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The sit-and-wait hypothesis predicts that bacteria can become more virulent when they survive and transmit outside of their hosts due to circumventing the costs of host mortality. While this hypothesis is largely supported theoretically and through comparative analysis, experimental validation is limited. Here we test this hypothesis in Streptococcus suis , an opportunistic zoonotic pig pathogen, where a pathogenic ecotype proliferated during the change to intensive pig farming that amplifies opportunities for fomite transmission. We show in an in vitro environmental survival experiment that pathogenic ecotypes survive for longer than commensal ecotypes, despite similar rates of decline. The presence of a polysaccharide capsule has no consistent effect on survival. Our findings suggest that extended survival in the food chain may augment the zoonotic capability of S. suis . Moreover, eliminating the long-term environmental survival of bacteria could be a strategy that will both enhance infection control and curtail the evolution of virulence.

We used phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of Klebsiella pneumoniae . We report the discovery of monoclonal antibodies (mAbs) binding to type 3 fimbrial proteins, including MrkA. We found that anti-MrkA mAbs were cross-reactive to a diverse panel of K. pneumoniae clinical isolates, representing different O-serotypes. mAbs binding to MrkA have previously been described and have been shown to provide prophylactic protection, although only modest protection when dosed therapeutically in vivo in a murine lung infection model. Here, we used a combination of binding and opsonophagocytic killing studies using a high-content imaging platform to provide a possible explanation for the modest therapeutic efficacy in vivo reported in that model. Our work shows that expression of K. pneumoniae type 3 fimbriae in in vitro culture is not homogenous within a bacterial population. Instead, sub-populations of bacteria that do, and do not, express type 3 fimbriae exist. In a high-content opsonophagocytic killing assay, we showed that MrkA-targeting antibodies initially promote killing by macrophages; however, over time, this effect is diminished. We hypothesize the reason for this is that bacteria not expressing MrkA can evade opsonophagocytosis. Our data support the fact that MrkA is a conserved, immunodominant protein that is antibody accessible on the surface of K. pneumoniae and suggest that additional studies should evaluate the potential of using anti-MrkA antibodies in different stages of K. pneumoniae infection (different sites in the body) as well as against K. pneumoniae biofilms in the body during infection and associated with medical devices.IMPORTANCEThere is an unmet, urgent need for the development of novel antimicrobial therapies for the treatment of Klebsiella pneumoniae infections. We describe the use of phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of K. pneumoniae . We discovered monoclonal antibodies (mAbs) binding to the type 3 fimbrial protein MrkA. The anti-MrkA mAbs were found to be highly cross-reactive, binding to all K. pneumoniae strains tested from a diverse panel of clinical isolates, and were active in an opsonophagocytic killing assay at pM concentrations. MrkA is important for biofilm formation; thus, our data support further exploration of the use of anti-MrkA antibodies for preventing and/or controlling K. pneumoniae in biofilms and during infection., Competing Interests: S.K.B., S.R., R.M., L.I., and P.W. are current/former employees of the AstraZeneca Group and may have/had stock options in AstraZeneca.

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis , a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized.

Staphylococcus aureus is a human commensal and opportunistic pathogen that also infects other animals. In humans and livestock, where S. aureus is most studied, strains are specialized for different host species. Recent studies have also found S. aureus in diverse wild animals. However, it remains unclear whether these isolates are also specialized for their hosts or whether their presence is due to repeated spillovers from source populations. This study focuses on S. aureus in fish, testing the spillover hypothesis in two ways. First, we examined 12 S. aureus isolates obtained from the internal and external organs of a farmed fish. While all isolates were from clonal complex 45, genomic diversity indicates repeated acquisition. The presence of a φSa3 prophage containing human immune evasion genes suggests that the source was originally human. Second, we tested for S. aureus in wild fish that were isolated from likely sources. In particular, we sampled 123 brown trout and their environment at 16 sites in the remote Scottish Highlands with variable levels of exposure to humans, birds, and livestock. This screen found no S. aureus infection in any of the wild populations or their environment. Together, these results support that the presence of S. aureus in fish and aquaculture is due to spillover from humans rather than specialization. Given the trends of increasing fish consumption, a better understanding of the dynamics of S. aureus spillover in aquaculture will mitigate future risks to fish and human health. IMPORTANCE Staphylococcus aureus is a human and livestock commensal but also an important pathogen responsible for high human mortality rates and economic losses in farming. Recent studies show that S. aureus is common in wild animals, including fish. However, we do not know whether these animals are part of the normal host range of S. aureus or whether infection is due to repeated spillover events from true S. aureus hosts. Answering this question has implications for public health and conservation. We find support for the spillover hypothesis by combining genome sequencing of S. aureus isolates from farmed fish and screens for S. aureus in isolated wild populations. The results imply that fish are unlikely to be a source of novel emergent S. aureus strains but highlight the prominence of the spillover of antibiotic-resistant bacteria from humans and livestock. This may affect both future fish disease potential and the risk of human food poisoning., Competing Interests: The authors declare no conflict of interest.

