Salmonella Genomic Island 1 Research Articles

Genomic Features and Phylogenetic Analysis of Antimicrobial-Resistant Salmonella Mbandaka ST413 Strains.

In recent years, Salmonella enterica subsp. enterica serovar Mbandaka (S. Mbandaka) has been increasingly isolated from laying hens and shell eggs around the world. Moreover, this serovar has been identified as the causative agent of several salmonellosis outbreaks in humans. Surprisingly, little is known about the characteristics of this emerging serovar, and therefore, we investigated antimicrobial resistance, virulence, and prophage genes of six selected Brazilian strains of Salmonella Mbandaka using Whole Genome Sequencing (WGS). Multi-locus sequence typing revealed that the tested strains belong to Sequence Type 413 (ST413), which has been linked to recent multi-country salmonellosis outbreaks in Europe. A total of nine resistance genes were detected, and the most frequent ones were aac(6')-Iaa, sul1, qacE, blaOXA-129, tet(B), and aadA1. A point mutation in ParC at the 57th position (threonine → serine) associated with quinolone resistance was present in all investigated genomes. A 112,960 bp IncHI2A plasmid was mapped in 4/6 strains. This plasmid harboured tetracycline (tetACDR) and mercury (mer) resistance genes, genes contributing to conjugative transfer, and genes involved in plasmid maintenance. Most strains (four/six) carried Salmonella genomic island 1 (SGI1). All S. Mbandaka genomes carried seven pathogenicity islands (SPIs) involved in intracellular survival and virulence: SPIs 1-5, 9, and C63PI. The virulence genes csgC, fimY, tcfA, sscA, (two/six), and ssaS (one/six) were absent in some of the genomes; conversely, fimA, prgH, and mgtC were present in all of them. Five Salmonella bacteriophage sequences (with homology to Escherichia phage phiV10, Enterobacteria phage Fels-2, Enterobacteria phage HK542, Enterobacteria phage ST64T, Salmonella phage SW9) were identified, with protein counts between 31 and 54, genome lengths of 24.7 bp and 47.7 bp, and average GC content of 51.25%. In the phylogenetic analysis, the genomes of strains isolated from poultry in Brazil clustered into well-supported clades with a heterogeneous distribution, primarily associated with strains isolated from humans and food. The phylogenetic relationship of Brazilian S. Mbandaka suggests the presence of strains with high epidemiological significance and the potential to be linked to foodborne outbreaks. Overall, our results show that isolated strains of S. Mbandaka are multidrug-resistant and encode a rather conserved virulence machinery, which is an epidemiological hallmark of Salmonella strains that have successfully disseminated both regionally and globally.

Pathogen genomics and phage-based solutions for accurately identifying and controlling Salmonella pathogens.

Salmonella is a food-borne pathogen often linked to poultry sources, causing gastrointestinal infections in humans, with the numbers of multidrug resistant (MDR) isolates increasing globally. To gain insight into the genomic diversity of common serovars and their potential contribution to disease, we characterized antimicrobial resistance genes, and virulence factors encoded in 88 UK and 55 Thai isolates from poultry; the presence of virulence genes was detected through an extensive virulence determinants database compiled in this study. Long-read sequencing of three MDR isolates, each from a different serovar, was used to explore the links between virulence and resistance. To augment current control methods, we determined the sensitivity of isolates to 22 previously characterized Salmonella bacteriophages. Of the 17 serovars included, Salmonella Typhimurium and its monophasic variants were the most common, followed by S. Enteritidis, S. Mbandaka, and S. Virchow. Phylogenetic analysis of Typhumurium and monophasic variants showed poultry isolates were generally distinct from pigs. Resistance to sulfamethoxazole and ciprofloxacin was highest in isolates from the UK and Thailand, respectively, with 14-15% of all isolates being MDR. We noted that >90% of MDR isolates were likely to carry virulence genes as diverse as the srjF, lpfD, fhuA, and stc operons. Long-read sequencing revealed the presence of global epidemic MDR clones in our dataset, indicating they are possibly widespread in poultry. The clones included MDR ST198 S. Kentucky, harboring a Salmonella Genomic Island-1 (SGI)-K, European ST34 S. 1,4,[5],12:i:-, harboring SGI-4 and mercury-resistance genes, and a S. 1,4,12:i:- isolate from the Spanish clone harboring an MDR-plasmid. Testing of all isolates against a panel of bacteriophages showed variable sensitivity to phages, with STW-77 found to be the most effective. STW-77 lysed 37.76% of the isolates, including serovars important for human clinical infections: S. Enteritidis (80.95%), S. Typhimurium (66.67%), S. 1,4,[5],12:i:- (83.3%), and S. 1,4,12: i:- (71.43%). Therefore, our study revealed that combining genomics and phage sensitivity assays is promising for accurately identifying and providing biocontrols for Salmonella to prevent its dissemination in poultry flocks and through the food chain to cause infections in humans.

