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Asset Tag: Antibiotic resistance

July 7, 2019

Rapid and consistent evolution of colistin resistance in XDR Pseudomonas aeruginosa during morbidostat culture.

Colistin is a last resort antibiotic commonly used against multidrug-resistant strains of Pseudomonas aeruginosa To investigate the potential for in-situ evolution of resistance against colistin and to map the molecular targets of colistin resistance, we exposed two P. aeruginosa isolates to colistin using a continuous culture device known as morbidostat. As a result, colistin resistance reproducibly increased 10-fold within ten days, and 100-fold within 20 days, along with highly stereotypic, yet strain specific mutation patterns. The majority of mutations hit the pmrAB two component signaling system and genes involved in lipopolysaccharide (LPS) synthesis, including lpxC, pmrE, and migA We tracked the frequencies of all arising mutations by whole genome deep sequencing every 3-4 days to provide a detailed picture of the dynamics of resistance evolution, including competition and displacement among multiple resistant sub-populations. In seven out of 18 cultures, we observed mutations in mutS along with a mutator phenotype that seemed to facilitate resistance evolution. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Evidence for the evolutionary steps leading to mecA-mediated ß-lactam resistance in staphylococci.

The epidemiologically most important mechanism of antibiotic resistance in Staphylococcus aureus is associated with mecA-an acquired gene encoding an extra penicillin-binding protein (PBP2a) with low affinity to virtually all ß-lactams. The introduction of mecA into the S. aureus chromosome has led to the emergence of methicillin-resistant S. aureus (MRSA) pandemics, responsible for high rates of mortality worldwide. Nonetheless, little is known regarding the origin and evolution of mecA. Different mecA homologues have been identified in species belonging to the Staphylococcus sciuri group representing the most primitive staphylococci. In this study we aimed to identify evolutionary steps linking these mecA precursors to the ß-lactam resistance gene mecA and the resistance phenotype. We sequenced genomes of 106 S. sciuri, S. vitulinus and S. fleurettii strains and determined their oxacillin susceptibility profiles. Single-nucleotide polymorphism (SNP) analysis of the core genome was performed to assess the genetic relatedness of the isolates. Phylogenetic analysis of the mecA gene homologues and promoters was achieved through nucleotide/amino acid sequence alignments and mutation rates were estimated using a Bayesian analysis. Furthermore, the predicted structure of mecA homologue-encoded PBPs of oxacillin-susceptible and -resistant strains were compared. We showed for the first time that oxacillin resistance in the S. sciuri group has emerged multiple times and by a variety of different mechanisms. Development of resistance occurred through several steps including structural diversification of the non-binding domain of native PBPs; changes in the promoters of mecA homologues; acquisition of SCCmec and adaptation of the bacterial genetic background. Moreover, our results suggest that it was exposure to ß-lactams in human-created environments that has driven evolution of native PBPs towards a resistance determinant. The evolution of ß-lactam resistance in staphylococci highlights the numerous resources available to bacteria to adapt to the selective pressure of antibiotics.

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July 7, 2019

Reclassification of the specialized metabolite producer Pseudomonas mesoacidophila ATCC 31433 as a member of the Burkholderia cepacia complex.

Pseudomonas mesoacidophila ATCC 31433 is a Gram-negative bacterium, first isolated from Japanese soil samples, that produces the monobactam isosulfazecin and the ß-lactam-potentiating bulgecins. To characterize the biosynthetic potential of P. mesoacidophila ATCC 31433, its complete genome was determined using single-molecule real-time DNA sequence analysis. The 7.8-Mb genome comprised four replicons, three chromosomal (each encoding rRNA) and one plasmid. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 was misclassified at the time of its deposition and is a member of the Burkholderia cepacia complex, most closely related to Burkholderia ubonensis The sequenced genome shows considerable additional biosynthetic potential; known gene clusters for malleilactone, ornibactin, isosulfazecin, alkylhydroxyquinoline, and pyrrolnitrin biosynthesis and several uncharacterized biosynthetic gene clusters for polyketides, nonribosomal peptides, and other metabolites were identified. Furthermore, P. mesoacidophila ATCC 31433 harbors many genes associated with environmental resilience and antibiotic resistance and was resistant to a range of antibiotics and metal ions. In summary, this bioactive strain should be designated B. cepacia complex strain ATCC 31433, pending further detailed taxonomic characterization.IMPORTANCE This work reports the complete genome sequence of Pseudomonas mesoacidophila ATCC 31433, a known producer of bioactive compounds. Large numbers of both known and novel biosynthetic gene clusters were identified, indicating that P. mesoacidophila ATCC 31433 is an untapped resource for discovery of novel bioactive compounds. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 is in fact a member of the Burkholderia cepacia complex, most closely related to the species Burkholderia ubonensis Further investigation of the classification and biosynthetic potential of P. mesoacidophila ATCC 31433 is warranted. Copyright © 2017 Loveridge et al.

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July 7, 2019

Synergistic interaction between phage therapy and antibiotics clears Pseudomonas aeruginosa infection in endocarditis and reduces virulence.

