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.     

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.     

Antiseptic susceptibility and distribution of antiseptic-resistance genes in methicillin-resistant Staphylococcus aureus

We examined the antiseptic susceptibilities and distribution of antiseptic-resistance genes qacA and smr in 98 isolates of methicillin-resistant Staphylococcus aureus obtained in 1992. Seventy-one strains were resistant to antiseptics. The qacA and smr genes were detected in 10 and 20 strains, respectively. The remaining 41 strains without qacA and smr were divided into two groups that exhibited low-level (n = 22) and high-level (n = 19) resistance to acriflavin. DNA cloning and sequencing suggested that norfloxacin-resistance gene norA was responsible for the high-level resistance to acriflavin. Our results indicated that four or more antiseptic-resistance genes exist in methicillin-resistant S. aureus and that antiseptic-resistant methicillin-resistant S. aureus strains without qacA and smr are widely spread in Japan.     

Resistance of Staphylococcus aureus strains to quaternary ammonium compounds and chlorhexidine

The level of susceptibility of 90 different Staphylococcus aureus strains to chosen quaternary ammonium compounds: cetyltrimethyl ammonium bromide, benzalkonium chloride and benzethonium chloride as well as to chlorhexidine digluconate were examined. The examined strains consist of three groups: hospital originated MRSA, hospital originated MSSA and non-hospital MSSA. The significant differences between these groups were observed in they susceptibility to the investigated disinfectants. The obtained MIC values showed that the most resistant were hospital MRSA strains, where 55% was estimated as resistant to cetyltrimethyl ammonium bromide, 72% were resistant to benzalkonium chloride and benzethonium chloride and 7% were resistant to chlorhexidine digluconate. Among hospital originated MSSA 3% of strains were resistant to cetyltrimethyl ammonium bromide and 6% were resistant to benzalkonium chloride and benzethonium chloride. 14% non-hospital S. aureus strains were resistant to benzalkonium chloride and benzethonium chloride. None were resistant to chlorhexidine digluconate or cetyltrimethyl ammonium bromide.     

西安市MRSA 耐消毒剂基因qacA/B 的检测

目的:了解西安市三甲综合医院耐甲氧西林金黄色葡萄球菌(MRSA)qacA/B基因的携带情况,以便掌握其对消毒剂的耐药情况。方法:收集临床分离的152株耐甲氧西林金黄色葡萄球菌,采用聚合酶链反应检测qacA/B基因,并将qacA/B基因扩增产物进行测序,测序结果进行比对。结果:152株耐甲氧西林金黄色葡萄球菌中有11株检出qacA/B阳性,qacA/B携带率7.2%;测序结果比对发现,4株qacA的基因编码序列同一位点均有一个碱基发生突变(C→T),苏氨酸突变为异亮氨酸。结论:该地区临床分离的耐甲氧西林金黄色葡萄球菌中qacA/B携带率相对较低,但也存在抵抗消毒剂的风险,临床应加强消毒剂使用的管理,阻止MRSA的传播,减少感染的发生。     

Efflux-mediated antiseptic resistance gene qacA from Staphylococcus aureus: common ancestry with tetracycline- and sugar-transport proteins

Resistance to intercalating dyes (ethidium, acriflavine) and other organic cations, such as quaternary ammonium-type antiseptic compounds, mediated by the Staphylococcus aureus plasmid pSK1 is specified by an energy-dependent export mechanism encoded by the qacA gene. From nucleotide sequence analysis, qacA is predicted to encode a protein of Mr 55017 containing 514 amino acids. The gene is likely to initiate with a CUG codon, and a 36 bp palindrome immediately preceding qacA, along with an upstream reading frame with homology to the TetR repressors, may be components of a regulatory circuit. The putative polypeptide specified by qacA has properties typical of a cytoplasmic membrane protein, and is indicated to be a member of a transport protein family that includes proteins responsible for export-mediated resistance to tetracycline and methylenomycin, and uptake of sugars and quinate. The analysis suggests that N- and C-terminal regions of these proteins are involved in energy coupling (proton translocation) and substrate transport, respectively. The last common ancestor of the qacA and related tet (tetracycline resistance) lineages is inferred to have been repressor controlled, as occurs for modern tet determinants from Gram-negative, but not those from Gram-positive, bacteria.     

