealed the occurrence of more than one entrance of ST1 strains in Rio de Janeiro. The tree shape and chronology allowed us to infer that the hospital-associated ST1-SCCmecIV from Brazil and the community-acquired USA400 from North America are not closely related and that they might have originated from different MSSA strains that independently acquired SCCmecIV cassettes. As expected, representatives of ST1 strains from Brazil showed lower cytotoxicity and a greater ability to survive inside human host cells. We suggest that Brazilian ST1-SCCmecIV strains have adapted to the hospital setting by reducing virulence and gaining the ability to persist and survive inside host cells. Possibly, these evolutionary strategies may balance the biologic cost of retaining multiple antibiotic resistance genes.

The Role of Subinhibitory Concentrations of Daptomycin and Tigecycline in Modulating Virulence in Staphylococcus aureus.

Staphylococcus aureus (S. aureus) infections are notoriously complicated by the ability of the organism to grow in biofilms and are difficult to eradicate with antimicrobial therapy. The purpose of the current study was to clarify the influence of sub-inhibitory concentrations (sub-MICs) of daptomycin and tigecycline antibiotics on biofilm adhesion factors and exoproteins expressions by S. aureus clinical isolates. Six clinical isolates representing positive biofilm S. aureus clones (3 methicillin-sensitive S. aureus (MSSA) and 3 methicillin-resistant S. aureus (MRSA)) were grown with sub-MICs (0.5 MIC) of two antibiotics (daptomycin and tigecycline) for 12 h of incubation. RNA extracted from culture pellets was used via relative quantitative real-time-PCR (qRT-PCR) to determine expression of specific adhesion (fnbA, fnbB, clfA, clfB, fib, ebps, cna, eno) and biofilm (icaADBC) genes. To examine the effect of sub-MIC of these antibiotics on the expression of extracellular proteins, samples from the culture supernatants of six isolates were collected after 12 h of treatment with or without tigecycline in order to profile protein production via 2D gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D gel-SDS-PAGE). Sub-MIC treatment of all clinical MRSA and MSSA strains with daptomycin or tigecycline dramatically induced or suppressed fnbA, fnbB, clfA, clfB, fib, ebps, cna, eno, and icaADBC gene expression. Furthermore, sub-MIC use of tigecycline significantly reduced the total number of separated protein spots across all the isolates, as well as decreasing production of certain individual proteins. Collectively, this study showed very different responses in terms of both gene expression and protein secretion across the various isolates. In addition, our results suggest that sub-MIC usage of daptomycin and tigecycline could signal virulence induction by S. aureus via the regulation of biofilm adhesion factor genes and exoproteins. If translating findings to the clinical treatment of S. aureus, the therapeutic regimen should be adapted depending on antibiotic, the virulence factor and strain type.

Status of Biofilm-Forming Genes among Jordanian Nasal Carriers of Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus.

Introduction:Biofilm formation in Staphylococcus aureus is a major virulence factor. Both MSSA and MRSA are common causes of community- and hospital-acquired infections and are associated with biofilm formation. The status of biofilm-forming genes has not been explored in Jordanian nasal carriers of S. aureus. This study investigates antibiotic resistance patterns and the prevalence of biofilm-forming genes between MSSA and MRSA in two distinct populations in Jordan. Methods:A total of 35 MSSA and 22 MRSA isolates were recovered from hospitalized patients and medical students at Prince Hamzah Hospital, Jordan. Antibiotic susceptibility was tested using disk diffusion method and Vitek 2 system. The phenotypic biofilm formation was tested using CRA and microtiter plate assays. The prevalence of the biofilm-forming genes was determined using multiplex PCR. Results:Among 57 S. aureus isolates, 22 (38.6%) isolates were MRSA and were highly resistant against benzylpenicillin, oxacillin, and imipenem. The frequencies of the icaADBC were 77.1%, 97.1%, 94.3%, and 97.1% respectively in MSSA compared to 86.4%, 100%, 100%, and 100% in MRSA isolates. On the other hand, the frequency of the fnbA, fnbB, clfA, fib, clfB, ebps, eno, and cna genes was 81.8%, 90.9%, 95.5%, 90.9%, 86.4%, 100%, 100%, and 40.9%, respectively in the MRSA isolates. Conclusion:In both groups, MRSA isolates, in comparison to MSSA, were significantly more resistant to cefoxitin, oxacillin, imipenem, tetracycline, clindamycin, and trimethoprim-sulfamethoxazole. Unexpectedly, biofilm formation and gene prevalence between MRSA and MSSA isolates showed no significant difference, suggesting other potential virulence mechanisms.

