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A standardised restriction fragment length polymorphism (RFLP) method for typing Mycobacterium avium isolates links IS901 with virulence for birds.

A standardised method for PvuII-PstI-IS901 restriction fragment length polymorphism (RFLP) typing was developed and evaluated against 173 isolates of Mycobacterium avium subsp. avium and M. avium subsp. silvaticum originating from birds (N=46) and their aviaries (N=5), pigs (N=85), cattle (N=18), reference serotype strains (N=9), humans (N=7), a horse (N=1), a nutria (N=1), and strain M. avium subsp. avium ST 18 (formerly M. avium subsp. paratuberculosis ST 18). PvuII-IS1245 RFLP typing was also performed on all isolates. DNA was digested in parallel by restriction endonucleases PvuII or PstI and hybridised to standard probes prepared by PCR. DNA fingerprints were scanned by CCD camera and analysed by the Gel Compar (Applied Maths, Version 4.1, Kortrijk, Belgium) software using a standard isolate control profile. A total of 52 PvuII-PstI RFLP profiles was described including 25 PvuII RFLP profiles designated A to Y and 25 PstI RFLP profiles designated A1-L3. Profiles were found to be stable in vivo and in vitro after multiple subcultures. High IS901 copy number was associated with a "bird" PvuII-IS1245 RFLP profile and low IS901 copy number with M. avium subsp. avium isolates from humans and the nutria. A virulence assay of 100 IS901-positive isolates using intramuscular infections of pullets showed 83 isolates differentiated into 32 RFLP types to be virulent and 17 isolates differentiated into 12 RFLP types as nonvirulent. Attenuation of virulence for pullets could be attributed to either multiple in vitro subculture, polyclonal infection or human passage and was not related to IS901 or IS1245 profiles.

Animals↗

Gene expression profile in Neisseria meningitidis and Neisseria lactamica upon host-cell contact: from basic research to vaccine development.

Differential gene regulation in the human pathogen Neisseria meningitidis group B (MenB) and in Neisseria lactamica, a human commensal species, was studied by whole genome microarray after bacterial interaction with epithelial cells. Host-cell contact induced changes in the expression of 347 and 285 genes in MenB and N. lactamica, respectively. Of these, only 167 were common to MenB and N. lactamica, suggesting that a different subset of genes is activated by pathogens and commensals. Change in gene expression was stable over time in N. lactamica, but short-lived in MenB. A large part (greater than 30%) of the regulated genes encoded proteins with unknown function. Among the known genes, those coding for pili, capsule, protein synthesis, nucleotide synthesis, cell wall metabolism, ATP synthesis, and protein folding were down-regulated in MenB. Transporters for iron, chloride and sulfate, some known virulence factors, GAPDH and the entire pathway of selenocysteine biosynthesis were upregulated. Gene expression profiling indicates that approximately 40% of the regulated genes encode putative surface-associated proteins, suggesting that upon cell contact Neisseria undergoes substantial surface remodeling. This was confirmed by FACS analysis of adhering bacteria using mouse sera against a subset of recombinant proteins. Finally, a few surface-located, adhesion-activated antigens were capable of inducing bactericidal antibodies, indicating that microarray technology can be exploited for the identification of new vaccine candidates.

Bacterial Adhesion↗

Phenotypic and genotypic characterization of virulence factors of Escherichia coli isolated from broiler chickens with simultaneous occurrence of cellulitis and other colibacillosis lesions.

The objective of this study was to characterize virulence factors of Escherichia coli isolates from broilers with simultaneous occurrence of cellulitis and other colibacillosis lesions. Thirty flocks were sampled and 237 birds with cellulitis were examined. Eighty-two (34.6%) of 237 birds condemned for cellulitis had gross lesions in the heart, air sacs, joints, or liver. In 58 chickens, E. coli was isolated from both the cellulitis and other lesions of colibacillosis, and 18.9% of the E. coli isolates from the 2 types of lesions belonged to the same O group. Escherichia coli of serogroups O78, O1, and O2 predominated. Isolates of the same serogroup that were derived from different lesions in the same birds had similar patterns of biotype, aerobactin production, serum sensitivity profile, antibiotic sensitivity, and K1 capsule production. Escherichia coli derived from cellulitis lesions produced virulence factors similar to those found in E. coli isolated from other colibacillosis lesions in poultry.

