Structural Variations and Rearrangements in Bacterial Type II Toxin-Antitoxin Systems.

Bacteria encode a wide range of survival and immunity systems, including CRISPR-Cas, restriction-modification systems, and toxin-antitoxin systems involved in defence against bacteriophages, as well as survival during challenging growth conditions or exposure to antibiotics. Toxin-antitoxin (TA) systems are small two- or three-gene cassettes consisting of a metabolic regulator (the "toxin") and its associated antidote (the "antitoxin"), which also often functions as a transcriptional regulator. TA systems are widespread in the genomes of pathogens but are also present in commensal bacterial species and on plasmids. For mobile elements such as plasmids, TA systems play a role in maintenance, and increasing evidence now points to roles of chromosomal toxin-antitoxin systems in anti-phage defence. Moreover, the widespread occurrence of toxin-antitoxin systems in the genomes of pathogens has been suggested to relate to survival during host infection as well as in persistence during antibiotic treatment. Upon repeated exposure to antibiotics, TA systems have been shown to acquire point mutations as well as more dramatic rearrangements such as in-frame deletions with potential relevance for bacterial survival and pathogenesis. In this review, we present an overview of the known functional and structural consequences of mutations and rearrangements arising in bacterial toxin-antitoxin systems and discuss their relevance for survival and persistence of pathogenic species.

In vivo cloning of PCR product via site-specific recombination in Escherichia coli.

Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and affordability have always been considered. The aim of this study, on the one hand, is to simplify gene cloning by skipping in vitro assembly reactions and, on the other hand, to reduce costs by eliminating relatively expensive materials. We investigated a cloning system using Escherichia coli harboring two plasmids, pLP-AmpR and pScissors-CmR. The pLP-AmpR contains a landing pad (LP) consisting of two genes (λ int and λ gam) that allow the replacement of the transformed linear DNA using site-specific recombination. After the replacement process, the inducible expressing SpCas9 and specific sgRNA from the pScissors-CmR (CRISPR/Cas9) vector leads to the removal of non-recombinant pLP-AmpR plasmids. The function of LP was explored by directly transforming PCR products. The pScissors-CmR plasmid was evaluated for curing three vectors, including the origins of pBR322, p15A, and pSC101. Replacing LP with a PCR product and fast-eradicating pSC101 origin-containing vectors was successful. Recombinant colonies were confirmed following gene replacement and plasmid curing processes. The results made us optimistic that this strategy may potentially be a simple and inexpensive cloning method. KEY POINTS: •The in vivo cloning was performed by replacing the target gene with the landing pad. •Fast eradication of non-recombinant plasmids was possible by adapting key vectors. •This strategy is not dependent on in vitro assembly reactions and expensive materials.

Dependence of post-segregational killing mediated by Type II restriction-modification systems on the lifetime of restriction endonuclease effective activity.

Plasmid-borne Type II restriction-modification (RM) systems mediate post-segregational killing (PSK). PSK is thought to be caused by the dilution of restriction and modification enzymes during cell division, resulting in accumulation of unmethylated DNA recognition sites and their cleavage by restriction endonucleases. PSK is the likely reason for stabilization of plasmids carrying RM systems in the absence of selection for plasmid maintenance. In this study, we developed a CRISPR interference-based method to eliminate RM-carrying plasmids and study PSK-related phenomena with minimal perturbation to the Escherichia coli host. Plasmids carrying the EcoRV, Eco29kI, and EcoRI RM systems were highly stable, and their loss resulted in SOS response and PSK. In contrast, plasmids carrying the Esp1396I system were poorly stabilized; their loss led to a temporary cessation of growth, followed by full recovery. We demonstrate that this unusual behavior is due to a limited lifetime of the Esp1396I restriction endonuclease activity, which, upon Esp1396I plasmid loss, disappears approximately after two cycles of cell division, i.e., before unmethylated sites appear in significant numbers. Our results indicate that whenever PSK induced by a loss of RM systems, and, possibly, other toxin-antitoxin systems, is considered, the lifetimes of individual system components and the growth rate of host cells shall be taken in account. Mathematical modeling shows, that unlike the situation with classical toxin-antitoxin systems, RM system-mediated PSK is possible when the lifetimes of restriction endonuclease and methyltransferase activities are similar, as long as the toxic restriction endonuclease activity persists for more than two chromosome replication cycles.IMPORTANCEIt is widely accepted that many Type II restriction-modification (RM) systems mediate post-segregational killing (PSK) if plasmids that encode them are lost. In this study, we harnessed an inducible CRISPR-Cas system to remove RM plasmids from Escherichia coli cells to study PSK while minimally perturbing cell physiology. We demonstrate that PSK depends on restriction endonuclease activity lifetime and is not observed when it is less than two replication cycles. We present a mathematical model that explains experimental data and shows that unlike the case of toxin-antitoxin-mediated PSK, the loss of an RM system induced PSK even when the RM enzymes have identical lifetimes.

