A vanG-type locus in Clostridium argentinense.

OBJECTIVES: The objective was to study a new vanG-type locus in Clostridium argentinense vanGCar and to determine its impact on glycopeptide susceptibility of the host. METHODS: The whole genome of C. argentinense NCIB 10714 was sequenced using Illumina single-reads sequencing technology. The presence of vanGCar in seven C. argentinense strains was tested by PCR and its expression was tested by quantitative RT-PCR (qRT-PCR). Glycopeptide susceptibility was determined by the Etest procedure. RESULTS: The vanGCar locus contained four genes encoding a carboxypeptidase, a d-alanine:d-serine ligase, a serine transporter and a serine racemase, and was present in the seven C. argentinense studied. An AraC-type transcriptional regulator was found upstream from the genes. C. argentinense NCIB 10714 was susceptible to vancomycin and to teicoplanin. qRT-PCR experiments revealed that vanGCar was not expressed without or with induction by a subinhibitory concentration of vancomycin. CONCLUSIONS: The new vanGCar locus was cryptic in C. argentinense and intrinsic to this species. Emergence of vancomycin resistance in C. argentinense due to decryptification of the vanGCar gene cluster could occur.

Identification of 50 class D ß-lactamases and 65 Acinetobacter-derived cephalosporinases in Acinetobacter spp.

Whole-genome sequencing of a collection of 103 Acinetobacter strains belonging to 22 validly named species and another 16 putative species allowed detection of genes for 50 new class D ß-lactamases and 65 new Acinetobacter-derived cephalosporinases (ADC). All oxacillinases (OXA) contained the three typical motifs of class D ß-lactamases, STFK, (F/Y)GN, and K(S/T)G. The phylogenetic tree drawn from the OXA sequences led to an increase in the number of OXA groups from 7 to 18. The topologies of the OXA and RpoB phylogenetic trees were similar, supporting the ancient acquisition of blaOXA genes by Acinetobacter species. The class D ß-lactamase genes appeared to be intrinsic to several species, such as Acinetobacter baumannii, Acinetobacter pittii, Acinetobacter calcoaceticus, and Acinetobacter lwoffii. Neither blaOXA-40/143- nor blaOXA-58-like genes were detected, and their origin remains therefore unknown. The phylogenetic tree analysis based on the alignment of the sequences deduced from blaADC revealed five main clusters, one containing ADC belonging to species closely related to A. baumannii and the others composed of cephalosporinases from the remaining species. No indication of blaOXA or blaADC transfer was observed between distantly related species, except for blaOXA-279, possibly transferred from Acinetobacter genomic species 6 to Acinetobacter parvus. Analysis of ß-lactam susceptibility of seven strains harboring new oxacillinases and cloning of the corresponding genes in Escherichia coli and in a susceptible A. baumannii strain indicated very weak hydrolysis of carbapenems. Overall, this study reveals a large pool of ß-lactamases in different Acinetobacter spp., potentially transferable to pathogenic strains of the genus.

Staphylococcus aureus VRSA-11B is a constitutive vancomycin-resistant mutant of vancomycin-dependent VRSA-11A.

Vancomycin-resistant Staphylococcus aureus VRSA-10 was isolated in 2009, whereas VRSA-11A and VRSA-11B were isolated from the same patient in 2010. Growth curves and determination of the nature of the peptidoglycan precursors and of the VanX d,d-dipeptidase activity in the absence and in the presence of vancomycin indicated that vancomycin resistance was inducible in VRSA-10, that VRSA-11A was partially dependent on glycopeptide for growth, and that VRSA-11B was constitutively resistant. Both VRSA-11A and -11B harbored an insertion sequence, ISEf1, at the same locus in the vanX-vanY intergenic region of Tn1546 and an S(183)A mutation in the chromosomal d-alanyl:d-alanine ligase (Ddl). This substitution has been shown to be responsible for a drastic diminution of the affinity of the enzyme for d-Ala at subsite 1 in Escherichia coli DdlB. VRSA-11B exhibited an additional mutation, P(216)T, in the transcriptional regulator VanR, most probably associated with constitutive expression of vancomycin resistance. It is thus likely that VRSA-11B is a constitutive derivative of VRSA-11A selected during prolonged vancomycin therapy. Synthesis of peptidoglycan precursors ending in d-Ala-d-lactate was responsible for oxacillin susceptibility of VRSA-11A and VRSA-11B despite the presence of a wild-type mecA gene in both strains.