Characterization of Extensively Drug-Resistant Salmonella enterica Serovar Kentucky Sequence Type 198 Isolates from Chicken Meat Products in Xuancheng, China.

The purpose of this study was to characterize extensively drug-resistant Salmonella enterica serovar Kentucky sequence type 198 (ST198) isolates from chicken meat products. Ten S. Kentucky strains obtained from chicken meat products in Xuancheng, China, carried 12 to 17 resistance genes, such as blaCTX-M-55, rmtB, tet(A), floR, and fosA3, combined with mutations within gyrA (S83F and D87N) and parC (S80I), resulting in resistance to numerous antimicrobial agents, including the clinically important antibiotics cephalosporin, ciprofloxacin, tigecycline, and fosfomycin. These S. Kentucky isolates shared a close phylogenetic relationship (21 to 36 single-nucleotide polymorphisms [SNPs]) and showed close genetic relatedness to two human clinical isolates from China. Three S. Kentucky strains were subjected to whole-genome sequencing using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) technology. All antimicrobial resistance genes were located on their chromosomes and clustered in one multiresistance region (MRR) and Salmonella genomic island (SGI) SGI1-K. The MRRs in three S. Kentucky strains were bounded by IS26 at both ends and were inserted downstream of the bcfABCDEFG cluster with 8-bp direct repeats. The MRRs were related to those of IncHI2 plasmids but differed by insertions, deletions, and rearrangements of multiple segments involving resistance genes and plasmid backbones. This finding suggests that the MRR fragment possibly originates from IncHI2 plasmids. Four SGI1-K variants with slight differences were identified in 10 S. Kentucky strains. Mobile elements, particularly IS26, play an essential role in forming distinct MRRs and SGI1-K structures. In conclusion, the emergence of extensively drug-resistant S. Kentucky ST198 strains containing numerous chromosomally located resistance genes is alarming and needs continued surveillance. IMPORTANCE Salmonella spp. are important foodborne pathogens, and multidrug-resistant (MDR) Salmonella strains have become a serious threat to clinical therapy. MDR S. Kentucky ST198 strains have been increasingly reported from various sources and have become a global risk. In this study, we described extensively drug-resistant S. Kentucky ST198 strains from chicken meat products from a city in China. Numerous resistance genes are clustered in the chromosomes of S. Kentucky ST198 strains, possibly acquired with the help of mobile elements. This would facilitate the spread of numerous resistance genes as intrinsic chromosomal genes within this global epidemic clone, with the potential to capture more resistance genes. The emergence and dissemination of extensively drug-resistant S. Kentucky ST198 pose a severe clinical and public health threat; therefore, continuous surveillance is warranted.

Conjugative IncC Plasmid Entry Triggers the SOS Response and Promotes Effective Transfer of the Integrative Antibiotic Resistance Element SGI1.

The broad-host-range IncC plasmid family and the integrative mobilizable Salmonella genomic island 1 (SGI1) and its derivatives enable the spread of medically important antibiotic resistance genes among Gram-negative pathogens. Although several aspects of the complex functional interactions between IncC plasmids and SGI1 have been recently deciphered regarding their conjugative transfer and incompatibility, the biological signal resulting in the hijacking of the conjugative plasmid by the integrative mobilizable element remains unknown. Here, we demonstrate that the conjugative entry of IncC/IncA plasmids is detected at an early stage by SGI1 through the transient activation of the SOS response, which induces the expression of the SGI1 master activators SgaDC, shown to play a crucial role in the complex biology between SGI1 and IncC plasmids. Besides, we developed an original tripartite conjugation approach to directly monitor SGI1 mobilization in a time-dependent manner following conjugative entry of IncC plasmids. Finally, we propose an updated biological model of the conjugative mobilization of the chromosomal resistance element SGI1 by IncC plasmids. IMPORTANCE Antimicrobial resistance has become a major public health issue, particularly with the increase of multidrug resistance (MDR) in both animal and human pathogenic bacteria and with the emergence of resistance to medically important antibiotics. The spread between bacteria of successful mobile genetic elements, such as conjugative plasmids and integrative elements conferring multidrug resistance, is the main driving force in the dissemination of acquired antibiotic resistances among Gram-negative bacteria. Broad-host-range IncC plasmids and their integrative mobilizable SGI1 counterparts contribute to the spread of critically important resistance genes (e.g., extended-spectrum β-lactamases [ESBLs] and carbapenemases). A better knowledge of the complex biology of these broad-host-range mobile elements will help us to understand the dissemination of antimicrobial resistance genes that occurred across Gammaproteobacteria borders.

Overview of Salmonella Genomic Island 1-Related Elements Among Gamma-Proteobacteria Reveals Their Wide Distribution Among Environmental Species.

