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July 7, 2019

Genetic and functional characterization of blaCTX-M-199, a novel tazobactam and sulbactam resistance-encoding gene located in a conjugative mcr-1-bearing IncI2 plasmid.

The study reported the genetic and functional characterization of a novel CTX-M-199 ß-lactamase, which was encoded by a blaCTX-M-64 variant gene found in a conjugative mcr-1-bearing IncI2 plasmid and exhibited resistance to ß-lactamase inhibitors, tazobactam and sulbactam. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Tripartite species interaction: eukaryotic hosts suffer more from phage susceptible than from phage resistant bacteria.

Evolutionary shifts in bacterial virulence are often associated with a third biological player, for instance temperate phages, that can act as hyperparasites. By integrating as prophages into the bacterial genome they can contribute accessory genes, which can enhance the fitness of their prokaryotic carrier (lysogenic conversion). Hyperparasitic influence in tripartite biotic interactions has so far been largely neglected in empirical host-parasite studies due to their inherent complexity. Here we experimentally address whether bacterial resistance to phages and bacterial harm to eukaryotic hosts is linked using a natural tri-partite system with bacteria of the genus Vibrio, temperate vibriophages and the pipefish Syngnathus typhle. We induced prophages from all bacterial isolates and constructed a three-fold replicated, fully reciprocal 75 × 75 phage-bacteria infection matrix.According to their resistance to phages, bacteria could be grouped into three distinct categories: highly susceptible (HS-bacteria), intermediate susceptible (IS-bacteria), and resistant (R-bacteria). We experimentally challenged pipefish with three selected bacterial isolates from each of the three categories and determined the amount of viable Vibrio counts from infected pipefish and the expression of pipefish immune genes. While the amount of viable Vibrio counts did not differ between bacterial groups, we observed a significant difference in relative gene expression between pipefish infected with phage susceptible and phage resistant bacteria.These findings suggest that bacteria with a phage-susceptible phenotype are more harmful against a eukaryotic host, and support the importance of hyperparasitism and the need for an integrative view across more than two levels when studying host-parasite evolution.

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July 7, 2019

Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli.

The mobile colistin resistance gene mcr-1 has attracted global attention, as it heralds the breach of polymyxins, one of the last-resort antibiotics for the treatment of severe clinical infections caused by multidrug-resistant Gram-negative bacteria. To date, six slightly different variants of mcr-1, and a second mobile colistin resistance gene, mcr-2, have been reported or annotated in the GenBank database. Here, we characterized a third mobile colistin resistance gene, mcr-3 The gene coexisted with 18 additional resistance determinants in the 261-kb IncHI2-type plasmid pWJ1 from porcine Escherichia colimcr-3 showed 45.0% and 47.0% nucleotide sequence identity to mcr-1 and mcr-2, respectively, while the deduced amino acid sequence of MCR-3 showed 99.8 to 100% and 75.6 to 94.8% identity to phosphoethanolamine transferases found in other Enterobacteriaceae species and in 10 Aeromonas species, respectively. pWJ1 was mobilized to an E. coli recipient by conjugation and contained a plasmid backbone similar to those of other mcr-1-carrying plasmids, such as pHNSHP45-2 from the original mcr-1-harboring E. coli strain. Moreover, a truncated transposon element, TnAs2, which was characterized only in Aeromonas salmonicida, was located upstream of mcr-3 in pWJ1. This ?TnAs2-mcr-3 element was also identified in a shotgun genome sequence of a porcine E. coli isolate from Malaysia, a human Klebsiella pneumoniae isolate from Thailand, and a human Salmonella enterica serovar Typhimurium isolate from the United States. These results suggest the likelihood of a wide dissemination of the novel mobile colistin resistance gene mcr-3 among Enterobacteriaceae and aeromonads; the latter may act as a potential reservoir for mcr-3IMPORTANCE The emergence of the plasmid-mediated colistin resistance gene mcr-1 has attracted substantial attention worldwide. Here, we examined a colistin-resistant Escherichia coli isolate that was negative for both mcr-1 and mcr-2 and discovered a novel mobile colistin resistance gene, mcr-3 The amino acid sequence of MCR-3 aligned closely with phosphoethanolamine transferases from Enterobacteriaceae and Aeromonas species originating from both clinical infections and environmental samples collected in 12 countries on four continents. Due to the ubiquitous profile of aeromonads in the environment and the potential transfer of mcr-3 between Enterobacteriaceae and Aeromonas species, the wide spread of mcr-3 may be largely underestimated. As colistin has been and still is widely used in veterinary medicine and used at increasing frequencies in human medicine, the continuous monitoring of mobile colistin resistance determinants in colistin-resistant Gram-negative bacteria is imperative for understanding and tackling the dissemination of mcr genes in both the agricultural and health care sectors. Copyright © 2017 Yin et al.

