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PubMed2006

Antibiotic resistance, integrons and Salmonella genomic island 1 among non-typhoidal Salmonella serovars in The Netherlands.

The objective of this study was to investigate the antimicrobial resistance patterns, integron characteristics and gene cassettes as well as the presence of Salmonella genomic island 1 (SGI1) in non-typhoidal Salmonella (NTS) isolates from human and animal origin. Epidemiologically unrelated Dutch NTS strains (n=237) originating from food-producing animals and human cases of salmonellosis were tested for their susceptibility to 15 antimicrobial agents. Resistance to 14 of these antimicrobials, including the third-generation cephalosporins, was detected. Resistance to sulphonamides, ampicillin, tetracycline, streptomycin, trimethoprim and nalidixic acid was common (>/=10% of the strains were resistant). Resistance against three or more antimicrobials was observed in 57 isolates. The same 237 strains were studied for the prevalence of class 1 integrons, their gene cassettes and the presence of SGI1. Thirty-six isolates (15.2%) carried class 1 integrons. These integrons had ten distinct profiles based on the size of the integron and restriction fragment length polymorphism analysis. Integrons were detected for the first time in serovars Indiana and Senftenberg. Multidrug resistance was strongly associated with the presence of class 1 integrons in which the aadA2, aadA1, bla(PSE-1), dfrA1, dfrA5, dfrA14 or sat genes were present, as determined by nucleotide sequence determination. The presence of gene cassettes or combinations of gene cassettes not previously found in integrons in Salmonella was observed. SGI1 or its variants (SGI-B, -C and -F) were present in 16 isolates belonging to either serovar Typhimurium, Derby or Albany. Regardless of whether the isolate was of human or animal origin, the same resistance phenotype, integron profile and SGI1 structure could be observed.

Animals↗
PubMed2006

Numerical analysis of DNA microarray data of Campylobacter jejuni strains correlated with survival, cytolethal distending toxin and haemolysin analyses.

Molecular epidemiological studies of the enteric pathogen Campylobacter jejuni have suggested that not all animal isolates are equally pathogenic to humans. We examined the use of numerical analysis of whole-genomotype data as a potential tool for evaluating C. jejuni virulence potential. Whole-genome microarray analysis was used to determine the gene-level complementarity of 12 Danish strains to the pathogenic, genome-sequenced strain NCTC 11168. Cytolethal distending toxin (CDT) and haemolysin activities, and survival characteristics under aerobic conditions at room temperature were also determined. Among the strains examined, 439 genes were polymorphic. Numerical analysis of these data by use of the squared Euclidean distance coefficient and Ward's clustering method clearly delineated strains into two clusters. CDT and haemolysin activities of cluster 1 strains were not statistically significantly different from cluster 2 strains. However, viability during aerobic incubation of cluster 1 strains was statistically significantly lower than corresponding estimates of cluster 2 strains. The number of missing or highly divergent genes in cluster 1 strains with respect to NCTC 11168 was also statistically significantly greater compared with those of cluster 2 strains. Sixty-seven genes present in NCTC 11168 were characteristically missing or divergent among cluster 1 strains. Of these, 53 genes were localised within 11 major gene clusters, of which eight were associated with surface structures and included flagellar, lipo-oligosaccharide, and membrane transport proteins. Our data indicate a correlation between C. jejuni genomic content, particularly in surface-coding regions, and its capacity for environmental survival, and may help explain why certain serotypes are more commonly reported in human disease.

Animals↗
PubMed2004

Inactivation of human adenovirus genome by different groups of disinfectants.

Human adenoviruses are model viruses for testing the virucidal efficacy of disinfectants. However, a recent study has shown that the chemical sensitivity of adenovirus serotypes varies significantly, possibly due to the composition of the viral capsid and/or the resistance of nucleic acids. We have investigated the effect of molecular changes in the viral genome of the human adenovirus subgenera C and D. A common oligonucleotide fragment within the hexon gene was amplified by polymerase chain reaction (PCR) after exposure to liposomal povidone-iodine (PVP-I), peracetic acid (PAA), and formaldehyde. The findings were compared with infectivity in cell cultures. PVP-I (0.125%) destroyed the infectivity of most serotypes within 60 min. However, PCR revealed no destruction of the adenoviral genome in most serotypes, even after exposure to higher PVP-I concentrations. PAA (0.5%) failed to inactivate the hexon gene of adenovirus types 22 and 44. Furthermore, the hexon gene of adenovirus type 22 was not altered by 0.7% formaldehyde. In conclusion, the genomes of human adenoviruses show considerably more chemical resistance than the complete viral particle. The molecular resistance of individual serotypes also varies. However, there was no clear correlation between the differences in the effect of disinfectants on infectivity of the complete viral particle and destruction of the viral genome.

