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BRIEF COMMUNICATION  
Year : 2017  |  Volume : 60  |  Issue : 4  |  Page : 546-549
Is diabetes mellitus an important risk factor for the antibiotic resistance in extraintestinal pathogenic Escherichia coli?


1 Department of Microbiology, Motilal Nehru Medical College, Allahabad, Uttar Pradesh, India
2 Department of Microbiology, Kasturba Medical College, Mangalore, Manipal University, Karnataka, India
3 Department of Medicine, Kasturba Medical College, Mangalore, Manipal University, Karnataka, India

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Date of Web Publication12-Jan-2018
 

   Abstract 


Escherichia coli is a major cause of extraintestinal infections in all age group. However, the infection becomes more severe when patients have some underlying condition such as Diabetes Mellitus. The aim of the study was to determine whether diabetic mellitus may act as an important risk factor for the E. coli to express drug resistance property. This descriptive study was carried out in a multi-specialty tertiary care hospital. One hundred and twenty-seven E. coli isolates from diabetic patients, and one hundred seventy-three isolates from nondiabetic patients were studied. Possession drug resistance genes were determined by multiplex polymerase chain reaction (PCR). Phylogenetic analysis was performed by triplex PCR. Antibiotic sensitivity testing was performed by Kirby-Bauer disk diffusion method. Among the study isolates from Diabetic patients maximum numbers were from phylogroup B2 (42.5%) and D (33%) similarly in case of nondiabetic patients B2 (29%) and D (38%) were the most common phylogroup. Presence of drug resistance genes among the diabetic and nondiabetic patient's isolates were as followed extended-spectrum beta-lactamase (70% and 70.5%) AmpC (9.5% and 14.5%) and NDM-1 ( 7% and 4.5%) and by disk diffusion methods susceptibility pattern were meropenem (94% and 94%), imipenem (92% and 92%), amikacin (76% and 74%), and ampicillin/sulbactam (68% and 69%), respectively. The proportion of diabetic patients strains with the drug resistance characteristics were not significantly different from that seen in nondiabetic patients strains, which indicating that in a predisposed host additional or subtraction bacterial aids for drug resistance property are not a necessity.

Keywords: Diabetes mellitus, drug resistance, Escherichia coli, phylogroup

How to cite this article:
Chakraborty A, Shenoy S, Adhikari P, Saralaya V, Rao S. Is diabetes mellitus an important risk factor for the antibiotic resistance in extraintestinal pathogenic Escherichia coli?. Indian J Pathol Microbiol 2017;60:546-9

How to cite this URL:
Chakraborty A, Shenoy S, Adhikari P, Saralaya V, Rao S. Is diabetes mellitus an important risk factor for the antibiotic resistance in extraintestinal pathogenic Escherichia coli?. Indian J Pathol Microbiol [serial online] 2017 [cited 2019 Jun 26];60:546-9. Available from: http://www.ijpmonline.org/text.asp?2017/60/4/546/222966





   Introduction Top


Diabetes mellitus (DM) is a clinical syndrome associated with deficiency of insulin secretion or action. In addition to classical complications of the disease, DM has also associated with the reduced response of T cells, and neutrophil function [1],[2] due to altered defense mechanisms Individuals with diabetes might be at higher risk for moderate or severe infection-related morbidity. Some of the common infections in diabetic people are foot infections, urinary tract infection, gangrenous cholecystitis, soft tissue infection.[3] Bacteremia can accompany infection at any of these sites. The treatment of such infections is increasingly becoming difficult because of the multidrug resistance (MDR) exhibited by the organism. As MDR has spread, we are already coming across infections that cannot be cured with any available antibiotics. Among the bacteria, the most common etiological agents found to be Escherichia coli followed by Klebsiella species, Proteus species, Pseudomonas species, Enterobacter species, and Gram-positive organisms include Staphylococcus aureus, group B Streptococcus, Enterococcus species.[4],[5] Several studies have reported that DM is one the vital risk factor for the MDR property of the isolates,[6],[7] similarly some comparison studies in between DM and non-DM patients isolates based of phenotypic test has failed to demonstrate the same.[8],[9] As E. coli is the most predominant organism causing infection in diabetic patients, in the present study, we want to assess whether DM may act as an important risk factor for the acquisitions of drug resistance property by phenotypic as well as genotypic techniques.


