Salmonella Genomic Island 1 Research Articles

Traveller-associated high-level ciprofloxacin-resistant Salmonella enterica Serovar Kentucky in the Republic of Korea

ObjectiveSalmonella enterica serotype Kentucky ST198 (S. Kentucky) is frequently associated with human infections and has been identified in travellers to North Africa, South Asia, Europe, and North America. The antimicrobial resistance of this serotype to multiple drugs, including ciprofloxacin (CIP), is a growing concern. However, little information is available regarding the occurrence and characterization of S. Kentucky in Korea. MethodsTo investigate the characteristics and possible origin of these infections, we characterized highly CIP-resistant S. Kentucky isolates collected through a national surveillance program. Single-nucleotide polymorphisms (SNPs) were identified by whole-genome sequencing (WGS), and genome sequencing was performed to investigate the genetic relationships and resistance mechanisms of the isolates. ResultsTen CIP-resistant S. Kentucky strains were isolated from diarrheal patients in Korea from 2008 to 2017. The travel histories of the patients indicated that seven had returned from Southeast Asia. WGS of all the isolates revealed the presence of Salmonella genomic island 1 (SGI1) and substitutions in the gyrA and parC genes, which are known to confer resistance to CIP. A multilocus sequence type (MLST) analysis revealed that the isolates belonged to ST198, which has been prevalent in Europe and Africa. Furthermore, a phylogenetic analysis showed that all 10 isolates shared close genetic relationships. ConclusionsWe report the identification of S. Kentucky in Korea through long-term surveillance. International travel, especially to Southeast Asia, has been a major risk factor for human infections of CIP-resistant S. Kentucky in Korea.

Draft genome sequence of a multidrug-resistant Salmonella enterica serotype Kentucky ST198 with chromosomal integration of blaCTX-M-14b isolated from a poultry slaughterhouse in China

The aim of this study was to characterise the draft genome sequence of a multidrug-resistant (MDR) Salmonella enterica serotype Kentucky strain (XJ9S) isolated from a poultry slaughterhouse in China. The genome was sequenced using an Illumina HiSeq platform and was assembled using SPAdes_3.12.0. The CGE Bacterial Analysis Pipeline was used to identify the sequence type (ST) as well as the presence of antimicrobial resistance genes (ARGs) and plasmids in strain XJ9S. Gaps among contigs that carried MDR Salmonella genomic island 1 (SGI1) fragments were filled in by PCR linkage and sequencing. The draft genome of strain XJ9S was assembled into 54 contigs with a total assembly size of 4 785 059 bp. XJ9S belonged to ST198 and harboured five acquired ARGs [blaCTX-M-14b, sul1, tetA(A), aacCA5 and aadA7]. The blaCTX-M-14b gene was located on a 2849-bp ISEcp1-mediated translocatable unit inserted in the chromosome. The other four acquired ARGs were carried by a new variant of SGI1 (SGI1-XJ9S; 38 593 bp) belonging to the SGI1-K group. Moreover, point mutations in the quinolone resistance-determining region (QRDR) were found at positions 83 (Ser83Phe) and 87 (Asp87Gly) of GyrA and at position 80 (Ser80Ile) of ParC. In this study, a new SGI1 variant (SGI1-XJ9S) was characterised for the first time. The draft genome sequence of S. Kentucky ST198 strain XJ9S isolated from a poultry slaughterhouse provides valuable information for tracing the potential spread of this MDR clone from poultry product processing to consumption, and even to humans.

Emergence of new variants of antibiotic resistance genomic islands among multidrug-resistant Salmonella enterica in poultry.

Non-typhoidal Salmonella enterica (NTS) are diverse and important bacterial pathogens consisting of more than 2600 different serovars, with varying host-specificity. Here, we characterized the poultry-associated serovars in Israel, analysed their resistome and illuminated the molecular mechanisms underlying common multidrug resistance (MDR) patterns. We show that at least four serovars including Infantis, Muenchen, Newport and Virchow present a strong epidemiological association between their temporal trends in poultry and humans. Worrisomely, 60% from all of the poultry isolates tested (n = 188) were multidrug resistant, mediated by chromosomal SNPs and different mobile genetics elements. A novel streptomycin-azithromycin resistance island and previously uncharacterized versions of the mobilized Salmonella genomic island 1 (SGI1) were identified and characterized in S. Blockley and S. Kentucky isolates respectively. Moreover, we demonstrate that the acquisition of SGI1 does not impose fitness cost during growth under nutrient-limited conditions or in the context of Salmonella infection in the mouse model. Overall, our data emphasize the role of the poultry production as a pool of specific epidemic MDR strains and autonomous genetic elements, which confer resistance to heavy metals and medically relevant antibiotics. These are likely to disseminate to humans via the food chain and fuel the increasing global antibiotic resistance crisis.

