Resistance Island Research Articles

Geographic Variation in Dominance of Spinosad Resistance in Colorado Potato Beetles (Coleoptera: Chrysomelidae).

Eastern New York State is frequently the site of Colorado potato beetle (Leptinotarsa decemlineata, Say) populations with the highest observed levels of insecticide resistance to a range of active ingredients. The dominance of a resistant phenotype will affect its rate of increase and the potential for management. On organic farms on Long Island, L. decemlineata evolved high levels of resistance to spinosad in a short period of time and that resistance has spread across the eastern part of the Island. Resistance has also emerged in other parts of the country as well. To clarify the level of dominance or recessiveness of spinosad resistance in different parts of the United States and how resistance differs in separate beetle populations, we sampled in 2010 beetle populations from Maine, Michigan, and Long Island. In addition, a highly resistant Long Island population was assessed in 2012. All populations were hybridized with a laboratory-susceptible strain to determine dominance. None of the populations sampled in 2010 were significantly different from additive resistance, but the Long Island population sampled in 2012 was not significantly different from fully recessive. Recessive inheritance of high-level resistance may help manage its increase.

Epigenomics, genomics, resistome, mobilome, virulome and evolutionary phylogenomics of carbapenem-resistant Klebsiella pneumoniae clinical strains.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) remains a major clinical pathogen and public health threat with few therapeutic options. The mobilome, resistome, methylome, virulome and phylogeography of CRKP in South Africa and globally were characterized. CRKP collected in 2018 were subjected to antimicrobial susceptibility testing, screening by multiplex PCR, genotyping by repetitive element palindromic (REP)-PCR, plasmid size, number, incompatibility and mobility analyses, and PacBio’s SMRT sequencing (n=6). There were 56 multidrug-resistant CRKP, having bla OXA-48-like and bla NDM-1/7 carbapenemases on self-transmissible IncF, A/C, IncL/M and IncX3 plasmids endowed with prophages, traT, resistance islands, and type I and II restriction modification systems (RMS). Plasmids and clades detected in this study were respectively related to globally established/disseminated plasmids clades/clones, evincing transboundary horizontal and vertical dissemination. Reduced susceptibility to colistin occurred in 23 strains. Common clones included ST307, ST607, ST17, ST39 and ST3559. IncFIIk virulent plasmid replicon was present in 56 strains. Whole-genome sequencing of six strains revealed least 41 virulence genes, extensive ompK36 mutations, and four different K- and O-loci types: KL2, KL25, KL27, KL102, O1, O2, O4 and O5. Types I, II and III RMS, conferring m6A (GATC, GATGNNNNNNTTG, CAANNNNNNCATC motifs) and m4C (CCWGG) modifications on chromosomes and plasmids, were found. The nature of plasmid-mediated, clonal and multi-clonal dissemination of blaOXA-48-like and blaNDM-1 mirrors epidemiological trends observed for closely related plasmids and sequence types internationally. Worryingly, the presence of both bla OXA-48 and bla NDM-1 in the same isolates was observed. Plasmid-mediated transmission of RMS, virulome and prophages influence bacterial evolution, epidemiology, pathogenicity and resistance, threatening infection treatment. The influence of RMS on antimicrobial and bacteriophage therapy needs urgent investigation.

Characterization of IncC Plasmids in Enterobacterales of Food-Producing Animals Originating From China.

Incompatibility group C (IncC) plasmids have received attention due to their broad host range and because they harbor key antibiotic resistance genes. Because these resistance genes can spread from food-producing animals to human, the proliferation of these plasmids represents a public health risk. In this study, a total of 20 IncC plasmids were collected from food-producing animals in China, and characterized by Oxford Nanopore Technologies long-read sequencing. Based on four key differences of the IncC backbone, 4 IncC plasmids were classified as type 1, 15 were classified as type 1/2 hybrid, and one was classified as type 2. The 15 type 1/2 hybrids were further divided into 13 type 1/2a and 2 type 1/2b, based on sequence differences arising from different homologous recombination events between type 1 and type 2 IncC backbones. Genome comparison of accessory resistance modules showed that different IncC plasmids exhibited various phenotypes via loss and gain of diverse modules, mainly within the blaCMY-carrying region, and two antibiotic resistance islands designated ARI-A and ARI-B. Interestingly, in addition to insertion and deletion events, IS26 or IS1294-mediated large sequence inversions were found in the IncC genome of the 4 type1/2a plasmids, suggesting that insertion sequence-mediated rearrangements also promote the diversity of the IncC genome. This study provides insight into the structural diversification and multidrug resistance of IncC plasmids identified from food-producing animals in China.

