Evolving origin-of-transfer sequences on staphylococcal conjugative and mobilizable plasmids-who's mimicking whom?

In Staphylococcus aureus, most multiresistance plasmids lack conjugation or mobilization genes for horizontal transfer. However, most are mobilizable due to carriage of origin-of-transfer (oriT) sequences mimicking those of conjugative plasmids related to pWBG749. pWBG749-family plasmids have diverged to carry five distinct oriT subtypes and non-conjugative plasmids have been identified that contain mimics of each. The relaxasome accessory factor SmpO, encoded by each conjugative plasmid, determines specificity for its cognate oriT. Here we characterized the binding of SmpO proteins to each oriT. SmpO proteins predominantly formed tetramers in solution and bound 5'-GNNNNC-3' sites within each oriT. Four of the five SmpO proteins specifically bound their cognate oriT. An F7K substitution in pWBG749 SmpO switched oriT-binding specificity in vitro. In vivo, the F7K substitution reduced but did not abolish self-transfer of pWBG749. Notably, the substitution broadened the oriT subtypes that were mobilized. Thus, this substitution represents a potential evolutionary intermediate with promiscuous DNA-binding specificity that could facilitate a switch between oriT specificities. Phylogenetic analysis suggests pWBG749-family plasmids have switched oriT specificity more than once during evolution. We hypothesize the convergent evolution of oriT specificity in distinct branches of the pWBG749-family phylogeny reflects indirect selection pressure to mobilize plasmids carrying non-cognate oriT-mimics.

Interplay between Amoxicillin Resistance and Osmotic Stress in Helicobacter pylori.

Rising antibiotic resistance rates are a growing concern for all pathogens, including Helicobacter pylori. We previously examined the association of specific mutations in PBP1 with amoxicillin resistance and fitness in H. pylori and found that V374L and N562Y mutations were associated with resistance, but also resulted in fitness defects. Furthermore, we found that hyperosmotic stress differentially altered the fitness of strains bearing these mutations; survival of the V374L strain was decreased by hyperosmotic stress, but the N562Y strain showed increased cell survival relative to that of wild-type G27. The finding that amoxicillin-resistant strains show environmentally dictated changes in fitness suggests a previously unexplored interaction between amoxicillin resistance and osmotic stress in H. pylori. Here, we further characterized the interaction between osmotic stress and amoxicillin resistance. Wild-type and isogenic PBP1 mutant strains were exposed to amoxicillin, various osmotic stressors, or combined antibiotic and osmotic stress, and viability was monitored. While subinhibitory concentrations of NaCl did not affect H. pylori viability, the combination of NaCl and amoxicillin resulted in synergistic killing; this was true even for the antibiotic-resistant strains. Moreover, similar synergy was found with other beta-lactams, but not with antibiotics that did not target the cell wall. Similar synergistic killing was also demonstrated when KCl was utilized as the osmotic stressor. Conversely, osmolar equivalent concentrations of sucrose antagonized amoxicillin-mediated killing. Taken together, our results support a previously unrecognized interaction between amoxicillin resistance and osmotic stress in H. pylori. These findings have interesting implications for the effectiveness of antibiotic therapy for this pathogen. IMPORTANCE Rising antibiotic resistance rates in H. pylori are associated with increased rates of treatment failure. Understanding how stressors impact antibiotic resistance may shed light on the development of future treatment strategies. Previous studies found that mutations in PBP1 that conferred resistance to amoxicillin were also associated with a decrease in bacterial fitness. The current study demonstrated that osmotic stress can enhance beta lactam-mediated killing of H. pylori. The source of osmotic stress was found to be important for these interactions. Given that relatively little is known about how H. pylori responds to osmotic stress, these findings fill important knowledge gaps on this topic and provide interesting implications for the effectiveness of antibiotic therapy for this pathogen.

Friend or Foe: Interbacterial Competition in the Nasal Cavity.

