Novel activator of mannose-specific phosphotransferase system permease expression in Listeria innocua, identified by screening for pediocin AcH resistance.

To identify genes that are important for class IIa bacteriocin interaction and resistance in Listeria species, transposon Tn917 knockout libraries were constructed for Listeria innocua strain Lin11 and screened for mutants that are resistant to pediocin AcH. A highly resistant mutant (G7) (MIC > 20 microg/ml; 1,000-fold less susceptible than the wild type), in which the transposon integrated into the putative promoter of the lin0142 gene, was isolated. lin0142 is located immediately upstream of the mpt operon (mptA/mptC/mptD) that encodes the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system permease EIItMan, which serves as a docking protein for class IIa bacteriocins. The transcription of the mpt operon is known to be positively controlled by sigma54 factor and ManR (a sigma54-associated activator). Transcripts for lin0142 and mpt were undetectable in the G7 mutant, based on quantitative real-time reverse transcriptase PCR analysis. When the wild-type lin0142 gene was expressed at a 7.9-fold-elevated level in the mutant via a multicopy-number plasmid, the level of mpt mRNA became 70% higher than that in the wild-type strain. In addition, the complementation strain reverted back to the pediocin AcH-susceptible phenotype. The levels of manR and rpoN (sigma54) mRNAs were not directly influenced by the level of lin0142 transcription. lin0142 is the only one of the three mpt regulatory genes whose transcription is induced, albeit slightly (1.2-fold), by glucose. The combined results show that the lin0142 gene encodes a novel activator of the mpt operon. The Lin0142 protein contains a winged-helix DNA-binding motif and is distantly related to the Crp-Fnr family of transcription regulators.

Prevalence and Biological Control of Bacteriocin-Producing Psychrotrophic Leuconostocs Associated with Spoilage of Vacuum-Packaged Processed Meats.

Low heat-processed vacuum-packaged commercial meat products are expected to have a relatively long shelf life at refrigerated temperature. However, temperature abuse often occurs and spoilage of these products is not uncommon. One common lype of spoilage is characterized by accumulation of gas and purge in the package wilh products having sour lo off odor. Although laclic acid bacteria as well as several nonlaclic acid bacteria are pre- dominant microflora in the fresh products, we isolated predominantly Leuconostoc camosum and Leuconostoc mesenteroides from the spoiled products. Many of these isolales produced bacteriocins lo which the predominanl gram-posilive bacteria normally found in these products are sensilive. As the leuconoslocs grew al low lemperalure and produce bacleriocins, they inhibiled growlh of associaled sensitive bacleria. Since they are helerofermenlalive, they produced CO2 (resulting in accumulalion of gas in the package) and acids (resulting in reduclion in pH, accumulalion of purge (liquid), and sour and off-odor). The bacleriocinogenic isolales were sensilive lo nisin or a bacteriocin (probably nisin) produced by Lactococcus UW1. An approach in which either nisin or a similar bacteriocin was added to the product formulation could be effective to control growth of the leuconostocs in the products. Alternately, a low level of homofermentative lactic acid bacteria, that can produce nisin or similar bacteriocin at low temperature (such as Lactococcus UW1) could be incorporated in the package to control heterofermentative leuconostocs associated with spoilage of the meat products.

Bacteriocins of Lactic Acid Bacteria in Combination Have Greater Antibacterial Activity.

Antibacterial efficiency of two bacteriocins from lactic acid bacteria, pediocin AcH, and nisin was tested individually and in combination against several gram-positive bacterial strains including some involved in food spoilage and foodborne diseases. Pediocin AcH and nisin were more antibacterial in combination than when they were used alone. The principles of this greater antibacterial spectrum have been proposed. Bacteriocins in combinations can be used advantageously to design efficient natural food biopreservative(s).

Viability Loss of Foodborne Pathogens by Starter Culture Metabolites.

Antibacterial metabolites produced by starter culture bacteria were tested for inhibitory properties against foodborne pathogens capable of growing in foods at refrigeration temperature. Three culture preparations containing the bacteriocins pediocin AcH, nisin, and sakacin A produced by Pediococcus acidilactici H, Lactococcus lactis ssp. lactis DL 16, and Lactobacillus sake 706, respectively, were studied in addition to four commercial preparations: Nisaplin (similar to nisin), Na-lactate, diacetyl and Microgard. All bacteriocin preparations were bactericidal to the Listeria strains tested including nine strains of L. monocytogenes , but not to any of the gram-negative pathogens studied. Some strains of Listeria were lysed by pediocin AcH. Storage studies at 4°C indicated that the bacteriocin preparations did not have bacteriostatic properties against the surviving cells. Diacetyl produced some bactericidal effect against strains of Yersinia enterocolitica , Aeromonas hydrophila , pathogenic Escherichia coli , and Salmonella anatum , but not against Listeria . Lactate had limited bacteriostatic effect against gram-negative pathogens and none against the gram-positive bacteria. Microgard had neither bactericidal nor bacteriostatic action against the bacteria used in this study.

