Antimicrobial Peptide Recognition Motif of the Substrate Binding Protein SapA from Nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen associated with respiratory diseases, including otitis media and exacerbations of chronic obstructive pulmonary disease. NTHi exhibits resistance to killing by host antimicrobial peptides (AMPs) mediated by SapA, the substrate binding protein of the sensitivity to antimicrobial peptides (Sap) transporter. However, the specific mechanisms by which SapA selectively binds various AMPs such as defensins and cathelicidin are unknown. In this study, we report mutational analyses of both defensin AMPs and the SapA binding pocket to define the specificity of AMP recognition. Bactericidal assays revealed that NTHi lacking SapA are more susceptible to human beta defensins and LL-37, while remaining highly resistant to a human alpha defensin. In contrast to homologues, our research underscores the distinct specificity of NTHi SapA, which selectively recognizes and binds to peptides containing the charged-hydrophobic motif PKE and RRY. These findings provide valuable insight into the divergence of SapA among bacterial species and NTHi SapA's ability to selectively interact with specific AMPs to mediate resistance.

The use of Escherichia coli strain Nissle 1917 shows promise for improving gastrointestinal and urinary health in dogs.

OBJECTIVES: To investigate the probiotic Escherichia coli Nissle 1917 (EcN) in canine idiopathic diarrhea and urinary tract infections. ANIMALS/SAMPLES: The utility of EcN was explored in a 3-phase study from March 2017 to June 2020. Eighty-nine dogs with idiopathic diarrhea were included in phase 1, 3 healthy dogs were included in phase 2, and uropathogenic E coli (UPEC) isolates from 38 dogs with urinary tract infections were included in phase 3. PROCEDURES: In phase 1, dogs with diarrhea were prospectively enrolled in a randomized study to receive EcN (108 EcN bacteria/mL; < 10 kg received 5 mL/dose, 10 to 25 kg received 10 mL/dose, or > 25 kg received 15 mL/dose) or placebo for 3 days, followed by a 15-day observation phase. In phase 2, healthy dogs received EcN as described in phase 1, with feces analyzed for E coli populations and microbiome composition at days 0, 3, and 7. In phase 3, EcN efficacy was tested by in vitro plate assay against UPEC isolates. RESULTS: Median duration of abnormal stool consistency, time to response, and duration of diarrhea were shorter for dogs that received EcN (5.0, 3.0, and 2.0 days, respectively) versus the placebo (7.0, 5.0, and 4.0 days, respectively) (P = .21, P = .05, and P = .039, respectively). EcN induced shifts in E coli diversity in healthy dogs while having minimal impact on overall microbiome structure. Furthermore, 68% of the canine UPEC isolates were susceptible to EcN in vitro. CLINICAL RELEVANCE: EcN improved the treatment of idiopathic diarrhea, colonized the gastrointestinal tract during the trial, and displayed in vitro competition with UPEC.

Escherichia coli probiotic exhibits in vitro growth-limiting effects on clinical feline uropathogenic E coli isolates.

OBJECTIVE: To characterize uropathogenic Escherichia coli (UPEC) in cases of clinical feline urinary tract infection (UTI) and subclinical bacteriuria and investigate the in vitro effects of E coli strain Nissle 1917 on isolate growth. ANIMALS: 40 cats with positive E coli culture results for urine collected during routine evaluation. PROCEDURES: Characterization of UPEC isolates was performed by PCR-based phylotype analysis and serotyping. Nissle 1917 effects on growth inhibition and competitive overgrowth against UPEC isolates were evaluated in vitro using a plate-based competition assay. RESULTS: Feline phylogroups were similar to previous human and feline UPEC studies, with most of the isolates belonging to phylogroup A (42.5%), B2 (37.5%), and D (15.0%). Fifty-two percent of isolates were found to be resistant to antimicrobials, with 19% of these being multidrug resistant (MDR). Nissle 1917 adversely affected the growth of 82.5% of all isolates and 100% of MDR isolates in vitro. The median zone of inhibition was 3.33 mm (range, 1.67 to 10.67 mm). Thirteen isolates were affected via competitive overgrowth and 20 via growth inhibition. CLINICAL RELEVANCE: UPEC isolates from cats were similar in phylogroup analysis to human and dog isolates. The in vitro effects of Nissle 1917 on UPEC warrant additional studies to determine if similar results can be duplicated in vivo.

Continuous Microevolution Accelerates Disease Progression during Sequential Episodes of Infection.

