Resistance Plasmid Research Articles

Dissemination of virulence and resistance genes among Klebsiella pneumoniae via outer membrane vesicle: An important plasmid transfer mechanism to promote the emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae.

Klebsiella pneumoniae is well-known opportunistic enterobacteria involved in complex clinical infections in humans and animals. The domestic animals might be a source of the multidrug-resistant virulent K. pneumoniae to humans. K. pneumoniae infections in domestic animals are considered as an emergent global concern. The horizontal gene transfer plays essential roles in bacterial genome evolution by spread of virulence and resistance determinants. However, the virulence genes can be transferred horizontally via K. pneumoniae-derived outer membrane vesicles (OMVs) remains to be unreported. In this study, we performed complete genome sequencing of two K. pneumoniae HvK2115 and CRK3022 with hypervirulent or carbapenem-resistant traits. OMVs from K. pneumoniae HvK2115 and CRK3022 were purified and observed. The carriage of virulence or resistance genes in K. pneumoniae OMVs was identified. The influence of OMVs on the horizontal transfer of virulence-related or drug-resistant plasmids among K. pneumoniae strains was evaluated thoroughly. The plasmid transfer to recipient bacteria through OMVs was identified by polymerase chain reaction, pulsed field gel electrophoresis and Southern blot. This study revealed that OMVs could mediate the intraspecific and interspecific horizontal transfer of the virulence plasmid phvK2115. OMVs could simultaneously transfer two resistance plasmids into K. pneumoniae and Escherichia coli recipient strains. OMVs-mediated horizontal transfer of virulence plasmid phvK2115 could significantly enhance the pathogenicity of human carbapenem-resistant K. pneumoniae CRK3022. The CRK3022 acquired the virulence plasmid phvK2115 could become a CR-hvKp strain. It was critically important that OMVs-mediated horizontal transfer of phvK2115 lead to the coexistence of virulence and carbapenem-resistance genes in K. pneumoniae, resulting in the emerging of carbapenem-resistant hypervirulent K. pneumoniae.

Leuconostoc mesenteroides LVBH107 Antibacterial Activity against Porphyromonas gingivalis and Anti-Inflammatory Activity against P. gingivalis Lipopolysaccharide-Stimulated RAW 264.7 Cells.

Probiotics, active microorganisms benefiting human health, currently serve as nutritional supplements and clinical treatments. Periodontitis, a chronic infectious oral disease caused by Porphyromonas gingivalis (P. gingivalis), activates the host immune response to release numerous proinflammatory cytokines. Here, we aimed to clarify Leuconostoc mesenterica (L. mesenteroides) LVBH107 probiotic effects based on the inhibition of P. gingivalis activities while also evaluating the effectiveness of an in vitro P. gingivalis lipopolysaccharide-stimulated RAW 264.7 cell-based inflammation mode. L. mesenteroides LVBH107 survived at acid, bile salts, lysozyme, and hydrogen peroxide conditions, auto-aggregated and co-aggregated with P. gingivalis, exhibited strong hydrophobicity and electrostatic action, and strongly adhered to gingival epithelial and HT-29 cells (thus exhibiting oral tissue adherence and colonization abilities). Moreover, L. mesenteroides LVBH107 exhibited sensitivity to antibiotics erythromycin, doxycycline, minocycline, ampicillin, and others (thus indicating it lacked antibiotic resistance plasmids), effectively inhibited P. gingivalis biofilm formation and inflammation (in vitro inflammation model), reduced the secretion of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) and inflammatory mediators (NO and PGE2), and decreased the expression levels of inflammation related genes. Thus, L. mesenterica LVBH107 holds promise as a probiotic that can inhibit P. gingivalis biofilm formation and exert anti-inflammatory activity to maintain oral health.