The aim of this study was to perform an in silico analysis of the available whole-genome sequencing data to detect syntenic genomic islands (GIs) having homology to Salmonella genomic island 1 (SGI1), analyze the genetic variations of their backbone, and determine their relatedness. Eighty-nine non-redundant SGI1-related elements (SGI1-REs) were identified among gamma-proteobacteria. With the inclusion of the thirty-seven backbones characterized to date, seven clusters were identified based on integrase homology: SGI1, PGI1, PGI2, AGI1 clusters, and clusters 5, 6, and 7 composed of GIs mainly harbored by waterborne or marine bacteria, such as Vibrio, Shewanella, Halomonas, Idiomarina, Marinobacter, and Pseudohongiella. The integrase genes and the backbones of SGI1-REs from clusters 6 and 7, and from PGI1, PGI2, and AGI1 clusters differed significantly from those of the SGI1 cluster, suggesting a different ancestor. All backbones consisted of two parts: the part from attL to the origin of transfer (oriT) harbored the DNA recombination, transfer, and mobilization genes, and the part from oriT to attR differed among the clusters. The diversity of SGI1-REs resulted from the recombination events between GIs of the same or other families. The oriT appeared to be a high recombination site. The multi-drug resistant (MDR) region was located upstream of the resolvase gene. However, most SGI1-REs in Vibrio, Shewanella, and marine bacteria did not harbor any MDR region. These strains could constitute a reservoir of SGI1-REs that could be potential ancestors of SGI1-REs encountered in pathogenic bacteria. Furthermore, four SGI1-REs did not harbor a resolvase gene and therefore could not acquire an integron. The presence of mobilization genes and AcaCD binding sites indicated that their conjugative transfer could occur with helper plasmids. The plasticity of SGI1-REs contributes to bacterial adaptation and evolution. We propose a more relevant classification to categorize SGI1-REs into different clusters based on their integrase gene similarity.

Long-Read Sequencing Reveals Evolution and Acquisition of Antimicrobial Resistance and Virulence Genes in Salmonella enterica.

Salmonella enterica is a significant and phylogenetically diverse zoonotic pathogen. To understand its genomic heterogeneity and antimicrobial resistance, we performed long-read sequencing on Salmonella isolated from retail meats and food animals. A collection of 134 multidrug-resistant isolates belonging to 33 serotypes were subjected to PacBio sequencing. One major locus of diversity among these isolates was the presence and orientation of Salmonella pathogenic islands (SPI), which varied across different serotypes but were largely conserved within individual serotypes. We also identified insertion of an IncQ resistance plasmid into the chromosome of fourteen strains of serotype I 4,[5],12:i:– and the Salmonella genomic island 1 (SGI-1) in five serotypes. The presence of various SPIs, SGI-1 and integrated plasmids contributed significantly to the genomic variability and resulted in chromosomal resistance in 55.2% (74/134) of the study isolates. A total of 93.3% (125/134) of isolates carried at least one plasmid, with isolates carrying up to seven plasmids. We closed 233 plasmid sequences of thirteen replicon types, along with twelve hybrid plasmids. Some associations between Salmonella isolate source, serotype, and plasmid type were seen. For instance, IncX plasmids were more common in serotype Kentucky from retail chicken. Plasmids IncC and IncHI had on average more than five antimicrobial resistance genes, whereas in IncX, it was less than one per plasmid. Overall, 60% of multidrug resistance (MDR) strains that carried >3 AMR genes also carried >3 heavy metal resistance genes, raising the possibility of co-selection of antimicrobial resistance in the presence of heavy metals. We also found nine isolates representing four serotypes that carried virulence plasmids with the spv operon. Together, these data demonstrate the power of long-read sequencing to reveal genomic arrangements and integrated plasmids with a high level of resolution for tracking and comparing resistant strains from different sources. Additionally, the findings from this study will help expand the reference set of closed Salmonella genomes that can be used to improve genome assembly from short-read data commonly used in One Health antimicrobial resistance surveillance.

Genomic diversity and resistome profiles of Salmonella enterica subsp. enterica serovar Kentucky isolated from food and animal sources in Ireland.

Salmonella enterica subsp. enterica serovar Kentucky is frequently isolated from poultry, dairy and beef cattle, the environment and people with clinical salmonellosis globally. However, the sources of this serovar and its diversity and antimicrobial resistance capacities remain poorly described in many regions. To further understand the genetic diversity and antimicrobial sensitivity patterns among S. Kentucky strains isolated from non-human sources in Ireland, we sequenced and analysed the genomes of 61 isolates collected from avian, bovine, canine, ovine, piscine, porcine, environmental and vegetation sources between 2000 and 2016. The majority of isolates (n=57, 93%) were sequence type (ST) 314, while only three isolates were ST198 and one was ST152. Several isolates were multidrug-resistant (MDR) and 14 carried at least one acquired antimicrobial resistance gene. When compared to a database of publicly available ST314, four distinct clades were identified (clades I-IV), with the majority of isolates from Ireland clustering together in Clade I. Two of the three ST198 isolates were characteristic of those originating outside of the Americas (Clade ST198.2), while one was distantly clustered with isolates from South and North America (Clade ST198.1). The genomes of the two clade ST198.2 isolates encoded Salmonella Genomic Island 1 (SGI1), were multidrug-resistant and encoded polymorphisms in the DNA gyrase (gyrA) and DNA topoisomerase (parC) known to confer resistance to fluoroquinolones. The single ST152 isolate was from raw beef, clustered with isolates from food and bovine sources in North America and was pan-susceptible. Results of this study indicate that most S. Kentucky isolates from non-human sources in Ireland are closely related ST314 and only a few isolates are antimicrobial-resistant. This study also demonstrates the presence of multidrug-resistant ST198 in food sources in Ireland.

