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Antimicrobial resistance is a critical issue that is no longer restricted to hospital settings, but also represents a growing problem involving intensive animal production systems. In this study, we have performed a microbiological and molecular investigation of priority pathogens carrying transferable resistance genes to critical antimicrobials in one-day-old chickens imported from Brazil to Uruguay. Bacterial identification was performed by MALDI-TOF mass spectrometry and antibiotic susceptibility was determined by Sensititre. Antimicrobial resistance genes were sought by polymerase chain reaction and clonality was assessed by PFGE. Four multidrug-resistant (MDR) representative strains were sequenced by Illumina and/or Oxford Nanopore Technologies. Twenty-eight MDR isolates identified as Escherichia coli (n= 14), Enterobacter cloacae (n= 11) and Klebsiella pneumoniae (n= 3). While resistance to oxyiminocephalosporins was due to blaCTX-M-2, blaCTX-M-8, blaCTX-M-15, blaCTX-M-55 and blaCMY-2, plasmid-mediated quinolone resistance was associated with qnrB19, qnrE1, and qnrB2 genes. Finally, resistance to aminoglycosides and fosfomycin was due to the presence of 16S rRNA methyltransferase rmtG and fosA-type genes, respectively. Short and long-read genome sequencing of E. cloacae ODC-Eclo3 strain revealed the presence of IncQ/rmtG (pUR-EC3.1, 7400-pb), IncHI2A/mcr-9.1/blaCTX-M-2 [pUR-EC3.2, ST16 (pMLST), 408,436-bp] and IncN2/qnrB19/aacC3/aph(3'')-Ib (pUR-EC3.3) resistance plasmids. Strikingly, the blaCTX-M-2 gene was carried by a novel Tn1696-like composite transposon designated Tn7337. In summary, we report that imported one-day-old chicks can act as Trojan horses for the hidden spread of WHO critical priority MDR pathogens harboring mcr-9, rmtG and extended-spectrum β-lactamase genes in poultry farms, which is a critical issue within a One Health perspective. Importance section Antimicrobial resistance is considered a significant problem for global health, including within the concept of "One Health", therefore, the food chain is a link that connects human and animal health directly. In this work, we searched for microorganisms resistant to antibiotics considered critical for human health in intestinal microbiota of one-day-old baby chicks imported to Uruguay from Brazil. We described antibiotic-resistant genes to antibiotics named as to watch or reserve for the WHO, such as rmtG or mcr9.1, which confers resistance to all the aminoglycosides and colistin, respectively, among others genes, and their presence in new mobile genetic elements that favor its dissemination. The sustained entry of these microorganisms evades the sanitary measures implemented by the countries and production establishments to reduce the selection of resistant microorganisms. These silently imported resistant microorganisms could explain a considerable part of the antimicrobial resistance problems found in the production stages of the system.

Since the initial discovery of mcr-1 in an Escherichia coli isolate from China, the gene has also been detected in Klebsiella pneumoniae and Salmonella enterica but is rarely reported in other Enterobacteriaceae . Here, we report the isolation and identification of a Shigella flexneri strain harboring mcr-1 from stool samples in a pig farm in China from 2009. The MIC of colistin for the isolate is 4 μg/ml. Conjugation assays showed that the donor S. flexneri strain has functional and transferable colistin resistance. Sequencing revealed that mcr-1 was present on a putative composite transposon flanked by inverted repeats of IS Apl1 . IMPORTANCE There are four species of Shigella , and Shigella flexneri is the most frequently isolated species in low- and middle-income countries (LMICs). In this study, we report a functional, transferable, plasmid-mediated mcr-1 gene in S. flexneri . We have shown that mcr-1 is located on a novel composite transposon which is flanked by inverted repeats of IS Apl1 . The host strain is multidrug resistant, and this multidrug resistance is also transferable. The finding of a functional mcr-1 gene in S. flexneri , a human-associated Enterobacteriaceae family member, is a cause for concern as infections due to S. flexneri are the main Shigella infections in most low- and middle-income countries.

