Sophia Kathariou

Resistance of Listeria monocytogenes Biofilms to Sanitizing Agents in a Simulated Food Processing Environment

ABSTRACT 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.

Survival of Clinical and Poultry-Derived Isolates of Campylobacter jejuni at a Low Temperature (4°C)

ABSTRACT Campylobacter jejuni is a leading cause of bacterial gastroenteritis in humans, and contamination of poultry has been implicated in illness. The bacteria are fastidious in terms of their temperature requirements, being unable to grow below ca. 31°C, but have been found to be physiologically active at lower temperatures and to tolerate exposure to low temperatures in a strain-dependent manner. In this study, 19 field isolates of C. jejuni (10 of clinical and 9 of poultry origin) were studied for their ability to tolerate prolonged exposure to low temperature (4°C). Although substantial variability was found among different strains, clinical isolates tended to be significantly more likely to remain viable following cold exposure than poultry-derived strains. In contrast, the relative degree of tolerance of the bacteria to freezing at −20°C and freeze-thawing was strain specific but independent of strain source (poultry versus clinical) and degree of cold (4°C) tolerance.

comK Prophage Junction Fragments as Markers for Listeria monocytogenes Genotypes Unique to Individual Meat and Poultry Processing Plants and a Model for Rapid Niche-Specific Adaptation, Biofilm Formation, and Persistence

ABSTRACT Different strains of Listeria monocytogenes are well known to persist in individual food processing plants and to contaminate foods for many years; however, the specific genotypic and phenotypic mechanisms responsible for persistence of these unique strains remain largely unknown. Based on sequences in comK prophage junction fragments, different strains of epidemic clones (ECs), which included ECII, ECIII, and ECV, were identified and shown to be specific to individual meat and poultry processing plants. The comK prophage-containing strains showed significantly higher cell densities after incubation at 30°C for 48 h on meat and poultry food-conditioning films than did strains lacking the comK prophage (P < 0.05). Overall, the type of strain, the type of conditioning film, and the interaction between the two were all highly significant (P < 0.001). Recombination analysis indicated that the comK prophage junction fragments in these strains had evolved due to extensive recombination. Based on the results of the present study, we propose a novel model in which the concept of defective comK prophage was replaced with the rapid adaptation island (RAI). Genes within the RAI were recharacterized as “adaptons,” as these genes may allow L. monocytogenes to rapidly adapt to different food processing facilities and foods. If confirmed, the model presented would help explain Listerias rapid niche adaptation, biofilm formation, persistence, and subsequent transmission to foods. Also, comK prophage junction fragment sequences may permit accurate tracking of persistent strains back to and within individual food processing operations and thus allow the design of more effective intervention strategies to reduce contamination and enhance food safety.

Genetic Characterization of Plasmid-Associated Benzalkonium Chloride Resistance Determinants in a Listeria monocytogenes Strain from the 1998-1999 Outbreak

ABSTRACT Quaternary ammonium compounds such as benzalkonium chloride (BC) are widely used as disinfectants in both food processing and medical environments. BC-resistant strains of Listeria monocytogenes have been implicated in multistate outbreaks of listeriosis and have been frequently isolated from food processing plants. However, the genetic basis for BC resistance in L. monocytogenes remains poorly understood. In this study, we have characterized a plasmid (pLM80)-associated BC resistance cassette in L. monocytogenes H7550, a strain implicated in the 1998-1999 multistate outbreak involving contaminated hot dogs. The BC resistance cassette (bcrABC) restored resistance to BC (MIC, 40 μg/ml) in a plasmid-cured derivative of H7550. All three genes of the cassette were essential for imparting BC resistance. The transcription of H7550 BC resistance genes was increased under sublethal (10 μg/ml) BC exposure and was higher at reduced temperatures (4, 8, or 25°C) than at 37°C. The level of transcription was higher at 10 μg/ml than at 20 or 40 μg/ml. In silico analysis suggested that the BC resistance cassette was harbored by an IS1216 composite transposon along with other genes whose functions are yet to be determined. The findings from this study will further our understanding of the adaptations of this organism to disinfectants such as BC and may contribute to the elucidation of possible BC resistance dissemination in L. monocytogenes.

