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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

Expanding landscapes of the diversified mcr-1-bearing plasmid reservoirs.

Polymyxin is a cationic polypeptide antibiotic that can disrupt bacterial cell membrane by interacting with its lipopolysaccharide molecules and is used as a last resort drug against lethal infections by the carbapenem-resistant superbugs (like NDM-1). However, global discovery of the MCR-1 colistin resistance dramatically challenges the newly renewed interest in colistin for clinical use.The mcr-1-harboring plasmids were acquired from swine and human Escherichia coli isolated in China, from 2015 to 2016, and subjected to Illumina PacBio RSII and Hi-Seq2000 for full genome sequencing. PCR was applied to close the gap of the assembled contigs. Ori-Finder was employed to predict the replication origin (oriC) in plasmids. The phenotype of MCR-1-producing isolates was evaluated on the LBA plates with various level of colistin. Genetic deletion was used to test the requirement of the initial “ATG” codon for the MCR-1 function.Here, we report full genomes of over 10 mcr-1-harboring plasmids with diversified replication incompatibilities. A novel hybrid IncI2/IncFIB plasmid pGD17-2 was discovered and characterized from a swine isolate with colistin resistance. Intriguingly, co-occurrence of two unique mcr-1-bearing plasmids (pGD65-3, IncI2, and pGD65-5, IncX4) was detected in a single isolate GD65, which might accelerate dissemination of the mcr-1 under environmental selection pressure. Genetic analyses of these plasmids mapped mobile elements in the context of antibiotic resistance and determined two insertion sequences (ISEcp1 and ISApl1) that are responsible for the mobilization of mcr-1. Gene deletion also proved that the first ATG codon is redundant in the mcr-1 gene.Collectively, our results extend landscapes of the diversified mcr-1-bearing plasmid reservoirs.

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July 7, 2019

Emergence and evolution of multidrug-resistant Klebsiella pneumoniae with both blaKPC and blaCTX-M integrated in the chromosome.

The extended-spectrum-ß-lactamase (ESBL)- and Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae represent serious and urgent threats to public health. In a retrospective study of multidrug-resistant K. pneumoniae, we identified three clinical isolates, CN1, CR14, and NY9, carrying both blaCTX-M and blaKPC genes. The complete genomes of these three K. pneumoniae isolates were de novo assembled by using both short- and long-read whole-genome sequencing. In CR14 and NY9, blaCTX-M and blaKPC were carried on two different plasmids. In contrast, CN1 had one copy of blaKPC-2 and three copies of blaCTX-M-15 integrated in the chromosome, for which the blaCTX-M-15 genes were linked to an insertion sequence, ISEcp1, whereas the blaKPC-2 gene was in the context of a Tn4401a transposition unit conjugated with a PsP3-like prophage. Intriguingly, downstream of the Tn4401a-blaKPC-2-prophage genomic island, CN1 also carried a clustered regularly interspaced short palindromic repeat (CRISPR)-cas array with four spacers targeting a variety of K. pneumoniae plasmids harboring antimicrobial resistance genes. Comparative genomic analysis revealed that there were two subtypes of type I-E CRISPR-cas in K. pneumoniae strains and suggested that the evolving CRISPR-cas, with its acquired novel spacer, induced the mobilization of antimicrobial resistance genes from plasmids into the chromosome. The integration and dissemination of multiple copies of blaCTX-M and blaKPC from plasmids to chromosome depicts the complex pandemic scenario of multidrug-resistant K. pneumoniae Additionally, the implications from this study also raise concerns for the application of a CRISPR-cas strategy against antimicrobial resistance. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

First report of Klebsiella oxytoca strain simultaneously producing NDM-1, IMP-4 and KPC-2 carbapenemases.

The nucleotide sequences of five plasmids from one Klebsiella oxytoca isolate were determined using the PacBio RS II system. Plasmid analysis revealed that blaNDM-1 was carried on an IncX3 plasmid. The blaIMP-4 and blaKPC-2 genes were located on IncN and IncP-6 plasmids, respectively. Comparative sequence analysis highlighted the successful spread of carbapenemase-harboring plasmids among different enterobacterial species. We report for the first time, to our knowledge, coproducing NDM-1, KPC-2, and IMP-4 carbapenemases on a K. oxytoca isolate. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Comparative genomic and phylogenetic analysis of a toxigenic clinical isolate of Corynebacterium diphtheriae strain B-D-16-78 from Malaysia.

