Characterization of Virulence Factors and Genetic Background of Staphylococcus aureus Isolated from Peking University People’s Hospital Between 2005 and 2009

The aim of this study was to investigate both the genetic features of MRSA strains and the occurrence of virulence factors produced by Staphylococcus aureus strains isolated from Peking University People’s Hospital in Beijing, China, between 2005 and 2009. A total of 179 S. aureus strains were isolated, 139 of which were MRSA. The MRSA strains were characterized epidemiologically by SCCmec typing, spa typing and agr typing, then were classified into different genetic groups. The prevalence of genes coding for 14 exotoxins and eight adhesion factors among the S. aureus samples was assessed via polymerase chain reaction. Cluster analysis based on virulence factors-encoding gene content was performed to divide the MRSA isolates into valid clusters. Correspondence analysis was done to analyze the correlation between virulence factors clusters and genetic groups. JCSC1716-agrI-t030 (67.6%), SCCmec-IIIA-agrI-t030 (14.4%), SCCmec-IIIA-agrI-t037 (8.6%) and SCCmecII-agrII-t002 (2.2%) were four predominant MRSA clones. PVL was positive only in MSSA strains, there were at least three superantigenic toxins in our HA-MRSA clones, the prevalence of 16 virulence factors genes (sea, seb, sec, sed, seg, sei, sej, pvl, lukE-lukD, eta, bbp, can, ebp, clfA, fib, fnbB) in MRSA and MSSA was found to be significantly different from MSSA (P < 0.05). Results of correspondence analysis among clusters based on virulence factors genes and groups based on genetic typing illustrated not only the correspondence relationship between groups and clusters overall (P < 0.001), but also the genetic diversity of MRSA strains with respect to virulence factors genes.     

Rare occurrence of methicillin-resistant Staphylococcus aureus CC130 with a novel mecA homologue in humans in Germany

MRSA CC130 containing the mecA homologue mecA(LGA251) were reported from the UK and from Denmark so far from cattle and humans. Here we report on 11 MRSA CC130 among a sample of 12691 isolates of human origin collected from January 2006 until June 2011. MRSA CC130 grew insufficiently on chromogernic agar plates for detection of MRSA; the agglutination test for presence of PBP2a was negative. We designed primers for specific detection of mecA(LGA251) as well as for concomitant detection of both, mec(LGA251) and mecA. As already described, the isolates exhibited spa-types t843, t1736, and t1773. The ccrA homologue indicated the presence SCCmec(XI). When subjected to further characterization by means of a commercially available microarray the isolates were negative for sak chp, and scn, and as expected positive for hla, untruncated hlb, and hld. They furthermore contained edinB, aur, slpA, slpB, slpE. From genes coding for surface and cell wall associated products the ica-operon, cap8, clfA, clfF, ebpS, fnbA, fnbB, sdrC were detected but not cna. The isolates were negative for enterotoxin genes and tst, as well as for eta, and etb; agr-type was III.     

Evidence for clonal evolution among highly polymorphic genes in methicillin-resistant Staphylococcus aureus

The evolution of Staphylococcus aureus has been described as predominantly clonal, based on evidence from seven housekeeping genes. We aimed to test if this was also true for more polymorphic genes. In a collection of 60 isolates including major European epidemic methicillin-resistant S. aureus (MRSA) and sporadic MRSA strains, we compared the partial gene sequences of seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL), six core adhesion genes (present in all strains) (clfA, clfB, fnbA, map, sdrC, and spa), and four accessory adhesion genes (not present in all strains) (ebpS, fnbB, sdrD, and sdrE). Nucleotide diversity of adhesion genes was 2- to 10-fold higher than genes used for multilocus sequence typing. All genes showed evidence for purifying selection with a weakly reduced level among accessory adhesion genes. Among these highly variable genes, there was no evidence for a difference in molecular evolution between epidemic and sporadic strains. Gene trees constructed from concatenated sequences of housekeeping, core adhesion, and accessory adhesion genes were highly congruent, indicating clonality, despite some evidence for homologous exchange. Further evidence for clonality was found with an overall positive correlation of allelic and nucleotidic divergence for both seven housekeeping genes and six core adhesion genes. However, for small allelic differences that fit the demarcations of clonal complexes (CCs) there was no such correlation, suggesting that recombination occurred. Therefore, despite an overall clonal population structure, recombination between related isolates within CCs might have contributed to S. aureus evolution.     