Acquisition of a genomic resistance island (AbGRI5) from global clone 2 through homologous recombination in a clinical Acinetobacter baumannii isolate.

To reconstruct the evolutionary history of the clinical Acinetobacter baumannii XH1056, which lacks the Oxford scheme allele gdhB. Susceptibility testing was performed using broth microdilution and agar dilution. The whole-genome sequence of XH1056 was determined using the Illumina and Oxford Nanopore platforms. MLST was performed using the Pasteur scheme and the Oxford scheme. Antibiotic resistance genes were identified using ABRicate. XH1056 was resistant to all antibiotics tested, apart from colistin, tigecycline and eravacycline. MLST using the Pasteur scheme assigned XH1056 to ST256. However, XH1056 could not be typed with the Oxford MLST scheme as gdhB is not present. Comparative analyses revealed that XH1056 contains a 52 933 bp region acquired from a global clone 2 (GC2) isolate, but is otherwise closely related to the ST23 A. baumannii XH858. The acquired region in XH1056 also contains a 34 932 bp resistance island that resembles AbGRI3 and contains the armA, msrE-mphE, sul1, blaPER-1, aadA1, cmlA1, aadA2, blaCARB-2 and ere(B) resistance genes. Comparison of the XH1056 chromosome to that of GC2 isolate XH859 revealed that the island in XH1056 is in the same chromosomal region as that in XH859. As this island is not in the standard AbGRI3 position, it was named AbGRI5. XH1056 is a hybrid isolate generated by the acquisition of a chromosomal segment from a GC2 isolate that contains a resistance island in a new location-AbGRI5. As well as generating ST256, it appears likely that a single recombination event is also responsible for the acquisition of AbGRI5 and its associated antibiotic resistance genes.

Scarless Removal of Large Resistance Island AbaR Results in Antibiotic Susceptibility and Increased Natural Transformability in Acinetobacter baumannii.

With a great diversity in gene composition, including multiple putative antibiotic resistance genes, AbaR islands are potential contributors to multidrug resistance in Acinetobacter baumannii However, the effective contribution of AbaR to antibiotic resistance and bacterial physiology remains elusive. To address this, we sought to accurately remove AbaR islands and restore the integrity of their insertion site. To this end, we devised a versatile scarless genome editing strategy. We performed this genetic modification in two recent A. baumannii clinical strains: the strain AB5075 and the nosocomial strain AYE, which carry AbaR11 and AbaR1 islands of 19.7 kbp and 86.2 kbp, respectively. Antibiotic susceptibilities were then compared between the parental strains and their AbaR-cured derivatives. As anticipated by the predicted function of the open reading frame (ORF) of this island, the antibiotic resistance profiles were identical between the wild type and the AbaR11-cured AB5075 strains. In contrast, AbaR1 carries 25 ORFs, with predicted resistance to several classes of antibiotics, and the AYE AbaR1-cured derivative showed restored susceptibility to multiple classes of antibiotics. Moreover, curing of AbaRs restored high levels of natural transformability. Indeed, most AbaR islands are inserted into the comM gene involved in natural transformation. Our data indicate that AbaR insertion effectively inactivates comM and that the restored comM is functional. Curing of AbaR consistently resulted in highly transformable and therefore easily genetically tractable strains. Emendation of AbaR provides insight into the functional consequences of AbaR acquisition.

Isolation and genomic characterization of a pathogenic Providencia rettgeri strain G0519 in turtle Trachemys scripta.