Genetic Markers Unique to Listeria monocytogenes Serotype 4b Differentiate Epidemic Clone II (Hot Dog Outbreak Strains) from Other Lineages

ABSTRACT A small number of closely related strains of Listeria monocytogenes serotype 4b, designated epidemic clone I (ECI), have been implicated in numerous outbreaks of food-borne listeriosis described during the past two decades in Europe and North America. In 1998 to 1999, a multistate outbreak traced to contaminated hot dogs involved a different strain type of serotype 4b, with genetic fingerprints rarely encountered before. In spite of the profound economic and public health impact of this outbreak, the implicated bacteria (designated epidemic clone II [ECII]) have remained poorly characterized genetically, and nucleotide sequences specific for these strains have not been reported. Using genome sequence information, PCR, and Southern blots, we identified DNA fragments which appeared to be either absent or markedly divergent in the hot dog outbreak strains but conserved among other serotype 4b strains. PCR with primers derived from these fragments as well as Southern blots with the amplicons as probes readily differentiated ECII from other serotype 4b strains. The serotype 4b-specific region harboring these fragments was adjacent to inlA, which encodes a well-characterized virulence determinant. The findings suggest that ECII strains have undergone divergence in portions of a serotype-specific region that is conserved in other serotype 4b strains. Although the mechanisms that drive this divergence remain to be identified, DNA-based tools from this region can facilitate the detection and further characterization of strains belonging to this lineage.

Listeria monocytogenes Strains Selected on Ciprofloxacin or the Disinfectant Benzalkonium Chloride Exhibit Reduced Susceptibility to Ciprofloxacin, Gentamicin, Benzalkonium Chloride, and Other Toxic Compounds

ABSTRACT Listeria monocytogenes is a leading agent for severe food-borne illness and death in the United States and other nations. Even though drug resistance has not yet threatened therapeutic interventions for listeriosis, selective pressure associated with exposure to antibiotics and disinfectants may result in reduced susceptibility to these agents. In this study, selection of several L. monocytogenes strains on either ciprofloxacin (2 μg/ml) or the quaternary ammonium disinfectant benzalkonium chloride (BC; 10 μg/ml) led to derivatives with increased MICs not only to these agents but also to several other toxic compounds, including gentamicin, the dye ethidium bromide, and the chemotherapeutic drug tetraphenylphosphonium chloride. The spectrum of compounds to which these derivatives exhibited reduced susceptibility was the same regardless of whether they were selected on ciprofloxacin or on BC. Inclusion of strains harboring the large plasmid pLM80 revealed that MICs to ciprofloxacin and gentamicin did not differ between the parental and plasmid-cured strains. However, ciprofloxacin-selected derivatives of pLM80-harboring strains had higher MICs than those derived from the plasmid-cured strains. Susceptibility to the antimicrobials was partially restored in the presence of the potent efflux inhibitor reserpine. Taken together, these data suggest that mutations in efflux systems are responsible for the multidrug resistance phenotype of strains selected on ciprofloxacin or BC.

Epidemic Clone I-Specific Genetic Markers in Strains of Listeria monocytogenes Serotype 4b from Foods

ABSTRACT Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.