Molecular Detection of Genes Involved in Biofilm Formation in Staphylococcus aureus Strains Isolates: Evidence From Shahid Motahari Hospital in Tehran

Background: Infections caused by Staphylococcus aureus strains are a major public health challenge worldwide, especially in specialized burn hospitals. Infections caused by S. aureus account for more than 50% of burn-related deaths. Objectives: Since data on characteristics of these isolates are not sufficient, the current study aimed to assess the prevalence of resistance to antibacterial agents and to analyze the distribution of biofilm, and adhesion encoding genes among S. aureus strains isolated from burn patients in Motahari Hospital, Tehran, Iran. Methods: A total of 83 S. aureus strains were collected from burn wounds of patients admitted to a referral burn center in Tehran for 10 months. In vitro antibacterial susceptibility of isolates was evaluated using the Kirby-Bauer disk diffusion method. Strains were subjected to polymerase chain reaction (PCR) to determine the presence of nucA, mecA, ebps, cna, bbp, fnbA, fnbB, clfA, and clfB genes. Results: The highest frequency of resistance was found to cephalexin and cefoxitin (87.9%), followed by clindamycin (75.9%), erythromycin (72.3%), and ciprofloxacin (60.2%). Five resistance patterns were identified in which cephalexin, cefoxitin, clindamycin, erythromycin, and ciprofloxacin had the most predominant resistance profile (36.1%). Biofilm gene detection indicated a markedly high prevalence of cna (74.7%), clfB (54.2%), clfA (50.6%), fnbA (42.1%), ebp (13.2%), and fnbB (12%). Six different biofilm genetic patterns were identified, wherein clfA, clfB, fnbA, ebp, and cna (30.1%), clfA, clfB, fnbA, fnbB, ebp, and cna (12%), and clfA, clfB, and cna (8.4%) were the top three most frequently identified patterns. Conclusions: The prevalence of biofilm encoding genes, which are associated with multidrug resistance in S. aureus strains isolated from burn patients, is high. Therefore, identification of epidemiology, molecular characteristics, and biofilm management of S. aureus infection in burn units would be helpful.

Vancomycin and linezolid resistance among multidrug-resistant Staphylococcus aureus clinical isolates and interaction with neutrophils

BackgroundDeveloped drug resistance against vancomycin and linezolid is a huge worrisome. The aim of our study was to investigate the rate of vancomycin and linezolid resistance in multidrug-resistant (MDR) Staphylococcus aureus (S. aureus) in Baghdad, Iraq. MethodsThree-hundred S. aureus were identified from various clinical origins in Baghdad, Iraq. Methicillin-and linezolid-resistant S. aureus (MRSA and LRSA, respectively) isolates were determined by disc diffusion, agar dilution and E-test methods. Biofilm formation was assayed using microtiter tissue plate (Mtp) assay. Polymerase chain reaction (PCR) was employed to detect the mecA, vanA, B, C, cfr and adhesin genes, followed by gene sequencing. Vancomycin-resistant S. aureus (VRSA) and LRSA strains interactions with neutrophils was assessed using flow-cytometry technique. ResultsTwo-hundred (67%) isolates were MRSA. One-hundred one (50.5%) and 44 (22%) MRSA were strong and moderate-level biofilm producers, respectively. All the strong biofilm-producer MRSA carried the clfA, clfB, fnbA, fnbB, fib and eno genes. Five (1.66%) and nine (3%) of the MRSA were VRSA and LRSA, respectively which were mostly isolated from blood and tissue infections. The vanA and cfr genes were detected in two and three MRSA, respectively. Three VRSA and two LRSA strains were strong biofilm-producers and contained adhesin genes (clfA, clfB, fnbA, fnbB, eno, fib, cna and ebps, but not bbp). The neutrophils mean apoptosis rate by the VRSA and LRSA strains included 80% and 66.7%, respectively. ConclusionVirulent MRSA rate was high. In addition, MRSA, VRSA and LRSA contained adhesin genes responsible for colonization and biofilm formation. Furthermore, these strains induced the neutrophils apoptosis. The evolution of pathogenic VRSA and LRSA is a concern towards eradication of MDR-S. aureus. More detailed works will be helpful to disclose the role of bacterial components in the biofilm formation and apoptosis and control strategies.