Providencia rettgeri infection has occurred occasionally in aquaculture, but is rare in turtles. Here, a pathogenic P. rettgeri strain G0519 was isolated from a diseased slider turtle (Trachemys scripta) in China, and qPCR assay was established for the RTX toxin (rtxD) gene. Histopathological examination showed that many inflammatory cells were infiltrated into heart, liver and intestine, as well as the necrosis of liver, kidney and spleen. The genome consisted of one circular chromosome (4.493Mb) and one plasmid (18.8kb), and predicted to contain 4170 and 19 protein-coding genes, respectively. Multiple pathogenic and virulence factors (e.g., fimbria, adhesion, invasion, toxin, hemolysin, chemotaxis, secretion system), multidrug-resistant genes (e.g., ampC, per-1, oxa-1, sul1, tetR) and a novel genomic resistance island PRI519 were identified. Comparative genome analysis revealed the closest relationship waswith P. rettgeri, and with P. heimbachae closerthan with other Providencia spp. To our knowledge, this was first report on genomic characterization of multidrug-resistant pathogenic P. rettgeri in cultured turtles.

Antibiotic Resistance in Vibrio cholerae: Mechanistic Insights from IncC Plasmid-Mediated Dissemination of a Novel Family of Genomic Islands Inserted at trmE.

Cholera remains a formidable disease, and reports of multidrug-resistant strains of the causative agent Vibrio cholerae have become common during the last 3 decades. The pervasiveness of resistance determinants has largely been ascribed to mobile genetic elements, including SXT/R391 integrative conjugative elements, IncC plasmids, and genomic islands (GIs). Conjugative transfer of IncC plasmids is activated by the master activator AcaCD whose regulatory network extends to chromosomally integrated GIs. MGIVchHai6 is a multidrug resistance GI integrated at the 3' end of trmE (mnmE or thdF) in chromosome 1 of non-O1/non-O139 V. cholerae clinical isolates from the 2010 Haitian cholera outbreak. In the presence of an IncC plasmid expressing AcaCD, MGIVchHai6 excises from the chromosome and transfers at high frequency. Herein, the mechanism of mobilization of MGIVchHai6 GIs by IncC plasmids was dissected. Our results show that AcaCD drives expression of GI-borne genes, including xis and mobIM , involved in excision and mobilization. A 49-bp fragment upstream of mobIM was found to serve as the minimal origin of transfer (oriT) of MGIVchHai6. The direction of transfer initiated at oriT was determined using IncC plasmid-driven mobilization of chromosomal markers via MGIVchHai6. In addition, IncC plasmid-encoded factors, including the relaxase TraI, were found to be required for GI transfer. Finally, in silico exploration of Gammaproteobacteria genomes identified 47 novel related and potentially AcaCD-responsive GIs in 13 different genera. Despite sharing conserved features, these GIs integrate at trmE, yicC, or dusA and carry a diverse cargo of genes involved in phage resistance.IMPORTANCE The increasing association of the etiological agent of cholera, Vibrio cholerae serogroup O1 and O139, with multiple antibiotic resistance threatens to deprive health practitioners of this effective tool. Drug resistance in cholera results mainly from acquisition of mobile genetic elements. Genomic islands conferring multidrug resistance and mobilizable by IncC conjugative plasmids were reported to circulate in non-O1/non-O139 V. cholerae clinical strains isolated from the 2010 Haitian cholera outbreak. As these genomic islands can be transmitted to pandemic V. cholerae serogroups, their mechanism of transmission needed to be investigated. Our research revealed plasmid- and genomic island-encoded factors required for the resistance island excision, mobilization, and integration, as well as regulation of these functions. The discovery of related genomic islands carrying diverse phage resistance genes but lacking antibiotic resistance-conferring genes in a wide range of marine dwelling bacteria suggests that these elements are ancient and recently acquired drug resistance genes.

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.

AbGRI4, a novel antibiotic resistance island in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates.