Animals↗

Use of plasmid profiles in epidemiologic surveillance of disease outbreaks and in tracing the transmission of antibiotic resistance.

Plasmids are circular deoxyribonucleic acid molecules that exist in bacteria, usually independent of the chromosome. The study of plasmids is important to medical microbiology because plasmids can encode genes for antibiotic resistance or virulence factors. Plasmids can also serve as markers of various bacterial strains when a typing system referred to as plasmid profiling, or plasmid fingerprinting is used. In these methods partially purified plasma deoxyribonucleic acid species are separated according to molecular size by agarose gel electrophoresis. In a second procedure, plasmid deoxyribonucleic acid which has been cleaved by restriction endonucleases can be separated by agarose gel electrophoresis and the resulting pattern of fragments can be used to verify the identity of bacterial isolates. Because many species of bacteria contain plasmids, plasmid profile typing has been used to investigate outbreaks of many bacterial diseases and to trace inter- and intra-species spread of antibiotic resistance.

Animals↗

Clonal relatedness of enterotoxigenic Escherichia coli strains isolated from a cohort of young children in Guinea-Bissau.

In a cohort study of 200 young children in Guinea-Bissau, it was previously found that some enterotoxigenic Escherichia coli (ETEC) strains were more pathogenic than others, depending on the type of toxin that they could produce, and that natural ETEC infections induced substantial protection against new infections with ETEC strains that had the same combination of toxins and colonization factors (CFs). We wanted to describe the clonal relatedness of the ETEC strains isolated during this study and to investigate whether the protective antigens and the virulence factors that were responsible for the pathogenic traits were common to strains that were clonally closely related or whether they were more likely to be encoded by the ETEC virulence plasmids that normally encode the toxins and the CFs. By performing repetitive sequence-based PCR analysis of strains representing 452 infections, we found that strains that had the same toxin-CF profile were usually closely related, although a few were unrelated. Strains that did not have the same toxin-CF profiles but that were positive for a given toxin or for a given CF were not consistently more closely related to each other than to strains that were negative for the same toxin or CF. Our results indicate that the pathogenic traits of ETEC were mainly attributed to genes carried on the ETEC virulence plasmids. Because most strains that had the same toxin-CF profile were closely related, it seems likely that the toxin-CF-specific protection was clonal and was not targeting antigens encoded by the virulence plasmids. These results are of relevance to the ETEC vaccine development effort.

Child↗

Virulence genes of the phytopathogen Rhodococcus fascians show specific spatial and temporal expression patterns during plant infection.

The phytopathogenic bacterium Rhodococcus fascians provokes shoot meristem formation and malformations on aerial plant parts, mainly at the axils. The interaction is accompanied by bacterial colonization of the plant surface and tissues. Upon infection, the two bacterial loci required for full virulence, fas and att, were expressed only at the sites of symptom development, although their expression profiles differed both spatially and temporally. The att locus was expressed principally in bacteria located on the plant surface at early stages of infection. Expression of the fas locus occurred throughout infection, mainly in bacteria that were penetrating, or had penetrated, the plant tissues and coincided with sites of meristem initiation and proliferation. The implications for the regulation of virulence genes of R. fascians during plant infection are discussed.

Bacterial Proteins↗

Identification of Mycoplasma pneumoniae proteins associated with hemadsorption and virulence.

Twenty-two mutants of Mycoplasma pneumoniae spontaneously deficient in hemadsorption were isolated. Examination of mutant protein profiles by one- and two-dimensional polyacrylamide gel electrophoresis permitted the grouping of these mutants into four classes. The largest class of mutants was deficient in four high-molecular-weight proteins (215,000, 210,000, 190,000, and 140,000). A second class of mutants lacked three proteins previously designated A, B, and C (72,000, 85,000, and 37,000, respectively). A single mutant, in addition to lacking proteins A, B, and C, was missing a fourth protein of 165,000 molecular weight. The remaining mutants exhibited protein profiles apparently identical to that of the wild-type strain. All mutant strains attached to the respiratory epithelium of hamster tracheal rings in vitro at reduced levels; however, mutants lacking proteins A, B, and C recognized only neuraminidase-insensitive receptors. None of the mutants tested produced detectable pneumonia in intranasally inoculated hamsters, although one mutant class demonstrated low-level survival in vivo.

Adhesiveness↗

Conservation of pathogenic mycoplasma antigens.

