The Ever-Expanding Pseudomonas Genus: Description of 43 New Species and Partition of the Pseudomonas putida Group

The genus Pseudomonas hosts an extensive genetic diversity and is one of the largest genera among Gram-negative bacteria. Type strains of Pseudomonas are well known to represent only a small fraction of this diversity and the number of available Pseudomonas genome sequences is increasing rapidly. Consequently, new Pseudomonas species are regularly reported and the number of species within the genus is constantly evolving. In this study, whole genome sequencing enabled us to define 43 new Pseudomonas species and provide an update of the Pseudomonas evolutionary and taxonomic relationships. Phylogenies based on the rpoD gene and whole genome sequences, including, respectively, 316 and 313 type strains of Pseudomonas, revealed sixteen groups of Pseudomonas and, together with the distribution of cyclic lipopeptide biosynthesis gene clusters, enabled the partitioning of the P. putida group into fifteen subgroups. Pairwise average nucleotide identities were calculated between type strains and a selection of 60 genomes of non-type strains of Pseudomonas. Forty-one strains were incorrectly assigned at the species level and among these, 19 strains were shown to represent an additional 13 new Pseudomonas species that remain to be formally classified. This work pinpoints the importance of correct taxonomic assignment and phylogenetic classification in order to perform integrative studies linking genetic diversity, lifestyle, and metabolic potential of Pseudomonas spp.

The Ever-Expanding Pseudomonas Genus: Description of 43 New Species and Partition of the Pseudomonas Putida Group

The genus Pseudomonas hosts an extensive genetic diversity and is one of the largest genera among Gram-negative bacteria. Type strains of Pseudomonas are well-known to represent only a small fraction of this diversity and the number of available Pseudomonas genome sequences is increasing rapidly. Consequently, new Pseudomonas species are regularly reported and the number of species within the genus is in constant evolution. In this study, whole genome se-quencing enabled us to define 43 new Pseudomonas species and to provide an update of the Pseu-domonas evolutionary and taxonomic relationships. Phylogenies based on the rpoD gene and whole genome sequences, including 316 and 313 type strains of Pseudomonas, respectively, re-vealed sixteen groups of Pseudomonas and justified the partitioning of the P. putida group into fifteen subgroups. Pairwise average nucleotide identities were calculated between type strains and a selection of 60 genomes of non-type strains of Pseudomonas. Forty-one strains were incor-rectly assigned at the species level and among those, 19 strains were shown to represent an addi-tional 13 new Pseudomonas species that remain to be formally classified. This work pinpoints the importance of correct taxonomic assignment and phylogenetic classification in order to perform integrative studies linking genetic diversity, lifestyle and metabolic potential of Pseudomonas spp.

Identification and differentiation of Pseudomonas species in field samples using an rpoD amplicon sequencing methodology

Species of the genus Pseudomonas are used for several biotechnological purposes, including plant biocontrol and bioremediation. To exploit the Pseudomonas genus in environmental, agricultural or industrial settings, the organisms must be profiled at species level as their bioactivity potential differs markedly between species. Standard 16S rRNA gene amplicon profiling does not allow for accurate species differentiation. Thus, the purpose of this study was to develop an amplicon-based high-resolution method targeting a 760 nt region of the rpoD gene enabling taxonomic differentiation of Pseudomonas species in soil samples. The method was benchmarked on a sixteen membered Pseudomonas species mock community. All 16 species were correctly and semi-quantitatively identified using rpoD gene amplicons, whereas 16S rRNA V3V4 amplicon sequencing only correctly identified one species. We analysed the Pseudomonas profile in thirteen soil samples in northern Zealand, Denmark, where samples were collected from grassland (3 samples) and agriculture soil (10 samples). Pseudomonas species represented up to 0.7% of the microbial community, of which each sampling site contained a unique Pseudomonas composition. Thirty culturable Pseudomonas strains were isolated from each grassland site and ten from each agriculture site and identified by Sanger sequencing of the rpoD gene. In all cases, the rpoD-amplicon approach identified more species than found by cultivation, including hard-to-culture non-fluorescent pseudomonads, as well as more than found by 16S rRNA V3V4 amplicon sequencing. Thus, rpoD profiling can be used for species profiling of Pseudomonas, and large scale prospecting of bioactive Pseudomonas may be guided by initial screening using this method.

