Listeria monocytogenes strains selected on ciprofloxacin or the disinfectant benzalkonium chloride exhibit reduced susceptibility to ciprofloxacin, gentamicin, benzalkonium chloride, and other toxic compounds

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.     

Screening of benzalkonium chloride resistance in Listeria monocytogenes strains isolated during cold smoked fish production

AIMS: To determine the susceptibility to disinfectants and cross-resistance to antibiotics in Listeria monocytogenes strains isolated from fish products and the fish-processing environment. METHODS AND RESULTS: Minimal inhibitory concentration assessment, using the agar dilution method, showed 108 of 255 L. monocytogenes isolates with low susceptibility to benzalkonium chloride (BC), commonly used in food industries. Most of them are from raw products of farmed fish during processing, while the remaining resistant isolates were mainly from the environment and finished products irrespective of the fish species. Two BC-resistant isolates were resistant to ethidium bromide (EB). The conservation of resistance after plasmid curing suggested that the resistance genes are not plasmid associated. EB accumulation assays demonstrated that the two BC(R) EB(R) isolates used an efflux pump to expel these substrates whereas a different mechanism was probably used by the majority of the strains with BC(R) EB(S) pattern. No cross-resistance was found with antibiotics. CONCLUSIONS: This study highlights the difference in susceptibilities to BC for L. monocytogenes strains isolated from fish-processing plants and in resistance mechanisms to BC developed by these bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of BC resistant L. monocytogenes strains could contribute to their adaptation and so explained their survival and persistence in the fish-processing environment.     

Role of efflux pumps in adaptation and resistance of Listeria monocytogenes to benzalkonium chloride

In this study, potential mechanisms underlying resistance and adaptation to benzalkonium chloride (BC) in Listeria monocytogenes were investigated. Two groups of strains were studied. The first group consisted of strains naturally sensitive to BC which could be adapted to BC. The second group consisted of naturally resistant strains. For all adapted isolates, there was a correlation between the resistance to BC and ethidium bromide, but this was not the case for the naturally resistant isolates. To investigate the role of efflux pumps in adaptation or resistance, reserpine, an efflux pump inhibitor, was added to the strains. Addition of reserpine to the sensitive and adapted strains resulted in a decrease in the MIC for BC, whereas no such decrease was observed for the resistant strains, indicating that efflux pumps played no role in the innate resistance of certain strains of L. monocytogenes to this compound. Two efflux pumps (MdrL and Lde) have been described in L. monocytogenes. Studies showed low and intermediate levels of expression of the genes encoding the efflux pumps for two selected resistant strains, H7764 and H7962, respectively. Adaptation to BC of sensitive isolates of L. monocytogenes resulted in significant increases in expression of mdrl (P < 0.05), but no such increase was observed for lde for two adapted strains of L. monocytogenes, LJH 381 (P = 0.91) and C719 (P = 0.11). This indicates that the efflux pump Mdrl is at least partly responsible for the adaptation to BC.     

Comparison of cold enrichment and U.S. Department of Agriculture methods for isolating Listeria monocytogenes from naturally contaminated foods. The Listeria Study Group.

We compared the cold enrichment (CE) and U.S. Department of Agriculture (USDA) methods for isolating Listeria monocytogenes by examining 402 food samples. The food samples were collected from refrigerators of listeriosis patients as part of a multistate active surveillance project to determine the role of foods in sporadic listeriosis in the United States. L. monocytogenes was isolated from 51 food samples (13%). The USDA method was significantly better (P less than 0.001) than the CE method. The isolation efficiencies of the USDA and CE methods were 96 and 59%, respectively. Quantitation of L. monocytogenes in the food samples revealed that many food samples containing less than 0.3 CFU/g were negative as determined by the CE method but positive as determined by the USDA method.     

Comparison of cold enrichment and U.S. Department of Agriculture methods for isolating Listeria monocytogenes from naturally contaminated foods. The Listeria Study Group.

