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A Simplified method of Three Dimensional Technique For The Detection Of AmpC Beta-Lactamases

R.K. Manojkumar Singh1, Nishith Kumar Pal2, Mandira Banerjee3, Soma Sarkar4, Puranjoy Saha4,Manideepa SenGupta4
1 Department of Microbiology,Jawaharlal Nehru Institute of Medical Sciences, Imphal; 2 Institute of Postgraduate Medical Education & Research, Kolkata. 3 Nilratan Sarkar Medical college, Kolkata. 4 Medical College & Hospital, Kolkata. Correspondence:

rkmksingh@gmail.com

Dr. R.K.Manojkumar Singh. Department of Microbiology, Jawaharlal Nehru Institute of Medical Sciences, Porompat, Imphal(East)-795005, India.

Abstract
Background: AmpC beta-lactamases are cephalosporinases that confer resistance to a wide variety of beta-lactam antibiotics. Detecting AmpC-mediated resistance in gram negative organisms is a challenge for laboratories due to misleading results in phenotypic tests. There is always a search for newer and reliable methods which will be more user-friendly to detect these enzymes in routine diagnostic laboratories.

Objective: To design a simplified three-dimensional test for detecting the occurrence of AmpC -lactamases.

Material and Methods: A total of 201 consecutive, non-duplicate, gram-negative clinical isolates of E.coli (n=65), Klebsiella spp.(n=70), Proteus spp.(n=4), citrobacter spp. (n=9), Enterobacter spp.( n=8), P.aeruginosa (n=27) and Acinetobacter spp.(n=18), obtained over a period of six months (July to December, 2011) from patients with nosocomial infections were screened for AmpC producers by Kirby Bauer disk diffusion method using cefoxitin (30 g). Isolates with zone diameter less than 18 mm were tested for inducible AmpC beta-lactamases by disk antagonism test, and plasmid mediated AmpC activity by indigenously developed simplified three dimensional and AmpC disk tests.

Results: Amongst the 201 gram-negative clinical isolates tested, the simplified
three dimensional test identified 60(29.8%) and AmpC disk test yielded 51(25.3%) as plasmid AmpC producers from 69 screening positive isolates. Production of inducible AmpC beta-lactamase was shown in 3(1.5%) isolates by disk antagonism test. Co-productions of extended spectrum beta-lactamases and metallo-betalactamases with AmpC beta-lactamases were observed in 3.9% and 4.9% isolates respectively.

Conclusion: This simplified three dimensional technique is simple, reproducible,

convenient, reliable and accurate means of detection of AmpC beta-lactamases. It can serve as a potentially useful diagnostic tool in those health care settings where polymerase chain reaction and inhibitor based methods for the detection of AmpC enzymes are costly and not readily available.

This article is available from: www.acmicrob.com

Keywords: AmpC, three-dimensional technique.

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Introduction
AmpC -lactamases are cephalosporinases that are poorly inhibited by -lactamase inhibitors such as clavulanic acid [1]. They can be differentiated from extended spectrum -lactamases (ESBLs) by their ability to hydrolyse cephamycins as well as other extended-spectrum cephalosporins [2]. Over the last few years, numerous outbreaks of infections with organisms producing AmpC -lactamase, have been observed worldwide. Coudron et al used the standard disk diffusion breakpoint for cefoxitin (zone diameter <18 mm) to screen isolates and used a three-dimensional extract test as a confirmatory test for isolates that harbour AmpC -lactamases [3]. The current Clinical and Laboratory Standards institute (CLSI) guidelines do not describe any method for detection of isolates producing AmpC -lactamases [4]. Various phenotypic techniques to identify AmpC -lactamase producing isolates are available but are still evolving and are not yet optimized for the clinical laboratory, which probably now underestimates this resistance mechanism. Several researchers have tried the three dimensional test with different modifications but it is laborious, time consuming and needs experience. Despite of the fact that multiplex PCR is the gold standard and highly accurate for testing, it is beyond the routine microbiology laboratories. The present study was conducted in a tertiary care hospital of eastern India with an attempt to develop a simplified threedimensional test and evaluate the effectiveness to be applied as a phenotypic method for detection of AmpC-harbouring gram negative organisms.

