| Abstract|| |
Background: Meropenem is empirically used as a last resort for the treatment of infections by non-fermenting gram-negative bacilli (NFGNB). Minimum inhibitory concentration (MIC) determined using agar or broth dilution methods is widely used for testing meropenem resistance. However, it is not possible in resource-poor settings. Aim: A prospective study was performed to evaluate the reliability of Kirby-Bauer disk diffusion (KBDD) method for detecting meropenem resistance among NFGNB. Materials and Methods: A total of 146 NFGNB consisting of 56 Acinetobacter baumannii, 24 Acinetobacter lwoffii, 48 Pseudomonas aeruginosa and 18 Pseudomonas spp. were included in the study. All the isolates were tested simultaneously by both KBDD method and agar dilution method. Results: Very major errors were not observed with A. baumannii, A. lwoffii and P. aeruginosa, while other Pseudomonas spp. showed a very major error rate of about 5.6%. The major error rates observed with A. baumannii, A. lwoffii, P. aeruginosa and Pseudomonas spp. were 1.8%, 0%, 2.1% and 28.6%, respectively. All the isolates showed a good correlation between zone diameters (KBDD method) and MICs (agar dilution method). The sensitivity and specificity of KBDD method for detecting meropenem resistance was above 90% for all the NFGNB except Pseudomonas spp. Conclusions: The KBDD method can be reliably used for routine testing of meropenem resistance in A. baumannii, A. lwoffii and P. aeruginosa. However, further studies are needed before employing this technique for detecting meropenem resistance in Pseudomonas spp.
Keywords: Acinetobacter , agar dilution, disk diffusion, minimum inhibitory concentration, Pseudomonas, zone diameter
|How to cite this article:|
Joseph NM, Sistla S, Dutta TK, Badhe AS, Rasitha D, Parija SC. Reliability of Kirby-Bauer disk diffusion method for detecting meropenem resistance among non-fermenting gram-negative bacilli. Indian J Pathol Microbiol 2011;54:556-60
|How to cite this URL:|
Joseph NM, Sistla S, Dutta TK, Badhe AS, Rasitha D, Parija SC. Reliability of Kirby-Bauer disk diffusion method for detecting meropenem resistance among non-fermenting gram-negative bacilli. Indian J Pathol Microbiol [serial online] 2011 [cited 2019 Dec 11];54:556-60. Available from: http://www.ijpmonline.org/text.asp?2011/54/3/556/85092
| Introduction|| |
Non-fermenting gram-negative bacilli (NFGNB) such as Pseudomonas spp. and Acinetobacter spp. are most frequently encountered pathogens in the health-care environment. They are also present as colonizers on the hands of health-care workers. , The inherent resistance of these bacterial agents to commonly used disinfectants and their tendency to colonize various surfaces have been pivotal in their emergence as important nosocomial pathogens.  They also have a prodigious capacity to acquire a wide range of new resistance determinants, which further compounds the problem. , Therefore, they are often multi-drug resistant (MDR), posing difficulty in successful management of the patients harboring infections caused by them.
The treatment of these pathogens is challenging as they are often resistant to a diverse group of antibiotics including β-lactams, aminoglycosides and fluoroquinolones. , Despite their resistance to most β-lactams, they may respond to carbapenems, which are not usually hydrolyzed by the extended spectrum β-lactamases or AmpC β-lactamases produced by these pathogens. Hence, carbapenems are being widely used as life-saving antibiotics for treatment of infections caused by these MDR organisms.  However, there has been a recent emergence of metallo-β-lactamase (MBL) producing strains of NFGNB, which can hydrolyze even carbapenems. ,, In addition, several other mechanisms such as efflux pumps and reduced outer membrane permeability due to loss of particular outer membrane proteins also mediate carbapenem resistance.  The MBL producing strains as well as those with other underlying mechanisms of drug resistance do not respond to the carbapenems. Therefore, there is a need to clearly differentiate the carbapenem resistant and susceptible strains for appropriate management of patients infected by them.
In the laboratory, meropenem is commonly used for testing carbapenem resistance. Meropenem resistance is reported either based on determination of minimum inhibitory concentration (MIC) using agar/broth dilution method or by measurement of zone of inhibition according to Kirby-Bauer disk diffusion (KBDD) method.  KBDD method is more economical and technically less demanding compared to the dilution methods.  In a study from Philadelphia, which compared the KBDD with agar dilution method for testing imipenem resistance among Pseudomonas aeruginosa, the results of agar dilution and KBDD method were found to have a strong correlation with a very few false susceptibilities.  However, in a recent study by Singh et al., about 85% of the isolates initially reported as meropenem resistant based on KBDD method were subsequently found to have MICs to meropenem in the sensitive zone, thereby questioning the reliability of the disk diffusion method.  There are only a few studies which have systematically evaluated the performance of KBDD in comparison to dilution methods for determining meropenem resistance.