Bacteria adapt to dynamic changes in the host during chronic and recurrent infections. Bacterial microevolution is one type of adaptation that imparts a selective advantage. We hypothesize that recurrent episodes of disease promote microevolution through genetic mutations that modulate disease severity. We use a pre-clinical model of otitis media (OM) to determine the potential role for microevolution of nontypeable Haemophilus influenzae (NTHI) during sequential episodes of disease. Whole genome sequencing reveals microevolution of hemoglobin binding and lipooligosaccharide (LOS) biosynthesis genes, suggesting that adaptation of these systems is critical for infection. These OM-adapted strains promote increased biofilm formation, inflammation, stromal fibrosis, and an increased propensity to form intracellular bacterial communities (IBCs). Remarkably, IBCs remain for at least one month following clinical resolution of infection, suggesting an intracellular reservoir as a nidus for recurrent OM. Additional approaches for therapeutic design tailored to combat this burdensome disease will arise from these studies.

Reprioritization of biofilm metabolism is associated with nutrient adaptation and long-term survival of Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHI) is a human-restricted pathogen with an essential requirement for heme-iron acquisition. We previously demonstrated that microevolution of NTHI promotes stationary phase survival in response to transient heme-iron restriction. In this study, we examine the metabolic contributions to biofilm formation using this evolved NTHI strain, RM33. Quantitative analyses identified 29 proteins, 55 transcripts, and 31 metabolites that significantly changed within in vitro biofilms formed by RM33. The synthesis of all enzymes within the tryptophan and glycogen pathways was significantly increased in biofilms formed by RM33 compared with the parental strain. In addition, increases were observed in metabolite transport, adhesin production, and DNA metabolism. Furthermore, we observed pyruvate as a pivotal point in the metabolic pathways associated with changes in cAMP phosphodiesterase activity during biofilm formation. Taken together, changes in central metabolism combined with increased stores of nutrients may serve to counterbalance nutrient sequestration.

Microevolution in response to transient heme-iron restriction enhances intracellular bacterial community development and persistence.

Bacterial pathogens must sense, respond and adapt to a myriad of dynamic microenvironmental stressors to survive. Adaptation is key for colonization and long-term ability to endure fluctuations in nutrient availability and inflammatory processes. We hypothesize that strains adapted to survive nutrient deprivation are more adept for colonization and establishment of chronic infection. In this study, we detected microevolution in response to transient nutrient limitation through mutation of icc. The mutation results in decreased 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in nontypeable Haemophilus influenzae (NTHI). In a preclinical model of NTHI-induced otitis media (OM), we observed a significant decrease in the recovery of effusion from ears infected with the icc mutant strain. Clinically, resolution of OM coincides with the clearance of middle ear fluid. In contrast to this clinical paradigm, we observed that the icc mutant strain formed significantly more intracellular bacterial communities (IBCs) than the parental strain early during experimental OM. Although the number of IBCs formed by the parental strain was low at early stages of OM, we observed a significant increase at later stages that coincided with absence of recoverable effusion, suggesting the presence of a mucosal reservoir following resolution of clinical disease. These data provide the first insight into NTHI microevolution during nutritional limitation and provide the first demonstration of IBCs in a preclinical model of chronic OM.

Transient Nutrient Deprivation Promotes Macropinocytosis-Dependent Intracellular Bacterial Community Development.

Nutrient limitation restricts bacterial growth in privileged sites such as the middle ear. Transient heme-iron restriction of nontypeable Haemophilus influenzae (NTHI), the major causative agent of chronic and recurrent otitis media (OM), promotes new and diverse phenotypes that can influence planktonic, biofilm, and intracellular lifestyles of NTHI. However, the bacterial responses to nutrient restriction that impact intracellular fate and survival of NTHI are unknown. In this work, we provide evidence for the role of transient heme-iron restriction in promoting the formation of intracellular bacterial communities (IBCs) of NTHI both in vitro and in vivo in a preclinical model of OM. We show that transient heme-iron restriction of NTHI results in significantly increased invasion and intracellular populations that escape or evade the endolysosomal pathway for increased intracellular survival. In contrast, NTHI continuously exposed to heme-iron traffics through the endolysosomal pathway for degradation. The use of pharmacological inhibitors revealed that prior heme-iron status does not appear to influence NTHI internalization through endocytic pathways. However, inhibition of macropinocytosis altered the intracellular fate of transiently restricted NTHI for degradation in the endolysosomal pathway. Furthermore, prevention of macropinocytosis significantly reduced the number of IBCs in cultured middle ear epithelial cells, providing evidence for the feasibility of this approach to reduce OM persistence. These results reveal that microenvironmental cues can influence the intracellular fate of NTHI, leading to new mechanisms for survival during disease progression.IMPORTANCE Otitis media is the most common bacterial infection in childhood. Current therapies are limited in the prevention of chronic or recurrent otitis media which leads to increased antibiotic exposure and represents a significant socioeconomic burden. In this study, we delineate the effect of nutritional limitation on the intracellular trafficking pathways used by nontypeable Haemophilus influenzae (NTHI). Moreover, transient limitation of heme-iron led to the development of intracellular bacterial communities that are known to contribute to persistence and recurrence in other diseases. New approaches for therapeutic interventions that reduce the production of intracellular bacterial communities and promote trafficking through the endolysosomal pathway were revealed through the use of pharmacological inhibition of macropinocytosis. This work demonstrates the importance of an intracellular niche for NTHI and provides new approaches for intervention for acute, chronic, and recurring episodes of otitis media.

