Gene amplifications cause high-level resistance against albicidin in gram-negative bacteria.

Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. The peptide antibiotic albicidin is such a promising candidate that, as a gyrase poison, shows bactericidal activity against a wide range of gram-positive and gram-negative bacteria. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against albicidin. RNA sequencing and proteomics data show that this novel mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of STM3175 (YgiV), a transcription regulator with a GyrI-like small molecule binding domain that traps albicidin with high affinity. X-ray crystallography and molecular docking reveal a new conserved motif in the binding groove of the GyrI-like domain that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in gram-negative bacteria, and our experiments confirm that STM3175 homologs can confer resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.

Salmonella Central Carbon Metabolism Enhances Bactericidal Killing by Fluoroquinolone Antibiotics.

The efficacy of killing by bactericidal antibiotics has been reported to depend in large part on the ATP levels, with low levels of ATP leading to increased persistence after antibiotic challenge. Here, we show that an atp operon deletion strain of Salmonella enterica serovar Typhimurium lacking the ATP synthase was at least 10-fold more sensitive to killing by the fluoroquinolone antibiotic ciprofloxacin and yet showed either increased survival or no significant difference compared with the wild-type strain when challenged with aminoglycoside or ß-lactam antibiotics, respectively. The increased cell killing and reduced bacterial survival (persistence) after fluoroquinolone challenge were found to involve metabolic compensation for the loss of the ATP synthase through central carbon metabolism reactions and increased NAD(P)H levels. We conclude that the intracellular ATP levels per se do not correlate with bactericidal antibiotic persistence to fluoroquinolone killing; rather, the central carbon metabolic pathways active at the time of challenge and the intracellular target of the antibiotic determine the efficacy of treatment.

Identification of Natural Mutations Responsible for Altered Infection Phenotypes of Salmonella enterica Clinical Isolates by Using Cell Line Infection Screens.

The initial steps of Salmonella pathogenesis involve adhesion to and invasion into host epithelial cells. While well-studied for Salmonella enterica serovar Typhimurium, the factors contributing to this process in other, host-adapted serovars remains unexplored. Here, we screened clinical isolates of serovars Gallinarum, Dublin, Choleraesuis, Typhimurium, and Enteritidis for adhesion to and invasion into intestinal epithelial cell lines of human, porcine, and chicken origins. Thirty isolates with altered infectivity were used for genomic analyses, and 14 genes and novel mutations associated with high or low infectivity were identified. The functions of candidate genes included virulence gene expression regulation and cell wall or membrane synthesis and components. The role of several of these genes in Salmonella adhesion to and invasion into cells has not previously been investigated. The genes dksA (encoding a stringent response regulator) and sanA (encoding a vancomycin high-temperature exclusion protein) were selected for further analyses, and we confirmed their roles in adhesion to and invasion into host cells. Furthermore, transcriptomic analyses were performed for S Enteritidis and S Typhimurium, with two highly infective and two marginally infective isolates for each serovar. Expression profiles for the isolates with altered infection phenotypes revealed the importance of type 3 secretion system expression levels in the determination of an isolate's infection phenotype. Taken together, these data indicate a new role in cell host infection for genes or gene variants previously not associated with adhesion to and invasion into the epithelial cells.IMPORTANCESalmonella is a foodborne pathogen affecting over 200 million people and resulting in over 200,000 fatal cases per year. Its adhesion to and invasion into intestinal epithelial cells represent one of the first and key steps in the pathogenesis of salmonellosis. Still, around 35 to 40% of bacterial genes have no experimentally validated function, and their contribution to bacterial virulence, including adhesion and invasion, remains largely unknown. Therefore, the significance of this study is in the identification of new genes or gene allelic variants previously not associated with adhesion and invasion. It is well established that blocking adhesion and/or invasion would stop or hamper bacterial infection; therefore, the new findings from this study could be used in future developments of anti-Salmonella therapy targeting genes involved in these key processes. Such treatment could be a valuable alternative, as the prevalence of antibiotic-resistant bacteria is increasing very rapidly.

Lectin-Mediated Bacterial Modulation by the Intestinal Nematode Ascaris suum.

