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International audience; Objectives: Pseudomonas aeruginosa occupies a central position in nosocomial infections and remains a significant cause of morbidity and mortality. The aim of this study was to characterize carbapenem resistance mechanisms in P. aeruginosa isolates from clinical specimens collected at the University Hospital of Oran, western Algeria. Materials and Methods: The identification of 214 nonduplicated P. aeruginosa isolates (collected from January to December 2016) was confirmed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Thirteen antibiotics were tested using the disc diffusion method. Carbapenemase-encoding genes were detected with the GeneXpert system and multiplex polymerase chain reaction (PCR). Clonal relatedness was determined using multilocus sequence typing (MLST) and the seven housekeeping genes were further used for phylogenetic analysis of imipenem-resistant P. aeruginosa using concatenated gene fragments. The flanking regions of the blaVIM-4 gene were analyzed by whole-genome sequencing. Results: Eleven isolates (5.39%) were resistant to carbapenems. PCR amplification and sequencing showed that six of these isolates (2.94%) harbored the blaVIM-4 gene that was carried on a novel class 1 integron. MLST analysis assigned the tested isolates to seven different sequence types (STs), of which two were new (ST3349 and ST3350) and five were previously described (ST244, ST499, ST709, ST809, and ST1239). Conclusion: In this study, we reported P. aeruginosa isolates producing VIM-4 in an Algerian hospital. The blaVIM-4 is harbored in class 1 integron with a new arrangement of genes cassettes.

Integrons are genetic systems conferring to bacteria a rapid adaptation capability. The integron integrase is able to capture, stockpile and shuffle novel functions embedded in cassettes. This involves the recognition of both substrates, theattIsite, and the cassette associatedattCsites. Integrons can be sedentary and chromosomally located (SCI) or, carried by conjugative plasmids (Mobile Integron, MI), hence favoring their dissemination among bacteria. Here, for the first time, we investigate the cassette recruitment in theVibrio choleraeSCI during conjugation and natural transformation. We demonstrated that horizontally transferred cassette can be recruited inside the chromosomal integron. The endogenous integrase expression is sufficiently triggered, after SOS response induction mediated by the entry of single-stranded cassettes during conjugation and natural transformation, to mediate significant cassette insertion. We demonstrate that theattIAinsertion is preferential, despite the presence of 180attCsites in the integron array. Thanks to the presence of a promoter in theattIAsite vicinity, all these newly inserted cassettes are expressed and prone to adaptive selection. We also show that the RecA protein is critical for cassette recruitment inV. choleraeSCI but not in MIs. Moreover,a contrarioto MIs, theV. choleraeSCI is not active in others bacterial hosts. MIs might have evolved from the SCIs by overcoming host factors, which would explain their large dissemination in bacteria and their role in the antibioresistance expansion.

Synthetic DNA design needs to harness the many information layers embedded in a DNA string. We previously developed the Evolutionary Landscape Painter (ELP), an algorithm that exploits the degeneracy of the code to increase protein evolvability. Here, we have used ELP to recode the integron integrase gene (intI1) in two alternative alleles. Although synonymous, both alleles yielded less IntI1 protein and were less active in recombination assays than intI1. We spliced the three alleles and mapped the activity decrease to the beginning of alternative sequences. Mfold predicted the presence of more stable secondary structures in the alternative genes. Using synonymous mutations, we decreased their stability and recovered full activity. Following a design-build-test approach, we have now updated ELP to consider such structures and provide streamlined alternative sequences. Our results support the possibility of modulating gene activity through the ad hoc design of 5′ secondary structures in synthetic genes. This article is protected by copyright. All rights reserved

Background The present study aimed to perform a deep phenotypic and genotypic analysis of 15 clinical carbapenem-resistant Acinetobacter baumannii (CRAb) strains isolated in Madagascar between 2008 and 2016 from diverse sources. Methods CRAb isolates collected from the Clinical Biology Centre of the Institut Pasteur of Madagascar, from the neonatal unit of Antananarivo military hospital, and from intensive care units of Mahajanga Androva and Antananarivo Joseph Ravoahangy Andrianavalona (HJRA) hospitals were subjected to susceptibility testing. Whole-genome sequencing allowed us to assess the presence of antibiotic-resistance determinants, insertion sequences, integrons, genomic islands and potential virulence factors in all strains. The structure of the carO porin gene and deduced protein (CarO) were also assessed in CRAb isolates. Results All isolates were found to be multidrug-resistant strains. Antibiotic-resistance genes against six classes of antimicrobial agents were described. The four carbapenem-resistance genes: bla OXA-51 like , bla OXA-23 , bla OXA-24 , and bla OXA-58 genes were detected in 100, 53.3, 13.3, and 6.6% of the isolates, respectively. Additionally, an ISAba1 located upstream of bla OXA-23 and bla ADC-like genes was observed in 53.3 and 66.7% of isolates, respectively. Further, Tn2006 and Tn2008 were found associated to the ISAba1-bla OXA-23 structure. An 8051-bp mobilizable plasmid harbouring the bla OXA-24 gene was isolated in two strains. In addition, 46.7% of isolates were positive for class 1 integrons. Overall, five sequences types (STs), with predominantly ST2, were detected. Several virulence genes were found in the CRAb isolates, among which two genes, epsA and ptk, responsible for the capsule-positive phenotype, were involved in A. baumannii pathogenesis. Conclusions This study revealed the presence of high-level carbapenem resistance in A. baumannii with the first description of OXA-24 and OXA-58 carbapenemases in Madagascar. This highlights the importance of better monitoring and controlling CRAb in Madagascan hospitals to avoid their spread.