Desulfitobacterium hafniense strain TCE1 is capable of metabolically reducing tetra- and trichloroethenes by organohalide respiration. A previous study revealed that the pce gene cluster responsible for this process is located on an active composite transposon, Tn- Dha1 . In the present work, we investigated the effects on the stability of the transposon during successive subcultivations of strain TCE1 in a medium depleted of tetrachloroethene. At the physiological level, an increased fitness of the population was observed after 9 successive transfers and was correlated with a decrease in the level of production of the PceA enzyme. The latter observation was a result of the gradual loss of the pce genes in the population of strain TCE1 and not of a regulation mechanism, as was postulated previously for a similar phenomenon described for Sulfurospirillum multivorans . A detailed molecular analysis of genetic rearrangements occurring around Tn- Dha1 showed two independent but concomitant events, namely, the transposition of the first insertion sequence, IS Dha1 -a, and homologous recombination across identical copies of IS Dha1 flanking the transposon. A new model is proposed for the genetic heterogeneity around Tn- Dha1 in D. hafniense strain TCE1, along with some considerations for the cleavage mechanism mediated by the transposase TnpA1 encoded by IS Dha1 .

The α-proteobacterial strain Sphingobium japonicum UT26 utilizes a highly chlorinated pesticide, γ-hexachlorocyclohexane (γ-HCH), as a sole source of carbon and energy, and haloalkane dehalogenase LinB catalyzes the second step of γ-HCH degradation in UT26. Functional complementation of a linB mutant of UT26, UT26DB, was performed by the exogenous plasmid isolation technique using HCH-contaminated soil, leading to our successful identification of a plasmid, pLB1, carrying the linB gene. Complete sequencing analysis of pLB1, with a size of 65,998 bp, revealed that it carries (i) 50 totally annotated coding sequences, (ii) an IS 6100 composite transposon containing two copies of linB , and (iii) potential genes for replication, maintenance, and conjugative transfer with low levels of similarity to other homologues. A minireplicon assay demonstrated that a 2-kb region containing the predicted repA gene and its upstream region of pLB1 functions as an autonomously replicating unit in UT26. Furthermore, pLB1 was conjugally transferred from UT26DB to other α-proteobacterial strains but not to any of the β- or γ-proteobacterial strains examined to date. These results suggest that this exogenously isolated novel plasmid contributes to the dissemination of at least some genes for γ-HCH degradation in the natural environment. To the best of our knowledge, this is the first detailed report of a plasmid involved in γ-HCH degradation.

A native composite transposon was isolated from Corynebacterium glutamicum ATCC 14751. This transposon comprises two functional copies of a corynebacterial IS 31831 -like insertion sequence organized as converging terminal inverted repeats. This novel 20.3-kb element, Tn 14751 , carries 17.4 kb of C. glutamicum chromosomal DNA containing various genes, including genes involved in purine biosynthesis but not genes related to bacterial warfare, such as genes encoding mediators of antibiotic resistance or extracellular toxins. A derivative of this element carrying a kanamycin resistance cassette, minicomposite Tn 14751 , transposed into the genome of C. glutamicum at an efficiency of 1.8 × 10 2 transformants per μg of DNA. Random insertion of the Tn 14751 derivative carrying the kanamycin resistance cassette into the chromosome was verified by Southern hybridization. This work paves the way for realization of the concept of minimum genome factories in the search for metabolic engineering via genome-scale directed evolution through a combination of random and directed approaches.

