Combining dense and sparse labeling in optical DNA mapping.

Optical DNA mapping (ODM) is based on fluorescent labeling, stretching and imaging of single DNA molecules to obtain sequence-specific fluorescence profiles, DNA barcodes. These barcodes can be mapped to theoretical counterparts obtained from DNA reference sequences, which in turn allow for DNA identification in complex samples and for detecting structural changes in individual DNA molecules. There are several types of DNA labeling schemes for ODM and for each labeling type one or several types of match scoring methods are used. By combining the information from multiple labeling schemes one can potentially improve mapping confidence; however, combining match scores from different labeling assays has not been implemented yet. In this study, we introduce two theoretical methods for dealing with analysis of DNA molecules with multiple label types. In our first method, we convert the alignment scores, given as output from the different assays, into p-values using carefully crafted null models. We then combine the p-values for different label types using standard methods to obtain a combined match score and an associated combined p-value. In the second method, we use a block bootstrap approach to check for the uniqueness of a match to a database for all barcodes matching with a combined p-value below a predefined threshold. For obtaining experimental dual-labeled DNA barcodes, we introduce a novel assay where we cut plasmid DNA molecules from bacteria with restriction enzymes and the cut sites serve as sequence-specific markers, which together with barcodes obtained using the established competitive binding labeling method, form a dual-labeled barcode. All experimental data in this study originates from this assay, but we point out that our theoretical framework can be used to combine data from all kinds of available optical DNA mapping assays. We test our multiple labeling frameworks on barcodes from two different plasmids and synthetically generated barcodes (combined competitive-binding- and nick-labeling). It is demonstrated that by simultaneously using the information from all label types, we can substantially increase the significance when we match experimental barcodes to a database consisting of theoretical barcodes for all sequenced plasmids.

Assessment of the plasmidome of an extremophilic microbial community from the Diamante Lake, Argentina.

Diamante Lake located at 4589 m.a.s.l. in the Andean Puna constitutes an extreme environment. It is exposed to multiple extreme conditions such as an unusually high concentration of arsenic (over 300 mg L-1) and low oxygen pressure. Microorganisms thriving in the lake display specific genotypes that facilitate survival, which include at least a multitude of plasmid-encoded resistance traits. Hence, the genetic information provided by the plasmids essentially contributes to understand adaptation to different stressors. Though plasmids from cultivable organisms have already been analyzed to the sequence level, the impact of the entire plasmid-borne genetic information on such microbial ecosystem is not known. This study aims at assessing the plasmidome from Diamante Lake, which facilitates the identification of potential hosts and prediction of gene functions as well as the ecological impact of mobile genetic elements. The deep-sequencing analysis revealed a large fraction of previously unknown DNA sequences of which the majority encoded putative proteins of unknown function. Remarkably, functions related to the oxidative stress response, DNA repair, as well as arsenic- and antibiotic resistances were annotated. Additionally, all necessary capacities related to plasmid replication, mobilization and maintenance were detected. Sequences characteristic for megaplasmids and other already known plasmid-associated genes were identified as well. The study highlights the potential of the deep-sequencing approach specifically targeting plasmid populations as it allows to evaluate the ecological impact of plasmids from (cultivable and non-cultivable) microorganisms, thereby contributing to the understanding of the distribution of resistance factors within an extremophilic microbial community.

Resistive pulse sensing device with embedded nanochannel (nanochannel-RPS) for label-free biomolecule and bionanoparticle analysis.