Review on Recent Advancement of Therapeutic Interventions in Combating Multidrug Resistant Bacteria

Antibiotics is a class of drug that plays an eminent role in combating the deadly infections caused by harmful pathogenic organisms. However, irrational use of these antibiotics leads to multidrug resistance in bacterial species which in turn contributes to the morbidity and mortality worldwide, due to which they are referred to as superbugs. Thus, multidrug resistance is known as the resistance of a microorganism to different initially sensitive antibiotics. Though research has led to the development of new antibiotics and its derivatives, the development of new antibiotics is challenged at every step due to the emergence of microbial resistance. The emergence of drug resistance resulted due to the presence of Drug Resistant Plasmid, transposon, antibiotic resistant cassette and further the resistant phenotype can be transferred from resistant to a sensitive microorganism making sensitive organism resistant. Some bacteria acquire the property of being Multi Drug Resistant due to the presence of efflux transporter proteins which can expel antibiotics in a nonspecific manner and overcoming such medical emergencies is the demand of the era. Thus, the lack of novel antibiotics has created the need for certain technological tools to combat this deadly phenomenon. Among them, anti-microbial peptide, anti-virulence compounds, phage therapy, nanotechnology and CRISPR-Cas system are the most commonly used techniques to combat multidrug resistance. The present review highlights different multidrug resistance tools such as the application of Phage therapy, use of nanotechnology, CRISPR-Cas system and antimicrobial peptides together, their working mechanism, utility, efficacy, credibility and relevance along with the modifications needed in the techniques for improvisation.

Co-transfer of mcr-8 with blaNDM-1 or tmexCD1-toprJ1 by plasmid hybridisation

Carbapenems, tigecycline and colistin are three important antimicrobial agents for the treatment of clinical infections caused by multidrug-resistant Enterobacteriaceae. Here we characterised the formation of hybrid plasmids containing mcr-8 and blaNDM-1 or tmexCD1-toprJ1 that could confer resistance to colistin and carbapenems or tigecycline. More specifically, these clinically important genes could be co-transferred through IS26- and ltrA-mediated plasmid fusion to clinical isolates during conjugation under single drug (colistin) selection, following which the recipient strains became carbapenem- or tigecycline-resistant. The transferability and stability of these hybrid multidrug resistance (MDR) plasmids depend on the bacterial host and the presence of antibiotics. Further evolution and adaptation of these hybrid plasmids may facilitate their emergence and spread, which is of great concern for clinical therapy.

Genomic Analysis of KPC-2-Producing Klebsiella pneumoniae ST11 Isolates at the Respiratory Department of a Tertiary Care Hospital in Beijing, China.

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is an important pathogen causing hospital-associated outbreaks worldwide. The spread of K. pneumoniae carbapenemase-2 (KPC-2)-producing CRKP is primarily associated with sequence type (ST) 11.MethodsA total of 152 KPC-2-producing K. pneumoniae ST11 isolates were collected from the respiratory department of a tertiary care hospital in Beijing, China between 2009 and 2018. The genome sequencing of these isolates was performed on the HiSeq X Ten sequencer. Multilocus sequence typing (MLST), capsular type, plasmid replicon types and resistance genes were identified. Fifteen isolates were selected for the subsequent single-molecule real-time (SMRT) sequencing on the PacBio RS II. Alignment of the complete sequences of the plasmids carrying blaKPC–2 and/or virulence genes was performed by using BRIG and Easyfig.ResultsFrom 2012 to 2018, the detection rate of the blaKPC–2-carrying CRKP rose rapidly from 3.3 to 28.1%. KPC-2-producing K. pneumoniae ST11 isolates were dominant in CRKP, which emerged in 2012 and caused several outbreaks. Most isolates exhibited multidrug-resistant to commonly used antibiotics, while all the isolates remained susceptible to tigecycline and polymyxin B. The single nucleotide polymorphism (SNP) analysis showed that all these 152 KPC-2-producing K. pneumoniae ST11 isolates could be divided into three genetically distinct clades (A, B, and C) and eleven subclades (A1–A9 and B1–B2). The majority belonged to clade A with KL47 serotype (n = 117, 77.0%), while KL64 and KL16 were identified in clades B and C, respectively. The blaKPC–2-carrying plasmids exhibited diverse types, namely, IncFII (pHN7A8)/IncR(6/15), IncFII (pHN7A8)/IncpA1763–KPC (5/15), IncFII (pHN7A8) (1/15), IncR (1/15), and IncpA1763–KPC (1/15). The genetic environment of blaKPC–2 showed nine IS26-based composite transposons, which had a basic core structure ISKpn27-blaKPC–2-ΔISKpn6. About 27.6% (42/152) isolates co-carried 2 to 4 virulence marker genes (namely, peg344, iucA, iroB, rmpA, and rmpA2) for hvKp strains. At least three isolates were identified to harbor virulence gene-carrying plasmids.ConclusionKPC-2-producing K. pneumoniae ST11 was highly heterogeneous in our hospital. Transmission of these strains was mainly mediated by twelve high-risk clones. The blaKPC–2-carrying plasmids and genetic environment of blaKPC–2 genes exhibited active evolution in K. pneumoniae ST11. More attention should be paid to the tendency of KPC-2-ST11 to acquire hypervirulent plasmids.

