Antibiotic resistance profile and co-production of extended spectrum beta lactamases and AmpC in Acinetobacter spp. in a level 1 trauma center from India

Introduction

Acinetobacter baumannii, named after Paul Baumann, is ubiquitous in soil and water and was earlier considered to be a normal commensal of the human flora.[1] However, it has now emerged as the most significant nosocomial pathogen in the health-care setting ESP in intensive care units (ICUs)[2] due to its virulence properties and its ability to acquire resistance to various antibiotics.[3] Reports of community-acquired Acinetobacter infections have increased over the past decade.[4] Acinetobacter spp. have been associated with a wide range of infections such as pneumonia, bloodstream infection, surgical site infection, and urinary tract infection.[5] The mortality of patients with A. baumannii infections in hospitals and in the ICU has ranged from 7.8% to 23% and from 10% to 43%, respectively.[6]

The extent of antimicrobial resistance is more severe in A. baumannii isolates from patients in Asian and European ICUs than from patients in American ICUs.[7] A significant increase in antimicrobial resistance has been noted worldwide from 2004 to 2009.[6] Acinetobacter spp. have a number of resistance mechanisms such as β lactamase enzyme production, aminoglycoside-modifying enzyme, porin mutation, and efflux pumps which confer resistance to large number of antibiotics. The first MDRAB isolate resistant to almost all available antibiotics in Taiwan was discovered in 1998.[8] A high level of carbapenem-resistant Acinetobacter spp. have also been reported from the Indian ICUs with the rate of resistance ranging from 76% to 90%.[910] The spread of multidrug-resistance determinants in A. baumannii is mostly through plasmid conjugation, transposon acquisition or integron mobilization to gain clusters of genes encoding resistance to several antibiotic families.[11]

Extended spectrum beta-lactamases (ESBLs) in A. baumannii are widely reported from many countries, such as India, France, Turkey, Korea, Belgium, Latin America, the United Kingdom, and the United States.[2] Acinetobacter inherently produces chromosomally mediated AmpC type cephalosporinases which are also known as Acinetobacter-derived cephalosporinases.[12] Co-production of ESBLs and AmpC β-lactamases is a major problem which is responsible for causing therapeutic failures with the use of most of the antibiotics. Rapidly growing resistance among clinical isolates suggests a need to detect resistance mechanisms in this organism. Various methods are available for detection of ESBL and AmpC production. However, none of these tests has been found to be the best in Acinetobacter spp.[13] Thus, the present study was designed to compare the various phenotypic tests of ESBL and AmpC production available with the gold standard of genotype.

Methodology

The study was conducted over a period of 3 years (2013–2015) at a 186 bedded level 1 trauma center of India. All consecutive strains of Acinetobacter spp. isolated from the various clinical samples were included in the study. All the strains isolated were identified using the Vitek 2 system, Biomerieux, and France (GNID ref 21341) and their antibiotic sensitivity was done using the disc diffusion method as per the CLSI guidelines. The antibiotics tested were: ceftazidime (CAZ) (30 μg), cefotaxime (CTX) (30 μg), ceftriaxone (CTR) (30 μg), cefepime (CPM) (30 μg), CPM/tazobactam (CPM/TZ) (30/10 μg), cefoperazone/sulbactam (SUL), imipenem (IMI) (10 μg), meropenem (MERO) (10 μg), amikacin (30 μg), netilmicin (30 μg), levofloxacin (5 μg), tigecycline (TGC) (15 μg), and colistin (CL) (10 μg).

All isolates found resistant to either CTX, CAZ, or CTR were tested for ESBL and AmpC production. ESBL detection was done using a double-disc synergy test (DDST) and E-test for ESBL detection using clavulanic acid or TZ as an inhibitor. AmpC detection was done using AmpC disc test and boronic acid inhibition test.

Results

A total of 154 strains of Acinetobacter spp. were isolated and identified. The antibiotic resistance pattern of the strains isolated is shown in Figure 1.

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Figure 1

Antibiotic resistance profile (% resistance) of Acinetobacter spp.

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Of the 154 strains tested, only 8 (5.1%), 2 (1.2%), and 3 (1.9%) strains, respectively, were sensitive to CAZ, CTX, and CTR. Thus, all the strains were tested for ESBL and AmpC production. Of the strains tested, 15 (9.7%), 17 (11%), 24 (15.6%), 27 (17.5%), 54 (35%), 67 (43.5%), and 72 (46.7%) strains showed ESBL production using CTX/CTX-clavulanate DDST, CTX/CTX-clavulanate E-test, CAZ/CAZ-clavulanate DDST, CAZ/CAZ-clavulanate E-test, Piperacillin/Piperacillin-TZ DDST, CTR/CTR-Sulbactum DDST, and Piperacillin/Piperacillin-TZ E-test, respectively. 20 (12.9%) and 19 (12.3%) of strains were positive for AmpC production using AmpC disc test and boronic acid inhibition test, respectively.

