Induction of the Viable-but-Nonculturable State in Salmonella Contaminating Dried Fruit.

Salmonella can become viable but nonculturable (VBNC) in response to environmental stressors, but the induction of the VBNC state in Salmonella contaminating ready-to-eat dried fruit is poorly characterized. Dried apples, strawberries, and raisins were mixed with a five-strain cocktail of Salmonella at 4% volume per weight of dried fruit at 109 CFU/g. The inoculated dried fruit were then dried in desiccators at 25°C until the water activity (aw) approximated that of the uninoculated dried fruit. However, Salmonella could not be recovered after drying, not even after enrichment, suggesting a population reduction of approximately 8 log CFU/g. To assess the potential impact of storage temperature on survival, dried apples were spot-inoculated with the Salmonella cocktail, dried under ambient atmosphere at 25°C, and stored at 4 and 25°C. Spot inoculation permitted recovery of Salmonella on dried apple after drying, with the population of Salmonella decreasing progressively on dried apples stored at 25°C until it was undetectable after about 46 days, even following enrichment. The population decline was noticeably slower at 4°C, with Salmonella being detected until 82 days. However, fluorescence microscopy and laser scanning confocal microscopy with the LIVE/DEAD BacLight bacterial viability system at time points at which no Salmonella could be recovered on growth media even following enrichment showed that a large proportion (56 to 85%) of the Salmonella cells on the dried fruit were viable. The data suggest that the unique combination of stressors in dried fruit can induce large numbers of VBNC cells of Salmonella. IMPORTANCE Salmonella is a leading foodborne pathogen globally causing numerous outbreaks of foodborne illnesses and remains the leading contributor to deaths attributed to foodborne disease in the United States and other industrialized nations. Therefore, efficient detection methods for Salmonella contaminating food are critical for public health and food safety. Culture-based microbiological methods are considered the gold standard for the detection and enumeration of Salmonella in food. Findings from this study suggest that unique stressors on dried fruit can induce the VBNC state in Salmonella, thus rendering it undetectable with culture-based methods even though the bacteria remain viable. Therefore, strong consideration should be given to using, in addition to culture-based methods, microscopic and molecular methods for the accurate detection of all viable and/or culturable cells of Salmonella contaminating dried fruit, as all of these cells have the potential to cause human illness.

Validation of an ampicillin selection protocol to enrich for mutants of Listeria monocytogenes unable to replicate on fresh produce.

Several outbreaks of listeriosis have implicated fresh produce but genetic factors required for growth of Listeria monocytogenes on produce remain poorly characterized. Based on the fact that ß-lactam antibiotics only kill bacterial cells that are growing, we hypothesized that ampicillin selection can enrich for L. monocytogenes mutants unable to grow on produce. For validation, we examined relative recovery of L. monocytogenes strain 2011L-2858 and its cold-sensitive mutant L1E4 following inoculation of cantaloupe rind fragments with 1:1 mixture of the strains and incubation at 4°C with or without ampicillin. Listeria monocytogenes from rind fragments inoculated with the mixed cultures and incubated in the presence of ampicillin were used to inoculate fresh rind fragments for a second round of enrichment. In the presence of ampicillin, the proportion of L1E4 increased from 55% on day 0 to 78% on day 14, with higher recovery (85% after 14 days) in the second round of enrichment. These data suggested that L1E4 was enriched on cantaloupe rind fragments while growing cells of the wildtype were killed by ampicillin. Application of this protocol to transposon mutant libraries from three L. monocytogenes strains yielded several mutants unable to grow on cantaloupe. Thus, ampicillin selection can facilitate discovery of genes essential for growth of L. monocytogenes on fresh produce.

Novel plasmid conferring kanamycin and tetracycline resistance in the turkey-derived Campylobacter jejuni strain 11601MD.

In Campylobacter spp., resistance to the antimicrobials kanamycin and tetracycline is frequently associated with plasmid-borne genes. However, relatively few plasmids of Campylobacter jejuni have been fully characterized to date. A novel plasmid (p11601MD; 44,095nt) harboring tet(O) was identified in C. jejuni strain 11601MD, which was isolated from the jejunum of a turkey produced conventionally in North Carolina. Analysis of the p11601MD sequence revealed the presence of a high-GC content cassette with four genes that included tet(O) and a putative aminoglycoside transferase gene (aphA-3) highly similar to kanamycin resistance determinants. Several genes putatively involved in conjugative transfer were also identified on the plasmid. These findings will contribute to a better understanding of the distribution of potentially self-mobilizing plasmids harboring antibiotic resistance determinants in Campylobacter spp. from turkeys and other sources.