In this study, we report the comparative genomics and phylogenetic analysis of Corynebacterium diphtheriae strain B-D-16-78 that was isolated from a clinical specimen in 2016. The complete genome of C. diphtheriae strain B-D-16-78 was sequenced using PacBio Single Molecule, Real-Time sequencing technology and consists of a 2,474,151-bp circular chromosome with an average GC content of 53.56%. The core genome of C. diphtheriae was also deduced from a total of 74 strains with complete or draft genome sequences and the core genome-based phylogenetic analysis revealed close genetic relationship among strains that shared the same MLST allelic profile. In the context of CRISPR-Cas system, which confers adaptive immunity against re-invading DNA, 73 out of 86 spacer sequences were found to be unique to Malaysian strains which harboured only type-II-C and/or type-I-E-a systems. A total of 48 tox genes which code for the diphtheria toxin were retrieved from the 74 genomes and with the exception of one truncated gene, only nucleotide substitutions were detected when compared to the tox gene sequence of PW8. More than half were synonymous substitution and only two were nonsynonymous substitutions whereby H24Y was predicted to have a damaging effect on the protein function whilst T262V was predicted to be tolerated. Both toxigenic and non-toxigenic toxin-gene bearing strains have been isolated in Malaysia but the repeated isolation of toxigenic strains with the same MLST profile suggests the possibility of some of these strains may be circulating in the population. Hence, efforts to increase herd immunity should be continued and supported by an effective monitoring and surveillance system to track, manage and control outbreak of cases. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Plasmid composition in Aeromonas salmonicida subsp. salmonicida 01-B526 unravels unsuspected type three secretion system loss patterns.

Aeromonas salmonicida subsp. salmonicida is a ubiquitous psychrophilic waterborne bacterium and a fish pathogen. The numerous mobile elements, especially insertion sequences (IS), in its genome promote rearrangements that impact its phenotype. One of the main virulence factors of this bacterium, its type three secretion system (TTSS), is affected by these rearrangements. In Aeromonas salmonicida subsp. salmonicida most of the TTSS genes are encoded in a single locus on a large plasmid called pAsa5, and may be lost when the bacterium is cultivated at a higher temperature (25 °C), producing non-virulent mutants. In a previous study, pAsa5-rearranged strains that lacked the TTSS locus on pAsa5 were produced using parental strains, including 01-B526. Some of the generated deletions were explained by homologous recombination between ISs found on pAsa5, whereas the others remained unresolved. To investigate those rearrangements, short- and long-read high-throughput sequencing technologies were used on the A. salmonicida subsp. salmonicida 01-B526 whole genome.Whole genome sequencing of the 01-B526 strain revealed that its pAsa5 has an additional IS copy, an ISAS5, compared to the reference strain (A449) sequence, which allowed for a previously unknown rearrangement to occur. It also appeared that 01-B526 bears a second large plasmid, named pAsa9, which shares 40 kbp of highly similar sequences with pAsa5. Following these discoveries, previously unexplained deletions were elucidated by genotyping. Furthermore, in one of the derived strains a fusion of pAsa5 and pAsa9, involving the newly discovered ISAS5 copy, was observed.The loss of TTSS and hence virulence is explained by one consistent mechanism: IS-driven homologous recombination. The similarities between pAsa9 and pAsa5 also provide another example of genetic diversity driven by ISs.

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July 7, 2019

Evidence for contemporary switching of the O-antigen gene cluster between Shiga toxin-producing Escherichia coli strains colonizing cattle.

Shiga toxin-producing Escherichia coli (STEC) comprise a group of zoonotic enteric pathogens with ruminants, especially cattle, as the main reservoir. O-antigens are instrumental for host colonization and bacterial niche adaptation. They are highly immunogenic and, therefore, targeted by the adaptive immune system. The O-antigen is one of the most diverse bacterial cell constituents and variation not only exists between different bacterial species, but also between individual isolates/strains within a single species. We recently identified STEC persistently infecting cattle and belonging to the different serotypes O156:H25 (n = 21) and O182:H25 (n = 15) that were of the MLST sequence types ST300 or ST688. These STs differ by a single nucleotide in purA only. Fitness-, virulence-associated genome regions, and CRISPR/CAS (clustered regularly interspaced short palindromic repeats/CRISPR associated sequence) arrays of these STEC O156:H25 and O182:H25 isolates were highly similar, and identical genomic integration sites for the stx converting bacteriophages and the core LEE, identical Shiga toxin converting bacteriophage genes for stx1a, identical complete LEE loci, and identical sets of chemotaxis and flagellar genes were identified. In contrast to this genomic similarity, the nucleotide sequences of the O-antigen gene cluster (O-AGC) regions between galF and gnd and very few flanking genes differed fundamentally and were specific for the respective serotype. Sporadic aEPEC O156:H8 isolates (n = 5) were isolated in temporal and spatial proximity. While the O-AGC and the corresponding 5′ and 3′ flanking regions of these aEPEC isolates were identical to the respective region in the STEC O156:H25 isolates, the core genome, the virulence associated genome regions and the CRISPR/CAS elements differed profoundly. Our cumulative epidemiological and molecular data suggests a recent switch of the O-AGC between isolates with O156:H8 strains having served as DNA donors. Such O-antigen switches can affect the evaluation of a strain’s pathogenic and virulence potential, suggesting that NGS methods might lead to a more reliable risk assessment.