Expression of the resistance-nodulation-cell division pump AdeIJK in Acinetobacter baumannii is regulated by AdeN, a TetR-type regulator.

Resistance-nodulation-division efflux system AdeIJK contributes to intrinsic resistance to various drug classes in Acinetobacter baumannii. By whole-genome sequencing, we have identified in clinical isolate BM4587 the adeN gene, located 813 kbp upstream from adeIJK, which encodes a TetR transcriptional regulator. In one-step mutant BM4666 overexpressing adeIJK, the deletion of cytosine 582 (C(582)) in the 3' portion of this gene was responsible for a frameshift mutation resulting in the deletion of the seven C-terminal residues. trans-Complementation of this BM4587 derivative with a plasmid expressing adeN restored antibiotic susceptibility to the host associated with the loss of adeJ overexpression. The inactivation of adeN in BM4587 led to a diminished susceptibility to antibiotics that are substrates for AdeIJK and to a 5-fold increase in adeJ expression. Taken together, these results indicate that AdeN represses AdeIJK expression. Quantitative reverse transcription-PCR (qRT-PCR) demonstrated that AdeN is constitutively expressed in BM4587 and does not regulate its own expression. Deletion of cytosine 582 and a 394-bp deletion of the 3' part of adeN were found in independent one-step adeIJK-overexpressing mutants selected from clinical isolates BM4667 and BM4651, respectively. The corresponding alterations were located in the α9 helix, which is known to be involved in dimerization, a process essential for the activity of TetR regulators. The adeN gene was detected in all of the 30 A. baumannii strains tested and in Acinetobacter calcoaceticus, Acinetobacter nosocomialis, and Acinetobacter pittii.

Detection of Streptococcus gallolyticus and Four Other CRC-Associated Bacteria in Patient Stools Reveals a Potential "Driver" Role for Enterotoxigenic Bacteroides fragilis.

Purpose: Streptococcus gallolyticus subspecies gallolyticus (SGG) is an opportunistic pathogen causing invasive infections in the elderly often associated with colon neoplasia. The prevalence of SGG in the stools of patients with normal colonoscopy (control) was compared with patients with colorectal adenomas (CRA) or with carcinomas (CRC) from stages I to IV. The presence of the pks island encoding colibactin as well as other CRC-associated bacteria such as toxicogenic Bacteroides fragilis, Fusobacterium nucleatum, and Parvimonas micra was also investigated. Patients and Methods: Fecal samples collected in France between 2011 and 2016 from patients with normal colonoscopy (n = 25), adenoma (n = 23), or colorectal cancer at different stages (n = 81) were tested by PCR for the presence of SGG, B. fragilis, F. nucleatum, P. micra, and the pks island. Relative quantification of SGG, F. nucleatum, and P. micra in stools was performed by qPCR. Results: SGG prevalence was significantly increased in the CRC group. Our results also revealed i) a strong and significant increase of toxinogenic B. fragilis in patients with early-stage adenoma and of pks island at late-stage CRC and ii) increased levels of F. nucleatum and P. micra in the stools of CRC patients. Furthermore, the simultaneous detection of these five bacterial markers was only found in CRC patients. Conclusions: Our results indicate that the prevalence or relative levels of CRC-associated bacteria vary during CRC development. Among them, B. fragilis (bft+) was singled out as the sole pathobiont detected at the early adenoma stage.

Efflux-mediated antibiotic resistance in Acinetobacter spp.

