A curated collection of Klebsiella metabolic models reveals variable substrate usage and gene essentiality.

The Klebsiella pneumoniae species complex (KpSC) is a set of seven Klebsiella taxa that are found in a variety of niches and are an important cause of opportunistic health care-associated infections in humans. Because of increasing rates of multi-drug resistance within the KpSC, there is a growing interest in better understanding the biology and metabolism of these organisms to inform novel control strategies. We collated 37 sequenced KpSC isolates isolated from a variety of niches, representing all seven taxa. We generated strain-specific genome-scale metabolic models (GEMs) for all 37 isolates and simulated growth phenotypes on 511 distinct carbon, nitrogen, sulfur, and phosphorus substrates. Models were curated and their accuracy was assessed using matched phenotypic growth data for 94 substrates (median accuracy of 96%). We explored species-specific growth capabilities and examined the impact of all possible single gene deletions using growth simulations in 145 core carbon substrates. These analyses revealed multiple strain-specific differences, within and between species, and highlight the importance of selecting a diverse range of strains when exploring KpSC metabolism. This diverse set of highly accurate GEMs could be used to inform novel drug design, enhance genomic analyses, and identify novel virulence and resistance determinants. We envisage that these 37 curated strain-specific GEMs, covering all seven taxa of the KpSC, provide a valuable resource to the Klebsiella research community.

In vivo selection of carbapenem resistance during persistent Klebsiella pneumoniae sequence type 395 bloodstream infection due to OmpK36 deletion.

Non-carbapenemase-producing carbapenem-resistant Enterobacterales (non-CP CRE) may be associated with a grave outcome. The common underlying mechanism is beta-lactamases and mutations in outer membrane porins. We report a case of a deep-seated infection caused by Klebsiella pneumoniae ST395 not amenable to source control, involving recurrent bloodstream infection, resulting in in vivo selection of carbapenem resistance under therapy. Three consecutive K. pneumoniae blood isolates were studied using short- and long-read sequencing. The genomes were subject to resistome and virulome, phylogenetic, and plasmid analyses. ompK36 porins were analyzed at the nucleotide and amino acid levels. Genomes were compared to 297 public ST395 K. pneumoniae genomes using cgMLST, resistome, and porin analyses and the EuSCAPE project. Relevant ompK36 and micF sequences were extracted and analyzed as above. The three sequential K. pneumoniae blood isolates belonged to the same clone. Subsequent CR isolates revealed a new large deletion of the ompK36 gene also involving the upstream region (deletion of micF). Comparison with public ST395 genomes revealed the study isolates belonged to clade B, representing a separate clone. N-terminal large ompK36 truncations were uncommon in both public data sets. In vivo selection of non-CP CRE K. pneumoniae could have substantial clinical implications. Such selection should be scrutinized through repeated cultures and frequent susceptibility testing during antimicrobial treatment, especially in the context of persistent or recurrent bloodstream infections and when adequate source control cannot be achieved. The occurrence of an unusually large deletion involving the ompK36 locus and upstream micF should be further studied.

Genomic diversity and antimicrobial resistance in clinical Klebsiella pneumoniae isolates from tertiary hospitals in Southern Ghana.

OBJECTIVES: Comprehensive data on the genomic epidemiology of hospital-associated Klebsiella pneumoniae in Ghana are scarce. This study investigated the genomic diversity, antimicrobial resistance patterns, and clonal relationships of 103 clinical K. pneumoniae isolates from five tertiary hospitals in Southern Ghana-predominantly from paediatric patients aged under 5 years (67/103; 65%), with the majority collected from urine (32/103; 31%) and blood (25/103; 24%) cultures. METHODS: We generated hybrid Nanopore-Illumina assemblies and employed Pathogenwatch for genotyping via Kaptive [capsular (K) locus and lipopolysaccharide (O) antigens] and Kleborate (antimicrobial resistance and hypervirulence) and determined clonal relationships using core-genome MLST (cgMLST). RESULTS: Of 44 distinct STs detected, ST133 was the most common, comprising 23% of isolates (n = 23/103). KL116 (28/103; 27%) and O1 (66/103; 64%) were the most prevalent K-locus and O-antigen types. Single-linkage clustering highlighted the global spread of MDR clones such as ST15, ST307, ST17, ST11, ST101 and ST48, with minimal allele differences (1-5) from publicly available genomes worldwide. Conversely, 17 isolates constituted novel clonal groups and lacked close relatives among publicly available genomes, displaying unique genetic diversity within our study population. A significant proportion of isolates (88/103; 85%) carried resistance genes for ≥3 antibiotic classes, with the blaCTX-M-15 gene present in 78% (n = 80/103). Carbapenem resistance, predominantly due to blaOXA-181 and blaNDM-1 genes, was found in 10% (n = 10/103) of the isolates. CONCLUSIONS: Our findings reveal a complex genomic landscape of K. pneumoniae in Southern Ghana, underscoring the critical need for ongoing genomic surveillance to manage the substantial burden of antimicrobial resistance.

Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing.

A perfect bacterial genome assembly is one where the assembled sequence is an exact match for the organism's genome-each replicon sequence is complete and contains no errors. While this has been difficult to achieve in the past, improvements in long-read sequencing, assemblers, and polishers have brought perfect assemblies within reach. Here, we describe our recommended approach for assembling a bacterial genome to perfection using a combination of Oxford Nanopore Technologies long reads and Illumina short reads: Trycycler long-read assembly, Medaka long-read polishing, Polypolish short-read polishing, followed by other short-read polishing tools and manual curation. We also discuss potential pitfalls one might encounter when assembling challenging genomes, and we provide an online tutorial with sample data (github.com/rrwick/perfect-bacterial-genome-tutorial).

Shigella Serotypes Associated With Carriage in Humans Establish Persistent Infection in Zebrafish.

Shigella represents a paraphyletic group of enteroinvasive Escherichia coli. More than 40 Shigella serotypes have been reported. However, most cases within the men who have sex with men (MSM) community are attributed to 3 serotypes: Shigella sonnei unique serotype and Shigella flexneri 2a and 3a serotypes. Using the zebrafish model, we demonstrate that Shigella can establish persistent infection in vivo. Bacteria are not cleared by the immune system and become antibiotic tolerant. Establishment of persistent infection depends on the O-antigen, a key constituent of the bacterial surface and a serotype determinant. Representative isolates associated with MSM transmission persist in zebrafish, while representative isolates of a serotype not associated with MSM transmission do not. Isolates of a Shigella serotype establishing persistent infections elicited significantly less macrophage death in vivo than isolates of a serotype unable to persist. We conclude that zebrafish are a valuable platform to illuminate factors underlying establishment of Shigella persistent infection in humans.

Polypolish: Short-read polishing of long-read bacterial genome assemblies.

Long-read-only bacterial genome assemblies usually contain residual errors, most commonly homopolymer-length errors. Short-read polishing tools can use short reads to fix these errors, but most rely on short-read alignment which is unreliable in repeat regions. Errors in such regions are therefore challenging to fix and often remain after short-read polishing. Here we introduce Polypolish, a new short-read polisher which uses all-per-read alignments to repair errors in repeat sequences that other polishers cannot. Polypolish performed well in benchmarking tests using both simulated and real reads, and it almost never introduced errors during polishing. The best results were achieved by using Polypolish in combination with other short-read polishers.

Impact of insertion sequences on convergent evolution of Shigella species.

Shigella species are specialised lineages of Escherichia coli that have converged to become human-adapted and cause dysentery by invading human gut epithelial cells. Most studies of Shigella evolution have been restricted to comparisons of single representatives of each species; and population genomic studies of individual Shigella species have focused on genomic variation caused by single nucleotide variants and ignored the contribution of insertion sequences (IS) which are highly prevalent in Shigella genomes. Here, we investigate the distribution and evolutionary dynamics of IS within populations of Shigella dysenteriae Sd1, Shigella sonnei and Shigella flexneri. We find that five IS (IS1, IS2, IS4, IS600 and IS911) have undergone expansion in all Shigella species, creating substantial strain-to-strain variation within each population and contributing to convergent patterns of functional gene loss within and between species. We find that IS expansion and genome degradation are most advanced in S. dysenteriae and least advanced in S. sonnei; and using genome-scale models of metabolism we show that Shigella species display convergent loss of core E. coli metabolic capabilities, with S. sonnei and S. flexneri following a similar trajectory of metabolic streamlining to that of S. dysenteriae. This study highlights the importance of IS to the evolution of Shigella and provides a framework for the investigation of IS dynamics and metabolic reduction in other bacterial species.