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July 7, 2019

Complete genome sequence of the cellulose-producing strain Komagataeibacter nataicola RZS01.

Komagataeibacter nataicola is an acetic acid bacterium (AAB) that can produce abundant bacterial cellulose and tolerate high concentrations of acetic acid. To globally understand its fermentation characteristics, we present a high-quality complete genome sequence of K. nataicola RZS01. The genome consists of a 3,485,191-bp chromosome and 6 plasmids, which encode 3,514 proteins and bear three cellulose synthase operons. Phylogenetic analysis at the genome level provides convincing evidence of the evolutionary position of K. nataicola with respect to related taxa. Genomic comparisons with other AAB revealed that RZS01 shares 36.1%~75.1% of sequence similarity with other AAB. The sequence data was also used for metabolic analysis of biotechnological substrates. Analysis of the resistance to acetic acid at the genomic level indicated a synergistic mechanism responsible for acetic acid tolerance. The genomic data provide a viable platform that can be used to understand and manipulate the phenotype of K. nataicola RZS01 to further improve bacterial cellulose production.

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July 7, 2019

Non-toxin-producing Bacillus cereus strains belonging to the B. anthracis clade isolated from the International Space Station.

In an ongoing Microbial Observatory investigation of the International Space Station (ISS), 11 Bacillus strains (2 from the Kibo Japanese experimental module, 4 from the U.S. segment, and 5 from the Russian module) were isolated and their whole genomes were sequenced. A comparative analysis of the 16S rRNA gene sequences of these isolates showed the highest similarity (>99%) to the Bacillus anthracis-B. cereus-B. thuringiensis group. The fatty acid composition, polar lipid profile, peptidoglycan type, and matrix-assisted laser desorption ionization-time of flight profiles were consistent with the B. cereus sensu lato group. The phenotypic traits such as motile rods, enterotoxin production, lack of capsule, and resistance to gamma phage/penicillin observed in ISS isolates were not characteristics of B. anthracis. Whole-genome sequence characterizations showed that ISS strains had the plcR non-B. anthracis ancestral “C” allele and lacked anthrax toxin-encoding plasmids pXO1 and pXO2, excluding their identification as B. anthracis. The genetic identities of all 11 ISS isolates characterized via gyrB analyses arbitrarily identified them as members of the B. cereus group, but traditional DNA-DNA hybridization (DDH) showed that the ISS isolates are similar to B. anthracis (88% to 90%) but distant from the B. cereus (42%) and B. thuringiensis (48%) type strains. The DDH results were supported by average nucleotide identity (>98.5%) and digital DDH (>86%) analyses. However, the collective phenotypic traits and genomic evidence were the reasons to exclude the ISS isolates from B. anthracis. Nevertheless, multilocus sequence typing and whole-genome single nucleotide polymorphism analyses placed these isolates in a clade that is distinct from previously described members of the B. cereus sensu lato group but closely related to B. anthracis. IMPORTANCE The International Space Station Microbial Observatory (Microbial Tracking-1) study is generating a microbial census of the space station’s surfaces and atmosphere by using advanced molecular microbial community analysis techniques supported by traditional culture-based methods and modern bioinformatic computational modeling. This approach will lead to long-term, multigenerational studies of microbial population dynamics in a closed environment and address key questions, including whether microgravity influences the evolution and genetic modification of microorganisms. The spore-forming Bacillus cereus sensu lato group consists of pathogenic (B. anthracis), food poisoning (B. cereus), and biotechnologically useful (B. thuringiensis) microorganisms; their presence in a closed system such as the ISS might be a concern for the health of crew members. A detailed characterization of these potential pathogens would lead to the development of suitable countermeasures that are needed for long-term future missions and a better understanding of microorganisms associated with space missions.

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July 7, 2019

Phenotypic and genomic survey on organic acid utilization profile of Pseudomonas mendocina strain S5.2, a vineyard soil isolate.