Among Acinetobacter spp., A. baumannii is the most frequently implicated in nosocomial infections, in particular in intensive care units. It was initially thought that multidrug resistance (MDR) in this species was due mainly to horizontal acquisition of resistance genes. However, it has recently become obvious that increased expression of chromosomal genes for efflux systems plays a major role in MDR. Among the five superfamilies of pumps, resistance-nodulation-division (RND) systems are the most prevalent in multiply resistant A. baumannii. RND pumps typically exhibit a wide substrate range that can include antibiotics, dyes, biocides, detergents, and antiseptics. Overexpression of AdeABC, secondary to mutations in the adeRS genes encoding a two-component regulatory system, constitutes a major mechanism of multiresistance in A. baumannii. AdeIJK, intrinsic to this species, is responsible for natural resistance, but since overexpression above a certain threshold is toxic for the host, its contribution to acquired resistance is minimal. The recently described AdeFGH, probably regulated by a LysR-type transcriptional regulator, also confers multidrug resistance when overexpressed. Non-RND efflux systems, such as CraA, AmvA, AbeM, and AbeS, have also been characterized for A. baumannii, as have AdeXYZ and AdeDE for other Acinetobacter spp. Finally, acquired narrow-spectrum efflux pumps, such as the major facilitator superfamily (MFS) members TetA, TetB, CmlA, and FloR and the small multidrug resistance (SMR) member QacE in Acinetobacter spp., have been detected and are mainly encoded by mobile genetic elements.

Characterization of a Four-Component Regulatory System Controlling Bacteriocin Production in Streptococcus gallolyticus.

Bacteriocins are natural antimicrobial peptides produced by bacteria to kill closely related competitors. The opportunistic pathogen Streptococcus gallolyticus subsp. gallolyticus was recently shown to outcompete commensal enterococci of the murine microbiota under tumoral conditions thanks to the production of a two-peptide bacteriocin named gallocin. Here, we identified four genes involved in the regulatory control of gallocin in S. gallolyticus subsp. gallolyticus UCN34 that encode a histidine kinase/response regulator two-component system (BlpH/BlpR), a secreted peptide (GSP [gallocin-stimulating peptide]), and a putative regulator of unknown function (BlpS). While BlpR is a typical 243-amino-acid (aa) response regulator possessing a phospho-receiver domain and a LytTR DNA-binding domain, BlpS is a 108-aa protein containing only a LytTR domain. Our results showed that the secreted peptide GSP activates the dedicated two-component system BlpH/BlpR to induce gallocin transcription. A genome-wide transcriptome analysis indicates that this regulatory system (GSP-BlpH/BlpR) is specific for bacteriocin production. Importantly, as opposed to BlpR, BlpS was shown to repress gallocin gene transcription. A conserved operator DNA sequence of 30 bp was found in all promoter regions regulated by BlpR and BlpS. Electrophoretic mobility shift assays (EMSA) and footprint assays showed direct and specific binding of BlpS and BlpR to various regulated promoter regions in a dose-dependent manner on this conserved sequence. Gallocin expression appears to be tightly controlled in S. gallolyticus subsp. gallolyticus by quorum sensing and antagonistic activity of 2 LytTR-containing proteins. Competition experiments in gut microbiota medium and 5% CO2 to mimic intestinal conditions demonstrate that gallocin is functional under these in vivo-like conditions.IMPORTANCEStreptococcus gallolyticus subsp. gallolyticus, formerly known as Streptococcus bovis biotype I, is an opportunistic pathogen causing septicemia and endocarditis in the elderly often associated with asymptomatic colonic neoplasia. Recent studies indicate that S. gallolyticus subsp. gallolyticus is both a driver and a passenger of colorectal cancer. We previously showed that S. gallolyticus subsp. gallolyticus produces a bacteriocin, termed gallocin, enabling colonization of the colon under tumoral conditions by outcompeting commensal members of the murine microbiota such as Enterococcus faecalis Here, we identified and extensively characterized a four-component system that regulates gallocin production. Gallocin gene transcription is activated by a secreted peptide pheromone (GSP) and a two-component signal transduction system composed of a transmembrane histidine kinase receptor (BlpH) and a cytosolic response regulator (BlpR). Finally, a DNA-binding protein (BlpS) was found to repress gallocin genes transcription, likely by antagonizing BlpR. Understanding gallocin regulation is crucial to prevent S. gallolyticus subsp. gallolyticus colon colonization under tumoral conditions.

