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Association of phylogenetic distribution and presence of integrons with multidrug resistance in Escherichia coli clinical isolates from children with diarrhoea
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Association of phylogenetic distribution and presence of integrons with multidrug resistance in Escherichia coli clinical isolates from children with diarrhoea Yesmi Patricia Ahumada-Santos a,1 , María Elena Báez-Flores a,1 , Sylvia Páz Díaz-Camacho b , Magdalena de Jesús Uribe-Beltrán a , Carlos Alberto Eslava-Campos c,d , Jesús Ricardo Parra-Unda a , Francisco Delgado-Vargas a,∗ a
School of Chemical and Biological Sciences, Autonomous University of Sinaloa, Ciudad Universitaria, Culiacan, Sinaloa, Mexico Research Unit in Environment and Health, Autonomous University of Occident, Culiacan, Sinaloa, Mexico School of Medicine, National Autonomous University of Mexico, Ciudad Universitaria, Coyoacan, 04510, Ciudad de Mexico, Mexico d Laboratory of Bacterial Pathogenicity, Hemato Oncology and Research Unit, Hospital Infantil de Mexico Federico Gomez 06720, Ciudad de Mexico, Mexico b c
a r t i c l e
i n f o
Article history: Received 29 July 2019 Received in revised form 29 October 2019 Accepted 26 November 2019 Keywords: Escherichia coli Phylogeny Integrons Antimicrobial resistance Diarrhoea
a b s t r a c t Background: Escherichia coli strains include both commensal and virulent clones distributed in different phylogenetic groups. Antimicrobial resistance is an increasingly serious public health threat at the global level and integrons are important mobile genetic elements involved in resistance dissemination. This paper aims to determine the phylogenetic groups and presence of class 1 (intl1) and 2 (intl2) integrons in E. coli clinical isolates from children with diarrhoea, and to associate these characteristics with their antimicrobial resistance. Methods: Phylogeny and presence of integrons (intl1 and intl2) were analysed by PCR and amplicon sequencing in 70 E. coli isolates from children with and without diarrhoea (35 of each group) from Sinaloa, Mexico; these variables were analysed for correlation with the antimicrobial resistance profile of the isolates. Results: The most frequent phylogroups were A (42.9%) and B2 (15.7%). The E. coli isolates from children with diarrhoea were distributed in all phylogroups; while strains from children without diarrhoea were absent from phylogroups C, E, and clade I. The 17.1% of the isolates carried integrons (15.7% intI1 and 1.4% intI2); 28.6% of the isolates from children with diarrhoea showed the class 1 integron. Strains of phylogroup A showed the highest frequency of integrons (33.3%). The association of multidrug resistance and the presence of integrons was identified in 58.3% of strains isolated from children with diarrhoea included in phylogroups A and B2. The sequence analysis of intl1 and intl2 showed silent point mutations and similarities with plasmids of some APEC and AIEC strains. Conclusion: Commensal E. coli strains are potential disseminators of antimicrobial resistance, and the improvement in the use of antimicrobials to treat childhood diarrhoea is essential for the control of such resistance. © 2019 The Author(s). Published by Elsevier Ltd on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/).
Introduction
Abbreviations: PCR, Polymerase Chain Reaction; APEC, Avian Pathogenic Escherichia coli; AIEC, Adherent-Invasive Escherichia coli; dNTP, deoxynucleotide triphosphates; UPEC, Uropathogenic Escherichia coli; EPEC, Enteropathogenic Escherichia coli; ExPEC, Extraintestinal Pathogenic Escherichia coli; UPGMA, Unweighted Pair Group Method with Arithmetic Mean. ∗ Corresponding author. E-mail address: [email protected] (F. Delgado-Vargas). 1 These authors contributed equally to this work.
Escherichia coli strains include both commensal and virulent clones; the former are bacteria of the human intestinal microbiota, while the virulent clones are pathogens that cause intestinal or extraintestinal diseases [1]. Considering the great bacterial diversity, their genomic analysis has been performed using different procedures. A multiplex PCR assay has been employed in order to describe eight different phylogroups, seven of which (A, B1, B2, C, D,
https://doi.org/10.1016/j.jiph.2019.11.019 1876-0341/© 2019 The Author(s). Published by Elsevier Ltd on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Ahumada-Santos YP, et al. Association of phylogenetic distribution and presence of integrons with multidrug resistance in Escherichia coli clinical isolates from children with diarrhoea. J Infect Public Health (2019), https://doi.org/10.1016/j.jiph.2019.11.019
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E, and F) belong to E. coli sensu stricto and the eight to cryptic clade I [2]. Different studies have shown that virulent strains belonging to phylogroups B2 and D are principally associated with extraintestinal infections, while those in other phylogroups are diarrhoeagenic and commensal [3]. However, it has been established that the commensal or pathogen concepts do not always show a relationship with the terms phylogroups [e.g., 4] or serotypes [e.g., 5]; and that diarrhoea does not only depends on the infection with a diarrheogenic strain but also of the genetic and nutritional characteristics of host [6]. Independently, E. coli strains are some of the most common etiological agents of moderate to severe diarrhoea, being responsible for the death of 525,000 children under the age of five in the world [7]. Antimicrobial resistance is an increasingly serious public health threat at the global level [8]. In this regard, the horizontal transfer of genes mediated by transposons and plasmids is one of the main mechanisms for the dissemination of antimicrobial resistance, and integrons are important mobile genetic elements involved in this dissemination, mostly in Gram-negative bacteria [9–11]. Integrons contain three essential elements: the gene intI encoding an integron integrase (IntI) which catalyses the excision and integration of the gene cassettes where the antibiotic resistance gene is located, a recombination site associated with the integron (attl), and a promoter (Pc) that directs the transcription of the captured gene. The amino acid sequences of IntIs have been used to classify them into five classes, and classes 1, 2, and 3 have been implicated in the distribution of resistance genes [9,11,12]. The aim of this study was to determine the phylogenetic groups and the presence of class 1 and 2 integrons in E. coli strains isolated from children with and without diarrhoea and their association with the previously evaluated antimicrobial resistance profiles.