Adenoviruses, Human↗
PubMed1999

Integrated pathway-genome databases and their role in drug discovery.

Integrated pathway-genome databases describe the genes and genome of an organism, as well as its predicted pathways, reactions, enzymes and metabolites. In conjunction with visualization and analysis software, these databases provide a framework for improved understanding of microbial physiology and for antimicrobial drug discovery. We describe pathway-based analyses of the genomes of a number of medically relevant microorganisms and a novel software tool that visualizes gene-expression data on a diagram showing the whole metabolic network of the microorganism.

Anti-Bacterial Agents↗
PubMed2006

Host-pathogen systems biology.

Unlike traditional biological research that focuses on a small set of components, systems biology studies the complex interactions between a large number of genes, proteins and other elements of biological networks and systems. Host-pathogen systems biology examines the interactions between the components of two distinct organisms, either a microbial or viral pathogen and its animal host or two different microbial species in a community. With the availability of complete genomic sequences of various hosts and pathogens, together with breakthroughs in proteomics, metabolomics and other experimental areas, the investigation of host-pathogen systems on a multitude of levels of detail has come within reach.

Animals↗
PubMed2000

The evolution of human pathogens: examples and clinical implications.

Recent advances in sequencing of complete bacterial genomes, molecular typing of micro-organisms, and research on microbial pathogenicity factors changed our view on the evolution of human bacterial pathogens. We review current evolutionary concepts on plague and meningococcal disease to illustrate the interplay of molecular phylogeny, epidemiology, and pathogenicity research. Furthermore, examples of the tremendous velocity of bacterial evolution under changing environmental conditions will be discussed.

Adaptation, Physiological↗
PubMed2026

Discovery of Glycosylated β-Amino Acid-Containing Macrolactams from Nonomuraea sp. 0L2P via Genome Mining.

β-Amino acid-containing macrolactams (β-AACMs) are a class of bioactive natural products characterized by nitrogen-containing starter units within polyketide-derived macrocycles. Here, we report four previously undescribed macrolactams, gruelactams A-D (1-4), from Nonomuraea sp. 0L2P, discovered through an integrated approach combining genome mining, 15N-labeling, and antibacterial screening. Their planar structures were elucidated by comprehensive spectroscopic analyses, including 1D and 2D NMR and HRESI-MS, and their configurations were partially assigned based on ROESY data and bioinformatic analysis. Genome sequencing and antiSMASH analysis identified a putative type I polyketide synthase (PKS) biosynthetic gene cluster, enabling the proposal of a biosynthetic pathway. Bioactivity assays showed that gruelactam D (4) exhibits antibacterial activity against Bacillus cereus and Staphylococcus aureus, with MIC values of 8 and 16 μg/mL, respectively. These findings expand the chemical diversity of β-AACMs and demonstrate the utility of genome-guided approaches for discovering bioactive natural products from rare actinomycetes.

Anti-Bacterial Agents↗
PubMed2002

Helicobacter pylori infection in mice: Role of outer membrane proteins in colonization and inflammation.

BACKGROUND & AIMS: Mouse models of Helicobacter pylori infection have yielded variable results with respect to colonization and inflammation. We examined whether outer membrane proteins (OMPs) or the cag pathogenicity island (PAI) was responsible for some of this variability. METHODS: C57BL/6 mice received clinical H. pylori isolates with different genotypes for the cag PAI and OMP gene switch status, as well as isogenic gene knockout mutants for cagE, oipA, babA2, hopZ, cagE/oipA, or oipA/hopZ. Bacterial density, histology, and mucosal cytokine/chemokine levels were measured after 4 and 12 weeks. RESULTS: oipA, hopZ, hopO, and hopP switch status influenced both H. pylori density and colonization ability in mice. When 2 or more of the genes were "off," colonization rates were markedly reduced compared with those for strains with all genes "on" (from 58% to 0% after 12 weeks). oipA knockout mutants caused reduced inflammation and decreased mucosal interleukin 6 messenger RNA and KC messenger RNA and protein levels. H. pylori density and colonization ability were reduced if hopO or hopP switch status was changed from "on" to "off." There was a close relationship (r > 0.7) between the H. pylori density of the gastric mucosa of humans and mice when using the same H. pylori strains. CONCLUSIONS: Many of the differences reported with mouse models may reflect subtle unrecognized differences (e.g., switch status of an OMP gene), emphasizing the necessity of characterizing isolates before and after experiments. The mouse model may be suitable for investigating factors related to colonization ability, H. pylori density, and gastric mucosal inflammation.

