Relationship of drug resistance to phylogenetic groups of E . coli isolates from wound infections

Background: Drug resistance is a major problem in Escherichia coli isolated from surgical wound infections. In this study, we evaluated relationship between phylogenicity and drug resistance. Methodology: A total of 29 multi-drug resistant (MDR) E. coli isolates of known drug resistance genes and integron profile were selected for the present study. Triplex PCR was conducted for phylogenetic classification of these isolates into four established phylogenetic groups: A, B1, B2 and D. Statistical analysis was done to determine the association of different drug resistance genes and integrons with the phylogenetic groups. Results: Most of the isolates (44.8%) belonged to phylogenetic group A followed by group B2 and D (24.1% each) and group B1 (6.9%). Conclusions: There is a definitive relationship between drug resistance and various phylogenetic groups of E. coli infecting wounds. A shift towards phylogenetic group A might be observed with an increasing drug resistance profile.


Introduction
Escherichia coli are primarily regarded as a cause of food-borne diarrhoeal diseases, but the importance of extraintestinal infections caused by E. coli cannot be underestimated.Extraintestinal E. coli mainly cause urinary tract infections (UTIs), surgical wound infections, and neonatal meningitis.Treatment of these infections has become a serious challenge for clinicians due to rapid development of antimicrobial drug resistance among these pathogens.Extraintestinal E. coli isolates causing surgical wound infections account for a loss of $94-252 million per annum in the United States alone [1] with a major share incurred on the treatment of resistant isolates.The losses in developing countries may be even more, but no comprehensive data is available to assess this.
Drug resistance in E. coli could predict its phylogenetic background and virulence profile in a complex fashion [2].The majority of extraintestinal E. coli isolates belong to the most virulent phylogenetic group B2 and to a lesser extent group D, whereas commensal E. coli mostly belong to the less virulent groups A or B1 [3].However, in some human E. coli isolates from UTIs, a shift away from the most virulent group B2 towards the less virulent groups A, B1 and D was observed with increasing drug resistance [4].
Much work has been done on the phylogenetic grouping of E. coli involved in UTIs, with special reference to their drug resistance profile [2,5].The data show evidence of a relationship between phylogenetic groups and drug resistance in E. coli from UTIs.However, no such data is available for E. coli infecting wounds.Thus, the current study was designed to investigate the relationship between drug resistance and phylogenetic grouping in locally isolated E. coli from surgical wound infections.

Sample collection and DNA extraction
A total of 29 multi-drug resistant (MDR) E. coli isolates from surgical wound infections were selected from stock cultures for the present study.These isolates were of known drug resistance genes and integron profiles (Table 1 revived in tryptic soy broth (TSB, Merck, Germany) by 18 hours of incubation at 37ºC.Total genomic DNA was extracted using the phenol-chloroform extraction method [6].The integrity of the DNA samples was checked by electrophoresis on 1% agarose gel and purity was determined by ratio of A260/A280 spectrophotometrically.DNA samples were quantified with a fluorometer (DyNA QUANT 200, USA).

Multiplex PCR for phylogenetic grouping
A triplex polymerase chain reaction (PCR) was conducted to determine the phylogenetic grouping of isolates by targeting two genes, chuA and yjaA, and an anonymous DNA fragment, TSPE4.C2 [3].Each 100 µl of PCR reaction mixture for multiplex PCR contained, in addition to 20 ng of DNA, 1.5 mM of MgCl 2, 70 µM of each dNTP, 1.0 µM of each primer and 10 U of Taq polymerase.Thermal cycler conditions were as follows: 94ºC for 5 minutes followed by 30 cycles of 94ºC for 30 seconds, 57ºC for 30 seconds, and 72ºC for 30 seconds.A final extension of 72ºC for 7 minutes was performed at the end of each PCR.

Phylogenetic grouping
The phylogenetic tree described by Clermont and colleagues was used to classify all E. coli isolates into four phylogenetic groups: A, B1, B2 and D. The grouping decision was made based on the presence or absence of specified amplifications [3].

