July 19, 2019  |  

Vertical transmission of highly similar bla CTX-M-1-harboring IncI1 plasmids in Escherichia coli with different MLST types in the poultry production pyramid.

The purpose of this study was to characterize sets of extended-spectrum ß-lactamases (ESBL)-producing Enterobacteriaceae collected longitudinally from different flocks of broiler breeders, meconium of 1-day-old broilers from theses breeder flocks, as well as from these broiler flocks before slaughter.Five sets of ESBL-producing Escherichia coli were studied by multi-locus sequence typing (MLST), phylogenetic grouping, PCR-based replicon typing and resistance profiling. The bla CTX-M-1-harboring plasmids of one set (pHV295.1, pHV114.1, and pHV292.1) were fully sequenced and subjected to comparative analysis.Eleven different MLST sequence types (ST) were identified with ST1056 the predominant one, isolated in all five sets either on the broiler breeder or meconium level. Plasmid sequencing revealed that bla CTX-M-1 was carried by highly similar IncI1/ST3 plasmids that were 105 076 bp, 110 997 bp, and 117 269 bp in size, respectively.The fact that genetically similar IncI1/ST3 plasmids were found in ESBL-producing E. coli of different MLST types isolated at the different levels in the broiler production pyramid provides strong evidence for a vertical transmission of these plasmids from a common source (nucleus poultry flocks).


July 7, 2019  |  

Molecular characterization of plasmid pMoma1of Moraxella macacae, a newly described bacterial pathogen of macaques.

We report the complete nucleotide sequence and characterization of a small cryptic plasmid of Moraxella macacae 0408225, a newly described bacterial species within the family Moraxellaceae and a causative agent of epistaxis in macaques. The complete nucleotide sequence of the plasmid pMoma1 was determined and found to be 5,375 bp in size with a GC content of 37.4 %. Computer analysis of the sequence data revealed five open reading frames encoding putative proteins of 54.4 kDa (ORF1), 17.6 kDa (ORF2), 13.3 kDa (ORF3), 51.6 kDa (ORF4), and 25.0 kDa (ORF5). ORF1, ORF2, and ORF3 encode putative proteins with high identity (72, 42, and 55 %, respectively) to mobilization proteins of plasmids found in other Moraxella species. ORF3 encodes a putative protein with similarity (about 40 %) to several plasmid replicase (RepA) proteins. The fifth open reading frames (ORF) was most similar to hypothetical proteins with unknown functions, although domain analysis of this sequence suggests it belongs to the Abi-like protein family. Upstream of the repA gene, a 470-bp intergenic region, was identified that contained an AT-rich section and two sets of tandem direct and indirect repeats, consistent with a putative origin of replication site. In contrast to other plasmids of Moraxella, the occurrence of pMoma1 in M. macacae isolates appears to be common as PCR testing of 14 clinical isolates from two different research institutions all contained the plasmid.


July 7, 2019  |  

Complete genome sequence of the Clostridium difficile laboratory strain 630¿ erm reveals differences from strain 630, including translocation of the mobile element CTn 5.

Background Clostridium difficile strain 630¿erm is a spontaneous erythromycin sensitive derivative of the reference strain 630 obtained by serial passaging in antibiotic-free media. It is widely used as a defined and tractable C. difficile strain. Though largely similar to the ancestral strain, it demonstrates phenotypic differences that might be the result of underlying genetic changes. Here, we performed a de novo assembly based on single-molecule real-time sequencing and an analysis of major methylation patterns.ResultsIn addition to single nucleotide polymorphisms and various indels, we found that the mobile element CTn5 is present in the gene encoding the methyltransferase rumA rather than adhesin CD1844 where it is located in the reference strain.ConclusionsTogether, the genetic features identified in this study may help to explain at least part of the phenotypic differences. The annotated genome sequence of this lab strain, including the first analysis of major methylation patterns, will be a valuable resource for genetic research on C. difficile.


July 7, 2019  |  

Complete genome sequence of BS49 and draft genome sequence of BS34A, Bacillus subtilis strains carrying Tn916.