International audience; Biologically functional DNA hairpins are found in archaea, prokaryotes and eukaryotes, playing essential roles in various DNA transactions. However, during DNA replication, hairpin formation can stall the polymerase and is therefore prevented by the single-stranded DNA binding protein (SSB). Here, we address the question how hairpins maintain their functional secondary structure despite SSB's presence. As a model hairpin, we used the recombinogenic form of the attC site, essential for capturing antibiotic-resistance genes in the integrons of bacteria. We found that attC hairpins have a conserved high GC-content near their apical loop that creates a dynamic equilibrium between attC fully opened by SSB and a partially structured attC-6-SSB complex. This complex is recognized by the recombinase IntI, which extrudes the hairpin upon binding while displacing SSB. We anticipate that this intriguing regulation mechanism using a base pair distribution to balance hairpin structure formation and genetic stability is key to the dissemination of antibiotic resistance genes among bacteria and might be conserved among other functional hairpins.

Integrons ensure a rapid and “on demand” response to environmental stresses driving bacterial adaptation. They are able to capture, store, and reorder functional gene cassettes due to site-specific recombination catalyzed by their integrase. Integrons can be either sedentary and chromosomally located or mobile when they are associated with transposons and plasmids. They are respectively called sedentary chromosomal integrons (SCIs) and mobile integrons (MIs). MIs are key players in the dissemination of antibiotic resistance genes. Here, we used in silico and in vivo approaches to study cassette excision dynamics in MIs and SCIs. We show that the orientation of cassette arrays relative to replication influences attC site folding and cassette excision by placing the recombinogenic strands of attC sites on either the leading or lagging strand template. We also demonstrate that stability of attC sites and their propensity to form recombinogenic structures also regulate cassette excision. We observe that cassette excision dynamics driven by these factors differ between MIs and SCIs. Cassettes with high excision rates are more commonly found on MIs, which favors their dissemination relative to SCIs. This is especially true for SCIs carried in the Vibrio genus, where maintenance of large cassette arrays and vertical transmission are crucial to serve as a reservoir of adaptive functions. These results expand the repertoire of known processes regulating integron recombination that were previously established and demonstrate that, in terms of cassette dynamics, a subtle trade-off between evolvability and genetic capacitance has been established in bacteria., IMPORTANCE The integron system confers upon bacteria a rapid adaptation capability in changing environments. Specifically, integrons are involved in the continuous emergence of bacteria resistant to almost all antibiotic treatments. The international situation is critical, and in 2050, the annual number of deaths caused by multiresistant bacteria could reach 10 million, exceeding the incidence of deaths related to cancer. It is crucial to increase our understanding of antibiotic resistance dissemination and therefore integron recombination dynamics to find new approaches to cope with the worldwide problem of multiresistance. Here, we studied the dynamics of recombination and dissemination of gene encoding cassettes carried on integrons. By combining in silico and in vivo analyses, we show that cassette excision is highly regulated by replication and by the intrinsic properties of cassette recombination sites. We also demonstrated differences in the dynamics of cassette recombination between mobile and sedentary chromosomal integrons (MIs and SCIs). For MIs, a high cassette recombination rate is favored and timed to conditions when generating diversity (upon which selection can act) allows for a rapid response to environmental conditions and stresses. In contrast, for SCIs, cassette excisions are less frequent, limiting cassette loss and ensuring a large pool of cassettes. We therefore confirm a role of SCIs as reservoirs of adaptive functions and demonstrate that the remarkable adaptive success of integron recombination system is due to its intricate regulation.

Integrons play a major role in the dissemination of antibiotic resistance genes among bacteria. Rearrangement of gene cassettes occurs by recombination between attI and attC sites, catalyzed by the integron integrase. Integron recombination uses an unconventional mechanism involving a folded single-stranded attC site. This site could be a target for several host factors and more precisely for proteins able to bind single-stranded DNA. One of these, Escherichia coli single-stranded DNA-binding protein (SSB), regulates many DNA processes. We studied the influence of this protein on integron recombination. Our results show the ability of SSB to strongly bind folded attC sites and to destabilize them. This effect was observed only in the absence of the integrase. Indeed, we provided evidence that the integrase is able to counterbalance the observed effect of SSB on attC site folding. We showed that IntI1 possesses an intrinsic property to capture attC sites at the moment of their extrusion, stabilizing them and recombining them efficiently. The stability of DNA secondary structures in the chromosome must be restrained to avoid genetic instability (mutations or deletions) and/or toxicity (replication arrest). SSB, which hampers attC site folding in the absence of the integrase, likely plays an important role in maintaining the integrity and thus the recombinogenic functionality of the integron attC sites. We also tested the RecA host factor and excluded any role of this protein in integron recombination.

