Classes 1 and 2 integrons in faecal Escherichia coli strains isolated from mother-child pairs in Nigeria

Background Antimicrobial resistance among enteric bacteria in Africa is increasingly mediated by integrons on horizontally acquired genetic elements. There have been recent reports of such elements in invasive pathogens across Africa, but very little is known about the faecal reservoir of integron-borne genes. Methods and findings We screened 1098 faecal Escherichia coli isolates from 134 mother-child pairs for integron cassettes by PCR using primers that anneal to the 5’ and 3’ conserved ends of the cassette regions and for plasmid replicons. Genetic relatedness of isolates was determined by flagellin and multi-locus sequence typing. Integron cassettes were amplified in 410 (37.5%) isolates and were significantly associated with resistance to trimethoprim and multiple resistance. Ten cassette combinations were found in class 1 and two in class 2 integrons. The most common class 1 cassette configurations were single aadA1 (23.4%), dfrA7 (18.3%) and dfrA5 (14.4%). Class 2 cassette configurations were all either dfrA1-satI-aadA1 (n = 31, 7.6%) or dfrA1-satI (n = 13, 3.2%). A dfr cassette was detected in 294 (31.1%) of trimethoprim resistant strains and an aadA cassette in 242 (23%) of streptomycin resistant strains. Strains bearing integrons carried a wide range of plasmid replicons of which FIB/Y (n = 169; 41.2%) was the most frequently detected. Nine isolates from five different individuals carried the dfrA17-aadA5-bearing ST69 clonal group A (CGA). The same integron cassette combination was identified from multiple distinct isolates within the same host and between four mother-child pairs. Conclusions Integrons are important determinants of resistance in faecal E. coli. Plasmids in integron-containing strains may contribute to dispersing resistance genes. There is a need for improved surveillance for resistance and its mechanisms of dissemination and persistence and mobility of resistance genes in the community and clinical settings.


Methods and findings
We screened 1098 faecal Escherichia coli isolates from 134 mother-child pairs for integron cassettes by PCR using primers that anneal to the 5' and 3' conserved ends of the cassette regions and for plasmid replicons. Genetic relatedness of isolates was determined by flagellin and multi-locus sequence typing. Integron cassettes were amplified in 410 (37.5%) isolates and were significantly associated with resistance to trimethoprim and multiple resistance. Ten cassette combinations were found in class 1 and two in class 2 integrons. The most common class 1 cassette configurations were single aadA1 (23.4%), dfrA7 (18.3%) and dfrA5 (14.4%). Class 2 cassette configurations were all either dfrA1-satI-aadA1 (n = 31, 7.6%) or dfrA1-satI (n = 13, 3.2%). A dfr cassette was detected in 294 (31.1%) of trimethoprim resistant strains and an aadA cassette in 242 (23%) of streptomycin resistant strains. Strains bearing integrons carried a wide range of plasmid replicons of which FIB/Y (n = 169; 41.2%) was the most frequently detected. Nine isolates from five different individuals carried the dfrA17-aadA5-bearing ST69 clonal group A (CGA). The same integron cassette combination was identified from multiple distinct isolates within the same host and between four mother-child pairs.

Conclusions
Integrons are important determinants of resistance in faecal E. coli. Plasmids in integroncontaining strains may contribute to dispersing resistance genes. There is a need for PLOS