Bacillus subtilis strains BS49 and BS34A, both derived from a common ancestor, carry one or more copies of Tn916, an extremely common mobile genetic element capable of transfer to and from a broad range of microorganisms. Here, we report the complete genome sequence of BS49 and the draft genome sequence of BS34A, which have repeatedly been used as donors to transfer Tn916, Tn916 derivatives or oriTTn916-containing plasmids to clinically important pathogens. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.


July 7, 2019  |  

Clonal dissemination of Enterobacter cloacae harboring blaKPC-3 in the upper midwestern United States.

Carbapenemase-producing, carbapenem-resistant Enterobacteriaceae, or CP-CRE, are an emerging threat to human and animal health, because they are resistant to many of the last-line antimicrobials available for disease treatment. Carbapenemase-producing Enterobacter cloacae harboring blaKPC-3 recently was reported in the upper midwestern United States and implicated in a hospital outbreak in Fargo, North Dakota (L. M. Kiedrowski, D. M. Guerrero, F. Perez, R. A. Viau, L. J. Rojas, M. F. Mojica, S. D. Rudin, A. M. Hujer, S. H. Marshall, and R. A. Bonomo, Emerg Infect Dis 20:1583-1585, 2014, http://dx.doi.org/10.3201/eid2009.140344). In early 2009, the Minnesota Department of Health began collecting and screening CP-CRE from patients throughout Minnesota. Here, we analyzed a retrospective group of CP-E. cloacae isolates (n = 34) collected between 2009 and 2013. Whole-genome sequencing and analysis revealed that 32 of the strains were clonal, belonging to the ST171 clonal complex and differing collectively by 211 single-nucleotide polymorphisms, and it revealed a dynamic clone under positive selection. The phylogeography of these strains suggests that this clone existed in eastern North Dakota and western Minnesota prior to 2009 and subsequently was identified in the Minneapolis and St. Paul metropolitan area. All strains harbored identical IncFIA-like plasmids conferring a CP-CRE phenotype and an additional IncX3 plasmid. In a single patient with multiple isolates submitted over several months, we found evidence that these plasmids had transferred from the E. cloacae clone to an Escherichia coli ST131 bacterium, rendering it as a CP-CRE. The spread of this clone throughout the upper midwestern United States is unprecedented for E. cloacae and highlights the importance of continued surveillance to identify such threats to human health. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

IncI1 plasmids encoding various blaCTX-Ms contributed to ceftriaxone resistance in Salmonella Enteritidis in China.

Resistance to extended spectrum ß-lactams in Salmonella, in particular serotypes such as S. Enteritidis that are frequently associated with clinical infections, is a serious public health concern. In this study, phenotypic characterization of 433 clinical S. Enteritidis strains obtained from a nationwide collection of China CDC during the period of 2005~2010 depicted an increasing trend of resistance to ceftriaxone from 2008 onwards. Seventeen (4%) of the strains were found to be resistant to ceftriaxone, 7% to ciprofloxacin and 0.7% to both ciprofloxacin and ceftriaxone. Most of the ceftriaxone-resistant S. Enteritidis strains (15/17) were genetically unrelated, and originated from Henan province. The complete sequence of an IncI1 plasmid pSE115 which belonged to a novel Sequence Type was obtained. This 87,255bp IncI1 plasmid was found to harbour a blaCTX-M-14 gene located in a novel Multidrug Resistance Region (MRR) within the tra locus. Although the majority of strains were also found to contain conjugative IncI1 plasmids of similar size to pSE115(~90kb) and harbor a variety of blaCTX-MGroup 1 and Group 9 elements, the novel MRR site at the tra locus in pSE115 was not detectable in the other IncI1 plasmids. Findings in this study show that cephalosporin resistance in S. Enteritidis strains collected in China was mainly due to dissemination of blaCTX-M-encoding IncI1 plasmids, resembling the situation in which IncI1 plasmids serve as major vectors of blaCTX-M variants in other members of Enterobacteriaceae. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

Complete sequence of conjugative IncA/C plasmid encoding CMY-2 ß-lactamase and RmtE 16S rRNA methyltransferase.