Materials and methods Sample collection Stool samples from 49 children under 5 years of age were collected using a rectal swab on Cary Blair medium, transported to the laboratory, and plated on MacConkey agar (Becton Dickinson). Typical E. coli colonies were identified using the automated VITEK system. The identified E. coli strains were cryopreserved at −70 ◦ C until use. The isolates included in this study were selected based on their resistance profile. The children were outpatients from two hospitals (The Civil Hospital of Culiacan and The ‘Dr. Rigoberto Aguilar Pico’ Paediatric Hospital of Sinaloa) in the city of Culiacan, Sinaloa, Mexico, during the autumn/winter of 2003/04.
DNA extraction Bacterial DNA was extracted by the boiling method [13]. DNA purity and concentration were measured by spectrophotometry, integrity was observed in agarose gels at 2% and the samples were kept at −20 ◦ C until use.
Determination of phylogenetic groups Classification of the E. coli isolates into phylogenetic groups was carried out by multiplex PCR [2]. The primer pairs used are listed in Table S1. In the negative control, water was used instead of the DNA template; and in the positive controls, the strains E. coli K12 (arpA and yjaA) and UPEC CFT073 (chuA, yjaA and TspE4.C2) were used. The amplified PCR products were analysed in 1.7% agarose gel stained with GelRed using a transilluminator.
PCR detection of integrase genes The presence of integrase genes (intIs) for the class 1 (intI1) and 2 (intI2) integrons was determinated [14]. The reaction mixture contained 3 L buffer (5X), 0.9 L MgCl2 (25 mM), 0.15 L dNTP (10 mM), 0.6 L of each primer (100 M), 1.05 U of Taq polymerase, 0.5 g of genomic DNA, and the volume was made up to 15 L with free nuclease water. The PCR amplification conditions were as follows: denaturation for 5 min at 94 ◦ C; 45 cycles of 1 min at 94 ◦ C, 1 min at 55 ◦ C (intI1) or 59 ◦ C (intI2) and 1 min at 72 ◦ C; and 5 min at 72 ◦ C as a final extension. The primer pairs used are shown in Table S1. Water instead of DNA template was used as the negative control, while E. coli strains positive for the intI1 (048) and intI2 (150) genes were the positive controls; these were obtained from the strain stock of the School of Medicine of the National Autonomous University of Mexico [15]. The amplicons were analysed in 1.7% agarose gel and purified using the Wizard® SV Gel and PCR CleanUp System (Promega). These amplicons were sequenced in both directions (Macrogen Inc.). The sequence analysis was carried out using CLUSTALW software, the GenBank database (Table S2), and the BLAST algorithm on the NCBI website. Evolutionary relationship analysis of intI1 and intI2 sequences Partial sequences of the intI1 and intI2 genes obtained in this study (Table S3) were aligned with those of E. coli strains of a different geographical/biological origin, which are available in GenBank (Table S2), using CLUSTAL W [16], MUSCLE algorithms [17] with MEGA7 (32-bit) MacOS, and Graphical software [18]. Phylogenetic reconstruction analysis was based on the UPGMA statistical test [19]; the conditions employed were 1000 bootstrap replications, the model of the number of differences applied by uniform rates among sites, a homogeneous pattern among lineages, partial detection for missing data treatment, and a 95% site coverage cut-off [20]. Statistical analysis The data were analysed using Stata version 14.0 software. The association between the phylogenetic groups and the presence of integrons for both groups of study as well as with the antimicrobial resistance profile was performed using the Chi-square test. The level of significance was set at p < 0.05. Results Seventy E. coli isolates of stool samples from 49 children were analysed: 35 isolates from 25 children with diarrhea and 35 isolates from 24 children without diarrhea as the control group. The group of children with diarrhea (48% female, 52% male) was an average age of 14.7 months and 56% under 1 year, while the group of control children (54.5% female, 45.5% male) was an average age of 26 months and 27.3% under 1 year. In all the stool samples, the presence of intestinal parasites, Shigella spp., Salmonella spp., Campylobacter spp., Yersinia spp., and Vibrio spp. as causative agents of diarrheal infection was discarded. In both groups, the frequency of resistance was higher for the beta-lactams family, 68.6% and 42.9% for children with and without diarrhoea, respectively [21]. The 70 analysed E. coli isolates were classified into every phylogroup defined up to the present time (Table 1). All of the phylogroups were identified in the E. coli isolates from children with diarrhoea, while phylogroups C, E, and clade I were absent in the isolates from children without diarrhoea. Phylogroup A was the most frequent in strains from both groups of children, 34.3% with and 51.4% without diarrhoea. The statistical analysis showed that
Please cite this article in press as: Ahumada-Santos YP, et al. Association of phylogenetic distribution and presence of integrons with multidrug resistance in Escherichia coli clinical isolates from children with diarrhoea. J Infect Public Health (2019), https://doi.org/10.1016/j.jiph.2019.11.019
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Table 1 Distribution of Escherichia coli isolates among phylogenetic groups. Phylogenetic Group
Children with diarrhoea n (%)
Children without diarrhoea n (%)
Total n (%)
A B1 B2 C D E F Clade I Unassignable Total
12 (34.3) 3 (8.6) 7 (20.0) 1 (2.9) 2 (5.7) 1 (2.9) 4 (11.4) 2 (5.7) 3 (8.6) 35 (100)
18 (51.4) 1 (2.9) 4 (11.4) 0 (0) 4 (11.4) 0 (0) 3 (8.6) 0 (0) 5 (14.3) 35 (100)
30 (42.9) 4 (5.7) 11 (15.7) 1 (1.4) 6 (8.6) 1 (1.4) 7 (10.0) 2 (2.9) 8 (11.4) 70 (100)
Table 2 Distribution of class 1 (intl1) and class 2 (intl2) integrons in the Escherichia coli isolated from children with and without diarrhoea. Integron
Children with diarrhoea n (%)
Children without diarrhoea n (%)
Total n (%)
intI1 intI2 None Total
10 (28.6) 1 (2.9) 24 (68.6) 35 (100)
1 (2.9) 0 (0) 34 (97.1) 35 (100)
11 (15.7) 1 (1.4) 58 (82.9) 70 (100)
