Hospital and urban wastewaters shape the matrix and active resistome of environmental biofilms.

Understanding the dynamics of antibiotic resistance gene (ARG) transfer and dissemination in natural environments remains challenging. Biofilms play a crucial role in bacterial survival and antimicrobial resistance (AMR) dissemination in natural environments, particularly in aquatic systems. This study focused on hospital and urban wastewater (WW) biofilms to investigate the potential for ARG dissemination through mobile genetic elements (MGEs). The analysis included assessing the biofilm extracellular polymeric substances (EPS), microbiota composition as well as metatranscriptomic profiling of the resistome and mobilome. We produced both in vitro and in situ biofilms and performed phenotypic and genomic analyses. In the in vitro setup, untreated urban and hospital WW was used to establish biofilm reactors, with ciprofloxacin added as a selective agent at minimal selective concentration. In the in situ setup, biofilms were developed directly in hospital and urban WW pipes. We first showed that a) the composition of EPS differed depending on the growth environment (in situ and in vitro) and the sampling origin (hospital vs urban WW) and that b) ciprofloxacin impacted the composition of the EPS. The metatranscriptomic approach showed that a) expression of several ARGs and MGEs increased upon adding ciprofloxacin for biofilms from hospital WW only and b) that the abundance and type of plasmids that carried individual or multiple ARGs varied depending on the WW origins of the biofilms. When the same plasmids were present in both, urban and hospital WW biofilms, they carried different ARGs.  We showed that hospital and urban wastewaters shaped the structure and active resistome of environmental biofilms, and we confirmed that hospital WW is an important hot spot for the dissemination and selection of antimicrobial resistance. Our study provides a comprehensive assessment of WW biofilms as crucial hotspots for ARG transfer. Hospital WW biofilms exhibited distinct characteristics, including higher eDNA abundance and expression levels of ARGs and MGEs, highlighting their role in antimicrobial resistance dissemination. These findings emphasize the importance of understanding the structural, ecological, functional, and genetic organization of biofilms in anthropized environments and their contribution to antibiotic resistance dynamics.

The expression of aminoglycoside resistance genes in integron cassettes is not controlled by riboswitches.

Regulation of gene expression is a key factor influencing the success of antimicrobial resistance determinants. A variety of determinants conferring resistance against aminoglycosides (Ag) are commonly found in clinically relevant bacteria, but whether their expression is regulated or not is controversial. The expression of several Ag resistance genes has been reported to be controlled by a riboswitch mechanism encoded in a conserved sequence. Yet this sequence corresponds to the integration site of an integron, a genetic platform that recruits genes of different functions, making the presence of such a riboswitch counterintuitive. We provide, for the first time, experimental evidence against the existence of such Ag-sensing riboswitch. We first tried to reproduce the induction of the well characterized aacA5 gene using its native genetic environment, but were unsuccessful. We then broadened our approach and analyzed the inducibility of all AgR genes encoded in integrons against a variety of antibiotics. We could not observe biologically relevant induction rates for any gene in the presence of several aminoglycosides. Instead, unrelated antibiotics produced mild but consistently higher increases in expression, that were the result of pleiotropic effects. Our findings rule out the riboswitch control of aminoglycoside resistance genes in integrons.

Novel Insights into blaGES Mobilome Reveal Extensive Genetic Variation in Hospital Effluents.

