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Novel high-throughput screening method using quantitative PCR to determine the antimicrobial susceptibility of Orientia tsutsugamushi clinical isolates.

ABSTRACT: Objectives:To develop a method to enable the large-scale antimicrobial susceptibility screening of Orientia tsutsugamushi clinical isolates, using one timepoint and one concentration of antibiotics to considerably speed up the time to result. Methods:Growth, harvesting, multiplicity of infection (moi) and the day to determine the MICs were optimized using five O. tsutsugamushi reference strains [susceptible (Karp, Kato and Gilliam) and putatively resistant (AFC-3 and AFSC-4)], one clinical isolate (UT76) and one rodent isolate (TA763). Subsequently, the MICs of azithromycin, chloramphenicol and doxycycline for these strains and 51 clinical isolates including AFSC-7 were determined. An optimal concentration was calculated using the epidemiological cut-off value. Results:The conditions for O. tsutsugamushi infection, growth and harvesting were determined to be an moi of 100:1 and trypsinization with the peak growth on day 10. The resulting MICs were in line with previously published susceptibility data for all reference strains, except for Karp and AFSC-4, which showed azithromycin MICs of 0.0156 and 0.0313?mg/L, compared with 0.0078 and 0.0156?mg/L, respectively, in previous reports. The MIC of doxycycline for AFC-3 was 0.125?mg/L compared with >4?mg/L in earlier reports. The final single screening concentrations were identified as: azithromycin, 0.125?mg/L; chloramphenicol, 8?mg/L; and doxycycline, 1?mg/L. Conclusions:This simplified procedure facilitates the simultaneous screening of 48 isolates for actively monitoring potential resistance of this important fever pathogen, with an 8-fold throughput improvement over early methods. The data do not support the existence of doxycycline- and chloramphenicol-resistant scrub typhus.

SUBMITTER: Phuklia W

PROVIDER: S-EPMC6293087 | biostudies-other | 2019 Jan

REPOSITORIES: biostudies-other

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Novel high-throughput screening method using quantitative PCR to determine the antimicrobial susceptibility of Orientia tsutsugamushi clinical isolates.

Phuklia Weerawat W Panyanivong Phonepasith P Sengdetka Davanh D Sonthayanon Piengchan P Newton Paul N PN Paris Daniel H DH Day Nicholas P J NPJ Dittrich Sabine S

The Journal of antimicrobial chemotherapy 20190101 1

<h4>Objectives</h4>To develop a method to enable the large-scale antimicrobial susceptibility screening of Orientia tsutsugamushi clinical isolates, using one timepoint and one concentration of antibiotics to considerably speed up the time to result.<h4>Methods</h4>Growth, harvesting, multiplicity of infection (moi) and the day to determine the MICs were optimized using five O. tsutsugamushi reference strains [susceptible (Karp, Kato and Gilliam) and putatively resistant (AFC-3 and AFSC-4)], one ...[more]

PMID: 30295746

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Project description:Orientia tsutsugamushi (Ots) frequently causes severe scrub typhus infections in the Asia-Pacific region. Korean investigators have demonstrated that Ots encodes five different autotransporter domain (ATD) proteins (ScaA-ScaE). ScaA functions as an adhesin and confers protective immunity in a lethal mouse model of Ots infection. Specific antibodies are detected against ScaA and ScaC in Korean scrub typhus patients. However, there is limited data on the distribution of the Sca protein genes in diverse isolates of Ots. By BLAST analysis with the conserved beta barrel autotransporter domain (ATD) regions of the sca proteins, we discovered a sixth gene scaF among 3 of 10 new partial Ots genome sequences available at NCBI GenBank (Sido, Karp, AFSC7). We designed two to seven specific TaqMan assays to detect the ATD for each of the six sca genes. The TaqMan assays among those for each sca gene which gave the greatest sensitivity and linearity with DNA log dilutions were then used for screening DNAs from Ots isolates grown in L929 mouse cells for sca genes. The sca prevalence survey was performed for all six sca genes with 178 DNAs from isolates from 12 countries. The survey results were confirmed by conventional PCR with primers from conserved regions of the passenger domains (PD) and ATD of the sca proteins. The ATD was highly conserved between the DNAs of different genotypes compared to the sca PD but each TaqMan assay was sca specific. The percentage positivity for 56 kDa and scaA genes in the 178 DNAs using Ha primers was 59.6% and 62.4%, respectively. Our scaA conventional ATD PCR assay was positive in 98.3% but scaA was present in all 178 DNAs (100%) by ATD TaqMan. scaB, scaC, scaD, scaE and scaF were detected in 33.7%, 97.8%, 93.8%, 97.2% and 43.3% isolates by ATD TaqMan, respectively. The ATDs of Ots sca genes are thus sufficiently conserved between different genotypes for molecular assay design. Four sca genes are widely distributed among diverse Ots isolates from diverse geographical areas. scaB and scaF were detected in fewer Ots isolates and absent from some available genome sequences. Whether the utility of the ScaA, ScaC, ScaD, and ScaE antigenic passenger protein domains exceeds that of the mixed 56 kDa type surface antigens of Ots now used in combination diagnostic assays needs to be determined before they can be considered as suitable alternative serological antigens for diagnosis of scrub typhus.

Dataset Information

Novel high-throughput screening method using quantitative PCR to determine the antimicrobial susceptibility of Orientia tsutsugamushi clinical isolates.

Publications

Novel high-throughput screening method using quantitative PCR to determine the antimicrobial susceptibility of Orientia tsutsugamushi clinical isolates.

Similar Datasets

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