Identification and Analysis of Antimicrobial Activities from a Model Moss Ceratodon purpureus.

The emergence of bacterial drug resistance is often viewed as the next great health crisis of our time. While more antimicrobial agents are urgently needed, very few new antibiotics are currently in the production pipeline. Here, we aim to identify and characterize novel antimicrobial natural products from a model dioicous moss, Ceratodon purpureus. We collected secreted moss exudate fractions from two C. purpureus strains, male R40 and female GG1. Exudates from the female C. purpureus strain GG1 did not exhibit inhibitory activity against any tested bacteria. However, exudates from the male moss strain R40 exhibited strong inhibitory properties against several species of Gram-positive bacteria, including Staphylococcus aureus and Enterococcus faecium, though they did not inhibit the growth of Gram-negative bacteria. Antibacterial activity levels in C. purpureus R40 exudates significantly increased over four weeks of moss cultivation in liquid culture. Size fractionation experiments indicated that the secreted bioactive compounds have a relatively low molecular weight of less than 1 kDa. Additionally, the R40 exudate compounds are thermostable and not sensitive to proteinase K treatment. Overall, our results suggest that the bioactive compounds present in C. purpureus R40 exudates can potentially add new options for treating infections caused by antibiotic-resistant Gram-positive bacteria.

Taxonomic and Metagenomic Analyses Define the Development of the Microbiota in the Chick.

Chicks are ideal to follow the development of the intestinal microbiota and to understand how a pathogen perturbs this developing population. Taxonomic/metagenomic analyses captured the development of the chick microbiota in unperturbed chicks and in chicks infected with Salmonella enterica serotype Typhimurium (STm) during development. Taxonomic analysis suggests that colonization by the chicken microbiota takes place in several waves. The cecal microbiota stabilizes at day 12 posthatch with prominent Gammaproteobacteria and Clostridiales. Introduction of S. Typhimurium at day 4 posthatch disrupted the expected waves of intestinal colonization. Taxonomic and metagenomic shotgun sequencing analyses allowed us to identify species present in uninfected chicks. Untargeted metabolomics suggested different metabolic activities in infected chick microbiota. This analysis and gas chromatography-mass spectrometry on ingesta confirmed that lactic acid in cecal content coincides with the stable presence of enterococci in STm-infected chicks. Unique metabolites, including 2-isopropylmalic acid, an intermediate in the biosynthesis of leucine, were present only in the cecal content of STm-infected chicks. The metagenomic data suggested that the microbiota in STm-infected chicks contained a higher abundance of genes, from STm itself, involved in branched-chain amino acid synthesis. We generated an ilvC deletion mutant (STM3909) encoding ketol-acid-reductoisomerase, a gene required for the production of l-isoleucine and l-valine. ΔilvC mutants are disadvantaged for growth during competitive infection with the wild type. Providing the ilvC gene in trans restored the growth of the ΔilvC mutant. Our integrative approach identified biochemical pathways used by STm to establish a colonization niche in the chick intestine during development. IMPORTANCE Chicks are an ideal model to follow the development of the intestinal microbiota and to understand how a pathogen perturbs this developing population. Using taxonomic and metagenomic analyses, we captured the development of chick microbiota to 19 days posthatch in unperturbed chicks and in chicks infected with Salmonella enterica serotype Typhimurium (STm). We show that normal development of the microbiota takes place in waves and is altered in the presence of a pathogen. Metagenomics and metabolomics suggested that branched-chain amino acid biosynthesis is especially important for Salmonella growth in the infected chick intestine. Salmonella mutants unable to make l-isoleucine and l-valine colonize the chick intestine poorly. Restoration of the pathway for biosynthesis of these amino acids restored the colonizing ability of Salmonella. Integration of multiple analyses allowed us to correctly identify biochemical pathways used by Salmonella to establish a niche for colonization in the chick intestine during development.

Serratia marcescens DUF1471-Containing Protein SrfN Is Needed for Adaptation to Acid and Oxidative Stresses.

