It is becoming increasingly apparent that innovations from the “golden age” of antibiotics are becoming ineffective, resulting in a pressing need for novel therapeutics. The bacteriocin family of antimicrobial peptides has attracted much attention in recent years as a source of potential alternatives. The most intensively studied bacteriocin is nisin, a broad spectrum lantibiotic that inhibits Gram-positive bacteria including important food pathogens and clinically relevant antibiotic resistant bacteria. Nisin is gene-encoded and, as such, is amenable to peptide bioengineering, facilitating the generation of novel derivatives that can be screened for desirable properties. It was to this end that we used a site-saturation mutagenesis approach to create a bank of producers of nisin A derivatives that differ with respect to the identity of residue 12 (normally lysine; K12). A number of these producers exhibited enhanced bioactivity and the nisin A K12A producer was deemed of greatest interest. Subsequent investigations with the purified antimicrobial highlighted the enhanced specific activity of this modified nisin against representative target strains from the genera Streptococcus, Bacillus, Lactococcus, Enterococcus and Staphylococcus.

New antibacterial agents are urgently needed for the elimination of biofilm-forming bacteria that are highly resistant to traditional antimicrobial agents. Proliferation of such bacteria can lead to significant economic losses in the agri-food sector. This study demonstrates the potential of the bacteriophage-derived peptidase, CHAPK, as a biocidal agent for the rapid disruption of biofilm-forming staphylococci, commonly associated with bovine mastitis. Purified CHAPK applied to biofilms of Staphylococcus aureus DPC5246 completely eliminated the staphylococcal biofilms within 4 h. In addition, CHAPK was able to prevent biofilm formation by this strain. The CHAPK lysin also reduced S. aureus in a skin decolonization model. Our data demonstrates the potential of CHAPK as a biocidal agent for prevention and treatment of biofilm-associated staphylococcal infections or as a decontaminating agent in the food and healthcare sectors.

Here, high-throughput sequencing was employed to reveal the highly diverse bacterial populations present in 62 Irish artisanal cheeses and, in some cases, associated cheese rinds. Using this approach, we revealed the presence of several genera not previously associated with cheese, including Faecalibacterium, Prevotella, and Helcococcus and, for the first time, detected the presence of Arthrobacter and Brachybacterium in goats' milk cheese. Our analysis confirmed many previously observed patterns, such as the dominance of typical cheese bacteria, the fact that the microbiota of raw and pasteurized milk cheeses differ, and that the level of cheese maturation has a significant influence on Lactobacillus populations. It was also noted that cheeses containing adjunct ingredients had lower proportions of Lactococcus species. It is thus apparent that high-throughput sequencing-based investigations can provide valuable insights into the microbial populations of artisanal foods.

Bacteriocins are an abundant and diverse group of ribosomally synthesized antimicrobial peptides produced by bacteria and archaea. Traditionally, bacteriocin production has been considered an important trait in the selection of probiotic strains, but until recently, few studies have definitively demonstrated the impact of bacteriocin production on the ability of a strain to compete within complex microbial communities and/or positively influence the health of the host. Although research in this area is still in its infancy, there is intriguing evidence to suggest that bacteriocins may function in a number of ways within the gastrointestinal tract. Bacteriocins may facilitate the introduction of a producer into an established niche, directly inhibit the invasion of competing strains or pathogens, or modulate the composition of the microbiota and influence the host immune system. Here we review the role of bacteriocin production in complex microbial communities and their potential to enhance human health.

ABSTRACT

The human appendix has historically been considered a vestige of evolutionary development with an unknown function. While limited data are available on the microbial composition of the appendix, it has been postulated that this organ could serve as a microbial reservoir for repopulating the gastrointestinal tract in times of necessity. We aimed to explore the microbial composition of the human appendix, using high-throughput sequencing of the 16S rRNA gene V4 region. Seven patients, 5 to 25 years of age, presenting with symptoms of acute appendicitis were included in this study. Results showed considerable diversity and interindividual variability among the microbial composition of the appendix samples. In general, however, Firmicutes was the dominant phylum, with the majority of additional sequences being assigned at various levels to Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria. Despite the large diversity in the microbiota found within the appendix, however, a few major families and genera were found to comprise the majority of the sequences present. Interestingly, also, certain taxa not generally associated with the human intestine, including the oral pathogens Gemella, Parvimonas, and Fusobacterium, were identified among the appendix samples. The prevalence of genera such as Fusobacterium could also be linked to the severity of inflammation of the organ. We conclude that the human appendix contains a robust and varied microbiota distinct from the microbiotas in other niches within the human microbiome. The microbial composition of the human appendix is subject to extreme variability and comprises a diversity of biota that may play an important, as-yet-unknown role in human health.