This paper reports an IC-compatible method for fabricating a PDMS-based resistive pulse sensing (RPS) device with embedded nanochannel (nanochannel-RPS) for label-free analysis of biomolecules and bionanoparticles, such as plasmid DNAs and exosomes. Here, a multilayer lithography process was proposed to fabricate the PDMS mold for the microfluidic device, comprising a bridging nanochannel, as the sensing gate. RPS was performed by placing the sensing and excitation electrodes symmetrically upstream and downstream of the sensing gate. In order to reduce the noise level, a reference electrode was designed and placed beside the excitation electrode. To demonstrate the feasibility of the proposed nanochannel-RPS device and sensing system, polystyrene micro- and nanoparticles with diameters of 1µm and 300 nm were tested by the proposed device with signal-to-noise ratios (SNR) ranging from 9.1-30.5 and 2.2-5.9, respectively. Furthermore, a nanochannel with height of 300 nm was applied for 4 kb plasmid DNA detection, implying the potential of the proposed method for label-free quantification of nanoscale biomolecules. Moreover, HeLa cell exosomes, known as a well-studied subtype of extracellular vesicles, were measured and analyzed by their size distribution. The result of the resistive pulse amplitude corresponded well to that of nanoparticle tracking analysis (NTA). The proposed nanochannel-RPS device and the sensing strategy are not only capable of label-free analysis for nanoscale biomolecules and bionanoparticles, but are also cost-effective for large-scale manufacturing.

Widespread Prevalence of Plasmid-Mediated Colistin Resistance Gene mcr-1 in Escherichia coli from Père David's Deer in China.

Père David's deer (Elaphurus davidianus or milu) is an endangered species, and the prevalence of antimicrobial resistance (AMR) such as mcr-1-positive strains among them has been unknown. In this study, we aimed to investigate the genomic characterizations of mcr-1-positive strains and provide insight into the dissemination of AMR in nature reserve settings. Sixty-seven mcr-1-positive Escherichia coli isolates from 97 fecal samples were identified by PCR and found resistant to colistin. The prevalence of ß-lactam resistance was very high, and there were 64 mcr-1-positive isolates containing ß-lactamase genes. Transconjugants of 66 mcr-1-positive isolates were acquired through conjugation experiments. PCR-based replicon typing (PBRT) showed that 44 strains harbored IncI2 mcr-1-bearing plasmids, eight strains harbored IncX4 mcr-1-carrying plasmids, and 14 strains harbored IncHI2 mcr-1-positive plasmids. Notably, mcr-1 was located in the chromosome of LD27-1. Clonal dissemination and horizontal dissemination of mcr-1 by plasmids coexist. We first report the prevalence of plasmid-mediated mcr-1 in E. coli from Père David's deer in China. mcr-1-bearing IncI2 plasmid was the most frequent plasmid type, and the first IncI2 plasmid harboring both blaCTX-M-132 and mcr-1 is characterized here. Our results support the implication of Père David's deer as a potential reservoir for MCR-1-producing E. coliIMPORTANCE The mcr-1 gene is widely reported around the world and has been identified on various plasmids with different replicon types. Resistance to the last-line antibiotic colistin mediated by mcr-1 still represents a threat to global public health. Père David's deer is a highly endangered species originating in China, and many deer are currently being raised in captivity for gradual reintroduction to the wild. If this species carries AMR bacteria, it will pose a potential threat to the environment. Therefore, research on the dissemination of mcr-1-positive E. coli from Père David's deer is of great significance. This is the first study to investigate the microbiological and genomic surveillance of MCR-1-producing bacteria colonized among Père David's deer in China, and we uncovered a high prevalence of MCR-1-producing E. coli The importance of constant surveillance for AMR bacteria in nature reserve settings is emphasized.

Whole-Genome Sequence Analysis of an Extensively Drug-Resistant Salmonella enterica Serovar Agona Isolate from an Australian Silver Gull (Chroicocephalus novaehollandiae) Reveals the Acquisition of Multidrug Resistance Plasmids.

