Isolation,Identification,Characterization And Genomic Analysis Of Proteus Mirabilis Producing KPC-2 Type Carbapenemase

Background Proteus bacteria belong to the family Enterobacteriaceae and are one of the clinically important conditional pathogens.The bacteria of this genus are widely distributed and can be detected in the soil,sewage,spoilage animals and plants,and the intestines of humans and animals in nature.In recent years,the widespread use of antibacterial drugs in the clinic,improper drinking of the contamination of water sources and ingestion of spoiled food have made the clinical infections caused by Proteus bacteria common,and the detection rate has increased year by year.At present,it is the Proteus mirabilis that has the highest clinical separation rate and is extremely harmful to humans.Proteus mirabilis has the flagellum of the whole body and is active.It is an important pathogen causing clinical urinary tract infection and also a common pathogen causing food poisoning,meningitis,otitis media,and chronic diarrhea in children.Carbapenem antibacterial drugs have a broad antibacterial spectrum and strong bactericidal effect,and are often used as the last line of defense for the infection of Enterobacteriaceae producing ESBL and Amp C enzyme.However,in recent years,clinical carbapenem antibiotics,including imipenem,meropenem,ertapenem and other unreasonable use and abuse,make the carbapenem-resistant Proteus mirabilis separation rate and infection rate increased,clinical treatment of its infection is facing a severe test.In 2008,the United States first reported Proteus mirabilis producing KPC-2 type carbapenemase.Later,China and Brazil discovered the presence of this type of enzyme in Proteus mirabilis in 2010 and 2015,respectively.The gene of KPC-2 type carbapenemase is localized to a plasmid and can be clonalized and gene-transformed horizontally by plasmid and transposable elements between bacterial species.In addition,infections caused by it can cause synergistic resistance to a variety of antimicrobial agents other than carbapenems and are often associated with high mortality in patients.It is well known that Proteus mirabilis relies on the flagella of the whole body to grow in the form of migration on the medium,but some Proteus mirabilis loses migratory growth characteristics after repeated passages.Related reports indicate that such strains have a higher resistance rate to carbapenems.At present,there are few reports on Proteus mirabilis producing KPC-2 type carbapenemase,and its regional epidemic characteristics and drug resistance characteristics are not clear.Therefore,the clinical research on it is still a hot topic.At present,the development of high-throughput sequencing technology and bioinformatics makes it possible to study the mechanism of bacterial drug resistance and transmission in depth.Through the second and third generation high-throughput sequencing of bacterial genome,we can obtain complete bacterial genomic data,and then use bioinformatics software to assemble and splicing,genomic annotation,gene sequence alignment,and phylogenetic analysis of sequencing data,which can deeply reveal the genetic characteristics of bacterial genome and evolutionary genetic characteristics of strains.Provide accurate and reliable laboratory data for the monitoring and treatment of resistant bacteria.Based on the above,we designed this topic to explore the characteristics of the resistance mechanism,molecular typing,genetic environment,transmission mechanism and micro-evolution of strains of Proteus mirabilis producing KPC-2 type carbapenemase separated in Hangzhou.Provide theoretical support and laboratory basis for clinical prevention and treatment of the bacteria.Methods and materials 1.Strain collection The experimental strains were isolated from the National Key Laboratory of Infectious Diseases Diagnosis and Treatment of the First Affiliated Hospital of Zhejiang University School of Medicine from January 2016 to June 2017,and the strains derived from the stool samples of clinical inpatients.According to mass spectrometry,6 strains of Proteus mirabilis were screened out from 365 carbapenem-resistant Enterobacteriaceae strains.2.Antimicrobial susceptibility test The susceptibility test was carried out by agar dilution method and micro broth dilution method,and the susceptibility results were interpreted according to CLSI 2018 and EUCAST 2018 standards.3.Detection of drug resistance genes and genotype identification The carbapenemase resistance gene amplification was performed by PCR test,and the gene subtype was identified by agarose gel electrophoresis and sequencing.4.Homology analysis of drug-resistant bacteria The homology of Proteus mirabilis producing KPC-2 type carbapenemase and successful transconjugants was analyzed by PFGE.5.Plasmid characterization analysis（1）The clonal transmission of Proteus mirabilis was analyzed using conjugation and transformation experiments.