Genotype analysis using PCR for TEM, SHV, CTXM, PER, and VEB genes showed that 69 (51.5%), 25 (21.1%), 40 (30.3%), 50 (44.2%), and 3 (4.3%) strains, respectively, were positive for TEM, SHV, CTXM, PER, and VEB genes. PCR for AmpC genes showed that only 24 (9.4%) strains produced DHAM gene. None of the strains showed production of MOXM, CITM, ACCM, EBCM, and FOXM. Using the SPSS software sensitivity and specificity of the tests was found which is given in Table 3. ESBL and AmpC co-production was seen in 18 (11.7%) strains.

Discussion

MDR Acinetobacter spp. have been isolated worldwide. The rate of carbapenem resistance in Acinetobacter spp. isolated from Europe has been increasing with the lowest rates being reported from Scandinavia.[17] According to MYSTIC surveillance study conducted in the year 2002–004 in 48 European hospitals, 26.9%, 30.2%, 67.6%, 66%, and 52.4% of the strains were resistant to MERO, IMI, CAZ, ciprofloxacin, and gentamicin respectively.[18] The prevalence of carbapenem-resistant Acinetobacter spp. from a burns unit in the USA was found to be substantially high, being 87% in the year 2007.[19] In the Asian subcontinent, the level of resistance to all the available antibiotics including TGC and CL is very high.[20] The level of CL resistance has been variable but is ever increasing ranging from; 2.7% from Europe,[20]2% in the UK,[21] and only sporadic cases from Slovakia.[22] Similarly, in our study also very high resistance was seen to all the available antibiotics.

ESBL detection in Acinetobacter spp. is difficult as standard guidelines for the same are not available unlike in Enterobacteraceae. Unlike Enterobacteriaceae, there are no zone diameter breakpoints for aztreonam and cefpodoxime in Acinetobacter spp.[23] Thus, in the present study, isolates showing resistance to CAZ, CTR, and CTX were tested for ESBL detection. ESBL detection was assessed using TZ, sulbactam, and clavulanate as inhibitors and was compared with PCR (taking PCR as the gold standard). In comparison, tests performed using SUL and TZ as inhibitors performed better than tests with clavulanic acid inhibitor. AmpC producing organisms act as hidden reservoirs for ESBLs. Such isolates, when tested using clavulanic acid inhibition test, are induced to produce high levels of AmpC enzymes which may antagonize the synergy arising from inhibition of ESBLsleading to false negative results. SUL and TZ are much less likely to induce AmpC β-lactamases and therefore, are preferable inhibitors for ESBL detection tests.[12] However, few studies using clavulanic acid as inhibitor for DDST have reported up to 28% ESBL production in Acinetobacter spp.[24]

The prevalence of AmpC β-lactamase producing Acinetobacter spp. appears to be increasing and they have been associated with increased nosocomial infections. However, in the present study, only 12% of strains showed AmpC β-lactamase production. Other authors also detected AmpC β-lactamases in 6 (50%) out of 12 isolates of the Acinetobacter species using the same method.[25]

Genotypically, TEM gene was found the most common gene in our hospital isolates. Safari et al. from Iran reported SHV gene as the most frequent gene in 58% of the strains.[26] Pragasam et al. from India reported TEM (54%), SHV (16%), and PER (1%) to be the most abundant in strains isolated from South India.[27]

Most of the ESBL producing strains in our study were resistant to almost all the antibiotics. This could be due to the expression of other resistance mechanisms such as production of carbapenemases, porin mutation, and outer membrane impermeability, The presence of resistance to beta-lactams is generally associated with resistance to other groups of antibiotics such as aminoglycosides and fluoroquinolones. Resistance mechanisms are generally transferable by way of plasmids.[2] According to the growing and threatening danger of these bacteria, the rapid identification and detection of this strain have an important role in preventing their spread. Furthermore, these findings illuminate the necessity of review in antimicrobial therapy, use of proper antibiotics, and the utilization of new antimicrobial elements to treat the infections resulting from these bacteria. This is a big lacuna of the current study as it was not clinically correlated with the response of the patient to the antibiotics.

Conclusion

Standard guidelines for ESBL detection in nil fermeners like Acinetobacter spp. must be laid down for ease of detection. Use of piperacillin/piperacillin-tazobactam E-test could be used as one of the standard methods.