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ABSTRACTThe fungal microbiota plays an important role in the pathogenesis of alcohol-associated liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD). In this study, we aimed to compare changes of the fecal fungal microbiota between patients with ALD and NAFLD and to elucidate patterns in different disease stages between the two conditions. We analyzed fungal internal transcribed spacer 2 (ITS2) sequencing using fecal samples from a cohort of 48 patients with ALD, 78 patients with NAFLD, and 34 controls. The fungal microbiota differed significantly between ALD and NAFLD. The genera Saccharomyces, Kluyveromyces, Scopulariopsis, and the species Candida albicans (C. albicans), Malassezia restricta (M. restricta), Scopulariopsis cordiae (S. cordiae) were significantly increased in patients with ALD, whereas the genera Kazachstania and Mucor were significantly increased in the NAFLD cohort. We identified the fungal signature consisting of Scopulariopsis, Kluyveromyces, M. restricta, and Mucor to have the highest discriminative ability to detect ALD vs NAFLD with an area under the curve (AUC) of 0.93. When stratifying the ALD and NAFLD cohorts by fibrosis severity, the fungal signature with the highest AUC of 0.92 to distinguish ALD F0-F1 vs NAFLD F0-F1 comprised Scopulariopsis, Kluyveromyces, Mucor, M. restricta, and Kazachstania. For more advanced fibrosis stages (F2-F4), the fungal signature composed of Scopulariopsis, Kluyveromyces, Mucor, and M. restricta achieved the highest AUC of 0.99 to differentiate ALD from NAFLD. This is the first study to identify a fungal signature to differentiate two metabolic fatty liver diseases from each other, specifically ALD from NAFLD. This might have clinical utility in unclear cases and might hence help shape treatment approaches. However, larger studies are required to validate this fungal signature in other populations of ALD and NAFLD.

Abstract Background Colistin is one of the last resort therapeutic options for treating carbapenemase-producing Enterobacterales, which are resistant to a broad range of beta-lactam antibiotics. However, the increased use of colistin in clinical and livestock farming settings in Thailand and China, has led to the inevitable emergence of colistin resistance. To better understand the rise of colistin-resistant strains in each of these settings, we characterized colistin-resistant Enterobacterales isolated from farmers, swine, and hospitalized patients in Thailand. Methods Enterobacterales were isolated from 149 stool samples or rectal swabs collected from farmers, pigs, and hospitalized patients in Thailand between November 2014–December 2017. Confirmed colistin-resistant isolates were sequenced. Genomic analyses included species identification, multilocus sequence typing, and detection of antimicrobial resistance determinants and plasmids. Results The overall colistin-resistant Enterobacterales colonization rate was 26.2% (n = 39/149). The plasmid-mediated colistin-resistance gene (mcr) was detected in all 25 Escherichia coli isolates and 9 of 14 (64.3%) Klebsiella spp. isolates. Five novel mcr allelic variants were also identified: mcr-2.3, mcr-3.21, mcr-3.22, mcr-3.23, and mcr-3.24, that were only detected in E. coli and Klebsiella spp. isolates from farmed pigs. Conclusion Our data confirmed the presence of colistin-resistance genes in combination with extended spectrum beta-lactamase genes in bacterial isolates from farmers, swine, and patients in Thailand. Differences between the colistin-resistance mechanisms of Escherichia coli and Klebsiella pneumoniae in hospitalized patients were observed, as expected. Additionally, we identified mobile colistin-resistance mcr-1.1 genes from swine and patient isolates belonging to plasmids of the same incompatibility group. This supported the possibility that horizontal transmission of bacterial strains or plasmid-mediated colistin-resistance genes occurs between humans and swine.

Abstract Alcohol use is a leading cause of chronic liver disease worldwide, and changes in the microbiome associated with alcohol use contribute to patients’ risk for liver disease progression. Less is known about the effects of alcohol use on the intestinal viral microbiome (virome) and interactions between bacteriophages and their target bacteria. We studied changes in the intestinal virome of 62 clinically well‐characterized patients with alcohol use disorder (AUD) during active alcohol use and after 2 weeks of alcohol abstinence, by extracting virus‐like particles and performing metagenomic sequencing. We observed decreased abundance of Propionibacterium, Lactobacillus, and Leuconostoc phages in patients with active AUD when compared with controls, whereas after 2 weeks of alcohol abstinence, patients with AUD demonstrated an increase in the abundance of Propionibacterium, Lactobacillus, and Leuconostoc phages. The intestinal virome signature was also significantly different in patients with AUD with progressive liver disease, with increased abundance of phages targeting Enterobacteria and Lactococcus species phages compared with patients with AUD with nonprogressive liver disease. By performing moderation analyses, we found that progressive liver disease is associated with changes in interactions between some bacteriophages and their respective target bacteria. In summary, active alcohol use and alcohol‐associated progressive liver disease are associated with changes in the fecal virome, some of which are partially reversible after a short period of abstinence. Progression of alcohol‐associated liver disease is associated with changes in bacteriophage–bacteria interactions.