Transmission of Klebsiella strains and plasmids within and between grey-headed flying fox colonies.

The grey-headed flying fox (Pteropus poliocephalus) is an endemic Australian fruit bat, known to carry zoonotic pathogens. We recently showed they harbour bacterial pathogen Klebsiella pneumoniae and closely related species in the K. pneumoniae species complex (KpSC); however, the dynamics of KpSC transmission and gene flow within flying fox colonies are poorly understood. High-resolution genome comparisons of 39 KpSC isolates from grey-headed flying foxes identified five putative strain transmission clusters (four intra- and one inter-colony). The instance of inter-colony strain transmission of K. africana was found between two flying fox populations within flying distance, indicating either direct or indirect transmission through a common food/water source. All 11 plasmids identified within the KpSC isolates showed 73% coverage (mean) and ≥95% identity to human-associated KpSC plasmids, indicating gene flow between human clinical and grey-headed flying fox isolates. Along with strain transmission, inter-species horizontal plasmid transmission between K. pneumoniae and Klebsiella africana was also identified within a flying fox colony. Finally, genome-scale metabolic models were generated to predict and compare substrate usage to previously published KpSC models, from human and environmental sources. These models indicated no distinction on the basis of metabolic capabilities. Instead, metabolic capabilities were consistent with population structure and ST/lineage.

A nationwide genomic study of clinical Klebsiella pneumoniae in Norway 2001-15: introduction and spread of ESBLs facilitated by clonal groups CG15 and CG307.

OBJECTIVES: To use the nationwide Norwegian surveillance programme on resistant microbes in humans (NORM) to address longitudinal changes in the population structure of Klebsiella pneumoniae isolates from 2001-15, focusing on the emergence and dissemination of ESBL-producing K. pneumoniae in Norway. METHODS: Among blood (n = 6124) and urinary tract (n = 5496) surveillance isolates from 2001-15, we used Illumina technology to whole genome sequence 201 ESBL-producing isolates from blood (n = 130) and urine (n = 71), and 667 non-ESBL isolates from blood. Complete genomes for four isolates were resolved with Oxford Nanopore sequencing. RESULTS: In a highly diverse collection, Klebsiella variicola ssp. variicola caused 24.5% of Klebsiella pneumoniae species complex (KpSC) bacteraemias. ESBL production was limited to K. pneumoniae sensu stricto (98.5%). A diverse ESBL population of 57 clonal groups (CGs) were dominated by MDR CG307 (17%), CG15 (12%), CG70 (6%), CG258 (5%) and CG45 (5%) carrying blaCTX-M-15. Yersiniabactin was significantly more common in ESBL-positive (37.8%) compared with non-ESBL K. pneumoniae sensu stricto isolates (12.7%), indicating convergence of virulence and resistance determinants. Moreover, we found a significantly lower prevalence of yersiniabactin (3.0%, 37.8% and 17.3%), IncFIB (58.7%, 87.9% and 79.4%) and IncFII plasmid replicons (40.5%, 82.8% and 54.2%) in K. variicola ssp. variicola compared with ESBL- and non-ESBL K. pneumoniae sensu stricto isolates, respectively. CONCLUSIONS: The increase in Norwegian ESBL-producing KpSC during 2010-15 was driven by CG307 and CG15 carrying blaCTX-M-15. K. variicola ssp. variicola was a frequent cause of invasive KpSC infection, but rarely carried ESBLs.

Epidemiology and genomic analysis of Klebsiella oxytoca from a single hospital network in Australia.