This study presents the first complete sequence of an IncU plasmid, pFBAOT6. This plasmid was originally isolated from a strain of Aeromonas caviae from hospital effluent (Westmorland General Hospital, Kendal, United Kingdom) in September 1997 (G. Rhodes, G. Huys, J. Swings, P. McGann, M. Hiney, P. Smith, and R. W. Pickup, Appl. Environ. Microbiol. 66: 3883-3890, 2000) and belongs to a group of related plasmids with global ubiquity. pFBAOT6 is 84,748 bp long and has 94 predicted coding sequences, only 12 of which do not have a possible function that has been attributed. Putative replication, maintenance, and transfer functions have been identified and are located in a region in the first 31 kb of the plasmid. The replication region is poorly understood but exhibits some identity at the protein level with replication proteins from the gram-positive bacteria Bacillus and Clostridium . The mating pair formation system is a virB homologue, type IV secretory pathway that is similar in its structural organization to the mating pair formation systems of the related broad-host-range (BHR) environmental plasmids pIPO2, pXF51, and pSB102 from plant-associated bacteria. Partitioning and maintenance genes are homologues of genes in IncP plasmids. The DNA transfer genes and the putative oriT site also exhibit high levels of similarity with those of plasmids pIPO2, pXF51, and pSB102. The genetic load region encompasses 54 kb, comprises the resistance genes, and includes a class I integron, an IS 630 relative, and other transposable elements in a 43-kb region that may be a novel Tn 1721 -flanked composite transposon. This region also contains 24 genes that exhibit the highest levels of identity to chromosomal genes of several plant-associated bacteria. The features of the backbone of pFBAOT6 that are shared with this newly defined group of environmental BHR plasmids suggest that pFBAOT6 may be a relative of this group, but a relative that was isolated from a clinical bacterial environment rather than a plant-associated bacterial environment.

Fifty-nine monophasic Salmonella enterica serovar Typhimurium isolates, collected in Belgium during the period from 2008 to 2011, have been serotyped as 4,[5]:i:− and shown to harbor an fljB coding sequence. The genetic differences between these strains and phenotypically biphasic Salmonella Typhimurium were analyzed through PCR and DNA sequencing. Genetic alterations in the fljB promoter region affecting expression of the phase 2 flagellin were observed in 53 isolates. Other genetic events in the invertible region carrying the fljB promoter were observed in 2 isolates. For the remaining 4 isolates, no molecular differences with a reference biphasic Salmonella Typhimurium strain could be observed. Next-generation sequencing of one representative isolate affected in the fljB promoter region revealed a 26-kb IS 26 composite transposon insertion along with a local genomic rearrangement. Several other IS 26 element-mediated alterations of this genomic region were observed. This group of monophasic Salmonella Typhimurium isolates was genetically heterogeneous, as revealed by multilocus variable-number tandem-repeat analysis (MLVA), PCR, and sequencing. Pigs and pork represented a major source of such monophasic isolates in Belgium, as reported in other countries. Three out of 5 isolates of human origin presented genetic profiles identical to those of food isolates, demonstrating the pathogenic potential of the newly characterized variants and potential dissemination along the food chain. This study highlighted the key role played by IS 26 insertions in the loss of phase 2 flagellin expression and the subsequent generation of multiple monophasic variant lineages from biphasic Salmonella Typhimurium ancestors.

The two haloacetate dehalogenase genes, dehH1 and dehH2 , on the 65-kb plasmid pUO1 from Delftia acidovorans strain B were found to be located on transposable elements. The dehH2 gene was carried on an 8.9-kb class I composite transposon (Tn Had1 ) that was flanked by two directly repeated copies of IS 1071 , IS 1071 L and IS 1071 R. The dehH1 gene was also flanked by IS 1071 L and a truncated version of IS 1071 (IS 1071 N). Tn Had1 , dehH1 , and IS 1071 N were located on a 15.6-kb class II transposon (Tn Had2 ) whose terminal inverted repeats and res site showed high homology with those of the Tn 21 -related transposons. Tn Had2 was defective in transposition because of its lacking the transposase and resolvase genes. Tn Had2 could transpose when the Tn 21 -encoded transposase and resolvase were supplied in trans . These results demonstrated that Tn Had2 is a defective Tn 21 -related transposon carrying another class I catabolic transposon.

Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl- N -methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine. Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene ( cehA ) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing cehA . No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of the cehA gene. Six open reading frames including the cehA gene were found in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon.

