Microbiology Section DOI : 10.7860/JCDR/2018/34462.11526
Year : 2018 | Month : May | Volume : 12 | Issue : 05 Page : DC12 - DC16

Changing Antibiogram Profile of Acinetobacter baumannii in Diabetic and Non-Diabetic Foot Ulcer Infections

Diwan Mahmood Khan1, MS Moosabba2, I Venkatakrishna Rao3

1 PhD Scholar, Department of Microbiology/General Surgery, Yenepoya Medical College Hospital, Mangalore, Karnataka, India.
2 Professor and Head, Department of General Surgery, Yenepoya Medical College Hospital, Mangalore, Karnataka, India.
3 Associate Professor, Department of Microbiology, Yenepoya Medical College Hospital, Mangalore, Karnataka, India.


NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR: Dr. I Venkatakrishna Rao, Associate Professor, Department of Microbiology, Yenepoya Medical College Hospital, Deralakatte, Dakshin Kannada, Mangalore-575018, Karnataka, India.
E-mail: veekayraoi656@gmail.com
Abstract

Introduction

Foot ulcer infection relies on the hosts immune status and pathophysiological condition. The differences between Diabetic Foot Ulcer (DFU) infections and non-DFU patients may alter the biofilm-forming capabilities of microorganism and thereby, play a key role in the regulation of ulcer healing. Extended Spectrum of β-Lactamase (ESBL) and Metallo-β-Lactamase (MBL) producing Acinetobacter baumannii isolates are reported as important causative agents of infection.

Aim

To determine the antimicrobial susceptibility, ESBL, MBL production, and biofilm formation in A. baumannii among diabetic and non-DFU patients.

Materials and Methods

A hospital-based prospective study was conducted from September 2016 to June 2017. Samples were collected from general surgery outpatients and inpatients suffering from foot ulcer infections and also include Wagner Grade II to V. Pus/tissue was collected and processed for standard methods of culture, antimicrobial susceptibility, biofilm formation assay, ESBL, MBL, Modified Hodge Test (MHT) and Minimum Inhibitory Concentration (MIC).

Results

A total of 70 bacterial isolates were obtained from 400 patients with DFU and non-DFU from pus and tissue specimens. Antibiogram profiles of DFU isolates were sensitive to colistin and resistant to all the major groups of antibiotics classes. Non-DFU isolates were sensitive to amikacin, ceftriaxone, gentamicin piperacillin/tazobactam, imipenem, meropenem, colistin and resistant to ceftazidime, cefotaxime, ciprofloxacin, co-trimoxazole, piperacillin and tetracycline. The results showed that out of 35 DFU isolates 13 (37.14%) produced ESBL, 10 (28.57%) produced MBL and 9 (25.71%) formed a strong biofilm. Further in 35 non-DFU isolates 8 (22.85%) produced ESBL, 5 (14.28%) produced MBL and 3 (8.57%) formed a strong biofilm. Almost all the isolates of Multi-Drug Resistance (MDR) are ESBL and MBL producer as well as biofilm formers.

Conclusion

Colistin is the drug of choice for the efficient treatment of multi-drug resistant isolates of foot ulcer patients. The rapid spreading of ESBL, MBL producers, and MDR require the implementation of not just surveillance study but also a proper and rational selection of antibiotics, especially for MDR for better clinical outcomes.

How to cite this article

Diwan Mahmood Khan, MS Moosabba, I Venkatakrishna Rao, .changing antibiogram profile of acinetobacter baumannii in diabetic and non-diabetic foot ulcer infections.Journal of Clinical and Diagnostic Research [serial online] 2018 May[cited:2019 Sep 22] 05 DC12 - DC16
 Available from
http://www.jcdr.net//back_issues.asp?issn=0973-709x&year=2018&month=May&volume=12&issue=05&page=DC12 - DC16&id=11526

Introduction

Diabetes mellitus is one of the major threats in the public health [1]. Almost 25% of diabetic patients develop foot ulcer infections during their lifespan due to poor glycolic control and low immune status. According to the International Diabetes Federation (IDF), approximately 69.2 million people have been affected nationwide and 415 million people worldwide are having diabetes [2]. DFU patients have 10 times more risk factors of infection than non-DFU patients and risk of infections are more common in male patients compared to female patients [3,4]. In non-DFU patients, once infection has developed, due to peripheral arterial disease and peripheral neuropathy of lower limb, it limits other secondary infections. However, it may lead to foot condition such as callosities, fissure, and trauma by a delay in treatment [5]. Foot ulcer infections include abscess, necrotizing fasciitis, gangrene, septic arthritis, tendonitis, cellulitis, and osteomyelitis [6].

