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Asset Tag: Bacterial genome

July 7, 2019

Complete genome sequencing and comparative genomic analysis of the thermotolerant acetic acid bacterium, Acetobacter pasteurianus SKU1108, provide a new insight into thermotolerance.

Acetobacter pasteurianus SKU1108 is a typical thermotolerant acetic acid bacterium. In this study, the complete genome sequence of the SKU1108 strain was elucidated, and information on genomic modifications due to the thermal adaptation of SKU1108 was updated. In order to obtain a clearer understanding of the genetic background responsible for thermotolerance, the SKU1108 genome was compared with those of two closely related complete genome strains, thermotolerant A. pasteurianus 386B and mesophilic A. pasteurianus NBRC 3283. All 24 “thermotolerant genes” required for growth at higher temperatures in the thermotolerant Acetobacter tropicalis SKU1100 strain were conserved in all three strains. However, these thermotolerant genes accumulated amino acid mutations. Some biased mutations, particularly those that occurred in xanthine dehydrogenase XdhA, may be related to thermotolerance. By aligning whole genome sequences, we identified ten SKU1108 strain-specific regions, three of which were conserved in the genomes of the two thermotolerant A. pasteurianus strains. One of the regions contained a unique paralog of the thermotolerant gene xdhA, which may also be responsible for conferring thermotolerance. Thus, comparative genomics of complete genome sequences may provide novel insights into the phenotypes of these thermotolerant strains.

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July 7, 2019

Use of WGS data for investigation of a long-term NDM-1-producing Citrobacter freundii outbreak and secondary in vivo spread of blaNDM-1 to Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca.

An outbreak of NDM-1-producing Citrobacter freundii and possible secondary in vivo spread of blaNDM-1 to other Enterobacteriaceae were investigated.From October 2012 to March 2015, meropenem-resistant Enterobacteriaceae were detected in 45 samples from seven patients at Aalborg University Hospital, Aalborg, Denmark. In silico resistance genes, Inc plasmid types and STs (MLST) were obtained from WGS data from 24 meropenem-resistant isolates (13 C. freundii, 6 Klebsiella pneumoniae, 4 Escherichia coli and 1 Klebsiella oxytoca) and 1 meropenem-susceptible K. oxytoca. The sequences of the meropenem-resistant C. freundii isolates were compared by phylogenetic analyses. In vitro susceptibility to 21 antimicrobial agents was tested. Furthermore, in vitro conjugation and plasmid characterization was performed.From the seven patients, 13 highly clonal ST18 NDM-1-producing C. freundii were isolated. The ST18 NDM-1-producing C. freundii isolates were only susceptible to tetracycline, tigecycline, colistin and fosfomycin (except for the C. freundii isolates from Patient 2 and Patient 7, which were additionally resistant to tetracycline). The E. coli and K. pneumoniae from different patients belonged to different STs, indicating in vivo transfer of blaNDM-1 in the individual patients. This was further supported by in vitro conjugation and detection of a 154 kb IncA/C2 plasmid with blaNDM-1. Patient screenings failed to reveal any additional cases. None of the patients had a history of recent travel abroad and the source of the blaNDM-1 plasmid was unknown.To our knowledge, this is the first report of an NDM-1-producing C. freundii outbreak and secondary in vivo spread of an IncA/C2 plasmid with blaNDM-1 to other Enterobacteriaceae.© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Full-length nucleotide sequences of mcr-1-harboring plasmids isolated from extended- spectrum-ß-lactamase-producing Escherichia coli isolates of different origins.

Here, we present the full sequences of three mcr-1-carrying plasmids isolated from extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli The plasmids belong to three different replicon types and are 34,640 bp, 209,401 bp, and 247,885 bp in size. We describe for the first time a composite transposon containing mcr-1 localized on a multidrug-resistant (MDR) IncHI2 plasmid harboring additional determinants of resistance to six different classes of antibiotics, including the ESBL gene blaCTX-M-1, and heavy metal resistance. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

The complete genome sequence of the nicotine-degrading bacterium Shinella sp. HZN7.

Nicotine is a natural alkaloid that is very toxic to humans. To eliminate the harmful effects of nicotine in the environment, biological methods employing microbes to degrade nicotine are required (Brandsch, 2006; Liu et al., 2015). Shinella sp. HZN7 can degrade nicotine efficiently via the variant of a pyridine and pyrrolidine pathways (VPP; Ma et al., 2013; Qiu et al., 2014, 2015). The main intermediates in this pathway include 6-hydroxy-nicotine, 6-hydroxy-N-methylmyosmine, 6-hydroxypseudooxynicotine, 6-hydroxy-3-succinoyl-pyridine, and 2,5-dihydroxypyridine. This strain is the first nicotine-degrading bacterium to be isolated from the genus Shinella.

