Recognition of Chlamydia trachomatis by Toll-like receptor 9 is altered during persistence.

Toll-like receptor 9 (TLR9) is an innate immune receptor that localizes to endosomes in antigen presenting cells and recognizes single stranded unmethylated CpG sites on bacterial genomic DNA (gDNA). Previous bioinformatic studies have demonstrated that the genome of the human pathogen Chlamydia trachomatis contains TLR9 stimulatory motifs, and correlative studies have implied a link between human TLR9 (hTLR9) genotype variants and susceptibility to infection. Here, we present our evaluation of the stimulatory potential of C. trachomatis gDNA and its recognition by hTLR9- and murine TLR9 (mTLR9)-expressing cells. Utilizing reporter cell lines, we demonstrate that purified gDNA from C. trachomatis can stimulate hTLR9 signaling, albeit at lower levels than gDNA prepared from other Gram-negative bacteria. Interestingly, we found that while C. trachomatis is capable of signaling through hTLR9 and mTLR9 during live infections in HEK293 reporter cell lines, signaling only occurs at later developmental time points. Chlamydia-specific induction of hTLR9 is blocked when protein synthesis is inhibited prior to the RB-to-EB conversion, exacerbated by the inhibition of lipooligosaccharide biosynthesis, and is significantly altered during the induction of aberrance/persistence. Our observations support the hypothesis that chlamydial gDNA is released during the conversion between the pathogen's replicative and infectious forms and during treatment with antibiotics targeting peptidoglycan assembly. Given that C. trachomatis inclusions do not co-localize with TLR9-containing vacuoles in the pro-monocytic cell line U937, our findings also hint that chlamydial gDNA is capable of egress from the inclusion, and traffics to TLR9-containing vacuoles via an as yet unknown pathway.

Listeria monocytogenes utilizes glutathione and limited inorganic sulfur compounds as sources of essential cysteine.

Listeria monocytogenes (Lm) is a Gram-positive facultative intracellular pathogen that leads a biphasic lifecycle, transitioning its metabolism and selectively inducing virulence genes when it encounters mammalian hosts. Virulence gene expression is controlled by the master virulence regulator PrfA, which is allosterically activated by the host- and bacterially derived glutathione (GSH). The amino acid cysteine is the rate-limiting substrate for GSH synthesis in bacteria and is essential for bacterial growth. Unlike many bacteria, Lm is auxotrophic for cysteine and must import exogenous cysteine for growth and virulence. GSH is enriched in the host cytoplasm, and previous work suggests that Lm utilizes exogenous GSH for PrfA activation. Despite these observations, the import mechanism(s) for GSH remains elusive. Analysis of known GSH importers predicted a homologous importer in Lm comprised of the Ctp ABC transporter and the OppDF ATPases of the Opp oligopeptide importer. Here, we demonstrated that the Ctp complex is a high-affinity GSH/GSSG importer that is required for Lm growth at physiologically relevant concentrations. Furthermore, we demonstrated that OppDF is required for GSH/GSSG import in an Opp-independent manner. These data support a model where Ctp and OppDF form a unique complex for GSH/GSSG import that supports growth and pathogenesis. In addition, we show that Lm utilizes the inorganic sulfur sources thiosulfate and H2S for growth in a CysK-dependent manner in the absence of other cysteine sources. These findings suggest a pathoadaptive role for partial cysteine auxotrophy in Lm, where locally high GSH/GSSG or inorganic sulfur concentrations may signal arrival to distinct host niches.

Persistence Alters the Interaction between Chlamydia trachomatis and Its Host Cell.

In response to stress, the obligate intracellular pathogen Chlamydia trachomatis stops dividing and halts its biphasic developmental cycle. The infectious, extracellular form of this bacterium is highly susceptible to killing by the host immune response, and by pausing development, Chlamydia can survive in an intracellular, "aberrant" state for extended periods of time. The relevance of these aberrant forms has long been debated, and many questions remain concerning how they contribute to the persistence and pathogenesis of the organism. Using reporter cell lines, fluorescence microscopy, and a dipeptide labeling strategy, we measured the ability of C. trachomatis to synthesize, assemble, and degrade peptidoglycan under various aberrance-inducing conditions. We found that all aberrance-inducing conditions affect chlamydial peptidoglycan and that some actually halt the biosynthesis pathway early enough to prevent the release of an immunostimulatory peptidoglycan component, muramyl tripeptide. In addition, utilizing immunofluorescence and electron microscopy, we determined that the induction of aberrance can detrimentally affect the development of the microbe's pathogenic vacuole (the inclusion). Taken together, our data indicate that aberrant forms of Chlamydia generated by different environmental stressors can be sorted into two broad categories based on their ability to continue releasing peptidoglycan-derived, immunostimulatory muropeptides and their ability to secrete effector proteins that are normally expressed at the mid- and late stages of the microbe's developmental cycle. Our findings reveal a novel, immunoevasive feature inherent to a subset of aberrant chlamydial forms and provide clarity and context to the numerous persistence mechanisms employed by these ancient, genetically reduced microbes.

