Journal of Chemotherapy
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
The genetic environment of the antiseptic resistance genes qacE∆1 and cepA in Klebsiella pneumoniae Abdulmonem A. Abuzaid & Sebastian G. B. Amyes To cite this article: Abdulmonem A. Abuzaid & Sebastian G. B. Amyes (2015) The genetic environment of the antiseptic resistance genes qacE∆1 and cepA in Klebsiella pneumoniae, Journal of Chemotherapy, 27:3, 139-144 To link to this article: http://dx.doi.org/10.1179/1973947814Y.0000000181
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Date: 01 April 2016, At: 22:11
Antimicrobial Original Research Paper
The genetic environment of the antiseptic resistance genes qacED1 and cepA in Klebsiella pneumoniae Abdulmonem A. Abuzaid, Sebastian G. B. Amyes
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Molecular Chemotherapy, Medical Microbiology, University of Edinburgh, UK The genetic environment of two antiseptic resistance genes (qacED1 and cepA) was examined in Klebsiella pneumoniae isolates obtained from the Royal Infirmary of Edinburgh between 2006 and 2008. In 4 of 34 isolates, which carried the qacED1 gene, the sul1 gene was located immediately downstream. In two of these, the orf5 gene of unknown function was found immediately downstream of the sul1 gene. In one case, this was substituted by the chrA gene. The cepA gene was carried by 56 (87.5%) isolates, and the pfkA gene was found directly downstream in 45 (70.3%) isolates, and in 40 (62.5%) of these isolates, the menG gene was found directly downstream. The cpxP gene was found in 47 (73.4%) isolates upstream of the cepA, and in 35 of these isolates, the cpxR gene was identified. These latter genes are transcription regulators, and reverse transcription polymerase chain reaction (RT-PCR) revealed that their presence was associated with cepA expression. Keywords: Antiseptic, Resistance, Klebsiella pneumoniae, Resistance island, cepA, qacED1
Introduction Klebsiella pneumoniae causes about 8% of hospitalacquired (nosocomial) infections including pneumonia, wound infections, urinary tract infections, diarrhea through enterotoxin release, and bacteremia in hospitalized patients.1–4 Moreover, K. pneumoniae has become a more serious pathogen as it can cause outbreaks and become multiple drug resistant, largely associated with the production of extended-spectrum beta-lactamases (ESBLs).4 ESBLs hydrolyze later generation cephalosporins, and their presence in Klebsiella spp. causing nosocomial infections leads to increased incidences of morbidity and mortality especially in intensive care units (ICUs), neonatal and surgical units.1–3,5–8 Patient-to-patient cross-infection is one of the most common modes of transmission of K. pneumoniae with hand contamination by hospital staff being a major contributory factor (17% ICU staff). Furthermore, about 50% of hospital outbreaks in neonatal ICUs are caused by contaminated instruments. Staff frequently wash their hands with cationic biocides (such as chlorhexidine), which have been associated with a 98–100% reduction in the number of patients infected with K. pneumoniae.9 Correspondence to: Sebastian G. B. Amyes, Molecular Chemotherapy, Medical Microbiology, University of Edinburgh, The Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK. Email: [email protected]
ß 2015 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947814Y.0000000181
Quaternary ammonium compounds are also used widely in hospitals as antiseptics and disinfectants.10 They are cationic biocides and those that contain alkyl chains (e.g. benzalkonium chloride) are particularly active. Resistance genes (qac) to these compounds are widespread, and qacE was originally identified in plasmid R751 in Klebsiella aerogenes.11 A variant of this gene, qacED1, is located on many integrons and consequently is widely distributed in Gram-negative bacteria.11–13 The mechanism of resistance to quaternary ammonium compounds mediated by the qac genes in K. pneumoniae is by proton motive force (PMF)-dependent efflux, though the exact role of the qacED1 is controversial, as it appears to be an incomplete gene.13,14 However, it is closely associated with reduced susceptibility to quaternary ammonium compounds, and it is widely distributed in Escherichia coli and other members of the Enterobacteriaceae.11,15 Chlorhexidine, a cationic antiseptic, is a biguanide compound and is used as a topical agent, which is active against many types of bacteria. It has been used extensively in European hospitals as a part of washing and cleaning procedures; such as surface cleaning, hand disinfection by surgical scrub, and skin preparation.10,16–20 Some hospital Gram-negative bacteria, including Klebsiella, can become resistant to chlorhexidine.21,22 The cationic component of chlorhexidine reacts with the anionic microbial cell surface, killing the bacteria by membrane damage followed by intracellular
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coagulation.10,23,24 The mechanisms of resistance to chlorhexidine in Gram-negative bacteria are ambiguous, but an association between cepA gene and chlorhexidine resistance has been identified in K. pneumoniae.25,26 The genes, cepA and qacED1, are closely associated with decreasing antiseptic susceptibility in strains of K. pneumoniae.26,27 Therefore, the aim of this study is to identify the genetic environment of these genes in K. pneumoniae and to investigate the diversity of the gene cassettes.
