scholarly journals Campylobacter jejuniColonization in the Crow Gut Involves Many Deletions within the Cytolethal Distending Toxin Gene Cluster

2018 ◽  
Vol 84 (6) ◽  
Author(s):  
Keya Sen ◽  
Jingrang Lu ◽  
Piyali Mukherjee ◽  
Tanner Berglund ◽  
Eunice Varughese ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Virulence Gene ◽  
Farm Animals ◽  
Toxin Gene ◽  
Broad Host Range ◽  
Cytolethal Distending Toxin ◽  
Gene Markers ◽  
Content Type ◽  
Control Programs ◽  
Geographical Regions

ABSTRACTCampylobacterspp. are major causes of gastroenteritis worldwide. The virulence potential ofCampylobactershed in crow feces obtained from a roost area in Bothell, Washington, was studied and compared with that from isolates from other parts of Washington and from a different crow species 7,000 miles away in Kolkata, India.Campylobacterorganisms were isolated from 61% and 69% of the fecal samples obtained from Washington and Kolkata, respectively, and were confirmed to beC. jejuni. The cytolethal distending toxin (CDT) gene cluster from these isolates revealed a truncated sequence of approximately 1,350 bp. Sequencing of the gene cluster revealed two types of mutations: a 668-bp deletion acrosscdtAandcdtBand a 51-bp deletion withincdtB. Some strains had additional 20-bp deletions incdtB. In either case, a functional toxin is not expected; a functional toxin is produced by the expression of three tandem genes,cdtA,cdtB, andcdtC. Reverse transcriptase PCR with total RNA extracted from the isolates showed no expression ofcdtB. A toxin assay performed with these isolates on HeLa cells failed to show cytotoxic effects on the cells. However, the isolates were able to colonize the chicken ceca for a period of at least 4 weeks, similar to that of a clinical isolate. Other virulence gene markers, flagellin A and CadF, were present in 100% of the isolates. Our study suggests that crows carry the bacteriumC. jejunibut with a dysfunctional toxin protein that is expected to drastically reduce its potential to cause diarrhea.IMPORTANCECampylobacters are a major cause of gastroenteritis in humans. Since outbreaks have most often been correlated with poultry or unpasteurized dairy products, contact with farm animals, or contaminated water, historically, the majority of the studies have been with campylobacter isolates from poultry, domestic animals, and human patients. However, the bacterium has a broad host range that includes birds. These reservoirs need to be investigated, because the identification of the source and a determination of the transmission routes for a pathogen are important for the development of evidence-based disease control programs. In this study, two species of the human-commensal crow, from two different geographical regions separated by 7,000 miles of land and water, have been examined for their ability to cause disease by shedding campylobacters. Our results show that the crow may not play a significant role in campylobacteriosis, because the campylobacter organisms they shed produce a nonfunctional toxin.

scholarly journals Prevalence and Distribution of Listeria monocytogenesinlAAlleles Prone to Phase Variation andinlAAlleles with Premature Stop Codon Mutations among Human, Food, Animal, and Environmental Isolates

2015 ◽  
Vol 81 (24) ◽  
pp. 8339-8345 ◽  
Author(s):  
Clyde S. Manuel ◽  
Anna Van Stelten ◽  
Martin Wiedmann ◽  
Kendra K. Nightingale ◽  
Renato H. Orsi
Keyword(s):  
Stop Codon ◽  
Premature Stop Codon ◽  
Phase Variation ◽  
Virulence Gene ◽  
Farm Animals ◽  
Nucleotide Substitutions ◽  
Content Type ◽  
Niche Adaptation ◽  
Frameshift Deletion ◽  
Animal Isolates

