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.
Listeria monocytogenes is a pathogen of significant concern in many ready to eat foods due to its ability to survive and multiply even under significant environmental stresses. Listeriosis in humans is a concern, especially to high-risk populations such as those who are immunocompromised or pregnant, due to the high rates of morbidity and mortality. Whole genome sequencing has become a routine part of assessing L. monocytogenes isolated from patients, and the frequency of different genetic subtypes associated with listeriosis is now being reported. The recent abundance of genome sequences for L. monocytogenes has provided a wealth of information regarding the variation in core and accessory genomic elements. Newly described accessory genomic regions have been linked to greater virulence capabilities as well as greater resistance to environmental stressors such as sanitizers commonly used in food processing facilities. This review will provide a summary of our current understanding of stress response and virulence phenotypes of L. monocytogenes, within the context of the genetic diversity of the pathogen.
cIn 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.
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.
ACKNOWLEDGMENTSI would like to thank Dr. Vera Katić for being my mentor, not only through PhD studies, but in my undergraduate years as well. Her objectivity, articulate thinking and dedication to work inspired me to keep moving forward even after long, tiresome days in the lab. I will forever appreciate Dr. Katic's encouragement to pursue personal happiness and her support in important life decisions.I am forever grateful to Dr. Sophia Kathariou for accepting me in her lab and making me feel at home while far from it. Her incredible ability to find the best solutions when things were not looking promising and open completely new horizons along with her creativity and knowledge made science so attractive. Looking at all the great, bright and skilful young people pursuing their dreams under Dr. Kathariou's guidance makes me very proud for being a part of it.My appreciation also goes to Dr. Lepšanović and Dr. Mišić for accepting to be members of the committee. I am thankful to all my lab mates and to Ms. Robin Siletzky for her solid support throughout my work. A special thanks to Dr. Vikrant Dutta for his patience, willingness to help and his much admired teaching skills.Lastly and foremost I would like to thank my family. To my dear parents Ljubo and Zora whose unconditional love, support and presence whenever I needed them have been and will always be my motive to be better. To my big brother Zoran, who has been, and is my role model, but still owes me German lessons. Finally, I want to thank my boys, my beloved husband Sergio and my precious son Gregory, who have been an endless source of energy, support and love. Thanks for being my wingmen guys! ABSTRACTAbility to contaminate ready-to-eat, highly processed, cold-stored food products, relatively high fatality rate and environmental adaptations define .Listeria monocytogenes as a challenging foodborne pathogen of high public health significance. Listeria's adaptations and prolonged persistence in food processing facilities are a key link to contamination of foods.In recent years, bacterial multidrug resistance has been recognized as an emerging health challenge. Exposure to antimicrobials (antibiotics, disinfectants or environmentally ubiquitous heavy metals), may provide selective pressures for resistance with accompanying cross-resistance and increase in environmental fitness.In this work I addressed some of the possible mechanisms of adaptations that may contribute to persistence of L. monocytogenes in the environment as well as to survival and growth in different foods. This includes adaptations to heavy metals such as cadmium, disinfectants such as benzalkonium chloride (BC), selected antibiotics (e.g. the fluoroquinolone ciprofloxacin) and other toxic compounds (e.g. tetraphenyl-phosponium, often used as an anti-carcinogenic and efflux pump substrate). In order to characterize the impact of multidrug resistance on survival and growth of L. monocytogenes in different foods, we tested mutants adapted to higher concentrations of antibiotic ciprofloxacin and ...
Poor sanitation of food contact surfaces has been a contributing factor in food borne disease outbreaks, especially those involving Listeria monocytogenes, Salmonella spp., Escherichia coli, Staphylococcus aureus etc. The objectives of this study were therefore to: 1. Determine the efficiency of a disinfectant based on chlorine dioxide in suspension in a closed system in a dairy plant. 2. Evaluate the possibility of disinfection of working surfaces with a disinfectant based on chlorine dioxide. In order to determine the germicidal effect of the disinfectant based on chlorine dioxide by suspension test (BSEN 1276:1997); the following test organisms were used: Listeria monocytogenes, Proteus mirabilis, Escherichia coli, Bacillus cereus, Staphylococcus aureus and Pseudomonas aeruginosa clinical isolate. The corrosive properties of the disinfectant based on chlorine dioxide were tested by IDF 077:1977 standard. The efficacy of this disinfectant was investigated in a closed system in a dairy plant. Results indicated a 100% reduction of >108 cfu/ml L. monocytogenes, E. coli, Proteus mirabilis, Pseudomonas aeruginosa, S. aureus, viable count after 1 minute of exposure to 100 ppm of the disinfectant based on chlorine dioxide and 400 ppm for Bacillus cereus. In the presence of 2% skim milk and 4 % skim milk concentrations of 200 and 250 ppm resulted in 100% reduction in numbers of the five of six test microorganisms, respectively. The spore former, Bacillus cereus is less susceptible to the disinfectant. Therefore, the efficient concentration for 100% reduction in viable count after 1 minute exposure was 500 ppm. The corrosive properties of the disinfectant were not determined. In the case of closed system disinfection in a dairy plant, reduction in viable count after 15 minute exposure to 100 ppm of disinfectant based on chlorine dioxide ranged from 80 to 100%
Most seasonal influenza vaccines are produced using hemagglutinin (HA) surface antigens from inactivated virions. However, virions are thought to be a suboptimal source for the less abundant NA surface antigen, which is also protective against severe disease. Here, we demonstrate that inactivated influenza virions are compatible with two modern approaches for improving protective NA antibody responses. Using a DBA/2J mouse model, we confirmed that the strong infection-induced NA inhibitory (NAI) antibody responses are only achieved by high dose immunizations of inactivated virions, likely due to low viral NA content. Based on this observation, we first produced virions with higher NA content by using reverse genetics to exchange the viral internal gene segments. Single immunizations with these inactivated virions enhanced NAI antibody responses, improved NA-based protection from a lethal viral challenge and allowed the development of natural immunity to the heterotypic challenge virus HA. Second, we combined inactivated virions with recombinant NA proteins. These mixtures increased NA-based protection following viral challenge and elicited stronger NA antibody responses than either component alone, especially when the NAs were homologous. Together, these results indicate that viral- and protein-based vaccines can be combined in a single syringe to improve protective antibody responses to influenza antigens.
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