Implementation of a novel in vitro model of infection of reconstituted human epithelium for expression of virulence genes in methicillin-resistant Staphylococcus aureus strains isolated from catheter-related infections in Mexico

Background

Methicillin-resistant Staphylococcus aureus (MRSA) are clinically relevant pathogens that cause severe catheter-related nosocomial infections driven by several virulence factors.

Methods

We implemented a novel model of infection in vitro of reconstituted human epithelium (RHE) to analyze the expression patterns of virulence genes in 21 MRSA strains isolated from catheter-related infections in Mexican patients undergoing haemodialysis. We also determined the phenotypic and genotypic co-occurrence of antibiotic- and disinfectant-resistance traits in the S. aureus strains, which were also analysed by pulsed-field-gel electrophoresis (PFGE).

Results

In this study, MRSA strains isolated from haemodialysis catheter-related infections expressed virulence markers that mediate adhesion to, and invasion of, RHE. The most frequent pattern of expression (present in 47.6% of the strains) was as follows: fnbA, fnbB, spa, clfA, clfB, cna, bbp, ebps, eap, sdrC, sdrD, sdrE, efb, icaA, and agr. Seventy-one percent of the strains harboured the antibiotic- and disinfectant-resistance genes ermA, ermB, tet(M), tet(K), blaZ, qacA, qacB, and qacC. PFGE of the isolated MRSA revealed three identical strains and two pairs of identical strains. The strains with identical PFGE patterns showed the same phenotypes and genotypes, including the same spa type (t895), suggesting hospital personnel manipulating the haemodialysis equipment could be the source of catheter contamination.

Conclusion

These findings help define the prevalence of MRSA virulence factors in catheter-related infections. Some of the products of the expressed genes that we detected in this work may serve as potential antigens for inclusion in a vaccine for the prevention of MRSA-catheter-related infections.     

Molecular population and virulence factor analysis of Staphylococcus aureus from bovine intramammary infection

Staphylococcus aureus isolates from cows in Ireland (n = 102) and the USA (n = 42) were characterized by RAPD-PCR and analysed for the production of a number of putative virulence factors. Of these strains 63 representative isolates were screened for the corresponding virulence factor genes by PCR or Southern hybridization or both. The isolates were divided into 12 distinct clonal types on the basis of their RAPD fingerprint profiles. Of the isolates, 107 (74.3%) tested positive for clumping factor in a slide agglutination test, all 24 RAPD type 7 isolates being negative for clumping factor. PCR analysis of region R, a repeat region of the clfA gene, revealed eight region-R sizes. There was a strong association between RAPD type and the clfA region-R genotype among Irish isolates. Of the RAPD type 7 isolates, 21 (87.5%) coproduced toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin C (SEC). Over 90% of isolates demonstrated haemolytic activity on sheep or rabbit red blood cells and all isolates harboured the gamma-haemolysin (hlg) locus. Of the Irish isolates, all those of RAPD type 7 were sensitive to penicillin G, whereas 86% of RAPD types 4 and 5 strains were resistant. Furthermore, RAPD types 5 and 7 were more likely to be associated with clinical mastitis whereas RAPD type 4 isolates were more often associated with a latent infection. The current study identifies some of the putative virulence factors produced by the predominant clonal types of bovine Staph. aureus that may be considered as components of a vaccine.     

Molecular nature of methicillin-resistant Staphylococcus aureus derived from explosive nosocomial outbreaks of the 1980s in Japan

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) with Panton-Valentine leukocidin (PVL) genes is increasing worldwide. Nosocomial outbreak-derived (hospital-acquired) MRSA (HA-MRSA) in Japan in the 1980s was also largely PVL(+). PVL(+) HA-MRSA and CA-MRSA shared the same multi-locus sequence type (ST30) and methicillin resistance cassette (SCCmecIV), but were divergent in oxacillin resistance, spa typing, PFGE analysis or clfA gene analysis. PVL(+) HA-MRSA, which probably originated in PVL(+)S. aureus ST30, was highly adhesive (carrying cna and bbp genes), highly-toxic (carrying luk(PV) and sea genes) and highly drug-resistant. PVL(+) HA-MRSA was once replaced by other PVL(-) HA-MRSA (e.g., ST5), and is re-emerging as CA-MRSA.     

Two different genes encode fibronectin binding proteins in Staphylococcus aureus. The complete nucleotide sequence and characterization of the second gene.