Many studies have been reported along the DFU patients who had been commonly encountered with predominant pathogens such as P. aeruginosa, E. coli, Staphylococcus spp., Klebsiella spp., Acinetobacter spp. and Proteus spp. Only few of them reported DFU and non-DFU patients with A. baumannii infections with threatening drug-resistant and limb amputation [7].

A. baumannii is the second most common isolated microorganism from non-fermented bacteria in foot ulcer infections. Although, Acinetobacter wound infection showed an isolation rate of 11.1%, it poses a problem to clinician whether to treat it aggressively as a pathogen or as a coloniser [8]. A. baumannii is regarded as a rare coloniser whose prevalence is 0.5% on human skin in temperate climates where it is commonly found in humid climates [9,10]. It is an opportunistic pathogen affecting patients with severe illnesses like immunodeficiency, diabetes prolong hospital stay due to a risk of developing a hospital acquired infection and prolonged exposure to antimicrobial agents [11,12].

Many factors lead to the emergence and survival of MDR A. baumannii in diabetic and non-DFU patients. Most common among them is biofilm production, that may lead to ineffective penetration of antibiotics due to the recruitment of leukocytes into the foot ulcers. Bacteria in the biofilm matrices possess antiphagocytic properties which can facilitate and alter the gene expression of intercellular communications [13]. Hence, peripheral arterial disease and neuropathy are often present in patients with foot ulcer infection and may contribute to poor penetration of antibiotics into the lower limb tissues which promotes resistance to bacteria [14]. More than 90% isolates of A. baumannii are resistant to all beta-lactams, fluoroquinolones, and aminoglycosides antibiotics. Several studies have reported the prevalence of ESBL and MBL producers which may promote the multi-drug resistance [7,15]. Colistin was the drug of choice for efficient handling of the non-healing ulcer with A. baumannii infections [16]. This study was conducted in order to understand the severity of the MDR and biofilm formation by A. baumannii in DFU compared to the non-DFU patients, which may lead to the development of secondary infections and non-healing ulcer and lead to amputation of lower limb extremities. This study was aimed to recognise the differences between the isolates among diabetic and non-DFUs. The present investigation was done in order to determine the prevalence, antibiotic susceptibility pattern, ESBL, MBL and biofilm formation of A. baumannii isolates in diabetic and non-DFU patients.

Materials and Methods

A hospital-based, prospective study was conducted over a period of 10 months from September 2016 to June 2017 at a Tertiary Care Hospital, Mangalore, Karnataka, India, after obtaining Institutional Ethics Committee approval (Reg. No- YU2016/172). Informed consent was obtained from all the participants.

Study design and sample collection: Samples (pus and exudates, tissues) were collected from patients who presented with foot ulcer infections. A total of 70 non-duplicate isolates were obtained from 400 patients screened. Thirty five isolates of A. baumannii were taken in each, among DFU and non-DFU patients. Inclusion criteria for DFU group was patient having DFU infection with age >18 years. Duplicate sample of the same patient were excluded. Inclusion criteria for non-DFU group included patient having ulcer with peripheral arterial disease, neuropathy and age >18 years. Patients with history of prior treatment and Meggitt Wagner classification system grading of foot ulcer: 0–I were excluded [17].

Bacteria Isolation and Identification

The collected pus/tissues were processed for Gram staining and cultured on 5% Sheep blood agar and MacConkey agar (Hi-Media Laboratories, India) for aerobic culture and incubated at 37°C overnight. Tissues were homogenised before inoculation and all isolates were confirmed using the BD Phoenix 100 system (Becton Dickinson, USA).

Antimicrobial Susceptibility Testing (AST)

AST was carried out by Kirby-Bauer method on Mueller Hinton Agar (MHA) and the results were interpreted according to CLSI 2016 guidelines [18]. Antibiotic disks tested were amikacin (30 μg), imipenem (10 μg), meropenem (10 μg), piperacillin (100 μg), piperacillin/tazobactam (110 μg), levofloxacin (5 μg), ciprofloxacin (5 μg), co-trimoxazole (Trimethoprim/sulfamethoxazole) (1.25/23.75 μg), ceftriaxone (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefepime (30 μg), gentamicin (10 μg), tobramycin (10 μg) and tetracycline (30 μg) (Hi-Media Laboratories, India). For control, E. coli (ATCC® 25922), E. coli (ATCC® 35218) and P. aeruginosa (ATCC® 27853) were used.

Minimum Inhibitory Concentration (MIC)

The MIC of colistin was determined for all the MDR A. baumannii isolates by using the E-test strips and agar methods according to the manufacturer’s instructions. The MIC breakpoints of ≤2 μg/L were regarded as susceptible and ≥4 μg/L as resistant [18,19].