ABSTRACTInListeria monocytogenes, 18 mutations leading to premature stop codons (PMSCs) in the virulence geneinlAhave been identified to date. While most of these mutations represent nucleotide substitutions, a frameshift deletion in a 5′ seven-adenine homopolymeric tract (HT) ininlAhas also been reported. This HT may play a role in phase variation and was first identified amongL. monocytogeneslineage II ribotype DUP-1039C isolates. In order to better understand the distribution of differentinlAmutations in this ribotype, a newly developed multiplex real-time PCR assay was used to screen 368 DUP-1039C isolates from human, animal, and food-associated sources for three known 5′inlAHT alleles: (i) wild-type (WT) (A7), (ii) frameshift (FS) (A6), and (iii) guanine interruption (A2GA4) alleles. Additionally, 228 DUP-1039C isolates were screened for allinlAPMSCs; data on the presence of allinlAPMSCs for the other 140 isolates were obtained from previous studies. The statistical analysis based on 191 epidemiologically unrelated strains showed that strains withinlAPMSC mutations (n= 41) were overrepresented among food-associated isolates, while strains encoding full-length InlA (n= 150) were overrepresented among isolates from farm animals and their environments. Furthermore, the A6allele was overrepresented and the A7allele was underrepresented among food isolates, while the A6allele was underrepresented among farm and animal isolates. Our results indicate that genetic variation ininlAcontributes to niche adaptation within the lineage II subtype DUP-1039C.

scholarly journals Cytolethal Distending Toxin Gene Cluster in Enterohemorrhagic Escherichiacoli O157:H− and O157:H7: Characterization and Evolutionary Considerations

2003 ◽  
Vol 71 (6) ◽  
pp. 3634-3638 ◽  
Author(s):  
Andreas Janka ◽  
Martina Bielaszewska ◽  
Ulrich Dobrindt ◽  
Lilo Greune ◽  
M. Alexander Schmidt ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Virulence Factor ◽  
Toxin Gene ◽  
Cytolethal Distending Toxin ◽  
Wild Type ◽  
Potential Virulence Factor

ABSTRACT We identified a cytolethal distending toxin (cdt) gene cluster in 87, 6, and 0% of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H−, EHEC O157:H7, and E. coli O55:H7/H− strains, respectively. The toxin was expressed by the wild-type EHEC O157 strains and by a cdt-containing cosmid from a library of SF EHEC O157:H− strain 493/89. The cdt flanks in strain 493/89 were homologous to bacteriophages P2 and lambda. Our data demonstrate that cdt, encoding a potential virulence factor, is present in the EHEC O157 complex and suggest that cdt may have been acquired by phage transduction.

scholarly journals An Aminotransferase Is Responsible for the Deamination of the N-Terminal Leucine and Required for Formation of Oxazolone Ring A in Methanobactin of Methylosinus trichosporium OB3b

2016 ◽  
Vol 83 (1) ◽  
Author(s):  
Wenyu Gu ◽  
Bipin S. Baral ◽  
Alan A. DiSpirito ◽  
Jeremy D. Semrau
Keyword(s):  
Gene Expression ◽  
Gene Cluster ◽  
Wilson Disease ◽  
Critical Role ◽  
Broad Host Range ◽  
Copper Binding ◽  
Copper Uptake ◽  
Methylosinus Trichosporium ◽  
Content Type ◽  
Oxazolone Ring

ABSTRACT Gene expression in methanotrophs has been shown to be affected by the availability of a variety of metals, most notably copper-regulating expression of alternative forms of methane monooxygenase. A copper-binding compound, or chalkophore, called methanobactin plays a key role in copper uptake in methanotrophs. Methanobactin is a ribosomally synthesized and posttranslationally modified peptide (RiPP) with two heterocyclic rings with an associated thioamide for each ring, formed from X-Cys dipeptide sequences that bind copper. The gene coding for the precursor polypeptide of methanobactin, mbnA, is part of a gene cluster, but the role of other genes in methanobactin biosynthesis is unclear. To begin to elucidate the function of these genes, we constructed an unmarked deletion of mbnABCMN in Methylosinus trichosporium OB3b and then homologously expressed mbnABCM using a broad-host-range cloning vector to determine the function of mbnN, annotated as coding for an aminotransferase. Methanobactin produced by this strain was found to be substantially different from wild-type methanobactin in that the C-terminal methionine was missing and only one of the two oxazolone rings was formed. Rather, in place of the N-terminal 3-methylbutanoyl-oxazolone-thioamide group, a leucine and a thioamide-containing glycine (Gly-Ψ) were found, indicating that MbnN is used for deamination of the N-terminal leucine of methanobactin and that this posttranslational modification is critical for closure of the N-terminal oxazolone ring in M. trichosporium OB3b. These studies provide new insights into methanobactin biosynthesis and also provide a platform for understanding the function of other genes in the methanobactin gene cluster. IMPORTANCE Methanotrophs, microbes that play a critical role in the carbon cycle, are influenced by copper, with gene expression and enzyme activity changing as copper levels change. Methanotrophs produce a copper-binding compound, or chalkophore, called methanobactin for copper uptake, and methanobactin plays a key role in controlling methanotrophic activity. Methanobactin has also been shown to be effective in the treatment of Wilson disease, an autosomal recessive disorder where the human body cannot correctly assimilate copper. It is important to characterize the methanobactin biosynthesis pathway to understand how methanotrophs respond to their environment as well as to optimize the use of methanobactin for the treatment of copper-related diseases such as Wilson disease. Here we show that mbnN, encoding an aminotransferase, is involved in the deamination of the N-terminal leucine and necessary for the formation of one but not both of the heterocyclic rings in methanobactin that are responsible for copper binding.

