Targeted gene disruption in Candida parapsilosis demonstrates a role for CPAR2_404800 in adhesion to a biotic surface and in a murine model of ascending urinary tract infection.

Candida parapsilosis is an emerging opportunistic pathogen, second in frequency only to C. albicans and commonly associated with both mucosal and systemic infections. Adhesion to biotic surfaces is a key step for the development of mycoses. The C. parapsilosis genome encodes 5 predicted agglutinin-like sequence proteins and their precise role in the adhesion process still remains to be elucidated. In this study, we focused on the putative adhesin Cpar2_404800, in view of its high homology to the most important adhesion molecule in C. albicans. Two independent lineages of C. parapsilosis CPAR2_404800 heterozygous and null mutants were obtained by site-specific deletion. CPAR2_404800 mutants did not differ from wild-type strain in terms of in vitro growth or in their ability to undergo morphogenesis. However, when compared for adhesion to a biotic surface, CPAR2_404800 null mutants exhibited a marked reduction in their adhesion to buccal epithelial cells (>60% reduction of adhesion index). Reintroduction of one copy of CPAR2_404800 gene in the null background restored wild type phenotype. A murine model of urinary tract infection was used to elucidate the in vivo contribution of CPAR2_404800. A 0.5 and 1 log10 reduction in colony forming unit numbers (per gram) was observed respectively in bladder and kidneys obtained from mice infected with null mutant compared to wild-type infected ones. Taken together, these findings provide the first evidence for a direct role of CPAR2_404800 in C. parapsilosis adhesion to host surfaces and demonstrate its contribution to the pathogenesis of murine urinary candidiasis.

Comparison of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis adhesive properties and pathogenicity.

Retrospective studies indicate that Candida metapsilosis and Candida orthopsilosis each represents 1-10% of the infections/colonisations attributed to C. parapsilosis by conventional biochemical tests. Little is known on the virulence properties of these fungi and on their role in the establishment/progression of the infection. In this study, the adhesive properties of clinical isolates belonging to the 'psilosis' species were assessed in an in vitro model of co-incubation with human buccal epithelial cells (HBECs). Ectophosphatase activity was also measured for all isolates, since the activity of this enzyme has previously been linked to adhesion properties in C. parapsilosis. The results indicate that whilst C. parapsilosis and C. orthopsilosis strains showed similar adhesion abilities, C. metapsilosis isolates displayed a significantly lower ability to adhere to HBECs (P<0.05). No evidence of a correlation between ectophosphatase activity and adhesion was observed, and this finding was also confirmed by phosphatase inhibition experiments. Experimental vaginal candidiasis induced in oestrogen-treated mice with representative isolates of the 3 species indicated that mice infected with C. metapsilosis displayed a reduced vaginal fungal burden, especially in the early stages of the infection. The overall findings confirm that C. orthopsilosis has a comparable behaviour to C. parapsilosis, whilst C. metapsilosis seems to possess a reduced virulence potential.

Characterization and PCR-based replicon typing of resistance plasmids in Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen, especially in intensive care units, and multidrug-resistant isolates have increasingly been reported during the last decade. Despite recent progress in knowledge of antibiotic resistance mechanisms in A. baumannii, little is known about the genetic factors driving isolates toward multidrug resistance. In the present study, the A. baumannii plasmids were investigated through the analysis and classification of plasmid replication systems and the identification of A. baumannii-specific mobilization and addiction systems. Twenty-two replicons were identified by in silico analysis, and five other replicons were identified and cloned from previously uncharacterized A. baumannii resistance plasmids carrying the OXA-58 carbapenem-hydrolyzing oxacillinase. Replicons were classified into homology groups on the basis of their nucleotide homology. A novel PCR-based replicon typing scheme (the A. baumannii PCR-based replicon typing [AB-PBRT] method) was devised to categorize the A. baumannii plasmids into homogeneous groups on the basis of the nucleotide homology of their respective replicase genes. The AB-PBRT technique was applied to a collection of multidrug-resistant A. baumannii clinical isolates carrying the bla(OXA-58) or bla(OXA-23) carbapenemase gene. A putative complete conjugative apparatus was identified on one plasmid whose self-conjugative ability was demonstrated in vitro. We showed that this conjugative plasmid type was widely diffused in our collection, likely representing the most important vehicle promoting the horizontal transmission of A. baumannii resistance plasmids.

Multilocus sequence typing of IncI1 plasmids carrying extended-spectrum beta-lactamases in Escherichia coli and Salmonella of human and animal origin.