Methods and Materials Bacterial strains and culture media
Pulsed-field gel electrophoresis (PFGE)
A total of 64 isolates of K. pneumoniae were isolated from a variety of infection sites at the Royal Infirmary Hospital in Edinburgh between 2006 and 2008.26 Originally, they had been isolated on MacConkey agar (Oxoid, Basingstoke, UK) and they had been initially characterized as K. pneumoniae by VITEK 2 and API 20E strips. The antibiograms and minimum inhibitory concentrations (MICs) of chlorhexidine were as previously described.26
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temperatures are described in Tables 1 and 2. DNA yield was estimated following electrophoresis in a 1.5% agarose gel (Fisher Scientific, Loughborough, UK). The gel was stained with GelRed, and the gel image was taken in tag image file format (JPEG files) with the Diversity Database software image capturing system (Bio-Rad, Hemel Hempstead, UK).26 The PCR fragments were then sequenced in the GenePool Genomics Facility at the University of Edinburgh (http://genepool.bio.ed.ac.uk/) to map the genes.28
Polymerase chain reaction (PCR) primers and sequence analysis The antiseptic resistance genes qacED1 and cepA detected by PCR with the primers and the annealing
Five isolates of K. pneumoniae containing the qacED1 gene were typed by PFGE in order to determine if they were closely related to each other.29 XbaI restriction endonuclease from Promega (Southampton, UK) was used to digest the agarose plug (Bio-Rad, Hertfordshire, UK). The 1% agarose gel was run at 6 V/cm using ramped pulse times from 5 to 45 seconds for 24 hours at 14uC in 0.56 Tris–borate–EDTA (TBE) buffer with a CHEF-DRII system (Bio-Rad, Hertfordshire, UK). A lambda-ladder PFGE marker was used as a size marker for PFGE (New England Biolabs, Hertfordshire, UK).
Table 1 Polymerase chain reaction (PCR) primers of the antiseptics resistance gene qacED1, expected size of product, and annealing temperature
Gene qacED1
qacED1 sul1
sul1 orf5
sul1 chrA
aadA1-sabg qacED1-sabg
IGR aadA1
dhfrA1 aadA1
Product size (bp)
PCR primers
Annealing temperature
No. of isolates (isolate numbers)
F 59-GGGAATTCGCCCTACACAAATTGGGAGA-39 R 59-AACACCGTCACCATGGCGTCGACGTCG-39 F 59-AGGCTGGTGGTTATGCACTC-39 R 59-CCGACTTCAGCTTTTGAAGG-39
370
49uC for 40 seconds
34
13
238
56uC for 40 seconds
4 (11, 17, 26, 34)
This work
F 59-AGGCTGGTGGTTATGCACTC-39 R 59-CGTATAGGCCACGCAGGTT-39
895
57.5uC for 40 seconds
2 (11, 17)
This work
F 59-ACGAGATTGTGCGGTTCTTC-39 R 59-GGGGTCATGCTCAACAACTT-39
974
56uC for 40 seconds
1 (26)
This work
F 59-TGAGGCGCTAAATGAAACCT-39 R 59-AACCAGGCAATGGCTGTAAT-39
1303
56.5uC for 40 seconds
4 (11, 17, 26, 39)
This work
F 59-ATGCCCGTTCCATACAGAAG-39 R 59-AGGTTTCATTTAGCGCCTCA-39
1836
56.5 for 40 seconds
2 (11, 17)
This work
F 59-CAATGGGAGCATTACCCAAC-39 R 59-TACTGCGCTGTACCAAATGC-39
844
56uC for 40 seconds
3 (11, 17, 26)
This work
bp: base pair; s: seconds; qac: quaternary ammonium compounds; IGR: intergenic region.
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Figure 1 Schematic representation of the genetic environment of the qacED1 and cepA antiseptic resistance genes in Klebsiella pneumoniae.
Analysis of gene expression RNA was extracted from rich broth cultures (Brain Heart Infusion, Oxoid, Basingstoke, UK) in the exponential growth phase at an optical density of 0.9–1.0 at OD600 (16109 cfu) using the RiboPure Bacteria kit (Ambion, Warrington, UK). The eluted RNA was treated with DNase 1 at 37uC for 30 minutes to remove trace amounts of genomic DNA. cDNA was synthesized from RNA by reverse transcription (RT)
according to the manufacturer’s instructions. Five microliters of cDNA was used in the PCR reactions (Access RT-PCR System kit, Promega, Southampton, UK). RT-PCR amplicons were run on an agarose gel and stained with GelRed for visualization and analyzed using the Bio-Rad Gel Doc 2000 software, Quantity One. RT-PCR products were performed on two separate occasions to assess the level of expression against a 16S amplicon used as a control.
Table 2 Polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) primers of the antiseptic resistance gene cepA, product size, and annealing temperature Product size (bp)
Gene
PCR primer
Annealing temperature
No. of isolates
cepA
F 59-CAACTCCTTCGCCTATCCCG-39 R 59-TCAGGTCAGACCAAACGGCG-39
1051
66uC for 40 seconds
56
24
cepA pfkA
F 59-CGCTGTTCCTGTTTCTCACC-39 R 59-CGCGCTTCTTCATGTTTTC-39
1099
66uC for 40 seconds
45
This work
pfkA menG
F 59-GTTCCTATATGGGGGCGATG-39 R 59-GAATGGCCGTGGTCATATTC-39
975
63.5uC for 40 seconds
40
This work
cpxP cepA
F 59-CCCCGTTAATGTTAGCGAAA-39 R 59-CTAACGAGGGCGATCAATGT-39
830
64.5uC for 40 seconds
47
This work
cpxR cpxP
F 59-ATCGAAATTGGCTTCCTGAC-39 R 59-GGCGTAAGCAGGTGGTACAT-39
830
66uC for 40 seconds
35
This work
RT-PCR primer 16S
F 59-CAGCCACACTGGAACTGAGA-39 R 59-GTTAGCCGGTGCTTCTTCTG-39
220
66uC for 40 seconds
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Figure 2 Cluster analysis of K. pneumoniae isolate numbers 11, 17, 26, 34, and 39 after pulsed-field gel electrophoresis (PFGE).