Genetic determinants for cadmium and arsenic resistance among Listeria monocytogenes serotype 4b isolates from sporadic human listeriosis patients.

In Listeria monocytogenes serotype 4b isolates from sporadic listeriosis, heavy metal resistance was primarily encountered in certain clonal groups (ECI, ECII, and ECIa). All arsenic-resistant isolates harbored the arsenic resistance cassette previously identified in pLI100; ECIa harbored additional arsenic resistance genes and a novel cadmium resistance determinant in a conserved chromosomal locus.

Chromosomal tet(O)-harboring regions in Campylobacter coli isolates from turkeys and swine.

In turkey-derived Campylobacter coli isolates of a unique lineage (cluster II), the tetracycline resistance determinant tet(O) was chromosomal and was part of a gene cassette (transposon) interrupting a Campylobacter jejuni-associated putative citrate transporter gene. In contrast, the swine-derived C. coli strain 6461 harbored a chromosomal tet(O) in a different genomic location.

Coselection of cadmium and benzalkonium chloride resistance in conjugative transfers from nonpathogenic Listeria spp. to other Listeriae.

Resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) may be an important contributor to the ability of Listeria spp. to persist in the processing plant environment. Although a plasmid-borne disinfectant resistance cassette (bcrABC) has been identified in Listeria monocytogenes, horizontal transfer of these genes has not been characterized. Nonpathogenic Listeria spp. such as L. innocua and L. welshimeri are more common than L. monocytogenes in food processing environments and may contribute to the dissemination of disinfectant resistance genes in listeriae, including L. monocytogenes. In this study, we investigated conjugative transfer of resistance to BC and to cadmium from nonpathogenic Listeria spp. to other nonpathogenic listeriae, as well as to L. monocytogenes. BC-resistant L. welshimeri and L. innocua harboring bcrABC, along with the cadmium resistance determinant cadA2, were able to transfer resistance to other nonpathogenic listeriae as well as to L. monocytogenes of diverse serotypes, including strains from the 2011 cantaloupe outbreak. Transfer among nonpathogenic Listeria spp. was noticeably higher at 25°C than at 37°C, whereas acquisition of resistance by L. monocytogenes was equally efficient at 25 and 37°C. When the nonpathogenic donors were resistant to both BC and cadmium, acquisition of cadmium resistance was an effective surrogate for transfer of resistance to BC, suggesting coselection between these resistance attributes. The results suggest that nonpathogenic Listeria spp. may behave as reservoirs for disinfectant and heavy metal resistance genes for other listeriae, including the pathogenic species L. monocytogenes.

Two novel type II restriction-modification systems occupying genomically equivalent locations on the chromosomes of Listeria monocytogenes strains.

Listeria monocytogenes is responsible for the potentially life-threatening food-borne disease listeriosis. One epidemic-associated clonal group of L. monocytogenes, epidemic clone I (ECI), harbors a Sau3AI-like restriction-modification (RM) system also present in the same genomic region in certain strains of other lineages. In this study, we identified and characterized two other, novel type II RM systems, LmoJ2 and LmoJ3, at this same locus. LmoJ2 and LmoJ3 appeared to recognize GCWGC (W = A or T) and GCNGC, respectively. Both RM systems consisted of genes with GC content below the genome average and were in the same genomic region in strains of different serotypes and lineages, suggesting site-specific horizontal gene transfer. Genomic DNA from the LmoJ2 and LmoJ3 strains grown at various temperatures (4 to 42°C) was resistant to digestion with restriction enzymes recognizing GCWGC or GCNGC, indicating that the methyltransferases were expressed under these conditions. Phages propagated in an LmoJ2-harboring strain exhibited moderately increased infectivity for this strain at 4 and 8°C but not at higher temperatures, while phages propagated in an LmoJ3 strain had dramatically increased infectivity for this strain at all temperatures. Among the sequenced Listeria phages, lytic phages possessed significantly fewer recognition sites for these RM systems than lysogenic phages, suggesting that in lytic phages sequence content evolved toward reduced susceptibility to such RM systems. The ability of LmoJ2 and LmoJ3 to protect against phages may affect the efficiency of phages as biocontrol agents for L. monocytogenes strains harboring these RM systems.

Effect of direct-fed microbials on performance and Clostridium perfringens colonization of turkey poults.