the frequency of E. coli isolates in the phylogroups was similar in both groups studied (X2 = 9.96, p = 0.286). In 17.1% of the E. coli isolates were identified class 1 (15.7%) or class 2 (1.4%) integrons. In the strains isolated from children with diarrhoea, the frequency of the class 1 integron was higher (28.6%) than that of the class 2 integron (2.9%). On the other hand, in the isolates of the control group was only identified the class 1 integron (2.9%). None of the strains showed the presence of both classes of integrons (Table 2). The integrase gene sequences of class 1 (intI1) and 2 (intI2) integrons of the E. coli isolates showed >99% identity to those reported in Genbank. Compared with published intl1 sequences, three of the 11 partial sequences of the E. coli isolates showed single-nucleotide changes at positions 60 (1/11) and 65 (2/11), permitting their classification by evolutionary relationship analysis into four different groups with similar sequence frequencies: 27.3% for three groups and 18.2% for the fourth group (Fig. 1). The same analysis of the partial sequences of intI2 showed >99% identity to sequences reported in plasmids and transposons, and evolutionary relationship analysis separated the sequences into three groups with the intl2 sequence as a member of the third group comprising six reference sequences (Fig. 2). For both analyses, the reference sequences selected as external controls (Table S2) are in separate branches (Figs. 1 and 2). Discussion The triplex PCR method is mostly used in phylogeny studies of commensal and diarrhoeic E. coli isolates but only considers phylogroups A, B1, B2, and D [22]; the quadruplex PCR method, used in this work, additionally includes phylogroups C, E, F, and cryptic clade I, which together classify approximately 13% of E. coli isolates [2]. In this study, 15.7% of the isolates were assigned to these phylogroups, and similar values have been reported previously [23,24]. The E. coli isolates were non-uniformly distributed among the eight phylogenetic groups, with A and B2 being the most frequent (Table 1). Similar results were obtained for E. coli isolates from faecal samples in India [24,25], Paris [23], and the United States [26]. Escherichia coli strains isolated from humans mainly belong to phylogenetic groups A and B2 [27], and the results in this study indicate
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that 58.6% of the isolates were in these phylogroups. In this regard, the prevalence of different phylogenetic groups among different human populations may differ [27–29]; this associated to socioeconomic factors, dietary habits, use of antibiotics, level of hygiene, geographical and climatic conditions, and the genetics of the host [23]. In this study, although the distribution of the E. coli isolates within the phylogroups was statistically similar between both groups of children, the frequency of strains included in phylogroup B2 was higher in the children with diarrhoea. Similar observations have been obtained in E. coli isolates from children in India [24], Pakistan [30], and Romania [31]. Phylogroup A has been reported as the most common group for isolates from children without diarrhoea [4]. Our results suggest a relationship between the E. coli isolates belonging to phylogroup B2 and their characteristic of diarrhoea induction, and an association of phylogroup A with commensal E. coli strains. The existence of E. coli isolates not classified into the defined phylogroups has been reported in other studies [24,30]; this phenomenon may be explained by the loss of specific genes due to the plasticity of the genome of E. coli, to the recombination of isolates belonging to two different phylogenetic groups, or to the existence of extremely rare phylogroups [2]. Moreover, cryptic clades are mainly associated with environmental E. coli, which are detected in human faecal samples with a frequency of 2-3% [32]; a similar value was found in this study (2.9%). The two isolates in clade I were from children with diarrhoea and the observed results may be related to a lack of good hygiene habits [3]. The prevalence of integrons in this study (17.1%) was lower than that reported in other studies (23.5%–78.2%) [10,24,25,33,34]. However, the higher prevalence of the class 1 integron (15.7%) compared to that of the class 2 integron (1.4%) corresponds to values previously reported; class 1 is the most identified integron in clinical isolates of Gram-negative and multidrug-resistant bacteria due to its recombination ability [9,11]. Although the presence of integrons was statistically similar in both groups of children, the frequency was higher in the children with diarrhoea (31.4%), suggesting that the presence of integrons in E. coli may be more closely associated with pathogenic than with commensal strains. This association has been observed in other regions of the world [33,35,36]. The susceptibility profile of the studied E. coli isolates was previously reported [21]. All isolates with integrons were resistant to at least one and up to six antimicrobials belonging to three different antimicrobial families, with 58.3% of them being classified as multidrug-resistant (two or more families of antimicrobials). As for the E. coli isolates without integrons, only 34.5% were classified as multidrug-resistant and 8.6% were susceptible to all antimicrobials. These values show the influence of the presence of integrons in the resistance of the studied isolates, as described in other works [10,24,34,36,37]. In this regard, this study shows a lower frequency of integrons in the commensal strains; however, these strains may be potential disseminators of antimicrobial resistance, mediated by horizontal gene transfer [10,29]. In the E. coli isolates with the class 1 integron from the children with diarrhoea, the frequency of resistance was higher for the beta-lactams family (carbenicillin 100%, ampicillin 80%, piperacillin 60%, ticarcillin/clavulanic acid 50%, and cefuroxime sodium 20%), followed by sulfonamides (trimethoprim/sulfamethoxazole 60%), and quinolones (nalidixic acid 10%), while the isolate with the class 1 integron from the children of the control group and the isolate with the class 2 integron from the children with diarrhoea were only resistant to beta-lactams (carbenicillin and cefuroxime sodium) (Table S4). Similar results have been reported [25,34,37]. It has been documented that resistance cassettes found in class 1 integrons confer resistance to all known beta-lactams [12], which corresponds to the resistance profile of the studied isolates (Table
Please cite this article in press as: Ahumada-Santos YP, et al. Association of phylogenetic distribution and presence of integrons with multidrug resistance in Escherichia coli clinical isolates from children with diarrhoea. J Infect Public Health (2019), https://doi.org/10.1016/j.jiph.2019.11.019
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Fig. 1. Evolutionary relationships of the Escherichia coli intl1 gene. 䊉 Sequences generated in the present study. Enterococcus faecalis, Staphylococcus aureus, Mus musculus, and Triticum aestivum were used as out-groups. The evolutionary history was inferred using the UPGMA method. The optimal tree with the sum of branch length = 723.65878788 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the number of differences method and are in the units of the number of base differences per sequence. The analysis involved 26 nucleotide sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Evolutionary analyses were conducted in MEGA7.
Fig. 2. Evolutionary relationships of the Escherichia coli intl2 gene. 䊉 Sequences generated in the present study. Mus musculus, Enterococcus faecalis, Bacillus thuringiensis tenebrionis, and Triticum aestivum were used as out-groups. The evolutionary history was inferred using the UPGMA method. The optimal tree with the sum of branch length = 129.97622549 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the number of differences method and are in the units of the number of base differences per sequence. The analysis involved 18 nucleotide sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Evolutionary analyses were conducted in MEGA7.