Although most of the approximately 94 million annual human cases of gastroenteritis due to Salmonella enterica resolve without medical intervention, antimicrobial therapy is recommended for patients with severe disease. Wild birds can be natural hosts of Salmonella that pose a threat to human health; however, multiple-drug-resistant serovars of S. enterica have rarely been described. In 2012, silver gull (Chroicocephalus novaehollandiae) chicks at a major breeding colony were shown to host Salmonella, most isolates of which were susceptible to antibiotics. However, multiple-drug-resistant (MDR) Escherichia coli with resistance to carbapenems, ceftazidime, and fluoroquinolones was reported from this breeding colony. In this paper, we describe a novel MDR Salmonella strain subsequently isolated from the same breeding colony. SG17-135, an isolate of S. enterica with phenotypic resistance to 12 individual antibiotics but only nine antibiotic classes including penicillins, cephalosporins, monobactams, macrolides, fluoroquinolones, aminoglycosides, dihydrofolate reductase inhibitors (trimethoprim), sulfonamides, and glycylcyclines was recovered from a gull chick in 2017. Whole-genome sequence (WGS) analysis of SG17-135 identified it as Salmonella enterica serovar Agona (S Agona) with a chromosome comprising 4,813,284 bp, an IncHI2 ST2 plasmid (pSG17-135-HI2) of 311,615 bp, and an IncX1 plasmid (pSG17-135-X) of 27,511 bp. pSG17-135-HI2 housed a complex resistance region comprising 16 antimicrobial resistance genes including blaCTX-M-55 The acquisition of MDR plasmids by S. enterica described here poses a serious threat to human health. Our study highlights the importance of taking a One Health approach to identify environmental reservoirs of drug-resistant pathogens and MDR plasmids.IMPORTANCE Defining environmental reservoirs hosting mobile genetic elements that shuttle critically important antibiotic resistance genes is key to understanding antimicrobial resistance (AMR) from a One Health perspective. Gulls frequent public amenities, parklands, and sewage and other waste disposal sites and carry drug-resistant Escherichia coli Here, we report on SG17-135, a strain of Salmonella enterica serovar Agona isolated from the cloaca of a silver gull chick nesting on an island in geographic proximity to the greater metropolitan area of Sydney, Australia. SG17-135 is closely related to pathogenic strains of S Agona, displays resistance to nine antimicrobial classes, and carries important virulence gene cargo. Most of the antibiotic resistance genes hosted by SG17-135 are clustered on a large IncHI2 plasmid and are flanked by copies of IS26 Wild birds represent an important link in the evolution and transmission of resistance plasmids, and an understanding of their behavior is needed to expose the interplay between clinical and environmental microbial communities.

Detection of Bacillus anthracis and Bacillus anthracis-like spores in soil from state of Rio de Janeiro, Brazil.

BACKGROUND: Bacillus anthracis is the aetiologic agent of anthrax, a re-emerging, septicaemic, haemorrhagic and lethal disease that affects humans, domestic ruminants and wildlife. Plasmids pXO1 and pXO2 are attributes that confer pathogenicity to B. anthracis strains. This bacterium was used as biological weapon in the World Wars and in the biological attack in the United States of America at 2001. B. anthracis is classified as a Tier 1 bioterrorism agent by the Centers for Diseases Control and Prevention. Anthrax is recognised as a re-emerging disease. Several studies concerning the dynamics of B. anthracis cycle in soil revealed that nonpathogenic B. anthracis strains due to lack of pXO2 plasmid are commonly found in some types of soil. OBJECTIVES: This study aimed isolation and identification of B. anthracis spores in soil samples of the state of Rio de Janeiro, Brazil. METHODS: Phenotypic and genotypic approaches were used to identify isolates including MALDI-TOF/MS, motility test, susceptibility to gamma phage and penicillin, survey for pag and cap genes as surrogates of pXO1 and pXO2 plasmids, respectively, and sequencing of 16SrRNA-encoding gene. Physicochemical analysis of the soil samples were carried out to describe soil characteristics. FINDINGS: We observed the presence of one B. anthracis pXO1+ and pXO2- isolated from clay loam soil; one B. anthracis-like strain pXO1+ and pXO2-isolated from loamy sand; and 10 Bacillus spp. strains sensitive to phage-gamma that need better characterisation to define which their species were recovered from loamy sand. MAIN CONCLUSIONS: This work showed promising results and it was the first study to report results from an active surveillance for B. anthracis in Brazil.

Construction and characterization of centromeric plasmids for Komagataella phaffii using a color-based plasmid stability assay.

The yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work, we designed a color-based genetic assay to investigate plasmid stability in K. phaffii and constructed vectors bearing K. phaffii centromeres and the ADE3 marker. These genetic tools were evaluated in terms of mitotic stability by transforming an ade2/ade3 auxotrophic strain and regarding plasmid copy number by quantitative PCR (qPCR). Our results confirmed that the centromeric plasmids were maintained at low copy numbers as a result of typical chromosome-like segregation during cell division. These features, combined with in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.

