Detection of AmpC β-lactamases production in Acinetobacter species by inhibitor (disk) based & modified three dimensional (enzyme extraction) methods

Sir, 
 
Among the nosocomial infections caused by Gram- negative bacteria, the Acinetobacter spp. is one of the established1 and predictable opportunistic pathogens in immunocompromised patients2,3. AmpC β-lactamases are class C or group I cephalosporinases and non susceptible to alpha methoxy β-lactams such as cefoxitin or cefotetan. The detection of these β-lactamases is clinically significant because these confer resistance to narrow, extended and broad spectrum cephalosporins as well as β-lactam/β-lactamase inhibitor combinations4. This study was undertaken to detect the presence of AmpC β-lactamase in clinical isolates of Acinetobacter species by two phenotypic methods. 
 
A total number of 136 non repetitive cefoxitin resistant clinical isolates of Acinetobacter spp. were obtained during January to December 2012 in the microbiology laboratory, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India. The isolates were identified only to the Genus level and speciation was not done. The zone size <18 mm around the cefoxitin disc was used as a screening test for the presence of AmpC β-lactamase production5. 
 
All cefoxitin resistant isolates were studied for the presence of AmpC enzyme by the modified three dimensional method5. In this method three kinds of results were recorded. Isolates that showed clear distortion of zone of inhibition of cefoxitin were considered as strong AmpC producers. Isolates with no distortion were considered AmpC non producers and isolates with little distortion were considered as weak or intermediate AmpC producer. 
 
The inhibitor (disc) based disc method6 was performed to confirm the AmpC producers. The test culture was swabbed on Mueller-Hinton agar (Hi-media, Mumbai) plates and cefoxitin (30 μg) and cefoxitin/boronic acid (30/400 μg) discs were placed at a distance of 20 mm from centre to centre. An increase of >0.5 mm around cefoxitin/boronic acid compared to cefoxitin alone was considered positive for the presence of AmpC production6. 
 
Among the 136 isolates screened, 82 (60.29%) were positive for the AmpC β-lactamase production by the inhibitor (disc) method. Of the total 136 isolates, 84 (61.76%) were strong AmpC producers, 16 (11.76%) intermediate or weak AmpC producers, and 36 (26.47%) were negative for the AmpC producers by the modified three dimensional (enzyme extract) method. 
 
The isolates harbouring AmpC β-lactamases are shown to be largely restricted to the hospitalized patients only5,7,8. The CLSI (Clinical and Laboratory Standards Institute) guidelines9 do not indicate the screening and confirmatory tests for detecting AmpC β-lactamases in Acinetobacter species. The modified three dimensional5 test is a confirmatory test for detecting both inducible and non inducible AmpC β-lactamases but is technically demanding. In case of inhibitor method using boronic acid with cefoxitin is simple, and this test is sensitive to detect the plasmid mediated AmpC β-lactamases10,11 as well as similar to the ESBL confirmatory test5. Among the cefoxitin resistant Gram-negative isolates, Sasirekha12 reported 20.4 per cent positive for AmpC production whereas Manoharan et al13 reported 36.5 per cent positivity. In a study form Kolkata, 32.77 per cent of isolates were reported positive for ampC by Amp disk test14. Several other studies have also reported AmpC β-lactamase positive Acinetobacter spp.14,15,16,17. 
 
In this study, 84 (61.76%) and 82 (60.29%) isolates were determined as AmpC producers by modified three dimensional and boronic acid inhibitor methods, respectively. When the two phenotypic methods were compared, the inhibitor method failed to detect the presence of AmpC in only two isolates; 38.23 per cent of cefoxitin resistant isolates were negative for AmpC production by both methods. The resistance to cefoxitin can also be mediated by certain class A beta lactamses, carbapenemases and decreased production of outer membrane porins18. In a study from Turkey, more positives (89.76%) were observed by three dimensional method than by boronic acid disk test (85.03%)19. However, Bakthavatchalu et al20 reported higher percentage of positives (93%) of AmpC producers by boronic acid inhibitor test than by the three dimensional method (91%). Lee et al21 compared modified Hodge test with boronic acid test and EDTA disk test to evaluate the presence of AmpC beta lactamase and reported the combination-disk test with boronic acid as a sensitive and efficient test for detecting AmpC producers. 
 