Virus host shifts, where a virus transmits to and infects a novel host species, are a major source of emerging infectious disease. Genetic similarity between eukaryotic host species has been shown to be an important determinant of the outcome of virus host shifts, but it is unclear if this is the case for prokaryotes where anti-virus defences can be transmitted by horizontal gene transfer and evolve rapidly. Here, we measure the susceptibility of 64 strains of Staphylococcaceae bacteria (48 strains of Staphylococcus aureus and 16 non-S. aureus species spanning 2 genera) to the bacteriophage ISP, which is currently under investigation for use in phage therapy. Using three methods-plaque assays, optical density (OD) assays, and quantitative (q)PCR-we find that the host phylogeny explains a large proportion of the variation in susceptibility to ISP across the host panel. These patterns were consistent in models of only S. aureus strains and models with a single representative from each Staphylococcaceae species, suggesting that these phylogenetic effects are conserved both within and among host species. We find positive correlations between susceptibility assessed using OD and qPCR and variable correlations between plaque assays and either OD or qPCR, suggesting that plaque assays alone may be inadequate to assess host range. Furthermore, we demonstrate that the phylogenetic relationships between bacterial hosts can generally be used to predict the susceptibility of bacterial strains to phage infection when the susceptibility of closely related hosts is known, although this approach produced large prediction errors in multiple strains where phylogeny was uninformative. Together, our results demonstrate the ability of bacterial host evolutionary relatedness to explain differences in susceptibility to phage infection, with implications for the development of ISP both as a phage therapy treatment and as an experimental system for the study of virus host shifts., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Walsh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Methicillin-resistant Staphylococcus aureus (MRSA) clonal-complex 398 (CC398) is the dominant livestock-associated (LA) MRSA lineage in European livestock and an increasing cause of difficult-to-treat human disease. LA-CC398 MRSA evolved from a diverse human-associated methicillin-sensitive population, and this transition from humans to livestock was associated with three mobile genetic elements (MGEs). In this study, we apply transposon-directed insertion site sequencing (TraDIS), a high-throughput transposon mutagenesis approach, to investigate genetic signatures that contribute to LA-CC398 causing disease in humans. We identified 26 genes associated with LA-CC398 survival in human blood and 47 genes in porcine blood. We carried out phylogenetic reconstruction on 1,180 CC398 isolates to investigate the genetic context of all identified genes. We found that all genes associated with survival in human blood were part of the CC398 core genome, while 2/47 genes essential for survival in porcine blood were located on MGEs. Gene SAPIG0966 was located on the previously identified Tn 916 transposon carrying a tetracycline resistance gene, which has been shown to be stably inherited within LA-CC398. Gene SAPIG1525 was carried on a phage element, which in part, matched phiSa2wa_st1, a previously identified bacteriophage carrying the Panton-Valentine leucocidin (PVL) virulence factor. Gene deletion mutants constructed in two LA-CC398 strains confirmed that the SAPIG0966 carrying Tn 916 and SAPIG1525 were important for CC398 survival in porcine blood. Our study shows that MGEs that carry antimicrobial resistance and virulence genes could have a secondary function in bacterial survival in blood and may be important for host adaptation. IMPORTANCE CC398 is the dominant type of methicillin-resistant Staphylococcus aureus (MRSA) in European livestock and a growing cause of human infections. Previous studies have suggested MRSA CC398 evolved from human-associated methicillin-sensitive Staphylococcus aureus and is capable of rapidly readapting to human hosts while maintaining antibiotic resistance. Using high-throughput transposon mutagenesis, our study identified 26 and 47 genes important for MRSA CC398 survival in human and porcine blood, respectively. Two of the genes important for MRSA CC398 survival in porcine blood were located on mobile genetic elements (MGEs) carrying resistance or virulence genes. Our study shows that these MGEs carrying antimicrobial resistance and virulence genes could have a secondary function in bacterial survival in blood and may be important for blood infection and host adaptation.