Mobile genetic elements contribute to the emergence and spread of multidrug-resistant bacteria by enabling the horizontal transfer of acquired antibiotic resistance among different bacterial species and genera. This study characterizes the genetic backbone of blaGES in Aeromonas spp. and Klebsiella spp. isolated from untreated hospital effluents. Plasmids ranging in size from 9 to 244 kb, sequenced using Illumina and Nanopore platforms, revealed representatives of plasmid incompatibility groups IncP6, IncQ1, IncL/M1, IncFII, and IncFII-FIA. Different GES enzymes (GES-1, GES-7, and GES-16) were located in novel class 1 integrons in Aeromonas spp. and GES-5 in previously reported class 1 integrons in Klebsiella spp. Furthermore, in Klebsiella quasipneumoniae, blaGES-5 was found in tandem as a coding sequence that disrupted the 3' conserved segment (CS). In Klebsiella grimontii, blaGES-5 was observed in two different plasmids, and one of them carried multiple IncF replicons. Three Aeromonas caviae isolates presented blaGES-1, one Aeromonas veronii isolate presented blaGES-7, and another A. veronii isolate presented blaGES-16. Multilocus sequence typing (MLST) analysis revealed novel sequence types for Aeromonas and Klebsiella species. The current findings highlight the large genetic diversity of these species, emphasizing their great adaptability to the environment. The results also indicate a public health risk because these antimicrobial-resistant genes have the potential to reach wastewater treatment plants and larger water bodies. Considering that they are major interfaces between humans and the environment, they could spread throughout the community to clinical settings. IMPORTANCE In the "One Health" approach, which encompasses human, animal, and environmental health, emerging issues of antimicrobial resistance are associated with hospital effluents that contain clinically relevant antibiotic-resistant bacteria along with a wide range of antibiotic concentrations, and lack regulatory status for mandatory prior and effective treatment. blaGES genes have been reported in aquatic environments despite the low detection of these genes among clinical isolates within the studied hospitals. Carbapenemase enzymes, which are relatively unusual globally, such as GES type inserted into new integrons on plasmids, are worrisome. Notably, K. grimontii, a newly identified species, carried two plasmids with blaGES-5, and K. quasipneumoniae carried two copies of blaGES-5 at the same plasmid. These kinds of plasmids are primarily responsible for multidrug resistance among bacteria in both clinical and natural environments, and they harbor resistant genes against antibiotics of key importance in clinical therapy, possibly leading to a public health problem of large proportion.

A qnrD-Plasmid Promotes Biofilm Formation and Class 1 Integron Gene Cassette Rearrangements in Escherichia coli.

Bacteria within biofilms may be exposed to sub-minimum inhibitory concentrations (sub-MICs) of antibiotics. Cell-to-cell contact within biofilms facilitates horizontal gene transfers and favors induction of the SOS response. Altogether, it participates in the emergence of antibiotic resistance. Aminoglycosides at sub-MICs can induce the SOS response through NO accumulation in E. coli carrying the small plasmid with the quinolone resistance qnrD gene (pDIJ09-518a). In this study, we show that in E. coli pDIJ09-518a, the SOS response triggered by sub-MICs of aminoglycosides has important consequences, promoting genetic rearrangement in class 1 integrons and biofilm formation. We found that the integrase expression was increased in E. coli carrying pDIJ09-518a in the presence of tobramycin, which was not observed for the WT isogenic strain that did not carry the qnrD-plasmid. Moreover, we showed that biofilm production was significantly increased in E. coli WT/pDIJ09-518a compared to the WT strain. However, such a higher production was decreased when the Hmp-NO detoxification pathway was fully functional by overexpressing Hmp. Our results showing that a qnrD-plasmid can promote biofilm formation in E. coli and potentiate the acquisition and spread of resistance determinants for other antibiotics complicate the attempts to counteract antibiotic resistance and prevention of biofilm development even further. We anticipate that our findings emphasize the complex challenges that will impact the decisions about antibiotic stewardship, and other decisions related to retaining antibiotics as effective drugs and the development of new drugs.

Unravelling complex transposable elements surrounding blaGES-16 in a Pseudomonas aeruginosa ExoU strain.

