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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2016  |  Volume : 59  |  Issue : 2  |  Page : 180-184
Virulence factor profiles, phylogenetic background, and antimicrobial resistance pattern of lactose fermenting and nonlactose fermenting Escherichia coli from extraintestinal sources


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

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Date of Web Publication9-May-2016
 

   Abstract 

Context: In recent years, nonlactose fermenting (NLF) Escherichia coli have been increasingly isolated in the microbiology laboratory, but their clinical significance has not yet been clearly elucidated. Aims: To characterize the lactose fermenting (LF) and NLF isolates on the basis of their virulence factors, phylogenetic background, and drug resistance property. Settings and Design: This descriptive study was carried out in a multi-specialty tertiary care hospital. Subjects and Methods: Three hundred nonrepeat E. coli isolates from inpatients were studied. Isolates were differentiated as LF and NLF on the basis of colony characteristics on MacConkey's agar. Possession of virulence and drug resistance genes was determined by multiplex polymerase chain reaction (PCR). Phylogenetic analysis was performed by triplex PCR methods. Antibiotic sensitivity testing was performed by disk diffusion method. Results: Of 300 isolates 39 (13%) were NLF isolates. Maximum number of NLF isolates belonged to phylogroups B2 and D when compared with LF isolates. The incidence of iutA, hlyA, and neuC genes were significantly higher in NLF isolates. The presence of drug resistance genes such as AmpC gene, SHV, and CTXM were higher in LF isolates. Conclusions: LF isolates demonstrated a higher antimicrobial resistance and NLF isolates possessed higher virulence properties. The microbiology laboratory should report lactose fermentation profile as it may help the physician to initiate appropriate treatment.

Keywords: Drug resistance, Escherichia coli, lactose fermenters, nonlactose fermenters, virulence

How to cite this article:
Chakraborty A, Adhikari P, Shenoy S, Saralaya V. Virulence factor profiles, phylogenetic background, and antimicrobial resistance pattern of lactose fermenting and nonlactose fermenting Escherichia coli from extraintestinal sources. Indian J Pathol Microbiol 2016;59:180-4

How to cite this URL:
Chakraborty A, Adhikari P, Shenoy S, Saralaya V. Virulence factor profiles, phylogenetic background, and antimicrobial resistance pattern of lactose fermenting and nonlactose fermenting Escherichia coli from extraintestinal sources. Indian J Pathol Microbiol [serial online] 2016 [cited 2019 Dec 10];59:180-4. Available from: http://www.ijpmonline.org/text.asp?2016/59/2/180/182032



   Introduction Top


Escherichia coli, one of the first enteric bacilli to be described and cultured, is a normal inhabitant of the intestinal tract of humans and animals.[1] In recent years, extraintestinal infections (EIs) due to E. coli are common in all age groups and can involve almost any organ or anatomical site.[2] Extraintestinal pathogenic E. coli (ExPEC) strains have acquired genes encoding diverse virulence factors (VFs) that enable them to cause infections outside of the gastrointestinal tract.[3] The treatment of E. coli infections is increasingly becoming difficult because of the multi-drug resistance exhibited by the organism. However, several studies have characterized these isolates based on their phenotypic, genotypic and clinical properties but little attention has been focused on nonlactose fermenting (NLF) E. coli isolates (atypical strains). Hence, the present study was undertaken to find out the VFs, phylogenetic background, and antimicrobial resistant pattern of NLF isolates in contrast to lactose fermenting (LF) isolates.


   Subjects and Methods Top


Participants and clinical isolates

The study was conducted during the period from August 2010 to January 2013, from patients of a tertiary care hospital in South India, after obtaining permission from the institutional ethical committee. Three hundred nonrepeated strains of E. coli were isolated from the specimen such as urine, blood, wound swab, pus, cerebrospinal fluid, ascites fluid, and intravascular devices from the study population.