Results
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Previous analysis of the strains had shown that there was a link between the carriage of efflux pump genes, cepA, qacDE1, and qacE, and reduced susceptibility to biocides. Most strains showed decreased susceptibility to chlorhexidine, Trigene, and benzalkonium chloride, and this correlated with the carriage of the cepA, qacED1, and qacE genes encoding antiseptic efflux. There was no correlation between the reduced susceptibility to biocides and antibiotic resistance in these clinical isolates of K. pneumoniae.26
Genetic environment of the qacED1 antiseptic resistance gene The qacED1 gene was amplified by PCR in 34 (53.1%) isolates. The sulfonamide resistance gene (sul1), encoding an additional dihydropteroate synthetase, was detected directly downstream of the qacED1 gene in 4 of these 34 isolates; specifically, these were in isolates 11, 17, 26, and 34 (Fig. 1). An open reading frame, orf5, with no known function was detected directly downstream of the sul1 gene in two isolates, numbers 11 and 17 (Fig. 1); however, isolate number 26 had the chromate resistance protein gene (chrA) downstream of the sul1 gene (Fig. 1). The aminoglycoside resistance gene (aadA1), conferring an aminoglycoside adenyltransferase, was found directly upstream of the qacED1 gene in 4 of 34 isolates possessing this gene; specifically, these were in isolates 11, 17, 26, and 39 (Fig. 1). Further analysis
Figure 3 Correlation between the minimum inhibitory concentration (MIC) of chlorhexidine and cepA gene expression 11464641 measured by reverse transcription polymerase chain reaction (RT-PCR). The correlation of the cepAcontaining isolates (N) is shown as a solid line, whereas the isolates lacking cepA (#) is shown as a dotted line for comparison.
revealed that three of these isolates (numbers 11, 17, and 26) had the trimethoprim-resistant dihydrofolate reductase dhfrA1 gene directly upstream of aadA1. Two of these isolates (numbers 11 and 17) had an intergenic region (IGR) between the aadA1 and the qacED1 genes, which was lacking in the other two isolates (26 and 34) (Fig. 1).
PFGE analysis When examined by PFGE, only two of the qacED1 isolates, 26 and 39, showed virtually identical patterns (.95%), suggesting that they were the same strain, whereas isolates 11 and 34 demonstrated similar, but not identical, band patterns (80%) indicating that they may be part of the same clonal group. Isolate 17 was an unrelated strain (Fig. 2). These results suggest that the qacED1 is, in part, being disseminated horizontally through the Klebsiella population and not just by the spread of a single strain.
cepA antiseptic resistance gene The cepA gene was amplified by PCR in 56 (87.5%) isolates. In 45 (70.3%) of these isolates, we have
Table 3 Reverse transcription polymerase chain reaction (RT-PCR) showed that the expression-level cepA correlates with the minimum inhibitory concentration (MIC) of chlorhexidine Isolates
MIC of chlorhexidine (mg/l)
cepA gene carriage
Level of expression (arbitrary units (AU))*
2 32 32 4 8 64 128 32 32 128 32 128 32
2 2 z 2 z 2 z z z z 2 z 2
100 108 142 114 119 107 147 137 169 174 106 222 103
16S control 7 11 14 16 17 24 26 34 37 39 52 55
* Expression of 100 AU units is taken as the baseline.
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identified the 6-phosphofructokinase-1 (pfkA) downstream of cepA gene. In 40 (62.5%) of these isolates, the methyltransferase gene (menG) was directly downstream of the pfkA gene (Fig. 1). The periplasmic repressor gene (cpxP) was identified upstream of cepA in 47 (73.4%) isolates. The transcription regulator (cpxR) was directly upstream of the cpxP gene in 35 (54.7%) of these isolates. This latter gene pattern is shown in Fig. 1.
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RT-PCR The MICs of these strains have been reported earlier,26 and the RT-PCR analysis of the cepA gene showed that it was expressed particularly at high MICs of chlorhexidine (Table 3). When these results were plotted against the log10 of the MIC, there was a correlation between the degree of expression and the MIC suggesting that cepA contributed to active efflux mechanisms (Fig. 3). On the other hand, in isolates that lacked the cepA, there was a comparable lack of expression.