ObjectivesTo investigate the genomic context of a novel resistance island (RI) in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates and global isolates.MethodsUsing a combination of long and short reads generated from the Oxford Nanopore and Illumina platforms, contiguous chromosomes and plasmid sequences were determined. BLAST-based analysis was used to identify the RI insertion target.ResultsGenomes of four multiply antibiotic-resistant A. baumannii clinical strains, from a US hospital system, belonging to prevalent MLST ST2 (Pasteur scheme) and ST281 (Oxford scheme) clade F isolates were sequenced to completion. A class 1 integron carrying aadB (tobramycin resistance) and aadA2 (streptomycin/spectinomycin resistance) was identified. The class 1 integron was 6.8 kb, bounded by IS26 at both ends, and embedded in a new target location between an α/β-hydrolase and a reductase. Due to its novel insertion site and unique RI composition, we suggest naming this novel RI AbGRI4. Molecular analysis of global A. baumannii isolates identified multiple AbGRI4 RI variants in non-ST2 clonal lineages, including variations in the resistance gene cassettes, integron backbone and insertion breakpoints at the hydrolase gene.ConclusionsA novel RI insertion target harbouring a class 1 integron was identified in a subgroup of ST2/ST281 clinical isolates. Variants of the RI suggested evolution and horizontal transfer of the RI across clonal lineages. Long- and short-read hybrid assembly technology completely resolved the genomic context of IS-bounded RIs, which was not possible using short reads alone.

Identification of Proteus genomic island 2 variants in two clonal Proteus mirabilis isolates with coexistence of a novel genomic resistance island PmGRI1

To characterize the MDR genomic islands (GIs) in Proteus mirabilis isolates. Two P. mirabilis strains (C55 and C74) of chicken origin were subjected to WGS (HiSeq and PacBio) and the MDR GIs were determined. P. mirabilis strains C55 and C74 are clonal strains and harbour different Proteus genomic island 2 (PGI2) variants (PGI2-C55 and PGI2-C74). The MDR region of PGI2-C55 is composed of two class 1 integrons, separated by a region containing seven copies of IS26 and eight resistance genes, including blaCTX-M-3 and fosA3. The region in PGI2-C74 is a complete In4-type class 1 integron, harbouring five gene cassettes (dfrA16, blaCARB-2, aadA2, cmlA1 and aadA1). In addition, C55 and C74 carry an SXT/R391 integrative and conjugative element (ICEPmiJpn1), harbouring blaCMY-2, and a novel 50.46 kb genomic resistance island named PmGRI1-C55. PmGRI1-C55 harbours a tyrosine-type recombinase/integrase that might be responsible for the integration of PmGRI1-C55 at the 3' end of tRNA-Sec. It carries an MDR region derived from Tn2670 that harbours a Tn21 region and carries six resistance genes (catA1, blaTEM-1b, aphA1a, sul2, strA and strB). Blast analysis showed diverse PmGRI1 variants in P. mirabilis and Escherichia coli strains. The finding of the two new PGI2 variants highlights that the homologous recombination between shared components of class 1 integrons and transposition by IS26 promote the diversity of MDR regions in PGI2. PmGRI1 is a new GI that carries various resistance genes identified in P. mirabilis and E. coli.

Inside environmental Clostridium perfringens genomes: antibiotic resistance genes, virulence factors and genomic features.

Until recently, research has focused on Clostridium perfringens in clinical settings without considering environmental isolates. In this study, environmental genomes were used to investigate possible antibiotic resistance and the presence of virulence traits in C. perfringens strains from raw surface water. In silico assembly of three C. perfringens strains, DNA generated almost complete genomes setting their length ranging from 3.4 to 3.6 Mbp with GC content of 28.18%. An average of 3,175 open reading frames was identified, with the majority associated with carbohydrate and protein metabolisms. The genomes harboured several antibiotic resistance genes for glycopeptides, macrolide-lincosamide-streptogramin B, β-lactam, trimethoprim, tetracycline and aminoglycosides and also the presence of several genes encoding for polypeptides and multidrug resistance efflux pumps and 35 virulence genes. Some of these encode for haemolysins, sialidase, hyaluronidase, collagenase, perfringolysin O and phospholipase C. All three genomes contained sequences indicating phage, antibiotic resistance and pathogenic islands integration sites. A genomic comparison of these three strains confirmed high similarity and shared core genes with clinical C. perfringens strains, highlighting their health security risks. This study provides a genomic insight into the potential pathogenicity of C. perfringens present in the environment and emphasises the importance of monitoring this niche in the future.

