Cna Genes Research Articles

The presence of both bone sialoprotein-binding protein gene and collagen adhesin gene as a typical virulence trait of the major epidemic cluster in isolates from orthopedic implant infections

Staphylococcus aureus is a major, highly clonal, pathogen causing implant infections. This study aimed at investigating the diverse distribution of bacterial adhesins in most prevalent S. aureus strain types causing orthopaedic implant infections. 200 S. aureus isolates, categorized into ribogroups by automated ribotyping, i.e. rDNA restriction fragment length polymorphism analysis, were screened for the presence of a panel of adhesins genes. Within the collection of isolates, automated ribotyping detected 98 distinct ribogroups. For many ribogroups, characteristic tandem genes arrangements could be identified. In the predominant S. aureus cluster, enlisting 27 isolates, the bbp gene encoding bone sialoprotein-binding protein appeared a typical virulence trait, found in 93% of the isolates. Conversely, the bbp gene was identified in just 10% of the remaining isolates of the collection. In this cluster, co-presence of bbp with the cna gene encoding collagen adhesin was a pattern consistently observed. These findings indicate a crucial role of both these adhesins, able to bind the most abundant bone proteins, in the pathogenesis of orthopaedic implant infections, there where biomaterials interface bone tissues. This study suggests that specific adhesins may synergistically act in the onset of implant infections and that anti-adhesin strategies should be targeted to adhesins conjointly present.

Panton-Valentine Leukocidin Gene Detected in a Staphylococcus Aureus Strain Isolated from a Knee Arthroprosthesis Infection

This report focuses on the molecular characterization of a Staphylococcus aureus strain isolated from a knee arthroprosthesis infection and recognized retrospectively as a carrier of the Panton-Valentine leukocidin gene. The stored microbiological isolate, which belonged to the strain collection of the Research Unit on Implant Infections of the Rizzoli Orthopaedic Institute, was retrieved for molecular analysis. Genotyping was carried out, revealing an interesting profile. In addition to the positivity for the Panton-Valentine toxin gene, the results indicated that the isolate belonged to the agr III group and was endowed with bbp and cna genes, both encoding for staphylococcal adhesins that bind bone proteins. The strain had the mecA gene for methicillin resistance, even though it was unable to resist any of the beta-lactam or other antibiotics. Its gene configuration matched that of other community-acquired methicillin-resistant and methicillin-susceptible Staphylococcus aureus(CA-MRSA and CA-MSSA) strains which have recently been reported worldwide. As far as we know,this is the first report on a PVL-positive S. aureus strain associated with an orthopedic implant (knee arthroprosthesis) infection.

Pheno- and Genotyping of Staphylococcus aureus Isolates of Sheep Origin

The aim of the study was to determine the prevalence of genes encoding virulence factors in Staphylococcus aureus strains isolated from raw sheep milk, sheep cheese and Bryndza cheese. Genes encoding staphylococcal enterotoxin (sea, seb, sec, sed and see), toxic shock syndrome toxin-1 (tst), exfoliative toxins (eta and etb) and collagen-binding protein (cna) were detected. In a total of 79 S. aureus isolates all assessed toxins encoding genes were found, except for see, eta and etb. Overall, 75.9% of S. aureus isolates were found to be positive for one or more toxin genes. The sec gene was found most frequently (24.1%), followed by tst (22.8%), seb (13.9%), sed (10.1%) and sea (5.1%). The cna gene was detected in 55.7% of S. aureus isolates. Based on tandem repeats in coa gene, five coa types were observed, further divided into 16 subtypes based on their RFLP pattern. Similarly tandem repeats in spa gene divided S. aureus isolates into 7 types. In the parallel antibiotic resistance study, 69.6% isolates were resistant to at least one of the 11 tested antibiotics. The pheno- and genotyping of S. aureus isolates of sheep origin presented in this work update the epidemiological data in Slovakia.