Ascariasis is a global health problem for humans and animals. Adult Ascaris nematodes are long-lived in the host intestine where they interact with host cells as well as members of the microbiota resulting in chronic infections. Nematode interactions with host cells and the microbial environment are prominently mediated by parasite-secreted proteins and peptides possessing immunomodulatory and antimicrobial activities. Previously, we discovered the C-type lectin protein AsCTL-42 in the secreted products of adult Ascaris worms. Here we tested recombinant AsCTL-42 for its ability to interact with bacterial and host cells. We found that AsCTL-42 lacks bactericidal activity but neutralized bacterial cells without killing them. Treatment of bacterial cells with AsCTL-42 reduced invasion of intestinal epithelial cells by Salmonella. Furthermore, AsCTL-42 interacted with host myeloid C-type lectin receptors. Thus, AsCTL-42 is a parasite protein involved in the triad relationship between Ascaris, host cells, and the microbiota.

Novel Avian Pathogenic Escherichia coli Genes Responsible for Adhesion to Chicken and Human Cell Lines.

Avian pathogenic Escherichia coli (APEC) is a major bacterial pathogen of commercial poultry contributing to extensive economic losses and contamination of the food chain. One of the initial steps in bacterial infection and successful colonization of the host is adhesion to the host cells. A random transposon mutant library (n = 1,300) of APEC IMT 5155 was screened phenotypically for adhesion to chicken (CHIC-8E11) and human (LoVo) intestinal epithelial cell lines. The detection and quantification of adherent bacteria were performed by a modified APEC-specific antibody staining assay using fluorescence microscopy coupled to automated VideoScan technology. Eleven mutants were found to have significantly altered adhesion to the cell lines examined. Mutated genes in these 11 "adhesion-altered mutants" were identified by arbitrary PCR and DNA sequencing. The genes were amplified from wild-type APEC IMT 5155, cloned, and transformed into the respective adhesion-altered mutants, and complementation was determined in adhesion assays. Here, we report contributions of the fdtA, rluD, yjhB, ecpR, and fdeC genes of APEC in adhesion to chicken and human intestinal cell lines. Identification of the roles of these genes in APEC pathogenesis will contribute to prevention and control of APEC infections.IMPORTANCE Avian pathogenic E. coli is not only pathogenic for commercial poultry but can also cause foodborne infections in humans utilizing the same attachment and virulence mechanisms. Our aim was to identify genes of avian pathogenic E. coli involved in adhesion to chicken and human cells in order to understand the colonization and pathogenesis of these bacteria. In contrast to the recent studies based on genotypic and bioinformatics data, we have used a combination of phenotypic and genotypic approaches for identification of novel genes contributing to adhesion in chicken and human cell lines. Identification of adhesion factors remains important, as antibodies elicited against such factors have shown potential to block colonization and ultimately prevent disease as prophylactic vaccines. Therefore, the data will augment the understanding of disease pathogenesis and ultimately in designing strategies against the infections.

Salmonella Co-opts Host Cell Chaperone-mediated Autophagy for Intracellular Growth.

Salmonella enterica are invasive intracellular pathogens that replicate within a membrane-bound compartment inside infected host cells known as the Salmonella-containing vacuole. How Salmonella obtains nutrients for growth within this intracellular niche despite the apparent isolation is currently not known. Recent studies have indicated the importance of glucose and related carbon sources for tissue colonization and intracellular proliferation within host cells during Salmonella infections, although none have been found to be essential. We found that wild-type Salmonella are capable of replicating within infected host cells in the absence of both exogenous sugars and/or amino acids. Furthermore, mutants defective in glucose uptake or dependent upon peptides for growth also showed no significant loss in intracellular replication, suggesting host-derived peptides can supply both carbon units and amino acids. Here, we show that intracellular Salmonella recruit the host proteins LAMP-2A and Hsc73, key components of the host protein turnover pathway known as chaperone-mediated autophagy involved in transport of cytosolic proteins to the lysosome for degradation. Host-derived peptides are shown to provide a significant contribution toward the intracellular growth of Salmonella The results reveal a means whereby intracellular Salmonella gain access to the host cell cytosol from within its membrane-bound compartment to acquire nutrients. Furthermore, this study provides an explanation as to how Salmonella evades activation of autophagy mechanisms as part of the innate immune response.

The Inflammatory Response to Enterotoxigenic E. coli and Probiotic E. faecium in a Coculture Model of Porcine Intestinal Epithelial and Dendritic Cells.