Clostridium perfringens is recognized as an enteric pathogen in humans, domestic animals, and livestock. This organism is associated with necrotic enteritis, gangrenous dermatitis, clostridial dermatitis (turkeys), and gizzard erosions in poultry. This study was conducted to evaluate the effectiveness of a direct-fed microbial (DFM), Primalac (Star Labs, Clarksdale, MO), in preventing intestinal colonization of turkey poults with C. perfringens. One-day-old turkey poults (n = 128) were randomly divided into 4 treatments with 4 replicates (8 birds/pen). Treatments were as follows: 1) basal diet without DFM (C); 2) basal diet supplemented with Primalac (1.5 kg/ton; PM); 3) basal diet with poults gavaged with C. perfringens (CCP); and 4) basal diet supplemented with Primalac and poults gavaged with C. perfringens (PMCP). Feed and water were provided ad libitum throughout the trials, and birds were inoculated with C. perfringens (10(8)cfu/mL) on d 3 and 7. On d 21, 2 birds/pen were killed, spleen and bursa of Fabricius were collected and weighed, and cecal contents were used for C. perfringens enumeration. Feed consumption, BW, and feed conversion were calculated throughout the trial (weekly and cumulatively). Data were analyzed using GLM of SAS (SAS Institute, Cary, NC; P < 0.05). Among the inoculated groups, birds fed the DFM-supplemented diet had significantly lower cecal C. perfringens counts than the birds fed the diet without the DFM. The C. perfringens (log(10) cfu/g) in ceca were as follows: C, 5.88; CCP, 7.26; PM, 5.35; PMCP, 6.19 ± 0.36. No differences were observed for BW (814 ± 11 g), feed conversion (1.33 ± 0.03), organ weights, or relative organ weights. Further studies are needed to fully ascertain the potential of using DFM to reduce the numbers of C. perfringens in the gastrointestinal tract of turkey poults.

Inactivation of a cold-induced putative rna helicase gene of Listeria monocytogenes is accompanied by failure to grow at low temperatures but does not affect freeze-thaw tolerance.

Freeze-thaw tolerance (cryotolerance) of Listeria monocytogenes is markedly influenced by temperature of growth of the bacteria, and may involve responses to low-temperature stresses encountered during freezing and thawing. A cold-sensitive mariner-based transposon mutant of L. monocytogenes F2365 was found to harbor a single insertion in LMOf2365_1746, encoding a putative RNA helicase, and earlier shown by other investigators to be induced during 4 degrees C growth of L. monocytogenes. The mutant had normal growth at 37 degrees C but completely failed to grow at either 4 or 10 degrees C, and had impaired growth and reduced swarming on soft agar at 25 degrees C. However, the mutation had no discernible influence on the ability of the bacteria to tolerate repeated freezing and thawing after growth at either 25 or 37 degrees C. The findings suggest that the transposon insertion in the putative helicase gene, in spite of the severely cold-sensitive phenotype that accompanies it, does not affect the ability of the bacteria to cope with cold-related stresses encountered during repeated freezing and thawing.

Conservation of genomic localization and sequence content of Sau3AI-like restriction-modification gene cassettes among Listeria monocytogenes epidemic clone I and selected strains of serotype 1/2a.

Listeria monocytogenes is a food-borne pathogen with a clonal population structure and apparently limited gene flow between strains of different lineages. Strains of epidemic clone I (ECI) have been responsible for numerous outbreaks and invariably have DNA that is resistant to digestion by Sau3AI, suggesting methylation of cytosine at GATC sites. A putative restriction-modification (RM) gene cassette has been identified in the genome of the ECI strain F2365 and all other tested ECI strains but is absent from other strains of the same serotype (4b). Homologous RM cassettes have not been reported among L. monocytogenes isolates of other serotypes. Furthermore, conclusive evidence for the involvement of this RM cassette in the Sau3AI resistance phenotype of ECI strains has been lacking. In this study, we describe a highly conserved RM cassette in certain strains of serotypes 1/2a and 4a that have Sau3AI-resistant DNA. In these strains the RM cassette was in the same genomic location as in the ECI reference strain F2365. The cassette included a gene encoding a putative recombinase, suggesting insertion via site-specific recombination. Deletion of the RM cassette in the ECI strain F2365 and the serotype 1/2a strain A7 rendered the DNA of both strains susceptible to Sau3AI digestion, providing conclusive evidence that the cassette includes a gene required for methylation of cytosine at GATC sites in both strains. The findings suggest that, in addition to its presence in ECI strains, this RM cassette and the accompanying genomic DNA methylation is also encountered among selected strains of other lineages.

DNA probes for unambiguous identification of Listeria monocytogenes epidemic clone II strains.

Listeria monocytogenes epidemic clone II (ECII) strains have been responsible for two major multistate outbreaks of food-borne listeriosis in the United States, but their prevalence and ecology remain poorly understood. In this study, we describe DNA probes that unambiguously identify this clonal group. These probes were able to differentiate ECII strains of outbreak, sporadic, or environmental origin from other L. monocytogenes strains of the same serotype (4b).

