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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 4  |  Issue : 4  |  Page : 248-252

Virulence property, phylogenetic background, and resistance pattern of Escherichia coli isolates from wound infections


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

Date of Web Publication11-Oct-2017

Correspondence Address:
Arindam Chakraborty
Moti Lal Nehru Medical College, Allahabad - 211 002, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cjhr.cjhr_48_17

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  Abstract 

Aims: The aim of the present study was to characterize the E. coli isolates from surgical wounds, traumatic wounds, and from foot ulcers on the basis of virulence and drug resistance. Subjects and Methods: A total of forty E. coli strains isolated from wound infections were studied. Phylogenetic background, virulence factors (VFs), and antibiotic resistance profiles were determined by phenotypic and genotypic methods. Correlation between phylogenetic groups, VFs, and drug resistance pattern were analyzed. Results: Analysis of virulence gene possession among the isolates indicated that a maximum number were carrying the fimH (39 strains; 97.5%) gene, followed by iutA (27; 67.5%), papC (16; 4%), hlyA (5; 12.5%), cnf1 ( 5; 12.5%), and neuC (1; 2.5%), respectively. The phylogroups B2 (32.5%) and D (42.5%) were more common. Thirty isolates (75%) were found to be positive for extended-spectrum β-lactamase genes. CIT type of plasmid-mediated AmpC was seen only in 6 (15%) isolates. Most effective antibiotics were carbapenem and sulfamethoxazole-trimethoprim groups of drugs. Conclusions: Our findings indicate that adherence factors and iron uptake systems are two of the more important CFs expressed by such isolates, and such strains are also observed to exhibit a higher degree of drug resistance. Carbapenems and co-trimoxazole may be considered as reliable and successful alternative medications for these isolates.

Keywords: Drug resistance, Escherichia coli, phylogroup, polymerase chain reaction, virulence factor, wound infection


How to cite this article:
Chakraborty A, Saralaya V, Adhikari P, Shenoy S. Virulence property, phylogenetic background, and resistance pattern of Escherichia coli isolates from wound infections. CHRISMED J Health Res 2017;4:248-52

How to cite this URL:
Chakraborty A, Saralaya V, Adhikari P, Shenoy S. Virulence property, phylogenetic background, and resistance pattern of Escherichia coli isolates from wound infections. CHRISMED J Health Res [serial online] 2017 [cited 2019 Oct 21];4:248-52. Available from: http://www.cjhr.org/text.asp?2017/4/4/248/216476


  Introduction Top


A wound is the result of physical distraction of the skin, one of the major obstacles to the establishment of infections by bacterial pathogens within internal tissues. Infection results when bacteria break through this barrier.[1] Escherichia More Details coli is one of the most common natural floras in the gut of humans and animals. However, outside of this normal habitat, it may cause serious infections. Contamination of wounds with such E. coli may lead to wound infection.[2] Various virulence factors (VFs) (P fimbriae, type I fimbriae, capsule, siderophores, hemolysin, and cytotoxic necrotizing factor) possessed by strains determine the pathogenic traits of these isolates.[3] Phylogenetic analysis has shown that medically important isolates mainly belong to four phylogroups (Group A, B1, B2, and D).[4] Antibiotic resistance exhibited by these E. coli strains is of major public health concern as they now have developed resistance to multiple groups of drugs.[5],[6] Due to the rapid spread of multidrug resistance among these strains, it is becoming increasingly difficult to treat such infections, and a chemotherapeutic endpoint may soon be reached wherein infections with these strains may not be cured with any of the presently available antibiotics. Hence, the present study was undertaken to analyze the possible interaction between possession of virulence markers, phylogenetic background, and antimicrobial resistance patterns exhibited by E. coli isolated from various wound infections.


  Subjects and Methods Top


This prospective study was conducted during the period from August 2010 to July 2014, from patients of the tertiary care hospitals in South India after obtaining permission from the institutional ethical committee (IEC/KMC/134/2010). The study population included hospitalized patients of all age groups whose wound sample grew E. coli and those participants who had received antimicrobial drugs during the past 1 month, and those who were discharged without treatment with antimicrobial drugs were excluded from the study.

Forty strains of E. coli were isolated from wound infections using standard sterile procedures from the study population. Isolates were identified based on colony morphology on blood agar and MacConkey agar and by standard biochemical identification tests.[7]

Antibiotic sensitivity testing was done using the modified Kirby-Bauer disk diffusion method in accordance with the Clinical and Laboratory Standards Institute guidelines.[8] The antibiotic disks (HiMedia, Mumbai) 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), co-trimoxazole (1.25/23.75 μg), cefoperazone + sulbactam (75/30 μg), imipenem (10 μg), meropenem (MRP; 10 μg), and ertapenem (10 μg). E. coli ATCC 25922 was used as quality control strain for antimicrobial susceptibility testing.