The gut epithelium constitutes an interface between the intestinal contents and the underlying gut-associated lymphoid tissue (GALT) including dendritic cells (DC). Interactions of intestinal epithelial cells (IEC) and resident DC are characterized by bidirectional crosstalk mediated by various factors, such as transforming growth factor-ß (TGF-ß) and thymic stromal lymphopoietin (TSLP). In the present study, we aimed (1) to model the interplay of both cell types in a porcine in vitro coculture consisting of IEC (cell line IPEC-J2) and monocyte-derived DC (MoDC) and (2) to assess whether immune responses to bacteria are altered because of the interplay between IPEC-J2 cells and MoDC. With regard to the latter, we focused on the inflammasome pathway. Here, we propose caspase-13 as a promising candidate for the noncanonical inflammasome activation in pigs. We conducted challenge experiments with enterotoxigenic Escherichia coli (ETEC) and probiotic Enterococcus faecium (E. faecium) NCIMB 10415. As potential mediators of IEC/DC interactions, TGF-ß and TSLP were selected for analyses. Cocultured MoDC showed attenuated ETEC-induced inflammasome-related and proinflammatory interleukin (IL)-8 reactions compared with MoDC monocultures. Caspase-13 was more strongly expressed in IPEC-J2 cells cocultured with MoDC and upon ETEC incubation. We found that IPEC-J2 cells and MoDC were capable of releasing TSLP. The latter cells secreted greater amounts of TSLP when cocultured with IPEC-J2 cells. TGF-ß was not modulated under the present experimental conditions in either cell types. We conclude that, in the presence of IPEC-J2 cells, porcine MoDC exhibited a more tolerogenic phenotype, which might be partially regulated by autocrine TSLP production. Noncanonical inflammasome signaling appeared to be modulated in IPEC-J2 cells. Our results indicate that the reciprocal interplay of the intestinal epithelium and GALT is essential for promoting balanced immune responses.

Characterization of Inflammasome Components in Pig Intestine and Analysis of the Influence of Probiotic Enterococcus Faecium during an Escherichia Coli Challenge.

The aim of the present study was to investigate systematically the expression of inflammasome components in pig intestine and to analyze the influence of age and long-term supplementation with the probiotic Enterococcus faecium NCIMB 10415 (E. faecium). In order to examine probiotic effects on the inflammasomes during a challenge with pathogens, enterotoxigenic Escherichia coli (ETEC) and E. faecium were directly added to pig jejunum in Ussing chambers. The mRNA expression of inflammasome components generally decreased in an oral-aboral direction in intestinal tissues. In 29-day-old piglets, the expression levels of NLRP3 were significantly higher and ASC (apoptotic speck-like protein containing a caspase recruitment domain) expression were lower compared with those in the ileum of 70-day-old pigs (p ≤ 0.05). Long-term supplementation with E. faecium significantly increased ASC expression levels in the jejunum and ileum of 29-day-old piglets compared to control animals (p ≤ 0.05). Ex vivo addition of ETEC or E. faecium did not affect mRNA expression of inflammasome components significantly, whereas IL-1ß protein release was significantly elevated in ETEC-incubated jejunum (p ≤ 0.05), providing evidence for the functional activation of the inflammasome, which was prevented by pre-incubation with E. faecium. We conclude that pre-incubation with E. faecium has a protective effect during ETEC challenge; this effect is probably not located at the inflammasome transcription level. The results of this study of the expression and regulation of inflammasome components in pigs are similar to those obtained in humans, reinforcing the use of pigs as a suitable model for translational inflammasome research.

Altered Cytokine Expression and Barrier Properties after In Vitro Infection of Porcine Epithelial Cells with Enterotoxigenic Escherichia coli and Probiotic Enterococcus faecium.

