Taru Singh1, Shukla Das 1*, VG Ramachandran1, Rumpa Saha1, Amir Maroof Khan 2, and Arvind Rai3
1Department of Microbiology, UCMS & Guru Teg Bahadur Hospital, Delhi, India
2Department of Community Medicine, UCMS & Guru Teg Bahadur Hospital, Delhi, India
3Divisioin of Biotechnology & Molecular Biology, National Centre for Disease Control, New Delhi, India
*Corresponding Author: Shukla Das, Professor, Department of Microbiology, UCMS & Guru Teg Bahadur Hospital, Dilshad Garden, Delhi - 110095, India.
Published: June 01, 2017
Citation: Shukla Das., et al. “Emergence of Extended Spectrum Beta Lactamases Producing Multi Drug Resistant Diarrheagenic Escherichia coli in Children Under Five Years”. Acta Scientific Medical Sciences 1.1 (2017).
Extended-spectrum b-lactamase producing Diarrhoeagenic Escherichia coli (DEC) have spread rapidly worldwide and impose a serious threat to human health, especially in children. The aim of our study was to compare phenotypic and genotypic methods for the detection of Extended-Spectrum Beta-Lactamases in children less than five years of age. A total of 120 Diarrhoeagenic E.coli isolates were subjected to antibiotic susceptibility testing by disc-diffusion method as per CLSI guidelines followed by combined disc test for confirmation. Further, molecular identification of ESBL genes were performed by multiplex PCR. All isolates were examined for the presence of CTX-M, TEM, OXA and SHV genes. Among them, 79/120 (65.83%) were resistant to Cefoxatime and Ceftazidime, of which 76/120 (63.33%) were ESBL positive by combined disc test. However, the numbers of isolates determined positive for ESBL by genotypic method were 77/120 (64.16%). One isolate showed the presence of all four ESBL genes. Eight out of 120 isolates (6.66%) were ESBL positive by PCR but negative by combined disc test. The results showed that some antibiotic sensitive isolates were also carrying ESBL genes. Such isolates have the potential to turn resistant later. The genotypic method for the detection of resistance genes was more consistent in comparison to the phenotypic methods.
Keywords: Extended Spectrum Beta Lactamases; diarrhoeagenic Escherichia coli ; multiplex PCR
One of the important resistant mechanisms in gram-negative bacteria against beta-lactam antibiotics is the production of beta-lactamase enzyme  . Evolution of beta lactamase enzyme was due to the use of new broad-spectrum antibiotics such as Cephalosporins which are used in the treatment of bacterial infections  Extended-spectrum Beta-lactamases (ESBLs) recognized in 1980s, in Klebsiella species and later in Escherichia coli and other gram-negative bacilli are currently spreading rapidly amongst other members of enterobacteriaceae, largely due to genes located on plasmids that can distribute across species barriers [3,4] . Infections with ESBL-producing bacteria are associated with nearly twice the mortality compared to non-ESBL producers  Carriage of resistance by commensal enterobacteriaceae strains in the gut may serve as a reservoir of resistance genes that may subsequently be acquired by strains that cause clinically significant infection  . More than 200 types of ESBLs have been found worldwide, which comprised of mainly 157 TEM, 101 SHV and 65 CTX-M variants [6,7] . The ESBL producing diarrhoeagenic E.coli can be detected either by phenotypic or genotypic methods. Phenotypic methods are not always consistent  , hence genotypic methods have proven to be a more useful option for detection. Moreover, carriage of ESBL producing diarrhoeagenic E.coli isolated from fecal samples in pediatric population can create a larger threat to the community through widespread transmission of these strains. Thus, the aim of our study was to detect the burden of ESBL producing diarrhoeagenic E.coli in fecal sample of children with diarrhea, children admitted to the hospital and on antibiotics for illness other than diarrhea and amongst healthy children, by phenotypic and genotypic methods.