Animals↗
PubMed2000

Molecular fingerprinting of Mycobacterium tuberculosis strains isolated in Vietnam using IS6110 as probe.

SETTING: Northern and Southern areas of Vietnam. OBJECTIVE: To study the correlation between DNA fingerprinting of 168 Mycobacterium tuberculosis strains isolated from patients with a particular historical past (political separation of Vietnam for 20 years) and data about geographical origin, drug susceptibility, HIV infection and BCG vaccination status. METHODS: Comparison of restriction fragment length polymorphism (RFLP) patterns produced by Southern hybridization of Pvull-digested chromosomal DNA. RESULTS: The number of IS6110 copies for the 168 strains ranges from 0 to 23. Strains originating from the North or the South differ strongly with respect to the number of copies of IS6110. Indeed, the strains originating from the north have predominantly from 3 to 14 IS6110 copies while the southern strains have predominantly from 15 to 23 IS6110 copies. Furthermore, strains isolated in the North are dispersed into 6 groups whereas 80% of the strains isolated in the South form a single group. Moreover, the prevalence of drug resistance is higher in strains isolated in the South than in the North. No noticeable correlation is observed between RFLP patterns, drug susceptibility, or HIV infection. CONCLUSION: The IS6110 fingerprints of 168 M. tuberculosis strains isolated in Vietnam showed a high range of polymorphism. Only a few strains have been found with no IS6110 (1.8%). The differences between the strains from the North and South, having more than six IS6110, suggests that they derived from ancestral strains that would be distinguishable by the number of IS6110 and their transposition sites throughout the genome. The genomic structure of the population of strains from South Vietnam resembles that of the Beijing strain population. This could account for a similar evolution of M. tuberculosis due to a selection by BCG-induced immunity in the two populations.

BCG Vaccine↗
PubMed1997

Diazaborine resistance in the yeast Saccharomyces cerevisiae reveals a link between YAP1 and the pleiotropic drug resistance genes PDR1 and PDR3.

We have investigated the mechanisms underlying resistance to the drug diazaborine in Saccharomyces cerevisiae. We used UV mutagenesis to generate resistant mutants, which were divided into three different complementation groups. The resistant phenotype in these groups was found to be caused by allelic forms of the genes AFG2, PDR1, and PDR3. The AFG2 gene encodes an AAA (ATPases associated to a variety of cellular activities) protein of unknown function, while PDR1 and PDR3 encode two transcriptional regulatory proteins involved in pleiotropic drug resistance development. The isolated PDR1-12 and PDR3-33 alleles carry mutations that lead to a L1044Q and a Y276H exchange, respectively. In addition, we report that overexpression of Yap1p, the yeast homologue of the transcription factor AP1, results in a diazaborine-resistant phenotype. The YAP1-mediated diazaborine resistance is dependent on the presence of functional PDR1 and PDR3 genes, although PDR3 had a more pronounced effect. These results provide the first evidence for a functional link between the Yap1p-dependent stress response pathway and Pdr1p/Pdr3p-dependent development of pleiotropic drug resistance.

Amino Acid Sequence↗
PubMed2001

A macrophage protein, Ym1, transiently expressed during inflammation is a novel mammalian lectin.

Oral infections of mice with Trichinella spiralis induce activation of peritoneal exudate cells to transiently express and secrete a crystallizable protein Ym1. Purification of Ym1 to homogeneity was achieved. It is a single chain polypeptide (45 kDa) with a strong tendency to crystallize at its isoelectric point (pI 5.7). Co-expression of Ym1 with Mac-1 and scavenger receptor pinpoints macrophages as its main producer. Protein microsequencing data provide information required for full-length cDNA cloning from libraries constructed from activated peritoneal exudate cells. A single open reading frame of 398 amino acids with a leader peptide (21 residues) typical of secretory protein was deduced and later deposited in GenBank (accession number M94584) in 1992. By means of surface plasmon resonance analyses, Ym1 has been shown to exhibit binding specificity to saccharides with a free amine group, such as GlcN, GalN, or GlcN polymers, but it failed to bind to other saccharides. The interaction is pH-dependent but Ca2+ and Mg2+ ion-independent. The binding avidity of Ym1 to GlcN oligosaccharides was enhanced by more than 1000-fold due to the clustering effect. Specific binding of Ym1 to heparin suggests that heparin/heparan sulfate may be its physiological ligand in vivo during inflammation and/or tissue remodeling. Although it shares approximately 30% homology with microbial chitinases, no chitinase activity was found associated with Ym1. Genomic Southern blot analyses suggest that Ym1 may represent a member of a novel lectin gene family.