Chlamydia trachomatis Oligopeptide Transporter Performs Dual Functions of Oligopeptide Transport and Peptidoglycan Recycling.

Peptidoglycan, the sugar-amino acid polymer that composes the bacterial cell wall, requires a significant expenditure of energy to synthesize and is highly immunogenic. To minimize the loss of an energetically expensive metabolite and avoid host detection, bacteria often recycle their peptidoglycan, transporting its components back into the cytoplasm, where they can be used for subsequent rounds of new synthesis. The peptidoglycan-recycling substrate binding protein (SBP) MppA, which is responsible for recycling peptidoglycan fragments in Escherichia coli, has not been annotated for most intracellular pathogens. One such pathogen, Chlamydia trachomatis, has a limited capacity to synthesize amino acids de novo and therefore must obtain oligopeptides from its host cell for growth. Bioinformatics analysis suggests that the putative C. trachomatis oligopeptide transporter OppABCDF (OppABCDF Ct ) encodes multiple SBPs (OppA1 Ct , OppA2 Ct , and OppA3 Ct ). Intracellular pathogens often encode multiple SBPs, while only one, OppA, is encoded in the E. coliopp operon. We hypothesized that the putative OppABCDF transporter of C. trachomatis functions in both oligopeptide transport and peptidoglycan recycling. We coexpressed the putative SBP genes (oppA1Ct , oppA2Ct , oppA3Ct ) along with oppBCDFCt in an E. coli mutant lacking the Opp transporter and determined that all three chlamydial OppA subunits supported oligopeptide transport. We also demonstrated the in vivo functionality of the chlamydial Opp transporter in C. trachomatis Importantly, we found that one chlamydial SBP, OppA3 Ct , possessed dual substrate recognition properties and is capable of transporting peptidoglycan fragments (tri-diaminopimelic acid) in E. coli and in C. trachomatis These findings suggest that Chlamydia evolved an oligopeptide transporter to facilitate the acquisition of oligopeptides for growth while simultaneously reducing the accumulation of immunostimulatory peptidoglycan fragments in the host cell cytosol. The latter property reflects bacterial pathoadaptation that dampens the host innate immune response to Chlamydia infection.

Extensive genome analysis of Coxiella burnetii reveals limited evolution within genomic groups.

BACKGROUND: Coxiella burnetii is a zoonotic pathogen that resides in wild and domesticated animals across the globe and causes a febrile illness, Q fever, in humans. An improved understanding of the genetic diversity of C. burnetii is essential for the development of diagnostics, vaccines and therapeutics, but genotyping data is lacking from many parts of the world. Sporadic outbreaks of Q fever have occurred in the United Kingdom, but the local genetic make-up of C. burnetii has not been studied in detail. RESULTS: Here, we report whole genome data for nine C. burnetii sequences obtained in the UK. All four genomes of C. burnetii from cattle, as well as one sheep sample, belonged to Multi-spacer sequence type (MST) 20, whereas the goat samples were MST33 (three genomes) and MST32 (one genome), two genotypes that have not been described to be present in the UK to date. We established the phylogenetic relationship between the UK genomes and 67 publically available genomes based on single nucleotide polymorphisms (SNPs) in the core genome, which confirmed tight clustering of strains within genomic groups, but also indicated that sub-groups exist within those groups. Variation is mainly achieved through SNPs, many of which are non-synonymous, thereby confirming that evolution of C. burnetii is based on modification of existing genes. Finally, we discovered genomic-group specific genome content, which supports a model of clonal expansion of previously established genotypes, with large scale dissemination of some of these genotypes across continents being observed. CONCLUSIONS: The genetic make-up of C. burnetii in the UK is similar to the one in neighboring European countries. As a species, C. burnetii has been considered a clonal pathogen with low genetic diversity at the nucleotide level. Here, we present evidence for significant variation at the protein level between isolates of different genomic groups, which mainly affects secreted and membrane-associated proteins. Our results thereby increase our understanding of the global genetic diversity of C. burnetii and provide new insights into the evolution of this emerging zoonotic pathogen.