IS26-Mediated Genetic Rearrangements in Salmonella Genomic Island 1 of Proteus mirabilis.

Salmonella genomic island 1 (SGI1) is an integrative mobilizable element integrated into the chromosome of bacteria, which plays an important role in the dissemination of antimicrobial resistance genes. Lots of SGI1 variants are found mainly in Salmonella enterica and Proteus mirabilis. In this study, a total of 157 S. enterica and 132 P. mirabilis strains were collected from food-producing animals in Sichuan Province of China between December 2016 and November 2017. Detection of the SGI1 integrase gene showed that three S. enterica and five P. mirabilis strains were positive for SGI1, which displayed different multidrug resistance profiles. Five different SGI1 variants, including two novel variants (SGI1-PmBC1123 and SGI1-PmSC1111), were characterized by whole genome sequencing and PCR linkage. In two novel SGI1 variants, IS26-mediated rearrangements resulted in large sequence inversions of the MDR regions extending outside the SGI1 backbone. The sul3-type III class 1 integron (5′CS-sat-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3) and gene cassettes aac(6′)-Ib-cr-blaOXA–1-catB3-arr-3 are found in SGI1-PmSC1111. Mobilization experiments indicated that three known variants were conjugally mobilized in trans to Escherichia coli with the help of a conjugative IncC plasmid. However, the two novel variants seemed to lose the mobilization, which might result from the sequence inversion of partial SGI1 backbone. The identification of the two novel SGI1 variants in this study suggested that IS26-mediated rearrangements promote the diversity of SGI1.

Salmonella Genomic Island 3 Is an Integrative and Conjugative Element and Contributes to Copper and Arsenic Tolerance of Salmonella enterica.

Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:- isolated in Japan was identical to that of a previously reported one across entire length of 81 kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3' end of the tRNA genes pheV or pheR The length of the target site was 52 or 55 bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4 compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4 compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.

Analysis of isolates from Bangladesh highlights multiple ways to carry resistance genes in Salmonella Typhi

BackgroundTyphoid fever, caused by Salmonella Typhi, follows a fecal-oral transmission route and is a major global public health concern, especially in developing countries like Bangladesh. Increasing emergence of antimicrobial resistance (AMR) is a serious issue; the list of treatments for typhoid fever is ever-decreasing. In addition to IncHI1-type plasmids, Salmonella genomic island (SGI) 11 has been reported to carry AMR genes. Although reports suggest a recent reduction in multidrug resistance (MDR) in the Indian subcontinent, the corresponding genomic changes in the background are unknown.ResultsHere, we assembled and annotated complete closed chromosomes and plasmids for 73 S. Typhi isolates using short-length Illumina reads. S. Typhi had an open pan-genome, and the core genome was smaller than previously reported. Considering AMR genes, we identified five variants of SGI11, including the previously reported reference sequence. Five plasmids were identified, including the new plasmids pK91 and pK43; pK43and pHCM2 were not related to AMR. The pHCM1, pPRJEB21992 and pK91 plasmids carried AMR genes and, along with the SGI11 variants, were responsible for resistance phenotypes. pK91 also contained qnr genes, conferred high ciprofloxacin resistance and was related to the H58-sublineage Bdq, which shows the same phenotype. The presence of plasmids (pHCM1 and pK91) and SGI11 were linked to two H58-lineages, Ia and Bd. Loss of plasmids and integration of resistance genes in genomic islands could contribute to the fitness advantage of lineage Ia isolates.ConclusionsSuch events may explain why lineage Ia is globally widespread, while the Bd lineage is locally restricted. Further studies are required to understand how these S. Typhi AMR elements spread and generate new variants. Preventive measures such as vaccination programs should also be considered in endemic countries; such initiatives could potentially reduce the spread of AMR.

Salmonella Genomic Island 1B Variant Found in a Sequence Type 117 Avian Pathogenic Escherichia coli Isolate.

