Erik C. Böttger

Towards a Phylogeny and Definition of Species at the Molecular Level within the Genus Mycobacterium

16S rRNA sequences from Mycobacterium tuberculosis, M. avium, M. gastri, M. kansasii, M. marinum, M. chelonae, M. smegmatis, M. terrae, M. gordonae, M. scrofulaceum, M. szulgai, M. intracellulare, M. nonchromogenicum, M. xenopi, M. malmoense, M. simiae, M. flavescens, M. fortuitum, and M. paratuberculosis were determined and compared. The sequence data were used to infer a phylogenetic tree, which provided the basis for a systematic phylogenetic analysis of the genus Mycobacterium. The groups of slow- and fast-growing mycobacteria could be differentiated as distinct entities. We found that M. simiae occupies phylogenetically an intermediate position between these two groups. The phylogenetic relatedness within the slow-growing species did not reflect the Runyon classification of photochromogenic, scotchromogenic, and nonchromogenic mycobacteria. In general, the phylogenetic units identified by using rRNA sequences confirmed the validity of phenotypically defined species; an exception was M. gastri, which was indistinguishable from M. kansasii when this kind of analysis was used.

Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot

Multidrug‐resistant strains of Mycobacterium tuberculosis have resulted in several recent outbreaks. Recognition of drug resistance is important both for treatment and to prevent further transmission. Here we use molecular biology techniques to study the basis of streptomycin resistance in single and multi‐drug‐resistant M. tuberculosis. We demonstrate that streptomycin resistance is associated with mutations implicated in ribosomal resistance. The mutations found either lead to amino acid changes in ribosomal protein SI2 or alter the primary structure of the 16S rRNA. The 16S rRNA region mutated perturbs a pseudoknot structure in a region which has been linked to ribosomal S12 protein.

Differentiation of Mycobacterium species by direct sequencing of amplified DNA

Nucleotide sequences specific for a range of Mycobacterium species were defined by computer-assisted sequence comparisons of small subunit ribosomal RNA. A polymerase chain reaction-based sequencing strategy was used to demonstrate that the 16S rRNA sequence can be used for the rapid identification of mycobacterial isolates. Identification at the species level can be obtained within 2 d, requiring less than 10,000 bacteria. This procedure reliably differentiates Mycobacterium spp. which are difficult to identify by classical methods, such as M. malmoense, M. szulgai and M. flavescens.

The determination and comparison of the 16S rRNA gene sequences of species of the genus Pseudomonas (sensu stricto) and estimation of the natural intrageneric relationships.

Summary As a consolidated effort on the part of several laboratories, partial and nearly complete sequence determinations of 165 rRNA genes have been applied as one of several analytical methods in a polyphasic study of the pseudomonads. Nearly-complete sequences have been determined of the PCR-amplified 16S rRNA genes of 21 species of the genus Pseudomonas (sensu stricto), including multiple strains of most species. Phylogenetic branching orders and the natural intrageneric relationships among the species have been inferred through sequence comparisons and cluster analysis and have not shown any obvious recognizable correlation with results derived through standard phenotypic criteria commonly used to group the species. This paper also focuses on the ability of 16S rRNA gene sequences, particularly the hypervariable sequence regions, to be used as nested identification markers and as target sites for the development of 16S rRNA sequence-based strategies for the identification of species of the genus Pseudomonas .

Fitness Cost of Chromosomal Drug Resistance-Conferring Mutations

ABSTRACT To study the cost of chromosomal drug resistance mutations to bacteria, we investigated the fitness cost of mutations that confer resistance to different classes of antibiotics affecting bacterial protein synthesis (aminocyclitols, 2-deoxystreptamines, macrolides). We used a model system based on an in vitro competition assay with defined Mycobacterium smegmatis laboratory mutants; selected mutations were introduced by genetic techniques to address the possibility that compensatory mutations ameliorate the resistance cost. We found that the chromosomal drug resistance mutations studied often had only a small fitness cost; compensatory mutations were not involved in low-cost or no-cost resistance mutations. When drug resistance mutations found in clinical isolates were considered, selection of those mutations that have little or no fitness cost in the in vitro competition assay seems to occur. These results argue against expectations that link decreased levels of antibiotic consumption with the decline in the level of resistance.

Identification of mutations in 23S rRNA gene of clarithromycin-resistant Mycobacterium intracellulare.

Clarithromycin is a potent macrolide that has been used for treating infections with nontuberculous mycobacteria. Pairs of susceptible and resistant Mycobacterium intracellulare strains were obtained from patients with chronic pulmonary M. intracellulare infections undergoing monotherapy with clarithromycin. Nucleotide sequence comparisons of the peptidyltransferase region in 23S rRNAs from parental and resistant strains revealed that in three of six resistant strains, for which the MIC was > 32 micrograms/ml, a single base was mutated (Escherichia coli equivalent, A-2058-->G, C, or U). As the modification of adenine 2058 by dimethylation is a frequent cause of macrolide resistance in a variety of different bacteria, we suggest that mutation of A-2058 confers acquired resistance to clarithromycin in M. intracellulare. Images

A Single 16S Ribosomal RNA Substitution Is Responsible for Resistance to Amikacin and Other 2-Deoxystreptamine Aminoglycosides in Mycobacterium abscessus and Mycobacterium chelonae