Bacteria can quickly adapt to constantly changing environments through a number of mechanisms, including secretion of secondary metabolites, peptides, and proteins. Serratia marcescens, an emerging pathogen with growing clinical importance due to its intrinsic resistance to several classes of antibiotics, can cause an array of infections in immunocompromised individuals. To better control the spread of S. marcescens infections, it is critical to identify additional targets for bacterial growth inhibition. We found that extracellular metabolites produced by the wild-type organism in response to peroxide exposure had a protective effect on an otherwise-H2O2-sensitive ΔmacAB indicator strain. Detailed analysis of the conditioned medium demonstrated that the protective effect was associated with a low-molecular-weight heat-sensitive and proteinase K-sensitive metabolite. Furthermore, liquid chromatography-tandem mass spectrometry analysis of the low-molecular-weight proteins present in the conditioned medium led to identification of the previously uncharacterized DUF1471-containing protein TBU67220 (SrfN). We found that loss of the srfN gene did not have an impact on the production of extracellular enzymes. However, the S. marcescens mutant lacking SrfN was significantly more sensitive to growth in medium with a low pH and to exposure to oxidative stress. Both defects were fully rescued by complementation. Thus, our results indicate that SrfN, a low-molecular-weight DUF1471-containing protein, is involved in S. marcescens SM6 adaptation to adverse environmental conditions. IMPORTANCE Serratia marcescens is ubiquitous in the environment and can survive in water, soil, plants, insects, and animals, and it can also cause infections in humans. In the face of disturbances such as oxidative or low-pH stress, bacteria adapt, survive, and recover through several mechanisms, including changes in their secretome. We show that a hydrogen peroxide-exposed S. marcescens milieu contains a small previously uncharacterized DUF1471-containing protein similar to the SrfN protein in Salmonella enterica serovar Typhimurium, and we illustrate the role of this protein in bacterial survival during acid and oxidative stresses.

Antimicrobial Activities of Secondary Metabolites from Model Mosses.

Plants synthetize a large spectrum of secondary metabolites with substantial structural and functional diversity, making them a rich reservoir of new biologically active compounds. Among different plant lineages, the evolutionarily ancient branch of non-vascular plants (Bryophytes) is of particular interest as these organisms produce many unique biologically active compounds with highly promising antibacterial properties. Here, we characterized antibacterial activity of metabolites produced by different ecotypes (strains) of the model mosses Physcomitrium patens and Sphagnum fallax. Ethanol and hexane moss extracts harbor moderate but unstable antibacterial activity, representing polar and non-polar intracellular moss metabolites, respectively. In contrast, high antibacterial activity that was relatively stable was detected in soluble exudate fractions of P. patens moss. Antibacterial activity levels in P. patens exudates significantly increased over four weeks of moss cultivation in liquid culture. Interestingly, secreted moss metabolites are only active against a number of Gram-positive, but not Gram-negative, bacteria. Size fractionation, thermostability and sensitivity to proteinase K assays indicated that the secreted bioactive compounds are relatively small (less than <10 kDa). Further analysis and molecular identification of antibacterial exudate components, combined with bioinformatic analysis of model moss genomes, will be instrumental in the identification of specific genes involved in the bioactive metabolite biosynthesis.

The ABC-Type Efflux Pump MacAB Is Involved in Protection of Serratia marcescens against Aminoglycoside Antibiotics, Polymyxins, and Oxidative Stress.