Prevalence and molecular characteristics of intestinal pathogenic Escherichia coli isolated from diarrheal pigs in Southern China.

Intestinal pathogenic Escherichia coli (InPEC) is one of the most common causes of bacterial diarrhea in farm animals, including profuse neonatal diarrhea and post weaning diarrhea (PWD) in piglets. In this study, we investigated the prevalence of InPEC and associated primary virulence factors among 543 non-duplicate E. coli isolates from diarrheal pigs from 15 swine farms in southern China. Six major virulence genes associated with InPEC were identified among 69 (12.71 %) E. coli isolates and included est (6.62 %), K88 (4.79 %), elt (3.68 %), eae (1.47 %), stx2 (0.92 %) and F18 (0.55 %). Three pathotypes of InPEC were identified including ETEC (8.10 %), EPEC (1.29 %) and STEC/ETEC (0.92 %). In particular, K88 was only found in ETEC from breeding farms, whereas F18 was only present in STEC/ETEC hybrid from finishing farms. Whole genome sequence analysis of 37 E. coli isolates revealed that InPEC strains frequently co-carried multiple antibiotic resistance gene (ARG). est, elt and F18 were also found to co-locate with ARGs on a single IncFIB/IncFII plasmid. InPEC isolates from different pathotypes also possessed different profiles of virulence genes and antimicrobial resistance genes. Population structure analysis demonstrated that InPEC isolates from different pathotypes were highly heterogeneous whereas those of the same pathotype were extremely similar. Plasmid analysis revealed that K88 and/or est/elt were found on pGX18-2-like/pGX203-2-like and pGX203-1-like IncFII plasmids, while F18 and elt/est, as well as diverse ARGs were found to co-locate on IncFII/IncFIB plasmids with a non-typical backbone. Moreover, these key virulence genes were flanked by or adjacent to IS elements. Our findings indicated that both clonal expansion and horizontal spread of epidemic IncFII plasmids contributed to the prevalence of InPEC and the specific virulence genes (F4, F18, elt and est) in the tested swine farms.

Genetics of resistance to trimethoprim in cotrimoxazole resistant uropathogenic Escherichia coli: integrons, transposons, and single gene cassettes.

Cotrimoxazole, the combined formulation of sulfamethoxazole and trimethoprim, is one of the treatments of choice for several infectious diseases, particularly urinary tract infections. Both components of cotrimoxazole are synthetic antimicrobial drugs, and their combination was introduced into medical therapeutics about half a century ago. In Gram-negative bacteria, resistance to cotrimoxazole is widespread, being based on the acquisition of genes from the auxiliary genome that confer resistance to each of its antibacterial components. Starting from previous knowledge on the genotype of resistance to sulfamethoxazole in a collection of cotrimoxazole resistant uropathogenic Escherichia coli strains, this work focused on the identification of the genetic bases of the trimethoprim resistance of these same strains. Molecular techniques employed included PCR and Sanger sequencing of specific amplicons, conjugation experiments and NGS sequencing of the transferred plasmids. Mobile genetic elements conferring the trimethoprim resistance phenotype were identified and included integrons, transposons and single gene cassettes. Therefore, strains exhibited several ways to jointly resist both antibiotics, implying different levels of genetic linkage between genes conferring resistance to sulfamethoxazole (sul) and trimethoprim (dfrA). Two structures were particularly interesting because they represented a highly cohesive arrangements ensuring cotrimoxazole resistance. They both carried a single gene cassette, dfrA14 or dfrA1, integrated in two different points of a conserved cluster sul2-strA-strB, carried on transferable plasmids. The results suggest that the pressure exerted by cotrimoxazole on bacteria of our environment is still promoting the evolution toward increasingly compact gene arrangements, carried by mobile genetic elements that move them in the genome and also transfer them horizontally among bacteria.