Extended Spectrum of β-Lactamase (ESBL)

Detection of the ESBL phenotype was performed by combined disk diffusion method on MHA plate using ceftazidime (30 μg), ceftazidime/clavulanic acid (30/10 μg) [18]. E. coli (ATCC® 25922) were used as the negative control and an in-house ESBL producing A. baumannii (ATCC® 19606) isolate was used as the positive control. The test was considered positive when an increase in the diameter of the zone of inhibition was ≥5 mm around ceftazidime/clavulanic acid against ceftazidime alone [19-21].

Metallo-β-Lactamase (MBL)

Detection of MBL was done by using Imipenem-EDTA combined disk test. The overnight broth cultures of test isolates along with standard control strains (opacity adjusted to 0.5 McFarland) was lawn cultured on MHA plate. After drying, 10 μg imipenem disks were placed on the lawn culture with 20 mm distance from the centre to centre of the disks. Another 10 μg imipenem impregnated with 750 μg of disodium EDTA was added to one of the imipenem disks and incubated overnight [18]. Isolates which showed ≥7 mm more inhibition zone (size) of imipenem – EDTA disk than compared to the imipenem disk alone, were considered as MBL producers. Positive control used was P. aeruginosa (ATCC® 27853) [22,23].

Modified Hodge Test (MHT)

Modified Hodge test was performed in all isolates. It is a screening test which helps in detection of carbapenemases. E. coli (ATCC® 25922) an indicator organism sensitive to carbapenems was cultured in peptone water to achieve 0.5 McFarland opacity standard and lawn cultured onto an MHA plate using a sterile cotton swab [18]. After drying, 10 μg imipenem disk was placed at the centre of the plate on the lawn culture, and an overnight growth of the test strain was heavily streaked from the edge of the imipenem disk outwards, to the periphery of the plate in one direction. After streaking positive and negative control from the edge of the disk outwards to the periphery of the plates, they were incubated at 37°C overnight and the presence of a distorted zone - clover-leaf shaped zone of inhibition was considered as a positive test [24,25].

Biofilm Formation Assay

Biofilm forming ability of all the isolates was performed as mentioned previously with certain modification [26]. Briefly, bacterial cells were grown overnight at 37°C in 5 mL Trypticase-Soy Broth (TSB) in a test tube. A 96-well flat bottomed polystyrene tissue culture plate was added with 200 μL of sterile TSB, inoculated with 10 μL of overnight culture and incubated at 37°C for 24 hours. After incubation, layer from each well was removed and washed carefully three times, with 200 μL of phosphate buffered saline (pH-7.2) in order to remove free-floating bacteria. Adherent bacteria were added with 200 μL of 99% methanol for 15 minutes. The plates were decanted, dried and stained for 7 minutes with 200 μL of 0.1% Hucker crystal violet. Excess stain was rinsed off with tap water. The plates were air dried and dye bound to the adherent cells was solubilised with 160 μL of 33% (v/v) glacial acetic acid per well. The Optical Density (OD) of each well was measured at 630 nm using ELISA reader (FLUOstar Omega, BMG LABTECH, Germany). The biofilm producing strain A. baumannii (ATCC® 19606) and P. aeruginosa (ATCC® 27853) were taken for Positive Control (PC). Wells inoculated with sterile broth were used as Negative Control (NC).

Statistical Analysis

Statistical analysis was performed using SPSS software version 23.0 (SPSS Inc., Chicago, IL). The chi-square test and Fisher’s-exact two-tailed test analysis were done in this study. Statistical significance was regarded as a p-value < 0.05.

Results

Out of the 70 isolates of A. baumannii obtained from 400-foot ulcer patients (17.50%), 35 (15.90%) isolates (35/220) were recovered from DFU and 35 (19.44%) isolates (35/180) from non-DFU patients. The mean age group of the patients was 56.57+10.34 years. Most of the isolates were recovered from the foot ulcer patients who were 44 to 60 years old [Table/Fig-1] and Wagner grading II to V [Table/Fig-2]. Infections on males were predominant 77.14% (27/35) compared to females 28.86% (8/35) among DFU and non-DFU patients. In present study, DFU patients were suffering from associated diseases such as hypertension, neuropathy, retinopathy, and nephropathy. Few patients also presented with ischaemic heart diseases. In non-DFU patients below the age of 30, had history of smoking/alcohol or drinking and above the age of 30, in addition to this had hypertension.

Distribution of age group and sex affected with DFU and NDFU in Acinetobacter baumannii infections.

Age (years)DFU (35/220=15.90%)NDFU (35/180=19.44%)
MaleFemaleMaleFemale
18-302153
31-433181
44-568471
≥5714273
Total278278

DFU: Diabetic foot ulcer; NDFU: Non-diabetic foot ulcer


Isolate distribution in different grades of ulcer in DFU and non-DFU patients.