Genomic Analysis of Ciprofloxacin-Resistant Salmonella enterica Serovar Kentucky ST198 From Spanish Hospitals.

Salmonella enterica serovar Kentucky (S. Kentucky) with sequence type (ST) 198 and highly resistant to ciprofloxacin (ST198-CipR) has emerged as a global MDR clone, posing a threat to public health. In the present study, whole genome sequencing (WGS) was applied to characterize all CipR S. Kentucky detected in five Spanish hospitals during 2009–2018. All CipR isolates (n = 13) were ST198 and carried point mutations in the quinolone resistance-determining regions (QRDRs) of both gyrA (resulting in Ser83Phe and Asp87Gly, Asp87Asn, or Asp87Tyr substitutions in GyrA) and parC (with Thr57Ser and Ser80Ile substitutions in ParC). Resistances to other antibiotics (ampicillin, chloramphenicol, gentamicin, streptomycin, sulfonamides, and tetracycline), mediated by the blaTEM–1B, catA1, aacA5, aadA7, strA, strB, sul1, and tet(A) genes, and arranged in different combinations, were also observed. Analysis of the genetic environment of the latter resistance genes revealed the presence of multiple variants of SGI1 (Salmonella genomic island 1)-K and SGI1-P, where all these resistance genes except catA1 were placed. IS26 elements, found at multiple locations within the SGI1 variants, have probably played a crucial role in their generation. Despite the wide diversity of SGI1-K- and SGI1-P-like structures, phylogenetic analysis revealed a close relationship between isolates from different hospitals, which were separated by a minimum of two and a maximum of 160 single nucleotide polymorphisms. Considering that S. enterica isolates resistant to fluoroquinolones belong to the high priority list of antibiotic-resistant bacteria compiled by the World Health Organization, continuous surveillance of the S. Kentucky ST198-CIPR clone is required.

Genomic islands targeting dusA in Vibrio species are distantly related to Salmonella Genomic Island 1 and mobilizable by IncC conjugative plasmids.

Salmonella Genomic Island 1 (SGI1) and its variants are significant contributors to the spread of antibiotic resistance among Gammaproteobacteria. All known SGI1 variants integrate at the 3' end of trmE, a gene coding for a tRNA modification enzyme. SGI1 variants are mobilized specifically by conjugative plasmids of the incompatibility groups A and C (IncA and IncC). Using a comparative genomics approach based on genes conserved among members of the SGI1 group, we identified diverse integrative elements distantly related to SGI1 in several species of Vibrio, Aeromonas, Salmonella, Pokkaliibacter, and Escherichia. Unlike SGI1, these elements target two alternative chromosomal loci, the 5' end of dusA and the 3' end of yicC. Although they share many features with SGI1, they lack antibiotic resistance genes and carry alternative integration/excision modules. Functional characterization of IMEVchUSA3, a dusA-specific integrative element, revealed promoters that respond to AcaCD, the master activator of IncC plasmid transfer genes. Quantitative PCR and mating assays confirmed that IMEVchUSA3 excises from the chromosome and is mobilized by an IncC helper plasmid from Vibrio cholerae to Escherichia coli. IMEVchUSA3 encodes the AcaC homolog SgaC that associates with AcaD to form a hybrid activator complex AcaD/SgaC essential for its excision and mobilization. We identified the dusA-specific recombination directionality factor RdfN required for the integrase-mediated excision of dusA-specific elements from the chromosome. Like xis in SGI1, rdfN is under the control of an AcaCD-responsive promoter. Although the integration of IMEVchUSA3 disrupts dusA, it provides a new promoter sequence and restores the reading frame of dusA for proper expression of the tRNA-dihydrouridine synthase A. Phylogenetic analysis of the conserved proteins encoded by SGI1-like elements targeting dusA, yicC, and trmE gives a fresh perspective on the possible origin of SGI1 and its variants.

