The Brucella suis IbpA heat-shock chaperone is not required for virulence or for expression of the VirB type IV secretion system VirB8 protein.

UNLABELLED: Brucella suis, facultative intracellular bacterial pathogen of mammals, and Agrobacterium tumefaciens, a plant pathogen, both use a VirB type IV secretion system (T4SS) to translocate effector molecules into host cells. HspL, an α-crystalline-type small heat-shock protein, acts as a chaperone for the Agrobacterium VirB8 protein, an essential component of the VirB system. An Agrobacterium mutant lacking hspL is attenuated due to a misfunctional T4SS. We have investigated whether IbpA (BRA0051), the Brucella HspL homologue, plays a similar role. Unlike HspL, IbpA does not interact with VirB8, and an IbpA mutant shows full virulence and no defect in VirB expression. These data show that the Brucella α-crystalline-type small heat-shock protein IbpA is not required for Brucella virulence. SIGNIFICANCE AND IMPACT OF STUDY: Many bacteria use type IV secretion systems (T4SS), multi-protein machines, to translocate DNA and protein substrates across their envelope. Understanding how T4SS function is important as they play major roles in the spread of plasmids carrying antibiotic resistance and in pathogenicity. In the plant pathogen Agrobacterium tumefaciens, HspL, an α-crystalline-type small heat-shock protein, acts as a chaperone for the essential type IV secretion system component VirB8. Here, we show that this is not the case for all T4SS; in the zoonotic pathogen Brucella suis, IbpA, the protein most related to HspL, does not play this role.

Membrane permeability, a pivotal function involved in antibiotic resistance and virulence in Enterobacter aerogenes clinical isolates.

Imipenem-susceptible E. aerogenes isolates exhibiting extended spectrum ß-lactamases, target mutations and a basal efflux expression, were identified in five patients. After imipenem treatment, imipenem-intermediate susceptible (IMI-I) or resistant (IMI-R) isolates emerged in these patients. Alteration in porin synthesis and increase in efflux expression were observed in the IMI-I isolates whereas complete loss of the porins, LPS alteration and efflux overexpression were observed in the IMI-R isolates. Bacterial virulence of the strains was investigated by the Caenorhabditis elegans model. The IMI-R isolates were shown to be significantly less virulent than the IMI-susceptible or IMI-I isolates. The pleiotropic membrane alteration and its associated fitness burden exhibited by E. aerogenes isolates influence their antibiotic resistance and their virulence behaviour. These findings highlight the balance between the low permeability-related resistance and virulence and their relationships with the treatment of resistant pathogens.

[Extended-spectrum beta-lactamases-producing Stenotrophomonas maltophilia strains: CTX-M enzymes detection and virulence study]. / Etude de souches de Stenotrophomonas maltophilia sécrétrices de BLSE : détection de CTX-M et étude de la virulence.

OBJECTIVE: To study the beta-lactamases content of Stenotrophomonas maltophilia strains and to evaluate the virulence potential of these strains with the in vivo Caenorhabditis elegans model. METHODOLOGY: From 1st January 2006 to 31st December 2006, a monitoring programme to study multidrug resistant Gram-negative bacteria including extended-spectrum beta-lactamases (ESBL)-producing S. maltophilia was conducted at Nîmes University Hospital and Perpignan Hospital. The ESBL production was confirmed by the double-disk synergy test using ceftazidime, cefotaxime and cefepime disks associated with clavulanic acid disk. The strains were characterized phenotypically (beta-lactamase[s] identification) and genotypically (pulsed-field gel electrophoresis, plasmid analysis) and evaluated for their virulence with the in vivo nematode C. elegans model (establishment of survival curves [LT50]). RESULTS: Twelve ESBL-producing S. maltophilia strains were isolated in eight patients (median age: 65 years+/-19) mainly during skin infections (41.7%). The ESBL content revealed the presence of four CTX-M-15-producing strains at the same patient. The analysis by ECP confirmed that the four strains were identical. The plasmid analysis demonstrated that the plasmid carrying CTX-M-15 in the worldwide clonal Escherichia coli O25-ST131 strain and S. maltophilia were different. The C. elegans model confirmed that S. maltophilia strains presented a low virulence potential (LT50=4.5days+/-0.5 according to the strains and nematode death in 10days+/-1) whatever their resistance. CONCLUSION: For the first time in France, a CTX-M-15-producing S. maltophilia strain has been identified. The in vivo model confirmed that these bacteria have a low potential virulence. However, these strains were isolated from "immunocompromised" and multihospital patients demonstrating the necessary monitoring of these patients. The CTX-M after diffusing in hospitals and community in E. coli strains seem to spread in other Gram-negative bacteria.