Salmonella genomic island 1 (SGI1) is an integrative genetic island first described in Salmonella enterica serovars Typhimurium DT104 and Agona in 2000. Variants of it have since been described in multiple serovars of S. enterica, as well as in Proteus mirabilis, Acinetobacter baumannii, Morganella morganii, and several other genera. The island typically confers resistance to older, first-generation antimicrobials; however, some variants carry blaNDM-1, blaVEB-6, and blaCTX-M15 genes that encode resistance to frontline, clinically important antibiotics, including third-generation cephalosporins. Genome sequencing studies of avian pathogenic Escherichia coli (APEC) identified a sequence type 117 (ST117) isolate (AVC96) with genetic features found in SGI1. The complete genome sequence of AVC96 was assembled from a combination of Illumina and single-molecule real-time (SMRT) sequence data. Analysis of the AVC96 chromosome identified a variant of SGI1-B located 18 bp from the 3' end of trmE, also known as the attB site, a known hot spot for the integration of genomic islands. This is the first report of SGI1 in wild-type E. coli The variant, here named SGI1-B-Ec1, was otherwise unremarkable, apart from the identification of ISEc43 in open reading frame (ORF) S023.IMPORTANCE SGI1 and variants of it carry a variety of antimicrobial resistance genes, including those conferring resistance to extended-spectrum β-lactams and carbapenems, and have been found in diverse S. enterica serovars, Acinetobacter baumannii, and other members of the Enterobacteriaceae SGI1 integrates into Gram-negative pathogenic bacteria by targeting a conserved site 18 bp from the 3' end of trmE For the first time, we describe a novel variant of SGI1 in an avian pathogenic Escherichia coli isolate. The presence of SGI1 in E. coli is significant because it represents yet another lateral gene transfer mechanism to enhancing the capacity of E. coli to acquire and propagate antimicrobial resistance and putative virulence genes. This finding underscores the importance of whole-genome sequencing (WGS) to microbial genomic epidemiology, particularly within a One Health context. Further studies are needed to determine how widespread SGI1 and variants of it may be in Australia.

Global phylogenomics of multidrug-resistant Salmonella enterica serotype Kentucky ST198.

Salmonella enterica serotype Kentucky can be a common causative agent of salmonellosis, usually associated with consumption of contaminated poultry. Antimicrobial resistance (AMR) to multiple drugs, including ciprofloxacin, is an emerging problem within this serotype. We used whole-genome sequencing (WGS) to investigate the phylogenetic structure and AMR content of 121 S. e nterica serotype Kentucky sequence type 198 isolates from five continents. Population structure was inferred using phylogenomic analysis and whole genomes were compared to investigate changes in gene content, with a focus on acquired AMR genes. Our analysis showed that multidrug-resistant (MDR) S. enterica serotype Kentucky isolates belonged to a single lineage, which we estimate emerged circa 1989 following the acquisition of the AMR-associated Salmonella genomic island (SGI) 1 (variant SGI1-K) conferring resistance to ampicillin, streptomycin, gentamicin, sulfamethoxazole and tetracycline. Phylogeographical analysis indicates this clone emerged in Egypt before disseminating into Northern, Southern and Western Africa, then to the Middle East, Asia and the European Union. The MDR clone has since accumulated various substitution mutations in the quinolone-resistance-determining regions (QRDRs) of DNA gyrase (gyrA) and DNA topoisomerase IV (parC), such that most strains carry three QRDR mutations which together confer resistance to ciprofloxacin. The majority of AMR genes in the S. e nterica serotype Kentucky genomes were carried either on plasmids or SGI structures. Remarkably, each genome of the MDR clone carried a different SGI1-K derivative structure; this variation could be attributed to IS26-mediated insertions and deletions, which appear to have hampered previous attempts to trace the clone’s evolution using sub-WGS resolution approaches. Several different AMR plasmids were also identified, encoding resistance to chloramphenicol, third-generation cephalosporins, carbapenems and/or azithromycin. These results indicate that most MDR S. e nterica serotype Kentucky circulating globally result from the clonal expansion of a single lineage that acquired chromosomal AMR genes 30 years ago, and has continued to diversify and accumulate additional resistances to last-line oral antimicrobials. This article contains data hosted by Microreact.

Entry Exclusion of Conjugative Plasmids of the IncA, IncC, and Related Untyped Incompatibility Groups.

