Evaluation of Different Phenotypic Methods for Detection of Amp C ...

ORIGINAL ARTICLE

Evaluation of Different Phenotypic Methods for Detection of Amp C Beta-Lactamase Producing Bacteria in Clinical Isolates

Afreenish Hassan, Javaid Usman, Fatima Kaleem, Maria Mushtaq Gill, Ali Khalid, Muhammad Iqbal and Paul Ingram

ABSTRACT

Objective: To compare the sensitivity and specificity of different phenotypic methods for detection of Amp C betalactamase producing bacteria. Study Design: Analytical study. Place and Duration of Study: Department of Microbiology, Army Medical College / National University of Sciences and Technology (NUST), Islamabad, Pakistan, from June 2010 to December 2010. Methodology: A total of 150 clinical isolates were screened for presence of Amp C beta-lactamase by using the cefoxitin disc. The confirmatory methods evaluated were inhibitor based assay (boronic acid), Amp C disc test and Amp C Etest. Three dimensional enzyme extract assay was used as the reference method for determining the sensitivity and specificity. Results: Among the total isolates tested, 62.8% bacteria showed the presence of Amp C beta-lactamase by standard three dimensional enzyme extract assay. Among the three methods compared, boronic acid disk test found out to be highly sensitive (88%) and specific (92%) for the detection of Amp C beta-lactamase producing bacteria. Conclusion: Detection of Amp C production is crucial in order to establish the antibiotic therapy and to attain the favourable clinical outcomes. Implementation of simple tests like boronic acid disk tests in the laboratories will help to alleviate the spread of Amp C beta-lactamase harboring organisms. Key Words: Amp C beta-lactamases. Drug resistance microbial. Enterobacteriaceae.

INTRODUCTION

Amp C beta-lactamases are of increasing clinical concern since the late 1970s as one of the mediators of antimicrobial resistance among the Gram negative bacteria.1 Amp C beta-lactamases belong to molecular class C in the Ambler structural classification of betalactamases while in the functional classification scheme of Bush et al. they were allocated to group-1.2 These are cephalosporinases which confer resistance to cephamycins, narrow-, expanded- and broad-spectrum cephalosporins, aztreonam and beta-lactam/betalactamases inhibitor combinations (ampicillin-clavulanic acid, pipericillin-tazobactam etc.).3 Amp C beta-lactamases are more sensitive to inhibition by sulbactam than by clavulanate or tazobactam.4 The genes for Amp C beta-lactamases are commonly found on chromosomes of several members of family Enterobacteriaceae including Enterobacter species, Shigella, Providencia, Citrobacter freundii, Morganella morganii, Serratia, Escherichia coli (E. coli). Since 1989 these inducible chromosomal genes were ascertained on plasmids, and were noticed in organisms which typically do not express these beta-lactamases such as E. coli, Klebsiella Department of Microbiology, Army Medical College, National University of Sciences and Technology (NUST), Islamabad. Correspondence: Dr. Afreenish Hassan, Department of Microbiology, Army Medical College, Rawalpindi. E-mail: [email protected] Received: August 15, 2012; Accepted: May 16, 2013.

pneumoniae, Salmonella spp, Citrobacter, Enterobacter, Proteus mirabilis.4-6

These plasmid mediated (or imported) Amp C betalactamases set hurdles for the clinical microbiologists working in the hospital laboratories. There are more than 20 different Amp C beta-lactamases which are mediated by plasmids.7 Amp C beta-lactamase production is associated with in vitro resistance to all beta-lactam antibiotics except carbapenems and cefepime.

Detection of such devious beta-lactamases is a challenge for the laboratories. Absence of a reliable method for detecting these resistant organisms leads to their rapid spread, leaving the patients and institutions at risk. Currently there are no Clinical and Laboratory Standards Institute (CLSI) recommended guidelines for detection of Amp C beta-lactamases and there is a need to focus on this issue as much as the detection of ESBLs (extended spectrum beta-lactamases).8 Researchers have used various test methods for Amp C betalactamase detection, namely the three dimensional extract test,9 modified double disk test,10 inhibitor based method using inhibitors like boronic acids,1 and cefoxitin agar method.11 The method used by Coudron et al.9 is considered standard phenotypic test in identification of Amp C beta-lactamases. He 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 identification of the isolates that harbor Amp C beta-lactamases.9

Journal of the College of Physicians and Surgeons Pakistan 2013, Vol. 23 (9): 629-632

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There is dearth of information on documentation of Amp C beta-lactamase producing bacteria in this region. Since their detection is thus imperative, this study was undertaken to find out the occurrence of these Amp C beta-lactamases by various phenotypic methods isolated from a tertiary care hospital of Pakistan.

METHODOLOGY

The study was carried out from June 2010 to December 2010 at the Department of Microbiology, Army Medical College/ National University of Sciences and Technology (NUST), Islamabad, Pakistan, affiliated with an 1100 bedded tertiary care hospital. Only those isolates were included in the study which were found positive for Amp C beta-lactamase production on screening test with cefoxitin disc. Duplicate samples from the same patients were not included in the study.