Molecular basis of vancomycin dependence in VanA-type Staphylococcus aureus VRSA-9.

The vancomycin-resistant Staphylococcus aureus VRSA-9 clinical isolate was partially dependent on glycopeptide for growth. The responsible vanA operon had the same organization as that of Tn1546 and was located on a plasmid. The chromosomal D-Ala:D-Ala ligase (ddl) gene had two point mutations that led to Q260K and A283E substitutions, resulting in a 200-fold decrease in enzymatic activity compared to that of the wild-type strain VRSA-6. To gain insight into the mechanism of enzyme impairment, we determined the crystal structure of VRSA-9 Ddl and showed that the A283E mutation induces new ion pair/hydrogen bond interactions, leading to an asymmetric rearrangement of side chains in the dimer interface. The Q260K substitution is located in an exposed external loop and did not induce any significant conformational change. The VRSA-9 strain was susceptible to oxacillin due to synthesis of pentadepsipeptide precursors ending in D-alanyl-D-lactate which are not substrates for the ß-lactam-resistant penicillin binding protein PBP2'. Comparison with the partially vancomycin-dependent VRSA-7, whose Ddl is 5-fold less efficient than that of VRSA-9, indicated that the levels of vancomycin dependence and susceptibility to ß-lactams correlate with the degree of Ddl impairment. Ddl drug targeting could therefore be an effective strategy against vancomycin-resistant S. aureus.

Screening and quantification of the expression of antibiotic resistance genes in Acinetobacter baumannii with a microarray.

An oligonucleotide-based DNA microarray was developed to evaluate expression of genes for efflux pumps in Acinetobacter baumannii and to detect acquired antibiotic resistance determinants. The microarray contained probes for 205 genes, including those for 47 efflux systems, 55 resistance determinants, and 35 housekeeping genes. The microarray was validated by comparative analysis of mutants overexpressing or deficient in the pumps relative to the parental strain. The performance of the microarray was also evaluated using in vitro single-step mutants obtained on various antibiotics. Overexpression, confirmed by quantitative reverse transcriptase PCR, of RND efflux pumps AdeABC, due to a G30D substitution in AdeS in a multidrug-resistant (MDR) strain obtained on gentamicin, and AdeIJK, in two mutants obtained on cefotaxime or tetracycline, was detected. A new efflux pump, AdeFGH, was found to be overexpressed in a mutant obtained on chloramphenicol. Study of MDR clinical isolates, including the AYE strain, whose entire sequence has been determined, indicated overexpression of AdeABC and of the chromosomally encoded cephalosporinase as well as the presence of several acquired resistance genes. The overexpressed and acquired determinants detected by the microarray could account for nearly the entire MDR phenotype of the isolates. The microarray is potentially useful for detection of resistance in A. baumannii and should allow detection of new efflux systems associated with antibiotic resistance.

Overexpression of resistance-nodulation-cell division pump AdeFGH confers multidrug resistance in Acinetobacter baumannii.