BACKGROUND: Infections caused by Klebsiella oxytoca are the second most common cause of Klebsiella infections in humans. Most studies have focused on K. oxytoca outbreaks and few have examined the broader clinical context of K. oxytoca. METHODS: Here, we collected all clinical isolates identified as K. oxytoca in a hospital microbiological diagnostic lab across a 15-month period (n = 239). Whole genome sequencing was performed on a subset of 92 isolates (all invasive, third-generation cephalosporin resistant (3GCR) and non-urinary isolates collected > 48 h after admission), including long-read sequencing on a further six isolates with extended-spectrum beta-lactamase or carbapenemase genes. RESULTS: The majority of isolates were sensitive to antimicrobials, however 22 isolates were 3GCR, of which five were also carbapenem resistant. Genomic analyses showed those identified as K. oxytoca by the clinical laboratory actually encompassed four distinct species (K. oxytoca, Klebsiella michiganensis, Klebsiella grimontii and Klebsiella pasteurii), referred to as the K. oxytoca species complex (KoSC). There was significant diversity within the population, with only 10/67 multi-locus sequence types (STs) represented by more than one isolate. Strain transmission was rare, with only one likely event identified. Six isolates had extended spectrum beta-lactamase (blaSHV-12 and/or blaCTX-M-9) or carbapenemase (blaIMP-4) genes. One pair of K. michiganensis and K. pasteurii genomes carried identical blaIMP-4 IncL/M plasmids, indicative of plasmid transmission. CONCLUSION: Whilst antimicrobial resistance was rare, the resistance plasmids were similar to those found in other Enterobacterales, demonstrating that KoSC has access to the same plasmid reservoir and thus there is potential for multi-drug resistance. Further genomic studies are required to improve our understanding of the KoSC population and facilitate investigation into the attributes of successful nosocomial isolates.

Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae.

Klebsiella pneumoniae has emerged as an important cause of two distinct public health threats: multi-drug resistant (MDR) healthcare-associated infections and drug susceptible community-acquired invasive infections. These pathotypes are generally associated with two distinct subsets of K. pneumoniae lineages or 'clones' that are distinguished by the presence of acquired resistance genes and several key virulence loci. Genomic evolutionary analyses of the most notorious MDR and invasive community-associated ('hypervirulent') clones indicate differences in terms of chromosomal recombination dynamics and capsule polysaccharide diversity, but it remains unclear if these differences represent generalised trends. Here we leverage a collection of >2200 K. pneumoniae genomes to identify 28 common clones (n ≥ 10 genomes each), and perform the first genomic evolutionary comparison. Eight MDR and 6 hypervirulent clones were identified on the basis of acquired resistance and virulence gene prevalence. Chromosomal recombination, surface polysaccharide locus diversity, pan-genome, plasmid and phage dynamics were characterised and compared. The data showed that MDR clones were highly diverse, with frequent chromosomal recombination generating extensive surface polysaccharide locus diversity. Additional pan-genome diversity was driven by frequent acquisition/loss of both plasmids and phage. In contrast, chromosomal recombination was rare in the hypervirulent clones, which also showed a significant reduction in pan-genome diversity, largely driven by a reduction in plasmid diversity. Hence the data indicate that hypervirulent clones may be subject to some sort of constraint for horizontal gene transfer that does not apply to the MDR clones. Our findings are relevant for understanding the risk of emergence of individual K. pneumoniae strains carrying both virulence and acquired resistance genes, which have been increasingly reported and cause highly virulent infections that are extremely difficult to treat. Specifically, our data indicate that MDR clones pose the greatest risk, because they are more likely to acquire virulence genes than hypervirulent clones are to acquire resistance genes.

Developmental patterns in the nasopharyngeal microbiome during infancy are associated with asthma risk.

BACKGROUND: Studies indicate that the nasal microbiome may correlate strongly with the presence or future risk of childhood asthma. OBJECTIVES: In this study, we tested whether developmental trajectories of the nasopharyngeal microbiome in early life and the composition of the microbiome during illnesses were related to risk of childhood asthma. METHODS: Children participating in the Childhood Origins of Asthma study (N = 285) provided nasopharyngeal mucus samples in the first 2 years of life, during routine healthy study visits (at 2, 4, 6, 9, 12, 18, and 24 months of age), and during episodes of respiratory illnesses, all of which were analyzed for respiratory viruses and bacteria. We identified developmental trajectories of early-life microbiome composition, as well as predominant bacteria during respiratory illnesses, and we correlated these with presence of asthma at 6, 8, 11, 13, and 18 years of age. RESULTS: Of the 4 microbiome trajectories identified, a Staphylococcus-dominant microbiome in the first 6 months of life was associated with increased risk of recurrent wheezing by age 3 years and asthma that persisted throughout childhood. In addition, this trajectory was associated with the early onset of allergic sensitization. During wheezing illnesses, detection of rhinoviruses and predominance of Moraxella were associated with asthma that persisted throughout later childhood. CONCLUSION: In infancy, the developmental composition of the microbiome during healthy periods and the predominant microbes during acute wheezing illnesses are both associated with the subsequent risk of developing persistent childhood asthma.