International audience; To investigate the possible role of the hospital environment in the dissemination of extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli isolates, 300 samples were taken during 2013 from abiotic surfaces (n = 250), healthcare worker hands (n = 27), and hands of patients (n = 23) in a Tunisian Hospital. ESBL-producing E. coli isolates were recovered in 3.7% of analyzed samples (4% abiotic surfaces; 4.3% hands of patients; 0% in healthcare worker hands), and one isolate/sample was further studied. The characterization of beta-lactamase genes, as well as the genetic environment of bla CTX-M gene, was performed by PCR and sequencing. The ESBL genes found were as follows: bla CTX-M-15 (eight isolates), bla CTX-M-15 +bla SHV-12 (two isolates), and bla SHV-12 (one isolate). The bla TEM-1b gene was detected in seven ESBL-positive isolates. The orf477 was found downstream of bla CTX-M-15 gene in 10 strains, whereas the ISEcp1 sequence was identified upstream of this gene in two isolates. The analysis of class 1 integrons by PCR and sequencing revealed five positive isolates with the following gene cassette arrangements: dfrA1-aadA1 (two isolates), aadA1 (two isolates), and aadA2 (one isolate). The virulence-encoding genes aer, eae, bfp, and hly were detected by PCR in six, four, four, and three isolates, respectively. The following sequence types and associated phylogroups were detected among ESBL-producing strains: ST167-phylogroup-A (six isolates) and ST131-phylogroup-B2 (two isolates). In conclusion, the hospital environment could be a reservoir of multiresistant bacteria, including ESBL-positive E. coli isolates, which could be acquired by the patient population, and strict control measures should be established to minimize this problem.

Analysis of two clonally related multiresistant Pseudomonas aeruginosa isolates led to the identification of a novel IMP-type metallo-β-lactamase. IMP-29 was significantly different from the other IMP variants (the closest variant being IMP-5 with 93% amino acid identity). The bla IMP-29 gene cassette was carried by a class 1 integron in strain 10.298, while in strain 10.266 it was located in a rearranged DNA region on a 30-kb conjugative plasmid. Biochemical analysis confirmed that IMP-29 efficiently hydrolyzed carbapenems.

International audience; Integrons are genetic elements able to acquire and rearrange open reading frames (ORFs) embedded in gene cassette units and convert them to functional genes by ensuring their correct expression. They were originally identified as a mechanism used by Gram-negative bacteria to collect antibiotic resistance genes and express multiple resistance phenotypes in synergy with transposons. More recently, their role has been broadened with the discovery of chromosomal integron (CI) structures in the genomes of hundreds of bacterial species. This review focuses on the resources carried in these elements, on their unique recombination mechanisms, and on the different mechanisms controlling the cassette dynamics. We discuss the role of the toxin/antitoxin (TA) cassettes for the stabilization of the large cassette arrays carried in the larger CIs, known as superintegrons. Finally, we explore the central role played by single-stranded DNA in the integron cassette dynamics in light of the recent discovery that the integron integrase expression is controlled by the SOS response.

International audience; OBJECTIVES: Integrons are bacterial genetic elements that can capture and express genes contained in mobile cassettes. Integrons have been described worldwide in Gram-negative bacteria and are a marker of antibiotic resistance. We developed a specific and sensitive Taqman probe-based real-time PCR method with three different primer-probe pairs for simultaneous detection of the three main classes of integron. METHODS: Sensitivity was assessed by testing mixtures of the three targets (intI integrase genes of each integron class) ranging from 10 to 10(8) copies. Specificity was determined with a panel of integron-containing and integron-free control strains. The method was then applied to clinical samples. RESULTS: The PCR method was specific and had a sensitivity of 10(2) copies for all three genes, regardless of their respective quantities. The method was quantitative from 10(3) to 10(7) copies, and was able to detect integrons directly in biological samples. CONCLUSIONS: We have developed a rapid, quantitative, specific and sensitive method that could prove useful for initial screening of Gram-negative isolates, or clinical samples, for likely multidrug resistance.

International audience; in the coding sequence. These mutations have previously been shown to result in increased azithromycin resistance due to induced expression of the MtrCDE efflux pump. 8 The genes erm(A), erm(B), erm(C) and erm(F), encoding rRNA methylases, and mef(A), encoding an efflux pump, were not present in these isolates. Molecular epidemiology analysis of the isolates was performed using the N. gonorrhoeae multiantigen sequence typing (NG-MAST) method, 9 which assigns an ST based on a combination of the highly variable por and tbpB alleles. The isolates were assigned ST3102 and ST1866, which are almost identical and differ only by one SNP in por. Thus far, the high-level azithromycin-resistant N. gonorrhoeae strains that have been isolated in various countries belong to 16 different STs, of which ST649 isolates appear to be most widespread. 5 – 7 The por and tbpB alleles of these STs differ by a total of 33–92 SNPs from the por and tbpB alleles of the strains described in this study, except for the Australian ST10133 isolate, 7 which contains an identical tbpB allele (33) and a por allele (2573) that only differs by 16 and 17 SNPs from the por alleles of ST3102 and ST1866, respectively. Interestingly, it was reported that this isolate was most likely contracted in China or Hong Kong, which might explain its closer genetic relationship. How widespread high-level azithromycin-resistant strains are in China remains to be determined. Several years earlier, in 2009, two ST1866 strains were isolated from patients in the city of Nanjing, 10 which is located in a neighbouring province. These strains were reported to have an azithromycin MIC of .64 mg/L. It is not known whether these strains actually are directly related to the ST1866 isolate described in this study, but if they are related it would indicate that this high-level azithromycin-resistant ST might be spreading in China. Azithromycin susceptibility testing is currently not the standard in China. However, the identification of high-level azithromycin-resistant N. gonorrhoeae isolates stresses the need for a more thorough overview of azithromycin susceptibility in China, particularly when ceftriaxone and azithromycin dual antimicrobial therapy is considered as a future first-line therapy. These high-level azithromycin-resistant isolates might have a major impact on the efficacy of this dual antimicrobial therapy.

