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Nisin resistance development is one of food safety challenges posed by Listeria monocytogenes , an important foodborne pathogen that causes human listeriosis. The GtcA flippase enzyme is functionally crucial in two separate pathways that glycosylate cell envelope wall teichoic acids (WTA) with N -acetylglucosamine (NAG) and lipoteichoic acids (LTA) with galactose, respectively. This study investigated phenotypic roles and molecular mechanisms underlying GtcA involvement in L. monocytogenes nisin resistance. A GtcA A65V mutation was linked with increased nisin resistance in a food processing environment associated L. monocytogenes strain. Examination of nisin stress survival and growth phenotypes among L. monocytogenes gtcA mutants in different genetic backgrounds showed that GtcA function promoted sensitivity and loss of its function through genetic deletion (Δ gtcA) and a natural GtcA A65V mutation increased nisin resistance. Individual contributions of GtcA WTA NAG and LTA galactose glycosylation functions to nisin resistance modulation were examined through nisin sensitivity analysis of genetic deletion mutants and L. monocytogenes strains complemented using functionally altered GtcA mutants. This revealed WTA NAG glycosylation to be the main functional mechanism that determines GtcA dependent nisin phenotypic sensitization. An examination for mechanisms underlying GtcA involvement in nisin sensitivity revealed that the loss of GtcA function induces changes in the cell envelope carbohydrate composition profiles reducing cell surface hydrophobicity. Overall, our results showed that cell envelope WTA NAG glycosylation promotes nisin susceptibility through facilitation of hydrophobic interactions between nisin and the Listeria cell envelope. There may be practical implications from our observations since nisin resistance could be gained in food associated L. monocytogenes strains that develop phage resistance through acquisition of mutations in genes that cause loss of cell envelope WTA NAG modifications. • GtcA induced cell envelope glycosylation sensitizes L. monocytogenes to nisin. • Loss of GtcA function increases L. monocytogenes nisin resistance. • GtcA impact on nisin sensitivity largely depends on cell envelope WTA NAG glycosylation. • WTA NAG devoid cell envelope reduce L. monocytogenes cell surface hydrophobicity. • Nisin resistance induced through reduced hydrophobicity interaction with Listeria cells. [ABSTRACT FROM AUTHOR]

An important step in the development of successful technological schemes for production of yogurt and other functional foods is the selection of appropriate Lactobacillus strains with useful properties that are resistant to antibiotics and bacteriocins. In the present study eleven Lactobacillus strains were isolated from fourteen homemade Bulgarian and Italian yogurts. The isolates were identified as Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) by 16S rDNA sequence analysis. The results from comparative 16S rRNA gene sequence-based phylogenetic analysis revealed 92-99% pairwise similarity of the isolates to the reference L. bulgaricus strains. The antimicrobial activity, antibiotic susceptibility and nisin resistance of the isolated L. bulgaricus strains were examined. Bulgarian L. bulgaricus strains 3-BG, 5-BG and 8-BG were characterized by highest antimicrobial activity against the Gram-positive bacteria Staphylococcus aureus ATCC 25923, Listeria monocytogenes NBIMCC 8632, Listeria ivanovii ATCC 19119, Listeria innocua ATCC 33090, Enterococcus faecalis ATCC 19433 and Enterococcus faecuim ATCC 19434. L. bulgaricus 8-BG was active also against the Gram-negative bacteria Pseudomonas aeruginosa ATCC 9027, Proteus vulgaris ATCC 6380, Salmonella enteritidis ATCC 13076, Salmonella abony NTCC 6017 and Escherichia coli ATCC 25922. In contrast, Italian L. bulgaricus strains demonstrated low antimicrobial activity. Bulgarian L. bulgaricus strains showed moderate sensitivity or resistance to most of the antibiotics used in the screening, while Italian L. bulgaricus strains were sensitive. Bulgarian L. bulgaricus strains 1-BG and 6-BG were resistant to 10 and 13 of a total of 24 antibiotics tested, respectively. Nisin resistance test showed that 10 of a total of 11 L. bulgaricus strains were highly sensitive to nisin (MIC values varying from 0.078 mg/mL to 0.156 mg/mL), except of Italian strain L. bulgaricus 6-IT which was resistant to nisin. [ABSTRACT FROM AUTHOR]