OBJECTIVES: Plasmids belonging to incompatibility group I1 (IncI1) are widespread in Enterobacteriaceae and are characterized by the presence of a cluster of genes encoding the type IV pili, contributing to the virulence of Shiga-toxigenic Escherichia coli. Recently, IncI1 plasmids were identified in E. coli and Salmonella strains of animal origin as responsible for the dissemination of beta-lactamase genes. Plasmid multilocus sequence typing (pMLST) was developed to discern naturally occurring IncI1 plasmids in homogeneous groups according to their allele assortment. METHODS: pMLST was developed by selecting multiple target genes on the available complete IncI1 plasmid DNA sequences. Sixteen plasmids, all assigned to the IncI1 group by the PCR-based replicon typing method, were included in this study. They were analysed for beta-lactamase genes and typed by restriction fragment length polymorphism (RFLP) and pMLST. RESULTS: Sixteen plasmids identified in E. coli and Salmonella isolated from animals and humans in different countries carried bla(CMY-2), bla(CTX-M-15), bla(CTX-M-1), bla(CTX-M-14), bla(TEM-52), bla(SHV-12) or bla(TEM-1) beta-lactamase genes. These plasmids were classified by RFLP in nine different groups corresponding to the nine sequence types determined by pMLST. CONCLUSIONS: The pMLST method was suitable for rapid and easy subtyping of IncI1 plasmids. This study demonstrates that the pMLST method can contribute to the epidemiological description of circulation of specific resistance plasmids among beta-lactamase producers isolated from animals and humans.

Comparative analysis of IncHI2 plasmids carrying blaCTX-M-2 or blaCTX-M-9 from Escherichia coli and Salmonella enterica strains isolated from poultry and humans.

Salmonella enterica bla(CTX-M-2) and bla(CTX-M-9) plasmid backbones from isolates from Belgium and France were analyzed. The bla(CTX-M-2-)plasmids from both human and poultry isolates were related to the IncHI2 pAPEC-O1-R plasmid, previously identified in the United States in avian Escherichia coli strains; the bla(CTX-M-9) plasmids were closely related to the IncHI2 R478 plasmid.

Outbreak of Acinetobacter baumannii producing the carbapenem-hydrolyzing oxacillinase OXA-58 in Rome, Italy.

In this study 45 epidemic and sporadic isolates of Acinetobacter baumannii were investigated by antimicrobial resistance, integron identifications and genotyping. Isolates were genotyped by random amplified polymorphism (RAPD) DNA and pulsed-field gel electrophoresis (PFGE). Four different RAPD patterns were observed among the isolates of our collection, further discerned in six PFGE types. Two prevalent genotypes were identified, one corresponding to a carbapenem resistant epidemic clone, causing an outbreak at the intensive care unit of a hospital of Rome. Two class 1 integrons, carrying different gene cassette arrays, were identified among the two prevalent genotypes. Nucleotide analysis of the integron-variable regions revealed the presence of the aacA4, orfO, bla(OXA-20), and aacC1, orfX, orfX', aadA1 gene cassette arrays, respectively. All the carbapenem resistant strains analyzed in this study carried the bla (OXA-58) gene located on plasmids.

Extended-spectrum beta-lactamase CTX-M-1 in Escherichia coli isolates from healthy poultry in France.

Genes encoding extended-spectrum beta-lactamase CTX-M-1 were detected in 12 Escherichia coli isolates recovered over a 7-month period from the ceca of healthy poultry in seven districts in France in 2005. Eleven of those strains were not clonally related and had a bla(CTX-M-1) gene located on transferable plasmids of different sizes and structures.

Multicopy blaOXA-58 gene as a source of high-level resistance to carbapenems in Acinetobacter baumannii.

The mechanisms at the origin of heterogeneous carbapenem resistance levels observed among Acinetobacter baumannii isolates collected in 2005 in a large University Hospital of Rome, Italy, were investigated. These isolates were related and possessed similar plasmids carrying the carbapenem-hydrolyzing oxacillinase gene bla(OXA-58) but showed variable levels of resistance to carbapenems. Analysis of sequences surrounding the bla(OXA-58) gene showed genetic variability, with the presence in several isolates of multiple copies of the bla(OXA-58) gene; this extra copy number was likely related to an IS26-mediated transposition or recombination process.

Dissemination of an extended-spectrum-beta-lactamase blaTEM-52 gene-carrying IncI1 plasmid in various Salmonella enterica serovars isolated from poultry and humans in Belgium and France between 2001 and 2005.

We report here the dissemination of a conjugative IncI1 plasmid carrying bla(TEM-52) on a Tn3 transposon conferring resistance to extended-spectrum cephalosporins in Salmonella enterica serovar Agona, Derby, Infantis, Paratyphi B dT(+), and Typhimurium isolates from poultry and humans in Belgium and France from 2001 to 2005. The most prevalent serovar spreading this resistance was serovar Infantis.

Replicon typing of plasmids encoding resistance to newer beta-lactams.

Polymerase chain reaction-based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems.

Identification of plasmids by PCR-based replicon typing.

The epidemiological importance of tracing plasmids conferring drug resistance prompted us to develop a PCR method based on replicons (inc/rep PCR) of the major plasmid incompatibility groups among Enterobacteriaceae. Eighteen pairs of primers were designed to perform 5 multiplex- and 3 simplex-PCRs, recognizing FIA, FIB, FIC, HI1, HI2, I1-Igamma, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA. The specificity of the method was tested on a collection of 61 reference plasmids and on 20 Salmonella enterica strains of different serotypes isolated in Italy. Results indicated that the inc/rep PCR method demonstrates high specificity and sensitivity in detecting replicons on reference plasmids and also revealed the presence of recurrent and common plasmids in epidemiologically unrelated Salmonella isolates of different serotypes. These results suggest that the method is potentially applicable to a large number of strains to trace the diffusion of specific multi-drug resistance plasmids in different environments.