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July 7, 2019

Whole genome sequence analysis indicates recent diversification of mammal-associated Campylobacter fetus and implicates a genetic factor associated with H2S production.

Campylobacter fetus (C. fetus) can cause disease in both humans and animals. C. fetus has been divided into three subspecies: C. fetus subsp. fetus (Cff), C. fetus subsp. venerealis (Cfv) and C. fetus subsp. testudinum (Cft). Subspecies identification of mammal-associated C. fetus strains is crucial in the control of Bovine Genital Campylobacteriosis (BGC), a syndrome associated with Cfv. The prescribed methods for subspecies identification of the Cff and Cfv isolates are: tolerance to 1 % glycine and H2S production.In this study, we observed the deletion of a putative cysteine transporter in the Cfv strains, which are not able to produce H2S from L-cysteine. Phylogenetic reconstruction of the core genome single nucleotide polymorphisms (SNPs) within Cff and Cfv strains divided these strains into five different clades and showed that the Cfv clade and a Cff clade evolved from a single Cff ancestor.Multiple C. fetus clades were observed, which were not consistent with the biochemical differentiation of the strains. This suggests the need for a closer evaluation of the current C. fetus subspecies differentiation, considering that the phenotypic differentiation is still applied in BGC control programs.

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July 7, 2019

Genome and plasmid analysis of blaIMP-4 -carrying Citrobacter freundii B38.

Sequencing of the blaIMP-4 -carrying C. freundii B38 using PacBio SMRT technique revealed that the genome contained a chromosome of 5,134,500 bp, and three plasmids, pOZ172 (127,005 bp), pOZ181 (277,592 bp), and pOZ182 (18,467 bp). Plasmid pOZ172 was identified as IncFIIY, like pP10164-NDM and pNDM-EcGN174. It carries a class 1 integron with four cassettes: blaIMP-4-qacG2-aacA4-aphA15, and a complete hybrid tni module (tniR-tniQ-tniB-tniA). The recombination of tniR from Tn402 (identical) with tniQBA (99%) from Tn5053 occurred within the res site of Tn402/5053. The Tn402/5053-like integron, named Tn6017, was inserted into Tn1722 at the res II site. The replication, partitioning and transfer systems of pOZ181 were similar to IncHI2 (e.g. R478) and contained a sul1-type class 1 integron with the cassette array: orf-dfrA1-orf-gcu37-aadA5 linked to an upstream Tn1696 tnpA-tnpR and to a downstream 3′ CS and ISCR1 A Tn2 transposon with a blaTEM-1b ß-lactamase was identified on pOZ182. Other interesting resistance determinants on the B38 chromosome included MDR efflux pumps, AmpC ß-lactamase, and resistances to Cu, Ag, As, and Zn. This is the first report of a complete tni module linked to a blaIMP- 4 carrying class 1 integron, and together with other recently reported non-sul1 integrons, represents the emergence of a distinct evolutionary lineage of class 1 integrons lacking a 3′ -CS (qacE?1-sul1). The unique cassette array, complete tni module of Tn6017, and incompatibility group of pOZ172 suggests a different blaIMP-4 evolutionary pathway in C. freundii B38 compared to other blaIMP-4 foundin Gram-negative bacteria in the Western Pacific Region. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Genomic studies of nitrogen-fixing rhizobial strains from Phaseolus vulgaris seeds and nodules.

Rhizobia are soil bacteria that establish symbiotic relationships with legumes and fix nitrogen in root nodules. We recently reported that several nitrogen-fixing rhizobial strains, belonging to Rhizobium phaseoli, R. trifolii, R. grahamii and Sinorhizobium americanum, were able to colonize Phaseolus vulgaris (common bean) seeds. To gain further insight into the traits that support this ability, we analyzed the genomic sequences and proteomes of R. phaseoli (CCGM1) and S. americanum (CCGM7) strains from seeds and compared them with those of the closely related strains CIAT652 and CFNEI73, respectively, isolated only from nodules.In a fine structural study of the S. americanum genomes, the chromosomes, megaplasmids and symbiotic plasmids were highly conserved and syntenic, with the exception of the smaller plasmid, which appeared unrelated. The symbiotic tract of CCGM7 appeared more disperse, possibly due to the action of transposases. The chromosomes of seed strains had less transposases and strain-specific genes. The seed strains CCGM1 and CCGM7 shared about half of their genomes with their closest strains (3353 and 3472 orthologs respectively), but a large fraction of the rest also had homology with other rhizobia. They contained 315 and 204 strain-specific genes, respectively, particularly abundant in the functions of transcription, motility, energy generation and cofactor biosynthesis. The proteomes of seed and nodule strains were obtained and showed a particular profile for each of the strains. About 82 % of the proteins in the comparisons appeared similar. Forty of the most abundant proteins in each strain were identified; these proteins in seed strains were involved in stress responses and coenzyme and cofactor biosynthesis and in the nodule strains mainly in central processes. Only 3 % of the abundant proteins had hypothetical functions.Functions that were enriched in the genomes and proteomes of seed strains possibly participate in the successful occupancy of the new niche. The genome of the strains had features possibly related to their presence in the seeds. This study helps to understand traits of rhizobia involved in seed adaptation.