（2）The S1-PFGE-Southern blot hybridization experiment was used to analyze whether the bla_KPC-2 resistance gene of Proteus mirabilis was located in the plasmid.6.Whole genome sequencing and bioinformatics analysis The second and third generation sequencing technologies were used to sequence the bacterial genome.Sequence alignment,genome annotation and phylogenetic tree construction were used to analyze the genome composition and evolutionary relationship of Proteus mirabilis,the collinear analysis and the construction of plasmid circle map were used to analyze the similarities and differences between the genomic structures of Proteus mirabilis.Results 1.Analysis of clinical data of strains In this study,6 strains of Proteus mirabilis producing KPC-2 type carbapenemase without migration growth were detected from 365 strains of carbapenem-resistant Enterobacteriaceae,which were strains L44,L49,L52,L71,L76 and L90.2.Analysis of drug susceptibility results The drug susceptibility results showed that 6 strains of Proteus mirabilis were sensitive to ceftazidime and amikacin,and some strains were sensitive to piperacillin/tazobactam.6 strains of Proteus mirabilis were intermediate or resistant to cefepime,cefpirome and doxycycline,and resistant to other drugs.The drug susceptibility results of transconjugants L44-3 and L52-10 were similar,and the drug susceptibility results of transconjugants L76-3 and L90-11 were similar.The transconjugants L44-3 and L52-10 were only sensitive to piperacillin/tazobactam,ceftazidime,ertapenem,amikacin and tigecycline;transconjugants L76-3 and L90-11 were sensitive to gentamicin,amikacin,levofloxacin,tetracycline,doxycycline,tigecycline,chloramphenicol,trimethoprim-sulfamethoxazole,nitrofurantoin,and polymyxin.Strain J53 were sensitive to all drugs.3.Detection results of carbapenemase resistance gene PCR amplification and agarose gel electrophoresis showed that 6 strains of Proteus mirabilis carried bla_KPC gene,and the bla_KPC-2 gene was confirmed by sequencing the PCR product containing bla_KPC gene.4.Results of homology analysis The PFGE results showed that the strains L44,L52 and L71 derived from the same patient had the same electrophoresis bands and were the same strain;the strains L49 and L76 derived from the same patient were the same electrophoresis bands as the strain L90,and were the same strain.The difference bands between the 6 strains of Proteus mirabilis were less than 3,and they were highly homologous.5.Conjugation and transformation assay and bla_KPC-2 gene localization results After conjugation and transformation experiments,the successfully ligated strains included strains L44,L52,L76 and L90,and their corresponding successful transconjugants were L44-3,L52-10,L76-3 and L90-11,respectively.Combined with S1-PFGE-Southern blot hybridization,strain homology analysis and three-generation sequencing of the strain L90,it was confirmed that strains L44,L52 and L71 all carried a plasmid and the plasmid contained bla_KPC-2 gene.The strains L49,L76 and L90 both carried two plasmids,only one plasmid contained the bla_KPC-2 gene.6.Analysis of genomics results（1）The NCBI and RAST databases were used to annotate the second-generation sequencing data of 6 strains of Proteus mirabilis.The results showed that strains L44,L52 and L71 were similar,and strains L49,L76 and L90 were similar.The number of encoding genes of strains L44,L52 and L71 ranged from 3700 to 3800,and there were three different CRISPR sequences;the number of encoding genes of strains L49,L76 and L90 reached more than 3800,and there were two different CRISPR sequences.（2）The CARD database was used to analyze the sequencing results of 6 strains of Proteus mirabilis in this study.It was found that the drug resistance genes carried by the strain include aminoglycoside resistance genes（aad A2,aad A5,aac（3）-IIb,aph（3’）-Ia,aph（3’’）-Ib,aph（6）-Id）,β-lactam(bla TEM-1,bla_KPC-2),sulfa resistance genes（sul1,sul2）,chloramphenicol resistance genes cat I,trimethoprim resistance genes（dfr A1,dfr A17）,and tetracycline resistance gene tet（J）.Among them,strains L49,L76 and L90 do not contain dfr A17.（3）After sequencing,in the plasmid of about 25 kb corresponding to 6 strains of Proteus mirabilis,the sequences of p L44,p L52 and p L71 were identical,and the sequences of p L491 and p L761 were identical.The sequence of p L901 was different from them.（4）In plasmid p L901,the upstream of bla_KPC-2 is the insertion sequence IS26,Tn3 dissociation enzyme gene tnp R and the insertion sequence ISKpn27,and the downstream of bla_KPC-2 is the insertion sequence ISKpn6,which is identical to the surrounding gene environment of bla_KPC-2 in plasmid p T211.However,in plasmid p T18,the partial sequence of the transposon Tn As1 was present upstream of the insertion sequence IS26,and the transposon Tn2 was downstream,which was slightly different from the surrounding gene environment of bla_KPC-2 in plasmid p L901.（5）The plasmid circle map of plasmids p L901,p L44,p L761 and p T211 were mapped,and the results showed that the four plasmid gene sequences were highly similar.The collinear analysis showed that the entire genome structure of the 6 strains of Proteus mirabilis was extremely similar,but partial gene rearrangement and inversion occurred.The result of collinear analysis was consistent with the phylogenetic tree,and the 6 strains of Proteus mirabilis were highly homologous.Conclusions 1.The six strains of Proteus mirabilis have high homology and the genome structure is very similar,suggesting that there is clonal transmission in the hospital.Therefore,hospitals in Hangzhou should pay great attention.2.Among the six strains of Proteus mirabilis,the genetic environment around bla_KPC-2 is completely identical,and its structure is IS26-tnp R（Tn3）-ISKpn27-bla_KPC-2-ISKpn6,suggesting that bla_KPC-2 is transferred and propagated in Proteus mirabilis in Hangzhou with this complete structure.3.Carrying bla_KPC-2 and other drug resistance genes at the same time is a molecular mechanism of strains showing multi-drug resistance and even pan-drug resistance.