Occurrence And Characteristics Of ESBLs-producing Laribacter Hongkongensis

Objectives1.To understand the prevalence and drug resistance of extended-spectrum(3-lactamase(ESBLs)-producing Laribacter hongkongensis(L.hongkongensis)from human,fish and frogs.2.To characterize the occurrence of genes encoding p-lactamases,other genes that confer resistance to sulfonamide(sull,sul2 and sul3),tetracycline(tetA,tetR,and tetAM),and fluoroquinolones(qnrA and aac(6,)-Ib-cr),as well as virulence factors among ESBLs-producing isolates.3.To characterize the ESBLs-producing isolates with respect to integrons and plasmid replicon types,provide evidence for the molecular mechanism of resistance transmission.Method1.A total of 112 L.hongkongensis isolates were selected for this study,including 2 from humen in Jaiangmen,1 from human in Guangzhou,1 from human in Hongkong,62 from Chinese tiger frogs and 46 from grass carps in Shenzhen,China.Of which,109 strains were isolated from April 2015 to May 2016,2 strains were isolated in October 2009,and 1 isolated in March 2009.All the isolates were identified by using a 16S rRNA gene-based PCR assay in combination with a series of biochemical tests2.All the confirmed L.hongkongensis isolates were tested for susceptibility against 25 antibiotics by the disc di□usion method on Mueller-Hinton agar according to the CLSI tentative standard for Enterobacteriaceae(except for erythromycin and rifampicin)and Staphylococcus spp.(for erythromycin and rifampicin).The double-disc synergy test(DDST)with cefotaxime or ceftazidime in proximity to amoxicillin-clavulanic acid was used to further confirmation of the isolates as ESBLs positive.3.The genes encoding TEM,CTX-M-1,CTX-M-9,SHV and OXA-1 typeβ-lactamases,other genes that confer resistance to sulfonamide(sull,sul2 and sul3),tetracycline(tetA,tetR,and tetM),and fluoroquinolones(qnrA and aac(6,)-Ib-cr)were screened by PCR.PCR amplification was also performed to screen for the presence of 18 plasmid replicons,integrons and the variable regions of the class 1 integrons,4.SPSS20.0 statistical software was used to analyze the distribution of drug resistance patterns,drug resistance genes,virulence genes,integrons,plasmid replicon types and the association among them.The comparison difference of the rate of compared with chi-square test or Fisher’s exact probability test,both bilateral inspection,inspection level a= 0.05.Results1.A total of 112 L.hongkongensis isolates were selected for this study,91(81.3%)of the isolates(3 humen,47 frogs and 41 fish)were confirmed as ESBLs producers.ESBLs-producing L.hongkongensis isolates showed significantly higher resistance rates to ampicillin(33.0%vs 9.5%),cefazolin(75.2%vs 38.1%),cefotaxime(85.7%vs 57.1%),ceftriaxone(87.9%vs 52.4%),cefoxitin(50.5%vs 19.0%),ciprofloxacin(18.7%vs 0%)and trimethoprim-sulfamethoxazole(29.7%vs 4.8%)than non-ESBLs-producing L.hongkongensis isolates(p<0.05).The incidence of multidrug resistance for ESBLs-producing L.hongkongensis isolates(58.9%)was higher than that observed for non-ESBLs-producing strains(38.1%)(p<0.05).2.The resistant rates of trimethoprim-sulfamethoxazole(57.4%vs 0.0%),tetracycline(53.2%vs 0.0%),ciprofloxacin(34.0%vs 2.4%),levofloxacin(19.1%vs 0.0%)and streptomycin(10.2%vs 0.0%)were significantly higher for the isolates from frog ESBLs-producing(72.3%)than for the isolates from fish(51.2%)(P<0.05).Multi-resistance strains were also more frequent among frog isolates than fish(p<0.05).Remarkably,multi-resistance frog isolates showed a higher average number of antibiotic resistance traits(5.44 traits)than did fish isolates with an average of 3.29 traits per strain.The dominant multi-resistance profile of frog isolates was CFP/CEP/CFZ/CTX/CAZ/CRO/FOX/RIF/TET/SXT,while the most frequent multi-resistance profile of multi-resistance fish and humen isolates was AMP/CFP/CEP/CFZ/CTX/CAZ/CRO/FOX/RIF and CFP/CEP/RIF/AMP/CAZ,respectively.3.PCR and sequencing revealed that all frog and fish ESBLs-producing strains were positive for at least one of the main β-lactamase gene groups.Of these,lacTX-M-9 was predominant(90.1%),followed by blaTEM(90.1%).The ESBLs-producing also contained genes conferring resistance to sulfonamides(sell,sul2),tetracycline(tetA,tetR)\and fluoroquinolones(aac(6’)-Ib-cr)(33.0%,20.9%,25.3%,26.4%and 9.9%,respectively).Genes that encode virulence determinants related to adhesin-related proteins(fimH,bfpA and eaeA),siderophore-related proteins(ireA and iucD),toxin gene hlyA,and tissue invasion,such as