Competition of Listeria monocytogenes Serotype 1/2a and 4b Strains in Mixed-Culture Biofilms

ABSTRACT The majority of Listeria monocytogenes isolates recovered from foods and the environment are strains of serogroup 1/2, especially serotypes 1/2a and 1/2b. However, serotype 4b strains cause the majority of human listeriosis outbreaks. Our investigation of L. monocytogenes biofilms used a simulated food-processing system that consisted of repeated cycles of growth, sanitation treatment, and starvation to determine the competitive fitness of strains of serotypes 1/2a and 4b in pure and mixed-culture biofilms. Selective enumeration of strains of a certain serotype in mixed-culture biofilms on stainless steel coupons was accomplished by using serotype-specific quantitative PCR and propidium monoazide treatment to prevent amplification of extracellular DNA or DNA from dead cells. The results showed that the serotype 1/2a strains tested were generally more efficient at forming biofilms and predominated in the mixed-culture biofilms. The growth and survival of strains of one serotype were not inhibited by strains of the other serotype in mixed-culture biofilms. However, we found that a cocktail of serotype 4b strains survived and grew significantly better in mixed-culture biofilms containing a specific strain of serotype 1/2a (strain SK1387), with final cell densities averaging 0.5 log10 CFU/cm2 higher than without the serotype 1/2a strain. The methodology used in this study contributed to our understanding of how environmental stresses and microbial competition influence the survival and growth of L. monocytogenes in pure and mixed-culture biofilms.

Host ranges of Listeria-specific bacteriophages from the turkey processing plant environment in the United States.

ABSTRACT Even though at least 400 Listeria phages have been isolated from various sources, limited information is available on phages from the food processing plant environment. Phages in the processing plant environment may play critical roles in determining the Listeria population that becomes established in the plant. In this study, we pursued the isolation of Listeria-specific phages from environmental samples from four turkey processing plants in the United States. These environmental samples were also utilized to isolate Listeria spp. Twelve phages were isolated and classified into three groups in terms of their host range. Of these, nine (group 1) showed a wide host range, including multiple serotypes of Listeria monocytogenes, as well as other Listeria spp. (L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii). The remaining phages mostly infected L. monocytogenes serotype 4b as well as L. innocua, L. ivanovii, and/or L. welshimeri. All but one of the strains of the serotype 4b complex (4b, 4d, 4e) from the processing plant environment could be readily infected by the wide-host-range phages isolated from the environment of the processing plants. However, many strains of other serotypes (1/2a [or 3a] and 1/2b [or 3b]), which represented the majority of L. monocytogenes strains isolated from the environmental samples, were resistant to infection by these phages. Experiments with two phage-resistant strains showed reduced phage adsorption onto the host cells. These findings suggest that phage resistance may be an important component of the ecology of L. monocytogenes in the turkey processing plants.

Antimicrobial Susceptibility Profiles and Strain Type Diversity of Campylobacter jejuni Isolates from Turkeys in Eastern North Carolina

ABSTRACT Campylobacter jejuni is one of the most common bacterial causes of human gastroenteritis, and recent findings suggest that turkeys are an important reservoir for this organism. In this study, 80 C. jejuni isolates from eastern North Carolina were characterized for resistance to nine antimicrobials, and strain types were determined by fla typing, pulsed-field gel electrophoresis (PFGE) with SmaI and KpnI, and (for 41 isolates) multilocus sequence typing (MLST). PFGE analysis suggested that many of the isolates (37/40 [ca. 93%]) in a major genomic cluster had DNA that was partially methylated at SmaI sites. Furthermore, 12/40 (30%) of the isolates in this cluster were completely resistant to digestion by KpnI, suggesting methylation at KpnI sites. MLST of 41 isolates identified 10 sequence types (STs), of which 4 were new. Three STs (ST-1839, ST-2132 and the new ST-2934) were predominant and were detected among isolates from different farms. The majority of the isolates (74%) were resistant to three or more antimicrobials, and resistance to ciprofloxacin was common (64%), whereas resistance to the other drug of choice for treatment of human campylobacteriosis, erythromycin, was never encountered. Most (33/34) of the kanamycin-resistant isolates were also resistant to tetracycline; however, only ca. 50% of the tetracycline-resistant isolates were also kanamycin resistant. Isolates with certain antimicrobial resistance profiles had identical or closely related strain types. Overall, the findings suggest dissemination of certain clonal groups of C. jejuni isolates in the turkey production industry of this region.