Parallel independent evolution of pathogenicity within the genus Yersinia.

The genus Yersinia has been used as a model system to study pathogen evolution. Using whole-genome sequencing of all Yersinia species, we delineate the gene complement of the whole genus and define patterns of virulence evolution. Multiple distinct ecological specializations appear to have split pathogenic strains from environmental, nonpathogenic lineages. This split demonstrates that contrary to hypotheses that all pathogenic Yersinia species share a recent common pathogenic ancestor, they have evolved independently but followed parallel evolutionary paths in acquiring the same virulence determinants as well as becoming progressively more limited metabolically. Shared virulence determinants are limited to the virulence plasmid pYV and the attachment invasion locus ail. These acquisitions, together with genomic variations in metabolic pathways, have resulted in the parallel emergence of related pathogens displaying an increasingly specialized lifestyle with a spectrum of virulence potential, an emerging theme in the evolution of other important human pathogens.

Prevalence and persistence of Escherichia coli in the airways of cystic fibrosis patients - an unrecognized CF pathogen?

Cystic fibrosis (CF) patients suffer from chronic recurrent bacterial airway infections, which eventually lead to reduced life expectancy. Escherichia coli has not been considered as a CF pathogen. A total of 176 patients were observed over 5.6 years on average from 2002 to 2009 in two CF centers in Muenster, Germany. Sputum and throat swab cultures were screened for E. coli. E. coli isolates were analyzed for clinical microbiologic characteristics as well as strain identity, clonal distribution and phenotypic variability. In 45 patients (25.6%) E. coli was cultured at least once, mostly at medium to high bacterial load and primarily from patients less than 5 and older than 8 years. In 19 patients (10.8%) the same E. coli strain was isolated at least 3 times within a period of more than 6 months, with a mean persistence of 29 months. Multi-locus sequence typing revealed a distinctively strong association of CF E. coli with the B2 major clonal group. During persistence, long-term colonizing strains exhibited phenotypic variability known for typical CF pathogens such as surface capsule overproduction and changes in colony size or hemolytic activity. E. coli was occasionally or persistently isolated in a quarter of CF patients, mostly in very young or older patients. The relatively high bacterial load of E. coli colonization, the distinct association with the highly virulent extra-intestinal B2 clonal group and phenotypic variability in the long-term colonizing strains suggests a previously unrecognized clinical significance of E. coli as a CF pathogen.

Recognition of Chlamydia trachomatis by Toll-Like Receptor 9 is altered during persistence.

Toll-like receptor 9 (TLR9) is an innate immune receptor that localizes to endosomes in antigen presenting cells and recognizes single stranded unmethylated CpG sites on bacterial genomic DNA. Previous bioinformatic studies have indicated that the genome of the human pathogen Chlamydia trachomatis contains TLR9 stimulatory motifs, and correlative studies have implied a link between human TLR9 (hTLR9) genotype variants and susceptibility to infection. Here we present our evaluation of the stimulatory potential of C. trachomatis gDNA and its recognition by hTLR9- and murine TLR9 (mTLR9)-expressing cells. We confirm that hTLR9 colocalizes with chlamydial inclusions in the pro-monocytic cell line, U937. Utilizing HEK293 reporter cell lines, we demonstrate that purified genomic DNA from C. trachomatis can stimulate hTLR9 signaling, albeit at lower levels than gDNA prepared from other Gram-negative bacteria. Interestingly, we found that while C. trachomatis is capable of signaling through hTLR9 and mTLR9 during live infections in non-phagocytic HEK293 reporter cell lines, signaling only occurs at later developmental time points. Chlamydia-specific induction of hTLR9 is blocked when protein synthesis is inhibited prior to the RB-to-EB conversion and exacerbated by the inhibition of lipooligosaccharide biosynthesis. The induction of aberrance / persistence also significantly alters Chlamydia-specific TLR9 signaling. Our observations support the hypothesis that chlamydial gDNA is released at appreciable levels by the bacterium during the conversion between its replicative and infectious forms and during treatment with antibiotics targeting peptidoglycan assembly.