Antimicrobial susceptibility pattern of A. baumannii among DFU and non-DFU patients is depicted in [Table/Fig-3]. All the DFU patients isolates of A. baumannii were resistant to major groups of antibiotics in comparison to non-DFU. DFU were sensitive to 100% colistin and resistant to ceftazidime (97.14%), cefotaxime (94.29%), 91.43% (cefepime, co-trimoxazole, and tetracycline), 88.57% (amikacin, ceftriaxone, ciprofloxacin, piperacillin), gentamicin (85.71%), 82.56% (levofloxacin, imipenem, piperacillin/tazobactam), tobramycin (80%) and meropenem (77.14%). NDFU were sensitive to 51.43% (amikacin, cefepime, gentamicin, tobramycin, piperacillin/tazobactam), 54.29% (ceftriaxone, imipenem), levofloxacin (57.14%), meropenem (68.57%), 100% colistin and resistant to 65.71% (tetracycline, ceftazidime, ciprofloxacin), cefotaxime (60%), and 51.43% (piperacillin, co-trimoxazole).

Antimicrobial susceptibility pattern of Acinetobacter baumannii in DFU and NDFU.

Antibiotic discAcinetobacterbaumannii*SensitiveResistantp-value*
AMKDFU4 (11.43%)31 (88.57%)0.00062
NDFU18 (51.43%)17 (48.57%)
CPMDFU3 (8.57%)32 (91.43%)0.000167
NDFU18 (51.43%)17 (48.57%)
CAZDFU1 (2.86%)34 (97.14%)0.000678
NDFU12 (34.29%)23 (65.71%)
CTXDFU2 (5.71%)33 (94.29%)0.001208
NDFU14 (40.00%)21 (60.00%)
CTRDFU4 (11.43%)31 (88.57%)0.000144
NDFU19 (54.29%)16 (45.71%)
CIPDFU4 (11.43%)31 (88.57%)0.026617
NDFU12 (34.29%)23 (65.71%)
LEVDFU6 (17.14%)29 (82.56%)0.001093
NDFU20 (57.14%)15 (42.86%)
COTDFU3 (8.57%)32 (91.43%)0.000402
NDFU17 (48.57%)18 (51.43%)
GENDFU5 (14.29%)30 (85.71%)0.001907
NDFU18 (51.43%)17(48.57%)
IPMDFU6 (17.14%)29 (82.56%)0.002409
NDFU19 (54.29%)16 (45.71%)
MRPDFU8 (22.86%)27 (77.14%)0.000144
NDFU24 (68.57%)11 (31.43%)
PIPDFU4 (11.43%)31 (88.57%)0.000402
NDFU17 (48.57%)18 (51.43%)
PITDFU6 (17.14%)29 (82.56%)0.005056
NDFU18 (51.43%)17 (48.57%)
TETDFU3 (8.57%)32 (91.43%)0.010239
NDFU12 (34.29%)23 (65.71%)
TOBDFU7 (20%)28 (80%)0.011862
NDFU18 (51.43%)17 (48.57%)

Notes: *Fisher exact two-tailed test (p-value < 0.05 and significant difference between DFU and non-DFU patients for all antibiotics).

*Acinetobacter baumannii isolated from diabetic and non-diabetic foot ulcer patients (Tissue, Pus and exudates)

AMK: Amikacin; CPM: Cefepime; CAZ: Ceftazidime; CTX: Cefotaxime; CTR: Ceftriaxone; CIP: Ciprofloxacin; LEV: Levofloxacin; COT: Co-trimoxazole; GEN: Gentamicin; IPM: Imipenem; MRP: Meropenem; PIP: Piperacillin; PIT: Piperacillin/tazobactam; TET: Tetracycline; TOB: Tobramycin; DFU: Diabetic foot ulcer; NDFU: Non-diabetic foot ulcer


ESBL and MBL producing isolates among A. baumannii were 37.14% (13/35) and 28.57% (10/35). Non-DFU isolates were ESBL 22.85% (8/35), and MBL 14.28% (5/35) respectively. All isolates of DFU and non-DFU isolates which are MBL positive were confirmed by MHT. MDR isolates of A. baumannii were 62.85% (22/35) in DFU and 28.57% (10/35) in non-DFU patients [Table/Fig-4]. Biofilm formation was seen in A. baumannii from DFU and more compared to non-DFU isolates [Table/Fig-5a,b]. Based on the tissue culture plate assay, the isolates were classified as strong {Optical Density (OD) >0.350}, moderate (OD between 0.200-0.350) or weak (OD 0.041-0.200) biofilm formation in A. baumannii. A chi-square (χ2) test showed that there was a significant difference between isolates from DFU and non-DFU patients with A. baumannii infections {p-value <0.05 (0.034); χ2 value=6.71 and degree of freedom (df)=2} which correlated with ESBL, MBL production and MDR with biofilm formation [Table/Fig-6a,b]. The hospital stays of patients in whom ESBL, MBL producer, biofilm formation, and MDR were isolated ranged between 15-40 days.

