Multidrug-resistant E. coli encoding high genetic diversity in carbohydrate metabolism genes displace commensal E. coli from the intestinal tract.

Extra-intestinal pathogenic Escherichia coli (ExPEC) can cause a variety of infections outside of the intestine and are a major causative agent of urinary tract infections. Treatment of these infections is increasingly frustrated by antimicrobial resistance (AMR) diminishing the number of effective therapies available to clinicians. Incidence of multidrug resistance (MDR) is not uniform across the phylogenetic spectrum of E. coli. Instead, AMR is concentrated in select lineages, such as ST131, which are MDR pandemic clones that have spread AMR globally. Using a gnotobiotic mouse model, we demonstrate that an MDR E. coli ST131 is capable of out-competing and displacing non-MDR E. coli from the gut in vivo. This is achieved in the absence of antibiotic treatment mediating a selective advantage. In mice colonised with non-MDR E. coli strains, challenge with MDR E. coli either by oral gavage or co-housing with MDR E. coli colonised mice results in displacement and dominant intestinal colonisation by MDR E. coli ST131. To investigate the genetic basis of this superior gut colonisation ability by MDR E. coli, we assayed the metabolic capabilities of our strains using a Biolog phenotypic microarray revealing altered carbon metabolism. Functional pangenomic analysis of 19,571 E. coli genomes revealed that carriage of AMR genes is associated with increased diversity in carbohydrate metabolism genes. The data presented here demonstrate that independent of antibiotic selective pressures, MDR E. coli display a competitive advantage to colonise the mammalian gut and points to a vital role of metabolism in the evolution and success of MDR lineages of E. coli via carriage and spread.

Validation testing to determine the sensitivity of lateral flow testing for asymptomatic SARS-CoV-2 detection in low prevalence settings: Testing frequency and public health messaging is key.

Lateral flow devices (LFDs) are quickly being implemented for use in large-scale population surveillance programs for SARS-CoV-2 infection in the United Kingdom. These programs have been piloted in city-wide screening in the city of Liverpool and are now being rolled out to support care home visits and the return home of University students for the Christmas break. Here, we present data on the performance of LFDs to test almost 8,000 students at the University of Birmingham between December 2 and December 9, 2020. The performance is validated against almost 800 samples using PCR performed in the University Pillar 2 testing lab and theoretically validated on thousands of Pillar 2 PCR testing results performed on low-prevalence care home testing samples. Our data show that LFDs do not detect infections presenting with PCR Ct values over 29 to 30 as determined using the Thermo Fisher TaqPath asssay. This may be of particular importance in detecting individuals that are either at the early, or late stages of infection, and reinforces the need for frequent, recurrent testing.

Klebsiella oxytoca Complex: Update on Taxonomy, Antimicrobial Resistance, and Virulence.

Klebsiella oxytoca is actually a complex of nine species-Klebsiella grimontii, Klebsiella huaxiensis, Klebsiella michiganensis, K. oxytoca, Klebsiella pasteurii, Klebsiella spallanzanii, and three unnamed novel species. Phenotypic tests can assign isolates to the complex, but precise species identification requires genome-based analysis. The K. oxytoca complex is a human commensal but also an opportunistic pathogen causing various infections, such as antibiotic-associated hemorrhagic colitis (AAHC), urinary tract infection, and bacteremia, and has caused outbreaks. Production of the cytotoxins tilivalline and tilimycin lead to AAHC, while many virulence factors seen in Klebsiella pneumoniae, such as capsular polysaccharides and fimbriae, have been found in the complex; however, their association with pathogenicity remains unclear. Among the 5,724 K. oxytoca clinical isolates in the SENTRY surveillance system, the rates of nonsusceptibility to carbapenems, ceftriaxone, ciprofloxacin, colistin, and tigecycline were 1.8%, 12.5%, 7.1%, 0.8%, and 0.1%, respectively. Resistance to carbapenems is increasing alarmingly. In addition to the intrinsic blaOXY, many genes encoding ß-lactamases with varying spectra of hydrolysis, including extended-spectrum ß-lactamases, such as a few CTX-M variants and several TEM and SHV variants, have been found. blaKPC-2 is the most common carbapenemase gene found in the complex and is mainly seen on IncN or IncF plasmids. Due to the ability to acquire antimicrobial resistance and the carriage of multiple virulence genes, the K. oxytoca complex has the potential to become a major threat to human health.