Identification of the organism

Isolates were identified based on colony morphology on blood agar, MacConkey's agar Four to five 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.[4] LF and NLF isolates were categorized based on lactose fermentation on MacConkey's agar.

E. coli ATCC 25922 was used as the quality control strains for antimicrobial susceptibility testing.

Phylotyping analysis

Phylogenetic analysis was performed by triplex polymerase chain reaction (PCR) based methods as described by Clermont et al.[5] 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).

Detection of virulence factor genes by multiplex polymerase chain reaction assay

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

  1. Set 1: A PCR assay was performed to detect papC, cnf1, and neuC genes as per primers and conditions described earlier with minor modification [6]
  2. Set 2: Another PCR assay was performed to detect hlyA, fimH, and iutA genes as per primers and conditions described earlier with minor modification.[6]


Antimicrobial susceptibility testing

Antibiotic susceptibility testing was done by the modified Kirby–Bauer disk diffusion method in accordance with Clinical and Laboratory Standards Institute guidelines.[7] The antibiotic disks (Hi-Media, 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), imipenem (IPM; 10 μg), meropenem (MRP; 10 μg), and ertapenem (ETP; 10 μg). Isolates were further tested for extended-spectrum beta-lactamase (ESBL) and AmpC activities by phenotypic methods, as described previously.[7],[8]

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.[9] Another PCR assay was performed to detect blaCTXM-15 variant of blaCTX-M as described earlier.[10]

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.[11]

Statistical analysis

Chi-square test was used to find association between the LF and NLF isolates. The analysis was performed using statistical package SPSS version 17 (USA).


   Results Top


A total of 300 nonrepeat E. coli isolates from patients with EIs were included in our study. These included 159 (53%) cases of urinary tract infection (UTI), 77 (25.6%) with bacteremia, 40 (13.3%) with wound infection, 19 (6.3%) with pneumonia, 3 (1%) intravascular device infection and 2 (0.6%) with meningitis. Of the 300 patients, 163 (54%) were males and 137 (46%) were females with the age group of ≤1 = 4 (1.3%), 2–18 = 8 (2.6%), 19–44 = 71 (23.6%), 45–59 = 87 (29%), and ≥60 = 130 (43%).

Of the 300 isolates, it was observed that on MacConkey's Agar, 261 (87%) isolates were LFs whereas 39 (13%) were NLFs. The clinical site of the isolation of LF and NLF isolates are summarized in [Table 1].
Table 1: Site of isolation, phylogeny and possession of virulence factors by LF and NLF strains of E. coli

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Sixty-one isolates were found to belong to phylogroup A and 27 strains to group B1, both phylogroups that are known to be commensal groups. Among the virulent groups (phylogroups B2 and D), 104 were from group B2 and 108 were from group D. Phylogenetic distribution of the LF and NLF isolates are summarized in [Table 1].

On analysis of virulence genes among the 300 isolates, we found maximum number were carrying fimH (90%) gene followed by iutA (68%), papC (45%), hlyA (23%), cnf1 ( 23%), and neuC (5%), respectively.

Of the 300 isolates, 70% isolates were found to be positive for ESBL genes, and CTXM types were the most predominant type. CIT type of plasmid-mediated AmpC was seen only in 12% of isolates.

On analysis of the NLF isolates and possession of VF genes among our ExPEC strains yielded the fact that there existed significant positive correlation between NLF E. coli isolates with iutA, hlyA, and neuC when compared to LF E. coli isolates [Table 1]. However, there were no significant difference in the distribution of β-lactamase genes and pAmpC (CIT type) gene [Table 2].
Table 2: Occurrence of ESBL and AmpC genes among E. coli strains