Co-occurring microflora and mucin drive Pseudomonas aeruginosa diversification and pathoadaptation.

While several environmental factors contribute to the evolutionary diversification of the pathogenic bacterium Pseudomonas aeruginosa during cystic fibrosis lung infections, relatively little is known about the impact of the surrounding microbiota. By using in vitro experimental evolution, we show that the presence of Stenotrophomonas maltophilia, Staphylococcus aureus, or them both, prevent the evolution of loss of virulence, which repeatedly occurs in the absence of these species due to mutations in regulators of the Pseudomonas Quinolone Signal quorum sensing system, vqsM and pqsR. Moreover, the strength of the effect of co-occurring species is attenuated through changes in the physical environment by the addition of mucin, resulting in selection for phenotypes resembling those evolved in the absence of the co-occurring species. Together, our findings show that variation in mucosal environment and the surrounding polymicrobial environment can determine the evolutionary trajectory of P. aeruginosa, partly explaining its diversification and pathoadaptation from acute to chronic phenotype during cystic fibrosis lung infections.

New concepts in antimicrobial resistance in cystic fibrosis respiratory infections.

In this review, we summarize the main points that were raised and highlighted during the pre-conference meeting to the 17th European Cystic Fibrosis Society Basic Science Conference, held from 30 March to 2 April, 2022 in Albufeira, Portugal. Keynote lectures provided an update on the latest information regarding the phenomenon of antimicrobial resistance (AMR) in cystic fibrosis (CF). Traditional themes such as in vitro antibiotic susceptibility testing and its clinical value, AMR evolution in persistent Pseudomonas aeruginosa infection and the impact of biofilm on AMR were discussed. In addition, the report gives an overview on very recent AMR-related topics that include an ecological view of AMR in CF lung, referred to as resistome, and novel anti-infective approaches in preclinical or early clinical research such as antibiofilm drugs and bacteriophages.

Interactions between host epithelial cells and Acinetobacter baumannii promote the emergence of highly antibiotic resistant and highly mucoid strains.

Acinetobacter baumannii is an important nosocomial pathogen. Upon colonizing a host, A. baumannii are subjected to selective pressure by immune defenses as they adapt to the host environment. However, the mechanism of this pathoadaptation is unknown. Here, we established an in vitro system to evolve A. baumannii driven by the continuous selective pressure exerted by epithelial cells, and we used a combination of experimental evolution, phenotypic characterization and multi-omics analysis to address the underlying mechanism. When continuously exposed to selective pressure by pulmonary epithelial cells, A. baumannii showed ptk mutation-mediated mucoid conversion (reduced adhesion and increased anti-phagocytic ability) by enhancement of capsular exopolysaccharide chain length; rsmG mutation-mediated deficiency of 7-methylguanosine modification in the 524th nucleotide of 16S rRNA, which increased ribosome translation efficiency; and rnaseI mutation-mediated changes in outer membrane permeability and efflux pump expression. Together, these mutations altered susceptibility to a variety of antimicrobial agents, including the novel antibiotic cefiderocol, by regulating siderophore and siderophore-receptor biosynthesis. In conclusion, pulmonary epithelial cells modulate A. baumannii pathoadaptation, implicating the host-microbe interaction in the survival and persistence of A. baumannii.

Persisting uropathogenic Escherichia coli lineages show signatures of niche-specific within-host adaptation mediated by mobile genetic elements.

Large-scale genomic studies have identified within-host adaptation as a hallmark of bacterial infections. However, the impact of physiological, metabolic, and immunological differences between distinct niches on the pathoadaptation of opportunistic pathogens remains elusive. Here, we profile the within-host adaptation and evolutionary trajectories of 976 isolates representing 119 lineages of uropathogenic Escherichia coli (UPEC) sampled longitudinally from both the gastrointestinal and urinary tracts of 123 patients with urinary tract infections. We show that lineages persisting in both niches within a patient exhibit increased allelic diversity. Habitat-specific selection results in niche-specific adaptive mutations and genes, putatively mediating fitness in either environment. Within-lineage inter-habitat genomic plasticity mediated by mobile genetic elements (MGEs) provides the opportunistic pathogen with a mechanism to adapt to the physiological conditions of either habitat, and reduced MGE richness is associated with recurrence in gut-adapted UPEC lineages. Collectively, our results establish niche-specific adaptation as a driver of UPEC within-host evolution.