We compared the cold enrichment (CE) and U.S. Department of Agriculture (USDA) methods for isolating Listeria monocytogenes by examining 402 food samples. The food samples were collected from refrigerators of listeriosis patients as part of a multistate active surveillance project to determine the role of foods in sporadic listeriosis in the United States. L. monocytogenes was isolated from 51 food samples (13%). The USDA method was significantly better (P less than 0.001) than the CE method. The isolation efficiencies of the USDA and CE methods were 96 and 59%, respectively. Quantitation of L. monocytogenes in the food samples revealed that many food samples containing less than 0.3 CFU/g were negative as determined by the CE method but positive as determined by the USDA method.     

Heavy Metal and Disinfectant Resistance of Listeria monocytogenes from Foods and Food Processing Plants

The persistence of Listeria monocytogenes in food processing plants and other ecosystems reflects its ability to adapt to numerous stresses. In this study, we investigated 138 isolates from foods and food processing plants for resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) and to heavy metals (cadmium and arsenic). We also determined the prevalence of distinct cadmium resistance determinants (cadA1, cadA2, and cadA3) among cadmium-resistant isolates. Most BC-resistant isolates were resistant to cadmium as well. Arsenic resistance was encountered primarily in serotype 4b and was an attribute of most isolates of the serotype 4b epidemic clonal group ECIa. Prevalence of the known cadmium resistance determinants was serotype associated: cadA1 was more common in isolates of serotypes 1/2a and 1/2b than 4b, while cadA2 was more common in those of serotype 4b. A subset (15/77 [19%]) of the cadmium-resistant isolates lacked the known cadmium resistance determinants. Most of these isolates were of serotype 4b and were also resistant to arsenic, suggesting novel determinants that may confer resistance to both cadmium and arsenic in these serotype 4b strains. The findings may reflect previously unrecognized components of the ecological history of different serotypes and clonal groups of L. monocytogenes, including exposures to heavy metals and disinfectants.     

The activity of ferulic and gallic acids in biofilm prevention and control of pathogenic bacteria

The activity of two phenolic acids, gallic acid (GA) and ferulic acid (FA) at 1000 μg ml(-1), was evaluated on the prevention and control of biofilms formed by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. In addition, the effect of the two phenolic acids was tested on planktonic cell susceptibility, bacterial motility and adhesion. Biofilm prevention and control were tested using a microtiter plate assay and the effect of the phenolic acids was assessed on biofilm mass (crystal violet staining) and on the quantification of metabolic activity (alamar blue assay). The minimum bactericidal concentration for P. aeruginosa was 500 μg ml(-1) (for both phenolic acids), whilst for E. coli it was 2500 μg ml(-1) (FA) and 5000 μg ml(-1) (GA), for L. monocytogenes it was >5000 μg ml(-1) (for both phenolic acids), and for S. aureus it was 5000 μg ml(-1) (FA) and >5000 μg ml(-1) (GA). GA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. FA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. Colony spreading of S. aureus was completely inhibited by FA. The interference of GA and FA with bacterial adhesion was evaluated by the determination of the free energy of adhesion. Adhesion was less favorable when the bacteria were exposed to GA (P. aeruginosa, S. aureus and L. monocytogenes) and FA (P. aeruginosa and S. aureus). Both phenolics had preventive action on biofilm formation and showed a higher potential to reduce the mass of biofilms formed by the Gram-negative bacteria. GA and FA promoted reductions in biofilm activity >70% for all the biofilms tested. The two phenolic acids demonstrated the potential to inhibit bacterial motility and to prevent and control biofilms of four important human pathogenic bacteria. This study also emphasizes the potential of phytochemicals as an emergent source of biofilm control products.     

Bacteriocin production and resistance to drugs are advantageous features for Lactobacillus acidophilus La-14, a potential probiotic strain

L. acidophilus La-14 produces bacteriocin active against L. monocytogenes ScottA (1600 AU/ml) in MRS broth at 30°C or 37°C. The bacteriocin proved inhibitory to different serological types of Listeria spp. Antimicrobial activity was completely lost after treatment of the cell-free supernatant with proteolytic enzymes. Addition of bacteriocin produced by L. acidophilus La-14 to a 3 h-old culture of L. monocytogenes ScottA repressed cell growth in the following 8h. Treatment of stationary phase cells of L. monocytogenes ScottA (107-108 CFU/ml) by the bacteriocin resulted in growth inhibition. Growth of L. acidophilus La-14 was not inhibited by commercial drugs from different generic groups, including nonsteroidal anti-inflammatory drugs (NSAID) containing diclofenac potassium or ibuprofen arginine. Only one non-antibiotic drug tested, Atlansil (an antiarrhythmic agent), had an inhibitory effect on L. acidophilus La-14 with MIC of 2.5 mg/ml. L. acidophilus La-14 was not affected by drugs containing sodium or potassium diclofenac. L. acidophilus La-14 shows a good resistance to several drugs and may be applied in combination for therapeutic use.     