Serratia marcescens is an emerging pathogen with increasing clinical importance due to its intrinsic resistance to several classes of antibiotics. The chromosomally encoded drug efflux pumps contribute to antibiotic resistance and represent a major challenge for the treatment of bacterial infections. The ABC-type efflux pump MacAB was previously linked to macrolide resistance in Escherichia coli and Salmonella enterica serovar Typhimurium. The role of the MacAB homolog in antibiotic resistance of S. marcescens is currently unknown. We found that an S. marcescens mutant lacking the MacAB pump did not show increased sensitivity to the macrolide antibiotic erythromycin but was significantly more sensitive to aminoglycoside antibiotics and polymyxins. We also showed that, in addition to its role in drug efflux, the MacAB efflux pump is required for swimming motility and biofilm formation. We propose that the motility defect of the ΔmacAB mutant is due, at least in part, to the loss of functional flagella on the bacterial surface. Furthermore, we found that the promoter of the MacAB efflux pump was active during the initial hours of growth in laboratory medium and that its activity was further elevated in the presence of hydrogen peroxide. Finally, we demonstrate a complete loss of ΔmacAB mutant viability in the presence of peroxide, which is fully restored by complementation. Thus, the S. marcescens MacAB efflux pump is essential for survival during oxidative stress and is involved in protection from polymyxins and aminoglycoside antibiotics.IMPORTANCE The opportunistic pathogen Serratia marcescens can cause urinary tract infections, respiratory infections, meningitis, and sepsis in immunocompromised individuals. These infections are challenging to treat due to the intrinsic resistance of S. marcescens to an extensive array of antibiotics. Efflux pumps play a crucial role in protection of bacteria from antimicrobials. The MacAB efflux pump, previously linked to efflux of macrolides in Escherichia coli and protection from oxidative stress in Salmonella enterica serovar Typhimurium, is not characterized in S. marcescens We show the role of the MacAB efflux pump in S. marcescens protection from aminoglycoside antibiotics and polymyxins, modulation of bacterial motility, and biofilm formation, and we illustrate the essential role for this pump in bacterial survival during oxidative stress. Our findings make the MacAB efflux pump an attractive target for inhibition to gain control over S. marcescens infections.

Helicobacter pylori and Its Antibiotic Heteroresistance: A Neglected Issue in Published Guidelines.

"Heteroresistance" is a widely applied term that characterizes most of the multidrug-resistant microorganisms. In microbiological practice, the word "heteroresistance" indicates diverse responses to specific antibiotics by bacterial subpopulations in the same patient. These resistant subpopulations of heteroresistant strains do not respond to antibiotic therapy in vitro or in vivo. Presently, there is no standard protocol available for the treatment of infections caused by heteroresistant Helicobacter pylori in clinical settings, at least according to recent guidelines. Thus, there is a definite need to open a new discussion on how to recognize, how to screen, and how to eliminate those problematic strains in clinical and environmental samples. Since there is great interest in developing new strategies to improve the eradication rate of anti-H. pylori treatments, the presence of heteroresistant strains/clones among clinical isolates of the bacteria should be taken into account. Indeed, increased knowledge of gastroenterologists about the existence of heteroresistance phenomena is highly required. Moreover, the accurate breakpoints should be examined/determined in order to have a solid statement of heteroresistance among the H. pylori isolates. The primary definition of heteroresistance was about coexistence of both resistant and susceptible isolates at the similar gastric microniche at once, while we think that it can be happened subsequently as well. The new guidelines should include a personalized aspect in the standard protocol to select a precise, effective antibiotic therapy for infected patients and also address the problems of regional antibiotic susceptibility profiles.

Genome Sequence of Pigmented Siderophore-Producing Strain Serratia marcescens SM6.

Here we present a draft genome sequence of laboratory strain Serratia marcescens SM6. Using the antiSMASH 5.0 prediction tool, we identified five biosynthetic gene clusters involved in secondary metabolite production (two siderophores and a biosurfactant serratamolide, a glucosamine derivative, and a thiopeptide). Whole-genome sequencing information will be useful for the detailed study of metabolites produced by Serratia marcescens.

Deciphering the Enzymatic Function of the Bovine Enteric Infection-Related Protein YfeJ from Salmonella enterica Serotype Typhimurium.

Non-typhoidal Salmonella can colonize the gastrointestinal system of cattle and can also cause significant food-borne disease in humans. The use of a library of single-gene deletions in Salmonella enterica serotype Typhimurium allowed identification of several proteins that are under selection in the intestine of cattle. STM2437 ( yfeJ) encodes one of these proteins, and it is currently annotated as a type I glutamine amidotransferase. STM2437 was purified to homogeneity, and its catalytic properties with a wide range of γ-glutamyl derivatives were determined. The catalytic efficiency toward the hydrolysis of l-glutamine was extremely weak with a kcat/ Km value of 20 M-1 s-1. γ-l-Glutamyl hydroxamate was identified as the best substrate for STM2437, with a kcat/ Km value of 9.6 × 104 M-1 s-1. A homology model of STM2437 was constructed on the basis of the known crystal structure of a protein of unknown function (Protein Data Bank entry 3L7N ), and γ-l-glutamyl hydroxamate was docked into the active site based on the binding of l-glutamine in the active site of carbamoyl phosphate synthetase. Acivicin was shown to inactivate the enzyme by reaction with the active site cysteine residue and the subsequent loss of HCl. Mutation of Cys91 to serine completely abolished catalytic activity. Inactivation of STM2437 did not affect the ability of this strain to colonize mice, but it inhibited the growth of S. enterica Typhimurium in bacteriologic media containing γ-l-glutamyl hydroxamate.