Discussion The presence of the qacED1 and cepA genes is considered to be linked to high-level MICs of biocides.26 This study investigated the genetic environment of the two antiseptic resistance genes, qacED1 and cepA, in K. pneumoniae. About half of the isolates carried the qacED1 gene, and this was usually located upstream of the sul1 sulfonamide resistance gene and directly downstream of the aminoglycoside adenyltransferase gene (aadA1), which was flanked by the dihydrofolate reductase gene dhfrA1. The qacED1 gene appears to be part of a small resistance island suggesting that this gene is linked and migrates with antibiotic resistance genes. The close linkage of qac genes to antibiotic resistance genes has been identified before in resistance islands; in the largest resistance island reported to date, in Acinetobacter baumannii strain AYE, there are four qac genes in the 45 gene resistance island,30 suggesting that there is strong selective pressure for these genes. The widespread carriage of qac genes in K. pneumoniae26 and their linkage to antibiotic resistance genes suggests that widespread use of biocides, particularly as antiseptics, could select antibiotic-resistant strains though there is no direct evidence for this so far. The cepA gene was much more prevalent than the qacED1; it was present in 88% of the isolates. This gene has only recently been reported25,26 and thus has not usually previously been associated with chlorhexidine resistance. Downstream of cepA, in many isolates there was a gene encoding 6-phosphofructokinase-1 (pfkA) and often a menG beyond that. More interestingly, in many of these isolates, the cepA gene was located behind cpxR/cpxP transcription regulator genes and these were responsible for the gene
Antiseptic resistance genes in Klebsiella pneumoniae
expression. The results show that as the MIC increases, so does the expression of cepA. The almost universal carriage of the cepA gene in K. pneumoniae (as this study indicates, the expression of which is linked to the chlorhexidine MIC) indicates that much of the K. pneumoniae population carries the potential for resistance. Our results do contrast in part with those of Naparstek et al.31 as they did not find a correlation between chlorhexidine susceptibility and cepA gene expression. The reason for this difference may be due to the fact that this study focused on the expression of cepA on just four strains. Our results indicate that a broader group of strains does show a correlation between the expression of this gene and the susceptibility to chlorhexidine. The different genetic environments and common distribution of the cepA gene strongly suggest that it may be disseminating by the presence of mobile genetic elements, such as integrons or transposons. An ongoing study is investigating the presence and nature of these elements. If biocides are used as recommended, the concentration of the biocide should far exceed the MIC of even the resistant strains. However, many biocides are not used appropriately and this will select out resistant strains, as indicated by the almost universal carriage of the cepA gene.
Disclaimer Statements Contributors Both authors were responsible for the design and execution of the research. Both authors were responsible for the drafting of the manuscript and have approved the final version. Funding A. Abuzaid and this project was funded by a grant provided by the General Department of Medical Services, Ministry of the Interior, Kingdom of Saudi Arabia. Conflicts of interest There are no conflicts of interest. Ethics approval There are no ethical issues related to this research.
Acknowledgements We would like to thank the General Department of Medical Services, Ministry of the Interior, Kingdom of Saudi Arabia for supporting this project.
References 1 Vincent WF. Infections caused by members of the genus Klebsiella. Quest Diagn. 2004;11:28–33. 2 Gupta A. Hospital-acquired infections in the neonatal intensive care unit – Klebsiella pneumoniae. Semin Perinatol. 2002;26: 340–5. 3 Jarvis WR, Munn VP, Highsmith AK, Culver DH, Hughes JM. The epidemiology of nosocomial infections caused by Klebsiella pneumoniae. Infect Control. 1985;6:68–74. 4 Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev. 1998;11:589–603.
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5 Otman J, Perugini ME, Tognim MCB, Vidotto MC. Atypical phenotypic characteristics of Klebsiella pneumoniae isolates from an outbreak in a neonatal intensive care unit in Brazil. Braz J Microbiol. 2007;38:273–7. 6 Marra AR, Wey SB, Castelo A, Gales AC, CAl G, Filho JR, et al. Nosocomial bloodstream infections caused by Klebsiella pneumoniae: impact of extended-spectrum beta-lactamase (ESBL) production on clinical outcome in a hospital with high ESBL prevalence. BMC Infect Dis. 2006;6:24. 7 Jacoby GA, Medeiros AA. More extended-spectrum betalactamases. Antimicrob Agents Chemother. 1991;35:1697–704. 8 Khanfar HS, Bindayna KM, Senok AC, Botta GA. Extended spectrum beta-lactamases (ESBL) in Escherichia coli and Klebsiella pneumoniae: trends in the hospital and community settings. J Infect Dev Ctries. 2009;3:295–9. 9 Casewell M, Phillips I. Hands as route of transmission for Klebsiella species. Br Med J. 1977;2:1315–7. 10 McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev. 1999;12:147–79. 11 Paulsen IT, Littlejohn TG, Ra˚dstro¨m P, Sundstrom L, Sko¨ld O, Swedberg G, et al. The 3’ conserved segment of integrons contains a gene associated with multidrug resistance to antiseptics and disinfectants. Antimicrob Agents Chemother. 1993;37:761–8. 12 Stokes HW, Hall RM. A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol Microbiol. 1989;3:1669–83. 13 Kazama H, Hamashima H, Sasatsu M, Arai T. Distribution of the antiseptic-resistance genes qacE and qacE delta 1 in Gramnegative bacteria. FEMS Microbiol Lett. 1998;159:173–8. 14 Paulsen IT, Brown MH, Skurray RA. Proton-dependent multidrug efflux systems. Microbiol Rev. 1996;60:575–608. 15 Ku¨cken D, Feucht H, Kaulfers P. Association of qacE and qacED1 with multiple resistances to antibiotics and antiseptics in clinical isolates of Gram-negative bacteria. FEMS Microbiol Lett. 2000;183:95–8. 16 Fraise AP. Susceptibility of antibiotic-resistant cocci to biocides. J Appl Microbiol. 2002;92(Suppl):158S–162S. 17 Fraise AP. Biocide abuse and antimicrobial resistance – a cause for concern? J Antimicrob Chemother. 2002;49:11–2. 18 Milstone AM, Passaretti CL, Perl TM. Chlorhexidine: expanding the armamentarium for infection control and prevention. Clin Infect Dis. 2008;46:274–81.