Percentage of ESBL, MBL producer and MDR Acinetobacter baumannii.

Comparison of biofilm formation in Acinetobacter baumannii among DFU and non-DFU isolates.

Biofilm FormationDFU (n=35)NDFU (n=35)p-value
Weak (0.041 – 0.200)03 (8.57%)10 (28.57%)χ2=6.71, df=2, p=0.034
Moderate (≥0.200 – 0.350)23 (65.71%)22 (62.86%)
Strong (≥0.350)09 (25.72%)03 (8.57%)

Chi-square (χ2) test; Significant p-value: <0.05, df: Degree of freedom; DFU: Diabetic foot ulcer; NDFU: Non-diabetic foot ulcer


Biofilm formation in Acinetobacter baumannii in DFU and NDFU isolates.

PC: Positive control, NC: Negative control; DFU: Diabetic foot ulcer; NDFU: Non-diabetic foot ulcer

Correlation of ESBL, MBL and MDR Acinetobacter baumannii with Biofilm formation in DFU

Acinetobacter baumanniiBiofilm Formation-DFU
Strong (09/35)Medium(23/35)Weak(03/35)
ESBL08050
MBL08020
MDR091102

ESBL: Extended spectrum of β-lactamase; MBL: Metallo-β-lactamase; MDR: Multi-drug resistant; DFU: Diabetic foot ulcer


Correlation of ESBL, MBL and MDR Acinetobacter baumannii with Biofilm formation in NDFU.

Acinetobacter baumanniiBiofilm Formation-NDFU
Strong (03/35)Medium (22/35)Weak (10/35)
ESBL020501
MBL02030
MDR03070

ESBL: Extended spectrum of β-lactamase; MBL: Metallo-β-lactamase; MDR: Multi-drug resistant; NDFU: Non-diabetic foot ulcer


Discussion

Foot ulcer infection complications are frequent clinical problems among non-communicable diseases leading to hospitalisation. Most commonly foot ulcer infection seen in patients who tend to have immune deficiency, mono-microbial, and poly-microbial infections. Foot ulcer infections are predominantly poly-microbial with the ability to form changing trends of susceptibility, ESBL, MBL, MDR and biofilm, are important causative agents resulting in treatment failure and increased risk of amputation.

This study shows A. baumannii infections in 15.90% DFU and 19.44% non-DFU patient, which is slightly higher than Murali TS et al., study which reported 14% in both isolates [13]. Another similar study by Karmaker M et al., reported the prevalence of 10% Acinetobacter spp. in DFU and non-DFU infections [27]. El-Din RA et al., reported 33% of A. baumannii isolated from DFU patients [15]. Jyothylekshmy V et al., reported the prevalence of 5.3% Acinetobacter spp. in DFU patients [28]. In present study, DFU isolates were 100% sensitive to colistin and resistant to ceftazidime (97.14%), cefotaxime (94.29%), 91.43% (cefepime, co-trimoxazole, and tetracycline), 88.57% (amikacin, ceftriaxone, ciprofloxacin, piperacillin), gentamicin (85.71%), 82.56% (levofloxacin, imipenem, piperacillin/tazobactam), tobramycin (80%) and meropenem (77.14%). Mendes JJ et al., reported sensitivity to 100% colistin and resistant to all routine antibiotics in DFU isolates [16]. Shanmugam P and Jeya M, reported antibiotic resistant were 83% ceftazidime, 67% ciprofloxacin, 50% (cefepime, gentamicin, tetracycline, tobramycin), 33% (amikacin, cotrimoxazole), 17% (imipenem, piperacillin/ tazobactam) in DFU isolates [7]. Another similar study, done by Murali TS et al., [13] reported DFU isolates resistant to amikacin (76%), cefotaxime (94%), ciprofloxacin (84%), and gentamicin (90%). Acinetobacter spp. were resistant to cefotaxime (88%), gentamicin (59%), amikacin (53%), ciprofloxacin (36%), and 29% (ceftazidime, piperacillin, piperacillin/tazobactam) in DFU isolates [29]. Similarly, Turhan V et al., in their study reported Acinetobacter spp. were resistant to piperacillin/tazobactam (88%), ciprofloxacin (84%), co-trimoxazole (75%), ceftazidime (63%), amikacin (53%), imipenem (29%) [30]. Akhi MT et al., reported antibiotic resistance to 100% (tetracycline, cefepime, ceftriaxone), and 50% (gentamicin, ciprofloxacin, imipenem, piperacillin/tazobactam) [31]. Results of present study indicated a significant difference between in DFU and non-DFU patients for all isolates, p-value <0.05 [Table/Fig-3].