Sequence Analysis of Plasmids in Vibrio anguillarum from Different Fish and Locations.

The genetic diversity of Vibrio anguillarum pJM1-like plasmids was investigated. Plasmids were isolated from 18 V. anguillarum serovar O1 strains collected from different geographic locations and fish species. The plasmids were sequenced and compared with the complete sequence of the published virulence plasmid pJM1. All 18 strains contained pJM1-like plasmids with approximately 65 kbp and all plasmids encoded the virulence genes responsible for the anguibactin iron sequestering system. The plasmids were highly conserved but minor differences were observed in some genes. A single nucleotide polymorphisms (SNPs) analysis showed 0-11 nucleotide variations between each of the 18 plasmids and the pJM1 plasmid. Compared with the sequence of pJM1, nonsynonymous SNPs were identified in fatC, angR, angL, pJM1_p19, and angE. In particular, a mutation found in 15 out of 18 sequenced plasmids in angR has previously been linked to hyperproduction of anguibactin and was found in all the European isolates. However, overall the pJM1-like plasmids isolated from V. anguillarum serovar O1 exhibited a high degree of conservation regardless of their geographical origin or fish species.

Wide genetic heterogeneity and low antimicrobial resistance of enteroaggregative Escherichia coli isolates from several rural communities.

Enteroaggregative Escherichia coli (EAEC), a highly heterogeneous pathotype of E. coli classified as typical and atypical, are an emerging cause of acute and persistent diarrhea. We aimed to investigate whether population living in rural geographic areas, impacts in the heterogeneity, dissemination and antimicrobial susceptibility of EAEC strains. EAEC isolates (n=73) were analysed for the presence of 23 putative virulence factors, plasmid and antimicrobial resistance profiles, biofilm formation, pulsedfield gel electrophoresis (PFGE) and by multilocus sequence typing (MLST). The agg3A, agg4A, agn43, aap, shf, astA, pet, pic/set1A and sat genes, biofilm forming and antimicrobial resistance were statistically associated with typical EAEC. A low frequency of all isolates was resistant or showed a multidrug-resistance profile. No isolate showed the same plasmid profile. In total, 58 different pulsotypes were observed. Sixteen isolates analysed by MLST belonged to 15 different sequence types (ST) and showed a different PFGE pattern and virulence-gene profile. The fact that the communities are semi-isolated did not impact on the peculiar heterogeneity of EAEC, being characterized as epidemiologically independent strains.

Detection of Bacillus anthracis and Bacillus anthracis-like spores in soil from state of Rio de Janeiro, Brazil

BACKGROUND Bacillus anthracis is the aetiologic agent of anthrax, a re-emerging, septicaemic, haemorrhagic and lethal disease that affects humans, domestic ruminants and wildlife. Plasmids pXO1 and pXO2 are attributes that confer pathogenicity to B. anthracis strains. This bacterium was used as biological weapon in the World Wars and in the biological attack in the United States of America at 2001. B. anthracis is classified as a Tier 1 bioterrorism agent by the Centers for Diseases Control and Prevention. Anthrax is recognised as a re-emerging disease. Several studies concerning the dynamics of B. anthracis cycle in soil revealed that nonpathogenic B. anthracis strains due to lack of pXO2 plasmid are commonly found in some types of soil. OBJECTIVES This study aimed isolation and identification of B. anthracis spores in soil samples of the state of Rio de Janeiro, Brazil. METHODS Phenotypic and genotypic approaches were used to identify isolates including MALDI-TOF/MS, motility test, susceptibility to gamma phage and penicillin, survey for pag and cap genes as surrogates of pXO1 and pXO2 plasmids, respectively, and sequencing of 16SrRNA-encoding gene. Physicochemical analysis of the soil samples were carried out to describe soil characteristics. FINDINGS We observed the presence of one B. anthracis pXO1+ and pXO2- isolated from clay loam soil; one B. anthracis-like strain pXO1+ and pXO2-isolated from loamy sand; and 10 Bacillus spp. strains sensitive to phage-gamma that need better characterisation to define which their species were recovered from loamy sand. MAIN CONCLUSIONS This work showed promising results and it was the first study to report results from an active surveillance for B. anthracis in Brazil.

