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Detection of AmpC β-lactamases in Escherichia coli, Klebsiella spp., Salmonella spp. and Proteus mirabilis in a regional clinical microbiology laboratory
ORIGINAL ARTICLE
10.1111/j.1469-0691.2009.02756.x
Detection of AmpC b-lactamases in Escherichia coli, Klebsiella spp., Salmonella spp. and Proteus mirabilis in a regional clinical microbiology laboratory J. D. D. Pitout1–3, P. G. Le1, K. L. Moore1, D. L. Church1,2,4 and D. B. Gregson1,2,4 1) Division of Microbiology, Calgary Laboratory Services, 2) Department of Pathology and Laboratory Medicine, 3) Department of Microbiology and Infectious Diseases and 4) Department of Medicine, University of Calgary, Calgary, AB, Canada
Abstract There are currently no standardized diagnostic tests available for the reliable detection of AmpC b-lactamases in Klebsiella spp., Escherichia coli, Proteus mirabilis and Salmonella spp. A study was designed to evaluate a confirmation disk test using cefotetan (CTT) and cefoxitin (FOX) with phenylboronic acid (PBA). It also investigated the most suitable screening concentrations of FOX, ceftriaxone (CRO) and ceftazidime (CAZ) for the detection of AmpC b-lactamases. A total of 126 control (consisting of 11 laboratory and 115 wellcharacterized clinical strains) and 29 840 non-repeat clinical isolates were included. FOX with PBA used in a confirmation test and CRO and CAZ as screening agents were found to be unreliable. FOX at ‡ 32 mg/L was the best screening agent and CTT with PBA was the best confirmation test. Of the clinical isolates 635 (2%) were found to be resistant to cefoxitin (MIC ‡ 32 ug/mL) and 332 (52%) were AmpC positive. E. coli was the most common organism with AmpC b-lactamases and was mostly present in urines from community patients. It is recommended that laboratories use FOX at 32 mg/L as a screening agent and perform a disk test with CTT and PBA to confirm the presence of an AmpC cephalosporinase in isolates of Klebsiella spp., E. coli, Salmonella spp. and P. mirabilis. This approach is convenient, practical and easy to incorporate into the workflow of a clinical laboratory. False-positive AmpC detection may occur with KPC-producing bacteria when inhibitor-based methods are used. Keywords: AmpC b-lactamases, disk test, laboratory detection Original Submission: 4 September 2008; Revised Submission: 17 November 2008; Accepted: 19 November 2008 Editor: D. Mack Article published online: 18 May 2009 Clin Microbiol Infect 2010; 16: 165–170 Corresponding author and reprint requests: J. D. D. Pitout, Division of Microbiology, Calgary Laboratory Services, #9, 3535 Research Road NW, Calgary, AB T2L 2K8, Canada E-mail: [email protected]
Introduction Enterobacteriaceae with inducible cephalosporinases, such as Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Morganella morganii and Providencia stuartii, may become resistant to oxyimino- (e.g. cefotaxime, ceftazidime) and 7-amethoxy (i.e. cephamycins) cephalosporins and monobactams by overproducing their chromosomal AmpC b-lactamases [1]. In Klebsiella pneumoniae, Salmonella spp. and Proteus mirabilis, which lack chromosomal b-lactamases, this type of resistance can be mediated by plasmid-encoded AmpC cephalosporinases [2]. Escherichia coli possesses genes encod-
ing chromosomal non-inducible AmpC b-lactamases that are regulated by weak promoters and strong attenuators, resulting in low amounts of the cephalosporinase [3]. Surveys of resistance mechanisms in cephamycin-resistant E. coli have identified a promoter or attenuator mutation, which results in the upregulation of naturally occurring chromosomal AmpC b-lactamase production [4]. Occasionally, cephamycin-resistant E. coli can also produce plasmid-mediated AmpC b-lactamases [2]. Plasmid-mediated AmpC cephalosporinases are derivatives of the chromosomally encoded AmpC b-lactamases of bacteria such as Enterobacter, C. feundii, M. morganii, Aeromonas spp. and Hafnia alvei [2]. These enzymes are not inhibited by commercial b-lactamase inhibitors, such as clavulanic acid, sulbactam and tazobactam, although different types of inhibitors, such as boronic acid and cloxacillin, have shown good inhibition. The genes are also typically encoded on large plasmids containing additional antibiotic resistance genes, which leaves few therapeutic options.
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A recent study from the Calgary Health Region has identified AmpC-producing E. coli as an emerging pathogen in the community that commonly causes urinary tract infections in older women [5]. A cost-effective, practical diagnostic approach was needed to establish accurate detection of organisms producing AmpC cephalosporinases. There are no guidelines from the CLSI available for the detection of bacteria with AmpC b-lactamases and the best approach to date is to screen for resistance to a cephamycin (e.g. cefoxitin) and/or oxyimino- (e.g. cefotaxime, ceftazidime) cephalosporins in isolates such as Klebsiella spp., E. coli, P. mirabilis and Salmonella spp. Such resistance, in combination with a negative CLSI extended-spectrum b-lactamase confirmatory test, is a possible, but non-specific, indicator for AmpC production [6]. The use of an AmpC inhibitor, boronic acid, has recently been described as an effective confirmation test for the detection of organisms with AmpC b-lactamases [7,8]. This method has not been extensively verified or validated for routine use in clinical laboratories. This study reports on the incorporation of a boronic acid disk confirmation test in the clinical laboratory. It describes the most appropriate screening criteria for the detection of AmpC cephalosporinases in Klebsiella spp., E. coli, P. mirabilis and Salmonella spp. It also investigates the prevalence of these enzymes among recent clinical isolates from the Calgary Health Region during 2006–2007.
