Non-replicative phage particles delivering CRISPR-Cas9 to target major blaCTX-M variants.

Cluster regularly interspaced short palindromic repeats and CRISPR associated protein 9 (CRISPR-Cas9) is a promising tool for antimicrobial re-sensitization by inactivating antimicrobial resistance (AMR) genes of bacteria. Here, we programmed CRISPR-Cas9 with common spacers to target predominant blaCTX-M variants in group 1 and group 9 and their promoter in an Escherichia coli model. The CRISPR-Cas9 was delivered by non-replicative phagemid particles from a two-step process, including insertion of spacer in CRISPR and construction of phagemid vector. Spacers targeting blaCTX-M promoters and internal sequences of blaCTX-M group 1 (blaCTX-M-15 and -55) and group 9 (blaCTX-M-14, -27, -65, and -90) were cloned into pCRISPR and phagemid pRC319 for spacer evaluation and phagemid particle production. Re-sensitization and plasmid clearance were mediated by the spacers targeting internal sequences of each group, resulting in 3 log10 to 4 log10 reduction of the ratio of resistant cells, but not by those targeting the promoters. The CRISPR-Cas9 delivered by modified ΦRC319 particles were capable of re-sensitizing E. coli K-12 carrying either blaCTX-M group 1 or group 9 in a dose-dependent manner from 0.1 to 100 multiplicity of infection (MOI). In conclusion, CRISPR-Cas9 system programmed with well-designed spacers targeting multiple variants of AMR gene along with a phage-based delivery system could eliminate the widespread blaCTX-M genes for efficacy restoration of available third-generation cephalosporins by reversal of resistance in bacteria.

Multidrug-resistant Escherichia coli causing canine pyometra and urinary tract infections are genetically related but distinct from those causing prostatic abscesses.

Despite extensive characterisation of uropathogenic Escherichia coli (UPEC) causing urinary tract infections (UTIs), the genetic background of non-urinary extraintestinal pathogenic E. coli (ExPEC) in companion animals remains inadequately understood. In this study, we characterised virulence traits of 104 E. coli isolated from canine pyometra (n = 61) and prostatic abscesses (PAs) (n = 38), and bloodstream infections (BSIs) in dogs (n = 2), and cats (n = 3). A stronger association with UPEC of pyometra strains in comparison to PA strains was revealed. Notably, 44 isolates exhibited resistance to third-generation cephalosporins and/or fluoroquinolones, 15 were extended-spectrum ß-lactamase-producers. Twelve multidrug-resistant (MDR) strains, isolated from pyometra (n = 4), PAs (n = 5), and BSIs (n = 3), along with 7 previously characterised UPEC strains from dogs and cats, were sequenced. Genomic characteristics revealed that MDR E. coli associated with UTIs, pyometra, and BSIs belonged to international high-risk E. coli clones, including sequence type (ST) 38, ST131, ST617, ST648, and ST1193. However, PA strains belonged to distinct lineages, including ST12, ST44, ST457, ST744, and ST13037. The coreSNPs, cgMLST, and pan-genome illustrated intra-clonal variations within the same ST from different sources. The high-risk ST131 and ST1193 (phylogroup B2) contained high numbers of ExPEC virulence genes on pathogenicity islands, predominating in pyometra and UTI. Hybrid MDR/virulence IncF multi-replicon plasmids, containing aerobactin genes, were commonly found in non-B2 phylogroups from all sources. These findings offer genomic insights into non-urinary ExPEC, highlighting its potential for invasive infections in pets beyond UTIs, particularly with regards to high-risk global clones.

Emergence and multi-lineages of carbapenemase-producing Acinetobacter baumannii-calcoaceticus complex from canine and feline origins.