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Results of Kirby–Bauer disk diffusion methods indicated that, of the 300 isolates, 291 (97%) were fully susceptible to ETP and similarly, 282 (94%), 276 (92%), and 267 (89%) isolates were susceptible to MRP, IPM, and nitrofurantoin, respectively. Among the isolates, 195 (65%) were resistant to ciprofloxacin, 144 (48%) were co-trimoxazole-resistant, and 138 (46%) were found to be gentamicin resistant. The resistance patterns exhibited by LF and NLF isolates against representative antimicrobials from different classes are shown in [Figure 1].
Figure 1: Resistance pattern of lactose fermenting and nonlactose fermenting isolates in the Kirby–Bauer sensitivity tests

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   Discussion Top


In our study, nearly 13% isolates were NLFs, and majority of them were from urinary tract followed by blood isolates. Altered phenotype could be due to an altered genetic makeup of the isolates. This finding is similar to other studies such as the one conducted by Raksha et al.[12] who studied 220 isolates of which 14 (6.36%) were NLF. Another study conducted by Sharma et al.[13] has also reported 12% of their isolates were NLF isolates.

ExPEC which routinely cause infections have been shown to belong to phylogroups B2 and D. Results of our study indicated that approximately 75% of the E. coli isolates from our patients belonged to phylogenetic group B2 and D which is in agreement with previous findings.[14],[15] A comparison of LF and NLF isolates in relation to their phylogroups detected the significant predominance of LF isolates in commensal phylogroups A and B1 and NLF isolates in the virulent phylogroups (B2 and D). This indicates that NLF isolates were more virulent in comparison to LF isolates.

In our study, we found a high prevalence of type-1 fimbriae producing isolates, 90% of the total isolates had the fimH gene which indicated their ability to attach to mucosal surfaces so as to initiate infection. However, our study failed to correlate fimH gene as a VF for both LF and NLF isolates.

In the present study, it was observed that two in three isolates were found to be positive with an iutA gene, which indicates the importance of its role in pathogenicity, we also observed that presence of iutA genes were significantly higher in NLF isolates when compared to LF isolates. Several investigators have reported that iutA was the most common VF trait among blood isolates.[16],[17]

We found 45% of the isolates to be positive for papC gene. PapC is one of the genes which is responsible for the assembly platform for the fimbrial growth and help the isolates for adherence to eukaryotic cells. Johnson et al. have suggested that P fimbriae contribute to the ability of E. coli strains to cause UTI, especially the more clinically severe forms.[18] Nevertheless, in our study, we failed to demonstrate the significance of papC in LF and/or NLF isolates as it was equally distributed in both type of isolates.

In our study, approximately 24% isolates were found to possess the hlyA gene. In contrast to LF isolates, we found the presence of hlyA gene was significantly higher in NLF isolates. It has been suggested that colonization with hemolytic strains of E. coli lead to a greater risk of developing UTI and such colonization may also contribute to tissue invasiveness and injury and even facilitate entry into the blood stream resulting in sepsis.[14],[17]

We also observed that approximately one in four isolates was carrying the cnf1 gene although there was no significant difference between LF and NLF isolates in the possession of the gene. The presence of cnf1 in E. coli may help them to escape from phagocytes as shown by Doye et al. who demonstrated that CNF-1 provokes an increased adherence of PMNL onto epithelial cells, and a decreased bacterial phagocytosis.[19]

Only 5% of our isolates possessed the neuC gene and majority of these were NLF isolates. Presences of neuC in isolates indicate their pathogenic character in that the capsulated strains basically inhibit the phagocytosis process due to their possession of capsular polysaccharide which has antiphagocytic action. Several studies reported that K1 isolates were responsible for meningitis, especially neonatal meningitis as capsular strains have the ability to cross the blood-brain barrier.[20],[21]

The rapid increase in the rate of antibiotic resistance of ExPEC isolates is a major cause of concern. In our study, isolates we observed a high degree of resistance pattern to commonly used antibiotics such as ampicillin, piperacillin, ciprofloxacin, and norfloxacin. However, around 50% isolates were resistant to ceftazidime. We also observed that isolates were resistant to drug combinations such as piperacillin/tazobactam (25%), and around 35% of the isolates were resistant to cefoperazone/sulbactam which is quite alarming. Several studies have also reported a high level of resistance to common antibiotics.[22],[23] Higher sensitivity was observed to amikacin (84%), nitrofurantoin (89%), ETP (97%) and other carbapenem group of drugs. However, while comparing the resistance pattern in between LF and NLF isolates we observed most of the LF isolates were multidrug resistant as they were less susceptible to aminoglycosides, fluoroquinolones, BL + BI, and carbapenem group of drugs.

