Comparative Analysis Of Plasmids Research Articles

Comparative genomics of IncQ1 plasmids carrying blaGES variants from clinical and environmental sources in Brazil

IncQ-type plasmids have become important vectors in the dissemination of blaGES among different bacterial genera and species from different environments around the world, and studies estimating the occurrence of Guiana extended-spectrum (GES)-type β-lactamases are gaining prominence. We analyzed the genetic aspects of two IncQ1 plasmids harboring different blaGES variants from human and environmental sources. The blaGES variants were identified using polymerase chain reaction (PCR) in Aeromonas veronii isolated from hospital effluent and Klebsiella variicola isolated from a rectal swab of a patient admitted to the cardiovascular intensive care unit in a different hospital. Antimicrobial-susceptibility testing and transformation experiments were performed for phenotypic analysis. Whole-genome sequencing was performed using Illumina and Oxford Nanopore platforms. The comparative analysis of plasmids was performed using BLASTn, and the IncQ1 plasmids showed a high identity and similar size. A. veronii harbored blaGES-7 in a class 1 integron (In2061), recently described by our group, and K. variicola carried blaGES-5 in the known class 1 integron. Both integrons showed a fused gene cassette that encodes resistance to aminoglycosides and fluoroquinolones, with an IS6100 truncating the 3′-conserved segment. The fused genes are transcribed together, although the attC site is disrupted. These gene cassettes can no longer be mobilized. This study revealed a mobilome that may contribute to the dissemination of GES-type β-lactamases in Brazil. Class 1 integrons are hot spots for bacterial evolution, and their insertion into small IncQ-like plasmids displayed successful recombination, allowing the spread of blaGES variants in various environments. Therefore, they can become prevalent across clinically relevant pathogens.

Complex evolutionary trajectories in vivo of two novel KPC variants conferring ceftazidime-avibactam resistance

More and more ceftazidime-avibactam resistant KPC-producing K. pneumoniae have been reported with its widespread use, and the detection rate of KPC variants has increased dramatically. However, the evolutionary mechanism and fitness effects during KPC mutation remained unknown. Here, we report the complex in vivo evolutionary trajectories of two novel KPC variants, KPC-155 (L169P/GT242A) and KPC-185 (D179Y/GT242A), from Klebsiella pneumoniae in the same patient. The novel variants were shown to confer ceftazidime-avibactam resistance but restore carbapenem susceptibility based on the results of plasmid transformation assays, cloning experiments, and enzyme kinetic measurements. In vitro competition experiments highlighted the adaptive advantage conferred by strains carrying these KPC variants, which could lead to the rapid spread of these ceftazidime-avibactam resistant strains. The growth curve indicated that blaKPC-185 had better growth conditions at lower avibactam concentration compared to blaKPC-155, which was consistent with ceftazidime-avibactam use in vivo. In addition, replicative transposition of the IS26-flanked translocatable unit (IS26-ISKpn6-blaKPC-ISKpn27-IS26) also contributes to the blaKPC amplification and formation of two copies (blaKPC-2 and blaKPC-185), conferring both carbapenem and ceftazidime-avibactam resistance. However, strains with double copies showed reduced competitive advantage and configuration stability. The comparative plasmid analysis of IS26 group (IS26-blaKPC-IS26) and Tn1721 group (Tn1721-blaKPC-IS26) revealed that IS26-insertion could influence the distribution of resistance genes and ability of self-conjugation. The dynamic changes in blaKPC configuration highlight the need for consistent monitoring including antimicrobial susceptibility testing and determination of blaKPC subtypes—during clinical treatment, especially when ceftazidime-avibactam is administered.

Genomic characterization of two carbapenem-resistant Serratia marcescens isolates causing bacteremia: Emergence of KPC-2-encoding IncR plasmids.

