Resistance of Listeria monocytogenes biofilms to sanitizing agents in a simulated food processing environment

The objective of this study was to evaluate the resistance of biofilms of Listeria monocytogenes to sanitizing agents under laboratory conditions simulating a food processing environment. Biofilms were initially formed on stainless steel and Teflon coupons using a five-strain mixture of L. monocytogenes. The coupons were then subjected to repeated 24-h daily cycles. Each cycle consisted of three sequential steps: (i) a brief (60 s) exposure of the coupons to a sanitizing agent (a mixture of peroxides) or saline as a control treatment, (ii) storage of the coupons in sterile plastic tubes without any nutrients or water for 15 h, (iii) and incubation of the coupons in diluted growth medium for 8 h. This regimen was repeated daily for up to 3 weeks and was designed to represent stresses encountered by bacteria in a food processing environment. The bacteria on the coupons were reduced in number during the first week of the simulated food processing (SFP) regimen, but then adapted to the stressful conditions and increased in number. Biofilms repeatedly exposed the peroxide sanitizer in the SFP regimen developed resistance to the peroxide sanitizer as well as other sanitizers (quaternary ammonium compounds and chlorine). Interestingly, cells that were removed from the biofilms on peroxide-treated and control coupons were not significantly different in their resistance to sanitizing agents. These data suggest that the resistance of the treated biofilms to sanitizing agents may be due to attributes of extracellular polymeric substances and is not an intrinsic attribute of the cells in the biofilm.     

Control of Listeria spp. by competitive-exclusion bacteria in floor drains of a poultry processing plant

In previous studies workers determined that two lactic acid bacterium isolates, Lactococcus lactis subsp. lactis C-1-92 and Enterococcus durans 152 (competitive-exclusion bacteria [CE]), which were originally obtained from biofilms in floor drains, are bactericidal to Listeria monocytogenes or inhibit the growth of L. monocytogenes both in vitro and in biofilms at 4 to 37 degrees C. We evaluated the efficacy of these isolates for reducing Listeria spp. contamination of floor drains of a plant in which fresh poultry is processed. Baseline assays revealed that the mean numbers of Listeria sp. cells in floor drains sampled on six different dates (at approximately biweekly intervals) were 7.5 log(10) CFU/100 cm(2) for drain 8, 4.9 log(10) CFU/100 cm(2) for drain 3, 4.4 log(10) CFU/100 cm(2) for drain 2, 4.1 log(10) CFU/100 cm(2) for drain 4, 3.7 log(10) CFU/100 cm(2) for drain 1, and 3.6 log(10) CFU/100 cm(2) for drain 6. The drains were then treated with 10(7) CE/ml in an enzyme-foam-based cleaning agent four times in 1 week and twice a week for the following 3 weeks. In samples collected 1 week after CE treatments were applied Listeria sp. cells were not detectable (samples were negative as determined by selective enrichment culture) for drains 4 and 6 (reductions of 4.1 and 3.6 log(10) CFU/100 cm(2), respectively), and the mean numbers of Listeria sp. cells were 3.7 log(10) CFU/100 cm(2) for drain 8 (a reduction of 3.8 log(10) CFU/100 cm(2)), <1.7 log(10) CFU/100 cm(2) for drain 1 (detectable only by selective enrichment culture; a reduction of 3.3 log(10) CFU/100 cm(2)), and 2.6 log(10) CFU/100 cm(2) for drain 3 (a reduction of 2.3 log(10) CFU/100 cm(2)). However, the aerobic plate counts for samples collected from floor drains before, during, and after CE treatment remained approximately the same. The results indicate that application of the two CE can greatly reduce the number of Listeria sp. cells in floor drains at 3 to 26 degrees C in a facility in which fresh poultry is processed.     

Evaluation of chromogenic media for the detection of Listeria species in food

AIMS: The aim of this study was to evaluate the performance of chromogenic agars, Agar Listeria according to Ottaviani and Agosti (ALOA) and Rapid L. mono agar, compared with Oxford agar for the enumeration and detection of Listeria species in food. METHODS AND RESULTS: A total of 170 food samples were examined using the three plating media. Listeria species were isolated from 63 samples. In contrast to Oxford agar, detection of Listeria colonies on chromogenic media was as good after 24 h of incubation of plates as after 48 h. While there was no significant difference in recovery of Listeria monocytogenes on the three media, recovery of other Listeria species was significantly poorer on Rapid L. mono agar compared with Oxford and ALOA agars. Recovery of species other than L. monocytogenes was significantly improved by including a secondary enrichment stage in the detection method. CONCLUSIONS: Using chromogenic agars, presumptive identification of L. monocytogenes is possible after 24 h, compared with 3-4 days using Oxford agar. However, the poor detection of species other than L. monocytogenes on Rapid L. mono agar is a disadvantage of this medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information regarding the isolation of Listeria species other than L. monocytogenes from food using chromogenic plating media. This is important, as non-pathogenic Listeria species act as markers for the likelihood of presence of L. monocytogenes and allow preventive action to be taken to avoid its presence.     