Analysis on epidemiological characteristics of multidrug-resistant Salmonella Kentucky in Beijing, 2010-2020

Objective: To investigate the epidemiological, drug resistance and molecular characteristics of Salmonella (S.) Kentucky strains isolated from diarrheal patients in Beijing. Methods: The drug susceptibility of 22 S. Kentucky strains isolated in Beijing during 2010-2020 was tested by using the micro broth dilution method. The multilocus sequence typing (MLST), drug resistance genes and Salmonella genomic island (SGI) identifications of the strains were performed by whole-genome sequencing (WGS).The pulsed field gel electrophoresis (PFGE) was performed to analyze the molecular epidemiological characteristics of the isolates. Results: The 22 strains were highly resistant to 8-22 kinds of antibiotics, especially to ciprofloxacin, cephalosporins and azithromycin, etc., showing a super high level of multidrug resistance and 21 strains were positive for extended-spetrum beta-lactamase (ESBL). The WGS analysis revealed that all the isolates belonged to ST198, carrying SGI1-K. The drug resistance genes tetA, sul1 and qacE were identified in all strains and Quinolone resistance-determining regions (QRDRs) showed 2 mutations in gyrA (S83F, D87 N) and 3 mutations in the parC gene (T57S, S80I, T255S). The resistance genes associated with β-lactam antibiotics (blaCTX-M-55, blaCTX-M-14b, blaTEM-141, blaTEM-206, blaTEM-209, blaTEM-214, blaTEM-1B), resistance genes associated with aminoglycosides [aac(3)-Id, aac(3)-IId, aac(6')-Iaa, aadA7, aadA17, aph(3')-Ia, aph(3'')-Ib, aph(6)-Id,rmtB] as well as floR, dfrA14, mphA and qnrS1 had significant differences in the strains of different years, which were highly consistent with the drug-resistant phenotype. Furthermore, phylogenetic analysis showed that the similarity of the 22 strains was more than 85%, and the strains were highly homologous to CIP-resistant ST198-X1 circulating in the world. In the process of spread, the drug resistance and PFGE spectrums had changed, two clusters had formed. Conclusions: The S. Kentucky strains isolated in Beijing shared high homology with the multidrug-resistant strain ST198-X1-SGI-1K isolated in the world, which has maintained a low level of spread since 2016, causing sporadic infections and clusters of diarrhea, and has serious resistances to fluoroquinolones, ESBLs and azithromycin. The surveillance for multidrug-resistant S. Kentucky should be strengthened.

Antimicrobial Resistance and Genomic Characterization of Two mcr-1-Harboring Foodborne Salmonella Isolates Recovered in China, 2016

The mcr-1 gene mediating mobile colistin resistance in Escherichia coli was first reported in China in 2016 followed by reports among different species worldwide, especially in E. coli and Klebsiella. However, data on its transmission in Salmonella are still lacking. This study analyzed the antimicrobial resistance (AMR) profiles and the mcr-1 gene presence in 755 foodborne Salmonella from 26 provinces of mainland, China in 2016. Genomic features of two mcr-1-carrying isolates, genome sequencing, serotypes and further resistance profiles were studied. Among the 755 Salmonella tested, 72.6% were found to be resistant to at least one antimicrobial agent and 10% were defined as multi-drug resistant (MDR). Salmonella Derby CFSA231 and Salmonella Typhimurium CFSA629 were mcr-1-harboring isolates. Both expressed an MDR phenotype and included a single circular chromosome and one plasmid. Among the 22 AMR genes identified in S. Derby CFSA231, only the mcr-1 gene was localized on the IncX4 type plasmid pCFSA231 while 20 chromosomal AMR genes, including four plasmid-mediated quinolone resistance (PMQR) genes, were mapped within a 64 kb Salmonella genomic island (SGI) like region. S. Typhimurium CFSA629 possessed 11 resistance genes including an mcr-1.19 variant and two ESBL genes. Two IS26-flanked composite-like transposons were identified. Additionally, 153 and 152 virulence factors were separately identified in these two isolates with secretion system and fimbrial adherence determinants as the dominant virulence classes. Our study extends our concern on mcr-1-carrying Salmonella in regards to antimicrobial resistance and virulence factors, and highlight the importance of surveillance to mitigate dissemination of mcr-encoding genes among foodborne Salmonella.

The Spatiotemporal Dynamics and Microevolution Events That Favored the Success of the Highly Clonal Multidrug-Resistant Monophasic Salmonella Typhimurium Circulating in Europe.

