Influence of Sublethal Concentrations of Common Disinfectants on Expression of Virulence Genes in Listeria monocytogenes

117

This study aimed to characterize physiological differences between persistent and presumed nonpersistent Listeria monocytogenes strains isolated at processing facilities and to investigate the molecular basis for this by transcriptomic sequencing. Full metabolic profiles of two strains, one persistent and one nonpersistent, were initially screened using Biolog's Phenotype MicroArray (PM) technology. Based on these results, in which major differences from selected antimicrobial agents were detected, another persistent strain and two nonpersistent strains were characterized using two antimicrobial PMs. Resistance to quaternary ammonium compounds (QACs) was shown to be higher among persistent strains. Growth of persistent and nonpersistent strains in various concentrations of the QACs benzethonium chloride (BZT) and cetylpyridinium chloride (CPC) was determined. Transcriptomic sequencing of a persistent and a presumed nonpersistent strain was performed to compare gene expression among these strains in the presence and absence of BZT. Two strains, designated "frequent persisters" because they were the most frequently isolated at the processing facility, showed overall higher resistance to QACs. Transcriptome analysis showed that BZT induced a complex peptidoglycan (PG) biosynthesis response, which may play a key role in BZT resistance. Comparison of persistent and nonpersistent strains indicated that transcription of many genes was upregulated among persistent strains. This included three gene operons: pdu, cob-cbi, and eut. These genes may play a role in the persistence of L. monocytogenes outside the human host.Listeria monocytogenes is a human food-borne pathogen that causes listeriosis in humans. Because infection is primarily food borne, control of the organism in food-processing environments, including dairy food production facilities, is important. There are many studies detailing the occurrence of L. monocytogenes in food-processing facilities (18,22,25,28). In order to better understand the ecology and transmission of L. monocytogenes strains through the processing facility, however, techniques such as molecular strain typing are necessary. Such studies not only play a crucial role in determining possible transmission vectors, they provide insights into the persistence of certain strains at the facility itself. This is of critical importance to the manufacturer, as strains that can successfully persist at the production facility have an increased chance of cross-contamination of the product and thus lead to an increased likelihood of product recall or human infection. A study published in 1995 by Lawrence and Gilmour (20) characterized strains isolated over a 12-month period at a poultryprocessing plant located in Ireland. This study identified 18 different randomly amplified polymorphic DNA (RAPD) profiles among the L. monocytogenes strains, and interestingly, one of these RAPD types was seen throughout the 12 months of the study. Similar evidence of persistent strains has subsequently been documented in o...

Section: Discussionmentioning

confidence: 65%

Physiological and Transcriptional Characterization of Persistent and Nonpersistent Listeria monocytogenes Isolates

Fox

Leonard

Jordan

2011

117

“…When different fish pathogens were exposed to the antimicrobial peptide cecropin B, V. anguillarum strain 775 displayed increased adhesion to CHSE-214 fish cells and an increased mortality rate in challenged medaka (Oryzias latipes) (67). Induction of virulence genes by antimicrobial compounds has also been reported for human pathogens such as Listeria monocytogenes (37,68) and Staphylococcus aureus (35,38). In the present study, none of the TDA-exposed clones was more infective toward CHSE-214 fish cells than were nonexposed clones and the wild-type strain.…”

Section: Discussionmentioning

confidence: 96%

Vibrio anguillarum Is Genetically and Phenotypically Unaffected by Long-Term Continuous Exposure to the Antibacterial Compound Tropodithietic Acid

Rasmussen

Grotkjær

D’Alvise

et al. 2016

Self Cite

Minimizing the use of antibiotics in the food production chain is essential for limiting the development and spread of antibioticresistant bacteria. One alternative intervention strategy is the use of probiotic bacteria, and bacteria of the marine Roseobacter clade are capable of antagonizing fish-pathogenic vibrios in fish larvae and live feed cultures for fish larvae. The antibacterial compound tropodithietic acid (TDA), an antiporter that disrupts the proton motive force, is key in the antibacterial activity of several roseobacters. Introducing probiotics on a larger scale requires understanding of any potential side effects of long-term exposure of the pathogen to the probionts or any compounds they produce. Here we exposed the fish pathogen Vibrio anguillarum to TDA for several hundred generations in an adaptive evolution experiment. No tolerance or resistance arose during the 90 days of exposure, and whole-genome sequencing of TDA-exposed lineages and clones revealed few mutational changes, compared to lineages grown without TDA. Amino acid-changing mutations were found in two to six different genes per clone; however, no mutations appeared unique to the TDA-exposed lineages or clones. None of the virulence genes of V. anguillarum was affected, and infectivity assays using fish cell lines indicated that the TDA-exposed lineages and clones were less invasive than the wild-type strain. Thus, long-term TDA exposure does not appear to result in TDA resistance and the physiology of V. anguillarum appears unaffected, supporting the application of TDA-producing roseobacters as probiotics in aquaculture. IMPORTANCEIt is important to limit the use of antibiotics in our food production, to reduce the risk of bacteria developing antibiotic resistance. We showed previously that marine bacteria of the Roseobacter clade can prevent or reduce bacterial diseases in fish larvae, acting as probiotics. Roseobacters produce the antimicrobial compound tropodithietic acid (TDA), and we were concerned regarding whether long-term exposure to this compound could induce resistance or affect the disease-causing ability of the fish pathogen. Therefore, we exposed the fish pathogen Vibrio anguillarum to increasing TDA concentrations over 3 months. We did not see the development of any resistance to TDA, and subsequent infection assays revealed that none of the TDA-exposed clones had increased virulence toward fish cells. Hence, this study supports the use of roseobacters as a non-risk-based disease control measure in aquaculture.

“…In the case of L. monocytogenes, the study by Crimmins et al (2) has provided clear evidence that MDR transporter systems may be involved in processes of fundamental importance to host-pathogen interactions, such as elicitation of the innate immune response. Furthermore, L. monocytogenes virulence genes prfA and inlA were shown to be upregulated in response to sublethal concentrations of quaternary ammonium compounds (13). It will be important to determine whether BC-or ciprofloxacin-selected strains are also impacted in their virulence in cell culture or animal models and whether attributes such as ability to persist in biofilms and persist in the environment may also be altered in such strains.…”

Section: Discussionmentioning

confidence: 99%

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

Rakić-Martinez

Drevets

Dutta

et al. 2011

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 g/ml) or the quaternary ammonium disinfectant benzalkonium chloride (BC; 10 g/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.