Proteomic and microscopic analysis of biofilms formed by Listeria monocytogenes 568

Biofilm formation may be important in the colonization of the food-processing environment by the food-borne pathogen Listeria monocytogenes. Listeria monocytogenes 568 formed adherent multicellular layers on a variety of test surfaces following growth at 37 degrees C with multiple transfers of the test surface into fresh medium. Microscopic examination of these adherent layers suggest that the cells were surrounded by extracellular material. The presence of a carbohydrate containing extracellular polymeric matrix was confirmed by labelling hydrated adherent layers with fluorescein-conjugated concanavalin A, indicating that these adherent layers are biofilms. To gain insight into the physiological state of cells in these biofilms, the proteomes from biofilm- and planktonic-grown cells from the same cultures were compared using 2-dimensional polyacrylamide gel electrophoresis. Nineteen proteins, which exhibited higher levels of expression in biofilm-grown cells, were successfully identified from the 2-D gels using a combination of MALDI-TOF and MS/MS. Proteins that were found to be more highly expressed in biofilm-grown cells were involved in stress response, envelope and protein synthesis, biosynthesis, energy generation, and regulatory functions. In biofilm-grown cells, many proteins in the pH range 4-6 ran as multiple spots arranged horizontally across the 2-D gels.     

Molecular analysis of microbial communities in endotracheal tube biofilms

Background

Ventilator-associated pneumonia is the most prevalent acquired infection of patients on intensive care units and is associated with considerable morbidity and mortality. Evidence suggests that an improved understanding of the composition of the biofilm communities that form on endotracheal tubes may result in the development of improved preventative strategies for ventilator-associated pneumonia.

Methodology/Principal Findings

The aim of this study was to characterise microbial biofilms on the inner luminal surface of extubated endotracheal tubes from ICU patients using PCR and molecular profiling. Twenty-four endotracheal tubes were obtained from twenty mechanically ventilated patients. Denaturing gradient gel electrophoresis (DGGE) profiling of 16S rRNA gene amplicons was used to assess the diversity of the bacterial population, together with species specific PCR of key marker oral microorganisms and a quantitative assessment of culturable aerobic bacteria. Analysis of culturable aerobic bacteria revealed a range of colonisation from no growth to 2.1×10⁸ colony forming units (cfu)/cm² of endotracheal tube (mean 1.4×10⁷ cfu/cm²). PCR targeting of specific bacterial species detected the oral bacteria Streptococcus mutans (n = 5) and Porphyromonas gingivalis (n = 5). DGGE profiling of the endotracheal biofilms revealed complex banding patterns containing between 3 and 22 (mean 6) bands per tube, thus demonstrating the marked complexity of the constituent biofilms. Significant inter-patient diversity was evident. The number of DGGE bands detected was not related to total viable microbial counts or the duration of intubation.

Conclusions/Significance

Molecular profiling using DGGE demonstrated considerable biofilm compositional complexity and inter-patient diversity and provides a rapid method for the further study of biofilm composition in longitudinal and interventional studies. The presence of oral microorganisms in endotracheal tube biofilms suggests that these may be important in biofilm development and may provide a therapeutic target for the prevention of ventilator-associated pneumonia.     

Molecular studies on the ecology of Listeria monocytogenes in the smoked fish processing industry