The European epidemic monophasic variant of Salmonella enterica serovar Typhimurium (S. 1,4,[5],12:i:-) characterized by the multi locus sequence type ST34 and the antimicrobial resistance ASSuT profile has become one of the most common serovars in Europe (EU) and the United States (US). In this study, we reconstructed the time-scaled phylogeny and evolution of this Salmonella in Europe. The epidemic S. 1,4,[5],12:i:- ST34 emerged in the 1980s by an acquisition of the Salmonella Genomic Island (SGI)-4 at the 3′ end of the phenylalanine phe tRNA locus conferring resistance to copper and arsenic toxicity. Subsequent integration of the Tn21 transposon into the fljAB locus gave resistance to mercury toxicity and several classes of antibiotics used in food-producing animals (ASSuT profile). The second step of the evolution occurred in the 1990s, with the integration of mTmV and mTmV-like prophages carrying the perC and/or sopE genes involved in the ability to reduce nitrates in intestinal contents and facilitate the disruption of the junctions of the host intestinal epithelial cells. Heavy metals are largely used as food supplements or pesticide for cultivation of seeds intended for animal feed so the expansion of the epidemic S. 1,4,[5],12:i:- ST34 was strongly related to the multiple-heavy metal resistance acquired by transposons, integrative and conjugative elements and facilitated by the escape until 2011 from the regulatory actions applied in the control of S. Typhimurium in Europe. The genomic plasticity of the epidemic S. 1,4,[5],12:i:- was demonstrated in our study by the analysis of the plasmidome. We were able to identify plasmids harboring genes mediating resistance to phenicols, colistin, and fluoroquinolone and also describe for the first time in six of the analyzed genomes the presence of two plasmids (pERR1744967-1 and pERR2174855-2) previously described only in strains of enterotoxigenic Escherichia coli and E. fergusonii.

Characterization of the emerging multidrug-resistant Salmonella enterica serotype Kentucky ST314 in China.

Salmonella enterica serotype Kentucky (S. Kentucky) is an important Salmonella serotype with multiple sequence types (ST) with a worldwide incidence. We identified 8 STs from 180 strains of S. Kentucky, and ST314 emerged as the most commonly encountered ST. Drug susceptibility testing revealed that ST314 had multiple resistance properties, and 75.5% of the strains were resistant to three or more classes of antimicrobials. The rate of resistance to chloramphenicol, florfenicol, sulfafurazole and tetracycline were greater than 60%. The rates of ST314 resistance to quinolones were as follows: ciprofloxacin, 32.1%; nalidixic acid, 16%; and ofloxacin, 7.5%. Investigating the mechanism of quinolone resistance of ST314 revealed that mutations in the quinolone resistance-determining regions were rare, and resistance mainly occurred due to the resistance genes carried by plasmids. Only 1.9% (2/106) of ST314 strains had mutations in the quinolone resistance-determining regions (QRDR). The drug resistance genes of ST314 were primarily of plasmid-mediated quinolone resistance (PMQR). The detection rate of Salmonella genomic island 1 (SGI1) in ST314 was 12.3%. XbaI-pulsed-field gel electrophoresis revealed that S.enterica Kentucky ST314 was capable of cross-regional and cross-host transmission in China. We found ST314 to be the dominant S. Kentucky ST in China, and it carried multidrug resistance. This is the first report about the emergence of quinolone-resistant S.enterica Kentucky ST314 in China, which is different from previous reports, and the findings of the present study suggest that the mechanism of quinolone resistance in these strains are plasmid-mediated. Notably, plasmids carrying resistance genes may promote the rapid spread of ciprofloxacin resistance.

Crucial role of Salmonella genomic island 1 master activator in the parasitism of IncC plasmids.

IncC conjugative plasmids and the multiple variants of Salmonella Genomic Island 1 (SGI1) are two functionally interacting families of mobile genetic elements commonly associated with multidrug resistance in the Gammaproteobacteria. SGI1 and its siblings are specifically mobilised in trans by IncC conjugative plasmids. Conjugative transfer of IncC plasmids is activated by the plasmid-encoded master activator AcaCD. SGI1 carries five AcaCD-responsive promoters that drive the expression of genes involved in its excision, replication, and mobilisation. SGI1 encodes an AcaCD homologue, the transcriptional activator complex SgaCD (also known as FlhDCSGI1) that seems to recognise and activate the same SGI1 promoters. Here, we investigated the relevance of SgaCD in SGI1′s lifecycle. Mating assays revealed the requirement for SgaCD and its IncC-encoded counterpart AcaCD in the mobilisation of SGI1. An integrative approach combining ChIP-exo, Cappable-seq, and RNA-seq confirmed that SgaCD activates each of the 18 AcaCD-responsive promoters driving the expression of the plasmid transfer functions. A comprehensive analysis of the activity of the complete set of AcaCD-responsive promoters of SGI1 and the helper IncC plasmid was performed through reporter assays. qPCR and flow cytometry assays revealed that SgaCD is essential to elicit the excision and replication of SGI1 and destabilise the helper IncC plasmid.

IncC helper dependent plasmid-like replication of Salmonella Genomic Island 1.