Twenty-six clinical isolates of Mycobacterium abscessus resistant to amikacin were identified. Most isolates were from patients with posttympanostomy tube placement otitis media or patients with cystic fibrosis who had received aminoglycoside therapy. Isolates were highly resistant (MICs > 1024 microg/mL) to amikacin, kanamycin, gentamicin, tobramycin, and neomycin (all 2-deoxystreptamine aminoglycosides) but not to streptomycin. Sequencing of their 16S ribosomal (r) RNA revealed that 16 (94%) of 17 had an A-->G mutation at position 1408. In vitro-selected amikacin-resistant mutants of M. abscessus and Mycobacterium chelonae had the same resistance phenotype, and 15 mutants all had the same A-->G substitution at position 1408. Introducing an rRNA operon from Mycobacterium smegmatis with a mutated A-->G at this position into a single functional allelic rRNA mutant of M. smegmatis produced the same aminoglycoside resistance phenotype. These studies demonstrate this 16S rRNA mutation is responsible for amikacin resistance in M. abscessus, which has only one copy of the rRNA operon.

RPSL+ : A DOMINANT SELECTABLE MARKER FOR GENE REPLACEMENT IN MYCOBACTERIA

Molecular genetic manipulations in mycobacteria would benefit from procedures which efficiently select for double‐crossover events by homologous recombination. Here we describe a vector‐host system for gene replacement in mycobacteria, the utility of which was investigated using functional inactivation of the pyrF gene in Mycobacterium smegmatis as a model. This system is based on the expression of the wild‐type rpsL gene coding for ribosomal protein S12 in a streptomycin‐resistant host. Owing to the absence of a mycobacterial origin the plasmids are unable to replicate autonomously in mycobacteria. The first selection for maintenance of cloned sequences is conferred by the kanamycin‐resistance gene. The second simultaneous selection by streptomycin is against maintenance of cloned sequences which contain the gene encoding the streptomycin‐sensitive allele of the rpsL gene. By placing the gene for positive selection and that used for negative selection within and outside the target gene of interest, respectively, gene replacement is obtained. A one‐step double selection procedure provides a means to distinguish strictly between gene replacement by double crossover versus homologous recombination by single crossover events. The system should have considerable potential for genera or species where single‐crossover events or even illegitimate recombination are the predominant recombination mechanisms. It should also be of wide use for the construction of mutants without a selectable phenotype.

Mycobacterium wolinskyi sp. nov. and Mycobacterium goodii sp. nov., two new rapidly growing species related to Mycobacterium smegmatis and associated with human wound infections : a cooperative study from the international working group on mycobacterial taxonomy

Previous investigations demonstrated three taxonomic groups among 22 clinical isolates of Mycobacterium smegmatis. These studies were expanded to 71 clinical isolates, of which 35 (49%) (group 1) were identical to five ATCC reference strains including the type strain ATCC 19420T. Twenty-eight isolates (39%) were group 2, and eight isolates (11%) were group 3. Isolates of groups 2 and 3 were most often associated with post-traumatic or post-surgical wound infections including osteomyelitis, were susceptible to sulfamethoxazole, amikacin, imipenem and the tetracyclines, variably resistant to clarithromycin, and susceptible (group 1), intermediately resistant (group 2) or resistant (group 3) to tobramycin. The three groups were similar by routine biochemical and growth characteristics, but had different mycolic acid dimethoxy-4-coumarinylmethyl ester elution patterns by HPLC and different PCR-restriction enzyme patterns of a 439 bp fragment of the hsp-65 gene. Group 3 isolates differed from group 1 by 18 bp by 16S rRNA sequencing and exhibited < 25% homology by DNA-DNA hybridization, being most closely related to Mycobacterium mageritense. The 16S rRNA of group 1 and group 2 isolates differed by only 3 bp, but by DNA-DNA hybridization they exhibited only 40% homology. The following names are proposed: Mycobacterium goodii sp. nov. for group 2 isolates (type strain ATCC 700504T = MO69T), Mycobacterium wolinskyi sp. nov. for group 3 isolates (type strain ATCC 700010T = MO739T) and Mycobacterium smegmatis sensu stricto for group 1 isolates.

Prolonged Outbreak of Mycobacterium chimaera Infection After Open-Chest Heart Surgery

BACKGROUND Invasive Mycobacterium chimaera infections were diagnosed in 2012 in 2 heart surgery patients on extracorporeal circulation. We launched an outbreak investigation to identify the source and extent of the potential outbreak and to implement preventive measures. METHODS We collected water samples from operating theaters, intensive care units, and wards, including air samples from operating theaters. Mycobacterium chimaera strains were characterized by randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). Case detection was performed based on archived histopathology samples and M. chimaera isolates since 2006, and the patient population at risk was prospectively surveyed. RESULTS We identified 6 male patients aged between 49 and 64 years with prosthetic valve endocarditis or vascular graft infection due to M. chimaera, which became clinically manifest with a latency of between 1.5 and 3.6 years after surgery. Mycobacterium chimaera was isolated from cardiac tissue specimens, blood cultures, or other biopsy specimens. We were able also to culture M. chimaera from water circuits of heater-cooler units connected to the cardiopulmonary bypass, and air samples collected when the units were in use. RAPD-PCR demonstrated identical patterns among M. chimaera strains from heater-cooler unit water circuits and air samples, and strains in 2 patient clusters. CONCLUSIONS The epidemiological and microbiological features of this prolonged outbreak provided evidence for the airborne transmission of M. chimaera from contaminated heater-cooler unit water tanks to patients during open-heart surgery.