Mobile genetic elements (MGEs) are agents of horizontal gene transfer in bacteria, but can also be vertically inherited by daughter cells. Establishing the dynamics that led to contemporary patterns of MGEs in bacterial genomes is central to predicting the emergence and evolution of novel and resistant pathogens. Methicillin-resistant Staphylococcus aureus (MRSA) clonal-complex (CC) 398 is the dominant MRSA in European livestock and a growing cause of human infections. Previous studies have identified three categories of MGEs whose presence or absence distinguishes livestock-associated CC398 from a closely related and less antibiotic-resistant human-associated population. Here, we fully characterise the evolutionary dynamics of these MGEs using a collection of 1180 CC398 genomes, sampled from livestock and humans, over 27 years. We find that the emergence of livestock-associated CC398 coincided with the acquisition of a Tn 916 transposon carrying a tetracycline resistance gene, which has been stably inherited for 57 years. This was followed by the acquisition of a type V SCC mec that carries methicillin, tetracycline, and heavy metal resistance genes, which has been maintained for 35 years, with occasional truncations and replacements with type IV SCC mec . In contrast, a class of prophages that carry a human immune evasion gene cluster and that are largely absent from livestock-associated CC398 have been repeatedly gained and lost in both human- and livestock-associated CC398. These contrasting dynamics mean that when livestock-associated MRSA is transmitted to humans, adaptation to the human host outpaces loss of antibiotic resistance. In addition, the stable inheritance of resistance-associated MGEs suggests that the impact of ongoing reductions in antibiotic and zinc oxide use in European farms on livestock-associated MRSA will be slow to be realised., Competing Interests: MM, GM, XB, RW, MH, LW No competing interests declared, (© 2022, Matuszewska, Murray et al.)

Mutation rates vary both within and between bacterial species, and understanding what drives this variation is essential for understanding the evolutionary dynamics of bacterial populations. In this study, we investigate two factors that are predicted to influence the mutation rate: ecology and genome size. We conducted mutation accumulation experiments on eight strains of the emerging zoonotic pathogen Streptococcus suis. Natural variation within this species allows us to compare tonsil carriage and invasive disease isolates, from both more and less pathogenic populations, with a wide range of genome sizes. We find that invasive disease isolates have repeatedly evolved mutation rates that are higher than those of closely related carriage isolates, regardless of variation in genome size. Independent of this variation in overall rate, we also observe a stronger bias towards G/C to A/T mutations in isolates from more pathogenic populations, whose genomes tend to be smaller and more AT-rich. Our results suggest that ecology is a stronger correlate of mutation rate than genome size over these timescales, and that transitions to invasive disease are consistently accompanied by rapid increases in mutation rate. These results shed light on the impact that ecology can have on the adaptive potential of bacterial pathogens., Competing Interests: The authors have declared that no competing interests exist.

