Resistance to β-lactams appears to be primarily caused by production of β-lactamases which include extended spectrum β-lactamases (ESBLs), metallo-β-lactamases, and most commonly, oxacillinases [3]. Acinetobacter inherently produces chromosomally mediated AmpC type cephalosporinases which are also known as Acinetobacter derived cephalosporinases (ADCs), which mediate resistance to cephalothin, cefazolin, cefoxitin, most of the penicillins and β-lactamase inhibitor β-lactam combinations. More than 25 varieties of AmpC β-lactamases that share ≥ 94% protein sequences have been described for Acinetobacterspp [4].
Materials and Methods
The present prospective study was conducted in the Department of Microbiology, Pt. B. D. Sharma PGIMS, Rohtak, during a period of one year (May 2010 to April 2011). A total of 100 strains of Acinetobacter species which were isolated from various clinical samples like blood, lower respiratory tract (LRT) samples (endotracheal aspirates, bronchoalveolar lavage, sputum), urine, pus, throat swabs, high vaginal swabs (HVS), CSF and other body fluids were included in the present study. All the isolates were identified by using standard microbiological procedures and antimicrobial susceptibility testing was done by Kirby-Bauer disc diffusion technique as per Clinical and Laboratory Standard Institute (CLSI) criteria [7]. Antibiotic discs used in the study were procured from Hi-media Laboratories, Mumbai, India and from BD Diagnostics, USA. American Type Culture Collection (ATCC) strain viz. E. coli. ATCC 25922 was employed as a control strain. Discs of the following antimicrobial agents, with their disc concentration, in brackets, were put up: ceftazidime (30μg), cefepime (30μg), ceftriaxone (30μg), cefotaxime (30μg), amoxycillin/clavulanic acid (20μg/10μg), imipenem (10μg), meropenem (10μg), piperacillin/tazobactam (100μg/10μg), ticarcillin/clavulanic acid (75μg/10μg), gentamicin (10μg), amikacin (30μg), netilmicin (30μg), ciprofloxacin (5μg), doxycycline (30μg), cotrimoxazole (25μg), aztreonam (30μg), polymyxin B (300 units), colistin (10μg) and cefoxitin (30μg).
ESBL Detection
Isolates showing reduced susceptibility to third generation cephalosporins were tested for ESBL production by CLSI double disc synergy method [7] and also by using sulbactam as an inhibitory agent [8].
Method in which sulbactam was used as an inhibitory agent: The test organism was inoculated on Mueller-Hinton agar (MHA) plate as per CLSI guidelines. One 30μg disc of ceftazidime, ceftriaxone and cefepime each were placed on surface of MHA plate and another 30/15μg disc of ceftazidime/sulbactam, ceftriaxone/sulbactam and cefepime/sulbactam each were placed on the same agar plate at a distance of approximately 15mm from the ceftazidime, cefotaxime and cefepime discs respectively. A > 5mm increase in zone diameter produced by antimicrobial agents which were tested in combination with sulbactam versus its zone when it was tested alone was considered as positive for ESBL production.
AmpC Detection
Isolates showing reduced susceptibility to cefoxitin were tested for AmpC detection by using AmpC disc test. A lawn culture of E. coli ATCC 25922 was grown on an MHA plate. Several colonies of test organism was inoculated on sterile discs (6mm) which were moistened with sterile saline (20μL). The inoculated disc was placed beside a cefoxitin disc on agar plate. The plates were incubated overnight at 35°C. A positive test was considered to be either flattening or indentation of the cefoxitin inhibition zone, which indicated enzymatic inhibition of cefoxitin. An undistorted zone was suggestive of a negative test [9].
Statistical Analysis
Two or more sets of variables were compared by using SPSS, version 17. If the p-value was <0.05, it was considered to be significant.
Results
Out of 100 clinical isolates of Acinetobacter, 82 were Acinetobacter baumannii and 18 were Acinetobacter lwoffii. Among isolates of A. baumannii, resistance against cefotaxime, ceftriaxone, cefepime and ceftazidime was observed in 97.56%, 96.34%, 95.12% and 93.9% strains respectively and among isolates of A. lwoffii, resistance to ceftriaxone was observed in 83.33% of isolates, 72.22% isolates showed resistance to ceftazidime and cefepime and resistance to cefotaxime was observed in 44.44% of isolates. ESBLs were detected in 4% of the Acinetobacter isolates and in 77% of the isolates by using clavulanic acid and sulbactam as inhibitory agents. Comparison of two methods with respect to ESBL detection has been shown in [Table/Fig-1]. AmpC β-lactamase production was detected in 64.63% isolates of A.baumannii and in 38.88% isolates of A. lwoffii [Table/Fig-2]. Coproduction of both ESBL and AmpC enzymes were seen in 29% of the Acinetobacter strains, out of which 23 were A. baumannii and six were A. lwoffii.