Serotype distribution and antimicrobial resistance of human Salmonella enterica in Bangui, Central African Republic, from 2004 to 2013.

BACKGROUND: Limited epidemiological and antimicrobial resistance data are available on Salmonella enterica from sub-Saharan Africa. We determine the prevalence of resistance to antibiotics in isolates in the Central African Republic (CAR) between 2004 and 2013 and the genetic basis for resistance to third-generation cephalosporin (C3G). METHODOLOGY/PRINCIPAL FINDINGS: A total of 582 non-duplicate human clinical isolates were collected. The most common serotype was Typhimurium (n = 180, 31% of the isolates). A randomly selected subset of S. Typhimurium isolates were subtyped by clustered regularly interspaced short palindromic repeat polymorphism (CRISPOL) typing. All but one invasive isolate tested (66/68, 96%) were associated with sequence type 313. Overall, the rates of resistance were high to traditional first-line drugs (18-40%) but low to many other antimicrobials, including fluoroquinolones (one resistant isolate) and C3G (only one ESBL-producing isolate). The extended-spectrum beta-lactamase (ESBL)-producing isolate and three additional ESBL isolates from West Africa were studied by whole genome sequencing. The blaCTX-M-15 gene and the majority of antimicrobial resistance genes found in the ESBL isolate were present in a large conjugative IncHI2 plasmid highly similar (> 99% nucleotide identity) to ESBL-carrying plasmids found in Kenya (S. Typhimurium ST313) and also in West Africa (serotypes Grumpensis, Havana, Telelkebir and Typhimurium). CONCLUSIONS/SIGNIFICANCE: Although the prevalence of ESBL-producing Salmonella isolates was low in CAR, we found that a single IncHI2 plasmid-carrying blaCTX-M-15 was widespread among Salmonella serotypes from sub-Saharan Africa, which is of concern.

Laboratory Cultivation and Storage of Shigella.

Shigella species, which are closely related to Escherichia coli, can easily be maintained and stored in the laboratory. This article includes protocols for preparation of routine growth conditions and media, for storage of the bacteria, and for monitoring of the presence of the virulence plasmid. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Growth of S. flexneri from frozen stocks or agar stabs Basic Protocol 2: Growth of S. flexneri in rich liquid medium Alternate Protocol 1: Growth of S. flexneri in rich defined medium Alternate Protocol 2: Growth of S. flexneri in minimal medium Basic Protocol 3: Storage of S. flexneri in frozen stocks Alternate Protocol 3: Storage of S. flexneri in agar stabs.

Occurrence and Genomic Characterization of Two MCR-1-Producing Escherichia coli Isolates from the Same Mink Farmer.

The spread of colistin resistance gene mcr-1 at the animal-human interface remains largely unknown. This work aimed to investigate the molecular characteristics of two extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli strains with mcr-1, i.e., strains H8 and H9, isolated from the same mink farmer. In this study, five mcr-positive E. coli strains were isolated from the mink farm. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) identified two genetically unrelated MCR-1 producers (H8 and H9) from the same farmer and two clonally related MCR-1-positive isolates (M5 and M6) from two different mink samples. Additionally, a mcr-1 variant, designated mcr-1.12, was identified in isolate M4. MIC determination revealed that all of the MCR-producing strains exhibited multiresistant phenotypes but showed susceptibility to imipenem, meropenem, amikacin, and tigecycline. Replicon typing showed that mcr-1 was associated with IncHI2 plasmids in 4 cases, while the gene was located on an IncI2 plasmid in 1 case. PacBio sequencing and plasmid analysis confirmed that the mcr-1 gene was located on an ∼204-kb IncHI2 plasmid in H8 and was carried by an ∼61-kb IncI2 plasmid in H9. To our knowledge, this work represents the first report of the occurrence of MCR-producing isolates from mink. Moreover, our report also describes the coexistence of two different MCR-1 producers in the same farmer. It highlights that fur farms can be reservoirs of mcr-1 genes. The identification of mcr-carrying plasmids on a fur farm is of potential public health importance, as it suggests that mcr is widespread in the animal husbandry industry.IMPORTANCE Colistin resistance is a real threat for both human and animal health. The mobile colistin resistance gene mcr has contributed to the persistence and transmission of colistin resistance at the interfaces of animals, humans, and ecosystems. Although mcr genes have usually been recovered from food animals, patients, and healthy humans, transmission of mcr genes at the animal-human interface remains largely unknown. This was the first study to isolate and characterize MCR-producing isolates from mink, as well as to report the coexistence of two different MCR-1 producers in the same farmer. The characterization and analysis of two MCR-1-producing E. coli isolates may have important implications for comprehension of the transmission dynamics of these bacteria. We emphasize the importance of improved multisectorial surveillance of colistin-resistant E. coli in this region.