The integron is a powerful system which, by capturing, stockpiling, and rearranging new functions carried by gene encoding cassettes, confers upon bacteria a rapid adaptation capability in changing environments. Chromosomally located integrons (CI) have been identified in a large number of environmental Gram-negative bacteria. Integron evolutionary history suggests that these sedentary CIs acquired mobility among bacterial species through their association with transposable elements and conjugative plasmids. As a result of massive antibiotic use, these so-called mobile integrons are now widespread in clinically relevant bacteria and are considered to be the principal agent in the emergence and rise of antibiotic multiresistance in Gram-negative bacteria. Cassette rearrangements are catalyzed by the integron integrase, a site-specific tyrosine recombinase. Central to these reactions is the single-stranded DNA nature of one of the recombination partners, the attC site. This makes the integron a unique recombination system. This review describes the current knowledge on this atypical recombination mechanism, its implications in the reactions involving the different types of sites, attC and attI , and focuses on the tight regulation exerted by the host on integron activity through the control of attC site folding. Furthermore, cassette and integrase expression are also highly controlled by host regulatory networks and the bacterial stress (SOS) response. These intimate connections to the host make the integron a genetically stable and efficient system, granting the bacteria a low cost, highly adaptive evolution potential “on demand”.

Multidrug-resistant Achromobacter xylosoxidans was recovered from the sputum of a patient with cystic fibrosis. The VEB-1 extended-spectrum β-lactamase was detected on a class 1 integron. This first report of a VEB-1–producing isolate in this population requires further investigation to determine its distribution.

Toxin-antitoxin (TA) systems have been reported in the genomes of most bacterial species, and their role when located on the chromosome is still debated. TA systems are particularly abundant in the massive cassette arrays associated with chromosomal superintegrons (SI). Here, we describe the characterization of two superintegron cassettes encoding putative TA systems. The first is the phd-doc SI system identified in Vibrio cholerae N16961. We determined its distribution in 36 V. cholerae strains and among five V. metschnikovii strains. We show that this cassette, which is in position 72 of the V. cholerae N16961 cassette array, is functional, carries its own promoter, and is expressed from this location. Interestingly, the phd-doc SI system is unable to control its own expression, most likely due to the absence of any DNA-binding domain on the antitoxin. In addition, this SI system is able to cross talk with the canonical P1 phage system. The second cassette that we characterized is the ccd Vfi cassette found in the V. fischeri superintegron. We demonstrate that CcdB Vfi targets DNA-gyrase, as the canonical CcB F toxin, and that ccd Vfi regulates its expression in a fashion similar to the ccd F operon of the conjugative plasmid F. We also establish that this cassette is functional and expressed in its chromosomal context in V. fischeri CIP 103206T. We tested its functional interactions with the ccdAB F system and found that CcdA Vfi is specific for its associated CcdB Vfi and cannot prevent CcdB F toxicity. Based on these results, we discuss the possible biological functions of these TA systems in superintegrons.

International audience; The complexity of even small gene networks makes them hardly amenable to rational design. Testing random combinations of genetic elements in a directed evolution procedure is thus of interest for many applications including metabolic engineering. Here we describe how the recombination machinery of class 1 integrons can be used to deliver and shuffle genetic elements at a chromosomal locus in E. coli.

International audience; ObjectivesTo determine the complete nucleotide sequence of the VIM-1-encoding plasmid pTC2, which was isolated from a Greek Providencia stuartii multiresistant strain.MethodsThe pTC2 plasmid was extracted and sequenced using shotgun and 3 kb paired-end DNA libraries and a 454 sequencing approach. Following assembly into a unique scaffold, gaps were closed by PCR followed by Sanger DNA sequencing. Gene predictions and annotations were performed using the CAAT-Box tool and the Conserved Domain Search service. Sequence comparisons were performed using the Artemis Comparison Tool.ResultsPlasmid pTC2 (180 184 bp) was found to be a multireplicon plasmid (IncA/C and IncR), with a large IncA/C backbone and a mosaic multidrug resistance (MDR) region, in which was inserted a 13 kb IncR fragment. Gene annotation allowed the identification of a complete IncA/C-type transfer system and of several putative maintenance modules, both on the IncA/C backbone and on the IncR fragment. The complex MDR region contained 9 insertion sequences (7 IS26, 1 IS1 and 1 IS6100), 10 resistance genes and a mercury resistance operon integrated into unit transposons, composite transposons or integrons.ConclusionsThe pTC2 combines a broad host range, transfer and maintenance capacities, plasticity of the MDR region and a wide variety of resistance genes, properties that may contribute to the spreading of resistance determinants.