   Subjects and Methods Top


Participants and clinical isolates

A total of 127 consecutive type 2 diabetic subjects with extraintestinal E. coli infections (UTI, Sepsis, Wound, Pneumonia) were studied from August 2010 to August 2013. The diagnosis of diabetes was made based on the WHO criteria.[10] As a control, one hundred seventy-three extraintestinal pathogenic E. coli (ExPEC) isolates from patients without DM were also studied. Subjects who received antimicrobial drugs during the past 1 month, pregnant women, and renal failure were excluded from the study. The study was approved by institutional ethical committee (IEC/KMC/134/2010).

Identification of the organism

Isolates were identified based on colony morphology on blood agar, MacConkey's agar 4–5 suspected colonies from each bacterial plate were picked, cultured, and then identified by the various biochemical tests. Biochemical tests were performed to confirm E. coli using Gram-staining, catalase test, indole, methyl red, Voges–Proskauer test, nitrate reduction, urease production, Simmon citrate agar, and various sugar fermentation tests.[11]

DNA extraction

Bacteria were harvested from tryptone soy agar, suspended in 250 μl of sterile water, incubated at 100°C for 5 min to release the DNA, and centrifuged.[12] The supernatant was used in the polymerase chain reaction (PCR) as described below.

Phylotyping analysis

Phylogenetic analysis was performed by triplex PCR based methods as described by Clermont et al.[13] Briefly, a combination of two genes (chuA and yjaA) and an anonymous DNA fragment (TSPE4.C2), (Primers obtained from Sigma Aldrich Pvt. Ltd., India). Allows the determination of the main phylogenetic groups of E. coli (these being A, B1, B2, and D).

Genotypic detection of extended-spectrum beta-lactamase encoding genes

A multiplex PCR assay was performed to detect and differentiate blaTEM, blaSHV and blaCTX-M genes. The primers were chosen from earlier published studies.[14],[15] Another PCR assay was performed to detect blaCTXM-15 variant of blaCTX-M as described earlier.[16]

Genotypic detection of plasmid-mediated AmpC β-lactamases

All isolates were tested by a multiplex PCR assay that identified six family specific AmpC genes carried on plasmids such as MOX, FOX, EBC, ACC, DHA, and CIT, using primers and conditions as described previously.[17]

Genotypic detection of blaNDM-1 gene

A PCR assay was performed to detect blaNDM-1 gene as per primer and conditions described earlier with minor modification.[18]

Antimicrobial susceptibility testing

Antibiotic susceptibility testing was done by the modified Kirby-Bauer disk diffusion method in accordance with the Clinical and Laboratory Standards Institute guidelines.[19] The antibiotic disks (HiMedia, Mumbai, India) used were ampicillin (10 μg), piperacillin (10 μg), piperacillin/tazobactam (100/10 μg), ceftriaxone (30 μg), cefotaxime (30 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), amikacin (30 μg), gentamicin (10 μg), cotrimoxazole (1.25/23.75 μg), cefoperazone + sulbactam (75/30 μg), imepenem (ipm; 10 μg), meropenem (mrp; 10 μg).

Statistical analysis

The analysis was performed using statistical package SPSS version 17.0 (IBM, USA).


   Results Top


One hundred and twenty-seven ExPEC isolates from diabetic patients and one hundred seventy-three isolates from nondiabetic patients were studied. Phylogenetic analysis of isolates carried out by triplex PCR indicated that maximum number of the isolates were from phylogroup B2 (54/127) and D (42/127), in case of the isolates from nondiabetic patients B2 (50/173) and D (66/173) were the most predominant phylogroup in compare to A and B1.