Spoilage of Vacuum-Packaged Refrigerated Beef by Clostridium.

A motile, gram-positive, spore forming, anaerobic, psychrotrophic bacterial species, probably from the genus Clostridium , was involved in spoilage of vacuum-packaged refrigerated fresh beef. The spoilage was associated with accumulation of large quantities of foul smelling gas and purge in the bag and loss of color and texture of the meat. Attempts to grow the organism in several laboratory media were not yet successful; however, inoculation of purge from a spoiled sample into a fresh beef, vacuum-packaging and refrigeration storage facilitated growth of this species and produced characteristic spoilage of beef.

Cellular Damage in Dried Lactobacillus acidophilus.

Lactobacillus acidophilus cells surviving freeze drying and vacuum drying became sensitive to oxgall and lysozyme probably from damage to the cell wall. The dried cells also became sensitive to NaCl and permeable to orthonitrophenol ß-galactoside from damage to the cytoplasmic membrane. Scanning electron microscopy indicated loss of some surface material from the damaged cells. Transmission electron microscopy (TEM) revealed partial loss of wall and membrane material, but these losses seemed to have resulted from the treatments given during fixation of cells for TEM and as a consequence of damage to the wall and membrane that occurred during drying. A surface protein of 46-kilodalton molecular weight, that is bound to the wall by hydrogen bonding, was also lost from the dried cells. It is postulated that drying adversely affects some weak bonds of the cellular macromolecules probably from the loss of bound water.

Impact of Bacterial Injury and Repair in Food Microbiology: its Past, Present and Future.

Studies done during the past 25 years revealed that microorganisms present in semipreserved foods can be injured by sublethal treatments. The injured cells, irrespective of differences in sublethal treatments, have similarities in their manifestation of injury and their repair. A simple resuscitation step incorporated into currently recommended isolation procedures would enable detection of these cells. In the future, microbial cell injury studies should be directed to include not only effective detection of index and pathogenic bacteria from foods, but also growth inhibition of spoilage microorganisms and preservation of lactic cultures.

Growth of Indicator, Pathogenic and Psychrotrophic Bacteria in Mechanically Separated Beef, Lean Ground Beef and Beef Bone Marrow 1.

Growth of Escherichia coli , Salmonella anatum , Staphylococcus aureus , Clostridium perfringens and naturally occurring psychrotrophs in mechanically separated beef (MSB), lean ground beef (LGB) and beef bone marrow (BBM) was studied. Six good grade steers were slaughtered and samples of MSB, LGB and BBM were prepared under proper sanitary care. Six hundred grams of each sample were collected; 100 g were used for chemical analysis. The remaining 500 g were divided into 10-g portions, each mixed with 10 ml of water and either frozen to -20°C or used immediately for bacteriological analysis. For growth studies, samples were inoculated with E. coli or one of the pathogens, incubated at 37°C up to 24 h and enumerated for colony forming units (CFU) on specific selective agar plates. During the first 8 h of incubation, E. coli and S. anatum multiplied rapidly in MSB and LGB but rather slowly in BBM. By 24 h, both species had multiplied to the same population level. Initial growth of S. aureus was rapid in MSB and LGB, but by 24 h its number was higher in LGB than in MSB or BBM. C. perfringens grew faster in LGB and slower in BBM during the 24-h period. Growth of psychrotrophs was determined by incubating the materials at 10, 7 and 3°C up to 10 d. The psychrotrophs grew fastest in MSB and slowest in LGB at all three temperatures, especially at 3°C. Rapid growth of various bacteria in MSB should be considered in its production, storage and subsequent use.

Bacteriology of Restructured Lamb Roasts Made with Mechanically Deboned Meat 1.

Composite samples of restructured lamb roast containing 10 or 30% mechanically deboned meat (MDM) were analyzed for bacteriological quality before and after cooking to 62.8°C (145°F). Uncooked samples had less than 3.0 × 104 colony forming units/g mesophilic and psychrotrophic aerobes and anaerobes including lactobacilli. In general, these groups, as well as coliforms and fecal coliforms, were present in higher numbers in uncooked roasts containing higher percentages of MDM. Staphylococcus aureus , Clostridium perfringens , Salmonella sp., Yersinia enterocolitica and Campylobacter jejuni were not detected in uncooked samples. Cooking reduced the number of aerobic and anaerobic spoilage bacteria and eliminated index bacteria (in 0.1 g) effectively.