Detection by PCR of adhesins genes and slime production in clinical Staphylococcus aureus

The presence of the ica loci and adhesins genes in clinical Staphylococcus aureus strains were considered important factors of virulence. In this study, 46 strains of Staphylococcus aureus were isolated from auricular infection, and were investigated for slime production using Congo Red Agar method (CRA). In order to detect the adhesins genes (ica A, ica D, fnb A, cna, Clf A) Polymerase Chain Reaction was used. Qualitative biofilm production of S. aureus using CRA plates revealed that 56.5% of strains were slime producers. In addition 78.26% of strains were ica A and ica D positive. While the fnbA gene was present in 76.1% of isolated strains. Furthermore, 56.5% of strains have the cna gene and 30.4% were clfA positives. Overall this study confirms the presence of fnb A and ica A/ica D genes in the majority of studies S. aureus strains isolated from Staphylococcal sepsis.

Presence of cna, emp and pls genes and pathogenicity of methicillin-resistant Staphylococcus aureus strains

Methicillin-resistant strains of Staphylococcus aureus (MRSA) are important etiological factors responsible for hospital-acquired infections. The aim of this study was to analyze the influence of the presence of emp, pls and cna genes on the pathogenicity of MRSA strains. The presence of these genes was tested by PCR in 302 MRSA strains isolated from hospitalized patients and from carriers. For each tested gene, proportions of positive and negative strains were similar among the infected patients and carriers. We did not find any obvious correlation between the presence of the three tested genes and the infectivity of strains. Our results may also suggest that a lack of emp and presence of pls may correlate with reduced virulence of these strains.

Lack of protection of mice against Staphylococcus aureus despite a significant immune response to immunization with a DNA vaccine encoding collagen-binding protein

Collagen-binding protein (CNA) is the major Staphylococcus aureus adhesin responsible for high affinity binding to collagen and is assumed to be a major virulence factor in infection and disease. Mutants lacking the cna gene are less virulent than the parent strain in models of septic arthritis, osteomyelitis, keratinitis, and endocarditis. In order to investigate the immunological and protective properties of a CNA-based DNA vaccine, a eukaryotic expression vector pCNA was constructed which expressed the collagen-binding domain of this adhesin in transfected cells. Three groups of 11 Balb/c mice received three injection of either pCNA, the empty expression vector (pCI) or PBS. Those injected with pCNA showed hi titre (64000) antibody and evidence of a cell-mediated immune response (CMI). The anti-CNA antibodies recognized the intact bacteria and prevented binding to collagen in vitro. However, the vaccination did not protect against bacterial challenge using the intra-peritoneal route of infection. Moreover, S. aureus that had been treated with sera from vaccinated mice caused a more severe infection than bacteria treated with sera from non-vaccinated mice. In summary, DNA vaccination against CNA produced a strong antibody and cellular response in mice but failed to protect from i.p. infection by S. aureus.

Key adhesin gene in community-acquired methicillin-resistant Staphylococcus aureus

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) possessing the Panton-Valentine leukocidin (PVL) gene ( luk PV ) is associated with skin and soft tissue infections, osteomyelitis, and necrotizing pneumonia. There are geographically two types of CA-MRSA: one (sequence type ST30) that is worldwide (pandemic) and the other (sequence types, e.g., ST1, ST8 or ST80) that is continent-specific. The pandemic type, but not continent-specific type, possessed the bone sialoprotein-adhesin gene ( bbp), which was associated with osteomyelitis. No recent hospital-acquired MRSA had the bbp gene, while past PVL-positive nosocomial outbreak-derived strains did possess it. The collagen-adhesin gene ( cna) was associated with pandemic CA-MRSA, though with positive cases even in continent-specific CA-MRSA and PVL-negative Japanese region-specific CA-MRSA. Thus, the pandemic type is characterized by the combination of luk PV and bbp (and cna) genes. A specific real-time PCR assay for the bbp gene was developed, and dual assay for bbp and luk PV in one test tube became possible.