KatG and KatE confer Acinetobacter resistance to hydrogen peroxide but sensitize bacteria to killing by phagocytic respiratory burst.

AIMS: Catalase catalyzes the degradation of H2O2. Acinetobacter species have four predicted catalase genes, katA, katE, katG, and katX. The aims of the present study seek to determine which catalase(s) plays a predominant role in determining the resistance to H2O2, and to assess the role of catalase in Acinetobacter virulence. MAIN METHODS: Mutants of Acinetobacter baumannii and Acinetobacter nosocomialis with deficiencies in katA, katE, katG, and katX were tested for sensitivity to H2O2, either by halo assays or by liquid culture assays. Respiratory burst of neutrophils, in response to A. nosocomialis, was assessed by chemiluminescence to examine the effects of catalase on the production of reactive oxygen species (ROS) in neutrophils. Bacterial virulence was assessed using a Galleria mellonella larva infection model. KEY FINDINGS: The capacities of A. baumannii and A. nosocomialis to degrade H2O2 are largely dependent on katE. The resistance of both A. baumannii and A. nosocomialis to H2O2 is primarily determined by the katG gene, although katE also plays a minor role in H2O2 resistance. Bacteria lacking both the katG and katE genes exhibit the highest sensitivity to H2O2. While A. nosocomialis bacteria with katE and/or katG were able to decrease ROS production by neutrophils, these cells also induced a more robust respiratory burst in neutrophils than did cells deficient in both katE and katG. We also found that A. nosocomialis deficient in both katE and katG was more virulent than the wildtype A. nosocomialis strain. SIGNIFICANCE: Our findings suggest that inhibition of Acinetobacter catalase may help to overcome the resistance of Acinetobacter species to microbicidal H2O2 and facilitate bacterial disinfection.

Comprehensive Proteomic and Metabolomic Signatures of Nontypeable Haemophilus influenzae-Induced Acute Otitis Media Reveal Bacterial Aerobic Respiration in an Immunosuppressed Environment.

A thorough understanding of the molecular details of the interactions between bacteria and host are critical to ultimately prevent disease. Recent technological advances allow simultaneous analysis of host and bacterial protein and metabolic profiles from a single small tissue sample to provide insight into pathogenesis. We used the chinchilla model of human otitis media to determine, for the first time, the most expansive delineation of global changes in protein and metabolite profiles during an experimentally induced disease. After 48 h of infection with nontypeable Haemophilus influenzae, middle ear tissue lysates were analyzed by high-resolution quantitative two-dimensional liquid chromatography-tandem mass spectrometry. Dynamic changes in 105 chinchilla proteins and 66 metabolites define the early proteomic and metabolomic signature of otitis media. Our studies indicate that establishment of disease coincides with actin morphogenesis, suppression of inflammatory mediators, and bacterial aerobic respiration. We validated the observed increase in the actin-remodeling complex, Arp2/3, and experimentally showed a role for Arp2/3 in nontypeable Haemophilus influenzae invasion. Direct inhibition of actin branch morphology altered bacterial invasion into host epithelial cells, and is supportive of our efforts to use the information gathered to modify outcomes of disease. The twenty-eight nontypeable Haemophilus influenzae proteins identified participate in carbohydrate and amino acid metabolism, redox homeostasis, and include cell wall-associated metabolic proteins. Quantitative characterization of the molecular signatures of infection will redefine our understanding of host response driven developmental changes during pathogenesis. These data represent the first comprehensive study of host protein and metabolite profiles in vivo in response to infection and show the feasibility of extensive characterization of host protein profiles during disease. Identification of novel protein targets and metabolic biomarkers will advance development of therapeutic and diagnostic options for treatment of disease.

