Resistance Gene Cluster Research Articles

Emergence of a floR-carrying plasmid in extended spectrum β-lactamase (ESBL) producing Pasteurella aerogenes, isolated from an avian species in China

Identification and analysis of the antimicrobial resistance of Pasteurella aerogenes (P. aerogenes) isolated from poultry. For susceptibility testing in accordance with the CLSI, plasmids were extracted via alkaline lysis and transferred by CaCl2 treatment. Genomic DNA of a representative P. aerogenes isolate was subjected to whole genome sequencing. CCCP was utilized to determine whether SF190908 contains an efflux pump. The blaVEB gene was ligated with the pET-28 plasmid and transferred to Escherichia coli to verify it as an ESBL gene. SF190908 isolated from poultry was identified as P. aerogenes based upon biochemical and 16s rRNA results. The isolate showed high MIC values for eight antimicrobials. Sequencing results showed that the mobile element-mediated antimicrobial resistance gene cluster conferred antimicrobial resistance on the strain, and a single 5,105-bp plasmid, designated pRCAD0752PA-1, was isolated. Four antimicrobial resistance gene clusters were identified in the SF190908 chromosome; one antimicrobial resistance gene cluster carried the blaVEB gene, which was verified as ESBL according to the CLSI and was detected in Pasteurellaceae for the first time, to the best of our knowledge. The efflux pump may confer antimicrobial resistance to SF190908. P. aerogenes isolated from poultry showed resistance genes encoded in mobile elements that confer multi-antimicrobial resistance to SF190908. The antimicrobial-resistant plasmid pRCAD0752PA-1 was isolated in SF190908 and conferred resistance to florfenicol. This study indicates an urgent need to increase efforts to monitor the spread of P. aerogenes multi-antimicrobial-resistant strains and plasmids, especially in newly discovered at-risk species such as poultry.

Abundant antibiotic resistance genes in rhizobiome of the human edible Moringa oleifera medicinal plant.

Moringa oleifera (or the miracle tree) is a wild plant species widely grown for its seed pods and leaves, and is used in traditional herbal medicine. The metagenomic whole genome shotgun sequencing (mWGS) approach was used to characterize antibiotic resistance genes (ARGs) of the rhizobiomes of this wild plant and surrounding bulk soil microbiomes and to figure out the chance and consequences for highly abundant ARGs, e.g., mtrA, golS, soxR, oleC, novA, kdpE, vanRO, parY, and rbpA, to horizontally transfer to human gut pathogens via mobile genetic elements (MGEs). The results indicated that abundance of these ARGs, except for golS, was higher in rhizosphere of M. oleifera than that in bulk soil microbiome with no signs of emerging new soil ARGs in either soil type. The most highly abundant metabolic processes of the most abundant ARGs were previously detected in members of phyla Actinobacteria, Proteobacteria, Acidobacteria, Chloroflexi, and Firmicutes. These processes refer to three resistance mechanisms namely antibiotic efflux pump, antibiotic target alteration and antibiotic target protection. Antibiotic efflux mechanism included resistance-nodulation-cell division (RND), ATP-binding cassette (ABC), and major facilitator superfamily (MFS) antibiotics pumps as well as the two-component regulatory kdpDE system. Antibiotic target alteration included glycopeptide resistance gene cluster (vanRO), aminocoumarin resistance parY, and aminocoumarin self-resistance parY. While, antibiotic target protection mechanism included RbpA bacterial RNA polymerase (rpoB)-binding protein. The study supports the claim of the possible horizontal transfer of these ARGs to human gut and emergence of new multidrug resistant clinical isolates. Thus, careful agricultural practices are required especially for plants used in circles of human nutrition industry or in traditional medicine.

Antibiotic and metal resistance of Stenotrophomonas maltophilia isolates from Eboling permafrost of the Tibetan Plateau.