Background: Antimicrobial resistance (AMR) is among the gravest threats to human health and food security worldwide. The use of antimicrobials in livestock production can lead to emergence of AMR, which can have direct effects on humans through spread of zoonotic disease. Pigs pose a particular risk as they are a source of zoonotic diseases and receive more antimicrobials than most other livestock. Here we use a large-scale genomic approach to characterise AMR in Streptococcus suis, a commensal found in most pigs, but which can also cause serious disease in both pigs and humans., Results: We obtained replicated measures of Minimum Inhibitory Concentration (MIC) for 16 antibiotics, across a panel of 678 isolates, from the major pig-producing regions of the world. For several drugs, there was no natural separation into 'resistant' and 'susceptible', highlighting the need to treat MIC as a quantitative trait. We found differences in MICs between countries, consistent with their patterns of antimicrobial usage. AMR levels were high even for drugs not used to treat S. suis, with many multidrug-resistant isolates. Similar levels of resistance were found in pigs and humans from regions associated with zoonotic transmission. We next used whole genome sequences for each isolate to identify 43 candidate resistance determinants, 22 of which were novel in S. suis. The presence of these determinants explained most of the variation in MIC. But there were also interesting complications, including epistatic interactions, where known resistance alleles had no effect in some genetic backgrounds. Beta-lactam resistance involved many core genome variants of small effect, appearing in a characteristic order., Conclusions: We present a large dataset allowing the analysis of the multiple contributing factors to AMR in S. suis. The high levels of AMR in S. suis that we observe are reflected by antibiotic usage patterns but our results confirm the potential for genomic data to aid in the fight against AMR., (© 2021. The Author(s).)

Emerging bacterial pathogens threaten global health and food security, and so it is important to ask whether these transitions to pathogenicity have any common features. We present a systematic study of the claim that pathogenicity is associated with genome reduction and gene loss. We compare broad-scale patterns across all bacteria, with detailed analyses of Streptococcus suis, an emerging zoonotic pathogen of pigs, which has undergone multiple transitions between disease and carriage forms. We find that pathogenicity is consistently associated with reduced genome size across three scales of divergence (between species within genera, and between and within genetic clusters of S. suis). Although genome reduction is also found in mutualist and commensal bacterial endosymbionts, genome reduction in pathogens cannot be solely attributed to the features of their ecology that they share with these species, that is, host restriction or intracellularity. Moreover, other typical correlates of genome reduction in endosymbionts (reduced metabolic capacity, reduced GC content, and the transient expansion of nonfunctional elements) are not consistently observed in pathogens. Together, our results indicate that genome reduction is a consistent correlate of pathogenicity in bacteria., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Staphylococcus aureus is an important human bacterial pathogen that has a cosmopolitan host range, including livestock, companion and wild animal species. Genomic and epidemiological studies show that S. aureus has jumped between host species many times over its evolutionary history. These jumps have involved the dynamic gain and loss of host-specific adaptive genes, usually located on mobile genetic elements. The same functional elements are often consistently gained in jumps into a particular species. Further sampling of diverse animal species is likely to uncover an even broader host range and greater genetic diversity of S. aureus than is already known, and understanding S. aureus host specificity in these hosts will mitigate the risks of emergent human and livestock strains., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Background: Glaesserella parasuis, the causative agent of Glӓsser's disease, is widespread in swine globally resulting in significant economic losses to the swine industry. Prevention of Glӓsser's disease in pigs has been plagued with an inability to design broadly protective vaccines, as many bacterin based platforms generate serovar or strain specific immunity. Subunit vaccines are of interest to provide protective immunity to multiple strains of G. parasuis. Selected proteins for subunit vaccination should be widespread, highly conserved, and surface exposed., Results: Two candidate proteins for subunit vaccination (RlpB and VacJ) against G. parasuis were identified using random mutagenesis and an in vitro organ culture system. Pigs were vaccinated with recombinant RlpB and VacJ, outer membrane proteins with important contributions to cellular function and viability. Though high antibody titers to the recombinant proteins and increased interferon-γ producing cells were found in subunit vaccinated animals, the pigs were not protected from developing systemic disease., Conclusions: It appears there may be insufficient RlpB and VacJ exposed on the bacterial surface for antibody to bind, preventing high RlpB and VacJ specific antibody titers from protecting animals from G. parasuis. Additionally, this work confirms the importance of utilizing the natural host species when assessing the efficacy of vaccine candidates.