Patients: The study population included three groups. Group 1 included 40 children with acute diarrhea (less than 72 hour duration) attending the Out Patient Department (OPD) and not receiving any antibiotic. Group-2 comprised of 40 children hospitalized and receiving antibiotic (oral or I/v for 72 hr or more or for conditions other than diarrhea). Group 3 included 40 healthy children below 5 years of age who were not suffering from diarrhea or any other disease, as controls. The study protocol was approved by institutional ethical committee.Processing of samples
Isolation: Fresh stool samples were inoculated on MacConkey agar plates and incubated aerobically at 37°C for 24 hours and lactose fermenting colonies were identified as E.coli by standard biochemical tests  . Two or three lactose fermenting colonies, were selected and inoculated (lawn culture) on Muller Hinton agar for antibiotic susceptibility testing and DNA extraction. Diarrhoeagenic E.coli was detected by multiplex pcr for virulent genes  .Antimicrobial Susceptibility Testing
Antimicrobial Susceptibility Testing was performed on Mueller-Hinton agar plates by the Kirby Bauer disc diffusion method as per CLSI guidelines  . All clinical isolates were screened for ESBL production by phenotypic method. Zone of inhibition < 22 mm with Ceftazidime (30 μg) and ≤ 27 mm with Cefotaxime (30 μg) were considered as a potential ESBL producer and were further confirmed by combination disc diffusion test  . E.coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as negative control and positive control respectively.ESBL confirmation
Double Disk Synergy Test (DDST) was performed on ceftazidime/cefotaxime resistant strains by placing disks of Ceftazidime (30 μg) or cefotaxime (30 μg) at a distance of 20 mm from the ceftazidime/clavunate (20/10 μg) disc on Mueller-Hinton agar plates. Enhanced zone of inhibition > 5 mm with ceftazidine/clavunate disc was considered as positive for ESBL production  . All diarrhoeagenic E.coli isolates irrespective of ESBL production by DDST were also tested for the presence of ESBL genes by multiplex PCR.
Primers used for amplifying the sequences were based on previously published literature [13,14] . Primer sequences for TEM F-AGT-GCTGCCATAACCATGAGG & R-CTGACTCCCCGTCGTGTAGATA (431bp), SHV-F-GATGAACGCTTTCCCATGATG & R-CGCTGTTATCGCTCATGG - TAA (214bp), OXA- F-ATTATCTACAGCAGCGCCAGTG & R-TGCATCCACGTCTTTGGTG (296bp), CTX-M- F-GACAAAGAGAGTGCAACGGATG & R-TCAGTGCGATCCAGACGAAA (501bp) and 16s RNA- F-CCCCCTGGACGAAGACTGAC & R-ACCGCTGGCAACAAAGGATA (401bp).Genotypic Assay
DNA extraction: Lactose fermenting colonies on MacConkey agar were isolated and confirmed as E.coli biochemically and genotypically as DEC by multiplex pcr. Genomic DNA extraction was done using RBC (Real Biotech Corporation) kit as per manufacturer’s instructions. All confirmed ESBL producing isolates by phenotypic method were subjected to molecular testing to detect ESBL producing genes.
PCR conditions : Each multiplex PCR test was performed in 0.2 ml pcr tube, containing total volume of 25 μl including, 2.5 μl buffer (10X), 1 μl dNTP’s (200 μM), MgCL 2 1.5 μl (1.5 mM), 1 μL primer (10 μM for each primer forward and reverse), 5ul of the DNA and water to make up the volume. All the four primer pairs of ESBL gene were added in the same tube along with 16sRNA gene primer .
PCR cycle: After an initial denaturation at 95°C for 10 mins the pcr tubes were subjected to 35 amplification cycles of 40 sec at 94°C, 30 sec at 60°C and 40 sec at 72°C, and final extension at 72°C for 7 mins  . Amplified PCR products were analyzed by gel electrophoresis on 1% agarose gel stained with ethidium bromide at 140 volts for 25 mins in a 10 well apparatus. A molecular marker of 100bp was used to determine the size of the amplicons. An internal control 16s RNA (401bp) was also used as amplification internal quality control  .
ESBL producing isolates were subjected to sequencing using same set of primers, as discussed earlier. Purification of the PCR products of ESBL genes and DNA sequencing was done commercially (Yaazh xenomics, Chennai). Analysis of nucleotide sequences was done by performing nucleotide BLAST in internet and CLUSTAL W2 & MEGA (Molecular Evolutionary Genetics Analysis) version 6.06 software was used for phylogenetic tree production.
DNA sequence analysis (DNA blast search) of the PCR product of each of the four tested primer pairs showed high identity levels ranging from 96 to 100% to the Gene Bank sequence database, confirming the specificity of the primers.
Statistical Analysis: Statistical Analysis was done using Statistical Package for Social Sciences (SPSS) (SPSS; Version 20.0). Chi square test and Fisher’s exact test were used to determine statistical significance of data. P value < 0.05 was considered significant. Multivariable logistic regression was done to determine the risk of predominant ESBL infection with increase in age in the three groups, of study subjects.
Total of 120 stool specimens, over a period of one year from July 2012 to July 2013 were collected from children from different categories as mentioned earlier. There was a male preponderance of 60.83% versus 39.16% of females with a mean age of 1.91 years (p = 0.348) in all the groups. DEC was detected in 106/120 (88.33%) isolates that included 39, 40 and 27 isolates in three groups respectively (data not shown). The combined disk test detected 76/120 DEC isolates as ESBL producer (p > 0.05) by phenotypic method.
