The Relevance Of Virulence Genes And Their Expression In Vivo And In Vitro Between APEC And UPEC Isolates And Knock-out Of Ftsk Gene In APEC Strain E058

Urinary tract infections (UTIs) are among the most common human diseases in the world, causing significant morbidity and mortality. Most community-acquired UTIs are due to uropathogenic E. coli (UPEC) infections. UPEC strains possess specialized virulence factors, enabling them to colonize and invade to the host, disrupt the host defense mechanisms, injure host tissues, and/or stimulate noxious host inflammatory response. The abilities of UPEC to grow extraintestinally may enable them to cause a variety of diseases, not just urinary tract ones. Avian colibacillosis refers to any localized or systemic infection such as an acute fatal septicemia or subacute pericarditis and airsacculitis caused entirely or partly by avian pathogenic Escherichia coli (APEC). It is responsible for large financial losses for the poultry industry each year due to mortality, lost production, and condemnations.Since APEC and UPEC may encounter similar challenges when establishing infection in extraintestinal locations, they may share a similar content of virulence genes and capacity to cause disease. It is becoming more and more apparent that the common presence of a set of virulence associated genes among APEC and UPEC strains as well as similar disease patterns and phylogenetic background indicate a genetic relationship between APEC and UPEC isolates. The potential whether APEC might serve as a reservoir of virulence genes for UPEC should be considered.1. Prevalence of virulence factors and antimicrobial resistance of UPEC isolates in Jiangsu area202 UPEC isolates were characterized for serogroups, virulence genes, the antimicrobial susceptibilities and the phylogenetic groups. Most of the typable strains belonged to O1, O6, O7, O15, O18, O26 and O25 serogroups, respectively. The most frequent phylogenetic groups were D and B2, which represented 42% and 38% of the isolates respectively, followed by A and B1 groups, which were 13% and 7%, respectively. Among 37 tested virulence genes, feoB and fimH were the most prevalent genes, which distributed in over 90% of the isolates. Above half of the isolates possessed one or more of the following 10 virulence genes—papA, papC, papEF, papG, kpsMTⅡ, ompT, iutA, fyuA, traT and irp-2. pap related genes were the most prevalent other than fimH among adhesin genes. kpsMT genes occurred in a high prevalence in protectin genes studied, and genes encoding toxins were at similar frequencies. All isolates showed over 50% resistance rate to all of 14 tested antimicrobials except for cefoxitin, nitrofurantoin and chloramphenicol, which were 22%, 23% and 36%, respectively. Among them, the resistance rate to nalidixic acid, mezlocillin, and ampicillin was higher than 90%. Importantly, all of the isolates were multi-resistant, and 73% of them were resistant to trimethoprim-sulfamethoxazole. The above evidence strongly suggests that the problem of antibiotic resistance for UPEC is rather serious in this area, and that empirical treatment of UTIs with all the above antibiotics is no longer appropriate in the same area. Our findings also showed that among human UPEC isolates originated from this area, cefotaxime-, cefoxitin-, chloramphenicol-, and nitrofurantoin-resistant isolates had a reduced virulence genes compared with those of susceptible strains. To our knowledge, this is the first report of a statistically significant reduction in virulence genes among nitrofurantoin-resistant isolates.2. Comparison of virulence genes and the expression of specific genes between APEC and UPEC isolates in murine urinary tract infection model and chicken challenge modelAs diversity, epidemiological sources, and evolutionary origins of ExPEC are so far only partially defined, 100 of APEC isolates and 202 of UPEC isolates were compared by their serogroups, content of virulence genes, phylogenetic groups, and the expression of specific virulence genes in the present study. The results showed that O1 and O18 were the dominant serogroups of UPEC isolates, which were also prevalent in APEC isolates. Besides the UPEC phylogenetic groups mentioned above, most of APEC isolates belong to A and B2 groups, which represented 42% and 36% of the isolates respectively, followed by D and B1 groups, which were 12% and 