Temperatures above 37°C increase virulence of a convergent Klebsiella pneumoniae sequence type 307 strain.

Background: Convergence of Klebsiella pneumoniae (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP. However, clinically used indicators for hypervirulent KP identification, such as hypermucoviscosity, appear to be differentially expressed in convergent KP, potential outbreak clones are difficult to identify. We aimed to fill such knowledge gaps by investigating the temperature dependence of hypermucoviscosity and virulence in a convergent KP strain isolated during a clonal outbreak and belonging to the high-risk sequence type (ST)307. Methods: Hypermucoviscosity, biofilm formation, and mortality rates in Galleria mellonella larvae were examined at different temperatures (room temperature, 28°C, 37°C, 40°C and 42°C) and with various phenotypic experiments including electron microscopy. The underlying mechanisms of the phenotypic changes were explored via qPCR analysis to evaluate plasmid copy numbers, and transcriptomics. Results: Our results show a temperature-dependent switch above 37°C towards a hypermucoviscous phenotype, consistent with increased biofilm formation and in vivo mortality, possibly reflecting a bacterial response to fever-like conditions. Furthermore, we observed an increase in plasmid copy number for a hybrid plasmid harboring carbapenemase and rmpA genes. However, transcriptomic analysis revealed no changes in rmpA expression at higher temperatures, suggesting alternative regulatory pathways. Conclusion: This study not only elucidates the impact of elevated temperatures on hypermucoviscosity and virulence in convergent KP but also sheds light on previously unrecognized aspects of its adaptive behavior, underscoring its resilience to changing environments.

The extent and characteristics of DNA transfer between plasmids and chromosomes.

Plasmids are extrachromosomal genetic elements that reside in prokaryotes. The acquisition of plasmids encoding beneficial traits can facilitate short-term survival in harsh environmental conditions or long-term adaptation of new ecological niches. Due to their ability to transfer between cells, plasmids are considered agents of gene transfer. Nonetheless, the frequency of DNA transfer between plasmids and chromosomes remains understudied. Using a novel approach for detection of homologous loci between genome pairs, we uncover gene sharing with the chromosome in 1,974 (66%) plasmids residing in 1,016 (78%) taxonomically diverse isolates. The majority of homologous loci correspond to mobile elements, which may be duplicated in the host chromosomes in tens of copies. Neighboring shared genes often encode similar functional categories, indicating the transfer of multigene functional units. Rare transfer events of antibiotics resistance genes are observed mainly with mobile elements. The frequent erosion of sequence similarity in homologous regions indicates that the transferred DNA is often devoid of function. DNA transfer between plasmids and chromosomes thus generates genetic variation that is akin to workings of endosymbiotic gene transfer in eukaryotic evolution. Our findings imply that plasmid contribution to gene transfer most often corresponds to transfer of the plasmid entity rather than transfer of protein-coding genes between plasmids and chromosomes.

Characterization of ST15-KL112 Klebsiella pneumoniae Co-Harboring Bla oxa-232 and rmtF in China.

Introduction: This study aimed to investigate the emergence and characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains that demonstrate resistance to multiple antibiotics, including aminoglycosides and tigecycline, in a Chinese hospital. Methods: A group of ten CRKP strains were collected from the nine patients in a Chinese hospital. Antimicrobial Susceptibility Testing (AST) and phenotypic inhibition assays precisely assess bacterial antibiotic resistance. Real-time quantitative PCR (RT-qPCR) was used to analyze the mRNA levels of efflux pump genes (acrA/acrB and oqxA/oqxB) and the regulatory gene (ramA). The core-genome tree and PFGE patterns were analyzed to assess the clonal and horizontal transfer expansion of the strains. Whole-genome sequencing was performed on a clinical isolate of K. pneumoniae named Kpn20 to identify key resistance genes and antimicrobial resistance islands (ARI). Results: The CRKP strains showed high resistance to carbapenems, aminoglycosides (CLSI, 2024), and tigecycline (EUCAST, 2024). The mRNA expression levels of efflux pump genes and regulatory genes were detected by RT-qPCR. All 10 isolates had significant differences compared to the control group of ATCC13883. The core-genome tree and PFGE patterns revealed five clusters, indicating clonal and horizontal transfer expansion. Three key resistance genes (blaoxa-232, blaCTX-M-15 , and rmtF) were observed in the K. pneumoniae clinical isolate Kpn20. Mobile antibiotic resistance islands were identified containing bla CTX-M-15 and rmtF, with multiple insertion sequences and transposons present. The coexistence of bla oxa-232 and rmtF in a high-risk K. pneumoniae strain was reported. Conjugation assay was utilized to investigate the transferability of bla oxa-232-encoding plasmids horizontally. Conclusion: The study highlights the emergence of ST15-KL112 high-risk CRKP strains with multidrug resistance, including to aminoglycosides and tigecycline. The presence of mobile ARI and clonal and horizontal transfer expansion of strains indicate the threat of transmission of these strains. Future research is needed to assess the prevalence of such isolates and develop effective control measures.

