Applied Microbiology: Open Access
Open Access

Description of a Clinical Strain of Mycobacterium cambodiensis sp. nov., a New Member of the Mycobacterium simiae Complex

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Introduction

In Cambodia, a country with high tuberculosis burden (incidence, for all clinical forms, estimated at 326 per 100,000 inhabitants in 2017, with 47,000 new cases reported for 16 million inhabitants[1-3], infections caused by non-tuberculous mycobacteria (NTM) also have been recorded. In 2011, the rate of NTM isolation among patients with presumptive multidrug-resistant tuberculosis and positive culture was 26.1% [4]. Among 128 (10.8%) NTM infections diagnosed in patients at Kampong Cham Provincial Reference Hospital between 2012 and 2014, the NTM species could not be identified in 22 cases. Moreover, four patients were co-infected by a NTM and a Mycobacterium tuberculosis complex species [5].

Here, we present the analysis of one isolate, initially referred as strain 716A, from a sputum specimen collected in a Cambodian patient with suspected clinical tuberculosis. We accumulated phenotypically and genetic evidences showing that this isolate belongs to a new species within the Mycobacterium simiae complex. We report the complete polyphasic characterization of strain 716A and its specific features relative to the other known species within the M. simiae complex.

Materials and Methods

Phenotypic characterization

Strain 716A was cultured on Middlebrook 7H10 agar medium (Becton Dickinson, Le Pont de Claix, France) supplemented with 10% OADC (Becton Dickinson) and in the mycobacterial Growth Indicator Tube (MGIT) liquid medium (BACTEC™ MGIT™ 960, Becton Dickinson). Enzyme activities and carbon source utilization were determined by inoculating API^® ZYM and API^® Coryne strips (bioMérieux, Bruz, France) [6], as described by the manufacturer, with an incubation time of four hours and 24 hours, respectively. The minimum inhibitory concentration (MIC) of the major antimycobacterial agents was determined using the ETEST® (bioMérieux, Craponnes, France).

Biolog phenotype microarray

The capacity of strain 716A to resist or not to 23 inhibitory chemicals and to metabolize 71 different carbon substrates was evaluated using Gen III Microplates Biolog® Phenotype MicroArray (Biolog Inc., Hayward, CA, USA) [7]. Strain 716A was cultured at 37°C on Middlebrook 7H10 agar medium (Becton Dickinson, Franklin Lakes, USA), 10% (v/v) OADC (Becton Dickinson) and 0.5% (v/v) glycerol for two weeks. Colonies were gently taken with sterile swabs, suspended in IF-C2 tubes, and adjusted to 65% transmittance using a turbidimeter (Biolog Inc). Suspensions were then poured into a sterile reservoir and 100 μL of each suspension were deposited in a well of a 96-well plate (with positive and negative control). Two plates (duplicate) were used and incubated in the OmniLog PM System (Biolog Inc.) at 37°C for four days. Results were expressed as area under the curve (AUC) by the Biolog parametric software.

Scanning electron microscopy

The shape and size of strain 716A were determined by scanning electron microscopy (TM4000, Hitachi, Tokyo, Japan) after negative staining at an operating voltage of 15 kV.

MALDI-TOF-MS

The full extraction protocol recommended by Bruker (Bruker Daltonics®, Bremen, Germany) was followed, using glass powder (G8772, Sigma-ALD), pure acetonitrile and formic acid diluted to 70%, as previously described [8]. Then, 1 μL of supernatant was deposited on a ground-steel MALDI target plate. After drying at room temperature, 1 μL of matrix solution (saturated α-cyano-4- hydroxycinnamic acid in 50% acetonitrile, 25% trifluoroacetic acid and 25% H₂O) (Sigma-Aldrich) was deposited on the sample. After drying at room temperature, the plate was loaded for analysis in the Microflex LT mass spectrometer (Bruker Daltonics). The results were obtained in the form of spectra and scores; spectra were recorded according to the previously described parameters [9] and were obtained using the MALDI Biotyper (MBT) Compass software, version 4.1.80. The identifications and their scores were obtained with the MALDI Biotyper software, version 4.1.80, and the Mycobacteria Library, version 4.0, database (contains 880 MSPs) (June version, 2017).

DNA preparation and genome sequencing

Total DNA of strain 716A was extracted by vortexing the suspension with glass powder (Sigma- Aldrich, St. Louis, MO, USA) using the FastPrep apparatus (MP Biomedicals, Santa Ana California, USA) and the Qiagen kit (Qiagen, Courtaboeuf, France), as previously described [10]. Then DNA was quantified with the Qubit™ dsDNA HS Assay Kit (Life technologies, Carlsbad, CA, USA), and 0.2 μg/μL of DNA was sequenced with the Illumina MiSeq system (Illumina Inc., San Diego, USA). Paired-end sequencing and automated cluster generation with dual indexed 2× 250-bp reads were performed in a 40-hour run [11].

Genome characterization and genome comparisons

The genome was assembled with SPAdes, version 3.12.0[12], and annotated with Prokka, version 1.13[13]. Emboss GC% and BlastN against the NCBI database were used for detecting the presence of plasmids. The Comprehensive Antibiotic Resistance Database (CARD) was used to identify antibiotic resistance genes. The rpoB gene, TYGS (Type strain genome server) based on the 16S rRNA gene, and Genome BLAST Distance Phylogeny (GBDP) was used to screen all species related to this strain. The similarity among genomes was estimated with the Genome to Genome Distance Calculator (GGDC), available at, and formula 2 was recommended to interpret the results of the analyzed draft genome. The mean levels of relatedness between strain 716A genome sequence and the sequences of other M. simiae complex members were measured with Orthologous Average Nucleotide Identity (OrthoANI). The DDH and OrthoANI values of this strain were calculated with all M. simiae complex members. The Roary pan-genome pipeline in the Galaxy software was used to release the pan-genomic comparison with other Mycobacterium species.