The aim of the present study was to elucidate the effects of the probiotic feed additive Enterococcus faecium NCIMB 10415 (E. faecium) on porcine jejunal epithelial cells (IPEC-J2) during an in vitro challenge with enterotoxigenic Escherichia coli (ETEC). Cells were incubated with E. faecium, ETEC, or both, and the effects on barrier function and structure and intra- and intercellular signaling were determined. Coincubation with E. faecium abolished the ETEC-induced decrease in transepithelial resistance (Rt) (p ≤ 0.05). No differences were seen in the expression levels of the intercellular connecting tight junction proteins examined. However, for the first time, a reorganization of the monolayer was observed in ETEC-infected cells but not in coincubated cells. ETEC induced an increase in cytotoxicity that was prevented by coincubation (p ≤ 0.05), whereas apoptosis rates were not affected by bacterial treatment. ETEC increased the mRNA expression and release of proinflammatory cytokines TNF-α, IL-1α, and IL-6 which could be prevented by coincubation for TNF-α mRNA expression and IL-6 protein (p ≤ 0.05). Likewise, cAMP concentrations elevated by ETEC were reduced in coincubated cells (p ≤ 0.05). These findings indicate a protective effect of the probiotic E. faecium on inflammatory responses during infection with ETEC.

Probiotic Escherichia coli Nissle 1917 reduces growth, Shiga toxin expression, release and thus cytotoxicity of enterohemorrhagic Escherichia coli.

Due to increased release or production of Shiga toxin by Enterohemorrhagic Escherichia coli (EHEC) after exposure to antimicrobial agents, the role of antimicrobial agents in EHEC mediated infections remains controversial. Probiotics are therefore rapidly gaining interest as an alternate therapeutic option. The well-known probiotic strain Escherichia coli Nissle 1917 (EcN) was tested in vitro to determine its probiotic effects on growth, Shiga toxin (Stx) gene expression, Stx amount and associated cytotoxicity on the most important EHEC strains of serotype O104:H4 and O157:H7. Following co-culture of EcN:EHEC in broth for 4 and 24 h, the probiotic effects on EHEC growth, toxin gene expression, Stx amount and cytotoxicity were determined using quantitative real time-PCR, Stx-ELISA and Vero cytotoxicity assays. Probiotic EcN strongly reduced EHEC numbers (cfu) of O104:H4 up to (68%) and O157:H7 to (72.2%) (p<0.05) in LB broth medium whereas the non-probiotic E. coli strain MG1655 had no effect on EHEC growth. The level of stx expression was significantly down-regulated, particularly for the stx2a gene. The stx down-regulation in EcN co-culture was not due to reduced numbers of EHEC. A significant inhibition in Stx amounts and cytotoxicity were also observed in sterile supernatants of EcN:EHEC co-cultures. These findings indicate that probiotic EcN displays strong inhibitory effects on growth, Shiga toxin gene expression, amount and cytotoxicity of EHEC strains. Thus, EcN may be considered as a putative therapeutic candidate, in particular against EHEC O104:H4 and O157:H7.

Enterococcus faecium NCIMB 10415 modulates epithelial integrity, heat shock protein, and proinflammatory cytokine response in intestinal cells.

Probiotics have shown positive effects on gastrointestinal diseases; they have barrier-modulating effects and change the inflammatory response towards pathogens in studies in vitro. The aim of this investigation has been to examine the response of intestinal epithelial cells to Enterococcus faecium NCIMB 10415 (E. faecium), a probiotic positively affecting diarrhea incidence in piglets, and two pathogenic Escherichia coli (E. coli) strains, with specific focus on the probiotic modulation of the response to the pathogenic challenge. Porcine (IPEC-J2) and human (Caco-2) intestinal cells were incubated without bacteria (control), with E. faecium, with enteropathogenic (EPEC) or enterotoxigenic E. coli (ETEC) each alone or in combination with E. faecium. The ETEC strain decreased transepithelial resistance (TER) and increased IL-8 mRNA and protein expression in both cell lines compared with control cells, an effect that could be prevented by pre- and coincubation with E. faecium. Similar effects were observed for the increased expression of heat shock protein 70 in Caco-2 cells. When the cells were challenged by the EPEC strain, no such pattern of changes could be observed. The reduced decrease in TER and the reduction of the proinflammatory and stress response of enterocytes following pathogenic challenge indicate the protective effect of the probiotic.

Role of F1C fimbriae, flagella, and secreted bacterial components in the inhibitory effect of probiotic Escherichia coli Nissle 1917 on atypical enteropathogenic E. coli infection.

