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ORIGINAL ARTICLE  
Year : 2015  |  Volume : 58  |  Issue : 1  |  Page : 22-26
Genomic analysis and clinical importance of Escherichia coli isolate from patients with sepsis


1 Department of Microbiology, Government Medical College, Ambedkar Nagar, Uttar Pradesh, India
2 Department of Medicine, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India
3 Department of Microbiology, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India

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Date of Web Publication11-Feb-2015
 

   Abstract 

Context: Escherichia coli is a major cause of bloodstream infections and death due to sepsis. Bacteremic isolates harbor a significantly greater repertoire of virulence factors (VFs) in contrast with commensal E. coli isolates. Aims: The aim was to determine the relationships between E. coli VFs, phylogenetic groups, and their clinical importance. Settings and Design: This descriptive study was carried out in a multi-specialty tertiary care hospital. Materials and Methods: Escherichia coli isolates from consecutive episodes of bacteremia in 100 patients were screened for their VFs, phylogenetic group, and their effect on patient's clinical outcome. Virulence genes of all isolates were determined by multiplex polymerase chain reaction (PCR). Phylogenetic analysis was performed by triplex PCR methods. Estimation of risk of death was calculated using APACHE score II calculator. Results: Of the 100 patients, the most common predisposing factors were diabetes (42%), followed by carcinoma (23%). On analysis of the VF genes of the isolates, a majority of strains (88%) were possessing the fimH gene followed by iutA (76%), papC (44%), cnf1 (16%), hlyA (16%) and neuC (5%) respectively. Phylogenetic analysis revealed that 25 (25%) isolates belonged to phylogroup A, 8(8%) strains to group B1, 30 (30%) were from group B2 and 37 (37%) were from group D. The incidence of iutA gene was significant in higher APACHE II score group. Conclusions: Our findings indicate that virulent as well as commensal strains are capable of causing sepsis. Host related predisposing factors, adherence factors, and iron uptake are essential for the survival of the sepsis inducing strains.

Keywords: Escherichia coli, iutA gene, sepsis, outcome, virulence factors

How to cite this article:
Chakraborty A, Adhikari P, Shenoy S, Saralaya V. Genomic analysis and clinical importance of Escherichia coli isolate from patients with sepsis. Indian J Pathol Microbiol 2015;58:22-6

How to cite this URL:
Chakraborty A, Adhikari P, Shenoy S, Saralaya V. Genomic analysis and clinical importance of Escherichia coli isolate from patients with sepsis. Indian J Pathol Microbiol [serial online] 2015 [cited 2020 Jul 2];58:22-6. Available from: http://www.ijpmonline.org/text.asp?2015/58/1/22/151161



   Introduction Top


Sepsis is a grave medical condition induced by an overwhelming infection of the bloodstream. Among the Gram-negative bacteria,  Escherichia More Details coli is the species that is most commonly associated with sepsis worldwide. [1] E. coli have acquired genes encoding diverse virulence factors (VFs) that enable them to cause infection in both normal and compromised hosts. Characteristic virulence traits that are present in most sepsis inducing E. coli isolates include various adhesins (e.g., P and type I fimbriae), factors to avoid or subvert host defense systems (e.g., capsule, lipopolysaccharide), mechanisms for nutrient acquisition (e.g., siderophores), and toxins (e.g., hemolysin, cytotoxic necrotizing factor). [2] Phylogenetic analysis has shown that sepsis inducing E. coli strains mainly belong to group B2 and to a lesser extent to group D. [3]

The clinical importance and the prevalence of these VFs has been studied in different parts of the world however in India there is insufficient data regarding the clinical importance, phylogroup and the distribution of the VFs among the sepsis inducing E. coli strains. Hence, the present study was undertaken to find out the clinical importance, phylogroups and distribution of the VFs among the blood isolates.


   Materials and Methods Top


Participants and clinical isolates: The study was conducted during the period from August 2010 to July 2013, from patients of tertiary care hospitals in south India after obtaining permission from the institutional ethical committee. One hundred nonrepeated sepsis inducing strains of E. coli were isolated from the study population. Study population included patients of all age groups and those subjects who had received antimicrobial drugs during the past 1-month were excluded. APACHE II scores were calculated for all patients based on the available data to quantify clinical virulence and severity of the infection clinical data from the patient's records were collected in a proforma. All patients were followed-up for the period of 1-year to monitor clinical outcome. Samples were processed immediately using standard procedures. The isolates were identified using automated biochemical system Vitek 2 (bioMerieux, France).