Conjugative plasmids of incompatibility group C (IncC), formerly known as A/C2, disseminate antibiotic resistance genes globally in diverse pathogenic species of Gammaproteobacteria. Salmonella genomic island 1 (SGI1) can be mobilized by IncC plasmids and was recently shown to reshape the conjugative type IV secretion system (T4SS) encoded by these plasmids to evade entry exclusion. Entry exclusion blocks DNA translocation between cells containing identical or highly similar plasmids. Here, we report that the protein encoded by the entry exclusion gene of IncC plasmids (eexC) mediates entry exclusion in recipient cells through recognition of the IncC-encoded TraGC protein in donor cells. Phylogenetic analyses based on EexC and TraGC homologs predicted the existence of at least three different exclusion groups among IncC-related conjugative plasmids. Mating assays using Eex proteins encoded by representative IncC and IncA (former A/C1) and related untyped plasmids confirmed these predictions and showed that the IncC and IncA plasmids belong to the C exclusion group, thereby explaining their apparent incompatibility despite their compatible replicons. Representatives of the two other exclusion groups (D and E) are untyped conjugative plasmids found in Aeromonas sp. Finally, we determined through domain swapping that the carboxyl terminus of the EexC and EexE proteins controls the specificity of these exclusion groups. Together, these results unravel the role of entry exclusion in the apparent incompatibility between IncA and IncC plasmids while shedding light on the importance of the TraG subunit substitution used by SGI1 to evade entry exclusion.IMPORTANCE IncA and IncC conjugative plasmids drive antibiotic resistance dissemination among several pathogenic species of Gammaproteobacteria due to the diversity of drug resistance genes that they carry and their ability to mobilize antibiotic resistance-conferring genomic islands such as SGI1 of Salmonella enterica While historically grouped as "IncA/C," IncA and IncC replicons were recently confirmed to be compatible and to abolish each other's entry into the cell in which they reside during conjugative transfer. The significance of our study is in identifying an entry exclusion system that is shared by IncA and IncC plasmids. It impedes DNA transfer to recipient cells bearing a plasmid of either incompatibility group. The entry exclusion protein of this system is unrelated to any other known entry exclusion proteins.

Distribution and characteristics of SGI1/PGI2 genomic island from Proteus strains in China

The emergence of multidrug-resistant Salmonella genomic island 1 (SGI1) and Proteus genomic island (PGI) bearing P. mirabilis present a serious threat to public health. In this study, we screened 288 Proteus isolates recovered from seven provinces in China. Fourteen strains (4.9%) all belonged to P. mirabilis were positive for SGI1/PGI2, including twelve from clinical samples (5.3%) and two from food (3.3%). A Blastn search against GenBank and phylogenetic analyses identified eight different SGI1 variants and one PGI2 variant from the fourteen SGI1/PGI2 variants. All SGI1 variants shared a common backbone and harbored different resistance gene(s), except the sul1 gene at its multidrug-resistant (MDR) region. Among the variants, three novel SGI1 variants, designated as SGI1-PmCA11, SGI1-PmCA14 and SGI1-PmCA46, contained different gene cassettes, which were similar to sequences in plasmids or class 1 integrons of Klebsiella pneumoniae, P. mirabilis, Escherichia coli and Salmonella. Moreover, one novel PGI2, designated as PGI2-PmCA72, had an identical gene cassette to the first class 1 integron from PGI2 (GenBank accession no. MG201402.1) in P. mirabilis, but varied due to missing, replaced, inserted and inverted gene clusters. The four novel SGI1/PGI2 variants contained the cmlA5, dfrA14, blaOXA-10, aadA15, blaOXA-1, catB3 and dfrA16 resistance genes, which have never been reported in SGI1/PGI2 variants. Phenotypically, all fourteen SGI1/PGI2-containing strains showed multidrug resistance. All except four strains were resistant to the first, or the second and/or-third generation cephalosporins. Considering the increasing number and the emergence of new SGI1/PGI2 variants, further surveillance is needed to prevent the spreading of the MDR genomic islands among Proteus isolates from human and food.

Identification and Characterization of New Resistance-Conferring SGI1s (Salmonella Genomic Island 1) in Proteus mirabilis.