A gene encoding a fibronectin binding protein (FnBP) has recently been isolated and sequenced from Staphylococcus aureus strain 8325-4. In the same bacterial strain, 682 bp downstream to the stop codon of this gene (fnbA), a second gene termed fnbB has not been discovered, encoding another FnBP (FnBPB). The two genes show in large parts striking sequence homologies. The complete amino acid sequence encoded by fnbB has been deduced and compared to that deduced from fnbA. In FnBPB a stretch of 66 amino acids downstream to the signal peptide has 75% identity with the corresponding region in FnBPA. At the C-terminal site another 394 amino acid stretch is almost identical in both gene products. This stretch contains the 38 amino acid long D repeats, the wall spanning Wr repeats and the hydrophobic membrane spanning domain. In FnBPA each of the three D repeats has been identified as a fibronectin binding structure. These structures are highly conserved in FnBPB and most likely represent the major Fn-binding domain of this protein. However, a subclone of gene fnbB lacking the coding region for the D repeats also clearly expresses fibronectin binding activity. This additional binding site is so far unique for FnBPB and interacts like the D domains with the N-terminal 24-31-kDa fragment of fibronectin. The purified recombinant FnBP fragment (not containing the D repeats) completely inhibits the binding of fibronectin to whole cells of S. aureus.     

Characterization of Staphylococcus aureus SarA binding sites

The staphylococcal accessory regulator locus (sarA) encodes a DNA-binding protein (SarA) that modulates expression of over 100 genes. Whether this occurs via a direct interaction between SarA and cis elements associated with its target genes is unclear, partly because the definitive characteristics of a SarA binding site have not been identified. In this work, electrophoretic mobility shift assays (EMSAs) were used to identify a SarA binding site(s) upstream of the SarA-regulated gene cna. The results suggest the existence of multiple high-affinity binding sites within the cna promoter region. Using a SELEX (systematic evolution of ligands by exponential enrichment) procedure and purified, recombinant SarA, we also selected DNA targets that contain a high-affinity SarA binding site from a random pool of DNA fragments. These fragments were subsequently cloned and sequenced. Randomly chosen clones were also examined by EMSA. These DNA fragments bound SarA with affinities comparable to those of recognized SarA-regulated genes, including cna, fnbA, and sspA. The composition of SarA-selected DNAs was AT rich, which is consistent with the nucleotide composition of the Staphylococcus aureus genome. Alignment of selected DNAs revealed a 7-bp consensus (ATTTTAT) that was present with no more than one mismatch in 46 of 56 sequenced clones. By using the same criteria, consensus binding sites were also identified upstream of the S. aureus genes spa, fnbA, sspA, agr, hla, and cna. With the exception of cna, which has not been previously examined, this 7-bp motif was within the putative SarA binding site previously associated with each gene.     

Host- and tissue-specific pathogenic traits of Staphylococcus aureus

Comparative genomics were used to assess genetic differences between Staphylococcus aureus strains derived from infected animals versus colonized or infected humans. A total of 77 veterinary isolates were genetically characterized by high-throughput amplified fragment length polymorphism (AFLP). Bacterial genotypes were introduced in a large AFLP database containing similar information for 1,056 human S. aureus strains. All S. aureus strains isolated from animals in close contact with humans (e.g., pet animals) were predominantly classified in one of the five main clusters of the AFLP database (cluster I). In essence, mastitis-associated strains from animals were categorized separately (cluster IVa) and cosegregated with bacteremia-associated strains from humans. Distribution of only 2 out of 10 different virulence genes differed across the clusters. The gene encoding the toxic shock syndrome protein (tst) was more often encountered among veterinary strains (P < 0.0001) and even more in the mastitis-related strains (P<0.0001) compared to human isolate results. The gene encoding the collagen binding protein (cna) was rarely detected among invasive human strains. The virulence potential, as indicated by the number of virulence genes per strain, did not differ significantly between the human- and animal-related strains. Our data show that invasive infections in pets and humans are usually due to S. aureus strains with the same genetic background. Mastitis-associated S. aureus isolated in diverse farm animal species form a distinct genetic cluster, characterized by an overrepresentation of the toxic shock syndrome toxin superantigen-encoding gene.     