A total of 150 bacteria were isolated from the clinical samples of urine, pus, blood, nasobronchial lavage, intravenous catheter tips and urinary catheter tips during the study period. Organisms were identified by standard microbiological procedures (Gram's stain appearance, colonial morphology, catalase test, cytochrome oxidase reaction, motility, routine biochemical tests) and by using API 20 E (Biomerieux, France).

Isolates were screened for Amp C production by disc diffusion method using cefoxitin disc (Oxoid, UK). A 30 µg cefoxitin disc was placed on inoculated MuellerHinton agar plates (Oxoid, UK). By following the CLSI criteria,12 isolates with zone diameter less than 18 mm were selected for Amp C beta-lactamase testing.9 E. coli 25922 was used as control strain.

Three dimensional extract method (3DET) as described by Coudron et al. was used as standard phenotypic method to detect Amp C production. 9 First, 0.5 McFarland bacterial suspensions were prepared from an overnight culture. 50 µl of each of which was inoculated in 10 ml of trypticase soy broth (TSB, Oxoid, UK). TSB along with organisms were incubated at 37°C for 4 hours. The bacterial cells were concentrated by centrifugation and crude enzyme preparations were made by freeze thawing the cell pellets 5 times. The surface of a Mueller-Hinton agar (Oxoid, UK) plate was inoculated with control strain of Escherichia coli ATCC 25922. A cefoxitin disc (30 µg) was placed in the centre of inoculated agar plates. By using a sterile scalpel blade, a slit beginning 5 mm from the edge of the disc was cut in the agar in outward radial direction. 30 µl of the enzyme preparation was dispensed into the slit, beginning near the disc and moving in an outward direction, by using a micropipette. Overfill of the slit was avoided. The inoculated agar plates were incubated at 37°C for 24 hours. Enhanced growth of surface organism at the point where the slit intersected the zone

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of inhibition was considered a positive three dimensional test and was interpreted as evidence for the presence of Amp C beta-lactamases.

All isolates subjected to three dimensional extract test were simultaneously checked by Amp C disk test.13,14,15 A lawn culture of 0.5 McFarland suspension of Escherichia coli ATCC 25922 was inoculated on surface of Mueller-Hinton agar (Oxoid, UK) plate. Sterile disk (6 mm) was moistened with sterile saline (20 µl) and inoculated with several colonies of test organism. The inoculated disk was then placed beside a 30 µg cefoxitin disk (Oxoid, UK), almost touching it, on the inoculated plate. After overnight incubation at 37°C, positive test was interpreted as a flattening or indentation of the cefoxitin inhibition zone in the vicinity of the test disk and negative test as absence of distortion.

All isolates subjected to three dimensional extract test were at the same time tested by boronic acid disk test.16 The stock solution was prepared by dissolving phenylboronic acid (benzeneboronic acid, Merck, UK) in dimethyl sulfoxide (Merck, UK) at a concentration of 20 mg/ml. 20 µl (containing 400 ug of phenylboronic acid) of this solution was dispensed on the disks containing 30 µg of cefoxitin. Test was performed by placing a cefoxitin disk (30 µg) and the disk containing 30 µg of cefoxitin plus 400 µg of phenylboronic acid on the Mueller- Hinton agar (Oxoid, UK) plate, inoculated with the test bacteria. After overnight incubation of the plates at 37°C, the isolate showing zone diameter around the disk containing cefoxitin and boronic acid more than 5 mm than the zone diameter around the disk containing cefoxitin alone was considered as Amp C beta-lactamase producer.

All isolates subjected to three dimensional extract test were simultaneously checked by Amp C Etest.17,18 Cefotetan/cefotetan+cloxacillin (CN/CNI, MIC range CN 0.5 – 32 µg/ml, CNI 0.5-32 µg/ml + cloxacillin, constant level) double sided strips were used. Briefly, a lawn of 0.5 McFarland suspension of the test isolate was inoculated on the Mueller-Hinton agar (Oxoid, UK) plate. Estrip was applied on the test organism according to manufacturer's instructions. After incubation for 16 – 20 hours at 37°C, results were interpreted. MIC ratio of ≥ 8 and/or the presence of a phantom zone or deformation of the ellipse were considered positive for Amp C betalactamases.

The comparative statistical analysis of these methods was done by using Statistical Package for Social Sciences (SPSS) version 17.0, 2 x 2 table. Data obtained from three dimensional extract method was considered as the gold standard9 for this study and compared with data from Amp C saline disk test, Amp C boronic acid test and Amp C Etest methods. Parameters like sensitivity, specificity, negative predictive value, positive predictive value and accuracy were determined.


Phenotypic methods for detection of Amp C beta-lactamase producing bacteria

True positives were Amp C beta-lactamase producers by 3DET, Amp C saline disk test, Amp C boronic acid test and Amp C Etest methods. False positive were Amp C beta-lactamase producers by Amp C saline disk test, Amp C boronic acid test and Amp C Etest methods and not by 3DET. False negative were the isolates which were non-Amp C beta-lactamase producers by Amp C saline disk test, Amp C boronic acid test and Amp C Etest methods but were producing Amp C betalactamases by 3DET. True negatives those which were non-Amp C beta-lactamase producers by all the four methods.