S4). The sul1 gene confers resistance to sulfonamides and is typically within the 3’-conserved-segment (3’-CS) of the base structure in the class 1 integron [11]. Of the studied isolates with the intI1,
only 54.5% were resistant to sulfonamide. This suggests the existence of another mechanism of resistance, the probable presence of the gene in another genetic context, or the existence of alleles
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intI1 n (%)
intI2 n (%)
None n (%)
Total with integrons n (%)
A B1 B2 C D E F Unassignable Clade I Total
4 (36.4) 0 (0) 2 (18.2) 1 (9.1) 1 (9.1) 0 (0) 2 (18.2) 1 (9.1) 0 (0) 11 (100)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (100) 1 (100)
26 (44.8) 4 (6.9) 9 (15.5) 0 (0) 5 (8.6) 1 (1.7) 5 (8.6) 7 (12.1) 1 (1.7) 58 (100)
4 (33.3) 0 (0) 2 (16.7) 1 (8.3) 1 (8.3) 0 (0) 2 (16.7) 1 (8.3) 1 (8.3) 12 (100)
[25]. The resistance cassettes in class 2 integrons show less diversity and usually contain genes conferring resistance to trimethoprim, streptothricin, and streptomycin [9]; in this regard, the studied isolate with intI2 was susceptible to trimethoprim, but resistant to the beta-lactams cefuroxime sodium and carbenicillin; this contrast could be due to resistance genes outside the context of the integron structure. The sequences of the intI1 and intI2 genes obtained from clinical samples are highly conserved [38], although some silent point mutations were found in the amplified sequences of the intI1 gene in this study. The intI1 and intI2 genes are associated with the Tn3 and Tn7 transposon families, respectively [9,11]; and these associations were recorded for the studied E. coli isolates (Fig. 1), showing that plasmids and transposons are vehicles for intra- and inter-species integron mobility [11]. The phylogenetic analysis of the intI1 sequences resulted in the formation of four groups; the sequences M4-5 (MK591027) and M21-3 (MK591034) in the first group showed 100% identity to the intI1 gene of the APEC and AIEC isolates, while those of the three remaining groups were highly identical to the intI1 sequences of E. coli strains isolated from animals used as food and from human faeces (Fig. 1). This association has been reported previously with extra-intestinal pathogenic E. coli (ExPEC) strains [39,40]; although the studied isolates were from intestinal origin, 27.3% of them were classified into phylogenetic groups B2 and D, as occurs with the ExPEC strains. As in other works, this similarity suggests that E. coli strains could be transmitted to humans from different niches (e.g., zoonotic or food) [40,41]. In the intI2 gene, the internal stop codon responsible for encoding a characteristic non-functional integrase was identified [11]; and the evolutionary relationship analysis did not show a clear separation between the intl2 sequences of different human and animal isolates, suggesting the circulation of E. coli strains between different ecological niches as for the intI1 gene (Fig. 2). It should be noted that the bioinformatic analysis of the E. coli intI1 and intI2 sequences showed high identity to those of other bacteria, suggesting that integrons are involved in the horizontal transfer of resistance genes across bacteria. The correlation analysis between the variables phylogroup and presence of integrons of the E. coli isolates showed that both groups of children are statistically similar (X2 = 6.7, p = 0.565). However, the isolates belonging to phylogroups B1 and E did not show integrons. Moreover, the highest frequency of integrons was found in the strains of group A (33.3%) (Table 3). Compared with our results, an association among the class 1 integron and phylogroups A, B1, and B2 was reported [24]; as well as between the class 2 integron and phylogroup B2 in EPEC isolates [24]. Similarly, in an adult Mexican population was reported the presence of class 1 integrons in E. coli isolates distributed in groups A, D, and B2; and class 2 integrons only in isolates belonging to phylogroup D [42]. This information contrasts with the results obtained in this study for the class 2 inte-
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gron. However, agrees with the prevalence of phylogroups A and B2 in the E. coli isolates containing integron genes. The commensal strains are associated with phylogroup A, so the presence of integrons primarily in this phylogroup suggests the great problem of resistance induced by the misuse and abuse of antibiotics in the studied population. The correlation analysis between the phylogenetic groups and the susceptibility profile by antimicrobial family of the studied E. coli isolates showed statistical similarity (X2 = 18.1, p = 0.798). Nevertheless, multidrug-resistant isolates were detected predominately in phylogroups A and B2 with resistance from one to 11 and 12 antibiotics, respectively; the isolates from children with diarrhoea showed a higher resistance. Several papers have reported no association between the susceptibility profile and the phylogenetic group assigned to E. coli isolates [23,24]; in this study, multidrugresistant bacteria have been identified within each phylogroup, indicating that the antibiotic resistance problem is found in both commensal and diarrhoeagenic strains and is independent of the phylogroup assigned.
Conclusions The phylogenetic distribution of the strains isolated from faeces of children with and without diarrhoea in the city of Culiacan, Sinaloa, Mexico was similar to that reported in other parts of the world, with a greater frequency of phylogroup A. For the E. coli isolates within phylogroup A, the frequencies of isolates with integrons and multidrug-resistant were high, suggesting that antibiotics are not properly used in children, and consequently that they are potential disseminators of resistance genes and can increase the risk of death. On the other hand, the association of APEC strains with detected integron sequences together with the tendency to classify the isolates of children with diarrhoea in phylogroup B2 suggests that the circulation of E. coli strains between different ecological niches, the existence of zoonotic potential, and the horizontal gene transfer are propagation mechanisms of antibiotic resistance. The information generated emphasises the importance of carrying out regular monitoring of phylogenetic distribution and antimicrobial susceptibility of commensal and pathogenic E. coli strains in each geographical area; such studies can be useful in the creation of proper guidelines for the management of antibiotics among the child population of the locality.
Funding This work was supported by the National Council for Science and Technology from Mexico (CONACYT); and the ‘Programa de Fomento y Apoyo a Proyectos de Investigacion’ (PROFAPI) of the Autonomous University of Sinaloa.
Conflict of interest None.
Acknowledgements Authors acknowledge to Dr. Ignacio Osuna-Ramírez by the statistical-analysis assistance; as well as to Diana Guadalupe Delgado-Solís, Solangel Osorio-Valle, and Stefany Margarita Tamayo-Felix by their assistance in performing different experimental assays.