Phenotypic and Genotypic Characterization Vibrio cholerae O139 of Clinical and Aquatic Isolates in China

To enhance the understanding of epidemiological impact of environmental Vibrio cholerae O139 strains, we characterized 10 clinical and 20 environmental isolates collected from human clinical samples and Pear River estuary during 2006 to 2008. Isolates were tested by PCR for eight virulence genes: cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and regulatory protein genes (tcpI). Genetic relatedness was assessed by pulsed-field gel electrophoresis (PFGE), and antibiotic susceptibility was determined using disk diffusion. Seven of eight virulence markers were detected in six clinical isolates and one environmental isolate. One clinical and one environmental isolate were positive for six virulence markers. 60% clinical isolates showed multi-drug resistance to tetracycline (TET), Nalidixic acid (NAL), chloramphenicol (CHL), and ampicillin (AMP), 70% were resistant to Trimethoprim + Sulfamethoxazole (SXT), while only 35% environmental strains were resistant to SXT. PFGE analysis revealed that the isolates in this study were formed three clusters. Cluster III was more related to strains from diarrheal patients than the strains in other clusters. Different from the clinical strains, most environmental strains lacked CTX and TCP gene clusters. Most environmental strains possess a single resistance profile, while most clinical isolates show multidrug resistant. PFGE analysis indicated the cluster III has more possibility to become a potential pathogenic clonal cluster.     

Molecular analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 strains: clonal relationships between clinical and environmental isolates

A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI and toxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA, zot, ace, tcpA, and tcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.     

Antibiotic resistance and virulence factors among clinical and food enterococci isolated in Slovakia

The resistance to antibiotics and the distribution of virulence factors in enterococci isolated from traditional Slovak sheep cheese bryndza was compared with strains from human infections. The occurrence of 4 enterococcal species was observed in 117 bryndza-cheese isolates. The majority of strains were identified as E. faecium (76 %) and E. faecalis (23 %). Several strains of E. durans and 1 strain of E. hirae were also present. More than 90 % of strains isolated from 109 clinical enterococci were E. faecalis, the rest belonged to E. faecium. The resistance to 6 antimicrobial substances (ampicillin, ciprofloxacin, higher concentration of gentamicin, nitrofurantoin, tetracycline and vancomycin) was tested in clinical and food enterococci. A higher level of resistance was found in clinical than in food strains and E. faecium had a higher resistance than E. faecalis; no resistance to vancomycin was detected. The occurrence of 3 virulence-associated genes, cylA (coding for hemolysin), gelE (coding for gelatinase) and esp (coding for surface protein) was monitored. Differences were found in the distribution of cylA gene between clinical and bryndza-cheese E. faecalis strains; in contrast to clinical strains (45 %), cylA gene was detected in 22 % of food isolates. The distribution of 2 other virulence factors, gelE and esp, was not significantly different in the two groups of E. faecalis strains. cylA and gelE genes were not detected in E. faecium but more than 70 % of clinical E. faecium were positive for esp, even thought none of the 79 E. faecium cheese isolates contained this gene.     

Detection of fibronectin-binding protein genes in staphylococcal strains from peri-prosthesis infections.

Several studies have been devoted to identify the adhesion mechanisms of Staphylococcus aureus and Staphylococcus epidermidis, which are the most frequent causes of prosthesis-associated infections. Recently, in particular for Staphylococcus aureus, considerable attention has been given to the host protein receptors as mediators for bacterial adherence. Fibronectin, in important matrix protein, seems to be a major ligand for bacterial adherence in the early stages of infection. To determine the importance of the fibronectin adhesin as virulence factor in Staphylococcus-induced prosthesis infection, a simple and reliable method using a polymerase chain reaction (PCR) was devised to identify fibronectin adhesin genes (fnbA and fnbB). Results obtained by this method were in accordance with those obtained by the in vitro phenotypic characterization of binding ability to fibronectin of Staphylococcus strains.     

Pheno- and genotyping of Staphylococcus aureus, isolated from bovine milk samples from São Paulo State, Brazil

In the present study, 87 Staphylococcus aureus isolates obtained from milk samples of 87 cows with mastitis in 6 different municipal districts of 2 regions of Sao Paulo State, Brazil, were compared pheno- and genotypically. Pulsed-field gel electrophoresis (PFGE) analysis of the strains was performed, and PCR was carried out to detect genes for a number of staphylococcal cell surface proteins, exoproteins, and 3 classes of agr genes. Nine distinct S. aureus lineages (LA-LI) were identified by PFGE. The lineages LA and LE, which accounted together for 63 strains (72.2%), were prevalent and had been collected from all of the 6 municipal districts, indicating a broad geographic distribution of these lineages; LB, LC, LD, LF, LG, LH, and LI, however, were isolated sporadically and accounted for 24 strains (27.8%). Some characteristics, like penicillin resistance and the presence of cap8 and agr class II genes, were associated with the prevalent lineages (LA and LE), and penicillin susceptibility and the presence of cna and cap5 genes were associated with sporadic lineages. According to the present results, some S. aureus lineages possess a combination of genes that confer the propensity to cause and disseminate infection, and only a limited number of clones are responsible for the cases of bovine mastitis on the various farms.     