Emergence and Comparative Genomics Analysis of Extended-Spectrum-ß-Lactamase-Producing Escherichia coli Carrying mcr-1 in Fennec Fox Imported from Sudan to China.

The aim of this study was to investigate the occurrence and genomic characteristics of extended-spectrum-ß-lactamase-producing Escherichia coli (ESBL-EC) in fennec fox imported from Sudan to China. We screened 88 fecal samples from fennec fox for ESBL-EC, using cefotaxime- and meropenem-supplemented selective medium. Antimicrobial susceptibility testing was performed by the agar dilution method except for colistin and tigecycline; for colistin and tigecycline, testing was conducted by the broth microdilution method. ESBL-EC bacteria were sequenced, and their genomes were characterized. Plasmid conjugation, S1 nuclease pulsed-field gel electrophoresis (PFGE), and Southern blotting were performed for a MCR-1-producing isolate. The genetic environment of mcr-1 and ESBL genes was also investigated. A total of 29 ESBL-EC bacteria were isolated from 88 fennec fox (32.9%), while no carbapenemase producers were found. The most prevalent genotypes were the blaCTX-M-55 and blaCTX-M-14 genes, followed by blaCTX-M-15 and blaCTX-M-64 We detected nine sequence types among 29 ESBL-EC. Furthermore, the mcr-1 gene was detected in isolate EcFF273. Conjugation analysis confirmed that the mcr-1 gene was transferable. S1 PFGE, Southern blotting, and whole-genome sequencing revealed that mcr-1 and blaCTX-M-64 were both located on a 65-kb IncI2 plasmid. This study reports for the first time the occurrence of ESBL-EC in fennec fox. The high prevalence of ESBL producers and the occurrence of MCR-1 producer in fennec fox imported into China from Sudan are unexpected. In addition, it clearly demonstrated that commensal E. coli strains can be reservoirs of blaCTX-M and mcr-1, potentially contributing to the dissemination and transfer of such genes to pathogenic bacteria among fennec fox. Our results support the implication of fennec fox as a biological vector for ESBL-producing members of the Enterobacteriaceae family.IMPORTANCE The extended-spectrum-ß-lactamase (ESBL)-producing members of the Enterobacteriaceae family are a global concern for both animal and human health. There is some information indicating a high prevalence of ESBL producers in food animals. Moreover, there have been an increasing number of reports on ESBL-producing strains resistant to the last-resort antibiotic colistin with the global dissemination of the plasmid-mediated mcr-1 gene, which is believed to have originated in animal breeding. However, little is known regarding the burden of ESBL-producing Enterobacteriaceae on wild animals. No data were available on the prevalence of antimicrobial resistance (AMR) among wild animals imported into China. This is the first study to investigate the microbiological and genomics surveillance investigation of ESBL colonization among fennec fox (Vulpes zerda) imported from Sudan to China, and we uncovered a high prevalence of ESBL-EC. Furthermore, the underlying mechanism of colistin resistance in an isolate that harbored mcr-1 was also investigated. Results of characterization and analysis of 29 ESBL-producing E. coli may have important implications on our understanding of the transmission dynamics of these bacteria. We emphasize the importance of improved multisectoral surveillance for colistin-resistant E. coli in this region.