Genetical studies

Preparation of template DNA

Five hundred microliters of sterile distilled water taken in a microcentrifuge tube was added 4–5 freshly subcultured identical colonies of the isolate. This suspension was heated in a water bath at 95°C for 10 min and then centrifuged at 10,000 rpm for 10 min.[9] The supernatant containing bacterial DNA was used as template for polymerase chain reaction (PCR). E. coli strains used as positive controls in the PCR assay were kindly provided by Ms. Lotte Jakobsen (Statens Serum Institut Microbiology and Infection Control 5 Artillerivej, build 46/202DK-2300 Copenhagen).

Phylogenetic analysis was performed by triplex PCR-based method as described by Clermont et al.[4] Briefly, a combination of two genes (chuA and yjaA) and an anonymous DNA fragment (TSPE4.C2) allows the determination of the main phylogenetic groups of E. coli (these being A, B1, B2, and D).

Two sets of multiplex PCR were developed to detect the following genes:

  1. Set 1: PCR assay was performed to detect papC, cnf1, and neuC genes as per primers and conditions described earlier with minor modification [10]
  2. Set 2: PCR assay was performed to detect hlyA, fimH, and iutA genes as per primers and conditions described earlier with minor modification.[10]


Another multiplex PCR assay was performed to detect and differentiate blaTEM, blaSHV, and blaCTXM genes using primers and conditions as described previously.[11],[12] Another PCR assay was also performed to detect blaCTXM-15 variant of blaCTXM as described previously.[13]

All isolates were tested by a multiplex PCR assay which identified six family-specific AmpC genes carried on plasmids, namely, MOX, FOX, EBC, ACC, DHA, and CIT, using primers and conditions as described previously.[14]

Statistical analysis

Chi-square test was used to analyze the association between virulence and drug resistance genes. Analysis was performed using statistical package SPSS version 17.0 (IBM, New York, USA).


  Results Top


A total of forty nonrepeat E. coli strains isolated from patients with wound infections were studied. Of the forty patients, 27 (67.5%) were males and 13 (32.5%) were females. The most common predisposing factor observed was diabetes (30%). Phylogenetic analysis revealed 8 (20%) isolates belonged to phylogroup A and 2 (5%) strains to group B1, both phylogroups which are known to be commensal groups. As might be expected, it was the isolates belonging to the virulent phylogroups, namely, B2 and D (13 [32.5%] and 17 [42.5%] isolates, respectively) that were observed to cause the majority of infections as compared to Group A and B1.

Regarding possession of virulence genes, maximum number of strains were observed to carry the fimH 39 (97.5%) gene, followed by iutA 27 (67.5%), papC 16 (40%), hlyA 5 (12.5%), cnf1 5 (12.5%), and neuC 1 (2.5%), respectively [Table 1]. Results of Kirby-Bauer disk diffusion methods indicated that, of the 40 isolates, 37 (92.5%) were resistant to ampicillin and 33 (82.5%) to piperacillin. The antibiotics which were found to be most effective were MRP (sensitive: 39 isolates), imipenem (sensitive: 38 isolates), and co-trimoxazole (sensitive: 37 isolates), respectively [Figure 1]. Multidrug resistance (resistance to three or more antimicrobial groups of drugs) profile was observed in 34 (85%) isolates.
Figure 1: Resistance pattern of the isolates in the Kirby–Bauer disk diffusion tests

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Table 1: Prevalence of virulence factor genes and drug resistance genes among the isolates

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Of the forty isolates, 30 (75%) isolates were found to be positive for extended-spectrum β-lactamase (ESBL) genes. A total of 27 (67.5%) strains were observed to possess the blaCTXM gene, 4 (10%) isolates were found to be positive for blaTEM, and none of them were found to contain the blaSHV. Approximately 26 (96%) of blaCTXM producers were found to be positive for blaCTXM-15. CIT type of plasmid-mediated AmpC carriage was seen only in 6 (15%) of isolates [Table 1].


  Discussion Top


E. coli is frequently isolated from wound infections; however, the virulence potential and resistance patterns of such strains have not yet been extensively studied.