IMPORTANCE

There are currently limited data available on the microbial composition of the human appendix. It has been suggested, however, that it may serve as a “safe house” for commensal bacteria that can reinoculate the gut at need. The present study is the first comprehensive view of the microbial composition of the appendix as determined by high-throughput sequencing. We have determined that the human appendix contains a wealth of microbes, including members of 15 phyla. Important information regarding the associated bacterial diversity of the appendix which will help determine the role, if any, the appendix microbiota has in human health is presented.

The objective of this study was to investigate if feeding genetically modified (GM) MON810 maize expressing the Bacillus thuringiensis insecticidal protein (Bt maize) had any effects on the porcine intestinal microbiota. Eighteen pigs were weaned at ∼28 days and, following a 6-day acclimatization period, were assigned to diets containing either GM (Bt MON810) maize or non-GM isogenic parent line maize for 31 days (n = 9/treatment). Effects on the porcine intestinal microbiota were assessed through culture-dependent and -independent approaches. Fecal, cecal, and ileal counts of total anaerobes, Enterobacteriaceae, and Lactobacillus were not significantly different between pigs fed the isogenic or Bt maize-based diets. Furthermore, high-throughput 16S rRNA gene sequencing revealed few differences in the compositions of the cecal microbiotas. The only differences were that pigs fed the Bt maize diet had higher cecal abundance of Enterococcaceae (0.06 versus 0%; P < 0.05), Erysipelotrichaceae (1.28 versus 1.17%; P < 0.05), and Bifidobacterium (0.04 versus 0%; P < 0.05) and lower abundance of Blautia (0.23 versus 0.40%; P < 0.05) than pigs fed the isogenic maize diet. A lower enzyme-resistant starch content in the Bt maize, which is most likely a result of normal variation and not due to the genetic modification, may account for some of the differences observed within the cecal microbiotas. These results indicate that Bt maize is well tolerated by the porcine intestinal microbiota and provide additional data for safety assessment of Bt maize. Furthermore, these data can potentially be extrapolated to humans, considering the suitability of pigs as a human model.

Caseicins A and B are low-molecular-weight antimicrobial peptides which are released by proteolytic digestion of sodium caseinate. Caseicin A (IKHQGLPQE) is a nine-amino-acid cationic peptide, and caseicin B (VLNENLLR) is a neutral eight-amino-acid peptide; both have previously been shown to exhibit antibacterial activity against a number of pathogens, including Cronobacter sakazakii. Previously, four variants of each caseicin which differed subtly from their natural counterparts were generated by peptide synthesis. Antimicrobial activity assays revealed that the importance of a number of the residues within the peptides was dependent on the strain being targeted. In this study, this engineering-based approach was expanded through the creation of a larger collection of 26 peptides which are altered in a variety of ways. The investigation highlights the generally greater tolerance of caseicin B to change, the fact that changes have a more detrimental impact on anti-Gram-negative activity, and the surprising number of variants which exhibit enhanced activity against Staphylococcus aureus.

The Listeria monocytogenes LiaSR two-component system (2CS) encoded by lmo1021 and lmo1022 plays an important role in resistance to the food preservative nisin. A nonpolar deletion in the histidine kinase-encoding component (ΔliaS) resulted in a 4-fold increase in nisin resistance. In contrast, the ΔliaS strain exhibited increased sensitivity to a number of cephalosporin antibiotics (and was also altered with respect to its response to a variety of other antimicrobials, including the active agents of a number of disinfectants). This pattern of increased nisin resistance and reduced cephalosporin resistance in L. monocytogenes has previously been associated with mutation of a second histidine kinase, LisK, which is a predicted regulator of liaS and a penicillin binding protein encoded by lmo2229. We noted that lmo2229 transcription is increased in the ΔliaS mutant and in a ΔliaS ΔlisK double mutant and that disruption of lmo2229 in the ΔliaS ΔlisK mutant resulted in a dramatic sensitization to nisin but had a relatively minor impact on cephalosporin resistance. We anticipate that further efforts to unravel the complex mechanisms by which LiaSR impacts on the antimicrobial resistance of L. monocytogenes could facilitate the development of strategies to increase the susceptibility of the pathogen to these agents.