Mobile Genetic Elements Harboring Antibiotic Resistance Determinants in Acinetobacter baumannii Isolates From Bolivia.

Using a combination of short- and long-read DNA sequencing, we have investigated the location of antibiotic resistance genes and characterized mobile genetic elements (MGEs) in three clinical multi-drug resistant Acinetobacter baumannii. The isolates, collected in Bolivia, clustered separately with three different international clonal lineages. We found a diverse array of transposons, plasmids and resistance islands related to different insertion sequence (IS) elements, which were located in both the chromosome and in plasmids, which conferred resistance to multiple antimicrobials, including carbapenems. Carbapenem resistance might be caused by a Tn2008 carrying the blaOXA–23 gene. Some plasmids were shared between the isolates. Larger plasmids were less conserved than smaller ones and they shared some homologous regions, while others were more diverse, suggesting that these big plasmids are more plastic than the smaller ones. The genetic basis of antimicrobial resistance in Bolivia has not been deeply studied until now, and the mobilome of these A. baumannii isolates, combined with their multi-drug resistant phenotype, mirror the transfer and prevalence of MGEs contributing to the spread of antibiotic resistance worldwide and require special attention. These findings could be useful to understand the antimicrobial resistance genetics of A. baumannii in Bolivia and the difficulty in tackling these infections.

The Novel Macrolide Resistance Genes mef(D), msr(F), and msr(H) Are Present on Resistance Islands in Macrococcus canis, Macrococcus caseolyticus, and Staphylococcus aureus.

Chromosomal resistance islands containing the methicillin resistance gene mecD (McRI mecD ) have been reported in Macrococcus caseolyticus Here, we identified novel macrolide resistance genes in Macrococcus canis on similar elements, called McRI msr These elements were also integrated into the 3' end of the 30S ribosomal protein S9 gene (rpsI), delimited by characteristic attachment (att) sites, and carried a related site-specific integrase gene (int) at the 5' end. They carried novel macrolide resistance genes belonging to the msr family of ABC subfamily F (ABC-F)-type ribosomal protection protein [msr(F) and msr(H)] and the macrolide efflux mef family [mef(D)]. Highly related mef(D)-msr(F) fragments were found on diverse McRI msr elements in M. canis, M. caseolyticus, and Staphylococcus aureus Another McRI msr -like element identified in an M. canis strain lacked the classical att site at the 3' end and carried the msr(H) gene but no neighboring mef gene. The expression of the novel resistance genes in S. aureus resulted in a low-to-moderate increase in the MIC of erythromycin but not streptogramin B. In the mef(D)-msr(F) operon, the msr(F) gene was shown to be the crucial determinant for macrolide resistance. The detection of circular forms of McRI msr and the mef(D)-msr(F) fragment suggested mobility of both the island and the resistance gene subunit. The discovery of McRI msr in different Macrococcus species and S. aureus indicates that these islands have a potential for dissemination of antibiotic resistance within the Staphylococcaceae family.

Novel Mobilizable Genomic Island GEI-D18A Mediates Conjugational Transfer of Antibiotic Resistance Genes in the Multidrug-Resistant Strain Rheinheimera sp. D18.

Aquatic environments act as reservoirs of antimicrobial-resistant bacteria and antimicrobial resistance (AMR) genes, and the dissemination of antibiotic resistance from these environments is of increasing concern. In this study, a multidrug-resistant bacterial strain, identified as Rheinheimera sp. D18, was isolated from the sea water of an industrial maricultural system in the Yellow Sea, China. Whole-genome sequencing of D18 revealed the presence of a novel 25.8 kb antibiotic resistance island, designated GEI-D18A, which carries several antibiotic resistance genes (ARGs), including aadA1, aacA3, tetR, tet(B), catA, dfrA37, and three sul1 genes. Besides, integrase, transposase, resolvase, and recombinase encoding genes were also identified in GEI-D18A. The transferability of GEI-D18A was confirmed by mating experiments between Rheinheimera sp. D18 and Escherichia coli 25DN, and efflux pump inhibitor assays also suggested that tet(B) in GEI-D18A was responsible for tetracycline resistance in both D18 and the transconjugant. This study represents the first characterization of a mobilizable antibiotic resistance island in a species of Rheinheimera and provides evidence that Rheinheimera spp. could be important reservoirs and vehicles for ARGs in the Yellow Sea area.