Root exudates are chemical compounds that are released from living plant roots and provide significant energy, carbon, nitrogen and phosphorus sources for microbes inhabiting the rhizosphere. The exudates shape the microflora associated with the plant, as well as influences the plant health and productivity. Therefore, a better understanding of the trophic link that is established between the plant and the associated bacteria is necessary. In this study, a comprehensive survey on the utilization of grapevine and rootstock related organic acids were conducted on a vineyard soil isolate which is Pseudomonas mendocina strain S5.2. Phenotype microarray analysis has demonstrated that this strain can utilize several organic acids including lactic acid, succinic acid, malic acid, citric acid and fumaric acid as sole growth substrates. Complete genome analysis using single molecule real-time technology revealed that the genome consists of a 5,120,146 bp circular chromosome and a 252,328 bp megaplasmid. A series of genetic determinants associated with the carbon utilization signature of the strain were subsequently identified in the chromosome. Of note, the coexistence of genes encoding several iron-sulfur cluster independent isoenzymes in the genome indicated the importance of these enzymes in the events of iron deficiency. Synteny and comparative analysis have also unraveled the unique features of D-lactate dehydrogenase of strain S5.2 in the study. Collective information of this work has provided insights on the metabolic role of this strain in vineyard soil rhizosphere.

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July 7, 2019

Genetic and biochemical characterization of HMB-1, a novel subclass B1 metallo-ß-lactamase found in a Pseudomonas aeruginosa clinical isolate.

To characterize a novel subclass B1 metallo-ß-lactamase (MBL) found in an MDR Pseudomonas aeruginosa clinical isolate.The isolate P. aeruginosa NRZ-03096 was recovered in 2012 from an anal swab from a patient hospitalized in Northern Germany and showed high MICs of carbapenems. MBL production was analysed by several phenotypic tests. Genetic characterization of the novel bla gene and MLST was performed by WGS. The novel bla gene was expressed in Escherichia coli TOP10 and the enzyme was subjected to biochemical characterization to determine the kinetic parameters K m and k cat .P. aeruginosa NRZ-03096 was resistant to all tested ß-lactams and showed an MBL phenotype. Shotgun cloning experiments yielded a clone producing a novel subclass B1 enzyme with only 74.3% identity to the next nearest relative, KHM-1. The novel MBL was named HMB-1 (for Hamburg MBL). Analysis of WGS data showed that the bla HMB-1 gene was chromosomally located as part of a Tn 3 family transposon that was named Tn 6345 . Expression of bla HMB-1 in E. coli TOP10 led to increased resistance to ß-lactams. Determination of K m and k cat revealed that HMB-1 had different hydrolytic characteristics compared with KHM-1, with lower hydrolytic rates for cephalosporins and a higher rate for imipenem.The identification of HMB-1 further underlines the ongoing spread and diversification of carbapenemases in Gram-negative human pathogens and especially in P. aeruginosa .

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July 7, 2019

Complete genome sequence of Stenotrophomonas sp. KCTC 12332, a biotechnological potential bacterium.

Hydroxy fatty acids are used in various industries due to their availability, and in particular, Stenotrophomonas sp. has been regarded as a potential candidate for biotechnological applications, including biotransformation that hydrate unsaturated fatty acids into their derivatives. Here we complete the genome sequence of Stenotrophomonas sp. KCTC 12332 which has a size of 4,541,594bp (G+C content of 63.83%) with 3790 coding DNA sequences (CDSs), 67 tRNA and 3 rRNA operons. The genome contains gene encoding oleate hydratase that can convert oleic acid into 10-hydroxyoctadecanoic acid. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Characterization and genome comparisons of three Achromobacter phages of the family Siphoviridae.

In this study, we present the characterization and genomic data of three Achromobacter phages belonging to the family Siphoviridae. Phages 83-24, JWX and JWF were isolated from sewage samples in Paris and Braunschweig, respectively, and infect Achromobacter xylosoxidans, an emerging nosocomial pathogen in cystic fibrosis patients. Analysis of morphology and growth parameters revealed that phages 83-24 and JWX have similar properties, both have nearly the same head and tail measurements, and both have a burst size between 85 and 100 pfu/cell. In regard to morphological properties, JWF had a much longer and more flexible tail compared to other phages. The linear double-stranded DNAs of all three phages are terminally redundant and not circularly permutated. The complete nucleotide sequences consist of 81,541 bp for JWF, 49,714 bp for JWX and 48,216 bp for 83-24. Analysis of the genome sequences showed again that phages JWX and 83-24 are quite similar. Comparison to the GenBank database via BLASTN revealed partial similarities to Roseobacter phage RDJL phi1 and Burkholderia phage BcepGomr. In contrast, BLASTN analysis of the genome sequence of phage JWF revealed only few similarities to non-annotated prophage regions in different strains of Burkholderia and Mesorhizobium.

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July 7, 2019

Starvation and recovery in the deep-sea methanotroph Methyloprofundus sedimenti.