Like other microbes that live on or in the human body, the bacteria that inhabit the upper respiratory tract, in particular the nasal cavity, have evolved to survive in an environment that presents a number of physical and chemical challenges; these microbes are constantly bombarded with nutritional fluctuations, changes in humidity, the presence of inhaled particulate matter (odorants and allergens), and competition with other microbes. Indeed, only a specialized set of species is able to colonize this niche and successfully contend with the host's immune system and the constant threat from competitors. To this end, bacteria that live in the nasal cavity have evolved a variety of approaches to outcompete contenders for the limited nutrients and space; broadly speaking, these strategies may be considered a type of "bacterial warfare." A greater molecular understanding of bacterial warfare has the potential to reveal new approaches or molecules that can be developed as novel therapeutics. As such, there are many studies within the last decade that have sought to understand the complex polymicrobial interactions that occur in various environments. Here, we review what is currently known about the age-dependent structure and interbacterial relationships within the nasal microbiota and summarize the molecular mechanisms that are predicted to dictate bacterial warfare in this niche. Although the currently described interactions are complex, in reality, we have likely only scratched the surface in terms of a true understanding of the types of interbacterial competition and cooperation that are thought to take place in and on the human body.

Analysis of fitness costs associated with metronidazole and amoxicillin resistance in Helicobacter pylori.

BACKGROUND: Increasing rates of antibiotic resistance are a major concern for all pathogens, including H. pylori. However, increased resistance often coincides with a decrease in relative fitness of the pathogen in the absence of the antibiotic, raising the possibility that increased resistance can be mitigated for some antibiotics by improved antibiotic husbandry. Therefore, a greater understanding of which types of antibiotic resistance create fitness defects in H. pylori may aid rational treatment strategies. MATERIALS AND METHODS: While a wealth of H. pylori literature reports mutations that correlate with increased resistance, few studies demonstrate causation for these same mutations. Herein, we examined fitness costs associated with metronidazole and amoxicillin resistance. Isogenic strains bearing literature reported point mutations in the rdxA and pbp1 genes were engineered and tested in in vitro competition assays to assess relative fitness. RESULTS: None of the metronidazole resistance mutations resulted in a fitness cost under the tested conditions. In contrast, amoxicillin-resistant mutant strains demonstrated a defect in competition by 24 hours. This change in fitness was further enhanced by moderate osmotic stress. However, under extreme osmotic stress, the amoxicillin-resistant N562Y PBP1 mutant strain showed enhanced fitness, suggesting that there are some pbp1 mutations that can give a conditional fitness advantage. CONCLUSIONS: Our results demonstrate the role of specific point mutations in rdxA and pbp1 in antibiotic resistance and suggest that amoxicillin-resistant strains of H. pylori show environmentally dictated changes in fitness. These later finding may be responsible for the relatively low rates of amoxicillin resistance seen in the United States.

Conjugative and replicative biology of the Staphylococcus aureus antimicrobial resistance plasmid, pC02.

Genetic transfer among bacteria propels rapid resistance to antibiotics and decreased susceptibility to antiseptics. Staphylococcus aureus is a common culprit of hospital and community acquired infections, and S. aureus plasmids have been shown to carry a multitude of antimicrobial resistance genes. We previously identified a novel conjugative, multidrug resistance plasmid, pC02, from the clinical S. aureus isolate C02. This plasmid contained the chlorhexidine resistance gene qacA, and we were able to demonstrate that conjugative transfer of pC02 imparted decreased chlorhexidine susceptibility to recipient strains. In silico sequence analysis of pC02 suggested that the plasmid is part of the pWBG749-family of conjugative plasmids and that it contains three predicted origins of transfer (oriT), two of which we showed were functional and could mediate plasmid transfer. Furthermore, depending on which oriT was utilized, partial transfer of pC02 was consistently observed. To define the ability of the pC02 plasmid to utilize different oriT sequences, we examined the mobilization ability of nonconjugative plasmid variants that were engineered to contain a variety of oriT family inserts. The oriT-OTUNa family was transferred at the highest frequency; additional oriT families were also transferred but at lower frequencies. Plasmid stability was examined, and the copy number of pC02 was defined using droplet digital PCR (ddPCR). pC02 was stably maintained at approximately 4 copies per cell. Given the conjugative plasticity of pC02, we speculate that this plasmid could contribute to the spread of antimicrobial resistance across Staphylococcal strains and species.