NisI confers immunity against nisin, with high substrate specificity to prevent a suicidal effect in nisin-producing Lactococcus lactis strains. However, the NisI maturation process as well as its influence on nisin resistance has not been characterized. Here, we report the roles of lipoprotein signal peptidase II (Lsp) and prolipoprotein diacylglyceryl transferase (Lgt) in NisI maturation and nisin resistance of L. lactis F44. We found that the resistance of nisin of an Lsp-deficient mutant remarkably decreased, while no significant differences in growth were observed. We demonstrated that Lsp could cleave signal peptide of NisI precursor in vitro. Moreover, diacylglyceryl modification of NisI catalyzed by Lgt played a decisive role in attachment of NisI on the cell envelope, while it exhibited no effects on cleavage of the signal peptides of NisI precursor. The dissociation constant (KD) for the interaction between nisin and NisI exhibited a 2.8-fold increase compared with that between nisin and pre-NisI with signal peptide by surface plasmon resonance (SPR) analysis, providing evidence that Lsp-catalyzed signal peptide cleavage was critical for the immune activity of NisI. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production. [ABSTRACT FROM AUTHOR]

The multi-resistant Staphylococcus capitis clone NRCS-A has recently been described as a major pathogen causing nosocomial, late-onset sepsis (LOS) in preterm neonates worldwide. NRCS-A representatives exhibit an atypical antibiotic resistance profile. Here, the complete closed genome (chromosomal and plasmid sequences) of NRCS-A prototype strain CR01 and the draft genomes of three other clinical NRCS-A strains from Australia, Belgium and the United Kingdom are annotated and compared to available non-NRCS-A S. capitis genomes. Our goal was to delineate the uniqueness of the NRCS-A clone with respect to antibiotic resistance, virulence factors and mobile genetic elements.We identified 6 antimicrobial resistance genes, all carried by mobile genetic elements. Previously described virulence genes present in the NRCS-A genomes are shared with the six non-NRCS-A S. capitis genomes. Overall, 63 genes are specific to the NRCS-A lineage, including 28 genes located in the methicillin-resistance cassette SCCmec. Among the 35 remaining genes, 25 are of unknown function, and 9 correspond to an additional type I restriction modification system (n=3), a cytosine methylation operon (n=2), and a cluster of genes related to the biosynthesis of teichoic acids (n=4). Interestingly, a tenth gene corresponds to a resistance determinant for nisin (nsr gene), a bacteriocin secreted by potential NRCS-A strain niche competitors in the gut microbiota.The genomic characteristics presented here emphasize the contribution of mobile genetic elements to the emergence of multidrug resistance in the S. capitis NRCS-A clone. No NRCS-A-specific known virulence determinant was detected, which does not support a role for virulence as a driving force of NRCS-A emergence in NICUs worldwide. However, the presence of a nisin resistance determinant on the NRCS-A chromosome, but not in other S. capitis strains and most coagulase-negative representatives, might confer a competitive advantage to NRCS-A strains during the early steps of gut colonization in neonates. This suggests that the striking adaptation of NRCS-A to the NICU environment might be related to its specific antimicrobial resistance and also to a possible enhanced ability to challenge competing bacteria in its ecological niche.

The aim of this study is the isolation of LAB tolerating nisin and identification within 16S rDNA sequencing ultimately. The microbiological quality of cheeses obtained from various Denizli local bazaars were found lower than the samples packaged and sold with commercial brands. The LAB counts grown in M17 and MRS were 8.38 and 7.29 log cfu/g, respectively. When 500 IU/mL nisin was inoculated to the M17 and MRS, the LAB counts in cheese samples were averagely 4.99 and 2.92 log cfu/g respectively. The 23 collected LAB tolerating nisin was found 97% similar within; Lactobacillus plantarum (4), Lactobacillus casei (3), Lactobacillus paracasei (3), Enterococcus faceium (3), Lactobacillus rhamnosus (2) and Lactobacillus fermentum (2). In conclusion, lactic cultures existed in white-brined cheese flora was affected from nisin significantly and potential lactic cultures which would be useful for the production of White-brined cheese production together with nisin were collected and identified. [ABSTRACT FROM AUTHOR]

Summary: Nisin is a bacteriocin that is widely used as a safe, natural preservative in food products. Nisin-controlled gene expression (NICE) systems and food-grade expression systems with nisin resistance as the selection marker are increasingly attracting attention owing to their food-grade statuses. However, the putative influence of nisin resistance on NICE systems deserves consideration when nisin is used as both the inducer and selection agent in lactococcal strains. In this paper, we described the cloning of the nisin resistance gene (nsr) and studied the effect of the encoded nisin resistance protein (NSR) on the efficiency of the NICE system in Lactococcus lactis, with the green fluorescence protein as the reporter protein. Results showed that NSR expression significantly weakened the inducing activity of nisin. Further studies have confirmed that this reduction in the inducing activity of nisin was a consequence of the proteolytic activity of NSR against nisin; the digested products showed drastically decreased inducing activities than native nisin. Conclusively, the expression of NSR imposes an adverse effect on the NICE system in L. lactis. [ABSTRACT FROM AUTHOR]