Mating pair stabilization mediates bacterial conjugation species specificity

Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating the spread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane (OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNA transfer. We show that TraN from the plasmid pKpQIL (Klebsiella pneumoniae) interacts with OmpK36, plasmids from R100-1 (Shigella flexneri) and pSLT (Salmonella Typhimurium) interact with OmpW, and the prototypical F plasmid (Escherichia coli) interacts with OmpA. Cryo-EM analysis revealed that TraNpKpQIL interacts with OmpK36 through the insertion of a β-hairpin in the tip of TraN into a monomer of the OmpK36 porin trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified a fourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classification scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNβ (OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect the distribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pair stabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emerging resistance plasmids in high-risk bacterial pathogens.

Fusion plasmid carrying the colistin resistance gene mcr of Escherichia coli isolated from healthy residents

The extensive spread of colistin resistance represents an enormous concern to infectious disease treatment, because colistin is one of the few effective antibiotics against multidrug-resistant bacterial infections, including carbapenem-resistant bacteria. This dissemination can be caused by plasmid transfer containing the colistin resistance gene mcr. Therefore, the plasmid host range affects horizontal gene transfer. This study reports a fusion plasmid of different incompatibility types, which could easily expand the plasmid host range, allowing widespread mcr prevalence in the microbial community. Genome sequences of colistin-resistant Escherichia coli isolates from stool specimens of healthy human residents in Ecuador were determined using the DNBSEQ and MinION platforms. Hybrid genome assembly was performed using Unicycler, and the genomes were annotated using DFAST. Genome analysis was performed using the Geneious Prime software. Two colistin-resistant E. coli strains isolated separately from different residents presented mcr-carrying plasmids with fused different incompatibility types, IncFIA, IncHIIA, and IncHIIB. The phylogenies of these host bacteria were different. The sizes of the mcr-carrying fusion plasmids pLR-06 and pLR-50 with the full Tn6330 mcr-transposon were 260 Kbp and 198 Kbp, respectively. Both fusion plasmids possessed other resistance genes, including tet(B), tet(M), blaTEM-1b, sul3, cmlA1, aadA1, aadA2, fosA3, and dfrA12. This is the first report of a fusion plasmid comprising different incompatibility types with mcr from colistin-resistant E. coli strains isolated from community residents. The mcr fusion plasmid may play a crucial role in achieving horizontal mcr transmission and the evolution of the multidrug resistance plasmid among hosts.

Large-Scale Analysis of Fitness Cost of tet(X4)-Positive Plasmids in Escherichia coli.