Salmonella genomic island 1 (SGI1) is a resistance-conferring chromosomal genomic island that contains an antibiotic resistance gene cluster. The international spread of SGI1-containing strains drew attention to the role of genomic islands in the dissemination of antibiotic resistance genes in Salmonella and other Gram-negative bacteria. In this study, five SGI1 variants conferring multidrug and heavy metal resistance were identified and characterized in Proteus mirabilis strains: SGI1-PmCAU, SGI1-PmABB, SGI1-PmJN16, SGI1-PmJN40, and SGI1-PmJN48. The genetic structures of SGI1-PmCAU and SGI1-PmABB were identical to previously reported SGI1s, while structural analysis showed that SGI1-PmJN16, SGI1-PmJN40, and SGI1-PmJN48 are new SGI1 variants. SGI1-PmJN16 is derived from SGI1-Z with the MDR region containing a new gene cassette array dfrA12-orfF-aadA2-qacEΔ1-sul1-chrA-orf1. SGI1-PmJN40 has an unprecedented structure that contains two right direct repeat sequences separated by a transcriptional regulator-rich DNA fragment, and is predicted to form two different extrachromosomal mobilizable DNA circles for dissemination. SGI1-PmJN48 lacks a common ORF S044, and its right junction region exhibits a unique genetic organization due to the reverse integration of a P. mirabilis chromosomal gene cluster and the insertion of part of a P. mirabilis plasmid, making it the largest known SGI1 to date (189.1 kb). Further mobility functional analysis suggested that these SGIs can be excised from the chromosome for transfer between bacteria, which promotes the horizontal transfer of antibiotic and heavy metal resistance genes. The identification and characterization of the new SGI1 variants in this work suggested the diversity of SGI1 structures and their significant roles in the evolution of bacteria.

Occurrence of Salmonella genomic island 1 (SGI1) in two African Proteus mirabilis strains isolated from diseased chicken flocks

Two P. mirabilis strains, PmSHR21 and PmSHR38, were collected from chicken flocks in Sharkia Governorate, Egypt in 2016. The two strains showed multidrug-resistance (MDR) phenotypes and were detected to harbour I) floR and sul1 genes conferring resistance to florfenicol and chloramphenicol, and sulfonamides, respectively, II) a ~1.9 kbp class 1 integron containing aadA2–lnuF genes conferring resistance to spectinomycin and streptomycin, and lincosamides, respectively. Interestingly, the two strains were detected to contain SGI1 variant, SGI1-W and inserted between the 3′ end of the chromosomal trmE gene and the hipB/hipA toxin/antitoxin homologue. Fingerprinting by ERIC-PCR of the two poultry strains identified in this study and the two human SGI1-carrying P. mirabilis strains described recently in our study showed identical ERIC-pattern between SGI1-W-carrying poultry and human strains, suggesting that they might be clonally related. The detection of SGI1 and its variants in P. mirabilis isolated from humans and chicken flocks in Egypt clarify the geographical and biological spreading through an inter-transmission pathway. To the best of our knowledge, this is the first study detecting SGI1-positive P. mirabilis isolated from chicken flocks in Africa.

\u201cDoes the Salmonella Genomic Island 1 (SGI1) confer invasiveness properties to human isolates?\u201d

BackgroundIn the eighties, a multidrug resistant clone of Salmonella Typhimurium DT104 emerged in UK and disseminated worldwide. This clone harbored a Salmonella genomic island 1 (SGI1) that consists of a backbone and a multidrug resistant region encoding for penta-resistance (ampicillin, chloramphenicol/florfenicol, streptomycin/spectinomycin, sulphonamides and tetracycline (ACSSuT)). Several authors suggested that SGI1 might have a potential role in enhancement of virulence properties of Salmonella enterica. The aim of this study was to investigate whether nontyphoidal S. enterica isolates carrying SGI1 cause more severe illness than SGI1 free ones in humans.MethodsFrom 2011 to 2016, all patients infected with nontyphoidal S. enterica in our hospital were retrospectively included. All nontyphoidal S. enterica isolates preserved in our University Hospital (Dijon, France) were screened for the presence of SGI1. Clinical and biological data of patients were retrospectively collected to evaluate illness severity. Statistical analysis of data was performed by Kruskal-Wallis test or Fisher’s exact test for univariate analysis, and by logistic regression for multivariate analysis.ResultsA total of 100 isolates of S. enterica (22 serovars) were collected. Twelve isolates (12%) belonging to 4 serovars harbored SGI1: S. Typhimurium, S. Infantis, S. Kentucky, S. St Paul. The severity of the disease was age-related (for invasive infection, sepsis and inflammatory response) and was associated with immunosuppression (for invasive infection, sepsis and bacteremia) but not with the presence of SGI1 or with antimicrobial resistance.ConclusionA rather high proportion (12%) of human clinical isolates belonging to various serovars (for the first time serovar St Paul) and harboring various antimicrobial resistance profile carried SGI1. Diseases due to SGI1-positive S. enterica or to antimicrobial resistant isolates were not more severe than the others. This first clinical observation should be confirmed by a multicenter and prospective study.