RESULTS

A total of 150 clinical isolates were included in the study. The isolates were obtained from the specimens of urine (n = 41), pus (n = 35), blood (n = 23), naso-bronchial lavage (n = 18), intravenous catheter tips (n = 15) and urinary catheter tips (n = 18). Majority of the isolates were recovered from the patients admitted in hospital (123/150), and among them 79 were from patients in intensive care unit. The screening test with cefoxitin disk (30 µg) detected 86/150 isolates as possible Amp C beta-lactamase producers. These 86 isolates were subjected to three dimensional extract test for Amp C beta-lactamase detection. Out of these 86, 54 (62.8%) showed the presence of Amp C beta-lactamase by standard phenotypic test. Majority of the organisms associated with Amp C beta-lactamase production were Escherichia coli 31 followed by Klebsiella pneumoniae 16, Klebsiella oxytoca 7.

The Amp C saline disk test detected 24 (28%) isolates as Amp C beta-lactamase producers. By boronic acid disc test Amp C beta-lactamase production was observed in 45 (52.3%) isolates. By Amp C Etest, only 18 (21%) isolates were found out to be Amp C betalactamase producers. Sensitivity and specificity of Amp C saline disk test was 79% and 86%, respectively. For Amp C boronic acid

test, sensitivity and specificity were 88% and 92%, respectively. Sensitivity and specificity of Amp C Etest method were 73% and 78%, respectively (Table I).

DISCUSSION

Detection of Amp C production is crucial in order to establish the antibiotic therapy and to attain the favourable clinical outcomes. Failure to trace these enzymes has contributed to their uncontrolled spread and sometimes to therapeutic failures.11 There is a need for a reliable phenotypic method that can be integrated into diagnostic laboratories for the detection of these resistant pathogens.

In this study, three phenotypic tests were tested against a standard phenotypic test. Boronic acid test detetcted the Amp C beta-lactamase carrying bacteria more reliably. Boronic acid compounds are known class-C enzyme inhibitors, which are not based on a βlactam structure. Disk tests based on their inhibitory activity have been firstly described for the detection of plasmid-mediated Amp C enzymes among enterobacterial pathogens.16,19 These tests were found to enhance considerably growth-inhibitory zone around disks of cefotetan, allowing the accurate differentiation of plasmid-mediated Amp C producing isolates. 16 Subsequently boronic acid tests using disks of cefoxitin, cefotaxime and ceftazidime were successfully evaluated for the detection of Amp C enzymes.20 Regional data from India showed that percentage of Amp C betalactamase producing bacteria as detected by boronic acid test was 47.3%, which is comparable to these study results (52.3%).1 Tsakris et al. from Iran found out the boronic acid test highly reliable and accurate for detection of Amp C beta-lactamase producing isolates, as it detetcted 88.9% of the tested isolates.21 In a study conducted by Coudron et al., boronic acid disk test detected 54 isolates out of 55 Amp C PCR positive isolates.16 With the Amp C saline disk test, we identified 28% isolates as Amp C beta-lactamase producers, comparable to Singhal et al. and Black et al. findings which

Table I: Comparison of Amp C saline disk test, Amp C boronic acid test and Amp C Etest with three dimensional extract test (3DET). Amp C saline disk test

Positive

Negative Amp C boronic acid test

Total

Positive

Negative

Amp C Etest

Total

Positive

Negative

Total

3DET

Positive

Negative

Total

5

53

58

Positive

Negative

19

24

40 5

45

Positive 13 5

18

PPV = Positive predictive value; NPV = Negative predictive value.

9

62 3

38

41

Negative 15 53

68

Sensitivity (%)

Specificity (%)

PPV (%)

NPV (%)

Accuracy (%)

88

92

93

88

91

73

78

46

91

78

28

86

Total 43

79

86

68

91

84

43

86

Total 28 58

86


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reported it as 36% and 31% respectively.13,14 In another study from India, prevalence of Amp C beta-lactamases was 16% as detected by Amp C saline disk test.22 Amp C saline disk test was found out to be 79% sensitive and 86% specific in this study, which is comparable to the results described by Ingram et al. in their study (86% sensitivity and 94% specificity).23 Etest strips with a gradient of cefotetan on one half and the same combined with a constant concentration of cloxacillin on the other half have been evaluated for Amp C detection.24 Either a reduction in cephamycin MIC of atleast three dilutions, deformation of the ellipse of inhibition, or a “phantom zone” was interpreted as a positive test. In this study, the sensitivity and specificity of Amp C Etest were 73 – 78% as against the findings of Ingram et al. (84% sensitivity and 94% specificity),23 Polsfuss et al. (77.4% sensitivity and 100% specificity)24 and Coyle et al. (61.6% sensitivity and 100% specificity).25

CONCLUSION

Implementation of simple tests like boronic acid disk tests in the laboratories can alleviate the spread of Amp C beta-lactamase harboring organisms. The potential benefits would include better patient outcomes in terms of avoiding inappropriate therapy, a reduction in the rise of antibiotic resistance and selection pressure through better infection control.

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