In the deep ocean, the conversion of methane into derived carbon and energy drives the establishment of diverse faunal communities. Yet specific biological mechanisms underlying the introduction of methane-derived carbon into the food web remain poorly described, due to a lack of cultured representative deep-sea methanotrophic prokaryotes. Here, the response of the deep-sea aerobic methanotroph Methyloprofundus sedimenti to methane starvation and recovery was characterized. By combining lipid analysis, RNA analysis, and electron cryotomography, it was shown that M. sedimenti undergoes discrete cellular shifts in response to methane starvation, including changes in headgroup-specific fatty acid saturation levels, and reductions in cytoplasmic storage granules. Methane starvation is associated with a significant increase in the abundance of gene transcripts pertinent to methane oxidation. Methane reintroduction to starved cells stimulates a rapid, transient extracellular accumulation of methanol, revealing a way in which methane-derived carbon may be routed to community members. This study provides new understanding of methanotrophic responses to methane starvation and recovery, and lays the initial groundwork to develop Methyloprofundus as a model chemosynthesizing bacterium from the deep sea.© 2016 John Wiley & Sons Ltd.

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July 7, 2019

Complete genome sequence of Microbulbifer sp. CCB-MM1, a halophile isolated from Matang Mangrove Forest, Malaysia.

Microbulbifer sp. CCB-MM1 is a halophile isolated from estuarine sediment of Matang Mangrove Forest, Malaysia. Based on 16S rRNA gene sequence analysis, strain CCB-MM1 is a potentially new species of genus Microbulbifer. Here we describe its features and present its complete genome sequence with annotation. The genome sequence is 3.86 Mb in size with GC content of 58.85%, harbouring 3313 protein coding genes and 92 RNA genes. A total of 71 genes associated with carbohydrate active enzymes were found using dbCAN. Ectoine biosynthetic genes, ectABC operon and ask_ect were detected using antiSMASH 3.0. Cell shape determination genes, mreBCD operon, rodA and rodZ were annotated, congruent with the rod-coccus cell cycle of the strain CCB-MM1. In addition, putative mreBCD operon regulatory gene, bolA was detected, which might be associated with the regulation of rod-coccus cell cycle observed from the strain.

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July 7, 2019

Genomic epidemiology of NDM-1-encoding plasmids in Latin American clinical isolates reveals insights into the evolution of multidrug resistance

Bacteria that produce the broad-spectrum Carbapenem antibiotic New Delhi Metallo-ß-lactamase (NDM) place a burden on health care systems worldwide, due to the limited treatment options for infections caused by them and the rapid global spread of this antibiotic resistance mechanism. Although it is believed that the associated resistance gene blaNDM-1 originated in Acinetobacter spp., the role of Enterobacteriaceae in its dissemination remains unclear. In this study, we used whole genome sequencing to investigate the dissemination dynamics of blaNDM-1-positive plasmids in a set of 21 clinical NDM-1-positive isolates from Colombia and Mexico (Providencia rettgeri, Klebsiella pneumoniae, and Acinetobacter baumannii) as well as six representative NDM-1-positive Escherichia coli transconjugants. Additionally, the plasmids from three representative P. rettgeri isolates were sequenced by PacBio sequencing and finished. Our results demonstrate the presence of previously reported plasmids from K. pneumoniae and A. baumannii in different genetic backgrounds and geographically distant locations in Colombia. Three new previously unclassified plasmids were also identified in P. rettgeri from Colombia and Mexico, plus an interesting genetic link between NDM-1-positive P. rettgeri from distant geographic locations (Canada, Mexico, Colombia, and Israel) without any reported epidemiological links was discovered. Finally, we detected a relationship between plasmids present in P. rettgeri and plasmids from A. baumannii and K. pneumoniae. Overall, our findings suggest a Russian doll model for the dissemination of blaNDM-1 in Latin America, with P. rettgeri playing a central role in this process, and reveal new insights into the evolution and dissemination of plasmids carrying such antibiotic resistance genes.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019

Genomic characterization of a large plasmid containing a bla NDM-1 gene carried on Salmonella enterica serovar Indiana C629 isolate from China.