Prevalence of Adhesion Genes fnbA &amp; fnbB of Staphylococcus aureus Isolated from Dental Patients Attending Dentistry Department of Tehsil Headquarter Hospital Pasrur, Pakistan

Introduction: The present study was intended to isolate, characterize and investigate the prevalence of virulence genes encoding fibronectin (fnbA and fnbB) adhesive molecules of S. aureus from dental patients attending outpatient department of THQ Hospital Pasur.&#x0D; Methodology: A total of 100 oral samples were collected from dental patients, pure cultures was segregated and identified through conventional microbiological methods to evaluate the prevalence of S. aureus. Isolates were further characterized by using specific primers for genotype fnbA and fnbB.&#x0D; Results: Results of the study declared that 68 samples were positive (68%) for staphylococcus aureus on the basis of growth on selective media and appearance of typical colonies supported by gram staining. These gram positive staphylococci were positive (86.7%) in coagulase and catalase testing. The results of polymerase chain reaction (PCR) revealed that 47 (69.1%) and 23 (33.8%) isolates showed amplification with type specific primer 23Sr RNA and NUC gene respectively. Furthermore, fnbA and fnbB type specific genes of S. aureus did not show any amplification in PCR reaction.&#x0D; Conclusion: Irrespectively the data in the present study showed the prevalence of S. aureus is significantly high in males and of age group of 20-40 years but no positive result was found for prevalence of fnbA and fnbB genes. All the S. aureus isolates were highly sensitive to vancomycin, linezolid and clindamycin.

Phenotypic and Molecular Detection of Biofilm Formation in Staphylococcus aureus Isolated from Different Sources in Algeria

Staphylococcus aureus is an opportunistic bacterium causing a wide variety of diseases. Biofilm formation of Staphylococcus aureus is of primary public and animal health concern. The purposes of the present study were to investigate the ability of Staphylococcus aureus isolated from animals, humans, and food samples to form biofilms and to screen for the presence of biofilm-associated and regulatory genes. In total, 55 Staphylococcus aureus isolated from sheep mastitis cases (n = 28), humans (n = 19), and from food matrices (n = 8) were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The ability of Staphylococcus aureus for slime production and biofilm formation was determined quantitatively. A DNA microarray examination was performed to detect adhesion genes (icaACD and biofilm-associated protein gene (bap)), genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), regulatory genes (accessory gene regulator (agr) and staphylococcal accessory regulator (sarA)), and the staphylococcal cassette chromosome mec elements (SCCmec). Out of 55 Staphylococcus aureus isolates, 39 (71.0%) and 23 (41.8%) were producing slime and biofilm, respectively. All Staphylococcus aureus strains isolated from food showed biofilm formation ability. 52.6% of the Staphylococcus aureus strains isolated from sheep with mastitis, and 17.9% of isolates from humans, were able to form a biofilm. Microarray analysis typed the Staphylococcus aureus into 15 clonal complexes. Among all Staphylococcus aureus isolates, four of the human isolates (21.1%) harbored the mecA gene (SCCmec type IV) typed into 2 clonal complexes (CC22-MRSA-IV and CC80-MRSA-IV) and were considered as methicillin-resistant, while two of them were slime-producing. None of the isolates from sheep with mastitis harbored the cna gene which is associated with biofilm production. The fnbB gene was found in 100%, 60% and 40% of biofilm-producing Staphylococcus aureus isolated from food, humans, and sheep with mastitis, respectively. Three agr groups were present and agr group III was predominant with 43.6%, followed by agr group I (38.2%), and agr group II (18.2%). This study revealed the capacity of Staphylococcus aureus isolates to form biofilms and highlighted the genetic background displayed by Staphylococcus aureus isolates from different sources in Algeria.

