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September 22, 2019

A comparison of genotypic and phenotypic methods for analyzing the susceptibility to sulfamethoxazole and trimethoprim in Edwardsiella piscicida.

In a study of 39 isolates of Edwardsiella piscicida made from Korean aquaculture sites, sul genes were detected in 16 isolates and dfr genes in 19. Ten isolates were shown to contain both sul and dfr genes. MIC and disc diffusion zones assays were performed to measure the phenotypic susceptibilities of the 39 isolates. Normalized resistance interpretation was applied to these data to categorize isolates as either fully susceptible or as manifesting reduced susceptibility. The standard CLSI protocols specify the use of a mixture of sulfamethoxazole/trimethoprim (20:1) in both MIC and disc diffusion tests. Using the CLSI MIC protocol, 100% of the isolates containing dfr genes, but only 75% of the isolates containing sul genes, were categorized as manifesting reduced susceptibility. Using the CLSI disc diffusion protocol, only 58% of the isolates containing dfr genes and 69% of those containing sul genes were categorized as manifesting reduced susceptibility. When the single agent trimethoprim was substituted for the combined mixture in both the MIC and disc diffusion protocols, 100% of the dfr-positive isolates were categorized as NWT. When the single-agent sulfamethoxazole was substituted, the analysis of the MIC characterized 100% and the disc zone data 94% of the sul-positive isolates as manifesting reduced susceptibility. It is argued that the use of trimethoprim and sulfamethoxazole as single agents in phenotypic susceptibility tests would provide more meaningful data than the currently recommended use of these two agents combined.

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September 22, 2019

Identification of the KPC plasmid pCT-KPC334: New insights on the evolutionary pathway of epidemic plasmids harboring fosA3-blaKPC-2 genes.

A novel, non-conjugative plasmid pKP1034 isolated from a fosfomycin-resistant, carbapenemase-producing Klebsiella pneumonia strain KP1034 was recently reported to carry fosA3, blaKPC-2, blaCTX-M-65, blaSHV-12 and rmtB genes, and was hypothesized to evolve from several recombination events of two closely related plasmids, pHN7A8 and pKPC-LK30 [1]. In this study, a plasmid pCT-KPC334 carrying fosA3, blaKPC-2, blaCTX-M-65, blaSHV-12, blaTEM-1, and rmtB genes was identified, providing evidence on the evolutionary pathway of plasmids harboring fosA3-blaKPC-2 genes.

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September 22, 2019

4.5 years within-patient evolution of a colistin resistant KPC-producing Klebsiella pneumoniae ST258.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) has emerged globally over the last decade as a major nosocomial pathogen that threatens patient care. These highly resistant bacteria are mostly associated with a single Kp clonal group, CG258, but the reasons for its host and hospital adaptation remain largely unknown.We analyzed the in vivo evolution of a colistin-resistant KPC-Kp CG258 strain that contaminated a patient following an endoscopy and was responsible for a fatal bacteremia 4.5 years later. Whole-genome sequencing was performed on 17 KPC-Kp isolates from this patient; single-nucleotide polymorphisms were analyzed and their implication in antimicrobial resistance and bacterial host adaptation investigated.The patient KPC-Kp strain diversified over 4.5 years at a rate of 7.5 substitutions per genome per year, resulting in broad phenotypic modifications. After 2 years of carriage, all isolates restored susceptibility to colistin. Higher expression of the fimbriae conferred the ability to produce more biofilm, and the isolate responsible for a bacteremia grew in human serum. The convergent mutations occurring in specific pathways, such as the respiratory chain and the cell envelope, revealed a complex long-term adaptation of KPC-Kp.Broad genomic and phenotypic diversification and the parallel selection of pathoadaptive mutations might contribute to long-term carriage and virulence of KPC-Kp CG258 strains and to the dissemination of this clone.

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September 22, 2019

Characterisation of a class 1 integron associated with the formation of quadruple blaGES-5 cassettes from an IncP-1ß group plasmid in Pseudomonas aeruginosa.