International audience; Site-specific recombination catalyzed by tyrosine recombinases follows a common pathway consisting of two consecutive strand exchanges. The first strand exchange generates a Holliday junction (HJ), which is resolved by a second strand exchange. In integrons, attC sites recombine as folded single-stranded substrates. Only one of the two attC site strands, the bottom one, is efficiently bound and cleaved by the integrase during the insertion of gene cassettes at the double-stranded attI site. Due to the asymmetry of this complex, a second strand exchange on the attC bottom strand (bs) would form linearized abortive recombination products. We had proposed that HJ resolution would rely on an uncharacterized mechanism, probably replication. Using an attC site carried on a plasmid with each strand specifically tagged, we followed the destiny of each strand after recombination. We demonstrated that only one strand, the one carrying the attC bs, is exchanged. Furthermore, we show that the recombination products contain the attC site bs and its entire de novo synthesized complementary strand. Therefore, we demonstrate the replicative resolution of single-strand recombination in integrons and rule out the involvement of a second strand exchange of any kind in the attC×attI reaction.

Bacterial resistance to β-lactams may rely on acquired β-lactamases encoded by class 1 integron-borne genes. Rearrangement of integron cassette arrays is mediated by the integrase IntI1. It has been previously established that integrase expression can be activated by the SOS response in vitro, leading to speculation that this is an important clinical mechanism of acquiring resistance. Here we report the first in vivo evidence of the impact of SOS response activated by the antibiotic treatment given to a patient and its output in terms of resistance development. We identified a new mechanism of modulation of antibiotic resistance in integrons, based on the insertion of a genetic element, the gcuF1 cassette, upstream of the integron-borne cassette bla OXA-28 encoding an extended spectrum β-lactamase. This insertion creates the fused protein GCUF1-OXA-28 and modulates the transcription, the translation, and the secretion of the β-lactamase in a Pseudomonas aeruginosa isolate (S-Pae) susceptible to the third generation cephalosporin ceftazidime. We found that the metronidazole, not an anti-pseudomonal antibiotic given to the first patient infected with S-Pae, triggered the SOS response that subsequently activated the integrase IntI1 expression. This resulted in the rearrangement of the integron gene cassette array, through excision of the gcuF1 cassette, and the full expression the β-lactamase in an isolate (R-Pae) highly resistant to ceftazidime, which further spread to other patients within our hospital. Our results demonstrate that in human hosts, the antibiotic-induced SOS response in pathogens could play a pivotal role in adaptation process of the bacteria., Author Summary The bacterial SOS response is a conserved regulatory network that is induced in response to DNA damage. Its activation in vitro leads to the emergence of resistance to antibiotics, leading to speculation that this is an important clinical mechanism of acquiring resistance. We found evidence here that antibiotic-induced SOS response plays a role in bacterial genome rearrangement in vivo within humans. The major classes of antibiotics can trigger the bacterial SOS response and our data raise questions about their wide use and their subsequent effect on the bacterial genetic adaptability. This suggests that emergence of antibiotic resistance during therapy could be reduced by inhibiting the bacterial SOS response. We showed that acquired resistance genes could spread latently in susceptible bacterial strains until needed. These findings could impact current policies for control of antibiotic resistance, which rely on the detection of resistant bacteria and on the assumption that resistance mechanisms have a functional cost to the bacteria. More generally, SOS response may spur changes in the behavior of bacteria and their faster adaptation to hostile environments, including humans.

International audience; Gene cassettes of class 1 integrons may be differently expressed depending on the Pc promoter variant as well as occasionally from a second promoter located downstream of Pc, named P2. So far, the distribution of the variants has only been described in an in silico study. In this study, the prevalence of these variants in vivo was analysed in a population of 85 Escherichia coli strains from a variety of phylogenetic groups isolated from healthy subjects and clinical samples in Spain and France from 2004 to 2007. The weakest variants (PcW and PcH1) prevailed (variants associated with the integrase having the most efficient excision activity), whilst the two strongest variants, PcW(TGN-10) and PcS, were less frequent. Furthermore, a new variant of P2 associated with PcW was characterised in one integron (harbouring the gene cassette bla(OXA-1)-aadA1) from a French strain of a healthy subject. This variant was hereafter named P2m3 and shows a G→A substitution in its -10 element (TACAGT to TACAAT), a mutation that doubled the strength of P2 and approached the level of expression of the strong PcW(TGN-10) variant. When the correlation between the Pc variants and the origin of the strains was analysed, no significant difference (P

International audience; Class 1 integrons are widespread genetic elements responsible for dissemination of antibiotic resistance among Gram-negative bacteria. Integrons allow bacteria to capture and express gene cassettes (GCs) via an integrase (IntI1) and a promoter (Pc) contained in the integron functional platform. GCs are transcribed from Pc, of which 13 variants of different strengths have been described, or, occasionally, from both Pc and a second promoter (P2). The intI1 promoter (PintI1) is repressed by LexA, which is the transcriptional repressor of the global regulatory SOS response network. Moreover, PintI1 lies face to face with Pc and overlaps P2, both configurations being propitious to transcriptional interference (TI). In this study, we analyzed possible transcriptional interference by quantifying transcripts produced from Pc, P2, and PintI1. We found that the Pc promoter interferes with the level of intI1 transcription but that this effect depends on the Pc variant: the strong Pc variant prevents intI1 expression, in contrast to the other variants. Although P2 formation results in LexA binding site disruption and thus prevents SOS regulation of intI1 expression, P2 does not interfere with PintI1. These findings reveal a tight relationship between GC and integrase expression.