Prevalence and clonal diversity of carbapenem-resistant Klebsiella pneumoniae causing neonatal infections: A systematic review of 128 articles across 30 countries.

BACKGROUND: Klebsiella pneumoniae is the most common pathogen causing neonatal infections, leading to high mortality worldwide. Along with increasing antimicrobial use in neonates, carbapenem-resistant K. pneumoniae (CRKP) has emerged as a severe challenge for infection control and treatment. However, no comprehensive systematic review is available to describe the global epidemiology of neonatal CRKP infections. We therefore performed a systematic review of available data worldwide and combined a genome-based analysis to address the prevalence, clonal diversity, and carbapenem resistance genes of CRKP causing neonatal infections. METHODS AND FINDINGS: We performed a systematic review of studies reporting population-based neonatal infections caused by CRKP in combination with a genome-based analysis of all publicly available CRKP genomes with neonatal origins. We searched multiple databases (PubMed, Web of Science, Embase, Ovid MEDLINE, Cochrane, bioRxiv, and medRxiv) to identify studies that have reported data of neonatal CRKP infections up to June 30, 2022. We included studies addressing the prevalence of CRKP infections and colonization in neonates but excluded studies lacking the numbers of neonates, the geographical location, or independent data on Klebsiella or CRKP isolates. We used narrative synthesis for pooling data with JMP statistical software. We identified 8,558 articles and excluding those that did not meet inclusion criteria. We included 128 studies, none of which were preprints, comprising 127,583 neonates in 30 countries including 21 low- and middle-income countries (LMICs) for analysis. We found that bloodstream infection is the most common infection type in reported data. We estimated that the pooled global prevalence of CRKP infections in hospitalized neonates was 0.3% (95% confidence interval [CI], 0.2% to 0.3%). Based on 21 studies reporting patient outcomes, we found that the pooled mortality of neonatal CRKP infections was 22.9% (95% CI, 13.0% to 32.9%). A total of 535 neonatal CRKP genomes were identified from GenBank including Sequence Read Archive, of which 204 were not linked to any publications. We incorporated the 204 genomes with a literature review for understanding the species distribution, clonal diversity, and carbapenemase types. We identified 146 sequence types (STs) for neonatal CRKP strains and found that ST17, ST11, and ST15 were the 3 most common lineages. In particular, ST17 CRKP has been seen in neonates in 8 countries across 4 continents. The vast majority (75.3%) of the 1,592 neonatal CRKP strains available for analyzing carbapenemase have genes encoding metallo-ß-lactamases and NDM (New Delhi metallo-ß-lactamase) appeared to be the most common carbapenemase (64.3%). The main limitation of this study is the absence or scarcity of data from North America, South America, and Oceania. CONCLUSIONS: CRKP contributes to a considerable number of neonatal infections and leads to significant neonatal mortality. Neonatal CRKP strains are highly diverse, while ST17 is globally prevalent and merits early detection for treatment and prevention. The dominance of blaNDM carbapenemase genes imposes challenges on therapeutic options in neonates and supports the continued inhibitor-related drug discovery.

The Effect of ß-Lactam Antibiotics on the Evolution of Ceftazidime/Avibactam and Cefiderocol Resistance in KPC-Producing Klebsiella pneumoniae.

