Study On Isolation And Identification Of Enterococcus From Dog And Cat In Northeast China And Specific Phage

Enterococcus is a Gram-positive coccus that is co-born with other bacteria in the intestinal tract of humans and animals and is also widely distributed in the natural environment.It is an opportunistic pathogen and is one of the most important pathogens causing hospital-acquired infections.The intimate relationship between dogs and cats and humans makes them an important source of pathogenic bacterial infections acquired by humans.This study focused on identifying Enterococcus in dogs and cats in the northeast region.We conducted isolation and identification of the bacteria,analyzed its antibiotic resistance level and virulence factor carriage,and assessed its potential public health risk.The purpose of this study was to provide surveillance and prevention measures for Enterococcus community infections.This study delves into the potential of using specific phages as a treatment for enterococcal infections in dogs and cats.The research involved isolating and purifying E.faecalis phages,followed by electron microscopic observation,biological characterization,lysis profile determination,and genome sequencing analysis.These findings provide a foundation for further exploration of the application of E.faecalis phages in the treatment of enterococcal infections.In this study,469(76.89%)enterococci strains were isolated from 610 fecal or anal swab samples of dogs and cats in northeastern China,mainly including 238(50.75%)E.faecium and128(27.29%)E.faecalis.The results of the analysis indicate that the prevalence of E.faecalis varies among different hosts and is statistically significant(P<0.05).The study found that police dogs had the lowest number of E.faecalis isolates,suggesting that the colonization and excretion of E.faecalis in dogs and cats may be influenced by factors such as feeding practices and environment.The results of the resistance phenotype analysis indicated that Enterococcus exhibited the highest resistance rates towards tetracycline and erythromycin,with rates reaching82.61% and 62.61%,respectively.Additionally,it was observed that 67.28% of the strains that exhibited tetracycline-resistant phenotypes were also resistant to erythromycin,showing the synergistic nature of antibiotic resistance,the resistance rates to rifampin,quinupristin/dalfopristin,amikacin,furantoin,ciprofloxacin and gentamicin were high(31.30%-48.26%),and low resistance rates(<15%)to ampicillin,linezolid,mupirocin,teicoplanin and vancomycin.Isolations were obtained from 145(30.92%)high level gentamicin resistant enterococci(HLGR)and 4(0.85%)vancomycin resistant enterococci(VRE).68.92% of the isolates were multidrug resistant(MDR)enterococci and 98.70% were resistant to at least one class of antibiotics.In addition,the resistance rates of E.faecium and E.faecalis to nine antimicrobials differed significantly(P<0.01),demonstrating that different species of enterococci have different resistance biases to antibiotics.The results of the virulence gene assay indicate that the two enterococci have different virulence patterns.E.faecalis carries a high number of virulence gene species,while the police dog strain has the least number of virulence genes compared to the strain source.Among E.faecalis,virulence genes detected above 40% included the adhesin gene ace(85.94%),the endocarditis antigen gene efa Afs(85.16%),the aggregated substance gene asa1(60.94%),the gelatinase gene gel E(51.56%),and the surface protein gene esp(42.97%).Among them,94.55% of the strains carrying esp gene also carried asa1 gene,and97.67% of the strains carrying hyl gene also carried gel E gene,reflecting the association of different virulence genes.This study confirmed that E.faecalis isolates can carry multiple virulence related genes and are resistant to multiple antibiotics,indicating that Enterococcus as a reservoir of drug resistance and virulence poses a great public health risk.Genomic framework mapping of 103 multi-virulent multidrug resistant E.faecalis isolates showed that more than half of the E.faecalis isolates carried multiple resistance genes for aminoglycosides(aph(3’)-III),tetracyclines(tet(M)),macrolides(erm(B)),and lincosamides(lsa(A)).The results of virulence gene matching showed that genes related to adhesins(Ace,Ebp pili,Efa A),exonucleases(Hyaluronidase),immunomodulation(Capsule)and biofilms(Bop D,Fibrinogen binding protein)were widely present,and more than 90% of the isolates carried the above virulence.Using the multilocus sequence typing(MLST)method,34 different ST types were obtained,including 4 groups and 25 independent types.Among them,ST16 was the dominant type,which belonged to the same complex clonal group CC16 as ST179,which is also the group more frequently seen in human clinical studies,indicating the cross-species transmission of E.faecalis between dogs and cats and humans.The combined sequencing analysis and PCR results of E.faecalis resistance and virulence genes show the complexity and severity of enterococcal virulence and resistance in dogs and cats.Therefore,monitoring the resistance and virulence genes of enterococci in dogs and cats can help in early warning of community transmission of enterococcal infections.In recent years,the use of clinical therapeutic antibiotics has become increasingly regulated to reduce bacterial resistance production,and the farming industry has begun to prohibit the addition of antibiotics to feed stocks.Phage preparations have received increasing attention as potential alternatives to antibiotics.Conducting biological characterization of specific phages and whole genome sequencing analysis can provide basic data information for phage therapy.In this study,a lysogenic phage of E.faecalis,named v B＿Efa S＿DEfc27b,was isolated from municipal wastewater using multi-virulent multidrug-resistant E.faecalis DEfc27 as the host.Transmission electron microscopy results showed that the head of phage DEfc27 b had an ortho-icosahedral structure with a diameter of about 55 nm and a tail length of about 185 nm,belonging to the Caudovirales order,Siphoviridae family.The phage could lyse 23%(30/128)of dog and cat E.faecalis isolates;the optimal multiplicity of infection(MOI)was 0.001;the one-step growth curve showed that the incubation period of DEfc27 b was 10 min,the outbreak period was 40 min,and the outbreak amount was 265 PFU/cell;the temperature tolerance was up to 50°C,and the p H tolerance range was p H 5-10.whole genome sequencing.The results showed that the genome of DEfc27 b is a linear double-stranded DNA with 41,054 bp,GC content 34.7%,and the whole genome sequence Gen Bank number is OQ269649.The genome of DEfc27 b has 63 coding sequences,no virulence gene,resistance gene and t RNA were found,30 coding sequences were annotated by comparison,and 33 putative proteins were identified.These predictable gene functions include DNA replication,encoding structural proteins,cleavage,phage assembly and other functions.After constructing evolutionary trees with terminal large subunit and capsid protein genes,the affinity of phage DEfc27 b to other phages was confirmed,and the closest affinity to clinical multidrug resistant E.faecalis isolate phage LY0322 was confirmed,proving that phage DEfc27 b is a new E.faecalis phage.In summary,this study isolated and identified E.faecalis from dogs and cats in Northeast China,systematically conducted the detection of drug resistance and virulence genes,and the whole genome sequencing analysis of multi-virulent multi-resistant strains,revealing the current situations of carrying virulence and drug resistance of E.faecalis from dogs and cats in Northeast China,providing a theoretical basis for the standardized use of veterinary antibiotics and the prevention and control of drug resistant pathogens.Meanwhile,a multi-virulence and multi-drug resistant strain of E.faecalis specific phage was isolated and obtained,which provided important basic data for phage application research.