Helicobacter pylori Genetic Polymorphisms in Gastric Disease Development.

Infecting half of the world's population, Helicobacter pylori is a medically important bacterium that induces a variety of gastric diseases, including gastritis, peptic ulcer disease, and gastric cancer. Sequencing of almost 1000 H. pylori isolates has revealed a diverse genome that contains abundant polymorphic genetic elements; many of these lie in factors likely to be associated with virulence. To ascertain the effect of these varying genetic elements, numerous epidemiological studies have investigated the contribution of the various polymorphisms to gastric disease development; particular focus has been placed on polymorphisms in the outer membrane proteins (OMPs), an effector protein, and a toxin produced by H. pylori. These studies have revealed geographic variation in the prevalence of various polymorphisms as well as in the associations between particular polymorphisms and gastric disease development. Furthermore, researchers have identified polymorphisms in multiple genes that frequently occur in combination. Though no single polymorphic genetic factor alone can fully account for gastric disease development in a population, the evaluation of multiple polymorphisms in a colonizing H. pylori strain can aid in the assessment of the pathogenic potential of the strain. Here we review specific H. pylori genetic polymorphisms (Bab proteins, Hom proteins, HopQ, SabA, SabB, OipA, IceA, VacA and CagA) that have been linked to disease outcome and discuss how geographic location and virulence factor polymorphisms together contribute to H. pylori-induced disease.

Certhrax Is an Antivirulence Factor for the Anthrax-Like Organism Bacillus cereus Strain G9241.

Bacillus cereus G9241 caused a life-threatening anthrax-like lung infection in a previously healthy human. This strain harbors two large virulence plasmids, pBCXO1 and pBC210, that are absent from typical B. cereus isolates. The pBCXO1 plasmid is nearly identical to pXO1 from Bacillus anthracis and carries genes (pagA1, lef, and cya) for anthrax toxin components (protective antigen [called PA1 in G9241], lethal factor [LF], and edema factor [EF], respectively). The plasmid also has an intact hyaluronic acid capsule locus. The pBC210 plasmid has a tetrasaccharide capsule locus, a gene for a PA1 homolog called PA2 (pagA2), and a gene (cer) for Certhrax, an ADP-ribosyltransferase toxin that inactivates vinculin. LF, EF, and Certhrax require PA for entry into cells. In this study, we asked what role PA1, PA2, LF, and Certhrax play in the pathogenicity of G9241. To answer this, we generated isogenic deletion mutations in the targeted toxin gene components and then assessed the strains for virulence in highly G9241-susceptible (A/J) and moderately G9241-sensitive (C57BL/6) mice. We found that full virulence of G9241 required PA1 and LF, while PA2 contributed minimally to pathogenesis of G9241 but could not functionally replace PA1 as a toxin-binding subunit in vivo Surprisingly, we discovered that Certhrax attenuated the virulence of G9241; i.e., a Δcer Δlef mutant strain was more virulent than a Δlef mutant strain following subcutaneous inoculation of A/J mice. Moreover, the enzymatic activity of Certhrax contributed to this phenotype. We concluded that Certhrax acts as an antivirulence factor in the anthrax-like organism B. cereus G9241.

Helicobacter pylori Biofilm Formation Is Differentially Affected by Common Culture Conditions, and Proteins Play a Central Role in the Biofilm Matrix.

