Plasmid Sequences Research Articles

Novel configurations of type I-E CRISPR-Cas system in Corynebacterium striatum clinical isolates

Clustered regularly interspaced short palindromic repeats (CRISPR) are a prokaryotic adaptive immune system that, through Cas proteins, promote the degradation of foreign nucleic acids such as phages and plasmids. We analyzed 10 genomes of Corynebacterium striatum clinical isolates from a public hospital in Rio de Janeiro, Brazil, the most emergent multidrug-resistant Corynebacterium species. All isolates were submitted to antimicrobial susceptibility testing. The occurrence and diversity of the CRISPR system were investigated by bioinformatics tools. Our analysis revealed that the isolates exhibited type I-E gene arrangements, and 3 more multidrug-resistant isolates, alternative type I-E gene arrangements, showing a divergent gene arrangement within the cas operon. Phylogenetic analysis of the cas1 gene of this type I-E CRISPR-Cas system alternative arrangement, termed here type I-E’, showed a cluster in a distinct clade of the type I-E CRISPR-Cas system. The systems’ guanine-cytosine (GC) content is lower than the genomic DNA’s GC content, and mobile genetic elements were found in some isolates near the CRISPR-Cas system. Most CRISPR spacers are unknown indicating that there is a reservoir of unexplored corynebacteriophages and plasmids. Some spacers showed perfect homologies with phage and plasmid sequences. Intact phage regions were found in 3 of our isolates, ranging from 9.1 to 43.8 kb, with regions showing similarity to Rhodococcus and Corynebacterium phages. Our results may contribute to research about the CRISPR-Cas system diversity in C. striatum, where there are no published data to date.

Detection and Typing of Plasmids in Acinetobacter baumannii Using rep Genes Encoding Replication Initiation Proteins.

Plasmids found in Acinetobacter species contribute to the spread of antibiotic resistance genes. They appear to be largely confined to this genus and cannot be typed with available tools and databases. Here, a method for distinguishing and typing these plasmids was developed using a curated, non-redundant set of 621 complete sequences of plasmids from Acinetobacter baumannii. Plasmids were separated into 3 groups based on the Pfam domains of the encoded replication initiation (Rep) protein and a fourth group that lack an identifiable Rep protein. The rep genes of each Rep-encoding group (n = 13 Rep_1, n = 107 RepPriCT_1, n = 351 Rep_3) were then clustered using a threshold of >95% nucleotide identity to define 80 distinct types. Five Rep_1 subgroups, designated R1_T1 to R1-T5, were identified and a sixth reported recently was added. Each R1 type corresponded to a conserved small plasmid sequence. The RepPriCT_1 plasmids fell into 5 subgroups, designated RP-T1 to RP-T5 and the Rep_3 plasmids comprised 69 distinct types (R3-T1 to R3-T69). Three R1, 2 RP and 32 R3 types are represented by only a single plasmid. Over half of the plasmids belong to the 4 most abundant types: the RP-T1 plasmids (n = 97), which include conjugation genes and are often associated with various acquired antibiotic resistance genes, and R3-T1, R3-T2 and R3-T3 (n = 95, 30 and 45, respectively). To facilitate typing and the identification of plasmids in draft genomes using this framework, we established the Acinetobacter Typing database containing representative nucleotide and protein sequences of the type markers (https://github.com/MehradHamidian/AcinetobacterPlasmidTyping). IMPORTANCE Though they contribute to the dissemination of genes that confer resistance to clinically important carbapenem and aminoglycoside antibiotics used to treat life-threatening Acinetobacter baumannii infections, plasmids found in Acinetobacter species have not been well studied. As these plasmids do not resemble those found in other Gram-negative pathogens, available typing systems are unsuitable. The plasmid typing system developed for A. baumannii plasmids with an identifiable rep gene will facilitate the classification and tracking of sequenced plasmids. It will also enable the detection of plasmid-derived contigs present in draft genomes that are widely ignored currently. Hence, it will assist in the tracking of resistance genes and other genes that affect survival in the environment, as they spread through the population. As identical or similar plasmids have been found in other Acinetobacter species, the typing system will also be broadly applicable in identifying plasmids in other members of the genus.