Materials and methods
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TABLE 1. Verification and validation of the AmpC disk test with control and recent clinical strains of Klebsiella spp., Escherichia coli, Proteus mirabilis and Salmonella spp.
Organisms
Control strains/lab no. E. coli A2 E. coli A3 E. coli A4 K. pneumoniae A5 K. pneumoniae A8 Salmonella spp. A6 K. pneumoniae A7 K. pneumoniae C2 E. coli CTX2 E. coli E. coli T1 Recent clinical isolates a E. coli (n = 40) b Salmonella spp. (n = 10) c K. pneumoniae (n = 10) c
P. mirabilis (n = 5) E. coli (n = 50)
a
Resistance mechanism(s)
AmpC disk test with FOX and PBA
AmpC disk test with CTT and PBA
MOX-1 FOX-1 MIR-1 ACT-1 DHA-1 CMY-2 ACC-1 KPC-2 CTX-M-15 SHV-2 TEM-3
Negative Negative Positive Negative Positive Positive Negative Positive Negative Negative Negative
Positive Positive Positive Positive Positive Positive Negative Positive Negative Negative Negative
CMY-like CMY-2
Positive (36/40) Positive (8/10)
Positive (40/40) Positive (10/10)
CMY-like FOX-like DHA-like CMY-like Cephamycin-resistant, AmpC-negative
Positive (7/10)
Positive (10/10)
Positive (4/5) Negative (50/50)
Positive (5/5) Negative (50/50)
a
Reference [5] and 35 E. coli isolated in 2004, 2005 at Calgary Laboratory Services (CLS) that tested AmpC negative with cefoxitin agar medium (CAM) method [12] and multiplex PCR [10] bReference [9]. c Recent isolates from CLS as identified with multiplex PCR [10]. FOX, cefoxitin; CTT, cefotetan; PBA, phenylboronic acid.
TABLE 2. Screening concentrations for control and recent clinical isolates (n = 72) of AmpC-producing Escherichia coli,
Overview of study
The first part of the study was designed to verify the boronic acid disk confirmation test using control strains and recent clinical isolates from previous studies as positive and negative controls [5,9] (Table 1). Cefotetan (CTT) and cefoxitin (FOX) with and without phenylboronic acid (PBA) as substrates for AmpC were initially included in the disk test. The second part of the study investigated the most suitable screening concentrations of FOX, ceftriaxone (CRO) and ceftazidime (CAZ) for the detection of AmpC b-lactamases in Klebsiella spp., E. coli, P. mirabilis and Salmonella spp., using control organisms and recent clinical isolates with known AmpC b-lactamases (Table 2). The third part of the study investigated the prevalence of these enzymes among recent clinical isolates of Klebsiella spp., E. coli, P. mirabilis and Salmonella spp. from the Calgary Health Region over a 1-year period from April 2006 to March 2007 using the best screening agent (as determined in Part 2) and confirming the result with the boronic acid-based disk test (as determined in Part 1).
Klebsiella spp., Salmonella spp. and Pneumoniae mirabilisa MIC, mg/L
FOX £8 16 32 > 32 Total CAZ £2 4 8 16 > 16 Total CRO £2 4 8 16 > 16 Total a
Isolates, n (%) (Microscan)
Isolates, n (%) (Vitek 2)
1 (1%) 0 26 (36%) 45 (63%) 72
1 (1%) 0 31 (43%) 40 (56%) 72
25 6 8 14 19 72
(35%) (8%) (11%) (19%) (26%)
25 8 10 16 13 72
(35%) (11%) (14%) (22%) (18%)
22 8 10 13 19 72
(31%) (11%) (14%) (18%) (26%)
22 11 13 15 11 72
(31%) (15%) (18%) (21%) (15%)
AmpC-producing isolates consisted of seven control strains (MOX-1, FOX-1, MIR-1, ACT-1, DHA-1, CMY-2, ACC-1) and the following recent clinical isolates: E. coli with CMY-2 (n = 40), Salmonella spp. with CMY-2 (n = 10), K. pneumoniae with CMY-like, FOX-like and DHA-like enzymes (n = 10) and P. mirabilis with CMY-like enzymes (n = 5). Refer to Table 1. FOX, cefoxitin; CRO, ceftriaxone; CAZ, ceftazidime.
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TABLE 3. Prevalence and susceptibility of AmpC b-lactamases among clinical bacterial isolates of Escherichia coli, Klebsiella spp., Salmonella spp. and Proteus mirabilis isolated at Calgary Laboratory Services from April 2006 to March 2007 Non-susceptibility to: n (%) a
Organism
Isolates, n
Screen positive, n
E. coli
b
PBA disk test positive, n
25 109
560
310
K. pneumoniae
2818
47
12
K. oxytoca P. mirabilis
505 1197
12 6
0 2
Salmonella spp. Total
211
10
8
29 840
635
332
SXT
GEN
TOB
NIT
CIP
FEP
IPM
77 (25%) 3 (25%) 0 1 (50%) 2 (25%) 83 (25%)
51 (16%) 1 (8%) 0 0
35 (11%) 1 (8%) 0 0
0
0
0
0 0
0 0
1 (13%) 37 (11%)
56 (18%) 2 (17%) 0 1 (50%) 1 (13%) 60 (18%)
0
1 (13%) 53 (16%)
10 (3%) 1 (8%) 0 1 (50%) 1 (13%) 13 (4%)
0
0
0
0
a
Using cefoxitin (FOX) at ‡ 32 mg/L with Vitek 2. Using cefotetan and phenylboronic acid (PBA) (see Materials and methods for details. Additional susceptibility testing in this study using the CLSI disk method shows resistance of 31% (103/332) to sulphisoazole, 23% (76/332) to chloramphenicol, 34% (114/ 332) to tetracycline and 20% (65/332) to streptomycin . SXT, trimethoprim-sulphamethoxazole; GEN, gentamicin; TOB, tobramycin; NIT, nitrofurantoin; CIP, ciprofloxacin; FEP, cefepime; IPM, imipenem.