Whole-genome sequencing of pathogenic bacteria Stenotrophomonas maltophilia from a less polluted environment of permafrost can help understand the intrinsic resistome of both antibiotics and metals. This study aimed to examine the maximum minimum inhibitory concentration (MIC) of both antibiotics and metals, as well as antibiotic resistance genes and metal resistance genes annotated from whole-genome sequences. The permafrost S. maltophilia was sensitive to ciprofloxacin, tetracycline, streptomycin, and bacitracin, and resistant to chloramphenicol, trimethoprim-sulfamethoxazole, erythromycin, Zn2+, Ni2+, Cu2+, and Cr6+, with a lower maximum MIC, compared with clinical S. maltophilia. The former strain belonged to the lower antibiotic resistance gene (ARG) and metal resistance gene (MRG) clusters compared with the latter ones. The permafrost strain contained no or only one kind of ARG or MRG on a single genomic island, which explained the aforementioned lower maximum MIC and less diversity of ARGs or MRGs. The result indicated that the co-occurrence of antibiotic and metal resistance was due to a certain innate ability of S. maltophilia. The continuous human use of antibiotics or metals induced selective pressure, resulting in higher MIC and more diverse ARGs and MRGs in human-impacted environments.

A reference-anchored oat linkage map reveals quantitative trait loci conferring adult plant resistance to crown rust (Puccinia coronata f. sp. avenae).

We mapped three adult plant resistance (APR) loci on oat chromosomes 4D and 6C and developed flanking KASP/PACE markers for marker-assisted selection and gene pyramiding. Using sequence orthology search and the available oat genomic and transcriptomic data, we surveyed these genomic regions for genes that may control disease resistance. Sources of durable disease resistance are needed to minimize yield losses in cultivated oat caused by crown rust (Puccinia coronata f. sp. avenae). In this study, we developed five oat recombinant inbred line mapping populations to identify sources of adult plant resistance from crosses between five APR donors and Otana, a susceptible variety. The preliminary bulk segregant mapping based on allele frequencies showed two regions in linkage group Mrg21 (Chr4D) that are associated with the APR phenotype in all five populations. Six markers from these regions in Chr4D were converted to high-throughput allele specific PCR assays and were used to genotype all individuals in each population. Simple interval mapping showed two peaks in Chr4D, named QPc.APR-4D.1 and QPc.APR-4D.2, which were detected in the OtanaA/CI4706-2 and OtanaA/CI9416-2 and in the Otana/PI189733, OtanaD/PI260616, and OtanaA/CI8000-4 populations, respectively. These results were validated by mapping two entire populations, Otana/PI189733 and OtanaA/CI9416, genotyped using Illumina HiSeq, in which polymorphisms were called against the OT3098 oat reference genome. Composite interval mapping results confirmed the presence of the two quantitative trait loci (QTL) located on oat chromosome 4D and an additional QTL with a smaller effect located on chromosome 6C. This mapping approach also narrowed down the physical intervals to between 5 and 19Mb, and indicated that QPc.APR-4D.1, QPc.APR-4D.2, and QPc.APR-6C explained 43.4%, 38.5%, and 21.5% of the phenotypic variation, respectively. In a survey of the gene content of each QTL, several clusters of disease resistance genes that may contribute to APR were found. The allele specific PCR markers developed for these QTL regions would be beneficial for marker-assisted breeding, gene pyramiding, and future cloning of resistance genes from oat.

Genetic Insight into Disease Resistance Gene Clusters by Using Sequencing-Based Fine Mapping in Sunflower (Helianthus annuus L.).

Rust and downy mildew (DM) are two important sunflower diseases that lead to significant yield losses globally. The use of resistant hybrids to control rust and DM in sunflower has a long history. The rust resistance genes, R13a and R16, were previously mapped to a 3.4 Mb region at the lower end of sunflower chromosome 13, while the DM resistance gene, Pl33, was previously mapped to a 4.2 Mb region located at the upper end of chromosome 4. High-resolution fine mapping was conducted using whole genome sequencing of HA-R6 (R13a) and TX16R (R16 and Pl33) and large segregated populations. R13a and R16 were fine mapped to a 0.48 cM region in chromosome 13 corresponding to a 790 kb physical interval on the XRQr1.0 genome assembly. Four disease defense-related genes with nucleotide-binding leucine-rich repeat (NLR) motifs were found in this region from XRQr1.0 gene annotation as candidate genes for R13a and R16. Pl33 was fine mapped to a 0.04 cM region in chromosome 4 corresponding to a 63 kb physical interval. One NLR gene, HanXRQChr04g0095641, was predicted as the candidate gene for Pl33. The diagnostic SNP markers developed for each gene in the current study will facilitate marker-assisted selections of resistance genes in sunflower breeding programs.