Five Years of GenoTyphi: Updates to the Global Salmonella Typhi Genotyping Framework.

In 2016, a whole-genome sequence (WGS)-based genotyping framework (GenoTyphi) was developed and provided a phylogenetically informative nomenclature for lineages of Salmonella Typhi, the etiological agent of typhoid fever. Subsequent surveillance studies have revealed additional epidemiologically important subpopulations, which require the definition of new genotypes and extension of associated software to facilitate the detection of antimicrobial resistance (AMR) mutations. Analysis of 4632 WGS provide an updated overview of the global S Typhi population structure and genotyping framework, revealing the widespread nature of haplotype 58 ([H58] 4.3.1) genotypes and the diverse range of genotypes carrying AMR mutations.

Rapid Genomic Characterization and Global Surveillance of Klebsiella Using Pathogenwatch.

BACKGROUND: Klebsiella species, including the notable pathogen K. pneumoniae, are increasingly associated with antimicrobial resistance (AMR). Genome-based surveillance can inform interventions aimed at controlling AMR. However, its widespread implementation requires tools to streamline bioinformatic analyses and public health reporting. METHODS: We developed the web application Pathogenwatch, which implements analytics tailored to Klebsiella species for integration and visualization of genomic and epidemiological data. We populated Pathogenwatch with 16 537 public Klebsiella genomes to enable contextualization of user genomes. We demonstrated its features with 1636 genomes from 4 low- and middle-income countries (LMICs) participating in the NIHR Global Health Research Unit (GHRU) on AMR. RESULTS: Using Pathogenwatch, we found that GHRU genomes were dominated by a small number of epidemic drug-resistant clones of K. pneumoniae. However, differences in their distribution were observed (eg, ST258/512 dominated in Colombia, ST231 in India, ST307 in Nigeria, ST147 in the Philippines). Phylogenetic analyses including public genomes for contextualization enabled retrospective monitoring of their spread. In particular, we identified hospital outbreaks, detected introductions from abroad, and uncovered clonal expansions associated with resistance and virulence genes. Assessment of loci encoding O-antigens and capsule in K. pneumoniae, which represent possible vaccine candidates, showed that 3 O-types (O1-O3) represented 88.9% of all genomes, whereas capsule types were much more diverse. CONCLUSIONS: Pathogenwatch provides a free, accessible platform for real-time analysis of Klebsiella genomes to aid surveillance at local, national, and global levels. We have improved representation of genomes from GHRU participant countries, further facilitating ongoing surveillance.

Evolution and genomic insight into methicillin-resistant Staphylococcus aureus ST9 in China.

OBJECTIVES: To reconstruct the evolutionary history and genomic epidemiology of Staphylococcus aureus ST9 in China. METHODS: Using WGS analysis, we described the phylogeny of 131 S. aureus ST9 isolates collected between 2002 and 2016 from 11 provinces in China, including six clinical samples from Taiwan. We also investigated the complex structure and distribution of the lsa(E)-carrying multiresistance gene cluster, and genotyped prophages in the genomes of the ST9 isolates. RESULTS: ST9 was subdivided into one major (n = 122) and one minor (n = 9) clade. Bayesian phylogeny predicted the divergence of ST9 isolates in pig farming in China as early as 1987, which then evolved rapidly in the following three decades. ST9 isolates shared similar multiresistance properties, which were likely acquired before the ST9 emergence in China. The accessory genome is highly conserved, and ST9 harboured similar sets of phages, but lacked certain virulence genes. CONCLUSIONS: Host exchange and regional transmission of ST9 have occurred between pigs and humans. Pig rearing and trading might have favoured gene exchanges between ST9 isolates. Resistance genes, obtained from the environment and other isolates, were stably integrated into the chromosomal DNA. The abundance of resistance genes among ST9 is likely attributed to the extensive use of antimicrobial agents in livestock. Phages are present in the genomes of ST9 and may play a role in the rapid evolution of this ST. Although human ST9 infections are rare, ST9 isolates may constitute a potential risk to public health as a repository of antimicrobial resistance genes.

A Bayesian approach for estimating typhoid fever incidence from large-scale facility-based passive surveillance data.

