G. Sundheim

Occurrence of and a possible mechanism for resistance to a quaternary ammonium compound in Listeria monocytogenes.

In a study of 200 Listeria monocytogenes isolates, 10% were determined to be resistant to benzalkonium chloride (BC). Serial subcultivation of initially BC sensitive (BC(S)) and BC resistant (BC(R)) isolates in sublethal concentrations of BC resulted in enhanced and approximately equal resistance of all strains to the compound. Fifty per cent of the BC(R) isolates showed resistance to ethidium bromide (EB) as well. A proton motive force (pmf)-dependent efflux of EB was demonstrated in BC(R) isolates, and in originally sensitive strains adapted to grow in BC. This efflux was not found in BC(S) strains. The result indicate that BC can induce a broad resistance mechanism based on a pmf-driven efflux pump. There was no indication that this type of resistance was related to resistance to antibiotics.

The qacG gene on plasmid pST94 confers resistance to quaternary ammonium compounds in staphylococci isolated from the food industry

The 2·3 kb resistance plasmid pST94 revealed a new gene (qacG) encoding resistance to benzalkonium chloride (BC), a commonly used quaternary ammonium disinfectant, and the intercalating dye ethidium bromide (Eb) in staphylococci isolated from the food industry. The 107 amino acid QacG protein showing 69·2% identity to the staphylococcal multi‐drug resistance protein Smr is a new member of the small multi‐drug resistance (SMR) protein family. QacG conferred resistance via proton dependent efflux. An additional ORF on pST94 encoded a protein with extensive similarity to replication proteins of other Gram‐positive bacteria. Gene constructs containing the qacG and smr gene region combined with the smr or qacG promoter, respectively, indicated that QacG is more efficient than Smr and that qacG has a weaker promoter. Resistant qacG‐ontaining cells could be adapted to withstand higher concentrations of BC. Adapted qacG‐containing cells showed increased resistance mainly to BC. In contrast, adaptation of sensitive cells showed cross‐resistance development to a range of compounds. Induction of proton‐dependent efflux was observed for BC‐adapted staphylococci cells not containing qacG. The ability of sublethal concentrations of BC to develop cross‐resistance and induce efflux mechanisms could be of practical significance; it should be considered before use of any new disinfectant and in the design of better disinfection procedures.

Bacterial resistance to disinfectants containing quaternary ammonium compounds

Abstract Quaternary ammonium compounds (QAC) are widely used as disinfectants in both medical and food environments. Microbial contaminants are, therefore, regularly exposed to their action and the isolation from clinical and food sources of resistant bacteria continues to be reported in many countries. Resistance to QAC in clinical strains of staphylococci is encoded by one of at least three resistance genes, designated qacA, qacB and qacC . Using hybridisation analysis, we have shown that these QAC resistance genes are also distributed among staphylococcal strains in the food industry. In addition, we have discovered two new resistance determinants in these food isolates, which are now being characterised and sequenced. Although the general level of resistance of pure cultures is low, the resistant strains have originally been isolated after exposure to the recommended user concentration of a commercial brand of QAC. We have also studied resistance to QAC in pseudomonads isolated from the food industry. Their level of resistance is much higher than that found in staphylococci. About 30% of the collected strains were able to grow in 200 μg·ml −1 benzalkonium chloride, the lowest recommended use concentration for this commonly used type of QAC.

Intrinsic and acquired resistance to quaternary ammonium compounds in food‐related Pseudomonas spp.

Aims: To determine the sensitivity of a strain used for disinfectants testing (Pseudomonas aeruginosa ATCC 15442) and food‐associated isolates to benzalkonium chloride and didecyl dimethylammonium chloride (DDAC). To determine whether the increase in bacterial resistance after adaptation to DDAC can be associated with phenotypic changes. To test the activity of alternative disinfectants to eliminate resistant Pseudomonas spp.