A recent study by Chakraborty et al. had reported a high prevalence of ESBL producing isolates, with CTX-M being the most predominant type, although, in the present study, there were no significant difference in possession of ESBL genes among the LF and NLF isolates.[24]

Plasmid-mediated AmpC β-lactamases belonging to Ambler class C are a new threat worldwide as they mediate resistance to a broad spectrum of antibiotics. In recent years, pAmpC is increasingly being identified in E. coli.[25] In the present study, we found around one in ten isolates were positive for the CIT type of plasmid-mediated AmpC gene. However, there were no significant difference between LF and NLF isolates in process of AmpC genes.


   Conclusion Top


Out study, findings suggest that LF isolates were less virulent but demonstrated higher resistance to antimicrobials whereas NLF isolates were more virulent albeit being less resistant. However, further investigations are required to explain the mechanisms at play behind these findings.

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 Lotte Jakobsen M. Sc. (biology), PhD Statens Serum Institute Microbiology and Infection Control 5 Artillerivej, build 46/202 DK-2300 Copenhagen for providing us the positive control isolates for the study.

Financial support and sponsorship

API Karnataka, India.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Burrows W. A Text of Medical Microbiology. 22nd ed. Philadelphia: W.B. Saunders Co.; 1985.  Back to cited text no. 1
    
2.
Johnson JR, Russo TA. Extraintestinal pathogenic Escherichia coli: “The other bad E coli”. J Lab Clin Med 2002;139:155-62.  Back to cited text no. 2
    
3.
Russo TA, Johnson JR. Medical and economic impact of extraintestinal infections due to Escherichia coli: Focus on an increasingly important endemic problem. Microbes Infect 2003;5:449-56.  Back to cited text no. 3
    
4.
Crichton PB. Enterobacteriaceae: Escherichia, Klebsiella, proteus and other genera. In: Collee JG, Fraser AG, Marmion BP, Siminons A, editors. Mackie and McCartney Practical Medical Microbiology. 14th ed. New York: Churchill Livingston; 1996. p. 361-4.  Back to cited text no. 4
    
5.
Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol 2000;66:4555-8.  Back to cited text no. 5
    
6.
Johnson JR, Stell AL. Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J InfectDis 2000;181:261-10.  Back to cited text no. 6
    
7.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. CLSI M100-S20U. Update June 2010. Wayne, PA: Clinical and Laboratory Standards Institute; 2010.  Back to cited text no. 7
    
8.
Black JA, Moland ES, Thomson KS. AmpC disk test for detection of plasmid-mediated AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal AmpC beta-lactamases. J Clin Microbiol 2005;43:3110-3.  Back to cited text no. 8
    
9.
Hosoglu S, Gündes S, Kolayli F, Karadenizli A, Demirdag K, Günaydin M, et al. Extended-spectrum beta-lactamases in ceftazidime-resistant Escherichia coli and Klebsiella pneumoniae isolates in Turkish hospitals. Indian J Med Microbiol 2007;25:346-50.  Back to cited text no. 9
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10.
Muzaheed DY, Adams-Haduch JM, Endimiani A, Sidjabat HE, Gaddad SM, et al. High prevalence of CTX-M-15-producing Klebsiella pneumoniae among inpatients and outpatients with urinary tract infection in Southern India. J Antimicrob Chemother 2008;61:1393-4.  Back to cited text no. 10
    
11.
Pérez-Pérez FJ, Hanson ND. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 2002;40:2153-62.  Back to cited text no. 11
    