(Hi-Media, Mumbai,India) and commercially available 6mm antimicrobial disks of cefoxitin (30g), cefotaxime (30g), ceftriaxone (30g), ceftazidime (30g), cefpodoxime (30g), aztreonam (30g) imipenem (10g), amoxicillin/clavulanate (20/10g), amikacin (30g), piperacillin (100g), piperacillintazobactam(100/10g), ciproploxacin (5g), and cotrimoxazole (1.25/23.75g) (Hi-Media, Mumbai, India). Screening for AmpC -lactamase production was performed by Cefoxitin disk test [3, 6]. Isolates showing inhibition zone diameter less than 18mm (screening positive) were further subjected to disk antagonism test for inducible AmpC enzyme, and simplified three dimensional and AmpC disk tests for the detection of plasmid AmpC -lactamases.

Detection of AmpC -lactamases 1. Inducible AmpC -lactamases [6]

The disk antagonism test was used for detection of inducible AmpC -lactamase in all the screening positive AmpC isolates. A 0.5 McFarland of test isolate was swabbed on Mueller-Hinton agar plate and cefotaxime (30g) or ceftazidime(30g) or ceftriaxone(30g) and cefoxitin (30g) disks were placed 20 mm apart from centre to centre. Isolates showing blunting of the cefotaxime or ceftazidime or ceftriaxone zone of inhibition adjacent to the cefoxitin disk were considered inducible AmpC -lactamase (Figure 1.).

2. Plasmid mediated AmpC -lactamases

I. Simplified three dimensional tests Briefly, fresh overnight growth from MuellerHinton agar was transferred to a pre-weighed sterile micro-centrifuge tube of 2ml capacity. The tube was weighed again to check the weight of the bacterial mass in order to obtain 10 mg of bacterial wet weight for each isolate. The growth was suspended in 1.5 ml of peptone water and incubated at 35oC for 4 hours. This was subsequently concentrated by centrifugation at 3000 rpm for 15 minutes and the supernatant was used as a crude enzyme extract for the test. Lawn culture of E. coli ATCC 25922 was prepared on MHA plates and cefoxitin (30 g) disk were placed on the plates. With a sterile surgical scapel blade (No. 15), linear slits (3 cm) beginning 3 mm from the edge of the cefoxitin disk were cut in an outward radial direction. At the other end of the slit a small triangular well was made using the same scapel blade after flaming and the enzyme extract was loaded for a total of 30 l by a micropipette using sterile tips. The plates were kept upright for 5 minutes until the liquid dried and incubated at 35C for 18-24hours.

Material and Methods

A total of 201 consecutive, non-duplicate gram-negative clinical isolates over a period of six months (July to December, 2011) were collected from clinical specimens such as urine, wound, blood, tracheal aspirates, endotracheal tube, endotracheal aspirates or central venous catheter of hospitalised patients suspected with nosocomial infections. The study included only the patients of all age groups and both sexes developing infections after 48 hours of hospital stay and those with infections at the time of admission or within 48 hours of hospitalization were excluded. The isolates were identified on the basis of conventional microbiological procedures [5]. Antibiotic susceptibility was determined by using the Kirby Bauers disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [4] using the Mueller Hinton agar

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Isolates showing clear distortion of the zone of inhibition of cefoxitin were considered as strong AmpC producers, minimal distortion as intermediate and no distortion as negative strains (Figure 2, 3, 4, 5). II. AmpC disk test [7] Sterile disks (6 mm) were moistened with sterile saline (20 l) and inoculated with several colonies of test organism was placed beside a cefoxitin disk (almost touching) on the MHA plate lawned with a culture of E. coli ATCC 25922. The plates were incubated overnight at 35C. A positive test appeared as a flattening or indentation of the cefoxitin inhibition zone in the vicinity of the test disk [8]. A negative test had an undistorted zone (Figure 6, 7).