Chronic alcohol consumption is associated with intestinal fungal dysbiosis, yet we understand little about how alterations of intestinal fungi (mycobiota) contribute to the pathogenesis of alcohol-associated liver disease. By reanalyzing internal transcribed spacer 2 amplicon sequencing of fecal samples from a cohort of 66 patients with alcohol use disorder for presence (as opposed to relative abundance) of fungal species, we observed that the presence of Malassezia restricta was associated with increased markers of liver injury. M. restricta exacerbates ethanol-induced liver injury both in acute binge and chronic ethanol-feeding models in mice. Using bone marrow chimeric mice, we found that the disease exacerbating effect by M. restricta was mediated by C-type lectin domain family 4, member N on bone marrow-derived cells. M. restricta induces inflammatory cytokines and chemokines in Kupffer cells through C-type lectin domain family 4, member N signaling. Targeting fungal pathobionts might be a therapeutic strategy for alcohol-associated liver disease.

Abstract Background Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) have continually grown as a global public health threat, with significant mortality rates observed across the world. We examined the clinical data from patients with CPE infections and their outcomes, concentrating on Klebsiella pneumoniae isolates. We analysed the clinical information, performed antimicrobial susceptibility testing, and conducted molecular epidemiological and genomic analyses on the isolates to identify patterns in the data. Methods The clinical characteristics of 33 hospitalised patients with confirmed CPE, including patient-related factors associated with the development of CPE infections, were examined. Patients were divided according to whether they were “colonised” or “infected” with CPE and by the timing and frequency of their rectal swab collections, from which 45 swabs were randomly selected for analysis. CPE isolates were purified, and antimicrobial susceptibility tests performed. Whole genome sequences of these isolates were determined and analysed to compute bacterial multilocus sequence types and plasmid replicon types, infer phylogenetic relationships, and identify antimicrobial resistance and virulence genes. Results Altogether, 88.9% (40/45) of the CPE isolates were K. pneumoniae. The most abundant carbapenemase gene family in the K. pneumoniae isolates (33/39) was bla OXA-232, with bla NDM-1 additionally identified in 19 of them. All CPE isolates carrying either bla OXA-232 or bla NDM-1 were resistant to meropenem, but only 40 from 45 were susceptible to colistin. Among the CPE-infected patients (n = 18) and CPE-colonised patients who developed CPE infections during the study (n = 3), all but one received standard colistin-based combination therapy. Phylogenetic analysis revealed the polyclonal spread of carbapenemase-producing K. pneumoniae (CPKP) within the patient population, with the following two major subclades identified: ST16 (n = 15) and ST231 (n = 14). CPKP-ST231 had the highest virulence score of 4 and was associated with primary bacteraemia. The siderophores yersiniabactin and aerobactin, considered to be important virulence factors, were only identified in the CPKP-ST231 genomes. Conclusions This study has revealed the genomic features of colonising CPE isolates, focusing on antimicrobial resistance and virulence determinants. This type of multi-layered analysis can be further exploited in Thailand and elsewhere to modify the regimes used for empirical antibiotic treatment and improve the management strategies for CPE infections in hospitalised patients.

Background & Aims: Fucosyltransferase 2 (Fut2)-mediated intestinal α1- 2-fucosylation is important for host–microbe interactions and has been associated with several diseases, but its role in obesity and hepatic steatohepatitis is not known. The aim of this study was to investigate the role of Fut2 in a Western-style diet–induced mouse model of obesity and steatohepatitis. Methods: Wild-type (WT) and Fut2-deficient littermate mice were used and features of the metabolic syndrome and steatohepatitis were assessed after 20 weeks of Western diet feeding. Results: Intestinal α1-2-fucosylation was suppressed in WT mice after Western diet feeding, and supplementation of α1-2-fucosylated glycans exacerbated obesity and steatohepatitis in these mice. Fut2-deficient mice were protected from Western diet–induced features of obesity and steatohepatitis despite an increased caloric intake. These mice have increased energy expenditure and thermogenesis, as evidenced by a higher core body temperature. Protection from obesity and steatohepatitis associated with Fut2 deficiency is transmissible to WT mice via microbiota exchange; phenotypic differences between Western diet–fed WT and Fut2-deficient mice were reduced with antibiotic treatment. Fut2 deficiency attenuated diet-induced bile acid accumulation by altered relative abundance of bacterial enzyme 7-α-hydroxysteroid dehydrogenases metabolizing bile acids and by increased fecal excretion of secondary bile acids. This also was associated with increased intestinal farnesoid X receptor/fibroblast growth factor 15 signaling, which inhibits hepatic synthesis of bile acids. Dietary supplementation of α1-2-fucosylated glycans abrogates the protective effects of Fut2 deficiency. Conclusions: α1-2-fucosylation is an important host-derived regulator of intestinal microbiota and plays an important role for the pathogenesis of obesity and steatohepatitis in mice.