Integrons are genetic platforms responsible for the dissemination of antimicrobial resistance genes among Gram-negative bacteria, primarily due to their association with transposable elements and conjugative plasmids. In this study, a cassette array containing four identical blaGES-5 genes embedded in a class 1 integron located on an IncP-1ß group plasmid from a clinical Pseudomonas aeruginosa strain was identified. Comparative genome analysis and conjugation assay showed that the plasmid pICP-4GES lacked the trbN, trbO and trbP genes but was conjugable. Antimicrobial susceptibility testing revealed that compared with single-copy blaGES-5 complementary strains, both the cloned and chromosome-targeted expression of four copies of blaGES-5 increased the minimum inhibitory concentration (MIC) by one to two dilutions for most of the selected antimicrobials. Quantitative real-time reverse transcription PCR (RT-qPCR) showed that the four consecutive cassettes increased blaGES-5 expression by approximately two-fold compared with the single-copy blaGES-5 strain, suggesting that the level of gene expression was not directly proportional to copy number. In addition, the gene cassette capture assay showed that the global blaGES-5 transfer frequency reached 5.38?×?10-4. Copyright © 2018. Published by Elsevier B.V.

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September 22, 2019

2,3-Butanediol production by the non-pathogenic bacterium Paenibacillus brasilensis.

2,3-Butanediol (2,3-BDO) is of considerable importance in the chemical, plastic, pharmaceutical, cosmetic, and food industries. The main bacterial species producing this compound are considered pathogenic, hindering large-scale productivity. The species Paenibacillus brasilensis is generally recognized as safe (GRAS) and is phylogenetically similar to P. polymyxa, a species widely used for 2,3-BDO production. Here, we demonstrate, for the first time, that P. brasilensis strains produce 2,3-BDO. Total 2,3-BDO concentrations for 15 P. brasilensis strains varied from 5.5 to 7.6 g/l after 8 h incubation at 32 °C in modified YEPD medium containing 20 g/l glucose. Strain PB24 produced 8.2 g/l of 2,3-BDO within a 12-h growth period, representing a yield of 0.43 g/g and a productivity of 0.68 g/l/h. An increase in 2,3-BDO production by strain PB24 was observed using higher concentrations of glucose, reaching 27 g/l of total 2,3-BDO in YEPD containing about 80 g/l glucose within a 72-h growth period. We sequenced the genome of P. brasilensis PB24 and uncovered at least six genes related to the 2,3-BDO pathway at four distinct loci. We also compared gene sequences related to the 2,3-BDO pathway in P. brasilensis PB24 with those of other spore-forming bacteria, and found strong similarity to P. polymyxa, P. terrae, and P. peoriae 2,3-BDO-related genes. Regulatory regions upstream of these genes indicated that they are probably co-regulated. Finally, we propose a production pathway from glucose to 2,3-BDO in P. brasilensis PB24. Although the gene encoding S-2,3-butanediol dehydrogenase (butA) was found in the genome of P. brasilensis PB24, only R,R-2,3- and meso-2,3-butanediol were detected by gas chromatography under the growth conditions tested here. Our findings can serve as a basis for further improvements to the metabolic capabilities of this little-studied Paenibacillus species in relation to production of the high-value chemical 2,3-butanediol.

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September 22, 2019

Lactobacillus rhamnosus LRB mediated inhibition of oral streptococci.

Lactobacillus rhamnosus is a lactic acid bacterium with a diverse ecological habitat. We recently isolated a L. rhamnosus strain (LRB) from a healthy baby-tooth that had naturally fallen out. We determined the whole genome sequence of LRB and found that the isolate is closely genetically related to an intestinal isolate, L. rhamnosus GG (ATCC 53103). However, the LRB genome had lost about a 75-kb segment and undergone a genomic rearrangement. We assessed LRB’s capacity to survive in the gut environment, at least temporarily. We found that LRB, like the intestinal isolate ATCC 53103, showed resistance to low pH but sensitive to bile salt. Surprisingly, we found that this oral isolate LRB showed strong antimicrobial activity against a variety of oral streptococci including Streptococcus mutans. The production of antimicrobial activity is dependent on media composition since some media supported the production while others did not. The production of antimicrobial activity is also dependent on growth temperature, with optimal production at 37°C. The antimicrobial activity was not restricted to streptococci, but effective against a variety of organisms, including ESKAPE pathogens.© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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September 22, 2019

Establishment of a dual-wavelength spectrophotometric method for analysing and detecting carbapenemase-producing Enterobacteriaceae.

The spread of carbapenemase-producing Enterobacteriaceae (CPE) is an increasing global public health concern. The development of simple and reliable methods for CPE detection is required in the clinical setting. This study aimed to establish a dual-wavelength measurement method using an ultraviolet-visible spectrophotometer to rapidly quantify imipenem hydrolysis in bacterial cell suspensions. The hydrolytic activities of 148 strains including various CPE strains (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter aerogenes containing the blaIMP, blaKPC, blaNDM, blaOXA, and blaVIM genes) were measured and analysed. A cut-off value was obtained for differentiation between CPE and non-CPE strains, and the method had high sensitivity (100%) and specificity (100%) within 60?min. Our system has potential clinical applications in detecting CPE.