10%, respectively. Some virulence genes, such as those encoding of adhesins, iron-related genes, and pTJ100-related genes were often carried by APEC and UPEC isolates. Althogh LD50 of UPEC strain U17 was slightly lower than that of APEC strain E058, similar lesions were observed in birds inoculated with either UPEC U17 or APEC E058. To gain further information of the correlation and pathogenesis of UPEC and APEC isolates, the in vivo expression of specific genes in both murine urinary tract infection (UTI) model and chicken challenge model were compared to that of UPEC U17 and APEC E058 grown statically to exponential phase in rich medium by DNA chips, respectively. In chicken challenge model, 89% of the T-test genes showed the similar tendency of expression in both APEC E058 and UPEC U17. In chicken challenge model, 5 genes (cvaC, neuC, ompT, iutA and iucCD) were differentially up-regulated in APEC E058, and two (cvaC and neuC) of them were also differentially up-regulated in UPEC U17, and the left three (ompT, iutA and iucCD) of them were up-regulated in UPEC U17 also. At the same time, 4 genes (aec-30, aes-8, gyrB and mdh) differentially down-regulated in APEC E058 were also down-regulated in UPEC U17. In murine UTI model, 82% of the T-test genes showed the similar tendency of expression in both APEC E058 and UPEC U17. Two differentially up-regulated genes (iucCD and tir) in UPEC U17 were also up-regulated in APEC E058, and 2 differentially down-regulated genes (sta and catI) in UPEC U17 were also down-regulated in APEC E058. The results show that in the same model (murine UTI model or chicken challenge model), most of the T-test genes of UPEC U17 and APEC E058 show the similar tendency of expression. Several iron-related genes were up-regulated in murine UTI model and /or chicken challenge model, revealing that iron acquisition is critical for E. coli to survive in blood or urinary tract. Based on these results, the potential for APEC to act as human UPEC or as a reservoir of virulence genes for UPEC should be considered. Further, this study represents the first comparison of APEC and UPEC transcriptome in vivo and provides specific insights into the mechanisms necessary for extraintestinal pathogenesis.3. Development and application of relative real time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for the analysis of the expression of ompT and ftsk genesTo relatively quantify the expression of ompT and ftsk genes of APEC strain E058 and UPEC strain U17, two-step real time quantitative RT-PCRs (qRT-PCR) were developed based on SYBR Green I, respectively. ompT and ftsk were as target genes and gapA as internal reference, respectively. Three standard curves were established using a series dilution of cDNA synthesized from the RNA of APEC E058 grown statically to exponential phase in rich medium. In chicken challenge model, the expression of ompT and ftsk in APEC E058 were up-regulated 6.69- and 23.57-fold compared to that of APEC E058 grown statically to exponential phase in rich medium, respectively. Meanwhile, the expression of ompT and ftsk were up-regulated 6.72- and 9.21-fold in UPEC U17 compared to that of UPEC U17 grown statically to exponential phase in rich medium, respectively. Previous study showed that the expression of ompT in APEC E058 was up-regulated 2.54-fold in chicken challenged model by using DNA chips, when compared to that of APEC E058 grown statically to exponential phase in rich medium. These data verified the results of DNA chips, and qRT-PCR analysis demonstrated a greater transcription level sensitivity than that of microarray analysis. The result also suggests that ompT and ftsk may be the important virulence genes of APEC.4. Construcion of ftsk knock-out mutant of APEC strain E058 and evaluation of its pathogenesis in chickensThe 709-bp fragment of ftsk1, 900-bp fragment of the ftsk2 and the gene encoding kanamycin resistance were generated by PCR respectively. All of these fragments were cloned into pBluescriptⅡS K (+) (pBSK) vector, termed pBSK-F2-Kan-F1. Fragment of F2-Kan-F1 amplified by PCR were transformed into APEC strain E058, and then the ftsk knock-out mutant of APEC strain E058 were generated by allelic replacement and named E058(?ftsk). The growth rate of E058(?ftsk) was slower than that of APEC E058. In chicken challenge model, the mutant was tested to determine the individual role of this gene for