organisms were inoculated on Mueller Hinton agar plates as recommended by the CLSI. Two 10 g imipenem disks were placed on the plate, and 10 L of EDTA solution was added to one of them to obtain the desired concentration (750 g). The inhibition zones of the imipenem and imipenem-EDTA disks were compared after 16 to 18 hours of incubation at 35C. An increase of 7 mm in zone inhibition diameter around the imipenem and EDTA disk in comparison to the imipenem disk alone was interpreted as a positive result for MBL production. Quality control: Quality control was achieved using K. pneumonia (ATCC 700603), E. coli (ATCC 25922) and known AmpC positive E.coli. Statistical Analysis: Data obtained from this study were analysed using descriptive statistics like percentage and proportion. Sensitivity value was calculated for comparing the results of three dimensional test performed by various researchers.

Detection of other -lactamases in presence of AmpC -lactamases:

1. ESBLs: The original double disk synergy test was modified for detecting ESBLs in AmpC-producing isolates by placing a disk of piperacillin-tazobactam (100/10 g) at a distance of 20 mm from cefepime (30 g) disc [9]. Briefly, a disk of amoxycillin/clavulanate (20/10g) was placed in the centre of Mueller Hinton agar; then 30 g disks of cefpodoxime, ceftazidime, cefotaxime and cefepime were kept at a distance of 20 mm from the amoxycillin/clavulanate disk (centre to centre), and a disk of piperacillin-tazobactam was placed at a distance of 20 mm from the cefepime disk. The organisms were considered to be producing ESBL when the zone of inhibition around cefepime or any of the extended-spectrum cephalosporin disks showed a clear-cut increase towards the piperacillin- tazobactam, or amoxicillin-clavulanate disks [9]. 2. MBLs: The combined disk test or the disk enhancement test was performed as described by Yong et al [10]. Test

Results
The isolated gram-negative organisms were E.coli (n = 65), Klebsiella spp.(n = 70),Proteus spp.(n=4),Citrobacter spp. (n = 9), Enterobacter spp.(n = 8), P.aeruginosa (n = 27) and Acinetobacter spp.(n = 18) . The potential AmpC -lactamase producers detected by the screening test were 69.

Inducible AmpC production

Among the 69 screening positive isolates, one each of Proteus spp, Pseudomonas aeruginosa and Acinetobacter spp (1.5%) revealed the presence of inducible AmpC -lactamases by disk antagonism test. (Table 1).

Table 1. Detection of AmpC -lactamases (Inducible & Plasmid).

Microorganisms ( No. of isolates) E.coli (65) Klebsiella spp. (70) Proteus spp. (4) Enterobacter spp. (8) Citrobacter spp. (9) Pseudomonas aeruginosa (27) Acinetobacter spp. (18) Total (202) Screening positive AmpC (%) 23(35.4) 22(31.4) 1(25) 2(25) 3(33.3) 8(29.6) 10(55.6) 69(34.2) Inducible AmpC Disk antagonism test (%) 0 0 1(25) 0 0 1(3.7) 1(5.6) 3(1.5) Plasmid mediated AmpC 3-DT(%) 21(32.3) 19(27.1) 0 2(25) 3(33.3) 7(25.9) 8(44.4) 60(29.7) AmpC disk test (%) 19(29.2) 16(22.9) 0 2(25) 2(22.2) 5(18.5) 7(38.9) 51(25.3)

AmpC: AmpC beta-lactamases, 3-DT: Three dimensional tests.

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Plasmid-mediated AmpC production

Plasmid-mediated AmpC -lactamase production was detected in 60(29.8%) isolates by the simplified three-dimensional test with the highest occurrence in Acinetobacter spp (46.4% ) followed by Citrobacter spp (33.3%), E.coli (32.2%), Klebsiella spp (27%), P.aeruginosa (25.9%) and Enterobacter spp (25). A clear distortion of the zone of inhibition of Cefoxitin was observed in 52(25.8%), minimal distortion in 8(3.9%) and no distortion in 9(4.4%) isolates ( Table 1, 2). AmpC disk test was able to show 51(25.3%) isolates as AmpC producers of which 38.9%, 29.2%, 25%, 22.9%, 22.2% and 18.5% are Acinetobacter spp, E.coli, Enterobacter spp,

Klebsiella spp, Citrobacter spp and P.aeruginosa respectively. Indentation indicating strong AmpC producer was observed in 40(19.8%) isolates and flattening (weak AmpC) was seen in 11(5.4) isolates ( Table 1, 2).