Co-occurrence of dual carbapenemases KPC-2 and OXA-48 with the mobile colistin resistance gene mcr-9.1 in Enterobacter xiangfangensis.

Bacterial infections with the genus Enterobacter are notoriously difficult to treat and often associated with resistance to penicillin, aminoglycosides, fluoroquinolones, and third-generation cephalosporins. Also, Enterobacter species have emerged as the third most common hosts for carbapenemases worldwide, forcing the use of colistin as a “last-resort” antibiotic for the treatment. Studies on the population structure of the genus Enterobacter repeatedly detect E. xiangfangensis as a common clinical species present worldwide. Here, we report on the characteristics of an extreme drug-resistant E. xiangfangensis isolate va18651 (ST88), obtained from a cervical swab of an expectant mother. The isolate was resistant to almost all the classes of antibiotics tested, including β-lactams (viz., penicillins, carbapenems, cephalosporin, monobactams, and their combinations), quinolone, aminoglycosides, and sulfonamide/dihydrofolate reductase inhibitor, and exhibited heteroresistance towards colistin. Analysis of its complete genome sequence revealed 37 antibiotic resistance genes (ARGs), including mcr-9.1, blaKPC-2 , and blaOXA-48 , encoded on three of the four different plasmids (cumulative plasmidome size 604,632 bp). An unusually high number of plasmid-based heavy metal resistance gene (HRG) clusters towards silver, arsenate, cadmium, copper, mercury, and tellurite were also detected. Virulence genes (VGs) for the lipopolysaccharide and capsular polysaccharide structures, iron acquisition (iroBCDEN, ent/fep/fes, sitABCD, iut, and fur), and a type VI secretion system, together with motility genes and Type IV pili, were encoded chromosomally. Thus, a unique combination of chromosomally encoded VGs, together with plasmid-encoded ARGs and HRGs, converged to result in an extreme drug-resistant, pathogenic isolate with survival potential in environmental settings. The use of a disinfectant, octenidine, led to its eradication; however, the existence of a highly antibiotic-resistant isolate with significant virulence potential is a matter of concern in public health settings and warrants further surveillance for extreme drug-resistant Enterobacter isolates.

Emergence of a Novel Plasmid-Mediated Tigecycline Resistance Gene Cluster, tmexCD4-toprJ4, in Klebsiella quasipneumoniae and Enterobacter roggenkampii.

ABSTRACTThe occurrence of transferable tigecycline resistance determinants, tmexCD1-toprJ1, tmexCD2-toprJ2, tmexCD3-toprJ1b, and multiple tet(A) and tet(X) variants, presents an unprecedented challenge to clinical therapeutic options. tmexCD-toprJ-like gene clusters can mediate multidrug resistance and have been detected in a variety of bacteria. Here, we characterized the fourth tmexCD-toprJ-like gene cluster, tmexCD4-toprJ4, identified on untypeable plasmids of Klebsiella quasipneumoniae and Enterobacter roggenkampii isolated from chicken meat and environmental samples from farm markets, respectively. TMexCD4-TOprJ4 was closely related (92 to 99% amino acid identity) to TMexCD1-TOprJ1, TMexCD2-TOprJ2, and TMexCD3-TOprJ1. Phylogenetic analysis revealed that tmexCD4-toprJ4 was not in the same branch as the other three variants. Expression of tmexCD4-toprJ4 increased tigecycline efflux in Escherichia coli and resulted in a 4- to 8-fold increase in MICs of tigecycline in E. coli and Klebsiella pneumoniae. Moreover, tmexCD4-toprJ4 can act synergistically with its upstream gene tet(A) to reduce the susceptibility of E. coli and K. pneumoniae strains to tigecycline. The tmexCD4-toprJ4-containing plasmid is a novel plasmid type and can be transferred to E. coli and K. pneumoniae only via electrotransformation. The increasing emergence of plasmid-mediated tigecycline resistance gene clusters suggests that the spread of tmexCD-toprJ-like gene clusters requires widespread attention.IMPORTANCE The plasmid-mediated tigecycline resistance gene cluster tmexCD1-toprJ1 and other variants have been detected in a variety of strains from multiple sources, including human-derived strains. In addition to tigecycline, these tmexCD-toprJ-like gene clusters reduce susceptibility of the host strain to many other antimicrobials. Here, we identified tmexCD4-toprJ4 in K. quasipneumoniae and E. roggenkampii, suggesting that this gene cluster is already present in the human-associated environment and the risk of transmission to humans is increased. Monitoring tigecycline-resistant Gram-negative bacteria is essential for understanding and addressing the spread of this gene cluster in agriculture and health care.