Phylotyping analysis

Phylogenetic analysis was performed by triplex polymerase chain reaction (PCR) based methods 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).

Detection of virulence factor genes by multiplex polymerase chain reaction assay:

Two sets of multiplex PCR were developed to detect following genes

Set 1

A PCR assay was performed to detect papC, cnf1 and neuC genes as per primers and conditions described earlier with minor modification. [5] Template DNA was amplified by Multiplex PCR with the use of oligonucleotide primers obtained from Sigma-Aldrich Pvt., Ltd. India. The PCR was performed in a final reaction volume of 50 μl containing 750 Mm Tris-HCl, 200 mM (NH 4 ) 2 SO 4 , 2.5 mM MgCl 2 0.2 mM each dNTP, 0.4 μM of papC primers and 0.6 μM of cnf1 and neuC primers, 1 U of Taq DNA polymerase and 4 μl template DNA. An Eppendorf thermocycler was used for amplification. The program for amplification included a step of initial denaturation at 95°C for 3 min, followed by 25 cycles of 94°C for 30 s, 61°C for 30 s and 68°C for 3 min and a final extension step at 72°C for 3 min.

The PCR products were loaded in 2% wt/vol agarose gel prepared in Tris-borate-ethylenediaminetetraacetic acid (EDTA) buffer at 120 V for 1 h and detected by ethidium bromide staining after electrophoresis.

Set 2

Another PCR assay was performed to detect hlyA, fimH and iutA genes as per primers and conditions described earlier with minor modification. [5] Template DNA was amplified by Multiplex PCR with the use of oligonucleotide primers obtained from Sigma-Aldrich Pvt. Ltd. India. The PCR was performed in a final reaction volume of 50 μl containing 750 Mm Tris-HCl, 200 mM (NH 4 ) 2 SO 4 , 2.5 mM MgCl 2 0.2 mM each dNTP, 0.6 μM of hlyA primers and 0.3 μM of iutA and fimH primers, 1 U of Taq DNA polymerase and 4 μl template DNA. An Eppendorf thermocycler was used for amplification. The program for amplification included a step of initial denaturation at 95°C for 3 min, followed by 25 cycles of 94°C for 30 s, 61°C for 30 s and 68°C for 3 min and a final extension step at 72°C for 3 min. The PCR products were loaded in 2% wt/vol agarose gel prepared in Tris-borate-EDTA buffer at 120 V for 1 h and detected by ethidium bromide staining after electrophoresis.

Statistical analysis

Chi-square test was used to find an association between the phylogroups, VF genes, and patient's clinical outcome. Analysis was performed using statistical package SPSS version 17.0 USA.


   Results Top


A total of 100 sepsis patients infected by E. coli were included in this study. Of the 100 patients, 67 (67%) were males and 33 (33%) were females with the age group of <1 = 1 (1%), 1-18 = 3 (3%), 19-44 = 16 (16%), 45-59 = 42 (33%) and >60 = 47 (47%). Estimation of risk of death was calculated by using APACHE score II calculator. Patients were grouped into 3 categories based on APACHE score (1-9 = I, 10-19 = II, 20-29 = III). Thirty-six patients were in the category I, 47 were category II and 17 were category III. The most common predisposing factors were diabetes (42%) followed by carcinoma (23%).

Phylogenetic grouping of the isolates was done by using the results of PCR amplification of the chuA and yjaA genes and DNA fragment TSPE4.C2 [Figure 1]. Twenty-five (25%) isolates were found to belong to phylogroup A and 8 (8%) strains to group B1, both phylogroups, that are known to be commensal groups. Among the virulent groups (phylogroups B2 and D), 30 (30%) were from group B2 and 37 (37%) were from group D.
Figure 1: Phylogenetic grouping of Uropathogenic Escherichia coli isolates: Phylogenetic Group A [(chu A– , yjaA– , TspE4.C2-) and (yjaA+, chu A– , TspE4.C2– )]; Group B1 [chu A– , yjaA– , TspE4.C2+]; Group B2 [(chuA+, yjaA+, TspE4.C2– ) and (chuA+, yjaA+, TspE4.C2+)]; and Group D [(chuA+, yjaA-,TspE4.C2– ) and (chuA+, yjaA– , TspE4.C2+)]


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As might be expected, it was the isolates belonging to the virulent phylogroups, namely B2 (38%) and D (35%) that were causing maximum number of infections as compared to Group A and B1 (statistically significant, P < 0.05).