OBJECTIVES: We characterised the complex surrounding regions of blaGES-16 in a Pseudomonas aeruginosa exoU+ strain (P-10.226) in Brazil. METHODS: Species identification was performed by MALDI-TOF MS, and the antimicrobial susceptibility profile was determined by broth microdilution based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. The whole genome sequencing (WGS) of P-10.226 strain was performed using both short-read paired-end sequencing on the Illumina MiSeq platform as well as the long-read Oxford Nanopore MinION. RESULTS: WGS analysis showed that P-10.226 carried blaGES-16, which was found as a gene cassette inserted into a novel class I integron, In1992 (aadB-blaOXA-56-blaGES-16-aadB-aadA6c), whose 3'-CS was truncated by a nested transposable element, IS5564::ISPa157. The structure was even more complex since IS6100-ΔIS6100 structure and a TnAs2-like harbouring the operon merRTPADE was found downstream In1992. Fragments of TnAs3 harbouring 25-bp imperfect inverted repeats were identified bordering the intl1 of In1992 and also flanking IS6100-ΔIS6100, which might be genetic marks of its previous presence in the genome. Interestingly, In1992 also shows a distinct cassette array from In581 (blaGES-16-dfrA22-aacA27-aadA1), which was previously reported in Serratia marcescens strains recovered in Brazil. Finally, exoU gene, which encodes a potent cytotoxin of type III secretion systems (T3SS) effector proteins from P. aeruginosa and is associated to severe infections, was also detected. CONCLUSION: We described the novel In1992 carrying blaGES-16 surrounded by complex transposition events in a XDR P. aeruginosa strain. The identification of many sets of direct repeats adjacent to TnAs3 fragments indicates a major past of transposition events that shaped the current genetic environment of In1992.

A qnr-plasmid allows aminoglycosides to induce SOS in Escherichia coli.

The plasmid-mediated quinolone resistance (PMQR) genes have been shown to promote high-level bacterial resistance to fluoroquinolone antibiotics, potentially leading to clinical treatment failures. In Escherichia coli, sub-minimum inhibitory concentrations (sub-MICs) of the widely used fluoroquinolones are known to induce the SOS response. Interestingly, the expression of several PMQR qnr genes is controlled by the SOS master regulator, LexA. During the characterization of a small qnrD-plasmid carried in E. coli, we observed that the aminoglycosides become able to induce the SOS response in this species, thus leading to the elevated transcription of qnrD. Our findings show that the induction of the SOS response is due to nitric oxide (NO) accumulation in the presence of sub-MIC of aminoglycosides. We demonstrated that the NO accumulation is driven by two plasmid genes, ORF3 and ORF4, whose products act at two levels. ORF3 encodes a putative flavin adenine dinucleotide (FAD)-binding oxidoreductase which helps NO synthesis, while ORF4 codes for a putative fumarate and nitrate reductase (FNR)-type transcription factor, related to an O2-responsive regulator of hmp expression, able to repress the Hmp-mediated NO detoxification pathway of E. coli. Thus, this discovery, that other major classes of antibiotics may induce the SOS response could have worthwhile implications for antibiotic stewardship efforts in preventing the emergence of resistance.

Bistable Expression of a Toxin-Antitoxin System Located in a Cryptic Prophage of Escherichia coli O157:H7.

Type II toxin-antitoxin (TA) systems are classically composed of two genes that encode a toxic protein and a cognate antitoxin protein. Both genes are organized in an operon whose expression is autoregulated at the level of transcription by the antitoxin-toxin complex, which binds operator DNA through the antitoxin's DNA-binding domain. Here, we investigated the transcriptional regulation of a particular TA system located in the immunity region of a cryptic lambdoid prophage in the Escherichia coli O157:H7 EDL933 strain. This noncanonical paaA2-parE2 TA operon contains a third gene, paaR2, that encodes a transcriptional regulator that was previously shown to control expression of the TA. We provide direct evidence that the PaaR2 is a transcriptional regulator which shares functional similarities to the lambda CI repressor. Expression of the paaA2-parE2 TA operon is regulated by two other transcriptional regulators, YdaS and YdaT, encoded within the same region. We argue that YdaS and YdaT are analogous to lambda Cro and CII and that they do not constitute a TA system, as previously debated. We show that PaaR2 primarily represses the expression of YdaS and YdaT, which in turn controls the expression of paaR2-paaA2-parE2 operon. Overall, our results show that the paaA2-parE2 TA is embedded in an intricate lambdoid prophage-like regulation network. Using single-cell analysis, we observed that the entire locus exhibits bistability, which generates diversity of expression in the population. Moreover, we confirmed that paaA2-parE2 is addictive and propose that it could limit genomic rearrangements within the immunity region of the CP-933P cryptic prophage. IMPORTANCE Transcriptional regulation of bacterial toxin-antitoxin (TA) systems allows compensation of toxin and antitoxin proteins to maintain a neutral state and avoid cell intoxication unless TA genes are lost. Such models have been primarily studied in plasmids, but TAs are equally present in other mobile genetic elements, such as transposons and prophages. Here, we demonstrate that the expression of a TA system located in a lambdoid cryptic prophage is transcriptionally coupled to the prophage immunity region and relies on phage transcription factors. Moreover, competition between transcription factors results in bistable expression, which generates cell-to-cell heterogeneity in the population, but without, however, leading to any detectable phenotype, even in cells expressing the TA system. We show that despite the lack of protein sequence similarity, this locus retains major lambda prophage regulation features.