scholarly journals A SilencedvanAGene Cluster on a Transferable Plasmid Caused an Outbreak of Vancomycin-Variable Enterococci

2016 ◽  
Vol 60 (7) ◽  
pp. 4119-4127 ◽  
Author(s):  
Audun Sivertsen ◽  
Torunn Pedersen ◽  
Kjersti Wik Larssen ◽  
Kåre Bergh ◽  
Torunn Gresdal Rønning ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Molecular Mechanisms ◽  
Genetic Difference ◽  
Brain Heart Infusion ◽  
Broad Host Range ◽  
S1 Nuclease ◽  
Content Type ◽  
Transcription Profiles

ABSTRACTWe report an outbreak of vancomycin-variablevanA+enterococci (VVE) able to escape phenotypic detection by current guidelines and demonstrate the molecular mechanisms forin vivoswitching into vancomycin resistance and horizontal spread of thevanAcluster. Forty-eightvanA+Enterococcus faeciumisolates and oneEnterococcus faecalisisolate were analyzed for clonality with pulsed-field gel electrophoresis (PFGE), and theirvanAgene cluster compositions were assessed by PCR and whole-genome sequencing of six isolates. The susceptible VVE strains were cultivated in brain heart infusion broth containing vancomycin at 8 μg/ml forin vitrodevelopment of resistant VVE. The transcription profiles of susceptible VVE and their resistant revertants were assessed using quantitative reverse transcription-PCR. Plasmid content was analyzed with S1 nuclease PFGE and hybridizations. Conjugative transfer ofvanAwas assessed by filter mating. The only genetic difference between thevanAclusters of susceptible and resistant VVE was an ISL3-family element upstream ofvanHAX, which silencedvanHAXgene transcription in susceptible VVE. Furthermore, the VVE had an insertion of IS1542betweenorf2andvanRthat attenuated the expression ofvanHAX. Growth of susceptible VVE occurred after 24 to 72 h of exposure to vancomycin due to excision of the ISL3-family element. ThevanAgene cluster was located on a transferable broad-host-range plasmid also detected in outbreak isolates with different pulsotypes, including oneE. faecalisisolate. Horizontally transferable silencedvanAable to escape detection and revert into resistance during vancomycin therapy represents a new challenge in the clinic. Genotypic testing of invasive vancomycin-susceptible enterococci byvanA-PCR is advised.

scholarly journals Plasmid Typing and Resistance Profiling of Escherichia fergusonii and Other Enterobacteriaceae Isolates from South Korean Farm Animals

2011 ◽  
Vol 77 (9) ◽  
pp. 3163-3166 ◽  
Author(s):  
Nabin Rayamajhi ◽  
Seung Bin Cha ◽  
Seung Won Shin ◽  
Byeong Yeal Jung ◽  
Suk-Kyung Lim ◽  
...  
Keyword(s):  
Virulence Genes ◽  
Farm Animals ◽  
Broad Host Range ◽  
Content Type ◽  
Zoonotic Pathogen ◽  
South Korean ◽  
Resistance Determinants ◽  
Broad Host Range Plasmid ◽  
High Degree ◽  
Plasmid Replicons

ABSTRACTIn this study, we focused on determining the distribution and prevalence of major plasmid replicons in β-lactam-resistantEscherichia fergusoniiandEnterobacteriaceaeof animal and human origin. A high degree of plasmid variability and multiple plasmid replicons were observed among the isolates. The IncF and IncI1 replicons were the most prevalent inE. fergusoniiandSalmonella entericaserovar Indiana isolated from swine and poultry in South Korea, respectively. The presence of broad-host-range plasmid replicons such as IncN, IncA/C, IncHI1, and IncHI2 that are associated with important virulence genes and toxins as well as antimicrobial resistance determinants indicates thatE. fergusoniihas the potential to become an important pig pathogen and possible emerging opportunistic zoonotic pathogen.

