Sensitivity of Listeria monocytogenes to sanitizers used in the meat processing industry

Nineteen Listeria monocytogenes strains were characterized by automated ribotyping, pulsed-field gel electrophoresis, and plasmid profiling to determine the relationship between genotype and sanitizer resistance. Isolates within a ribogroup had a consistent sensitivity or resistance phenotype except for ribogroup C isolates. All isolates with resistance phenotypes harbored two plasmids. The sensitivity of L. monocytogenes strains to quaternary ammonium compounds (QACs) was correlated with sensitivity to sanitizers and antibiotics with other modes of action. All isolates tested contained the mdrL gene, which encodes an efflux pump that confers resistance to QACs and is both chromosome and plasmid borne.     

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.     

A multidrug efflux transporter in Listeria monocytogenes

A chromosomal gene (mdrL) was found in Listeria monocytogenes L028, showing a high degree of similarity with multidrug efflux transporters of the major facilitator superfamily (family 2). An allele-substituted mutant of this gene failed to pump out ethidium bromide and presented lower minimal inhibitory concentrations of macrolides, cefotaxime and heavy metals. This is the first multidrug efflux pump described in Listeria.     

A two-component multidrug efflux pump, EbrAB, in Bacillus subtilis

Genes (ebrAB) responsible for ethidium resistance were cloned from chromosomal DNA of Bacillus subtilis ATCC 9372. The recombinant plasmid produced elevated resistance against ethidium bromide, acriflavine, pyronine Y, and safranin O not only in Escherichia coli but also in B. subtilis. It also caused an elevated energy-dependent efflux of ethidium in E. coli. EbrA and EbrB showed high sequence similarity with members of the small multidrug resistance (SMR) family of multidrug efflux pumps. Neither ebrA nor ebrB was sufficient for resistance, but introduction of the two genes carried on different plasmids conferred drug resistance. Thus, both EbrA and EbrB appear to be necessary for activity of the multidrug efflux pump. In known members of the SMR family, only one gene produces drug efflux. Thus, EbrAB is a novel SMR family multidrug efflux pump with two components.     

Detection of smr and qacA genes in Staphylococcus aureus strains using PCR

The 31 strains of Staphylococcus aureus were examined for the presence of smr and qacA determinants. The smr gene was found in 15 strains. Fourteen of them were MRSA resistant to quaternary ammonium compounds, ethidium bromide, and acriflavine. One was MSSA strain resistant to ethidium bromide and acriflavine. The qacA gene was found in two MRSA strains resistant to quaternary ammonium compounds, ethidium bromide, chlorhexidine and acriflavine. One of these two strains possessed both smr and qacA genes.     

Discarding multidrug resistance inducers, the possible role of a biosensing reporter in antimicrobial discovery.

The aim of the present work was to determine whether the luminescence-based reporter plasmid pQacLux could be applied to drug discovery in order to discard compounds with defined properties. Non-pathogenic Staphylococcus aureus RN4220 cells bearing pQacLux were incubated with different concentrations of a disinfectant of common use in hospitals. The in vivo light emission response of the plasmid to the given stimuli was then quantified and compared to a negative control for the construction of dose-response curves. The selected disinfectant provided a convenient model for the activity of quaternary ammonium compounds. In spite of the use of a raw model solution, the system revealed high levels of sensitivity. According to the results obtained, pQacLux could be conveniently used in the first steps of drug development in order to discard all possible multidrug resistance inducers.     

Involvement of the smeAB multidrug efflux pump in resistance to plant antimicrobials and contribution to nodulation competitiveness in Sinorhizobium meliloti

The contributions of multicomponent-type multidrug efflux pumps to antimicrobial resistance and nodulation ability in Sinorhizobium meliloti were comprehensively analyzed. Computational searches identified genes in the S. meliloti strain 1021 genome encoding 1 pump from the ATP-binding cassette family, 3 pumps from the major facilitator superfamily, and 10 pumps from the resistance-nodulation-cell division family, and subsequently, these genes were deleted either individually or simultaneously. Antimicrobial susceptibility tests demonstrated that deletion of the smeAB pump genes resulted in increased susceptibility to a range of antibiotics, dyes, detergents, and plant-derived compounds and, further, that specific deletion of the smeCD or smeEF genes in a ΔsmeAB background caused a further increase in susceptibility to certain antibiotics. Competitive nodulation experiments revealed that the smeAB mutant was defective in competing with the wild-type strain for nodulation. The introduction of a plasmid carrying smeAB into the smeAB mutant restored antimicrobial resistance and nodulation competitiveness. These findings suggest that the SmeAB pump, which is a major multidrug efflux system of S. meliloti, plays an important role in nodulation competitiveness by mediating resistance toward antimicrobial compounds produced by the host plant.     