Clostridium difficile is an important nosocomial pathogen associated particularly with diarrheal disease in elderly individuals in hospitals and long-term care facilities. We examined the carriage rate of Clostridium difficile by culture as a function of fecal microbiota composition in elderly subjects recruited from the community, including outpatient, short-term respite, and long-term hospital stay subjects. The carriage rate ranged from 1.6% (n = 123) for subjects in the community, to 9.5% (n = 43) in outpatient settings, and increasing to 21% (n = 151) for patients in short- or long-term care in hospital. The dominant 072 ribotype was carried by 43% (12/28) of subjects, while the hypervirulent strain R027 (B1/NAP1/027) was isolated from 3 subjects (11%), 2 of whom displayed C. difficile associated diarrhea (CDAD) symptoms at the time of sampling. Emerging ribotypes with enhanced virulence (078 and 018) were also isolated from two asymptomatic subjects. Pyrosequencing of rRNA gene amplicons was used to determine the composition of the fecal microbiota as a surrogate for the microbial population structure of the distal intestine. Asymptomatic subjects (n = 20) from whom C. difficile was isolated showed no dramatic difference at the phylum or family taxonomic level compared to those that were culture negative (n = 252). However, in contrast, a marked reduction in microbial diversity at genus level was observed in patients who had been diagnosed with CDAD at the time of sampling and from whom C. difficile R027 was isolated.

Ltnα and Ltnβ are individual components of the two-peptide lantibiotic lacticin 3147 and are unusual in that, although ribosomally synthesized, they contain d-amino acids. These result from the dehydration of l-serine to dehydroalanine by LtnM and subsequent stereospecific hydrogenation to d-alanine by LtnJ. Homologues of LtnJ are rare but have been identified in silico in Staphylococcus aureus C55 (SacJ), Pediococcus pentosaceus FBB61 (PenN), and Nostoc punctiforme PCC73102 (NpnJ, previously called NpunJ [P. D. Cotter et al., Proc. Natl. Acad. Sci. U. S. A. 102:18584–18589, 2005]). Here, the ability of these enzymes to catalyze d-alanine formation in the lacticin 3147 system was assessed through heterologous enzyme production in a ΔltnJ mutant. PenN successfully incorporated d-alanines in both peptides, and SacJ modified Ltnα only, while NpnJ was unable to modify either peptide. Site-directed mutagenesis was also employed to identify residues of key importance in LtnJ. The most surprising outcome from these investigations was the generation of peptides by specific LtnJ mutants which exhibited less bioactivity than those generated by the ΔltnJ strain. We have established that the reduced activity of these peptides is due to the inability of the associated LtnJ enzymes to generate d-alanine residues in a stereospecific manner, resulting in the presence of both d- and l-alanines at the relevant locations in the lacticin 3147 peptides.

Lactobacillus ruminis is one of at least twelve motile but poorly characterized species found in the genus Lactobacillus. Of these, only L. ruminis has been isolated from mammals, and this species may be considered as an autochthonous member of the gastrointestinal microbiota of humans, pigs and cows. Nine L. ruminis strains were investigated here to elucidate the biochemistry and genetics of Lactobacillus motility. Six strains isolated from humans were non-motile while three bovine isolates were motile. A complete set of flagellum biogenesis genes was annotated in the sequenced genomes of two strains, ATCC25644 (human isolate) and ATCC27782 (bovine isolate), but only the latter strain produced flagella. Comparison of the L. ruminis and L. mali DSM20444T motility loci showed that their genetic content and gene-order were broadly similar, although the L. mali motility locus was interrupted by an 11.8 Kb region encoding rhamnose utilization genes that is absent from the L. ruminis motility locus. Phylogenetic analysis of 39 motile bacteria indicated that Lactobacillus motility genes were most closely related to those of motile carnobacteria and enterococci. Transcriptome analysis revealed that motility genes were transcribed at a significantly higher level in motile L. ruminis ATCC27782 than in non-motile ATCC25644. Flagellin proteins were isolated from L. ruminis ATCC27782 and from three other Lactobacillus species, while recombinant flagellin of aflagellate L. ruminis ATCC25644 was expressed and purified from E. coli. These native and recombinant Lactobacillus flagellins, and also flagellate L. ruminis cells, triggered interleukin-8 production in cultured human intestinal epithelial cells in a manner suppressed by short interfering RNA directed against Toll-Like Receptor 5. This study provides genetic, transcriptomic, phylogenetic and immunological insights into the trait of flagellum-mediated motility in the lactobacilli.