Enhancing recombinant antibody yield in Chinese hamster ovary cells.

A range of recombinant monoclonal antibodies (rMAbs) have found application in treating diverse diseases, spanning various cancers and immune system disorders. Chinese hamster ovary (CHO) cells have emerged as the predominant choice for producing these rMAbs due to their robustness, ease of transfection, and capacity for posttranslational modifications akin to those in human cells. Transient transfection and/or stable expression could be conducted to express rMAbs in CHO cells. To bolster the yield of rMAbs in CHO cells, a multitude of approaches have been developed, encompassing vector optimization, medium formulation, cultivation parameters, and cell engineering. This review succinctly outlines these methodologies when also addressing challenges encountered in the production process, such as issues with aggregation and fucosylation.

Supercoiled DNA percentage: A key in-process control of linear DNA template for mRNA drug substance manufacturing.

The development of messenger RNA (mRNA) vaccines and therapeutics necessitates the production of high-quality in vitro-transcribed mRNA drug substance with specific critical quality attributes (CQAs), which are closely tied to the uniformity of linear DNA template. The supercoiled plasmid DNA is the precursor to the linear DNA template, and the supercoiled DNA percentage is commonly regarded as a key in-process control (IPC) during the manufacturing of linear DNA template. In this study, we investigate the influence of supercoiled DNA percentage on key mRNA CQAs, including purity, capping efficiency, double-stranded RNA (dsRNA), and distribution of poly(A) tail. Our findings reveal a significant impact of supercoiled DNA percentage on mRNA purity and in vitro transcription yield. Notably, we observe that the impact on mRNA purity can be mitigated through oligo-dT chromatography, alleviating the tight range of DNA supercoiled percentage to some extent. Overall, this study provides valuable insights into IPC strategies for DNA template chemistry, manufacturing, and controls (CMC) and process development for mRNA drug substance.

PlasmidHunter: accurate and fast prediction of plasmid sequences using gene content profile and machine learning.

Plasmids are extrachromosomal DNA found in microorganisms. They often carry beneficial genes that help bacteria adapt to harsh conditions. Plasmids are also important tools in genetic engineering, gene therapy, and drug production. However, it can be difficult to identify plasmid sequences from chromosomal sequences in genomic and metagenomic data. Here, we have developed a new tool called PlasmidHunter, which uses machine learning to predict plasmid sequences based on gene content profile. PlasmidHunter can achieve high accuracies (up to 97.6%) and high speeds in benchmark tests including both simulated contigs and real metagenomic plasmidome data, outperforming other existing tools.

Short-chain fatty acids inhibit bacterial plasmid transfer through conjugation in vitro and in ex vivo chicken tissue explants.

The animal gut acts as a potent reservoir for spreading and maintaining conjugative plasmids that confer antimicrobial resistance (AMR), fitness, and virulence attributes. Interventions that inhibit the continued emergence and expansion of AMR and virulent strains in agricultural and clinical environments are greatly desired. This study aims to determine the presence and efficacy of short-chain fatty acids (SCFA) inhibitory effects on the conjugal transfer of AMR plasmids. In vitro broth conjugations were conducted between donor Escherichia coli strains carrying AMP plasmids and the plasmid-less Escherichia coli HS-4 recipient strain. Conjugations were supplemented with ddH2O or SCFAs at 1, 0.1, 0.01, or 0.001 molar final concentration. The addition of SCFAs completely inhibited plasmid transfer at 1 and 0.1 molar and significantly (p < 0.05) reduced transfer at 0.01 molar, regardless of SCFA tested. In explant models for the chicken ceca, either ddH2O or a final concentration of 0.025 M SCFAs were supplemented to the explants infected with donor and recipient E. coli. In every SCFA tested, significant decreases in transconjugant populations compared to ddH2O-treated control samples were observed with minimal effects on donor and recipient populations. Finally, significant reductions in transconjugants for plasmids of each incompatibility type (IncP1ε, IncFIß, and IncI1) tested were detected. This study demonstrates for the first time the broad inhibition ability of SCFAs on bacterial plasmid transfer and eliminates AMR with minimal effect on bacteria. Implementing interventions that increase the concentrations of SCFAs in the gut may be a viable method to reduce the risk, incidence, and rate of AMR emergence in agricultural and human environments.