Localized Peptidoglycan Biosynthesis in Chlamydia trachomatis Conforms to the Polarized Division and Cell Size Reduction Developmental Models.

Cell size regulation in bacteria is a function of two basic cellular processes: the expansion of the cell envelope and its constriction at spatially defined points at what will eventually become the division plane. In most bacterial species, both cell wall expansion and restriction are dependent on peptidoglycan (PG), a structural polymer comprised of sugars and amino acids that imparts strength and rigidity to bacterial membranes. Pathogenic Chlamydia species are unique in that their cell walls contain very little PG, which is restricted almost entirely to the apparent division plane of the microbe's replicative forms. Very little is known about the degree to which PG affects the size and shape of C. trachomatis during its division process, and recent studies suggest the process is initiated via a polarized mechanism. We conducted an imaging study to ascertain the dimensions, orientation, and relative density of chlamydial PG throughout the organism's developmental cycle. Our analysis indicates that PG in replicating C. trachomatis can be associated with four, broad structural forms; polar/septal disks, small/thick rings, large rings, and small/thin rings. We found that PG density appeared to be highest in septal disks and small/thick rings, indicating that these structures likely have high PG synthesis to degradation ratios. We also discovered that as C. trachomatis progresses through its developmental cycle PG structures, on average, decrease in total volume, indicating that the average cell volume of chlamydial RBs likely decreases over time. When cells infected with C. trachomatis are treated with inhibitors of critical components of the microbe's two distinct PG synthases, we observed drastic differences in the ratio of PG synthesis to degradation, as well as the volume and shape of PG-containing structures. Overall, our results suggest that C. trachomatis PG synthases differentially regulate the expansion and contraction of the PG ring during both the expansion and constriction of the microbe's cell membrane during cell growth and division, respectively.

Achromobacter xylosoxidans and Stenotrophomonas maltophilia: Emerging Pathogens Well-Armed for Life in the Cystic Fibrosis Patients' Lung.

In patients with cystic fibrosis (CF), the lung is a remarkable ecological niche in which the microbiome is subjected to important selective pressures. An inexorable colonization by bacteria of both endogenous and environmental origin is observed in most patients, leading to a vicious cycle of infection-inflammation. In this context, long-term colonization together with competitive interactions among bacteria can lead to over-inflammation. While Pseudomonas aeruginosa and Staphylococcus aureus, the two pathogens most frequently identified in CF, have been largely studied for adaptation to the CF lung, in the last few years, there has been a growing interest in emerging pathogens of environmental origin, namely Achromobacter xylosoxidans and Stenotrophomonas maltophilia. The aim of this review is to gather all the current knowledge on the major pathophysiological traits, their supporting mechanisms, regulation and evolutionary modifications involved in colonization, virulence, and competitive interactions with other members of the lung microbiota for these emerging pathogens, with all these mechanisms being major drivers of persistence in the CF lung. Currently available research on A. xylosoxidans complex and S. maltophilia shows that these emerging pathogens share important pathophysiological features with well-known CF pathogens, making them important members of the complex bacterial community living in the CF lung.

LasR-regulated proteases in acute vs. chronic lung infection: a double-edged sword.

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen capable of causing both acute and chronic infections, particularly in individuals with compromised host defenses. The quorum sensing transcriptional activator LasR is widely recognized for its role in regulating the expression of acute virulence factors, notably several secreted proteases which cause direct host damage and subvert host immunity in acute infections. Paradoxically, lung infections caused by LasR-deficient variants, which are found in at least a third of cystic fibrosis (CF) patients with chronic P. aeruginosa infections, are associated with accelerated lung disease and increased markers of inflammation compared to infections caused by strains with a functional LasR system. While the loss of LasR function often (although not always) results in impaired production of LasR-controlled acute virulence factors, the implication of this pathoadaptation on host-pathogen interactions and chronic disease pathology is less well recognized. We recently observed that loss of LasR function in lasR variants, which results in impaired secreted protease production, led to increased expression of the membrane-bound surface adhesion molecule mICAM-1 in the airway epithelium, and increased neutrophilic inflammation. Specifically, human airway epithelial cells stimulated with lasR variants had higher mICAM-1 expression and greater neutrophil binding in vitro compared to stimulation with wild-type P. aeruginosa. In a subacute non-lethal P. aeruginosa lung infection model, lasR variant infection also induced higher mICAM-1 expression in the murine airway epithelium and was associated with increased neutrophilic pulmonary inflammation in vivo. Here, we discuss how (loss of) LasR function and LasR-regulated proteases affect host immunity, inflammation and tissue pathology in acute vs. chronic P. aeruginosa lung infection.