Physical and biochemical characterization of the qacA gene encoding antiseptic and disinfectant resistance in Staphylococcus aureus

We have previously cloned a 3.5 kb fragment from the Staphylococcus aureus multiresistance plasmid pSK1 which carries the qacA determinant responsible for linked resistance to acriflavine (Acr), ethidium bromide (Ebr), quaternary ammonium compounds (Qar), propamidine isethionate (Pir), and diamidinodiphenylamine dihydrochloride (Ddr). This report presents a biochemical and physical analysis of qacA and shows the widespread carriage of this gene on S. aureus resistance plasmids. Tn5 insertion mutagenesis defined the extent of qacA to within 2.40 kb of pSK1 DNA. Examination of the expression of insertion and deletion mutants of the cloned qacA sequences in both maxicells and minicells led to the association of a 50 kDa protein, designated QacA, with the AcrEbrQarPirDdr phenotype. Based on fluorimetric and isotopic assays used to determine the extent of accumulation of ethidium bromide by S. aureus strains harbouring pSK1, we propose that the basis of AcrEbrQarPirDdr in S. aureus is a qacA-mediated efflux system.     

Comparative sensitivity to antibiotics and biocides of methicillin-resistant Staphylococcus aureus strains isolated from Saudi Arabia and Great Britain

Methicillin-resistant Staphylococcus aureus (MRSA) isolated in Saudi Arabia and Great Britain were examined for susceptibility to antibiotics and biocides. The strains differed in their sensitivity patterns. None of the Saudi strains showed resistance to propamidine isethionate, but most of the British gentamicin methicillin-resistant Staph. aureus (GMRSA) strains were highly resistant to this compound and to some other nucleic acid-binding (NAB) compounds. Both groups showed a low level of resistance towards quaternary ammonium compounds (QACs), but resistance to these compounds was not associated with resistance to gentamicin in the Saudi strains. The aminoglycoside-resistant determinants were non-conjugative in these strains. Natural MRSA strains were good recipients for pWG613, but transferred this plasmid in reciprocal crosses at significantly lower rates.     

Multiresistance genes of Rhizobium etli CFN42

Multidrug efflux pumps of bacteria are involved in the resistance to various antibiotics and toxic compounds. In Rhizobium etli, a mutualistic symbiont of Phaseolus vulgaris (bean), genes resembling multidrug efflux pump genes were identified and designated rmrA and rmrB. rmrA was obtained after the screening of transposon-generated fusions that are inducible by bean-root released flavonoids. The predicted gene products of rmrAB shared significant homology to membrane fusion and major facilitator proteins, respectively. Mutants of rmrA formed on average 40% less nodules in bean, while mutants of rmrA and rmrB had enhanced sensitivity to phytoalexins, flavonoids, and salicylic acid, compared with the wild-type strain. Multidrug resistance genes emrAB from Escherichia coli complemented an rmrA mutant from R. etli for resistance to high concentrations of naringenin.     

High-level resistance to ethidium bromide and antiseptics in Staphylococcus aureus

A gene encoding high-level resistance to ethidium bromide (EB) and antiseptics was isolated from a transferable plasmid, pTZ22, in Staphylococcus aureus. DNA sequencing revealed that the plasmid has two copies of the ebr gene that normally mediates low-level resistance to EB and antiseptics. The efflux rate for EB of strains with duplicated ebr genes was twice the rate of strains with a single ebr gene. It was concluded that the duplication of ebr is responsible for the high-level resistance to EB and antiseptics.     