The enzyme triphenylmethane reductase (TMR) reduces toxic triphenylmethane dyes into colorless, nontoxic derivatives, and TMR-producing microorganisms have been proposed as bioremediation tools. Analysis of the genome of Listeria monocytogenes H7858 (1998-1999 hot dog outbreak) revealed that the plasmid (pLM80) of this strain harboring a gene cassette ( bcrABC ) conferring resistance to benzalkonium chloride (BC) and other quaternary ammonium disinfectants also harbored a gene ( tmr ) highly homologous to TMR-encoding genes from diverse Gram-negative bacteria. The pLM80-associated tmr was located two genes downstream of bcrABC as part of a putative IS 1216 composite transposon. To confirm the role of tmr in triphenylmethane dye detoxification, we introduced various tmr -harboring fragments of pLM80 in a pLM80-cured derivative of strain H7550, from the same outbreak as H7858, and assessed the resistance of the constructs to the triphenylmethane dyes crystal violet (CV) and malachite green. Transcriptional and subcloning data suggest that the regulation of TMR is complex. Constructs harboring fragments spanning bcrABC and tmr were CV resistant, and in such constructs tmr transcription was induced by sublethal levels of either BC or CV. However, constructs harboring only tmr and its upstream intergenic region could also confer resistance to CV, albeit at lower levels. Screening a panel of BC-resistant L. monocytogenes strains revealed that all those harboring bcrABC and adjacent pLM80 sequences, including tmr , were resistant to CV and decolorized this dye. The findings suggest a potential role of TMR as a previously unknown adaptive attribute for environmental persistence of L. monocytogenes .

The conjugative transposon CTnDOT is virtually identical over most of its length to another conjugative transposon, CTnERL, except that CTnDOT carries an ermF gene that is not found on CTnERL. In this report, we show that the region containing ermF appears to consist of a 13-kb chimera composed of at least one class I composite transposon and a mobilizable transposon (MTn). Although the ermF region contains genes also carried on Bacteroides transposons Tn 4351 and Tn 4551 , it does not contain the IS 4351 element which is found on these transposons. In CTnDOT, insertion of the ermF region occurred near a stem-loop structure at the end of orf2 , an open reading frame located immediately downstream of the integrase ( int ) gene of CTnDOT, and in a region known to be important for excision of CTnERL and CTnDOT. The chimera that comprises the ermF region can apparently no longer excise and circularize, but it contains a functional mobilization region related to that described for the Bacteroides MTn Tn 4399 . Analysis of 19 independent Bacteroides isolates showed that the ermF region is located in the same position in all of the strains analyzed and that the compositions of the ermF region are almost identical in these strains. Therefore, it appears that CTnDOT-like elements present in community and clinical isolates of Bacteroides were derived from a common ancestor and proliferated in the diverse Bacteroides population.

Alcaligenes eutrophus ( Ralstonia eutropha ) NH9, isolated in Japan, utilizes 3-chlorobenzoate as its sole source of carbon and energy. Sequencing of the relevant region of plasmid pENH91 from strain NH9 revealed that the genes for the catabolic enzymes were homologous to the genes of the modified ortho -cleavage pathway. The genes from strain NH9 ( cbnR-ABCD ) showed the highest homology (89 to 100% identity at the nucleotide level) to the tcbR-CDEF genes on plasmid pP51 of the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51, which was isolated in The Netherlands. The structure of the operon, including the lengths of open reading frames and intervening sequences, was completely conserved between the cbn and tcb genes. Most nucleotide substitutions were localized within and proximal to the cbnB ( tcbD ) gene. The difference in the chloroaromatics that the two strains could use as growth substrates seemed to be due to differences in enzymes that convert substrates to chlorocatechols. The restriction map of plasmid pENH91 was clearly different from that of pP51 except in the regions that contained the cbnR-ABCD and tcbR-CDEF genes, respectively, suggesting that the chlorocatechol gene clusters might have been transferred as units. Two homologous sequences, present as direct repeats in both flanking regions of the cbnR-ABCD genes on pENH91, were found to be identical insertion sequences (ISs), designated IS 1600 , which formed a composite transposon designated Tn 5707 . Although the tcbR-CDEF genes were not associated with similar ISs, a DNA fragment homologous to IS 1600 was cloned from the chromosome of strain P51. The sequence of the fragment suggested that it might be a remnant of an IS. The two sequences, together with IS 1326 and nmoT , formed a distinct cluster on a phylogenetic tree of the IS 21 family. The diversity of the sources of these IS or IS-like elements suggests the prevalence of ISs of this type.