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September 22, 2019

Complete genomic analysis of a kingdom crossing Klebsiella variicola isolate.

Bacterial isolate X39 was isolated from a community-acquired pneumonia patient in Beijing, China. A phylogenetic tree based on rpoB genes and average nucleotide identity data confirmed that isolate X39 belonged to Klebsiella variicola. The genome of K. variicola X39 contained one circular chromosome and nine plasmids. Comparative genomic analyses with other K. variicola isolates revealed that K. variicola X39 contained the most unique genes. Of these unique genes, many were prophages and transposases. Many virulence factors were shared between K. variicola X39 and Klebsiella pneumoniae F1. The pathogenicity of K. variicola X39 was compared with that of K. pneumoniae F1 in an abdominal infection model. The results indicated that K. variicola X39 was less virulent than typical clinical K. pneumoniae F1. The genome of K. variicola X39 also contained some genes involved in plant colonization, nitrogen fixation, and defense against oxidative stress. GFP-labeled K. variicola X39 could colonize maize as an endophytic bacterium. We concluded that K. variicola X39 was a kingdom-crossing strain.

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September 22, 2019

Discovery of mcr-1-mediated colistin resistance in a highly virulent Escherichia coli lineage.

Resistance to last-line polymyxins mediated by the plasmid-borne mobile colistin resistance gene (mcr-1) represents a new threat to global human health. Here we present the complete genome sequence of an mcr-1-positive multidrug-resistant Escherichia coli strain (MS8345). We show that MS8345 belongs to serotype O2:K1:H4, has a large 241,164-bp IncHI2 plasmid that carries 15 other antibiotic resistance genes (including the extended-spectrum ß-lactamase blaCTX-M-1) and 3 putative multidrug efflux systems, and contains 14 chromosomally encoded antibiotic resistance genes. MS8345 also carries a large ColV-like virulence plasmid that has been associated with E. coli bacteremia. Whole-genome phylogeny revealed that MS8345 clusters within a discrete clade in the sequence type 95 (ST95) lineage, and MS8345 is very closely related to the highly virulent O45:K1:H4 clone associated with neonatal meningitis. Overall, the acquisition of a plasmid carrying resistance to colistin and multiple other antibiotics in this virulent E. coli lineage is concerning and might herald an era where the empirical treatment of ST95 infections becomes increasingly more difficult.IMPORTANCEEscherichia coli ST95 is a globally disseminated clone frequently associated with bloodstream infections and neonatal meningitis. However, the ST95 lineage is defined by low levels of drug resistance amongst clinical isolates, which normally provides for uncomplicated treatment options. Here, we provide the first detailed genomic analysis of an E. coli ST95 isolate that has both high virulence potential and resistance to multiple antibiotics. Using the genome, we predicted its virulence and antibiotic resistance mechanisms, which include resistance to last-line antibiotics mediated by the plasmid-borne mcr-1 gene. Finding an ST95 isolate resistant to nearly all antibiotics that also has a high virulence potential is of major clinical importance and underscores the need to monitor new and emerging trends in antibiotic resistance development in this important global lineage. Copyright © 2018 Forde et al.

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September 22, 2019

Comparative analysis of blaKPC-2- and rmtB-carrying IncFII-family pKPC-LK30/pHN7A8 hybrid plasmids from Klebsiella pneumoniae CG258 strains disseminated among multiple Chinese hospitals.