Adaptation and genomic erosion in fragmented Pseudomonas aeruginosa populations in the sinuses of people with cystic fibrosis.

Pseudomonas aeruginosa notoriously adapts to the airways of people with cystic fibrosis (CF), yet how infection-site biogeography and associated evolutionary processes vary as lifelong infections progress remains unclear. Here we test the hypothesis that early adaptations promoting aggregation influence evolutionary-genetic trajectories by examining longitudinal P. aeruginosa from the sinuses of six adults with CF. Highly host-adapted lineages harbored mutator genotypes displaying signatures of early genome degradation associated with recent host restriction. Using an advanced imaging technique (MiPACT-HCR [microbial identification after passive clarity technique]), we find population structure tracks with genome degradation, with the most host-adapted, genome-degraded P. aeruginosa (the mutators) residing in small, sparse aggregates. We propose that following initial adaptive evolution in larger populations under strong selection for aggregation, P. aeruginosa persists in small, fragmented populations that experience stronger effects of genetic drift. These conditions enrich for mutators and promote degenerative genome evolution. Our findings underscore the importance of infection-site biogeography to pathogen evolution.

Phase Variation in HMW1A Controls a Phenotypic Switch in Haemophilus influenzae Associated with Pathoadaptation during Persistent Infection.

Genetic variants arising from within-patient evolution shed light on bacterial adaptation during chronic infection. Contingency loci generate high levels of genetic variation in bacterial genomes, enabling adaptation to the stringent selective pressures exerted by the host. A significant gap in our understanding of phase-variable contingency loci is the extent of their contribution to natural infections. The human-adapted pathogen nontypeable Haemophilus influenzae (NTHi) causes persistent infections, which contribute to underlying disease progression. The phase-variable high-molecular-weight (HMW) adhesins located on the NTHi surface mediate adherence to respiratory epithelial cells and, depending on the allelic variant, can also confer high epithelial invasiveness or hyperinvasion. In this study, we characterize the dynamics of HMW-mediated hyperinvasion in living cells and identify a specific HMW binding domain shared by hyperinvasive NTHi isolates of distinct pathological origins. Moreover, we observed that HMW expression decreased over time by using a longitudinal set of persistent NTHi strains collected from chronic obstructive pulmonary disease (COPD) patients, resulting from increased numbers of simple-sequence repeats (SSRs) downstream of the functional P2hmw1A promoter, which is the one primarily driving HMW expression. Notably, the increased SSR numbers at the hmw1 promoter region also control a phenotypic switch toward lower bacterial intracellular invasion and higher biofilm formation, likely conferring adaptive advantages during chronic airway infection by NTHi. Overall, we reveal novel molecular mechanisms of NTHi pathoadaptation based on within-patient lifestyle switching controlled by phase variation. IMPORTANCE Human-adapted bacterial pathogens have evolved specific mechanisms to colonize their host niche. Phase variation is a contingency strategy to allow adaptation to changing conditions, as phase-variable bacterial loci rapidly and reversibly switch their expression. Several NTHi adhesins are phase variable. These adhesins are required for colonization but also immunogenic, in such a way that bacteria with lower adhesin levels are better equipped to survive an immune response, making their contribution to natural infections unclear. We show here that the major NTHi adhesin HMW1A displays allelic variation, which can drive a phase-variable epithelial hyperinvasion phenotype. Over time, hmw1A phase variation lowers adhesin expression, which controls an NTHi lifestyle switch from high epithelial invasiveness to lower invasion and higher biofilm formation. This reversible loss of function aligns with the previously stated notion that epithelial infection is essential for NTHi infection establishment, but once established, persistence favors gene inactivation, in this case facilitating biofilm growth.

Comparative and Evolutionary Genomics of Isolates Provide Insight into the Pathoadaptation of Aeromonas.