Introduction
The level of antibiotic resistance among pathogenic and commensal bacteria has steadily increased and has become a global health concern [1]. Much of the problem has been attributed to mobile genetic elements such as plasmids and transposons [2]. Integrons are genetic elements that also contribute to the prevalence and horizontal transmission of antibiotic resistance [3,4,5]. Integrons are genetic elements with a site-specific recombination system for capturing, expressing and exchanging gene cassettes [6]. They are characterized by conserved features, namely an intI gene encoding an integrase, a recombination site (attI), a promoter (P) and the ability to integrate gene cassettes comprise of a single open reading frame (orf) and a specific recombination site, attC [4,7]. Intracellularly, gene cassettes exist either in a linear form inserted into an integron or as a free circular cassette that is not dependent on an integron [7,8]. At least nine classes of integrons have been described and class 1and 2 integrons are the most predominantly associated with antibiotic resistance in clinical isolates [9]. The capture of resistance genes is especially important when these integrons are disseminated by broad-host-range conjugative plasmids or transposons. So far, more than 8000 gene cassette arrays have been identified in class 1 integrons (http:// integrall.bio.ua.pt/). Yet, less than 10 array compositions are commonly reported [9,10,11]. These commonly reported arrays are found in a wide variety of hosts and environments, highlighting a high level of horizontal dissemination of these elements among bacterial populations and species. Clonal expansion also contributes to the current prevalence of interregional spread of integron-carrying bacterial species. However, the extent to which each contributes is unknown [12]. In Nigeria, there have been reports of integrons in strains isolated from the clinical and environmental settings [13,14,15,16]. However, very little is known about the faecal reservoir of integron-borne genes. In this study, we investigated prevalence of classes 1 and 2 integrons, their association with antimicrobial resistance and how resistance cassettes are disseminated in faecal, Escherichia coli strains isolated from children with diarrhoea and their apparently healthy mothers.,

Isolation and identification of Escherichia coli strains
All faecal samples were inoculated onto Eosin Methylene Blue (EMB) agar plates (Oxoid Ltd., Basingstoke, Hampshire, England) and incubated for 24 hours aerobically at 37˚C. From each sample, up to five morphologically distinct colonies typical of E. coli were selected and identified by standard biochemical testing [17].

DNA extraction
All isolates were grown overnight in 5 ml of peptone broth (Oxoid, England). A 1 ml aliquot of the culture was centrifuged at 10,000 rpm for two minutes in a microcentrifuge (BioRad, USA). DNA was extracted from each isolate using the Promega Wizard genomic extraction kit (Promega, corporation, Madison, USA) according to the manufacturer's instructions.

Detection of class 1 and 2 integrons
All the isolates were screened for class 1 and 2 integrons by polymerase chain reaction (PCR). Class 1 integron was detected and amplified by Levesque 5CS and 3CS primers which bind the 5' and 3' conserved ends respectively [19]. Class 2 integron was detected and amplified by White hep74 and White hep51 to hybridize attI2 and orfX respectively [20] (S1 Table).
The following PCR cycle, adapted from proposed by Lévesque et al. (1995, with modifications) was used to amplify the variable regions of both class 1 and class 2 integrons. following a 2 minute hot start at 94˚C, 40 cycles of denaturation 94˚C for 30s, annealing 57˚C for 30s, extension 72˚C for 1 min per kb were performed. E. coli strains 042 (carrying a class 1 integron) and 17-2 (carrying a class 2 integron) were used as positive controls in the integron PCRs and E. coli K-12 strain DH5α, lacking integrons, was used as a negative control. 5μl of each PCR reaction was electrophoresed on a 1% agarose gel in 1X Tris Acetate EDTA buffer. Gels were stained with ethidium bromide for 15 minutes, destained in distilled water for 30 minutes, and visualized under ultraviolet (UV) light.

Identification of resistance cassettes and cassette combinations in integrons
Unique resistance cassettes and cassette combinations within class 1 and 2 integrons were delineated by performing restriction fragment length polymorphism (RFLP) with Mbo1 (Biolab, England) and Alu1 (Biolab, England) as previously described [21]. Up to three amplicons from each unique profile were ligated into a pGEMT-Easy vector (Promega, USA) and sequenced. The identity of contained cassettes was determined using BLAST [22].

Flagellin typing
The entire coding sequence of the variable chromosomal gene fliC was amplified by PCR using the primers F-FLIC (5'-ATG GCA CAA GTA ATT AAT AAC CAA C-3') and R-FLIC (5' CTA ACC CTG CAG CAG AGA CA-3') [28]. The cycling conditions were 30s at 95˚C, 1 min at 60˚C, and 2 min at 72˚C for 35 cycles. PCR product was digested with Rsal (Promega, USA) as described by Fields et al. (1997) [29]. The restriction profiles were compared after electrophoresis on 1.5% agarose gel. The H-type of flagellin RFLPs of interest was determined by sequencing and BLAST analysis.