Enteropathogenic Escherichia coli (EPEC) is recognized as an important intestinal pathogen that frequently causes acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probiotic E. coli strain Nissle 1917 (EcN) is known to be effective in the treatment of several gastrointestinal disorders. While both in vitro and in vivo studies have described strong inhibitory effects of EcN on enteropathogenic bacteria, including pathogenic E. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenic E. coli (aEPEC) with respect to single infection steps, including adhesion, microcolony formation, and the attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagella, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific to EcN, but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of the inhibitory factors. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence-associated proteins of EPEC, but not their expression.

Concurrent Ascaris infection modulates host immunity resulting in impaired control of Salmonella infection in pigs.

Ascaris is one of the most widespread helminth infections, leading to chronic morbidity in humans and considerable economic losses in pig farming. In addition, pigs are an important reservoir for the zoonotic salmonellosis, where pigs can serve as asymptomatic carriers. Here, we investigated the impact of an ongoing Ascaris infection on the immune response to Salmonella in pigs. We observed higher bacterial burdens in experimentally coinfected pigs compared to pigs infected with Salmonella alone. The impaired control of Salmonella in the coinfected pigs was associated with repressed interferon gamma responses in the small intestine and with the alternative activation of gut macrophages evident in elevated CD206 expression. Ascaris single and coinfection were associated with a rise of CD4-CD8α+FoxP3+ Treg in the lymph nodes draining the small intestine and liver. In addition, macrophages from coinfected pigs showed enhanced susceptibility to Salmonella infection in vitro and the Salmonella-induced monocytosis and tumor necrosis factor alpha production by myeloid cells was repressed in pigs coinfected with Ascaris. Hence, our data indicate that acute Ascaris infection modulates different immune effector functions with important consequences for the control of tissue-invasive coinfecting pathogens.IMPORTANCEIn experimentally infected pigs, we show that an ongoing infection with the parasitic worm Ascaris suum modulates host immunity, and coinfected pigs have higher Salmonella burdens compared to pigs infected with Salmonella alone. Both infections are widespread in pig production and the prevalence of Salmonella is high in endemic regions of human Ascariasis, indicating that this is a clinically meaningful coinfection. We observed the type 2/regulatory immune response to be induced during an Ascaris infection correlates with increased susceptibility of pigs to the concurrent bacterial infection.

The broader context of antibiotic resistance: zinc feed supplementation of piglets increases the proportion of multi-resistant Escherichia coli in vivo.

Following the Europe-wide ban of antimicrobial growth promoters, feed supplementation with zinc has increased in livestock breeding. In addition to possible beneficial effects on animal health, feed supplementation with heavy metals is known to influence the gut microbiota and might promote the spread of antimicrobial resistance via co-selection or other mechanisms. As Escherichia coli is among the most important pathogens in pig production and often displays multi-resistant phenotypes, we set out to investigate the influence of zinc feed additives on the composition of the E. coli populations in vivo focusing on phylogenetic diversity and antimicrobial resistance. In a piglet feeding trial, E. coli were isolated from ileum and colon digesta of high dose zinc-supplemented (2500ppm) and background dose (50ppm) piglets (control group). The E. coli population was characterized via pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) for the determination of the phylogenetic background. Phenotypic resistance screening via agar disk diffusion and minimum inhibitory concentration testing was followed by detection of resistance genes for selected clones. We observed a higher diversity of E. coli clones in animals supplemented with zinc compared to the background control group. The proportion of multi-resistant E. coli was significantly increased in the zinc group compared to the control group (18.6% vs. 0%). For several subclones present both in the feeding and the control group we detected up to three additional phenotypic and genotypic resistances in the subclones from the zinc feeding group. Characterization of these subclones suggests an increase in antimicrobial resistance due to influences on plasmid uptake by zinc supplementation, questioning the reasonability of zinc feed additives as a result of the ban of antimicrobial growth promoters.

Feeding the probiotic Enterococcus faecium strain NCIMB 10415 to piglets specifically reduces the number of Escherichia coli pathotypes that adhere to the gut mucosa.

