Molecular Profiling of Shiga Toxin-Producing Escherichia coli and Enteropathogenic E. coli Strains Isolated from French Coastal Environments

Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains may be responsible for food-borne infections in humans. Twenty-eight STEC and 75 EPEC strains previously isolated from French shellfish-harvesting areas and their watersheds and belonging to 68 distinguishable serotypes were characterized in this study. High-throughput real-time PCR was used to search for the presence of 75 E. coli virulence-associated gene targets, and genes encoding Shiga toxin (stx) and intimin (eae) were subtyped using PCR tests and DNA sequencing, respectively. The results showed a high level of diversity between strains, with 17 unique virulence gene profiles for STEC and 56 for EPEC. Seven STEC and 15 EPEC strains were found to display a large number or a particular combination of genetic markers of virulence and the presence of stx and/or eae variants, suggesting their potential pathogenicity for humans. Among these, an O26:H11 stx1a eae-β1 strain was associated with a large number of virulence-associated genes (n = 47), including genes carried on the locus of enterocyte effacement (LEE) or other pathogenicity islands, such as OI-122, OI-71, OI-43/48, OI-50, OI-57, and the high-pathogenicity island (HPI). One O91:H21 STEC strain containing 4 stx variants (stx1a, stx2a, stx2c, and stx2d) was found to possess genes associated with pathogenicity islands OI-122, OI-43/48, and OI-15. Among EPEC strains harboring a large number of virulence genes (n, 34 to 50), eight belonged to serotype O26:H11, O103:H2, O103:H25, O145:H28, O157:H7, or O153:H2.

Staphylococcus aureus CC398: host adaptation and emergence of methicillin resistance in livestock

UNLABELLED Since its discovery in the early 2000s, methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) has become a rapidly emerging cause of human infections, most often associated with livestock exposure. We applied whole-genome sequence typing to characterize a diverse collection of CC398 isolates (n = 89), including MRSA and methicillin-susceptible S. aureus (MSSA) from animals and humans spanning 19 countries and four continents. We identified 4,238 single nucleotide polymorphisms (SNPs) among the 89 core genomes. Minimal homoplasy (consistency index = 0.9591) was detected among parsimony-informative SNPs, allowing for the generation of a highly accurate phylogenetic reconstruction of the CC398 clonal lineage. Phylogenetic analyses revealed that MSSA from humans formed the most ancestral clades. The most derived lineages were composed predominantly of livestock-associated MRSA possessing three different staphylococcal cassette chromosome mec element (SCCmec) types (IV, V, and VII-like) including nine subtypes. The human-associated isolates from the basal clades carried phages encoding human innate immune modulators that were largely missing among the livestock-associated isolates. Our results strongly suggest that livestock-associated MRSA CC398 originated in humans as MSSA. The lineage appears to have undergone a rapid radiation in conjunction with the jump from humans to livestock, where it subsequently acquired tetracycline and methicillin resistance. Further analyses are required to estimate the number of independent genetic events leading to the methicillin-resistant sublineages, but the diversity of SCCmec subtypes is suggestive of strong and diverse antimicrobial selection associated with food animal production. IMPORTANCE Modern food animal production is characterized by densely concentrated animals and routine antibiotic use, which may facilitate the emergence of novel antibiotic-resistant zoonotic pathogens. Our findings strongly support the idea that livestock-associated MRSA CC398 originated as MSSA in humans. The jump of CC398 from humans to livestock was accompanied by the loss of phage-carried human virulence genes, which likely attenuated its zoonotic potential, but it was also accompanied by the acquisition of tetracycline and methicillin resistance. Our findings exemplify a bidirectional zoonotic exchange and underscore the potential public health risks of widespread antibiotic use in food animal production.