Conjugation is one mechanism for intra- and inter-species horizontal gene transfer among bacteria. Conjugative elements have been instrumental in many bacterial species to face the threat of antibiotics, by allowing them to evolve and adapt to these hostile conditions. Conjugative plasmids are transferred to plasmidless recipient cells as single-stranded DNA. We used lacZ and gfp fusions to address whether conjugation induces the SOS response and the integron integrase. The SOS response controls a series of genes responsible for DNA damage repair, which can lead to recombination and mutagenesis. In this manuscript, we show that conjugative transfer of ssDNA induces the bacterial SOS stress response, unless an anti-SOS factor is present to alleviate this response. We also show that integron integrases are up-regulated during this process, resulting in increased cassette rearrangements. Moreover, the data we obtained using broad and narrow host range plasmids strongly suggests that plasmid transfer, even abortive, can trigger chromosomal gene rearrangements and transcriptional switches in the recipient cell. Our results highlight the importance of environments concentrating disparate bacterial communities as reactors for extensive genetic adaptation of bacteria., Author Summary Bacteria exchange DNA in their natural environments. The process called conjugation consists of DNA transfer by cell contact from one bacterium to another. Conjugative circular plasmids have been identified as shuttles and reservoirs for adaptive genes. It is now established that such lateral gene transfer plays an essential role, especially for the antibiotic resistance development and dissemination among bacteria. Moreover, integrons, platforms of mobile gene cassettes, have been instrumental in this phenomenon, through their successful association with conjugative resistance plasmids. We demonstrate in this study that the conjugative transfer of plasmids triggers a bacterial stress response—the SOS response—in recipient cells and can impact the cassette content of integrons. The SOS response is already known to induce various genome modifications. Human and animal pathogens cohabit with environmental bacteria, in niches which will favor DNA exchange. SOS induction during conjugation is thus most probably able to impact a wide range of genomes. Bacterial SOS response could then be a suitable target for co-treatment of infections in order to prevent exchange of antibiotic resistance/adaptation genes.

Erratum in EMBO J. 2010 Nov 3;29(21):3745.; International audience; By mobilizing small DNA units, integrons have a major function in the dissemination of antibiotic resistance among bacteria. The acquisition of gene cassettes occurs by recombination between the attI and attC sites catalysed by the IntI1 integron integrase. These recombination reactions use an unconventional mechanism involving a folded single-stranded attC site. We show that cellular bacterial processes delivering ssDNA, such as conjugation and replication, favour proper folding of the attC site. By developing a very sensitive in vivo assay, we also provide evidence that attC sites can recombine as cruciform structures by extrusion from double-stranded DNA. Moreover, we show an influence of DNA superhelicity on attC site extrusion in vitro and in vivo. We show that the proper folding of the attC site depends on both the propensity to form non-recombinogenic structures and the length of their variable terminal structures. These results draw the network of cell processes that regulate integron recombination.

Background: Integrons have a well-established role in the dissemination of resistance among Gram-negative pathogens and are thus a useful marker of antibiotic resistance. Shigellae are noteworthy for their multiple drug resistance, having gradually acquired resistance to most widely use and inexpensive antimicrobial drugs. Methodology: A total of 32 Shigella strains belonging to serotypes flexneri , dysenteriae , and boydii 20, a new Shigella serovar, resistant to at least four antibiotics were analyzed by molecular techniques. Results: Class 1 integrons were the most prevalent (92.8%); class 2 integrons were found in 16 strains (57.1%). Fifty percent of the strains harboured both class 1 and 2 integrons ( intI1 and intI2 genes); this combination of integrase genes was most prevalent in S. boydii 20 and S . dysenteriae strains. The class 1 integrons detected contained dfr and aadA cassettes, alone or in combination ( dfrA5 / dfrA15 , or dfrA15 - aadA1 , dfrA1 - aadA2 ), and an atypical cassette array with an insertion sequence ( oxa30 - aadA1- IS 1 ). For class 2 integrons, we detected either the same cassettes as those found in Tn 7 ( dfrA1 - sat1 - aadA1 - orfX ) or truncated class 2 integrons without aadA1 or orfX . The tns genes were absent from all class 2 integrons. The distribution of integrons among RAPD profiles and serotypes revealed a clonal spread of integrons into serotypes and a transfer of integrons between different serotypes. Conclusions: The detection of integrons in a new Shigella serovar, in addition with a high integron prevalence among Shigella strains, confirms the propensity of shigellae to acquire and disseminate resistance determinants.