The concept of Helicobacter pylori biofilm formation is relatively new. To help provide a foundation for future biofilm studies, we characterized the biofilm formation ability of a common H. pylori lab strain, G27. The goal of this study was to evaluate biofilm formation by G27 in response to common culture conditions and to explore the biofilm matrix. Our results indicate that while various types of growth media did not dramatically affect biofilm formation, surface selection had a significant effect on the final biofilm mass. Furthermore, enzymatic assays and confocal microscopy revealed that proteins appear to be the primary structural component of the H. pylori extracellular matrix; extracellular DNA (eDNA) and polysaccharides were also present but appear to play a secondary role. Finally, we found that two well-characterized antibiofilm cationic peptides differentially affected early and late-stage biofilms. Together these results provide interesting avenues for future investigations that will seek to understand H. pylori biofilm formation.IMPORTANCE The study of H. pylori biofilm formation is still in its infancy. As such, there is great variability in how biofilm assays are performed across labs. While several groups have begun to investigate factors that influence H. pylori biofilm formation, it is not yet understood how H. pylori biofilm formation may vary based on commonly used conditions. These inconsistencies lead to difficulties in interpretation and comparison between studies. Here, we set out to characterize biofilm formation by a commonly available lab strain, G27. Our findings provide novel insight into optimal biofilm conditions, the biofilm matrix, and possible mechanisms to block or disrupt biofilm formation.

Structure and dynamics of Helicobacter pylori nickel-chaperone HypA: an integrated approach using NMR spectroscopy, functional assays and computational tools.

Helicobacter pylori HypA (HpHypA) is a metallochaperone necessary for maturation of [Ni,Fe]-hydrogenase and urease, the enzymes required for colonization and survival of H. pylori in the gastric mucosa. HpHypA contains a structural Zn(II) site and a unique Ni(II) binding site at the N-terminus. X-ray absorption spectra suggested that the Zn(II) coordination depends on pH and on the presence of Ni(II). This study was performed to investigate the structural properties of HpHypA as a function of pH and Ni(II) binding, using NMR spectroscopy combined with DFT and molecular dynamics calculations. The solution structure of apo,Zn-HpHypA, containing Zn(II) but devoid of Ni(II), was determined using 2D, 3D and 4D NMR spectroscopy. The structure suggests that a Ni-binding and a Zn-binding domain, joined through a short linker, could undergo mutual reorientation. This flexibility has no physiological effect on acid viability or urease maturation in H. pylori. Atomistic molecular dynamics simulations suggest that Ni(II) binding is important for the conformational stability of the N-terminal helix. NMR chemical shift perturbation analysis indicates that no structural changes occur in the Zn-binding domain upon addition of Ni(II) in the pH 6.3-7.2 range. The structure of the Ni(II) binding site was probed using 1H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals around the paramagnetic metal ion. On this basis, two possible models were derived using quantum-mechanical DFT calculations. The results provide a comprehensive picture of the Ni(II) mode to HpHypA, important to rationalize, at the molecular level, the functional interactions of this chaperone with its protein partners.

Comparative analysis of the Hom family of outer membrane proteins in isolates from two geographically distinct regions: The United States and South Korea.

BACKGROUND: Helicobacter pylori encodes numerous outer membrane proteins (OMPs), but only a few have been characterized in depth. Deletion, duplication, and allelic variation of many of the H. pylori OMPs have been reported, which suggests that these proteins may play key roles in host adaptation. Herein, we characterize the variation observed within the Hom family of OMPs in H. pylori obtained from two geographically distinct populations. MATERIALS AND METHODS: PCR genotyping of the hom genes was carried out using clinical isolates from South Korea and the United States. A combination of statistical, phylogenetic, and protein modeling analyses was conducted to further characterize the hom variants. RESULTS: Variations in the closely related hom genes, homA and homB, occur in regions that are predicted to encode environmentally exposed loops. A similar phenomenon is true for homCS as compared to homCL . Conversely, little variation was observed in homD. Certain variants of the Hom family of proteins were more prominent in isolates from the Korean population as compared to isolates from the United States. CONCLUSION: En masse, our data show that the homA, homB, and homC profiles vary based upon the geographic origin of the strain; however, the fourth member of the hom family, homD, is more highly conserved. Additionally, protein topology modeling showed that many of the less well-conserved regions between homA and homB and between homCS and homCL corresponded to predicted environmentally exposed loops, suggesting that the divergence of the Hom family may be due to host adaptation/pressure.

Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori.

The human pathogen Helicobacter pylori requires nickel for colonization of the acidic environment of the stomach. HypA, a Ni metallochaperone that is typically associated with hydrogenase maturation, is also required for urease maturation and acid survival of H. pylori. There are two proposed Ni site structures for HypA; one is a paramagnetic six-coordinate site characterized by X-ray absorption spectroscopy (XAS) in unmodified HypA, while another is a diamagnetic four-coordinate planar site characterized by solution nuclear magnetic resonance in an N-terminally modified HypA construct. To determine the role of the N-terminal amine in Ni binding of HypA, an N-terminal extension variant, L2*-HypA, in which a leucine residue was inserted into the second position of the amino acid sequence in the proposed Ni-binding motif, was characterized in vitro and in vivo. Structural characterization of the Ni site using XAS showed a coordination change from six-coordinate in wild-type HypA (WT-HypA) to five-coordinate pyramidal in L2*-HypA, which was accompanied by the loss of two N/O donor protein ligands and the addition of an exogenous bromide ligand from the buffer. The magnetic properties of the Ni sites in WT-HypA compared to those of the Ni sites in L2*-HypA confirmed that a spin-state change from high to low spin accompanied this change in structure. The L2*-HypA H. pylori strain was shown to be acid sensitive and deficient in urease activity in vivo. In vitro characterization showed that L2*-HypA did not disrupt the HypA-UreE interaction that is essential for urease maturation but was at least 20-fold weaker in Ni binding than WT-HypA. Characterization of the L2*-HypA variant clearly demonstrates that the N-terminal amine of HypA is involved in proper Ni coordination and is necessary for urease activity and acid survival.

Characterization of Key Helicobacter pylori Regulators Identifies a Role for ArsRS in Biofilm Formation.

UNLABELLED: Helicobacter pylori must be able to rapidly respond to fluctuating conditions within the stomach. Despite this need for constant adaptation, H. pylori encodes few regulatory proteins. Of the identified regulators, the ferric uptake regulator (Fur), the nickel response regulator (NikR), and the two-component acid response system (ArsRS) are each paramount to the success of this pathogen. While numerous studies have individually examined these regulatory proteins, little is known about their combined effect. Therefore, we constructed a series of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS. A growth curve analysis revealed minor variation in growth kinetics across the strains; these were most pronounced in the triple mutant and in strains lacking ArsS. Visual analysis showed that strains lacking ArsS formed large aggregates and a biofilm-like matrix at the air-liquid interface. Biofilm quantification using crystal violet assays and visualization via scanning electron microscopy (SEM) showed that all strains lacking ArsS or containing a nonphosphorylatable form of ArsR (ArsR-D52N mutant) formed significantly more biofilm than the wild-type strain. Molecular characterization of biofilm formation showed that strains containing mutations in the ArsRS pathway displayed increased levels of cell aggregation and adherence, both of which are key to biofilm development. Furthermore, SEM analysis revealed prevalent coccoid cells and extracellular matrix formation in the ArsR-D52N, ΔnikR ΔarsS, and Δfur ΔnikR ΔarsS mutant strains, suggesting that these strains may have an exacerbated stress response that further contributes to biofilm formation. Thus, H. pylori ArsRS has a previously unrecognized role in biofilm formation. IMPORTANCE: Despite a paucity of regulatory proteins, adaptation is key to the survival of H. pylori within the stomach. While prior studies have focused on individual regulatory proteins, such as Fur, NikR, and ArsRS, few studies have examined the combined effect of these factors. Analysis of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS revealed a previously unrecognized role for the acid-responsive two-component system ArsRS in biofilm formation.

