JAC Advance Access published online on August 15, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn327
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Original research |
First report of qacG, qacH and qacJ genes in Staphylococcus haemolyticus human clinical isolates
1 Departamento de Microbiología, Inmunología y Parasitología, Facultad de Medicina, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina 2 Instituto de Investigaciones Alfredo Lanari, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina
* Corresponding author. Tel: +54-11-5950-9500 ext. 2180; Fax: +54-11-4964-2554; E-mail: paolajeric{at}yahoo.com
Received 10 April 2008; returned 14 June 2008; revised 11 July 2008; accepted 18 July 2008
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Abstract |
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Objectives: To investigate phenotypically and genotypically the presence of MDR efflux pumps in 21 clinical isolates of Staphylococcus haemolyticus collected over a period of 10 years.
Methods: MICs of different antibiotics and biocides were determined by the broth dilution method in the presence/absence of carbonyl cyanide-m-chlorophenylhydrazone (CCCP), an efflux pump inhibitor. PCR followed by sequencing was performed to detect the qac genes that encode for antiseptic resistance. Clonal relationships were determined by PFGE SmaI patterns using a standard protocol.
Results: All the isolates were resistant to gentamicin, 15 to erythromycin, 18 to ciprofloxacin, 7 to chloramphenicol and 1 to tetracycline. They showed higher susceptibility to antibiotics when they were exposed to CCCP. The MICs of ethidium bromide, SDS and benzalkonium chloride were also decreased, whereas the MIC of triclosan was decreased in only four isolates in the presence CCCP. Of the 21 isolates, qacA/B was detected in 5 isolates, smr in all of the isolates, qacG in 11 isolates, qacH in 10 isolates and qacJ in 4 isolates. PFGE analysis of the 21 isolates clustered them into 14 clones at 90% similarity corresponding to differences of between 7 and 16 bands among the clones.
Conclusions: The efflux mechanism seems to be an important mechanism to confer resistance to antibiotics and biocides through MDR pumps. It was observed that several qac genes coexist in some of the isolates and seem to act simultaneously in the removal of different compounds out of the bacterial cell. The qac genes are horizontally spread among different clones.
Key Words: S. haemolyticus , multidrug resistance , efflux pumps
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Introduction |
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Staphylococcus haemolyticus is an opportunistic bacterial pathogen found in the normal skin flora, commonly isolated from the axillae, perineum and inguinal areas from humans.1 Among coagulase-negative staphylococci, S. haemolyticus is second only to Staphylococcus epidermidis in its frequency of isolation from human blood cultures and may also cause septicaemia, peritonitis, otitis and urinary tract infections.1
One of the mechanisms used by staphylococci to resist the action of antibiotics and biocides is the expression of an efflux system. These pumps can be specific for one substrate,2 such as TetK and TetL for tetracycline, or be able to transport multiple drugs (MDR pumps).3 Among the five MDR families of efflux systems currently described in bacteria, the ABC (ATP binding cassette) and MFS (major facilitator superfamily) include the majority of the efflux systems described in Staphylococcus spp., Enterococcus spp. and Streptococcus spp. NorA, NorB, NorC, MdeA and SepA are multidrug transporters described in Staphylococcus aureus that confer resistance to quinolones, quaternary ammonium compounds (QACs), dyes and ethidium bromide amid other compounds.
Disinfectants based on QACs have various applications in veterinary medicine and are used in hygienic handwashes.4–6 The genes that encode the loss of susceptibility to QAC by efflux found in staphylococci, qacA, qacB, smr, qacG, qacH and qacJ, are, in general, plasmid-borne and confer less susceptibility to cationic antiseptic agents including dyes (acriflavine, ethidium bromide), QACs (benzalkonium chloride) and biguanides (chlorhexidine digluconate, only qacA). Here we report for the first time the presence of qacG, qacH and qacJ in S. haemolyticus clinical isolates from humans.
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Materials and methods |
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Twenty-one S. haemolyticus clinical isolates were collected during a period of 10 years from two different hospitals in the city of Buenos Aires. The sources of the isolates appear in Tables 1 and 2. The criteria of selection were that they had to be resistant to two or more antibiotics that are structurally different. The presence of a putative multidrug efflux system was assessed by the determination of the MICs of ciprofloxacin, tetracycline, chloramphenicol, gentamicin, erythromycin and biocides ethidium bromide, SDS, benzalkonium chloride and triclosan (Johnson Diversey, Buenos Aires) using the broth dilution method according to the CLSI guidelines7–9 in the presence/absence of carbonyl cyanide-m-chlorophenylhydrazone (CCCP; Sigma-Aldrich, USA). S. aureus 29 213 and Escherichia coli AG112 (a mutant that expresses the efflux pump AcrAB) were used as controls to test antibiotic and biocide susceptibilities, respectively. It was considered that a decrease in the MIC value of 3-fold or more in the presence of CCCP indicated the presence of an efflux mechanism (Tables 1 and 2).
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With regard to antiseptic resistance, the presence of qacA, qacB, smr, qacG, qacH and qacJ genes was determined by PCR using a standard protocol with primers previously described4–6 using S. aureus Mu50 (for qacA, qacB and smr), S. aureus RN4220 (pSK265) qacG, RN4220 (pSK265) qacH and RN4220 (pSK265) qacJ as controls; the S. aureus RN4220 strains were kindly provided by Jostein Bjorland (Norwegian School of Veterinary Science). The annealing temperatures were: qacA/B, 40°C; smr, 60°C; qacG and qacH, 49°C; qacJ, 47°C. The PCR products were sequenced using the ABI3730XL system (Macrogen, Korea).