We performed a study to evaluate the reliability of KBDD for detecting meropenem resistance among NFGNB.
| Materials and Methods|| |
Study Design and Setting
A prospective comparative study was conducted over a period 15 months from October 2006 to December 2007, at an 860-bedded tertiary care hospital and Institution of National Importance in India. A total of 146 NFGNB were included in this study. These bacterial isolates were recovered from patients on mechanical ventilation, who were either colonized or actively infected by these bacteria. , The demographic data of the patients who harbored these NFGNB, have been described in detail in our previously published articles. , The isolates were identified based on standard bacteriological techniques. 
All the isolates were tested for meropenem resistance by using KBDD method according to the Clinical Laboratory Standards Institute (CLSI) guidelines.  The test organism was picked up with sterile loop and suspended in peptone water and incubated at 37°C for 2 hours. The turbidity of the suspension was adjusted to 0.5 McFarland's standard (1.5 x 10 8 CFU/mL).  It was then spread on the surface of a cation-adjusted Mueller-Hinton agar (MHA) plate using sterile cotton swab. The meropenem (10 mg) disk was placed on the agar. The plates were incubated at 37°C overnight. The zone of inhibition was measured and interpreted as per the CLSI guidelines.  Zone diameter ≥16 mm was interpreted as sensitive, while a zone ≤13 mm was considered as resistant, and a zone size in between the two cut-offs was considered as intermediate.
Agar Dilution Method
The MIC of meropenem was determined using agar dilution method according to the CLSI guidelines. Doubling dilutions for meropenem ranging from 0.25 mg/mL through 128 mg/mL were tested. Isolates with MIC ≥16 mg/mL were interpreted as resistant, while those with MIC ≤4 mg/mL were considered as sensitive, and those with values between the two cut-offs were regarded as intermediate. 
Escherichia More Details coli (ATCC 25922) and P. aeruginosa (ATCC 27853), were used as quality control strains for both the susceptibility testing methods.
Definition of Errors
The discrepancy between the two methods was referred to as errors. According to the CLSI guidelines, the errors were defined as very major error (sensitive according to KBDD method but resistant by agar dilution method), major error (resistant according to KBDD method but sensitive by agar dilution method) and minor error (sensitive or resistant by KBDD method and intermediate by agar dilution method or vice versa).  In accordance with CLSI, occurrence of ≥1.5% very major errors and ≥3% major errors was considered as unacceptable. 
Data entry and analysis were done using statistical software SPSS for Windows Version 16.0 (SPSS Inc., Chicago, IL, USA). Percentages were calculated for categorical variables. Scatter plots were generated and Pearson's correlation coefficient was calculated for measuring the linear association between the zone diameter obtained by KBDD method and the MICs determined by agar dilution method. Sensitivity, specificity, positive and negative predictive values were calculated using GraphPad InStat version 3.00 for Windows 95, GraphPad Software (San Diego, CA, USA) for determining the diagnostic value of KBDD method in detecting meropenem resistance. P values <0.05 were considered statistically significant.
| Results|| |
A total of 146 NFGNB, including 56 Acinetobacter baumannii, 24 Acinetobacter lwoffii, 48 P. aeruginosa and 18 Pseudomonas spp. were studied. Overall, 73 out of the 146 (50%) isolates were resistant to meropenem according to agar dilution method. The percentages of meropenem susceptibility of the individual NFGNB are shown in [Table 1]. The MICs at which 50% of the isolates were inhibited (MIC50 values), the MICs at which 90% of the isolates were inhibited (MIC90 values), and the ranges of MICs for the different NFGNB are also summarized in [Table 1].
|Table 1: Activity of meropenem against the non-fermenting, gramnegative bacilli|
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The rates of discrepancy between MIC (agar dilution method) and KBDD method are summarized in [Table 2]. The discrepancies observed with A. baumannii , A. lwoffii and P. aeruginosa were within the acceptable limits, while Pseudomonas spp. showed discrepant results above the acceptable limit recommended by CLSI.
|Table 2: Rates of discrepancy between MIC and Kirby-Bauer disk diffusion method|
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The scatter plots generated for the individual NFGNB, showing the linear association between the zone diameter obtained by KBDD method and the MICs determined by agar dilution method, are depicted in [Figure 1]. The MICs and zone diameters were observed to have a significant negative linear association for all the NFGNB.
