Efflux pump activity in fluoroquinolone and tetracycline resistant Salmonella and E. coli implicated in reduced susceptibility to household antimicrobial cleaning agents

Abstract

It has been shown that the inappropriate use of antimicrobial household agents selects for organisms with resistance mechanisms (e.g. efflux pumps), which could lead to the development of antibiotic resistance. The reverse hypothesis, that antibiotic-resistant organisms become tolerant to other antibacterial agents (e.g. disinfectants) due to the action of efflux pumps, has however not been extensively examined. The objective of this study was to establish whether there is a link between antibiotic resistance in potential gastrointestinal pathogens and reduced sensitivity of these organisms to commonly used household antimicrobial agents. In this study, tetracycline and ofloxacin sensitive and resistant Escherichia coli (9 strains) and Salmonella spp. (8 strains) were isolated from poultry and clinical samples. In order to assess whether these bacteria had active efflux pumps, ethidium bromide accumulation assays were performed. Extrusion of the active components of three commercial household agents (triclosan, sodium salicylate, and ortho-phenylphenol) by efflux pumps was tested using spectrophotometric accumulation assays. In order to simulate the kitchen environment, in-use disinfectant testing using the commercial household agents was performed to determine changes in their efficacy due to antibiotic resistance. Active efflux pump activity and extrusion of all three active ingredients was observed only in the antibiotic resistant organisms. The antibiotic sensitive bacteria were also more susceptible than the resistant isolates to the household antimicrobial agents at concentrations below that recommended by the manufacturer. These resistant bacteria could potentially be selected for and result in hard to treat infections.

Introduction

The occurrence of bacteria resistant to multiple antibiotics presents a serious problem in the treatment of bacterial infections (Bolhuis et al., 1995). One of the underlying mechanisms for this multidrug resistance phenotype is the expression and increased activity of efflux pumps, which facilitate the transfer of toxic substances from within cells to the external environment (Bohn and Bouloc, 1998, Webber and Piddock, 2003). Efflux systems that contribute to antibiotic resistance have already been described in a number of clinically important bacteria, including Salmonella typhimurium (Webber and Piddock, 2003), and Escherichia coli (Poole, 2000). Efflux pumps can transport a variety of structurally dissimilar compounds or may be specific for a single substrate (Poole, 2000, Webber and Piddock, 2003).

There is little doubt that the inappropriate clinical application of antimicrobial agents has led to the increased rates of multidrug resistant bacteria (Cruchaga et al., 2001), and the use of antibiotics in the veterinary and farming arena has recently come under scrutiny (Singer et al., 2003). The introduction of antibiotics in livestock rearing has resulted in a variety of developments in animal husbandry practices (Manie et al., 1998, Butaye et al., 2003). These include the breeding of animals in batteries/pens that are filled to capacity at optimal temperature and low light intensity to enhance growth rates and increase body mass (Manie et al., 1998). Many antibiotics are administered therapeutically for disease prevention, but subtherapeutic doses are also administered to improve feed efficiency and accelerate weight gain in poultry, swine, sheep and cattle (Manie et al., 1998, Chopra and Roberts, 2001, Geornaras et al., 2001, White et al., 2001, McEwen and Fedorka-Cray, 2002, Butaye et al., 2003).

Although not everyone is in agreement as to whether subtherapeutic doses lead to the development of antibiotic resistance in bacteria, there is extensive evidence that they result in significant selection pressures on animal pathogens and commensals (Quednau et al., 1998, McEwen and Fedorka-Cray, 2002). The antibiotic resistance that has arisen in zoonotic organisms such as Salmonella, Listeria and E. coli poses a threat to both animal and human health (Chopra and Roberts, 2001). Since potential pathogens can be transferred from animals to humans via the food chain (Parsonnet and Kass, 1987), they could result in diseases that are difficult to treat or transfer their resistance genes to other bacteria (Chopra and Roberts, 2001).

Several brands of household disinfectant products are marketed as being superior to others due to the addition of one or more antibacterial compounds, since it is claimed that these products kill or inactivate bacteria more efficiently than the regular items (Kusumaningrum et al., 2002). Researchers have recently started to express concern regarding the use of antimicrobial chemicals in the domestic setting due to possible selection of resistant organisms (McDonnell and Russell, 1999).

This study was undertaken to establish whether there is a relationship between antibiotic resistant organisms and reduced susceptibility to the antimicrobial agents found in commonly used household disinfectant products, and whether this could be due to the action of efflux pumps.