Alcohol-induced liver disease is closely related to translocation of bacterial products and bacteria from the intestine to the liver. However, it is not known whether bacterial translocation to the liver depends on certain intestinal microbiota changes that would predispose bacteria to translocate to the liver. In this study, we investigated the microbiota in the jejunum, ileum, cecum, feces and liver of mice subjected to chronic ethanol feeding using a Lieber DeCarli diet model of chronic ethanol feeding for 8 weeks. We demonstrate that chronic ethanol administration changes alpha diversity in the ileum and the liver and leads to compositional changes especially in the ileum. This is largely driven by an increase in gram-negative phyla – the source of endotoxins. Moreover, gram-negative Prevotella not only increased in the mucus layer of the ileum but also in liver samples. These results suggest that bacterial translocation to the liver might be associated with microbiota changes in the distal gastrointestinal tract.

Shigellosis is a leading global cause of diarrheal disease and travelers’ diarrhea now being complicated by the dissemination of antibiotic resistance, necessitating the development of alternative antibacterials such as therapeutic bacteriophages (phages). Phages with lytic activity against Shigella strains were isolated from sewage. The genomes of 32 phages were sequenced, and based on genomic comparisons belong to seven taxonomic genera: Teetrevirus, Teseptimavirus, Kayfunavirus, Tequatrovirus, Mooglevirus, Mosigvirus and Hanrivervirus. Phage host ranges were determined with a diverse panel of 95 clinical isolates of Shigella from Southeast Asia and other geographic regions, representing different species and serotypes. Three-phage mixtures were designed, with one possessing lytic activity against 89% of the strain panel. This cocktail exhibited lytic activity against 100% of S. sonnei isolates, 97.2% of S. flexneri (multiple serotypes) and 100% of S. dysenteriae serotypes 1 and 2. Another 3-phage cocktail composed of two myophages and one podophage showed both a broad host range and the ability to completely sterilize liquid culture of a model virulent strain S. flexneri 2457T. In a Galleria mellonella model of lethal infection with S. flexneri 2457T, this 3-phage cocktail provided a significant increase in survival.

BackgroundAlcohol-associated liver disease (ALD) is an important cause of morbidity and mortality worldwide. The intestinal microbiota is involved in the development and progression of ALD; however, little is known about commensal fungi therein.MethodsWe studied the dynamic changes of the intestinal fungal microbiome, or mycobiome, in 66 patients with alcohol use disorder (AUD) and after 2 weeks of alcohol abstinence using internal transcribed spacer 2 (ITS2) amplicon sequencing of fecal samples.ResultsPatients with AUD had significantly increased abundance of the genera Candida, Debaryomyces, Pichia, Kluyveromyces, and Issatchenkia, and of the species Candida albicans and Candida zeylanoides compared with control subjects. Significantly improved liver health markers caspase-cleaved and intact cytokeratin 18 (CK18-M65) levels and controlled attenuation parameter (CAP) in AUD patients after 2 weeks of alcohol abstinence were associated with significantly lower abundance of the genera Candida, Malassezia, Pichia, Kluyveromyces, Issatchenkia, and the species C. albicans and C. zeylanoides. This was mirrored by significantly higher specific anti-C. albicans immunoglobulin G (IgG) and M (IgM) serum levels in AUD patients in relation to control participants, and significantly decreased anti-C. albicans IgG levels in AUD subjects after 2 weeks of abstinence. The intestinal abundance of the genus Malassezia was significantly higher in AUD subjects with progressive liver disease compared with non-progressive liver disease.ConclusionIn conclusion, improved liver health in AUD patients after alcohol abstinence was associated with lower intestinal abundances of Candida and Malassezia, and lower serum anti-C. albicans IgG levels. Intestinal fungi might serve as a therapeutic target to improve the outcome of patients in ALD.