|Figure 1: Scatier plots showing the linear association between the zone diameters obtained by KBDD method and the MICs determined by agar dilution method, (a) Acinetobacter baumannii ), (b) Acinetobacter lwoffi i (n=24), (c) Pseudomonas aeruginosa =48), (d) Pseudomonas spp. (n=18)|
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The sensitivity, specificity, positive predictive value and negative predictive value of KBDD method for detecting meropenem resistance among the different species of NFGNB are summarized in [Table 3].
|Table 3: Diagnostic value of Kirby-Bauer disk diffusion method for detecting meropenem resistance|
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| Discussion|| |
The occurrence of MDR NFGNB is increasing over the past few decades. , Meropenem is being empirically used as a last resort for the treatment of infections by these MDR pathogens.  However, the recent emergence of meropenem resistant strains emphasizes the need to routinely test the isolates for meropenem susceptibility, in order to decide whether the empirically started meropenem can be continued or a change in antibiotic regimen is necessary. This gains more importance with the availability of alternative treatment options such as combination of colistin with rifampin and/or tigecycline, which has been shown to successfully treat infections caused by meropenem resistant strains. 
Most of the treating physicians prefer to modify the treatment based on the MIC values determined by either agar or broth dilution methods, as these methods are considered to be very accurate. However, these techniques may not be feasible in all settings and they require more technical expertise and/or specialized equipments. Therefore, there is a need for simple and accurate methods which can be performed even in resource-poor settings. KBDD method is a well-standardized technique used for routine testing of the susceptibility to most antibiotics. But whether this technique can be reliably used for testing of meropenem resistance was mostly unanswered. There are only a few studies that have evaluated this technique for routine testing of meropenem resistance among NFGNB. In a study by Singh et al., an unacceptably high rate of major error (85%) was observed when KBDD method was used to detect meropenem resistance among Acinetobacter.  However, they used only 21 isolates to compare KBDD and agar dilution methods, which could have been responsible for the high error rates. Moreover, they compared only the resistant isolates using both the techniques.
In the present study undertaken to systematically evaluate the KBDD method, 50% of the isolates were resistant to meropenem based on MIC, while the others were sensitive. This equal distribution of the isolates is a positive aspect of our study, as it ensured adequate testing of both sensitive and resistant isolates by KBDD method. We observed that KBDD method performed well for testing meropenem resistance among the Acinetobacter spp. as there was no very major error and only 1.8% major errors were noticed with A. lwoffii, which was well within the acceptable limit. In a similar study, which compared disk diffusion method and broth microdilution method for detecting meropenem resistance among 193 Acinetobacter spp., only 0.5% major errors and 6.7% minor errors were observed.  In our study, we observed a significant negative linear association between zone diameters and MICs when tested with Acinetobacter spp., which further confirms that KBDD method correlates well with MICs and therefore can be reliably used in settings where MIC determination is not feasible. Moreover, this technique had an acceptable sensitivity, specificity, positive predictive value and negative predictive value for detecting meropenem resistance among Acinetobacter spp.
In the present study, the accuracy of KBDD method for detecting meropenem resistance among P. aeruginosa was also good as only 2.1% major errors and 8.3% minor errors were observed. Moreover, there was a very good correlation between MICs and zone diameters, and the sensitivity and specificity were also reasonably good. In a similar study, which compared disk diffusion method and MICs determined by E-test, only 4.5% of the 157 P. aeruginosa tested showed discordance and most of this discordance was due to minor errors.  Therefore, KBDD method can be reliably used for testing meropenem resistance in P. aeruginosa.
When the KBDD method was used for testing meropenem resistance among other Pseudomonas spp., though a good correlation was observed between zone diameters and MICs, we observed 5.6% very major errors and 28.6% major errors, which is considered unacceptable according to the CLSI guidelines.  Moreover, the sensitivity and specificity were not good enough to suggest the routine use of KBDD for testing meropenem resistance in this group of NFGNB.
A major limitation of our study is that we have tested only a small number of isolates. The unacceptably high error rates observed with Pseudomonas spp. in our study could be due to the small number of isolates tested. Therefore, further studies are needed to confirm the usefulness of the KBDD method in this group of NFGNB.
In conclusion, the KBDD method can be reliably used for routine testing of meropenem resistance among A. baumannii, A. lwoffii and P. aeruginosa. However, further studies are needed before employing this technique for detecting meropenem resistance among other Pseudomonas spp.
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Noyal M Joseph
No. 98, 8th Cross, Nanbargal Nagar, Oulgaret, Pondicherry - 605 010
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]