Diagnosis of leaf bacterial diseases of coffee reveals the prevalence of halo blight

ABSTRACT The diagnosis of foliar bacterial diseases in coffee (Coffea arabica), such as halo blight (Pseudomonas syringae pv. garcae), bacterial leaf spot (P. syringae pv. tabaci), bacterial blight (P. cichorii), and dark leaf spot (Robbsia andropogonis), is considered a challenge for plant pathologists. The misidentification has been occurring when the diagnosis is solely based on symptoms and biochemical properties. Thus, the objective of this study was to identify and differentiate species and pathovars of Pseudomonas pathogenic to coffee plants, enabling a survey of the occurrence of these bacteria in the main producing regions of Minas Gerais state, Brazil. Firstly, the pathogenicity of the isolates was confirmed by inoculation in C. arabica cv. Catuaí Vermelho IAC 99. Then, biochemical analyses, combined with, repetitive element-polymerase chain reaction (rep-PCR) and phylogeny based on rpoD gene sequences were used to characterize 84 Pseudomonas isolates from coffee crops and nurseries. Based on rpoD-phylogeny, 73 isolates were identified as P. syringae pv. garcae, five as P. syringae pv. tabaci and six as P. cichorii. The rep-PCR results suggest a high genetic variability in populations of Pseudomonas syringae pv. garcae and P. cichorii. This is the first report of the occurrence of bacterial leaf spot (P. syringae pv. tabaci) in the coffee-producing filed in Minas Gerais State. The findings confirmed the prevalence of P. syringae pv. garcae in coffee production fields in the State and the generated knowledge will contribute for the development of species-specific primers for the identification and detection of this pathogen.

Reliable Identification of Environmental Pseudomonas Isolates Using the rpoD Gene

The taxonomic affiliation of Pseudomonas isolates is currently assessed by using the 16S rRNA gene, MultiLocus Sequence Analysis (MLSA), or whole genome sequencing. Therefore, microbiologists are facing an arduous choice, either using the universal marker, knowing that these affiliations could be inaccurate, or engaging in more laborious and costly approaches. The rpoD gene, like the 16S rRNA gene, is included in most MLSA procedures and has already been suggested for the rapid identification of certain groups of Pseudomonas. However, a comprehensive overview of the rpoD-based phylogenetic relationships within the Pseudomonas genus is lacking. In this study, we present the rpoD-based phylogeny of 217 type strains of Pseudomonas and defined a cutoff value of 98% nucleotide identity to differentiate strains at the species level. To validate this approach, we sequenced the rpoD of 145 environmental isolates and complemented this analysis with whole genome sequencing. The rpoD sequence allowed us to accurately assign Pseudomonas isolates to 20 known species and represents an excellent first diagnostic tool to identify new Pseudomonas species. Finally, rpoD amplicon sequencing appears as a reliable and low-cost alternative, particularly in the case of large environmental studies with hundreds or thousands of isolates.

A Case of Aeromonas trota in an Immunocompromised Patient with Diarrhea

According to recent literature, 95.4% of the Aeromonas strains associated with human clinical cases correspond to four species: Aeromonas caviae, Aeromonas dhakensis, Aeromonas veronii and Aeromonas hydrophila. However, other less prevalent species such as Aeromonas trota, are also described from clinical samples. Based on its low incidence, the latter species can be regarded as rare and it is the only Aeromonas species susceptible to ampicillin. From the taxonomic point of view, A. trota is considered a synonym of the species Aeromonas enteropelogenes. The objective of this study is to present a new clinical case associated with A. trota in order to increase the knowledge about this species. The strain was recovered from the feces of a 69-year-old patient with a diarrheal syndrome and peritoneal psammocarcinoma. The preliminary identification as Aeromonas sp. was obtained with the API 20E, but it was characterized as Aeromonas jandei and also as Aeromonas enteropelogenes with different scores with the matrix-assisted laser desorption ionization time of flight (MALDI-TOF). Based on the sequence of the rpoD gene, it was confirmed to be A. trota. The antimicrobial resistance pattern showed that the strain was susceptible to ampicillin, penicillins in combination with beta-lactamase inhibitors, quinolones, carbapenems, aminoglycosides and cephalosporins, except cephalothin. In conclusion, the recognition of an Aeromonas strain susceptible to ampicillin should alert the clinical microbiologist of the possible involvement of this rare species. Furthermore, the MALDI-TOF database should be updated indicating that the species A. enteropelogenes, is a synonym of A. trota.