Tigecycline is one of important antimicrobial agents for the treatment of infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However, the emergence and prevalence of plasmid-mediated tigecycline resistance gene tet(X4) are threatening human and animal health. Fitness cost elicited by resistance plasmids is a key factor affecting the maintenance and transmission of antibiotic resistance genes (ARGs) in the host. A comparative analysis of the fitness cost of different types of tet(X4)-positive plasmids is helpful to understand and predict the prevalence of dominant plasmids. In this study, we performed a large-scale analysis of fitness cost of tet(X4)-positive plasmids origin from clinical isolates. These plasmids were successfully electroporated into a reference strain Escherichia coli TOP10, and a series of transformants carrying the tet(X) gene were obtained. The effects of tet(X4)-positive plasmids on the growth rate, plasmid stability, relative fitness, biofilm formation, and virulence in a Galleria mellonella model were evaluated. Consequently, we found that these plasmids resulted in varying degrees of fitness cost on TOP10, including delayed bacterial growth and attenuated virulence. Out of these plasmids, tet(X4)-harboring IncFII plasmids showed the lowest fitness cost on the host. Furthermore, by means of experimental evolution in the presence of commonly used drugs in clinic, the fitness cost of tet(X4)-positive plasmids was substantially alleviated, accompanied by increased plasmid stability. Collectively, our data reveal the differential fitness cost caused by different types of tet(X4)-positive plasmids and suggest that the wide use of tetracycline antibiotics may promote the evolution of plasmids.

How heavy metal stress promotes dissemination of antibiotic resistance genes in the activated sludge process

Heavy metals have been recently revealed as promoters to antibiotic resistance gene (ARG) dissemination in water environment, but their influence on ARG transfer in the activated sludge process has not been clear. In this study, a set of sequencing batch reactors (SBRs) and micro-scale microfluidic chips were established to quantify the impacts of heavy metals (0.5 mM of Pb, 0.1 mM of As, and 0.005 mM of Hg) on the ARG spreading in the activated sludge consortium. Under heavy metal stress, transfer frequencies were 1.7–3.6 folds increase compared to the control. Gram-negative bacteria increased significantly after heavy metal added, which were more prone to receiving resistant plasmid from donors. Meanwhile, the relative expression of genes related to conjugation changed in activated sludge, especially the expression of outer membrane protein and oxidative stress regulatory genes increased by 2.9–7.4 folds and 7.8–13.1 folds, respectively. Furthermore, using microfluidic chips, the dynamics of ARG transfer was observed at single cell level under heavy metal pressure. Heavy metals firstly promoted conjugation and then vertical gene transfer played an important part for ARG spreading. The results provided in-depth understanding of the influence of heavy metals on ARG behavior in the environment.

Genomic and Phenotypic Characterisation of Multi-Drug ResistantEscherichia coli carried by meat handlers and slaughterhouse meat from North-Western Tanzania

Our research is part of a larger project Supporting the National Action Plan on Antimicrobial Resistance (SNAP-AMR) in Tanzania. The aspect of this work is to determine the AMR carriage and associated mobile genetic elements of extended spectrum β-lactamase (ESBL) producing Escherichia coli within the community of Mwanza, in North-Western Tanzania. From 54 slaughterhouses a total of 322 E. coli isolates were obtained from raw meat, with 210 originating from meat handlers. Initial antimicrobial susceptibility testing identified 71 (22%) and 27 (13%) of the meat and meat handler isolates, respectively, as being ESBL E. coli. ST2852 was the most abundant MLST, followed by globally disseminated and ESBL associated ST38. Whole genome sequencing and phenotypic analysis revealed 88% of our isolates were resistant to three or more antimicrobial classes. On average, our isolates carried at least 10 different AMR associated genes, with 80% having one or more AMR conferring chromosomal mutation. In relation to ESBL, 88 out of 98 of the isolates carried the globally disseminated allotype blaCTX-M-15, with over 55% of the isolates encoding blaCTX-M-15on multi-drug resistant (MDR) plasmids. The remaining strains encoded one of three different blaCTX-Mallotypes. Our data highlights high prevalence of MDR ESBL E. coli within the community and its potential risk of transmission through the food chain. In our future work, we will combine these community carriage data with our work on ESBL Enterobacteriaceae carriage and infection within healthcare settings, to evaluate the genomic epidemiology of ESBL organisms in this region.

Studies on the Transmission of a Tigecycline Resistance-Mediating tet(A) Gene Variant from Enterobacter hormaechei via a Two-Step Recombination Process.