Amino Acid Sequence↗
PubMed2000

Analysis of candidate-host immunogenetic determinants in herpes simplex virus-associated Mollaret's meningitis.

Infection due to herpes simplex virus (HSV) is associated with recurrent aseptic meningitis (Mollaret's meningitis); however, the neuropathogenesis of this disease remains unknown. We collected 20 cerebrospinal fluid (CSF) specimens that were positive for HSV DNA by using polymerase chain reaction (PCR) assay from patients with a clinical diagnosis of Mollaret's meningitis. Patients were predominantly female (female:male, 22:1), with an average age of 32.8 years (range, 18-46 years). Using direct sequence analysis of HSV PCR products obtained from the CSF, we determined that all of the patients were infected with HSV type 2. In addition, we evaluated polymorphisms in 2 human genomic loci, which are associated with either severe or recurrent microbial infections (interferon-gamma receptor [IFN-gammaR] and mannose binding lectin [MBL]); these host genes were also amplified directly from the CSF specimens. No mutations were found in exons 2 or 3 of the IFN-gammaR gene (n=20). In contrast, there were 4 (20%), 4 (20%), and 0 mutations found in codons 52, 54, and 57, respectively, in exon 1 of MBL (n=20). A significantly higher frequency of codon 52 mutations (P=.04) was observed, compared with racially matched control patients.

Adolescent↗
PubMed2025

Genomic structure of class 1 and 2 integrons in non-typhoidal Salmonella isolated from food animals and related meat products in the USA.

OBJECTIVES: Integrons facilitate the capture and expression of exogenous genes, including antimicrobial resistance (AMR) genes. This study aimed to detect the presence of integrons, examine their genomic structure and location, and analyse integron-associated AMR, virulence and stress response genes in Salmonella using WGS. METHODS: WGS data from 193 Salmonella strains, representing 38 serotypes isolated from food animals and related meat products (2001-2019), were analysed using bioinformatic tools to assess integron presence and characterize their genomic architectures. RESULTS: Of 193 isolates, 116 (60.1%) harboured class 1 and/or class 2 integrons. Class 1 integrons alone were detected in 105 isolates, with some containing multiple copies. One S. Infantis isolate harboured only class 2 integrons, whereas 10 others contained both classes. No class 3-5 integrons were found. Twenty-seven class 1 integrons were chromosomal; the rest were plasmid-associated, linked to various plasmid incompatibility (Inc) types. Sixty-nine distinct AMR genes conferring resistance to 11 antimicrobial classes were found in integron cassettes or integron-associated plasmids. Genes linked to resistance to quaternary ammonium compounds and heavy metals, as well as ISs and transposons, were also identified. Significant virulence and stress response genes and proteins such as groES-groEL, LysR and EAL (glutamate, alanine and leucine) were common in integron cassettes. CONCLUSIONS: Class 1 integrons are prevalent in MDR Salmonella isolates from food animals and related meat products and are linked to diverse plasmid types. Their association with AMR, virulence and stress response genes underscores their role in AMR dissemination, and bacterial adaptation and pathogenicity.

Integrons↗
PubMed2026

Molecular surveillance of foodborne bacterial pathogens and resistome in food products from Hong Kong.

Foodborne infections pose an increasing public health challenge worldwide. The problem has been aggravated by the dissemination of antimicrobial resistance genes among zoonotic pathogens, which results in a sharp increase in antibiotic resistance rate recorded among the major foodborne pathogens. To obtain an overview of the extent to which food products purchased in the markets in Hong Kong were contaminated by foodborne pathogens, we collected 95 raw meat samples from wet markets and isolated 236 bacterial strains of various species, with Escherichia coli being the most dominant species (131 strains). Contamination of food products by multiple foodborne pathogens was commonly observed. These include both Gram-positive and Gram-negative bacteria that exhibit various levels of resistance, with some possessing multiple clinically important antibiotic resistance genes. Seventeen bacterial strains of various species isolated from three food samples were comprehensively analysed by the Oxford Nanopore R10.4 technology. Novel conjugative plasmids carrying antimicrobial resistance gene-bearing mobile genetic elements were commonly detectable in the test strains. Some of the plasmids were shown to have originated from other environmental sources or other bacterial species, indicating that raw foods in the local market may serve as a reservoir of resistance-encoding genetic elements from which such elements are disseminated to various microbial pathogens. These findings suggest a need to perform periodic but comprehensive surveillance of multidrug-resistant bacterial pathogens and the major antimicrobial resistance genes in common food products, so as to disrupt the transmission routes of such organisms and the resistance-encoding genetic elements that they harbour.