Salmonella Pathogenicity Island 1 Is Expressed in the Chicken Intestine and Promotes Bacterial Proliferation.

Salmonella enterica serovar Enteritidis is a common cause of foodborne illness in the United States. The bacterium can be transmitted to humans via contaminated chicken meat and eggs, and virulence in humans requires type III secretion system 1 (TTSS-1), encoded on Salmonella pathogenicity island 1 (SPI-1). Chickens often carry S Enteritidis subclinically, obscuring the role of SPI-1 in facilitating bacterial colonization. To evaluate the role of SPI-1 in the infection of chicks by Salmonella, we created and utilized strains harboring a stable fluorescent reporter fusion designed to quantify SPI-1 expression within the intestinal tracts of animals. Using mutants unable to express TTSS-1, we demonstrated the important role of the secretion system in facilitating bacterial colonization. We further showed that coinoculation of an SPI-1 mutant with the wild-type strain increased the number of mutant organisms in intestinal tissue and contents, suggesting that the wild type rescues the mutant. Our results support the hypothesis that SPI-1 facilitates S Enteritidis colonization of the chicken and make SPI-1 an attractive target in preventing Salmonella carriage and colonization in chickens to reduce contamination of poultry meat and eggs by this foodborne pathogen.

The Salmonella type-3 secretion system-1 and flagellar motility influence the neutrophil respiratory burst.

Neutrophils are innate immune response cells designed to kill invading microorganisms. One of the mechanisms neutrophils use to kill bacteria is generation of damaging reactive oxygen species (ROS) via the respiratory burst. However, during enteric salmonellosis, neutrophil-derived ROS actually facilitates Salmonella expansion and survival in the gut. This seeming paradox led us to hypothesize that Salmonella may possess mechanisms to influence the neutrophil respiratory burst. In this work, we used an in vitro Salmonella-neutrophil co-culture model to examine the impact of enteric infection relevant virulence factors on the respiratory burst of human neutrophils. We report that neutrophils primed with granulocyte-macrophage colony stimulating factor and suspended in serum containing complement produce a robust respiratory burst when stimulated with viable STm. The magnitude of the respiratory burst increases when STm are grown under conditions to induce the expression of the type-3 secretion system-1. STm mutants lacking the type-3 secretion system-1 induce less neutrophil ROS than the virulent WT. In addition, we demonstrate that flagellar motility is a significant agonist of the neutrophil respiratory burst. Together our data demonstrate that both the type-3 secretion system-1 and flagellar motility, which are established virulence factors in enteric salmonellosis, also appear to directly influence the magnitude of the neutrophil respiratory burst in response to STm in vitro.

De novo pyrimidine synthesis is necessary for intestinal colonization of Salmonella Typhimurium in chicks.

pyrE (STM3733) encodes orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10), the fifth enzyme of the de novo pyrimidine biosynthetic pathway. We identified a ΔpyrE mutant as under selection in screening of a Salmonella mutant library in 4-day old chicks. Here, we confirm that a ΔpyrE mutant colonizes 4-day old chicks poorly in competitive infection with isogenic wild type, and that the ability of this mutant to colonize chicks could be restored by providing a copy of pyrE in trans. We further show that our ΔpyrE mutant grows poorly in nutrient poor conditions in vitro, and that the ability of this mutant to grow is restored, both in vitro and in chicks, when precursors to the pyrimidine salvage pathway were provided. This finding suggests that the environment in the chick intestine during our infections lacks sufficient precursors of the pyrimidine salvage pathway to support Salmonella growth. Finally, we show that the colonization defect of a ΔpyrE mutant during infection occurs in to chicks, but not in CBA/J mice or ligated ileal loops in calves. Our data suggest that de novo pyrimidine synthesis is necessary for colonization of Salmonella Typhimurium in the chick, and that the salvage pathway is not used in this niche.