Molecular Characterization of Salmonella Genomic Island 1 in Proteus mirabilis Isolates from Chungcheong Province, Korea.

The emergence and dissemination of Salmonella genomic island 1 (SGI1) are strongly associated with the occurrence of multidrug-resistant (MDR) enterobacteria in humans and animals. Diverse SGI1s have been reported among Salmonella enterica and Proteus mirabilis in several countries. We aimed to characterize SGI1 in P. mirabilis isolates from humans and chickens in Chungcheong Province, Korea. A total of 44 P. mirabilis isolates were recovered from humans (n = 20) and chickens (n = 24). Antimicrobial susceptibility was determined by disk diffusion assay. To detect and characterize SGI1s, PCR amplification and PCR mapping experiments were performed. Repetitive extragenic palindromic-PCR (REP-PCR) was performed to assess the clonality of the isolates. The four P. mirabilis strains (16.7%) from chicken harbored a SGI1, whereas none of the isolates from clinical specimens contained SGI1. The SGI1s detected in our study were all confirmed as SGI1-PmABB harboring aminoglycoside-resistant genes (aacCA5 and aadA7). In P. mirabilis isolates, the presence of SGI1-PmABB was significantly correlated with high resistance rates of the isolates to antimicrobial agents, such as gentamicin, streptomycin, and spectinomycin. Moreover, the four SGI1-bearing isolates showed the same REP-PCR patterns and that suggested both horizontal and clonal spread of the isolates. This study is the first attempt to determine SGI1s in P. mirabilis isolates in Korea. We confirmed that P. mirabilis isolates carrying SGI1-PmABB were distributed at poultry farms in Korea. The present study emphasizes the need for continuous monitoring of SGI1s to prevent spreading of the MDR genomic islands among P. mirabilis isolates from humans and animals.

Salmonella genomic island 1 (SGI1) reshapes the mating apparatus of IncC conjugative plasmids to promote self-propagation.

IncC conjugative plasmids and Salmonella genomic island 1 (SGI1) and relatives are frequently associated with multidrug resistance of clinical isolates of pathogenic Enterobacteriaceae. SGI1 is specifically mobilized in trans by IncA and IncC plasmids (commonly referred to as A/C plasmids) following its excision from the chromosome, an event triggered by the transcriptional activator complex AcaCD encoded by these helper plasmids. Although SGI1 is not self-transmissible, it carries three genes, traNS, traHS and traGS, coding for distant homologs of the predicted mating pore subunits TraNC, TraHC and TraGC, respectively, encoded by A/C plasmids. Here we investigated the regulation of traNS and traHGS and the role of these three genes in the transmissibility of SGI1. Transcriptional fusion of the promoter sequences of traNS and traHGS to the reporter gene lacZ confirmed that expression of these genes is inducible by AcaCD. Mating experiments using combinations of deletion mutants of SGI1 and the helper IncC plasmid pVCR94 revealed complex interactions between these two mobile genetic elements. Whereas traNC and traHGC are essential for IncC plasmid transfer, SGI1 could rescue null mutants of each individual gene revealing that TraNS, TraHS and TraGS are functional proteins. Complementation assays of individual traC and traS mutants showed that not only do TraNS/HS/GS replace TraNC/HC/GC in the mating pore encoded by IncC plasmids but also that traGS and traHS are both required for SGI1 optimal transfer. In fact, remodeling of the IncC-encoded mating pore by SGI1 was found to be essential to enhance transfer rate of SGI1 over the helper plasmid. Furthermore, traGS was found to be crucial to allow DNA transfer between cells bearing IncC helper plasmids, thereby suggesting that by remodeling the mating pore SGI1 disables an IncC-encoded entry exclusion mechanism. Hence traS genes facilitate the invasion by SGI1 of cell populations bearing IncC plasmids.