Multi locus sequence typing (MLST)
Greater resolution of similarity between chromosomes of selected isolates was provided by multilocus sequence typing. This was done by amplifying seven housekeeping genes namely adenylate kinase (adk), fumarate hydratase (fumC), gyrase B (gyrB), isocitrate dehydrogenase (icd), malate dehydrogenase (mdh), adenylosuccinate synthetase (purA) and recombinase A (recA) with primers described by Wirth et al [30] (S2 Table). Cycling conditions were as follows: 95˚C for 2 minutes, followed by 35 cycles of 94˚C for 1 minute, 56˚C for 1 minute and 72˚C for 1 minute. A final, 3 minute elongation step was performed at 72˚C. Amplified DNA products of the housekeeping genes were sequenced and alleles were assigned by using an online E. coli MLST database at http://www.mlst.net.

Statistical analysis
The Chi-square (χ2) and Fischer's exact tests (two-tailed) of R statistical software package (version 3.3.0) were used to determine the statistical significance of the data. Pearson Product Moment Correlation Coefficient, r, calculated using SPSS (SPSS, Inc. Chicago, Illinois) was used to identify significant correlations. All reported p-values were two-sided and a p-value of less than or equal to 0.05 was considered to be statistically significant.

Subjects and faecal Escherichia coli isolates
A total of 1098 Escherichia coli strains was isolated from the stool samples of 134 mother and child pairs recruited into the study. These comprised of 542 isolates from children aged up to 60 months (13.36±2.12) and 556 isolates from their mothers' ages of 15-46 years (25.88 ±7.07).

Antimicrobial resistance rates in the faecal Escherichia coli isolates
As shown in Table 1, the majority of the E. coli isolates from both mothers and their children were resistant to streptomycin (n = 1050, 95.8%), sulphonamide (n = 1038, 94.2%), ampicillin (n = 1014, 92.5%), tetracycline (n = 1025, 93.3%) and trimethoprim (n = 947, 85.9%) except to ciprofloxacin (n = 94, 8.4%). Isolates that were resistant to ciprofloxacin (χ 2 = 6.498; p = 0.009) were more frequently isolated from healthy mothers than their children. The differences in resistance to other antimicrobials were not significantly different between isolates obtained from children and those from mothers.

Plasmid replicon types and integrons
Plasmid replicons belonging to all incompatibility groups sought except A, C, K, B and W were identified among integron-containing strains. (The protocol we used does not delineate IncY from the more common IncFIB replicon). As shown in supplementary Table 4, the majority of the integron containing isolates (n = 330, 80.5%) carried one or more of ten different plasmid-replicon-types and at least 133 carried more than one type. IncFIB/Y (n = 169; 41.2%) was the most frequently detected plasmid replicon in the isolates (S4 Table). Pearson regressions did not reveal any association between integron cassette combinations and specific plasmid replicons, pointing to a role for plasmids other than those sought in this study and/or transposons in disseminating integrons (Fig 1). In 34 instances, the same integron cassette array was detected in at least one E. coli isolate from a child as well as an isolate from that child's mother (Table 4), leading us to hypothesize transmission of integron-bearing strains and/or mobile elements within families.

Diversity among strains bearing the dfrA5 cassette
We have previously identified a widely disseminated transposon as being responsible for disseminating integron-borne dfrA7 cassettes among commensal E. coli isolates in Nigeria and elsewhere in Africa [21]. dfrA7 was highly prevalent in the current study and associated with a wide variety of plasmid replicons (S4 Table). A single dfrA5 cassette was the second most common single dfr cassette in this study and has been associated with successful invasive nontyphoidal Salmonella lineages across Africa [31]. As the context for this cassette among commensals remains unknown, we used a simple PCR marker to define one chromosomal feature and one plasmid feature in each of the strains dfrA5 isolates obtained in this study to determine whether clonal expansion and/or successful plasmids showed associations with the presence of dfrA5. As shown in Figs 2 and 3, fourteen flagellin types were identified and arbitrarily labelled A-N. Type C (23%) was predominantly identified followed by type E (16%) and type B (11%). Multiple distinct isolates with the dfrA5 cassette were identified in the same host in six instances. In three of those instances, (pairs 246, 250 and 289) the same fliC allele was not identified in the mother and the infant whereas in the other three (pairs 130, 174 and 195), identical fliC allele although no single allele type was seen in more than one pair. Two of the types seen in pairs were the most common overall (C and E). Of interest is that for four of the six pairs with dfrA5-positive isolates, isolates from both the mother and the child carried FIB/Y replicons in four cases and this replicon represents 42.4% of dfrA5 isolates overall (S5 Table).