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July 7, 2019

Whole-genome comparative analysis of Salmonella enterica serovar Newport strains reveals lineage-specific divergence.

Salmonella enterica subsp. enterica serovar Newport has been associated with various foodborne outbreaks in humans and animals. Phylogenetically, serovar Newport is one of several Salmonella serovars that are polyphyletic. To understand more about the polyphyletic nature of this serovar, six food, environment, and human isolates from different Newport lineages were selected for genome comparison analyses. Whole genome comparisons demonstrated that heterogeneity mostly occurred in the prophage regions. Lineage-specific characteristics were also present in the Salmonella pathogenicity islands and fimbrial operons. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2017. This work is written by US Government employees and is in the public domain in the US.

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July 7, 2019

Free-living Enterobacterium Pragia fontium 24613: complete genome sequence and metabolic profiling.

Pragia fontium is one of the few species that belongs to the group of atypical hydrogen sulfide-producing enterobacteria. Unlike other members of this closely related group, P. fontium is not associated with any known host and has been reported as a free-living bacterium. Whole genome sequencing and metabolic fingerprinting confirmed the phylogenetic position of P. fontium inside the group of atypical H2S producers. Genomic data have revealed that P. fontium 24613 has limited pathogenic potential, although there are signs of genome decay. Although the lack of specific virulence factors and no association with a host species suggest a free-living style, the signs of genome decay suggest a process of adaptation to an as-yet-unknown host.

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July 7, 2019

Whole genome sequencing and analysis of Campylobacter coli YH502 from retail chicken reveals a plasmid-borne type VI secretion system.

Campylobacter is a major cause of foodborne illnesses worldwide. Campylobacter infections, commonly caused by ingestion of undercooked poultry and meat products, can lead to gastroenteritis and chronic reactive arthritis in humans. Whole genome sequencing (WGS) is a powerful technology that provides comprehensive genetic information about bacteria and is increasingly being applied to study foodborne pathogens: e.g., evolution, epidemiology/outbreak investigation, and detection. Herein we report the complete genome sequence of Campylobacter coli strain YH502 isolated from retail chicken in the United States. WGS, de novo assembly, and annotation of the genome revealed a chromosome of 1,718,974 bp and a mega-plasmid (pCOS502) of 125,964 bp. GC content of the genome was 31.2% with 1931 coding sequences and 53 non-coding RNAs. Multiple virulence factors including a plasmid-borne type VI secretion system and antimicrobial resistance genes (beta-lactams, fluoroquinolones, and aminoglycoside) were found. The presence of T6SS in a mobile genetic element (plasmid) suggests plausible horizontal transfer of these virulence genes to other organisms. The C. coli YH502 genome also harbors CRISPR sequences and associated proteins. Phylogenetic analysis based on average nucleotide identity and single nucleotide polymorphisms identified closely related C. coli genomes available in the NCBI database. Taken together, the analyzed genomic data of this potentially virulent strain of C. coli will facilitate further understanding of this important foodborne pathogen most likely leading to better control strategies. The chromosome and plasmid sequences of C. coli YH502 have been deposited in GenBank under the accession numbers CP018900.1 and CP018901.1, respectively.

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July 7, 2019

Insight into potential probiotic markers predicted in Lactobacillus pentosus MP-10 genome sequence.