Abstract Background The development of high-throughput sequencing and analysis has accelerated multi-omics studies of thousands of microbial species, metagenomes, and infectious disease pathogens. Omics studies are enabling genotype-phenotype association studies which identify genetic determinants of pathogen virulence and drug resistance, as well as phylogenetic studies designed to track the origin and spread of disease outbreaks. These omics studies are complex and often employ multiple assay technologies including genomics, metagenomics, transcriptomics, proteomics, and metabolomics. To maximize the impact of omics studies, it is essential that data be accompanied by detailed contextual metadata (e.g., specimen, spatial-temporal, phenotypic characteristics) in clear, organized, and consistent formats. Over the years, many metadata standards developed by various metadata standards initiatives have arisen; the Genomic Standards Consortium’s minimal information standards (MIxS), the GSCID/BRC Project and Sample Application Standard. Some tools exist for tracking metadata, but they do not provide event based capabilities to configure, collect, validate, and distribute metadata. To address this gap in the scientific community, an event based data-driven application, OMeta, was created that allows users to quickly configure, collect, validate, distribute, and integrate metadata. Results A data-driven web application, OMeta, has been developed for use by researchers consisting of a browser-based interface, a command-line interface (CLI), and server-side components that provide an intuitive platform for configuring, capturing, viewing, and sharing metadata. Project and sample metadata can be set based on existing standards or based on projects goals. Recorded information includes details on the biological samples, procedures, protocols, and experimental technologies, etc. This information can be organized based on events, including sample collection, sample quantification, sequencing assay, and analysis results. OMeta enables configuration in various presentation types: checkbox, file, drop-box, ontology, and fields can be configured to use the National Center for Biomedical Ontology (NCBO), a biomedical ontology server. Furthermore, OMeta maintains a complete audit trail of all changes made by users and allows metadata export in comma separated value (CSV) format for convenient deposition of data into public databases. Conclusions We present, OMeta, a web-based software application that is built on data-driven principles for configuring and customizing data standards, capturing, curating, and sharing metadata.

Background and Aims Alcoholic hepatitis is the most severe form of alcohol-related liver disease. While the gut microbiome is known to play a role in disease development and progression, less is known about specific compositional changes of the gut bacterial microbiome associated with disease severity. Therefore, the aim of our study was to correlate gut microbiota features with disease severity in alcoholic hepatitis patients. Methods We used 16S rRNA gene sequencing on fecal samples from 74 alcoholic hepatitis patients, which were enrolled at 9 centers in Europe, the United States, and Mexico in a multi-center observational study. The relative abundance of gut bacterial taxa on genus level, as well as the microbiome diversity, was correlated to various clinical, laboratory, and histologic parameters. Results We observed a negative correlation between the model for end-stage liver disease score and Shannon diversity, independent of potentially confounding factors (Padjust = 0.046). Alcoholic hepatitis patients with more severe disease had significantly decreased relative abundances of Akkermansia while the relative abundance of Veillonella was increased. We observed a reduction in the Bacteroides abundance (Padjust = 0.048) and Shannon diversity (Padjust = 0.018) in antibiotic-treated patients and patients receiving steroids had an increase in Veillonella abundance (Padjust = 0.005), which was both independent of potentially confounding factors. Conclusion We observed distinct changes in the gut bacterial microbiome of alcoholic hepatitis patients with more severe disease. The gut bacterial microbiome might be an attractive target to prevent and treat this deadly disease.

Providencia rettgeri is an emerging opportunistic Gram-negative pathogen with reports of increasing antibiotic resistance. Pan-drug resistant (PDR) P. rettgeri infections are a growing concern, demonstrating a need for the development of alternative treatment options which is fueling a renewed interest in bacteriophage (phage) therapy. Here, we identify and characterize phage vB_PreP_EPr2 (EPr2) with lytic activity against PDR P. rettgeri MRSN 845308, a clinical isolate that carries multiple antibiotic resistance genes. EPr2 was isolated from an environmental water sample and belongs to the family Autographiviridae, subfamily Studiervirinae and genus Kayfunavirus, with a genome size of 41,261 base pairs. Additional phenotypic characterization showed an optimal MOI of 1 and a burst size of 12.3 ± 3.4 PFU per bacterium. EPr2 was determined to have a narrow host range against a panel of clinical P. rettgeri strains. Despite this fact, EPr2 is a promising lytic phage with potential for use as an alternative therapeutic for treatment of PDR P. rettgeri infections.

Alcoholic hepatitis is a major health care burden in the United States due to significant morbidity and mortality. Early identification of patients with alcoholic hepatitis at greatest risk of death is extremely important for proper treatments and interventions to be instituted. In this study, we used gradient boosting, random forest, support vector machine and logistic regression analysis of laboratory parameters, fecal bacterial microbiota, fecal mycobiota, fecal virome, serum metabolome and serum lipidome to predict mortality in patients with alcoholic hepatitis. Gradient boosting achieved the highest AUC of 0.87 for both 30-day mortality prediction using the bacteria and metabolic pathways dataset and 90-day mortality prediction using the fungi dataset, which showed better performance than the currently used model for end-stage liver disease (MELD) score.