Increasing antibiotic resistance warrants therapeutic alternatives. Here we investigated the efficacy of bacteriophage-therapy (phage) alone or combined with antibiotics against experimental endocarditis (EE) due to Pseudomonas aeruginosa, an archetype of difficult-to-treat infection.In vitro fibrin clots and rats with aortic EE were treated with an antipseudomonas phage cocktail alone or combined with ciprofloxacin. Phage pharmacology, therapeutic efficacy, and resistance were determined.In vitro, single-dose phage therapy killed 7 log colony-forming units (CFUs)/g of fibrin clots in 6 hours. Phage-resistant mutants regrew after 24 hours but were prevented by combination with ciprofloxacin (2.5 × minimum inhibitory concentration). In vivo, single-dose phage therapy killed 2.5 log CFUs/g of vegetations in 6 hours (P < .001 vs untreated controls) and was comparable with ciprofloxacin monotherapy. Moreover, phage/ciprofloxacin combinations were highly synergistic, killing >6 log CFUs/g of vegetations in 6 hours and successfully treating 64% (n = 7/11) of rats. Phage-resistant mutants emerged in vitro but not in vivo, most likely because resistant mutations affected bacterial surface determinants important for infectivity (eg, the pilT and galU genes involved in pilus motility and LPS formation).Single-dose phage therapy was active against P. aeruginosa EE and highly synergistic with ciprofloxacin. Phage-resistant mutants had impaired infectivity. Phage-therapy alone or combined with antibiotics merits further clinical consideration.

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July 7, 2019

Staphylococcus aureus CC395 harbours a novel composite staphylococcal cassette chromosome mec element.

CoNS species are likely reservoirs of the staphylococcal cassette chromosome mec (SCC mec ) in Staphylococcus aureus . S . aureus CC395 is unique as it is capable of exchanging DNA with CoNS via bacteriophages, which are also known to mediate transfer of SCC mec .To analyse the structure and putative origin of the SCC mec element in S . aureus CC395.The only MRSA CC395 strain described in the literature, JS395, was subjected to WGS, and its SCC mec element was compared with those found in CoNS species and other S. aureus strains.JS395 was found to carry an unusually large 88 kb composite SCC mec element. The 33 kb region downstream of orfX harboured a type V SCC mec element and a CRISPR locus, which was most similar to those found in the CoNS species Staphylococcus capitis and Staphylococcus schleiferi . A 55 kb SCC element was identified downstream of the type V SCC mec element and contained a mercury resistance region found in the composite SCC element of some Staphylococcus epidermidis and S . aureus strains, an integrated S . aureus plasmid containing genes for the detoxification of cadmium and arsenic, and a stretch of genes that was partially similar to the type IVg SCC mec element found in a bovine S . aureus strain.The size and complexity of the SCC mec element support the idea that CC395 is highly prone to DNA uptake from CoNS. Thus CC395 may serve as an entry point for SCC mec and SCC structures into S . aureus .

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July 7, 2019

Chromosomal 16S ribosomal RNA methyltransferase RmtE1 in Escherichia coli sequence type 448.

We identified rmtE1, an uncommon 16S ribosomal methyltransferase gene, in an aminoglycoside- and cephalosporin-resistant Escherichia coli sequence type 448 clinical strain co-harboring blaCMY-2. Long-read sequencing revealed insertion of a 101,257-bp fragment carrying both resistance genes to the chromosome. Our findings underscore E. coli sequence type 448 as a potential high-risk multidrug-resistant clone.

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July 7, 2019

Whole-genome sequence of Acinetobacter pittii HUMV-6483 isolated from human urine.

Acinetobacter pittii strain HUMV-6483 was obtained from urine from an adult patient. We report here its complete genome assembly using PacBio single-molecule real-time sequencing, which resulted in a chromosome with 4.07 Mb and a circular contig of 112 kb. About 3,953 protein-coding genes are predicted from this assembly. Copyright © 2017 Chapartegui-González et al.

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July 7, 2019

Virulence and genomic feature of a virulent Klebsiella pneumoniae sequence type 14 strain of serotype K2 harboring blaNDM-5 in China.

The objective of this study was to reveal the molecular mechanism involved in carbapenem resistance and virulence of a K2 Klebsiella pneumoniae clinical isolate 24835. The virulence of the strain was determined by in vitro and in vivo methods. The de novo whole-genome sequencing technology and molecular biology methods were used to analyze the genomic features associated with the carbapenem resistance and virulence of K. pneumoniae 24835. Strain 24835 was highly resistant to carbapenems and belonged to ST14, exhibited hypermucoviscous and unique K2-aerobactin-kfu-rmpA positive phenotype. As the only carbapenemase gene in strain 24835, blaNDM-5 was located on a 46-kb IncX3 self-transmissible plasmid, which is a very close relation of pNDM-MGR194 from India. Genetic context of blaNDM-5 in strain 24835 was closely related to those on IncX3 plasmids in various Enterobacteriaceae species in China. The combination of multiple virulence genes may work together to confer the relative higher virulence in K. pneumoniae 24835. Significantly increased resistance to serum killing and mice mortality were found in the virulent New Delhi metallo-ß-lactamase (NDM)-producing K. pneumoniae strain compared to the other NDM-producing K. pneumoniae strain. Our study provides basic information of phenotypic and genomic features of K. pneumoniae 24835, a strain displaying carbapenem resistance and relatively high level of virulence. These findings are concerning for the potential of NDM-like genes to disseminate among virulent K. pneumoniae isolates.