protectins(traT and iss)(100%,28.6%,1.1%,23.1%,7.7%,2.2%,92.3%and 42.9%,respectively).4.The resistance genes sull,sul2,tetA,tetR and aac(6’)-Ib-cr were detected more frequently in frog ESBLs-producing isolates(55.3%,40.4%,40.4%,42.6%,17.0%,respectively)than in fish isolates(9.8%,0.0%,9.8%,9.8%,2.4%,respectively)(p<0.05).The average number of virulence factors traits were higher among frog strains(3.40)than the fish strains(3.02),and the distribution of three virulence-associated determinants for the type IV bundle-forming pill gene(bfpA)(40.4%vs 17.1%),ferric aerobactin receptor gene(iucD)(34.0%vs 12.2%)and iron-responsive element gene(ireA)(12.8%vs 2.4%)were markedly higher in strains isolated from frogs than fish(p<0.05).Among the 47 ESBL-positive frog strains,18 distinct combination patterns of virulence genes were found,and each included only a few strains.The four most frequent patterns(n≥5)fimH-traT-iss,fimH-traT-iss-bfpA,fimH-traT-bfpA,and fimH-traT-ireA were present in 10,7,7,and 6 isolates,respectively.The fish strains exhibited 8 different profiles,and 30 strains belonged to either the fimH-traT-iss(25 isolates)profile or the frmH-traT(5 isolates)profile.In total,these 2 major profiles accounted for 73.2%of the fish strains.5.Among the 112 L.hongkongensis isolates,10 replicon types were detected,including IncFrep(83.9%),IncFIA(69.6%),IncFIB(33.0%),IncL/M(25.9%),IncI1(24.1%),IncFIC(23.2%),IncHI2(11.6%),IncA/C(11.6%),IncK/B(5.4%),and IncX(4.5%)groups.The disstribution of replicon types were different among ESBL-producing/non-ESBL-producing L.hongkongensis isolates,and MDR/non-MDR strains.6.The integron integrase genes intll and intI3 were detected in 24.1%(27/112)and 22.3%(25/112)of 112 L.hongkongensis isolates,respectively.Class 1 integrons were more frequently observed among ESBL-producing strains(28.6%,26/91)than among non-ESBL-producing strains(4.8%,1/21)(P<0.05).Fish ESBLs-producing strains were distinguished by a significantly lower prevalence(14.6%,6/41)of class 1 integrons than the frog strains(42.6%,20/88)(P<0.05).After PCR amplification of the variable regions(VR)of the intl1-positive L.hongkongensis isolates,22 isolates(81.5%,22/27)tested positive for the presence of the variable region.All the variable regions demonstrated the presence of five di□erent cassette combinations for five di□erent genes,which harbored genes that confer resistance to trimethoprim(dfrAl and dfrA27),streptomycin or spectinomycin(aadA1),kanamycin and gentamicin(aacA4)and rifampicin(arr3).Class 1 integron-positive frog ESBLs-producing isolates harboring more kinds of gene cassettes than fish.7.A statistically significant(P<0.05)association was detected between the incidence of antimicrobial resistance(genes)and class 1 integrons among the collection of frog ESBLs-producing L.hongkongensis isolates.The resistance to trimethoprim-sulfamethoxazole and tetracycline was significantly associated with the presence of integrons.Moreover,the associations between the class 1 integrons and the resistance genes sull,sul2,tetA and tetR were clear.Conclusion1.A very high occurrence of ESBLs-producing L.hongkongensis(81.3%)was observed in Human and aquatic animals,and blacrx-M-9(90.1%)and blaTEm(75.8%)were the two major ESBLs-encoding genes in the studied isolates,indicating multiple genes mediate the production of ESBLs.2.A large diversity of drug resiatance genes,integrons,virulence genes,plasmid replicons and resistance phenotypes was observed among ESBLs producers.More importantly,the higher detection rates of resistance phenotypes,drug reistance genes,virulence markers,class 1 integrons and gene cassettes were observed among frog ESBLs-positive L.hongkongensis,which is a serious public health concern3.Ten replicon types were detected among the 112 L.hongkongensis isolates,including IncFrep,IncFIA,IncFIB,IncL/M,IncI1,IncFIC,IncHI2,IncA/C,IncK/B,and IncX groups,with the IncFrep froup most dominant.Th.e disstribution of replicon types were different among ESBL-producing/non-ESBL-producing L hongkongensis isolates,and MDR/non-MDR strains.4.The resistance to trimethoprim-sulfamethoxazole and tetracycline was significantly associated with the presence of integrons.Moreover,the associations between the class 1 integrons and the resistance genes sul1,sul2,tetA and tetR were clear.The high occurrence of sul1 gene,dfrA and aadA gene cassettes which confer resistance to sulfamethoxazole and trimethoprim,might contribute to sulfonamide resistance among the frog ESBLs producers in this study.