Glaesserella ( Haemophilus ) parasuis is a commensal bacterium of the upper respiratory tract in pigs and also the causative agent of Glässer's disease, which causes significant morbidity and mortality in pigs worldwide. Isolates are characterized into 15 serovars by their capsular polysaccharide, which has shown a correlation with isolate pathogenicity. To investigate the role the capsule plays in G. parasuis virulence and host interaction, a capsule mutant of the serovar 5 strain HS069 was generated (HS069Δcap) through allelic exchange following natural transformation. HS069Δcap was unable to cause signs of systemic disease during a pig challenge study and had increased sensitivity to complement killing and phagocytosis by alveolar macrophages. Compared with the parent strain, HS069Δcap produced more robust biofilm and adhered equivalently to 3D4/31 cells; however, it was unable to persistently colonize the nasal cavity of inoculated pigs, with all pigs clearing HS069Δcap by 5 days postchallenge. Our results indicate the importance of the capsular polysaccharide to G. parasuis virulence as well as nasal colonization in pigs., (Copyright © 2020 American Society for Microbiology.)

We report here the draft genome sequences of the type strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319). These NAD-dependent bacterial species are frequently found in the upper respiratory tract of pigs and are occasionally associated with lung pathology., (Copyright © 2020 Bossé et al.)

Our understanding of the ecological and evolutionary context of novel infections is largely based on viral diseases, even though bacterial pathogens may display key differences in the processes underlying their emergence. For instance, host-shift speciation, in which the jump of a pathogen into a novel host species is followed by the specialization on that host and the loss of infectivity of previous host(s), is commonly observed in viruses, but less often in bacteria. Here, we suggest that the extent to which pathogens evolve host generalism or specialism following a jump into a novel host will depend on their level of adaptation to dealing with different environments, their rates of molecular evolution and their ability to recombine. We then explore these hypotheses using a formal model and show that the high levels of phenotypic plasticity, low rates of evolution and the ability to recombine typical of bacterial pathogens should reduce their propensity to specialize on novel hosts. Novel bacterial infections may therefore be more likely to result in transient spillovers or increased host ranges than in host shifts. Finally, consistent with our predictions, we show that, in two unusual cases of contemporary bacterial host shifts, the bacterial pathogens both have small genomes and rapid rates of substitution. Further tests are required across a greater number of emerging pathogens to assess the validity of our hypotheses. This article is part of the theme issue 'Dynamic and integrative approaches to understanding pathogen spillover'.

Bacterial endosymbionts are very common in terrestrial arthropods, but infection levels vary widely among populations. Experiments and within-species comparisons suggest that environmental temperature might be important in explaining this variation. To investigate the importance of temperature, at broad geographical and taxonomic scales, we extended a global database of terrestrial arthropods screened for Wolbachia and Cardinium. Our final dataset contained data from more than 117 000 arthropods (over 2500 species) screened for Wolbachia and more than 18 000 arthropods (over 800 species) screened for Cardinium, including samples from 137 different countries, with mean temperatures varying from -6.5 to 29.2°C. In insects and relatives, Cardinium infection showed a clear and consistent tendency to increase with temperature. For Wolbachia, a tendency to increase with temperature in temperate climates is counteracted by reduced prevalence in the tropics, resulting in a weak negative trend overall. We discuss the implications of these results for natural and introduced symbionts in regions affected by climate change.

Streptococcus suis is a major pathogen of swine, responsible for a number of chronic and acute infections, and is also emerging as a major zoonotic pathogen, particularly in South-East Asia. Our study of a diverse population of S. suis shows that this organism contains both Type I and Type III phase-variable methyltransferases. In all previous examples, phase-variation of methyltransferases results in genome wide methylation differences, and results in differential regulation of multiple genes, a system known as the phasevarion (phase-variable regulon). We hypothesized that each variant in the Type I and Type III systems encoded a methyltransferase with a unique specificity, and could therefore control a distinct phasevarion, either by recombination-driven shuffling between different specificities (Type I) or by biphasic on-off switching via simple sequence repeats (Type III). Here, we present the identification of the target specificities for each Type III allelic variant from S. suis using single-molecule, real-time methylome analysis. We demonstrate phase-variation is occurring in both Type I and Type III methyltransferases, and show a distinct association between methyltransferase type and presence, and population clades. In addition, we show that the phase-variable Type I methyltransferase was likely acquired at the origin of a highly virulent zoonotic sub-population.

