Antimicrobial Susceptibility of Mycobacterium abscessus Complex Clinical Isolates from a Chinese Tertiary Hospital

Introduction

Mycobacterium abscessus complex (MABC) is a group of pathogens that account for 80% of rapidly growing mycobacteria (RGM) isolates. MABC strains are ubiquitous environmental bacteria often found in dust, soil, and water.¹ They often cause pulmonary infections (which can occur in patients with cystic fibrosis), complicated infections of the skin and soft tissues, and disseminated infections, leading to high mortality rates.² MABC strains can be classified into three clearly divergent subspecies: M. abscessus subsp. Abscessus(M. abscessus), M. abscessus subsp. Massiliense(M. massiliense), and M. abscessus subsp. bolletii (M. bolletii) based on genomic analysis, which has dramatically increased our knowledge regarding MABC.³ The high rates of antimicrobial resistance of MABC strains can render even combination antibiotic treatment ineffective.⁴ Consequently, identifying novel treatment approaches is imperative.

Combination drug regimens, which can involve clarithromycin (CLA), amikacin (AMK), azithromycin (AZM), the cephalosporin (a type of β-lactam) cefoxitin (FOX), and the carbapenem (another type of β-lactam) imipenem (IPM), are recommended by the American Thoracic Society and the Infectious Disease Society of America, but less than half of patients with MABC infection can be cured with these treatments.^5,6 One report indicated that M. abscessus and M. bolletii isolates often exhibit inducible resistance to CLA, while M. massiliense isolates were mostly susceptible.⁷ However, another study reported that M. abscessus is less responsive to CLA.⁸ Because of the high rates of resistance, treatment for MABC infections is often time-consuming and costly.

Recently, several molecular mechanisms underlying CLA resistance were identified. The primary innate mechanism underlying CLA resistance in MABC involves an increase in expression of the erythromycin ribosomal methylase gene, erm(41). Induction of erm(41) is mediated by the transcription factor whiB7,⁹ and rifabutin suppresses inducible CLA resistance by preventing induction of whiB7 and erm(41) expression.¹⁰ Inducible resistance to CLA was identified in a sequevar with an intact erm(41) but with a single-nucleotide polymorphism (C to T) at position 28¹¹ (C at position 28 in M. abscessus results in CLA susceptibility). Notably, M. massiliense strains with a CLA susceptible phenotype have a nonfunctional erm(41) gene.⁷ Acquired high-level resistance to CLA can be caused by a point mutation (A2058G or A2059G) in the rrl gene (which encodes domain V of 23S rRNA).¹¹

In addition, there are some mechanisms of resistance of MABC to carbapenems. Resistance to β-lactams (such as the carbapenem IPM) was found to be related to the initial characterization of the MABC β-lactamase (Bla_Mab) and transpeptidases.^12,13 The development of new antimicrobials is important to overcome the emergence of carbapenem resistance;¹⁴ currently, there are several active pharmaceutical programs developing β-lactamase inhibitors. Previous studies reported that the β-lactamase inhibitor avibactam (AVI) could effectively inhibit Bla_Mab and thus improve the effects of β-lactams in MABC infection.^13,15 Recent evidence suggests that dual β-lactam therapy may be more effective than using a single β-lactam.¹⁶

In this study, MABC isolates were assessed for susceptibility to various antimicrobials, and the relationship between the erm(41) gene and inducible CLA resistance in each subspecies of MABC was explored. The results provide guidance for the empirical therapy of RGM infections. Additionally, we assessed the individual and combined effects of two β-lactams (imipenem [IPM] and ceftazidime [CAZ]) and a β-lactamase inhibitor (avibactam [AVI]), and the results provide further insights into the treatment options.

Materials and Methods

Collection of MABC Isolates, Culture Conditions, and Informed Consent

Between 2014 and 2018, 129 MABC isolates were randomly isolated from the sputum and bronchoalveolar fluids of patients with clinical signs of lower respiratory tract infections in three tertiary hospitals (Shanghai Pulmonary Hospital, the Second Affiliated Hospital of Nanchang University, and the Second Affiliated Hospital of Suzhou University). All isolates were cultured in Middlebrook 7H10broth (BD, France) supplemented with 10% (vol/vol) oleic acid-albumin-dextrose-catalase (Thermo Fisher Scientific, USA). The cultures were incubated at 37°C for 7 days.

The Ethics Committee of Shanghai Pulmonary Hospital, Tongji University School of Medicine, exempted this study from ethical review because the assessment of the bacteria was part of routine hospital laboratory procedures. Verbal informed consent was obtained from all participants.