Comparison of the two methods for ESBL detection in 100 isolates of Acinetobacter species
| Acinetobacter species | Number of ESBL producing isolates by CLSI method n(%) | Number of ESBL producing isolates by sulbactam n(%) | p-value |
|---|
| A. baumannii (82) | 4 (4.8) | 65 (79.2) | <0.001 |
| A. lwoffii (18) | 0 | 12 (66.6) | <0.001 |
| Total (100) | 4 (4) | 77 (77) | <0.001 |
Distribution of AmpC β-lactamase producing Acinetobacter isolates
| Acinetobacter species | Number of cefoxitin resistant isolates | Number of isolates showing flattening | Number of isolates showing indentation | Total number of AmpC producing isolates |
|---|
| n | % | n | % | n | % |
|---|
| A. baumannii(82) | 82 | 27 | 50.94 | 26 | 49.05 | 53 | 64.63 |
| A. lwoffii(18) | 18 | 5 | 71.42 | 2 | 28.57 | 7 | 38.88 |
| Total | 100 | 32 | 53.33 | 28 | 46.66 | 60 | 60.0 |
Discussion
ESBL producing Acinetobacter isolates continue to be a major problem in clinical setups worldwide. So, knowledge on their prevalence is essential, to guide clinicians towards providing appropriate antibiotic therapies. In the present study, by using clavulanic acid as inhibitory agent, ESBLs were detected in 4% of the Acinetobacter isolates. Various studies done in past by using clavulanic acid as inhibitory agent had reported ESBL production in 2.08%, 21.4%, 28% and 44% isolates of Acinetobacter species [10–13].
As is evident from the results of present study and data of other authors, the prevalence of ESBL producing Acinetobacter species varies greatly in different geographical areas and also from hospital to hospital. By using sulbactam as an inhibitory agent, we detected ESBLs in 77% of the Acinetobacter isolates. Our results were in accordance with those of other authors, who reported presence of ESBLs in 75% of the Acinetobacter isolates with use of sulbactam [14].
On comparing the two methods of ESBL detection, a highly significant difference (p value <0.01) in the rate of ESBL production was observed in our study. This may be due to the reason that Acinetobacter species also contain additional resistance mechanisms to β-lactam antibiotics, which can mask the presence of ESBL activity. This organism inherently possess chromosomally encoded inducible AmpC cephalosporinases which can hydrolyze all β-lactam antibiotics. AmpC producing organisms act as hidden reservoirs for ESBLs. Such isolates, when they are tested by clavulanic acid inhibition test, are induced to produce high levels of AmpC enzymes which may antagonize the synergy arising from inhibition of ESBLs, which produces a false negative result. Sulbactam and tazobactam are much less likely to induce AmpC β-lactamases and therefore, are preferable inhibitors for ESBL detection tests [15].
As very few studies have been carried out, which have reported ESBL production in Acinetobacter species on using phenotypic methods, only limited data is available for comparison. Various past studies which used PCR analysis and isoelectric focusing, have reported PER-1 type of ESBLs in 54.6% and 46% of isolates of Acinetobacter species [16,17]. However, Naas et al reported VEB-1 type of ESBLs in 95% of Acinetobacter isolates, which was higher than that seen in the present study [18]. An outbreak occurred in France, in which VEB-1 type of ESBLs was detected in all the 12 isolates of A. baumannii which were studied [19]. These authors detected genes which were responsible for production of ESBLs, but many a times, there might be presence of silent genes which are not expressed phenotypically and this could be the reason for lower rate of ESBL production in our isolates.
In the current study, on comparing the antimicrobial resistance patterns of ESBL producing and ESBL non producing Acinetobacter isolates, a highly significant difference (p value <0.001) was observed for resistance to aminoglycosides and fluoroquinolones. Resistance to ciprofloxacin, netilmicin, gentamicin, amikacin and meropenem was observed in 83.11%, 74.02%, 84.41%, 77.92% and 70.12% of ESBL producing isolates of Acinetobacter species respectively, whereas resistance to same drugs was observed in 34.78%, 13.04%, 60.86%, 21.73% and 4.34% non ESBL producing isolates of Acinetobacter species respectively [Table/Fig-3]. Similar findings have been reported by other authors [14,17,19].This may be due to the reason that genes coding for ESBLs reside on plasmids and that these plasmids carrying ESBL genes also carry resistant genes for other antibiotics. The most frequent co-resistance found in ESBL producing organisms was that for aminoglycosides, fluoroquinolones, tetracyclines, chloramphenicol and trimethoprim-sulfamethoxazole [20].