Convergent evolution of PICIs-mediated phage interference

Staphylococcal pathogenicity islands (SaPIs), the prototype members of the family of phage inducible chromosomal islands (PICIs), are extremely mobile phage satellites, which are transferred between bacterial hosts after their induction by a helper phage. The intimate relationship between SaPIs and their helper phages is one of the most studied examples of virus satellite interactions in prokaryotic cells. SaPIs encode and disseminate virulence and fitness factors, representing a driving force for bacterial adaptation and pathogenesis. Many SaPIs encode a conserved morphogenetic operon, including a core set of genes whose function allows them to parasitize and exploit the phage life cycle. One of the central mechanisms of this molecular piracy is the specific packaging of the SaPI genomes into reduced sized capsid structures derived from phage proteins. Pac phages were classically thought to be the only phages involved in the mobilisation of phage-mediated virulence genes, including the transfer of SaPIs within related and non-related bacteria. This study presents the involvement of S. aureus cos phages in the intra- and intergeneric transfer of cos SaPIs for the first time. A novel example of molecular parasitism is shown, by which this newly characterised group of cos SaPIs uses two distinct and complementary mechanisms to take over the helper phage packaging machinery for their own reproduction. SaPIbov5, the prototype of the cos SaPIs, does not encode the characteristic morphogenetic operon found in pac SaPIs. However, cos SaPIs features both pac and cos phage cleavage sequences in their genome, ensuring SaPI packaging in small- and full-sized phage particles, depending on the helper phage. Moreover, cos-site packaging in S. aureus was shown to require the activity of a phage HNH nuclease. The HNH protein functions together with the large terminase subunit, triggering cleavage and melting of the cos-site sequence. In addition, a novel piracy strategy, severely interfering with the helper phage reproduction, was identified in cos SaPIs and characterised. This mechanism of piracy depends on the cos SaPI-encoded ccm gene, which encodes a capsid protein involved in the formation of small phage particles, modifying the assembling process via a scaffolding mechanism. This strategy resembles the ones described for pac SaPIs and represents a remarkable example of convergent evolution. A further convergent mechanism of capsid size-reduction was identified and characterised for the Enterococcus faecalis EfCIV583 pathogenicity island, another member of the PICI family. In this case, the self-encoded CpmE conducts this molecular piracy through a putative scaffolding function. Similar to cos SaPIs, EfCIV583 carries the helper phage cleavage sequence in its genome enabling its mobilisation by the phage terminase complex. The results presented in this thesis show how two examples of non-related members of the PICI family follow the same evolutionary convergent strategy to interfere with their helper phage. These findings could indicate that the described strategies might be widespread among PICIs and implicate a significant impact of PICIs mediated-virulence gene transfer in bacterial evolution and the emergence of pathogenic bacteria.

Genes that encodes NAGT, MIF1 and MIF2 are not virulence factors for kala-azar caused by Leishmania infantum

IntroductionKala-azar is a disease resulting from infection by Leishmania donovani and Leishmania infantum. Most patients with the disease exhibit prolonged fever, wasting, anemia and hepatosplenomegaly without complications. However, some patients develop severe disease with hemorrhagic manifestations, bacterial infections, jaundice, and edema dyspnea, among other symptoms, followed by death. Among the parasite molecules that might influence the disease severity are the macrophage migration inhibitory factor-like proteins (MIF1 and MIF2) and N-acetylglucosamine-1-phosphotransferase (NAGT), which act in the first step of protein N-glycosylation. This study aimed to determine whether MIF1, MIF2 and NAGT are virulence factors for severe kala-azar.MethodsTo determine the parasite genotype in kala-azar patients from Northeastern Brazil, we sequenced the NAGT genes of L. infantum from 68 patients as well as the MIF1 and MIF2 genes from 76 different subjects with diverse clinical manifestations. After polymerase chain reaction (PCR), the fragments were sequenced, followed by polymorphism identification.ResultsThe nucleotide sequencing of the 144 amplicons revealed the absence of genetic variability of the NAGT, MIF1 and MIF2 genes between the isolates. The conservation of these genes suggests that the clinical variability of kala-azar does not depend upon these genes. Additionally, this conservation suggests that these genes may be critical for parasite survival.ConclusionsNAGT, MIF1 and MIF2 do not alter the severity of kala-azar. NAGT, MIF1 and MIF2 are highly conserved among different isolates of identical species and exhibit potential for use in phylogenetic inferences or molecular diagnosis.

Influence of a subinhibitory concentration of vancomycin on the in vitro expression of virulence-related genes in the vancomycin-resistant Enterococcus faecalis

ABSTRACTINTRODUCTION:Exposure to subinhibitory concentrations (SICs) of antimicrobials may alter the bacterial transcriptome.METHODS: Here, we evaluated the expression of nine virulence-related genes in vancomycin-resistant enterococci (VRE) urinary tract infection isolates grown at SICs of vancomycin.RESULTS:A Subinhibitory concentrations of vancomycin interferes with gene modulation, but does not affect the phenotype of a VRE strain in vitro .CONCLUSIONS:Subinhibitory concentrations of vancomycin may regulate the expression of virulence factors in vivo or contribute to the selection of vancomycin-resistant strains.