ABSTRACTTo investigate the contribution of a tet(A) variant to tigecycline resistance in Enterobacter hormaechei and the recombination events that occurred during transmission of this variant. MICs were determined by broth microdilution. E. hormaechei G17 was characterized by PCR, transfer assay, S1-PFGE, Southern blot hybridization, and WGS analysis. A tet(A) variant conferring resistance to tigecycline was present in E. hormaechei G17. This strain harbored two resistance plasmids (pG17-1, 264,084 bp and pG17-2, 68,610 bp) and its E. coli transformant Tm-G17TGC one resistance plasmid (pTm-G17, 93,013 bp). The comparative analysis of pG17-1, pG17-2, and pTm-G17 showed that a tet(A) variant-carrying multiresistance gene cluster (~23 kb) originating from pG17-1 had integrated into pG17-2, forming the novel plasmid pTm-G17. In a first step, this multiresistance gene cluster was excised from pG17-1 by recombination of homologous sequences, including △TnAs1 at both termini, thereby generating an unconventional circularizable structure (UCS). In a second step, this UCS integrated into pG17-2 via recombination between homologous sequences, including IS26 present on both, the UCS and pG17-2, thereby giving rise to the new plasmid pTm-G17. In summary, a tet(A) variant conferring resistance to tigecycline was reported in E. hormaechei. Transfer of a tet(A) variant-carrying multiresistance gene cluster between plasmids occurred in a two-step recombination process, in which homologous sequences, including either △TnAs1 or IS26, were involved.IMPORTANCE Tigecycline is an important last-resort broad spectrum antimicrobial agent. This study describes the two-step recombination processes resulting in the transfer of the tet(A) variant gene between different plasmids in E. hormaechei, which depicts the role of recombination processes in the generation of UCSs and new plasmids, both carrying a tet(A) variant conferring resistance to tigecycline. Such processes enhance the dissemination of resistance genes, which is of particular relevance for resistance genes, such as the tet(A) variant. The presence and transmission of a tet(A) variant in E. hormaechei will compromise the efficacy of tigecycline treatment for E. hormaechei associated infection.

Genetic Resistance Determinants in Clinical Acinetobacter pittii Genomes.

Antimicrobial-resistant pathogenic bacteria are an increasing problem in public health, especially in the healthcare environment, where nosocomial infection microorganisms find their niche. Among these bacteria, the genus Acinetobacter which belongs to the ESKAPE pathogenic group harbors different multi-drug resistant (MDR) species that cause human nosocomial infections. Although A. baumannii has always attracted more interest, the close-related species A. pittii is the object of more study due to the increase in its isolation and MDR strains. In this work, we present the genomic analysis of five clinically isolated A. pittii strains from a Spanish hospital, with special attention to their genetic resistance determinants and plasmid structures. All the strains harbored different genes related to β-lactam resistance, as well as different MDR efflux pumps. We also found and described, for the first time in this species, point mutations that seem linked with colistin resistance, which highlights the relevance of this comparative analysis among the pathogenic species isolates.

A Small KPC-2-Producing Plasmid in Klebsiella pneumoniae: Implications for Diversified Vehicles of Carbapenem Resistance.

ABSTRACTThe convergence of hypervirulence to carbapenem-resistant K. pneumoniae (CRKP) in a highly transmissible ST11 clone poses a great challenge to public health and anti-infection therapy. Recently, we revealed that an expanding repertoire of diversified KPC-2-producing plasmids occurs in these high-risk clones. Here, we report a clinical case infected with a rare isolate of ST437 CRKP, K186, which exhibited KPC-2 production. Apart from its 5,322,657-bp long chromosome, whole-genome sequencing of strain K186 elucidated three distinct resistance plasmids (designated pK186_1, pK186_2, and pK186_KPC, respectively). Unlike the prevalently larger form of KPC-2-producing plasmids (~120 to ~170 kb) earlier we observed, pK186_KPC is an IncN-type, small plasmid of 26,012bp in length. Combined with the colinear alignment of plasmid genome, the analyses of insertion sequences further suggested that this carbapenem-resistant pK186_KPC might arise from the cointegration of its ancestral IncN and IncFII plasmids, exclusively relying on IS26-based transposition events. Taken together, the result represents an unusual example of blaKPC-2-bearing small plasmids, and highlights an ongoing arsenal of diversified carriers benefiting the transferability of KPC-2 carbapenem resistance.IMPORTANCE A rare ST437 isolate termed K186 was clinically determined which was unlike ST11, the dominant sequence type of CRKP. Whole-genome sequencing enabled us to discover three distinct resistance plasmids, namely, pK186_1, pK186_2, and pK186_KPC. Among them, pK186_KPC appears as a unique plasmid ~26 kb in size, much smaller than the prevalent forms (~120 to ~170 kb). Intriguingly, genetic analysis suggests that it might originate from Proteus mirabilis. This result constitutes an additional example of differentiated plasmid vehicles dedicated to the emergence and dissemination of KPC-2 carbapenem resistance.

