Subspecies Distribution And Drug Resistance Characteristics Of Mycobacterium Abscessus Complex Clinical Isolates

Background: Mycobacterium abscessus complex(MABC)is currently the most common rapid-growing non-tuberculosis mycobacterium(NTM)and is one of the important pathogenic bacteria causing various infections.Epidemiological investigations have found that the incidence of MABC infection is increasing year by year in China,and it is naturally resistant to many antibiotics.Therefore,the main focus of its prevention and treatment is on the subtypes and resistance characteristics of this strain.In recent years,studies have shown that the drug resistance of MABC in China has certain regional differences and epidemic characteristics,and the resistance characteristics of this strain are also constantly changing.Research indicates that the production rate of broad-spectrum β-lactamases has been increasing year by year in China,which has become an important issue in clinical treatment.Therefore,in-depth research on the subtype distribution and resistance characteristics of clinically isolated MABC in China will help to better understand the epidemiology and resistance mechanisms of this pathogen,and provide more effective drug choices and personalized treatment plans for clinical treatment.Objective: To understand the distribution of subtypes of clinically isolated MABC in Guangzhou Chest Hospital,compare the resistance characteristics of different subtypes and phenotypes of MABC clinically isolated strains in the region,and verify the relationship between in vitro drug sensitivity of clinically isolated MABC strains to clarithromycin and amikacin and reported gene mutations.Methods:In this retrospective study,a total of 200 MABC clinical isolates were collected from Guangzhou Chest Hospital from March 2019 to September 2019 and identified by biochip technology.Whole genome sequencing was performed on the isolates and in vitro drug susceptibility testing was carried out using 15 common antibiotics(Amikacin,Moxifloxacin,Linezolid,Ceftriaxone,Cefepime,Cefoxitin,Tigecycline,Tigecycline,Minocycline,Doxycycline,Ciprofloxacin,Amoxicillin/clavulanic acid 2:1 ratio,Timethoprim/sulfamethoxazole,Imipenem,and Clarithromycin)and 4 anti-tuberculosis drugs(Bedaquiline,Clofazimine,Delamanid,and Capreomycin)by microplate method.The reference genome sequences of Mycobacterium abscessus subsp.abscessus(Mab,strain: ATCC 19977,ASM6918v1),Mycobacterium abscessus subsp.massiliense(Mma,strain: JCM 15300,ASM49726v2 FLAC047,ASM214003v1),and Mycobacterium abscessus subsp.bolletii(Mbo,strain: BD,ASM360971v1)were downloaded from NCBI according to the type strains provided by LPSN database.Data quality control and filtering were performed using the fastp software in the Linux system,MABC subspecies identification was carried out using Burrow-Wheeler Aligner(bwa)software,and gene variation detection was performed using SAMtools and BCFtools software.Results: 1.The distribution of MABC clinical isolates subtypesIn this study,a total of 196 clinically isolated MABC strains were collected from 195 patients,of which 124 patients had a long-term residency in Guangdong Province,with half of them from Guangzhou(62/124,50.0%).The most common subtype was Mab(103/200,51.5%),followed by Mma(89/200,44.5%),and the least common was Mbo(2/200,1%).In addition,2 clinical isolates were identified as both Mab and Mma(2/200,1%),and 4 clinical isolates were identified as non-MABC,so they were not included in the analysis.Among the 196 MABC strains,193 completed drug susceptibility testing,and 3 Mab strains were contaminated by other bacteria and could not complete drug susceptibility testing,and were therefore excluded.2.Antimicrobial susceptibility results of different subspecies of clinical MABC isolates.Firstly,among the tested drugs,Mab demonstrated the lowest resistance rates to tigecycline(0%)and amikacin(2%),followed by clarithromycin(27%),linezolid (50%),and cefoxitin(71%).Secondly,Mma exhibited the lowest resistance rates to amikacin(1%)and tigecycline(6%),with slightly higher rates observed for clarithromycin(17%),linezolid(44%),cefoxitin(63%),and minocycline(74%).Moreover,the remaining drugs showed resistance rates exceeding 92%.Importantly,Mab displayed significantly higher resistance rates to clarithromycin(p=0.001)and minocycline(p<0.001)compared to Mma.Notably,in vitro testing revealed that bedaquiline and clofazimine exhibited effective antibacterial activity against MABC clinical isolates,while delamanie and capreomycin failed to inhibit their growth.3.Susceptibility to Clarithromycin and rrl Genomic Variations in MABCThe resistance rate to clarithromycin in Mab was 27%(27/100),with 36 Mab isolates showing inducible resistance to clarithromycin.The resistance rate to clarithromycin in Mma was 17%(15/89),with no Mma isolates showing inducible resistance.When the MIC of clarithromycin was >16μg/ml,rrl gene mutations at position 2270 occurred in 24%(5/21)of Mab isolates and 57%(8/14)of Mma isolates,where the original A base was replaced by G.No mutations were observed in isolates with an MIC <16μg/ml.4.Analysis of Relationship between erm(41)Gene and Clarithromycin Susceptibility in MABCAmong the clarithromycin-sensitive Mab isolates,72%(18/25)had a C base at position 28 of the erm(41)gene,while only 1 isolate of inducibly resistant or resistant Mab isolates had a C base at this position.In addition,there was a significant difference in the mutation status at position 255 of the erm(41)gene between clarithromycin-sensitive and acquired resistant Mab isolates(χ2=14.584,P=0.001).47%(17/36)of the acquired resistant Mab isolates had a mutation at position 255(A>G),while only 8%(2/25)of the sensitive isolates had this mutation.The erm(41)gene was deficient in all 89 Mma isolates.5.Susceptibility to Amikacin and rrs Genomic Variations in MABCThe resistance rate to amikacin in MABC was only 2%(3/193),with 67%(2/3)of resistant isolates having a mutation at position 1375 of the rrs gene(A>G),while no mutations were observed in other isolates.Detection of a mutation at position 1375 of the rrs gene helped indicate resistance to amikacin.No mutations were observed in the rrs gene when the MIC of amikacin was ≤1μg/m L.6.The correlation between MABC infection and clinical manifestationAmong the patients from whom MABC was isolated after 2 months of anti-tuberculosis treatment,only 3 cases(3/35)of tuberculosis progression were observed.In contrast,among the patients who were not receiving anti-tuberculosis treatment or in whom MABC was isolated within 2 months,even those who received more than 2 months of anti-NTM treatment,70%(42/60)showed disease progression.Conclusion:1.In our study,the most common subspecies of MABC is Mab,followed by Mma and the least common is Mbo.2.This study found that MABC clinical isolates were most sensitive to tigecycline and amikacin,followed by clarithromycin and linezolid.Bedaquiline and clofazimine both had good antibacterial activity against MABC clinical isolates,while doramapimod and capreomycin were not effective in inhibiting its growth.There were differences in the in vitro susceptibility results of MABC clinical isolates among different subtypes and phenotypes.3.Detection of the erm(41)gene mutation at position 28 and the rrl gene mutation at position 2270 in clinical isolates of MABC may help indicate inducible and acquired resistance to clarithromycin.Detection of the rrs gene mutation at position 1375 may help indicate resistance to amikacin.