Identification of Subspecies and Detection of erm(41) and rrl Mutations

Genomic DNA was extracted from the MABC cultures for identification based on the sequences of the genes 16S rRNA, rpoB, and hsp65. The 16S rRNA gene was amplified using primers F (5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ) and R (5ʹ-ACGGGCGGTGTCTACAA-3ʹ), as previously described.¹⁷ A 723-bp fragment of the rpoB gene was amplified using primers rpoBF (5ʹ-GGCAAGGTCACCCCGAAGGG-3ʹ) and rpoBR (5ʹ-AGCGGCTGCTGGGTGATCATC-3ʹ), as previously described.¹⁸ The hsp65 gene was amplified using primers hsp65F (5ʹ-ACCAACGATGGTGTGTCCAT-3ʹ) and hsp65R (5ʹ-CTTGTCGAACCGCATACCCT-3ʹ), as previously described.¹⁹ The erm(41) gene (F:5ʹ-TGGTATCCGCTCACTGATGA-3ʹ and R:5ʹ-GCGGTGGATGATGGAAAG-3ʹ) and rrl gene (F: 5ʹ–CCTGCACGAATGGCGTAACG-3ʹ and R: 5ʹ-CACCAGAGGTTCGTCCGTC-3ʹ) were amplified as described by Maurer et al.²⁰ The Basic Local Alignment Search Tool (BLAST) program (https://blast.ncbi.nlm.nih.gov) was used for gene sequence comparisons.

Antibiotics

The carbapenem β-lactam imipenem (IPM; Sigma), the cephalosporin β-lactam ceftazidime (CAZ; Sigma), and the β-lactamase inhibitor avibactam (AVI; IHMA Inc.) were used alone or in combination. AVI was kindly provided by Prof. Chen Liang from Newark University (USA). Stock solutions of IPM and AVI were prepared in sterile water, while a stock solution of CAZ was prepared in dimethyl sulfoxide (DMSO). The stock concentrations of AVI, IPM, and CAZ were 1, 10, and 10 mg/mL, respectively. These stock solutions were diluted to the desired working concentrations with 7H9 medium.

Antimicrobial Susceptibility Tests

The susceptibility of all isolates to the following 15 antimicrobials (Thermo Fisher Scientific) were assessed according to the manufacturer’s instructions: amikacin (AMK), ciprofloxacin (CIP), moxifloxacin (MXF), trimethoprim–sulfamethoxazole (SXT), linezolid (LZD), ceftriaxone (CRO), cefepime (FEP), cefoxitin (FOX), tobramycin (TOB), tigecycline (TGC), minocycline (MIN), doxycycline (DOX), amoxicillin/clavulanic acid (AMC), imipenem (IPM) and clarithromycin (CLA). The minimum inhibitory concentrations (MICs) of the 15 antimicrobials for the 129 MABC isolates were determined using Sensititre RAPMYCOI MIC plates (Thermo Fisher Scientific) and the broth microdilution method, as recommended in the Clinical and Laboratory Standards Institute (CLSI)(M24-A2). The MIC ranges tested and CLSI M24-A2²¹ breakpoints for the 15 antimicrobials are listed in Table 2. We assessed the MICs of the 15 antimicrobials after 72 h of incubation, while we assessed the MICs of CLA after 3 and 14 days of incubation, in order to assess inducible CLA resistance. M. abscessus ATCC19,977 was used as the reference strain to compare the MICs.

Table 1 Antimicrobial Susceptibility of 129 Mycobacterium abscessus Complex (MABC) Isolates

Table 2 Antimicrobial Concentration Ranges for Drug Susceptibility Testing and MIC Breakpoints of Antimicrobial Agents

Synergistic Antimicrobial Susceptibility Tests

The MIC values of IPM, CAZ, and AVI were also assessed, alone and in combination (involving 100μg/mL CAZ and 4μg/mL AVI). The MICs were determined for each of the 129 MABC isolates using the broth microdilution method in 96-well plates after incubation at 37°C for 48 h, according to CLSI (M24-A2) and Chen et al.²²

Statistical Analysis

Differences between groups were compared using Pearson’sχ² test. A two-tailed P<0.05 was considered statistically significant. All computations were performed using Graph Pad Prism (version 7.0, La Jolla, CA, USA).

Results

MABC Subspecies Identification

M. abscessus, M. bolletii and M. massiliense accounted for 75.2% (97/129), 14.7% (19/129), and 10.1% (13/129) of the MABC isolates.

Antimicrobial Susceptibility Profiles

The antimicrobial susceptibilities of the 129 MABC isolates of the three subspecies are summarized in Table 1. The drug sensitivity results of each isolates were provided in the supplementary data (the file called 16_Apr_2020_16_Apr_2020_data.xlsx). In general, the antimicrobial resistance rates were very high. The MABC isolates exhibited >90% resistance to nine of the 15 antimicrobials tested (FEP, CRO AMC, IMI, MIN, CIP, MXF, SXT, and TOB), and the rates of resistance to DOX and LZD were >70%. But the isolates were most susceptible to TGC (87.6%) and AMK (65.1%).