Non-DFU patient’s isolates were susceptible to 100% colistin, meropenem (68.57%), levofloxacin (57.14%), 54.29% (ceftriaxone, imipenem), 51.43% (amikacin, cefepime, gentamicin, tobramycin, piperacillin/tazobactam) and resistant to 65.71% (tetracycline, ceftazidime, ciprofloxacin), cefotaxime (60%), and 51.43% (piperacillin, co-trimoxazole). Murali TS et al., reported antibiotic sensitivity to non-DFU isolates were 25% (amikacin, ciprofloxacin, gentamicin) and resistance to 100% cefotaxime, and 75% (amikacin, ciprofloxacin) [13].

ESBL and MBL production in A. baumannii was reported on many studies but unfortunately, CLSI guidelines do not support or advocate. A. baumannii producing ESBL was first reported from Turkey in 2001 and India in 2007. Similarly, MBL production in A. baumannii was reported from South Korea in 2001 and India in 2011. Few studies are reported on diabetic foot ulcer infection in A. baumannii. This study shows that among DFU isolates, 37.14% (13/35) A. baumannii were ESBL producer and MBL 28.57% (10/35). Shanmugam P et al., reported 33.33% were ESBL and 16.6% MBL producers [7]. Another study by El-Din RA et al., reported 34.61% MBL producers [15]. The present study showed that, non-DFU isolates were producing ESBL 22.85% (8/35) and 14.28% (5/35) MBL. Surprisingly, none of the studies has reported ESBL and MBL production in non-DFU isolates of A. baumannii. Some studies has reported, but it is not clear whether specimens/sample should be taken from the patient of peripheral arterial and peripheral neuropathy or not. Hence, this study is first which reports ESBL and MBL production in A. baumannii among non-DFU.

Almost all MDR isolates of A. baumannii produce biofilm in DFU and non-DFU patients. Biofilm formation was categorised into strong, moderate, and weak. In this study 25.72% (9/35), 65.71% (23/35), 8.57% (03/35) prevalence was reported as strong, moderate and weak respectively. A significant difference was observed between diabetic and non-diabetic groups [Table/Fig-5a,b]. Similar to our study, Swarna SR and Gomathi S, reported 23.07% strong biofilm formation and 15.38% moderate biofilm formation which was much lower than the biofilm formation observed in present study and 38.46% weak which was higher compared to our study [32]. Murali TS et al., reported biofilm formation in DFU isolates was 39.13% [13]. Another study by Zubair M et al., reported 60% strong and 40% weak biofilm formation in DFU isolates [33]. Similarly, Vatan A et al., reported 59.25% strong and 40.75% weak biofilm formation [34]. Di Domenico EG et al., reported 8.9% strong biofilm formation in DFU isolates [35].

Biofilm formation in non-DFU patients of A. baumannii in present study showed 8.57% (03/35) strong, 62.86% (22/35) moderate, and 28.57% (10/35) weak biofilm formation. Amazingly, we found that biofilm formation in DFU isolates correlating with ESBL, MBL and MDR A baumannii were more predominant than non-DFU isolates [Table/Fig-6a,b]. Murali TS et al., reported the correlation of biofilm and MDR isolates in DFU and non-DFU patients that were more virulent compared to non-DFU isolates leading to limb amputation [13]. In present study, we found wide variation and changing trend of antimicrobial susceptibility pattern compared to other studies. DFU patients compared to non-DFU patients had significant difference of healing ulcer due to their compromised immune status. There is a need to understand the characteristics of A. baumannii in non-healing ulcer infections among DFU and non-DFU isolates.

Limitation

In the present study, the MIC of netilmicin and polymixin B was not determined as the duration of sample collection was limited. The study did not include any molecular approach to find out ESBL, MBL, and drug resistance genes.

Conclusion

This study highlights the need to establish antimicrobial susceptibility surveillance for A. baumannii to determine the appropriate empirical treatment regimen. Periodical checkup in a tertiary care hospital with the help of infection control and wound/ulcer management committee will help to inhibit the spreading of A. baumannii infections. The biofilm formation in DFU compared to non-DFU patients is the most severe threat to the non-healing ulcers. The rapid spreading of ESBL, MBL producers, and MDR require the implementation of not just surveillance study but also a proper and rational selection of antibiotics, especially for MDR which help the clinician in the treatment of diabetic and non-DFU infection patients.