The Salmonella genomic island 1 (SGI1) and its variants are mobilized by IncA and IncC conjugative plasmids. SGI1-family elements and their helper plasmids are effective transporters of multidrug resistance determinants. SGI1 exploits the transfer apparatus of the helper plasmid and hijacks its activator complex, AcaCD, to trigger the expression of several SGI1 genes. In this way, SGI1 times its excision from the chromosome to the helper entry and expresses mating pore components that enhance SGI1 transfer. The SGI1-encoded T4SS components and the FlhDC-family activator proved to be interchangeable with their IncC-encoded homologs, indicating multiple interactions between SGI1 and its helpers. As a new aspect of this crosstalk, we report here the helper-induced replication of SGI1, which requires both activators, AcaCD and FlhDCSGI1, and significantly increases the stability of SGI1 when coexists with the helper plasmid. We have identified the oriVSGI1 and shown that S004-repA operon encodes for a translationally coupled leader protein and an IncN2/N3-related RepA that are expressed under the control of the AcaCD-responsive promoter PS004. This replicon transiently maintains SGI1 as a 4–8-copy plasmid, not only stabilizing the island but also contributing to the fast displacement of the helper plasmid.

Whole-genome characterisation of TEM-1 and CMY-2 β-lactamase-producing Salmonella Kentucky ST198 in Lebanese broiler chain

ObjectivesSalmonella enterica subsp. enterica serovar Kentucky has been associated with the worldwide ciprofloxacin-resistant (CIPR) Salmonella Kentucky sequence type 198 (ST198) epidemic clone, mostly recovered from poultry farms and products. The aim of this study was to examine whether this expanding clone exists in the Lebanese broiler chain. MethodsEight CIPR and extended-spectrum cephalosporin-resistant Salmonella Kentucky isolates previously recovered from Lebanese broilers were genetically characterised by whole-genome sequencing. ResultsSeven of the eight isolates belonged to ST198 and were phylogenetically closely related. They all harboured mutations in the chromosomal quinolone resistance genesgyrA and parC with double and single substitutions, respectively. The blaTEM-1B and blaCMY-2 genes were both detected in six isolates. Insertion sequence ISEcp1 was located upstream of blaCMY-2, harboured by IncI1 plasmids in four strains. An IS10 transposition coupled to homologous recombination at transposition sites mediated CMY-2 plasmid integration into the chromosome of one strain. Resistance genes to aminoglycosides [aadA7 and aac(3)-Id], tetracyclines [tet(A)] and sulfonamides (sul1) were detected in five strains, among which four were positive for the presence of Salmonella genomic island 1 (SGI1) variant SGI1-K. All studied isolates harboured a variety of Salmonella pathogenicity islands (SPIs) as well as common regulatory and virulence genes. ConclusionHere we report for the first time in Lebanon the detection and dissemination of the emerging highly drug-resistantSalmonella Kentucky ST198. Our findings shed new light on this clone as a potential public-health threat.

Replication of theSalmonella Genomic Island 1 (SGI1) triggered by helper IncC conjugative plasmids promotes incompatibility and plasmid loss.

The mobilizable resistance island Salmonella genomic island 1 (SGI1) is specifically mobilized by IncA and IncC conjugative plasmids. SGI1, its variants and IncC plasmids propagate multidrug resistance in pathogenic enterobacteria such as Salmonella enterica serovars and Proteus mirabilis. SGI1 modifies and uses the conjugation apparatus encoded by the helper IncC plasmid, thus enhancing its own propagation. Remarkably, although SGI1 needs a coresident IncC plasmid to excise from the chromosome and transfer to a new host, these elements have been reported to be incompatible. Here, the stability of SGI1 and its helper IncC plasmid, each expressing a different fluorescent reporter protein, was monitored using fluorescence-activated cell sorting (FACS). Without selective pressure, 95% of the cells segregated into two subpopulations containing either SGI1 or the helper plasmid. Furthermore, FACS analysis revealed a high level of SGI1-specific fluorescence in IncC+ cells, suggesting that SGI1 undergoes active replication in the presence of the helper plasmid. SGI1 replication was confirmed by quantitative PCR assays, and extraction and restriction of its plasmid form. Deletion of genes involved in SGI1 excision from the chromosome allowed a stable coexistence of SGI1 with its helper plasmid without selective pressure. In addition, deletion of S003 (rep) or of a downstream putative iteron-based origin of replication, while allowing SGI1 excision, abolished its replication, alleviated the incompatibility with the helper plasmid and enabled its cotransfer to a new host. Like SGI1 excision functions, rep expression was found to be controlled by AcaCD, the master activator of IncC plasmid transfer. Transient SGI1 replication seems to be a key feature of the life cycle of this family of genomic islands. Sequence database analysis revealed that SGI1 variants encode either a replication initiator protein with a RepA_C domain, or an alternative replication protein with N-terminal replicase and primase C terminal 1 domains.

Two New SGI1-LK Variants Found in Proteus mirabilis and Evolution of the SGI1-HKL Group of Salmonella Genomic Islands.