The antibiotic resistance pattern of DEC isolates is depicted in Table 1. The antibiotic resistant pattern of the rest of non diarrhogenic E.coli was almost similar as that of DEC but it cannot be compared because of its small number.
|Antibiotic resistant||Group 1-39 n**(%)||Group 2-40 n**(%)||Group 3-27 n**(%)||TOTAL-106 n**(%)||P value|
|Norfloxacin (NOR)||9 (23.07)||7 (17.5)||10 (37.03)||26 (24.52)||0.709|
|Cefotaxime (CTX)||27 (69.23)||33 (82.5)||7 (25.92)||67 (63.2)||0.01*|
|Imipenem (IMP)||5 (12.82)||3 (7.5)||0||8 (7.54)||0.095|
|Meropenem (MEM)||2 (5.12)||1 (2.5)||0||3 (2.83)||0.772|
|Ceftazidime (CAZ)||8 (20.51)||4 (10)||0||12 (11.32)||0.006*|
|Aztreonam (ATM)||5 (12.82)||6 (15)||0||11 (10.37)||0.046*|
|Nalidixic acid (NAL)||8 (20.51)||0||0||14 (13.20)td>||0.01*|
|Amoxicillin (AMX)||1 (2.56)||2 (5)||0||3 (0.94)||0.772|
|Gentamicin (GEN)||15 (38.46)||14 (35)||2 (7.4)||31 (29.24)||0.001*|
|Ciprofloxacin (CIP)||20 (51.28)||17 (42.5)||20 (74.07)||57 (53.77)||0.740|
|Ampicillin (AMP)||2 (5.12)||2 (5)||0||4 (3.77)||0.544|
|Amikacin (AMK)||9 (23.07)||14 (35)||0||23 (21.69)||0.01*|
|Polymixin B (PMB)||1 (2.56)||0||0||1 (0.94)td>||1.000|
|Cefotaxamine+clauvinic acid (CCA)||0||1 (2.5)||0||1 (0.94)||1.000|
|Ceftriazone (CRO)||0||1 (2.5)||1 (3.7)||2 (1.88)||1.000|
|Piperacillin+tazobactam (TZP)||10 (25.64)||9 (22.5)||2 (7.4)||21 (19.81)||0.037*|
Table 1: Frequency of resistance to antimicrobial agents of DEC isolates from three study groups.
**Antibiotic frequencies are presented as absolute numbers (n) with percentage in parentheses.
Age and sex wise distribution of ESBL production by phenotypic and genotypic methods is shown in Table 2 The odds of presence of ESBL infection in group 1 was 2.455 times higher when compared to the odds of presence of ESBL infection in group 3.Similarly, the odds of presence of ESBL infection in group 2 was 1.909 times higher in comparison to the odds of the presence of ESBL infection in group 3 as shown in Table 3.
|Groups||Sex||Average Age (Years)||Phenotypic||Genotypic|
|Male||Female||ESBL Positive by Screening||ESBL Confirmation by DDST||ESBL by PCR|
|Group 1 (n = 40)||24 (60)||16 (40)||2.17||28 (70)||27 (67.5)||27 (67.5)|
|Group 2 (n = 40)||20 (50)||20 (50)||1.74||29 (72.5)||27 (67.5)||27 (67.5)|
|Group 3 (n = 40)||29 (72.5)||11 (27.5)||3.88||22 (55)||22 (55)||23 (57.5)|
|Total (n = 120)||73 (60.83)||47 (39.16)||2.59||79 (65.83)||76 (63.33)||77 (64.16)|
|P value (Group 1,3)||0.091||0.061||0.311||0.251||0.485|
|P value (Group 2,3)||0.011*||0.166||0.311||0.251||0.485|
Comparison of ESBL genes by phenotypic and genotypic methods.
*significant P values Gene frequencies are presented as absolute numbers with percentage in parentheses.
|***ESBL infection||Age (1-29 months) (30-60) months||P value||Odds ratio 95% CI** (lower-upper)|
|(Group 1 and 3)||ESBL+||30||10||0.061||2.455 (0.950-6.339)|
|(Group 2 and 3)||ESBL+||28||12||0.166||1.909 (0.761-4.788)|
Association of ESBL production with different age groups.