RmtE is a rare 16S-RMTase which was first reported in an aminoglycoside-resistant Escherichia coli strain of calf origin (1). Subsequently, we reported the first human case of infection caused by RmtE-producing E. coli (2). The rmtE gene is carried on a self-conjugative plasmid (pYDC637) in the latter strain. The present work aimed to elucidate the genetic context of rmtE. The sequencing approach has been described previously (3). In brief, the plasmid was extracted from an E. coli TOP10 transformant carrying pYDC637 and sequenced on a PacBio RS II sequencing instrument (Pacific Biosciences, Menlo Park, CA). Assembly was also conducted using the HGAP pipeline (Pacific Biosciences) as previously described (3).


July 7, 2019  |  

vanG element insertions within a conserved chromosomal site conferring vancomycin resistance to Streptococcus agalactiae and Streptococcus anginosus.

Three vancomycin-resistant streptococcal strains carrying vanG elements (two invasive Streptococcus agalactiae isolates [GBS-NY and GBS-NM, both serotype II and multilocus sequence type 22] and one Streptococcus anginosus [Sa]) were examined. The 45,585-bp elements found within Sa and GBS-NY were nearly identical (together designated vanG-1) and shared near-identity over an ~15-kb overlap with a previously described vanG element from Enterococcus faecalis. Unexpectedly, vanG-1 shared much less homology with the 49,321-bp vanG-2 element from GBS-NM, with widely different levels (50% to 99%) of sequence identity shared among 44 related open reading frames. Immediately adjacent to both vanG-1 and vanG-2 were 44,670-bp and 44,680-bp integrative conjugative element (ICE)-like sequences, designated ICE-r, that were nearly identical in the two group B streptococcal (GBS) strains. The dual vanG and ICE-r elements from both GBS strains were inserted at the same position, between bases 1328 and 1329, within the identical RNA methyltransferase (rumA) genes. A GenBank search revealed that although most GBS strains contained insertions within this specific site, only sequence type 22 (ST22) GBS strains contained highly related ICE-r derivatives. The vanG-1 element in Sa was also inserted within this position corresponding to its rumA homolog adjacent to an ICE-r derivative. vanG-1 insertions were previously reported within the same relative position in the E. faecalis rumA homolog. An ICE-r sequence perfectly conserved with respect to its counterpart in GBS-NY was apparent within the same site of the rumA homolog of a Streptococcus dysgalactiae subsp. equisimilis strain. Additionally, homologous vanG-like elements within the conserved rumA target site were evident in Roseburia intestinalis. Importance: These three streptococcal strains represent the first known vancomycin-resistant strains of their species. The collective observations made from these strains reveal a specific hot spot for insertional elements that is conserved between streptococci and different Gram-positive species. The two GBS strains potentially represent a GBS lineage that is predisposed to insertion of vanG elements. Copyright © 2014 Srinivasan et al.


July 7, 2019  |  

Genome sequencing of two Neorhizobium galegae strains reveals a noeT gene responsible for the unusual acetylation of the nodulation factors.

The species Neorhizobium galegae comprises two symbiovars that induce nodules on Galega plants. Strains of both symbiovars, orientalis and officinalis, induce nodules on the same plant species, but fix nitrogen only in their own host species. The mechanism behind this strict host specificity is not yet known. In this study, genome sequences of representatives of the two symbiovars were produced, providing new material for studying properties of N. galegae, with a special interest in genomic differences that may play a role in host specificity.The genome sequences confirmed that the two representative strains are much alike at a whole-genome level. Analysis of orthologous genes showed that N. galegae has a higher number of orthologs shared with Rhizobium than with Agrobacterium. The symbiosis plasmid of strain HAMBI 1141 was shown to transfer by conjugation under optimal conditions. In addition, both sequenced strains have an acetyltransferase gene which was shown to modify the Nod factor on the residue adjacent to the non-reducing-terminal residue. The working hypothesis that this gene is of major importance in directing host specificity of N. galegae could not, however, be confirmed.Strains of N. galegae have many genes differentiating them from strains of Agrobacterium, Rhizobium and Sinorhizobium. However, the mechanism behind their ecological difference is not evident. Although the final determinant for the strict host specificity of N. galegae remains to be identified, the gene responsible for the species-specific acetylation of the Nod factors was identified in this study. We propose the name noeT for this gene to reflect its role in symbiosis.