We recently reported the complete sequence of a blaKPC-2- and rmtB-carrying IncFII-family plasmid p675920-1 with the pKPC-LK30/pHN7A8 hybrid structure. Comparative genomics of additional sequenced plasmids with similar hybrid structures and their prevalence in blaKPC-carrying Klebsiella pneumoniae strains from China were investigated in this follow-up study.A total of 51 blaKPC-carrying K. pneumoniae strains were isolated from 2012 to 2016 from five Chinese hospitals and genotyped by multilocus sequence typing. The blaKPC-carrying plasmids from four representative strains were sequenced and compared with p675920-1 and pCT-KPC. Plasmid transfer, carbapenemase activity determination, and bacterial antimicrobial susceptibility test were performed to characterize resistance phenotypes mediated by these plasmids. The prevalence of pCT-KPC-like plasmids in these blaKPC-carrying K. pneumoniae strains was screened by PCR.The six KPC-encoding plasmids p1068-KPC, p20049-KPC, p12139-KPC and p64917-KPC (sequenced in this study) and p675920-1 and pCT-KPC slightly differed from one another due to deletion and acquisition of various backbone and accessory regions. Two major accessory resistance regions, which included the blaKPC-2 region harboring blaKPC-2 (carbapenem resistance) and blaSHV-12 (ß-lactam resistance), and the MDR region carrying rmtB (aminoglycoside resistance), fosA3 (fosfomycin resistance), blaTEM-1B (ß-lactam resistance) and blaCTX-M-65 (ß-lactam resistance), were found in each of these six plasmids and exhibited several parallel evolution routes. The pCT-KPC-like plasmids were present in all the 51 K. pneumoniae isolates, all of which belonged to CG258.There was clonal dissemination of K. pneumoniae CG258 strains, harboring blaKPC-2- and rmtB-carrying IncFII-family pKPC-LK30/pHN7A8 hybrid plasmids, among multiple Chinese hospitals.

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September 22, 2019

Endogenous rRNA sequence variation can regulate stress response gene expression and phenotype.

Prevailing dogma holds that ribosomes are uniform in composition and function. Here, we show that nutrient limitation-induced stress in E. coli changes the relative expression of rDNA operons to alter the rRNA composition within the actively translating ribosome pool. The most upregulated operon encodes the unique 16S rRNA, rrsH, distinguished by conserved sequence variation within the small ribosomal subunit. rrsH-bearing ribosomes affect the expression of functionally coherent gene sets and alter the levels of the RpoS sigma factor, the master regulator of the general stress response. These impacts are associated with phenotypic changes in antibiotic sensitivity, biofilm formation, and cell motility and are regulated by stress response proteins, RelA and RelE, as well as the metabolic enzyme and virulence-associated protein, AdhE. These findings establish that endogenously encoded, naturally occurring rRNA sequence variation can modulate ribosome function, central aspects of gene expression regulation, and cellular physiology. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Antimicrobial resistance profile of mcr-1 positive clinical isolates of Escherichia coli in China From 2013 to 2016.

Multidrug-resistant (MDR) Escherichia coli poses a great challenge for public health in recent decades. Polymyxins have been reconsidered as a valuable therapeutic option for the treatment of infections caused by MDR E. coli. A plasmid-encoded colistin resistance gene mcr-1 encoding phosphoethanolamine transferase has been recently described in Enterobacteriaceae. In this study, a total of 123 E. coli isolates obtained from patients with diarrheal diseases in China were used for the genetic analysis of colistin resistance in clinical isolates. Antimicrobial resistance profile of polymyxin B (PB) and 11 commonly used antimicrobial agents were determined. Among the 123 E. coli isolates, 9 isolates (7.3%) were resistant to PB and PCR screening showed that seven (5.7%) isolates carried the mcr-1 gene. A hybrid sequencing analysis using single-molecule, real-time (SMRT) sequencing and Illumina sequencing was then performed to resolve the genomes of the seven mcr-1 positive isolates. These seven isolates harbored multiple plasmids and are MDR, with six isolates carrying one mcr-1 positive plasmid and one isolate (14EC033) carrying two mcr-1 positive plasmids. These eight mcr-1 positive plasmids belonged to the IncX4, IncI2, and IncP1 types. In addition, the mcr-1 gene was the solo antibiotic resistance gene identified in the mcr-1 positive plasmids, while the rest of the antibiotic resistance genes were mostly clustered into one or two plasmids. Interestingly, one mcr-1 positive isolate (14EC047) was susceptible to PB, and we showed that the activity of MCR-1-mediated colistin resistance was not phenotypically expressed in 14EC047 host strain. Furthermore, three isolates exhibited resistance to PB but did not carry previously reported mcr-related genes. Multilocus sequence typing (MLST) showed that these mcr-1 positive E. coli isolates belonged to five different STs, and three isolates belonged to ST301 which carried multiple virulence factors related to diarrhea. Additionally, the mcr-1 positive isolates were all susceptible to imipenem (IMP), suggesting that IMP could be used to treat infection caused by mcr-1 positive E. coli isolates. Collectively, this study showed a high occurrence of mcr-1 positive plasmids in patients with diarrheal diseases of Guangzhou in China and the abolishment of the MCR-1 mediated colistin resistance in one E. coli isolate.