scholarly journals First Report of the Local Spread of Vancomycin-Resistant Enterococci Ascribed to the Interspecies Transmission of a vanA Gene Cluster-Carrying Linear Plasmid

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Yusuke Hashimoto ◽  
Izumi Kita ◽  
Masato Suzuki ◽  
Hidetada Hirakawa ◽  
Hirofumi Ohtaki ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Gene Cluster ◽  
Vancomycin Resistance ◽  
Linear Plasmid ◽  
Broad Host Range ◽  
Content Type ◽  
Local Spread ◽  
Vancomycin Resistant Enterococci ◽  
Antimicrobial Resistance Genes ◽  
Vancomycin Resistant

ABSTRACT Vancomycin-resistant enterococci pose a threat in the clinical setting and have been linked to hospital outbreaks worldwide. In 2017, a local spread of VanA-type vancomycin-resistant enterococci (VRE) occurred in Japan, and 25 enterococcal isolates, including 14 Enterococcus faecium, 8 E. raffinosus, and 3 E. casseliflavus isolates, were identified from four inpatients. Molecular analysis of the multispecies of VanA-type VRE revealed the involvement of both the dissemination of clonally related VRE strains between patients and the horizontal transfer of plasmids harboring the vanA gene cluster between Enterococcus spp. Pulsed-field gel electrophoresis showed that the plasmid DNAs without S1 nuclease treatment were able to migrate into the gel, suggesting that the topology of the plasmid was linear. Whole-genome sequencing revealed that this plasmid, designated pELF2, was 108,102 bp long and encoded multiple antimicrobial resistance genes, including ermA and ant(9). The amino acid sequences of putative replication- and transfer-related genes were highly conserved between pELF2 and pELF1, the latter of which was the first identified enterococcal conjugative linear plasmid. On comparing the genomic structure, pELF2 showed the presence of a backbone similar to that of pELF1, especially with respect to the nucleotide sequences of both terminal ends, indicating a hybrid-type linear plasmid, possessing two different terminal structures. pELF2 possessed a broad host range and high conjugation frequencies for enterococci. The easy transfer of pELF2 to different Enterococcus spp. in vitro might explain this local spread of multiple species, highlighting the clinical threat from the spread of antimicrobial resistance by an enterococcal linear plasmid. IMPORTANCE Increasing multidrug resistance, including vancomycin resistance, in enterococci is a major concern in clinical settings. Horizontal gene transfer, such as via plasmids, has been shown to play a crucial role in the acquisition of vancomycin resistance. Among vancomycin resistance types, the VanA type is one of the most prevalent, and outbreaks caused by VanA-type vancomycin-resistant enterococci (VRE) have occurred worldwide. Here, we describe an enterococcal linear plasmid responsible for multispecies local spread of VanA-type VRE. Such a study is important because although hospital outbreaks caused by mixed enterococcal species have been reported, this particular spread indicates plasmid transfer across species. This is a crucial finding because the high risk for such a spread of antimicrobial resistance calls for regular monitoring and surveillance.

scholarly journals Campylobacter hyointestinalis Isolated from Pigs Produces Multiple Variants of Biologically Active Cytolethal Distending Toxin

2015 ◽  
Vol 83 (11) ◽  
pp. 4304-4313 ◽  
Author(s):  
Kazumasa Kamei ◽  
Noritoshi Hatanaka ◽  
Masahiro Asakura ◽  
Srinuan Somroop ◽  
Worada Samosornsuk ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Gene Clusters ◽  
Amino Acid Sequences ◽  
Biologically Active ◽  
Cytolethal Distending Toxin ◽  
Content Type ◽  
M Phase ◽  
Important Virulence Factor ◽  
Animal Isolates ◽  
Gel Diffusion