Integrative mobilizable elements belonging to the SGI1-H, -K, and -L Salmonella genomic island 1 (SGI1) variant groups are distinguished by the presence of an alteration in the backbone (IS1359 replaces 2.8 kb of the backbone extending from within traN [S005] to within S009). Members of this SGI1-HKL group have been found in Salmonella enterica serovars and in Proteus mirabilis Two novel variants from this group, designated SGI1-LK1 and SGI1-LK2, were found in the draft genomes of antibiotic-resistant P. mirabilis isolates from two French hospitals. Both variants can be derived from SGI1-PmGUE, a configuration found previously in another P. mirabilis isolate from France. SGI1-LK1 could arise via an IS26-mediated inversion in the complex class 1 integron that duplicated the IS26 element and the target site in IS6100 SGI1-LK1 also has a larger 8.59-kb backbone deletion extending from traN to within S013 and removing traG and traH. However, SGI1-LK1 was mobilized by an IncC plasmid. SGI1-LK2 can be derived from a hypothetical progenitor, SGI1-LK0, that is related to SGI1-PmGUE but lacks the aphA1 gene and one copy of IS26. The integron of SGI1-LK2 could arise via deletion of DNA adjacent to an IS26 and a deletion occurring via homologous recombination between duplicated copies of part of the integron 3'-conserved segment. SGI1-K can also be derived from SGI1-LK0. This would involve an IS26-mediated deletion and an inversion via homologous recombination of a segment between inversely oriented IS26s. Similar events can explain the configuration of the integrons in other SGI1-LK variants.IMPORTANCE Members of the SGI1-HKL subgroup of SGI1-type integrative mobilizable elements have a characteristic alteration in their backbone. They are widely distributed among multiply antibiotic-resistant Salmonella enterica serovars and Proteus mirabilis isolates. The SGI1-K type, found in the globally disseminated multiply antibiotic-resistant Salmonella enterica serovar Kentucky clone ST198 (sequence type 198), and various configurations in the original SGI1-LK group, found in other multiresistant S. enterica serovars and Proteus mirabilis isolates, have complex and highly plastic resistance regions due to the presence of IS26 However, how these complex forms arose and the relationships between them had not been analyzed. Here, a hypothetical progenitor, SGI1-LK0, that can be formed from the simpler SGI1-H is proposed, and the pathways to the formation of new variants, SGI1-LK1 and SGI1-LK2, found in P. mirabilis and other reported configurations via homologous recombination and IS26-mediated events are proposed. This led to a better understanding of the evolution of the SGI1-HKL group.

Salmonella Genomic Island 1 is Broadly Disseminated within Gammaproteobacteriaceae.

Salmonella genomic island 1 (SGI1) is an integrative mobilisable element that plays an important role in the capture and spread of multiple drug resistance. To date, SGI1 has been found in clinical isolates of Salmonella enterica serovars, Proteus mirabilis, Morganella morganii, Acinetobacter baumannii, Providencia stuartii, Enterobacter spp, and recently in Escherichia coli. SGI1 preferentially targets the 3´-end of trmE, a conserved gene found in the Enterobacteriaceae and among members of the Gammaproteobacteria. It is, therefore, hypothesised that SGI1 and SGI1-related elements (SGI1-REs) may have been acquired by diverse bacterial genera. Here, Bitsliced Genomic Signature Indexes (BIGSI) was used to screen the NCBI Sequence Read Archive (SRA) for putative SGI1-REs in Gammaproteobacteria. Novel SGI-REs were identified in diverse genera including Cronobacter spp, Klebsiella spp, and Vibrio spp and in two additional isolates of Escherichia coli. An extensively drug-resistant human clonal lineage of Klebsiella pneumoniae carrying an SGI1-RE in the United Kingdom and an SGI1-RE that lacks a class 1 integron were also identified. These findings provide insight into the origins of this diverse family of clinically important genomic islands and expand the knowledge of the potential host range of SGI1-REs within the Gammaproteobacteria.

Ciprofloxacin-Resistant Salmonella enterica Serovar Kentucky ST198 in Broiler Chicken Supply Chain and Patients, China, 2010-2016.

Salmonella enterica serovar Kentucky (S. Kentucky) sequence type 198 has emerged as a global zoonotic pathogen. We explored Salmonella enterica serovar Kentucky ST198 samples from the broiler chicken supply chain and patients between 2010 and 2016. Here, we collected 180 S. Kentucky isolates from clinical cases and the poultry supply chain. We performed XbaI pulsed-field gel electrophoresis and multilocus sequence typing. We assessed mutations in the quinolone resistance-determining regions and screened for the presence of the Salmonella genomic island 1 (SGI1). We determined that 63 (35.0%) of the 180 isolates were S. Kentucky ST198. Chinese strains of S. Kentucky ST198 have a high transmission of ciprofloxacin resistance (38/63, 60.3%) and a high risk of multidrug resistance. The quinolone resistance of the S. Kentucky ST198 strain found in China may be due to mutations in its quinolone resistance-determining region. Our study firstly revealed that ciprofloxacin-resistant S. Kentucky ST198 strains can undergo cross-host transmission, thereby causing a serious foodborne public health problem in China.