Virulence characteristic and MLST-agr genetic background of high-level mupirocin-resistant, MRSA isolates from Shanghai and Wenzhou, China

The emergence and prevalence of high-level mupirocin-resistant, methicillin-resistant Staphylococcus aureus (MuH MRSA) is challenging the eradication of MRSA nasal carriage and the treatment of skin and soft tissue infections. To understand the potentially pathogenetic capacity and the genetic basis of MuH MRSA, it is important to have a detailed knowledge of the molecular traits of this organism. Fifty three MuH MRSA isolates were gathered from Shanghai (28 isolates) and Wenzhou (25 isolates) in China. These isolates, consisting of 27 different PFGE-SCCmec-spa patterns, were examined by PCR for 35 virulence genes and further typed using agr (accessory gene regulator) typing and MLST (multilocus sequence typing). All 53 strains were positive for the genes hlg/hlg variant and icaD, and negative for seb, sed, see, seh, eta, etb, hld, cap-5, and ACME-arcA. Compared with Wenzhou isolates, Shanghai isolates were more likely to carry seg (P?=?0.002) and several other genes which were not found in Wenzhou strains such as sec, sei, tst (P<0.001 each), and pvl (P?=?0.012), and less likely to contain sea (P<0.001), cna (P?=?0.031), and efb (P?=?0.045). MLST and agr typing showed that ST239-agr1, ST5-agr1, and ST239-agr2 were the common lineages in MuH MRSA isolates from these two different regions. Our results indicated that MuH MRSA strains from two different geographic regions of China have differences in distribution of some virulence genes, while their major MLST-agr genetic backgrounds were accordant.     

Application of monoclonal antibodies generated against Panton-Valentine Leukocidin (PVL-S) toxin for specific identification of community acquired methicillin resistance Staphylococcus aureus

Panton-Valentine Leukocidin (PVL) produced by community acquired methicillin Staphylococcus aureus (CA-MRSA) involved in skin and soft-tissue infections and necrotizing pneumonia comprised of two fractions, namely PVL S and PVL F. In the present study, three monoclonal antibodies designated as MAb1, MAb9 and MAb10 were generated against recombinant PVL-S (35 kDa) protein of S. aureus. All the three MAbs specifically reacted to confirm PVL-S positive strains of S. aureus recovered from clinical samples in Western blot analysis. Similarly all the three MAbs did not show any binding to other tested 14 different pathogenic bacteria belonging to other genera and species in Western blot analysis. Furthermore, a simple dot-ELISA method was standardized for the identification of PVL-S toxin containing S. aureus strains. Initially in dot-ELISA, Protein A (Spa) of S. aureus posed background noise problems due to the non-specific binding of antibodies resulting in false positive reactions. With the addition of 10 mM diethylpyrocarbonate (DEPC) along with 5% milk in PBS in the blocking step prevented this non-specific binding of Spa to mouse anti-PVL monoclonal antibodies in dot-ELISA. Once standardized, this simple dot-ELISA was evaluated with nine PVL positive strains recovered from food, environmental and clinical samples and the results were compared with PCR assay for the presence of PVL toxin genes and also with Western blot analysis. A 100% correlation was found between dot-ELISA, PCR assay and Western blot analysis. Collectively our results suggest the newly developed simple dot-ELISA system can be of immense help in providing, rapid detection of the PVL toxin containing S. aureus strains at a relatively low cost and will be a valuable tool for the reliable identification of CA-MRSA.     

Phenotypic and genotypic characterization of Staphylococci causing breast peri-implant infections in oncologic patients

Background

Staphylococcus epidermidis and S. aureus have been identified as the most common bacteria responsible for sub-clinical and overt breast implant infections and their ability to form biofilm on the implant as been reported as the essential factor in the development of this type of infections. Biofilm formation is a complex process with the participation of several distinct molecules, whose relative importance in different clinical settings has not yet been fully elucidated. To our knowledge this is the first study aimed at characterizing isolates causing breast peri-implant infections.