On analysis of the VF genes of the 100 isolates, a majority of strains (88; 88%) were possessing the fimH gene and a very few isolates (5; 5%) were harboring neuC genes [Figure 2] and [Figure 3]. Distribution of other VFs genes is summarized in [Table 1].
Figure 2: Multiplex polymerase chain reaction assays for neuC, cnf1, papC genes: Lane 1: 100bp DNA ladder; Lane 2: positive control; Lane 3: negative control. Lane 4 and 5: Test isolates


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Figure 3: Multiplex polymerase chain reaction assays for hlyA, fimH, iutA genes: Lane 1:100bp DNA ladder; Lane 2: negative control; Lane 3: positive control; Lane 5 and 6: Test isolates


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Table 1: Prevalence of virulence factor genes among the isolates


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A study of the possession of multiple VF genes revealed that 8 isolates possessed five VF genes, 25 isolates were observed to possess 4 VF genes, 46 strains contained 3 VF genes, 37 with 2 and 14 isolates were positive with 1 VF gene.

Study of APACHE II scores with the possession of virulence genes, revealed that the incidence of iutA was significant in higher APACHE II score [P < 0.05; [Table 2].
Table 2: Correlation of genotypic characteristics of infecting strains of ExPEC with APACHE score II and clinical outcome


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Of our study population, the maximum number of patients (59%) recovered with appropriate antibiotic treatment. Relapses were seen in 7% patients. On the analysis of the distribution of virulence genes among outcome groups, there were no significant different in distribution of virulence genes [Table 2].


   Discussion Top


In our study population, we found that age was an important risk factor for susceptibility to infection with E. coli. Elderly patients (>60 years) were more susceptible to infection, when compared with any other age group. Several investigators have also reported the same. [6],[7] Among study population, the proportion of infected males was significantly higher than females.

Regarding the factors that predispose to E. coli infections, we found that nearly 50% of our sepsis patients had diabetes, and nearly one-third of our patients had carcinoma. This finding is similar to other investigators who all reported that diabetes and malignancy were the two common underlying conditions associated with bacteremia. [7],[8],[9]

In the present study, we also observed that diabetes was associated with significant lower mortality while the carcinoma emerged as the most significant risk factor associated with sepsis indicating that host immunity is an important factor in causing invasive infection.

Strains which routinely cause infections have been shown to belong to phylogroups B2 and D. Results of our study indicated that approximately 70% of E. coli isolates from our patients belonged to phylogenetic group B2 and D, which is in agreement with previous findings. [10],[11] We also observed around one in four isolates were from phylo-group A, which indicate that virulent, as well as commensal strains, are equally capable of causing sepsis. The least frequently isolated phylogenetic group in our study was group B1 that is also in accordance with similar studies done elsewhere. [10],[11],[12]

In our study, we found a high prevalence of type-1 fimbriae producing isolates, 88% of the isolates had the fimH gene, which indicated their ability to attach on to mucosal surfaces so as to initiate infection. Rijavec et al. also had reported high prevalence of fimH genes (95%) among the blood isolates. 10 Several recent studies have also demonstrated a high prevalence of fimH genes in E. coli from extra intestinal infections. [12],[13],[14]

In the present study, it was observed that two in three isolates were found to possess the iut gene, which indicates the importance of its role in pathogenicity. This evidence suggests that for blood isolates iutA may act as a significant virulent trait. This finding is similar to other studies where several investigators have reported that iutA was the most common VF trait among blood isolates when compared to other extraintestinal E. coli isolates. [5],[13],[14]

We found that approximately 1 in 2 of our isolates were positive for the papC genes, 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. This finding is supported by other investigator's findings where they found about half of their study isolates carried the papC gene. [14],[15]

In our study, we found approximately one in five blood isolates were carrying the cnf1 gene, a finding which is similar to another study done by Ananias and Yano where they reported around 21% of blood isolates were positive for the cnf1 gene. [14] Presence of cnf1 in isolates may help them to escape from phagocytes as shown by Doye et al. who demonstrated that CNF-1 provokes an increased adherence of Polymorphonuclear Leukocyte (PMNL) onto epithelial cells and a decreased bacterial phagocytosis. [16]

Only 5% of our isolates were found to possess the neuC gene. Presences of neuC in blood isolates indicate their pathogenic character; in that, the capsulated strains basically inhibit the phagocytosis process due to their expression of capsular polysaccharide that has antiphagocytic action. [17] However, our study failed to correlate neuC gene as a VF as the incidence was very low when compared with other virulence traits.