Molecular mechanisms of gastric cancer initiation and progression by Helicobacter pylori.

PURPOSE OF REVIEW: Infection with the Gram-negative, microaerophilic pathogen Helicobacter pylori results in gastric cancer in a subset of infected individuals. As such, H. pylori is the only WHO classified bacterial class I carcinogen. Numerous studies have identified mechanisms by which H. pylori alters host cell signaling pathways to cause disease. The purpose of this review is to highlight recent studies that explore mechanisms associated with induction of gastric cancer. RECENT FINDINGS: Over the last year and a half, new mechanisms contributing to the etiology of H. pylori-associated gastric cancer development have been discovered. In addition to utilizing the oncogenic CagA toxin to alter host cell signaling pathways, H. pylori also induces host DNA damage and alters DNA methylation to perturb downstream signaling. Furthermore, H. pylori activates numerous host cell pathways and proteins that result in epithelial-to-mesenchymal transition and induction of cell survival and proliferation. SUMMARY: Mounting evidence suggests that H. pylori promotes gastric carcinogenesis using a multifactorial approach. Intriguingly, many of the targeted pathways and mechanisms show commonality with diverse forms of cancer.

Heterologous Pseudomonas aeruginosa O-antigen delivery using a Salmonella enterica serovar Typhimurium wecA mutant strain.

There is a broad interest in adapting live vaccine strains (LVS) for use as recombinant vaccines that can deliver heterologous antigens. The Salmonella enterica serovar Typhimurium SL1344 ΔwecA LVS contains a mutation in wecA that abrogates production of Enterobacterial common antigen. This ΔwecA strain is attenuated in vivo, persistently colonizes the host, and protects against both wild type and cross-Salmonella serovar lethal challenge in a murine model of salmonellosis. Given these characteristics, we hypothesized that the SL1344 ΔwecA strain could be used as a carrier for heterologous antigen expression. To test this hypothesis, SL1344 ΔwecA was engineered to express the Pseudomonas aeruginosa O11 O-antigen gene cluster. Intraperitoneal (IP) but not oral immunization of BALB/c mice with the heterologous expression strain protected against lethal P. aeruginosa intranasal (IN) challenge. Furthermore, IP immunization resulted in P. aeruginosa O11-specific Ig and IgG antibody production. Functional analysis of sera collected from the IP immunized mice showed antibody-mediated agglutination and opsonophagocytic activity against P. aeruginosa. En masse, these results indicate that the S. Typhimurium SL1344 ΔwecA strain expressing the P. aeruginosa O11 O-antigen gene cluster is able to induce a humoral immune response and to protect against lethal P. aeruginosa challenge. As such, the S. Typhimurium SL1344 ΔwecA LVS can likely serve as a vehicle for expression of a wide variety of heterologous antigens as a means to create recombinant vaccines.

Copper Resistance of the Emerging Pathogen Acinetobacter baumannii.