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September 22, 2019

pYR4 from a Norwegian isolate of Yersinia ruckeri is a putative virulence plasmid encoding both a type IV pilus and a type IV secretion system

Enteric redmouth disease caused by the pathogen Yersinia ruckeri is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of Y. ruckeri have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian Y. ruckeri isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic Yersiniae, pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other Y. ruckeri plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the Y. ruckeri NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system (tra). In contrast to other Yersinia plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other Y. ruckeri plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the Y. ruckeri 150 and Y. ruckeri ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for de novo genome sequencing. As the tra operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to Y. ruckeri virulence.

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September 22, 2019

Detection of mcr-1 plasmids in Enterobacteriaceae isolates from human specimens: Comparison with those in Escherichia coli isolates from livestock in Korea.

The emerging mobile colistin resistance gene, mcr-1, is an ongoing worldwide concern and an evaluation of clinical isolates harboring this gene is required in Korea. We investigated mcr-1-possessing Enterobacteriaceae among Enterobacteriaceae strains isolated in Korea, and compared the genetic details of the plasmids with those in Escherichia coli isolates from livestock.Among 9,396 Enterobacteriaceae clinical isolates collected between 2010 and 2015, 1,347 (14.3%) strains were resistant to colistin and those were screened for mcr-1 by PCR. Colistin minimum inhibitory concentrations (MICs) were determined by microdilution, and conjugal transfer of the mcr-1-harboring plasmids was assessed by direct mating. Whole genomes of three mcr-1-positive Enterobacteriaceae clinical isolates and 11 livestock-origin mcr-1-positive E. coli isolates were sequenced.Two E. coli and one Enterobacter aerogenes clinical isolates carried carried IncI2 plasmids harboring mcr-1, which conferred colistin resistance (E. coli MIC, 4 mg/L; E. aerogenes MIC, 32 mg/L). The strains possessed the complete conjugal machinery except for E. aerogenes harboring a truncated prepilin peptidase. The E. coli plasmid transferred more efficiently to E. coli than to Klebsiella pneumoniae or Enterobacter cloacae recipients. Among the three bacterial hosts, the colistin MIC was the highest for E. coli owing to the higher mcr-1-plasmid copy number and mcr-1 expression levels. Ten mcr-1-positive chicken-origin E. coli strains also possessed mcr-1-harboring IncI2 plasmids closely related to that in the clinical E. aerogenes isolate, and the remaining one porcine-origin E. coli possessed an mcr-1-harboring IncX4 plasmid.mcr-1-harboring IncI2 plasmids were identified in clinical Enterobacteriaceae isolates. These plasmids were closely associated with those in chicken-origin E. coli strains in Korea, supporting the concept of mcr-1 dissemination between humans and livestock.© The Korean Society for Laboratory Medicine.

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September 22, 2019

Isolation, characterization, genomic sequencing, and GFP-marked insertional mutagenesis of a high-performance nitrogen-fixing bacterium, Kosakonia radicincitans GXGL-4A and visualization of bacterial colonization on cucumber roots.

A gram-negative bacterium GXGL-4A was originally isolated from maize roots. It displayed nitrogen-fixing (NF) ability under nitrogen-free culture condition, and had a significant promotion effect on cucumber growth in the pot inoculation test. The preliminary physiological and biochemical traits of GXGL-4A were characterized. Furthermore, a phylogenetic tree was constructed based on 16S ribosomal DNA (rDNA) sequences of genetically related species. To determine the taxonomic status of GXGL-4A and further utilize its nitrogen-fixing potential, genome sequence was obtained using PacBio RS II technology. The analyses of average nucleotide identity based on BLAST+ (ANIb) and correlation indexes of tetra-nucleotide signatures (Tetra) showed that the NF isolate GXGL-4A is closely related to the Kosakonia radicincitans type strain DSM 16656. Therefore, the isolate GXGL-4A was eventually classified into the species of Kosakonia radicincitans and designated K. radicincitans GXGL-4A. A high consistency in composition and gene arrangement of nitrogen-fixing gene cluster I (nif cluster I) was found between K. radicincitans GXGL-4A and other Kosakonia NF strains. The mutants tagged with green fluorescence protein (GFP) were obtained by transposon Tn5 mutagenesis, and then, the colonization of gfp-marked K. radicincitans GXGL-4A cells on cucumber seedling root were observed under fluorescence microscopy. The preferential sites of the labeled GXGL-4A cell population were the lateral root junctions, the differentiation zone, and the elongation zone. All these results should benefit for the deep exploration of nitrogen fixation mechanism of K. radicincitans GXGL-4A and will definitely facilitate the genetic modification process of this NF bacterium in sustainable agriculture.

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