Feed supplementation with the probiotic Enterococcus faecium for piglets has been found to reduce pathogenic gut microorganisms. Since Escherichia coli is among the most important pathogens in pig production, we performed comprehensive analyses to gain further insight into the influence of E. faecium NCIMB 10415 on porcine intestinal E. coli. A total of 1,436 E. coli strains were isolated from three intestinal habitats (mucosa, digesta, and feces) of probiotic-supplemented and nonsupplemented (control) piglets. E. coli bacteria were characterized via pulsed-field gel electrophoresis (PFGE) for clonal analysis. The high diversity of E. coli was reflected by 168 clones. Multilocus sequence typing (MLST) was used to determine the phylogenetic backgrounds, revealing 79 sequence types (STs). Pathotypes of E. coli were further defined using multiplex PCR for virulence-associated genes. While these analyses discerned only a few significant differences in the E. coli population between the feeding groups, analyses distinguishing clones that were uniquely isolated in either the probiotic group only, the control group only, or both groups (shared group) revealed clear effects at the habitat level. Interestingly, extraintestinal pathogenic E. coli (ExPEC)-typical clones adhering to the mucosa were significantly reduced in the probiotic group. Our data show a minor influence of E. faecium on the overall population of E. coli in healthy piglets. In contrast, this probiotic has a profound effect on mucosa-adherent E. coli. This finding further substantiates a specific effect of E. faecium strain NCIMB 10415 in piglets against pathogenic E. coli in the intestine. In addition, these data question the relevance of data based on sampling fecal E. coli only.

Prophage Gifsy-1 Induction in Salmonella enterica Serovar Typhimurium Reduces Persister Cell Formation after Ciprofloxacin Exposure.

Persister cells are drug-tolerant bacteria capable of surviving antibiotic treatment despite the absence of heritable resistance mechanisms. It is generally thought that persister cells survive antibiotic exposure through the implementation of stress responses and/or energy-sparing strategies. Exposure to DNA gyrase-targeting antibiotics could be particularly detrimental for bacteria that carry prophages integrated in their genomes. Gyrase inhibitors are known to induce prophages to switch from their dormant lysogenic state into the lytic cycle, causing the lysis of their bacterial host. However, the influence of resident prophages on the formation of persister cells has only been recently appreciated. Here, we evaluated the effect of endogenous prophage carriage on the generation of bacterial persistence during Salmonella enterica serovar Typhimurium exposure to both gyrase-targeting antibiotics and other classes of bactericidal antibiotics. Results from the analysis of strain variants harboring different prophage combinations revealed that prophages play a major role in limiting the formation of persister cells during exposure to DNA-damaging antibiotics. In particular, we present evidence that prophage Gifsy-1 (and its encoded lysis proteins) are major factors limiting persister cell formation upon ciprofloxacin exposure. Resident prophages also appear to have a significant impact on the initial drug susceptibility, resulting in an alteration of the characteristic biphasic killing curve of persister cells into a triphasic curve. In contrast, a prophage-free derivative of S. Typhimurium showed no difference in the killing kinetics for ß-lactam or aminoglycoside antibiotics. Our study demonstrates that induction of prophages increased the susceptibility toward DNA gyrase inhibitors in S. Typhimurium, suggesting that prophages have the potential for enhancing antibiotic efficacy. IMPORTANCE Bacterial infections resulting from antibiotic treatment failure can often be traced to nonresistant persister cells. Moreover, intermittent or single treatment of persister cells with ß-lactam antibiotics or fluoroquinolones can lead to the formation of drug-resistant bacteria and to the emergence of multiresistant strains. It is therefore important to have a better understanding of the mechanisms that impact persister formation. Our results indicate that prophage-associated bacterial killing significantly reduces persister cell formation in lysogenic cells exposed to DNA-gyrase-targeting drugs. This suggests that therapies based on gyrase inhibitors should be favored over alternative strategies when dealing with lysogenic pathogens.

A virulence factor as a therapeutic: the probiotic Enterococcus faecium SF68 arginine deiminase inhibits innate immune signaling pathways.