Comamonas testosteroni T-2 degrades p-toluenesulfonate (TSA) via p-sulfobenzoate (PSB) and protocatechuate and degrades toluenecarboxylate via terephthalate (TER) and protocatechuate. The appropriate genes are expressed in at least five regulatory units, some of which are also found in C. testosteroni PSB-4 (F. Junker, R. Kiewitz, and A. M. Cook, J. Bacteriol. 179:919-927, 1997). C. testosteroni T-2 was found to contain two plasmids, pTSA (85 kbp) and pT2T (50 kbp); a TSA- mutant (strain TER-1) contained only plasmid pT2T. C. testosteroni PSB-4, which does not degrade TSA, contained one plasmid, pPSB (85 kbp). The type strain contained no plasmids. Conjugation experiments showed that plasmid pTSA (possibly in conjunction with pT2T) was conjugative, and the single copy of the TSA operon (tsaMBCD) with its putative regulator gene (tsaR) in strain T-2 was found on plasmid pTSA, which also carried the PSB genes (psbAC) and presumably transport for both substrates. Plasmid pTSA was assigned to the IncP1 beta group and was found to carry two copies of insertion element IS1071. Plasmid pPSB (of strain PSB-4), which could be maintained in strains with plasmid pTSA or pT2T, was also conjugative and was found to carry the PSB genes as well as to contain two copies of IS1071. In attempted conjugations with the type strain, no plasmid was recovered, but the PSB+ transconjugant carried two copies of IS1071 in the chromosome. We presume the PSB genes to be located in a composite transposon. The genes encoding the putative TER operon and degradation of protocatechuate, with the meta cleavage pathway, were attributed a chromosomal location in strains T-2 and PSB-4.

Growth on 3-chlorobenzoate was found to induce the enzymes of the protocatechuate meta ring fission pathway in Alcaligenes sp. strain BR60. The chlorobenzoate catabolic genes, designated cba, were localized to a 3.7-kb NotI-EcoRI fragment within the nonrepeated region of the composite transposon Tn5271. The cba genes were cloned onto two broad-host-range vectors and expressed in Escherichia coli and Alcaligenes sp. strain BR6024. In E. coli, expression of the cba genes with the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible tac promoter of the IncQ vector pMMB66HE resulted in the production of protocatechuate and chlorodihydroxybenzoate metabolites of 3-chlorobenzoate. Expression of this construct in one orientation resulted in the formation of two polypeptides 51 and 42 kDa in size. This result was confirmed by subcloning into pGEM3Zf and then incorporating L-35S-methionine into newly synthesized proteins, using the thermally regulated T7 polymerase-promoter system. Introduction of the NotI-EcoRI fragment into Alcaligenes sp. strain BR6024 (Cba-P), using the IncP broad-host-range, mobilizable plasmid pBW13, restored the 3-chlorobenzoate-degradative phenotype and resulted in the accumulation of protocatechuate and chlorodihydroxybenzoate intermediates. The data indicate that a two-component dioxygenase specified by Tn5271 oxidizes 3-chlorobenzoate at the 3,4- or 4,5-positions. This activity extends the range of pathways for chloroaromatic compounds known to be functional in the environment. The new pathway avoids the toxicity attributed to the accumulation of chlorocatechol metabolites in bacteria degrading chlorobenzoates.