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19 Larson EL. APIC guideline for handwashing and hand antisepsis in health care settings. Am J Infect Control. 1995;23:251–69. 20 Russell AD, Day MJ. Antibacterial activity of chlorhexidine. J Hosp Infect. 1993;25:229–38. 21 Hammond SA, Morgan JR, Russell AD. Comparative susceptibility of hospital isolates of Gram-negative bacteria to antiseptics and disinfectants. J Hosp Infect. 1987;9:255–64. 22 Stickler DJ, Thomas B, Chawla JC. Antiseptic and antibiotic resistance in Gram-negative bacteria causing urinary tract infection in spinal cord injured patients. Paraplegia. 1981;19:50–8. 23 Russell AD. Chlorhexidine: antibacterial action and bacterial resistance. Infection. 1986;14:212–5. 24 Kuyyakanond T, Quesnel LB. The mechanism of action of chlorhexidine. FEMS Microbiol Lett. 1992;79:211–5. 25 Fang CT, Chen HC, Chuang YP, Chang SC, Wang JT. Cloning of a cation efflux pump gene associated with chlorhexidine resistance in Klebsiella pneumoniae. Antimicrob Agents Chemother. 2002;46:2024–8. 26 Abuzaid A, Hamouda A, Amyes SGB. Klebsiella pneumoniae susceptibility to biocides and its association with cepA, qacDeltaE and qacE efflux pump genes and antibiotic resistance. J Hosp Infect. 2012;81:87–91. 27 Russell AD. Biocide use and antibiotic resistance: the relevance of laboratory findings to clinical and environmental situations. Lancet Infect Dis. 2003;3:794–803. 28 Dimude JU, Amyes SGB. Molecular characterisation and diversity in Enterobacter cloacae from Edinburgh and Egypt carrying blaCTX-M-14 and blaVIM-4 b-lactamase genes. Int J Antimicrob Agents. 2013;41:574–7. 29 Miranda G, Kelly C, Solorzano F, Leanos B, Coria R, Patterson JE. Use of pulsed-field gel electrophoresis typing to study an outbreak of infection due to Serratia marcescens in a neonatal intensive care unit. J Clin Microbiol. 1996;34:3138–41. 30 Fournier PE, Vallenet D, Barbe V, Audic S, Ogata H, Poirel L, et al. Comparative genomics of multidrug resistance in Acinetobacter baumannii. PLoS Genet. 2006;2:62–72. 31 Naparstek L, Carmeli Y, Chmelnitsky I, Banin E, NavonVenezia S. Reduced susceptibility to chlorhexidine among extremely-drug-resistant strains of Klebsiella pneumoniae. J Hosp Infect. 2012;81:15–9.
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
The genetic environment of the antiseptic resistance genes qacE∆1 and cepA in Klebsiella pneumoniae Abdulmonem A. Abuzaid & Sebastian G. B. Amyes To cite this article: Abdulmonem A. Abuzaid & Sebastian G. B. Amyes (2015) The genetic environment of the antiseptic resistance genes qacE∆1 and cepA in Klebsiella pneumoniae, Journal of Chemotherapy, 27:3, 139-144 To link to this article: http://dx.doi.org/10.1179/1973947814Y.0000000181
Published online: 11 Mar 2014.
Submit your article to this journal
Article views: 22
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yjoc20 Download by: [University of Exeter]
Date: 01 April 2016, At: 22:11
Antimicrobial Original Research Paper
The genetic environment of the antiseptic resistance genes qacED1 and cepA in Klebsiella pneumoniae Abdulmonem A. Abuzaid, Sebastian G. B. Amyes
Downloaded by [University of Exeter] at 22:11 01 April 2016
Molecular Chemotherapy, Medical Microbiology, University of Edinburgh, UK The genetic environment of two antiseptic resistance genes (qacED1 and cepA) was examined in Klebsiella pneumoniae isolates obtained from the Royal Infirmary of Edinburgh between 2006 and 2008. In 4 of 34 isolates, which carried the qacED1 gene, the sul1 gene was located immediately downstream. In two of these, the orf5 gene of unknown function was found immediately downstream of the sul1 gene. In one case, this was substituted by the chrA gene. The cepA gene was carried by 56 (87.5%) isolates, and the pfkA gene was found directly downstream in 45 (70.3%) isolates, and in 40 (62.5%) of these isolates, the menG gene was found directly downstream. The cpxP gene was found in 47 (73.4%) isolates upstream of the cepA, and in 35 of these isolates, the cpxR gene was identified. These latter genes are transcription regulators, and reverse transcription polymerase chain reaction (RT-PCR) revealed that their presence was associated with cepA expression. Keywords: Antiseptic, Resistance, Klebsiella pneumoniae, Resistance island, cepA, qacED1