Isolation and characterization of the novel bacteriophage vB_SmaS_BUCT626 against Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia has been recognized as an emerging global opportunistic pathogen, and it is intrinsically resistant to most antibiotics, which makes the limited choice for treating S. maltophilia infections. Bacteriophage with the proper characterization is considered as a promising alternative treatment option to control S. maltophilia infections. In this study, we isolated a novel Siphoviridae bacteriophage vB_SmaS_BUCT626 with lytic activity against S. maltophilia. Phage vB_SmaS_BUCT626 can lysis 10 of 20 S. maltophilia and was relatively stable at a wide range of temperatures (4-70°C) and pH values (3.0-13.0) and exhibited good tolerance to chloroform. The genome of phage vB_SmaS_BUCT626 was a 61,662-bp linear double-stranded DNA molecule with a GC content of 56.2%, and contained 100 open-reading frames. It carried no antibiotic resistance, toxin, virulence-related genes, or lysogen-formation gene clusters. Together, these characteristics make phage vB_SmaS_BUCT626, a viable candidate as a biocontrol agent against S. maltophilia infection.

Complete Genome Analysis of Highly Pathogenic Non-O1/O139 Vibrio cholerae Isolated From Macrobrachium rosenbergii Reveals Pathogenicity and Antibiotic Resistance-Related Genes.

Non-O1/O139 Vibrio cholerae is a highly virulent pathogen that causes mass mortalities of various aquatic animals. In the present study, we sequenced the whole genome of non-O1/O139 V. cholerae GXFL1-4, isolated from Macrobrachium rosenbergii, to reveal the pathogenicity and antibiotic resistance. The result showed its genome contained two circular chromosomes and one plasmid with a total size of 4,282,243 bp, which harbored 3,869 coding genes. Among them, 3,047, 2,659, and 3,661 genes were annotated in the Clusters of Orthologous Genes (COG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG), respectively. In addition, 372 potential virulence genes were predicted based on the Virulence Factor Database (VFDB) database, such as type II, III, IV, and VI secretion systems related genes, flagella genes, and pilus formation or motility-related genes. Blast results in the Comprehensive Antibiotic Resistance Database (CARD) database showed that the strain contained 148 antibiotic resistance-related genes belonging to 27 categories, such as efflux pump complex antibiotic resistance genes and antibiotic resistance gene cluster genes. The Pathogen-Host Interaction (PHI) database annotated 320 genes related to pathogen-host interaction, such as T3SS, virulence regulatory factors, transcriptional regulators, and two-component response regulator related genes. The whole-genome analysis suggested that the pathogenic non-O1/O139 V. cholerae strain GXFL1-4 might have a complex molecular mechanism of pathogenicity and antibiotic resistance. This study provides a wealth of information about non-O1/O139 V. cholerae genes related to its pathogenicity and drug resistance and will facilitate the understanding of its pathogenesis as well as the development of prevention and treatment strategies for the pathogen.

Geographical resistome profiling in the honeybee microbiome reveals resistance gene transfer conferred by mobilizable plasmids