Nisin U is a member of the extended nisin family of lantibiotics. Here we identify the presence of nisin U immunity gene homologues in Streptococcus infantarius subsp. infantarius BAA-102. Heterologous expression of these genes in Lactococcus lactis subsp. cremoris HP confers protection to nisin U and other members of the nisin family, thereby establishing that the recently identified phenomenon of resistance through immune mimicry also occurs with respect to nisin.

Bacteriocins produced by Lactobacillus salivarius isolates derived from a gastrointestinal origin have previously demonstrated efficacy for in vivo protection against Listeria monocytogenes infection. In this study, comparative genomic analysis was employed to investigate the intraspecies diversity of seven L. salivarius isolates of human and porcine intestinal origin, based on the genome of the well-characterized bacteriocin-producing strain L. salivarius UCC118. This revealed a highly conserved megaplasmid-borne gene cluster in these strains involved in the regulation and secretion of two-component class IIb bacteriocins. However, considerable intraspecific variation was observed in the structural genes encoding the bacteriocin peptides. They ranged from close relatives of abp118, such as salivaricin P, which differs by 2 amino acids, to completely novel bacteriocins, such as salivaricin T, which is characterized in this study. Salivaricin T inhibits closely related lactobacilli and bears little homology to previously characterized salivaricins. Interestingly, the two peptides responsible for salivaricin T activity, SalTα and SalTβ, share considerable identity with the component peptides of thermophilin 13, a bacteriocin produced by Streptococcus thermophilus. Furthermore, the salivaricin locus of strain DPC6488 also encodes an additional novel one-component class IId anti-listerial bacteriocin, salivaricin L. These findings suggest a high level of redundancy in the bacteriocins that can be produced by intestinal L. salivarius isolates using the same enzymatic production and export machinery. Such diversity may contribute to their ability to dominate and compete within the complex microbiota of the mammalian gut.

The draft genome of Bifidobacterium breve DPC 6330, isolated from an elderly patient, was determined. B. breve DPC 6330 was previously identified to synthesize the beneficial metabolite conjugated linoleic acid from free linoleic acid. The sequence will allow identification and characterization of the genetic determinants of its putative beneficial properties.

In this study, whole-genome microarrays were used to gain insights into the global molecular response of Lactococcus lactis subsp. lactis IL1403 at an early stage of infection with the lytic phage c2. The bacterium differentially regulated the expression of 61 genes belonging to 14 functional categories, including cell envelope processes (12 genes), regulatory functions (11 genes), and carbohydrate metabolism (7 genes). The nature of these genes suggests a complex response involving four main mechanisms: (i) induction of membrane stress proteins, (ii) d-alanylation of cell wall lipoteichoic acids (LTAs), (iii) maintenance of the proton motive force (PMF), and (iv) energy conservation. The phage presence is sensed as a membrane stress in L. lactis subsp. lactis IL1403, which activated a cell wall-targeted response probably orchestrated by the concerted action of membrane phage shock protein C-like homologues, the global regulator SpxB, and the two-component system CesSR. The bacterium upregulated genes (ddl and dltABCD) responsible for incorporation of d-alanine esters into LTAs, an event associated with increased resistance to phage attack in Gram-positive bacteria. The expression of genes (yshC, citE, citF) affecting both PMF components was also regulated to restore the physiological PMF, which was disrupted following phage infection. While mobilizing the response to the phage-mediated stress, the bacterium activated an energy-saving program by repressing growth-related functions and switching to anaerobic respiration, probably to sustain the PMF and the overall cell response to phage. To our knowledge, this represents the first detailed description in L. lactis of the molecular mechanisms involved in the host response to the membrane perturbations mediated by phage infection.