b
Phenylboronic acid disk confirmation test
Screening for AmpC-producing bacteria
The disks were prepared as follows: 120 mg of PBA (also known as benzeneboronic acid/anhydride) (Alfa Aesar, Ward Hill, MA, USA) was dissolved in 3 mL dimethyl sulphoxide (Alfa Aesar). Three millilitres of sterile distilled water was added to this solution to create a stock solution; 20 lL of the stock solution was dispensed onto each of 30 lg CTT, 30 lg FOX and blank disks (6 mm in diameter). These disks had been obtained from Oxoid Inc. (Nepean, ON, Canada). The disks were allowed to dry for 30 min and used either immediately or stored in airtight vials with desiccant at 4 C. An increase of ‡ 5 mm in zone diameter in the presence of PBA compared with either CTT or FOX tested alone was considered to represent a positive test for the presence of an AmpC b-lactamase [7,8].
To determine the most appropriate screening concentrations of FOX, CAZ and CRO that would detect AmpC-producing Klebsiella spp., E. coli, P. mirabilis and Salmonella spp., the following isolates were included in Part 2 of the study: seven control strains (with MOX-1, FOX-1, MIR-1, ACT-1, DHA1, CMY-2, ACC-1) and 65 recent clinical isolates (E. coli with CMY-like (n = 40), Salmonella spp. with CMY-2 (n = 10), K. pneumoniae with CMY-like, FOX-like and DHA-like enzymes (n = 10) and P. mirabilis with CMY-like enzymes (n = 5)). These bacteria were the same as those used in Part 1 of the study (Tables 1 and 2). The minimum inhibitory concentrations (MICs) were determined with Microscan gram-negative (NMIC30; Dade Behring Canada Inc., Mississauga, ON, Canada) and confirmed with Vitek 2 using ASTN019 panels (Vitek AMS; bioMe´rieux Vitek Systems Inc., Hazelwood, MO, USA). The quality control strain used for this part of the study was E. coli ATCC 25922.
Verification of the test
The accuracy of the AmpC disk test was determined using 11 strains with well-described resistance mechanisms (Table 1); reproducibility was investigated by performing the assay in triplicate on consecutive days. Recent clinical AmpC-producing E. coli and Salmonella isolates described in previous studies were also included to test the performance of the assay [5,9]. These included 50 isolates with CMY types of b-lactamases and 50 that were resistant to FOX but tested AmpC negative (Table 1). We also selected 10 K. pneumoniae with CMY-like (n = 2), FOX-like (n = 7), DHA-like (n = 1), and five P. mirabilis with CMY-like enzymes. These bacteria were previously isolated at Calgary Laboratory Services (CLS) during 2000–2005 and the cephalosporinases were characterized with a multiplex PCR as previously described [10].
Bacterial isolates
Consecutive non-duplicate isolates of Klebsiella spp., E. coli, P. mirabilis and Salmonella spp. (one per patient) collected at CLS during April 2006 to March 2007 were included in Part 3 of this study (Table 3). Strains were identified to species level with Vitek 2 (Vitek AMS; bioMe´rieux Vitek Systems Inc.). MICs of the following drugs were determined with Vitek 2 using AST-N019 panels (Vitek AMS; bioMe´rieux Vitek Systems Inc.): ceftriaxone (CRO); ceftazidime (CAZ); cefepime (FEP); cefoxitin (FOX); imipenem (IPM); gentamicin (GEN); tobramycin (TOB); trimethoprim-sulphamethoxazole (SXT); nitrofurantoin (NIT), and ciprofloxacin (CIP). Throughout this study, results were interpreted using CLSI
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criteria for broth dilution [11]. The quality control strains used for this part of the study were E. coli ATCC 25922 and E. coli ATCC 35218.