Resistant pandemic lineage in the fecal reservoir of mothers and infants
Fourteen strains out of 1098 E. coli strains isolated from mother and child pairs carried dfrA17-aadA5 cassette that is normally found in clonal group A (CGA), a multi-resistant E. coli belonging to multilocus sequence type (MLST) 69 [32]. These strains comprised of 12 strains with only dfrA17-aadA5 cassette and two strains with a dfrA17-aadA5 cassette and dfrA1-sat1, a class 2 Integron cassettes ( Table 3). As CGA possess a chromosomally located dfrA17-aadA5-bearing integron, we sought to determine whether any of the 14 dfrA17-aadA5positive strains identified in this study belong to this clone. CGA strains carry an H18 flagellin (pattern A in our study) and belong to the MLST type 69 whose allele profile includes fumC69. We identified the fliC, fumC and recA allele types for the dfrA17-aadA5-positive strains. As shown in Table 5, nine isolates from five different individuals carried the CGA-associated H18  [26] scheme confirmed that all nine strains belong to ST69. In one instance, independent dfrA17-aadA5-positive isolates were obtained from a mother and her infant.

Discussion
This study examined integron cassette content in faecal E. coli from children with diarrhea and their apparently healthy mothers. Integrons are gene exchange systems and are known to play a significant role in the acquisition and dissemination of antimicrobial resistance genes and to be selected by antimicrobial pressure [6]. While our study had the limitation of only detecting cassette regions under 4 Kb in size that had intact 5' and 3' ends, we still amplified integron cassettes from as many as 37.3% of isolates. Our detection of 340 (31%) class 1 integron containing strains and 44(4%) class 2 integron containing strains is comparable to data from studies using a similar methodology, which reported a prevalence of integrons to be above 30% [21,33,34,35,36]. Integrons are clearly widespread in faecal E. coli in this environment and may be responsible for high rates of resistance observed in this study. This is true for trimethoprim where a significant association between the presence of resistance and amplification of integron cassettes was seen and for which eight different cassette arrays including a resistance-conferring dfrA gene were detected. The preponderance of the cassette arrays with a dfrA gene may be associated with the recent and current intensive use of trimethoprim in many common infections in the study environment as well as to prevent opportunistic infections in HIV-positive patients [21,37,38,39]. Resistance to chloramphenicol and nalidixic acid was also associated with the presence of integrons even though cassettes conferring resistance to these agents were not recovered. This points to integrons as markers of multiresistant strains and the potential that integrons are physically linked to other resistance genes.
Unlike class 2 integrons, the diversity of class 1 integron cassettes/cassette combinations is low when compared with studies performed elsewhere [40,41]. The low diversity of class 1 cassettes led us to hypothesize that a few strains or mobile elements may be disseminating class 1 integron-mediated resistance. Integrons are not themselves mobile but can be rapidly disseminated when contained within mobile elements such as plasmids. We detected known plasmid replicons in a large number (81.7%) of integron containing isolates examined. In particular, IncF plasmids (predominantly IncFIB/Y replicons but also IncFIA and IncFIC) were commonly found in association with the integron-borne cassettes identified. IncF plasmids represent one of the most prevalent incompatibility types and have been identified worldwide in Enterobacteriaceae from different origins and sources [42,43]. These plasmids appear to significantly contribute to the dissemination of antibiotic resistance in Enterobacteriaceae and some have been associated with specific genes conferring resistance to β-lactams, quinolones, and aminoglycosides [42,44,45,46]. Therefore the preponderance of IncFIB in integron containing isolates may indicate its contribution to the dissemination of integron-borne cassettes in this environment. The study also points to a significant prevalence of IncO plasmids, which have not previously been described from Nigeria.