Analysis of the first genetic engineering attribution challenge

The ability to identify the designer of engineered biological sequences—termed genetic engineering attribution (GEA)—would help ensure due credit for biotechnological innovation, while holding designers accountable to the communities they affect. Here, we present the results of the first Genetic Engineering Attribution Challenge, a public data-science competition to advance GEA techniques. Top-scoring teams dramatically outperformed previous models at identifying the true lab-of-origin of engineered plasmid sequences, including an increase in top-1 and top-10 accuracy of 10 percentage points. A simple ensemble of prizewinning models further increased performance. New metrics, designed to assess a model’s ability to confidently exclude candidate labs, also showed major improvements, especially for the ensemble. Most winning teams adopted CNN-based machine-learning approaches; however, one team achieved very high accuracy with an extremely fast neural-network-free approach. Future work, including future competitions, should further explore a wide diversity of approaches for bringing GEA technology into practical use.

The endogenous Coxiella burnetii plasmid encodes a functional toxin-antitoxin system.

Coxiella burnetii is the causative agent of Q fever. All C. burnetii isolates encode either an autonomously replicating plasmid (QpH1, QpDG, QpRS, or QpDV) or QpRS-like chromosomally integrated plasmid sequences. The role of the ORFs present in these sequences is unknown. Here, the role of the ORFs encoded on QpH1 was investigated. Using a new C. burnetii shuttle vector (pB-TyrB-QpH1ori), we cured the C. burnetii Nine Mile Phase II strain of QpH1. The ΔQpH1 strain grew normally in axenic media but had a significant growth defect in Vero cells, indicating QpH1 was important for C. burnetii virulence. We developed an inducible CRISPR interference system to examine the role of individual QpH1 plasmid genes. CRISPRi of cbuA0027 resulted in significant growth defects in axenic media and THP-1 cells. The cbuA0028/cbuA0027 operon encodes CBUA0028 (ToxP) and CBUA0027 (AntitoxP), which are homologous to the HigB2 toxin and HigA2 antitoxin, respectively, from Vibrio cholerae. Consistent with toxin-antitoxin systems, overexpression of toxP resulted in a severe intracellular growth defect that was rescued by co-expression of antitoxP. ToxP inhibited protein translation. AntitoxP bound the toxP promoter (PtoxP) and ToxP, with the resulting complex binding also PtoxP. In summary, our data indicate that C. burnetii maintains an autonomously replicating plasmid because of a plasmid-based toxin-antitoxin system.

SourceFinder: a Machine-Learning-Based Tool for Identification of Chromosomal, Plasmid, and Bacteriophage Sequences from Assemblies.

High-throughput genome sequencing technologies enable the investigation of complex genetic interactions, including the horizontal gene transfer of plasmids and bacteriophages. However, identifying these elements from assembled reads remains challenging due to genome sequence plasticity and the difficulty in assembling complete sequences. In this study, we developed a classifier, using random forest, to identify whether sequences originated from bacterial chromosomes, plasmids, or bacteriophages. The classifier was trained on a diverse collection of 23,211 chromosomal, plasmid, and bacteriophage sequences from hundreds of bacterial species. In order to adapt the classifier to incomplete sequences, each complete sequence was subsampled into 5,000 nucleotide fragments and further subdivided into k-mers. This three-class classifier succeeded in identifying chromosomes, plasmids, and bacteriophages using k-mer distributions of complete and partial genome sequences, including simulated metagenomic scaffolds with minimum performance of 0.939 area under the receiver operating characteristic curve (AUC). This classifier, implemented as SourceFinder, has been made available as an online web service to help the community with predicting the chromosomal, plasmid, and bacteriophage sources of assembled bacterial sequence data (https://cge.food.dtu.dk/services/SourceFinder/). IMPORTANCE Extra-chromosomal genes encoding antimicrobial resistance, metal resistance, and virulence provide selective advantages for bacterial survival under stress conditions and pose serious threats to human and animal health. These accessory genes can impact the composition of microbiomes by providing selective advantages to their hosts. Accurately identifying extra-chromosomal elements in genome sequence data are critical for understanding gene dissemination trajectories and taking preventative measures. Therefore, in this study, we developed a random forest classifier for identifying the source of bacterial chromosomal, plasmid, and bacteriophage sequences.