Results Part 1: verification of the PBA disk test
The one-off process of determining the accuracy of the test was performed using the 11 strains with well-described resistance mechanisms and the recent clinical isolates (Table 1). Most of the control strains were accurately detected by CTT with PBA. However, A-7 (ACC-1) tested negative and C2 (KPC-2) tested positive. FOX with PBA did not detect the following control strains: A2 (MOX-1); A3 (FOX-1); A5 (ACT-1), and A7 (AAC-1), whereas C2 (KPC-2) tested positive (Table 1). The CTT with PBA test performed adequately with all the recent clinical isolates with AmpC cephalosporinases (n = 65) that tested positive, whereas those isolates resistant to cefoxitin but AmpC-negative (n = 50) tested negative (Table 1). FOX with PBA did not detect 10 of 65 recent clinical isolates with AmpC cephalosporinases. Overall, the sensitivities of CTT with PBA, and FOX with PBA, were 99% and 81%, respectively, and the specificity of CTT with PBA, and FOX with PBA, was 98% in both cases. The performance of the inhibitor test was compared with another recently described AmpC detection method called the cefoxitin agar medium (CAM) test and showed identical results with positive and negative control organisms [12]. Reproducibility (determined by performing the assay in triplicate on consecutive days with the 11 control strains) showed the same results with CTT and FOX on each occasion. On this basis, CTT and PBA were used for the confirmation of AmpC b-lactamases during Part 3 of the study. Part 2: screening agents for AmpC-producing bacteria
MICs determinations using Microscan panels with FOX, CAZ and CRO showed the following: a total of 71/72 (99%) had MICs ‡ 32 mg/L of FOX; 47/72 (65%) had MICs > 2 mg/L of CAZ, and 50/72 (69%) had MICs > 2 mg/L of CRO (Table 2). MICs determined with Vitek 2 showed similar results (there was a one-dilution difference with FOX in five isolates, with CAZ in six and with CRO in eight) (Table 2). It was interesting to note that none of the control strains or recent clinical isolates had an MIC of 16 mg/L of FOX (the one isolate with an MIC £ 8 mg/L was K. pneumoniae A7, producing ACC-1). We performed AmpC testing (using the PBA confirmation disk test) on 101 clinical isolates of E. coli, Klebsiella spp. Salmonella spp. and P. mirabilis (isolated at CLS during
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2000–2005) with a FOX MIC of 16 mg/L and none tested positive. Therefore, we decided to use FOX at ‡ 32 mg/L as an adequate concentration to screen for AmpC producing E. coli, Klebsiella spp., Salmonella spp. and P. mirabilis. Part 3: prevalence among the clinical bacterial isolates
A total of 29 840 non-repeat bacteria (i.e. one isolate per patient) were isolated at CLS over a 12-month period from April 2006 to March 2007 and were included in Part 3 of the study (Table 3). These consisted of 25 109 (84%) E. coli, 2818 (9%) K. pneumoniae, 505 (2%) Klebsiella oxytoca, 1197 (4%) P. mirabilis, and 211 (1%) Salmonella species. Of these, 635 (2%) were found to be resistant to cefoxitin (MIC ‡ 32 ug/mL) and 332/635 (52%) were positive for AmpC cephalosporinases using the confirmation disk test with CTT and PBA. The majority were E. coli (310/332, 93%), followed by K. pneumoniae (12/332, 4%), Salmonella spp. (8/332, 2%), and P. mirabilis (2/332, 1%). The majority of isolates (206/332, 62%) were submitted from community collection sites. Of the 332 AmpC b-lactamase-producing isolates, 83 (25%) were non-susceptible to SXT, 37 (11%) to TOB, 53 (16%) to GEN, 13 (4%) to NIT and 60 (18%) to CIP. No resistance was detected to IPM or FEP.
Discussion Organisms with plasmid-mediated AmpC enzymes are mostly responsible for nosocomial outbreaks on a worldwide basis. A study reported by Pai et al., showed that patients infected with plasmid-mediated AmpC-producing K. pneumoniae had similar clinical features and outcomes as patients infected with TEMor SHV-related extended-spectrum b-lactamase producers [13]. Furthermore, if combined with decreased outer membrane permeability, bacteria with such enzymes can become resistant to carbapenems [14]. Therefore, it is important to detect AmpC-producing organisms to ensure effective therapeutic intervention and optimal clinical outcome, as well as to control the spread of these organisms. However, most current methods for detection of these enzymes are technically demanding and time-consuming and therefore unsuitable for clinical laboratories to perform on a routine basis [6]. This study showed that an inhibitor-based PBA disk confirmation test with cefotetan is a simple and reliable confirmation method for use in clinical laboratories. This assay uses methodology similar to that of the CLSI Xxx [extended-spectrum b-lactamase (ESBL)] phenotypic confirmatory disk test. This test was easy to perform, interpret and incorporate into the workflow of our high-volume laboratory. Although CTT with PBA gave the best performance overall for detection of AmpC
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 165–170
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Pitout et al. Laboratory detection of AmpC b-lactamases
enzymes, it was unable to detect ACC-1 (Table 1). ACC-1 originated from the chromosomal cephalosporinase of H. alvei and is different from most other plasmid-mediated AmpC b-lactamases in that isolates with this enzyme are sensitive to the cephamycins (i.e. cefoxitin), but show decreased susceptibility to ceftazidime [15]. Bacteria with ACC-1 are relatively rare and have only been reported from countries such as the UK [16], Germany [17] and France [18]. Isolates (i.e. E. coli, Klebsiella spp. and P. mirabilis) that are intermediate to ceftazidime and sensitive to cefoxitin, combined with a negative ESBL confirmatory test, can be further tested with the cefoxitin double-disk test to confirm the presence of ACC-1 [15]. Phenylboronic acid inhibited K. pneumoniae C2 producing KPC-2 and gave a false-positive AmpC disk test result (Table 1). A false-positive reaction has also been previously described with different AmpC inhibitors in KPC-producing strains [19,20]. KPC-producing bacteria are causing a pandemic and have been shown to be widespread in enterobacterial species including Klebsiella spp. and E. coli from certain areas of the USA and Europe [21]. Clinical laboratories need to be aware of the fact that KPC causes resistance to the cephamycins and can test positive with inhibitor-based AmpC disk confirmation tests. Klebsiella spp., E. coli, Salmonella spp. and P. mirabilis that test positive for AmpC cephalosporinases with subsequent decreased susceptibility to the carbapenems (e.g. ertapenem) should be referred to a reference laboratory to investigate for possible KPC production [22]. In summary, clinical laboratories should routinely screen for AmpC cephalosporinases in Klebsiella spp., E. coli, Salmonella spp. and P. mirabilis with a cefoxitin MIC ‡ 32 mg/L and confirm the presence of these enzymes using the PBA disk confirmation test with cefotetan described herein. This approach is convenient, practical and easy to incorporate into the workflow of a clinical laboratory. Laboratories should also be aware that KPC-producing bacteria can test false-positive for AmpC b-lactamases with inhibitor-based methods.
Acknowledgements We thank T. Ross and Y. Wie, Calgary Laboratory Services, Calgary, AB, for their technical support of this study, and E. Smith Moland and G. Arlet for providing control strains.