*significant P values
** CI = confidence interval
***ESBL = extended spectrum beta lactamases.
The genotyping results of ESBL producing isolates obtained by multiplex PCR are shown in Figure 1. Of the total 77 ESBL producers (27, 27 and 23 from group 1, 2 and 3 respectively) were detected from 120 E.coli isolates, TEM gene alone was present in 49 (40.83%), SHV alone in 39 (32.5%), CTX-M alone in 23 (19.16%) and OXA alone in 20 (16.66%) samples as shown in Table 4 (a,b).
|Group-1 (n = 40)||Phenotypic methods (ESBL)||TEM||SHV||CTX||OXA||TEM + SHtV||TEM + CTX||TEM + OXA||SHV + CTX||SHV + OXA||CTX + OXA||TEM + SHV + CTX + OXA|
|Positive (n = 27)||17 (62.96)||12 (44.44)||7 (25.92)||6 (22.22)||7 (25.92)||4 (14.81)||4 (14.81)||3 (11.11)||2 (7.4)||0||0|
|negative (n = 13)||2 (15.38)||2 (15.38)||0||0||0||0||0||0||0||0||0|
|Odds ratio 95% CI (lower-upper)||1.357 (0.559- 3.292)||1.269 (0.487- 3.311)||0.848 (0.275- 2.613)||0.832 (0.253- 2.737)||1.000 (0.316- 3.169)||1.370 (0.286- 6.559)||0.778 (0.193- 3.137)||0.730 (0.152- 3.492)||0.474 (0.082- 2.746)||2.111 (1.666- 2.676)||2.026 (1.620- 2.533)|
(a)- Between group 1 and 3.
|Group-2 (n = 40)||Phenotypic methods (ESBL)||TEM||SHV||CTX||OXA||TEM + SHtV||TEM + CTX||TEM + OXA||SHV + CTX||SHV + OXA||CTX + OXA||TEM + SHV + CTX + OXA|
|Positive (n = 27)||14 (51.85)||12 (44.44)||8 (29.62)||7 (25.92)||8 (29.62)||4 (14.81)||2 (7.4)||4 (14.81)||3 (11.11)||3 (11.33)||0|
|negative (n = 13)||0||2 (15.38)||0||0||0||0||0||0||0||0||0|
|Odds ratio 95% CI (lower-upper)||0.808 (0.326- 2.000)||1.420 (0.549- 3.673)||1.517 (0.536- 4.293)||1.000 (0.316- 3.169)||1.347 (0.434- 4.180)||1.370 (0.286- 6.559)||0.368 (0.067- 2.023)||1.000 (0.232- 4.310)||0.730 (0.152- 3.492)||0.730 (0.152- 3.492)||2.026 (1.620- 2.533)|
(b)- Between group 2 and 3.
Presence/absence of TEM, SHV, CTX-M and OXA genes in E.coli isolates by Multiplex PCR
*significant P values
** CI = confidence interval
Figure 1: Multiplex PCR products of SHV, TEM, CTX-M and OXA genes run on 1.5% agarose gel. Lanes 2-6: E. coli isolated from patients; lane 1 & 7:100 bp size marker. An internal control 16S RNA was also used as internal control (401bp).
Eight isolates of E.coli were (6.5%) positive for ESBL production by PCR but negative by combined disc test. Specimens that revealed ESBL production by multiplex PCR were further confirmed by uniplex (Figure 2).
Co production of TEM and SHV was detected in 22 isolates followed by TEM and CTX in 11 isolates; TEM and OXA in 11 isolates and SHV with CTX also in 11 isolates Table 4. The presence of TEM, SHV and CTX genes alone were also detected in ESBL negative isolates (by combined disk test).
DNA sequence analysis (DNA blast search) of the PCR product of each of the tested primer pairs showed high identity levels ranging from 96 to 100% to the Gene Bank sequence database. We obtained the following accession numbers KP973433, KP973432, KP973428, KP973427 etc. (Figure 3) No mutations were detected in these genes, when we compared our sequences with already existing sequence in NCBI database.
Figure 2: A Uniplex PCR: of SHV, TEM, CTX-M and OXA genes run on 1.5% agarose gel. Lanes 2-6: E. coli isolated from patients; lane 1 & 7: 100 bp size marker.
Figure 3: Phylogenetic tree showing evolutionary history using the Neighbor-Joining method.The evolutionary distances were computed using the Maximum Composite Likelihood method.
Data regarding the prevalence and composition of various types of extended-spectrum beta lactamases (ESBL) in children under five is sparsely available. Current prevalence of ESBL producing organisms is widely variable globally and reported to be between < 1 percent to 74 percent  . This variation may be due to the presence of mobile genetic elements. CTX-M type ESBL is associated with a highly complex genetic structure and harbor ESBL genes and mobile elements which are also found in a variety of plasmids and often carry antibiotic resistance genes.