BackgroundThe spread of antibiotic resistance genes (ARGs) has been of global concern as one of the greatest environmental threats. The gut microbiome of animals has been found to be a large reservoir of ARGs, which is also an indicator of the environmental antibiotic spectrum. The conserved microbiota makes the honeybee a tractable and confined ecosystem for studying the maintenance and transfer of ARGs across gut bacteria. Although it has been found that honeybee gut bacteria harbor diverse sets of ARGs, the influences of environmental variables and the mechanism driving their distribution remain unclear.ResultsWe characterized the gut resistome of two closely related honeybee species, Apis cerana and Apis mellifera, domesticated in 14 geographic locations across China. The composition of the ARGs was more associated with host species rather than with geographical distribution, and A. mellifera had a higher content of ARGs in the gut. There was a moderate geographic pattern of resistome distribution, and several core ARG groups were found to be prevalent among A. cerana samples. These shared genes were mainly carried by the honeybee-specific gut members Gilliamella and Snodgrassella. Transferrable ARGs were frequently detected in honeybee guts, and the load was much higher in A. mellifera samples. Genomic loci of the bee gut symbionts containing a streptomycin resistance gene cluster were nearly identical to those of the broad-host-range IncQ plasmid, a proficient DNA delivery system in the environment. By in vitro conjugation experiments, we confirmed that the mobilizable plasmids could be transferred between honeybee gut symbionts by conjugation. Moreover, “satellite plasmids” with fragmented genes were identified in the integrated regions of different symbionts from multiple areas.ConclusionsOur study illustrates that the gut microbiota of different honeybee hosts varied in their antibiotic resistance structure, highlighting the role of the bee microbiome as a potential bioindicator and disseminator of antibiotic resistance. The difference in domestication history is highly influential in the structuring of the bee gut resistome. Notably, the evolution of plasmid-mediated antibiotic resistance is likely to promote the probability of its persistence and dissemination.Aghy2aoGk5W_8F1dgySKf9Video

High burden and diversity of carbapenemase-producing Enterobacterales observed in wastewater of a tertiary care hospital in Germany.

Hospitals are one of the main reservoirs of multi-resistant Enterobacterales (MRE). As MRE are resistant to the most frequently used antibiotics, therapy for patients with MRE infections is challenging. It has been previously described that MRE from hospital wastewater can pass into municipal wastewater and even surface water. In this study, we investigated the diversity and epidemiology of MRE in the wastewater of a large tertiary care hospital. Wastewater samples were collected for a four-day period and tested for the presence of Enterobacterales resistant to 3rd gen. cephalosporins. Representative isolates were further characterized by whole genome sequencing. In 120 β-glucuronidase-producing isolates, 68 Escherichia coli and, interestingly, also 52 Citrobacter freundii were identified. In 120 β-glucosidase-producing isolates 45 Serratia marcescens, 34 Klebsiella oxytoca, 32 Enterobacter cloacae and 9 Klebsiella pneumoniae were observed. For all species various MLST sequence types and different clusters of resistance genes were determined, showing a great diversity within the different Enterobacterales, further corroborated by clonal analysis performed by cgMLST. The most prominent clone was wastewater associated E. coli ST635, which accounted for 47.1% of all E. coli isolates. Interestingly, 45.6% of E. coli, 88.5% of C. freundii, 95.6% of S. marcescens, 91.2% of K. oxytoca, 96.9% of E. cloacae and 88.9% of K. pneumoniae isolates carried a carbapenemase gene, indicating a high burden with carbapenemase-producing Enterobacterales. Comparison with clinical isolates from the same hospital displayed few clonal matches. One wastewater isolate of K. pneumoniae was identified to be closely related compared to a clone that had been introduced into the hospital during an outbreak four years earlier. One E. coli isolate was identified as identical to an isolate from a patient, with inpatient stay during the sampling period. The data obtained in this study highlight the problem of antibiotic resistance of Enterobacterales in hospital wastewater. In particular, the clustered occurrence of carbapenemase genes is of great concern and underscores the problem of increasingly scarce antibiotic options against these bacteria.

Inhibition of the Vancomycin Resistance in Staphylococcus aureus in Egypt Using Silver Nanoparticles.

Staphylococcus aureus is a major human pathogen that is sometimes resistant to vancomycin. In this study, the prevalence of vancomycin-resistant Staphylococcus aureus (VRSA) was studied. 100 isolates of S. aureus were identified based on biochemical and molecular evidence. The antibiotic susceptibility of the studied isolates was tested against 13 antibiotics by the disc diffusion method that showed 24 vancomycin-resistant isolates. The minimum inhibitory concentrations (MICs) were estimated by the agar dilution method to determine vancomycin intermediate-resistant S. aureus (VISA) and VRSA. The resistance gene cluster (vanA, vanR, vanH, and vanY) was amplified by PCR and then sequenced. Amplification of vanA and vanR genes showed that they are present in 21.4% and 14.3% of VRSA isolates, respectively, whereas none of the studied genes has been detected in VISA strains. A significant antimicrobial effect toward VRSA isolates using silver nanoparticles (AgNPs) synthesized from S. aureus and rosemary leaves was recorded. This study confirmed the existence of VRSA strains in Egypt. Furthermore, the use of silver nanoparticles inhibits these vancomycin-resistant S. aureus strains in vitro.