Please cite this article in press as: Ahumada-Santos YP, et al. Association of phylogenetic distribution and presence of integrons with multidrug resistance in Escherichia coli clinical isolates from children with diarrhoea. J Infect Public Health (2019), https://doi.org/10.1016/j.jiph.2019.11.019
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Association of phylogenetic distribution and presence of integrons with multidrug resistance in Escherichia coli clinical isolates from children with diarrhoea Yesmi Patricia Ahumada-Santos a,1 , María Elena Báez-Flores a,1 , Sylvia Páz Díaz-Camacho b , Magdalena de Jesús Uribe-Beltrán a , Carlos Alberto Eslava-Campos c,d , Jesús Ricardo Parra-Unda a , Francisco Delgado-Vargas a,∗ a
School of Chemical and Biological Sciences, Autonomous University of Sinaloa, Ciudad Universitaria, Culiacan, Sinaloa, Mexico Research Unit in Environment and Health, Autonomous University of Occident, Culiacan, Sinaloa, Mexico School of Medicine, National Autonomous University of Mexico, Ciudad Universitaria, Coyoacan, 04510, Ciudad de Mexico, Mexico d Laboratory of Bacterial Pathogenicity, Hemato Oncology and Research Unit, Hospital Infantil de Mexico Federico Gomez 06720, Ciudad de Mexico, Mexico b c
a r t i c l e
i n f o
Article history: Received 29 July 2019 Received in revised form 29 October 2019 Accepted 26 November 2019 Keywords: Escherichia coli Phylogeny Integrons Antimicrobial resistance Diarrhoea
a b s t r a c t Background: Escherichia coli strains include both commensal and virulent clones distributed in different phylogenetic groups. Antimicrobial resistance is an increasingly serious public health threat at the global level and integrons are important mobile genetic elements involved in resistance dissemination. This paper aims to determine the phylogenetic groups and presence of class 1 (intl1) and 2 (intl2) integrons in E. coli clinical isolates from children with diarrhoea, and to associate these characteristics with their antimicrobial resistance. Methods: Phylogeny and presence of integrons (intl1 and intl2) were analysed by PCR and amplicon sequencing in 70 E. coli isolates from children with and without diarrhoea (35 of each group) from Sinaloa, Mexico; these variables were analysed for correlation with the antimicrobial resistance profile of the isolates. Results: The most frequent phylogroups were A (42.9%) and B2 (15.7%). The E. coli isolates from children with diarrhoea were distributed in all phylogroups; while strains from children without diarrhoea were absent from phylogroups C, E, and clade I. The 17.1% of the isolates carried integrons (15.7% intI1 and 1.4% intI2); 28.6% of the isolates from children with diarrhoea showed the class 1 integron. Strains of phylogroup A showed the highest frequency of integrons (33.3%). The association of multidrug resistance and the presence of integrons was identified in 58.3% of strains isolated from children with diarrhoea included in phylogroups A and B2. The sequence analysis of intl1 and intl2 showed silent point mutations and similarities with plasmids of some APEC and AIEC strains. Conclusion: Commensal E. coli strains are potential disseminators of antimicrobial resistance, and the improvement in the use of antimicrobials to treat childhood diarrhoea is essential for the control of such resistance. © 2019 The Author(s). Published by Elsevier Ltd on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/).
Introduction
Abbreviations: PCR, Polymerase Chain Reaction; APEC, Avian Pathogenic Escherichia coli; AIEC, Adherent-Invasive Escherichia coli; dNTP, deoxynucleotide triphosphates; UPEC, Uropathogenic Escherichia coli; EPEC, Enteropathogenic Escherichia coli; ExPEC, Extraintestinal Pathogenic Escherichia coli; UPGMA, Unweighted Pair Group Method with Arithmetic Mean. ∗ Corresponding author. E-mail address: [email protected] (F. Delgado-Vargas). 1 These authors contributed equally to this work.
Escherichia coli strains include both commensal and virulent clones; the former are bacteria of the human intestinal microbiota, while the virulent clones are pathogens that cause intestinal or extraintestinal diseases [1]. Considering the great bacterial diversity, their genomic analysis has been performed using different procedures. A multiplex PCR assay has been employed in order to describe eight different phylogroups, seven of which (A, B1, B2, C, D,
https://doi.org/10.1016/j.jiph.2019.11.019 1876-0341/© 2019 The Author(s). Published by Elsevier Ltd on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Ahumada-Santos YP, et al. Association of phylogenetic distribution and presence of integrons with multidrug resistance in Escherichia coli clinical isolates from children with diarrhoea. J Infect Public Health (2019), https://doi.org/10.1016/j.jiph.2019.11.019
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E, and F) belong to E. coli sensu stricto and the eight to cryptic clade I [2]. Different studies have shown that virulent strains belonging to phylogroups B2 and D are principally associated with extraintestinal infections, while those in other phylogroups are diarrhoeagenic and commensal [3]. However, it has been established that the commensal or pathogen concepts do not always show a relationship with the terms phylogroups [e.g., 4] or serotypes [e.g., 5]; and that diarrhoea does not only depends on the infection with a diarrheogenic strain but also of the genetic and nutritional characteristics of host [6]. Independently, E. coli strains are some of the most common etiological agents of moderate to severe diarrhoea, being responsible for the death of 525,000 children under the age of five in the world [7]. Antimicrobial resistance is an increasingly serious public health threat at the global level [8]. In this regard, the horizontal transfer of genes mediated by transposons and plasmids is one of the main mechanisms for the dissemination of antimicrobial resistance, and integrons are important mobile genetic elements involved in this dissemination, mostly in Gram-negative bacteria [9–11]. Integrons contain three essential elements: the gene intI encoding an integron integrase (IntI) which catalyses the excision and integration of the gene cassettes where the antibiotic resistance gene is located, a recombination site associated with the integron (attl), and a promoter (Pc) that directs the transcription of the captured gene. The amino acid sequences of IntIs have been used to classify them into five classes, and classes 1, 2, and 3 have been implicated in the distribution of resistance genes [9,11,12]. The aim of this study was to determine the phylogenetic groups and the presence of class 1 and 2 integrons in E. coli strains isolated from children with and without diarrhoea and their association with the previously evaluated antimicrobial resistance profiles.
Materials and methods Sample collection Stool samples from 49 children under 5 years of age were collected using a rectal swab on Cary Blair medium, transported to the laboratory, and plated on MacConkey agar (Becton Dickinson). Typical E. coli colonies were identified using the automated VITEK system. The identified E. coli strains were cryopreserved at −70 ◦ C until use. The isolates included in this study were selected based on their resistance profile. The children were outpatients from two hospitals (The Civil Hospital of Culiacan and The ‘Dr. Rigoberto Aguilar Pico’ Paediatric Hospital of Sinaloa) in the city of Culiacan, Sinaloa, Mexico, during the autumn/winter of 2003/04.
