Prevalence of metallo-β-lactamases producers among carbapenem-resistant Acinetobacter baumannii strains isolated from diabetic foot ulcers

The aim of this work was to detect the prevalence of metallo-β-lactamases (MBL) producers among carbapenem-resistant Acinetobacter baumannii strains isolated from diabetic foot ulcers. Out of 290 samples of diabetic foot ulcers, 99 strains of A. baumannii (33%) were isolated and identified by conventional culture methods. Antibiotic sensitivity pattern of the isolated A. baumanni strains was done by disc diffusion method. For A. baumanni strains that was resistant to imipenem and meropenem, MBL production was screened by potentiated disc test and confirmed by multiplex polymerase chain reaction for blaIMP and blaVIM genes. Out of 99 strains of A. baumannii, 26 (26%) were found resistant to imipenem and/or meropenem. Of these 26 carpabenem resistant A. baumannii strains 9 (34.61%) were positive for MBL by potentiated disc test, and 6 strains (23.07%) were positive for blaVIM or blaIMP by multiplex PCR; where blaVIM gene was detected in 4 strains (15.38%) and blaIMP was detected in 2 strains (7.69%). The antimicrobial susceptibility profile for the isolated A. baumanni strains showed that the highest sensitivity was to meropenem (74.4%), imepenem (76.55%), amikacin (65%) and the lowest sensitivity was to ceftazidime (11%) and ciprofloxacin (12%). Rapid dissemination of carbapenemresistant isolates in diabetic foot ulcers is worrisome and calls for judicious use of antibiotics. blaVIM and blaIMP genes have a role in carbapenem-resistant in the community. More studies are needed to differentiate MBL from non-metalloenzymes producers.


INTRODUCTION
Foot ulcers are among the leading causes of morbidity in diabetics and are the most common indication for admission in this population (Azer et al., 1999).
Devitalized tissue is the site where the bacteria responsible for the non-healing ulcers inflict damage.Infectious agents are associated with amputation of the *Corresponding author.E-mail: almrasha@yahoo.com.Tel.01225210409.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License infected foot if not treated promptly (Logerfo et al., 1984).Acinetobacter baumannii has emerged as an important opportunistic Gram-negative bacteria in health care institutions globally, as it resists desiccation, is hard to eradicate and has numerous intrinsic and acquired mechanisms of drug resistance.Production of carbapenamases, which hydrolyse carbapenems, the drugs with high efficacy and broad spectrum of activity against this organism, has been a cause of worry to the clinician and the microbiologist (Peleg et al., 2008).
MBL production is typically associated with resistance to aminoglycosides and fluoroquinolones, further compromising therapeutic options.Among the seven types of MBL genes described throughout the World, bla IMP and bla VIM are the most common (Karthika et al., 2009).
The genes responsible for MBL production may be chromosomal or plasmid mediated and pose a threat of horizontal transfer.The introduction of carbapenems into clinical practice was of great help in the treatment of serious bacterial infections caused by β-lactam resistant bacteria and is the drug of choice for the infection caused by cephalosporin-resistant Gram negative infections (Varaiya et al., 2008).
The aim of the present study was to detect the prevalence of MBL producers among carpabenemresistant A. baumannii strains isolated from diabetic foot ulcers with antimicrobial susceptibility pattern of A. baumannii strains isolated from diabetic foot ulcers.

MATERIALS AND METHODS
Over a one-year period from June 2012 to May 2013, 99 isolates of A. baumannii were isolated from patients with diabetic foot ulcers admitted to surgery department in Tanta University Hospital.The specimens were collected, transported and processed in microbiology laboratory without any delay.This work had the approval of the ethical committee in Faculty of Medicine, Tanta University and a written consent from all participants.

Inclusion criteria
Diabetic patients with duration of 10-15 days of diabetic foot infections, with no antibiotic therapy for one week before the study were used.

Exclusion criteria
Patients treated with antibiotics before admission for this attack of infection and chronic diseases other than diabetes.

Microbiological study
Culture specimens were obtained at the time of admission, after the surface of the wound had been washed vigorously with saline, and followed by debridement of superficial exudates.Specimens were obtained by scraping the ulcer base or the deep portion of the wound edge with a sterile curette.The soft tissue specimens were promptly sent to the laboratory for microbiological study (Shanker et al., 2005).

MBL production
Screening for the detection of MBL was done by disc potentiation test with EDTA-impregnated imipenem discs and EDTAimpregnated meropenem discs and confirmed by multiplex PCR (Hemalatha et al., 2005).

