The status of the species Bacillus aerophilus and Bacillus stratosphericus. Request for an Opinion.

During a study assessing the diversity of the Bacillus pumilus group it became apparent that the type strains of both Bacillus aerophilus and Bacillus stratosphericus were not available from any established culture collection, nor from the authors who originally described them. Therefore, type strains of these species cannot be included in any further scientific studies. It is therefore proposed that the Judicial Commission of the International Committee of Systematics of Prokaryotes place the names Bacillus aerophilus and Bacillus stratosphericus on the list of rejected names, if suitable replacements for the type strains are not found or if neotype strains are not proposed within two years following the publication of this Request for an Opinion.

Differentiation of Bacillus pumilus and Bacillus safensis using MALDI-TOF-MS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) despite being increasingly used as a method for microbial identification, still present limitations in which concerns the differentiation of closely related species. Bacillus pumillus and Bacillus safensis, are species of biotechnological and pharmaceutical significance, difficult to differentiate by conventional methodologies. In this study, using a well-characterized collection of B. pumillus and B. safensis isolates, we demonstrated the suitability of MALDI-TOF-MS combined with chemometrics to accurately and rapidly identify them. Moreover, characteristic species-specific ion masses were tentatively assigned, using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and primary literature. Delineation of B. pumilus (ions at m/z 5271 and 6122) and B. safensis (ions at m/z 5288, 5568 and 6413) species were supported by a congruent characteristic protein pattern. Moreover, using a chemometric approach, the score plot created by partial least square discriminant analysis (PLSDA) of mass spectra demonstrated the presence of two individualized clusters, each one enclosing isolates belonging to a species-specific spectral group. The generated pool of species-specific proteins comprised mostly ribosomal and SASPs proteins. Therefore, in B. pumilus the specific ion at m/z 5271 was associated with a small acid-soluble spore protein (SASP O) or with 50S protein L35, whereas in B. safensis specific ions at m/z 5288 and 5568 were associated with SASP J and P, respectively, and an ion at m/z 6413 with 50S protein L32. Thus, the resulting unique protein profile combined with chemometric analysis, proved to be valuable tools for B. pumilus and B. safensis discrimination, allowing their reliable, reproducible and rapid identification.

Phylogenetic and clonality analysis of Bacillus pumilus isolates uncovered a highly heterogeneous population of different closely related species and clones.

Bacillus pumilus is a Gram-positive bacterium with a wide range of attributed applications, namely as a plant growth promoting rhizobacteria (PGPR), animal, and human probiotic. However, a rare putative role in human diseases has been reported, namely in food poisoning or as anthrax-like cutaneous infectious agent. This species is difficult to distinguish from its closely related species on the basis of phenotypic or biochemical characteristics and 16S rRNA gene sequences. In this study, the phylogenetic analysis of gyrB and rpoB gene sequences of a collection of isolates previously identified as B. pumilus, assigned most of them (93%, 38 of 41 isolates) to B. safensis or to the new recently described B. invictae. Moreover, we extended the previously reported recognized habitats of these species and unveiled a human health or biotechnological relevance (e.g. as implicated in food poisoning or PGPR) for them. Additionally, we demonstrated that both B. safensis and B. invictae species encompass a clonally diverse population, which can justify their great adaptation ability to different niches, with evidence of clonal-host specificity.

Bacillus invictae sp. nov., isolated from a health product.