We have applied molecular approaches, including PCR-based detection strategies and DNA fingerprinting methods, to study the ecology of Listeria monocytogenes in food processing environments. A total of 531 samples, including raw fish, fish during the cold-smoking process, finished product, and environmental samples, were collected from three smoked fish processing facilities during five visits to each facility. A total of 95 (17.9%) of the samples tested positive for L. monocytogenes using a commercial PCR system (BAX for Screening/Listeria monocytogenes), including 57 (27.7%) environmental samples (n = 206), 8 (7.8%) raw material samples (n = 102), 23 (18.1%) samples from fish in various stages of processing(n = 127), and 7 (7.3%) finished product samples (n = 96). L. monocytogenes was isolated from 85 samples (16.0%) using culture methods. Used in conjunction with a 48-h enrichment in Listeria Enrichment Broth, the PCR system had a sensitivity of 91.8% and a specificity of 96.2%. To track the origin and spread of L. monocytogenes, isolates were fingerprinted by automated ribotyping. Fifteen different ribotypes were identified among 85 isolates tested. Ribotyping data established possible contamination patterns, implicating raw materials and the processing environment as potential sources of finished product contamination. Analysis of the distribution of ribotypes revealed that each processing facility had a unique contamination pattern and that specific ribotypes persisted in the environments of two facilities over time (P < or = 0.0006). We conclude that application of molecular approaches can provide critical information on the ecology of different L. monocytogenes strains in food processing environments. This information can be used to develop practical recommendations for improved control of this important food-borne pathogen in the food industry.     

Microbial characterization of biofilms in domestic drains and the establishment of stable biofilm microcosms

We have used heterotrophic plate counts, together with live-dead direct staining and denaturing gradient gel electrophoresis (DGGE), to characterize the eubacterial communities that had formed as biofilms within domestic sink drain outlets. Laboratory microcosms of these environments were established using excised biofilms from two separate drain biofilm samples to inoculate constant-depth film fermentors (CDFFs). Drain biofilms harbored 9.8 to 11.3 log(10) cells of viable enteric species and pseudomonads/g, while CDFF-grown biofilms harbored 10.6 to 11.4 log(10) cells/g. Since live-dead direct staining revealed various efficiencies of recovery by culture, samples were analyzed by DGGE, utilizing primers specific for the V2-V3 region of eubacterial 16S rDNA. These analyses showed that the major PCR amplicons from in situ material were represented in the microcosms and maintained there over extended periods. Sequencing of amplicons resolved by DGGE revealed that the biofilms were dominated by a small number of genera, which were also isolated by culture. One drain sample harbored the protozoan Colpoda maupasi, together with rhabtidid nematodes and bdelloid rotifers. The microcosm enables the maintenance of stable drain-type bacterial communities and represents a useful tool for the modeling of this ecosystem.     

Antibiotic resistance in Listeria species isolated from catfish fillets and processing environment

Aims: To investigate the susceptibility of 221 Listeria spp. (86 Listeria monocytogenes, 41 Listeria innocua and 94 Listeria seeligeri‐Listeria welshimeri‐Listeria ivanovii) isolated from catfish fillets and processing environment to 15 antibiotics. Methods and Results: Listeria isolates were analysed by disc‐diffusion assay for their resistance to 15 drugs. All isolates were resistant to cefotaxime and clindamycin but were sensitive to ampicillin, cephalothin, chloramphenicol, erythromycin, gentamycin, kanamycin, rifampin, streptomycin, sulfamethoxazole/trimethoprim and vancomycin. Unlike L. monocytogenes and L. seeligeri‐L. welshimeri‐L. ivanovii isolates, 22% of L. innocua isolates displayed tetracycline/oxytetracycline resistance. Screening of tet genes by PCR identified tet(M) gene in the chromosome of all tetracycline/oxytetracycline‐resistant L. innocua. However, this gene was not associated with the integrase gene of Tn1545. Repetitive extragenic palindromic‐ and enterobacterial repetitive intergenic consensus‐PCR typing methods showed no genotype‐specific tetracycline resistance in the tet(M)‐positive strains. Conclusions: Catfish fillets and processing environment were currently free of L. monocytogenes resistant to antibiotics commonly used in human listeriosis treatment. However, the presence of tet(M) gene in L. innocua raises the possibility of future acquisition of resistance by L. monocytogenes. Significance and Impact of the Study: These data will be helpful in improving background data on antibiotics resistance strains isolated from food and processing environment.     

Molecular and physiological characterization of dominant bacterial populations in traditional mozzarella cheese processing

The development of the dominant bacterial populations during traditional Mozzarella cheese production was investigated using physiological analyses and molecular techniques for strain typing and taxonomic identification. Analysis of RAPD fingerprints revealed that the dominant bacterial community was composed of 25 different biotypes, and the sequence analysis of 16S rDNA demonstrated that the isolated strains belonged to Leuconostoc mesenteroides subsp. mesenteroides , Leuc. lactis , Streptococcus thermophilus , Strep. bovis , Strep. uberis, Lactococcus lactis subsp. lactis , L. garviae, Carnobacterium divergens , C. piscicola, Aerococcus viridans , Staphylococcus carnosus, Staph. epidermidis , Enterococcus faecalis , Ent. sulphureus and Enterococcus spp. The bacterial populations were characterized for their physiological properties. Two strains, belonging to Strep. thermophilus and L. lactis subsp. lactis , were the most acidifying; the L. lactis subsp. lactis strain was also proteolytic and eight strains were positive to citrate fermentation. Moreover, the molecular techniques allowed the identification of potential pathogens in a non-ripened cheese produced from raw milk.     