Decisions about typhoid fever prevention and control are based on estimates of typhoid incidence and their uncertainty. Lack of specific clinical diagnostic criteria, poorly sensitive diagnostic tests, and scarcity of accurate and complete datasets contribute to difficulties in calculating age-specific population-level typhoid incidence. Using data from the Strategic Typhoid Alliance across Africa and Asia program, we integrated demographic censuses, healthcare utilization surveys, facility-based surveillance, and serological surveillance from Malawi, Nepal, and Bangladesh to account for under-detection of cases. We developed a Bayesian approach that adjusts the count of reported blood-culture-positive cases for blood culture detection, blood culture collection, and healthcare seeking-and how these factors vary by age-while combining information from prior published studies. We validated the model using simulated data. The ratio of observed to adjusted incidence rates was 7.7 (95% credible interval [CrI]: 6.0-12.4) in Malawi, 14.4 (95% CrI: 9.3-24.9) in Nepal, and 7.0 (95% CrI: 5.6-9.2) in Bangladesh. The probability of blood culture collection led to the largest adjustment in Malawi, while the probability of seeking healthcare contributed the most in Nepal and Bangladesh; adjustment factors varied by age. Adjusted incidence rates were within or below the seroincidence rate limits of typhoid infection. Estimates of blood-culture-confirmed typhoid fever without these adjustments results in considerable underestimation of the true incidence of typhoid fever. Our approach allows each phase of the reporting process to be synthesized to estimate the adjusted incidence of typhoid fever while correctly characterizing uncertainty, which can inform decision-making for typhoid prevention and control.

Genomic surveillance of antimicrobial resistant bacterial colonisation and infection in intensive care patients.

BACKGROUND: Third-generation cephalosporin-resistant Gram-negatives (3GCR-GN) and vancomycin-resistant enterococci (VRE) are common causes of multi-drug resistant healthcare-associated infections, for which gut colonisation is considered a prerequisite. However, there remains a key knowledge gap about colonisation and infection dynamics in high-risk settings such as the intensive care unit (ICU), thus hampering infection prevention efforts. METHODS: We performed a three-month prospective genomic survey of infecting and gut-colonising 3GCR-GN and VRE among patients admitted to an Australian ICU. Bacteria were isolated from rectal swabs (n = 287 and n = 103 patients ≤2 and > 2 days from admission, respectively) and diagnostic clinical specimens between Dec 2013 and March 2014. Isolates were subjected to Illumina whole-genome sequencing (n = 127 3GCR-GN, n = 41 VRE). Multi-locus sequence types (STs) and antimicrobial resistance determinants were identified from de novo assemblies. Twenty-three isolates were selected for sequencing on the Oxford Nanopore MinION device to generate completed reference genomes (one for each ST isolated from ≥2 patients). Single nucleotide variants (SNVs) were identified by read mapping and variant calling against these references. RESULTS: Among 287 patients screened on admission, 17.4 and 8.4% were colonised by 3GCR-GN and VRE, respectively. Escherichia coli was the most common species (n = 36 episodes, 58.1%) and the most common cause of 3GCR-GN infection. Only two VRE infections were identified. The rate of infection among patients colonised with E. coli was low, but higher than those who were not colonised on admission (n = 2/33, 6% vs n = 4/254, 2%, respectively, p = 0.3). While few patients were colonised with 3GCR- Klebsiella pneumoniae or Pseudomonas aeruginosa on admission (n = 4), all such patients developed infections with the colonising strain. Genomic analyses revealed 10 putative nosocomial transmission clusters (≤20 SNVs for 3GCR-GN, ≤3 SNVs for VRE): four VRE, six 3GCR-GN, with epidemiologically linked clusters accounting for 21 and 6% of episodes, respectively (OR 4.3, p = 0.02). CONCLUSIONS: 3GCR-E. coli and VRE were the most common gut colonisers. E. coli was the most common cause of 3GCR-GN infection, but other 3GCR-GN species showed greater risk for infection in colonised patients. Larger studies are warranted to elucidate the relative risks of different colonisers and guide the use of screening in ICU infection control.

GeneMates: an R package for detecting horizontal gene co-transfer between bacteria using gene-gene associations controlled for population structure.