First report in Africa of two clinical isolates of Proteus mirabilis carrying Salmonella genomic island (SGI1) variants, SGI1-PmABB and SGI1-W

Two Proteus mirabilis strains, designated PmTAN59 and PmKAF126, were isolated from two different Egyptian cities in 2014 and 2015, respectively. PmTAN59 was isolated from a sputum swab from a pneumonia patient in Tanta University Teaching Hospital. PmKAF126 was isolated from a patient with a diabetic foot infection in a hospital in the city of Kafr El-Sheikh. The two isolates were identified with bacterial small ribosomal RNA (16S rRNA) gene amplification and sequencing and tested for antimicrobial sensitivity with a Kirby–Bauer disk diffusion assay. The two strains were resistant to amoxicillin/clavulante, ampicillin, cefotaxime, cefoxitin, ceftriaxone, chloramphenicol, ciprofloxacin, colistin, gentamicin, kanamycin, nalidixic acid, spectinomycin, streptomycin, sulfamethoxazole/trimethoprime, and tetracycline, but sensitive to aztreonam, imipenem, and meropenem. Molecular characterization was used to map the entire backbone, including the multiple antibiotic resistance (MDR) region, of Salmonella genomic island 1 (SGI1). Both isolates carried a structure similar to SGI1, with two different MDR regions corresponding to SGI1-PmABB in PmTAN59 and SGI1-W in PmKAF126. SGI1-PmABB carried an integron of ~1.5kb with a two-gene cassette, aacCA5–aadA7, which confers resistance to gentamicin, streptomycin, and spectinomycin, whereas SGI1-W carried an integron of ~1.9kb containing aadA2–lnuF, which confers resistance to spectinomycin, streptomycin, and lincosamides. PmKAF126 carried the entire SGI1 sequence, however PmTAN59 carried a SGI1 structure with a deletion in the region from ORF S005 to ORF S009 and accompanied by insertion of IS1359 (1258bp). Furthermore, PmTAN59 carried class 2 integron of ~2.2kb containing dfrA1-sat2-aadA1. An ERIC-PCR analysis detected no clonal relationship between the two strains. Molecular screening for other antimicrobial resistance genes and a plasmid analysis indicated that PmTAN59 carried an IncFIB plasmid type. This strain also carried blaTEM-1 and the plasmid-mediated quinolone-resistance gene qnrA1. However, PmKAF126 carried no plasmids and no resistance gene other than that contained in the MDR region of SGI1 and floR gene conferring resistance to florfenicol. To the best of our knowledge, this is the first report of an SGI1-positive P. mirabilis strain in Egypt or on the entire African continent.

Characterization of the Genetic Environment of theblaVEB-4Gene, Associated with a Transposable Region in aProteus mirabilisClinical Isolate

Proteus mirabilis is the second most common cause of urinary tract infections and is also an important cause of nosocomial infections. TEM-type and CTX-M-type extended-spectrum β-lactamases (ESBLs) are the most widely distributed in this bacterial species, but minor ESBLs such as the VEB-type have also been identified. The aim of this study was to analyze the genetic environment of the blaVEB-4 gene found in a P. mirabilis clinical isolate recovered in Spain. P. mirabilis N2231 showed resistance to penicillins, cephalosporins, and aminoglycosides, remaining susceptible to imipenem, cefoxitin, β-lactamases inhibitors, and quinolones. Southern blot analysis revealed that blaVEB-4 was located in the chromosome. Analysis of the blaVEB-4 genetic context revealed a 15 kb segment 98% identical to the multidrug resistance (MDR) region of a Salmonella genomic island 1 (SGI1), which included a class 1 integron belonging to the In104 family, previously described in blaVEB-6-producing P. mirabilis VB1248. blaVEB-4 was surrounded by repeat elements, transposon Tn1721, and located on a class 1 integron containing aacA4-aadB-dfrA1-orfC genes. The blaVEB-4 gene was inserted in a complex structure of a class 1 integron, which is part of an MDR region of an SGI1, possibly involved in the mobilization of the gene and homologous recombination.