In-vitro and in-vivo evidence of dose-dependent decrease of uropathogenic Escherichia coli virulence after consumption of commercial Vaccinium macrocarpon (cranberry) capsules.

This study evaluated the antibacterial efficacy of the consumption of cranberry capsules vs. placebo in the urine of healthy volunteers. A first double-blind, randomised, crossover trial involved eight volunteers who had followed three regimens, with or without cranberry, with a wash-out period of at least 6 days between each regimen. Twelve hours after consumption of cranberry or placebo hard capsules, the first urine of the morning was collected. Different Escherichia coli strains were cultured in the urine samples. Urinary antibacterial adhesion activity was measured in vitro using the human T24 epithelial cell-line, and in vivo using the Caenorhabditis elegans killing model. With the in-vitro model, 108 mg of cranberry induced a significant reduction in bacterial adherence to T24 cells as compared with placebo (p <0.001). A significant dose-dependent decrease in bacterial adherence in vitro was noted after the consumption of 108 and 36 mg of cranberry (p <0.001). The in-vivo model confirmed that E. coli strains had a reduced ability to kill C. elegans after growth in the urine of patients who consumed cranberry capsules. Overall, these in-vivo and in-vitro studies suggested that consumption of cranberry juice represents an interesting new strategy to prevent recurrent urinary tract infection.

[Cranberry (Vaccinium macrocarpon) and urinary tract infections: study model and review of literature]. / Cranberry (Vaccinium macrocarpon) et infections urinaires: étude et revue de la littérature.

Cranberries (Vaccinium macrocarpon) have long been the focus of interest for their beneficial effects in preventing urinary tract infections. Among cranberry compounds, a group of proanthocyanidins (PACs) with A-type linkages were isolated which exhibit bacterial anti-adhesion activity against uropathogenic Escherichia coli strains. These PAC inhibit P-fimbriae synthesis and induce a bacterial deformation. This activity was demonstrated on both antibiotic susceptible and resistant bacteria. This review focused on the last discoveries in the knowledge of cranberry effects.

Virulence genotype and nematode-killing properties of extra-intestinal Escherichia coli producing CTX-M beta-lactamases.

This study evaluated the virulence potential of Escherichia coli isolates producing CTX-M beta-lactamases. During a 24-month period, 33 extended-spectrum beta-lactamase (ESBL)-producing E. coli, including 14 CTX-M-producers, were isolated from urinary tract infections at Nîmes University Hospital, France. The prevalence of 14 major virulence factors (VFs) was investigated by PCR and compared with the prevalence in a group of 99 susceptible E. coli isolates. Ten VFs were less prevalent (p <0.05) in the ESBL isolates than the susceptible E. coli, while iutA and traT were more prevalent in ESBL isolates (p <0.05). Moreover, the CTX-M-producing isolates had significantly fewer VFs than TEM-producing isolates. A novel infection model using the nematode Caenorhabditis elegans was developed to assess the virulence properties of extra-intestinal pathogenic E. coli (ExPEC) strains in vivo. C. elegans infection assays, using 14 ESBL-producing E. coli and ten susceptible E. coli isolates, indicated that the ability to kill nematodes correlated with the presence of VFs, and that CTX-M-producing isolates had relatively low virulence in vivo. Overall, the results suggested that hospital-acquired CTX-M-producing E. coli, although adapted for survival in an antibiotic-rich environment such as the hospital milieu, have a relatively low intrinsic virulence potential.

[Caenorhabditis elegans: in vivo study model of bacterial virulence]. / Caenorhabditis elegans: modèle d'étude in vivo de la virulence bactérienne.

