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 blaCTX-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

A new role for Zinc limitation in bacterial pathogenicity: modulation of α-hemolysin from uropathogenic Escherichia coli.

Metal limitation is a common situation during infection and can have profound effects on the pathogen's success. In this report, we examine the role of zinc limitation in the expression of a virulence factor in uropathogenic Escherichia coli. The pyelonephritis isolate J96 carries two hlyCABD operons that encode the RTX toxin α-hemolysin. While the coding regions of both operons are largely conserved, the upstream sequences, including the promoters, are unrelated. We show here that the two hlyCABD operons are differently regulated. The hly II operon is efficiently silenced in the presence of zinc and highly expressed when zinc is limited. In contrast, the hly I operon does not respond to zinc limitation. Genetic studies reveal that zinc-responsive regulation of the hly II operon is controlled by the Zur metalloregulatory protein. A Zur binding site was identified in the promoter sequence of the hly II operon, and we observe direct binding of Zur to this promoter region. Moreover, we find that Zur regulation of the hly II operon modulates the ability of E. coli J96 to induce a cytotoxic response in host cell lines in culture. Our report constitutes the first description of the involvement of the zinc-sensing protein Zur in directly modulating the expression of a virulence factor in bacteria

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 blaIMP–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 blaIMP–63, among Pseudomonas species

An efficient system for the generation of marked genetic mutants in members of the genus Burkholderia

To elucidate the function of a gene in bacteria it is vital that targeted gene inactivation (allelic replacement) can be achieved. Allelic replacement is often carried out by disruption of the gene of interest by insertion of an antibiotic-resistance marker followed by subsequent transfer of the mutant allele to the genome of the host organism in place of the wild-type gene. However, due to their intrinsic resistance to many antibiotics only selected antibiotic-resistance markers can be used in members of the genus Burkholderia, including the Burkholderia cepacia complex (Bcc). Here we describe the construction of improved antibiotic-resistance cassettes that specify resistance to kanamycin, chloramphenicol or trimethoprim effectively in the Bcc and related species. These were then used in combination with and/or to construct a series enhanced suicide vectors, pSHAFT2, pSHAFT3 and pSHAFT-GFP to facilitate effective allelic replacement in the Bcc. Validation of these improved suicide vectors was demonstrated by the genetic inactivation of selected genes in the Bcc species Burkholderia cenocepacia and B. lata, and in the non-Bcc species, B. thailandensis

Inverse Correlation between Promoter Strength and Excision Activity in Class 1 Integrons

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

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), β-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

Diversity of class 1 integron gene cassette Pc promoter variants in clinical Escherichia coli strains and description of a new P2 promoter variant.

International audienceGene 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<0.05) was observed in the Pc variant distribution according to the geographic origin or clinical setting

High-Level Gene Cassette Transcription Prevents Integrase Expression in Class 1 Integrons▿

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

Identification and analysis of integrons and cassette arrays in bacterial genomes

International audienceIntegrons recombine gene arrays and favor the spread of antibiotic resistance. Their broader roles in bacterial adaptation remain mysterious, partly due to lack of computational tools. We made a program – IntegronFinder – to identify integrons with high accuracy and sensitivity. IntegronFinder is available as a standalone program and as a web application. It searches for attC sites using covari-ance models, for integron-integrases using HMM profiles , and for other features (promoters, attI site) using pattern matching. We searched for integrons, integron-integrases lacking attC sites, and clusters of attC sites lacking a neighboring integron-integrase in bacterial genomes. All these elements are especially frequent in genomes of intermediate size. They are missing in some key phyla, such as-Proteobacteria, which might reflect selection against cell lineages that acquire integrons. The similarity between attC sites is proportional to the number of cassettes in the integron, and is particularly low in clusters of attC sites lacking integron-integrases. The latter are unexpectedly abundant in genomes lacking integron-integrases or their remains, and have a large novel pool of cassettes lacking homologs in the databases. They might represent an evolutionary step between the acquisition of genes within inte-grons and their stabilization in the new genome