The capacity for some pathogens to jump into different host-species populations is a major threat to public health and food security. Staphylococcus aureus is a multi-host bacterial pathogen responsible for important human and livestock diseases. Here, using a population-genomic approach, we identify humans as a major hub for ancient and recent S. aureus host-switching events linked to the emergence of endemic livestock strains, and cows as the main animal reservoir for the emergence of human epidemic clones. Such host-species transitions are associated with horizontal acquisition of genetic elements from host-specific gene pools conferring traits required for survival in the new host-niche. Importantly, genes associated with antimicrobial resistance are unevenly distributed among human and animal hosts, reflecting distinct antibiotic usage practices in medicine and agriculture. In addition to gene acquisition, genetic diversification has occurred in pathways associated with nutrient acquisition, implying metabolic remodelling after a host switch in response to distinct nutrient availability. For example, S. aureus from dairy cattle exhibit enhanced utilization of lactose-a major source of carbohydrate in bovine milk. Overall, our findings highlight the influence of human activities on the multi-host ecology of a major bacterial pathogen, underpinned by horizontal gene transfer and core genome diversification.

Colistin represents one of the few available drugs for treating infections caused by carbapenem-resistant Enterobacteriaceae. As such, the recent plasmid-mediated spread of the colistin resistance gene mcr-1 poses a significant public health threat, requiring global monitoring and surveillance. Here, we characterize the global distribution of mcr-1 using a data set of 457 mcr-1-positive sequenced isolates. We find mcr-1 in various plasmid types but identify an immediate background common to all mcr-1 sequences. Our analyses establish that all mcr-1 elements in circulation descend from the same initial mobilization of mcr-1 by an ISApl1 transposon in the mid 2000s (2002-2008; 95% highest posterior density), followed by a marked demographic expansion, which led to its current global distribution. Our results provide the first systematic phylogenetic analysis of the origin and spread of mcr-1, and emphasize the importance of understanding the movement of antibiotic resistance genes across multiple levels of genomic organization.

Bacteria that cause serious disease often have smaller genomes, and fewer genes, than their nonpathogenic, or less pathogenic relatives. Here, we review evidence for the generality of this association, and summarise the various reasons why the association might hold. We focus on the population genetic processes that might lead to reductive genome evolution, and show how several of these could be connected to pathogenicity. We find some evidence for most of the processes having acted in bacterial pathogens, including several different modes of genome reduction acting in the same lineage. We argue that predictable processes of genome evolution might not reflect any common underlying process., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

By providing pollination services, bees are among the most important insects, both in ecological and economical terms. Combined next-generation and classical sequencing approaches were applied to discover and study new insect viruses potentially harmful to bees. A bioinformatics virus discovery pipeline was used on individual Illumina transcriptomes of 13 wild bees from three species from the genus Halictus and 30 ants from six species of the genera Messor and Aphaenogaster. This allowed the discovery and description of three sequences of a new virus termed Halictus scabiosae Adlikon virus (HsAV). Phylogenetic analyses of ORF1, RNA-dependent RNA-polymerase (RdRp) and capsid genes showed that HsAV is closely related to (+)ssRNA viruses of the unassigned Sinaivirus genus but distant enough to belong to a different new genus we called Halictivirus. In addition, our study of ant transcriptomes revealed the first four sinaivirus sequences from ants (Messor barbarus, M. capitatus and M. concolor). Maximum likelihood phylogenetic analyses were performed on a 594 nt fragment of the ORF1/RdRp region from 84 sinaivirus sequences, including 31 new Lake Sinai viruses (LSVs) from honey bees collected in five countries across the globe and the four ant viral sequences. The phylogeny revealed four main clades potentially representing different viral species infecting honey bees. Moreover, the ant viruses belonged to the LSV4 clade, suggesting a possible cross-species transmission between bees and ants. Lastly, wide honey bee screening showed that all four LSV clades have worldwide distributions with no obvious geographical segregation.