The rate of FOX resistance was significantly lower than the rates of resistance to the two other cephalosporins (FEP and CRO) (P<0.05). The rates of FOX resistance for M. abscessus, M. massiliense, and M. bolletii were 34.0%, 46.2%, and 63.2%, respectively (P<0.05). Additionally, the rate of IPM resistance was significantly lower for M. massiliense (23.1%) than M. abscessus (41.2%) and M. bolletii (47.4%) (P<0.05).

CLA Susceptibility Testing and erm Genotyping in MABC Isolates

Out of all the MABC isolates, 72 (74.2%) were susceptible to CLA on day 3 but resistant on day 14 (indicating inducible resistance). Although the CLA resistance rate was higher on day 14 than on day 3 for M. abscessus (88.7% versus 14.4%), the corresponding rates were similar for M. massiliense (7.7% versus 7.7%), with none of the M. massiliense isolates exhibiting inducible resistance. Of the 19 M. bolletii isolates studied, 68.4% had inducible resistance, which could not be attributed to the presence of the erm(41) T28 polymorphism. In contrast, the majority of M. abscessus isolates (86 isolates) belonged to the erm(41) T28 sequevar. Of the 129 isolates, 24 (18.6%) were susceptible to CLA on day 14. Among these, 14 (10.9%) had an erm(41) deletion (comprising all 13 M. massiliense isolates and one M. abscessus isolates) and the remaining 10 (7.8%) were M. abscessus isolates with the erm(41) C28 mutation. Among the 129 MABC isolates, an rrl point mutation was found in only one isolate (an M. massiliense isolate). There were no erm(41) or rrl mutations in the M. bolletii isolates.

Susceptibility of MABC Isolates to β-Lactams (IPM and CAZ) and a β-Lactamase Inhibitor (AVI)

All 129 MABC isolates underwent susceptibility testing regarding IPM, CAZ, and AVI used alone or in combination. The average MIC₅₀ and MIC₉₀values are shown in Table 3. The drug sensitivity results of each isolates were provided in the supplementary data (the file called 16_Apr_2020_16_Apr_2020_data.xlsx). The MIC values for CAZ alone ranged from 128 to >1024 μg/mL, with average MIC₉₀ and MIC₅₀values of 1024 and 512 μg/mL, respectively; after adding AVI, the MIC values for CAZdid not change. The MIC values for IPM alone ranged from 2 to >64 μg/mL, with average MIC₉₀ and MIC₅₀ values of 32 and 16 μg/mL, respectively; there were also no significant changes when IPM was combined with AVI. However, after adding CAZ (100 μg/mL) only to IPM, there was a 4-fold reduction in the MIC₅₀ and MIC₉₀ values for IPM compared to when IPM was used alone. Additionally, after adding both CAZ (100 μg/mL) and AVI (4 μg/mL), the MIC range of IPM for the MABC isolates decreased from 2–>64 to 0.5–16 μg/mL; there was a 4-fold reduction in the MIC₅₀ and MIC₉₀ values compared to when IPM was used with AVI only, which also occurred in the reference strain (M. abscessus ATCC 19977).

Table 3 Antimicrobial Activities Imipenem (IPM), Ceftazidime (CAZ), and Avibactam (AVI) Alone or in Combination, Against 129 Mycobacterium abscessus Complex (MABC) Isolates

Discussion

In this study, we assessed the antimicrobial susceptibility of 129 MABC strains belonging to three subspecies and examined the association between the erm(41) gene and inducible CLA resistance. The MABC subspecies exhibited varied resistance rates to antimicrobials. When the antimicrobial resistance rates obtained in the present study were compared with the results of previous studies, we found that there were high rates of resistance to multiple antimicrobials among the three MABC subspecies. The resistance rates regarding fluoroquinolones (CIP and MXF) and other broad-spectrum antibiotics (such as DOX, SXT, and MIN) were similar (all >70%) to those previously reported for MABC isolates in Taiwan Province of China,²³ as well as in Japan,²⁴ Thailand,²⁵ the USA,²⁶ and France.²⁷ In contrast, the rate of IPM resistance varied significantly among different studies. It was 40.9% in our study, which was in agreement with the rates reported in Beijing²⁸ and Japan.²⁴ However, studies conducted in 2017 in Taiwan (61%),²³ 2015 in Australia (68%),²⁹ and 2018 in Shanghai (65%)³⁰ reported higher rates of IPM resistance. Notably, the M. abscessus isolates in our study had a higher resistance rate to FOX (34.02%) than the M. abscessus strains in a study in Japan (16.7%).³¹ In Korea³² and Japan,³³ the TOB resistance rate was 30–32%, while it was 90% in our study. These variations may be due to differences among the diverse studies in patient treatment histories or in the isolates.