Overlapping and complementary oxidative stress defense mechanisms in nontypeable Haemophilus influenzae.

The Gram-negative commensal bacterium nontypeable Haemophilus influenzae (NTHI) can cause respiratory tract diseases that include otitis media, sinusitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During colonization and infection, NTHI withstands oxidative stress generated by reactive oxygen species produced endogenously, by the host, and by other copathogens and flora. These reactive oxygen species include superoxide, hydrogen peroxide (H2O2), and hydroxyl radicals, whose killing is amplified by iron via the Fenton reaction. We previously identified genes that encode proteins with putative roles in protection of the NTHI isolate strain 86-028NP against oxidative stress. These include catalase (HktE), peroxiredoxin/glutaredoxin (PgdX), and a ferritin-like protein (Dps). Strains were generated with mutations in hktE, pgdX, and dps. The hktE mutant and a pgdX hktE double mutant were more sensitive than the parent to killing by H2O2. Conversely, the pgdX mutant was more resistant to H2O2 due to increased catalase activity. Supporting the role of killing via the Fenton reaction, binding of iron by Dps significantly mitigated the effect of H2O2-mediated killing. NTHI thus utilizes several effectors to resist oxidative stress, and regulation of free iron is critical to this protection. These mechanisms will be important for successful colonization and infection by this opportunistic human pathogen.

Bacterial differentiation, development, and disease: mechanisms for survival.

Bacteria have the exquisite ability to maintain a precise diameter, cell length, and shape. The dimensions of bacteria size and shape are a classical metric in the distinction of bacterial species. Much of what we know about the particular morphology of any given species is the result of investigations of planktonic cultures. As we explore deeper into the natural habitats of bacteria, it is increasingly clear that bacteria can alter their morphology in response to the environment in which they reside. Specific morphologies are also becoming recognized as advantageous for survival in hostile environments. This is of particular importance in the context of both colonization and infection in the host. There are multiple examples of bacterial pathogens that use morphological changes as a mechanism for evasion of host immune responses and continued persistence. This review will focus on two systems where specific morphological changes are essential for persistence in animal models of human disease. We will also offer insight into the mechanism underlying the morphological changes and how these morphotypes aid in persistence. Additional examples of morphological changes associated with survival will be presented.

HrrF is the Fur-regulated small RNA in nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) are Gram-negative commensal bacteria that reside in the nasopharynx. NTHi can also cause multiple upper and lower respiratory tract diseases that include sinusitis, conjunctivitis, bronchitis, and otitis media. In numerous bacterial species the ferric uptake regulator (Fur) acts as a global regulator of iron homeostasis by negatively regulating the expression of iron uptake systems. However in NTHi strain 86-028NP and numerous other bacterial species there are multiple instances where Fur positively affects gene expression. It is known that many instances of positive regulation by Fur occur indirectly through a small RNA intermediate. However, no examples of small RNAs have been described in NTHi. Therefore we used RNA-Seq analysis to analyze the transcriptome of NTHi strain 86-028NPrpsL and an isogenic 86-028NPrpsLΔfur strain to identify Fur-regulated intergenic transcripts. From this analysis we identified HrrF, the first small RNA described in any Haemophilus species. Orthologues of this small RNA exist only among other Pasteurellaceae. Our analysis showed that HrrF is maximally expressed when iron levels are low. Additionally, Fur was shown to bind upstream of the hrrF promoter. RNA-Seq analysis was used to identify targets of HrrF which include genes whose products are involved in molybdate uptake, deoxyribonucleotide synthesis, and amino acid biosynthesis. The stability of HrrF is not dependent on the RNA chaperone Hfq. This study is the first step in an effort to investigate the role small RNAs play in altering gene expression in response to iron limitation in NTHi.

Small substrate transport and mechanism of a molybdate ATP binding cassette transporter in a lipid environment.

Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. To understand the mechanism of transport in the biological membrane as a whole, the effects of the lipid bilayer on transport needed to be addressed. Continuous wave-electron paramagnetic resonance and in vivo molybdate uptake studies were used to test the impact of the lipid environment on the mechanism and function of MolBC-A. Working with the bacterium Haemophilus influenzae, we found that MolBC-A functions as a low affinity molybdate transporter in its native environment. In periods of high extracellular molybdate concentration, H. influenzae makes use of parallel molybdate transport systems (MolBC-A and ModBC-A) to take up a greater amount of molybdate than a strain with ModBC-A alone. In addition, the movement of the translocation pathway in response to nucleotide binding and hydrolysis in a lipid environment is conserved when compared with in-detergent analysis. However, electron paramagnetic resonance spectroscopy indicates that a lipid environment restricts the flexibility of the MolBC translocation pathway. By combining continuous wave-electron paramagnetic resonance spectroscopy and substrate uptake studies, we reveal details of molybdate transport and the logistics of uptake systems that employ multiple transporters for the same substrate, offering insight into the mechanisms of nutrient uptake in bacteria.

Host Antimicrobial Peptides in Bacterial Homeostasis and Pathogenesis of Disease.

Innate immune responses function as a first line of host defense against the development of bacterial infection, and in some cases to preserve the sterility of privileged sites in the human host. Bacteria that enter these sites must counter host responses for colonization. From the host's perspective, the innate immune system works expeditiously to minimize the bacterial threat before colonization and subsequent dysbiosis. The multifactorial nature of disease further challenges predictions of how each independent variable influences bacterial pathogenesis. From bacterial colonization to infection and through disease, the microenvironments of the host are in constant flux as bacterial and host factors contribute to changes at the host-pathogen interface, with the host attempting to eradicate bacteria and the bacteria fighting to maintain residency. A key component of this innate host response towards bacterial infection is the production of antimicrobial peptides (AMPs). As an early component of the host response, AMPs modulate bacterial load and prevent establishment of infection. Under quiescent conditions, some AMPs are constitutively expressed by the epithelium. Bacterial infection can subsequently induce production of other AMPs in an effort to maintain sterility, or to restrict colonization. As demonstrated in various studies, the absence of a single AMP can influence pathogenesis, highlighting the importance of AMP concentration in maintaining homeostasis. Yet, AMPs can increase bacterial virulence through the co-opting of the peptides or alteration of bacterial virulence gene expression. Further, bacterial factors used to subvert AMPs can modify host microenvironments and alter colonization of the residential flora that principally maintain homeostasis. Thus, the dynamic interplay between host defense peptides and bacterial factors produced to quell peptide activity play a critical role in the progression and outcome of disease.

Ferric uptake regulator and its role in the pathogenesis of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is a commensal microorganism of the human nasopharynx, and yet is also an opportunistic pathogen of the upper and lower respiratory tracts. Host microenvironments influence gene expression patterns, likely critical for NTHi persistence. The host sequesters iron as a mechanism to control microbial growth, and yet iron limitation influences gene expression and subsequent production of proteins involved in iron homeostasis. Careful regulation of iron uptake, via the ferric uptake regulator Fur, is essential in multiple bacteria, including NTHi. We hypothesized therefore that Fur contributes to iron homeostasis in NTHi, is critical for bacterial persistence, and likely regulates expression of virulence factors. Toward this end, fur was deleted in the prototypic NTHi clinical isolate, 86-028NP, and we assessed gene expression regulated by Fur. As expected, expression of the majority of genes that encode proteins with predicted roles in iron utilization was repressed by Fur. However, 14 Fur-regulated genes encode proteins with no known function, and yet may contribute to iron utilization or other biological functions. In a mammalian model of human otitis media, we determined that Fur was critical for bacterial persistence, indicating an important role for Fur-mediated iron homeostasis in disease progression. These data provide a profile of genes regulated by Fur in NTHi and likely identify additional regulatory pathways involved in iron utilization. Identification of such pathways will increase our understanding of how this pathogen can persist within host microenvironments, as a common commensal and, importantly, as a pathogen with significant clinical impact.

Haemophilus influenzae and oxidative stress.

Haemophilus influenzae is a commensal of the human upper respiratory tract. H. influenzae can, however, move out of its commensal niche and cause multiple respiratory tract diseases. Such diseases include otitis media in young children, as well as exacerbations of chronic obstructive pulmonary disease (COPD), sinusitis, conjunctivitis, and bronchitis. During the course of colonization and infection, H. influenzae must withstand oxidative stress generated by multiple reactive oxygen species produced endogenously, by other co-pathogens and by host cells. H. influenzae has, therefore, evolved multiple mechanisms that protect the cell against oxygen-generated stresses. In this review, we will describe these systems relative to the well-described systems in Escherichia coli. Moreover, we will compare how H. influenzae combats the effect of oxidative stress as a necessary phenotype for its roles as both a successful commensal and pathogen.