Comparative genomic analysis and molecular examination of the diversity of enterotoxigenic Escherichia coli isolates from Chile.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common diarrheal pathogens in the low- and middle-income regions of the world, however a systematic examination of the genomic content of isolates from Chile has not yet been undertaken. Whole genome sequencing and comparative analysis of a collection of 125 ETEC isolates from three geographic locations in Chile, allowed the interrogation of phylogenomic groups, sequence types and genes specific to isolates from the different geographic locations. A total of 80.8% (101/125) of the ETEC isolates were identified in E. coli phylogroup A, 15.2% (19/125) in phylogroup B, and 4.0% (5/125) in phylogroup E. The over-representation of genomes in phylogroup A was significantly different from other global ETEC genomic studies. The Chilean ETEC isolates could be further subdivided into sub-clades similar to previously defined global ETEC reference lineages that had conserved multi-locus sequence types and toxin profiles. Comparison of the gene content of the Chilean ETEC identified genes that were unique based on geographic location within Chile, phylogenomic classifications or sequence type. Completion of a limited number of genomes provided insight into the ETEC plasmid content, which is conserved in some phylogenomic groups and not conserved in others. These findings suggest that the Chilean ETEC isolates contain unique virulence factor combinations and genomic content compared to global reference ETEC isolates.

Plasmid Dissemination and Selection of a Multidrug-Resistant Klebsiella pneumoniae Strain during Transplant-Associated Antibiotic Therapy.

Antibiotics, which are used both to prevent and to treat infections, are a mainstay therapy for lifesaving procedures such as transplantation. For this reason, and many others, increased antibiotic resistance among human-associated pathogens, such as the carbapenem-resistant Enterobacteriaceae species, is of grave concern. In this study, we report on a hematopoietic stem cell transplant recipient in whom cultures detected the emergence of carbapenem resistance and spread across five strains of bacteria that persisted for over a year. Carbapenem resistance in Citrobacter freundii, Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae was linked to a pair of plasmids, each carrying the Klebsiella pneumoniae carbapenemase gene (blaKPC). Surveillance cultures identified a carbapenem-susceptible strain of Citrobacter freundii that may have become resistant through horizontal gene transfer of these plasmids. Selection of a multidrug-resistant Klebsiella pneumoniae strain was also detected following combination antibiotic therapy. Here we report a plasmid carrying the blaKPC gene with broad host range that poses the additional threat of spreading to endogenous members of the human gut microbiome.IMPORTANCE Antibiotic-resistant bacteria are a serious threat to medically fragile patient populations. The spread of antibiotic resistance through plasmid-mediated mechanisms is of grave concern as it can lead to the conversion of endogenous patient-associated strains to difficult-to-treat pathogens.

Genetic Features of Antarctic Acinetobacter radioresistens Strain A154 Harboring Multiple Antibiotic-Resistance Genes.

While antibiotic-resistant bacteria have been detected in extreme environments, including Antarctica, to date there are no reports of Acinetobacter species isolated from this region. Here, we characterized by whole-genome sequencing (WGS) the genetic content of a single antibiotic-resistant Acinetobacter spp. isolate (A154) collected in Antarctica. The isolate was recovered in 2013 from soil samples at Fildes Peninsula, Antarctica, and was identified by detection of the intrinsic OXA-23 gene, and confirmed by Tetra Correlation Search (TCS) and WGS. The antibiotic susceptibility profile was determined by disc diffusion, E-test, and broth microdilution methods. From WGS data, the acquired resistome and insertion sequence (IS) content were identified by in silico analyses. Plasmids were studied by the alkaline lysis method followed by pulsed-field gel electrophoresis and conventional PCR. The A154 isolate was identified as A. radioresistens by WGS analysis and displayed >99.9 of similarity by TCS in relation with the databases. Moreover, it was resistant to ampicillin, ceftriaxone, ceftazidime, cefepime, cefotaxime, streptomycin, and kanamycin. Likewise, in addition to the intrinsic blaOXA-23-like gene, A154 harbored the plasmid-encoded antibiotic-resistance genes blaPER-2, tet(B), aph(3')-Vla, strA, and strB, as well as a large diversity of ISs. This is the first report of antibiotic-resistant A. radioresistens in Antarctica. Our findings show the presence of several resistance genes which could be either intrinsic or acquired in the region.

Emergence of diversity in carbapenemase-producing Escherichia coli ST131, England, January 2014 to June 2016.