Functional and Safety Characterization of Weissella paramesenteroides Strains Isolated from Dairy Products through Whole-Genome Sequencing and Comparative Genomics

Strains belonging to the Weissella genus are frequently recovered from spontaneously fermented foods. Their functional, microbial-modulating, and probiotic traits enhance not only the sensorial properties but also the nutritional value, beneficial effects, and safety of fermented products. Sporadic cases of opportunistic pathogenicity and antibiotic resistance have deprived safety status from all Weissella species, which thus remain understudied. Our study increased the number of available high-quality and taxonomically accurate W. paramesenteroides genomes by 25% (9 genomes reported, leading to a total of 36 genomes). We conducted a phylogenetic and comparative genomic analysis of the most dominant Weissella species (W. cibaria, W. paramesenteroides, W. viridescens, W. soli, W. koreensis, W. hellenica and W. thailadensis). The phylogenetic tree corroborated species assignment but also revealed phylogenetic diversity within the Weissella species, which is likely related to the adaptation of Weissella in different niches. Using robust alignment criteria, we showed the overall absence of resistance and virulence genes in Weissella spp., except for one W. cibaria isolate carrying blaTEM-181. Enrichment analysis showed the association of Weissella species several CAZymes, which are essential for biotechnological applications. Additionally, the combination of CAZyme metabolites with probiotics can potentially lead to beneficial effects for hosts, such as the inhibition of inflammatory processes and the reduction of cholesterol levels. Bacteriocins and mobile genetic elements MGEs (Inc11 plasmid and ISS1N insertion sequence) were less abundant, however W. thailadensis and W. viridescens showed significant association with specific bacteriocin-encoding genes. Lastly, an analysis of phenotypic traits underlined the need to carefully evaluate W. cibaria strains before use as food additives and suggested the possibility of employing W. paramesenteroides and W. hellenica in the fermentation process of vegetable products. More studies providing high-resolution characterization of Weissella strains from various sources are necessary to elucidate the safety of Weissella spp. and exploit their beneficial characteristics.

Plasmids of the urinary microbiota.

Studies of the last decade have identified a phylogenetically diverse community of bacteria within the urinary tract of individuals with and without urinary symptoms. Mobile genetic elements (MGEs), including plasmids and phages, within this niche have only recently begun to be explored. These MGEs can expand metabolic capacity and increase virulence, as well as confer antibiotic resistance. As such, they have the potential to contribute to urinary symptoms. While plasmids for some of the bacterial taxa found within the urinary microbiota (urobiome) have been well characterized, many urinary species are under-studied with few genomes sequenced to date. Using a two-pronged bioinformatic approach, we have conducted a comprehensive investigation of the plasmid content of urinary isolates representative of 102 species. The bioinformatic tools plasmidSPAdes and Recycler were used in tandem to identify plasmid sequences from raw short-read sequence data followed by manual curation. In total, we identified 603 high-confidence plasmid sequences in 20 different genera of the urobiome. In total, 70 % of these high-confidence plasmids exhibit sequence similarity to plasmid sequences from the gut. This observation is primarily driven by plasmids from E. coli , which is found in both anatomical niches. To confirm our bioinformatic predictions, long-read sequencing was performed for 23 of the E. coli isolates in addition to two E. coli strains that were sequenced as part of a prior study. Overall, 66.95 % of these predictions were confirmed highlighting the strengths and weaknesses of current bioinformatic tools. Future studies of the urobiome, especially concerning under-studied species in the urobiome, should employ long-read sequencing to expand the catalogue of plasmids for this niche.