Diverse cadmium resistance determinants in Listeria monocytogenes isolates from the turkey processing plant environment.

Two different cadA cadmium resistance determinants (cadA1, first identified in Tn5422, and cadA2, associated with pLM80) were detected among cadmium-resistant Listeria monocytogenes strains from turkey processing plants. Prevalence of cadA1 versus cadA2 was serotype associated. Cadmium-resistant isolates that were also resistant to benzalkonium chloride (BC) were more likely to harbor cadA2 alone or together with cadA1 than isolates that were cadmium resistant but BC susceptible.

Role of growth temperature in freeze-thaw tolerance of Listeria spp.

The food-borne pathogen Listeria monocytogenes can grow in a wide range of temperatures, and several key virulence determinants of the organism are expressed at 37 degrees C but are strongly repressed below 30 degrees C. However, the impact of growth temperature on the ability of the bacteria to tolerate environmental stresses remains poorly understood. In other microorganisms, cold acclimation resulted in enhanced tolerance against freezing and thawing (cryotolerance). In this study, we investigated the impact of growth temperature (4, 25, and 37 degrees C) on the cryotolerance of 14 strains of L. monocytogenes from outbreaks and from food processing plant environments and four strains of nonpathogenic Listeria spp. (L. welshimeri and L. innocua). After growth at different temperatures, cells were frozen at -20 degrees C, and repeated freeze-thaw cycles were applied every 24 h. Pronounced cryotolerance was exhibited by cells grown at 37 degrees C, with a <1-log decrease after 18 cycles of freezing and thawing. In contrast, freeze-thaw tolerance was significantly reduced (P < 0.05) when bacteria were grown at either 4 or 25 degrees C, with log decreases after 18 freeze-thaw cycles ranging from 2 to >4, depending on the strain. These findings suggest that growth at 37 degrees C, a temperature required for expression of virulence determinants of L. monocytogenes, is also required for protection against freeze-thaw stress. The negative impact of growth at low temperature on freeze-thaw stress was unexpected and has not been reported before with this or other psychrotrophic microorganisms.

Longitudinal study of prevalence of Campylobacter jejuni and Campylobacter coli from turkeys and swine grown in close proximity.

Eastern North Carolina is a major contributor to both turkey and swine production in the United States. In this region, turkeys and swine are frequently grown in close proximity and by common growers. To further characterize colonization of turkeys and swine with Campylobacter in such a setting, we investigated the prevalence of thermophilic campylobacters in eight paired operations involving turkey farms in close proximity to finishing swine farms. All 15 surveyed flocks and 15 herds were Campylobacter positive at one or more sampling times. Campylobacter was isolated from 1,310 (87%) of the 1,512 turkey samples and 1,116 (77%) of the 1,448 swine samples. Most (> 99%) campylobacters from swine samples were Campylobacter coli, found in 59 to 97% of the samples from the different herds. Both Campylobacterjejuni and C. coli were recovered from the turkey flocks (overall prevalences of 52 and 35%, respectively). Prevalence among flocks ranged from 31 to 86% for C. jejuni and 0 to 67% for C. coli, and both species were recovered from most flocks. Relative prevalence of C. coli was higher in young birds (brooders), whereas C. jejuni predominated in grow-out birds (P < 0.0001). The prevalence of C. coli in a swine herd was generally not a good predictor for prevalence of this species in the corresponding turkey flock. These findings indicate that even though turkeys and swine grown in proximity to each other were commonly colonized with thermophilic campylobacters, the relative prevalences of C. jejuni and C. coli appear to be host associated.

Differential effects of temperature on natural transformation to erythromycin and nalidixic acid resistance in Campylobacter coli.

Campylobacter jejuni and Campylobacter coli are naturally competent, but limited information exists on the impact of environmental conditions on transformation. In this study, we investigated the impact of temperature and microaerobic versus aerobic atmosphere on transformation of C. coli to erythromycin and nalidixic acid resistance. Frequency of transformation was not significantly different between microaerobic (5 to 10% CO(2)) and aerobic conditions. However, C. coli was transformed to erythromycin resistance at a significantly higher frequency at 42 degrees C than at 25 degrees C (P < 0.05), and few or no transformants were obtained at 25 degrees C. In contrast, transformation to nalidixic acid resistance was highly efficient at both 42 degrees C and 25 degrees C and was similar or, at the most, fourfold higher at 42 degrees C than at 25 degrees C. DNase I treatment experiments suggested that steps both prior and subsequent to internalization of DNA were influenced by temperature in the case of transformation of C. coli to erythromycin resistance. However, the moderately increased (fourfold) frequency of transformation to nalidixic acid resistance at 42 degrees C compared to that at 25 degrees C was exclusively associated with steps prior to DNA internalization. These findings suggest that transformation to erythromycin resistance may be significantly more frequent in the gastrointestinal tract of hosts such as poultry (at 42 degrees C) than in other habitats characterized by lower temperatures, whereas transformation to nalidixic acid resistance may be highly efficient both within and outside the animal hosts.