ESBL and MBL production in presence of AmpC -lactamases:

The co-existence of ESBL and AmpC was detected by modified double disk synergy test in 8(3.9%) isolates whereas coproduction of MBL and AmpC was shown by 10(4.9%) isolates. All the MBL producers were found to show resistance to imipenem ( Table 3).

Table 2. Detection of AmpC -lactamases by Simplified three dimensional & AmpC disk tests.
Plasmid mediated AmpC -lactamases Screening positive AmpC isolates (%) Simplified three dimensional test (%) Minimal distortion (%) 2(3) 1(1.4) 0 0 1(11) 2(7.4) 2(11) 8(3.9) No. distortion (%) 2(3) 3(4.3) 1(25) 0 0 1(3.7) 2(11) 9(4.4) AmpC disk test(%) No. Distortion (%) 4(6.2) 6(8.6) 1(25) 0 1(11) 3(11) 3(16.7) 18(8.9)

Microorganisms ( No. of isolates)

Distortion (%) 19(29.2) 18(25.7) 0 2(25) 2(22.2) 5(18.5) 6(33.3) 52(25.7)

Indentation (%) 15(23) 13(18.6) 0 2(25) 1(11) 4(14.8) 5(27.8) 40(19.8)

Flattening (%) 4(6.2) 3(4.3) 0 0 1(11) 1(3.7) 2(11) 11(5.4)

E.coli (65) Klebsiella spp. (70) Proteus spp. (4) Enterobacter spp. (8) Citrobacter spp. (9) Pseudomonas aeruginosa (27) Acinetobacter spp. (18) Total (202)

23(35.4) 22(31.4) 1(25) 2(25) 3(33.3) 8(29.6) 10(55.6) 69(34.2)

Table 3. Detection of ESBLs,MBLs & pure AmpC in screening positive AmpC isolates.
Microorganisms (No. of isolates) E.coli (65) Klebsiella spp. (70) Proteus spp. (4) Enterobacter spp. (8) Citrobacter spp. (9) Pseudomonas aeruginosa (27) Acinetobacter spp. (18) Total (202) Screening positive AmpC (%) 23(35.4) 22(31.4) 1(25) 2(25) 3(33.3) 8(29.6) 10(55.6) 69(34.2) AmpC+ ESBLs(%) 4(6.2) 3(4.3) 0 0 0 1(3.7) 0 8(3.9) AmpC+ MBLs(%) 2(3) 1(1.4) 0 0 0 3(11) 4(22.2) 10(4.9) Pure AmpC(%) 15(23) 15(21.4) 1(25) 2(25) 3(33.3) 4(14.8) 5(27.8) 45(22.3)

AmpC: AmpC beta-lactamases, ESBLs: Extended spectrum beta-lactamases, MBLs: Metallo-beta-lactamases.

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Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 1. Positive disk antagonism test. (Proteus spp.) [cefotaxime(R) showing blunting & cefoxitin (L)]. Figure 2. Simplified three dimensional showing distortion in two isolates of E.coli. Figure 3. Simplified three dimensional showing distortion in two isolates of Klebsiella spp. Figure 4. Simplified three dimensional showing minimal distortion (E. coli ). Figure 5. Negative three dimensional tests. Figure 6. AmpC disk test showing indentation (R) & flattening(L). Figure 7. AmpC disk test showing indentation (R) & negative(L).

Figure 7.