Results

Strain 716A formed orange, smooth and scotochromogenic colonies on Middlebrook 7H10 agar medium, containing 10% (v/v) OADC and 0.5% (v/v) glycerol, after 12-day incubation at 37°C. Biochemical characterization of strain 716A showed a positive reaction to the catalase test, but the oxidase test was negative at room temperature. Investigation of the strain enzymatic activity, using the API ZYM and API CORYNE strips (bioMérieux, Craponne, France), gave positive results for esterase (C4), lipase esterase (C8), lipase (C14), leucine arylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, and alkaline phosphatase. These observations suggested that strain 716A was a Gram-positive bacterium, and Ziehl-Neelsen staining showed pink acid-fast bacilli. Scanning electron microscopy analysis of 100 bacilli indicated that these rod-shaped bacilli measured 1.2 ± 0.29 μm in length and 0.58 ± 0.07 μm in width (Figure 1). The reproducible matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) profile of strain 716A did not match any of the existing profiles in the Bruker database (version 4.1.80), with an identification score <1.38 (Figure 2). This suggested that strain 716A could be an undescribed species. Strain 716A was deposited in the Collection de Souches de l’Unité des Rickettsies (CSUR) with the number CSURP9652. Moreover, in vitro antibiotic susceptibility testing showed that strain 716A was susceptible to isoniazid (minimum inhibiting concentration, MIC=0.47 μg/mL), amikacin (MIC=1 μg/mL) and trimethoprim-sulfamethoxazole (MIC=0.19 μg/mL), and resistant to linezolid (MIC>256 μg/ mL), minocycline (MIC>256 μg/mL), doxycycline (MIC>256 μg/mL), rifampicin (MIC>256 μg/mL) and chloramphenicol (MIC>256 μg/mL). Additional phenotypic analyses with the Biolog® Phenotype MicroArray technology (Hayward, California, United States of America) indicated that strain 716A metabolized four carbon sources: Tween 40 (from 24 hours to 7 days), methyl pyruvate and citric acid (from 50 hours to 7 days) and glycerol (only from 90 hours to 7 days). Conversely, strain 716A was not altered by tetrazolium violet during the entire incubation period (from 10 hours to 7 days) (Table 1).

Figure 1: Scanning electron microscopy image of the Mycobacterium cambodiensis strain CSUR9652. Scale bar, 5.00 µm.

Figure 2: MALDI-TOF-MS spectrum of the Mycobacterium cambodiensis strain CSUR9652.

Table 1: Scanning electron microscopy image of the Mycobacterium cambodiensis strain CSUR9652. Scale bar, 5.00 µm.

Whole-genome sequencing yielded 128 scaffolds corresponding to 5,703,981 bp with a GC content of 69.3% (GCA_902652685.1). The genome was predicted to encode 5,263 genes, including 5,207 protein-coding genes and 56 RNAs (52 tRNA, three rRNA, and one tmRNA). Nucleotide BLAST (blastn suite) against the standard nucleotide collection (nr/nt) database using Megablast (optimized for highly similar sequences) of the 3,453-bp rpoB gene showed that strain 716A was related to the M. simiae complex, with 93.86% of sequence similarity to the homologous rpoB sequence of Mycobacterium parascrofulaceum. In addition, the 16S rRNA gene sequence indicated that Mycobacterium saskatchewanense DSM 44616, Mycobacterium interjectum ATCC 51457, Mycobacterium paraense IEC26 and Mycobacterium palustreDSM 44572 were the closest species to strain 716A, but with low percentage of identity (26.6%, 25.8%, 25.4% and 25.3%, respectively). In the phylogenetic tree built using Genome BLAST Distance Phylogeny (GBDP), strain 716A was closest to M. saskatchewanense among the 19 members of the M. simiae complex (Figures 3). In silico DNA- DNA hybridization (DDH) analysis of strain 716A yielded less than 30% of identity with the different members of the M. simiae complex (Table 2). Moreover, the overall similarity between the strain 716A sequence and those of M. simiae complex members, measured with the OrthoANI algorithm, ranged between 83.3% and 78.19% (Table 3). Pan-genome analysis of strain 716A within the M. simiae complex yielded 36,451 genes, including 1,103 core genes, 523 soft core genes, 4,941 shell genes shared by several species, and 29,884 cloud genes unique to one species. The pan- genome tree showed that strain 716A was more closely related to M. saskatchewanense (Figures 4).

Figure 3: Phylogenetic tree based on the DDH values of Mycobacterium cambodiensis CSURP9652 with M. simiae complex members. Tree inferred with FastME 2.1.6.1 [14] from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance, formula d5. The numbers above branches are GBDP pseudo-bootstrap support values >95% from 100 replications, with a mean branch support of 95.9%. The tree was rooted at the midpoint [15].

Figure 4: Pan-genome tree incorporating whole genome sequence data of Mycobacterium cambodiensis CSURP9652 and of 20 Mycobacterium simiae complex members.

Table 2: Comparison of Mycobacterium cambodiensis CSURP9652 with Mycobacterium simiae complex members using GGDC, formula 2 (DDH estimates based on identities/high-scoring segment pair length).