Transparency Declaration J. D. D. Pitout, D. L. Church and D. B. Gregson had previously received funding from Merck (Canada) and Wyeth (Canada) for research projects.
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10.1111/j.1469-0691.2009.02756.x
Detection of AmpC b-lactamases in Escherichia coli, Klebsiella spp., Salmonella spp. and Proteus mirabilis in a regional clinical microbiology laboratory J. D. D. Pitout1–3, P. G. Le1, K. L. Moore1, D. L. Church1,2,4 and D. B. Gregson1,2,4 1) Division of Microbiology, Calgary Laboratory Services, 2) Department of Pathology and Laboratory Medicine, 3) Department of Microbiology and Infectious Diseases and 4) Department of Medicine, University of Calgary, Calgary, AB, Canada
Abstract There are currently no standardized diagnostic tests available for the reliable detection of AmpC b-lactamases in Klebsiella spp., Escherichia coli, Proteus mirabilis and Salmonella spp. A study was designed to evaluate a confirmation disk test using cefotetan (CTT) and cefoxitin (FOX) with phenylboronic acid (PBA). It also investigated the most suitable screening concentrations of FOX, ceftriaxone (CRO) and ceftazidime (CAZ) for the detection of AmpC b-lactamases. A total of 126 control (consisting of 11 laboratory and 115 wellcharacterized clinical strains) and 29 840 non-repeat clinical isolates were included. FOX with PBA used in a confirmation test and CRO and CAZ as screening agents were found to be unreliable. FOX at ‡ 32 mg/L was the best screening agent and CTT with PBA was the best confirmation test. Of the clinical isolates 635 (2%) were found to be resistant to cefoxitin (MIC ‡ 32 ug/mL) and 332 (52%) were AmpC positive. E. coli was the most common organism with AmpC b-lactamases and was mostly present in urines from community patients. It is recommended that laboratories use FOX at 32 mg/L as a screening agent and perform a disk test with CTT and PBA to confirm the presence of an AmpC cephalosporinase in isolates of Klebsiella spp., E. coli, Salmonella spp. and P. mirabilis. This approach is convenient, practical and easy to incorporate into the workflow of a clinical laboratory. False-positive AmpC detection may occur with KPC-producing bacteria when inhibitor-based methods are used. Keywords: AmpC b-lactamases, disk test, laboratory detection Original Submission: 4 September 2008; Revised Submission: 17 November 2008; Accepted: 19 November 2008 Editor: D. Mack Article published online: 18 May 2009 Clin Microbiol Infect 2010; 16: 165–170 Corresponding author and reprint requests: J. D. D. Pitout, Division of Microbiology, Calgary Laboratory Services, #9, 3535 Research Road NW, Calgary, AB T2L 2K8, Canada E-mail: [email protected]
Introduction Enterobacteriaceae with inducible cephalosporinases, such as Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Morganella morganii and Providencia stuartii, may become resistant to oxyimino- (e.g. cefotaxime, ceftazidime) and 7-amethoxy (i.e. cephamycins) cephalosporins and monobactams by overproducing their chromosomal AmpC b-lactamases [1]. In Klebsiella pneumoniae, Salmonella spp. and Proteus mirabilis, which lack chromosomal b-lactamases, this type of resistance can be mediated by plasmid-encoded AmpC cephalosporinases [2]. Escherichia coli possesses genes encod-
ing chromosomal non-inducible AmpC b-lactamases that are regulated by weak promoters and strong attenuators, resulting in low amounts of the cephalosporinase [3]. Surveys of resistance mechanisms in cephamycin-resistant E. coli have identified a promoter or attenuator mutation, which results in the upregulation of naturally occurring chromosomal AmpC b-lactamase production [4]. Occasionally, cephamycin-resistant E. coli can also produce plasmid-mediated AmpC b-lactamases [2]. Plasmid-mediated AmpC cephalosporinases are derivatives of the chromosomally encoded AmpC b-lactamases of bacteria such as Enterobacter, C. feundii, M. morganii, Aeromonas spp. and Hafnia alvei [2]. These enzymes are not inhibited by commercial b-lactamase inhibitors, such as clavulanic acid, sulbactam and tazobactam, although different types of inhibitors, such as boronic acid and cloxacillin, have shown good inhibition. The genes are also typically encoded on large plasmids containing additional antibiotic resistance genes, which leaves few therapeutic options.
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A recent study from the Calgary Health Region has identified AmpC-producing E. coli as an emerging pathogen in the community that commonly causes urinary tract infections in older women [5]. A cost-effective, practical diagnostic approach was needed to establish accurate detection of organisms producing AmpC cephalosporinases. There are no guidelines from the CLSI available for the detection of bacteria with AmpC b-lactamases and the best approach to date is to screen for resistance to a cephamycin (e.g. cefoxitin) and/or oxyimino- (e.g. cefotaxime, ceftazidime) cephalosporins in isolates such as Klebsiella spp., E. coli, P. mirabilis and Salmonella spp. Such resistance, in combination with a negative CLSI extended-spectrum b-lactamase confirmatory test, is a possible, but non-specific, indicator for AmpC production [6]. The use of an AmpC inhibitor, boronic acid, has recently been described as an effective confirmation test for the detection of organisms with AmpC b-lactamases [7,8]. This method has not been extensively verified or validated for routine use in clinical laboratories. This study reports on the incorporation of a boronic acid disk confirmation test in the clinical laboratory. It describes the most appropriate screening criteria for the detection of AmpC cephalosporinases in Klebsiella spp., E. coli, P. mirabilis and Salmonella spp. It also investigates the prevalence of these enzymes among recent clinical isolates from the Calgary Health Region during 2006–2007.