Results

Thirteen S. aureus and seven S. epidermidis causing breast peri-implant infections were studied.Using the broth microdilution method and the E-test, the majority of the strains were susceptible to all antibiotics tested. Methicillin resistance was detected in two S. epidermidis. All strains had different RAPD profiles and were able to produce biofilms in microtitre plate assays but, while all S. aureus carried and were able to express icaA and icaD genes, this was only true for one S. epidermidis. Biofilm development was glucose- and NaCl-induced (5 S. aureus and 1 S. epidermidis) or glucose-induced (the remaining strains). Proteinase K and sodium metaperiodate treatment had different effects on biofilms dispersion revealing that the strains studied were able to produce chemically different types of extracellular matrix mediating biofilm formation.All S. aureus strains harboured and expressed the atlA, clfA, FnA, eno and cna genes and the majority also carried and expressed the sasG (10/13), ebpS (10/13) genes.All S. epidermidis strains harboured and expressed the atlE, aae, embp genes, and the majority (six strains) also carried and expressed the fbe, aap genes.Genes for S. aureus capsular types 5 and 8 were almost equally distributed. The only leukotoxin genes detected were lukE/lukD (6/13).

Conclusions

S. aureus and S. epidermidis breast peri-implant infections are caused by heterogeneous strains with different biofilm development mechanisms.Since the collagen adhesin (cna) gene is not ubiquitously distributed among S. aureus, this protein could have an important role in the cause of breast peri-implant infections.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0368-x) contains supplementary material, which is available to authorized users.     

Phenotypic and genotypic analysis of pathogenic Escherichia coli virulence genes recovered from Riyadh,Saudi Arabia

The current study was carried out to evaluate the phenotypic and genotypic characterization of avian pathogenic Escherichia coli recovered from Riyadh, Saudi Arabia. During the period of 10th February–30th May 2015, 70 E. coli strains were isolated from chicken farms located in Riyadh, Saudi Arabia. All strains were tested phenotypically by standard microbiological techniques, serotyped and the virulence genes of such strains were detected by polymerase chain reaction (PCR). Most of the recovered strains from chickens belonged to serotype O111:K58 25 strains (35.7%), followed by serotype O157:H7 13 strains (18.57%), followed by serotype O114:K90 10 strains (14.29%), then serotype O126:K71 9 strains (12.9%), serotype O78:K80 8 strains (11.43%) and in lower percentage serotype O114:K90 and O119:K69 5 strains (7.14%). The virulence genotyping of E. coli isolates recovered from broilers revealed the presence of the uidA gene in all the field isolates (6 serovars) examined in an incidence of 100%, as well as the cvaC gene was also present in all field isolates (6 serovars), while the iutA gene and the iss gene were detected in 5 out of 6 field serovars in an incidence of 81.43% and 64.29%, respectively. Phenotypical examination of the other virulence factors revealed that 65 isolates were hemolytic (92.9%), as well as 15 isolates (21.42%) were positive for enterotoxin production. Meanwhile, 21 isolates (30%) were positive for verotoxin production, 58 isolates (82.86%) for the invasiveness and 31 isolates (44.29%) for Congo red binding activities of the examined serotypes.     

A recA-LexA-dependent pathway mediates ciprofloxacin-induced fibronectin binding in Staphylococcus aureus

Subinhibitory concentrations of ciprofloxacin (CPX) raise the fibronectin-mediated attachment of fluoroquinolone-resistant Staphylococcus aureus by selectively inducing fnbB coding for one of two fibronectin-binding proteins: FnBPB. To identify candidate regulatory pathway(s) linking drug exposure to up-regulation of fnbB, we disrupted the global response regulators agr, sarA, and recA in the highly quinolone-resistant strain RA1. Whereas agr and sarA mutants of RA1 exposed to CPX still displayed increased adhesion to fibronectin, the CPX-triggered response was abolished in the uvs-568 recA mutant, but was restored following complementation with wild type recA. Steady-state levels of recA and fnbB, but not fnbA, mRNA were co-coordinately increased >3-fold in CPX-exposed strain RA1. Electrophoretic mobility shift assays revealed specific binding of purified S. aureus SOS-repressor LexA to recA and fnbB, but not to fnbA or rpoB promoters. DNase I footprint analysis showed LexA binding overlapping the core promoter elements in fnbB. We conclude that activation of recA and derepression of lexA-regulated genes by CPX may represent a response to drug-induced damage that results in a novel induction of a virulence factor leading to increased bacterial tissue adherence.