Nosocomial transmission and rearrangement of large resistance-virulence hybrid plasmids between two bacteremic ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae strains with low fitness cost

ObjectivesTo characterize nosocomial transmission and rearrangement of the resistance-virulence plasmid between two ST11-K64 carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strains (JX-CR-hvKP-10 and JX-CR-hvKP-9) with low fitness. MethodsPhenotypic tests were used to assess the virulence of JX-CR-hvKP-10 and JX-CR-hvKP-9. Whole-genome sequencing was used to analyze JX-CR-hvKP-10 and JX-CR-hvKP-9 chromosomes and plasmids. Fitness and conjugation experiments were also conducted using these two CR-hvKP isolates. ResultsPhenotypic tests indicated that both JX-CR-hvKP-10 and JX-CR-hvKP-9 were multidrug-resistant and hypervirulent K. pneumoniae. Whole-genome sequencing and clinical information demonstrated that the super large resistance-virulence fusion plasmid pJX10-1 formed precisely by the fusion of pJX9-1 and pJX9-2 via the nosocomial transmission. Interestingly pJX9-1 itself was also a classic resistance-virulence fusion plasmid by way of the blaKPC-carrying resistance plasmid and pLVPK-like virulence plasmid. Compared with classic K. pneumoniae ATCC700603, fitness analysis revealed no significant difference in growth was observed between JX-CR-hvKP-10 and JX-CR-hvKP-9. ConclusionNosocomial transmission and rearrangement of a blaKPC-harboring plasmid and a pLVPK-like virulence plasmid with a low fitness cost in ST11 K. pneumoniae enhances drug resistance and virulence simultaneously. Thus, active surveillance of this hybrid plasmid is needed to prevent these efficient resistance-virulence plasmids from disseminating in hospital settings.

A widespread family of WYL-domain transcriptional regulators co-localizes with diverse phage defence systems and islands.

Bacteria are under constant assault by bacteriophages and other mobile genetic elements. As a result, bacteria have evolved a multitude of systems that protect from attack. Genes encoding bacterial defence mechanisms can be clustered into ‘defence islands’, providing a potentially synergistic level of protection against a wider range of assailants. However, there is a comparative paucity of information on how expression of these defence systems is controlled. Here, we functionally characterize a transcriptional regulator, BrxR, encoded within a recently described phage defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a combination of reporters and electrophoretic mobility shift assays, we discovered that BrxR acts as a repressor. We present the structure of BrxR to 2.15 Å, the first structure of this family of transcription factors, and pinpoint a likely binding site for ligands within the WYL-domain. Bioinformatic analyses demonstrated that BrxR-family homologues are widespread amongst bacteria. About half (48%) of identified BrxR homologues were co-localized with a diverse array of known phage defence systems, either alone or clustered into defence islands. BrxR is a novel regulator that reveals a common mechanism for controlling the expression of the bacterial phage defence arsenal.

Submarine Outfalls of Treated Wastewater Effluents are Sources of Extensively- and Multidrug-Resistant KPC- and OXA-48-Producing Enterobacteriaceae in Coastal Marine Environment.