Genomic analysis of VIM-2-producing Enterobacter hormaechei subsp. steigerwaltii.

Carbapenemase-producing Enterobacterales (CPE) is a major public-health concern. Here we describe the occurrence of blaVIM-2 in three isolates of Enterobacter hormaechei subsp. steigerwaltii. The blaVIM-2 gene was part of a class II transposon Tn1332 and was embedded in a remnant of a class 1 integron. Tn1332 was carried by a large, conjugative, non-typeable plasmid. The three isolates belonged to sequence type 90 (ST90). Two isolates (90H2 and 90H3) were highly related [<10 single nucleotide polymorphisms (SNPs)], whereas isolate 104D2 exhibited more than 50 SNPs and Tn1332 was inserted in a different place in the plasmid. Another IncHI-type plasmid carrying the extended-spectrum ß-lactamase (ESBL) gene blaCTX-M-15 was identified in 90H2 and 90H3. Among the three isolates, isolate 104D2 was negative for detection of carbapenemase activity using the biochemical Carba NP test, despite the presence of Tn1332 on the same plasmid. Mutants of 104D2 with higher minimum inhibitory concentrations (MICs) for carbapenems were obtained and one mutant (m104D2) was analysed. In contrast to 104D2, mutant m104D2 gave a positive Carba NP test. The mutant possessed two copies of Tn1332 per cell and a nonsense mutation in WecA, an enzyme involved in enterobacterial common antigen and peptidoglycan intermediate biosynthesis. This study describes the first occurrence of Tn1332 in Enterobacterales and the phenotypic diversity of VIM-2-producing E. hormaechei.

Characterisation of plasmids harbouring extended-spectrum cephalosporin resistance genes in Escherichia coli from French rivers.

Antimicrobial resistance is a "One Health" issue that requires improved knowledge of the presence and abundance of resistant bacteria in the environment. Extended-spectrum cephalosporins (ESCs) are critically important antibiotics (CIAs), and resistance to these CIAs is often encoded by beta-lactamase genes borne on conjugative plasmids. We thus decided to characterise 21 plasmids of ESC-resistant Escherichia coli randomly selected from isolates previously obtained from river water collected in a rural area in western France. The plasmids encoding ESC resistance were sequenced to investigate the diversity of the genes encoding ESC resistance and their genetic context. Sequences revealed that eleven IncI1 pMLST3 plasmids carried the blaCTX-M-1 and sul2 genes, and some of them also had the tet(A), aadA5 or dfrA17 genes. The blaCTX-M-1 gene was also detected on an IncN plasmid. Five plasmids obtained from four rivers contained blaCTX-M-14, either on IncI1 or on IncFII plasmids. Two strains from two rivers contained blaCTX-M-15 on IncN pMLST7 plasmids, with qnrS1 and dfrA14 genes. One plasmid contained the blaCTX-M-55, a blaTEM-1B-like, and fosA genes. One plasmid contained the blaCMY-2 gene. The diversity of the genes and plasmids of the resistant bacteria isolated from French rivers is probably related to the various animal and human origins of the isolated bacteria.

First Occurrence of the OXA-198 Carbapenemase in Enterobacterales.