BackgroundEscherichia coli ST131, a global, high-risk clone, comprises fluoroquinolone resistance (FQ-R) mutations and CTX-M extended-spectrum beta-lactamases associated with the fimH30-encoding clades, C1 and C2. Further carbapenem resistance development in ST131 is a public health concern.AimThis observational study aimed to probe the diversity of carbapenemase-producing E. coli (CP E. coli) ST131 across England.MethodsST131 isolates were identified using whole-genome sequencing (WGS) data generated for all non-duplicate CP E. coli from human samples submitted to the national reference laboratory from January 2014 to June 2016. Antimicrobial resistance (AMR) gene content and single nucleotide polymorphism (SNP) data were compared against a published ST131 phylogeny and analysed alongside patient metadata.ResultsThirty-nine genetically diverse ST131 CP E. coli, from eight of nine regions, represented 10% of CP E. coli isolates sequenced. Ten and eight isolates were from the FQ-susceptible (FQ-S) clades A and B, while eight and 15 isolates belonged to the FQ-R clades C1 or C2, respectively. Seven distinct carbapenemases were identified: KPC-2 (21 isolates, 6 regions) frequently occurred among clade C2 isolates (n = 10). OXA-48-producers (10 isolates, 3 regions) were often from clade A (n = 5). NDM-1 (n = 4), NDM-5 (n = 1), VIM-1 (n = 1), VIM-4 (n = 1) and OXA-181 (n = 1) were also identified. Clade C2 isolates encoded more AMR genes than those from clades A (p = 0.02), B (p = 9.6 x 10-3) or C1 (p = 0.03).ConclusionWhen compared with its global predominance among ESBL-E. coli, ST131 represented a fraction of the CP E. coli received, belonging to diverse clades and encoding diverse carbapenemases. The greater accumulation of resistance genes in clade C2 isolates highlights the need for ongoing monitoring of this high-risk lineage.

Tracking Carbapenem-Producing Klebsiella pneumoniae Outbreak in an Intensive Care Unit by Whole Genome Sequencing.

The presence of carbapenem-producing Klebsiella pneumoniae (CP-Kp) is a serious threat to the control of nosocomial infections. Plasmid-mediated horizontal transfer of the resistance gene makes it difficult to control hospital-acquired CP- Kp infections. Nine CP- Kp strains were isolated during an outbreak in the intensive care unit of Shanghai Huashan hospital in east China. We conducted a retrospective study to identify the origin and route of transmission of this CP-Kp outbreak. Whole-genome sequencing (WGS) analysis was performed on 9 clinical isolates obtained from 8 patients, and the results were compared to clinical and epidemiological records. All isolates were ST11 CP-Kp. Single-nucleotide polymorphisms and the presence and structure of plasmids indicated that this CP-Kp outbreak had different origins. These 9 isolates were partitioned into two clades according to genetic distance. Four plasmids, CP002474.1, CP006799.1, CP018455.1, and CP025459.1, were detected among the 9 isolates. The plasmid phylogeny and antibiotic resistance (AR) gene profile results were consistent with the sequencing results. We found that two clades of CP-Kp were responsible for this nosocomial outbreak and demonstrated the transmission route from two index patients. Plasmid carriage and phylogeny are a useful tool for identifying clades involved in disease transmission.

Increase and diversity of carbapenemase-producing Escherichia coli isolates, Italy.

Aim: This study reports on a surveillance in an Italian hospital focused on carbapenemase-producing Escherichia coli (CP-Ec). Materials & methods: Eighteen isolates (nine from clinical specimens and nine from rectal swab) were characterized for antibiotic susceptibilities, typing features, main carbapenemase, extended-spectrum ß-lactamases (ESBLs) and other bla genes, and their transferability by conjugation and transformation. Results: An increase in CP-Ec isolates was observed during 3-year surveillance period. Compared with the clinical isolates, all belonging to one sequence type (ST), ST131, those from rectal swab were very heterogeneous and belonged to eight STs. Transfer data confirmed the role of conjugative plasmids in the spreading of carbapenemase genes. Conclusion: The prevalence of CP-Ec in Italy has risen, with a substantial increase over the last year.