Clonal relationships among Clostridium perfringens of porcine origin as determined by multilocus sequence typing

Clostridium perfringens is ubiquitous in the environment and the intestinal tracts of most mammals, but this organism also causes gas gangrene and enteritis in human and animal hosts. While expression of specific toxins correlates with specific disease in certain hosts, the other factors involved in commensalism and host pathogenesis have not been clearly identified. A multilocus sequence typing (MLST) scheme was developed for C. perfringens with the aim of grouping isolates with respect to disease presentation and/or host preference. Sequence data were obtained from one virulence and seven housekeeping genes for 132 C. perfringens isolates that comprised all five toxin types and were isolated from 10 host species. Eighty sequence types (STs) were identified, with the majority (75%) containing only one isolate. eBURST analysis identified three clonal complexes, which contained 59.1% of the isolates. Clonal complex (CC) 1 contained 31, predominantly type A isolates from diverse host species. Clonal complex 2 contained 75% of the bovine type E isolates examined in this study. Clonal complex 3 consisted predominantly of porcine type A and type C isolates. Interestingly, these porcine isolates ( n = 32) all carried consensus cpb2 and cna genes, encoding beta2 toxin and CpCna, a collagen binding protein, respectively. This compares to carriage of both these genes by only 3.6% of porcine isolates not present in clonal complex 3 ( n = 28). The data obtained indicates that MLST may be used to identify host species relationships with respect to these C. perfringens isolates.

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 (SCC mecIV), 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.

An immunogenicity study of a newly fusion protein Cna-FnBP vaccinated against Staphylococcus aureus infections in a mice model

Adhesins are considered the most important virulence factors during early phase Staphylococcus aureus infection. The present report describes a newly fusion protein, named Cna-FnBP that was constructed by fusion of the fnb and cna genes of S. aureus and expressed in E. coli. The recombinant protein was designed to broaden the function spectrum of block binding activity to S. aureus adhesion. Vaccination of the recombinant protein induced a strong and specific humoral response to Cna-FnBP in mice. In addition, splenocyte proliferation was provoked by in vitro stimulation with recombinant Cna-FnBP, thus, indicating direct implication of these cells in the immune response. These pre-incubated bacteria were phagocytosed by neutrophils at an increased level in vitro in a mouse model. Mice immunized with Cna-FnBP survived significantly longer following the challenge with S. aureus than nonimmunized mice did. These results indicate that Cna-FnBP is a promising vaccine for the prevention of S. aureus infections. Overall, the results suggest that fusion compounds which elicted from ECM-binding proteins (ECMBPs) were used to immunize against adhesins represents a valuable approach to combat S. aureus infections.

Association of genes encoding beta2 toxin and a collagen binding protein in Clostridium perfringens isolates of porcine origin

Clostridium perfringens is a cause of economically significant enteritis in livestock. Beta2 toxin, encoded by one of two cpb2 alleles, is implicated as a virulence factor in this disease. Previous studies determined that the consensus cpb2 allele is preferentially associated with C. perfringens isolated from pigs. In C. perfringens strain 13, the consensus cpb2 allele is found on the plasmid pCP13, which also carries cna, encoding a putative collagen binding protein, CpCna. This protein was shown to be a bona fide collagen adhesin, as recombinant, HIS-tagged CpCna bound collagen type I as determined by Far Western blotting. Genomic DNA from C. perfringens isolated from a variety of host species were subjected to PCR to determine the prevalence of cna in these strains and correlate its carriage with the presence and type of cpb2 allele. The cna gene was found in 55.8% of isolates from all host species ( n = 208) and 68.1% of porcine isolates ( n = 119). In cpb2+ isolates, cna was present in 69.9% of isolates from all hosts ( n = 153), but was found in 98.7% of porcine isolates ( n = 75). Furthermore in porcine isolates, the consensus cpb2 allele and cna were absolutely correlated with the presence of pcp12, a pCP13-encoded gene, and pcp12 was never found in any isolate that lacks either cpb2 allele. The finding that CpCna binds collagen and that the cna gene is associated with the consensus cpb2 allele implicates CpCna as a potential virulence factor in porcine enteritis caused by C. perfringens.

