INNO-LiPA DNA line probe assay misidentification of M. smegmatis as Mycobacterium fortuitum complex.

Seven weeks after being kicked in the face by a cow, a 34-year-old male patient developed a posttraumatic mycobacterial lymphadenitis. A rapidly growing mycobacterial isolate cultured from a surgically drained lymphadenitis pus specimen was identified as Mycobacterium smegmatis by matrix-assisted laser desorption/ionization mass spectrometry and a combination of ITS-, hsp65-, and 16S rRNA-DNA sequence analysis, but as Mycobacterium fortuitum complex using the commercial INNO-LiPA Mycobacteria v2 line probe assay. As it is unclear if the misidentification of this strain is an exception, more research is required.

WGS more accurately predicts susceptibility of Mycobacterium tuberculosis to first-line drugs than phenotypic testing.

BACKGROUND: Drug-susceptibility testing (DST) of Mycobacterium tuberculosis complex (MTBC) isolates by the Mycobacteria Growth Indicator Tube (MGIT) approach is the most widely applied reference standard. However, the use of WGS is increasing in many developed countries to detect resistance and predict susceptibility. We investigated the reliability of WGS in predicting drug susceptibility, and analysed the discrepancies between WGS and MGIT against the first-line drugs rifampicin, isoniazid, ethambutol and pyrazinamide. METHODS: DST by MGIT and WGS was performed on MTBC isolates received in 2016/2017. Nine genes and/or their promotor regions were investigated for resistance-associated mutations: rpoB, katG, fabG1, ahpC, inhA, embA, embB, pncA and rpsA. Isolates that were discrepant in their MGIT/WGS results and a control group with concordant results were retested in the MGIT, at the critical concentration and a lower concentration, and incubated for up to 45 days after the control tube became positive in the MGIT. RESULTS: In total, 1136 isolates were included, of which 1121 were routine MTBC isolates from the Netherlands. The negative predictive value of WGS was ≥99.3% for all four first-line antibiotics. The majority of discrepancies for isoniazid and ethambutol were explained by growth at the lower concentrations, and for rifampicin by prolonged incubation in the MGIT, both indicating low-level resistance. CONCLUSIONS: Applying WGS in a country like the Netherlands, with a low TB incidence and low prevalence of resistance, can reduce the need for phenotypic DST for ∼90% of isolates and accurately detect mutations associated with low-level resistance, often missed in conventional DST.

Prevalence and Characterization of Heterogeneous Variable-Number Tandem-Repeat Clusters Comprising Drug-Susceptible and/or Variable Resistant Mycobacterium tuberculosis Complex Isolates in the Netherlands from 2004 to 2016.

The variable-number tandem-repeat (VNTR) typing method is used to study tuberculosis (TB) transmission. Clustering of Mycobacterium tuberculosis isolates with identical VNTR patterns is assumed to reflect recent transmission. Hence, clusters are thought to be homogeneous regarding antibiotic resistance. In practice, however, heterogeneous clusters are also identified. This study investigates the prevalence and characteristics of heterogeneous VNTR clusters and assesses whether isolates in these clusters remain clustered when subjected to whole-genome sequencing (WGS). In the period from 2004 to 2016, 9,072 isolates were included. Demographic and epidemiological linkage data were obtained from the Netherlands Tuberculosis Register. VNTR clusters were defined as homogeneous when isolates shared identical resistance profiles or as heterogeneous if both susceptible and (variable) resistant isolates were found. Multivariate logistic regression analysis was performed to identify factors associated with heterogeneous clustering. Isolates from 2016 were subjected to WGS, and a genetic distance of 12 single nucleotide polymorphisms (SNPs) was used as the cutoff for WGS clustering. In total, 4,661/9,072 (51%) isolates were clustered into 985 different VNTR clusters, of which 217 (22%) were heterogeneous. Patient characteristics associated with heterogeneous clustering were non-Dutch ethnicity (odds ratio [OR], 1.46 [95% confidence interval {CI}, 1.22 to 1.75]), asylum seeker (OR, 1.51 [95% CI, 1.24 to 1.85]), extrapulmonary TB (OR, 1.26 [95% CI, 1.09 to 1.46]), previous TB diagnosis (OR, 1.38 [95% CI, 1.04 to 1.82]), and not being a contact of a TB patient (OR, 1.35 [95% CI, 1.08 to 1.69]). With WGS, 34% of heterogeneous and 78% of homogeneous isolates from 2016 remained clustered. Heterogeneous VNTR clusters are common but seem to be explained by a substantial degree of false clustering by VNTR typing compared to WGS.