The probiotic bacterial strain Enterococcus faecium SF68 has been shown to alleviate symptoms of intestinal inflammation in human clinical trials and animal feed supplementation studies. To identify factors involved in immunomodulatory effects on host cells, E. faecium SF68 and other commensal and clinical Enterococcus isolates were screened using intestinal epithelial cell lines harboring reporter fusions for NF-κB and JNK(AP-1) activation to determine the responses of host cell innate immune signaling pathways when challenged with bacterial protein and cell components. Cell-free, whole-cell lysates of E. faecium SF68 showed a reversible, inhibitory effect on both NF-κB and JNK(AP-1) signaling pathway activation in intestinal epithelial cells and abrogated the response to bacterial and other Toll-like receptor (TLR) ligands. The inhibitory effect was species-specific, and was not observed for E. avium, E. gallinarum, or E. casseliflavus. Screening of protein fractions of E. faecium SF68 lysates yielded an active fraction containing a prominent protein identified as arginine deiminase (ADI). The E. faecium SF68 arcA gene encoding arginine deiminase was cloned and introduced into E. avium where it conferred the same NF-κB inhibitory effects on intestinal epithelial cells as seen for E. faecium SF68. Our results indicate that the arginine deiminase of E. faecium SF68 is responsible for inhibition of host cell NF-κB and JNK(AP-1) pathway activation, and is likely to be responsible for the anti-inflammatory and immunomodulatory effects observed in prior clinical human and animal trials. The implications for the use of this probiotic strain for preventive and therapeutic purposes are discussed.

Metabolic Characteristics of Porcine LA-MRSA CC398 and CC9 Isolates from Germany and China via Biolog Phenotype MicroArrayTM.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an important zoonotic pathogen, often multi-resistant to antimicrobial agents. Among swine, LA-MRSA of clonal complex (CC) 398 dominates in Europe, Australia and the Americas, while LA-MRSA-CC9 is the main epidemic lineage in Asia. Here, we comparatively investigated the metabolic properties of rare and widespread porcine LA-MRSA isolates from Germany and China using Biolog Phenotype MicroArray technology to evaluate if metabolic variations could have played a role in the development of two different epidemic LA-MRSA clones in swine. Overall, we were able to characterize the isolates' metabolic profiles and show their tolerance to varying environmental conditions. Sparse partial least squares discriminant analysis (sPLS-DA) supported the detection of the most informative substrates and/or conditions that revealed metabolic differences between the LA-MRSA lineages. The Chinese LA-MRSA-CC9 isolates displayed unique characteristics, such as a consistently delayed onset of cellular respiration, and increased, reduced or absent usage of several nutrients. These possibly unfavorable metabolic properties might promote the ongoing gradual replacement of the current epidemic LA-MRSA-CC9 clone in China with the emerging LA-MRSA-CC398 lineage through livestock trade and occupational exposure. Due to the enhanced pathogenicity of the LA-MRSA-CC398 clone, the public health risk posed by LA-MRSA from swine might increase further.

Closely related Campylobacter jejuni strains from different sources reveal a generalist rather than a specialist lifestyle.

BACKGROUND: Campylobacter jejuni and Campylobacter coli are human intestinal pathogens of global importance. Zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. However, little is known about their general and host-specific mechanisms of colonization, or virulence and pathogenicity factors. In certain hosts, Campylobacter species colonize persistently and do not cause disease, while they cause acute intestinal disease in humans. RESULTS: Here, we investigate putative host-specificity using phenotypic characterization and genome-wide analysis of genetically closely related C. jejuni strains from different sources. A collection of 473 fresh Campylobacter isolates from Germany was assembled between 2006 and 2010 and characterized using MLST. A subset of closely related C. jejuni strains of the highly prevalent sequence type ST-21 was selected from different hosts and isolation sources. PCR typing of strain-variable genes provided evidence that some genes differed between these strains. Furthermore, phenotypic variation of these strains was tested using the following criteria: metabolic variation, protein expression patterns, and eukaryotic cell interaction. The results demonstrated remarkable phenotypic diversity within the ST-21 group, which however did not correlate with isolation source. Whole genome sequencing was performed for five ST-21 strains from chicken, human, bovine, and food sources, in order to gain insight into ST-21 genome diversity. The comparisons showed extensive genomic diversity, primarily due to recombination and gain of phage-related genes. By contrast, no genomic features associated with isolation source or host were identified. CONCLUSIONS: The genome information and phenotypic data obtained in vitro and in a chicken infection model provided little evidence of fixed adaptation to a specific host. Instead, the dominant C. jejuni ST-21 appeared to be characterized by phenotypic flexibility and high genetic microdiversity, revealing properties of a generalist. High genetic flexibility might allow generalist variants of C. jejuni to reversibly express diverse fitness factors in changing environments.