Whole-genome sequence of a putative pathogenic Bacillus sp. strain SD-4 isolated from cattle feed

ObjectivesThe present study describes the draft genome sequence of a novel Bacillus sp. strain SD-4 isolated from animal feed. The study aims to get a deeper insight into antimicrobial resistance and secondary metabolite biosynthetic gene clusters (BGCs) and the association between them. MethodsThe strain SD-4 was preliminarily evaluated for antibacterial activities, motility, biofilm formation, and enterotoxin production using in vitro assays. The genome of strain SD-4 was sequenced using the Illumina HiSeq 2500 platform with paired-end reads. The reads were assembled and annotated using SPAdes and PGAP, respectively. The genome was further analysed using several bioinformatics tools, including TYGS, AntiSMASH, RAST, PlasmidFinder, VFDB, VirulenceFinder, CARD, PathogenFinder, MobileElement finder, IslandViewer, and CRISPRFinder. ResultsIn vitro assays showed that the strain is motile, synthesises biofilm, and produces an enterotoxin and antibacterial metabolites. The genome analysis revealed that the strain SD-4 carries antimicrobial resistance genes (ARGs), virulence factors, and beneficial secondary metabolite BGCs. Further genome analysis showed interesting genome architectures containing several mobile genetic elements, including two plasmid replicons (repUS22 and rep20), five prophages, and at least four genomic islands (GIs), including one Listeria pathogenicity island LIPI-1. Moreover, the strain SD-4 is identified as a putative human pathogen. ConclusionThe genome of strain SD-4 harbours several BGCs coding for biologically active metabolites. It also contains antimicrobial resistance genes and is identified as a potential human pathogen. These results can be used to better comprehend antibiotic resistance in environmental bacteria that are not influenced by human intervention.

Functional genome mining and taxono-genomics reveal eco-physiological traits and species distinctiveness of aromatic-degrading Pseudomonas bharatica sp. nov.

Assistive eco-physiological traits are necessary for microbes to adapt and colonize at polluted niches, enabling efficient clean-up. To demarcate species distinctiveness and eco-physiological traits of aromatic compounds metabolizing Pseudomonas sp. CSV86T (earlier identified as Pseudomonas putida), an Indian isolate from a petrol station soil, comparative genome mining, taxono-genomic, and physiological analyses were performed. A 6.79 Mbp genome (62.72 G+C mol%) of CSV86T encodes 6798 CDS and 238 unique genes. Naphthalene metabolism and Co-Zn-Cd resistance gene clusters were part of distinct genomic islands. Abundance of transporters (aromatics, organic acids, amino acids, and metals) and mobile elements (integrases, transposases, conjugative proteins) differentiated CSV86T from its closest relatives. Enhanced siderophore production for Fe-uptake during aromatic metabolism, indole acetic acid production, and fusaric acid resistance wasvalidated by genomic attributes. Full-length 16S-rRNA phylogeny revealed Pseudomonas japonica WLT as a closest relative of CSV86T . However, lower genomic indices (<97% gyrB-rpoB-rpoD homology, <90% ANI, <50% DNA-DNA relatedness) and taxonomic differences (assimilation of organic acids, amino acids, fatty acids composition) substantially differentiated CSV86T from its closest relatives, indicating it to be a novel species as Pseudomonas bharatica. Preferential metabolism of aromatics with advantageous eco-physiological traits renders CSV86T an ideal candidate for bioremediation and host for metabolic engineering.

Large-scale WGS of carbapenem-resistant Acinetobacter baumannii isolates reveals patterns of dissemination of ST clades associated with antibiotic resistance.