The nematode Caenorhabditis elegans presents many advantages as a model system. The worm has recently emerged as a potentially useful tool for the study of host-pathogen interactions. This paper presents advantages and inconveniences of this model, the variety of bacterial pathogens studied, and its use to monitor virulence of Extraintestinal Escherichia coli strains.

Aromatic compound-dependent Brucella suis is attenuated in both cultured cells and mouse models.

The aroC gene of the facultative intracellular pathogen Brucella suis was cloned and sequenced. The cloned aroC gene complements Escherichia coli and Salmonella enterica serovar Typhimurium aroC mutants. A B. suis aroC mutant was found to be unable to grow in a defined medium without aromatic compounds. The mutant was highly attenuated in tissue culture (THP1 macrophages and HeLa cells) and murine virulence models.

Identification of Brucella suis genes affecting intracellular survival in an in vitro human macrophage infection model by signature-tagged transposon mutagenesis.

Bacteria of the genus Brucella are facultative intracellular pathogens which have developed the capacity to survive and multiply in professional and nonprofessional phagocytes. The genetic basis of this aspect of Brucella virulence is still poorly understood. To identify new virulence factors, we have adapted signature-tagged transposon mutagenesis, which has been used essentially in animal models, to an in vitro human macrophage infection model. A library of 1,152 Brucella suis 1330 tagged mini-Tn5 Km2 mutants, in 12 pools, was screened for intracellular survival and multiplication in vitamin D(3)-differentiated THP1 cells. Eighteen mutants were identified, and their attenuation was confirmed in THP1 macrophages and HeLa cells. For each avirulent mutant, a genomic fragment containing the transposon was cloned. The genomic DNA sequence flanking the transposon allowed us to assign functions to all of the inactivated genes. Transposon integration had occurred in 14 different genes, some of which were known virulence genes involved in intracellular survival or biosynthesis of smooth lipopolysaccharide (the virB operon and manB), thus validating the model. Other genes identified encoded factors involved in the regulation of gene expression and enzymes involved in biosynthetic or metabolic pathways. Possible roles in the virulence of Brucella for the different factors identified are discussed.

A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis.

Analysis of a TnblaM mutant of Brucella suis 1330, identified as being unable to multiply in Hela cells, allowed us to identify a 11 860 bp region of the B. suis genome encoding a type IV secretion system, homologous to the VirB system of Agrobacterium tumefaciens and the Ptl system of Bordetella pertussis. DNA sequence revealed 12 open reading frames (ORFs) encoding homologues of the 11 VirB proteins present in the pTi plasmid of Agrobacterium with a similar genetic organization, and a twelfth ORF encoding a putative lipoprotein, homologous to a protein involved in mating pair formation during bacterial conjugation and to adhesins used by Pseudomonas species to bind to plant roots. Phylogenetic trees based on the sequences of VirB4 and VirB9 protein homologues suggest that evolution of the systems from DNA transfer towards protein secretion did not stem from a single event but that the protein secretion systems have evolved independently. Four independent mutants in virB5, virB9 or virB10 were highly attenuated in an in vitro infection model with human macrophages. The virulence was restored by complementation with a plasmid containing the full virB region. The virB region appears to be essential for the intracellular survival and multiplication of B. suis.

Unconventional genomic organization in the alpha subgroup of the Proteobacteria.

Pulsed-field gel electrophoresis was used to analyze the genomic organization of 16 bacteria belonging or related to the family Rhizobiaceae of the alpha subgroup of the class Proteobacteria. The number and sizes of replicons were determined by separating nondigested DNA. Hybridization of an rrn gene probe was used to distinguish between chromosomes and plasmids. Members of the genus Agrobacterium all possess two chromosomes, and each biovar has a specific genome size. As previously demonstrated for Agrobacterium tumefaciens C58, the smaller chromosomes of Agrobacterium biovar 1 and Agrobacterium rubi strains appear to be linear. The genomes of Rhizobium strains were all of similar sizes but were seen to contain either one, two, or three megareplicons. Only one chromosome was present in the member of the related genus Phyllobacterium. We found one or two chromosomes in Rhodobacter and Brucella species, two chromosomes in Ochrobactrum anthropi, and one chromosome in Mycoplana dimorpha and Bartonella quintana; all of these genera are related to the Rhizobiaceae. The presence of multiple chromosomes is discussed from a phylogenetic and taxonomic point of view.