Effects of Bacillus Serine Proteases on the Bacterial Biofilms.

Serratia marcescens is an emerging opportunistic pathogen responsible for many hospital-acquired infections including catheter-associated bacteremia and urinary tract and respiratory tract infections. Biofilm formation is one of the mechanisms employed by S. marcescens to increase its virulence and pathogenicity. Here, we have investigated the main steps of the biofilm formation by S. marcescens SR 41-8000. It was found that the biofilm growth is stimulated by the nutrient-rich environment. The time-course experiments showed that S. marcescens cells adhere to the surface of the catheter and start to produce extracellular polymeric substances (EPS) within the first 2 days of growth. After 7 days, S. marcescens biofilms maturate and consist of bacterial cells embedded in a self-produced matrix of hydrated EPS. In this study, the effect of Bacillus pumilus 3-19 proteolytic enzymes on the structure of 7-day-old S. marcescens biofilms was examined. Using quantitative methods and scanning electron microscopy for the detection of biofilm, we demonstrated a high efficacy of subtilisin-like protease and glutamyl endopeptidase in biofilm removal. Enzymatic treatment resulted in the degradation of the EPS components and significant eradication of the biofilms.

Contribution of Asparagine Catabolism to Salmonella Virulence.

Salmonellae are pathogenic bacteria that cause significant morbidity and mortality in humans worldwide. Salmonellae establish infection and avoid clearance by the immune system by mechanisms that are not well understood. We previously showed that l-asparaginase II produced by Salmonella enterica serovar Typhimurium (S Typhimurium) inhibits T cell responses and mediates virulence. In addition, we previously showed that asparagine deprivation such as that mediated by l-asparaginase II of S Typhimurium causes suppression of activation-induced T cell metabolic reprogramming. Here, we report that STM3997, which encodes a homolog of disulfide bond protein A (dsbA) of Escherichia coli, is required for l-asparaginase II stability and function. Furthermore, we report that l-asparaginase II localizes primarily to the periplasm and acts together with l-asparaginase I to provide S Typhimurium the ability to catabolize asparagine and assimilate nitrogen. Importantly, we determined that, in a murine model of infection, S Typhimurium lacking both l-asparaginase I and II genes competes poorly with wild-type S Typhimurium for colonization of target tissues. Collectively, these results indicate that asparagine catabolism contributes to S Typhimurium virulence, providing new insights into the competition for nutrients at the host-pathogen interface.

Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks.

Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection.

Multicopy Single-Stranded DNA Directs Intestinal Colonization of Enteric Pathogens.

Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking its retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate, but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules.

Identification of novel factors involved in modulating motility of Salmonella enterica serotype typhimurium.

Salmonella enterica serotype Typhimurium can move through liquid using swimming motility, and across a surface by swarming motility. We generated a library of targeted deletion mutants in Salmonella Typhimurium strain ATCC14028, primarily in genes specific to Salmonella, that we have previously described. In the work presented here, we screened each individual mutant from this library for the ability to move away from the site of inoculation on swimming and swarming motility agar. Mutants in genes previously described as important for motility, such as flgF, motA, cheY are do not move away from the site of inoculation on plates in our screens, validating our approach. Mutants in 130 genes, not previously known to be involved in motility, had altered movement of at least one type, 9 mutants were severely impaired for both types of motility, while 33 mutants appeared defective on swimming motility plates but not swarming motility plates, and 49 mutants had reduced ability to move on swarming agar but not swimming agar. Finally, 39 mutants were determined to be hypermotile in at least one of the types of motility tested. Both mutants that appeared non-motile and hypermotile on plates were assayed for expression levels of FliC and FljB on the bacterial surface and many of them had altered levels of these proteins. The phenotypes we report are the first phenotypes ever assigned to 74 of these open reading frames, as they are annotated as 'hypothetical genes' in the Typhimurium genome.