Abstract Background Bacterial pan-genomes, comprised of conserved and variable genes across multiple sequenced bacterial genomes, allow for identification of genomic regions that are phylogenetically discriminating or functionally important. Pan-genomes consist of large amounts of data, which can restrict researchers ability to locate and analyze these regions. Multiple software packages are available to visualize pan-genomes, but currently their ability to address these concerns are limited by using only pre-computed data sets, prioritizing core over variable gene clusters, or by not accounting for pan-chromosome positioning in the viewer. Results We introduce PanACEA (Pan-genome Atlas with Chromosome Explorer and Analyzer), which utilizes locally-computed interactive web-pages to view ordered pan-genome data. It consists of multi-tiered, hierarchical display pages that extend from pan-chromosomes to both core and variable regions to single genes. Regions and genes are functionally annotated to allow for rapid searching and visual identification of regions of interest with the option that user-supplied genomic phylogenies and metadata can be incorporated. PanACEA’s memory and time requirements are within the capacities of standard laptops. The capability of PanACEA as a research tool is demonstrated by highlighting a variable region important in differentiating strains of Enterobacter hormaechei. Conclusions PanACEA can rapidly translate the results of pan-chromosome programs into an intuitive and interactive visual representation. It will empower researchers to visually explore and identify regions of the pan-chromosome that are most biologically interesting, and to obtain publication quality images of these regions.

Proton pump inhibitors (PPIs) reduce gastric acid secretion and modulate gut microbiota composition. Here Llorente et al. show that PPIs induce bacterial overgrowth of enterococci, which, in turn, exacerbate ethanol-induced liver disease both in mice and humans.

ABSTRACT Stenotrophomonas maltophilia is a Gram-negative, nonfermenting, environmental bacillus that is an important cause of nosocomial infections, primarily associated with the respiratory tract in the immunocompromised population. Aiming to understand the population structure, microbiological characteristics and impact of allelic variation on β-lactamase structure and function, we collected 130 clinical isolates from across the United States. Identification of 90 different sequence types (STs), of which 63 are new allelic combinations, demonstrates the high diversity of this species. The majority of the isolates (45%) belong to genomic group 6. We also report excellent activity of the ceftazidime-avibactam and aztreonam combination, especially against strains recovered from blood and respiratory infections for which the susceptibility is higher than the susceptibility to trimethoprim-sulfamethoxazole, considered the “first-line” antibiotic to treat S. maltophilia. Analysis of 73 blaL1 and 116 blaL2 genes identified 35 and 43 novel variants of L1 and L2 β-lactamases, respectively. Investigation of the derived amino acid sequences showed that substitutions are mostly conservative and scattered throughout the protein, preferentially affecting positions that do not compromise enzyme function but that may have an impact on substrate and inhibitor binding. Interestingly, we detected a probable association between a specific type of L1 and L2 and genomic group 6. Taken together, our results provide an overview of the molecular epidemiology of S. maltophilia clinical strains from the United States. In particular, the discovery of new L1 and L2 variants warrants further study to fully understand the relationship between them and the β-lactam resistance phenotype in this pathogen. IMPORTANCE Multiple antibiotic resistance mechanisms, including two β-lactamases, L1, a metallo-β-lactamase, and L2, a class A cephalosporinase, make S. maltophilia naturally multidrug resistant. Thus, infections caused by S. maltophilia pose a big therapeutic challenge. Our study aims to understand the microbiological and molecular characteristics of S. maltophilia isolates recovered from human sources. A highlight of the resistance profile of this collection is the excellent activity of the ceftazidime-avibactam and aztreonam combination. We hope this result prompts controlled and observational studies to add clinical data on the utility and safety of this therapy. We also identify 35 and 43 novel variants of L1 and L2, respectively, some of which harbor novel substitutions that could potentially affect substrate and/or inhibitor binding. We believe our results provide valuable knowledge to understand the epidemiology of this species and to advance mechanism-based inhibitor design to add to the limited arsenal of antibiotics active against this pathogen.

ABSTRACT Outbreaks of infection occur more often than they are reported in most developing countries, largely due to poor diagnostic services. A Klebsiella species bacteremia outbreak in a newborn unit with high mortality was recently encountered at a location being surveilled for childhood bacteremia. These surveillance efforts offered the opportunity to determine the cause of this neonatal outbreak. In this report, we present the whole-genome sequences of New Delhi metallo-β-lactamase (NDM-5)-containing Klebsiella quasipneumoniae subsp. similipneumoniae bloodstream isolates from a neonatal bacteremia outbreak at a tertiary hospital in Nigeria and as part of the largest collection of K. pneumoniae bloodstream isolates from children in Africa. Comparative analysis of the genetic environment surrounding the NDM-5 genes revealed nearly perfect sequence identity to blaNDM-5-bearing IncX3-type plasmids from other members of the Enterobacteriaceae. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae is of global health importance, yet there is a paucity of genome-based studies in Africa. Here we report fatal blood-borne NDM-5-producing K. quasipneumoniae subsp. similipneumoniae infections from Nigeria, Africa. New Delhi metallo-β-lactamase (NDM)-producing Klebsiella spp. are responsible for high mortality and morbidity, with the NDM-5 variant showing elevated carbapenem resistance. The prevalence of NDM-5 in Klebsiella has been limited primarily to K. pneumoniae, with only one isolate being collected from Africa. During an outbreak of sepsis in a teaching hospital in Nigeria, five NDM-5-producing K. quasipneumoniae subsp. similipneumoniae sequence type 476 isolates were identified. Given the increased resistance profile of these strains, this study highlights the emerging threat of blaNDM-5 dissemination in hospital environments. The observation of these NDM-5-producing isolates in Africa stresses the urgency to improve monitoring and clinical practices to reduce or prevent the further spread of resistance.