Complete Genome and Plasmids Sequences of a Clinical Proteus mirabilis Isolate Producing Plasmid Mediated NDM-1 From Italy

Background: The spread of carbapenemase genes, such as blaNDM-1, in Proteus mirabilis poses a public health threat. The aim of the study was to characterize the genome and plasmids sequences of an NDM-1-positive strain (IBCRE14), which was isolated in 2019 from a catheterized patient hospitalized in Italy. Methods: Whole genome sequencing (WGS) of IBCRE14 was performed on extracted genomic DNA using Sequel I platform. Genome assembly was performed using “Microbial Assembly”. Genomic analysis was conducted by uploading the contigs to ResFinder and PlasmidFinder databases from the Center for Genomic Epidemiology. Results: IBCRE14 had a genome size of 4,018,329 bp and harboured genes coding for resistance to aminoglycosides (aadA1), phenicol (cat), tetracycline (tetJ), and trimethoprim (dfrA1). A large plasmid (pIB_NDM_1) harboured antibiotic resistance genes against sulphonamide (sul1), trimethoprim (dfrA14), tetracycline (tetB), rifampicin (arr-2), aminoglycosides (aadA1, aph3-VI), and beta-lactams (blaOXA-10, blaNDM-1). Furthermore, a small plasmid (pIB_COL3M) harboured a qnrD1 gene coding for quinolone resistance. Conclusion: The ability to conjugate and the presence of a composite antibiotic resistance island suggests that pIB_NDM_1 could both acquire more resistance genes and easily disseminate. To our knowledge, this is the first report on an untypable plasmid harbouring blaNDM-1 in P. mirabilis, in Italy.

Transcriptomic analysis of gene expression of Verticillium dahliae upon treatment of the cotton root exudates

BackgroundCotton Verticillium wilt is one of the most devastating diseases for cotton production in the world. Although this diseases have been widely studied at the molecular level from pathogens, the molecular basis of V. dahliae interacted with cotton has not been well examined.ResultsIn this study, RNA-seq analysis was carried out on V. dahliae samples cultured by different root exudates from three cotton cultivars (a susceptible upland cotton cultivar, a tolerant upland cotton cultivar and a resistant island cotton cultivar) and water for 0 h, 6 h, 12 h, 24 h and 48 h. Statistical analysis of differentially expressed genes revealed that V. dahliae responded to all kinds of root exudates but more strongly to susceptible cultivar than to tolerant and resistant cultivars. Go analysis indicated that ‘hydrolase activity, hydrolyzing O-glycosyl compounds’ related genes were highly enriched in V. dahliae cultured by root exudates from susceptible cotton at early stage of interaction, suggesting genes related to this term were closely related to the pathogenicity of V. dahliae. Additionally, ‘transmembrane transport’, ‘coenzyme binding’, ‘NADP binding’, ‘cofactor binding’, ‘oxidoreductase activity’, ‘flavin adenine dinucleotide binding’, ‘extracellular region’ were commonly enriched in V. dahliae cultured by all kinds of root exudates at early stage of interaction (6 h and 12 h), suggesting that genes related to these terms were required for the initial steps of the roots infections.ConclusionsBased on the GO analysis results, the early stage of interaction (6 h and 12 h) were considered as the critical stage of V. dahliae-cotton interaction. Comparative transcriptomic analysis detected that 31 candidate genes response to root exudates from cotton cultivars with different level of V. dahliae resistance, 68 response to only susceptible cotton cultivar, and 26 genes required for development of V. dahliae. Collectively, these expression data have advanced our understanding of key molecular events in the V. dahliae interacted with cotton, and provided a framework for further functional studies of candidate genes to develop better control strategies for the cotton wilt disease.