A carbapenem-resistant Citrobacter sp. was recovered from routine screening of multidrug-resistant bacteria. This isolate coproduced OXA-48 and OXA-198. OXA-48 was carried by the prototypical IncL plasmid, whereas OXA-198 was carried by a peculiar IncHI-type plasmid. This carbapenemase gene was inserted within a class 1 integron located on a conjugative plasmid. This report describes the first occurrence of OXA-198 in Enterobacterales.

Genomic Analysis of Multidrug-Resistant Escherichia coli from Surface Water in Northeast Georgia, United States: Presence of an ST131 Epidemic Strain Containing blaCTX-M-15 on a Phage-Like Plasmid.

Surface water is suspected of playing a role in the development and spread of antimicrobial-resistant (AR) bacteria, including human pathogens. In our previous study, 496 Escherichia coli isolates were recovered from water samples collected over a 2-year period from the Upper Oconee watershed, Athens, GA, United States, of which 34 (6.9%) were AR isolates. Of these, six isolates were selected based on their multidrug resistance (MDR) phenotypes, the presence of mobile genetic elements, and their pathogenic potential and were subjected to whole-genome sequence (WGS) analysis to enhance our understanding of environmental MDR E. coli isolates. This study is the first report on genomic characterization of MDR E. coli from environmental water in the United States through a WGS approach. The sequences of the six MDR E. coli isolates were analyzed and the locations of their AR genes were identified. One of the E. coli isolates was an ST131 epidemic strain, which also produced an extended-spectrum ß-lactamase encoded by the blaCTX-M-15 gene, carried on a plasmid that is a member of a very rarely reported family of phage-like plasmids. This is the first time an in-depth sequence analysis has been done on a blaCTX-M-15- containing phage-like plasmid, the presence of which suggests a new emerging mechanism of AR gene transmission.

Acquisition of class C ß-lactamase PAC-1 by ST664 strains of Pseudomonas aeruginosa.

Four ST664 (serotype O:5) strains of Pseudomonas aeruginosa highly resistant to antibiotics including ceftolozane/tazobactam and ceftazidime/avibactam but susceptible to colistin, were found to harbor the rare class C ß-lactamase PAC-1 encoding gene on a chromosomally-located Tn1721-like transposon. Gene bla PAC-1 was associated with the 16S rRNA methylase determinant rmtF2, that confers pan-aminoglycoside resistance. These genotypically-related strains were isolated in repatriated patients from Mauricius and Afghanistan and close to a lineage reported in Nepal, Pakistan and India.

Antimicrobial Resistance Genes, Cassettes, and Plasmids Present in Salmonella enterica Associated With United States Food Animals.

The ability of antimicrobial resistance (AR) to transfer, on mobile genetic elements (MGEs) between bacteria, can cause the rapid establishment of multidrug resistance (MDR) in bacteria from animals, thus creating a foodborne risk to human health. To investigate MDR and its association with plasmids in Salmonella enterica, whole genome sequence (WGS) analysis was performed on 193 S. enterica isolated from sources associated with United States food animals between 1998 and 2011; 119 were resistant to at least one antibiotic tested. Isolates represented 86 serotypes and variants, as well as diverse phenotypic resistance profiles. A total of 923 AR genes and 212 plasmids were identified among the 193 strains. Every isolate contained at least one AR gene. At least one plasmid was detected in 157 isolates. Genes were identified for resistance to aminoglycosides (n = 472), ß-lactams (n = 84), tetracyclines (n = 171), sulfonamides (n = 91), phenicols (n = 42), trimethoprim (n = 8), macrolides (n = 5), fosfomycin (n = 48), and rifampicin (n = 2). Plasmid replicon types detected in the isolates were A/C (n = 32), ColE (n = 76), F (n = 43), HI1 (n = 4), HI2 (n = 20), I1 (n = 62), N (n = 4), Q (n = 7), and X (n = 35). Phenotypic resistance correlated with the AR genes identified in 95.4% of cases. Most AR genes were located on plasmids, with many plasmids harboring multiple AR genes. Six antibiotic resistance cassette structures (ARCs) and one pseudo-cassette were identified. ARCs contained between one and five resistance genes (ARC1: sul2, strAB, tetAR; ARC2: aac3-iid; ARC3: aph, sph; ARC4: cmy-2; ARC5: floR; ARC6: tetB; pseudo-ARC: aadA, aac3-VIa, sul1). These ARCs were present in multiple isolates and on plasmids of multiple replicon types. To determine the current distribution and frequency of these ARCs, the public NCBI database was analyzed, including WGS data on isolates collected by the USDA Food Safety and Inspection Service (FSIS) from 2014 to 2018. ARC1, ARC4, and ARC5 were significantly associated with cattle isolates, while ARC6 was significantly associated with chicken isolates. This study revealed that a diverse group of plasmids, carrying AR genes, are responsible for the phenotypic resistance seen in Salmonella isolated from United States food animals. It was also determined that many plasmids carry similar ARCs.