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July 7, 2019

Comparative genomics reveals Lysinibacillus sphaericus group comprises a novel species.

Early in the 1990s, it was recognized that Lysinibacillus sphaericus, one of the most popular and effective entomopathogenic bacteria, was a highly heterogeneous group. Many authors have even proposed it comprises more than one species, but the lack of phenotypic traits that guarantee an accurate differentiation has not allowed this issue to be clarified. Now that genomic technologies are rapidly advancing, it is possible to address the problem from a whole genome perspective, getting insights into the phylogeny, evolutive history and biology itself.The genome of the Colombian strain L. sphaericus OT4b.49 was sequenced, assembled and annotated, obtaining 3 chromosomal contigs and no evidence of plasmids. Using these sequences and the 13 other L. sphaericus genomes available on the NCBI database, we carried out comparative genomic analyses that included whole genome alignments, searching for mobile elements, phylogenomic metrics (TETRA, ANI and in-silico DDH) and pan-genome assessments. The results support the hypothesis about this species as a very heterogeneous group. The entomopathogenic lineage is actually a single and independent species with 3728 core genes and 2153 accessory genes, whereas each non-toxic strain seems to be a separate species, though without a clear circumscription. Toxin-encoding genes, binA, B and mtx1, 2, 3 could be acquired via horizontal gene transfer in a single evolutionary event. The non-toxic strain OT4b.31 is the most related with the type strain KCTC 3346.The current L. sphaericus is actually a sensu lato due to a sub-estimation of diversity accrued using traditional non-genomics based classification strategies. The toxic lineage is the most studied with regards to its larvicidal activity, which is a greatly conserved trait among these strains and thus, their differentiating feature. Further studies are needed in order to establish a univocal classification of the non-toxic strains that, according to our results, seem to be a paraphyletic group.

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July 7, 2019

Complete sequencing of plasmids containing blaOXA-163 and blaOXA-48 in Escherichia coli ST131.

OXA-48-like enzymes have emerged as important extended-spectrum ß-lactamases/carbapenemases in E. coli ST131. We report the structure of the first fully sequenced blaOXA-163 plasmid, and of two other blaOXA-48 plasmids in this lineage. blaOXA-163 was located on a 71kb IncN plasmid with other resistance genes. blaOXA-48 was present on IncL/M plasmids, genetically similar to other blaOXA-48 plasmid sequences, and consistent with inter-species/inter-lineage spread. The presence of blaOXA-48-like genes on epidemic plasmids in ST131 is of concern. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Plasmids from Shiga toxin-producing Escherichia coli strains with rare enterohemolysin gene (ehxA) subtypes reveal pathogenicity potential and display a novel evolutionary path.

Most Shiga toxin-producing Escherichia coli (STEC) strains associated with severe disease, such as hemolytic-uremic syndrome (HUS), carry large enterohemolysin-encoding (ehxA) plasmids, e.g., pO157 and pO103, that contribute to STEC clinical manifestations. Six ehxA subtypes (A through F) exist that phylogenetically cluster into eae-positive (B, C, F), a mix of eae-positive (E) and eae-negative (A), and a third, more distantly related, cluster of eae-negative (D) STEC strains. While subtype B, C, and F plasmids share a number of virulence traits that are distinct from those of subtype A, sequence data have not been available for subtype D and E plasmids. Here, we determined and compared the genetic composition of four subtype D and two subtype E plasmids to establish their evolutionary relatedness among ehxA subtypes and define their potential role in pathogenicity. We found that subtype D strains carry one exceptionally large plasmid (>200 kbp) that carries a variety of virulence genes that are associated with enterotoxigenic and enterohemorrhagic E. coli, which, quite possibly, enables these strains to cause disease despite being food isolates. Our data offer further support for the hypothesis that this subtype D plasmid represents a novel virulence plasmid, sharing very few genetic features with other plasmids; we conclude that these plasmids have evolved from a different evolutionary lineage than the plasmids carrying the other ehxA subtypes. In contrast, the 50-kbp plasmids of subtype E (pO145), although isolated from HUS outbreak strains, carried only few virulence-associated determinants, suggesting that the clinical presentation of subtype E strains is largely a result of chromosomally encoded virulence factors.Bacterial plasmids are known to be key agents of change in microbial populations, promoting the dissemination of various traits, such as drug resistance and virulence. This study determined the genetic makeup of virulence plasmids from rare enterohemolysin subtype D and E Shiga toxin-producing E. coli strains. We demonstrated that ehxA subtype D plasmids represent a novel E. coli virulence plasmid, and although subtype D plasmids were derived from nonclinical isolates, they encoded a variety of virulence determinants that are associated with pathogenic E. coli In contrast, subtype E plasmids, isolated from strains recovered from severely ill patients, carry only a few virulence determinants. The results of this study reemphasize the plasticity and vast diversity among E. coli plasmids. This work demonstrates that, although E. coli strains of certain serogroups may not be frequently associated with disease, they should not be underestimated in protecting human health and food safety. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

IncHI2 plasmids are the key vectors responsible for oqxAB transmission among Salmonella species.