Results
All isolates showed different combinations of amplification products (Figure 1).Phylogenetic grouping revealed that group A was most prevalent with 13/29 (44.8%) isolates; seven (24.1%) isolates were found to belong to each of B2 and D; and B1 was least prevalent in these wound-infecting MDR E. coli as only two (6.9%) isolates belonged to this group.
The distribution of drug resistance genes and integrons in different phylogenetic groups is shown in Table 1.The most salient results were that all B2 group isolates carried mutated gyrA and catA1 genes while all D group isolates were positive for catP gene.Among group A isolates, TEM and mutated gyrA genes were the most prevalent (76.9%).The comparison of intra-group percentages revealed that class 1 integrons were most prevalent (57.1%) in group D isolates, followed by group A (38.5%) and group B2 (28.6%).Contrary to this, class 2 integrons were equally distributed in groups B2 and D (42.9% each).Statistical analyses demonstrated that the distribution of class 1 and class 2 integrons among various phylogenetic groups (B2, D and A) had statistical significance (p < 0.05) with the exception of class 2 integrons in group A (P > 0.05).

Discussion
Millions of health and economic losses occur every year due to extraintestinal infections by E. coli [1].Antimicrobial drug resistance is a major causative factor for these losses.E. coli antimicrobial drug resistance is strongly related to phylogenetic Lane 5: yjaA amplified product (211 bp).Lane 3: chuA amplified product (279 bp); yjaA amplified product (211 bp); TSPE4.C2 amplified product (152 bp).
grouping [2].Extraintestinal E. coli isolates typically belong to phylogenetic groups B2 and D respectively, whereas commensal isolates belong to group A. The current study was designed to elucidate the effect of drug resistance on phylogenetic grouping in E. coli isolates from surgical wound origins.
To our knowledge, no work has previously been done in this area.
We studied 29 MDR E. coli isolates of known drug resistance genes and integron profile.As extraintestinal E. coli (from surgical wound origins), our isolates were expected to belong mostly to group B2 [3]; however, this was not the case.The majority of our isolates (44.8%) belonged to the reportedly less virulent group A, followed by groups B2 and D (24.1% each), and group B1 (6.9%).This pattern is comparable to those noted in some previous studies of extraintestinal E. coli associated with UTIs in Russian females which reported that 55% of isolates belonged to group A, 23% belonged to groups B2 and D each, and no isolate represented group B1 [5].In other studies, the resistant E. coli involved in UTIs showed a phylogenetic shift from group B2 toward group A [7,8].The highest prevalence of group A in the present study demonstrated a similar phylogenetic shift in wound infecting E. coli isolates.We observed a general shift towards non-B2 phylogenetic groups.This phenomenon may be due to the presence of high drug resistance, as all of our E. coli were MDR isolates.Similar results have been reported in UTIs [8].
The relationship of drug resistance genes and integrons with all phylogenetic groups was statistically analyzed (Table 1).Statistical analyses demonstrated that the distribution of class 1 and class 2 integrons among various phylogenetic groups (B2, D and A) had statistical significance with the exception of class 2 integrons in group A. There are some reports available on E. coli from UTIs revealing that integrons are less frequent in B2 than in non-B2 isolates [7].Our isolates showed the same pattern regarding class 1 integrons as only 28.6% of B2 isolates carried class 1 integrons in comparison to 57.1% and 38.5% isolates of groups D and A respectively.However, class 2 integrons were equally distributed among group B2 and D isolates (42.9% each) while only 7.7% isolates of group A carried class 2 integrons.
The relationship between phylogenetic groups and drug resistance genes has not previously been reported.We found that all B2 group isolates were positive for mutated gyrA and catA1 genes while all D group isolates were positive for the catP gene.Though most of the drug resistance genes were found in all phylogenetic groups (Table 1), there was no phylogenetic group found harboring a specific gene in a significantly higher proportion as compared with other groups.
We conclude that there is a definitive relationship between drug resistance and various phylogenetic groups of E. coli infecting wounds.A shift towards phylogenetic group A might be observed with an increasing drug resistance profile.

Table 1 .
Distribution of antimicrobial resistance genes among phylogenetic groups.
*The p value could not be calculated due to the low number of isolates.** Due to the low number of isolates, the percentage cannot be compared to that of other groups.