Haemophilus parasuis is a diverse bacterial species that is found in the upper respiratory tracts of pigs and can also cause Glässer's disease and pneumonia. A previous pangenome study of H. parasuis identified 48 genes that were associated with clinical disease. Here, we describe the development of a generalized linear model (termed a pathotyping model) to predict the potential virulence of isolates of H. parasuis based on a subset of 10 genes from the pangenome. A multiplex PCR (mPCR) was constructed based on these genes, the results of which were entered into the pathotyping model to yield a prediction of virulence. This new diagnostic mPCR was tested on 143 field isolates of H. parasuis that had previously been whole-genome sequenced and a further 84 isolates from the United Kingdom from cases of H. parasuis- related disease in pigs collected between 2013 and 2014. The combination of the mPCR and the pathotyping model predicted the virulence of an isolate with 78% accuracy for the original isolate collection and 90% for the additional isolate collection, providing an overall accuracy of 83% (81% sensitivity and 93% specificity) compared with that of the "current standard" of detailed clinical metadata. This new pathotyping assay has the potential to aid surveillance and disease control in addition to serotyping data., (Copyright © 2017 American Society for Microbiology.)

Antimicrobial resistance in Streptococcus suis, a global zoonotic pathogen of pigs, has been mostly studied only in diseased animals using surveys that have not evaluated changes over time. We compared patterns of resistance between S. suis isolates from clinical cases of disease (CC) and non-clinical case (NCC) pigs in England, collected over two discrete periods, 2009-2011 and 2013-2014. Minimum inhibitory concentrations (MIC) of 17 antimicrobials (nine classes) were determined on 405 S. suis isolates categorised by sampling period and disease association to assess changes in resistance over time and association with disease. First, isolates were characterized as resistant or susceptible using published clinical breakpoints. Second, epidemiological cut-offs (ECOFF) were derived from MIC values, and isolates classified as wild type (WT) below the ECOFF and non-wild type (NWT) above the ECOFF. Finally, isolate subsets were analysed for shifts in MIC distribution. NCC isolates were more resistant than CC isolates to cephalosporins, penams, pleuromutilins, potentiated sulphonamides and tetracyclines in both study periods. Resistance levels among CC isolates increased in 2013-2014 relative to 2009-2011 for antimicrobials including aminoglycosides, cephalosporins, fluoroquinolones, pleuromutilins, potentiated sulphonamides and tetracyclines. The prevalence of isolates categorised as NWT for five or more classes of antimicrobials was greater among NCC than CC isolates for both time periods, and increased with time. This study used standardised methods to identify significant shifts in antimicrobial resistance phenotypes of S. suis isolated from pigs in England, not only over time but also between isolates from known clinical cases or disease-free pigs., (Copyright © 2017. Published by Elsevier B.V.)

Streptococcus suis is a zoonotic swine pathogen and a major public health concern in Asia, where it emerged as an important cause of bacterial meningitis in adults. While associated with food-borne transmission in Asia, zoonotic S. suis infections are mainly occupational hazards elsewhere. To identify genomic differences that can explain zoonotic potential, we compared whole genomes of 98 S. suis isolates from human patients and pigs with invasive disease in the Netherlands, and validated our observations with 18 complete and publicly available sequences. Zoonotic isolates have smaller genomes than non-zoonotic isolates, but contain more virulence factors. We identified a zoonotic S. suis clone that diverged from a non-zoonotic clone by means of gene loss, a capsule switch, and acquisition of a two-component signalling system in the late 19th century, when foreign pig breeds were introduced. Our results indicate that zoonotic potential of S. suis results from gene loss, recombination and horizontal gene transfer events.