To describe the population genetics and antibiotic resistance gene distribution of carbapenem-resistant Acinetobacter baumannii (CRAB) isolates causing infections in three Mediterranean countries. Isolates were collected during the 2013-17 AIDA clinical trial in six hospitals in Israel, Greece and Italy. WGS, bioinformatic characterization and antibiotic resistance profiling were performed. In the 247 CRAB isolates characterized in this study, ST distribution varied by country: 29/31 (93.5%) Greek isolates, 34/41 (82.9%) Italian isolates and 70/175 (40.0%) Israeli isolates belonged to ST2. The identified ST2 isolates included eight distinct clades: 2C, 2D and 2H were significantly more common in Italy, while 2F was unique to Greece. The uncommon ST3 was not present among Greek isolates and constituted only 5/41 (12%) Italian isolates. On the other hand, it was much more common among Israeli isolates: 78/175 (44.6%) belonged to ST3. The vast majority of isolates, 240/247 (97.2%), were found to harbour acquired carbapenemases, primarily blaOXA-23. The chromosomal oxaAb (blaOXA-51-like) and ampC genes characteristic of this organism were also ubiquitous. Most (96.4%) ST3 isolates carried a broad-host-range plasmid IncP1α. The geographical differences in CRAB populations support the theory that clonal spread of CRAB leads to endemicity in hospitals and regions. The close association between antibiotic resistance genes and clades, and between plasmids and STs, suggest that de novo creation of MDR A. baumannii is rare. The clustering of antibiotic resistance genes and plasmids that is unique to each clade/ST, and nearly uniform within clades/STs, suggests that horizontal transmission is rare but crucial to the clade's/ST's success.

Extensively resistant Acinetobacter baumannii isolate RCH52 carries several resistance genes derived from an IncC plasmid.

To identify the origins of resistance in a sporadic extensively resistant Acinetobacter baumannii isolate. The complete genome of RCH52 was determined by combining available Illumina short reads with MinION (Oxford Nanopore) long reads using Unicycler. Bioinformatic searches were used to identify features of interest. The complete genome of RCH52 revealed an unusual chromosomal region containing all of the antibiotic resistance genes, except tet39, which is in a plasmid. A 129 585 bp segment was bounded by inversely oriented copies of ISAba1 and included two groups of resistance genes separated by the large segment of the backbone of type 1 IncC plasmids that lies between the ARI-A and ARI-B resistance islands but does not include the replication region. The ISAba1-bounded segment was located in a novel integrative element that had integrated into the chromosomal thyA gene but provided a replacement thyA gene. Several resistance genes are derived from either the ARI-A or the ARI-B resistance islands found in IncC plasmids that have been brought together by an IS26-mediated deletion of the original plasmid. This non-replicating circular molecule (or translocatable unit) has been incorporated into a smaller ISAba1-bounded unit that includes oxa23 in Tn2008B via homologous recombination between sul2-CR2-floR segments found in both. The plasmids shared by most Gram-negative pathogens, including the broad host range IncC plasmids, have not been detected in Acinetobacter species. However, it seems likely that they can conjugate into members of this genus and contribute pre-existing clusters of antibiotic resistance genes.

Novel SCCmec type XV (7A) and two pseudo-SCCmec variants in foodborne MRSA in China.

Staphylococcal cassette chromosome mec (SCCmec) elements are highly diverse and have been classified into 14 types. Novel SCCmec variants have been frequently detected from humans and animals but rarely from food. To characterize a novel SCCmec type and two SCCmec variants identified from food-associated MRSA in China. Three MRSA (NV_1, NT_611 and NT_8) collected from retail foods in China were subjected to WGS and the SCCmec elements were determined. The novel SCCmecXV identified in NV_1 carried the mec gene complex class A (mecI-mecR1-mecA-IS431) and the ccr gene complex 7 (ccrA1B6), and a Tn558-mediated phenicol exporter gene fexA was detected in this SCCmecXV cassette. The pseudo-SCCmec elements ΨSCCmecNT_611 and ΨSCCmecNT_8 showed a truncated SCCmec pattern, carrying the class C2 mec gene complex but missing the ccr genes. The ΨSCCmecNT_611 element shared more similarities with those of Staphylococcus haemolyticus (AB478934.1) and carried a heavy metal resistance gene cluster cadD-cadX-arsC-arsB-arsR-copA. The ΨSCCmecNT_8 MRSA exhibited a highly resistant phenotype, showing the absence of a 19.3 kb segment compared with the reference SCCmecXII element (CP019945.1). Notably, a 46 kb region containing multiple transposons encoding antimicrobial or metal resistance genes flanked by IS431 or IS256 was identified ∼30 kb downstream from the mec gene complex in ΨSCCmecNT_8, which might explain such high resistance in MRSA NT_8. Our finding of novel and pseudo-SCCmec elements reflected the ongoing intra/interspecies genetic rearrangements in staphylococci. Further study will be needed to investigate the biological significance and prevalence of those SCCmec variants along the food chain.