Introduction Klebsiella pneumoniae causes about 8% of hospitalacquired (nosocomial) infections including pneumonia, wound infections, urinary tract infections, diarrhea through enterotoxin release, and bacteremia in hospitalized patients.1–4 Moreover, K. pneumoniae has become a more serious pathogen as it can cause outbreaks and become multiple drug resistant, largely associated with the production of extended-spectrum beta-lactamases (ESBLs).4 ESBLs hydrolyze later generation cephalosporins, and their presence in Klebsiella spp. causing nosocomial infections leads to increased incidences of morbidity and mortality especially in intensive care units (ICUs), neonatal and surgical units.1–3,5–8 Patient-to-patient cross-infection is one of the most common modes of transmission of K. pneumoniae with hand contamination by hospital staff being a major contributory factor (17% ICU staff). Furthermore, about 50% of hospital outbreaks in neonatal ICUs are caused by contaminated instruments. Staff frequently wash their hands with cationic biocides (such as chlorhexidine), which have been associated with a 98–100% reduction in the number of patients infected with K. pneumoniae.9 Correspondence to: Sebastian G. B. Amyes, Molecular Chemotherapy, Medical Microbiology, University of Edinburgh, The Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK. Email: [email protected]
ß 2015 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947814Y.0000000181
Quaternary ammonium compounds are also used widely in hospitals as antiseptics and disinfectants.10 They are cationic biocides and those that contain alkyl chains (e.g. benzalkonium chloride) are particularly active. Resistance genes (qac) to these compounds are widespread, and qacE was originally identified in plasmid R751 in Klebsiella aerogenes.11 A variant of this gene, qacED1, is located on many integrons and consequently is widely distributed in Gram-negative bacteria.11–13 The mechanism of resistance to quaternary ammonium compounds mediated by the qac genes in K. pneumoniae is by proton motive force (PMF)-dependent efflux, though the exact role of the qacED1 is controversial, as it appears to be an incomplete gene.13,14 However, it is closely associated with reduced susceptibility to quaternary ammonium compounds, and it is widely distributed in Escherichia coli and other members of the Enterobacteriaceae.11,15 Chlorhexidine, a cationic antiseptic, is a biguanide compound and is used as a topical agent, which is active against many types of bacteria. It has been used extensively in European hospitals as a part of washing and cleaning procedures; such as surface cleaning, hand disinfection by surgical scrub, and skin preparation.10,16–20 Some hospital Gram-negative bacteria, including Klebsiella, can become resistant to chlorhexidine.21,22 The cationic component of chlorhexidine reacts with the anionic microbial cell surface, killing the bacteria by membrane damage followed by intracellular
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coagulation.10,23,24 The mechanisms of resistance to chlorhexidine in Gram-negative bacteria are ambiguous, but an association between cepA gene and chlorhexidine resistance has been identified in K. pneumoniae.25,26 The genes, cepA and qacED1, are closely associated with decreasing antiseptic susceptibility in strains of K. pneumoniae.26,27 Therefore, the aim of this study is to identify the genetic environment of these genes in K. pneumoniae and to investigate the diversity of the gene cassettes.
Methods and Materials Bacterial strains and culture media
Pulsed-field gel electrophoresis (PFGE)
A total of 64 isolates of K. pneumoniae were isolated from a variety of infection sites at the Royal Infirmary Hospital in Edinburgh between 2006 and 2008.26 Originally, they had been isolated on MacConkey agar (Oxoid, Basingstoke, UK) and they had been initially characterized as K. pneumoniae by VITEK 2 and API 20E strips. The antibiograms and minimum inhibitory concentrations (MICs) of chlorhexidine were as previously described.26
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temperatures are described in Tables 1 and 2. DNA yield was estimated following electrophoresis in a 1.5% agarose gel (Fisher Scientific, Loughborough, UK). The gel was stained with GelRed, and the gel image was taken in tag image file format (JPEG files) with the Diversity Database software image capturing system (Bio-Rad, Hemel Hempstead, UK).26 The PCR fragments were then sequenced in the GenePool Genomics Facility at the University of Edinburgh (http://genepool.bio.ed.ac.uk/) to map the genes.28
Polymerase chain reaction (PCR) primers and sequence analysis The antiseptic resistance genes qacED1 and cepA detected by PCR with the primers and the annealing
Five isolates of K. pneumoniae containing the qacED1 gene were typed by PFGE in order to determine if they were closely related to each other.29 XbaI restriction endonuclease from Promega (Southampton, UK) was used to digest the agarose plug (Bio-Rad, Hertfordshire, UK). The 1% agarose gel was run at 6 V/cm using ramped pulse times from 5 to 45 seconds for 24 hours at 14uC in 0.56 Tris–borate–EDTA (TBE) buffer with a CHEF-DRII system (Bio-Rad, Hertfordshire, UK). A lambda-ladder PFGE marker was used as a size marker for PFGE (New England Biolabs, Hertfordshire, UK).