DNA extraction Bacterial DNA was extracted by the boiling method [13]. DNA purity and concentration were measured by spectrophotometry, integrity was observed in agarose gels at 2% and the samples were kept at −20 ◦ C until use.
Determination of phylogenetic groups Classification of the E. coli isolates into phylogenetic groups was carried out by multiplex PCR [2]. The primer pairs used are listed in Table S1. In the negative control, water was used instead of the DNA template; and in the positive controls, the strains E. coli K12 (arpA and yjaA) and UPEC CFT073 (chuA, yjaA and TspE4.C2) were used. The amplified PCR products were analysed in 1.7% agarose gel stained with GelRed using a transilluminator.
PCR detection of integrase genes The presence of integrase genes (intIs) for the class 1 (intI1) and 2 (intI2) integrons was determinated [14]. The reaction mixture contained 3 L buffer (5X), 0.9 L MgCl2 (25 mM), 0.15 L dNTP (10 mM), 0.6 L of each primer (100 M), 1.05 U of Taq polymerase, 0.5 g of genomic DNA, and the volume was made up to 15 L with free nuclease water. The PCR amplification conditions were as follows: denaturation for 5 min at 94 ◦ C; 45 cycles of 1 min at 94 ◦ C, 1 min at 55 ◦ C (intI1) or 59 ◦ C (intI2) and 1 min at 72 ◦ C; and 5 min at 72 ◦ C as a final extension. The primer pairs used are shown in Table S1. Water instead of DNA template was used as the negative control, while E. coli strains positive for the intI1 (048) and intI2 (150) genes were the positive controls; these were obtained from the strain stock of the School of Medicine of the National Autonomous University of Mexico [15]. The amplicons were analysed in 1.7% agarose gel and purified using the Wizard® SV Gel and PCR CleanUp System (Promega). These amplicons were sequenced in both directions (Macrogen Inc.). The sequence analysis was carried out using CLUSTALW software, the GenBank database (Table S2), and the BLAST algorithm on the NCBI website. Evolutionary relationship analysis of intI1 and intI2 sequences Partial sequences of the intI1 and intI2 genes obtained in this study (Table S3) were aligned with those of E. coli strains of a different geographical/biological origin, which are available in GenBank (Table S2), using CLUSTAL W [16], MUSCLE algorithms [17] with MEGA7 (32-bit) MacOS, and Graphical software [18]. Phylogenetic reconstruction analysis was based on the UPGMA statistical test [19]; the conditions employed were 1000 bootstrap replications, the model of the number of differences applied by uniform rates among sites, a homogeneous pattern among lineages, partial detection for missing data treatment, and a 95% site coverage cut-off [20]. Statistical analysis The data were analysed using Stata version 14.0 software. The association between the phylogenetic groups and the presence of integrons for both groups of study as well as with the antimicrobial resistance profile was performed using the Chi-square test. The level of significance was set at p < 0.05. Results Seventy E. coli isolates of stool samples from 49 children were analysed: 35 isolates from 25 children with diarrhea and 35 isolates from 24 children without diarrhea as the control group. The group of children with diarrhea (48% female, 52% male) was an average age of 14.7 months and 56% under 1 year, while the group of control children (54.5% female, 45.5% male) was an average age of 26 months and 27.3% under 1 year. In all the stool samples, the presence of intestinal parasites, Shigella spp., Salmonella spp., Campylobacter spp., Yersinia spp., and Vibrio spp. as causative agents of diarrheal infection was discarded. In both groups, the frequency of resistance was higher for the beta-lactams family, 68.6% and 42.9% for children with and without diarrhoea, respectively [21]. The 70 analysed E. coli isolates were classified into every phylogroup defined up to the present time (Table 1). All of the phylogroups were identified in the E. coli isolates from children with diarrhoea, while phylogroups C, E, and clade I were absent in the isolates from children without diarrhoea. Phylogroup A was the most frequent in strains from both groups of children, 34.3% with and 51.4% without diarrhoea. The statistical analysis showed that
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Table 1 Distribution of Escherichia coli isolates among phylogenetic groups. Phylogenetic Group
Children with diarrhoea n (%)
Children without diarrhoea n (%)
Total n (%)
A B1 B2 C D E F Clade I Unassignable Total
12 (34.3) 3 (8.6) 7 (20.0) 1 (2.9) 2 (5.7) 1 (2.9) 4 (11.4) 2 (5.7) 3 (8.6) 35 (100)
18 (51.4) 1 (2.9) 4 (11.4) 0 (0) 4 (11.4) 0 (0) 3 (8.6) 0 (0) 5 (14.3) 35 (100)
30 (42.9) 4 (5.7) 11 (15.7) 1 (1.4) 6 (8.6) 1 (1.4) 7 (10.0) 2 (2.9) 8 (11.4) 70 (100)
Table 2 Distribution of class 1 (intl1) and class 2 (intl2) integrons in the Escherichia coli isolated from children with and without diarrhoea. Integron
Children with diarrhoea n (%)
Children without diarrhoea n (%)
Total n (%)
intI1 intI2 None Total
10 (28.6) 1 (2.9) 24 (68.6) 35 (100)
1 (2.9) 0 (0) 34 (97.1) 35 (100)
11 (15.7) 1 (1.4) 58 (82.9) 70 (100)
the frequency of E. coli isolates in the phylogroups was similar in both groups studied (X2 = 9.96, p = 0.286). In 17.1% of the E. coli isolates were identified class 1 (15.7%) or class 2 (1.4%) integrons. In the strains isolated from children with diarrhoea, the frequency of the class 1 integron was higher (28.6%) than that of the class 2 integron (2.9%). On the other hand, in the isolates of the control group was only identified the class 1 integron (2.9%). None of the strains showed the presence of both classes of integrons (Table 2). The integrase gene sequences of class 1 (intI1) and 2 (intI2) integrons of the E. coli isolates showed >99% identity to those reported in Genbank. Compared with published intl1 sequences, three of the 11 partial sequences of the E. coli isolates showed single-nucleotide changes at positions 60 (1/11) and 65 (2/11), permitting their classification by evolutionary relationship analysis into four different groups with similar sequence frequencies: 27.3% for three groups and 18.2% for the fourth group (Fig. 1). The same analysis of the partial sequences of intI2 showed >99% identity to sequences reported in plasmids and transposons, and evolutionary relationship analysis separated the sequences into three groups with the intl2 sequence as a member of the third group comprising six reference sequences (Fig. 2). For both analyses, the reference sequences selected as external controls (Table S2) are in separate branches (Figs. 1 and 2). Discussion The triplex PCR method is mostly used in phylogeny studies of commensal and diarrhoeic E. coli isolates but only considers phylogroups A, B1, B2, and D [22]; the quadruplex PCR method, used in this work, additionally includes phylogroups C, E, F, and cryptic clade I, which together classify approximately 13% of E. coli isolates [2]. In this study, 15.7% of the isolates were assigned to these phylogroups, and similar values have been reported previously [23,24]. The E. coli isolates were non-uniformly distributed among the eight phylogenetic groups, with A and B2 being the most frequent (Table 1). Similar results were obtained for E. coli isolates from faecal samples in India [24,25], Paris [23], and the United States [26]. Escherichia coli strains isolated from humans mainly belong to phylogenetic groups A and B2 [27], and the results in this study indicate