The rapid and ongoing spread of carbapenemase-producing Enterobacteriaceae has led to a global health threat. However, a limited number of studies have addressed this problem in the marine environment. We investigated their emergence in the coastal waters of the central Adriatic Sea (Croatia), which are recipients of submarine effluents from two wastewater treatment plants. Fifteen KPC-producing Enterobacteriaceae (nine Escherichia coli, four Klebsiella pneumoniae and two Citrobacter freundii) were recovered, and susceptibility testing to 14 antimicrobials from 10 classes showed that four isolates were extensively drug resistant (XDR) and two were resistant to colistin. After ERIC and BOX-PCR typing, eight isolates were selected for whole genome sequencing. The E. coli isolates belonged to serotype O21:H27 and sequence type (ST) 2795, while K. pneumoniae isolates were assigned to STs 37 and 534. Large-scale genome analysis revealed an arsenal of 137 genes conferring resistance to 19 antimicrobial drug classes, 35 genes associated with virulence, and 20 plasmid replicons. The isolates simultaneously carried 43–90 genes encoding for antibiotic resistance, while four isolates co-harbored carbapenemase genes blaKPC-2 and blaOXA-48. The blaOXA-48 was associated with IncL-type plasmids in E. coli and K. pneumoniae. Importantly, the blaKPC-2 in four E. coli isolates was located on ~40 kb IncP6 broad-host-range plasmids which recently emerged as blaKPC-2 vesicles, providing first report of these blaKPC-2-bearing resistance plasmids circulating in E. coli in Europe. This study also represents the first evidence of XDR and potentially virulent strains of KPC-producing E. coli in coastal waters and the co-occurrence of blaKPC-2 and blaOXA-48 carbapenemase genes in this species. The leakage of these strains through submarine effluents into coastal waters is of concern, indicating a reservoir of this infectious threat in the marine environment.

Whole-genome sequencing analysis of a rare Salmonella diarizonae clinical strain carrying multiple plasmids and novel gene cassettes

Human infection caused by an uncommon Salmonella enterica subsp. diarizonae (hereafter S. diarizonae) is rising. However, knowledge concerning S. diarizonae is still limited. This study aimed to investigate the genomic features of S. diarizonae S499 isolated from a child patient with gastroenteritis symptom in China. The antimicrobial susceptibility of S. diarizonae S499 was determined by microdilution broth assay. Whole genome was sequenced using Illumina HiSeq X-10 and PacBio RS II platforms and was de novo assembled using Unicycler and SPAdes. Conjugation experiment was performed by a broth mating method. S. diarizonae S499 was a multi-drug resistant (MDR) isolate and showed resistance to all cephalosporin drugs tested. Six plasmids (pS0499A, pS0499B, pS0499C, pS0499D, pS0499E and pS0499F) were identified. A rare gene cassette IS26-blaCTX-M-55-wbuc-△blaTEM-1-IS26-intI1 was repeatedly inserted into pS0499A three times in one locus and reversely inserted into plasmid pS0499D. That enhanced cephalosporin resistance. To the best of our knowledge, this finding has not been reported previously. Both pS0499A and pS0499B contained multiple resistance genes and could transfer to recipient strain E. coli EC600. This article reported the genome features of S. diarizonae S499, which contained four resistant plasmids including a novel plasmid pS0499A with a novel gene cassette rearrangement. These data could contribute to a better understanding of the antimicrobial resistance mechanisms and transmission dynamics of S. diarizonae.