Genotyping was performed with SmaI PFGE by a standard protocol using the CHEF DR-III system (Bio-Rad, Hercules, CA, USA). Percentage similarity was estimated by the simple matching coefficient, and the matrix was clustered by the unweighted pair group method. In this study, a 90% similarity level was considered, corresponding to differences of between 7 and 16 bands among the clones. Patterns were analysed using TREECON software.10
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Results and discussion |
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All of the isolates were resistant to gentamicin (MIC between 32 and 256 mg/L), 15 to erythromycin (MIC
8 mg/L), 18 to ciprofloxacin (MIC 4 mg/L), 7 to chloramphenicol (MIC 32 mg/L) and 1 to tetracycline (MIC = 32 mg/L). The MICs of ethidium bromide ranged between 2 and 16 mg/L, the MICs of SDS ranged between 1 and 128 mg/L, the MICs of benzalkonium chloride ranged between 1 and 8 mg/L and the MICs of triclosan ranged between 0.008 and 0.25 mg/L (Tables 1 and 2). The isolates that were resistant to gentamicin, erythromycin and chloramphenicol showed a significant decrease in susceptibility when they were exposed to CCCP (Table 1), indicating that for these antibiotics an efflux system could be the primary mechanism of resistance. In the case of ciprofloxacin, only 50% of the isolates resistant to this antibiotic showed a decrease in the MIC in the presence of CCCP denoting that efflux confers resistance along with other mechanisms such as mutations in the GyrA or GrlA proteins.11
All the isolates showed a significant decrease in the MICs of ethidium bromide, SDS and benzalkonium chloride in the presence of CCCP, whereas the MIC of triclosan was decreased in only four isolates in the presence of the same compound. The phenotype observed indicates that ethidium bromide, SDS and benzalkonium chloride could be substrates of a common pump. All the isolates that showed a decrease in the MIC of ciprofloxacin also showed a decrease to biocides but not vice versa, which could indicate a different efflux mechanism for these components. The decrease in the MIC of triclosan in only four isolates could denote that an additional efflux system which was different from the ones investigated was present in them.
Of the 21 isolates, qacA/B was detected in 5 isolates, whereas all of the isolates showed the presence of smr. qacG was present in 11 isolates, qacH in 10 isolates and qacJ in 4 isolates (Tables 1 and 2) that also harboured qacH indicating that qacJ and qacH could reside on common plasmids. It was observed that one isolate (SH 11) harboured qacG, qacH and qacJ at the same time. The PCR product sizes were 203 bp (qacG), 225 bp (qacH) and 242 bp (qacJ), and the nucleotide sequence analysis demonstrated 98% (qacG), 99% (qacJ) and 100% (qacH) similarity with their respective genes in the database. GenBank accession numbers for qacG, qacH and qacJ are EU622633 [GenBank] , EU622634 [GenBank] and EU622635 [GenBank] , respectively. In those isolates that carried smr, qacA/B and/or qacG, it was observed that SDS could be a substrate of any of them since the isolates that showed a decrease in the MIC of SDS in the presence of CCCP harboured one or all of the genes named above.
PFGE analysis of the 21 isolates clustered them into 14 clones (Figure 1). Cluster II (isolates 4 and 6), which appeared in the year 2004 from a bloodstream infection, acquired the gene qacA/B during 2005 and lost the gene qacH. Also the MIC of triclosan was not decreased with the addition of CCCP in isolate 4 compared with isolate 6, which could mean that the gene that encoded the ‘efflux pump’ for triclosan may have been lost or was inactivated producing an isolate more susceptible to this component. The ‘efflux’ of triclosan was an acquisition during recent years since the four isolates whose MICs of this biocide were decreased by the addition of CCCP were isolated between 2004 and 2007. The presence of qacJ was an event that took place between 1998 and 2001 since only four isolates from those years harboured it.
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Although the isolation of S. haemolyticus as a primary pathogen is not a frequent event, the remarkable fact is that the strains collected for this study were relevant for many reasons since they were resistant to several antibiotics and caused significant infections over the last 10 years. Another fact is that they harboured a myriad of antiseptic resistance genes that could act synergistically to produce the phenotype observed. The qacG, qacH and qacJ genes were described in coagulase-negative staphylococci in equines, dairy cattle and goat herds as a result of both intra- and inter-species spread of QAC resistance plasmids,6 and qacH was recently found in 3% of MRSA isolates in the UK.12 But, to our knowledge, this is the first time that the three genes qacG, qacH and qacJ have been encountered in S. haemolyticus of human origin. Taking into account that S. haemolyticus could act as a reservoir of resistance determinants and serve as a donor to more virulent staphylococci,5 this finding gives a relevant significance to this study. The notable issue that the genes qacG, qacH and qacJ were found for the first time in isolates from human origin demonstrates the broad spreading of antiseptic resistance, a fact that emphasizes the importance of the rational use of both antiseptics and antibiotics.
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This work was supported in part by grants PICT 05-10648 and PICT 05-32577 from ANPCyT (Agencia Nacional de Promoción de la Ciencia y la Técnica), Buenos Aires, Argentina, to D. O. S. and by grant M-009 from UBACyT (Universidad de Buenos Aires, Ciencia y Técnica), Argentina, to D. O. S.
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None to declare.
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Acknowledgements |
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We thank Pablo Arduino from Johnson Diversey for providing the benzalkonium chloride and triclosan. We thank Roemmers laboratories for providing the antibiotics used in this study.
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References |
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