The carbapenemase-producing Acinetobacter baumannii is an important opportunistic bacterium and frequently causes hospital-acquired infections in humans. It also has increasingly been reported in veterinary medicine. This study illustrates multiple clones of carbapenemase-producing A. baumannii disseminating and causing diseases in dogs and cats in Thailand. Between 2016 and 2020, 44 A. baumannii and two A. pittii isolates exhibiting imipenem resistance (MIC≥16 µg/mL) from diagnostic samples were characterized by Pasteur multilocus sequence typing (MLST), sequence grouping (SG), repetitive extragenic palindromic element (rep)-PCR fingerprint analysis and antimicrobial resistance (AMR) profiling. All isolates contained blaOXA-23 in the Tn2006 family, and A. baumannii showed the sequence type (ST) 16 (14/44), ST149 (12/44), ST25 (6/44), ST2 (4/44), ST1581 (3/44), ST23 (2/44), ST1575 (1/44) and ST1576 (1/44). DNA fingerprint analysis and SG illustrated clonal relationships in the STs and its single locus variants, and AMR gene profiles, including tetracycline and aminoglycoside resistance genes, showed minor variations in the clones. The findings suggest that blaOXA-23 has been spread in multiple clones of A. baumannii and A. pittii from canine and feline hosts. With the collection of multiple AMR genes and intrinsic resistance, antimicrobial options are limited for treatment, and pets can be a potential reservoir of extensively drug-resistant, carbapenemase-producing A. baumannii in the community. Epidemiological tracking by passive and active surveillance in animals, veterinary personnel and hospital environment and preventive measurements should be promoted to decrease the risk of infection and transmission to humans.

Multiple and High-Risk Clones of Extended-Spectrum Cephalosporin-Resistant and blaNDM-5-Harbouring Uropathogenic Escherichia coli from Cats and Dogs in Thailand.

Resistance to extended-spectrum cephalosporins (ESC) and carbapenems in Escherichia coli (E. coli), increasingly identified in small animals, indicates a crisis of an antimicrobial resistance situation in veterinary medicine and public health. This study aimed to characterise the genetic features of ESC-resistant E. coli isolated from cats and dogs with urinary tract infections in Thailand. Of 72 ESC-resistant E. coli isolated from diagnostic samples (2016-2018), blaCTX-M including group 1 (CTX-M-55, -15 and -173) and group 9 (CTX-M-14, -27, -65 and -90) variants were detected in 47 isolates (65.28%) using PCR and DNA sequencing. Additional antimicrobial resistance genes, including plasmid-mediated AmpC (CIT and DHA), blaNDM-5, mcr-3, mph(A) and aac(6')-Ib-cr, were detected in these isolates. Using a broth microdilution assay, all the strains exhibited multidrug-resistant phenotypes. The phylogroups were F (36.11%), A (20.83%), B1 (19.44%), B2 (19.44%) and D (4.17%), with several virulence genes, plasmid replicons and an integrase gene. The DNA fingerprinting using a repetitive extragenic palindromic sequence-PCR presented clonal relationships within phylogroups. Multiple human-associated, high-risk ExPEC clones associated with multidrug resistance, including sequence type (ST) 38, ST131, ST224, ST167, ST354, ST410, ST617 and ST648, were identified, suggesting clonal dissemination. Dogs and cats are a potential reservoir of ESC-resistant E. coli and significant antimicrobial resistance genes.

Enhanced efficacy of immersion vaccination in tilapia against columnaris disease by chitosan-coated "pathogen-like" mucoadhesive nanovaccines.