Comparison of the antimicrobial resistance pattern of ESBL producing and ESBL non-producing Acinetobacter isolate to antibiotics other than β-lactams
| Antimicrobial drugs | ESBL producers (n=77) | ESBL non-producers (n=23) | p-value |
|---|
| n | % | n | % |
|---|
| Ciprofloxacin | 64 | 83.11 | 8 | 34.78 | <0.001 |
| Netilmicin | 57 | 74.02 | 3 | 13.04 | <0.001 |
| Gentamicin | 65 | 84.41 | 14 | 60.86 | <0.05 |
| Amikacin | 60 | 77.92 | 5 | 21.73 | <0.001 |
| Meropenem | 54 | 70.12 | 1 | 4.34 | <0.001 |
| Imipenem | 55 | 71.42 | 15 | 65.21 | >0.05 |
| Cotrimoxazole | 64 | 83.11 | 15 | 65.21 | >0.05 |
| Doxycycline | 51 | 66.23 | 15 | 65.21 | >0.05 |
AmpC β-lactamase enzymes confer resistance to a wide variety of β-lactam antibiotics, except carbapenems. The prevalence of AmpC β-lactamase producing Acinetobacterspp. appears to be increasing and they have been associated with increased nosocomial infections. In the present study, AmpC β-lactamases were detected in 60% of the Acinetobacter isolates by AmpC disc method. Other authors also detected AmpC β-lactamases in 6 (50%) out of 12 isolates of the Acinetobacter species by using same method [21].The results of our study were in accordance with those of these studies. However, some studies have described a lower rate of AmpC β-lactamase production in Acinetobacter spp., which ranged from 20% to 45% [9,22,23]. This could be explained by the fact that the number of isolates which were included in these studies was much lower than that which was included in the current study. Different selection criteria used for Acinetobacter isolates could be another reason for obtaining different rates of AmpC β-lactamase production in current study than those seen in the above mentioned studies. In the present study, we performed AmpC disc test only for those isolates which were resistant to cefoxitin, whereas in other studies, tests for detection of AmpC β-lactamase were done for all the isolates which were included in study, irrespective of cefoxitin resistance. As no CLSI recommendations exist, regarding the method which has to be used for detection of AmpC β-lactamases, different methods have been used by different authors. This could be another reason for obtaining a different and higher rate of AmpC production in our study.
The current study showed that no AmpC β-lactamase enzymes were detected in 40.4% of cefoxitin resistant Acinetobacter isolates. One previous study had reported no AmpC β-lactamase activity in 55.55% of the cefoxitin resistant isolates of Acinetobacter species which were studied [9]. Other authors have also reported that 8 (80%) out of 10 cefoxitin resistant isolates of Acinetobacter species did not show any production of AmpC β-lactamase enzymes [23].This could be due to the reason that cefoxitin resistance could occur in AmpC non-producing isolates because of mechanisms other than AmpC production, such as lack of permeation of porins or there could have been a low level expression of ampC genes, which had not been detected by the present method.
In the present study, coproduction of ESBL and AmpC enzymes was detected in 29% of the Acinetobacter isolates. During literature search, it was observed that only limited studies had been conducted on coproduction of β-lactamases in Acinetobacter spp. Rajini et al reported coproduction of these enzymes in 2 (20%) out of 10 isolates of Acinetobacter species which were studied [15]. Nagano et al studied three Acinetobacter isolates for the production of β-lactamases and they reported coproduction of both ESBL and AmpC enzymes in all three (100%) isolates [24].One study showed coproduction of metallo- β-lactamases and AmpC β-lactamases in 54% of the Acinetobacter isolates which were studied [25].Another study reported ESBL production in 17.95% isolates and production of AmpC β-lactamases in 56.67% of Acinetobacter isolates which had been studied, but this study did not highlight the prevalence of Acinetobacter isolates which had shown coproduction of β-lactamases [26].The major lacunae in past studies which showed coproduction, was the lesser number of Acinetobacter strains which were studied. Hence, these could not be considered as statistically significant. It has been suggested that such studies should be conducted on more isolates obtained from geographically diverse areas, with molecular confirmation of these enzymes, so that suitable conclusions can be made regarding this aspect.
Conclusion
β-lactams are the most widely used antimicrobials worldwide, which are favoured because of their efficacies, broad spectra and low toxicities. Acinetobacter is a pathogen which is well known for its high antimicrobial resistance and it most commonly shows resistance to β-lactams, as it produces β-lactamases. Rapid phenotypic detection of combined mechanisms of antimicrobial resistance, such as ESBL and AmpC expressions, is crucial for epidemiological purposes and for implementing appropriate antimicrobial therapies and infection control measures.