Table 1 Polymerase chain reaction (PCR) primers of the antiseptics resistance gene qacED1, expected size of product, and annealing temperature
Gene qacED1
qacED1 sul1
sul1 orf5
sul1 chrA
aadA1-sabg qacED1-sabg
IGR aadA1
dhfrA1 aadA1
Product size (bp)
PCR primers
Annealing temperature
No. of isolates (isolate numbers)
F 59-GGGAATTCGCCCTACACAAATTGGGAGA-39 R 59-AACACCGTCACCATGGCGTCGACGTCG-39 F 59-AGGCTGGTGGTTATGCACTC-39 R 59-CCGACTTCAGCTTTTGAAGG-39
370
49uC for 40 seconds
34
13
238
56uC for 40 seconds
4 (11, 17, 26, 34)
This work
F 59-AGGCTGGTGGTTATGCACTC-39 R 59-CGTATAGGCCACGCAGGTT-39
895
57.5uC for 40 seconds
2 (11, 17)
This work
F 59-ACGAGATTGTGCGGTTCTTC-39 R 59-GGGGTCATGCTCAACAACTT-39
974
56uC for 40 seconds
1 (26)
This work
F 59-TGAGGCGCTAAATGAAACCT-39 R 59-AACCAGGCAATGGCTGTAAT-39
1303
56.5uC for 40 seconds
4 (11, 17, 26, 39)
This work
F 59-ATGCCCGTTCCATACAGAAG-39 R 59-AGGTTTCATTTAGCGCCTCA-39
1836
56.5 for 40 seconds
2 (11, 17)
This work
F 59-CAATGGGAGCATTACCCAAC-39 R 59-TACTGCGCTGTACCAAATGC-39
844
56uC for 40 seconds
3 (11, 17, 26)
This work
bp: base pair; s: seconds; qac: quaternary ammonium compounds; IGR: intergenic region.
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Figure 1 Schematic representation of the genetic environment of the qacED1 and cepA antiseptic resistance genes in Klebsiella pneumoniae.
Analysis of gene expression RNA was extracted from rich broth cultures (Brain Heart Infusion, Oxoid, Basingstoke, UK) in the exponential growth phase at an optical density of 0.9–1.0 at OD600 (16109 cfu) using the RiboPure Bacteria kit (Ambion, Warrington, UK). The eluted RNA was treated with DNase 1 at 37uC for 30 minutes to remove trace amounts of genomic DNA. cDNA was synthesized from RNA by reverse transcription (RT)
according to the manufacturer’s instructions. Five microliters of cDNA was used in the PCR reactions (Access RT-PCR System kit, Promega, Southampton, UK). RT-PCR amplicons were run on an agarose gel and stained with GelRed for visualization and analyzed using the Bio-Rad Gel Doc 2000 software, Quantity One. RT-PCR products were performed on two separate occasions to assess the level of expression against a 16S amplicon used as a control.
Table 2 Polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) primers of the antiseptic resistance gene cepA, product size, and annealing temperature Product size (bp)
Gene
PCR primer
Annealing temperature
No. of isolates
cepA
F 59-CAACTCCTTCGCCTATCCCG-39 R 59-TCAGGTCAGACCAAACGGCG-39
1051
66uC for 40 seconds
56
24
cepA pfkA
F 59-CGCTGTTCCTGTTTCTCACC-39 R 59-CGCGCTTCTTCATGTTTTC-39
1099
66uC for 40 seconds
45
This work
pfkA menG
F 59-GTTCCTATATGGGGGCGATG-39 R 59-GAATGGCCGTGGTCATATTC-39
975
63.5uC for 40 seconds
40
This work
cpxP cepA
F 59-CCCCGTTAATGTTAGCGAAA-39 R 59-CTAACGAGGGCGATCAATGT-39
830
64.5uC for 40 seconds
47
This work
cpxR cpxP
F 59-ATCGAAATTGGCTTCCTGAC-39 R 59-GGCGTAAGCAGGTGGTACAT-39
830
66uC for 40 seconds
35
This work
RT-PCR primer 16S
F 59-CAGCCACACTGGAACTGAGA-39 R 59-GTTAGCCGGTGCTTCTTCTG-39
220
66uC for 40 seconds
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Figure 2 Cluster analysis of K. pneumoniae isolate numbers 11, 17, 26, 34, and 39 after pulsed-field gel electrophoresis (PFGE).
Results
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Previous analysis of the strains had shown that there was a link between the carriage of efflux pump genes, cepA, qacDE1, and qacE, and reduced susceptibility to biocides. Most strains showed decreased susceptibility to chlorhexidine, Trigene, and benzalkonium chloride, and this correlated with the carriage of the cepA, qacED1, and qacE genes encoding antiseptic efflux. There was no correlation between the reduced susceptibility to biocides and antibiotic resistance in these clinical isolates of K. pneumoniae.26
Genetic environment of the qacED1 antiseptic resistance gene The qacED1 gene was amplified by PCR in 34 (53.1%) isolates. The sulfonamide resistance gene (sul1), encoding an additional dihydropteroate synthetase, was detected directly downstream of the qacED1 gene in 4 of these 34 isolates; specifically, these were in isolates 11, 17, 26, and 34 (Fig. 1). An open reading frame, orf5, with no known function was detected directly downstream of the sul1 gene in two isolates, numbers 11 and 17 (Fig. 1); however, isolate number 26 had the chromate resistance protein gene (chrA) downstream of the sul1 gene (Fig. 1). The aminoglycoside resistance gene (aadA1), conferring an aminoglycoside adenyltransferase, was found directly upstream of the qacED1 gene in 4 of 34 isolates possessing this gene; specifically, these were in isolates 11, 17, 26, and 39 (Fig. 1). Further analysis
Figure 3 Correlation between the minimum inhibitory concentration (MIC) of chlorhexidine and cepA gene expression 11464641 measured by reverse transcription polymerase chain reaction (RT-PCR). The correlation of the cepAcontaining isolates (N) is shown as a solid line, whereas the isolates lacking cepA (#) is shown as a dotted line for comparison.
revealed that three of these isolates (numbers 11, 17, and 26) had the trimethoprim-resistant dihydrofolate reductase dhfrA1 gene directly upstream of aadA1. Two of these isolates (numbers 11 and 17) had an intergenic region (IGR) between the aadA1 and the qacED1 genes, which was lacking in the other two isolates (26 and 34) (Fig. 1).