ABSTRACT The population structure of health care-associated pathogens reflects patterns of diversification, selection, and dispersal over time. Empirical data detailing the long-term population dynamics of nosocomial pathogens provide information about how pathogens adapt in the face of exposure to diverse antimicrobial agents and other host and environmental pressures and can inform infection control priorities. Extensive sequencing of clinical isolates from one hospital spanning a decade and a second hospital in the Cleveland, OH, metropolitan area over a 3-year time period provided high-resolution genomic analysis of the Acinetobacter baumannii metapopulation. Genomic analysis demonstrated an almost complete replacement of the predominant strain groups with a new, genetically distinct strain group during the study period. The new group, termed clade F, differs from other global clone 2 (GC2) strains of A. baumannii in several ways, including its antibiotic resistance and lipooligosaccharide biosynthesis genes. Clade F strains are part of a large phylogenetic group with broad geographic representation. Phylogenetic analysis of single-nucleotide variants in core genome regions showed that although the Cleveland strains are phylogenetically distinct from those isolated from other locations, extensive intermixing of strains from the two hospital systems was apparent, suggesting either substantial exchange of strains or a shared, but geographically restricted, external pool from which infectious isolates were drawn. These findings document the rapid evolution of A. baumannii strains in two hospitals, with replacement of the predominant clade by a new clade with altered lipooligosaccharide loci and resistance gene repertoires. IMPORTANCE Multidrug-resistant (MDR) A. baumannii is a difficult-to-treat health care-associated pathogen. Knowing the resistance genes present in isolates causing infection aids in empirical treatment selection. Furthermore, knowledge of the genetic background can assist in tracking patterns of transmission to limit the spread of infections in hospitals. The appearance of a new genetic background in A. baumannii strains with a different set of resistance genes and cell surface structures suggests that strong selective pressures exist, even in highly MDR pathogens. Because the new strains have levels of antimicrobial resistance similar to those of the strains that were displaced, we hypothesize that other features, including host colonization and infection, may confer additional selective advantages and contribute to their increased prevalence.

The siderophore–antibiotic conjugate LCB10-0200 (a.k.a. GT-1) has been developed to combat multidrug-resistant Gram-negative bacteria. In this study, the in vitro activity of LCB10-0200 and LCB10-0200/avibactam (AVI) has been investigated against carbapenem-resistant Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Minimal inhibitory concentrations (MICs) of LCB10-0200, LCB10-0200/AVI, aztreonam, aztreonam/AVI, ceftazidime, ceftazidime/AVI, and meropenem were measured using the agar dilution method. Whole genome sequencing was performed using Illumina and the resistome was analyzed. LCB10-0200 displayed stronger activity than the comparator drugs in meropenem-resistant E. coli and K. pneumoniae, and the addition of AVI enhanced the LCB10-0200 activity to MIC ≤ 0.12 mg/L for 90.5% of isolates. In contrast, whereas LCB10-0200 alone showed potent activity against meropenem-resistant A. baumannii and P. aeruginosa at MIC ≤ 4 mg/L for 84.3% of isolates, the combination with AVI did not improve its activity. LCB10-0200/AVI was active against CTX-M-, SHV-, CMY-, and KPC- producing E. coli and K. pneumoniae, while LCB10-0200 alone was active against ADC-, OXA-, and VIM- producing A. baumannii and P. aeruginosa. Both LCB10-0200 and LCB10-0200/AVI displayed low activity against IMP- and NDM- producing strains. LCB10-0200 alone exhibited strong activity against selected strains. The addition of AVI significantly increased LCB10-0200 activity against carbapenem-resistant E. coli, K. pneumoniae.