The bla NDM-1 gene in Salmonella species is mostly reported in clinical cases, but is rarely isolated from red and white meat in China.A Salmonella Indiana (S. Indiana) isolate was cultured from a chicken carcass procured from a slaughterhouse in China. Antimicrobial susceptibility was tested against a panel of agents. Whole-genome sequencing of the isolate was carried out and data was analyzed.A large plasmid, denoted as plasmid pC629 (210,106 bp), containing a composite cassette, consisting of IS26-bla NDM-1-ble MBL -?trpF-tat-cutA-ISCR1-sul1-qacE?1-aadA2-dfrA12-intI1-IS26 was identified. The latter locus was physically linked with bla OXA-1, bla CTX-M-65, bla TEM-1-encoding genes. A mercury resistance operon merACDEPTR was also identified; it was flanked on the proximal side, among IS26 element and the distally located on the bla NDM-1 gene. Plasmid pC629 also contained 21 other antimicrobial resistance-encoding genes, such as aac(6′)-Ib-cr, aac(3)-VI, aadA5, aph(4)-Ia, arr-3, blmS, brp, catB3, dfrA17, floR, fosA, mph(A), mphR, mrx, nimC/nimA, oqxA, oqxB, oqxR, rmtB, sul1, sul2. Two virulence genes were also identified on plasmid pC629.To the best of our knowledge, this is the first report of bla NDM-1 gene being identified from a plasmid in a S. Indiana isolate cultured from chicken carcass in China.

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July 7, 2019

Detection of an Escherichia coli sequence type 167 strain with two tandem copies of blaNDM-1 in the chromosome.

New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Enterobacteriaceae has disseminated rapidly throughout the world and poses an urgent threat to public health. Previous studies confirmed that the blaNDM-1 gene is typically carried in plasmids but rarely in chromosome. We discovered a multidrug-resistant Escherichia coli strain Y5, originating from a urine sample and containing the blaNDM-1 gene, which did not transfer by either conjugation or electrotransformation. We confirmed the possibility of its chromosome location by S1-pulsed-field gel electrophoresis (PFGE) and XbaI-PFGE, followed by Southern blotting. To determine the genomic background of blaNDM-1, the genome of Y5 was completely sequenced and compared to other reference genomes. The results of our study revealed that this isolate consists of a 4.8-Mbp chromosome and three plasmids, it is an epidemic clone of sequence type (ST) 167, and it shows 99% identity with Escherichia coli 6409 (GenBank accession no. CP010371), which lacks the same blaNDM-1 gene-surrounding structure as Y5. The blaNDM-1 gene is embedded in the chromosome along with two tandem copies of an insertion sequence common region 1 (ISCR1) element (sul1-ARR-3-cat-blaNDM-1-bleo-ISCR1), which appears intact in the plasmid from Proteus mirabilis (GenBank accession no. KP662515). The genomic context indicates that the ISCR1 element mediated the blaNDM-1 transposition from a single source plasmid to the chromosome. Our study is the first report of an Enterobacteriaceae strain harboring a chromosomally integrated blaNDM-1, which directly reveals the vertical spreading pattern of the gene. Close surveillance is urgently needed to monitor the emergence and potential spread of ST167 strains that harbor blaNDM-1. Copyright © 2016 American Society for Microbiology.

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July 7, 2019

Emergence of Klebsiella variicola positive for NDM-9, a variant of New Delhi metallo-ß-lactamase, in an urban river in South Korea.

To examine the presence of pathogenic bacteria carrying New Delhi metallo-ß-lactamase in the environment and to characterize the genome structures of these strains.Phenotypic screening of antimicrobial susceptibility and WGS were conducted on three Klebsiella variicola strains possessing NDM-9 isolated from an urban river.Three carbapenem-resistant K. variicola isolated from Gwangju tributary were found to possess bla NDM-9 genes. Antimicrobial susceptibility testing indicated resistance of these strains to aminoglycosides, carbapenems, cephems, folate pathway inhibitors, fosfomycin and penicillins, but susceptibility to fluoroquinolones, phenicols, tetracyclines and miscellaneous agents. WGS revealed that the 108 kb IncFII(Y)-like plasmids carry bla NDM-9 sandwiched between IS 15 for the GJ1 strain, IS 26 for the GJ2 strain, IS 15D1 for the GJ3 strain and IS Vsa3 , and further bracketed by IS 26 and Tn AS3 along with the mercury resistance operon upstream and the class 1 integron composed of gene cassettes of aadA2 , dfrA12 and sul1 downstream. An aph(3′)-Ia gene conferring resistance to aminoglycosides is located after the integrons. Chromosomally encoded bla LEN-13 , fosA , aqxA and oqxB genes, as well as plasmid-mediated bla TEM-1B and bla CTX-M-65 encoding ESBL, ant(3′)-Ia and mph (A) genes, were also identified.The findings of the present study provide us with the information that NDM-9 has been spreading into the environment. Dissemination of NDM-9 in the environment has raised a health risk alarm as this variant of NDM carries MDR genes with highly transferable mobile genetic elements, increasing the possibility of resistance gene transfer among microorganisms in the environment.

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