The four-plasmid complement of the raw milk cheese isolate Lactococcus lactis subsp. lactis biovar diacetylactis DPC3901 was sequenced, and some genetic features were functionally analyzed. The complete sequences of pVF18 (18,977 bp), pVF21 (21,739 bp), pVF22 (22,166 bp), and pVF50 (53,876 bp) were obtained. Each plasmid contained genes not previously described for Lactococcus, in addition to genes associated with plant-derived lactococcal strains. Most of the novel genes were found on pVF18 and encoded functions typical of bacteria associated with plants, such as activities of plant cell wall modification (orf11 and orf25). In addition, a predicted high-affinity regulated system for the uptake of cobalt was identified (orf19 to orf21 [orf19-21]), which has a single database homolog on a plant-derived Leuconostoc plasmid and whose functionality was demonstrated following curing of pVF18. pVF21 and pVF22 encode additional metal transporters, which, along with orf19-21 of pVF18, could enhance host ability to uptake growth-limiting amounts of biologically essential ions within the soil. In addition, vast regions from pVF50 and pVF21 share significant homology with the plant-derived lactococcal plasmid pGdh442, which is indicative of extensive horizontal gene transfer and recombination between these plasmids and suggests a common plant niche for their hosts. Phenotypes associated with these regions include glutamate dehydrogenase activity and Na+ and K+ transport. The presence of numerous plant-associated markers in L. lactis DPC3901 suggests a plant origin for the raw milk cheese isolate and provides for the first time the genetic basis to support the concept of the plant-milk transition for Lactococcus strains.

The objective of this study was to investigate the in vivo activity of the lantibiotic lacticin 3147 against the luminescent Staphylococcus aureus strain Xen 29 using a murine model. Female BALB/c mice (7 weeks old, 17 g) were divided into groups (n = 5) and infected with the Xen 29 strain via the intraperitoneal route at a dose of 1 × 106 cfu/animal. After 1.5 hr, the animals were treated subcutaneously with doses of phosphate-buffered saline (PBS; negative control) or lacticin 3147. Luminescent imaging was carried 3 and 5 hours postinfection. Mice were then sacrificed, and the levels of S. aureus Xen 29 in the liver, spleen, and kidneys were quantified. Notably, photoluminescence and culture-based analysis both revealed that lacticin 3147 successfully controlled the systemic spread of S. aureus in mice thus indicating that lacticin 3147 has potential as a chemotherapeutic agent for in vivo applications.

Caseicin A (IKHQGLPQE) and caseicin B (VLNENLLR) are antimicrobial peptides generated through the bacterial fermentation of sodium caseinate, and on the basis of this and previous studies, they are active against many Gram-negative pathogens (Cronobacter sakazakii, Cronobacter muytjensii, Salmonella enterica serovar Typhimurium, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas fluorescens) as well as the Gram-positive organism Staphylococcus aureus. Here we describe further studies with the aim of establishing the importance of specific (charged and nonpolar aliphatic) residues within the caseicin peptides and the effects that they have on the bacteria listed above. In order to achieve our objective, we created four derivatives of each caseicin (A1 to A4 and B1 to B4) in which specific residues were altered, and results obtained with these derivatives were compared to wild-type caseicin activity. Although conversion of cationic residues to alanine in caseicins B1 (R8A change), A1 (K2A), A2 (H3A), and A3 (K2A-H3A) generally resulted in their activity against microbial targets being reduced or unaltered, C. sakazakii DPC6440 was unusual in that it displayed enhanced sensitivity to three peptides (caseicins A1, A3, and B2) in which positively charged residues had been eliminated. While the replacement of leucine with alanine in selected variants (B3 and B4) resulted in reduced activity against a number of strains of Cronobacter and, in some cases, S. Typhimurium, these changes enhanced the activities of these peptides against DPC6440 and a number of S. aureus strains. It is thus apparent that the importance of specific residues within the caseicin peptides is dependent on the strain being targeted.