First report of Klebsiella pneumoniae co-producing OXA-181, CTX-M-55, and MCR-8 isolated from the patient with bacteremia.

The worldwide spread of carbapenem-resistant Enterobacteriaceae (CRE) has led to a major challenge to human health. In this case, colistin is often used to treat the infection caused by CRE. However, the coexistence of genes conferring resistance to carbapenem and colistin is of great concern. In this work, we reported the coexistence of blaOXA-181, blaCTX-M-55, and mcr-8 in an ST273 Klebsiella pneumoniae isolate for the first time. The species identification was performed using MALDI-TOF MS, and the presence of various antimicrobial resistance genes (ARGs) and virulence genes were detected by PCR and whole-genome sequencing. Antimicrobial susceptibility testing showed that K. pneumoniae 5589 was resistant to aztreonam, imipenem, meropenem, ceftriaxone, cefotaxime, ceftazidime, levofloxacin, ciprofloxacin, gentamicin, piperacillin-tazobactam, cefepime, and polymyxin B, but sensitive to amikacin. S1-pulsed-field gel electrophoresis (PFGE) and Southern blotting revealed the mcr-8 gene was carried on a ~ 138 kb plasmid with a conserved structure (IS903B-ymoA-inhA-mcr-8-copR-baeS-dgkA-ampC). In addition, blaOXA-181 was found on another ~51 kb plasmid with a composite transposon flanked by insertion sequence IS26. The in vitro conjugation experiments and plasmid sequence probe indicated that the plasmid p5589-OXA-181 and the p5589-mcr-8 were conjugative, which may contribute to the propagation of ARGs. Relevant detection and investigation measures should be taken to control the prevalence of pathogens coharboring blaOXA-181, blaCTX-M-55 and mcr-8.

Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA.

Recent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in bacterial isolates. WGS is particularly useful for the clinical characterization of fastidious and slow-growing bacteria. Despite its potential, several challenges should be addressed before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical laboratory. Here, with three distinct ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), different approaches were compared to identify best practices for detecting AMR genes, including: total genomic DNA and plasmid DNA extractions, the solo assembly of Illumina short-reads and of Oxford Nanopore Technologies (ONT) long-reads, two hybrid assembly pipelines, and three in silico AMR databases. We also determined the susceptibility of each strain to 21 antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also detectable in total genomic DNA, indicating that, at least in these three enterobacterial genera, the purification of plasmid DNA was not necessary to detect plasmid-borne AMR genes. Illumina short-reads used with ONT long-reads in either hybrid or polished assemblies of total genomic DNA enhanced the sensitivity and accuracy of AMR gene detection. Phenotypic susceptibility closely corresponded with genotypes identified by sequencing; however, the three AMR databases differed significantly in distinguishing mobile dedicated AMR genes from non-mobile chromosomal housekeeping genes in which rare spontaneous resistance mutations might occur. This study indicates that each method employed in a WGS workflow has an impact on the detection of AMR genes. A combination of short- and long-reads, followed by at least three different AMR databases, should be used for the consistent detection of such genes. Further, an additional step for plasmid DNA purification and sequencing may not be necessary. This study reveals the need for standardized biochemical and informatic procedures and database resources for consistent, reliable AMR genotyping to take full advantage of WGS in order to expedite patient treatment and track AMR genes within the hospital and community.