The ability of L. monocytogenes cells to adapt to a variety of stressors contributes to its growth in a wide range of foods. The present study examines the effect of acid and of the acid tolerance response (ATR) on membrane fluidity and on the organism’s resistance to acid and to the bacteriocin nisin. When ATR was induced in wild-type cells, these cells also became resistant to nisin. ATR(+) cells also had lower membrane rigidities than control ATR(−) cells that had not been subjected to the acid tolerance response. However, cells that were genetically resistant to nisin did not show any significant ( P < 0.05) change in rigidity when grown in the presence of nisin. These studies suggest that the use of acid and nisin for L. monocytogenes control in ready-to-eat foods may be compromised if cross-resistance emerges. [ABSTRACT FROM AUTHOR]

Lactococcus lactis is a food-grade microorganism of major commercial importance in food industry. For commercial application of genetically modified L. lactis, a food-grade expression system is strongly recommended. In this study, two food-grade selection markers, nisin immunity gene nisI and nisin resistance gene nsr, were evaluated as dominant markers for the L. lactis food-grade expression system. By using an efficient PSlpAl promoter fused to a signal peptide from subtilisin YaB (SPYAB), a functional recombinant Ganoderma lucidium immunomodulatory protein rLZ8 was expressed extracellularly in L. lactis. Replacing the antibiotic marker gene into the proper food-grade selection marker nsr gene, the rLZ8 was expressed extracellularly in the food-grade L. lactis system. This study provides a rationale basis for a food-grade system to express functional peptides extracellularly as an important tool for oral administration of genetically modified L. lactis. [ABSTRACT FROM AUTHOR]

Abstract: The antibiotic susceptibility of wild Listeria monocytogenes strains and their corresponding nisin resistant variants was assessed. The resistant strains were more sensitive to most of the tested antibiotics than their wild-type counterparts. A slight increase in MIC was observed for a few antibiotics including the membrane disturbing polymixin B. Cross-resistance was detected with two synthetic antimicrobial peptides. A lower C15/C17 ratio in the membrane fatty acid composition of the nisin resistant strains was found, and one strain pair showed a significant difference in surface hydrophobicity. As judged by these results, no clear correlation could be established between resistance to nisin and to worldwide-used antibiotics. [Copyright &y& Elsevier]

Streptococcus thermophilus INIA 463 became nisin-resistant after exposure in skim milk to subminimal inhibitory concentrations of nisin (1–3 IU/ml) for less than 2 h. Addition of 20 IU/ml caused a 4 log unit decrease in S. thermophilus counts of a culture not exposed previously to nisin, whereas no decrease was observed in the culture exposed to nisin for 2 h. Transfer of immunity genes as responsible for nisin resistance was discarded. The presence of extracellular or intracellular specific nisin-degrading enzymes was not detected in the nisin-resistant variant of S. thermophilus INIA 463. Nisin resistance was caused by the induction of a resistance mechanism. Transmission electron microscopy (TEM) revealed that the nisin-resistant variant of S. thermophilus INIA 463 had a thickened cell wall compared to the wild strain. Resistance to nisin was lost after one transfer (4 h growth) in nisin-free skim milk. [Copyright &y& Elsevier]

The FDA-approved antimicrobial peptide nisin has been used to prevent contamination of food products from Gram-positive pathogens including Listeria monocytogenes (L. monocytogenes). However, some L. monocytogenes strains are tolerant or resistant to nisin treatment at a dose that would normally be lethal. Therefore, alternative or combined antimicrobial treatment strategies are required to effectively control L. monocytogenes contaminations. In this study, we investigate whether combination treatment with nisin and fatty acids can overcome the nisin resistance of L. monocytogenes by monitoring cell survival and biofilm inactivation of two strains (ATCC15313 serotype 1/2a and ATCC19115 serotype 4b). Lauric acid and N-tridecanoic acid were chosen because they exhibit antimicrobial activities. First, we observed that nisin (22.5 μg/mL) rapidly killed cells with up to a 6-log reduction after 3 h, but treatment for prolonged periods (i.e. , up to 24 h) caused the cells to recover growth via the development of either inherent nisin resistance or temporary nisin tolerance. However, co-treatment of nisin and each fatty acid significantly decreased the cell survival and hindered the development of nisin resistance. In addition, the co-treatment effectively inactivated cells in biofilms. The combined antimicrobial treatment may be promising approach in the food processing environment, where Listeria contamination occurs. Image 1 • Prolonged nisin treatment leads to resistance or tolerance of L. monocytogenes. • Co-treatment of nisin and lauric acid/N-tridecanoic acid rapidly inactivates cells. • The co-treatment impairs the development of nisin resistance. • The co-treatment of nisin and fatty acids efficiently inactivates cells in biofilms. [ABSTRACT FROM AUTHOR]