Identification of Diverse Integron and Plasmid Structures Carrying a Novel Carbapenemase Among Pseudomonas Species.

A novel carbapenem-hydrolyzing beta-lactamase, called IMP-63, was identified in three clonally distinct strains of Pseudomonas aeruginosa and two strains of Pseudomonas putida isolated within a 4 year timeframe in three French hospitals. The bla IMP-63 gene that encodes this carbapenemase turned out to be located in the variable region of four integrons (In1297, In1574, In1573, and In1572) and to coexist with novel or rare gene cassettes (fosM, gcu170, gcuF1) and insertion elements (ISPsp7v, ISPa16v). All these integrons except one (In1574) were flanked by a copy of insertion sequence ISPa17 next to the orf6 putative gene, and were carried by non-conjugative plasmids (pNECK1, pROUSS1, pROUSS2, pROUE1). These plasmids exhibit unique modular structures and partial sequence homologies with plasmids previously identified in various non-fermenting environmental Gram-negative species. Lines of evidence suggest that ISPa17 promoted en bloc the transposition of IMP-63-encoding integrons on these different plasmids. As demonstrated by genotyping experiments, isolates of P. aeruginosa harboring the 28.9-kb plasmid pNECK1 and belonging to international "high-risk" clone ST308 were responsible for an outbreak in one hospital. Collectively, these data provide an insight into the complex and unpredictable routes of diffusion of some resistance determinants, here bla IMP-63, among Pseudomonas species.

Proteae: a reservoir of class 2 integrons?

OBJECTIVES: Our aim was to confirm with a large panel of clinical isolates that class 2 integrons are highly prevalent in Proteae and to analyse their genetic characteristics. METHODS: Proteae (Proteus spp., Morganella spp. and Providencia spp.) isolates were collected from clinical samples during 2013 at Limoges University Hospital, France. The presence of class 1, 2 and 3 integrons was investigated by quantitative PCR. The presence of a stop codon in the intI2 gene was determined by Sanger sequencing. The gene cassette arrays of class 2 integrons were determined by PCR-RFLP and Sanger sequencing or next-generation sequencing when needed. RESULTS: Of the 327 Proteae collected, 103 (31.5%) harboured a class 2 integron and 45 (13.8%) a class 1 integron. No class 3 integrons were detected. One functional IntI2 integrase was detected in a Morganella morganii isolate. Six different gene cassette arrays were detected. Four had already been described in the literature: dfrA1-sat2-aadA1 (72 isolates), dfrA1-catB2-sat2-aadA1 (17), sat2-aadA1 (6) and lnu(F), dfrA1, aadA1 (1). We identified two new gene cassette arrays: (i) a new variant of the dfrA1 gene cassette (one isolate; the one with the functional IntI2); and (ii) the array dfrA1-gcu115-sat2 harbouring the new gcu115 gene cassette with two ORFs encoding proteins of unknown functions (five isolates). CONCLUSIONS: We showed a high frequency of class 2 integrons, as well as a diversity of gene cassette arrays, among Proteae. This work highlights that the Proteae tribe plays an important role as a reservoir of class 2 integrons.

Class 1 integrons in Acinetobacter baumannii: a weak expression of gene cassettes to counterbalance the lack of LexA-driven integrase repression.