Before a probiotic bacterium can even begin to fulfill its biological role, it must survive a battery of environmental stresses imposed during food processing and passage through the gastrointestinal tract (GIT). Food processing stresses include extremes in temperature, as well as osmotic, oxidative and food matrix stresses. Passage through the GIT is a hazardous journey for any bacteria with deleterious lows in pH encountered in the stomach to the detergent-like properties of bile in the duodenum. However, bacteria are equipped with an array of defense mechanisms to counteract intracellular damage or to enhance the robustness of the cell to withstand lethal external environments. Understanding these mechanisms in probiotic bacteria and indeed other bacterial groups has resulted in the development of a molecular toolbox to augment the technological and gastrointestinal performance of probiotics. This has been greatly aided by studies which examine the global cellular responses to stress highlighting distinct regulatory networks and which also identify novel mechanisms used by cells to cope with hazardous environments. This review highlights the latest studies which have exploited the bacterial stress response with a view to producing next-generation probiotic cultures and highlights the significance of studies which view the global bacterial stress response from an integrative systems biology perspective.

Due to the severity of the food-borne infection listeriosis, strict legislation governs the detectable and permissible limits at which Listeria monocytogenes is permitted in foods. These requirements, coupled with the ubiquitous nature of L. monocytogenes strains and the potential for epidemic outbreaks, mean that the pathogen can devastate affected sectors of the food industry. Although almost all L. monocytogenes strains have the potential to cause listeriosis, those implicated in the vast majority of epidemics belong to a subset of strains belonging to evolutionary lineage I. It has been established that a significant proportion of these strains, including those implicated in the majority of outbreaks, produce an additional hemolysin, designated listeriolysin S (LLS), which may be responsible for the enhanced virulence of these strains. In order to ultimately establish this definitively, it is important to first be able to rapidly discriminate between LLS-positive and -negative strains. Here, after essential genes within the LLS-encoding cluster, Listeria pathogenicity island 3, were identified by deletion mutagenesis, a real-time PCR assay which targets one such gene, llsX, was developed as a means of identifying LLS-positive L. monocytogenes. The specificity of the assay was validated against a panel of 40 L. monocytogenes strains (20 of which were LLS positive) and 25 strains representative of other Listeria species. Furthermore, 1 CFU of an LLS-positive strain per 25 g/ml of spiked foods was detected in less than 30 h when the assay was coupled with culture enrichment. The detection limit of this assay was 10 genome equivalents.

The aim of this study was to use comparative modeling to predict the three-dimensional structure of the CHAPK protein (cysteine, histidine-dependent amidohydrolase/peptidase domain of the LysK endolysin, derived from bacteriophage K). Iterative PSI-BLAST searches against the Protein Data Bank (PDB) and nonredundant (nr) databases were used to populate a multiple alignment for analysis using the T-Coffee Expresso server. A consensus Maximum Parsimony phylogenetic tree with a bootstrap analysis setting of 1,000 replicates was constructed using MEGA4. Structural templates relevant to our target (CHAPK) were identified, processed in Expresso and used to generate a 3D model in the alignment mode of SWISS-MODEL. These templates were also processed in the I-TASSER web server. A Staphylococcus saprophyticus CHAP domain protein, 2K3A, was identified as the structural template in both servers. The I-TASSER server generated the CHAPK model with the best bond geometries when analyzed using PROCHECK and the most logical organization of the structure. The predicted 3D model indicates that CHAPK has a papain-like fold. Circular dichroism spectropolarimetry also indicated that CHAPK has an αβ fold, which is consistent with the model presented. The putative active site maintained a highly conserved Cys54-His117-Glu134 charge relay and an oxyanion hole residue Asn136. The residue triplet, Cys-His-Glu, is known to be a viable proteolytic triad in which we predict the Cys residue is used in a nucleophilic attack on peptide bonds at a specific site in the pentaglycine cross bridge of staphylococcal cell wall peptidoglycan. Use of comparative modeling has allowed approximation of the 3D structure of CHAPK giving information on the structure and an insight into the binding and active site of the catalytic domain. This may facilitate its development as an alternative antibacterial agent.