Molecular Identification and Virulence Potential of the Genus Aeromonas Isolated from Wild Rainbow Trout (Oncorhynchus mykiss) in Mexico

ABSTRACT The members of the Aeromonas genus are important foodborne pathogens, with a worldwide distribution. Wild rainbow trout, from the national protected area Santuario del Agua State Park, Corral de Piedra, were analyzed. Species of Aeromonas were isolated from the trout, and their pathogenic potential was analyzed based on different pathogenicity and virulence factors. The isolates were identified as A. allosaccharophila (n = 15), A. sobria (n = 8), A. veronii (n = 3), A. rivipollensis (n = 2), A. piscicola (n = 2), and A. popoffii (n = 1), by RNA polymerase sigma factor (rpoD) gene sequencing. Sequence similarity with the type strain was 92.2 to 99.6% for A. sobria isolates, 97.8 to 98.0% for A. allosaccharophila isolates, 99.2% for the A. popoffii isolate, 99.2 to 100% for A. piscicola isolates, and 98.2 to 99.2% for A. veronii isolates. Notably, isolates A30T2–gills and A30T2–spleen showed sequence similarity of 98.0% with strain A. media CECT 4232T and 99.0% with strain A. rivipollensis P2G1T. Virulence genes were detected by PCR at the following frequencies: fla and serine protease, 96.77%; aerA, 93.54%; aexT, 87.09%; lipases, 74.19%; ascV and ahyB, 67.74%; exu, 61.29%; act, 41.93%; ascF-G, 38.70%; lafA, 32.26%; alt, 6.46%; aopP, 9.67%; and ast, 3.23%. These results indicate that several Aeromonas species had the potential pathogenicity to infect wild rainbow trout in the waterway created by the Corral de Piedra dam, suggesting they could be an emerging zoonotic pathogen.

Biodegradation of Petroleum Hydrocarbons by Bacillus subtilis BL-27, a Strain with Weak Hydrophobicity

The biodegradation of petroleum hydrocarbons has many potential applications and has attracted much attention recently. The hydrocarbon-degrading bacterium BL-27 was isolated from petroleum-polluted soil and was compounded with surfactants to improve biodegradation. Its 16S rDNA and rpoD gene sequences indicated that it was a strain of Bacillus subtilis. Strain BL-27 had extensive adaptability and degradability within a broad range of temperatures (25–50 °C), pH (4.0–10.0) and salinity (0–50 g/L NaCl). Under optimal conditions (45 °C, pH 7.0, 1% NaCl), the strain was able to degrade 65% of crude oil (0.3%, w/v) within 5 days using GC-MS analysis. Notably, strain BL-27 had weak cell surface hydrophobicity. The adherence rate of BL-27 to n-hexadecane was 29.6% with sucrose as carbon source and slightly increased to 33.5% with diesel oil (0.3%, w/v) as the sole carbon source, indicating that the cell surface of BL-27 is relatively hydrophilic. The strain was tolerant to SDS, Tween 80, surfactin, and rhamnolipids at a concentration of 500 mg/L. The cell surface hydrophobicity reduced more with the addition of surfactants, while the chemical dispersants, SDS (50–100 mg/L) and Tween 80 (200–500 mg/L), significantly increased the strain’s ability to biodegrade, reaching 75–80%. These results indicated that BL-27 has the potential to be used for the bioremediation of hydrocarbon pollutants and could have promising applications in the petrochemical industry.