The occurrence and transmission of carbapenemase-producing-Enterobacterales (CPE) on a global scale has become a major issue. Clinical reports are rarely providing information on the genomic and plasmid features of carbapenem-resistant Serratia marcescens. Our objective was to investigate the resistance and transmission dynamics of two carbapenem-resistant S. marcescens that are resistant to carbapenem and have caused bacteremia in China. Blood specimens were taken from two individuals with bacteremia. Multiplex PCR was employed to identify genes that code for carbapenemase. Antimicrobial susceptibility tests and plasmid analysis were conducted on S. marcescens isolates SM768 and SM4145. The genome of SM768 and SM4145 were completely sequenced using NovaSeq 6000-PE150 and PacBio RS II platforms. Antimicrobial resistance genes (ARGs) were predicted using the ResFinder tool. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and southern blotting were employed to analyze plasmids. Two S. marcescens that produced KPC-2 were identified from bloodstream infections. The antimicrobial susceptibility testing demonstrated that both of the isolates had a resistance to various antibiotics. The whole-genome sequence (WGS) and plasmid analysis revealed the presence of bla KPC-2-bearing IncR plasmids and multiple plasmid-borne antimicrobial resistance genes in the isolates. Our comparative plasmid analysis suggested that the two IncR plasmids identified in this study could be derived from a common ancestor. Our findings revealed the emergence of bla KPC-2-bearing IncR plasmid in China, which could be a hindrance to the transmission of KPC-2-producing S. marcescens in clinical settings.

Carbapenem resistance in Acinetobacter pittii isolates mediated by metallo-β-lactamases.

To characterize the genetic environment of metallo-β-lactamases (MBL) in carbapenem-resistant clinical Acinetobacter pittii isolates. Seventeen carbapenem-resistant A. pittii isolates harbouring an MBL were collected between 2010 and 2015 in Germany. Antimicrobial susceptibility testing was performed using agar dilution. Presence of MBLs was confirmed by PCR and their genetic location determined by S1-pulsed-field gel electrophoresis followed by Southern blot hybridization. Whole-genome sequencing was performed using the Miseq and MinION platforms. Isolates were typed using an ad hoc core genome MLST scheme. Conjugation into A. baumannii was tested by broth mating. In 10 isolates the MBL was plasmid-encoded and in seven isolates chromosomally encoded. blaGIM-1 and blaVIM-2 were plasmid-encoded, blaVIM-4 was chromosomally encoded, while blaNDM-1 was chromosomally encoded in four and plasmid-encoded in three isolates. Seven of ten plasmids were conjugative into A. baumannii. Although most isolates were unrelated, the backbones of the MBL-encoding plasmid showed >99% similarity and only differed in the MBL-encoding area. blaNDM-1-harbouring plasmids were highly similar to other plasmids from Acinetobacter isolates worldwide while the blaVIM-2- and blaGIM-1-encoding plasmids have not been described. These data show the existence of a promiscuous plasmid circulating in A. pittii isolates in Germany that differs only in the MBL-encoding region. Its plasmid backbone has been found globally among multiple Acinetobacter spp. These data should raise awareness of an epidemic conjugative plasmid that has independently acquired MBLs. We should also consider that future comparative plasmid analysis will look beyond solely the resistome and include the mobile elements carrying the resistance genes.

Intra- and Interspecies Spread of a Novel Conjugative Multidrug Resistance IncC Plasmid Coharboring blaOXA-181 and armA in a Cystic Fibrosis Patient.