Materials and methods
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TABLE 1. Verification and validation of the AmpC disk test with control and recent clinical strains of Klebsiella spp., Escherichia coli, Proteus mirabilis and Salmonella spp.
Organisms
Control strains/lab no. E. coli A2 E. coli A3 E. coli A4 K. pneumoniae A5 K. pneumoniae A8 Salmonella spp. A6 K. pneumoniae A7 K. pneumoniae C2 E. coli CTX2 E. coli E. coli T1 Recent clinical isolates a E. coli (n = 40) b Salmonella spp. (n = 10) c K. pneumoniae (n = 10) c
P. mirabilis (n = 5) E. coli (n = 50)
a
Resistance mechanism(s)
AmpC disk test with FOX and PBA
AmpC disk test with CTT and PBA
MOX-1 FOX-1 MIR-1 ACT-1 DHA-1 CMY-2 ACC-1 KPC-2 CTX-M-15 SHV-2 TEM-3
Negative Negative Positive Negative Positive Positive Negative Positive Negative Negative Negative
Positive Positive Positive Positive Positive Positive Negative Positive Negative Negative Negative
CMY-like CMY-2
Positive (36/40) Positive (8/10)
Positive (40/40) Positive (10/10)
CMY-like FOX-like DHA-like CMY-like Cephamycin-resistant, AmpC-negative
Positive (7/10)
Positive (10/10)
Positive (4/5) Negative (50/50)
Positive (5/5) Negative (50/50)
a
Reference [5] and 35 E. coli isolated in 2004, 2005 at Calgary Laboratory Services (CLS) that tested AmpC negative with cefoxitin agar medium (CAM) method [12] and multiplex PCR [10] bReference [9]. c Recent isolates from CLS as identified with multiplex PCR [10]. FOX, cefoxitin; CTT, cefotetan; PBA, phenylboronic acid.
TABLE 2. Screening concentrations for control and recent clinical isolates (n = 72) of AmpC-producing Escherichia coli,
Overview of study
The first part of the study was designed to verify the boronic acid disk confirmation test using control strains and recent clinical isolates from previous studies as positive and negative controls [5,9] (Table 1). Cefotetan (CTT) and cefoxitin (FOX) with and without phenylboronic acid (PBA) as substrates for AmpC were initially included in the disk test. The second part of the study investigated the most suitable screening concentrations of FOX, ceftriaxone (CRO) and ceftazidime (CAZ) for the detection of AmpC b-lactamases in Klebsiella spp., E. coli, P. mirabilis and Salmonella spp., using control organisms and recent clinical isolates with known AmpC b-lactamases (Table 2). The third part of the study investigated the prevalence of these enzymes among recent clinical isolates of Klebsiella spp., E. coli, P. mirabilis and Salmonella spp. from the Calgary Health Region over a 1-year period from April 2006 to March 2007 using the best screening agent (as determined in Part 2) and confirming the result with the boronic acid-based disk test (as determined in Part 1).
Klebsiella spp., Salmonella spp. and Pneumoniae mirabilisa MIC, mg/L
FOX £8 16 32 > 32 Total CAZ £2 4 8 16 > 16 Total CRO £2 4 8 16 > 16 Total a
Isolates, n (%) (Microscan)
Isolates, n (%) (Vitek 2)
1 (1%) 0 26 (36%) 45 (63%) 72
1 (1%) 0 31 (43%) 40 (56%) 72
25 6 8 14 19 72
(35%) (8%) (11%) (19%) (26%)
25 8 10 16 13 72
(35%) (11%) (14%) (22%) (18%)
22 8 10 13 19 72
(31%) (11%) (14%) (18%) (26%)
22 11 13 15 11 72
(31%) (15%) (18%) (21%) (15%)
AmpC-producing isolates consisted of seven control strains (MOX-1, FOX-1, MIR-1, ACT-1, DHA-1, CMY-2, ACC-1) and the following recent clinical isolates: E. coli with CMY-2 (n = 40), Salmonella spp. with CMY-2 (n = 10), K. pneumoniae with CMY-like, FOX-like and DHA-like enzymes (n = 10) and P. mirabilis with CMY-like enzymes (n = 5). Refer to Table 1. FOX, cefoxitin; CRO, ceftriaxone; CAZ, ceftazidime.
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TABLE 3. Prevalence and susceptibility of AmpC b-lactamases among clinical bacterial isolates of Escherichia coli, Klebsiella spp., Salmonella spp. and Proteus mirabilis isolated at Calgary Laboratory Services from April 2006 to March 2007 Non-susceptibility to: n (%) a
Organism
Isolates, n
Screen positive, n
E. coli
b
PBA disk test positive, n
25 109
560
310
K. pneumoniae
2818
47
12
K. oxytoca P. mirabilis
505 1197
12 6
0 2
Salmonella spp. Total
211
10
8
29 840
635
332
SXT
GEN
TOB
NIT
CIP
FEP
IPM
77 (25%) 3 (25%) 0 1 (50%) 2 (25%) 83 (25%)
51 (16%) 1 (8%) 0 0
35 (11%) 1 (8%) 0 0
0
0
0
0 0
0 0
1 (13%) 37 (11%)
56 (18%) 2 (17%) 0 1 (50%) 1 (13%) 60 (18%)
0
1 (13%) 53 (16%)
10 (3%) 1 (8%) 0 1 (50%) 1 (13%) 13 (4%)
0
0
0
0
a
Using cefoxitin (FOX) at ‡ 32 mg/L with Vitek 2. Using cefotetan and phenylboronic acid (PBA) (see Materials and methods for details. Additional susceptibility testing in this study using the CLSI disk method shows resistance of 31% (103/332) to sulphisoazole, 23% (76/332) to chloramphenicol, 34% (114/ 332) to tetracycline and 20% (65/332) to streptomycin . SXT, trimethoprim-sulphamethoxazole; GEN, gentamicin; TOB, tobramycin; NIT, nitrofurantoin; CIP, ciprofloxacin; FEP, cefepime; IPM, imipenem.