Occurrence and Nature of Double Alleles in Variable-Number Tandem-Repeat Patterns of More than 8,000 Mycobacterium tuberculosis Complex Isolates in The Netherlands.

Since 2004, variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates has been applied on a structural basis in The Netherlands to study the epidemiology of tuberculosis (TB). Although this technique is faster and technically less demanding than the previously used restriction fragment length polymorphism (RFLP) typing, reproducibility remains a concern. In the period from 2004 to 2015, 8,532 isolates were subjected to VNTR typing in The Netherlands, with 186 (2.2%) of these exhibiting double alleles at one locus. Double alleles were most common in loci 4052 and 2163b. The variables significantly associated with double alleles were urban living (odds ratio [OR], 1.503; 95% confidence interval [CI], 1.084 to 2.084; P = 0.014) and pulmonary TB (OR, 1.703; 95% CI, 1.216 to 2.386; P = 0.002). Single-colony cultures of double-allele strains were produced and revealed single-allele profiles; a maximum of five single nucleotide polymorphisms (SNPs) was observed between the single- and double-allele isolates from the same patient when whole-genome sequencing (WGS) was applied. This indicates the presence of two bacterial populations with slightly different VNTR profiles in the parental population, related to genetic drift. This observation is confirmed by the fact that secondary cases from TB source cases with double-allele isolates sometimes display only one of the two alleles present in the source case. Double alleles occur at a frequency of 2.2% in VNTR patterns in The Netherlands. They are caused by biological variation rather than by technical aberrations and can be transmitted either as single- or double-allele variants.

Biphasic kinetics of a methanotrophic community is a combination of growth and increased activity per cell.

Because methane-oxidizing bacteria (MOB) are the only biological sink for the greenhouse gas methane, knowledge of the functioning of these bacteria in various ecosystems is needed to understand the dynamics observed in global methane emission. The activity of MOB is commonly assessed by methane oxidation assays. The resulting methane depletion curves often follow a biphasic pattern of initial and induced methane oxidation activity, often interpreted as representing the in situ active and total MOB community, respectively. The application of quantitative-PCR on soil incubations, which were stopped before, at and after the transition point in the methane-depletion curve, demonstrated that both pmoA-mRNA was produced as well as substantial cell growth took place already in the initial phase. In addition, type Ia and II MOB displayed markedly different behaviour, which can be interpreted as ecologically different strategies. For the correct interpretation of methane oxidation assays, the use of small time windows is recommended to calculate methane oxidation activities to avoid substantial cell growth.

Whole-community genome amplification (WCGA) leads to compositional bias in methane-oxidizing communities as assessed by pmoA-based microarray analyses and QPCR.

Whole-genome amplification (WGA) using multiple displacement amplification (MDA) has recently been introduced to the field of environmental microbiology. The amplification of single-cell genomes or whole-community metagenomes decreases the minimum amount of DNA needed for subsequent molecular community analyses. The resolution of profiling methods of environmental microbial communities will increase substantially by the use of the whole-community genome amplification (WCGA) procedure, assuming that the original community composition is not affected qualitatively as well as quantitatively. The present study aims to test if WCGA introduces a bias when applied to aerobic proteobacterial methanotrophic communities. For this, first, we subjected samples from freshwater lake sediment to WCGA, and amplified using primers targeting the pmoA gene coding for the α-subunit of the methane monooxygenase enzyme. Second, we analysed community composition using a diagnostic microarray and quantitative PCR (QPCR) assays. These methods clearly demonstrated that the WCGA amplification introduced a bias. Thus, numbers of γ-proteobacterial methanotrophs ('type Ia') increased significantly while the α-proteobacterial methanotrophs ('type II') were not amplified by the WCGA procedure. It is hypothesized that this bias is caused by the differences in GC content, which may compromise the efficiency of the MDA reaction.