Rickettsia is a genus of intracellular bacteria whose hosts and transmission strategies are both impressively diverse, and this is reflected in a highly dynamic genome. Some previous studies have described the evolutionary history of Rickettsia as non-tree-like, due to incongruity between phylogenetic reconstructions using different portions of the genome. Here, we reconstruct the Rickettsia phylogeny using whole-genome data, including two new genomes from previously unsampled host groups. We find that a single topology, which is supported by multiple sources of phylogenetic signal, well describes the evolutionary history of the core genome. We do observe extensive incongruence between individual gene trees, but analyses of simulations over a single topology and interspersed partitions of sites show that this is more plausibly attributed to systematic error than to horizontal gene transfer. Some conflicting placements also result from phylogenetic analyses of accessory genome content (i.e., gene presence/absence), but we argue that these are also due to systematic error, stemming from convergent genome reduction, which cannot be accommodated by existing phylogenetic methods. Our results show that, even within a single genus, tests for gene exchange based on phylogenetic incongruence may be susceptible to false positives., (© The Author(s) 2015. Published by Oxford University Press, on behalf of the Society of Systematic Biologists.)

Termites are eusocial insects related to cockroaches that feed on lignocellulose. These insects are key species in ecosystems since they recycle a large amount of nutrients but also are pests, exerting major economic impacts. Knowledge on the molecular pathways underlying reproduction, caste differentiation or lignocellulose digestion would largely benefit from additional transcriptomic data. This study focused on transcriptomes of secondary reproductive females (nymphoid neotenics). Thirteen transcriptomes were used: 10 of Reticulitermes flavipes and R. grassei sequenced from a previous study, and two transcriptomes of R. lucifugus sequenced for the present study. After transcriptome assembly and read mapping, we examined interspecific variations of genes expressed by termites or gut microorganisms. A total of 18,323 orthologous gene clusters were detected. Functional annotation and taxonomic assignment were performed on a total of 41,287 predicted contigs in the three termite species. Between the termite species studied, functional categories of genes were comparable. Gene ontology (GO) terms analysis allowed the discovery of 9 cellulases and a total of 79 contigs potentially involved in 11 enzymatic activities used in wood metabolism. Altogether, results of this study illustrate the strong potential for the use of comparative interspecific transcriptomes, representing a complete resource for future studies including differentially expressed genes between castes or SNP analysis for population genetics.

Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 10(5) ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Streptococcus suis is a major porcine and zoonotic pathogen responsible for significant economic losses in the pig industry and an increasing number of human cases. Multiple isolates of S. suis show marked genomic diversity. Here, we report the analysis of whole genome sequences of nine pig isolates that caused disease typical of S. suis and had phenotypic characteristics of S. suis, but their genomes were divergent from those of many other S. suis isolates. Comparison of protein sequences predicted from divergent genomes with those from normal S. suis reduced the size of core genome from 793 to only 397 genes. Divergence was clear if phylogenetic analysis was performed on reduced core genes and MLST alleles. Phylogenies based on certain other genes (16S rRNA, sodA, recN, and cpn60) did not show divergence for all isolates, suggesting recombination between some divergent isolates with normal S. suis for these genes. Indeed, there is evidence of recent recombination between the divergent and normal S. suis genomes for 249 of 397 core genes. In addition, phylogenetic analysis based on the 16S rRNA gene and 132 genes that were conserved between the divergent isolates and representatives of the broader Streptococcus genus showed that divergent isolates were more closely related to S. suis. Six out of nine divergent isolates possessed a S. suis-like capsule region with variation in capsular gene sequences but the remaining three did not have a discrete capsule locus. The majority (40/70), of virulence-associated genes in normal S. suis were present in the divergent genomes. Overall, the divergent isolates extend the current diversity of S. suis species but the phenotypic similarities and the large amount of gene exchange with normal S. suis gives insufficient evidence to assign these isolates to a new species or subspecies. Further, sampling and whole genome analysis of more isolates is warranted to understand the diversity of the species.

The complete nucleotide sequence of a 7.7kb mobilisable plasmid (pM3446F), isolated from a florfenicol resistant isolate of Actinobacillus pleuropneumoniae, showed extended similarity to plasmids found in other members of the Pasteurellaceae containing the floR gene as well as replication and mobilisation genes. Mobilisation into other Pasteurellaceae species confirmed that this plasmid can be transferred horizontally., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England., Methods: Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids., Results: A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene., Conclusions: This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)