Detection of SGI1/PGI1 Elements and Resistance to Extended-Spectrum Cephalosporins in Proteae of Animal Origin in France

Proteae, and especially Proteus mirabilis, are often the cause of urinary tract infections (UTIs) in humans. They were reported as carriers of extended-spectrum β-lactamase (ESBL) genes, and recently of carbapenemases, mostly carried by the Salmonella genomic island 1 (SGI1) and Proteus genomic island 1 (PGI1). Proteae have also lately become an increasing cause of UTIs in companion animals, but antimicrobial susceptibility data in animals are still scarce. Here, we report the characterization of 468 clinical epidemiologically unrelated Proteae strains from animals collected between 2013 and 2015 in France. Seventeen P. mirabilis strains (3.6%) were positive for SGI1/PGI1 and 18 Proteae (3.8%) were resistant to extended-spectrum cephalosporins (ESC). The 28 isolates carrying SGI1/PGI1 and/or ESC-resistance genes were isolated from cats, dogs, and horses. ESBL genes were detected in six genetically related P. mirabilis harboring blaV EB-6 on the SGI1-V variant, but also independently of the SGI1-V, in 3 P. mirabilis strains (blaVEB-6 and blaCTX-M-15) and 1 Providencia rettgeri strain (blaCTX-M-1). The AmpC resistance genes blaCMY -2 and/or blaDHA-16 were detected in 9 P. mirabilis strains. One strain presented both an ESBL and AmpC gene. Interestingly, the majority of the ESBL/AmpC resistance genes were located on the chromosome. In conclusion, multiple ESC-resistance genetic determinants are circulating in French animals, even though SGI1-V-carrying P. mirabilis seems to be mainly responsible for the spread of the ESBL gene blaVEB-6 in dogs and horses. These results are of public health relevance and show that companion animals in close contact with humans should be regarded as a potential reservoir of ESC-resistant bacteria as well as a reservoir of ESC-resistance genes that could further disseminate to human pathogens.

Diversity of antibiotic-resistance genes in Canadian isolates of Aeromonas salmonicida subsp. salmonicida: dominance of pSN254b and discovery of pAsa8.

The bacterium Aeromonas salmonicida subsp. salmonicida is a common pathogen in fish farms worldwide. Since the antibiotic resistance of this bacterial species is on the increase, it is important to have a broader view on this issue. In the present study, we tested the presence of known plasmids conferring multi-drug resistance as well as antibiotic resistance genes by a PCR approach in 100 Canadian A. salmonicida subsp. salmonicida isolates. Our study highlighted the dominance of the conjugative pSN254b plasmid, which confers multi-drug resistance. We also identified a new multi-drug plasmid named pAsa8, which has been characterized by a combination of sequencing technologies (Illumina and Oxford nanopore). This new plasmid harbors a complex class 1 integron similar to the one of the Salmonella genomic island 1 (SGI1) found in Salmonella enterica and Proteus mirabilis. Consequently, in addition to providing an update on the A. salmonicida subsp. salmonicida isolates that are resistant to antibiotics, our data suggest that this bacterium is potentially an important reservoir of drug resistance genes and should consequently be monitored more extensively. In addition, we describe a screening method that has the potential to become a diagnostic tool that is complementary to other methods currently in use.

Destabilization of IncA and IncC plasmids by SGI1 and SGI2 type Salmonella genomic islands

Both the Salmonella genomic islands (SGI) and the conjugative IncC plasmids are known to contribute substantially to the acquisition of resistance to multiple antibiotics, and plasmids in the A/C group are known to mobilize the Salmonella genomic island SGI1, which also carries multiple antibiotic resistance genes. Plasmid pRMH760 (IncC; A/C2) was shown to mobilize SGI1 variants SGI1-I, SGI1-F, SGI1-K and SGI2 from Salmonella enterica to Escherichia coli where it was integrated at the preferred location, at the end of the trmE (thdF) gene. The plasmid was transferred at a similar frequency. However, we observed that co-transfer of the SGI and the plasmid was rarer. In E. coli to E. coli transfer, the frequency of transfer of the IncC plasmid pRMH760 was at least 1000-fold lower when the donor carried SGI1-I or SGI1-K, indicating that the SGI suppresses transfer of the plasmid. In addition, pRMH760 was rapidly lost from both E. coli and S. enterica strains that also carried SGI1-I, SGI1-F or SGI2. However, plasmid loss was not seen when the SGI1 variant was SGI1-K, which lacks two segments of the SGI1 backbone. The complete sequence of the SGI1-I and SGI1-F were determined and SGI1-K also carries two single base substitutions relative to SGI1-I. The IncA (A/C1) plasmid RA1 was also shown to mobilize SGI2-A and though there are significant differences between the backbones of IncA and IncC plasmids, RA1 was also rapidly lost when SGI2-A was present in the same cell. We conclude that there are multiple interactions, both cooperative and antagonistic, between an IncA or IncC plasmid and the SGI1 and SGI2 family genomic islands.