Due to high correlation of isolates within birds that manifested in highly variable covariate effect estimates data not shown , a random effect for birds was not used. Instead, isolates were collapsed across birds i. Therefore, final mixed models for both poultry and humans included a random intercept only for the household. Year of sample collection can influence the presence of a small-scale production operation and levels of AMR.
Diarrheal case status of humans might be causally linked to AMR levels: If a person is sick and takes antibiotics, that might then select for AMR organisms adjusting for a variable that is causally linked only to the outcome can increase statistical efficiency Model adjustments, therefore, included year of sample collection for isolates from birds and year of sample collection and original diarrheal case status for isolates from humans.
In addition to the latter adjustment for humans in model 1 , because MGEs might be related to both small-scale production operations and AMR, we constructed a second model controlling for MGEs model 2. We collected 1, isolates from chickens small-scale production birds and household birds in the 20 villages.
Higher prevalence of AMR for all markers was seen in isolates from small-scale production birds compared with those from household birds Table 1.
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Unadjusted and adjusted odds ratios from mixed models showed significantly elevated odds for the presence of all MGE markers among small-scale production birds, and significantly elevated odds of resistance for 7 of 12 antibiotics tested as well as MDR , compared with household birds Table 2.
Table 1. Abbreviations: int1 , class-1 integron; int2 , class-2 integron; MDR, multiple-drug resistance; tetA , tetracycline class A; tetB , tetracycline class B. Models for 5 of 12 antibiotics not shown due to low frequencies. Table 3. Mixed models accounted for clustering at the household level; isolates were collapsed across birds due to high within-bird correlation. Reference group was household birds in villages without small-scale production operations. There were 45 humans associated with production birds and associated with household birds.
The prevalences of AMR to 8 of 12 antibiotics and 4 of 4 MGE markers were higher for isolates from humans associated with production birds compared with those associated with household birds Table 1.
Odds ratios from nested mixed models are shown in Table 4. As with the poultry data, there was low AMR prevalence for a subset of antibiotics, and these models are not reported. There was also low prevalence of int2 and tetB ; these were combined with int1 and tetA for a single category for integrons and one for tetracycline genes Table 4. Table 4. Models for 7 of 12 antibiotics not shown due to low frequencies.
Mixed models accounted for clustering at the household level. Reference group is humans associated with household birds in villages without small-scale production operations. Elevated levels of markers for MGEs, particularly isolates positive for int1 and tetA , were consistently observed in small-scale production birds and associated humans compared with household birds and associated humans.
To explore how MGEs might facilitate AMR in isolates from poultry and humans, the prevalence of phenotypic AMR by small-scale production exposure was further stratified by the presence or absence of MGEs results for int1 are shown in Figure 1. Three distinct groups of phenotypic AMR emerged when using all isolates from poultry, which we refer to here as group 1, group 2, and group 3.
In group 2 Figure 1 B , both the presence of small-scale production operations and int1 increased the prevalence of AMR.
Resistance to group 3 antibiotics was high with production exposure, but sometimes both production and integron exposures were required Figure 1 C. Similar grouping patterns were also seen when stratifying by int2 , tetA , and tetB data not shown. In humans, there were low frequencies of resistance to group 2 and 3 antibiotics, and so similar patterns failed to emerge for these groups data not shown. Prevalence of antimicrobial resistance in E.
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A For poultry isolates in group 1, the association of small-scale production operations appeared to be driven largely by the presence or absence of int1. B For poultry isolates in group 2, the association of small-scale production operations and int1 was roughly additive. C For poultry isolates in group 3, while small-scale production operations resulted in high resistance levels, both int1 and small-scale production operation exposures were often needed to reach high levels of resistance. D Resistance patterns in isolates from humans associated with small-scale production or household birds for group 1 antibiotics mirrored those seen in isolates from poultry see A.
White: household birds, int1 -negative; dark gray: household birds, int1 -positive; light gray: small-scale production birds, int1 -negative; black: small-scale production birds, int1 -positive. This study suggests that production operations on a smaller scale, and not just industrial-scale poultry operations, are associated with increases in resistant isolates in poultry and humans associated with these operations. In addition, the MGE and phenotypic data presented here highlighted a potentially important role of small-scale production operations within the study site, namely an elevated occurrence of MGEs in E.
Integrons house a wide array of resistance genes 20 , which can result in the expression of resistant phenotypes not directly related to antibiotics used in production farming.