Methicillin-resistant Staphylococcus aureus genotyping using a small set of polymorphisms

The aim of this study was to identify a set of genetic polymorphisms that efficiently divides methicillin-resistant Staphylococcus aureus (MRSA) strains into groups consistent with the population structure. The rationale was that such polymorphisms could underpin rapid real-time PCR or low-density array-based methods for monitoring MRSA dissemination in a cost-effective manner. Previously, the authors devised a computerized method for identifying sets of single nucleotide polymorphisms (SNPs) with high resolving power that are defined by multilocus sequence typing (MLST) databases, and also developed a real-time PCR method for interrogating a seven-member SNP set for genotyping S. aureus. Here, it is shown that these seven SNPs efficiently resolve the major MRSA lineages and define 27 genotypes. The SNP-based genotypes are consistent with the MRSA population structure as defined by eBURST analysis. The capacity of binary markers to improve resolution was tested using 107 diverse MRSA isolates of Australian origin that encompass nine SNP-based genotypes. The addition of the virulence-associated genes cna, pvl and bbp/sdrE, and the integrated plasmids pT181, pI258 and pUB110, resolved the nine SNP-based genotypes into 21 combinatorial genotypes. Subtyping of the SCCmec locus revealed new SCCmec types and increased the number of combinatorial genotypes to 24. It was concluded that these polymorphisms provide a facile means of assigning MRSA isolates into well-recognized lineages.

Community-acquired Methicillin-resistantStaphylococcus aureus, Uruguay

A novel, methicillin-resistant Staphylococcus aureus clone (Uruguay clone) with a non–multidrug-resistant phenotype caused a large outbreak, including 7 deaths, in Montevideo, Uruguay. The clone was distinct from the highly virulent community clone represented by strain MW2, although both clones carried Panton-Valentine leukocidin gene and cna gene.

Prevalence of cna, fnbA and fnbB adhesin genes among Staphylococcus aureus isolates from orthopedic infections associated to different types of implant

Here are reported data on virulence determinants of Staphylococcus aureus from orthopedic surgical infections, emphasizing on the genes encoding fibronectin ( fnbA, fnbB) and collagen ( cna) adhesins. 191 S. aureus strains from orthopedic infections (53 from internal fixation devices, 29 external fixation devices, 15 knee arthroprostheses, 30 hip arthroprostheses, 45 surgical reconstruction and 19 non-associated to medical devices) were investigated for the presence of the genes of the collagen-binding protein Cna and of the two fibronectin-binding proteins, FnbA and FnbB. 87 (46%) strains were found to be cna+ without significant variations across the different surgical categories considered. Conversely, the fnbA and the fnbB genes were almost always present in all surgical categories. The finding that, among the investigated adhesins, fibronectin-adhesins are present in the majority of the implant associated S. aureus clinical isolates encourages the development of strategies to specifically block the interaction of bacteria with matrix fibronectin by antagonist ligands.

Relationship between some Staphylococcus aureus pathogenic factors and growth rates and somatic cell counts

Staphylococcus aureus isolates produce several pathogenic factors. The combination of these products influences the pathogenic role of different isolates, but their specific effects are well known in the pathogenesis of udder infections. This study focused on the association of polymorphism of the coagulase gene, protein A gene, collagen-binding protein gene, and of fibrinogen-binding protein gene on somatic cell count (SCC) and on Staph. aureus growth rate. Fifty Staph. aureus isolates from 13 dairy cow herds, located in seven different provinces, were considered. The results showed a low frequency of cna gene, similar to the one observed in human isolates. Meanwhile, the high frequency of efb gene indirectly confirmed the role of this factor in bacterial pathogenesis, being associated with adhesion to epithelia. The association of these two single genes with SCC and growth rate showed to be not significant. The polymorphism of spa gene was confirmed to be significantly associated with inflammatory response and growth rate, albeit with a pattern different from the one suggested for human isolates. Sorting of isolates based on the clusters obtained by combining polymorphisms of spa and coa genes and the presence of cna and efb genes, showed that a single cluster (cluster V) was prevalent in the different herds and provinces, while the other six clusters identified were widely spread among the remaining 60% of the isolates. Results showed that clusters VI and VII had significantly higher growth rates at 3, 4, and 6 h in comparison with the other clusters. Meanwhile, quarters infected with these strains showed significantly lower SCC levels. The frequency of isolates from cluster V, suggested that they should possess pathogenic factors increasing their invasiveness, even if in the presence of a stronger inflammatory response. These results indirectly confirm previous findings on the different interactions between isolates and the udder immune system. They also suggest that isolates with higher growth rates and inducing a lower inflammatory response have better chances to spread among the herd. The relatively simple genomic method proposed in this study could be applied by an increasing number of diagnostic laboratories and could be useful in studying the epidemiology of Staph. aureus intra-mammary infections in dairy herds when collecting data from the field.