Lactobacillus pentosus MP-10 is a potential probiotic lactic acid bacterium originally isolated from naturally fermented Aloreña green table olives. The entire genome sequence was annotated to in silico analyze the molecular mechanisms involved in the adaptation of L. pentosus MP-10 to the human gastrointestinal tract (GIT), such as carbohydrate metabolism (related with prebiotic utilization) and the proteins involved in bacteria-host interactions. We predicted an arsenal of genes coding for carbohydrate-modifying enzymes to modify oligo- and polysaccharides, such as glycoside hydrolases, glycoside transferases, and isomerases, and other enzymes involved in complex carbohydrate metabolism especially starch, raffinose, and levan. These enzymes represent key indicators of the bacteria’s adaptation to the GIT environment, since they involve the metabolism and assimilation of complex carbohydrates not digested by human enzymes. We also detected key probiotic ligands (surface proteins, excreted or secreted proteins) involved in the adhesion to host cells such as adhesion to mucus, epithelial cells or extracellular matrix, and plasma components; also, moonlighting proteins or multifunctional proteins were found that could be involved in adhesion to epithelial cells and/or extracellular matrix proteins and also affect host immunomodulation. In silico analysis of the genome sequence of L. pentosus MP-10 is an important initial step to screen for genes encoding for proteins that may provide probiotic features, and thus provides one new routes for screening and studying this potentially probiotic bacterium.

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July 7, 2019

Comparative genomics of all three Campylobacter sputorum biovars and a novel cattle-associated C. sputorum clade.

Campylobacter sputorum is a non-thermotolerant campylobacter that is primarily isolated from food animals such as cattle and sheep. C. sputorum is also infrequently associated with human illness. Based on catalase and urease activity, three biovars are currently recognized within C. sputorum: bv. sputorum (catalase negative, urease negative), bv. fecalis (catalase positive, urease negative), and bv. paraureolyticus (catalase negative, urease positive). A multi-locus sequence typing (MLST) method was recently constructed for C. sputorum. MLST typing of several cattle-associated C. sputorum isolates suggested that they are members of a divergent C. sputorum clade. Although catalase positive, and thus technically bv. fecalis, the taxonomic position of these strains could not be determined solely by MLST. To further characterize C. sputorum, the genomes of four strains, representing all three biovars and the divergent clade, were sequenced to completion. Here we present a comparative genomic analysis of the four C. sputorum genomes. This analysis indicates that the three biovars and the cattle-associated strains are highly-related at the genome level with similarities in gene content. Furthermore, the four genomes are strongly syntenic with one or two minor inversions. However, substantial differences in gene content were observed among the three biovars. Finally, although the strain representing the cattle-associated isolates was shown to be C. sputorum, it is possible that this strain is a member of a novel C. sputorum subspecies; thus, these cattle-associated strains may form a second taxon within C. sputorum. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2017. This work is written by US Government employees and is in the public domain in the US.

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July 7, 2019

Characterization of NDM-5-positive extensively resistant Escherichia coli isolates from dairy cows.

The aim of this study was to investigate the prevalence of blaNDM-5 gene in Escherichia coli isolates from dairy cows and to characterize the molecular traits of the blaNDM-5-positive isolates. A total of 169 cows were sampled (169 feces and 169 raw milk samples) in three dairy farms in Jiangsu Province and 203 E. coli isolates were recovered. Among these strains, three isolates carried blaNDM-5 gene, including one co-harboring mcr-1, which belonged to sequence type 446 and the other two belonged to ST2. Susceptibility testing revealed that the three blaNDM-5-positive isolates showed extensive resistance to antimicrobials. The blaNDM-5 gene was located on a ~46-kb IncX3 transferrable pNDM-MGR194-like plasmid in all three isolates, while mcr-1 was located on a ~260-kb IncHI2 plasmid pXGE1mcr. Competition experiments revealed that acquisition of blaNDM-5 or mcr-1-bearing plasmid can incur fitness cost of bacterial host, however, plasmid stability testing showed that both blaNDM-5 and mcr-1-carrying plasmid maintained stable in the hosts after ten passages without antimicrobial selection. Whole genome sequencing revealed that the mcr-1 gene coexisted with multiple resistance genes in pXGE1mcr and the backbone of this plasmid was similar to that of previously reported mcr-1-positive plasmid pHNSHP45-2. Moreover, pXGE1mcr could be conjugated into clinical NDM-5-positive E. coli isolates in vitro, thereby generating strains that approached pan-resistance. Active surveillance efforts are imperative to monitor the prevalence of blaNDM-5 and mcr-1 in carbapenem-resistant Enterobacteriaceae from dairy farms throughout China. Copyright © 2017 Elsevier B.V. All rights reserved.

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