Disc potentiation test methods
Test organism was inoculated onto plates of Mueller-Hintonagar plate (opacity adjusted to 0.5 McFarland opacity standards).A 0.5m EDTA solution was prepared by dissolving 186.1 g of disodium EDTA 2H 2 O in 1000 ml of distilled water and adjusting it to pH 8.0 by using NaOH.The mixture was sterilized by autoclaving.Two 10mg imipenem discs and meropenem discs were placed on the plate; 5 ml of EDTA solution was added to one of the disceach.The inhibition zones of the imipenem and imipenem-EDTA discs and meropenem and meropenem-EDTA discs were compared after 16-18 h of incubation at 35 8°C.An increase in the zone size of at least 7 mm around the imipenem-EDTA disc and meropenem-EDTA discs was recorded as an MBL-positive strain (Leek et al., 2003).
A total of 4-5 identical colonies of A. baumannii were resuspended in 500 μl of sterile saline in 1.5 ml Eppendorf tube.This was boiled at 100°C for 10 min, centrifuged at 8000 rpm for 5 min and the supernatant containing DNA was used for further processing.The PCR mixture used was as follows: 1 μl DNA template in a 49 μl mixture containing 10 mMTris/HCl (pH 8.8), 50 mMKCl, 4 mM MgCl 2 , 200 μM each dNTP (FermentasGenetix Biotech Pvt. Ltd., New Delhi), 1 μl of each of the forward and reverse primers (Bangalore genei) and 1 unit Taq DNA polymerase (FermentasGenetix Biotech Pvt.Ltd.,NewDelhi).The PCR conditions included: initial denaturation at 94°C for 5 min followed by 33 cycles each of 94°C for 25 s, 53°C for 40 s and 72°C for 50 s, followed by a single final elongation step at 72°C for 6 min.The PCR product of 188 bp for bla-IMP and 382 bp for bla-VIM was visualized by 1.5% agarose gel electrophoresis containing ethidium bromide, 0.5 μg/ml (Bangalore Genei).

RESULTS
Out of 290 cases of diabetic foot ulcers attending surgery department, Tanta university hospital; 99 A. baumannii strains were isolated and were eligible for the study including 51 (51.51%) male patients and 48 (48.48%) with mean age±SD (49 ± 16.8 years).The mean duration of diabetes in the patients of the study was 12 ± 10.2 years and the mean duration of diabetic foot ulcers was The result of the study shows that 26 (26.26%) A. baumanii strains out of 99 strains were resistant to at least one or both carbapenem tested.Out of these, 22 (84.61%) were resistant to both imipenem and meropenem, 3 (11.53%) to meropenem and one strain (3.84%) to imipenem alone.Among 99 A. baumanni strains isolated from diabetic foot ulcers, Amikacin showed the highest level of sensitivity (65.6%), followed by colistin (43.3%).9.09% only were sensitive to cefoperazone, 23.33% were sensitive to piperacillin/tazobactam. Table 2 shows antibiotic sensitivity pattern of A. baumanni strains isolated from diabetic foot ulcers.
The results of the study show that out of 26 carpabenem resistant A. baumannii strains 9 strains (34.61%) were positive for MBL by potentiated disc test, and 6 strains (23.07%) were positive for bla VIM or bla IMP by multiplex PCR; where bla VIM gene was detected in 4 strains (15.38%) and bla IMP was detected in 2 strains (7.69%).Figure 1 shows Agarose gel electrophoresis showing positive amplification of 382 and 188 base fragments specific for bla VIM and bla IMP respectively.