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Discussion
The three-dimensional test was designed to detect both AmpC and ESBL production. In the indirect form used first for AmpC detection by Thomson KS et al, a conventional disk diffusion susceptibility assay is carried out with a susceptible strain, such as E. coli ATCC 25922, as the lawn and a suspension of the test organism, which is added to a circular slit in the agar 3 mm from a disk containing cefoxitin or some other agent. Distortion of the zone of inhibition indicates a positive test, as cefoxitin is hydrolyzed by the presence of an AmpC enzyme. This technique has its own limitations, despite being increasingly sensitive [11]. In subsequent modification by Coudron et al, a radial slit was employed, and rather than using intact cells, the test organisms were concentrated by centrifugation, and the pellet was freeze-thawed five to seven times to release -lactamase [3]. The method could overcome the need for rotator, but filling these slits without spillage was not solved. Manchanda V et al further modified the procedure by creating a well at the outer edge of the slit and the enzyme extract was put into the well [2]. The method overcomes all the problems of three-dimensional test but it is technically demanding and needs experience. A simplified method of three dimension test was developed with few modifications from the previous one. We prepared the enzyme extract by centrifugation after allowing incubating the organisms for 4 hours and it was not freeze thawed. A small triangular well was made at the end of the slit through which the enzyme extract was loaded using micropipette with sterile tips so that any spillage while loading an enzyme extract was far from the area where distortion of zone had to be observed and filling the slits through these wells was easy and rapid. This technique had advantages in terms of less expertise and minimal equipment required, but great care had to be taken while filling the slits and incubating the plates to prevent spillover of the suspension. In our study, simplified three dimensional test detected 60 AmpC producers from 69 screening positive isolates showing a sensitivity of 86.9%. This suggests that the technique can be used for routine detection of the AmpC enzyme in a clinical laboratory. Manchanda V et al, Coudron et al, Singhal S et al, Bosak S et al, Mohamudha PR et al and Rudresh SM et al found 75.6%, 35.5%, 36%, 100%, 93.6% and 70% sensitivity rate for modified three dimensional test respectively [2-3, 8, 12-14]. It was found that the sensitivity of the three dimensional test was more (86.9%) when compared to Amp C disk test (73.9%). However, Amp C disk test was relatively easier to perform when compared to three dimensional test. We employed disk antagonism test to identify inducible expression

of chromosomal AmpC -lactamases where three isolates were positive ( one each of Proteus spp, Pseudomonas aeruginosa and Acinetobacter spp), thus revealing the presence of only plasmid mediated resistance in E.coli and Klebsiella spp, and this finding is well correlated with previous Indian studies [6,15] . A limitation of methods used to detect the AmpC enzyme is that an increasing number of clinical isolates have multiple beta-lactamases, which in turn can make inhibition patterns complex and difficult to interpret [16]. Our study demonstrated the co-existence phenotype of both ESBL and AmpC in 6.2%, 4.3% and 3.7% isolates of E. coli, Klebsiella spp. and Pseudomonas aeruginosa respectively. This could be due to the fact that plasmid-mediated AmpC enzymes have been shown to disseminate among Enterobacteriaceae , sometimes in combination with ESBLs. In our study, cefepime was the best and reliable cephalosporin in detecting ESBL in presence of AmpC producer as high level AmpC production has minimal effect on the activity of cefepime, and tazobactam turned out to be the best -lactamase inhibitor in detecting ESBL production followed by clavulanic acid [17]. It has been reported that clavulanic acid may induce expression of high level AmpC production in organisms producing both ESBL and AmpC together, and may antagonize rather than protect the antibacterial activity of the partner -lactam, thereby making any synergy arising from inhibition of an ESBL, and much better inhibition is achieved with the sulphones, such as tazobactam and sulbactam which are preferable inhibitors for ESBL detection tests in AmpC producers [16, 18-19]. Our study also demonstrated the presence of MBL (4.9%) among AmpC producing isolates and imipenem was found to be the most effective drug, showing maximum susceptibility of 93.2% which is in agreement with earlier studies [6, 20]. Isoelectric focusing and genotypic characterization based on multiplex PCR which are considered gold standard could not carried out due to lack of infrastructure in our institution.

Conclusion
This three-dimensional test was easier to perform and interpret. It was found as sensitive and specific as other modifications and the additional cost is minimal. Clinical laboratories which lack the infrastructure of advanced molecular methods may use this technique to confirm the presence of AmpCmediated resistance.

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