ABSTRACTA novel multidrug resistance conjugative 177,859-bp IncC plasmid pJEF1-OXA-181 coharboring the carbapenemase-coding blaOXA181 and the aminoglycoside resistance 16S rRNA methyltransferase-coding armA genes was detected in two unrelated Escherichia coli gut isolates of ST196 and ST648, as well as two ST35 Klebsiella pneumoniae gut and sputum isolates of a cystic fibrosis patient. The armA gene was located within the antimicrobial resistance island ARI-A and the blaOXA181 gene, which was preceded by IS903 and ISEcp1Δ was inserted within the transfer genes region without affecting conjugation ability. Comparative plasmid analysis with other related IncC plasmids showed the presence of blaOXA181, as well as its integration site, are thus far unique for these types of plasmids. This study illustrates the potential of a promiscuous multidrug resistance plasmid to acquire antibiotic resistance genes and to disseminate in the gut of the same host.IMPORTANCE Colocalization of carbapenemases and aminoglycoside resistance 16S rRNA methylases on a multidrug resistance conjugative plasmid poses a serious threat to public health. Here, we describe the novel IncC plasmid pJEF1-OXA-181 cocarrying blaOXA-181 and armA as well as several other antimicrobial resistance genes (ARGs) in different Enterobacterales isolates of the sputum and gut microbiota of a cystic fibrosis patient. IncC plasmids are conjugative, promiscuous elements which can incorporate accessory antimicrobial resistance islands making them key players in ARGs spread. This plasmid was thus far unique among IncC plasmids to contain a blaOXA-181 which was integrated in the transfer gene region without affecting its conjugation ability. This study highlights that new plasmids may be introduced into a hospital through different species hosted in one single patient. It further emphasizes the need of continuous surveillance of multidrug-resistant bacteria in patients at risk to avoid spread of such plasmids in the health care system.

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.

Complete genome sequencing and comparative plasmid analysis of KPC-2-producing Klebsiella pneumoniae isolated from hospital sewage water in Japan

ObjectivesThe Klebsiella pneumoniae carbapenemase (blaKPC) gene is one of the most widespread carbapenemase genes in the world. However, there are few reports on KPC-producing bacteria in Japan. The aim of this study was therefore to investigate KPC-producing K. pneumoniae in Japan. MethodsA KPC-2-producingK. pneumoniae strain (KAM260) was isolated from hospital sewage water in Japan in 2018. The complete genome was determined by whole-genome sequencing. Subsequent comparative sequence analysis of the blaKPC-2-carrying plasmid was performed. ResultsKlebsiella pneumoniae KAM260, belonging to sequence type 3026 (ST3026), harboured the blaKPC-2 gene in 114.6-kbp plasmid pKAM260_2 with IncFIB(pQIL) and IncFII(K) replicons. pKAM260_2 was highly identical to pKpQIL-like plasmids, which carry blaKPC genes and have spread worldwide. pKAM260_2 had functional conjugation-associated genes and was transferable to Escherichia coli. ConclusionpKAM260_2, the self-transmissible plasmid carrying theblaKPC-2 gene, was detected from hospital sewage water in Japan and was characterised as a pKpQIL-like plasmid. This plasmid needs to be monitored in Japan in the future owing to its high diffusivity.

Whole-genome sequencing reveals resistome of highly drug-resistant retail meat and human Salmonella Dublin.

Non-typhoidal Salmonella (NTS) are a significant source of foodborne illness worldwide, with disease symptoms most often presenting as self-limiting gastroenteritis; however, occasionally the infection spreads and becomes invasive, frequently requiring anti-microbial treatment. The cattle-adapted Dublin serovar of NTS has commonly been associated with invasive illness and anti-microbial resistance (AMR). Here, the enhanced resolution conferred by whole-genome sequencing was utilized to elucidate and compare the resistome and genetic relatedness of 14 multidrug-resistant (MDR) and one pan-susceptible S. Dublin, isolated primarily in Pennsylvania, from fresh retail meat (one isolate) and humans (14 isolates). Twelve different genetic AMR determinants, including both acquired and chromosomal, were identified. Furthermore, comparative plasmid analysis indicated that AMR was primarily conferred by a putative IncA/C2 plasmid. A single pan-susceptible S. Dublin isolate, collected from the same timeframe and geographical region as the MDR isolates, did not carry an IncA/C2 replicon sequence within its genome. Moreover, the pan-susceptible isolate was genetically distinct from its MDR counterparts, as it was separated by ≥267 single nucleotide polymorphisms (SNPs), whereas there was a ≤38 SNP distance between the MDR isolates. Collectively, this data set advances our understanding of the genetic basis of the highly drug-resistant nature of S. Dublin, a serovar with significant public health implications.