Emergence of a Predominant Clone of Community-Acquired Staphylococcus aureus Among Children in Houston, Texas

Community-acquired (CA), methicillin-resistant Staphylococcus aureus (MRSA) infections among children are increasing in the United States. At Texas Children's Hospital (TCH), surveillance has been in place since August 2001. The objectives of this study were to describe the distribution of CA S. aureus among patients at TCH and to study genomic relationships of isolates collected between August 2001 and July 2003. Genomic relationships were determined with repetitive element-polymerase chain reaction (PCR). Multilocus sequence typing was performed for selected strains representing major clones. Molecular characterization of CA-MRSA was performed with PCR, including staphylococcal cassette chromosome (SCCmec), pvl (lukS-PV plus lukF-PV), hla, hlb and selected microbial surface components recognizing adhesive matrix molecule genes, ie, cna, clfA, fnbA and fnbB. A 62% increase was observed in CA S. aureus infections from year 1 (2001-2002) to year 2 (2002-2003), whereas the annual number of hospital admissions was unchanged. CA methicillin-sensitive S. aureus isolates were more likely to be associated with invasive infections than were CA-MRSA isolates (P < 0.01). TCH clone A, sequence type (ST) 8, was responsible for approximately 94% of all CA-MRSA isolated from children in the greater Houston area. Clone A differed from clones B (ST30) and C (ST1) by lacking the cna gene while carrying the fnbB gene. One CA-MRSA clone, TCH clone A, has become the predominant cause of CA S. aureus infections among children in the Houston area. It causes a wide spectrum of diseases, including complicated pneumonia.

Pheno and genotyping ofStaphylococcus 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.

Distribution of virulence genes of Staphylococcus aureus isolated from stable nasal carriers

In the present study, we report data on virulence determinants of Staphylococcus aureus from stable nasal carriers, emphasizing on the genes encoding fibronectin ( fnbA, fnbB) and collagen ( cna) adhesive molecules. Of the 44 S. aureus isolates included, 32 isolates (16 pairs) were cultured from the anterior nares of 16 healthy carriers, eight isolates (four pairs) were collected from the nose of four patients with recurrent skin infections and four isolates were obtained from the infection site of these patients. The period between the two nasal swabs taken was 3–5 days. The persistency of carriage could be demonstrated by the indistinguishable genotypic chracteristics of the S. aureus isolates in each pair. This could be shown by determination of gene polymorphisms of coa gene and the X-region and IgG-binding region encoding segments of spa gene. In addition, the isolates within the pairs showed identical toxin patterns. This was determined by PCR amplification of the genes encoding staphylococcal enterotoxins (SEA to SEJ) and TSST-1. The genotypic properties also yielded an identity between persistent nasal carriage isolates and the corresponding skin infection isolates of the four patients. In addition, all S. aureus nasal and skin infection isolates were positive for gene fnbA, fnbB and cna could be found with a high frequency. Among the 44 isolates investigated, 16 isolates (36.7%) harbored gene fnbB and 21 isolates (47.7%) gene cna. The data in the present study showed a relatively wide distribution of the genes fnb and cna among the investigated isolates, indicating that the persistent carriage of strains harboring these virulence determinants may increase the risk for subsequent invasive infections in carriers.

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.

The VirR/VirS regulatory cascade affects transcription of plasmid-encoded putative virulence genes in Clostridium perfringens strain 13

We analyzed the transcriptional regulation of the putative virulence genes encoded on the plasmid pCP13 from Clostridium perfringens strain 13. The transcription of the beta2-toxin ( cpb2) and possible collagen adhesin ( cna) genes were regulated in both a positive and negative manner, respectively, by the two-component VirR/VirS system. The secondary regulator of the VirR/VirS system, VR-RNA, also affects the expression of both of these genes in the same fashion as the VirR/VirS system. This indicates that the global regulatory cascade of the VirR/VirS system controls the expression of virulence genes located on the plasmid, as well as those found chromosomally in C. perfringens strain 13.