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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

Whole genome characterization of a naturally occurring vancomycin-dependent Enterococcus faecium from a patient with bacteremia.

Vancomycin-dependent enterococci are a relatively uncommon phenotype recovered in the clinical laboratory. Recognition and recovery of these isolates are important, to provide accurate identification and susceptibility information to treating physicians. Herein, we describe the recovery of a vancomycin-dependent and revertant E. faecium isolates harboring vanB operon from a patient with bacteremia. Using whole genome sequencing, we found a unique single nucleotide polymorphism (S186N) in the D-Ala-D-Ala ligase (ddl) conferring vancomycin-dependency. Additionally, we found that a majority of in vitro revertants mutated outside ddl, with some strains harboring mutations in vanS, while others likely containing novel mechanisms of reversion. Copyright © 2017 Elsevier B.V. All rights reserved.

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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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July 7, 2019

Tracing the Enterococci from Paleozoic origins to the hospital.

We examined the evolutionary history of leading multidrug resistant hospital pathogens, the enterococci, to their origin hundreds of millions of years ago. Our goal was to understand why, among the vast diversity of gut flora, enterococci are so well adapted to the modern hospital environment. Molecular clock estimation, together with analysis of their environmental distribution, phenotypic diversity, and concordance with host fossil records, place the origins of the enterococci around the time of animal terrestrialization, 425-500 mya. Speciation appears to parallel the diversification of hosts, including the rapid emergence of new enterococcal species following the End Permian Extinction. Major drivers of speciation include changing carbohydrate availability in the host gut. Life on land would have selected for the precise traits that now allow pathogenic enterococci to survive desiccation, starvation, and disinfection in the modern hospital, foreordaining their emergence as leading hospital pathogens. Copyright © 2017 Elsevier Inc. All rights reserved.

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July 7, 2019

Genomic analysis of factors associated with low prevalence of antibiotic resistance in extraintestinal pathogenic Escherichia coli sequence type 95 strains.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains belonging to multilocus sequence type 95 (ST95) are globally distributed and a common cause of infections in humans and domestic fowl. ST95 isolates generally show a lower prevalence of acquired antimicrobial resistance than other pandemic ExPEC lineages. We took a genomic approach to identify factors that may underlie reduced resistance. We fully assembled genomes for four ST95 isolates representing the four major fimH-based lineages within ST95 and also analyzed draft-level genomes from another 82 ST95 isolates, largely from the western United States. The fully assembled genomes of antibiotic-resistant isolates carried resistance genes exclusively on large (>90-kb) IncFIB/IncFII plasmids. These replicons were common in the draft genomes as well, particularly in antibiotic-resistant isolates, but we also observed multiple instances of a smaller (8.3-kb) ampicillin resistance plasmid that had been previously identified in Salmonella enterica. Among ST95 isolates, pansusceptibility to antibiotics was significantly associated with the fimH6 lineage and the presence of homologs of the previously identified 114-kb IncFIB/IncFII plasmid pUTI89, both of which were also associated with reduced carriage of other plasmids. Potential mechanistic explanations for lineage- and plasmid-specific effects on the prevalence of antibiotic resistance within the ST95 group are discussed. IMPORTANCE Antibiotic resistance in bacterial pathogens is a major public health concern. This work was motivated by the observation that only a small proportion of ST95 isolates, a major pandemic lineage of extraintestinal pathogenic E. coli, have acquired antibiotic resistance, in contrast to many other pandemic lineages. Understanding bacterial genetic factors that may prevent acquisition of resistance could contribute to the development of new biological, medical, or public health strategies to reduce antibiotic-resistant infections.

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July 7, 2019

A novel disrupted mcr-1 gene and a lysogenized phage P1-like sequence detected from a large conjugative plasmid, cultured from a human atypical enteropathogenic Escherichia coli (aEPEC) recovered in China.

Sir,The recent description of the plasmid-mediated colistin resistance gene, mcr-1, in bacterial isolates cultured in China has triggered several retrospective studies investigating this gene.1The mcr-1 gene has so far been reported to be associated with various plas- mid replicon types, and was found only rarely to be chromoso- mally encoded.2,3However, no report of a directly inactivated mcr-1 gene has been described to date. In this study, we present the complete nucleotide sequence of an ESBL-producing atypical enteropathogenic Escherichia coli (aEPEC) isolate, SLK172, one of whose plasmids carried a uniquely disrupted mcr-1 gene, being inactivated following the insertion of an ISApl1 element (Figure1a).

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