DFU: Diabetic foot ulcer; NDFU: Non-diabetic foot ulcerNotes: *Fisher exact two-tailed test (p-value < 0.05 and significant difference between DFU and non-DFU patients for all antibiotics).*Acinetobacter baumannii isolated from diabetic and non-diabetic foot ulcer patients (Tissue, Pus and exudates)AMK: Amikacin; CPM: Cefepime; CAZ: Ceftazidime; CTX: Cefotaxime; CTR: Ceftriaxone; CIP: Ciprofloxacin; LEV: Levofloxacin; COT: Co-trimoxazole; GEN: Gentamicin; IPM: Imipenem; MRP: Meropenem; PIP: Piperacillin; PIT: Piperacillin/tazobactam; TET: Tetracycline; TOB: Tobramycin; DFU: Diabetic foot ulcer; NDFU: Non-diabetic foot ulcerChi-square (χ2) test; Significant p-value: <0.05, df: Degree of freedom; DFU: Diabetic foot ulcer; NDFU: Non-diabetic foot ulcerESBL: Extended spectrum of β-lactamase; MBL: Metallo-β-lactamase; MDR: Multi-drug resistant; DFU: Diabetic foot ulcerESBL: Extended spectrum of β-lactamase; MBL: Metallo-β-lactamase; MDR: Multi-drug resistant; NDFU: Non-diabetic foot ulcer

References

[1]Esmat MM, Al Islam A, Diabetic foot infection: Bacteriological causes and antimicrobial therapy Am J Sci 2012 8(10):389-93.  [Google Scholar]

[2]Atlas ID, International Diabetes Federation 2015 7th Edition:9-17.Available from: https://www.idf.org/e-library/epidemiology-research/diabetes-atlas/13-diabetes-atlas-seventh-edition.html  [Google Scholar]

[3]Shakil S, Khan AU, Infected foot ulcers in male and female diabetic patients: a clinico-bioinformative study Ann Clin Microbiol Antimicrob 2010 9(1):2  [Google Scholar]

[4]Chadwick P, Edmonds M, McCardle J, Armstrong D, Best practice guidelines: Wound management in diabetic foot ulcers Wounds Int 2013   [Google Scholar]

[5]Zaine NH, Hitos K, Vicaretti M, Fletcher JP, Begg L, Burns J, Characteristics of non-diabetic foot ulcers in Western Sydney, Australia J Foot Ankle Res 2016 9(1):6  [Google Scholar]

[6]Lipsky BA, Berendt AR, Cornia PB, Pile JC, Peters EJ, Armstrong DG, Infectious diseases society of Aamerica clinical practice guideline for the diagnosis and treatment of diabetic foot infections Clin Infect Dis 2012 54(12):e132-73.  [Google Scholar]

[7]Shanmugam P, Jeya M, The bacteriology of diabetic foot ulcers, with a special reference to multidrug resistant strains J Clin Diagn Res 2013 7(3):441-45.  [Google Scholar]

[8]Lahiri KK, Mani NS, Purai SS, Acinetobacter spp as nosocomial pathogen: Clinical significance and antimicrobial sensitivity Med J Armed Forces India 2004 60(1):07-10.  [Google Scholar]

[9]Bali NK, Fomda BA, Bashir H, Zahoor D, Lone S, Koul PA, Emergence of carbapenem-resistant Acinetobacter in a temperate north Indian State Br J Biomed Sci 2013 70(4):156-60.  [Google Scholar]

[10]Seifert H, Dijkshoorn L, Gerner-Smidt P, Pelzer N, Tjernberg I, Vaneechoutte M, Distribution of Acinetobacter species on human skin: comparison of phenotypic and genotypic identification methods J Clin Microbiol 1997 35(11):2819-25.  [Google Scholar]

[11]Bergogne-Berezin E, Towner KJ, Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features Clin Microbiol Rev 1996 9(2):148  [Google Scholar]

[12]Peleg AY, Seifert H, Paterson DL, Acinetobacter baumannii: emergence of a successful pathogen Clin Microbiol Rev 2008 21(3):538-82.  [Google Scholar]

[13]Murali TS, Kavitha S, Spoorthi J, Bhat DV, Prasad AS, Upton Z, Characteristics of microbial drug resistance and its correlates in chronic diabetic foot ulcer infections J Med Microbiol 2014 63(10):1377-85.  [Google Scholar]

[14]Hartemann-Heurtier A, Robert J, Jacqueminet S, Ha Van G, Golmard JL, Jarlier V, Diabetic foot ulcer and multidrug-resistant organisms: risk factors and impact Diabetic Medicine 2004 21(7):710-15.  [Google Scholar]

[15]El-Din RA, El-Bassat H, El-Bedewy M, El-Mohamady H, Prevalence of metallo-lactamases producers among carbapenem-resistant Acinetobacter baumannii strains isolated from diabetic foot ulcers Afr J Microbiol Res 2014 8(15):1580-85.  [Google Scholar]

[16]Mendes JJ, Marques-Costa A, Vilela C, Neves J, Candeias N, Cavaco-Silva P, Clinical and bacteriological survey of diabetic foot infections in Lisbon Diabetes Res Clin Pract 2012 95(1):153-61.  [Google Scholar]