In this study, we aimed to clarify the evolutionary trajectory of a Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) population during ß-lactam antibiotic therapy. Five KPC-Kp isolates were collected from a single patient. Whole-genome sequencing and a comparative genomics analysis were performed on the isolates and all blaKPC-2-containing plasmids to predict the population evolution process. Growth competition and experimental evolution assays were conducted to reconstruct the evolutionary trajectory of the KPC-Kp population in vitro. Five KPC-Kp isolates (KPJCL-1 to KPJCL-5) were highly homologous, and all harbor an IncFII blaKPC-containing plasmid (pJCL-1 to pJCL-5). Although the genetic structures of these plasmids were almost identical, distinct copy numbers of the blaKPC-2 gene were detected. A single copy of blaKPC-2 was presented in pJCL-1, pJCL-2, and pJCL-5, two copies of blaKPC (blaKPC-2 and blaKPC-33) were presented in pJCL-3, and three copies of blaKPC-2 were presented in pJCL-4. The blaKPC-33-harboring KPJCL-3 isolate presented resistance to ceftazidime-avibactam and cefiderocol. The blaKPC-2 multicopy strain KPJCL-4 had an elevated ceftazidime-avibactam MIC. The patient had been exposed to ceftazidime, meropenem, and moxalactam, after which KPJCL-3 and KPJCL-4 were isolated, which both showed a significant competitive advantage under antimicrobial pressure in vitro. Experimental evolution assays revealed that blaKPC-2 multicopy-containing cells were increased in the original single-copy blaKPC-2-harboring KPJCL-2 population under selection with ceftazidime, meropenem, or moxalactam, generating a low-level ceftazidime-avibactam resistance phenotype. Moreover, blaKPC-2 mutants with a G532T substitution, G820 to C825 duplication, G532A substitution, G721 to G726 deletion, and A802 to C816 duplication increased in the blaKPC-2 multicopy-containing KPJCL-4 population, generating high-level ceftazidime-avibactam resistance and reduced cefiderocol susceptibility. Ceftazidime-avibactam and cefiderocol resistance can be selected by ß-lactam antibiotics other than ceftazidime-avibactam. Notably, blaKPC-2 gene amplification and mutation are important in KPC-Kp evolution under antibiotic selection.

E. coli ST11 (O157:H7) does not encode a functional AcrF efflux pump.

Escherichia coli is a facultative anaerobe found in a wide range of environments. Commonly described as the laboratory workhorse, E. coli is one of the best characterized bacterial species to date, however much of our understanding comes from studies involving the laboratory strain E. coli K-12. Resistance-nodulation-division efflux pumps are found in Gram-negative bacteria and can export a diverse range of substrates, including antibiotics. E. coli K-12 has six RND pumps; AcrB, AcrD, AcrF, CusA, MdtBC and MdtF, and it is frequently reported that all E. coli strains possess these six pumps. However, this is not true of E. coli ST11, a lineage of E. coli, which is primarily composed of the highly virulent important human pathogen, E. coli O157:H7. Here we show that acrF is absent from the pangenome of ST11 and that this lineage of E. coli has a highly conserved insertion within the acrF gene, which when translated encodes 13 amino acids and two stop codons. This insertion was found to be present in 97.59 % of 1787 ST11 genome assemblies. Non-function of AcrF in ST11 was confirmed in the laboratory as complementation with acrF from ST11 was unable to restore AcrF function in E. coli K-12 substr. MG1655 ΔacrB ΔacrF. This shows that the complement of RND efflux pumps present in laboratory bacterial strains may not reflect the situation in virulent strains of bacterial pathogens.

SARS-CoV-2 Testing in the Community: Testing Positive Samples with the TaqMan SARS-CoV-2 Mutation Panel To Find Variants in Real Time.

Genome sequencing is a powerful tool for identifying SARS-CoV-2 variant lineages; however, there can be limitations due to sequence dropout when used to identify specific key mutations. Recently, ThermoFisher Scientific has developed genotyping assays to help bridge the gap between testing capacity and sequencing capability to generate real-time genotyping results based on specific variants. Over a 6-week period during the months of April and May 2021, we set out to assess the ThermoFisher TaqMan mutation panel genotyping assay, initially for three mutations of concern and then for an additional two mutations of concern, against SARS-CoV-2-positive clinical samples and the corresponding COVID-19 Genomics UK Consortium (COG-UK) sequencing data. We demonstrate that genotyping is a powerful in-depth technique for identifying specific mutations, is an excellent complement to genome sequencing, and has real clinical health value potential, allowing laboratories to report and take action on variants of concern much more quickly.

Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent.

Klebsiella pneumoniae is recognized as an urgent threat to human health due to the increasing isolation of multidrug resistant strains. Hypervirulent strains are a major concern due to their ability to cause life-threating infections in healthy hosts. The type VI secretion system (T6SS) is widely implicated in microbial antagonism, and it mediates interactions with host eukaryotic cells in some cases. In silico search for genes orthologous to T6SS component genes and T6SS effector genes across 700 K. pneumoniae genomes shows extensive diversity in T6SS genes across the K. pneumoniae species. Temperature, oxygen tension, pH, osmolarity, iron levels, and NaCl regulate the expression of the T6SS encoded by a hypervirulent K. pneumoniae strain. Polymyxins and human defensin 3 also increase the activity of the T6SS. A screen for regulators governing T6SS uncover the correlation between the transcription of the T6SS and the ability to kill E. coli prey. Whereas H-NS represses the T6SS, PhoPQ, PmrAB, Hfq, Fur, RpoS and RpoN positively regulate the T6SS. K. pneumoniae T6SS mediates intra and inter species bacterial competition. This antagonism is only evident when the prey possesses an active T6SS. The PhoPQ two component system governs the activation of K. pneumoniae T6SS in bacterial competitions. Mechanistically, PhoQ periplasmic domain, and the acid patch within, is essential to activate K. pneumoniae T6SS. Klebsiella T6SS also mediates anti-fungal competition. We have delineated the contribution of each of the individual VgrGs in microbial competition and identified VgrG4 as a T6SS effector. The DUF2345 domain of VgrG4 is sufficient to intoxicate bacteria and yeast. ROS generation mediates the antibacterial effects of VgrG4, and the antitoxin Sel1E protects against the toxic activity of VgrG4. Our findings provide a better understanding of the regulation of the T6SS in bacterial competitions, and place ROS as an early event in microbial competition.

S-Variant SARS-CoV-2 Lineage B1.1.7 Is Associated With Significantly Higher Viral Load in Samples Tested by TaqPath Polymerase Chain Reaction.

A SARS-CoV-2 variant B1.1.7 containing mutation Δ69/70 has spread rapidly in the United Kingdom and shows an identifiable profile in ThermoFisher TaqPath RT-qPCR, S gene target failure (SGTF). We analyzed recent test data for trends and significance. Linked cycle threshold (Ct) values for respiratory samples showed that a low Ct for ORF1ab and N were clearly associated with SGTF. Significantly more SGTF samples had higher inferred viral loads between 1×107 and 1×108. Our conclusion is that patients whose samples exhibit the SGTF profile are more likely to have high viral loads, which may explain higher infectivity and rapidity of spread.

Transferable Acinetobacter baumannii plasmid pDETAB2 encodes OXA-58 and NDM-1 and represents a new class of antibiotic resistance plasmids.

OBJECTIVES: To characterize a blaOXA-58- and blaNDM-1-containing MDR plasmid from a rare Acinetobacter baumannii lineage and compare it with related plasmids to explore the distribution and evolution of a new plasmid group. METHODS: A. baumannii DETAB-P2 was isolated from a rectal swab of an intensive care patient. Antibiotic susceptibility was determined using broth microdilution. DETAB-P2 was mated with A. baumannii ATCC 17978 and putative transconjugants were characterized by S1/PFGE and Southern hybridization. WGS was performed on the Illumina and Oxford Nanopore platforms. MLST was performed with the Pasteur and Oxford schemes. Antibiotic resistance genes were identified with ABRicate. Plasmid sequence annotation was performed manually. Complete plasmids in GenBank with the same rep gene were used for comparative analyses. RESULTS: A. baumannii DETAB-P2 was ST138 by the Pasteur scheme and a novel Oxford type, ST2209. It transferred blaOXA-58 and blaNDM-1 to ATCC 17978 in the 100 072 bp plasmid pDETAB2 that also carried bleMBL, sul2, aacC2d, tet(39), msr(E)-mph(E) and putative mercury resistance and RND efflux system determinants. pDETAB2 represents a new plasmid type, GR34, and contained 16 pdif sites and several novel dif modules. Only a 10 kbp core sequence is shared amongst pDETAB2 and 18 further GR34 plasmids in GenBank, with diverse accessory regions comprised of various dif modules. CONCLUSIONS: GR34 plasmids are found in several Acinetobacter species from diverse environments. They display considerable variation in accessory content owing to the presence of pdif sites and an array of dif modules, some of which contain antibiotic resistance genes.