Red tilapia (Oreochromis sp.) has become one of the most important fish in aquaculture. Bacterial infection caused by Flavobacterium columnare, the causative agent of columnaris disease, has been now identified as one of the most serious infectious diseases in farmed red tilapia and cause major financial damage to the producers. Among the effective prevention and control strategies, vaccination is one of the most effective approach. As the surface of living fish is covered by mucus and directly associated with the mucosal immunity, we therefore hypothesized that better adsorption on mucosal surfaces and more efficient vaccine efficacy could be enhanced biomimetic nanoparticles mimicking the mucoadhesive characteristic of live F. columnare. In this work, we describe an effective approach to targeted antigen delivery by coating the surface of nanoparticles with mucoadhesive chitosan biopolymer to provide "pathogen-like" properties that ensure nanoparticles binding on fish mucosal membrane. The physiochemical properties of nanovaccines were analyzed, and their mucoadhesive characteristics and immune response against pathogens were also evaluated. The prepared vaccines were nano-sized and spherical as confirmed by scanning electron microscope (SEM). The analysis of hydrodynamic diameter and zeta-potential also suggested the successful modification of nanovaccines by chitosan as indicated by positively charged and the overall increased diameter of chitosan-modified nanovaccines. In vivo mucoadhesive study demonstrated the excellent affinity of the chitosan-modified nanovaccines toward fish gills as confirmed by bioluminescence imaging, fluorescent microscopy, and spectrophotometric quantitative measurement. Following vaccination with the prepared nanovaccines by immersion 30 min, the challenge test was then carried out 30 and 60 days post-vaccination and resulted in high mortalities in the control. The relative percent survival (RPS) of vaccinated fish was greater than 60% for mucoadhesive nanovaccine. Our results also suggested that whole-cell vaccines failed to protect fish from columnaris infection, which is consistent with the mucoadhesive assays showing that whole-cell bacteria were unable to bind to mucosal surfaces. In conclusion, we could use this system to deliver antigen preparation to the mucosal membrane of tilapia and obtained a significant increase in survival compared to controls, suggesting that targeting mucoadhesive nanovaccines to the mucosal surface could be exploited as an effective method for immersion vaccination.

The potential of mucoadhesive polymer in enhancing efficacy of direct immersion vaccination against Flavobacterium columnare infection in tilapia.

Vaccination is the most effective approach for prevention of infectious diseases in aquaculture. Although immersion vaccination is more applicable compared to in-feed/oral administration and injection, this method suffers from low potency as the efficiency of uptake of antigens through mucosal membranes is limited. In this study, we have successfully developed a mucoadhesive vaccine delivery system to enhance the efficacy of direct immersion vaccination against Flavobacterium columnare, the causative agent of columnaris disease in red tilapia. A formalin-killed negatively charged, bacterial cell suspension was used to prepare a mucoadhesive vaccine by electrostatic coating with positively charged chitosan. Our results demonstrate that the chitosan-complexed vaccine greatly increases its mucoadhesiveness, thus increasing the chances of vaccine uptake by the gill mucosa and improving the protection obtained against columnaris infection. The surface charge of the chitosan-complexed vaccine was altered from anionic to cationic after chitosan modification. Tilapia were vaccinated with the prepared chitosan-complexed vaccine by immersion. The challenge test was then carried out 30 and 60 days post vaccination, which resulted in a high level of mortalities in the non-vaccinated and uncomplexed vaccine groups. A high relative percentage survival (RPS) of vaccinated fish was noted with the mucoadhesive vaccine. Our results indicated that the naked vaccine failed to protect the fish from columnaris infection, which is consistent with the mucoadhesive assays performed during the study showing that the naked vaccine was unable to bind to mucosal surfaces. This system is therefore an effective method for immersion vaccination in order to deliver the antigen preparation to the mucosal surface membrane of the fish.

Thermoresponsive Bacteriophage Nanocarrier as a Gene Delivery Vector Targeted to the Gastrointestinal Tract.

The use of the gastrointestinal tract as a site for the local delivery of DNA is an exciting prospect. In order to obtain an effective vector capable of delivering a gene of interest to target cells to achieve sufficient and sustained transgene expression, with minimal toxicity, we developed a new generation of filamentous bacteriophage. This particular bacteriophage was genetically engineered to display an arginine-glycine-aspartic acid (RGD) motif (an integrin-binding peptide) on the major coat protein pVIII and carry a mammalian DNA cassette. One unanticipated observation is the thermoresponsive behavior of engineered bacteriophage. This finding has led us to simplify the isolation method to purify bacteriophage particles from cell culture supernatant by low-temperature precipitation. Our results showed that, in contrast to non-surface modified, the RGD-modified bacteriophage was successfully used to deliver a transgene to mammalian cells. Our in vitro model of the human intestinal follicle-associated epithelium also demonstrated that bacteriophage particles were stable in simulated gastrointestinal fluids and able to cross the human intestinal barrier. In addition, we confirmed an adjuvant property of the engineered bacteriophage to induce nitric oxide production by macrophages. In conclusion, our study demonstrated the possibility of using bacteriophage for gene transfer in the gastrointestinal tract.