Class 1 integrons are widespread genetic elements that allow bacteria to capture and express gene cassettes that are usually promoterless. These integrons play a major role in the dissemination of antibiotic resistance among Gram-negative bacteria. They typically consist of a gene (intI) encoding an integrase (that catalyzes the gene cassette movement by site-specific recombination), a recombination site (attI1), and a promoter (Pc) responsible for the expression of inserted gene cassettes. The Pc promoter can occasionally be combined with a second promoter designated P2, and several Pc variants with different strengths have been described, although their relative distribution is not known. The Pc promoter in class 1 integrons is located within the intI1 coding sequence. The Pc polymorphism affects the amino acid sequence of IntI1 and the effect of this feature on the integrase recombination activity has not previously been investigated. We therefore conducted an extensive in silico study of class 1 integron sequences in order to assess the distribution of Pc variants. We also measured these promoters' strength by means of transcriptional reporter gene fusion experiments and estimated the excision and integration activities of the different IntI1 variants. We found that there are currently 13 Pc variants, leading to 10 IntI1 variants, that have a highly uneven distribution. There are five main Pc-P2 combinations, corresponding to five promoter strengths, and three main integrases displaying similar integration activity but very different excision efficiency. Promoter strength correlates with integrase excision activity: the weaker the promoter, the stronger the integrase. The tight relationship between the aptitude of class 1 integrons to recombine cassettes and express gene cassettes may be a key to understanding the short-term evolution of integrons. Dissemination of integron-driven drug resistance is therefore more complex than previously thought., Author Summary Integrons are widespread bacterial genetic elements able to capture and express gene cassettes that often encode antibiotic resistance determinants. Gene cassettes are usually promoterless and are transcribed from a common promoter, Pc. Pc is located within the coding sequence of the integron integrase, IntI, which is the key element catalyzing the integration and excision of gene cassettes. Several Pc variants, associated with different integrase amino acid sequences, have been described, but the influence of these differences on integrase activity has never been investigated. Here, we show that Pc is highly polymorphic, conferring a wide range of antibiotic resistance. Furthermore, we found that different Pc variants are associated with different integrase excision activities: the weaker the Pc variant, the more active the integrase. These results point to evolutionary compromises between the expression and mobility of drug resistance determinants located on integrons.

International audience; Integrons are found in the genome of hundreds of environmental bacteria but are mainly known for their role in the capture and spread of antibiotic resistance determinants among Gram-negative pathogens. We report a direct link between this system and the ubiquitous SOS response. We found that LexA controlled expression of most integron integrases and consequently regulated cassette recombination. This regulatory coupling enhanced the potential for cassette swapping and capture in cells under stress, while minimizing cassette rearrangements or loss in constant environments. This finding exposes integrons as integrated adaptive systems and has implications for antibiotic treatment policies.

Received 25 September 2008; returned 4 November 2008; revised 13 November 2008; accepted 17 November 2008 Objectives: Clinical isolates of Salmonella enterica serovar Haifa and Newport, which displayed extended multidrug resistance phenotypes, were investigated for the presence of Salmonella genomic island 1 (SGI1) and the genetic organization of its antibiotic resistance gene clusters. Methods: The S. enterica strains were isolated from humans in France in 2003 and 2004. Antibiotic susceptibility tests and various molecular techniques were used for detection and characterization of SGI1. Results: We identified SGI1 integrated in the 3 0 end of the chromosomal thdF gene in six multidrugresistant serovar Haifa and Newport strains. Two strains, of serovar Haifa and Newport, harboured the previously described SGI1-H variant. A new variant of the novel SGI1-Ks group, named SGI1-K6, revealed IS26-mediated rearrangements of the antibiotic resistance gene cluster in two serovar Newport strains. Two other serovar Newport strains harboured the SGI1-L complex class 1 integron containing the dfrA15 and blaPSE-1 resistance gene cassettes. In addition, these variants of SGI1 also contained large IS26-composite transposons inserted by a transposition event in the SGI1 backbone. These IS26-composite transposons showed a similar genetic structure to the SGI1-K variants containing an In4-type integron, a mercury resistance operon and parts of Tn1721 and Tn5393. These extended resistance gene clusters containing up to 10 antibiotic resistance genes were named SGI1-L1 and -L2. Conclusions: The serovar Haifa represents the 16th S. enterica serovar in which SGI1 has been identified. The genomic island SGI1 appears to be a hotspot of acquisition of antibiotic resistance genes by the transposition of In4-type integrons and large IS26-composite transposons.