Acinetobacter baumannii is an important emerging pathogen that is capable of causing many types of severe infection, especially in immunocompromised hosts. Since A. baumannii can rapidly acquire antibiotic resistance genes, many infections are on the verge of being untreatable, and novel therapies are desperately needed. To investigate the potential utility of copper-based antibacterial strategies against Acinetobacter infections, we characterized copper resistance in a panel of recent clinical A. baumannii isolates. Exposure to increasing concentrations of copper in liquid culture and on solid surfaces resulted in dose-dependent and strain-dependent effects; levels of copper resistance varied broadly across isolates, possibly resulting from identified genotypic variation among strains. Examination of the growth-phase-dependent effect of copper on A. baumannii revealed that resistance to copper increased dramatically in stationary phase. Moreover, A. baumannii biofilms were more resistant to copper than planktonic cells but were still susceptible to copper toxicity. Exposure of bacteria to subinhibitory concentrations of copper allowed them to better adapt to and grow in high concentrations of copper; this copper tolerance response is likely achieved via increased expression of copper resistance mechanisms. Indeed, genomic analysis revealed numerous putative copper resistance proteins that share amino acid homology to known proteins in Escherichia coli and Pseudomonas aeruginosa Transcriptional analysis revealed significant upregulation of these putative copper resistance genes following brief copper exposure. Future characterization of copper resistance mechanisms may aid in the search for novel antibiotics against Acinetobacter and other highly antibiotic-resistant pathogens. IMPORTANCE: Acinetobacter baumannii causes many types of severe nosocomial infections; unfortunately, some isolates have acquired resistance to almost every available antibiotic, and treatment options are incredibly limited. Copper is an essential nutrient but becomes toxic at high concentrations. The inherent antimicrobial properties of copper give it potential for use in novel therapeutics against drug-resistant pathogens. We show that A. baumannii clinical isolates are sensitive to copper in vitro, both in liquid and on solid metal surfaces. Since bacterial resistance to copper is mediated though mechanisms of efflux and detoxification, we identified genes encoding putative copper-related proteins in A. baumannii and showed that expression of some of these genes is regulated by the copper concentration. We propose that the antimicrobial effects of copper may be beneficial in the development of future therapeutics that target multidrug-resistant bacteria.

Dietary choice affects Shiga toxin-producing Escherichia coli (STEC) O157:H7 colonization and disease.

The likelihood that a single individual infected with the Shiga toxin (Stx)-producing, food-borne pathogen Escherichia coli O157:H7 will develop a life-threatening sequela called the hemolytic uremic syndrome is unpredictable. We reasoned that conditions that enhance Stx binding and uptake within the gut after E. coli O157:H7 infection should result in greater disease severity. Because the receptor for Stx, globotriaosylceramide, is up-regulated in the presence of butyrate in vitro, we asked whether a high fiber diet (HFD) that reportedly enhances butyrate production by normal gut flora can influence the outcome of an E. coli O157 infection in mice. To address that question, groups of BALB/c mice were fed high (10%) or low (2%) fiber diets and infected with E. coli O157:H7 strain 86-24 (Stx2+). Mice fed an HFD exhibited a 10- to 100-fold increase in colonization, lost 15% more body weight, exhibited signs of morbidity, and had 25% greater mortality relative to the low fiber diet (LFD)-fed group. Additionally, sections of intestinal tissue from HFD-fed mice bound more Stx1 and expressed more globotriaosylceramide than did such sections from LFD-fed mice. Furthermore, the gut microbiota of HFD-fed mice compared with LFD-fed mice contained reduced levels of native Escherichia species, organisms that might protect the gut from colonization by incoming E. coli O157:H7. Taken together, these results suggest that susceptibility to infection and subsequent disease after ingestion of E. coli O157:H7 may depend, at least in part, on individual diet and/or the capacity of the commensal flora to produce butyrate.

Correlation between nasal microbiome composition and remote purulent skin and soft tissue infections.

The incidence of skin and soft tissue infections (SSTIs) has increased dramatically over the past decade, resulting in significant morbidity in millions of otherwise healthy individuals worldwide. Certain groups, like military personnel, are at increased risk for SSTI development. Although nasal colonization with Staphylococcus aureus is an important risk factor for the development of SSTIs, it is not clear why some colonized individuals develop disease while others do not. Recent studies have revealed the importance of microbial diversity in human health. Therefore, we hypothesized that the nasal microbiome may provide valuable insight into SSTI development. To examine this hypothesis, we obtained anterior-naris samples from military trainees with cutaneous abscesses and from asymptomatic (non-SSTI) participants. We also obtained samples from within abscess cavities. Specimens were analyzed by culture, and the microbial community within each sample was characterized using a 16S sequencing-based approach. We collected specimens from 46 non-SSTI participants and from 40 participants with abscesses. We observed a significantly higher abundance of Proteobacteria in the anterior nares in non-SSTI participants (P < 0.0001) than in participants with abscesses. Additionally, we noted a significant inverse correlation between Corynebacterium spp. and S. aureus (P = 0.0001). The sensitivity of standard microbiological culture for abscesses was 71.4%. These data expand our knowledge of the complexity of the nasal and abscess microbiomes and potentially pave the way for novel therapeutic and prophylactic countermeasures against SSTI.