Acinetobacter baumannii is a major nosocomial pathogen which frequently develops multidrug resistance by acquisition of antibiotic resistance genes and overexpression of intrinsic efflux systems, such as the RND efflux pumps AdeABC and AdeIJK. A third RND system was characterized by studying spontaneous mutants BM4663 and BM4664, which were selected in the presence of chloramphenicol and norfloxacin, respectively, from the AdeABC- and AdeIJK-defective derivative A. baumannii BM4652. They exhibited enhanced resistance to fluoroquinolones, tetracycline-tigecycline, chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, sodium dodecyl sulfate, and dyes such as ethidium bromide, safranin O, and acridine orange. Comparison of transcriptomes of mutants with that of their parental strain, using a microarray technology, demonstrated the overexpression of three genes that encoded an RND efflux system, named AdeFGH. Inactivation of AdeFGH in BM4664 restored an antibiotic susceptibility profile identical to that of BM4652, indicating that AdeFGH was cryptic in BM4652 and responsible for multidrug resistance in its mutants. RNA analysis demonstrated that the three genes were cotranscribed. The adeFGH operon was found in 36 out of 40 A. baumannii clinical isolates, but none of the 22 isolates tested overexpressed the pump genes. Spontaneous MDR mutant BM4684, overexpressing adeFGH, was obtained from clinical isolate BM4587, indicating that adeFGH can be overexpressed in a strain harboring adeABC-adeIJK. An open reading frame, coding a LysR-type transcriptional regulator, named adeL, was located upstream from the adeFGH operon and transcribed in the opposite direction. Mutations in adeL were found in the three adeFGH-overexpressing mutants, suggesting that they were responsible for overexpression of AdeFGH.

An original infection model identifies host lipoprotein import as a route for blood-brain barrier crossing.

Pathogens able to cross the blood-brain barrier (BBB) induce long-term neurological sequelae and death. Understanding how neurotropic pathogens bypass this strong physiological barrier is a prerequisite to devise therapeutic strategies. Here we propose an innovative model of infection in the developing Drosophila brain, combining whole brain explants with in vivo systemic infection. We find that several mammalian pathogens are able to cross the Drosophila BBB, including Group B Streptococcus (GBS). Amongst GBS surface components, lipoproteins, and in particular the B leucine-rich Blr, are important for BBB crossing and virulence in Drosophila. Further, we identify (V)LDL receptor LpR2, expressed in the BBB, as a host receptor for Blr, allowing GBS translocation through endocytosis. Finally, we show that Blr is required for BBB crossing and pathogenicity in a murine model of infection. Our results demonstrate the potential of Drosophila for studying BBB crossing by pathogens and identify a new mechanism by which pathogens exploit the machinery of host barriers to generate brain infection.

VanA-type vancomycin-resistant Staphylococcus aureus.

Since 2002, nine methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) strains that are also resistant to vancomycin (VRSA) have been reported in the United States, including seven clinical isolates from Michigan. The strains harbor a plasmid-borne Tn1546 element following conjugation from a glycopeptide-resistant Enterococcus strain. In the second step, Tn1546 transposed to a resident plasmid in five strains; the acquired plasmid behaved as a suicide gene delivery vector, and the incoming DNA had been rescued by illegitimate recombination. Surprisingly, combination of a glycopeptide with a beta-lactam has a strong synergistic effect against VRSA, both in vitro and in an animal model, despite resistance of the strains to both drug classes when administered separately. This results from the fact that the late peptidoglycan precursors ending in D-alanine-D-lactate (D-Ala-D-Lac) that are mainly synthesized in the presence of glycopeptide inducers are not substrates for PBP2', which is the only transpeptidase that remains active in the presence of oxacillin. One VRSA strain is partially dependent on vancomycin for growth due to a mutation in the host D-Ala:D-Ala ligase, thus having to rely on the inducible resistance pathway for cell wall synthesis. Competition growth experiments in the absence of inducer between the MRSA recipient and isogenic VRSA transconjugant revealed a disadvantage for the transconjugant, accounting, in part, for the low level of dissemination of the VRSA clinical isolates. The association of multiple molecular and environmental factors has been implicated in the regional emergence of VRSA in Michigan.

VanA-type Staphylococcus aureus strain VRSA-7 is partially dependent on vancomycin for growth.