This study reported and analysed the complete sequences of two oqxAB-bearing IncHI2 plasmids harboured by a clinical S. Typhimurium strain and an S. Indiana strain of animal origin, respectively. Particularly, pA3T recovered from S. Indiana comprised the resistance determinants oqxAB, aac(6′)Ib-cr, fosA3 and blaCTX-M-14 Further genetic screening of 63 oqxAB-positive Salmonella spp. isolates revealed that the majority carried IncHI2 plasmids, confirming that such plasmids play a pivotal role in dissemination of oqxAB in Salmonella spp.. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Multiplication of blaOXA-23 is common in clinical Acinetobacter baumannii, but does not enhance carbapenem resistance.

To investigate the copy number of blaOXA-23 and its correlation with carbapenem resistance in carbapenem-resistant Acinetobacter baumannii (CRAB).A total of 113 blaOXA-23-positive clinical CRAB isolates were collected from two hospitals in Zhejiang province, China. Their genetic relatedness was determined by MLST. The MIC of imipenem was determined using the agar diffusion method and the copy number of blaOXA-23 was measured using quantitative real-time PCR (qRT-PCR). The complete genomes of five clinical CRAB strains were sequenced using PacBio technology to investigate the multiplication mechanism of blaOXA-23.Most of the isolates (100/113) belonged to global clone II and the MIC of imipenem ranged from 16 to 96 mg/L. The gene blaOXA-23 resided exclusively in Tn2006 or Tn2009. Approximately 38% of the isolates carried two or more copies of blaOXA-23. The copy number of blaOXA-23 was not correlated with the MIC of imipenem. Within the five sequenced strains, multiple copies of blaOXA-23 were either tandemly clustered or independently inserted at different genomic sites.Multiplication of blaOXA-23 is common in CRAB, but does not enhance carbapenem resistance. Multiplication can be present in the form of either tandem amplifications or independent insertions at different sites.© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

MALDI-TOF mass spectrometry enables a comprehensive and fast analysis of dynamics and qualities of stress responses of Lactobacillus paracasei subsp. paracasei F19.

Lactic acid bacteria (LAB) are widely used as starter cultures in the manufacture of foods. Upon preparation, these cultures undergo various stresses resulting in losses of survival and fitness. In order to find conditions for the subsequent identification of proteomic biomarkers and their exploitation for preconditioning of strains, we subjected Lactobacillus (Lb.) paracasei subsp. paracasei TMW 1.1434 (F19) to different stress qualities (osmotic stress, oxidative stress, temperature stress, pH stress and starvation stress). We analysed the dynamics of its stress responses based on the expression of stress proteins using MALDI-TOF mass spectrometry (MS), which has so far been used for species identification. Exploiting the methodology of accumulating protein expression profiles by MALDI-TOF MS followed by the statistical evaluation with cluster analysis and discriminant analysis of principle components (DAPC), it was possible to monitor the expression of low molecular weight stress proteins, identify a specific time point when the expression of stress proteins reached its maximum, and statistically differentiate types of adaptive responses into groups. Above the specific result for F19 and its stress response, these results demonstrate the discriminatory power of MALDI-TOF MS to characterize even dynamics of stress responses of bacteria and enable a knowledge-based focus on the laborious identification of biomarkers and stress proteins. To our knowledge, the implementation of MALDI-TOF MS protein profiling for the fast and comprehensive analysis of various stress responses is new to the field of bacterial stress responses. Consequently, we generally propose MALDI-TOF MS as an easy and quick method to characterize responses of microbes to different environmental conditions, to focus efforts of more elaborate approaches on time points and dynamics of stress responses.

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July 7, 2019

Novel methyltransferase recognition motif identified in Chania multitudinisentens RB-25(T) gen. nov., sp. nov.

DNA methylation, defined by the addition of a methyl group to adenine or cytosine bases in DNA catalyzed by DNA methyltransferases (MTases), is one of the most studied post-replicative DNA modification mechanism in bacteria (Roberts et al., 2003b). The three forms of nucleotide methylation identified to date are: N6-methyladenine(m6A), N4-methylcytosine (m4C), and 5-methylcytosine (m5C) (Gromova and Khoroshaev, 2003).

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