Recurrent Methicillin-Resistant Staphylococcus aureus Cutaneous Abscesses and Selection of Reduced Chlorhexidine Susceptibility during Chlorhexidine Use.

We describe the selection of reduced chlorhexidine susceptibility during chlorhexidine use in a patient with two episodes of cutaneous USA300 methicillin-resistant Staphylococcus aureus abscess. The second clinical isolate harbors a novel plasmid that encodes the QacA efflux pump. Greater use of chlorhexidine for disease prevention warrants surveillance for resistance.

An enterobacterial common antigen mutant of Salmonella enterica serovar Typhimurium as a vaccine candidate.

Due to increasing rates of invasive Salmonella enterica serovar Typhimurium infection, there is a need for an effective vaccine to prevent this disease. Previous studies showed that a mutation in the first gene of the Enterobacterial common antigen biosynthetic pathway, wecA, resulted in attenuation of S. Typhimurium in a murine model of salmonellosis. Furthermore, immunization with a wecA(-) strain protected against lethal challenge with the parental wild type S. Typhimurium strain. Herein, we examined whether the S. Typhimurium wecA(-) strain could also provide cross-protection against non-parental strains of S. Typhimurium and S. Enteritidis. We found that intraperitoneal immunization (IP) with S. Typhimurium SL1344 wecA(-) resulted in a significant increase in survival compared to control mice for all Salmonella challenge strains tested. Oral immunization with SL1344 wecA(-) also resulted in increased survival; however, protection was less significant than with intraperitoneal immunization. The increase in survival of SL1344 wecA(-) immunized mice was associated with a Salmonella-specific IgG antibody response. Furthermore, analysis of sera from IP and orally immunized animals revealed cross-reactive antibodies to numerous Salmonella isolates. Functional analysis of antibodies found within the sera from IP immunized animals revealed agglutination and opsonophagocytic activity against all tested O:4 Salmonella serovars. Together these results indicate that immunization with a S. Typhimurium wecA(-) strain confers protection against lethal challenge with wild type S. Typhimurium and S. Enteritidis and that immunization correlates with functional antibody production.

Analysis of a single Helicobacter pylori strain over a 10-year period in a primate model.

Helicobacter pylori from different individuals exhibits substantial genetic diversity. However, the kinetics of bacterial diversification after infection with a single strain is poorly understood. We investigated evolution of H. pylori following long-term infection in the primate stomach; Rhesus macaques were infected with H. pylori strain USU101 and then followed for 10 years. H. pylori was regularly cultured from biopsies, and single colony isolates were analyzed. At 1-year, DNA fingerprinting showed that all output isolates were identical to the input strain; however, at 5-years, different H. pylori fingerprints were observed. Microarray-based comparative genomic hybridization revealed that long term persistence of USU101 in the macaque stomach was associated with specific whole gene changes. Further detailed investigation showed that levels of the BabA protein were dramatically reduced within weeks of infection. The molecular mechanisms behind this reduction were shown to include phase variation and gene loss via intragenomic rearrangement, suggesting strong selective pressure against BabA expression in the macaque model. Notably, although there is apparently strong selective pressure against babA, babA is required for establishment of infection in this model as a strain in which babA was deleted was unable to colonize experimentally infected macaques.