Evaluation of adherence, hemagglutination, and presence of genes codifying for virulence factors of Acinetobacter baumannii causing urinary tract infection

Acinetobacter baumannii is a strictly aerobic bacterium which causes severe infections, however its pathogenic characteristics are not well defined. Thirteen A. baumannii strains isolated from urine of hospitalized and nonhospitalized patients with different ages were investigated for the presence of virulence factors. The isolates belonged to biotypes 2, 6, and 9 and were sensitive to imipenem. The majority of them showed resistance to amikacin, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, norfloxacin, and trimethoprim-sulfamethoxazole. None of A. baumannii strains presented genes codifying for 17 different virulence factors previously described in uropathogenic Escherichia coli, when tested by polymerase chain reaction (PCR). Nine isolates agglutinated human group AB erythrocytes, in presence of mannose, but none of them agglutinated group O erythrocytes. Adherence to polystyrene was observed in 7 isolates, and this result did not correlate with that obtained in hemagglutination assay. All the isolates were able to grow in iron-limiting conditions, showing that A. baumannii produces some type of siderophore. However, the genes iutA and fyuA, from iron uptake system of E. coli and Yersinia sp., respectively, were not present in the isolates, suggesting the presence of a different type of siderophore. The fimbriae of A. baumannii strains that mediates the adherence are possibly mannose-resistant, eventhough the mechanism of adherence to human epithelial cells still remains to be elucidated.

Contribution of CgPDR1-Regulated Genes in Enhanced Virulence of Azole-Resistant Candida glabrata.

In Candida glabrata, the transcription factor CgPdr1 is involved in resistance to azole antifungals via upregulation of ATP binding cassette (ABC)-transporter genes including at least CgCDR1, CgCDR2 and CgSNQ2. A high diversity of GOF (gain-of-function) mutations in CgPDR1 exists for the upregulation of ABC-transporters. These mutations enhance C. glabrata virulence in animal models, thus indicating that CgPDR1 might regulate the expression of yet unidentified virulence factors. We hypothesized that CgPdr1-dependent virulence factor(s) should be commonly regulated by all GOF mutations in CgPDR1. As deduced from transcript profiling with microarrays, a high number of genes (up to 385) were differentially regulated by a selected number (7) of GOF mutations expressed in the same genetic background. Surprisingly, the transcriptional profiles resulting from expression of GOF mutations showed minimal overlap in co-regulated genes. Only two genes, CgCDR1 and PUP1 (for PDR1upregulated and encoding a mitochondrial protein), were commonly upregulated by all tested GOFs. While both genes mediated azole resistance, although to different extents, their deletions in an azole-resistant isolate led to a reduction of virulence and decreased tissue burden as compared to clinical parents. As expected from their role in C. glabrata virulence, the two genes were expressed as well in vitro and in vivo. The individual overexpression of these two genes in a CgPDR1-independent manner could partially restore phenotypes obtained in clinical isolates. These data therefore demonstrate that at least these two CgPDR1-dependent and -upregulated genes contribute to the enhanced virulence of C. glabrata that acquired azole resistance.

Classification of antimicrobial resistance using artificial neural networks and the relationship of 38 genes associated with the virulence of Escherichia coli isolates from broilers

Avian pathogenic Escherichia coli (APEC) is responsible for various pathological processes in birds and is considered as one of the principal causes of morbidity and mortality, associated with economic losses to the poultry industry. The objective of this study was to demonstrate that it is possible to predict antimicrobial resistance of 256 samples (APEC) using 38 different genes responsible for virulence factors, through a computer program of artificial neural networks (ANNs). A second target was to find the relationship between (PI) pathogenicity index and resistance to 14 antibiotics by statistical analysis. The results showed that the RNAs were able to make the correct classification of the behavior of APEC samples with a range from 74.22 to 98.44%, and make it possible to predict antimicrobial resistance. The statistical analysis to assess the relationship between the pathogenic index (PI) and resistance against 14 antibiotics showed that these variables are independent, i.e. peaks in PI can happen without changing the antimicrobial resistance, or the opposite, changing the antimicrobial resistance without a change in PI.