ABSTRACTCampylobacter hyointestinalisisolated from swine with proliferative enteritis often is considered to be pathogenic. While the precise virulence mechanisms of this species remain unclear, we have recently identified a cytolethal distending toxin (cdt) gene cluster inC. hyointestinalisisolated from a patient with diarrhea (W. Samosornsuk et al., J Med Microbiol, 27 July 2015,http://dx.doi.org/10.1099/jmm.0.000145). However, the sequences of thecdtgenes inC. hyointestinaliswere found to be significantly different and the gene products are immunologically distinct from those of otherCampylobacterspecies. In this study, we demonstrate the presence of a second variant of thecdtgene cluster inC. hyointestinalis, designatedcdt-II, while the former is namedcdt-I. Sequencing of thecdt-IIgene cluster and deduced amino acid sequences revealed that homologies between the subunits CdtA, CdtB, and CdtC of ChCDT-I and ChCDT-II are 25.0, 56.0, and 24.8%, respectively. Furthermore, the CdtB subunit of ChCDT-II was found to be immunologically unrelated to that of ChCDT-I by Ouchterlony double gel diffusion test. Recombinant ChCDT-II also induced cell distention and death of HeLa cells by blocking the cell cycle at G2/M phase. Interestingly, thecdt-IIgenes were detected in all 23 animal isolates and in 1 human isolate ofC. hyointestinalis, and 21 of these strains carried bothcdt-Iandcdt-IIgene clusters. Altogether, our results indicate that ChCDT-II is an important virulence factor ofC. hyointestinalisin animals.

scholarly journals Looking for the X Factor in Bacterial Pathogenesis: Association of orfX-p47 Gene Clusters with Toxin Genes in Clostridial and Non-Clostridial Bacterial Species

Toxins ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 19 ◽  
Author(s):  
Maria B. Nowakowska ◽  
François P. Douillard ◽  
Miia Lindström
Keyword(s):  
Gene Cluster ◽  
In Silico ◽  
Botulinum Neurotoxin ◽  
Bacterial Species ◽  
Gene Clusters ◽  
Bacterial Pathogenesis ◽  
Toxin Gene ◽  
Toxin Complex ◽  
Analytical Tools ◽  
Bacillus Isolate

The botulinum neurotoxin (BoNT) has been extensively researched over the years in regard to its structure, mode of action, and applications. Nevertheless, the biological roles of four proteins encoded from a number of BoNT gene clusters, i.e., OrfX1-3 and P47, are unknown. Here, we investigated the diversity of orfX-p47 gene clusters using in silico analytical tools. We show that the orfX-p47 cluster was not only present in the genomes of BoNT-producing bacteria but also in a substantially wider range of bacterial species across the bacterial phylogenetic tree. Remarkably, the orfX-p47 cluster was consistently located in proximity to genes coding for various toxins, suggesting that OrfX1-3 and P47 may have a conserved function related to toxinogenesis and/or pathogenesis, regardless of the toxin produced by the bacterium. Our work also led to the identification of a putative novel BoNT-like toxin gene cluster in a Bacillus isolate. This gene cluster shares striking similarities to the BoNT cluster, encoding a bont/ntnh-like gene and orfX-p47, but also differs from it markedly, displaying additional genes putatively encoding the components of a polymorphic ABC toxin complex. These findings provide novel insights into the biological roles of OrfX1, OrfX2, OrfX3, and P47 in toxinogenesis and pathogenesis of BoNT-producing and non-producing bacteria.

scholarly journals Whole-Genome Single-Nucleotide-Polymorphism Analysis for Discrimination of Clostridium botulinum Group I Strains

2014 ◽  
Vol 80 (7) ◽  
pp. 2125-2132 ◽  
Author(s):  
Narjol Gonzalez-Escalona ◽  
Ruth Timme ◽  
Brian H. Raphael ◽  
Donald Zink ◽  
Shashi K. Sharma
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Clostridium Botulinum ◽  
Toxin Gene ◽  
Polymorphism Analysis ◽  
Snp Analysis ◽  
Whole Genome ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type ◽  
Group I

ABSTRACTClostridium botulinumis a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA+OrfX−) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA−OrfX+) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producingC. botulinumstrains: two strains with the HA+OrfX−cluster (69A and 32A) and one strain with the HA−OrfX+cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly availableC. botulinumgroup I strains revealed five distinct lineages. Strains 69A and 32A clustered with theC. botulinumtype A1 Hall group, and strain CDC297 clustered with theC. botulinumtype Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination ofC. botulinumgroup I strains and demonstrates the utility of this analysis in quickly differentiatingC. botulinumstrains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.

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