Interplay between Amino Acid Substitution in GyrA and QnrB19: Elevating Fluoroquinolone Resistance in Salmonella Typhimurium.

Globally, there have been increasing reports of antimicrobial resistance in nontyphoidal Salmonella (NTS), which can develop into severe and potentially life-threatening diarrhea. This study focuses on the synergistic effects of DNA gyrase mutations and plasmid-mediated quinolone resistance (PMQR) genes, specifically qnrB19, on fluoroquinolone (FQ) resistance in Salmonella Typhimurium. By utilizing recombinant mutants, GyrAS83F and GyrAD87N, and QnrB19's, we discovered a significant increase in fluoroquinolones resistance when QnrB19 is present. Specifically, ciprofloxacin and moxifloxacin's inhibitory concentrations rose 10- and 8-fold, respectively. QnrB19 was found to enhance the resistance capacity of mutant DNA gyrases, leading to high-level FQ resistance. Additionally, we observed that the ratio of QnrB19 to DNA gyrase played a critical role in determining whether QnrB19 could protect DNA gyrase against FQ inhibition. Our findings underscore the critical need to understand these resistance mechanisms, as their coexistence enables bacteria to withstand therapeutic FQ levels, posing a significant challenge to treatment efficacy.

Large-Scale Culture and Plasmid Preparation Procedure for Low-Yield Bacmids Containing Full-Length SARS-CoV-2 cDNAs.

Reverse genetic methods to manipulate viral genomes are key tools in modern virological experimentation. They allow for the generation of reporter virus genomes to simplify the assessment of virus growth and for the analysis of the impact of specific mutations in the genome on virus phenotypes. For SARS-CoV-2, reverse genetic systems are complicated by the large size of the viral genome and the instability of certain genomic sections in bacteria requiring the use of low-copy number bacterial artificial chromosome plasmids (bacmids). However, even with the use of bacmids, faithfully amplifying SARS-CoV-2 bacmids is often challenging. In this chapter, we describe a detailed protocol to grow SARS-CoV-2 bacmids and highlight the challenges and optimal techniques to produce large quantities of SARS-CoV-2 bacmids that are free of deletions and mutations. Overall, this chapter has recapitulated an overview of the maxi-preparation procedure for large unstable bacmids like SARS-CoV-2 to facilitate downstream applications.

Innovative Delivery System Combining CRISPR-Cas12f for Combatting Antimicrobial Resistance in Gram-Negative Bacteria.

Antimicrobial resistance poses a significant global challenge, demanding innovative approaches, such as the CRISPR-Cas-mediated resistance plasmid or gene-curing system, to effectively combat this urgent crisis. To enable successful curing of antimicrobial genes or plasmids through CRISPR-Cas technology, the development of an efficient broad-host-range delivery system is paramount. In this study, we have successfully designed and constructed a novel functional gene delivery plasmid, pQ-mini, utilizing the backbone of a broad-host-range Inc.Q plasmid. Moreover, we have integrated the CRISPR-Cas12f system into the pQ-mini plasmid to enable gene-curing in broad-host of bacteria. Our findings demonstrate that pQ-mini facilitates the highly efficient transfer of genetic elements to diverse bacteria, particularly in various species in the order of Enterobacterales, exhibiting a broader host range and superior conjugation efficiency compared to the commonly used pMB1-like plasmid. Notably, pQ-mini effectively delivers the CRISPR-Cas12f system to antimicrobial-resistant strains, resulting in remarkable curing efficiencies for plasmid-borne mcr-1 or blaKPC genes that are comparable to those achieved by the previously reported pCasCure system. In conclusion, our study successfully establishes and optimizes pQ-mini as a broad-host-range functional gene delivery vector. Furthermore, in combination with the CRISPR-Cas system, pQ-mini demonstrates its potential for broad-host delivery, highlighting its promising role as a novel antimicrobial tool against the growing threat of antimicrobial resistance.