[17]Oyibo SO, Jude EB, Tarawneh I, Nguyen HC, Harkless LB, Boulton AJ, A comparison of two diabetic foot ulcer classification systems: the Wagner and the University of Texas wound classification systems Diabetes Care 2001 24(1):84-88.  [Google Scholar]

[18]Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 26th edition CLSI supplements M100S. 2016  [Google Scholar]

[19]Litake GM, Ghole VS, Niphadkar KB, Joshi SG, Phenotypic ESBL detection in Acinetobacter baumannii: a real challenge Am J Infect Dis 2015 11(3):48  [Google Scholar]

[20]Safari M, Nejad AS, Bahador A, Jafari R, Alikhani MY, Prevalence of ESBL and MBL encoding genes in Acinetobacter baumannii isolated from patients of intensive care units (ICU) Saudi J Biol Sci 2015 22(4):424-29.  [Google Scholar]

[21]Sinha M, Srinivasa H, Macaden R, Antibiotic resistance profile & extended spectrum beta-lactamase (ESBL) production in Acinetobacter species Indian J Med Res 2007 126(1):63  [Google Scholar]

[22]Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y, Imipenem-EDTA disk method for differentiation of metallo–β–lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp J Clin Microbiol 2002 40(10):3798-801.  [Google Scholar]

[23]Shivaprasad A, Antony B, Shenoy P, Comparative evaluation of four phenotypic tests for detection of metallo–β–lactamase and carbapenemase production in Acinetobacter baumannii J Clin Diagn Res 2014 8(5):DC05-08.  [Google Scholar]

[24]Lee K, Chong Y, Shin HB, Kim YA, Yong D, Yum JH, Modified Hodge and EDTA–disk synergy tests to screen metallo–β–lactamase-producing strains of Pseudomonas and Acinetobacter species Clin Microbiol Infect 2001 7(2):88-91.  [Google Scholar]

[25]Amudhan SM, Sekar U, Arunagiri K, Sekar B, OXA beta-lactamase-mediated carbapenem resistance in Acinetobacter baumannii Indian J Med Microbiol 2011 29(3):269  [Google Scholar]

[26]Babapour E, Haddadi A, Mirnejad R, Angaji SA, Amirmozafari N, Biofilm formation in clinical isolates of nosocomial Acinetobacter baumannii and its relationship with multidrug resistance Asian Pac J Trop Biomed 2016 6(6):528-33.  [Google Scholar]

[27]Karmaker M, Sanyal SK, Sultana M, Hossain MA, Association of bacteria in diabetic and non-diabetic foot infection–An investigation in patients from Bangladesh J Infect Public Health 2016 9(3):267-77.  [Google Scholar]

[28]Jyothylekshmy V, Menon AS, Abraham S, Epidemiology of diabetic foot complications in a podiatry clinic of a tertiary hospital in South India KLE Univ Health Sci J 2015 8(1):48  [Google Scholar]

[29]Banashankari GS, Rudresh HK, Harsha AH, Prevalence of gram negative bacteria in diabetic foot-a clinico-microbiological study Al Ameen J Med Sci 2012 5(3):224  [Google Scholar]

[30]Turhan V, Mutluoglu M, Acar A, Hatipoglu M, Onem Y, Uzun G, Increasing incidence of Gram-negative organisms in bacterial agents isolated from diabetic foot ulcers J Infect Dev Ctries 2013 7(10):707-12.  [Google Scholar]

[31]Akhi MT, Ghotaslou R, Asgharzadeh M, Varshochi M, Pirzadeh T, Memar MY, Bacterial etiology and antibiotic susceptibility pattern of diabetic foot infections in Tabriz, Iran GMS Hyg Infect Control 2015 :10  [Google Scholar]

[32]Swarna SR, Gomathi S, Biofilm production in carbapenem resistant isolates from chronic wound infections Int J Res Med Sci 2017 6(2):61-67.  [Google Scholar]

[33]Zubair M, Malik A, Ahmad J, Rizvi M, Farooqui KJ, Rizvi MW, A study of biofilm production by gram negative organisms isolated from diabetic foot ulcer patients Biology and Medicine 2011 3(2):147-57.  [Google Scholar]

[34]Vatan A, Saltoglu N, Yemisen M, Balkan II, Surme S, Demiray T, Association between biofilm and multi/extensive drug resistance in diabetic foot infection Int J Clin Pract Suppl 2018 doi: 10.1111/ijcp.13060. [Epub ahead of print]  [Google Scholar]

[35]Di Domenico EG, Farulla I, Prignano G, Gallo MT, Vespaziani M, Cavallo I, Biofilm is a major virulence determinant in bacterial colonization of chronic skin ulcers independently from the multidrug resistant phenotype Int J Mol Sci 2017 18(5):1077  [Google Scholar]