The blue diatom Haslea ostrearia from the Indian Ocean coast of South Africa, with comparative analysis of Haslea organellar genomes

Haslea ostrearia represents the model species of blue diatoms, a cluster of benthic marine species all belonging to the genus Haslea, noticeable for producing a blue pigment called marennine famous for its greening activity on the gills of bivalves but also for its potential in biotechnology. The exact distribution of H. ostrearia is unknown. It has been long considered a cosmopolitan diatom, but recent studies provided evidence for cryptic diversity and the existence of several other blue species, some of them inhabiting places where diatoms described as H. ostrearia had previously been observed. Recently, a marine diatom with blue tips was isolated into clonal culture from a plankton net sample from Kei Mouth on the Indian Ocean coast of South Africa. It was identified as H. ostrearia through a combination of LM/SEM microscopy and molecular analysis. This constitutes the first established record of this species from South Africa and the Indian Ocean and the second record for the southern hemisphere. Molecular barcoding clearly discriminated the South African strain from an Australian strain and cox1 based molecular phylogeny associated it instead with strains from the French Atlantic Coast, raising questions about the dispersal of this species. The complete mitochondrial and plastid genomes were compared to those of Haslea nusantara and Haslea silbo. Multigene phylogenies performed with all protein-coding genes of the plastome and the mitogenome associated H. ostrearia with H. silbo. In addition, complete sequences of circular plasmids were obtained and one of them showed an important conservation with a plasmid found in H. silbo.

Comprehensive Genome and Plasmidome Analysis of Antimicrobial Resistant Bacteria in Wastewater Treatment Plant Effluent of Tokyo.

To characterize environmental antimicrobial resistance (AMR) in urban areas, extended-spectrum β-lactamase- (ESBL)/carbapenemase-producing bacteria (EPB/CPB, respectively) from urban wastewater treatment plant effluents in Tokyo were isolated on CHROMagar ESBL plate. Complete genome sequence analysis, including plasmids, indicated that 126 CTX-M-positive isolates (31%) were identified among the 404 obtained isolates. The CTX-M-9 group was predominant (n = 65, 52%), followed by the CTX-M-1 group (n = 44, 35%). Comparative genome analysis revealed that CTX-M-27-positive E. coli O16:H5-ST131-fimH41 exhibited a stable genome structure and clonal-global dissemination. Plasmidome network analysis revealed that 304 complete plasmid sequences among 85 isolates were grouped into 14 incompatibility (Inc) network communities (Co1 to Co14). Co10 consisted of primarily IncFIA/IncFIB plasmids harboring blaCTX-M in E. coli, whereas Co12 consisted primarily of IncFIA(HI1)/Inc FIB(K) plasmids harboring blaCTX-M, blaKPC, and blaGES in Klebsiella spp. Co11 was markedly located around Co10 and Co12. Co11 exhibited blaCTX-M, blaKPC, and blaNDM, and was mainly detected in E. coli and Klebsiella spp. from human and animal sources, suggesting a mutual role of Co11 in horizontal gene transfer between E. coli and Klebsiella spp. This comprehensive resistome analysis uncovers the mode of relational transfer among bacterial species, highlighting the potential source of AMR burden on public health in urban communities.

Chromosome and Plasmid Sequences of Pantoea sp. Strain SOD02 Isolated from an Urban Freshwater Stream.

ABSTRACTAfter isolating Pantoea sp. strain SOD02 from an urban freshwater stream in Providence, RI, we used PacBio RSII data for de novo assembly and Illumina MiSeq data for polishing. This yielded complete circular sequences for a 4,227,027-bp chromosome with 54.7% GC and a 926,844-bp plasmid with 54.0% GC.

A Plasmid Carrying blaIMP-56 in Pseudomonas aeruginosa Belonging to a Novel Resistance Plasmid Family.