Differences in chromosome number and genome rearrangements in the genus Brucella.

We have studied the genomic structure and constructed the SpeI, PacI and I-CeuI restriction maps of the four biovars of the pathogenic bacterium Brucella suis. B. suis biovar 1 has two chromosomes of 2.1 Mb and 1.15 Mb, similar to those of the other Brucella species: B. melitensis, B. abortus, B. ovis and B. neotomae. Two chromosomes were also observed in the genome of B. suis biovars 2 and 4, but with sizes of 1.85 Mb and 1.35 Mb, whereas only one chromosome with a size of 3.1 Mb was found in B. suis biovar 3. We show that the differences in chromosome size and number can be explained by rearrangements at chromosomal regions containing the three rrn genes. The location and orientation of these genes confirmed that these rearrangements are due to homologous recombination at the rrn loci. This observation allows us to propose a scheme for the evolution of the genus Brucella in which the two chromosome-containing strains can emerge from an hypothetical ancestor with a single chromosome, which is probably similar to that of B. suis biovar 3. As the genus Brucella is certainly monospecific, this is the first time that differences in chromosome number have been observed in strains of the same bacterial species.

Genome structure and phylogeny in the genus Brucella.

PacI and SpeI restriction maps were obtained for the two chromosomes of each of the six species of the genus Brucella: B. melitensis, B. abortus, B. suis, B. canis, B. ovis, and B. neotomae. Three complementary techniques were used: hybridization with the two replicons as probes, cross-hybridization of restriction fragments, and a new mapping method. For each type strain, a unique I-SceI site was introduced in each of the two replicons, and the location of SpeI sites was determined by linearization at the unique site, partial digestion, and end labeling of the fragments. The restriction and genetic maps of the six species were highly conserved. However, numerous small insertions or deletions, ranging from 1 to 34 kb, were observed by comparison with the map of the reference strain of the genus, B. melitensis 16M. A 21-kb Spel fragment specific to B. ovis was found in the small chromosome of this species. A 640-kb inversion was demonstrated in the B. abortus small chromosome. All of these data allowed the construction of a phylogenetic tree, which reflects the traditional phenetic classification of the genus.

Investigation of the Actinobacillus actinomycetemcomitans genome by pulsed field gel electrophoresis.

Pulsed field gel electrophoresis was used to investigate nineteen strains of Actinobacillus actinomycetemcomitans. The genome was found to contain a single chromosome whose size we estimate to be 2300 kb from the sum of restriction fragments generated with rare cutting endonucleases. We detected the presence of large plasmids with sizes ranging from 35 to 300 kb. In some strains, extrachromosomal elements constitute over 20% of the total genome. Comparison of the profiles of ApaI digests of the 19 strains showed a high degree of polymorphism with 13 different profiles, providing a new tool for epidemiological studies.

Epidemiological study by pulsed-field gel electrophoresis of an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a geriatric hospital.

Twelve cases of infections caused by extended-spectrum beta-lactamase (ESBla)-producing Klebsiella pneumoniae were reported between August 1991 and March 1993 in the Geriatric Department of the Nimes University Hospital, where these bacterial had not been previously isolated. Restriction profiles of total genomic DNAs cleaved by XbaI and SpeI were compared by pulsed-field gel electrophoresis. The strains that were tested included the 12 isolates from K. pneumoniae-infected patients, strains recovered from rectal swabs of asymptomatic patients in the same ward, and strains isolated in other hospitals in Nîmes at the same time. The restriction profiles of the 12 isolates and those recovered from asymptomatic patients in the same ward were very similar. Over a period of more than 1 year, extended-spectrum beta-lactamases were not detected in K. pneumoniae isolates with restriction patterns different from that of the epidemic strain. It seems, therefore, that there was no transfer of a plasmid or a gene coding for ESBla to strains of K. pneumoniae that were different from the epidemic strain. At the same time, ESBla-producing K. pneumoniae isolates exhibiting restriction endonuclease profiles very different from that of the epidemic strain were isolated from other hospitals in Nîmes. None of these strains caused an outbreak. Pulsed-field gel electrophoresis, which allows precise characterization of strains beyond the species level, is a useful tool for studying the ESBla-producing K. pneumoniae strains involved in nosocomial outbreaks.