Natural transformation of Vibrio natriegens with large genetic cluster enables alginate assimilation for isopentenol production.

Alginate is a major component of brown macroalgae, and its efficient utilization is critical for developing sustainable technologies. Vibrio natriegens is a fast-growing marine bacterium that has gained massive attention due to its potential as an alternative industrial chassis. However, V. natriegens cannot naturally metabolize alginate, limiting its usage in marine biomass conversion. In this study, V. natriegens was engineered to utilize marine biomass, kelp, as a carbon source. A total of 33.8 kb of the genetic cluster for alginate assimilation from Vibrio sp. dhg was integrated into V. natriegens by natural transformation. Engineered V. natriegens was further modified to produce 1.8 mg/L of isopentenol from 16 g/L of crude kelp powder. This study not only presents the very first case in which V. natriegens can be naturally transformed with large DNA fragments but also highlights the potential of this strain for converting marine biomass into valuable products.

Genomic characterization of carbapenem and colistin-resistant Klebsiella pneumoniae isolates from humans and dogs.

Introduction: Carbapenem and colistin-resistant Enterobacteriaceae, including Klebsiella pneumoniae, have become a growing global concern, posing a significant threat to public health. Currently, there is limited information about the genetic background of carbapenem and colistin-resistant K. pneumoniae isolates infecting humans and dogs in Thailand. This study aimed to characterize carbapenem and colistin-resistant genes in six resistant K. pneumoniae clinical isolates (three from humans and three from dogs) which differed in their pulse field gel electrophoresis profiles. Methods: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), antimicrobial susceptibility testing, and whole-genome sequencing were employed to identify and analyze the isolates. Results and discussion: All six isolates were carbapenemase-producing K. pneumoniae isolates with chromosomally carried blaSHV, fosA, oqxA and oqxB genes, as well as nine to 21 virulence genes. The isolates belonged to five multilocus sequence types (STs): one isolate from a human and one from a dog belonged to ST16, with the other two human isolates being from ST340 and ST1269 and the other two dog isolates were ST147 and ST15. One human isolate and two dog isolates harbored the same blaOXA-232 gene on the ColKP3 plasmid, and one dog isolate carried the blaOXA-48 gene on the IncFII plasmid. Notably, one human isolate exhibited resistance to colistin mediated by the mcr-3.5 gene carried on the IncFII plasmid, which co-existed with resistance determinants to other antibiotics, including aminoglycosides and quinolones. In conclusion, this study provides a comprehensive characterization of both chromosome- and plasmid-mediated carbapenem and colistin resistance in a set of K. pneumoniae clinical isolates from unrelated humans and dogs in Thailand. The similarities and differences found contribute to our understanding of the potential widescale dissemination of these important resistance genes among clinical isolates from humans and animals, which in turn may contribute to outbreaks of emerging resistant clones in hospital settings.

Antifungal potential of multi-drug-resistant Pseudomonas aeruginosa: harnessing pyocyanin for candida growth inhibition.

Introduction: Pseudomonas aeruginosa is notorious for its multidrug resistance and its involvement in hospital-acquired infections. In this study, 20 bacterial strains isolated from soil samples near the Hindan River in Ghaziabad, India, were investigated for their biochemical and morphological characteristics, with a focus on identifying strains with exceptional drug resistance and pyocyanin production. Methods: The isolated bacterial strains were subjected to biochemical and morphological analyses to characterize their properties, with a particular emphasis on exopolysaccharide production. Strain GZB16/CEES1, exhibiting remarkable drug resistance and pyocyanin production. Biochemical and molecular analyses, including sequencing of its 16S rRNA gene (accession number LN735036.1), plasmid-curing assays, and estimation of plasmid size, were conducted to elucidate its drug resistance mechanisms and further pyocynin based target the Candida albicans Strain GZB16/CEES1 demonstrated 100% resistance to various antibiotics used in the investigation, with plasmid-curing assays, suggesting plasmid-based resistance gene transmission. The plasmid in GZB16/CEES1 was estimated to be approximately 24 kb in size. The study focused on P. aeruginosa's pyocyanin production, revealing its association with anticandidal activity. The minimum inhibitory concentration (MIC) of the bacterial extract against Candida albicans was 50 µg/ml, with a slightly lower pyocyanin-based MIC of 38.5 µg/ml. Scanning electron microscopy illustrated direct interactions between P. aeruginosa strains and Candida albicans cells, leading to the destruction of the latter. Discussion: These findings underscore the potential of P. aeruginosa in understanding microbial interactions and developing strategies to combat fungal infections. The study highlights the importance of investigating bacterial-fungal interactions and the role of pyocyanin in antimicrobial activity. Further research in this area could lead to the development of novel therapeutic approaches for combating multidrug-resistant infections.

