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BackgroundStenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains.ResultsAll S. maltophilia CF isolates tested in the present study were able, although at different levels, to adhere to and form biofilm on IB3-1 cell monolayers. Scanning electron and confocal microscopy revealed S. maltophilia structures typical of biofilm formation on bronchial IB3-1 cells. The loss of flagella significantly (P < 0.001) decreased bacterial adhesiveness, if compared to that of their parental flagellated strains. S. maltophilia CF isolates were also able to invade IB3-1 cells, albeit at a very low level (internalization rate ranged from 0.01 to 4.94%). Pre-exposure of IB3-1 cells to P. aeruginosa PAO1 significantly increased S. maltophilia adhesiveness. Further, the presence of S. maltophilia negatively influenced P. aeruginosa PAO1 adhesiveness.ConclusionsThe main contribution of the present study is the finding that S. maltophilia is able to form biofilm on and invade CF-derived IB3-1 bronchial epithelial cells, thus posing a rationale for the persistence and the systemic spread of this opportunistic pathogen in CF patients. Experiments using in vivo models which more closely mimic CF pulmonary tissues will certainly be needed to validate the relevance of our results.

The colonization and development of gut microbiota immediately after birth is highly variable and depends on several factors, such as delivery mode and modality of feeding during the first months of life. A cohort of 31 mother and neonate pairs, including 25 at-term caesarean (CS) and 6 vaginally (V) delivered neonates (DNs), were included in this study and 121 meconium/faecal samples were collected at days 1 through 30 following birth. Operational taxonomic units (OTUs) were assessed in 69 stool samples by phylogenetic microarray HITChip and inter- and intra-individual distributions were established by inter-OTUs correlation matrices and OTUs co-occurrence or co-exclusion networks. 1H-NMR metabolites were determined in 70 stool samples, PCA analysis was performed on 55 CS DNs samples, and metabolome/OTUs co-correlations were assessed in 45 CS samples, providing an integrated map of the early microbiota OTUs-metabolome. A microbiota “core” of OTUs was identified that was independent of delivery mode and lactation stage, suggesting highly specialized communities that act as seminal colonizers of microbial networks. Correlations among OTUs, metabolites, and OTUs-metabolites revealed metabolic profiles associated with early microbial ecological dynamics, maturation of milk components, and host physiology.

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A conjugative IncL/M plasmid (pSEM) conferring resistance to gentamicin, amikacin, kanamycin, sulfonamides, and expanded-spectrum cephalosporins was found in pathogenic strains of Salmonella enterica serotype Typhimurium. Resistance to aminoglycosides was encoded by a sul1-type class 1 integron (In-t3). An extendedspectrum beta-lactamase gene, bla SHV-5 , was identified 3.5 kb downstream of the integrase (intI1) gene of In-t3. Nucleotide sequence analysis of the 5.3-kb bla SHV-5 -In-t3 region of pSEM highlighted striking similarities with IncL/M plasmids isolated from nosocomial gram-negative pathogens, conferring resistance to expandedspectrum cephalosporins and aminoglycosides.Although antibiotic resistance is becoming a major threat to human health worldwide, information concerning the dissemination and geographical distribution of antibiotic-resistant bacterial pathogens remains scattered (8,14,26). During the last decade many hospital outbreaks caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae have been reported, and most of the ESBL-producing strains were found to carry the bla TEM-1 , bla TEM-2 , and bla SHV-1 gene derivatives (5,6,9,11,19,27). These genes are prevalently located on large conjugative plasmids of the incompatibility (Inc) groups IncC, IncFI, IncH12, and IncL/M (3,5,7,13,18,20,23,28).More recent reports have highlighted the emergence of ESBL-producing strains endowed with an extremely wide spectrum of antibiotic resistance, including resistance to sulfonamides, trimethoprim, streptomycin, kanamycin, gentamicin, and amikacin (17,21,23). However, the molecular mechanisms involved in the acquisition and/or transmission of ESBL-encoding genes are poorly understood.In previous work we identified an IncL/M plasmid, referred to as pSEM, in eight epidemiologically unrelated, multipledrug-resistant strains of Salmonella enterica serotype Typhimurium. This plasmid contains a sul1-type class 1 integron (In-t3) carrying the aacA4, aacC1, and aadA1 gene cassettes and conferring resistance to sulfonamides, kanamycin, gentamicin, and amikacin (29). The streptomycin and spectinomycin-resistance determinant, aadA1, is poorly expressed in In-t3 because it is located in a distal position relative to the main integron promoter P ant (29).The Salmonella strains harboring pSEM were analyzed in more detail with regard to their antibiotic resistance profile. Microdilution susceptibility tests demonstrated that all eight strains were resistant to ceftazidime (Ն16 g/ml), cefotaxime (Ն32 g/ml), ceftriaxone (Ն64 g/ml), cefamandole (Ն32 g/ ml), cefoxitin (Ն8 g/ml), and aztreonam (Ն16 g/ml) but sensitive to imipenem (Ͻ4 g/ml).To clone the gene responsible for resistance to expandedspectrum cephalosporins, an EcoRI genomic library was constructed in the pUC18 vector using total DNA extracted from the prototypic Salmonella 202 strain. The library was introduced by transformation in Escherichia coli DH5␣ competent cells, and selection was performed on Luria-Bertani agar plates containing ceftaz...