blaIMP and blaVIM are the most detected plasmid-encoded carbapenemase genes in Pseudomonas aeruginosa. Previous studies have reported plasmid sequences carrying blaIMP variants, except blaIMP-56. In this study, we aimed to characterize a plasmid carrying blaIMP-56 in a P. aeruginosa strain isolated from a Mexican hospital. The whole genome of P. aeruginosa strain PE52 was sequenced using Illumina Miseq 2 × 150 bp, with 5 million paired-end reads. We characterized a 27 kb plasmid (pPE52IMP) that carried blaIMP-56. The phylogenetic analysis of RepA in pPE52IMP and 33 P. aeruginosa plasmids carrying resistance genes reported in the GenBank revealed that pPE52IMP and four plasmids (pMATVIM-7, unnamed (FDAARGOS_570), pD5170990, and pMRVIM0713) were in the same clade. These closely related plasmids belonged to the MOBP11 subfamily and had similar backbones. Another plasmid (p4130-KPC) had a similar backbone to pPE52IMP; however, its RepA was truncated. In these plasmids, the resistance genes blaKPC-2, blaVIM variants, aac(6′)-Ib4, blaOXA variants, and blaIMP-56 were inserted between phd and resolvase genes. This study describes a new family of plasmids carrying resistance genes, with a similar backbone, the same RepA, and belonging to the MOBP11 subfamily in P. aeruginosa. In addition, our characterized plasmid harboring blaIMP-56 (pPE52IMP) belongs to this family.

Characterization of carbapenem-resistant Acinetobacter baumanniiST540 and Klebsiella pneumoniaeST2237 isolates in a pneumonia case from China.

This study aimed to characterize the chromosome and plasmid sequences, and determine the transferability of plasmids in carbapenem-resistance Acinetobacter baumannii DD520 and Klebsiella pneumoniae DD521 isolates from the same patient who was co-infected in a hospital in China. Both isolates DD520 and DD521 exhibited multidrug resistance phenotype, especially the former isolate which was resistant to nine classes of antimicrobials including carbapenems, quinolones, penicillins, cephalosporins, tetracyclines, phenicols, fosfomycins, sulfanilamides and aminoglycosides. Carbapenem resistance genes of blaOXA-23 and blaOXA-66 were identified on the chromosome of A. baumannii DD520, and blaKPC-2 was found in the plasmid pDD521.2 from K. pneumoniae DD521. Phylogenetic analysis revealed that A. baumannii DD520 belonged to the ST540 clone, and K. pneumoniae DD521 belonged to the ST2237 clone. Plasmid analysis suggested that blaKPC-2 was embedded into plasmid pDD521.2, which might be resulted from IS26- and Tn1721-mediated transposition. Plasmid pDD521.2 carrying blaKPC-2 successfully transferred from K. pneumoniae DD521 into Escherichia coli C600, and carbapenems resistance also transferred in the conjugation. To our knowledge, it was the first report of A. baumannii ST540 and K. pneumoniae ST2237 in the same patient in China. Both these two isolates exhibited resistance to carbapenem, which was likely to have resulted from carbapenem-resistance genes blaOXA-23 -blaOXA-66 on the chromosome of A. baumannii ST540, and blaKPC-2 in the plasmid of K. pneumoniae ST2237. Our study highlighted that effective measures were urgent to prevent and control the co-infection caused by two or more carbapenem-resistance pathogens in the same patient.

Genome Sequence Resource for Strains of Pseudomonas syringae Phylogroup 2b and P. viridiflava Phylogroup 7a Causing Bacterial Stem Blight of Alfalfa.

Genome Sequence Resource for Strains of Pseudomonas syringae Phylogroup 2b and P. viridiflava Phylogroup 7a Causing Bacterial Stem Blight of Alfalfa.

Characterization of blaNDM-5-and blaCTX-M-199-Producing ST167 Escherichia coli Isolated from Shared Bikes.

Shared bikes as a public transport provide convenience for short-distance travel. Whilst they also act as a potential vector for antimicrobial resistant (AR) bacteria and antimicrobial resistance genes (ARGs). However, the understanding of the whole genome sequence of AR strains and ARGs-carrying plasmids collected from shared bikes is still lacking. Here, we used the HiSeq platform to sequence and analyze 24 Escherichia coli isolated from shared bikes around Metro Stations in Beijing. The isolates from shared bikes showed 14 STs and various genotypes. Two blaNDM-5 and blaCTX-M-199-producing ST167 E. coli have 16 resistance genes, four plasmid types and show >95% of similarities in core genomes compared with the ST167 E. coli strains from different origins. The blaNDM-5- or blaCTX-M-199-carrying plasmids sequencing by Nanopore were compared to plasmids with blaNDM-5- or blaCTX-M-199 originated from humans and animals. These two ST167 E. coli show high similarities in core genomes and the plasmid profiles with strains from hospital inpatients and farm animals. Our study indicated that ST167 E. coli is retained in diverse environments and carried with various plasmids. The analysis of strains such as ST167 can provide useful information for preventing or controlling the spread of AR bacteria between animals, humans and environments.