Identification and characterization of quaternary ammonium compound resistant staphylococci from the food industry

The distribution of known genes conferring resistance to quaternary ammonium compounds (QACs) among different species of staphylococci isolated from the food industry was investigated. Twenty-four isolates hosting one of the genes qacA/qacB, smr, qacG or qacH, were subjected to species identification. Species determination was performed by biochemical analyses (API STAPH), comparative 16S rDNA sequence analysis and tDNA intergenic spacer length polymorphism analysis. Good correlation was obtained between the different methods. The isolates belonged to six different species of coagulase-negative Staphylococcus. The most commonly found species were Staphylococcus epidermidis and Staphylococcus saprophyticus. The results also indicated the possible spread of specific isolates of staphylococci which may reflect the dominance of certain strains in environments were QACs are used on a regular basis. The isolates were further characterized by the resistance phenotype to antimicrobial agents including antibiotics and disinfectants. Resistance to ampicillin, penicillin G and dyes was prevalent in strains harbouring the qacA or qacB genes, features also common among clinical staphylococci containing qacA/qacB. One QAC resistant strain harbouring the smr gene showed resistance to ampicillin, penicillin, tetracycline, erythromycin and trimethoprim. No enterotoxin production was detected among the QAC resistant strains.

Factors contributing to the survival of poultry associated Pseudomonas spp. exposed to a quaternary ammonium compound

Resistance to benzalkonium chloride (BC) among Pseudomonas spp. isolated from poultry carcasses was determined and strategies for elimination of resistant strains evaluated. This investigation showed that resistance was quite common, about 30% of the isolates being able to grow in 200 μg ml−1 BC. Pseudomonas fluorescens strains were generally less susceptible than strains of Ps. lundensis and Ps. fragi. An overnight incubation in medium containing 200 μg ml−1 BC was sufficient to reduce the susceptibility of two Pseudomonas strains to the lethal effect of BC significantly. Adding EDTA enhanced the lethal effect of BC, but the effect was reduced after growing cells in medium containing BC and EDTA. Growth in medium with a quaternary ammonium compound (QAC) rendered the cells more susceptible to chlorine, phenolics and alkylaminoacetate. These results indicate that alternating use of QACs with these compounds can be used to avoid build‐up of resistant strains. In addition, increased temperatures improved the lethal effect of BC and should be considered when planning disinfection routines.

Cross‐resistance to antibiotics of Escherichia coli adapted to benzalkonium chloride or exposed to stress‐inducers

Aims:  To study the effects of adaptation and stress on the resistance to benzalkonium chloride (BC) and cross‐resistance to antibiotics in Escherichia coli.

Factors influencing a suspension test method for antimicrobial activity of disinfectants.

S LANGSRUD AND G. SUNDHEIM. 1998. Factors influencing the numbers of Escherichia coli DSM 682 and Staphylococcus aureus ATCC 6538 surviving exposure to disinfectants were evaluated by factorial design. Aerobic conditions during precultivation rendered E. coli more resistant to the lethal activity of benzalkonium chloride (BC) and a disinfectant containing grape fruit extract (GSE), whereas Staph, aureus became more sensitive. The degree of shaking and the pre‐growth medium (tryptone soy broth or Mueller‐Hinton broth) did not influence the result of the bactericidal test. The number of E. coli surviving BC treatment was significantly lower if the neutralizing broth contained thiosulphate, plate pouring was used instead of plate spreading, or the plates were incubated at 37 instead of 30 °C. The negative effect of plate pouring was also found with Staph, aureus. The use of filtration without prior neutralization of the disinfectant decreased the numbers of chlorine‐treated, but not BC‐treated, E. coli. The results showed that rigorous standardization is necessary to obtain good reproducibility of bactericidal suspension tests.

Characterization of Serratia marcescens surviving in disinfecting footbaths.

Aim: To determine if disinfecting footbaths in the food industry were contaminated with bacteria and to characterize some of the bacteria present.

Identification of pseudomonads from fresh and chill-stored chicken carcasses

Results of carbon source assimilation tests (17 carbon compounds) led to 88% of pseudomonads from cold-stored chicken carcasses being assigned to one of 17 groups. Of these groups, 13 had combinations of properties identical to, or with readily recognizable degrees of similarity to those of published species/biovars. Two of the four groups having carbon assimilation patterns dissimilar to any known species had cellular fatty acid composition corresponding to Pseudomonas fluorescens, and two to Pseudomonas lundensis or Pseudomonas fragi. The P. fluorescens biovars all had higher amounts of 16:1 cis 9 (21-37%) and 18:1 cis 11 (10-19%), than of 17:0 cyclo (1-17%) and 19:0 cyclo (0-1%). In contrast, for P. lundensis and P. fragi, the relative amounts of these unsaturated acids and cyclopropane acids were reversed. Both the carbon source assimilation tests and the cellular fatty acid composition led to the conclusion that none of the species were dominant, although the P. fluorescens biovars constituted about 50% of the isolated pseudomonads.