Association of qacE and qacEDelta1 with multiple resistance to antibiotics and antiseptics in clinical isolates of Gram-negative bacteria

Clinical isolates of Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia were tested for resistance to antibiotics and to the antiseptics benzalkonium chloride and cetyltrimethylammonium bromide. Furthermore, they were examined for the presence of the resistance genes qacE and qacEDelta1. qacEDelta1 was detected by PCR in 10% of all (n=103) and in 81% of multiply antibiotic-resistant strains (n=15). qacE was found in only one of 37 P. aeruginosa strains. The minimum inhibitory concentrations of benzalkonium chloride, cetyltrimethylammonium bromide, and ethidium bromide were not significantly different for qacEDelta1/qacE-positive or -negative strains. Our data indicate that multiply antibiotic-resistant Gram-negative bacteria are not necessarily more resistant to quaternary ammonium compounds than antibiotic-sensitive strains even though qacE or qacEDelta1 is present.     

Sensitivity of methicillin-resistant Staphylococcus aureus strains to some antibiotics, antiseptics and disinfectants

The effects of antibiotics, antiseptics and disinfectants against some methicillin resistant (MRSA) and methicillin-sensitive (MSSA) Staphylococcus aureus strains have been studied. The MRSA and MSSA strains were equally sensitive to phenols, esters of para(4)-hydroxybenzoit acid and chlorhexidine but MRSA strains were slightly more resistant to quaternary ammonium compounds and considerably more so to dibromopropamidine isothionate. Some MRSA strains were also resistant to phenylmercuric nitrate (but not another organomercurial, thiomersal), mercuric chloride and cadmium chloride. All MRSA strains produced β-lactamase. Strains from the Royal Free Hospital, London were highly resistant to β-lactam antibiotics, erythromycin, trimethoprim and tetracyctines but were sensitive to other antibiotics. One strain from the University Hospital of Wales, Cardiff was resistant to gentamicin but sensitive to tetracycline and trimethoprim.     

Sensitivity of methicillin-resistant Staphylococcus aureus strains to some antibiotics, antiseptics and disinfectants

The effects of antibiotics, antiseptics and disinfectants against some methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) Staphylococcus aureus strains have been studied. The MRSA and MSSA strains were equally sensitive to phenols, esters of para(4)-hydroxybenzoic acid and chlorhexidine but MRSA strains were slightly more resistant to quaternary ammonium compounds and considerably more so to dibromopropamidine isothionate. Some MRSA strains were also resistant to phenylmercuric nitrate (but not another organomercurial, thiomersal), mercuric chloride and cadmium chloride. All MRSA strains produced beta-lactamase. Strains from the Royal Free Hospital, London were highly resistant to beta-lactam antibiotics, erythromycin, trimethoprim and tetracyclines but were sensitive to other antibiotics. One strain from the University Hospital of Wales, Cardiff was resistant to gentamicin but sensitive to tetracycline and trimethoprim.     

The NorM efflux pump of Neisseria gonorrhoeae and Neisseria meningitidis recognizes antimicrobial cationic compounds

In Neisseria gonorrhoeae and Neisseria meningitidis, we identified a gene that would encode a protein highly similar to NorM of Vibrio parahaemolyticus (Y. Morita et al., Antimicrob. Agents Chemother. 42:1778-1782, 1998). A nonpolar insertional mutation in either the gonococcal or meningococcal norM gene resulted in increased bacterial sensitivity to compounds harboring a quaternary ammonium on an aromatic ring (e.g., ethidium bromide, acriflavine hydrochloride, 2-N-methylellipticinium, and berberine). The presence of point mutations within the -35 region of a putative norM promoter or a likely ribosome binding site resulted in an increased resistance of gonococci and meningococci to the same compounds, as well as to norfloxacin and ciprofloxacin. Structure-activity relationship studies with putative NorM substrates have found that a cationic moiety is essential for NorM recognition.     

Contribution of the multidrug efflux pump LfrA to innate mycobacterial drug resistance

Multidrug resistance (MDR) in bacteria has been associated with efflux pumps that export structurally unrelated compounds and decrease cytoplasmic drug accumulation. To investigate MDR in mycobacteria, we studied the Mycobacterium smegmatis mutant mc(2)11, which is resistant to doxorubicin, tetracycline, rhodamine, ethidium bromide and the hydrophilic fluoroquinolones. A genomic library constructed from this mutant was used to select clones conferring resistance to doxorubicin. Surprisingly, the clone selected encodes the efflux pump LfrA, which has been reported to confer resistance to hydrophilic fluoroquinolones, ethidium bromide, rhodamine, and acriflavine. To define the contribution of LfrA to the innate mycobacterial drug resistance and to the MDR phenotype in mc(2)11, the lfrA gene was disrupted in both the mc(2)11 mutant and the mc(2)155 wild-type parent. LfrA disruption of the wild-type strain decreased resistance to ethidium bromide and acriflavine, and increased accumulation of ethidium bromide. However, disruption of lfrA gene results only in a 2-fold decrease in minimal inhibitory concentrations (MICs) for ciprofloxacin, doxorubicin, rhodamine, and accumulation of [(14)C]ciprofloxacin was unchanged. LfrA disruption of the MDR strain mc(2)11 produced a similar phenotype. Thus, LfrA contributes significantly to the intrinsic MICs of M. smegmatis for ethidium bromide and acriflavine, but not for ciprofloxacin, doxorubicin or rhodamine.     