July 7, 2019  |  

Complete sequence of a conjugative IncN plasmid harboring blakpc-2, blashv-12, and qnrS1 from an Escherichia coli sequence type 648 strain

We sequenced a novel conjugative blaKPC-2-harboring IncN plasmid, pYD626E, from an Escherichia coli sequence type 648 strain previously identified in Pittsburgh, Pennsylvania. pYD626E was 72,800 bp long and carried four ß-lactamase genes, blaKPC-2, blaSHV-12, blaLAP-1, and blaTEM-1. In addition, it harbored qnrS1 (fluoroquinolone resistance) and dfrA14 (trimethoprim resistance). The plasmid profile and clinical history supported the in vivo transfer of this plasmid between Klebsiella pneumoniae and Escherichia coli. Copyright © 2014, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

Plasmid characterization and chromosome analysis of two netF+ Clostridium perfringens isolates associated with foal and canine necrotizing enteritis.

The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and unique plasmid-encoded locus.


July 7, 2019  |  

Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation.

Butanol is currently one of the most discussed biofuels. Its use provides many benefits in comparison to bio-ethanol, but the price of its fermentative production is still high. Genetic improvements could help solve many problems associated with butanol production during ABE fermentation, such as its toxicity, low concentration achievable in the cultivation medium, the need for a relatively expensive substrate, and many more. Clostridium pasteurianum NRRL B-598 is non-type strain producing butanol, acetone, and a negligible amount of ethanol. Its main benefits are high oxygen tolerance, utilization of a wide range of carbon and nitrogen sources, and the availability of its whole genome sequence. However, there is no established method for the transfer of foreign DNA into this strain; this is the next step necessary for progress in its use for butanol production.We have described functional protocols for conjugation and transformation of the bio-butanol producer C. pasteurianum NRRL B-598 by foreign plasmid DNA. We show that the use of unmethylated plasmid DNA is necessary for efficient transformation or successful conjugation. Genes encoding DNA methylation and those for restriction-modification systems and antibiotic resistance were searched for in the whole genome sequence and their homologies with other clostridial bacteria were determined. Furthermore, activity of described novel type I restriction system was proved experimentally. The described electrotransformation protocol achieved an efficiency 1.2 × 10(2) cfu/µg DNA after step-by-step optimization and an efficiency of 1.6 × 10(2) cfu/µg DNA was achieved by the sonoporation technique using a standard laboratory ultrasound bath. The highest transformation efficiency was achieved using a combination of these approaches; sono/electroporation led to an increase in transformation efficiency, to 5.3 × 10(2) cfu/µg DNA.Both Dam and Dcm methylations are detrimental for transformation of C. pasteurianum NRRL B-598. Methods for conjugation, electroporation, sonoporation, and a combined method for sono/electroporation were established for this strain. The methods described could be used for genetic improvement of this strain, which is suitable for bio-butanol production.


July 7, 2019  |  

Complete genome sequence of MIDG2331, a genetically tractable serovar 8 clinical isolate of Actinobacillus pleuropneumoniae.

We report here the complete annotated genome sequence of a clinical serovar 8 isolate Actinobacillus pleuropneumoniae MIDG2331. Unlike the serovar 8 reference strain 405, MIDG2331 is amenable to genetic manipulation via natural transformation as well as conjugation, making it ideal for studies of gene function. Copyright © 2016 Bossé et al.


July 7, 2019  |  

Complete genome sequence of UV-resistant Campylobacter jejuni RM3194, including an 81.08-kilobase plasmid.

Campylobacter jejuni strain RM3194 was originally isolated from a human with enteritis and contains a novel 81,079-bp plasmid. RM3194 has exhibited superior survival compared to other Campylobacter jejuni strains when challenged with UV light. The chromosome of RM3194 was determined to be 1,651,183 bp, with a G+C content of 30.5%. Copyright © 2016 Gunther et al.


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