Efficacy and advantage of immunotherapy for melanoma via intramuscular co-expression of plasmid-encoded PD-1 and CTLA-4 scFvs.

Immunotherapy, in the shape of immune checkpoint inhibitors (ICIs), has completely changed the treatment of cancer. However, the increasing expense of treatment and the frequency of immune-related side effects, which are frequently associated with combination antibody therapies and Fc fragment of antibody, have limited the patient's ability to benefit from these treatments. Herein, we presented the therapeutic effects of the plasmid-encoded PD-1 and CTLA-4 scFvs (single-chain variable fragment) for melanoma via an optimized intramuscular gene delivery system. After a single injection, the plasmid-encoded ICI scFv in mouse sera continued to be above 150 ng/mL for 3 weeks and reached peak amounts of 600 ng/mL. Intramuscular delivery of plasmid encoding PD-1 and CTLA-4 scFvs significantly changed the tumor microenvironment, delayed tumor growth, and prolonged survival in melanoma-bearing mice. Furthermore, no significant toxicity was observed, suggesting that this approach could improve the biosafety of ICIs combination therapy. Overall, the expression of ICI scFvs in vivo using intramuscular plasmid delivery could potentially develop into a reliable, affordable, and safe immunotherapy technique, expanding the range of antibody-based gene therapy systems that are available.

Genomic characterisation of Escherichia coli isolated from poultry at retail through Sink Surveillance in Dhaka, Bangladesh reveals high levels of multi-drug resistance.

The surveillance of antimicrobial resistance (AMR) in commensal Escherichia coli from livestock at slaughter is widely employed to assess the potential for risk to humans. There is currently a limited understanding of AMR in Bangladesh poultry at retail in live bird markets, with studies focussing solely on phenotypic characterisation of resistance. To address this evidence gap we performed antimicrobial susceptibility testing and whole genome sequencing on E. coli obtained from chickens from live bird markets in Dhaka in 2018 (n = 38) and 2020 (n = 45). E. coli were isolated from caeca samples following ISO guidelines and sequenced using short and long read methods. Multidrug resistance was extremely common (n = 77) and there was excellent concordance between AMR phenotype and the presence of corresponding AMR genes or mutations. There was considerable genomic diversity, with 43 different sequence types detected. Public health considerations included the high occurrence of resistance to ciprofloxacin (n = 75) associated with plasmid-residing qnrS or mutations in the gyrA and parC chromosomal genes; and the detection of a tigecycline resistant isolate harbouring tet(X4) on an IncHI1A/B-IncFIA mosaic plasmid. Thirty-nine isolates were resistant to azithromycin and harboured mphA, with a significant increase in the incidence of resistance between 2018 and 2020. Although azithromycin is banned for veterinary use in Bangladesh it remains an important treatment option for humans. Interestingly, mphA confers high-level resistance to azithromycin and erythromycin, and the latter is commonly used on poultry farms in Bangladesh. Seven isolates were colistin resistant and carried mcr1. For two isolates hybrid assemblies revealed that mcr1 resided on a highly conserved IncHI2 plasmid that had 93% nucleotide identity to a plasmid from the published genome of an E. coli isolate of Bangladeshi human origin. Six isolates had resistance to third generation cephalosporins, associated with plasmid-residing bla CTX-M-55, bla CTX-M-65, or bla DHA-1. By employing phenotypic and genomic approaches for AMR surveillance we have provided new insights into the potential for One Health AMR linkages in Bangladesh. Employing similar approaches in human and environmental sectors will help inform the One Health approach to addressing AMR, and generate evidence to support mitigation measures such as improved antimicrobial stewardship.