virulence and persistence in 1-day old SPF chicks. At 6, 12 and 24 hours after inoculation, the number of colony forming unit (CFU) recovered from the liver, spleen, lung and blood of chickens inoculated with E058(?ftsk) were significantly less than that of chickens inoculated with the parent strain APEC E058 (p<0.05), respectively. All birds were died during 48 hours post-inoculation with E058, however, only 20% of the birds were died within the same period post-inoculation with E058(?ftsk). All data showed that E058(?ftsk) had a reduced virulence in 1-day old chickens. It was hypothesized that ftsk may be an important virulence gene of APEC E058. The actual function of ftsk gene needs further studying.In summary, the primary conclusions were drawn as following:1) 202 UPEC isolates were collected from patients with UTIs in Jiangsu Province. Among those isolates, O1, O6, O7, O15, O18, O25 and O26 were the prevalent serogroups, and D and B2 were the most frequent phylogenetic groups.2) Among 37 tested virulence genes, feoB and fimH were the most prevalent genes, which distributed in over 90% of the isolates. Above half of the isolates possessed one or more of the following 10 virulence genes, papA, papC, papEF, papG, kpsMTⅡ, ompT, iutA, fyuA, traT and irp-2.3) All UPEC strains showed over 50% resistance rate to all of 14 tested antimicrobials except for cefoxitin, nitrofurantoin and chloramphenicol, which were 22%, 23% and 36%, respectively. Among them, the resistance rate to nalidixic acid, mezlocillin, and ampicillin was higher than 90%. Importantly, all of the isolates were multi-resistant, and 73% of them were resistant to trimethoprim-sulfamethoxazole.4) Of the UPEC isolates originated from Jiangsu area, cefotaxime-, cefoxitin-, chloramphenicol-, and nitrofurantoin-resistant isolates have a reduced virulence genes compared with those of susceptible strains. And this is the first report of a statistically significant reduction in virulence genes among nitrofurantoin-resistant isolates.5) UPEC and APEC isolates showed substantial overlap in terms of their serogroups, phylogenetic groups, and virulence genotypes including their possession of certain genes associated with large transmissible plasmids of APEC.6) No host specificity could be observed in both APEC and UPEC isolates, since both avian- and human-originted isolates were highly pathogenic to chickens as well as caused similar lesions in the birds inoculated.7) In chicken challenge model, 89% of the T-test genes showed the similar tendency of expression in both APEC E058 and UPEC U17. 5 genes (cvaC, neuC, ompT, iutA and iucCD) were differentially up-regulated in APEC E058, and two (cvaC and neuC) of them were also differentially up-regulated in UPEC U17, and the left three (ompT, iutA and iucCD) of them were up-regulated in UPEC U17 also. 4 genes (aec-30, aes-8, gyrB and mdh) differentially down-regulated in APEC E058 were also down-regulated in UPEC U17.8) In murine UTI model, 82% of the T-test genes showed the similar tendency of expression in both APEC E058 and UPEC U17. Two differentially up-regulated genes (iucCD and tir) in UPEC U17 were also up-regulated in APEC E058, and 2 differentially down-regulated genes (sta and catI) in UPEC U17 were also down-regulated in APEC E058.9) Several iron-related genes were up-regulated in UTI model and/or chicken challenge model, revealing that iron acquisition is critical for E. coli to survive in blood or urinary tract.10) Two-step qRT-PCRs were developed based on SYBR Green I for relatively quantifying the expression level of ompT and ftsk genes of APEC strain E058 and UPEC strain U17. Three standard curves were established using a series dilution of cDNA synthesized from the RNA of APEC E058 grown in vitro.11) The expression of ompT and ftsk of UPEC U17 and APEC E058 were up-regulated in chicken challenge model compared to that of APEC E058 and UPEC U17 grown statically to exponential phase in rich medium, respectively. These data verify the results of DNA chips, and qRT-PCR analysis demonstrate a greater transcription level sensitivity than that of microarray analysis, and suggest that ompT and ftsk may be the important virulence genes of APEC.12) All data show that E058(?ftsk) has a reduced virulence in 1-day old chickens. It is reasonable that ftsk gene screened in SSH and confirmed in qRT-PCR might be an important virulence gene of APEC E058.