Aeromonads are ubiquitous aquatic bacteria that cause opportunistic infections in humans, but their pathogenesis remains poorly understood. A pathogenomic approach was undertaken to provide insights into the emergence and evolution of pathogenic traits in aeromonads. The genomes of 64 Aeromonas strains representative of the whole genus were analyzed to study the distribution, phylogeny, and synteny of the flanking sequences of 13 virulence-associated genes. The reconstructed evolutionary histories varied markedly depending on the gene analyzed and ranged from vertical evolution, which followed the core genome evolution (alt and colAh), to complex evolution, involving gene loss by insertion sequence-driven gene disruption, horizontal gene transfer, and paraphyly with some virulence genes associated with a phylogroup (aer, ser, and type 3 secretion system components) or no phylogroup (type 3 secretion system effectors, Ast, ExoA, and RtxA toxins). The general pathogenomic overview of aeromonads showed great complexity with diverse evolution modes and gene organization and uneven distribution of virulence genes in the genus; the results provided insights into aeromonad pathoadaptation or the ability of members of this group to emerge as pathogens. Finally, these findings suggest that aeromonad virulence-associated genes should be examined at the population level and that studies performed on type or model strains at the species level cannot be generalized to the whole species.

Transcriptomic analysis of longitudinal Burkholderia pseudomallei infecting the cystic fibrosis lung.

The melioidosis bacterium, Burkholderia pseudomallei, is increasingly being recognised as a pathogen in patients with cystic fibrosis (CF). We have recently catalogued genome-wide variation of paired, isogenic B. pseudomallei isolates from seven Australasian CF cases, which were collected between 4 and 55 months apart. Here, we extend this investigation by documenting the transcriptomic changes in B. pseudomallei in five cases. Following growth in an artificial CF sputum medium, four of the five paired isolates exhibited significant differential gene expression (DE) that affected between 32 and 792 genes. The greatest number of DE events was observed between the strains from patient CF9, consistent with the hypermutator status of the latter strain, which is deficient in the DNA mismatch repair protein MutS. Two patient isolates harboured duplications that concomitantly increased expression of the ß-lactamase-encoding gene penA, and a 35 kb deletion in another abolished expression of 29 genes. Convergent expression profiles in the chronically-adapted isolates identified two significantly downregulated and 17 significantly upregulated loci, including the resistance-nodulation-division (RND) efflux pump BpeEF-OprC, the quorum-sensing hhqABCDE operon, and a cyanide- and pyocyanin-insensitive cytochrome bd quinol oxidase. These convergent pathoadaptations lead to increased expression of pathways that may suppress competing bacterial and fungal pathogens, and that enhance survival in oxygen-restricted environments, the latter of which may render conventional antibiotics less effective in vivo. Treating chronically adapted B. pseudomallei infections with antibiotics designed to target anaerobic infections, such as the nitroimidazole class of antibiotics, may significantly improve pathogen eradication attempts by exploiting this Achilles heel.

Phylogenomic, Pan-genomic, Pathogenomic and Evolutionary Genomic Insights into the Agronomically Relevant Enterobacteria Pantoea ananatis and Pantoea stewartii.

Pantoea ananatis is ubiquitously found in the environment and causes disease on a wide range of plant hosts. By contrast, its sister species, Pantoea stewartii subsp. stewartii is the host-specific causative agent of the devastating maize disease Stewart's wilt. This pathogen has a restricted lifecycle, overwintering in an insect vector before being introduced into susceptible maize cultivars, causing disease and returning to overwinter in its vector. The other subspecies of P. stewartii subsp. indologenes, has been isolated from different plant hosts and is predicted to proliferate in different environmental niches. Here we have, by the use of comparative genomics and a comprehensive suite of bioinformatic tools, analyzed the genomes of ten P. stewartii and nineteen P. ananatis strains. Our phylogenomic analyses have revealed that there are two distinct clades within P. ananatis while far less phylogenetic diversity was observed among the P. stewartii subspecies. Pan-genome analyses revealed a large core genome comprising of 3,571 protein coding sequences is shared among the twenty-nine compared strains. Furthermore, we showed that an extensive accessory genome made up largely by a mobilome of plasmids, integrated prophages, integrative and conjugative elements and insertion elements has resulted in extensive diversification of P. stewartii and P. ananatis. While these organisms share many pathogenicity determinants, our comparative genomic analyses show that they differ in terms of the secretion systems they encode. The genomic differences identified in this study have allowed us to postulate on the divergent evolutionary histories of the analyzed P. ananatis and P. stewartii strains and on the molecular basis underlying their ecological success and host range.