BACKGROUND: Horizontal gene transfer contributes to bacterial evolution through mobilising genes across various taxonomical boundaries. It is frequently mediated by mobile genetic elements (MGEs), which may capture, maintain, and rearrange mobile genes and co-mobilise them between bacteria, causing horizontal gene co-transfer (HGcoT). This physical linkage between mobile genes poses a great threat to public health as it facilitates dissemination and co-selection of clinically important genes amongst bacteria. Although rapid accumulation of bacterial whole-genome sequencing data since the 2000s enables study of HGcoT at the population level, results based on genetic co-occurrence counts and simple association tests are usually confounded by bacterial population structure when sampled bacteria belong to the same species, leading to spurious conclusions. RESULTS: We have developed a network approach to explore WGS data for evidence of intraspecies HGcoT and have implemented it in R package GeneMates ( github.com/wanyuac/GeneMates ). The package takes as input an allelic presence-absence matrix of interested genes and a matrix of core-genome single-nucleotide polymorphisms, performs association tests with linear mixed models controlled for population structure, produces a network of significantly associated alleles, and identifies clusters within the network as plausible co-transferred alleles. GeneMates users may choose to score consistency of allelic physical distances measured in genome assemblies using a novel approach we have developed and overlay scores to the network for further evidence of HGcoT. Validation studies of GeneMates on known acquired antimicrobial resistance genes in Escherichia coli and Salmonella Typhimurium show advantages of our network approach over simple association analysis: (1) distinguishing between allelic co-occurrence driven by HGcoT and that driven by clonal reproduction, (2) evaluating effects of population structure on allelic co-occurrence, and (3) direct links between allele clusters in the network and MGEs when physical distances are incorporated. CONCLUSION: GeneMates offers an effective approach to detection of intraspecies HGcoT using WGS data.

Novel Subclone of Carbapenem-Resistant Klebsiella pneumoniae Sequence Type 11 with Enhanced Virulence and Transmissibility, China.

We aimed to clarify the epidemiologic and clinical importance of evolutionary events that occurred in carbapenem-resistant Klebsiella pneumoniae (CRKP). We collected 203 CRKP causing bloodstream infections in a tertiary hospital in China during 2013-2017. We detected a subclonal shift in the dominant clone sequence type (ST) 11 CRKP in which the previously prevalent capsular loci (KL) 47 had been replaced by KL64 since 2016. Patients infected with ST11-KL64 CRKP had a significantly higher 30-day mortality rate than other CRKP-infected patients. Enhanced virulence was further evidenced by phenotypic tests. Phylogenetic reconstruction demonstrated that ST11-KL64 is derived from an ST11-KL47-like ancestor through recombination. We identified a pLVPK-like virulence plasmid carrying rmpA and peg-344 in ST11-KL64 exclusively from 2016 onward. The pLVPK-like-positive ST11-KL64 isolates exhibited enhanced environmental survival. Retrospective screening of a national collection identified ST11-KL64 in multiple regions. Targeted surveillance of this high-risk CRKP clone is urgently needed.

Systems biology and big data in asthma and allergy: recent discoveries and emerging challenges.

Asthma is a common condition caused by immune and respiratory dysfunction, and it is often linked to allergy. A systems perspective may prove helpful in unravelling the complexity of asthma and allergy. Our aim is to give an overview of systems biology approaches used in allergy and asthma research. Specifically, we describe recent "omic"-level findings, and examine how these findings have been systematically integrated to generate further insight.Current research suggests that allergy is driven by genetic and epigenetic factors, in concert with environmental factors such as microbiome and diet, leading to early-life disturbance in immunological development and disruption of balance within key immuno-inflammatory pathways. Variation in inherited susceptibility and exposures causes heterogeneity in manifestations of asthma and other allergic diseases. Machine learning approaches are being used to explore this heterogeneity, and to probe the pathophysiological patterns or "endotypes" that correlate with subphenotypes of asthma and allergy. Mathematical models are being built based on genomic, transcriptomic and proteomic data to predict or discriminate disease phenotypes, and to describe the biomolecular networks behind asthma.The use of systems biology in allergy and asthma research is rapidly growing, and has so far yielded fruitful results. However, the scale and multidisciplinary nature of this research means that it is accompanied by new challenges. Ultimately, it is hoped that systems medicine, with its integration of omics data into clinical practice, can pave the way to more precise, personalised and effective management of asthma.