Of all the cassettes detected in integrons, aadA1 (n = 96, 23.4%) which encodes resistance to spectinomycin/streptomycin was the commonest. This is in line with various reports of high prevalence of aadA1 among integron-positive isolates in the literature [20,47]. This was followed by dfrA7 (n = 75, 18.3%), a cassette that has also been reported worldwide as a commonly identified cassette combination in integrons [48,49,50,51]. In our previous research, focused on different populations and strains, we demonstrated that a single dfrA7 cassette is the predominant amplifiable cassette combination from fecal E. coli in Nigeria and elsewhere in Africa [21]. This was also the case in this study. Previously, we found that a widely disseminated transposon accounted for the high frequency of this particular allele [21]. While there were seven other dfr-containing configurations, the second most common one, a single dfrA5 cassette was also greatly over-represented in the study. Kingsley et al [51] and Okoro et al [31] have reported this cassette in Salmonella enterica Typhimurium in the context of a transposon that is similar to the one we found associated with dfrA7. S. Typhimurium strains carrying this transposon represent a successful lineage that has expanded at multiple sites on the African continent. We wanted to know whether overrepresentation of dfrA5 in this study was due to clonal expansion of one or a few E. coli lineages or mobility of the cassette through the commensal flora by virtue of a transposon or other mobile element. Flagellin typing of the 59 isolates that carried a single dfrA5 cassette revealed that while some flagellin types were very common, none represented more than 23% of the set and there were 14 flagellin types overall. As each flagellin type is seen in multiple E. coli lineages, it is unlikely that a significant expansion of any dfrA5-bearing clone has occurred. Dissemination of dfrA5 across the commensal flora could be due to a successful plasmid or a more modular element such as a transposon that can associate with different plasmids or the chromosome [52]. In the case of the former, a single plasmid marker would be overrepresented in the set. However PCR screening for eleven plasmid replicons identified 49 among dfrA5 strains and different replicons were associated with different flagellin types. Altogether the data point to diverse strain backgrounds and possible over-representation of FIB/Y plasmids in integron bearing strains but little indication of clonal expansion of a distinct dfrA5-bearing lineage. The dissemination of dfrA5 could also be due to a more modular mechanism potentially similar to what we have previously seen for dfrA7. Venturini et al. [52] recently reported a role for IS26 elements in disseminating dfrA5, which could very well be under play in our own study environment. In addition to pointing to flexible contexts for more common integron arrays, this study also provided a little insight into circulation of a successful pandemic lineage, the dfrA17-aadA5-bearing ST69 CGA in the faecal microflora. Multi-resistant E. coli belonging to multilocus sequence type (MLST) 69 have been termed CGA and implicated in invasive infections in different parts of the world, including Nigeria [32]. The epidemiology of CGA in Nigeria is understudied since pandemic lineages are rarely sought here. As CGA possess a chromosomally located dfrA17-aadA5-bearing integron, we sought to determine whether any of the 14 dfrA17-aadA5-positive strains identified in this study belong to this clone. Likely members of this lineage represented a small minority of the integron-bearing strains detected in this study. Verified ST69 strains were detected in 5 (1.9%) of 264 individuals including both members of one mother-infant pair. Our data suggest that CGA may be shared among individuals in the same household and its presence may facilitate the dissemination of dfrA17-aadA5 cassette in this environment.

Conclusions
In conclusion, this study reveals the presence of integrons in fecal E. coli isolated from apparently healthy mothers and their sick children in Nigeria. The identified integrons contain antibiotic-resistant gene cassettes and while the variety of cassette combinations is very limited, the backgrounds of strains carrying these elements and the repertoire of plasmids associated with them are reasonably diverse. The detection of different replicons in association with different flagellin types in dfrA5 strains, points to a successful plasmid or a more modular element that is borne on plasmids or located on the chromosome for its dissemination. In view of the findings of this study, there is a need for improved surveillance, which can provide information for the persistence and mobility of resistance genes between the community and clinical settings.
Supporting information S1