PFGE analysis When examined by PFGE, only two of the qacED1 isolates, 26 and 39, showed virtually identical patterns (.95%), suggesting that they were the same strain, whereas isolates 11 and 34 demonstrated similar, but not identical, band patterns (80%) indicating that they may be part of the same clonal group. Isolate 17 was an unrelated strain (Fig. 2). These results suggest that the qacED1 is, in part, being disseminated horizontally through the Klebsiella population and not just by the spread of a single strain.
cepA antiseptic resistance gene The cepA gene was amplified by PCR in 56 (87.5%) isolates. In 45 (70.3%) of these isolates, we have
Table 3 Reverse transcription polymerase chain reaction (RT-PCR) showed that the expression-level cepA correlates with the minimum inhibitory concentration (MIC) of chlorhexidine Isolates
MIC of chlorhexidine (mg/l)
cepA gene carriage
Level of expression (arbitrary units (AU))*
2 32 32 4 8 64 128 32 32 128 32 128 32
2 2 z 2 z 2 z z z z 2 z 2
100 108 142 114 119 107 147 137 169 174 106 222 103
16S control 7 11 14 16 17 24 26 34 37 39 52 55
* Expression of 100 AU units is taken as the baseline.
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identified the 6-phosphofructokinase-1 (pfkA) downstream of cepA gene. In 40 (62.5%) of these isolates, the methyltransferase gene (menG) was directly downstream of the pfkA gene (Fig. 1). The periplasmic repressor gene (cpxP) was identified upstream of cepA in 47 (73.4%) isolates. The transcription regulator (cpxR) was directly upstream of the cpxP gene in 35 (54.7%) of these isolates. This latter gene pattern is shown in Fig. 1.
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RT-PCR The MICs of these strains have been reported earlier,26 and the RT-PCR analysis of the cepA gene showed that it was expressed particularly at high MICs of chlorhexidine (Table 3). When these results were plotted against the log10 of the MIC, there was a correlation between the degree of expression and the MIC suggesting that cepA contributed to active efflux mechanisms (Fig. 3). On the other hand, in isolates that lacked the cepA, there was a comparable lack of expression.
Discussion The presence of the qacED1 and cepA genes is considered to be linked to high-level MICs of biocides.26 This study investigated the genetic environment of the two antiseptic resistance genes, qacED1 and cepA, in K. pneumoniae. About half of the isolates carried the qacED1 gene, and this was usually located upstream of the sul1 sulfonamide resistance gene and directly downstream of the aminoglycoside adenyltransferase gene (aadA1), which was flanked by the dihydrofolate reductase gene dhfrA1. The qacED1 gene appears to be part of a small resistance island suggesting that this gene is linked and migrates with antibiotic resistance genes. The close linkage of qac genes to antibiotic resistance genes has been identified before in resistance islands; in the largest resistance island reported to date, in Acinetobacter baumannii strain AYE, there are four qac genes in the 45 gene resistance island,30 suggesting that there is strong selective pressure for these genes. The widespread carriage of qac genes in K. pneumoniae26 and their linkage to antibiotic resistance genes suggests that widespread use of biocides, particularly as antiseptics, could select antibiotic-resistant strains though there is no direct evidence for this so far. The cepA gene was much more prevalent than the qacED1; it was present in 88% of the isolates. This gene has only recently been reported25,26 and thus has not usually previously been associated with chlorhexidine resistance. Downstream of cepA, in many isolates there was a gene encoding 6-phosphofructokinase-1 (pfkA) and often a menG beyond that. More interestingly, in many of these isolates, the cepA gene was located behind cpxR/cpxP transcription regulator genes and these were responsible for the gene
Antiseptic resistance genes in Klebsiella pneumoniae
expression. The results show that as the MIC increases, so does the expression of cepA. The almost universal carriage of the cepA gene in K. pneumoniae (as this study indicates, the expression of which is linked to the chlorhexidine MIC) indicates that much of the K. pneumoniae population carries the potential for resistance. Our results do contrast in part with those of Naparstek et al.31 as they did not find a correlation between chlorhexidine susceptibility and cepA gene expression. The reason for this difference may be due to the fact that this study focused on the expression of cepA on just four strains. Our results indicate that a broader group of strains does show a correlation between the expression of this gene and the susceptibility to chlorhexidine. The different genetic environments and common distribution of the cepA gene strongly suggest that it may be disseminating by the presence of mobile genetic elements, such as integrons or transposons. An ongoing study is investigating the presence and nature of these elements. If biocides are used as recommended, the concentration of the biocide should far exceed the MIC of even the resistant strains. However, many biocides are not used appropriately and this will select out resistant strains, as indicated by the almost universal carriage of the cepA gene.
Disclaimer Statements Contributors Both authors were responsible for the design and execution of the research. Both authors were responsible for the drafting of the manuscript and have approved the final version. Funding A. Abuzaid and this project was funded by a grant provided by the General Department of Medical Services, Ministry of the Interior, Kingdom of Saudi Arabia. Conflicts of interest There are no conflicts of interest. Ethics approval There are no ethical issues related to this research.
Acknowledgements We would like to thank the General Department of Medical Services, Ministry of the Interior, Kingdom of Saudi Arabia for supporting this project.
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