Molecular analysis of tet(W) gene-mediated tetracycline resistance in dominant intestinal Bifidobacterium species from healthy humans

tet(W) was found responsible for tetracycline resistance (MICs, 4 to > or =32 microg ml(-1)) in dominant bifidobacterial species from the gastrointestinal tracts of healthy humans. The gene from Bifidobacterium longum H66 proved to be identical over a 2.6-kbp region to the recently described tet(W) determinant of Butyrivibrio fibrisolvens.     

Molecular phylogenetic analysis of dominant microbial populations in aged refuse

The phylogenetic analysis of dominant microbial populations in 8-year-old refuse samples was done in terms of the whole Bacterial and Archaeal domains. The results indicated that the Bacterial 16S rRNA genes sequences from the aged refuse were largely affiliated with the genus Bacillus, and that more than 60 % of the Archaeal sequences were closely related to the methanogenic archaeon. Some inferentially identified extremophilic organisms, particularly alkaliphiles and/or halophiles, were noted to be present in the aged refuse. Moreover, molecular evidence for the occurrence of ammonia-oxidizing Archaea in aged refuse was reported, which opens up avenues for elucidating its role in ammonia transformation in landfill systems. It seems reasonable to assume that the highly complex environment within the landfill systems may select for microbial populations with versatile metabolism and strong adaptation. These findings underline the need for further biochemical and ecological study of these organisms in aged refuse.     

Quantitative detection of Listeria monocytogenes in biofilms by real-time PCR

A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 x 10(2) CFU/cm2.     

A shift in the dominant toxin-producing algal species in central California alters phycotoxins in food webs

In California, the toxic algal species of primary concern are the dinoflagellate Alexandrium catenella and members of the pennate diatom genus Pseudo-nitzschia, both producers of potent neurotoxins that are capable of sickening and killing marine life and humans. During the summer of 2004 in Monterey Bay, we observed a change in the taxonomic structure of the phytoplankton community—the typically diatom-dominated community shifted to a red tide, dinoflagellate-dominated community. Here we use a 6-year time series (2000–2006) to show how the abundance of the dominant harmful algal bloom (HAB) species in the Bay up to that point, Pseudo-nitzschia, significantly declined during the dinoflagellate-dominated interval, while two genera of toxic dinoflagellates, Alexandrium and Dinophysis, became the predominant toxin producers. This change represents a shift from a genus of toxin producers that typically dominates the community during a toxic bloom, to HAB taxa that are generally only minor components of the community in a toxic event. This change in the local HAB species was also reflected in the toxins present in higher trophic levels. Despite the small contribution of A. catenella to the overall phytoplankton community, the increase in the presence of this species in Monterey Bay was associated with an increase in the presence of paralytic shellfish poisoning (PSP) toxins in sentinel shellfish and clupeoid fish. This report provides the first evidence that PSP toxins are present in California's pelagic food web, as PSP toxins were detected in both northern anchovies (Engraulis mordax) and Pacific sardines (Sardinops sagax). Another interesting observation from our data is the co-occurrence of DA and PSP toxins in both planktivorous fish and sentinel shellfish. We also provide evidence, based on the statewide biotoxin monitoring program, that this increase in the frequency and abundance of PSP events related to A. catenella occurred not just in Monterey Bay, but also in other coastal regions of California. Our results demonstrate that changes in the taxonomic structure of the phytoplankton community influences the nature of the algal toxins that move through local food webs and also emphasizes the importance of monitoring for the full suite of toxic algae, rather than just one genus or species.     