The protein profiles of different mycoplasmas are generally species-specific; however, several pathogenic species share certain biologic properties and/or virulence factors. We compared species of human, avian and animal origin to determine if there was evidence for conservation of key antigens. Log-phase cultures of Mycoplasma pneumoniae, M. genitalium, M. gallisepticum and M. pulmonis were subjected to SDS-gel electrophoretic analysis. Protein profiles were compared with silver-stained gels. To examine antigenic cross-reactivity, Western blots were prepared by transferring separated proteins to nitrocellulose filters and incubating these with homotypic and heterotypic antisera. Localization of cross-reacting antigens was done by immunoradioautography using 125I-labeled antiglobulins. Several two-way cross-reactions were observed among M. pneumoniae, M. genitalium, and M. gallisepticum; M. pulmonis shared no significant cross-reactions with the other three species. Preliminary evidence suggests that the cross-reactivity was due to conservation of certain antigenic determinants, although the overall protein composition of the four species was different.

Antigens, Bacterial↗

Stability of plasmids in five strains of Salmonella maintained in stab culture at different temperatures.

Four strains of Salmonella berta and one of Salm. enteritidis were stored as stab cultures in sugar-free agar at 5 degrees, 22 degrees and 30 degrees C and in 15% glycerol at -80 degrees C. The stability of the plasmid profiles in each of the strains was monitored over a period of 2.5 years. Plasmid profiles were stable in all strains stored at -80 degrees C, and only six of 450 colonies examined from strains kept in sugar-free agar at 5 degrees C had lost plasmid molecules. Seventy of 440 colonies from stab cultures that were kept at 22 degrees C, and 71 of 440 colonies at 30 degrees C showed changed plasmid profiles. The total number of plasmids lost increased with time, and occasionally, more than one plasmid molecule was lost in the same strain. The virulence associated plasmid of Salm. enteritidis was remarkably stable as it was maintained in all colonies examined at all temperatures investigated. Likewise, no change in SmaI restriction profile was observed in this plasmid molecule at any temperature.

Plasmids↗

Pleiotropic role of quorum-sensing autoinducer 2 in Photorhabdus luminescens.

Bacterial virulence is an integrative process that may involve quorum sensing. In this work, we compared by global expression profiling the wild-type entomopathogenic Photorhabdus luminescens subsp. laumondii TT01 to a luxS-deficient mutant unable to synthesize the type 2 quorum-sensing inducer AI-2. AI-2 was shown to regulate more than 300 targets involved in most compartments and metabolic pathways of the cell. AI-2 is located high in the hierarchy, as it controls the expression of several transcriptional regulators. The regulatory effect of AI-2 appeared to be dose dependent. The luxS-deficient strain exhibited decreased biofilm formation and increased type IV/V pilus-dependent twitching motility. AI-2 activated its own synthesis and transport. It also modulated bioluminescence by regulating the synthesis of spermidine. AI-2 was further shown to increase oxidative stress resistance, which is necessary to overcome part of the innate immune response of the host insect involving reactive oxygen species. Finally, we showed that the luxS-deficient strain had attenuated virulence against the lepidopteran Spodoptera littoralis. We concluded that AI-2 is involved mainly in early steps of insect invasion in P. luminescens.

Bacterial Proteins↗

Detection of additional restriction fragment length polymorphisms among the weakly virulent (nonaggressive) and highly virulent (aggressive) isolates of Leptosphaeria maculans.

Isolates of Leptosphaeria maculans were analyzed for their genetic relatedness based on DNA restriction fragment length polymorphisms (RFLPs), employing as Southern hybridization probes a combination of heat shock responsive genes (hsp70 and hsp80 from Neurospora crassa), the cutinase gene of Magnaporthe grisea, and cloned genomic DNA sequences from a virulent strain. Southern hybridization analysis revealed a high frequency of DNA polymorphism. Restriction fragments generated by each enzyme-probe combination resulted in distinct banding patterns, clearly separating the isolates into two groups. The cutinase gene probe did not reveal any polymorphisms. Although the majority of the probes used displayed RFLP profiles unique to each group, a nonaggressive isolate, LmA, showed additional genetic characteristics in common with the virulent pathotype.

Ascomycota↗

Elevated levels of Legionella pneumophila stress protein Hsp60 early in infection of human monocytes and L929 cells correlate with virulence.