Metabolic redundancy in bacteria is a common feature of microbial ecosystems. For example, multiple biochemical pathways encoded by genetically divergent operons may function in the degradation of single, relatively simple organic compounds. Among the aerobic members of the class Proteobacteria alone, toluene degradation is initiated by a range of dioxygenase (tod) and monooxygenase (xyl, tmo, tbm, tbu, and tou) catabolic operons (2, 5, 15, 33, 45). Two or more of these operons may be expressed in a single strain (16). Similarly, two nonhomologous phenol degradation operons occur in aerobic members of the Proteobacteria: the multicomponent phenol hydroxylases represented by the dmp operon (30, 31) and the single-component phenol hydroxylases represented by the pheA or tbuD genes (20, 22). For chlorobenzoic acid (CBA) degradation, there are at least three distinct biochemical pathways found in isolates collected from around the world (Fig. ​(Fig.1)1) (9, 10, 27, 29, 37, 42). The clc, cba, and fcb operons are nonhomologous and are found at a variety of different loci including chromosomes, plasmids, and transposons in different bacteria (8, 25, 32, 41, 46). A fourth pathway via gentisate has recently been characterized at the biochemical level in Alcaligenes sp. strain L6 (21). FIG. 1 Representative metabolic pathways encoded by the nonhomologous CBA-degradative operons clcABD, cbaABC, and fcbBAC. The genes that make up these operons are indicated in open boxes below the metabolic steps they encode. Schematic representations of the ... While redundancy of metabolic pathways in microbial ecosystems is recognized, few studies have been designed to show whether pollutants in natural environments select a single, dominant genotype or multiple, redundant genotypes. In addition, we know little about how genotypes found at polluted sites differ from those that occur at pristine sites. These were questions we addressed in this study of the selection of clc, cba, and fcb genotypes at the Hyde Park chemical landfill site. In recent studies of the genotypes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation in 2,4-D-amended soils, it was found that selection of divergent families of 2,4-D-α-ketoglutarate dioxygenase (tfdA) genes occurred (12, 17, 24). tfdA gene families I and III, representative of the Sphingomonas and broad-host-range pJP4-like operons, respectively, were most commonly selected following long-term 2,4-D exposure. In other work, the distribution of the nonhomologous phenol hydroxylase genes was studied in isolates from freshwater and marine samples (31, 36). Genes homologous to the multicomponent phenol hydroxylase dmp operon were most often detected in isolates from both of these environments. The single-component phenol hydroxylase operon pheBA, which is part of a composite transposon structure, occurred rarely. This property allowed Peters et al. (36) to infer that pheBA had transferred horizontally between bacteria in river water following the introduction of this genotype for bioremediation of phenol contaminants. In recent studies of enrichments from pristine soils, it was found that the ability to degrade 3-chlorobenzoate (3-CBA) was widespread and that the predominant pathway was via chlorocatechol intermediates (12, 13, 23). Nevertheless, genomic DNA of isolates from these pristine soils failed to hybridize with DNA probes derived from two of the known CBA catabolic operons (clc and cba). Studies using gene probes representative of the range of known CBA metabolic pathways have not been done on samples from environments chronically contaminated by CBA. This is surprising given the central role of CBA degradation in the aerobic metabolism of polychlorinated biphenyls. In a previous study, we determined the 3-CBA, 4-CBA, and 3,4-dichlorobenzoate (3,4-DCBA) degradation potentials of contaminated and noncontaminated freshwater sediment samples taken from three streams at the Hyde Park, Niagara Falls, chemical landfill site (34, 35). The previous study showed that contaminants leaching from the site, which include CBA congeners, chlorobiphenyls (CBPs), chlorinated hydrocarbons, and phenols, have significantly increased the CBA degradation potential relative to that of a noncontaminated control stream nearby. The previous study also revealed seasonal and year-to-year variation in degradation potentials. In the study reported here, we used the same sites to determine the incidence of the clcABD, cbaABC, and fcbB catabolic genotypes among isolates enriched from samples of both contaminated and noncontaminated sources at Hyde Park. Genetic variation at the clc, cba, and fcb loci; the incidence of IS1071, which flanks the cba operon; and phenotypic relationships between the hosts of these genes were determined.