On investigating the presence of drug resistance genes (blaCTXM, blaTEM, blaSHV, blaCTXM15, blaNDM-1 and pAmpC) among the study group isolates there was no substantial differences were observed [Table 1].
Table 1: Phylogeny and the presence of drug resistance genes among diabetic and nondiabetic patients Escherichia coli isolates

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Results of Kirby-Bauer disk diffusion methods indicated that, of the diabetic and nondiabetic study group isolates, (94% and 94%) were fully susceptible to meropenem and similarly, 92% and 92%, 76% and 74% and 68% and 69% isolates were susceptible to imipenem, amikacin, and ampicillin/sulbactam, respectively. Resistance pattern of the antibiotics was summarized in [Figure 1].
Figure 1: Antimicrobial sensitivity pattern (in percentage) to different classes of antibiotics among the diabetic and nondiabetic patients Escherichia coli isolates

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   Discussion Top


In this study, we have tried to determine whether there are differences in the phylogroup and the antibiotic sensitivity patterns of the ExPEC isolates concerned with diabetic and nondiabetic patients. The study was carried out on a large series of adult diabetic and nondiabetic patients admitted in a tertiary care hospital.

ExPEC which routinely cause infections have been shown to belong to phylogroups B2 and D. Results of our study indicated that approximately three in four isolates belonged to phylogenetic group B2 and D which is in agreement with previous findings.[20],[21] A comparison of diabetic and nondiabetic patient's isolates in relation to their phylogroups we found B2 group isolates was higher in diabetic patients.

The results of our study have also shown that around 70% of both the study group isolates were harboring extended-spectrum beta-lactamase (ESBL) genes. On analysis of the prevalence of different ESBL genes (blaTEM, blaSHV and blaCTX) we found blaCTX-M is the most common in both the study group isolates and blaCTXM15 is most predominant subtype among them. Several other investigators were also reported high prevalence of ESBL producing isolates from diabetic as well as nondiabetic patients.[22],[23]

In recent years, the incidence of pAmpC positive E. coli has been reported from various part of the country. Almost all types of pAmpC (CIT, DHA, MOX, ACC, and CMY) are common in India.[24],[25] In our study, we found that only the CIT family of pAmpC was present in the isolates; however, it was not statistically significant among the study group isolates.

While comparing the presence of blaNDM-1 genes among the study group isolates we did not find any noteworthy differences. Although very few isolates were blaNDM-1 positive, it is alarming as such infections may result in mortality or chronic persistence leading to repeated hospitalization.

The rapid increase in the rate of antibiotic resistance is a major cause of concern. In our study group isolates, we observed a high degree of resistance pattern to commonly used antibiotics such as ampicillin, piperacillin, ciprofloxacin, and norfloxacin. We also observed that approximately one in three isolates were resistant to piperacillin/tazobactam, cefoperazone/sulbactam and ampicillin/sulbactam which are quite alarming. Several investigators from India also reported high resistance rate of commonly uses antibiotic among their study isolates.[23],[26]

We also observed a high degree of antibiotic resistance in that approximately 2 of 3 strains of the study group isolates showed a resistance profile to >3 groups of antibiotics, which indicated a high degree of MDR. None of the isolates were 100% susceptible to any of the antimicrobials tested.

Several investigators have reported high level of antibiotic resistance among the DM patients isolates [6],[7] nevertheless in our study we have found DM, as well as non-DM patients isolates, were equally showing a high degree of resistance pattern which is in concurrence with the studies done by Meiland et al.[8] and Bonadio et al.[9]


   Conclusions Top


Our study found that both the study group isolates B2 and D were most predominant phylotype and for the acquisitions of drug resistance property predisposing factor such as DM were not an essential risk factor for E. coli as both the study group isolates were equally hauling the drug-resistant genes. However, high blood sugar level may help these isolates to rapid multiplication and can establish more severe form of the infection.

Acknowledgment

We are grateful to Manipal University, Manipal, India and Association of Physicians, Karnataka, for providing infrastructure and financial support respectively, to conduct the study. We would like to thanks Ms. Lotte Jakobsen MSc (biology), PhD Statens Serum Institut Microbiology and Infection Control 5 Artillerivej, build 46/202 DK-2300 Copenhagen S for providing us the positive control isolates for the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Dr. Arindam Chakraborty
Department of Microbiology, Motilal Nehru Medical College, Allahabad - 211 002, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_261_16

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