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In a study of livestock farms with heavy use of antibiotics, all types of resistance genes were enriched, and such enrichment was highly correlated with the abundance of MGEs such as transposases 31 , often linked with integrons The ability of class-1 integrons to carry AMR genes is the most likely explanation for our observed relationship between int1 , small-scale production operations, and AMR Figure 1.
This finding can be explained by physical links to MGEs. All class-1 integrons incorporate a sulfonamide sul resistance gene as part of their structure, and often carry dihydrofolate reductase dfr genes conveying resistance to trimethoprim In addition, tetracycline resistance genes are often found on the same conjugative plasmids as integrons 34 , In contrast to group 1, for group 2 antibiotics, the presence of int1 contributed to some increases of resistance in isolates from poultry, while additional exposure to small-scale production operations resulted in even higher resistance levels.
This finding is plausible because, while genes conferring resistance to the group 2 antibiotics have been found on class-1 integrons, some are less common than the sul and dfr genes For group 3 antibiotics, exposures to small-scale production operations or int1 genes alone tended to result in minor increases in resistance. Only when both small-scale production operations and integrons were present did we observe high levels of resistance, suggesting that even antibiotics with overall low AMR levels in a community can achieve higher levels given the presence of both these exposures.
The selection of MGEs due to small-scale production operations might be an evolutionary response to exposure to a large variety of antibiotics. In this study site, various types of antibiotics were detected in feed used in production coops Such broad antibiotic use in this setting might select bacterial isolates in birds carrying MGEs, which in turn might influence AMR in bacterial isolates from humans.
This agrees with recent evidence showing that transmission of AMR from animals to humans might be driven more by MGEs than cross-colonization by resistant strains The higher prevalence of tetA and tetB genes in isolates from small-scale production birds could also be explained by antibiotic use, because tetracycline has been previously detected in feed used in small-scale production operations in this area After adjusting for the presence of MGEs, the relationships between small-scale production operations and phenotypic resistance were attenuated, indicating overlap in the variance in the outcome AMR explained by presence of MGEs and these operations.
One plausible explanation for this overlap is that small-scale production operations increase the prevalence of isolates containing MGEs, which in turn increases prevalence of AMR isolates. In addition, humans associated with household birds in small-scale production villages had elevated odds of tetracycline resistance Table 5. Together, these results are indirect evidence of environmental spread of AMR at the community level from small-scale production operations, possibly through soil, air, water, or meat.
We add a new perspective by sampling E. In this region of Ecuador, high levels of AMR in isolates from these groups were observed despite the small-scale nature of the operations, suggesting that high levels of AMR might be found in small-scale production operations elsewhere in the developing world. Given the growing interest in utilizing poultry as a tool for economic development 36 — 38 , the results presented here are important when considering policy implications, especially in nations that have not yet enacted or enforced regulations on nontherapeutic antibiotic use.
Moser, Ian Spicknall, Carl F. The target-site duplications at the extremities of both IS and IS suggest there were no further rearrangements after these insertions. By contrast, all three other IS 3 elements in E. IS Thus, the IS element in B may have resulted from an IS 3 transposition near ycdV , followed by a recombination event between the new copy and the one near ycdT , which led to the deletion of the intervening sequence. The sequence adjacent to one side of the IS element in B corresponds to sgcR However, the other adjacent sequence is similar to a region of a Shigella flexneri pathogenicity island that contains the remnants of various IS elements [ 31 ].
No target-site duplication was found at the extremities of IS , which suggests that a further rearrangement occurred at this locus. Our data also indicate that this chromosomal region contains several other IS elements in B, suggesting that this region may have been acquired by horizontal gene transfer. This region was previously reported to harbor horizontally transferred DNA in other E. As noted above, hok-sok loci are derived from plasmid stabilization systems, but most have been inactivated by IS elements in laboratory strains.
Five such loci were reported in K [ 29 ]. A sixth one, hokX - sokX , was reported in E. Previous research has shown that E. Multi-locus enzyme electrophoresis puts both of these strains in the A group [ 37 ], whereas that same approach places OH7 in group E [ 38 ]. The nucleotide sequences for the regions adjacent to the IS elements in our E.
The first is that of sequence similarity. Travisano pers.