The role of potentiating mutations in the evolution of pandemic Escherichia coli clones.

The Escherichia coli species exhibits a vast array of variable lifestyles, including environmental, commensal, and pathogenic organisms. Many of these E. coli contribute significantly to the global threat of antimicrobial resistance (AMR). Multidrug-resistant (MDR) clones of E. coli have arisen multiple times over varying timescales. The repeated emergence of successful pandemic clones, including the notorious ST131 lineage, highlights a desperate need to further study the evolutionary processes underlying their emergence and success. Here, we review the evolutionary emergence of E. coli ST131 pandemic clones and draw parallels between their evolutionary trajectories and those of other lineages. From colonization and expansion to the acquisition of multidrug resistance plasmids, potentiating mutations are present at each stage, leading to a proposed sequence of events that may result in the formation of an antimicrobial-resistant pandemic clone.

NDM Metallo-ß-Lactamases and Their Bacterial Producers in Health Care Settings.

New Delhi metallo-ß-lactamase (NDM) is a metallo-ß-lactamase able to hydrolyze almost all ß-lactams. Twenty-four NDM variants have been identified in >60 species of 11 bacterial families, and several variants have enhanced carbapenemase activity. Klebsiella pneumoniae and Escherichia coli are the predominant carriers of blaNDM, with certain sequence types (STs) (for K. pneumoniae, ST11, ST14, ST15, or ST147; for E. coli, ST167, ST410, or ST617) being the most prevalent. NDM-positive strains have been identified worldwide, with the highest prevalence in the Indian subcontinent, the Middle East, and the Balkans. Most blaNDM-carrying plasmids belong to limited replicon types (IncX3, IncFII, or IncC). Commonly used phenotypic tests cannot specifically identify NDM. Lateral flow immunoassays specifically detect NDM, and molecular approaches remain the reference methods for detecting blaNDM Polymyxins combined with other agents remain the mainstream options of antimicrobial treatment. Compounds able to inhibit NDM have been found, but none have been approved for clinical use. Outbreaks caused by NDM-positive strains have been reported worldwide, attributable to sources such as contaminated devices. Evidence-based guidelines on prevention and control of carbapenem-resistant Gram-negative bacteria are available, although none are specific for NDM-positive strains. NDM will remain a severe challenge in health care settings, and more studies on appropriate countermeasures are required.

Interaction networks, ecological stability, and collective antibiotic tolerance in polymicrobial infections.

Polymicrobial infections constitute small ecosystems that accommodate several bacterial species. Commonly, these bacteria are investigated in isolation. However, it is unknown to what extent the isolates interact and whether their interactions alter bacterial growth and ecosystem resilience in the presence and absence of antibiotics. We quantified the complete ecological interaction network for 72 bacterial isolates collected from 23 individuals diagnosed with polymicrobial urinary tract infections and found that most interactions cluster based on evolutionary relatedness. Statistical network analysis revealed that competitive and cooperative reciprocal interactions are enriched in the global network, while cooperative interactions are depleted in the individual host community networks. A population dynamics model parameterized by our measurements suggests that interactions restrict community stability, explaining the observed species diversity of these communities. We further show that the clinical isolates frequently protect each other from clinically relevant antibiotics. Together, these results highlight that ecological interactions are crucial for the growth and survival of bacteria in polymicrobial infection communities and affect their assembly and resilience.

Bacterial Microcompartment-Mediated Ethanolamine Metabolism in Escherichia coli Urinary Tract Infection.