Sir, Integrons are genetic elements that acquire gene cassettes by integrase-catalysed site-specific recombination. Integrons contain various combinations of gene cassettes encoding antibiotic resistance determinants and are widespread among Gram-negative bacteria. Five classes of integrons have been described to date, based on the integrase coding sequence. Classes 1 and 2 are the most frequent. Class 2 was found in transposon Tn7 and its derivatives (Tn1825, Tn1826 and Tn4132), and its 30 segment contains five tns genes involved in transposon movements. The intI2 integrase gene of class 2 integrons contains a premature stop codon at position 179, rendering the integrase non-functional. Therefore, class 2 integrons usually contain the same array of four gene cassettes, three antibiotic resistance gene cassettes (dfrA1, sat and aadA1, conferring resistance to trimethoprim, streptothricin and spectinomycin/streptomycin, respectively) and an orfX of unknown function. However, variations of the cassette contents have been described, resulting from Intl1 integrase-catalysed co-integrate formation between a class 1 and a class 2 integron, or from an RecA-dependent homologous recombination between two copies of the same cassette found in both classes of integron. Indeed, the dfrA1 and aadA1 cassettes, initially described in class 2 integrons, have also been found in class 1 integrons. Class 1 and 2 integrons have frequently been found in Shigella strains, and class 2 integrons predominate in Shigella sonnei and Shigella flexneri. – 5 We have previously described integron structures in S. sonnei strains isolated in Senegal; they included a class 2 integron of 4 kb found in two epidemiologically related isolates. Here, we describe the organization of this class 2 integron. The following primers were used for PCR mapping of the class 2 integron: hep74 in attI2, ORFX1 (CTCGTACTTG CGATGGCATC) in ORFX, and int2CS2 and int7S, both located in tnsE. Amplification was performed with the Expand Long Template PCR System (Roche Molecular Biochemicals, Roche Diagnostics GmbH, Germany). With primers hep74 and ORFX1, agarose gel electrophoresis identified a 4074 bp DNA fragment (data not shown) in both isolates, instead of the expected 3095 bp product. No PCR product was obtained with primers int2CS2 and int7S, suggesting a different arrangement in the 30 region of this class 2 integron. The entire class 2 integron was amplified with primer int2S in intI2 and with primer ORFX3 located in Tn7, downstream of the ORFX cassette. A PCR product of 4174 bp was purified with the QIAquick kit (Qiagen SA, Courtaboeuf, France), cloned with the pGEM-T vector system (Promega, Madison, WI, USA), transformed into XL1-Blue competent cells (Stratagene, Garden Grove, CA, USA) and sequenced. Sequence analysis of the 4174 bp insert of the recombinant plasmid showed that this integron harboured the four gene cassettes usually found in class 2 integrons, namely dfrA1, sat1, aadA1 and orfX (accession no. EU732664). However, the sat1 cassette was interrupted at position 186 by a complete copy of the insertion sequence IS911, a member of the IS3 family. IS911 was initially isolated from Shigella dysenteriae, but has since been detected (10–20 copies per cell) in the four Shigella species. As usually described for IS911, its insertion generated a 3 bp duplication (TAT) in the target sat1 sequence. To our knowledge, two ISs have previously been described in class 2 integrons, but not within the array of gene cassettes. Dubois et al. described a class 2 integron with an IS630 element in a strain of S. flexneri, and Biskri and Mazel described a class 2 integron with an IS1 in a strain of Escherichia coli. Both were located between intI2 and the first gene cassette. To determine whether the resistance determinants carried by the integron were transferable, we performed a conjugation experiment on MH agar plates from S. sonnei to an E. coli strain resistant to nalidixic acid. We used a selective medium containing 50 mg/L nalidixic acid plus 100 mg/L trimethoprim. No transconjugants were obtained, suggesting the chromosomal location of this Tn7-containing class 2 integron as previously described in Shigella isolates. Pan et al. described a class 2 integron on transposon Tn7 inserted into the Tn7-specific target attTn7 of the S. sonnei chromosome downstream of the glmUS operon. Thus, to confirm the chromosomal insertion of Tn7, we successfully performed a PCR with primers Tn7R-F and glmS-R as described by Pan et al. Another feature of this atypical class 2 integron is the deletion of the tns genes, described in Tn7 and its derivatives. A class 2 integron lacking the tns genes has previously been described in Acinetobacter baumannii, but the 30 segment of the class 2 integron contained the ORFs found in the 30 segment of class 1 integrons (qacED1, sul1 and ORF5). The PCR method previously used to detect such a chimeric integron with primers aadAL and ORFR was unsuccessful, suggesting that the class 2 integron that we describe here is not a hybrid class 2/class 1 integron. In conclusion, we describe an unusual class 2 integron in a S. sonnei strain isolated in Africa. This new class 2 integron is characterized by the presence of one complete copy of the IS911 element inserted within the sat1 gene cassette. Journal of Antimicrobial Chemotherapy 62, 843–851 doi:10.1093/jac/dkn264 Advance Access publication 18 June 2008

We describe the first IMP metallo-β-lactamase in Aeromonas caviae : IMP-19, which differed from IMP-2 by a single amino acid change (Arg to Ala at position 38). bla IMP-19 was found within a class 1 integron located on a 35-kb plasmid. This is also the first description of an IMP producer in France.

A qnrB2 determinant was described for a new complex sul1 -type integron from Salmonella enterica serovar Keurmassar. The genetic structure contained two class 1 integrons surrounding two common regions (CRs) separated by a partial 3′ conserved segment. The qnrB2 gene is adjacent to the first CR.

International audience; A multiresistant strain of Pseudomonas aeruginosa, PA2345, belonging to serotype O:1, was isolated at the Teaching Hospital of Besançon, France. Resistance to beta-lactams, including third-generation cephalosporins, depended upon a chromosomally-located composite transposon carrying the bla(PER-1) gene encoding extended-spectrum beta-lactamase PER-1. PA2345 was unrelated genotypically to two previous PER-1-producing isolates of P. aeruginosa. Sequence analysis of the transposon in PA2345 revealed the presence of two insertion sequences (ISPa23 and ISPa24) with very different predicted transposases (TnpA1, TnpA2), which were both bordered by closely related 16-bp inverted repeats. High resistance of PA2345 to aminoglycosides was caused, in part, by a chromosomal class-I integron containing gene cassettes aadB, encoding an ANT(2'') enzyme, and aadA11, encoding a new ANT(3'') enzyme with 281 amino-acids that conferred elevated resistance to streptomycin and spectinomycin. Stable overproduction of efflux system MexXY contributed to resistance to amikacin, while mutations in the quinolone resistance-determining regions of gyrA and parC accounted for the high resistance of PA2345 to fluoroquinolones. The study indicates that multidrug resistance in P. aeruginosa might arise from sequential acquisition of a variety of mechanisms provided by both horizontal gene transfers and mutations in chromosomal genes.