Survival and heat resistance of Listeria monocytogenes after exposure to alkali and chlorine

A strain of Listeria monocytogenes isolated from a drain in a food-processing plant was demonstrated, by determination of D values, to be more resistant to the lethal effect of heat at 56 or 59 degrees C following incubation for 45 min in tryptose phosphate broth (TPB) at pH 12.0 than to that of incubation for the same time in TPB at pH 7.3. Cells survived for at least 6 days when they were suspended in TPB at pHs 9.0, 10.0, and 11.0 and stored at 4 or 21 degrees C. Cells of L. monocytogenes incubated at 37 degrees C for 45 min and then stored for 48 or 144 h in TPB at pH 10.0 were more resistant to heat treatment at 56 degrees C than were cells stored in TPB at pH 7.3. The alkaline-stress response in L. monocytogenes may induce resistance to otherwise lethal thermal-processing conditions. Treatment of cells in 0.05 M potassium phosphate buffer (pH 7.00 +/- 0.05) containing 2.0 or 2.4 mg of free chlorine per liter reduced populations by as much as 1.3 log(10) CFU/ml, while treatment with 6.0 mg of free chlorine per liter reduced populations by as much as 4.02 log(10) CFU/ml. Remaining subpopulations of chlorine-treated cells exhibited some injury, and cells treated with chlorine for 10 min were more sensitive to heating at 56 degrees C than cells treated for 5 min. Contamination of foods by L. monocytogenes cells that have survived exposure to processing environments ineffectively cleaned or sanitized with alkaline detergents or disinfectants may have more severe implications than previously recognized. Alkaline-pH-induced cross-protection of L. monocytogenes against heat has the potential to enhance survival in minimally processed as well as in heat-and-serve foods and in foods on holding tables, in food service facilities, and in the home. Cells surviving exposure to chlorine, in contrast, are more sensitive to heat; thus, the effectiveness of thermal processing in achieving desired log(10)-unit reductions is not compromised in these cells.     

Real-time PCR assay to differentiate Listeriolysin S-positive and -negative strains of Listeria monocytogenes

Due to the severity of the food-borne infection listeriosis, strict legislation governs the detectable and permissible limits at which Listeria monocytogenes is permitted in foods. These requirements, coupled with the ubiquitous nature of L. monocytogenes strains and the potential for epidemic outbreaks, mean that the pathogen can devastate affected sectors of the food industry. Although almost all L. monocytogenes strains have the potential to cause listeriosis, those implicated in the vast majority of epidemics belong to a subset of strains belonging to evolutionary lineage I. It has been established that a significant proportion of these strains, including those implicated in the majority of outbreaks, produce an additional hemolysin, designated listeriolysin S (LLS), which may be responsible for the enhanced virulence of these strains. In order to ultimately establish this definitively, it is important to first be able to rapidly discriminate between LLS-positive and -negative strains. Here, after essential genes within the LLS-encoding cluster, Listeria pathogenicity island 3, were identified by deletion mutagenesis, a real-time PCR assay which targets one such gene, llsX, was developed as a means of identifying LLS-positive L. monocytogenes. The specificity of the assay was validated against a panel of 40 L. monocytogenes strains (20 of which were LLS positive) and 25 strains representative of other Listeria species. Furthermore, 1 CFU of an LLS-positive strain per 25 g/ml of spiked foods was detected in less than 30 h when the assay was coupled with culture enrichment. The detection limit of this assay was 10 genome equivalents.     

Epidemic clone I-specific genetic markers in strains of Listeria monocytogenes serotype 4b from foods

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.     

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.