12.
Raksha R, Srinivasa H, Macaden RS. Occurrence and characterisation of uropathogenic Escherichia coli in urinary tract infections. Indian J Med Microbiol 2003;21:102-7.  Back to cited text no. 12
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13.
Sharma S, Bhat GK, Shenoy S. Virulence factors and drug resistance in Escherichia coli isolated from extraintestinal infections. Indian J Med Microbiol 2007;25:369-73.  Back to cited text no. 13
[PUBMED]  Medknow Journal  
14.
Rijavec M, Müller-Premru M, Zakotnik B, Zgur-Bertok D. Virulence factors and biofilm production among Escherichia coli strains causing bacteraemia of urinary tract origin. J Med Microbiol 2008;57(Pt 11):1329-34.  Back to cited text no. 14
    
15.
Johnson JR, Scheutz F, Ulleryd P, Kuskowski MA, O'Bryan TT, Sandberg T. Phylogenetic and pathotypic comparison of concurrent urine and rectal Escherichia coli isolates from men with febrile urinary tract infection. J Clin Microbiol 2005;43:3895-900.  Back to cited text no. 15
    
16.
Mora A, López C, Dabhi G, Blanco M, Blanco JE, Alonso MP, et al. Extraintestinal pathogenic Escherichia coli O1:K1:H7/NM from human and avian origin: Detection of clonal groups B2 ST95 and D ST59 with different host distribution. BMC Microbiol 2009;9:132.  Back to cited text no. 16
    
17.
Johnson JR, Stell AL. Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J Infect Dis 2000;181:261-9.  Back to cited text no. 17
    
18.
Johnson JR, Kuskowski MA, Owens K, Gajewski A, Winokur PL. Phylogenetic origin and virulence genotype in relation to resistance to fluoroquinolones and/or extended-spectrum cephalosporins and cephamycins among Escherichia coli isolates from animals and humans. J Infect Dis 2003;188:759-68.  Back to cited text no. 18
    
19.
Doye A, Mettouchi A, Bossis G, Clément R, Buisson-Touati C, Flatau G, et al. CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. Cell 2002;111:553-64.  Back to cited text no. 19
    
20.
Obata-Yasuoka M, Ba-Thein W, Tsukamoto T, Yoshikawa H, Hayashi H. Vaginal Escherichia coli share common virulence factor profiles, serotypes and phylogeny with other extraintestinal E. coli. Microbiology 2002;148(Pt 9):2745-52.  Back to cited text no. 20
    
21.
Korczak B, Frey J, Schrenzel J, Pluschke G, Pfister R, Ehricht R, et al. Use of diagnostic microarrays for determination of virulence gene patterns of Escherichia coli K1, a major cause of neonatal meningitis. J Clin Microbiol 2005;43:1024-31.  Back to cited text no. 21
    
22.
Jadhav S, Hussain A, Devi S, Kumar A, Parveen S, Gandham N, et al. Virulence characteristics and genetic affinities of multiple drug resistant uropathogenic Escherichia coli from a semi urban locality in India. PLoS One 2011;6:e18063.  Back to cited text no. 22
    
23.
Banu A, Kabbin SJ, Anand M. Extraintestinal infections due to Escherichia coli: An emerging issue. J Clin Diagn Res 2011;5:486-90.  Back to cited text no. 23
    
24.
Chakraborty A, Adhikari P, Shenoy S, Saralaya V. Clinical significance and phylogenetic background of extended spectrum ß-lactamase producing Escherichia coli isolates from extra-intestinal infections. J Infect Public Health 2015;8:248-53.  Back to cited text no. 24
    
25.
Chakraborty A, Adhikari P, Shenoy S, Saralaya V. Characterization of plasmid mediated AmpC producing Escherichia coli clinical isolates from a tertiary care hospital in South India. Indian J Pathol Microbiol 2014;57:255-8.  Back to cited text no. 25
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Correspondence Address:
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: 10.4103/0377-4929.182032

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