Analysis of genotypic variation in genes associated with virulence in Aggregatibacter actinomycetemcomitans clinical isolates

Background and Objective: Although certain serotypes of Aggregatibacter actinomycetemcomitans are associated more with aggressive periodontitis than are other serotypes, the correlation between distinct lineages and virulence traits in this species is poorly understood. This study aimed to evaluate the polymorphism of genes encoding putative virulence factors of clinical isolates, and to correlate these findings with A. actinomycetemcomitans serotypes, genotypes and periodontal status of the hosts. Material and Methods: Twenty-six clinical isolates from diverse geographic populations with different periodontal conditions were evaluated. Genotyping was performed using pulse-field gel electrophoresis. Polymorphisms in the genes encoding leukotoxin, Aae, ApaH and determinants for serotype-specific O polysaccharide were investigated. Results: The isolates were classified into serotypes a-f, and exhibited three apaH genotypes, five aae alleles and 25 macrorestriction profiles. Two serotype b isolates (7.7%), obtained from Brazilian patients with aggressive periodontitis, were associated with the highly leukotoxic genotype; these isolates showed identical fingerprint patterns and aae and apaH genotypes. Serotype c, obtained from various periodontal conditions, was the most prevalent among Brazilian isolates, and isolates were distributed in two aae alleles, but formed a genetically distinct group based on apaH analysis. Cluster analysis showed a close relationship between fingerprinting genotypes and serotypes/apaH genotypes, but not with aae genotypes. Conclusion: Apart from the deletion in the ltx promoter region, no disease-associated markers were identified. Non-JP2-like strains recovered from individuals with periodontal disease exhibited considerable genetic variation regarding aae/apaH genotypes, serotypes and XhoI DNA fingerprints.

Expression of the sigma35 and cry2AB genes involved in Bacillus thuringiensis virulence

Muitos genes estão envolvidos nos mecanismos de esporulação da bactéria Bacillus thuringiensis. A regulação e expressão desses genes resultam em uma produção massiva da proteína Cry, responsável pela morte das larvas de muitos insetos. Neste trabalho monitorou-se a expressão de genes de Bacillus thuringiensis, ao longo de três fases de seu desenvolvimento. Foram construídos macroarrays de DNA dos genes selecionados, cujas seqüências estão disponibilizadas no GenBank. Estes genes foram hibridizados com cDNAs obtidos de B. thuringiensis kurstaki HD-1. As sondas de cDNA foram sintetizadas a partir da transcrição reversa do RNA da bactéria, extraído durante as fases de crescimento logarítmico, estacionária e esporulativa, marcadas com 33PadCTP. A expressão diferencial encontrada foi significativa para dois genes de B.thuringiensis, um relacionado aos fatores sigma (sigma35) e outro ao gene cry (cry2Ab). Detectaram-se diferenças entre as médias de expressão do fator sigma e do gene cry2Ab. Os valores máximos de expressão diferencial foram obtidos para o gene codificador do fator sigma35 na fase log e na fase esporulativa. Na análise de médias observou-se expressão do gene cry2Ab apenas na fase log; no entanto, de forma bem mais baixa quando comparado com a expressão de sigma35, nas três fases.

The contribution of genes required for anaerobic respiration to the virulence of Salmonella enterica serovar Gallinarum for chickens

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Salmonella enterica serovar typhimurium colonizing the lumen of the chicken intestine grows slowly and upregulates a unique set of virulence and metabolism genes

The pattern of global gene expression in Salmonella enterica serovar Typhimurium bacteria harvested from the chicken intestinal lumen (cecum) was compared with that of a late-log-phase LB broth culture using a whole-genome microarray. Levels of transcription, translation, and cell division in vivo were lower than those in vitro. S. Typhimurium appeared to be using carbon sources, such as propionate, 1,2-propanediol, and ethanolamine, in addition to melibiose and ascorbate, the latter possibly transformed to D-xylulose. Amino acid starvation appeared to be a factor during colonization. Bacteria in the lumen were non- or weakly motile and nonchemotactic but showed upregulation of a number of fimbrial and Salmonella pathogenicity island 3 (SPI-3) and 5 genes, suggesting a close physical association with the host during colonization. S. Typhimurium bacteria harvested from the cecal mucosa showed an expression profile similar to that of bacteria from the intestinal lumen, except that levels of transcription, translation, and cell division were higher and glucose may also have been used as a carbon source. © 2011, American Society for Microbiology.