In the present study, it was observed that, of the infected population, the number of infected males was significantly higher than females and the most important predisposing factor was diabetes (one in three patients). E. coli which routinely causes extraintestinal infections belongs mainly to phylogroups B2 and D. Results of our study indicated that roughly 75% of the E. coli isolates from our patients' belonged to phylogenetic groups B2 and D which are in concurrence with previous findings.[15]

In our study, we detected a very high prevalence of type-1 fimbriae producing gene with 97.5% of the isolates possessing the fimH gene which might indicate their enhanced ability to attach onto host surfaces so as to initiate infection. Several recent studies such as that of Kudinha et al. and Mora et al. have also demonstrated a high prevalence of fimH genes in E. coli from extraintestinal infections although none of the studies involved wound isolates.[16],[17]

In the present study, it was observed that two of every three isolates were found to possess the iutA gene, which probably signifies the importance of this gene in pathogenicity. This evidence suggests that iutA may act as a significant virulent trait. Similar findings have been observed by other workers wherein several studies have reported that iutA was the most common VF trait among the extraintestinal pathogenic E. coli isolates.[10],[17] Studies have also reported that the possession of iutA genes was the most common virulence trait markers among blood isolates.[18] Thus, our study results indicate that our isolates may also have the potential to cause sepsis. PapC is one of the genes which is responsible for the assembly platform for fimbrial growth, helping the bacterium in adherence to eukaryotic cells so as to initiate infection. In the present study, we found 40% of isolates to be positive for the papC gene, a finding which is supported by other studies, wherein it has been found that nearly half of the study isolates carried the papC gene.[19] We also observed that only 12.5% of our isolates were found to possess the hlyA gene which indicated that hlyA may not be a significant virulence trait in our isolates although a study by Petkovsek et al.[20] conducted on E. coli isolates from skin and soft tissue infections has reported that around 30% of such isolates were harboring hlyA gene.

Results of our study indicated that approximately one in every ten isolates was carrying the cnf1 g ene. The presence of cnf1 in isolates and their expression may help such strains to escape from phagocytes as shown by Doye et al. who demonstrated that cnf1 provokes an increased adherence of polymorphonuclear leukocyte onto epithelial cells and a decreased rate of bacterial phagocytosis.[21] The study by Petkovsek et al. also reported that around 32% of their isolates were harboring the cnf1 gene.[20] However, these findings are not in agreement with the results of our investigation. The neuC gene was found to occur in only one of our isolates, indicating that it might not play an important role in the pathogenesis of wound infections although a much larger study is required to confirm any such finding.

Among all the antibiotics tested, the highest degree of sensitivity was observed with MRP (98%), imipenem (95%), and co-trimoxazole (97%) drugs. Several studies have reported the prevalence of a high level of resistance by E. coli to commonly used antibiotics.[5],[6] A study by Banu et al. in patients with E. coli infections reported that around 96% strains were resistant to ampicillin, 74% to co-trimoxazole, 44% to ciprofloxacin, 56% to gentamicin, and 35% were resistant to amikacin, respectively.[22] Regarding multidrug resistance among our isolates, it was disheartening to observe that a majority of our isolates (85%) were multidrug resistant. In light of this finding, it is evident that traditional antibiotics used for therapeutic reasons are fast becoming obsolete due to the rise of MDR bacteria or the so-called “superbugs.”

In our study, we observed a high rate of genotypically ESBL-positive isolates (75%), indicating a high prevalence of ESBL producers in our setup, and further, we found CTXM as the most predominant type of plasmid among the ESBL producers (60%). Only 10% were TEM positive and none of them were found to harbor the SHV gene. Among the subtypes, CTXM-15 was the most predominant (96%) resistance marker observed among the isolates. Several recent Indian studies have reported high prevalence of ESBL producers, and CTXM-15 has been considered to be the most predominant subtype.[22],[23],[24]

Our study detected the presence of the CIT type of AmpC gene in approximately 15% of isolates. This was the only type of plasmid-mediated AmpC gene possessed by our isolates. However, some Indian studies have reported a prevalence of up to 30% of isolates harboring AmpC genes, although in those studies, the majority of strains were isolates from urine and blood.[25],[26]

Limitation of our study

  1. Clinical outcome can be influenced by host factors, time of presentation, and antibiotic choice in addition to the phenotypical and genotypical characters of the infecting E. coli strains
  2. The presence of only plasmid-mediated AmpC β-lactamase genes was targeted, and other possible mechanisms of cefoxitin resistance such as chromosomal hyperproducers or porin loss mutants were not detected.



  Conclusions Top


The results of our study would be helpful in guiding early appropriate empirical therapy for wound infections. Based on our finding, we recommend that carbapenems and/or co-trimoxazole may preferably be used for treatment. As a majority of the isolates were found to belong to the virulent phylogroups and the virulence genes fimH and iutA occurred at a significantly higher rate among these strains, it may be inferred that these strains are more capable to initiate infection and if not successfully treated in the early stages, may lead to life-threatening infections such as sepsis.

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 thank 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

This study was supported by API Karnataka, India.

Conflicts of interest

There are no conflicts of interest.

 
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