Leptospirosis is a worldwide zoonosis, responsible for more than 1 million cases and 60,000 deaths every year. Among the 13 pathogenic species of the genus Leptospira, serovars belonging to L. interrogans serogroup Icterohaemorrhagiae are considered to be the most virulent strains, and responsible for majority of the reported severe cases. Serovars Copenhageni and Icterohaemorrhagiae are major representatives of this serogroup and despite their public health relevance, little is known regarding the genetic differences between these two serovars. In this study, we analyzed the genome sequences of 67 isolates belonging to L. interrogans serovars Copenhageni and Icterohaemorrhagiae to investigate the influence of spatial and temporal variations on DNA sequence diversity. Out of the 1072 SNPs identified, 276 were in non-coding regions and 796 in coding regions. Indel analyses identified 258 indels, out of which 191 were found in coding regions and 67 in non-coding regions. Our phylogenetic analyses based on SNP dataset revealed that both serovars are closely related but showed distinct spatial clustering. However, likelihood ratio test of the indel data statistically confirmed the presence of a frameshift mutation within a homopolymeric tract of lic12008 gene (related to LPS biosynthesis) in all the L. interrogans serovar Icterohaemorrhagiae strains but not in the Copenhageni strains. Therefore, this internal indel identified can genetically distinguish L. interrogans serovar Copenhageni from serovar Icterohaemorrhagiae with high discriminatory power. To our knowledge, this is the first study to identify global sequence variations (SNPs and Indels) in L. interrogans serovars Copenhageni and Icterohaemorrhagiae.

Background: The predominant species in clinical Enterobacter isolates is E. hormaechei. Many articles, clinicians, and GenBank submissions misname these strains as E. cloacae. The lack of sequenced type strains or named species/subspecies for some clades in the E. cloacae complex complicate the issue. Methods: The genomes of the type strains for Enterobacter hormaechei subsp. oharae, E. hormaechei subsp. steigerwaltii, and E. xiangfangensis, and two strains from Hoffmann clusters III and IV of the E. cloacae complex were sequenced. These genomes, the E. hormaechei subsp. hormaechei type strain, and other available Enterobacter type strains were analysed in conjunction with all extant Enterobacter genomes in NCBI’s RefSeq using Average Nucleotide Identity (ANI). Results: There were five recognizable subspecies of E. hormaechei: E. hormaechei subsp. hoffmannii subsp. nov., E. hormaechei subsp. xiangfangensis comb. nov., and the three previously known subspecies. One of the strains sequenced from the E. cloacae complex was not a novel E. hormaechei subspecies but rather a member of a clade of a novel species: E. roggenkampii sp. nov.. E. muelleri was determined to be a later heterotypic synonym of E. asburiae which should take precedence. Conclusion: The phylogeny of the Enterobacter genus, particularly the cloacae complex, was re-evaluated based on the type strain genome sequences and all other available Enterobacter genomes in RefSeq.

Leptospirosis, caused by pathogenic spirochetes belonging to the genus Leptospira, is a zoonosis with important impacts on human and animal health worldwide. Research on the mechanisms of Leptospira pathogenesis has been hindered due to slow growth of infectious strains, poor transformability, and a paucity of genetic tools. As a result of second generation sequencing technologies, there has been an acceleration of leptospiral genome sequencing efforts in the past decade, which has enabled a concomitant increase in functional genomics analyses of Leptospira pathogenesis. A pathogenomics approach, by coupling of pan-genomic analysis of multiple isolates with sequencing of experimentally attenuated highly pathogenic Leptospira, has resulted in the functional inference of virulence factors. The global Leptospira Genome Project supported by the U.S. National Institute of Allergy and Infectious Diseases to which key scientific contributions have been made from the international leptospirosis research community has provided a new roadmap for comprehensive studies of Leptospira and leptospirosis well into the future. This review describes functional genomics approaches to apply the data generated by the Leptospira Genome Project towards deepening our knowledge of virulence factors of Leptospira using the emerging discipline of pathogenomics.