Silver nanoparticles embedded in zeolite membranes: release of silver ions and mechanism of antibacterial action.

BACKGROUND: The focus of this study is on the antibacterial properties of silver nanoparticles embedded within a zeolite membrane (AgNP-ZM). METHODS AND RESULTS: These membranes were effective in killing Escherichia coli and were bacteriostatic against methicillin-resistant Staphylococcus aureus. E. coli suspended in Luria Bertani (LB) broth and isolated from physical contact with the membrane were also killed. Elemental analysis indicated slow release of Ag(+) from the AgNP-ZM into the LB broth. The E. coli killing efficiency of AgNP-ZM was found to decrease with repeated use, and this was correlated with decreased release of silver ions with each use of the support. Gene expression microarrays revealed upregulation of several antioxidant genes as well as genes coding for metal transport, metal reduction, and ATPase pumps in response to silver ions released from AgNP-ZM. Gene expression of iron transporters was reduced, and increased expression of ferrochelatase was observed. In addition, upregulation of multiple antibiotic resistance genes was demonstrated. The expression levels of multicopper oxidase, glutaredoxin, and thioredoxin decreased with each support use, reflecting the lower amounts of Ag(+) released from the membrane. The antibacterial mechanism of AgNP-ZM is proposed to be related to the exhaustion of antioxidant capacity. CONCLUSION: These results indicate that AgNP-ZM provide a novel matrix for gradual release of Ag(+).

Identification of Haemophilus ducreyi genes expressed during human infection.

To identify Haemophilus ducreyi transcripts that are expressed during human infection, we used selective capture of transcribed sequences (SCOTS) with RNA isolated from pustules obtained from three volunteers infected with H. ducreyi, and with RNA isolated from broth-grown bacteria used to infect volunteers. With SCOTS, competitive hybridization of tissue-derived and broth-derived sequences identifies genes that may be preferentially expressed in vivo. Among the three tissue specimens, we identified 531 genes expressed in vivo. Southern blot analysis of 60 genes from each tissue showed that 87 % of the identified genes hybridized better with cDNA derived from tissue specimens than with cDNA derived from broth-grown bacteria. RT-PCR on nine additional pustules confirmed in vivo expression of 10 of 11 selected genes in other volunteers. Of the 531 genes, 139 were identified in at least two volunteers. These 139 genes fell into several functional categories, including biosynthesis and metabolism, regulation, and cellular processes, such as transcription, translation, cell division, DNA replication and repair, and transport. Detection of genes involved in anaerobic and aerobic respiration indicated that H. ducreyi likely encounters both microenvironments within the pustule. Other genes detected suggest an increase in DNA damage and stress in vivo. Genes involved in virulence in other bacterial pathogens and 32 genes encoding hypothetical proteins were identified, and may represent novel virulence factors. We identified three genes, lspA1, lspA2 and tadA, known to be required for virulence in humans. This is the first study to broadly define transcripts expressed by H. ducreyi in humans.

Sequence and functional analyses of Haemophilus spp. genomic islands.

BACKGROUND: A major part of horizontal gene transfer that contributes to the diversification and adaptation of bacteria is facilitated by genomic islands. The evolution of these islands is poorly understood. Some progress was made with the identification of a set of phylogenetically related genomic islands among the Proteobacteria, recognized from the investigation of the evolutionary origins of a Haemophilus influenzae antibiotic resistance island, namely ICEHin1056. More clarity comes from this comparative analysis of seven complete sequences of the ICEHin1056 genomic island subfamily. RESULTS: These genomic islands have core and accessory genes in approximately equal proportion, with none demonstrating recent acquisition from other islands. The number of variable sites within core genes is similar to that found in the host bacteria. Furthermore, the GC content of the core genes is similar to that of the host bacteria (38% to 40%). Most of the core gene content is formed by the syntenic type IV secretion system dependent conjugative module and replicative module. GC content and lack of variable sites indicate that the antibiotic resistance genes were acquired relatively recently. An analysis of conjugation efficiency and antibiotic susceptibility demonstrates that phenotypic expression of genomic island-borne genes differs between different hosts. CONCLUSION: Genomic islands of the ICEHin1056 subfamily have a longstanding relationship with H. influenzae and H. parainfluenzae and are co-evolving as semi-autonomous genomes within the 'supragenomes' of their host species. They have promoted bacterial diversity and adaptation through becoming efficient vectors of antibiotic resistance by the recent acquisition of antibiotic resistance transposons.