Teichoic acid glycosylation mediated by gtcA is required for phage adsorption and susceptibility of Listeria monocytogenes serotype 4b.

An insertion mutant of gtcA, responsible for serotype-specific glycosylation of the cell wall teichoic acid in serotype 4b strains of Listeria monocytogenes, was also resistant to both Listeria genus- and serotype 4b-specific phages. The sugar substituents on teichoic acid appeared essential for the adsorption of phages A500 (serotype 4b specific) and A511 (Listeria genus specific) to serotype 4b L. monocytogenes.

Heavy-metal and benzalkonium chloride resistance of Listeria monocytogenes isolates from the environment of turkey-processing plants.

The resistance of Listeria monocytogenes to cadmium and arsenic has been used extensively for strain subtyping. However, limited information is available on the prevalence of such resistance among isolates from the environment of food-processing plants. In addition, it is not known whether the resistance of such isolates to heavy metals may correlate with resistance to quaternary ammonium compounds extensively used as disinfectants in the food-processing industry. In this study, we characterized 192 L. monocytogenes isolates (123 putative strains) from the environment of turkey-processing plants in the United States for resistance to cadmium and arsenic and to the quaternary ammonium disinfectant benzalkonium chloride (BC). Resistance to cadmium was significantly more prevalent among strains of serotypes 1/2a (or 3a) and 1/2b (or 3b) (83% and 74%, respectively) than among strains of the serotype 4b complex (19%). Resistance to BC was encountered among 60% and 51% of the serotype 1/2a (or 3a) and 1/2b (or 3b) strains, respectively, and among 7% of the strains of the serotype 4b complex. All BC-resistant strains were also resistant to cadmium, although the reverse was not always the case. In contrast, no correlation was found between BC resistance and resistance to arsenic, which overall was low (6%). Our findings suggest that the processing environment of turkey-processing plants may constitute a reservoir for L. monocytogenes harboring resistance to cadmium and to BC and raise the possibility of common genetic elements or mechanisms mediating resistance to quaternary ammonium disinfectants and to cadmium in L. monocytogenes.

Absence of serotype-specific surface antigen in laboratory variants of epidemic-associated Listeria monocytogenes strains.

Variants that lacked reactivity with the serotype 4b-specific monoclonal antibody c74.22 and that lost susceptibility to certain Listeria- or serotype 4b-specific phages were identified in the course of genetic studies with serotype 4b Listeria monocytogenes strains H7550 and F2381L (epidemic clones I and II, respectively). Our findings suggest that such variants can become inadvertently established under laboratory conditions and suggest caution in work involving serotype 4b strains and genetic constructs thereof.

Clonal population structure and specific genotypes of multidrug-resistant Campylobacter coli from Turkeys.

Commercial turkey flocks in North Carolina have been found to be colonized frequently with Campylobacter coli strains that are resistant to several antimicrobials (tetracycline, streptomycin, erythromycin, kanamycin, and ciprofloxacin/nalidixic acid). Such strains have been designated multidrug resistant (MDR). However, the population structure of MDR C. coli from turkeys remains poorly characterized. In this study, an analysis of multilocus sequence typing (MLST)-based sequence types (STs) of 59 MDR strains from turkeys revealed that the majority of these strains corresponded to one of 14 different STs, with three STs accounting for 41 (69%) of the strains. The major STs were turkey specific, and most (87%) of the strains with these STs were resistant to the entire panel of antibiotics mentioned above. Some (13%) of the strains with these STs were susceptible to just one or two of the antibiotics in this panel. Further subtyping using fla typing and pulsed-field gel electrophoresis with SmaI and KpnI revealed that the major MDR STs corresponded to strains of related but distinct subtypes, providing evidence for genomic diversification within these STs. These findings suggest that MDR strains of C. coli from turkeys have a clonal population structure characterized by the presence of a relatively small number of clonal groups that appear to be disseminated in the turkey production system. In addition, the observed correlation between STs and the MDR profiles of the microbes indicates that MLST-based typing holds potential for source-tracking applications specific to the animal source (turkeys) and the antimicrobial resistance profile (MDR status) of C. coli.

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.