Nisin is a class I bacteriocin (lantibiotic), which is employed by the food and veterinary industries and exhibits potent activity against numerous pathogens. However, this activity could be further improved through the targeting and inhibition of factors that contribute to innate nisin resistance. Here we describe a novel locus, lmo1967, which is required for optimal nisin resistance in Listeria monocytogenes. The importance of this locus, which is a homologue of the tellurite resistance gene telA, was revealed after the screening of a mariner random mutant bank of L. monocytogenes for nisin-susceptible mutants. The involvement of telA in nisin resistance was confirmed through an analysis of a nonpolar deletion mutant. In addition to being 4-fold-more susceptible to nisin, the ΔtelA strain was also 8-fold-more susceptible to gallidermin and 2-fold-more susceptible to cefuroxime, cefotaxime, bacitracin, and tellurite. This is the first occasion upon which telA has been investigated in a Gram-positive organism and also represents the first example of a link being established between a telA gene and resistance to cell envelope-acting antimicrobials.

This study investigated the effect of bacteriophages (phages) e11/2 and e4/1c against Escherichia coli O157:H7 in an ex vivo rumen model and in cattle in vivo. In the ex vivo rumen model, samples were inoculated with either 103 or 106 CFU/ml inoculum of E. coli O157:H7 and challenged separately with each bacteriophage. In the presence of phage e11/2, the numbers of E. coli O157:H7 bacteria were significantly (P < 0.05) reduced to below the limit of detection within 1 h. Phage e4/1c significantly (P < 0.05) reduced E. coli O157:H7 numbers within 2 h of incubation, but the number of surviving E. coli O157:H7 bacteria then remained unchanged over a further 22-h incubation period. The ability of a phage cocktail of e11/2 and e4/1c to reduce the fecal shedding of E. coli O157:H7 in experimentally inoculated cattle was then investigated in two cattle trials. Cattle (yearlings, n = 20 for trial one; adult fistulated cattle, n = 2 for trial two) were orally inoculated with 1010 CFU of E. coli O157:H7. Animals (n = 10 for trial one; n = 1 for trial two) were dosed daily with a bacteriophage cocktail of 1011 PFU for 3 days postinoculation. E. coli O157:H7 and phage numbers in fecal and/or rumen samples were determined over 7 days postinoculation. E. coli O157:H7 numbers rapidly declined in all animals within 24 to 48 h; however, there was no significant difference (P > 0.05) between the numbers of E. coli O157:H7 bacteria shed by the phage-treated or control animals. Phages were recovered from the rumen but not from the feces of the adult fistulated animal in trial two but were recovered from the feces of the yearling animals in trial one. While the results from the rumen model suggest that phages are effective in the rumen, further research is required to improve the antimicrobial effectiveness of phages for the elimination of E. coli O157:H7 in vivo.

A mariner transposon bank was used to identify loci that contribute to the innate resistance of Listeria monocytogenes to the lantibiotic nisin. In addition to highlighting the importance of a number of loci previously associated with nisin resistance (mprF, virRS, and telA), a nisin-sensitive phenotype was associated with the disruption of anrB (lmo2115), a gene encoding the permease component of an ABC transporter. The contribution of anrB to nisin resistance was confirmed by the creation of nonpolar deletion mutants. The loss of this putative multidrug resistance transporter also greatly enhanced sensitivity to bacitracin, gallidermin, and a selection of β-lactam antibiotics. A comparison of the relative antimicrobial sensitivities of a number of mutants established the ΔanrB strain as being one of the most bacitracin-sensitive L. monocytogenes strains identified to date.

Analysis of a complete set of glutamate decarboxylase (gad) mutants of Listeria monocytogenes strain LO28 (ΔgadD1, ΔgadDT1, ΔgadD2, ΔgadT2, and ΔgadD3 mutants) revealed that the ΔgadD1 mutant is impaired in its ability to tolerate exposure to both sublethal and lethal levels of the lantibiotic nisin. gadD1 is strain variable and is found only in approximately 50% of L. monocytogenes strains. Growth and survival experiments revealed that possession of gadD1 correlates with a higher degree of tolerance to nisin. Significantly, a similar finding using a gadB mutant of L. lactis IL1403 implies that this may be a general phenomenon in Gram-positive bacteria. Our findings thus suggest that the specific inhibition of GAD activity or a reduction in the levels of free glutamate may prevent the growth of otherwise resistant GAD+ bacteria in foods where low pH and/or nisin is used as a preservative.