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.

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.

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.

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.

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.

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).

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.

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.

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.

Involvement of closely related strains of a new clonal group of Listeria monocytogenes in the 1998-99 and 2002 multistate outbreaks of foodborne listeriosis in the United States.

In 1998-99, a multistate outbreak of listeriosis in the United States was associated with contaminated hot dogs and was caused by a strain of Listeria monocytogenes serotype 4b that had been only rarely encountered before in the national PulseNet database. Upon further characterization, the strains from this outbreak were designated as Epidemic Clone II (ECII). ECII isolates exhibited diversification in a genomic region ("region 18") that was otherwise conserved among L. monocytogenes of serotype 4b. Additional unique genetic markers were identified through genome sequencing of one of the isolates from the 1998-99 outbreak. In 2002, another multistate outbreak of listeriosis also involved bacteria of serotype 4b and was attributed to contaminated turkey deli meats. Molecular subtyping data revealed that the macrorestriction patterns of the isolates from the 1998-99 and 2002 outbreaks were closely related. In addition, the 2002 outbreak isolates harbored chromosomal genetic markers found to be unique to, and typical of, the 1998-99 outbreak isolates, including diversification in genomic region 18. Macroarray- based subtyping using chromosomal sequences confirmed the close genetic relatedness between the isolates from the two outbreaks. Genomic content was highly conserved among isolates from each outbreak, with differences detected only in prophage and internalin-like gene sequences. However, since these differences were observed among isolates from each of the outbreaks, they did not differentiate the 1998-99 isolates as a group from those of the 2002 outbreak. Two of 15 randomly chosen serotype 4b clinical isolates from a non-outbreak period (calendar year 2003) appeared to be closely related to the 1998-99 and 2002 outbreak isolates. These findings suggest that both multistate outbreaks of listeriosis in the United States involved closely related members of a single clonal group (ECII) that had not been identified in outbreaks prior to 1998. Since the outbreaks involved different food vehicles and processing plants, the findings suggest establishment of ECII in a still unidentified reservoir in the United States, from which the organisms were introduced to different processing plants.