Complete Nucleotide Sequence of Plasmids of Two Escherichia coli Strains Carrying bla NDM- 5 and bla NDM - 5 and bla OXA - 181 From the Same Patient.

Aim of this study was to genetically characterize two carbapenemase-producing Escherichia coli strains obtained from a pediatric patient affected by diarrhea, expressing OXA-181 and/or NDM-5 type enzymes. The above microorganisms were collected in the same Desenzano hospital (Northern Italy) where the blaNDM–5 gene was detected for the first time in Italy 3 years ago. One strain (5P), belonged to sequence type ST405/ST477 (according to Pasture/Oxford schemes) and serotype O102:H6. It was characterized by a 130562 bp multi-replicon plasmid IncFII/IncFIA/IncFIB (pVSI_NDM-5) enclosing two main antibiotic resistance islands: (i) ARI-I, 10030 bp in size, carried genes coding for β-lactam- (blaOXA–1, blaCTX–M–15), fluoroquinolone/aminoglycoside- (aac(6′)-lb-cr) and phenicol- resistance (catB3), (ii) ARI-II, 15326 bp in size, carried genes coding for sulfonamide- (sul1), β-lactam- (blaNDM–5, blaTEM–1B), phenicol- (catB3), trimethoprim- (dfrA17), antiseptic- (qacEΔ1), and aminoglycoside- (aadA5, rmtB) resistance. The other isolate (5M), belonged to sequence type ST2659/ST759 and serotype O50/02:H18, and carried four plasmids: a 153866 bp multi-replicon IncFII/IncFIA/IncFIB (pISV_IncFII_NDM-5), an 89866 bp IncI1 plasmid, a 51480 bp IncX3 plasmid (pISV_IncX3_OXA181), and a 41143 bp IncI plasmid (pISV_IncI_CMY-42). pISV_IncFII_NDM-5 carried two main antibiotic resistance islands: (i) ARI-III, 12220 bp in size, carried genes coding for β-lactam- (blaOXA–1), fluoroquinolone/aminoglycoside- (aac(6′)-lb-cr), tetracycline- (tet(B)) and phenicol- resistance (catB3, catA1), and ii) ARI-IV, 26527 bp in size, carried determinants coding for macrolide- (erm(B), mph(A)), sulfonamide- (sul1), beta-lactam- (blaNDM–5, blaTEM–1B), trimethoprim- (dfrA14, dfrA12), antiseptic- (qacEΔ1), and aminoglycoside- resistance (aadA5). pISV_IncI_CMY-42 harbored the blaCMY–42 gene coding for beta-lactam resistance, pISV_IncX3_OXA181 harbored genes encoding fluoroquinolone- (qnrS1) and beta-lactams- resistance (blaOXA–181). In conclusion, the detection of two different NDM-5 E. coli strains from a pediatric patient with a history of travel to the Far East countries strongly highlight an increasing trend and risk of importation from such areas.

Molecular characterization of blaKHM-1 encoding plasmid in an Enterobacter hormaechei subsp. hoffmannii isolate from blood culture.

KHM-1 was first reported in 1997 in Japan as a novel metallo-β-lactamase mediated by Citrobacter freundii carrying pKHM-1 plasmid. There have been few reports in the clinical field since then. A blaKHM-1–positive Enterobacter hormaechei subsp. hoffmannii in E. cloacae complex, isolate OIPH-N069 was isolated from an inpatient blood culture in 2016. The isolate was characterized by whole-genome sequencing, comparative analysis of the blaKHM-1 encoding plasmid, antimicrobial susceptibility tests, and bacterial conjugation. OIPH-N069 was classified into ST78 of E. cloacae complex, and was multidrug resistant because of the presence of antimicrobial resistance genes in addition to blaKHM-1 on its chromosome and plasmids. blaKHM-1 was located on 136,816 bp of the IncA/C2 plasmid pN069-1, which could be transferred to different bacterial species. The backbone structure, genetic arrangement of the class 1 integron cassette, and the blaKHM-1 gene located downstream of the IncA/C2 antibiotic resistance island, ARI-A, in pN069-1 and pKHM-1 were identical. Horizontal gene transfer of the blaCTX-M-2–ISEcp1 resistance gene module only occurred with pN069-1. The study findings indicate not only the structural conservation of blaKHM-1 encoding plasmids over time and across species, but also the risk of the spread of blaKHM-1 encoding plasmids to other bacterial species and the accumulation of additional resistance genes.