Integrons recruit resistance genes through integrase-driven recombination events that are regulated by the bacterial SOS response and require the repressor LexA. Class 1 integrons genes are expressed from a common promoter, Pc, of which at least 5 predominant variants, classified from weak to strong, have been described. In Escherichia coli, there is an intertwined regulation between gene cassette expression and integrase activity: the stronger the promoter, the weaker the integrase. Class 1 integrons have been frequently described in Acinetobacter baumannii. However, Acinetobacter spp. lack the LexA repressor, suggesting that the integrase is constitutively expressed. We characterized the integron content of 83 clinical and environmental A. baumannii strains. We found a predominance of Pc variants described as strong in E. coli. The Pc expression level was 2- to 4-fold lower in A. baumannii than in E. coli, and the diversity of the gene cassette array was low. In A. baumannii, integrons with a PcS promoter might have been selected to enable sufficient resistance while avoiding the toxicity of a highly active integrase. Furthermore, a transcriptional interference between PcS and PintI1 (as shown in E. coli) may limit the expression of the integrase and thus counterbalance the lack of LexA-driven integrase repression to prevent the cost of the integrase.

The Role of ISCR1-Borne POUT Promoters in the Expression of Antibiotic Resistance Genes.

The ISCR1 (Insertion sequence Common Region) element is the most widespread member of the ISCR family, and is frequently present within γ-proteobacteria that occur in clinical settings. ISCR1 is always associated with the 3'Conserved Segment (3'CS) of class 1 integrons. ISCR1 contains outward-oriented promoters POUT, that may contribute to the expression of downstream genes. In ISCR1, there are two POUT promoters named PCR1-1 and PCR1-2. We performed an in silico analysis of all publically available ISCR1 sequences and identified numerous downstream genes that mainly encode antibiotic resistance genes and that are oriented in the same direction as the POUT promoters. Here, we showed that both PCR1-1 and PCR1-2 significantly increase the expression of the downstream genes bla CTX-M-9 and dfrA19. Our data highlight the role of ISCR1 in the expression of antibiotic resistance genes, which may explain why ISCR1 is so frequent in clinical settings.

Characterization of plasmids harboring blaCTX-M genes in Escherichia coli from French pigs.

Resistance to extended-spectrum cephalosporins is prevalent in French pig E. coli isolates. The aim of this study was to characterize the plasmids and genes present in pathogenic and commensal extended-spectrum cephalosporins -resistant isolates. The resistance plasmids of 26 strains were sequenced and then analyzed to identify resistance and virulence genes. Results showed that resistance to extended-spectrum cephalosporins in French pig E. coli isolates is-as in other food animals in France-mainly carried by highly similar blaCTX-M-1 IncI1/ST3 plasmids. These plasmids very often bear other resistance genes such as resistance to sulphonamides (sul2), trimethoprim (dfrA17) and aminoglycosides (aadA5), and occasionally to tetracycline (tet(A)), macrolides (mph(A) and erm genes), phenicols (floR) or streptomycin (strA, strB). Few virulence genes were detected, including colicins, heat-stable enterotoxins, adhesins or temperature-sensitive hemagglutinins. The other cefotaximases detected were blaCTX-M-27 and blaCTX-M-14, the latter being on an IncF plasmid which showed very close identity to a human epidemic plasmid. Importantly, resistance genes for quinolones or polymyxins were never detected on the extended-spectrum cephalosporins resistance plasmids. These results are helpful to evidence the risk of co-selecting cephalosporins -resistance using antibiotics outside this group. They also highlight the occasional presence in pigs of human epidemic plasmids.

Dissemination of Multidrug-Resistant Proteus mirabilis Clones Carrying a Novel Integron-Borne blaIMP-1 in a Tertiary Hospital.

This study aimed to characterize multidrug-resistant Proteus mirabilis clones carrying a novel class 1 integron-borne blaIMP-1 In1359 was inserted into a large conjugative plasmid that also carried blaCTX-M-2 The production of carbapenemases in Enterobacteriaceae that are intrinsically resistant to polymyxins and tigecycline is very worrisome, representing a serious challenge to clinicians and infection control teams.