b
Phenylboronic acid disk confirmation test
Screening for AmpC-producing bacteria
The disks were prepared as follows: 120 mg of PBA (also known as benzeneboronic acid/anhydride) (Alfa Aesar, Ward Hill, MA, USA) was dissolved in 3 mL dimethyl sulphoxide (Alfa Aesar). Three millilitres of sterile distilled water was added to this solution to create a stock solution; 20 lL of the stock solution was dispensed onto each of 30 lg CTT, 30 lg FOX and blank disks (6 mm in diameter). These disks had been obtained from Oxoid Inc. (Nepean, ON, Canada). The disks were allowed to dry for 30 min and used either immediately or stored in airtight vials with desiccant at 4 C. An increase of ‡ 5 mm in zone diameter in the presence of PBA compared with either CTT or FOX tested alone was considered to represent a positive test for the presence of an AmpC b-lactamase [7,8].
To determine the most appropriate screening concentrations of FOX, CAZ and CRO that would detect AmpC-producing Klebsiella spp., E. coli, P. mirabilis and Salmonella spp., the following isolates were included in Part 2 of the study: seven control strains (with MOX-1, FOX-1, MIR-1, ACT-1, DHA1, CMY-2, ACC-1) and 65 recent clinical isolates (E. coli with CMY-like (n = 40), Salmonella spp. with CMY-2 (n = 10), K. pneumoniae with CMY-like, FOX-like and DHA-like enzymes (n = 10) and P. mirabilis with CMY-like enzymes (n = 5)). These bacteria were the same as those used in Part 1 of the study (Tables 1 and 2). The minimum inhibitory concentrations (MICs) were determined with Microscan gram-negative (NMIC30; Dade Behring Canada Inc., Mississauga, ON, Canada) and confirmed with Vitek 2 using ASTN019 panels (Vitek AMS; bioMe´rieux Vitek Systems Inc., Hazelwood, MO, USA). The quality control strain used for this part of the study was E. coli ATCC 25922.
Verification of the test
The accuracy of the AmpC disk test was determined using 11 strains with well-described resistance mechanisms (Table 1); reproducibility was investigated by performing the assay in triplicate on consecutive days. Recent clinical AmpC-producing E. coli and Salmonella isolates described in previous studies were also included to test the performance of the assay [5,9]. These included 50 isolates with CMY types of b-lactamases and 50 that were resistant to FOX but tested AmpC negative (Table 1). We also selected 10 K. pneumoniae with CMY-like (n = 2), FOX-like (n = 7), DHA-like (n = 1), and five P. mirabilis with CMY-like enzymes. These bacteria were previously isolated at Calgary Laboratory Services (CLS) during 2000–2005 and the cephalosporinases were characterized with a multiplex PCR as previously described [10].
Bacterial isolates
Consecutive non-duplicate isolates of Klebsiella spp., E. coli, P. mirabilis and Salmonella spp. (one per patient) collected at CLS during April 2006 to March 2007 were included in Part 3 of this study (Table 3). Strains were identified to species level with Vitek 2 (Vitek AMS; bioMe´rieux Vitek Systems Inc.). MICs of the following drugs were determined with Vitek 2 using AST-N019 panels (Vitek AMS; bioMe´rieux Vitek Systems Inc.): ceftriaxone (CRO); ceftazidime (CAZ); cefepime (FEP); cefoxitin (FOX); imipenem (IPM); gentamicin (GEN); tobramycin (TOB); trimethoprim-sulphamethoxazole (SXT); nitrofurantoin (NIT), and ciprofloxacin (CIP). Throughout this study, results were interpreted using CLSI
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criteria for broth dilution [11]. The quality control strains used for this part of the study were E. coli ATCC 25922 and E. coli ATCC 35218.
Results Part 1: verification of the PBA disk test
The one-off process of determining the accuracy of the test was performed using the 11 strains with well-described resistance mechanisms and the recent clinical isolates (Table 1). Most of the control strains were accurately detected by CTT with PBA. However, A-7 (ACC-1) tested negative and C2 (KPC-2) tested positive. FOX with PBA did not detect the following control strains: A2 (MOX-1); A3 (FOX-1); A5 (ACT-1), and A7 (AAC-1), whereas C2 (KPC-2) tested positive (Table 1). The CTT with PBA test performed adequately with all the recent clinical isolates with AmpC cephalosporinases (n = 65) that tested positive, whereas those isolates resistant to cefoxitin but AmpC-negative (n = 50) tested negative (Table 1). FOX with PBA did not detect 10 of 65 recent clinical isolates with AmpC cephalosporinases. Overall, the sensitivities of CTT with PBA, and FOX with PBA, were 99% and 81%, respectively, and the specificity of CTT with PBA, and FOX with PBA, was 98% in both cases. The performance of the inhibitor test was compared with another recently described AmpC detection method called the cefoxitin agar medium (CAM) test and showed identical results with positive and negative control organisms [12]. Reproducibility (determined by performing the assay in triplicate on consecutive days with the 11 control strains) showed the same results with CTT and FOX on each occasion. On this basis, CTT and PBA were used for the confirmation of AmpC b-lactamases during Part 3 of the study. Part 2: screening agents for AmpC-producing bacteria
MICs determinations using Microscan panels with FOX, CAZ and CRO showed the following: a total of 71/72 (99%) had MICs ‡ 32 mg/L of FOX; 47/72 (65%) had MICs > 2 mg/L of CAZ, and 50/72 (69%) had MICs > 2 mg/L of CRO (Table 2). MICs determined with Vitek 2 showed similar results (there was a one-dilution difference with FOX in five isolates, with CAZ in six and with CRO in eight) (Table 2). It was interesting to note that none of the control strains or recent clinical isolates had an MIC of 16 mg/L of FOX (the one isolate with an MIC £ 8 mg/L was K. pneumoniae A7, producing ACC-1). We performed AmpC testing (using the PBA confirmation disk test) on 101 clinical isolates of E. coli, Klebsiella spp. Salmonella spp. and P. mirabilis (isolated at CLS during