A clinical Pseudomonas juntendi strain with bla IMP-1 carried by an integrative and conjugative element in China.

ObjectiveTo precisely determine the species of a carbapenem-resistant Pseudomonas strain 1809276 isolated from the urine of a Chinese patient and analyze its integrative and conjugative element (ICE) 1276 formation mechanism.MethodsSingle-molecule real-time (SMRT) sequencing was carried out on strain 18091276 to obtain the complete chromosome and plasmid (pCN1276) sequences, and average nucleotide identity (ANI) was used for precise species identification. The ICEs in GenBank with the same integrase structure as ICE 1276 were aligned. At the same time, the transfer ability of blaIMP−1 and the antibiotic sensitivity of Pseudomonas juntendi 18091276 were tested.ResultsThis bacterium was P. juntendi, and its drug resistance mechanism is the capture of the accA4' gene cassette by the Tn402-like type 1 integron (IntI1-blaIMP−1) to form In1886 before its capture by the ΔTn4662a-carrying ICE 1276. The acquisition of blaIMP−1 confers carbapenem resistance to P. juntendi 18091276.ConclusionThe formation of blaIMP−1-carrying ICE 1276, its further integration into the chromosomes, and transposition and recombination of other elements promote bacterial gene accumulation and transmission.

Functional comparison of anti-restriction and anti-methylation activities of ArdA, KlcA, and KlcAHS from Klebsiella pneumoniae.

Anti-restriction proteins are typically encoded by plasmids, conjugative transposons, or phages to improve their chances of entering a new bacterial host with a type I DNA restriction and modification (RM) system. The invading DNA is normally destroyed by the RM system. The anti-restriction proteins ArdA, KlcA, and their homologues are usually encoded on plasmid of carbapenemase-resistant Klebsiella pneumoniae. We found that the plasmid sequence and restriction proteins affected horizontal gene transfer, and confirmed the anti-restriction and anti-methylation activities of ArdA and KlcA during transformation and transduction. Among the three anti-restriction proteins, ArdA shows stronger anti-restriction and anti-methylation effects, and KlcAHS was weaker. KlcA shows anti-methylation only during transformation. Understanding the molecular mechanism underlying the clinical dissemination of K. pneumoniae and other clinically resistant strains from the perspective of restrictive and anti-restrictive systems will provide basic theoretical support for the prevention and control of multidrug-resistant bacteria, and new strategies for delaying or even controlling the clinical dissemination of resistant strains in the future.

Increased Expression and Amplification of blaKPC-2 Contributes to Resistance to Ceftazidime/Avibactam in a Sequence Type 11 Carbapenem-Resistant Klebsiella pneumoniae Strain.

