Spontaneous bacteriocin resistance in Listeria monocytogenes as a susceptibility screen for identifying different mechanisms of resistance and modes of action by bacteriocins of lactic acid bacteria.

A practical system was devised for grouping bacteriocins of lactic acid bacteria (LAB) based on mode of action as determined by changes in inhibitory activity to spontaneously-acquired bacteriocin resistance (Bac(R)). Wild type Listeria monocytogenes 39-2 was sensitive to five bacteriocins produced by 3 genera of LAB: pediocin PA-1 and pediocin Bac3 (Pediococcus), lacticin FS97 and lacticin FS56 (Lactococcus), and curvaticin FS47 (Lactobacillus). A spontaneous Bac(R) derivative of L. monocytogenes 39-2 obtained by selective recovery against lacticin FS56 provided complete resistance to the bacteriocin made by Lactococcus lactis FS56. The lacticin FS56-resistant strain of L. monocyotgenes 39-2 was also cross-resistant to curvaticin FS47 and pediocin PA-1, but not to lacticin FS97 or pediocin Bac3. The same pattern of cross-resistance was also observed with Bac(R) isolates obtained with L. monocytogenes Scott A-2. A spontaneous mutation that renders a strain cross-resistant to different bacteriocins indicates that they share a common mechanism of resistance due to similar modes of action of the bacteriocins. Spontaneous resistance was acquired to other bacteriocins (in aggregate) by following the same procedure against which the Bac(R) strain was still sensitive. In subsequent challenge assays, mixtures of bacteriocins of different modes of action provided greater inhibition than mixtures of bacteriocins of the same mode of action (as determined by our screening method). This study identifies a methodical approach to classify bacteriocins into functional groups based on mechanism of resistance (i.e., mode of action) that could be used for identifying the best mixture of bacteriocins for use as biopreservatives.

A 'bacteriocin PCR array' for identification of bacteriocin-related structural genes in lactic acid bacteria.

Bacteriocins have been identified in many strains of lactic acid bacteria (LAB) which are a source of natural food preservatives and microbial inhibitors. Our objectives were to use a PCR array of primers to identify bacteriocin structural genes in Bac(+) LAB. DNA sequence homology at the 5'- and 3'-ends of the various structural genes indicated that non-specific priming may allow PCR amplification of heterologous bacteriocin genes. Successful amplification was obtained by real-time PCR and confirmed by melting curve and agarose gel analysis. Sequence information specific to targeted bacteriocin structural genes from the intra-primer regions of amplimers was compared to sequences residing in GenBank. The bacteriocin PCR array allowed the successful amplification of bacteriocin structural genes from strains of Lactobacillus, Lactococcus, and Pediococcus including one whose amino acid sequence was unable to be determined by Edman degradation analysis. DNA sequence analysis identified as many as 3 bacteriocin structural genes within a given strain, identifying ten unique bacteriocin sequences that were previously uncharacterized (partial homology) and one that was 100% identical to sequences in GenBank. This study provides a rapid approach to sequence and identify bacteriocin structural genes among Bac(+) LAB using a microplate bacteriocin PCR array.

Effect of inhibitory extracts derived from liquid smoke combined with postprocess pasteurization for control of Listeria monocytogenes on ready-to-eat meats.

Surface pasteurization was examined in combination with low-phenolic antimicrobial extracts derived from liquid smoke to inhibit and prevent the growth of Listeria monocytogenes during the shelf life of ready-to-eat meats. In preliminary trials with retail frankfurters, one smoke derivative (2-min dip) produced a 0.3-log reduction of L. monocytogenes and a 1-min in-bag pasteurization (73.9 degrees C) produced a 2.9-log reduction, whereas a combination of the two treatments produced a 5.3-log reduction that resulted in no detectable Listeria by week 3 under accelerated shelf-life conditions (10 degrees C). In trials with frankfurters manufactured without lactate or diacetate that were treated with a shortened 1-s dip, this smoke extract and one with reduced smoke flavor and color both produced a > 4.5-log reduction of L. monocytogenes on frankfurters when heated at 73.9 degrees C for 1 min, with no recoverable Listeria detected for 10 weeks when stored at 6.1 degrees C. When deli turkey breast chubs manufactured without lactate, diacetate, or nitrite were treated with a 1-s dip in combination with radiant-heat pasteurization (270 degrees C), growth of L. monocytogenes was retarded but not prevented. However, in a similar study in which smoke extract treatment of deli turkey breast was combined with in-bag postpackage pasteurization (water submersion at 93.3 degrees C), a 60-, 45-, or even 30-s heat treatment resulted in a 2- to 3-log reduction of L. monocytogenes, with no growth on the meat during 10 weeks of storage at 6.1 degrees C. These findings indicate that reduced-acid low-phenolic antimicrobial liquid smoke derivatives combined with surface pasteurization are capable of reducing or preventing growth of L. monocytogenes to meet the criteria for the U.S. Department of Agriculture Food Safety and Inspection Service Alternative 1 process for ready-to-eat deli meat products manufactured without lactate or diacetate.

Negative subtraction hybridization: an efficient method to isolate large numbers of condition-specific cDNAs.

BACKGROUND: The construction of cDNA libraries is a useful tool to understand gene expression in organisms under different conditions, but random sequencing of unbiased cDNA collections is laborious and can give rise to redundant EST collections. We aimed to isolate cDNAs of messages induced by switching Aspergillus nidulans from growth on glucose to growth on selected polysaccharides. Approximately 4,700 contigs from 12,320 ESTs were already available from a cDNA library representing transcripts isolated from glucose-grown A. nidulans during asexual development. Our goals were to expand the cDNA collection without repeated sequencing of previously identified ESTs and to find as many transcripts as possible that are specifically induced in complex polysaccharide metabolism. RESULTS: We have devised a Negative Subtraction Hybridization (NSH) method and tested it in A. nidulans. NSH entails screening a plasmid library made from cDNAs prepared from cells grown under a selected physiological condition with labeled cDNA probes prepared from another physiological condition. Plasmids with inserts that failed to hybridize to cDNA probes through two rounds of screening (i.e. negatives) indicate that they are transcripts present at low concentration in the labeled probe pool. Thus, these transcripts will be predominantly condition-specific, along with some rare transcripts. In a screen for transcripts induced by switching the carbon source from glucose to 12 selected polysaccharides, 3,532 negatives were isolated from approximately 100,000 surveyed colonies using this method. Negative clones were end-sequenced and assembled into 2,039 contigs, of which 1,722 were not present in the previously characterized glucose-grown cDNA library. Single-channel microarray hybridization experiments confirmed that the majority of the negatives represented genes that were differentially induced by a switch from growth in glucose to one or more of the polysaccharides. CONCLUSIONS: The Negative Subtraction Hybridization method described here has several practical benefits. This method can be used to screen any existing cDNA library, including full-length and pooled libraries, and does not rely on PCR or sequence information. In addition, NSH is a cost-effective method for the isolation of novel, full-length cDNAs for differentially expressed transcripts or enrichment of rare transcripts.