Identification and sequence analysis of IS6501, an insertion sequence in Brucella spp.: relationship between genomic structure and the number of IS6501 copies.

An insertion sequence (IS) element of Brucella ovis, named IS6501, was isolated and its complete nucleotide sequence determined. IS6501 is 836 bp in length and occurs 20-35 times in the B. ovis genome and 5-15 times in other Brucella species. Analysis of the junctions at the sites of insertion revealed a small target site duplication of four bases and inverted repeats of 17 bp with one mismatch. IS6501 presents significant similarity (53.4%) with IS427 identified in Agrobacterium tumefaciens, suggesting a common ancestral sequence. A long ORF of 708 bp was identified encoding a protein with a predicted molecular mass of 26 kDa and sharing sequence identity with the hypothetical protein 1 of A. tumefaciens and with the transposase of Mycobacterium tuberculosis. IS6501 is present in all Brucella strains we have tested. Restriction fragment length polymorphism of reference and field strains of two species (B. melitensis and B. ovis) was studied using either pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA or hybridization of EcoRI-digested DNA using IS6501 as a probe. The genome of B. melitensis biovar 3 contains about 10 IS copies per genome and field strains of the same species could not be distinguished either by IS hybridization or by XbaI (PFGE) restriction patterns. In contrast, the number of IS copies in the B. ovis genome is around 30 and the different field strains can be differentiated by both methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of two independent chromosomes in the Brucella melitensis 16M genome.

Mapping the restriction fragments of the Brucella melitensis 16M genome with a new restriction endonuclease, PacI, which cut the DNA into only eight fragments, indicated that this species contains two unique and independent replicons of about 2,100 and 1,150 kb. Pulsed-field gel electrophoresis of intact DNA revealed two bands migrating the expected distances. These replicons were identified as two unique and independent chromosomes by the presence of rRNA operons and genes for heat shock proteins mapping to separate replicons.

Use of pulsed-field gel electrophoresis for investigation of hospital outbreaks of Acinetobacter baumannii.

Genomic DNAs from taxonomically and epidemiologically well-defined strains of Acinetobacter baumannii were digested with restriction endonucleases that cleave with low frequency, and the fragments were separated by pulse-field gel electrophoresis. Restriction fragment length polymorphisms were observed. Restriction fragment length polymorphism analysis can be used as an epidemiological tool to delineate outbreaks of nosocomial infections caused by A. baumannii.

DNA polymorphism in strains of Listeria monocytogenes.

DNA polymorphism in 35 Listeria monocytogenes strains belonging to serovars 1/2a, 1/2b, 1/2c, and 4b was studied by genomic DNA digestion. The restriction endonucleases ApaI and NotI, which cleave DNA at rare sequences, were used, and DNA fragments were analyzed by pulsed-field gel electrophoresis. Restriction fragment length polymorphism varied among different serovars and was used for epidemiological studies, but serovar 1/2c isolates could not be analyzed because their restriction patterns were indistinguishable. The genome sizes were calculated by addition of the sizes of the ApaI fragments and were found to be about 2,660 kb for serovar 1/2a strains, 2,640 kb for serovar 1/2b strains, and 2,710 kb for serovar 4b strains but only 2,340 kb for serovar 1/2c strains. This last group therefore appears to differ from the other serovar strains by the absence of restriction fragment length polymorphism and a chromosome that is 15% shorter, suggesting that strains of serovar 1/2c have quite recently emerged.

Physical map of the Brucella melitensis 16 M chromosome.

We present the first restriction map of the Brucella melitensis 16 M chromosome obtained by Southern blot hybridization of SpeI, XhoI, and XbaI fragments separated by pulsed-field gel electrophoresis. All restriction fragments (a total of 113) were mapped into an open circle. The main difficulty in mapping involved the exceedingly high number of restriction fragments, as was expected considering the 59% G + C content of the Brucella genome. Several cloned genes were placed on this map, especially rRNA operons which are repeated three times. The size of the B. melitensis chromosome, estimated as 2,600 kb long in a previous study, appeared longer (3,130 kb) by restriction mapping. This restriction map is an initial approach to achieve a genetic map of the Brucella chromosome.