emergence of the OXA-23 epidemic lineage could result from selective advantage conferred by the bla(OXA-23)-like determinant, which provides increased resistance to carbapenems.

The role in virulence of the Shigella flexneri ospB-phoN2 operon has been evaluated. Here we confirm that OspB is an effector and show that apyrase, the product of phoN2, may be a virulence factor, since it is required for efficient intercellular spreading. Apyrase may be important in a deoxynucleoside triphosphate-hydrolyzing activity-independent manner, suggesting that it may act as an interaction partner in the process of IcsA localization.ospB and phoN2 (apy) are genes located on the virulence plasmid (pINV) of Shigella species and of related enteroinvasive Escherichia coli (EIEC) strains (3,6,35). Indirect evidence indicates that ospB and phoN2 may contribute to pathogenicity (1,3,5,8,17,23,25,35). ospB encodes a protein of unknown function (OspB) surmised to be an effector secreted by the type III secretion (TTS) apparatus of Shigella flexneri (6,20). The two genes are organized in a single highly conserved bicistronic operon. Although phoN2 encodes a periplasmic protein (5), their expression is regulated by the VirF-VirB cascade (35) and by MxiE in concert with IpgC so that they are expressed in a VirB-dependent, MxiE-independent manner under conditions of nonactivated secretion and are up-regulated, in a MxiEdependent manner, under conditions of activated secretion (8,17,20,26,30), indicating that these genes may be important in postinvasion events related to virulence.phoN2 encodes apyrase, a periplasmic enzyme which belongs to the family of ATP-hydrolyzing enzymes (3, 5, 18) able to sequentially hydrolyze nucleoside triphosphates to diphosphates and then to monophosphates. However, it is not active against monophosphates, a distinguishing feature from acid phosphatases (1,3,41). Due to its catalytic activity and primary structure (apyrase presents an exposed N-terminal poly-proline sequence), it has been implicated in the decrease of host cell intracellular ATP, a characteristic metabolic event following S. flexneri infection, as well as in the S. flexneri-induced actin-polymerization process (1,3,5,25,35). Thus, these findings strongly suggest that OspB and apyrase may be virulence factors. The purpose of this study was to investigate the role of both genes in the mechanism of pathogenicity of S. flexneri. OspB is an effector secreted by the TTS apparatus of S. flexneri. To confirm and extend previous reports indicatingOspB as an effector secreted by the TTS apparatus (6, 20), we transduced a nonpolar deletion encompassing mxiA into the S. flexneri M90T derivative strain HND549 (ospB::3ϫFLAG), thus generating HND5311 (mxiA ospB::3ϫFLAG) ( Table 1). C-terminal 3ϫ FLAG tagging was achieved essentially as described by Uzzau et al. (42) using the specific primer pair shown in Table S1 in the supplemental material. Whole-cell extracts and supernatants of exponentially growing bacteria were analyzed by immunoblotting using monoclonal antibodies against the 3ϫ FLAG epitope (Sigma) and mouse polyclonal antibodies preparation directed against apyrase (Fig. 1). Apyrase antiserum was obtained by immunizing BALB/...

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