In conclusion, our findings suggest that the inhibitor based disc method can be used in routine clinical microbiology laboratories to confirm the presence of AmpC in Acinetobacter species.

A total number of 136 non repetitive cefoxitin resistant clinical isolates of Acinetobacter spp. were obtained during January to December 2012 in the microbiology laboratory, Sri venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India. The isolates were identified only to the Genus level and speciation was not done. The zone size <18 mm around the cefoxitin disc was used as a screening test for the presence of AmpC β-lactamase production 5 .
All cefoxitin resistant isolates were studied for the presence of AmpC enzyme by the modified three dimensional method 5 . In this method three kinds of results were recorded. Isolates that showed clear distortion of zone of inhibition of cefoxitin were considered as strong AmpC producers. Isolates with no distortion were considered AmpC non producers and isolates with little distortion were considered as weak or intermediate AmpC producer.
The inhibitor (disc) based disc method 6 was performed to confirm the AmpC producers. The test culture was swabbed on Mueller-Hinton agar (Himedia, Mumbai) plates and cefoxitin (30 µg) and cefoxitin/boronic acid (30/400 µg) discs were placed at a distance of 20 mm from centre to centre. An increase of >0.5 mm around cefoxitin/boronic acid compared to cefoxitin alone was considered positive for the presence of AmpC production 6 .
The isolates harbouring AmpC β-lactamases are shown to be largely restricted to the hospitalized patients only 5,7,8 . The CLSI (Clinical and Laboratory Standards Institute) guidelines 9 do not indicate the screening and confirmatory tests for detecting AmpC β-lactamases in Acinetobacter species. The modified three dimensional 5 test is a confirmatory test for detecting both inducible and non inducible AmpC β-lactamases but is technically demanding. In case of inhibitor method using boronic acid with cefoxitin is simple, and this test is sensitive to detect the plasmid mediated AmpC β-lactamases 10,11 as well as similar to the ESBL confirmatory test 5 . Among the cefoxitin resistant Gram-negative isolates, Sasirekha 12 reported 20.4 per cent positive for AmpC production whereas Manoharan et al 13 reported 36.5 per cent positivity. In a study form Kolkata, 32.77 per cent of isolates were reported positive for ampC by Amp disk test 14 . Several other studies have also reported AmpC β-lactamase positive Acinetobacter spp. 14-17 .

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In this study, 84 (61.76%) and 82 (60.29%) isolates were determined as AmpC producers by modified three dimensional and boronic acid inhibitor methods, respectively. When the two phenotypic methods were compared, the inhibitor method failed to detect the presence of AmpC in only two isolates; 38.23 per cent of cefoxitin resistant isolates were negative for AmpC production by both methods. The resistance to cefoxitin can also be mediated by certain class A beta lactamses, carbapenemases and decreased production of outer membrane porins 18 . In a study from Turkey, more positives (89.76%) were observed by three dimensional method than by boronic acid disk test (85.03%) 19 . However, Bakthavatchalu et al 20 reported higher percentage of positives (93%) of AmpC producers by boronic acid inhibitor test than by the three dimensional method (91%). Lee et al 21 compared modified Hodge test with boronic acid test and EDTA disk test to evaluate the presence of AmpC beta lactamase and reported the combination-disk test with boronic acid as a sensitive and efficient test for detecting AmpC producers.
In conclusion, our findings suggest that the inhibitor based disc method can be used in routine clinical microbiology laboratories to confirm the presence of AmpC in Acinetobacter species.