In our study, multiple VF genes were observed in several isolates. It was detected that out of the six virulence traits that were targeted, around one in ten isolates were positive with at least five VFs, and at least one VF gene was observed in one in seven isolates. Several other investigators also reported the presence of multiple VFs among the isolates. [12],[13],[14],[15],[18]

In our study, we found a significant association between the iutA gene and the isolates, which indicated that iron metabolism acts as one of the important virulence property of these isolates. We also found the presence of a higher percentage of fimH and papC genes among the isolates that indicated that adhesion was also an important factor for invasiveness of the isolates. We also found that iutA also plays a significant role in higher APACHE II score, which indicates the severity of infections. By using the suppression subtractive hybridization technique, Mokady et al. subtracted the genome of E. coli K12 from septicemia inducing strains of E. coli to determine potential VFs that may be involved in septicemia, they found iron uptake systems (aerobactin), serum resistance and adhesins (type 1 pili, p pili) were important putative VFs of the septicemia inducing strains. [19] Yet another study by Ananias and Yano in blood isolates found that iut, fimH and papC were significantly higher. [14]

In the present study, no correlation was observed regarding clinical outcome and the possession of virulence genes in stains of E. coli.

In conclusion, our findings indicate that possession of virulence genes such as papC and fimH help the isolates to attach onto mucosal surfaces so as to initiate infection also iron uptake mechanisms are essential for survival of sepsis inducing E. coli strains. Indeed host related predisposing factors such as age, diabetes, malignancy and other underlying conditions are also equally responsible for the development of infections.

 
   References Top

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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. 4
    
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Jauréguy F, Carbonnelle E, Bonacorsi S, Clec'h C, Casassus P, Bingen E, et al. Host and bacterial determinants of initial severity and outcome of Escherichia coli sepsis. Clin Microbiol Infect 2007;13:854-62.  Back to cited text no. 8
    
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Siegman-Igra Y, Fourer B, Orni-Wasserlauf R, Golan Y, Noy A, Schwartz D, et al. Reappraisal of community-acquired bacteremia: A proposal of a new classification for the spectrum of acquisition of bacteremia. Clin Infect Dis 2002;34:1431-9.  Back to cited text no. 9
    
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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. 10
    
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Bukh AS, Schønheyder HC, Emmersen JM, Søgaard M, Bastholm S, Roslev P. Escherichia coli phylogenetic groups are associated with site of infection and level of antibiotic resistance in community-acquired bacteraemia: A 10 year population-based study in Denmark. J Antimicrob Chemother 2009;64:163-8.  Back to cited text no. 11
    
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Cooke NM, Smith SG, Kelleher M, Rogers TR. Major differences exist in frequencies of virulence factors and multidrug resistance between community and nosocomial Escherichia coli bloodstream isolates. J Clin Microbiol 2010;48:1099-104.  Back to cited text no. 12
    
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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. 13
    
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Ananias M, Yano T. Serogroups and virulence genotypes of Escherichia coli isolated from patients with sepsis. Braz J Med Biol Res 2008;41:877-83.  Back to cited text no. 14
    
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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. 16
    
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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. 17
    
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Arisoy M, Aysev D, Ekim M, Ozel D, Köse SK, Ozsoy ED, et al. Detection of virulence factors of Escherichia coli from children by multiplex polymerase chain reaction. Int J Clin Pract 2006;60:170-3.  Back to cited text no. 18
    
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Mokady D, Gophna U, Ron EZ. Extensive gene diversity in septicemic Escherichia coli strains. J Clin Microbiol 2005;43:66-73.  Back to cited text no. 19
    

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Correspondence Address:
Dr. Arindam Chakraborty
Department of Microbiology Government Medical College, Ambedkar Nagar, Uttar Pradesh
India
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Source of Support: API Karnataka, India, Conflict of Interest: None


DOI: 10.4103/0377-4929.151161

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