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The low similarity here may reflect rapid divergence owing to the absence of any selective constraint on this inactivated gene. This abrupt transition in sequence similarity suggests that one of these two strains, since they diverged from their common ancestor, underwent a recombination event in this region with some more distantly related strain [ 36 ]. Overall, there is striking contrast between the high level of gene-sequence similarity of E. A second level of comparison is the gene order along the chromosome. While the genetic map for K is well established [ 39 ], we have only local sequences in the vicinity of IS elements for E.
Nonetheless, in 14 cases, an IS element in B was located between two genes. Except for one case IS , discussed above in which we found a Shigella flexneri -related sequence on one side, the same pair of genes are immediately adjacent, and have the same relative orientation, in K as in B.
Several IS insertions found in this study appear to be responsible for three phenotypes that either distinguish E. First, the IS element in B is associated with a deletion of the upstream promoter and first bp of ompC This deletion also eliminates micF , which negatively controls expression of ompF [ 40 , 41 ]. The corresponding outer-membrane proteins, OmpC and OmpF, are the two major porins of K, with their relative expression regulated by osmolarity [ 42 , 43 ].
By contrast, E. Second, the particular B strain with which we work was isolated by Seymour Lederberg [ 27 ] as a restriction-modification deficient mutant following treatment with the mutagen 1-methylnitronitrosoguanidine. This mutation was called rm and genetic mapping indicated that it was a few minutes counterclockwise to the thr - ara - leu region 0.
We found the IS element at That gene is involved in the 5-methylcytosine restriction system, and this position fits with Lederberg's mapping of the rm mutation, which together suggest that this element is responsible for the restriction-modification deficiency. Third, as described in detail elsewhere [ 17 ], this strain of E. This genetic instability is a consequence of the IS element located immediately upstream of rbsD In this study, we mapped most of the IS elements present in the E.
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No IS 5 element is present. We compared the resulting data with two other E. The most striking common feature is the presence of IS elements, often identical, in multiple hok - sok loci in both B and K By contrast with this situation in these two laboratory strains, IS elements are absent from these loci in strains more recently isolated from nature, including OH7. This association indicates that these regions of the chromosome underwent chromosomal rearrangements, in particular either the insertion or deletion of the DNA regions that distinguish the different strains; in the case of insertions, the large-scale differences also imply horizontal transfer.
IS elements are well known, of course, to play important roles in gene deletion events and in the incorporation of foreign DNA into the chromosome during and following horizontal gene transfer. One such B-island contains a gene, wbbD , that is identical to a gene involved in O-antigen synthesis from O7:K1, but the IS element precludes this function in B. The available evidence also indicates that local gene order along the two chromosomes is largely the same, except for islands of strain-specific DNA. The strain used in this study is an E.
For cloning experiments, we used E. Genomic DNA was prepared from 1. Plasmid DNA was prepared from 1. All restriction enzymes were purchased from Life Technologies. Sequences adjacent to the various IS elements were cloned as described previously [ 25 ]. Briefly, genomic DNA of the E. Fragments were separated on a 0. The primer pairs used for inverse-PCR to amplify sequences adjacent to the different IS elements are listed in Table 2. All primer pairs are near the extremities of the corresponding IS and are directed outward.
The fragments containing adjacent sequences were used as probes in subsequent hybridization experiments to confirm that the right fragments were cloned. These cloned adjacent sequences were then sequenced using the same primers as for the PCR experiments. All map positions are reported based on the genome sequence of E. DNA fragments used as probes were cold-labeled and hybridizations were performed with the DIG-labeling and detection kit sold by Roche.
Sequences adjacent to IS elements were used to probe reference membranes that had been previously probed with the corresponding IS elements themselves. The membranes were probed with the IS elements, stripped and re-probed with adjacent sequences to demonstrate that the correct sequences were cloned. Mahillon J, Chandler M: Insertion sequences. Microbiol Mol Biol Rev. Mol Gen Genet. The mean length of the rosette loops was 1. Both relaxed folded and supercoiled folded forms of DNA were observed on the preparation. Sometimes the rosettes were connected with large aggregates of DNA possibly the material of bacterial chromosomes and had the appearance of thick fibers with numerous lateral loops.
Linear, cyclic and various replicative forms of DNA have also been observed. It is assumed that rosettes of the extrachromosomal elements of E. How does Europe PMC derive its citations network? Protein Interactions. Protein Families. Nucleotide Sequences.
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