Legionella pneumophila 2064 was selectively radiolabelled in mouse L929 cells and human monocytes to identify proteins expressed early in the course of infection. Polypeptide profiles (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography) of adherent or intracellular bacteria indicated that a 60-kDa stress protein (Hsp60) was preferentially synthesized. Hsp60 synthesis was not induced by medium alone. The synthesis of many polypeptides, including OmpS (major outer membrane protein), diminished over the 1-h period postinfection. However, by 17 h postinfection OmpS and Hsp60 were the dominant proteins synthesized by 2064. To establish whether induction of Hsp60 was a correlate of virulence, an isogenic avirulent strain (2064M) of 2064 was isolated following selection on a nonpermissive medium. 2064M did not exhibit a stress response when adherent or intracellular in L929 cells or in human monocytes and failed to abrogate phagosome-lysosome fusion. When grown in vitro, 2064M exhibited no deficiencies in the heat shock response and its polypeptide profile resembled that of 2064. Immunogold electron microscopy was used to localize Hsp60 in L. pneumophila-infected L929 cells. There was an increase in the number of gold particles associated with phagosomes for phagosomes harboring single 2064 bacteria compared with those harboring 2064M. Moreover, by 1 h postinfection, a sixfold increase in the number of gold spheres associated with the membranes of phagosomes was observed for phagosomes harboring 2064 compared with those harboring 2064M. These studies indicate that virulent, but not NaCl-tolerant avirulent, strains of L. pneumophila respond to host-cell-associated environmental signals early in the course of infection. This response includes increased synthesis and possibly extracellular secretion of Hsp60 concomitant with repression of the expression of other genes, including ompS.

Animals↗

Characterization of verocytotoxin-producing Escherichia coli O157 isolates from patients with haemolytic uraemic syndrome in Western Europe.

Fifty verocytotoxin (VT)-producing Escherichia coli (VTEC) strains of serogroup O157 were characterized by phage typing, polymerase chain reaction (PCR) for VT genes and the E. coli attaching and effacing (eae) gene, and random amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting. The collection represented isolates obtained from patients with diarrhoea-associated haemolytic-uraemic syndrome (D+ HUS) and their family contacts, isolated in the Netherlands, Belgium and Germany between 1989 and 1993. Based on isolates from separate families (n = 27) seven different phage types were identified, types 2 (44%) and 4 (33%) were predominant. Eighty-five percent of the strains contained only VT2 gene sequences and 15% both VT1 and VT2. All strains of the dominant phage types 2 and 4 carried the VT2 gene. Strains that belonged to the minor phage types 8, 14, 32 carried both VT1 and VT2 genes, with the exception of two isolates identified as phage types 49 and 54 which contained only VT2 genes. All O157 VTEC strains possessed the chromosomally-located eae gene, which indicates its usefulness as virulence marker. RAPD-PCR fingerprinting identified four distinct banding patterns, with one profile found among 79% of the strains. Based on the combined results of all typing methods used in this study, the collection of 50 O157 VTEC strains could be divided into nine distinct groups. Strains isolated from different persons within one family could not be distinguished by any of these methods. The data suggest that O157 VTEC strains are members of one clone that has become widely distributed.

Adult↗

The role of Helicobacter pylori in paediatrics.

Helicobacter pylori is a bacterium that colonizes the human stomach, especially during childhood. Although it has been studied intensively during the past decade, many controversies still exist on many important issues, including the clinical relevance of virulence factors, indications for treatment, recommended procedures for diagnosis and the typical symptom profile, among others. The lack of double-blind, placebo-controlled studies in children illustrates the paucity of hard scientific data, and is the major reason for differences in opinion. Therefore, the present review of literature published during the past 2 years raises many questions.

Adolescent↗

Live attenuated AIDS viruses as vaccines: promise or peril?

Live attenuated viruses can provide vaccine protection against various viral illnesses. A number of live attenuated strains of the simian immunodeficiency virus (SIV) or related lentiviruses have been evaluated in primate models as vaccine candidates against AIDS. Impressive efficacy was observed for some viruses, most notably SIV strains with deletions in the nef-gene. Sterilizing immunity was seen against homologous and heterologous virus challenge, against cell-free and cell-associated challenge, against intravenous and mucosal challenge, and against challenge as early as 3 weeks and as late as 2.25 years after just one immunization. However, these promising efficacy results are overshadowed by safety problems, such as reversion of the vaccine strain to a pathogenic virus encoding full-length nef or residual virulence of multiply deleted vaccine strains. Strategies aimed at decreasing the replicative capacity of nef-deleted vaccine strains to increase the safety profile have significantly curtailed vaccine efficacy. Nevertheless, studies of live attenuated vaccine strains should proceed and should focus on determining the correlates of vaccine protection and the molecular determinants for virulence and attenuation.