Comparative sensitivity to antibiotics and biocides of methicillin-resistant Staphylococcus aureus strains isolated from Saudi Arabia and Great Britain

S.B. AL-MASAUDI, A.D. RUSSELL AND M.J. DAY. 1991. Methicillin-resistant Staphylococcus aureus (MRSA) isolated in Saudi Arabia and Great Britain were examined for susceptibility to antibiotics and biocides. The strains differed in their sensitivity patterns. None of the Saudi strains showed resistance to propamidine isethionate, but most of the British gentamicin methicillin-resistant Staph. aureus (GMRSA) strains were highly resistant to this compound and to some other nucleic acid-binding (NAB) compounds. Both groups showed a low level of resistance towards quaternary ammonium compounds (QACs), but resistance to these compounds was not associated with resistance to gentamicin in the Saudi strains. The aminoglycoside-resistant determinants were non-conjugative in these strains. Natural MRSA strains were good recipients for pWG613, but transferred this plasmid in reciprocal crosses at significantly lower rates.     

Insights into the environmental resistance gene pool from the genome sequence of the multidrug-resistant environmental isolate Escherichia coli SMS-3-5

The increasing occurrence of multidrug-resistant pathogens of clinical and agricultural importance is a global public health concern. While antimicrobial use in human and veterinary medicine is known to contribute to the dissemination of antimicrobial resistance, the impact of microbial communities and mobile resistance genes from the environment in this process is not well understood. Isolated from an industrially polluted aquatic environment, Escherichia coli SMS-3-5 is resistant to a record number of antimicrobial compounds from all major classes, including two front-line fluoroquinolones (ciprofloxacin and moxifloxacin), and in many cases at record-high concentrations. To gain insights into antimicrobial resistance in environmental bacterial populations, the genome of E. coli SMS-3-5 was sequenced and compared to the genome sequences of other E. coli strains. In addition, selected genetic loci from E. coli SMS-3-5 predicted to be involved in antimicrobial resistance were phenotypically characterized. Using recombinant vector clones from shotgun sequencing libraries, resistance to tetracycline, streptomycin, and sulfonamide/trimethoprim was assigned to a single mosaic region on a 130-kb plasmid (pSMS35_130). The remaining plasmid backbone showed similarity to virulence plasmids from avian-pathogenic E. coli (APEC) strains. Individual resistance gene cassettes from pSMS35_130 are conserved among resistant bacterial isolates from multiple phylogenetic and geographic sources. Resistance to quinolones was assigned to several chromosomal loci, mostly encoding transport systems that are also present in susceptible E. coli isolates. Antimicrobial resistance in E. coli SMS-3-5 is therefore dependent both on determinants acquired from a mobile gene pool that is likely available to clinical and agricultural pathogens, as well, and on specifically adapted multidrug efflux systems. The association of antimicrobial resistance with APEC virulence genes on pSMS35_130 highlights the risk of promoting the spread of virulence through the extensive use of antibiotics.     

Active efflux,a common mechanism for biocide and antibiotic resistance

Energy-driven drug efflux systems are increasingly recognized as mechanisms of antibiotic resistance. Chromosomally located or acquired by bacteria, they can either be activated by environmental signals or by a mutation in a regulatory gene. Two major categories exist: those systems energized by proton motive force and those dependent on ATP. The pumps may have limited or broad substrates, the so-called multiple drug resistance pumps, which themselves form a number of related families. The multiple antibiotic resistance (mar) locus and mar regulon in Escherichia coli and other members of the Enterobacteriaceae is a paradigm for a generalized response locus leading to increased expression of efflux pumps. One such pump, the AcrAB pump extrudes biocides such as triclosan, chlorhexidine and quaternary ammonium compounds as well as multiple antibiotics. In Pseudomonas aeruginosa, a number of multidrug efflux pumps export a broad range of substrates. Since bacteria expressing these pumps thwart the efficacy of both kinds of therapeutic agents which control infectious diseases--biocides which prevent transmission of infectious disease agents and antibiotics which treat and cure infectious diseases--they are of particular concern. The prudent use of antibiotics and biocides will guard against the selection and propagation of drug-resistant mutants and preserve the efficacy of these valuable anti-infective agents.     

The presence of qacA/B gene in Brazilian methicillin-resistant Staphylococcus aureus

A total of 74 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from three government hospitals in 2002 and 2003 were examined concerning the distribution of qacA/B gene, which is the determinant of resistance to quaternary ammonium compounds largely employed in hospital disinfection. By polymerase chain reaction the qacA/B gene was found in 80% of the isolates, which is a significant result considering it is the first time that qacA/B gene is being reported for Brazilian MRSA strains and it is presented at a high rate.     

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-containing 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.