Identification of novel tetracycline resistance gene tet(X14) and its co-occurrence with tet(X2) in a tigecycline-resistant and colistin-resistant Empedobacter stercoris

ABSTRACT Tigecycline is one of the last-resort antibiotics to treat severe infections. Recently, tigecycline resistance has sporadically emerged with an increasing trend, and Tet(X) family represents a new resistance mechanism of tigecycline. In this study, a novel chromosome-encoded tigecycline resistance gene, tet(X14), was identified in a tigecycline-resistant and colistin-resistant Empedobacter stercoris strain ES183 recovered from a pig fecal sample in China. Tet(X14) shows 67.14-96.39% sequence identity to the other variants [Tet(X) to Tet(X13)]. Overexpression of Tet(X14) in Escherichia coli confers 16-fold increase in tigecycline MIC (from 0.125 to 2 mg/L), which is lower than that of Tet(X3), Tet(X4) and Tet(X6). Structural modelling predicted that Tet(X14) shared a high homology with the other 12 variants with RMSD value from 0.003 to 0.055, and Tet(X14) can interact with tetracyclines by a similar pattern as the other Tet(X)s. tet(X14) and two copies of tet(X2) were identified on a genome island with abnormal GC content carried by the chromosome of ES183, and no mobile genetic elements were found surrounding, suggesting that tet(X14) might be heterologously obtained by ES183 via recombination. Blasting in Genbank revealed that Tet(X14) was exclusively detected on the chromosome of Riemerella anatipestifer, mainly encoded on antimicrobial resistance islands. E. stercoris and R. anatipestifer belong to the family Flavobacteriaceae, suggesting that the members of Flavobacteriaceae maybe the major reservoir of tet(X14). Our study reports a novel chromosome-encoded tigecycline resistance gene tet(X14). The expanded members of Tet(X) family warrants the potential large-scale dissemination and the necessity of continuous surveillance for tet(X)-mediated tigecycline resistance.

Multidrug resistance genes are associated with a 42-kb island TGI1 carrying a complex class 1 integron in Trueperella pyogenes

ObjectivesThis research was conducted to ascertain the context and location of the antibiotic resistance determinants in a multiple antibiotic-resistant Trueperella pyogenes isolate TP1. MethodsThe genome was sequenced using PacBio RS II, and the filtered data were assembled using Canu. Sequences were annotated on the basis of those in GenBank, and the genomic island (GI) of the TP1 was predicted by IslandPath-DIMOB. ResultsTP1 as a multiple antibiotic-resistant isolate was recovered at Jilin Province (China) in 2017 from a dairy cow with pneumonia. TP1 exhibited resistance to aminoglycosides (gentamicin and amikacin), macrolides (erythromycin), lincosamides (clindamycin), sulfonamides (sulfamonomethoxine), tetracyclines (tetracycline and doxycycline) and chloramphenicols (chloramphenicol and florfenicol). An antibiotic resistance gene clustered together with the aadB, aadA1, cmlA5 and cmlA6 resistance genes located on a 7-kilobase (kb) multidrug-resistant (MDR) region, constituting a complex class 1 integron. The MDR region was located at one end of a 42-kb GI, and IS6100Δ1 mediated a genetic rearrangement with the complex class 1 integron-like SGI1 and formed a composite transposon. Furthermore, the tetW gene was located outside the four GIs consistent with tetracycline and doxycycline resistance. The ermD gene positioned in the front end of the 42-kb GI played an important role in mediating acquired erythromycin and clindamycin resistance. ConclusionsMultiple resistance genes are located in a complex class 1 integron within a 42-kb T. pyogenes genomic island (TGI1), leading to TP1 multiple drug resistance. In comparison with SG1 families, TGI1 possesses versatile gene distribution and specific gene context for it upstream and downstream, and it represents a new lineage of genomic resistance islands.