AIDS Vaccines↗

Population structure and antibiotic resistance of Salmonella isolates from diseased poultry in Jiangxi Province, China.

Salmonella poses a significant threat to human and animal health. However, the relationship among population diversity, antibiotic resistance, and infection risk remains largely unexplored. In this study, 69 Salmonella strains were isolated from diseased poultry in Jiangxi Province from 2021 to 2024. Using whole-genome sequencing, serotype prediction, MLST, virulence and resistance gene analysis, antibiotic susceptibility testing, and mobile genetic element annotation, we characterized the diversity, resistance profiles, and transmission mechanisms of these strains. The results showed high diversity, with Salmonella enterica subsp. enterica serovar Typhimurium (>60%) and ST19 (62.31%) as the dominant serovar and sequence type, respectively. Several avian isolates were genomically similar to human isolates, indicating potential zoonotic risk. All strains harbored conserved core virulence modules, whereas accessory modules (e.g., cdtB, astA, pefA) varied and may affect pathogenicity. The multidrug resistance rate was 97.1%, with 100% resistance to erythromycin, tilmicosin and tiamulin, and resistance rates of 91.3%, 84.1%, and 71.0% to sulfonamides, enrofloxacin, and ceftiofur, respectively. Sixty-eight resistance genes were identified. Highly conserved antimicrobial resistance gene (ARG) modules (e.g., sul2-aph(3″)-Ib-aph(6')-Id-tet(A)) were shared between chromosomes and plasmids and were flanked by mobile elements such as Tn3 and IS3. Genomic islands (GIs) and plasmids in some strains carried resistance gene clusters highly homologous to those in pathogens from humans, pigs, and chickens, suggesting active horizontal transfer of resistance genes across hosts. This study revealed high diversity, prevalent multidrug resistance, and active horizontal transfer of resistance genes in avian-derived Salmonella from Jiangxi Province, emphasizing the need for cross-host resistance monitoring and antibiotic management within the 'One Health' framework.

Horizontal gene transfer↗

Nucleoid remodeling by an altered HU protein: reorganization of the transcription program.

Bacterial nucleoid organization is believed to have minimal influence on the global transcription program. Using an altered bacterial histone-like protein, HUalpha, we show that reorganization of the nucleoid configuration can dynamically modulate the cellular transcription pattern. The mutant protein transformed the loosely packed nucleoid into a densely condensed structure. The nucleoid compaction, coupled with increased global DNA supercoiling, generated radical changes in the morphology, physiology, and metabolism of wild-type K-12 Escherichia coli. Many constitutive housekeeping genes involved in nutrient utilization were repressed, whereas many quiescent genes associated with virulence were activated in the mutant. We propose that, as in eukaryotes, the nucleoid architecture dictates the global transcription profile and, consequently, the behavior pattern in bacteria.

Bacterial Proteins↗

Global regulation of Staphylococcus aureus genes by Rot.

Staphylococcus aureus produces a wide array of cell surface and extracellular proteins involved in virulence. Expression of these virulence factors is tightly controlled by numerous regulatory loci, including agr, sar, sigB, sae, and arl, as well as by a number of proteins with homology to SarA. Rot (repressor of toxins), a SarA homologue, was previously identified in a library of transposon-induced mutants created in an agr-negative strain by screening for restored protease and alpha-toxin. To date, all of the SarA homologues have been shown to act as global regulators of virulence genes. Therefore, we investigated the extent of transcriptional regulation of staphylococcal genes by Rot. We compared the transcriptional profile of a rot agr double mutant to that of its agr parental strain by using custom-made Affymetrix GeneChips. Our findings indicate that Rot is not only a repressor but a global regulator with both positive and negative effects on the expression of S. aureus genes. Our data also indicate that Rot and agr have opposing effects on select target genes. These results provide further insight into the role of Rot in the regulatory cascade of S. aureus virulence gene expression.

Bacterial Proteins↗