ABSTRACTCeftazidime/avibactam (CAZ/AVI) is regarded as an effective alternative antibiotic for the clinical treatment of Klebsiella pneumoniae carbapenemase (KPC)-producing isolates. As resistance has been reported in some strains, it is critical to understand the key mechanisms contributing to the acquired resistance to CAZ/AVI. From January 2018 to April 2020, 127 KPC-producing carbapenem-resistant Klebsiella pneumoniae strains (CRKPs) were isolated at a university hospital in Chongqing, China, and 25 strains showed reduced susceptibility to CAZ/AVI. All reduced-susceptibility CRKPs were deficient in Ompk35 and Ompk36 porins, and 24 strains had a premature termination at amino acid position 63 in Ompk35 and 134 to 135 glycine and aspartic acid (GD) insertion in OmpK36, while the blaKPC-2 expression level showed no significant difference compared to that of strain BAA-1705. Four reduced-susceptibility strains evolved resistance under selective pressure of CAZ/AVI with the blaKPC-2 expression level increased, and two of these strains had mutations in the Ω-loop. The study found a strain of CRKP55 with changes in the resistance phenotype during conjugation, evolving from reduced sensitivity to high-level resistance to CAZ/AVI. Through plasmid sequencing and reverse transcription-quantitative PCR, it was speculated that insertion sequence (IS)26-mediated blaKPC-2 gene amplification caused the MIC value change in the conjugant JKP55. Our findings illustrated the potential of CAZ/AVI resistance under antibiotic stress and demonstrated that IS26 may mediate blaKPC-2 replication transposition, leading to high-level resistance during horizontal gene transfer. Investigation of CAZ/AVI resistance mechanisms may offer a unique opportunity to study the horizontal evolutionary trajectories of K. pneumoniae high-risk clones.IMPORTANCE Klebsiella pneumoniae carbapenemase (KPC) production is the most common mechanism of K. pneumoniae resistance to carbapenems in China. Currently, CAZ/AVI is considered a potential alternative therapeutic option for infections caused by these isolates. However, there have been increasing reports of resistant or reduced-sensitivity strains since the approval of this agent. In this study, resistance to CAZ/AVI was induced under drug-selective pressure and was caused by blaKPC-2 overexpression and/or substitutions in the Ω-loop of KPC. Additionally, it was demonstrated that a conjugative plasmid carrying blaKPC-2 could transfer horizontally between species, and perhaps, IS26-derived tandem amplification of blaKPC-2 during this period led to high-level resistance to CAZ/AVI. Our research suggests that IS26-mediated resistance evolution may have important implications in guiding clinical antibiotic use.

Molecular characterization of florfenicol and oxazolidinone resistance in Enterococcus isolates from animals in China.

Florfenicol is widely used for the treatment of bacterial infections in domestic animals. The aim of this study was to analyze the molecular mechanisms of florfenicol and oxazolidinone resistance in Enterococcus isolates from anal feces of domestic animals. The minimum inhibitory concentration (MIC) levels were determined by the agar dilution method. Polymerase chain reaction (PCR) was performed to analyze the distribution of the resistance genes. Whole-genome sequencing and comparative plasmid analysis was conducted to analyze the resistance gene environment. A total of 351 non-duplicated enteric strains were obtained. Among these isolates, 22 Enterococcus isolates, including 19 Enterococcus. faecium and 3 Enterococcus. faecalis, were further studied. 31 florfenicol resistance genes (13 fexA, 3 fexB, 12 optrA, and 3 poxtA genes) were identified in 15 of the 19 E. faecium isolates, and no florfenicol or oxazolidinone resistance genes were identified in 3 E. faecalis isolates. Whole-genome sequencing of E. faecium P47, which had all four florfenicol and oxazolidinone resistance genes and high MIC levels for both florfenicol (256 mg/L) and linezolid (8 mg/L), revealed that it contained a chromosome and 3 plasmids (pP47-27, pP47-61, and pP47-180). The four florfenicol and oxazolidinone resistance genes were all related to the insertion sequences IS1216 and located on two smaller plasmids. The genes fexB and poxtA encoded in pP47-27, while fexA and optrA encoded in the conjugative plasmid pP47-61. Comparative analysis of homologous plasmids revealed that the sequences with high identities were plasmid sequences from various Enterococcus species except for the Tn6349 sequence from a Staphylococcus aureus chromosome (MH746818.1). The current study revealed that florfenicol and oxazolidinone resistance genes (fexA, fexB, poxtA, and optrA) were widely distributed in Enterococcus isolates from animal in China. The mobile genetic elements, including the insertion sequences and conjugative plasmid, played an important role in the horizontal transfer of florfenicol and oxazolidinone resistance.