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2000–2005) with a FOX MIC of 16 mg/L and none tested positive. Therefore, we decided to use FOX at ‡ 32 mg/L as an adequate concentration to screen for AmpC producing E. coli, Klebsiella spp., Salmonella spp. and P. mirabilis. Part 3: prevalence among the clinical bacterial isolates
A total of 29 840 non-repeat bacteria (i.e. one isolate per patient) were isolated at CLS over a 12-month period from April 2006 to March 2007 and were included in Part 3 of the study (Table 3). These consisted of 25 109 (84%) E. coli, 2818 (9%) K. pneumoniae, 505 (2%) Klebsiella oxytoca, 1197 (4%) P. mirabilis, and 211 (1%) Salmonella species. Of these, 635 (2%) were found to be resistant to cefoxitin (MIC ‡ 32 ug/mL) and 332/635 (52%) were positive for AmpC cephalosporinases using the confirmation disk test with CTT and PBA. The majority were E. coli (310/332, 93%), followed by K. pneumoniae (12/332, 4%), Salmonella spp. (8/332, 2%), and P. mirabilis (2/332, 1%). The majority of isolates (206/332, 62%) were submitted from community collection sites. Of the 332 AmpC b-lactamase-producing isolates, 83 (25%) were non-susceptible to SXT, 37 (11%) to TOB, 53 (16%) to GEN, 13 (4%) to NIT and 60 (18%) to CIP. No resistance was detected to IPM or FEP.
Discussion Organisms with plasmid-mediated AmpC enzymes are mostly responsible for nosocomial outbreaks on a worldwide basis. A study reported by Pai et al., showed that patients infected with plasmid-mediated AmpC-producing K. pneumoniae had similar clinical features and outcomes as patients infected with TEMor SHV-related extended-spectrum b-lactamase producers [13]. Furthermore, if combined with decreased outer membrane permeability, bacteria with such enzymes can become resistant to carbapenems [14]. Therefore, it is important to detect AmpC-producing organisms to ensure effective therapeutic intervention and optimal clinical outcome, as well as to control the spread of these organisms. However, most current methods for detection of these enzymes are technically demanding and time-consuming and therefore unsuitable for clinical laboratories to perform on a routine basis [6]. This study showed that an inhibitor-based PBA disk confirmation test with cefotetan is a simple and reliable confirmation method for use in clinical laboratories. This assay uses methodology similar to that of the CLSI Xxx [extended-spectrum b-lactamase (ESBL)] phenotypic confirmatory disk test. This test was easy to perform, interpret and incorporate into the workflow of our high-volume laboratory. Although CTT with PBA gave the best performance overall for detection of AmpC
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Pitout et al. Laboratory detection of AmpC b-lactamases
enzymes, it was unable to detect ACC-1 (Table 1). ACC-1 originated from the chromosomal cephalosporinase of H. alvei and is different from most other plasmid-mediated AmpC b-lactamases in that isolates with this enzyme are sensitive to the cephamycins (i.e. cefoxitin), but show decreased susceptibility to ceftazidime [15]. Bacteria with ACC-1 are relatively rare and have only been reported from countries such as the UK [16], Germany [17] and France [18]. Isolates (i.e. E. coli, Klebsiella spp. and P. mirabilis) that are intermediate to ceftazidime and sensitive to cefoxitin, combined with a negative ESBL confirmatory test, can be further tested with the cefoxitin double-disk test to confirm the presence of ACC-1 [15]. Phenylboronic acid inhibited K. pneumoniae C2 producing KPC-2 and gave a false-positive AmpC disk test result (Table 1). A false-positive reaction has also been previously described with different AmpC inhibitors in KPC-producing strains [19,20]. KPC-producing bacteria are causing a pandemic and have been shown to be widespread in enterobacterial species including Klebsiella spp. and E. coli from certain areas of the USA and Europe [21]. Clinical laboratories need to be aware of the fact that KPC causes resistance to the cephamycins and can test positive with inhibitor-based AmpC disk confirmation tests. Klebsiella spp., E. coli, Salmonella spp. and P. mirabilis that test positive for AmpC cephalosporinases with subsequent decreased susceptibility to the carbapenems (e.g. ertapenem) should be referred to a reference laboratory to investigate for possible KPC production [22]. In summary, clinical laboratories should routinely screen for AmpC cephalosporinases in Klebsiella spp., E. coli, Salmonella spp. and P. mirabilis with a cefoxitin MIC ‡ 32 mg/L and confirm the presence of these enzymes using the PBA disk confirmation test with cefotetan described herein. This approach is convenient, practical and easy to incorporate into the workflow of a clinical laboratory. Laboratories should also be aware that KPC-producing bacteria can test false-positive for AmpC b-lactamases with inhibitor-based methods.
Acknowledgements We thank T. Ross and Y. Wie, Calgary Laboratory Services, Calgary, AB, for their technical support of this study, and E. Smith Moland and G. Arlet for providing control strains.
Transparency Declaration J. D. D. Pitout, D. L. Church and D. B. Gregson had previously received funding from Merck (Canada) and Wyeth (Canada) for research projects.
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