Hong Kong↗
PubMed2007

The proteomic profile of Fusobacterium nucleatum is regulated by growth pH.

Fusobacterium nucleatum is a saccharolytic Gram-negative anaerobic organism believed to play an important role in the microbial succession associated with the development of periodontal disease. Its genome contains niche-specific genes shared with the other inhabitants of dental plaque, which may help to explain its ability to survive and grow in the changing environmental conditions experienced in the gingival sulcus during the transition from health to disease. The pH of the gingival sulcus increases during the development of periodontitis and this is thought to occur by the metabolism of nutrients supplied by gingival crevicular fluid. In comparison with other plaque inhabitants, F. nucleatum has the greatest ability to neutralize acidic environments. The differential expression of soluble cytoplasmic proteins induced by acidic (pH 6.4) or basic (pH 7.4 and 7.8) conditions, during long-term anaerobic growth in a chemostat, was identified by two-dimensional gel electrophoresis and image analysis software. Twenty-two proteins, found to have altered expression in response to external pH, were identified by tryptic digestion and mass spectrometry. Eight differentially expressed proteins associated with increased energy (ATP) production via the 2-oxoglutarate and Embden-Meyerhof pathways appeared to be directed towards either cellular biosynthesis or the maintenance of internal homeostasis. Overall, these results represent the first proteomic investigation of F. nucleatum and the identification of gene products which may be important in the organism's persistence during the transition from health to disease in vivo.

Anaerobiosis↗
PubMed2004

Differentiate to thrive: lessons from the Legionella pneumophila life cycle.

When confronted by disparate environments, microbes routinely alter their physiology to tolerate or exploit local conditions. But some circumstances require more drastic remodelling of the bacterial cell, as sporulation by the Bacillus and Streptomyces species of soil bacteria vividly illustrates. Cellular differentiation is also crucial for pathogens, the challenge for which is to colonize one host, then be transmitted to the next. Using the Gram-negative Legionella pneumophila as a model intracellular pathogen, we describe how biogenesis of the replication vacuole is determined by the developmental state of the bacterium. Subsequently, when replicating bacteria have exhausted the nutrient supply, the pathogens couple their conversion to stationary phase physiology with expression of traits that promote transmission to a new host. The cellular differentiation of L. pneumophila is co-ordinated by a regulatory circuit that integrates several elements that are broadly conserved in the microbial world. The alarmone (p)ppGpp promotes transcription directed by the alternative sigma factors RpoS, FliA and, probably, RpoN, and also post-transcriptional control mediated by a two-component regulatory system, LetA/S (GacA/S), and an mRNA-binding protein, CsrA (RsmA). By applying knowledge of microbial differentiation in combination with tools to screen the complete genomes of pathogens, experiments can be designed to identify two distinct classes of virulence traits: factors that promote replication and those dedicated to transmission.

Amoeba↗
PubMed2004

Subgingival plaque microbiota in Saudi Arabians after use of miswak chewing stick and toothbrush.

BACKGROUND: The chewing stick, the miswak, is used in many developing countries as the traditional means for oral hygiene. It is prepared from the roots, twigs and stem of Salvadora persica or other alternative local plants. OBJECTIVES: To compare the effects of the chewing stick miswak (from S. persica) and toothbrush on subgingival plaque microflora among Saudi Arabian individuals. Further, to investigate whether components extracted from S. persica may interfere with the subgingival plaque micro-organisms. MATERIAL AND METHODS: Fifteen healthy Saudi Arabian male volunteers aged 21-36 years were included in a single-blind, randomized cross-over study. The participants were taught how to use each device properly. Plaque sampling for DNA test was performed at the baseline, 1 week after professional tooth cleaning, and after 3 weeks of either miswak or toothbrush use. Identification and quantification of microbial species were performed by the checkerboard method, using whole genomic, digoxigenin-labelled DNA probes. Inhibition zones around miswak were examined on agar plates with Actinobacillus actinomycetemcomitans and the leukotoxicity of this bacterium was analyzed in a bioassay with macrophages+/-extracts of miswak. RESULTS: Miswak and toothbrushing had a similar influence on the levels of the subgingival microbiota. However, A. actinomycetemcomitans was significantly more reduced by miswak (p<0.05) than by toothbrushing. These results were supported by our in vitro results which, indicated that extracts from S. persica might interfere with the growth and leukotoxicity of A. actinomycetemcomitans. CONCLUSIONS: In contrast to toothbrush use, miswak use significantly reduced the amount of A. actinomycetemcomitans in the subgingival plaque.

Adult↗