Prevalence of Viable Lactobacillus acidophilus in Dried Commercial Products.

Samples of 27 dried acidophilus products used as dietary adjuncts for Lactobacillus acidophilus in humans, were enumerated for viable cells on plate count agar (PCA), MRS broth plus 1.5% agar (MRSA) and MRSA plus 0.15 % oxgall (MRSOA). Colony-forming units did not differ greatly on plating media because most viable cells formed colonies aerobically, anaerobically or in the presence of bile salts. Health food samples had very low numbers of viable cells and counts varied between samples from different lots. Only one of four brands from the health food group had viable L. acidophilus cells. Samples from this group had organisms other than lactobacilli, including coliforms and gram-negative, lactose-negative, motile rods. Most pharmaceutical and milk culture samples had high numbers of viable cells, but only two brands from pharmaceutical samples had viable cells of L. acidophilus . L. acidophilus strains were susceptible to drying, with vacuum drying being more lethal than freeze drying.

Cryoprotectants Protect Lipopolysaccharide Molecules of the Outer Membrane of Escherichia coli B from Freeze-Damage.

Lipopolysaccharide (LPS) molecules of the outer membrane (OM) of Escherichia coli B were damaged by freezing and thawing as evidenced by lysozyme lysis of cells frozen in water and their inability to adsorb LPS-specific phages. Permeating cryoprotectants, i.e., glycerol and dimethylsulfoxide (DMSO), protected this damage effectively, as the frozen cells were resistant against lysis by lysozyme and were able to adsorb phages. In contrast, non-permeating cryoprotectants, i.e., polyvinylpyrrolidone (PVP) and dextran, protected LPS molecules so that frozen cells were resistant against the lytic effect of lysozyme but were not able to adsorb specific phages effectively. Although all four cryoprotectants protected cells against viability loss due to freezing, survival was much higher with glycerol and DMSO than with PVP and dextran. The non-permeating cryoprotectants likely formed a physical barrier around the cell surface, whereas the permeating cryoprotectants did not form such a barrier.

Occurrence of Enteric Bacteria and Viruses in Oysters.

Enteric bacteria and virus levels were determined in oysters from paired stations that were opened or closed for commercial shellfishing on the basis of total coliform levels in the water. Six pairs of stations were sampled quarterly over a 1-year period. Enteric viruses were found in 3 of 24 50-g oyster samples from closed areas and in none of 23 samples from open areas. Salmonella was found in 2 of 47 samples of 40 g each, one from an open and the other from a closed area. Although enteric pathogens of fecal origin were found only in oysters that exceeded the recommended market limit of 230 fecal coliforms per 100 g of meat, the fecal coliform levels in some virus-positive samples were much lower than those in Salmonella -positive samples. Vibrio parahemolyticus levels were similar in oysters from both open and closed beds, indicating no particular association with fecal pollution. However, there was a marked seasonal variation in V. parahemolyticus levels. Total but not fecal coliform levels in oysters from open beds correlated with the occurrence of rainfall 1 or 2 days before sample collection. Neither total nor fecal coliform levels in oysters from closed beds correlated with rainfall. These findings suggest that fecal coliforms levels in oysters are less influenced by rainfall than are total coliforms, and therefore may be a more specific indicator of recent fecal pollution.

Methods to Detect Stressed Microorganisms 1.

A population of surviving bacteria, after a sublethal physical or chemical treatment, is composed of the stressed or injured and the uninjured or normal cells. Injured cells develop sensitivity to many chemical compounds due to damage to their permeability barriers in the cell wall and the cell membrane. They also fail to repair this damage in the presence of selective compounds and consequently fail to multiply. However, the cells can repair this damage relatively rapidly in a nutritionally adequate environment in the absence of selective agents. Repaired cells regain their resistance to the selective compound and can multiply in a selective environment. Many nonsterile or semipreserved foods are exposed to different kinds of sublethal treatments during processing, handling and storage and thus can contain indicator and pathogenic bacteria in the injured state. These organisms, because of their resistance to many selective compounds, are normally enumerated and isolated with selective media. The injured cells, due to their developed sensitivity will not be detected in these media. However, repaired cells can be detected in these media. Based on these principles two methods, designated as 'liquid-repair' and 'solid-repair' methods, have been developed in our laboratory. The liquid-repair method is effective for enumeration by the MPN technique and isolation of pathogenic and indicator bacteria from different types of semipreserved foods. The solid-repair method in principle can be used for direct enumeration of any organism which is usually enumerated by the selective plating procedure.