In the early 2000s, when LZD was first used clinically, it was reported to be active against many species of RGM.³⁴ However, we found that the rate of resistance to LZD was high, similar to rates reported in Taiwan (70%)²³ and the UK (96%),³⁵ but higher than the 5% rate reported by a study in Korea.³⁶ Notably, there have been few reports of a rate of LZD resistance as high as that found in M. bolletii in our study (73.7%). With the exception of the resistance rate for LZD, the resistance rates of M. bolletii were higher than those of M. abscessus and M. massiliense. Therefore, precise differentiation between these subspecies is important for clinical purposes.

Bastian et al reported that MABC infections were usually poorly responsive to CLA because of acquired and/or inducible resistance.³⁷ Mutations in the rrl gene confer acquired CLA resistance, while a single-nucleotide polymorphism (T28) in erm(41) at position 28 leads to inducible CLA resistance.⁷ Notably, the acquired CLA resistance rate (on day 3) in the M. abscessus isolates of 14.4% (14/97) was similar to the rate reported in South Korea (15.84%), higher than those reported in France (9.09%) and the USA (2.51%), and lower than that reported in China (33.95%).^27,38,39 We hypothesize that the geographic diversity in the population structure of MABC may be a major reason why researchers from various regions observed contradictory results. In addition, the high rate of inducible resistance that we identified is consistent with those reported by other studies.^38,40 This supports the need to modify the CLA susceptibility test recommended by the CLSI,²¹ by assessing the MIC of CLA after an additional longer period of incubation (eg, 14 days).

In our study, most of the mutations identified involved the erm(41) gene (75.1%) rather than the rrl gene (0.8%). We concluded that the erm(41) mutations may be associated with the treatment failure that occurs in many cases of MABC infection. Nash et al and Kim et al studied the molecular profiles of MABC isolates and found that the erm(41) C28 polymorphism was related to susceptibility.^7,41 Of our129 isolates, 23 (17.8%) were susceptible to CLA. Among these, 14 (10.9%) had an erm(41) deletion (comprising all 13 M. massiliense isolates and one M. abscessus isolate) and10 (7.8%) were M. abscessus isolates with the erm(41) C28 mutation. Of the 19 M. bolletii isolates studied, 68.4% exhibited inducible CLA resistance, which could not be attributed to the presence of the erm(41) T28 polymorphism. This indicates that there is another mechanism of resistance to CLA in M. bolletii. In addition, it has been reported that acquisition of CLA resistance is 100% mediated by structural 50S ribosomal subunit mutations for the M. abscessus erm(41) C28 sequevar and M. massiliense, whereas it is less common for the M. abscessus erm(41) T28 sequevar and M. bolletii(other mechanisms may be responsible for CLA resistance in these strains).⁴² Hence, we hypothesize that detecting an erm(41) deletion may differentiate M. massiliense from M. abscessus and M. bolletii, but more extensive research is needed to accurately define the reliability of using erm(41) deletions to identify M. massiliense.

Given the increasing prevalence of multidrug-resistant MABC infections, the development of novel treatment regimens is imperative. Although knowledge regarding the efficacy of β-lactams and β-lactamase inhibitors in MABC remains limited, several studies have used the available data to develop synergistic treatment regimens for treating MABC infections.^22,43 In this study, we evaluated the in vitro susceptibility of the 129 MABC isolates to IPM in the presence of CAZ and/or AVI. The MIC₅₀ and MIC₉₀values of IPM after the addition of CAZ plus AVI, compared to the values after the addition of AVI only, decreased 4-fold to 4 and 8μg/mL, respectively. Similarly, the addition of 100μg/mL CAZ only led to 4-fold decreases in the MIC₅₀ and MIC₉₀values of IPM (while there were no significant changes in the MIC of IPM when IPM was combined with 4 μg/mL AVI only). The initial resistance or intermediate resistance to IPM changed to sensitivity. Interestingly, CAZ alone had poor activity against MABC but combining IPM and CAZ led to effective activity, indicating that the effect of triple therapy may be driven primarily by CAZ rather than AVI. In contrast, Lefebvre et al.¹³ reported that inhibition of the β-lactamase Bla_Mab by AVI improves the in vitro effects of IPM against MABC and Pandey et al.²² reported that 4μg/mL AVI enhanced the bactericidal activity of the β-lactam ceftaroline; it can be deduced that the β-lactamase of MABC strains probably hydrolyzes IPM (though this was not confirmed by our study). Future studies will need to identify effective measures for reducing exposure to these difficult-to-treat pathogens.

Conclusion

The MABC isolates exhibited varied resistance rates to antimicrobials. The antimicrobial susceptibility profile and the characteristics of the erm(41) gene associated with inducible CLA resistance were different among the three MABC subspecies. Additionally, there was synergy between the β-lactam IPM and 100μg/mL of the β-lactam CAZ against MABC infection.