Characterization of a novel bla NDM-5-harboring IncFII plasmid and an mcr-1-bearing IncI2 plasmid in a single Escherichia coli ST167 clinical isolate.

Background and purposeThe spread of the plasmid-mediated, colistin-resistance gene mcr-1 into New Delhi metallo-β-lactamase (NDM)-producing bacterial isolates can cause untreatable infections. In this study, we conducted a molecular characterization of a novel, conjugative, blaNDM-5-positive IncFII plasmid (pNDM-EC16-50) together with an mcr-1-bearing IncI2 plasmid in a single Escherichia coli ST167 clinical isolate EC16-50.Methods and resultsEC16-50, which belongs to the E. coli strain ST167 and phylogroup A, was identified to co-produce NDM-5 and MCR-1. S1-PFGE and Southern blotting showed that blaNDM-5 and mcr-1 genes were located oñ95 kb and ~65 kb plasmids, respectively. A conjugation assay revealed that both blaNDM-5- and mcr-1-bearing plasmids were self-transmissible. Comparative plasmid analysis suggested that blaNDM-5-harboring F2:A-:B-plasmid might have evolved from the well-reported NDM-carrying pMC-NDM-like plasmid via recombination with a locally emerged pSJ_94 plasmid, whereas the mcr-1-carrying IncI2 plasmid was similar to previously reported mcr-1-bearing plasmids in China.Conclusion and impactThis study represents the first report of the NDM-5 carrying Inc-FII- but not IncX3-type plasmid in an MCR-1-producing E. coli isolate. More striking was the dissemination of mcr-1 in a successful epidemic NDM-5-producing E. coli clone ST167, which could facilitate the spread of colistin resistance in carbapenemase-producing E. coli isolates.

Primordial origin and diversification of plasmids in Lyme disease agent bacteria

BackgroundWith approximately one-third of their genomes consisting of linear and circular plasmids, the Lyme disease agent cluster of species has the most complex genomes among known bacteria. We report here a comparative analysis of plasmids in eleven Borreliella (also known as Borrelia burgdorferi sensu lato) species.ResultsWe sequenced the complete genomes of two B. afzelii, two B. garinii, and individual B. spielmanii, B. bissettiae, B. valaisiana and B. finlandensis isolates. These individual isolates carry between seven and sixteen plasmids, and together harbor 99 plasmids. We report here a comparative analysis of these plasmids, along with 70 additional Borreliella plasmids available in the public sequence databases. We identify only one new putative plasmid compatibility type (the 30th) among these 169 plasmid sequences, suggesting that all or nearly all such types have now been discovered. We find that the linear plasmids in the non-B. burgdorferi species have undergone the same kinds of apparently random, chaotic rearrangements mediated by non-homologous recombination that we previously discovered in B. burgdorferi. These rearrangements occurred independently in the different species lineages, and they, along with an expanded chromosomal phylogeny reported here, allow the identification of several whole plasmid transfer events among these species. Phylogenetic analyses of the plasmid partition genes show that a majority of the plasmid compatibility types arose early, most likely before separation of the Lyme agent Borreliella and relapsing fever Borrelia clades, and this, with occasional cross species plasmid transfers, has resulted in few if any species-specific or geographic region-specific Borreliella plasmid types.ConclusionsThe primordial origin and persistent maintenance of the Borreliella plasmid types support their functional indispensability as well as evolutionary roles in facilitating genome diversity. The improved resolution of Borreliella plasmid phylogeny based on conserved partition-gene clusters will lead to better determination of gene orthology which is essential for prediction of biological function, and it will provide a basis for inferring detailed evolutionary mechanisms of Borreliella genomic variability including homologous gene and plasmid exchanges as well as non-homologous rearrangements.