Limitation in the detection of Listeria monocytogenes in food in the presence of competing Listeria innocua

Aims: Detectability of Listeria monocytogenes at 10⁰ CFU per food sample in the presence of Listeria innocua using standard microbiological detection was evaluated and compared with the real‐time PCR‐based method. Methods and Results: Enrichment in half‐Fraser broth followed by subculture in Fraser broth according to EN ISO 11290‐1 was used. False‐negative detection of 10⁰ CFU L. monocytogenes was obtained in the presence of 10¹ CFU L. innocua per sample using the standard detection method in contrast to more than 10⁵ CFU L. innocua per sample using real‐time PCR. Identification of L. monocytogenes on the chromogenic medium by the standard procedure was impossible if L. innocua was able to overgrow L. monocytogenes by more than three orders of magnitude after the enrichment in model samples. These results were confirmed using naturally contaminated food samples. Conclusions: Standard microbiological method was insufficient for the reliable detection of 10⁰ CFU L. monocytogenes in the presence of more than 10⁰ CFU of L. innocua per sample. On the other hand, if the growth of L. monocytogenes was sufficient to reach the concentration equal to the detection limit of PCR, the amount of the other microflora present in the food sample including L. innocua was not relevant for success of the PCR detection of L. monocytogenes. Significance and Impact of the Study: After the enrichment, the PCR detection is more convenient than the standard one as PCR detection is not compromised by other present microflora.     

Plasmids in Listeria monocytogenes and other Listeria species.

One hundred and twenty-two food, clinical, and veterinary strains of Listeria monocytogenes were examined for the presence of plasmids. Twenty-five (20%) contained plasmids, which varied from 1.3 to 66 MDa in size. Of 10 strains of other Listeria species (L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, L. grayi, and L. murrayi) examined, seven (70%) contained plasmids, varying from 38 to 53 MDa. No strains with multiple plasmids were found. Plasmids of identical size were isolated from related strains in some, although not all, cases. The presence of a plasmid in a strain was not related to phenotypic characters of known extrachromosomal inheritance.     

The characterization of Listeria spp. isolated from food products and the food‐processing environment

Aim: To enhance the information pertaining to the epidemiology of a collection of 378 Listeria spp. isolates obtained from several food‐processing plants in Ireland over a 3‐ year period (2004–2007). Methods and results: The collection was characterized by pulsed‐field gel electrophoresis (PFGE). The most prevalent pulse‐type was PFGE profile I (n = 14·5%) that consisted mainly of environmental Listeria spp. samples. Serotyping of 145 Listeria monocytogenes isolates was performed. The most common serovar was 1/2a and comprised 57·4% (n = 77) of the L. monocytogenes collection. The other serovars were as follows: 4b (14·1%, n = 19), 1/2b (9·7%, n = 13), 4c (4·4%, n = 6) and 1/2c (6·7%, n = 9), respectively. Eleven isolates were identified as non‐Listeria spp., the remaining ten L. monocytogenes isolates were nontypeable. The antimicrobial susceptibility testing revealed the antibiotic that isolates displayed the most resistance to was gentamicin (5%) followed by sulfamethoxazole‐trimethoprim (2%), tetracycline and ciprofloxacin (1·5%). Conclusions: The subtyping has indicated the diversity of the Listeria spp. The presence of serotype 1/2a, 1/2b and 4b in both raw and cooked ready‐to‐eat food products is a public health concern, as these serotypes are frequently associated with foodborne outbreaks and sporadic cases of human listeriosis. In addition, the emergence of antimicrobial‐resistant L. monocytogenes isolates could have serious therapeutic consequences. Significance and Impact of Study: The molecular subtyping and the further characterization of these isolates may be valuable particularly in the context of a suspected common source outbreak in the future.     

Molecular cytogenetic analysis of the Vigna species distributed in Korea

Vigna plants distributed in Korea were analyzed by molecular cytogenetic fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and rDNA ITS/NTS sequences. FISH revealed that variable 45S rRNA gene loci (one to four) were localized on the terminal regions of chromosomes, while two conserved 5S rRNA gene loci from all species examined, except for rice bean (single locus), were detected. FISH and GISH showed the characteristic organization of rRNA gene loci and genomic homology on the chromosomes, indicating their cytogenetic relatationships. ITS sequence revealed that there was considerable variation in length (190–207 bp in ITS1, 205–221 bp in ITS2) and nucleotide composition (7–67 bp). The 5S rRNA gene unit comprised coding region (118 bp) and extensive sequence heterogeneity (97–221 bp). Phylogenetic analysis of the ITS and NTS sequences demonstrated that the Vigna species are divided into two groups: angularis (V. angularis, V. umbellata, V. nakashimae and V. nipponensis) and unguiculata (V. unguiculata, V. sesquipedalis and V. vexillata). Sequence data also showed that mung bean was closer to the angularis group.