Majority of isolates in our study were multidrug resistant. The definition most frequently used for multidrug resistant bacteria is ‘resistant to three or more antimicrobial classes’ [18-23] Most DEC isolates were predominantly sensitive to polymixin B, ceftriazone, amoxicillin & cefotaxamine + clauvinic acid. Resistance was seen with cefotaxime (69.23%, 82.5%, 25.9%) followed by ciprofloxacin (51.28%, 42.5%, and 74.07%) and norfloxacin (23.07%, 17.5%, and 37.03%) (p < 0.05) in isolates from three groups respectively. High rates of ESBL-positive isolates are recorded in different parts of India, and not restricted to any single city or region  . Our study also showed high ESBL production (67.5 percent) in group 1 and 2 which was slightly higher than our healthy group  . It was observed that, children admitted to the hospital receiving antibiotics harbored high ESBL producing E.coli similar to children with acute diar - rhea, suggesting the survival of resistant strains in the community both as pathogen as well as commensals . Further, it also indicates that the existing system of extensive use of antibiotic in pediatric population will provide opportunity for the resistant strains to estab - lish their population in the gut. Our observation suggest, that high ESBL producing strains are likely to emerge, due to indiscriminate use of these antibiotics as indicated by high cefotaxime resistance. Among neighboring countries, ESBL production in Islamabad and Rawalpindi was found to be 48 and 35 % respectively among E. coli isolates ; 9.2 % in Korea and 10.3 % in Arabia [26,27] .
Molecular epidemiology of carriage of ESBL-producing DEC, in 40 healthy children depicted 57.5 percent as ESBL-producers which was higher than cited in other limited studies [28,29] . TEM gene was found in 47.5%, 40% and 35% in groups 1, 3 and 2 respectively. The existence of SHV gene was higher in group 1 and 2 (35%) in comparison to healthy group (27.55%). The production of CTX and OXA genes was more frequent in group 2 and 3 with 20% and 17.5% as compared to 17.5% and 15% in group 3. This pattern of occur - rence of ESBL genes (tem followed by shv, ctx and oxa) is similar to other studies [30,31] Presence of possibility of other ESBL genes cannot be ignored, as we detected only four common ESBL genes among other 200-300 known genes. Detection of ESBL production by phenotypic and genotypic methods showed marginal difference. The genotypic method has a higher reproducibility as compared to the phenotypic methods and proves to be a better tool for detecting ESBL producing isolates of diarrhoeagenic E.coli .
Studies from different countries report erratic prevalence of ESBL producing E.coli colonization [32-37] . Enhanced pathogenicity of the bacteria carrying beta-lactamase genes increases the mortality risk of infected individuals and pose a threat to the community [38,39] . TEM and SHV genes have a high rate of occurrence in E.coli as seen in our study  . The chance of occurrence of both the genes is almost similar, since SHV-1 shares 68 percent similarity of its amino acids with TEM-1 and has a similar overall structure [41,42] . Therefore, TEM and SHV genes can be used for the molecular screening of ESBL positive samples in Indian isolates. Incorrect identification of antibiotic resistance may lead to uncontrolled spread of resistant genes; hence a continuous surveillance of antibiotic resistant pathogens in hospitals, should become a priority.
In order to increase the accuracy of the results, sequencing with both forward and reverse primers of ESBL genes obtained by chromatograms and predicted amino acid sequences, showed no mutations.
ESBL-producing DEC is a major concern in children suffering from diarrhea. The pediatric population is at a higher risk of acquiring multidrug resistance flora, due to limitation in therapeutic options and restricted prescription policy of harsher antibiotics especially the use of cephalosporins. The results of this study demonstrated a need for heightened awareness regarding the increasing frequency of these highly resistant isolates as reservoirs in pediatric population and their potential impact on transmission to community and hospital environment. This observation also underscores the need to improve microbiological diagnostic facilities and antibiotic resistance surveillance in resource-poor settings; to be able to effectively revise antibiotic regimens and avoid emergence of resistance.
This work was supported in part by the Council of Scientific and Industrial Research, Library Avenue, Pusa, New Delhi 110012 India, (projects 08/532 (0007)/2011-EMR-I. Special thanks to all children and their parents) that participated in the research. We also thank all the staff members of our department for their support.
Council of Scientific and Industrial Research, Library Avenue, Pusa, New Delhi 110012 India. The work is attributed to the Department of Microbiology, UCMS and GTB Hospital, Dilshad Garden, Delhi – 110095, India.
Copyright: © 2017 Shukla Das., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.