DISCUSSION
Diabetic foot ulcer is the most common complication requiring hospitalization among diabetic patients (Logerfo et al., 1984 andBridges et al., 1994).It is also the most common cause of non-traumatic lower extremity amputations (El-Tahawy et al., 2000).Physicians have an important role in prevention, early diagnosis, and management of diabetic foot complications.Management, however, entails an extensive knowledge of the major risk factors for amputation and preventive maintenance with special reference to drug resistance in bacteria (Logerfo et al., 1984;El-Tahawy et al., 2000).
MBLs have been identified from clinical isolates in members of Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp.over the past few years (Walsh et al., 2005;Kohlenberg et al., 2009).Strains producing these enzymes have been responsible for nosocomial outbreaks that have been accompanied by serious and prolonged infections.MBLs are powerful carbapenemases and can hydrolyze a wide variety of βlactams, including penicillins, cephalosporins and carbapenems.Since the initial isolation of carbapenem resistant A. baumannii producing bla-IMP-1 and bla-VIM-1 type MBL in Japan and Italy, respectively (Lee et al., 2003).Clinical isolates of these strains have been identified worldwide.CLSI document (2009) has no guidelines for detecting MBLs, however, it has recommended modified Hodge test for detection of carbapenamases but in members of Enterobacteriaceae only.
The present work was carried out over a period of one year that extended from June 2012 to May 2013 on 290 patients with diabetic foot ulcers attending department of surgery in Tanta University Hospital.Out of 290 clinical samples, A. baumanni strains could be isolated from 99 (34.13%)cases.This percentage was in agreement with Gadepalli et al. (2006) who could isolate A. baumanni from 40% of cases of diabetic foot ulcers.
The results of antimicrobial susceptibility profile for the 99 A. baumanni strains isolated in this study showed that the highest sensitivity was to Meropenem (74.4%),Imepenem (76.55%),Amikacin (65%) and the lowest sensitivity was to Cefoperazone (9.09%), Ceftazidime (11%) and Ciprofloxacin (12%).The results showed that 9 strains (9.09%) were only sensitive to cefoperazone, and 23 strains (23.33%) were sensitive to piperacillin/tazobactam.The study of Umadevi et al. (2011) showed that the majority of Acinetobacter spp.were susceptible to piperacillin-tazobactam (83%), imipenem (67%) and trimethoprim-sulfamethoxazole (67%), while being less susceptible to gentamicin (17%), amikacin (50%), ciprofloxacin (67%), tetracycline (50%), ceftriaxone (33%) and ceftazidime (33%).In accordance with the results of this study, the result of Shanker et al. (2005) showed that 76.4% of the isolated A. baumanni strains in their study were sensitive to Amikacin, but in reverse to the results of this study they found that 100% of the isolated A. baumanni strains were sensitive to Imepenem and ciprofloxacin and 76.5% of the strains were sensitive to piperacillin/tazobactam Carbapenems are used for treating serious infections caused by multidrug-resistant Gram-negative bacilli.Resistance to carbapenems is due to decreased outer membrane permeability, increased efflux systems, alteration of penicillin-binding proteins, and the production of carbapenem hydrolyzing enzymes, that is, carbapenemases.The resistance may also be due to the production of metallo-β-lactamases (MBL); such resistance can be chromosomally encoded or plasmid mediated (Gladstone et al., 2005) The results of the present study showed that 26 (26.26%) A. baumanii strains out of 99 strains were resistant to at least one or both carbapenem tested.Out of these 26 carpabenem resistant strains, 22 (84.61%) were resistant to both imipenem and meropenem, 3 (11.53%) to meropenem only and one strain (3.84%) to imipenem alone.In various studies across the world, varying rates of resistance (4-60%) have been reported for imipenem and meropenem.Among the Indian workers, Gladstone et al. (2005) reported 14.2%, whereas Taneja et al. (2003) reported 36.4%.
As regard the EDTA-imipenem-microbiological assay, which differentiates metalloenzymes from non-metalloenzymes, in A. baumanii strains, by this assay, we could confirm 9 isolates (9.3%) as MBL producers and 15 (32.56%) as non-metalloenzyme producers among the 26 screen test positives.However, of the 9 MBL positives only 4 (15.38%)showed presence of bla-VIM and 2 showed presence of bla-IMP (7.69%) and none of the 15 non-metalloenzyme producers showed presence of either bla-IMP or bla-VIM.Karthika et al. (2009) Singh et al. (2009)."False" positivity of disc potentiation test method (three isolates) against the PCR, could be due to presence of either other uncommon MBL encoding gene like SIM, SPM, GIM, AIM or variants of IMP and VIM (Singh et al., 2009).Very high MBL positivity of 95.2 and 88% in P. aeruginosa using disc potentiation test method has been reported by Singh et al. (2009) and Jain et al. (2011), respectively. However, Quinones-Falconi et al. (2009) using PCR as a gold standard reported only 3.5% MBL positivity in P. aeruginosa using disc potentiation test method.Galicia et al. (2009) have reported 3.4% MBL positivity in P. aeruginosa using disc potentiation test method with excellent specificity but poor sensitivity of the test.We did not come across any study on EIM in A. baumannii

Conclusion
Rapid dissemination of carbapenem-resistant isolates in diabetic foot ulcers is worrisome and calls for judicious use of antibiotics .The.bla VIM and bla IMP genes have a role in carbapenem resistance in the community.More studies are needed to differentiate MBL from nonmetalloenzymes producers.

Table 1 .
Demographic and clinical characteristics of patients with diabetic foot infections.

Table 2 .
Antibiotic sensitivity pattern of A. baumanni strains isolated from diabetic foot ulcers.
12 ± 10.2 days.Table1shows demographic and clinical characteristics of patients of the study.
who found bla-VIM MBL gene only in 7 (16.28%) of the 43 screen test positive isolates reported bla-IMP-1 in 42% of A. baumannii.while Amudhan et al. (2001) reported bla-VIM in 46.55% with and both bla-IMP and bla-VIM in only one isolate of A. baumannii.The non-demonstration of IMP and VIM genes in 20 of our 26 screen test positive isolates could be either due to presence of unidentified MBL gene, limitation of the primer set used either with regards to picking up the variant IMP/VIM gene or because of presence of MBL genes other than IMP/VIM, presence of other enzymes [OXA like (Ambler class D) carbapenamases AmpC β-lactamases] or other mechanism of carbapenem resistance, namely; loss of porins, increase in efflux pump activity alteration in penicillin binding proteins (PBPs),