VanA-type Staphylococcus aureus strain VRSA-7 was partially dependent on glycopeptides for growth. The vanA gene cluster, together with the erm(A) and the ant(9)-Ia resistance genes, was carried by the ca. 35- to 40-kb conjugative plasmid pIP848 present at five copies per cell. The chromosomal ddl gene had a mutation that led to a N308K substitution in the d-Ala:d-Ala ligase that resulted in a 1,000-fold decrease in activity relative to that of strain VRSA-6. Strain VRSA-7 grown in the absence or in the presence of vancomycin mainly synthesized precursors ending in d-Ala-d-Lac, indicating that the strain relied on the vancomycin resistance pathway for peptidoglycan synthesis. Greatly enhanced growth in the presence of glycopeptides and the absence of mutations in the genes for VanR and VanS indicated the inducible expression of resistance. Thus, a combination of loose regulation of the vanA operon by the two-component system and a gene dosage effect accounts for the partial glycopeptide dependence of VRSA-7. Since peptidoglycan precursors ending in D-Ala-D-Lac are not processed by PBP 2', the strain was fully susceptible to oxacillin, despite the production of a wild-type PBP 2'.

Antitumor and antibacterial properties of virally encoded cationic sequences.

Objective: The objective of this study was to test our Viral Quinta Columna Strategy (VQCS), a new biological hypothesis predicting that specific multifunctional virally encoded cationic domains may have the capacity to penetrate human cells and interact with PP2A proteins to deregulate important human intracellular pathways, and may display LL37 cathelicidin-like antagonistic effects against multiple pathogens such as bacteria or viruses. Methods: We comparatively analyzed the host defense properties of adenodiaphorins and of some specific cationic sequences encoded by different viruses using two distinct biological models: U87G, a well-characterized cell tumor model; and a group B Streptococcus agalactiae NEM316 ΔdltA, highly sensitive to LL37 cathelicidin. Results: We found that the adenovirus type 2 E4orf4 is a cell-permeable protein containing a new E4orf464-95 protein transduction domain, named large adenodiaphorin or LadD64-95. Interestingly, the host defense LL37 peptide is the unique cathelicidin in humans. In this context, we also demonstrated that similarly to LL37 LadD64-95, several virally encoded cationic sequences including the C-terminus HIV-1 89.6 Vpr77-92, shorter adenodiaphorins AdD67-84/AdD/69-84/AdD69-83, as well as HIV-2 Tat67-90 and JC polyomavirus small t115-134, displayed similar toxicity against Gram-positive S. agalactiae NEM316 ΔdltA strain. Finally, LadD64-95, adenodiaphorin AdD67-84, AdD69-84, and LL37 and LL17-32 cathelicidin peptides also inhibited the survival of human U87G glioblastoma cells. Conclusion: In this study, we demonstrated that specific cationic sequences encoded by four different viruses displayed antibacterial activities against S. agalactiae NEM316 ΔdltA strain. In addition, HIV-1 Vpr71-92 and adenovirus 2 E4orf464-95, two cationic penetrating sequences that bind PP2A, inhibited the survival of U87G glioblastoma cells. These results illustrate the host defense properties of virally encoded sequences and could represent an initial step for future complete validation of the VQCS hypothesis.

Insights into Streptococcus agalactiae PI-2b pilus biosynthesis and role in adherence to host cells.

The core PI-2b pilus present in "hypervirulent" ST-17 Streptococcus agalactiae strains consists of three pilin subunits (Spb1, Ap1 and Ap2) assembled by sortase SrtC1 and cell-wall anchored by Srt2. Spb1 was shown to be the major pilin and Ap2 the anchor pilin. Ap1 is a putative adhesin. Two additional genes, orf and lep, are part of this operon. The contribution of Lep and Ap1 to the biogenesis of the PI-2b pilus was investigated. Concerning the role of PI-2b, we found that higher PI-2b expression resulted in higher adherence to human brain endothelial cells and higher phagocytosis by human THP1 macrophages.

Sequence of conjugative plasmid pIP1206 mediating resistance to aminoglycosides by 16S rRNA methylation and to hydrophilic fluoroquinolones by efflux.