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.     

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.     

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.     

Plasmids and bacterial resistance to biocides

Plasmid-encoded fu1 bacterial resistance to antibiotics and to anions and cations (including important mercurial and silver compounds) has been widely studied. Plasmid-mediated resistance to organic cationic agents which are important biocides has been described for chlorhexidine and quaternary ammonium compounds (and also for the less important acridines, diamidines and ethidium bromide) in antibiotic-resistant Staphylococcus aureus and Staph. epidermidis strains. Plasmids may also encode reduced biocide susceptibility of Gram-negative bacteria, but intrinsic resistance is likely to be of greater significance. Antibiotic resistance and biocide resistance may be linked but this is not always found clinically.     

mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌

目的 应用mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌（methicillin resistant staphylococcus aureus,MRSA）。方法 临床分离的70株金黄色葡萄球菌,应用mecA基因PCR扩增法鉴定MRSA,并与苯唑西林纸片扩散法进行比较。结果 70株金黄色葡萄球菌用PCR扩增法和纸片扩散法有6株鉴定有差异,4株。mecA基因阳性而纸片扩散法鉴定为敏感,1株mecA基因阳性纸片扩散法鉴定为临界耐药,1株mecA基因阴性却表现为苯唑西林耐药,2种方法符合率为91．43％。结论 mecA基因PCR扩增法可以准确、快速判定MRSA,特别是对隐匿型或低水平耐药菌株的检出有重要的价值。     

Ethidium Bromide-Resistant Mutant of Bacillus subtilis

An ethidium bromide-resistant mutant (EB8) derived from a Marburg strain of Bacillus subtilis was found to be conditionally resistant to 10 mug of ethidium bromide per ml. Expression of resistance is complete only during vegetative growth at incubation temperatures above 30 C in complex medium or minimal medium supplemented with Casamino Acids. Strain EB8 is cross-resistant to acriflavine and proflavine. The ethidium bromide resistance marker is co-transduced with hisA1 at a frequency of 6% and is located to the right of hisA1 on the B. subtilis chromosome as it is usually represented on the map. Incorporation of [5-(3)H] uridine by strain EB8 showed that ribonucleic acid synthesis in both whole cells and protoplasts is ethidium bromide-resistant.     

Ethidium bromide resistance in a rat liver epithelial cell line: Association with enhanced drug efflux

Ethidium bromide-resistant cell strains were obtained by continuous selection of an adult rat liver-derived cell line (ARL6T) grown in the continuous presence of 200 ngl ml ethidium bromide. Comparison of resistant strains and parental (sensitive) cells was made for uptake and binding of ethidium bromide, visualized as fluorescent ethidium bromide-nucleic acid complexes. Although uptake of ethidium bromide was similar in parental and resistant cells, efflux kinetics were markedly different. Over a three-hour period, parental (sensitive) cells maintained fluorescence following a short ethidium bromide pulse (100 g/ ml ethidium bromide). In contrast, ethidium bromide-resistant cell lines eliminated photographically detectable fluorescent complexes within three hours following pulse exposure to ethidium bromide. The rapid elimination of ethidium bromide fluorescent complexes in all (5) resistant cell strains examined supports an efflux mechanism as contributing to the resistance of ethidium bromide cytotoxicity in these cells.Abbreviations EtBr ethidium bromide - HBSS Hanks' balanced salt solution     

The gene that determines resistance to tioconazole and to acridine derivatives in Aspergillus nidulans may have a corresponding gene in Trichophyton rubrum

Understanding the genetic mechanisms involved in resistance to antifungal agents is important in the fight against pathogenic fungi. In the present investigation we studied a strain of the model fungus Aspergillus nidulans which presents resistance to tioconazole and behaves as the wild strain in the presence of other azole derivatives. Genetic analysis revealed that this resistance is due to a mutation in a single gene located on chromosome II, closely linked to the allele responsible for resistance to acriflavine and other acridine derivatives, i.e., acrA1. This result suggests that a multidrug resistance (MDR)-type mechanism may be involved. Two tioconazole-resistant strains of the pathogenic fungus Trichophyton rubrum obtained after mutagenic treatment also became simultaneously resistant to acriflavine and ethidium bromide, suggesting the existence of a resistance mechanism similar to that observed with the acrA1 mutation in A. nidulans. This revised version was published online in June 2006 with corrections to the Cover Date.