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that 58.6% of the isolates were in these phylogroups. In this regard, the prevalence of different phylogenetic groups among different human populations may differ [27–29]; this associated to socioeconomic factors, dietary habits, use of antibiotics, level of hygiene, geographical and climatic conditions, and the genetics of the host [23]. In this study, although the distribution of the E. coli isolates within the phylogroups was statistically similar between both groups of children, the frequency of strains included in phylogroup B2 was higher in the children with diarrhoea. Similar observations have been obtained in E. coli isolates from children in India [24], Pakistan [30], and Romania [31]. Phylogroup A has been reported as the most common group for isolates from children without diarrhoea [4]. Our results suggest a relationship between the E. coli isolates belonging to phylogroup B2 and their characteristic of diarrhoea induction, and an association of phylogroup A with commensal E. coli strains. The existence of E. coli isolates not classified into the defined phylogroups has been reported in other studies [24,30]; this phenomenon may be explained by the loss of specific genes due to the plasticity of the genome of E. coli, to the recombination of isolates belonging to two different phylogenetic groups, or to the existence of extremely rare phylogroups [2]. Moreover, cryptic clades are mainly associated with environmental E. coli, which are detected in human faecal samples with a frequency of 2-3% [32]; a similar value was found in this study (2.9%). The two isolates in clade I were from children with diarrhoea and the observed results may be related to a lack of good hygiene habits [3]. The prevalence of integrons in this study (17.1%) was lower than that reported in other studies (23.5%–78.2%) [10,24,25,33,34]. However, the higher prevalence of the class 1 integron (15.7%) compared to that of the class 2 integron (1.4%) corresponds to values previously reported; class 1 is the most identified integron in clinical isolates of Gram-negative and multidrug-resistant bacteria due to its recombination ability [9,11]. Although the presence of integrons was statistically similar in both groups of children, the frequency was higher in the children with diarrhoea (31.4%), suggesting that the presence of integrons in E. coli may be more closely associated with pathogenic than with commensal strains. This association has been observed in other regions of the world [33,35,36]. The susceptibility profile of the studied E. coli isolates was previously reported [21]. All isolates with integrons were resistant to at least one and up to six antimicrobials belonging to three different antimicrobial families, with 58.3% of them being classified as multidrug-resistant (two or more families of antimicrobials). As for the E. coli isolates without integrons, only 34.5% were classified as multidrug-resistant and 8.6% were susceptible to all antimicrobials. These values show the influence of the presence of integrons in the resistance of the studied isolates, as described in other works [10,24,34,36,37]. In this regard, this study shows a lower frequency of integrons in the commensal strains; however, these strains may be potential disseminators of antimicrobial resistance, mediated by horizontal gene transfer [10,29]. In the E. coli isolates with the class 1 integron from the children with diarrhoea, the frequency of resistance was higher for the beta-lactams family (carbenicillin 100%, ampicillin 80%, piperacillin 60%, ticarcillin/clavulanic acid 50%, and cefuroxime sodium 20%), followed by sulfonamides (trimethoprim/sulfamethoxazole 60%), and quinolones (nalidixic acid 10%), while the isolate with the class 1 integron from the children of the control group and the isolate with the class 2 integron from the children with diarrhoea were only resistant to beta-lactams (carbenicillin and cefuroxime sodium) (Table S4). Similar results have been reported [25,34,37]. It has been documented that resistance cassettes found in class 1 integrons confer resistance to all known beta-lactams [12], which corresponds to the resistance profile of the studied isolates (Table
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Fig. 1. Evolutionary relationships of the Escherichia coli intl1 gene. 䊉 Sequences generated in the present study. Enterococcus faecalis, Staphylococcus aureus, Mus musculus, and Triticum aestivum were used as out-groups. The evolutionary history was inferred using the UPGMA method. The optimal tree with the sum of branch length = 723.65878788 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the number of differences method and are in the units of the number of base differences per sequence. The analysis involved 26 nucleotide sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Evolutionary analyses were conducted in MEGA7.
Fig. 2. Evolutionary relationships of the Escherichia coli intl2 gene. 䊉 Sequences generated in the present study. Mus musculus, Enterococcus faecalis, Bacillus thuringiensis tenebrionis, and Triticum aestivum were used as out-groups. The evolutionary history was inferred using the UPGMA method. The optimal tree with the sum of branch length = 129.97622549 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the number of differences method and are in the units of the number of base differences per sequence. The analysis involved 18 nucleotide sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Evolutionary analyses were conducted in MEGA7.