Urinary tract infections (UTIs) are common and in general are caused by intestinal uropathogenic Escherichia coli (UPEC) ascending via the urethra. Microcompartment-mediated catabolism of ethanolamine, a host cell breakdown product, fuels the competitive overgrowth of intestinal E. coli, both pathogenic enterohemorrhagic E. coli and commensal strains. During a UTI, urease-negative E. coli bacteria thrive, despite the comparative nutrient limitation in urine. The role of ethanolamine as a potential nutrient source during UTIs is understudied. We evaluated the role of the metabolism of ethanolamine as a potential nitrogen and carbon source for UPEC in the urinary tract. We analyzed infected urine samples by culture, high-performance liquid chromatography, reverse transcription-quantitative PCR, and genomic sequencing. The ethanolamine concentration in urine was comparable to the concentration of the most abundant reported urinary amino acid, d-serine. Transcription of the eut operon was detected in the majority of urine samples containing E. coli screened. All sequenced UPEC strains had conserved eut operons, while metabolic genotypes previously associated with UTI (dsdCXA, metE) were mainly limited to phylogroup B2. In vitro ethanolamine was found to be utilized as a sole source of nitrogen by UPEC strains. The metabolism of ethanolamine in artificial urine medium (AUM) induced metabolosome formation and provided a growth advantage at the physiological levels found in urine. Interestingly, eutE (which encodes acetaldehyde dehydrogenase) was required for UPEC strains to utilize ethanolamine to gain a growth advantage in AUM, suggesting that ethanolamine is also utilized as a carbon source. These data suggest that urinary ethanolamine is a significant additional carbon and nitrogen source for infecting E. coli strains.

Genomic and Functional Analysis of Emerging Virulent and Multidrug-Resistant Escherichia coli Lineage Sequence Type 648.

The pathogenic extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli lineage ST648 is increasingly reported from multiple origins. Our study of a large and global ST648 collection from various hosts (87 whole-genome sequences) combining core and accessory genomics with functional analyses and in vivo experiments suggests that ST648 is a nascent and generalist lineage, lacking clear phylogeographic and host association signals. By including large numbers of ST131 (n = 107) and ST10 (n = 96) strains for comparative genomics and phenotypic analysis, we demonstrate that the combination of multidrug resistance and high-level virulence are the hallmarks of ST648, similar to international high-risk clonal lineage ST131. Specifically, our in silico, in vitro, and in vivo results demonstrate that ST648 is well equipped with biofilm-associated features, while ST131 shows sophisticated signatures indicative of adaption to urinary tract infection, potentially conveying individual ecological niche adaptation. In addition, we used a recently developed NFDS (negative frequency-dependent selection) population model suggesting that ST648 will increase significantly in frequency as a cause of bacteremia within the next few years. Also, ESBL plasmids impacting biofilm formation aided in shaping and maintaining ST648 strains to successfully emerge worldwide across different ecologies. Our study contributes to understanding what factors drive the evolution and spread of emerging international high-risk clonal lineages.

Wastewater used for urban agriculture in West Africa as a reservoir for antibacterial resistance dissemination.

State of art metagenomics were used to investigate the microbial population, antibiotic resistance genes and plasmids of medical interest in wastewater used for urban agriculture in Ouagadougou (Burkina Faso). Wastewater samples were collected from three canals near agricultural fields in three neighbourhoods. Assessment of microbial population diversity revealed different microbial patterns among the different samples. Sequencing reads from the wastewaters revealed different functional specializations of microbial communities, with the predominance of carbohydrates and proteins metabolism functions. Eleven pathogen-specific and 56 orthologous virulence factor genes were detected in the wastewater samples. These virulence factors are usually found in human pathogens that cause gastroenteritis and/or diarrhoea. A wide range of antibiotic resistance genes was identified; 81 are transmissible by mobile genetic elements. These included seven different extended spectrum ß-lactamase genes encoding synthesis of four enzyme families, including two metallo-ß-lactamases (blaAIM-1 and blaGES-21). Ten different incompatibility groups of Enterobacteriaceae plasmid replicons (ColE, FIB, FIC, FII, P, Q, R, U, Y, and A/C), and 30 plasmid replicon types from Gram-positive bacteria. All are implicated in the wide distribution of antibiotic resistance genes. We conclude that wastewater used for urban agriculture in the city represents a high risk for spreading bacteria and antimicrobial resistance among humans and animals.