Prevalence and characterization of Staphylococcus aureus isolates from subclinical bovine mastitis in southern Xinjiang, China

Staphylococcus aureus is one of the major pathogens causing mastitis in dairy herds. The colonization of dairy cows and subsequent contamination of raw milk by S. aureus, especially strains exhibiting multidrug resistance and biofilm-forming and toxin-producing abilities, remains an important issue for both dairy farmers and public health. In this study, we investigated the prevalence, antimicrobial susceptibility, biofilm formation, and genetic diversity of S. aureus from subclinical bovine mastitis in dairy farms located in southern Xinjiang, China. Sixty-five isolates from 84 subclinical mastitic milk samples were identified as S. aureus. The resistance rates to penicillin, erythromycin, clindamycin, tetracycline, gentamicin, linezolid, rifampicin, quinupudin-dafupudin, ciprofloxacin, norfloxacin, and chloramphenicol were 58.5, 44.6, 40.0, 18.5, 12.3, 10.8, 9.2, 6.2, 4.6, 4.6, and 1.5%, respectively. All isolates were susceptible to cefoxitin, sulfamethoxazole-trimethoprim, and vancomycin. Isolates from farm A showed a significantly higher resistance rate to tetracycline (16.9%) than those from farm B (1.5%). The most frequently detected virulence factors were hla (96.9%, 63/65) and hlb (100.0%, 65/65). The percentage rates of the staphylococcal enterotoxin genes sea, sec, sed, seg, seh, sei, and sej in S. aureus isolates were 4.6, 33.8, 27.7, 3.1, 41.5, 41.5, and 7.7%, respectively. The percentage rate of the sec gene in isolates from farm B (30.8%) was significantly higher than that of farm A (3.1%). The percentage rates of the tsst and pvl genes in S. aureus isolates were 26.2 and 40.0%. The percentage rate of the pvl gene in isolates from farm B (32.3%) was significantly higher than that of farm A (7.7%). The adhesion molecules fnbA, fnbB, clfA, clfB, and cna were detected in 21 (32.3%), 23 (35.4%), 65 (100.0%), 65 (100.0%), and 65 (100.0%) isolates, respectively. The percentage rates of the icaA, sarA, tcaR, ccp, luxS, and sigB genes in S. aureus isolates were 69.2, 100.0, 86.2, 95.4, 84.6, and 100.0%, respectively. The fnbB and icaA genes were more frequently detected in isolates from farm A (29.2 and 40.0%, respectively) than those from farm B (6.2 and 29.2%, respectively). The luxS gene was more often found in isolates from farm B (50.8%) than those from farm A (33.8%). Using the microplate method, 61.5, 26.2, and 10.8% of the isolates showed weak, moderate, and strong biofilm-forming abilities, respectively. Different clonal complex (CC) and spa-types were identified, including CC81, CC398, CC88, CC5405, and CC5406. Importantly, in this study we report for the first time 41 new sequence types (ST) among 44 distinct ST. These results indicated high genetic diversity of S. aureus involved in subclinical bovine mastitis in southern Xinjiang, China. The results also showed that S. aureus from subclinical bovine mastitis cases in southern Xinjiang, China, were mainly resistant to β-lactams, erythromycin, and clindamycin. Also, biofilm- and adhesion-related genes, which are increasingly known as important virulence factors in the pathogenesis of S. aureus infections, were detected at a high rate. This study could help identify predominant clones and provide surveillance measures to decrease or eliminate S. aureus contamination in raw milk of dairy cows with subclinical mastitis.

Does biofilm formation have different pathways in Staphylococcus aureus?

Objective(s):Biofilm formation is one of the most important factors in the development of infections caused by Staphylococcus aureus. In this study, the expression levels of genes responsible for biofilm formation were studied in methicillin sensitive and methicillin resistant S. aureus.Materials and Methods:A total of 100 meticillin-resistant s.aureus (MRSA) and meticillin-sensetive s.aureus (MSSA) isolates were studied. Bacterial biofilm formation was evaluated phenotypically using microtiter plate method. Real-time PCR tests were conducted to determine the expression levels of genes involved in biofilm formation.Results:Quantitative biofilm formation test was repeated three times for each specimen. The prevalence of weak, medium, and strong biofilm producers were 16%, 49%, and 35%, respectively. In MSSA isolates, expression levels of ica genes increased compared to the fnbA, fnbB, clfA and clfB genes. These results were different in MRSA isolates, and ica genes showed a decreased gene expression levels compared to the aforementioned genes. Conclusion:Considering the results of this study, clf genes probably contribute to the same extent in both MRSA and MSSA isolates, and there is probably no significant difference in the role of these genes in these isolates. In addition, the results of this study indicated that MRSA may not use the conventional route for biofilm formation and may use independent pathways through Polysaccharide intercellular adhesion (PIA).