Self-transferable IncFI plasmid pIP1206, isolated from an Escherichia coli clinical isolate, carries two new resistance determinants: qepA, which confers resistance to hydrophylic fluoroquinolones by efflux, and rmtB, which specifies a 16S rRNA methylase conferring high-level aminoglycoside resistance. Analysis of the 168,113-bp sequence (51% G+C) revealed that pIP1206 was composed of several subregions separated by copies of insertion sequences. Of 151 open reading frames, 56 (37%) were also present in pRSB107, isolated from a bacterium in a sewage treatment plant. pIP1206 contained four replication regions (RepFIA, RepFIB, and two partial RepFII regions) and a transfer region 91% identical with that of pAPEC-O1-ColBM, a plasmid isolated from an avian pathogenic E. coli. A putative oriT region was found upstream from the transfer region. The antibiotic resistance genes tet(A), catA1, bla(TEM-1), rmtB, and qepA were clustered in a 33.5-kb fragment delineated by two IS26 elements that also carried a class 1 integron, including the sulI, qacEDelta1, aad4, and dfrA17 genes and Tn10, Tn21, and Tn3-like transposons. The plasmid also possessed a raffinose operon, an arginine deiminase pathway, a putative iron acquisition gene cluster, an S-methylmethionine metabolism operon, two virulence-associated genes, and a type I DNA restriction-modification (R-M) system. Three toxin/antitoxin systems and the R-M system ensured stabilization of the plasmid in the host bacteria. These data suggest that the mosaic structure of pIP1206 could have resulted from recombination between pRSB107 and a pAPEC-O1-ColBM-like plasmid, combined with structural rearrangements associated with acquisition of additional DNA by recombination and of mobile genetic elements by transposition.

Regulation of PI-2b Pilus Expression in Hypervirulent Streptococcus agalactiae ST-17 BM110.

The widely spread Streptococcus agalactiae (also known as Group B Streptococcus, GBS) "hypervirulent" ST17 clone is strongly associated with neonatal meningitis. The PI-2b locus is mainly found in ST17 strains but is also present in a few non ST17 human isolates such as the ST-7 prototype strain A909. Here, we analysed the expression of the PI-2b pilus in the ST17 strain BM110 as compared to the non ST17 A909. Comparative genome analyses revealed the presence of a 43-base pair (bp) hairpin-like structure in the upstream region of PI-2b operon in all 26 ST17 genomes, which was absent in the 8 non-ST17 strains carrying the PI-2b locus. Deletion of this 43-bp sequence in strain BM110 resulted in a 3- to 5-fold increased transcription of PI-2b. Characterization of PI-2b promoter region in A909 and BM110 strains was carried out by RNAseq, primer extension, qRT-PCR and transcriptional fusions with gfp as reporter gene. Our results indicate the presence of a single promoter (Ppi2b) with a transcriptional start site (TSS) mapped 37 bases upstream of the start codon of the first PI-2b gene. The large operon of 16 genes located upstream of PI-2b codes for the group B carbohydrate (also known as antigen B), a major constituent of the bacterial cell wall. We showed that the hairpin sequence located between antigen B and PI-2b operons is a transcriptional terminator. In A909, increased expression of PI-2b probably results from read-through transcription from antigen B operon. In addition, we showed that an extended 5' promoter region is required for maximal transcription of gfp as a reporter gene in S. agalactiae from Ppi2b promoter. Gene reporter assays performed in Lactococcus lactis strain NZ9000, a related non-pathogenic Gram-positive species, revealed that GBS-specific regulatory factors are required to drive PI-2b transcription. PI-2b expression is up-regulated in the BM110ΔcovR mutant as compared to the parental BM110 strain, but this effect is probably indirect. Collectively, our results indicate that PI-2b expression is regulated in GBS ST17 strains, which may confer a selective advantage in the human host either by reducing host immune responses and/or increasing their dissemination potential.

Comparison of four methods, including semi-automated rep-PCR, for the typing of vancomycin-resistant Enterococcus faecium.