Combined Analysis of Variation in Core, Accessory and Regulatory Genome Regions Provides a Super-Resolution View into the Evolution of Bacterial Populations.

The use of whole-genome phylogenetic analysis has revolutionized our understanding of the evolution and spread of many important bacterial pathogens due to the high resolution view it provides. However, the majority of such analyses do not consider the potential role of accessory genes when inferring evolutionary trajectories. Moreover, the recently discovered importance of the switching of gene regulatory elements suggests that an exhaustive analysis, combining information from core and accessory genes with regulatory elements could provide unparalleled detail of the evolution of a bacterial population. Here we demonstrate this principle by applying it to a worldwide multi-host sample of the important pathogenic E. coli lineage ST131. Our approach reveals the existence of multiple circulating subtypes of the major drug-resistant clade of ST131 and provides the first ever population level evidence of core genome substitutions in gene regulatory regions associated with the acquisition and maintenance of different accessory genome elements.

Sequence Type 273 Carbapenem-Resistant Klebsiella pneumoniae Carrying blaNDM-1 and blaIMP-4.

A carbapenem-resistant Klebsiella pneumoniae isolate was recovered from human blood. Its whole-genome sequence was obtained using Illumina and long-read MinION sequencing. The strain belongs to sequence type 273 (ST273), which was found recently and caused an outbreak in Southeast Asia. It has two carbapenemase genes, blaNDM-1 (carried by an ST7 IncN self-transmissible plasmid) and blaIMP-4 (located on a self-transmissible IncHI5 plasmid). Non-KPC-producing ST237 may represent a lineage of carbapenem-resistant K. pneumoniae, which warrants further monitoring.

Coexistence of Two blaNDM-5 Genes on an IncF Plasmid as Revealed by Nanopore Sequencing.

In a carbapenem-resistant Escherichia coli clinical isolate of sequence type 167, two copies of blaNDM-5 were found on a 144,225-bp IncF self-transmissible plasmid of the F36:A4:B- type. Both blaNDM-5 genes were located in 11,065-bp regions flanked by two copies of IS26 The two regions were identical in sequence but were present at different locations on the plasmid, suggesting a duplication of the same region. This study highlights the complex genetic contexts of blaNDM-5.

blaNDM-21, a new variant of blaNDM in an Escherichia coli clinical isolate carrying blaCTX-M-55 and rmtB.

Objectives: New Delhi MBL (NDM) is a type of carbapenemase; 20 variants of NDM have been identified to date. We have found a new variant of NDM, NDM-21, and describe it here. Methods: A carbapenem-resistant Escherichia coli was subjected to WGS using an Illumina X10 sequencer to identify the antimicrobial resistance genes and its ST. The gene encoding the new variant of NDM was cloned into E. coli DH5α, with blaNDM-5 being cloned as the control. Transformants were tested for susceptibility to carbapenems. Mating was performed to obtain the plasmid carrying the new blaNDM gene and the complete plasmid sequence was obtained using long-read MinION sequencing. Results: The E. coli isolate belonged to ST617 and phylogenetic group A. It had a gene encoding NDM-21, a new NDM variant. NDM-21 differs from NDM-5 by a Gly-to-Ser amino acid substitution at position 69 (G69S). NDM-21 retains the same activity against carbapenems as NDM-5. blaNDM-21 is carried by a 46.1 kb IncX3 plasmid, which is self-transmissible, and is located in a complex genetic context as blaNDM-5. The isolate also carried blaCTX-M-55, which encodes an ESBL conferring resistance to aztreonam (which completed its resistance to all clinically available ß-lactams), and rmtB, which mediates high-level resistance to aminoglycosides, on an IncFII plasmid. Conclusions: A new NDM variant has been identified and blaNDM-21 has evolved from blaNDM-5 on an IncX3 plasmid.