A Gram-positive, rod-shaped, endospore-forming Bacillus isolate, Bi.(FFUP1) (T), recovered in Portugal from a health product was subjected to a polyphasic study and compared with the type strains of Bacillus pumilus, Bacillus safensis, Bacillus altitudinis and Bacillus xiamenensis, the phenotypically and genotypically most closely related species. Acid production from cellobiose, D-glucose and D-mannose and absence of acid production from D-arabinose, erythritol, inositol, maltose, mannitol, raffinose, rhamnose, sorbitol, starch and L-tryptophan discriminated this new isolate from the type strains of the most closely related species. Additionally, a significant different protein and carbohydrate signature was evidenced by spectroscopic techniques, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and Fourier transform IR spectroscopy with attenuated total reflectance. Using a chemometric approach, the score plot generated by principal component analysis clearly delineated the isolate as a separate cluster. The quinone system for strain Bi.(FFUP1) (T) comprised predominantly menaquinone MK-7 and major polar lipids were diphosphatidylglycerol, an unidentified phospholipid and an unidentified glycolipid. Strain Bi.(FFUP1) (T) showed ≥ 99% 16S rRNA gene sequence similarity to B. safensis FO-036b(T), B. pumilus (7061(T) and SAFR-032), B. altitudinis 41KF2b(T) and B. xiamenensis HYC-10(T). Differences in strain Bi.FFUP1 (T) gyrB and rpoB sequences in comparison with the most closely related species and DNA-DNA hybridization experiments with Bi.FFUP1 (T) and B. pumilus ATCC 7061(T), B. safensis FO-036b(T), B. altitudinis 41KF2b(T) and B. xiamenensis HYC-10(T) gave relatedness values of 39.6% (reciprocal 38.0%), 49.9% (reciprocal 42.9%), 61.9% (reciprocal 52.2%) and 61.7% (reciprocal 49.2%), respectively, supported the delineation of strain Bi.(FFUP1) (T) as a representative of a novel species of the genus Bacillus, for which the name Bacillus invictae sp. nov. is proposed, with strain Bi.(FFUP1) (T) ( =DSM 26896(T) =CCUG 64113(T)) as the type strain.

Successful application of the DiversiLab repetitive-sequence-based PCR typing system for confirmation of the circulation of a multiresistant Pseudomonas aeruginosa clone in different hospital wards.

The applicability of the repetitive-sequence-based PCR (rep-PCR)-based DiversiLab system was tested compared with the pulsed field gel electrophoresis (PFGE) to type a phenotypically similar subset of a large collection of multiresistant Pseudomonas aeruginosa strains isolated during a 17-month period from patients treated in different wards including 4 intensive care units (ICUs). Five environmental P. aeruginosa isolates obtained from one of the ICUs were also included. The DiversiLab system and the PFGE demonstrated the genetic relationship among the isolates with the same efficacy. One of the environmental isolates had the same rep-PCR type as the circulating clone. Multilocus sequence typing of one of the clinical isolates of the circulating clone proved that it is a member of a clonal complex of P. aeruginosa that has not been previously described in clinical samples.

Diversity of Tn1546 and its role in the dissemination of vancomycin-resistant enterococci in Portugal.

We characterized the molecular diversity of vanA vancomycin-resistant enterococci (VRE; 176 isolates/87 pulsed-field gel electrophoresis types) from different sources and cities in Portugal (1996 to 2004): (i) food animals (FA; n = 38 isolates out of 31 samples), hospitalized humans (HH; n = 101/101), healthy human volunteers (HV; n = 7/4), and environmental sources (n = 30/10). Some strains were isolated from different hosts and persistently recovered for years. Twenty-four Tn1546 variants were identified, all located on plasmids (30 to 250 kb). Some Tn1546 variants were associated with specific sources such as FA (3 types), HH (11 types), or HV (1 type), while others were recovered from isolates of different origins (8 types). Polymorphisms in the central vanRSHA region of Tn1546 were scarcely detected, while alterations upstream of vanR and downstream of vanA were frequently identified involving mutations (vanS and vanX), deletions (vanY), insertions (IS1216V, ISEf1, and IS19; sequences with or without homology with others available in GenBank databases), and different genetic rearrangements. Most Tn1546 variants contained IS1216V (14 types) or ISEf1 (6 types). IS1216V was found alone or associated with an IS3-like element at different orientations and positions in Tn1546 from human, animal, and environmental samples. ISEf1 was located within vanX-vanY region at nucleotide 9044 of Tn1546 variants mostly associated with clinical isolates, suggesting a common genetic platform. IS19 was observed within the vanX-vanY region in one Tn1546 variant from poultry. Recent spread of VRE in Portugal reflects a complex epidemiology involving both clonal spread and plasmid dissemination containing a variety of Tn1546 types. Apparent Tn1546 heterogeneity among enterococci from human, animal, and environmental sources might reflect frequent genetic exchange events and evolution of particular widely disseminated genetic elements.

Molecular characterization of glycopeptide-resistant Enterococcus faecium isolates from Portuguese hospitals.

Fifty-one pulsed-field gel electrophoresis types and 17 Tn1546 variants were identified among 101 Enterococcus faecium isolates recovered in three distant Portuguese hospitals. Intra- and interhospital dissemination of specific strains and Tn1546 types was detected, which might largely contribute to the endemicity of vancomycin-resistant E. faecium in Portuguese hospitals, as happened previously in other geographical locations.