S4). The sul1 gene confers resistance to sulfonamides and is typically within the 3’-conserved-segment (3’-CS) of the base structure in the class 1 integron [11]. Of the studied isolates with the intI1,
only 54.5% were resistant to sulfonamide. This suggests the existence of another mechanism of resistance, the probable presence of the gene in another genetic context, or the existence of alleles
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Y.P. Ahumada-Santos et al. / Journal of Infection and Public Health xxx (2019) xxx–xxx Table 3 Distribution of class 1 (intl1) and class 2 (intl2) integrons among the phylogenetic groups. Phylogroup
intI1 n (%)
intI2 n (%)
None n (%)
Total with integrons n (%)
A B1 B2 C D E F Unassignable Clade I Total
4 (36.4) 0 (0) 2 (18.2) 1 (9.1) 1 (9.1) 0 (0) 2 (18.2) 1 (9.1) 0 (0) 11 (100)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (100) 1 (100)
26 (44.8) 4 (6.9) 9 (15.5) 0 (0) 5 (8.6) 1 (1.7) 5 (8.6) 7 (12.1) 1 (1.7) 58 (100)
4 (33.3) 0 (0) 2 (16.7) 1 (8.3) 1 (8.3) 0 (0) 2 (16.7) 1 (8.3) 1 (8.3) 12 (100)
[25]. The resistance cassettes in class 2 integrons show less diversity and usually contain genes conferring resistance to trimethoprim, streptothricin, and streptomycin [9]; in this regard, the studied isolate with intI2 was susceptible to trimethoprim, but resistant to the beta-lactams cefuroxime sodium and carbenicillin; this contrast could be due to resistance genes outside the context of the integron structure. The sequences of the intI1 and intI2 genes obtained from clinical samples are highly conserved [38], although some silent point mutations were found in the amplified sequences of the intI1 gene in this study. The intI1 and intI2 genes are associated with the Tn3 and Tn7 transposon families, respectively [9,11]; and these associations were recorded for the studied E. coli isolates (Fig. 1), showing that plasmids and transposons are vehicles for intra- and inter-species integron mobility [11]. The phylogenetic analysis of the intI1 sequences resulted in the formation of four groups; the sequences M4-5 (MK591027) and M21-3 (MK591034) in the first group showed 100% identity to the intI1 gene of the APEC and AIEC isolates, while those of the three remaining groups were highly identical to the intI1 sequences of E. coli strains isolated from animals used as food and from human faeces (Fig. 1). This association has been reported previously with extra-intestinal pathogenic E. coli (ExPEC) strains [39,40]; although the studied isolates were from intestinal origin, 27.3% of them were classified into phylogenetic groups B2 and D, as occurs with the ExPEC strains. As in other works, this similarity suggests that E. coli strains could be transmitted to humans from different niches (e.g., zoonotic or food) [40,41]. In the intI2 gene, the internal stop codon responsible for encoding a characteristic non-functional integrase was identified [11]; and the evolutionary relationship analysis did not show a clear separation between the intl2 sequences of different human and animal isolates, suggesting the circulation of E. coli strains between different ecological niches as for the intI1 gene (Fig. 2). It should be noted that the bioinformatic analysis of the E. coli intI1 and intI2 sequences showed high identity to those of other bacteria, suggesting that integrons are involved in the horizontal transfer of resistance genes across bacteria. The correlation analysis between the variables phylogroup and presence of integrons of the E. coli isolates showed that both groups of children are statistically similar (X2 = 6.7, p = 0.565). However, the isolates belonging to phylogroups B1 and E did not show integrons. Moreover, the highest frequency of integrons was found in the strains of group A (33.3%) (Table 3). Compared with our results, an association among the class 1 integron and phylogroups A, B1, and B2 was reported [24]; as well as between the class 2 integron and phylogroup B2 in EPEC isolates [24]. Similarly, in an adult Mexican population was reported the presence of class 1 integrons in E. coli isolates distributed in groups A, D, and B2; and class 2 integrons only in isolates belonging to phylogroup D [42]. This information contrasts with the results obtained in this study for the class 2 inte-
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gron. However, agrees with the prevalence of phylogroups A and B2 in the E. coli isolates containing integron genes. The commensal strains are associated with phylogroup A, so the presence of integrons primarily in this phylogroup suggests the great problem of resistance induced by the misuse and abuse of antibiotics in the studied population. The correlation analysis between the phylogenetic groups and the susceptibility profile by antimicrobial family of the studied E. coli isolates showed statistical similarity (X2 = 18.1, p = 0.798). Nevertheless, multidrug-resistant isolates were detected predominately in phylogroups A and B2 with resistance from one to 11 and 12 antibiotics, respectively; the isolates from children with diarrhoea showed a higher resistance. Several papers have reported no association between the susceptibility profile and the phylogenetic group assigned to E. coli isolates [23,24]; in this study, multidrugresistant bacteria have been identified within each phylogroup, indicating that the antibiotic resistance problem is found in both commensal and diarrhoeagenic strains and is independent of the phylogroup assigned.
Conclusions The phylogenetic distribution of the strains isolated from faeces of children with and without diarrhoea in the city of Culiacan, Sinaloa, Mexico was similar to that reported in other parts of the world, with a greater frequency of phylogroup A. For the E. coli isolates within phylogroup A, the frequencies of isolates with integrons and multidrug-resistant were high, suggesting that antibiotics are not properly used in children, and consequently that they are potential disseminators of resistance genes and can increase the risk of death. On the other hand, the association of APEC strains with detected integron sequences together with the tendency to classify the isolates of children with diarrhoea in phylogroup B2 suggests that the circulation of E. coli strains between different ecological niches, the existence of zoonotic potential, and the horizontal gene transfer are propagation mechanisms of antibiotic resistance. The information generated emphasises the importance of carrying out regular monitoring of phylogenetic distribution and antimicrobial susceptibility of commensal and pathogenic E. coli strains in each geographical area; such studies can be useful in the creation of proper guidelines for the management of antibiotics among the child population of the locality.
Funding This work was supported by the National Council for Science and Technology from Mexico (CONACYT); and the ‘Programa de Fomento y Apoyo a Proyectos de Investigacion’ (PROFAPI) of the Autonomous University of Sinaloa.
Conflict of interest None.
Acknowledgements Authors acknowledge to Dr. Ignacio Osuna-Ramírez by the statistical-analysis assistance; as well as to Diana Guadalupe Delgado-Solís, Solangel Osorio-Valle, and Stefany Margarita Tamayo-Felix by their assistance in performing different experimental assays.
Please cite this article in press as: Ahumada-Santos YP, et al. Association of phylogenetic distribution and presence of integrons with multidrug resistance in Escherichia coli clinical isolates from children with diarrhoea. J Infect Public Health (2019), https://doi.org/10.1016/j.jiph.2019.11.019
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Please cite this article in press as: Ahumada-Santos YP, et al. Association of phylogenetic distribution and presence of integrons with multidrug resistance in Escherichia coli clinical isolates from children with diarrhoea. J Infect Public Health (2019), https://doi.org/10.1016/j.jiph.2019.11.019