Defined single-gene and multi-gene deletion mutant collections in Salmonella enterica sv Typhimurium.

We constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (KanR) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (CamR) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a KanR cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a CamR cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available.

The ABC-type efflux pump MacAB protects Salmonella enterica serovar typhimurium from oxidative stress.

UNLABELLED: Multidrug efflux pumps are integral membrane proteins known to actively excrete antibiotics. The macrolide-specific pump MacAB, the only ABC-type drug efflux pump in Salmonella, has previously been linked to virulence in mice. The molecular mechanism of this link between macAB and infection is unclear. We demonstrate that macAB plays a role in the detoxification of reactive oxygen species (ROS), compounds that salmonellae are exposed to at various stages of infection. macAB is induced upon exposure to H2O2 and is critical for survival of Salmonella enterica serovar Typhimurium in the presence of peroxide. Furthermore, we determined that macAB is required for intracellular replication inside J774.A1 murine macrophages but is not required for survival in ROS-deficient J774.D9 macrophages. macAB mutants also had reduced survival in the intestine in the mouse colitis model, a model characterized by a strong neutrophilic intestinal infiltrate where bacteria may experience the cytotoxic actions of ROS. Using an Amplex red-coupled assay, macAB mutants appear to be unable to induce protection against exogenous H2O2 in vitro, in contrast to the isogenic wild type. In mixed cultures, the presence of the wild-type organism, or media preconditioned by the growth of the wild-type organism, was sufficient to rescue the macAB mutant from peroxide-mediated killing. Our data indicate that the MacAB drug efflux pump has functions beyond resistance to antibiotics and plays a role in the protection of Salmonella against oxidative stress. Intriguingly, our data also suggest the presence of a soluble anti-H2O2 compound secreted by Salmonella cells through a MacAB-dependent mechanism. IMPORTANCE: The ABC-type multidrug efflux pump MacAB is known to be required for Salmonella enterica serovar Typhimurium virulence after oral infection in mice, yet the function of this pump during infection is unknown. We show that this pump is necessary for colonization of niches in infected mice where salmonellae encounter oxidative stress during infection. MacAB is required for growth in cultured macrophages that produce reactive oxygen species (ROS) but is not needed in macrophages that do not generate ROS. In addition, we show that MacAB is required to resist peroxide-mediated killing in vitro and for the inactivation of peroxide in the media. Finally, wild-type organisms, or supernatant from wild-type organisms grown in the presence of peroxide, rescue the growth defect of macAB mutants in H2O2. MacAB appears to participate in the excretion of a compound that induces protection against ROS-mediated killing, revealing a new role for this multidrug efflux pump.

Novel determinants of intestinal colonization of Salmonella enterica serotype typhimurium identified in bovine enteric infection.

Cattle are naturally infected with Salmonella enterica serotype Typhimurium and exhibit pathological features of enteric salmonellosis that closely resemble those in humans. Cattle are the most relevant model of gastrointestinal disease resulting from nontyphoidal Salmonella infection in an animal with an intact microbiota. We utilized this model to screen a library of targeted single-gene deletion mutants to identify novel genes of Salmonella Typhimurium required for survival during enteric infection. Fifty-four candidate mutants were strongly selected, including numerous mutations in genes known to be important for gastrointestinal survival of salmonellae. Three genes with previously unproven phenotypes in gastrointestinal infection were tested in bovine ligated ileal loops. Two of these mutants, STM3602 and STM3846, recapitulated the phenotype observed in the mutant pool. Complementation experiments successfully reversed the observed phenotypes, directly linking these genes to the colonization defects of the corresponding mutant strains. STM3602 encodes a putative transcriptional regulator that may be involved in phosphonate utilization, and STM3846 encodes a retron reverse transcriptase that produces a unique RNA-DNA hybrid molecule called multicopy single-stranded DNA. The genes identified in this study represent an exciting new class of virulence determinants for further mechanistic study to elucidate the strategies employed by Salmonella to survive within the small intestines of cattle.