Environmental emission of multiresistant Escherichia coli carrying the colistin resistance gene mcr-1 from German swine farms.

Pigs have been the focus of the worldwide spread of colistin resistance. However, there is little information on the transmission of mcr-1 -containing bacteria into the environment of pig farms. We therefore rescreened environmental Escherichia coli isolates from the surrounding farm areas of three previously mcr-1 -positive swine herds in Germany. Thirty-five mixed bacterial cultures obtained from boot swabs, flies, dog faeces and manure from three pig farms in Germany in 2011-12 were non-selectively recultivated and the presence of the mcr-1 gene was checked by real-time PCR. After separation, single E. coli colonies were subsequently isolated and the presence of mcr-1 was confirmed by PCR and sequencing. In addition, phenotypic antimicrobial resistance screening and WGS followed by phylogenetic analysis and resistance genotyping as well as plasmid typing were performed. Seven mcr-1 -positive E. coli strains originating from environmental boot swabs, dog faeces, stable flies and manure were found. The isolates belonged to five different STs (ST10, ST1011, ST1140, ST5281 and ST342) and harboured extensive additional resistance genes. Comparative plasmid analysis predominantly located mcr-1 on IncX4 plasmids, which are strongly related to a recently described plasmid of human clinical origin (pICBEC72Hmcr). WGS-based analysis of the environmental E. coli isolates of farm surroundings showed clear links to mcr-1 -harbouring E. coli recovered from pig production in Europe as well as from human clinical isolates worldwide, presenting another piece of the puzzle, which further complicates the rapidly evolving epidemiology of plasmid-mediated colistin-resistant E. coli strains.

The Complete Sequence and Comparative Analysis of a Multidrug-Resistance and Virulence Multireplicon IncFII Plasmid pEC302/04 from an Extraintestinal Pathogenic Escherichia coli EC302/04 Indicate Extensive Diversity of IncFII Plasmids.

Extraintestinal pathogenic Escherichia coli (ExPEC) that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA, and FIB) with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as blaTEM−1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR) to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD) which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM)-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA)-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok) and a plasmid partitioning system, ParAB, and PsiAB, which are important for plasmid maintenance were also found. Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of clinical importance. Such phenomenon is bothersome when the plasmids are transmissible, facilitating the spread of virulence and resistance plasmids among pathogenic bacteria. Notably, certain TA systems are more commonly found in particular ExPEC plasmid types, indicating the possible relationships between certain TA systems and ExPEC pathogenesis.

Structural and functional analysis of mitochondrial plasmids in Claviceps purpurea.

Several strains of Claviceps purpurea, a phytopathogenic Ascomycete, contain mitochondrial (mt) plasmids in high molar excess relative to mtDNA. Comparative analysis of plasmids of four strains of different geographic origin revealed that all the plasmids are structurally related (size; linearity; restriction map; probably 5'-linked terminal protein; terminal inverted repeats, TIRs); two of them are even identical, indicating a possible mobility of these genetic entities. In strain K it was shown that plasmid titres are comparably high in axenic cultures and in parasitic structures (sclerotia). Detailed analysis of plasmid pClK1 proved the existence of a perfect TIR of 327 bp; the plasmid's structure and details of its nucleotide sequence indicate a replication modus comparable to that of adenoviruses. pClK1 is almost completely transcribed resulting in two major transcripts of 3.5 and 3.15 kb, respectively. In plasmid-free strains (cured by ethidium bromide treatment) these mRNAs are not detectable; nevertheless they show no significant difference in phenotype. As judged from their structural properties they could be derived from viral ancestors. In this context the plasmids' close relationship to mt plasmids of higher plants may be of special interest.