We have assessed the performance of semi-automated rep-PCR (Diversilab®) and multilocus sequence typing (MLST) in comparison to pulsed-field gel electrophoresis (PFGE) for typing a collection of 29 epidemiologically characterized vancomycin-resistant Enterococcus faecium (VRE). Sixteen strains that harbored the Tn1546 element were typed by PCR mapping. The discriminative power of the typing methods was calculated by the Simpson's index of diversity, and the concordance between methods was evaluated by the Kendall's coefficient of concordance. Semi-automated rep-PCR appeared as discriminative as PFGE and was further compared with PFGE for typing 67 VRE isolated during a hospital outbreak. Rep-PCR appeared to be more discriminative than PFGE for this second set of strains. Reproducibility of DiversiLab® was also tested against 35 selected isolates. Only three showed less than 97% similarity, indicating high reproducibility at this level of discrimination. In conclusion, semi-automated rep-PCR is a useful tool for rapid screening of VRE isolates during an outbreak, although cost of the system may be limiting for routine implementation. PFGE, which remains the reference method, should be used for confirmation and evaluation of the genetic relatedness of epidemic isolates.

Genetic and biochemical characterization of CAD-1, a chromosomally encoded new class A penicillinase from Carnobacterium divergens.

Carnobacterium divergens clinical isolates BM4489 and BM4490 were resistant to penicillins but remained susceptible to combinations of amoxicillin-clavulanic acid and piperacillin-tazobactam. Cloning and sequencing of the responsible determinant from BM4489 revealed a coding sequence of 912 bp encoding a class A beta-lactamase named CAD-1. The bla(CAD-1) gene was assigned to a chromosomal location in the two strains that had distinct pulsed-field gel electrophoresis patterns. CAD-1 shared 53% and 42% identity with beta-lactamases from Bacillus cereus and Staphylococcus aureus, respectively. Alignment of CAD-1 with other class A beta-lactamases indicated the presence of 25 out of the 26 isofunctional amino acids in class A beta-lactamases. Escherichia coli harboring bla(CAD-1) exhibited resistance to penams (benzylpenicillin and amoxicillin) and remained susceptible to amoxicillin in combination with clavulanic acid. Mature CAD-1 consisted of a 34.4-kDa polypeptide. Kinetic analysis indicated that CAD-1 exhibited a narrow substrate profile, hydrolyzing benzylpenicillin, ampicillin, and piperacillin with catalytic efficiencies of 6,600, 3,200, and 2,900 mM(-1) s(-1), respectively. The enzyme did not interact with oxyiminocephalosporins, imipenem, or aztreonam. CAD-1 was inhibited by tazobactam (50% inhibitory concentration [IC(50)] = 0.27 microM), clavulanic acid (IC(50) = 4.7 microM), and sulbactam (IC(50) = 43.5 microM). The bla(CAD-1) gene is likely to have been acquired by BM4489 and BM4490 as part of a mobile genetic element, since it was not found in the susceptible type strain CIP 101029 and was adjacent to a gene for a resolvase.

Transferable resistance to aminoglycosides by methylation of G1405 in 16S rRNA and to hydrophilic fluoroquinolones by QepA-mediated efflux in Escherichia coli.

Plasmid pIP1206 was detected in Escherichia coli strain 1540 during the screening of clinical isolates of Enterobacteriaceae for high-level resistance to aminoglycosides. The sequence of this IncFI conjugative plasmid of ca. 100 kb was partially determined. pIP1206 carried the rmtB gene for a ribosome methyltransferase that was shown to modify the N7 position of nucleotide G1405, located in the A site of 16S rRNA. It also contained the qepA (quinolone efflux pump) gene that encodes a 14-transmembrane-segment putative efflux pump belonging to the major facilitator superfamily of proton-dependent transporters. Disruption of membrane proton potential by the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone in a transconjugant harboring the qepA gene resulted in elevation of norfloxacin accumulation. The transporter conferred resistance to the hydrophilic quinolones norfloxacin and ciprofloxacin.