Specific Inhibition of VanZ-Mediated Resistance to Lipoglycopeptide Antibiotics.

Teicoplanin is a natural lipoglycopeptide antibiotic with a similar activity spectrum as vancomycin; however, it has with the added benefit to the patient of low cytotoxicity. Both teicoplanin and vancomycin antibiotics are actively used in medical practice in the prophylaxis and treatment of severe life-threatening infections caused by gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, Enterococcus faecium and Clostridium difficile. The expression of vancomycin Z (vanZ), encoded either in the vancomycin A (vanA) glycopeptide antibiotic resistance gene cluster or in the genomes of E. faecium, as well as Streptococcus pneumoniae and C. difficile, was shown to specifically compromise the antibiotic efficiency through the inhibition of teicoplanin binding to the bacterial surface. However, the exact mechanisms of this action and protein structure remain unknown. In this study, the three-dimensional structure of VanZ from E. faecium EnGen0191 was predicted by using the I-TASSER web server. Based on the VanZ structure, a benzimidazole based ligand was predicted to bind to the VanZ by molecular docking. Importantly, this new ligand, named G3K, was further confirmed to specifically inhibit VanZ-mediated resistance to teicoplanin in vivo.

Prevalence, Serotypes, and Antimicrobial Resistance of Salmonella from Mink Feces and Feed in the United States.

Nontyphoidal Salmonella is a leading cause of foodborne illnesses, and concentrated animal production such as commercial mink farming can be a reservoir. The objective of this study was to determine the prevalence, serotypes, antimicrobial resistance, virulence genes and mobile genetic elements, and phylogenetic characterization of Salmonella from mink fecal (n = 42) and feed (n = 8) samples at a commercial mink farm in the United States. Salmonella was detected from 11 (26.2%) fecal and 1 (12.5%) feed samples. Twelve fecal isolates obtained from the positive samples were identified into four serotypes: Salmonella Uganda (eight isolates; two isolates were obtained from one sample), Salmonella Reading (two isolates), Salmonella Heidelberg (one isolate), and Salmonella Orion (one isolate). Two isolates from the positive feed sample were identified as Salmonella Cerro. Twelve isolates, one isolate per positive sample, were whole genome sequenced for further characterization. The two Salmonella Reading isolates were multidrug resistant (MDR) with the classical ASSuT (ampicillin, streptomycin, sulfisoxazole, and tetracycline) phenotype. Genotypically, the isolates were correspondingly positive, both by polymerase chain reaction and by whole-genome sequencing (WGS), for blaTEM-1, aph(3″)-Ib and aph(6)-Id, sul2, and tet(A). WGS additionally identified trimethoprim resistance gene dfrA8 in Salmonella Reading, and fosfomycin resistance gene fosA7 in Salmonella Heidelberg. All isolates carried Salmonella-specific multidrug resistance (mdsA/B), gold resistance, and intimin-like virulence genes. In addition, Salmonella Orion carried copper and silver resistance gene clusters. The two Salmonella Reading isolates also carried a cytolethal distension toxin (cdtB) gene. Salmonella Reading isolates belonged to ST412, a predominant sequence type among turkey and human isolates obtained in relation to recent salmonellosis outbreaks in North America. Phylogenetically, Salmonella Uganda, Salmonella Heidelberg, and Salmonella Reading were mostly associated with historic human/clinical, and poultry and swine source isolates. This study indicates that mink production can be a reservoir for foodborne pathogens such as Salmonella with MDR serovars commonly associated with human illnesses.

Novel vancomycin resistance gene cluster in Enterococcus faecium ST1486, Belgium, June 2021.

We identified a novel van gene cluster in a clinical Enterococcus faecium isolate with vancomycin minimum inhibitory concentration (MIC) of 4 µg/mL. The ligase gene, vanP, was part of a van operon cluster of 4,589 bp on a putative novel integrative conjugative element located in a ca 98 kb genomic region presumed to be acquired by horizontal gene transfer from Clostridium scidens and Roseburia sp. 499. Screening for van genes in E. faecium strains with borderline susceptibility to vancomycin is important.