Characterization of Putative Transcriptional Regulator Orf90

The rapid emergence of antibiotic resistant bacteria is a pressing concern, threatening public health and food security worldwide. Antibiotic resistance commonly originates through conjugative plasmid transfer (CPT), where plasmids are transferred and replicated between bacteria. Although the overall mechanism of CPT is known, many plasmid‐encoded proteins essential for CPT remain unstudied. One such unstudied plasmid encoded protein is orf90, a putative transcriptional regulator located on the multiresistant plasmid pSK41 from Staphylococcus aureus. Methicillin resistant S. aureus(MRSA) is considered a serious threat by the Centers for Disease Control and Prevention (CDC), and how it acquires resistance plasmids is still an open area of study. In this investigation, we sought to gain valuable insight into the structure and character of orf90. We successfully expressed and purified two orf90 constructs, an untagged version (His‐orf90) and a maltose binding protein tagged version (His‐MBP‐orf90). We hypothesize that orf90 will bind the pSK41 origin of transfer and that one of our constructs will yield usable crystals for structure solution. Crystallography trials for His‐Orf90 did not yield macromolecular crystals, possibly due to the sample purity. Binding of His‐Orf90 to a 205 bp region of pSK41 containing the conserved origin of transfer (oriT) was confirmed via EMSA (Electrophoretic Mobility Shift Assay). Additionally, crystallization screens with His‐MBP‐orf90 identified three favorable conditions for crystallization. We have shown that orf90 binds to the pSK41 oriT and that His‐MBP‐orf90 is a promising crystallization construct. Characterizing pSK41 Orf90’s structure and function will further enhance our understanding of CPT, potentially yielding useful data for research aimed at combating antibiotic resistance.

A mathematical model of plasmid-carried antibiotic resistance transmission in two types of cells

Abstract Antibiotic resistance is a significant public health problem. When resistance genes are being carried on plasmids, the spread can be greatly accelerated. In this paper, the transmission of antibiotic resistance in two types of cells is discussed. A mathematical model is established to describe the dynamics of the transmission of plasmids. The effects of different parameters on the stable solution and sensitivity analysis are studied by numerical simulation. The conclusions show that the concentration of antibiotics must reach a certain level to kill the pathogenic bacteria. If the concentration of antibiotics is not high to a certain extent, the treatment becomes ineffective. If the cost of cells carried on plasmids and the rate of resistance plasmids segregation too high, the drug-resistant cells will gradually die out in the system. The rate of horizontal transfer of resistance plasmids is directly related to the spread of drug resistance. With the increase in the horizontal transfer rate of resistance plasmids, cells in the body gradually turn into cells with antibiotic resistance, which causes substantial difficulties in the treatment of diseases.

Genomic Analysis of Multidrug-Resistant Hypervirulent (Hypermucoviscous) Klebsiella pneumoniae Strain Lacking the Hypermucoviscous Regulators (rmpA/rmpA2).

Hypervirulent K. pneumoniae (hvKP) strains possess distinct characteristics such as hypermucoviscosity, unique serotypes, and virulence factors associated with high pathogenicity. To better understand the genomic characteristics and virulence profile of the isolated hvKP strain, genomic data were compared to the genomes of the hypervirulent and typical K. pneumoniae strains. The K. pneumoniae strain was isolated from a patient with a recurrent urinary tract infection, and then the string test was used for the detection of the hypermucoviscosity phenotype. Whole-genome sequencing was conducted using Illumina, and bioinformatics analysis was performed for the prediction of the isolate resistome, virulome, and phylogenetic analysis. The isolate was identified as hypermucoviscous, type 2 (K2) capsular polysaccharide, ST14, and multidrug-resistant (MDR), showing resistance to ciprofloxacin, ceftazidime, cefotaxime, trimethoprim-sulfamethoxazole, cephalexin, and nitrofurantoin. The isolate possessed four antimicrobial resistance plasmids (pKPN3-307_type B, pECW602, pMDR, and p3K157) that carried antimicrobial resistance genes (ARGs) (blaOXA-1, blaCTX-M-15, sul2, APH(3″)-Ib, APH(6)-Id, and AAC(6′)-Ib-cr6). Moreover, two chromosomally mediated ARGs (fosA6 and SHV-28) were identified. Virulome prediction revealed the presence of 19 fimbrial proteins, one aerobactin (iutA) and two salmochelin (iroE and iroN). Four secretion systems (T6SS-I (13), T6SS-II (9), T6SS-III (12), and Sci-I T6SS (1)) were identified. Interestingly, the isolate lacked the known hypermucoviscous regulators (rmpA/rmpA2) but showed the presence of other RcsAB capsule regulators (rcsA and rcsB). This study documented the presence of a rare MDR hvKP with hypermucoviscous regulators and lacking the common capsule regulators, which needs more focus to highlight their epidemiological role.