ABSTRACT Knowledge regarding the genomic structure of Enterobacter spp., the second most prevalent carbapenemase-producing Enterobacteriaceae, remains limited. Here we sequenced 97 clinical Enterobacter species isolates that were both carbapenem susceptible and resistant from various geographic regions to decipher the molecular origins of carbapenem resistance and to understand the changing phylogeny of these emerging and drug-resistant pathogens. Of the carbapenem-resistant isolates, 30 possessed blaKPC-2, 40 had blaKPC-3, 2 had blaKPC-4, and 2 had blaNDM-1. Twenty-three isolates were carbapenem susceptible. Six genomes were sequenced to completion, and their sizes ranged from 4.6 to 5.1 Mbp. Phylogenomic analysis placed 96 of these genomes, 351 additional Enterobacter genomes downloaded from NCBI GenBank, and six newly sequenced type strains into 19 phylogenomic groups—18 groups (A to R) in the Enterobacter cloacae complex and Enterobacter aerogenes. Diverse mechanisms underlying the molecular evolutionary trajectory of these drug-resistant Enterobacter spp. were revealed, including the acquisition of an antibiotic resistance plasmid, followed by clonal spread, horizontal transfer of blaKPC-harboring plasmids between different phylogenomic groups, and repeated transposition of the blaKPC gene among different plasmid backbones. Group A, which comprises multilocus sequence type 171 (ST171), was the most commonly identified (23% of isolates). Genomic analysis showed that ST171 isolates evolved from a common ancestor and formed two different major clusters; each acquiring unique blaKPC-harboring plasmids, followed by clonal expansion. The data presented here represent the first comprehensive study of phylogenomic interrogation and the relationship between antibiotic resistance and plasmid discrimination among carbapenem-resistant Enterobacter spp., demonstrating the genetic diversity and complexity of the molecular mechanisms driving antibiotic resistance in this genus. IMPORTANCE Enterobacter spp., especially carbapenemase-producing Enterobacter spp., have emerged as a clinically significant cause of nosocomial infections. However, only limited information is available on the distribution of carbapenem resistance across this genus. Augmenting this problem is an erroneous identification of Enterobacter strains because of ambiguous typing methods and imprecise taxonomy. In this study, we used a whole-genome-based comparative phylogenetic approach to (i) revisit and redefine the genus Enterobacter and (ii) unravel the emergence and evolution of the Klebsiella pneumoniae carbapenemase-harboring Enterobacter spp. Using genomic analysis of 447 sequenced strains, we developed an improved understanding of the species designations within this complex genus and identified the diverse mechanisms driving the molecular evolution of carbapenem resistance. The findings in this study provide a solid genomic framework that will serve as an important resource in the future development of molecular diagnostics and in supporting drug discovery programs.

In the original PDF version of this Article, which was published on 16 October 2017, the publication date was incorrectly given as 10 October 2017. This has now been corrected in the PDF; the HTML version of the paper was correct from the time of publication.

The plant pathogenic species Pseudomonas syringae is divided into numerous pathovars based on host specificity. For example, P. syringae pv. tomato DC3000 is pathogenic on tomato and Arabidopsis, whereas P. syringae pv. syringae 61 is pathogenic on bean. The ability of P. syringae strains to elicit the hypersensitive response (HR) in non-hosts or be pathogenic (or parasitic) in hosts is dependent on the Hrp (type III secretion) system and effector proteins this system is thought to inject into plant cells. To test the role of the Hrp system in determining host range, the hrp/hrc gene cluster (hrpK through hrpR) was deleted from DC3000 and complemented in trans with the orthologous cluster from strain 61. Mutant CUCPB5114 expressing the bean pathogen Hrp system on plasmid pCPP2071 retained the ability of wild-type DC3000 to elicit the HR in bean, to grow and cause bacterial speck in tomato, and to elicit a cultivar-specific (gene-for-gene) HR in tomato plants carrying the Pto resistance gene. However, the symptoms produced in compatible tomato plants involved markedly reduced chlorosis, and CUCPB5114(pCPP2071) did not grow or produce symptoms in Arabidopsis Col-0 although it was weakly virulent in NahG Arabidopsis. A hypersensitive-like collapse was produced by CUCPB5114(pCPP2071) in Arabidopsis Col-0 at 1 × 107 CFU/ml, but only if the bacteria also expressed AvrB, which is recognized by the RPM1 resistance gene in Col-0 and confers incompatibility. These observations support the concept that the P. syringae effector proteins, rather than secretion system components, are the primary determinants of host range at both the species and cultivar levels of host specificity.

Background: The predominant species in clinical Enterobacter isolates is E. hormaechei. Many articles, clinicians, and GenBank submissions misname these strains as E. cloacae. The lack of sequenced type strains or named species/subspecies for some clades in the E. cloacae complex complicate the issue. Methods: The genomes of the type strains for Enterobacter hormaechei subsp. oharae, E. hormaechei subsp. steigerwaltii, and E. xiangfangensis, and two strains from Hoffmann clusters III and IV of the E. cloacae complex were sequenced. These genomes, the E. hormaechei subsp. hormaechei type strain, and other available Enterobacter type strains were analysed in conjunction with all extant Enterobacter genomes in NCBI’s RefSeq using Average Nucleotide Identity (ANI). Results: There were five recognizable subspecies of E. hormaechei: E. hormaechei subsp. hoffmannii subsp. nov., E. hormaechei subsp. xiangfangensis comb. nov., and the three previously known subspecies. One of the strains sequenced from the E. cloacae complex was not a novel E. hormaechei subspecies but rather a member of a clade of a novel species: E. roggenkampii sp. nov.. E. muelleri was determined to be a later heterotypic synonym of E. asburiae which should take precedence. Conclusion: The phylogeny of the Enterobacter genus, particularly the cloacae complex, was re-evaluated based on the type strain genome sequences and all other available Enterobacter genomes in RefSeq.