https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-0691.2008.02070.x

Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories

The class Alphaproteobacteria is comprised of a diverse assemblage of Gram-negative bacteria that includes organisms of varying morphologies, physiologies and habitat preferences many of which are of clinical and ecological importance. Alphaproteobacteria classification has proved to be difficult, not least when taxonomic decisions rested heavily on a limited number of phenotypic features and interpretation of poorly resolved 16S rRNA gene trees. Despite progress in recent years regarding the classification of bacteria assigned to the class, there remains a need to further clarify taxonomic relationships. Here, draft genome sequences of a collection of genomes of more than 1000 Alphaproteobacteria and outgroup type strains were used to infer phylogenetic trees from genome-scale data using the principles drawn from phylogenetic systematics. The majority of taxa were found to be monophyletic but several orders, families and genera, including taxa recognized as problematic long ago but also quite recent taxa, as well as a few species were shown to be in need of revision. According proposals are made for the recognition of new orders, families and genera, as well as the transfer of a variety of species to other genera and of a variety of genera to other families. In addition, emended descriptions are given for many species mainly involving information on DNA G+C content and (approximate) genome size, both of which are confirmed as valuable taxonomic markers. Similarly, analysis of the gene content was shown to provide valuable taxonomic insights in the class. Significant incongruities between 16S rRNA gene and whole genome trees were not found in the class. The incongruities that became obvious when comparing the results of the present study with existing classifications appeared to be caused mainly by insufficiently resolved 16S rRNA gene trees or incomplete taxon sampling. Another probable cause of misclassifications in the past is the partially low overall fit of phenotypic characters to the sequence-based tree. Even though a significant degree of phylogenetic conservation was detected in all characters investigated, the overall fit to the tree varied considerably.

/pdf/analysis-of-1-000-type-strain-genomes-substantially-improves-57hicbb77a.pdf

Analysis of 1,000+ Type-Strain Genomes Substantially Improves Taxonomic Classification of Alphaproteobacteria.

The family Rhodobacteraceae can be considered a paradigm of modern taxonomy of prokaryotes. Taking into account the number of species and genera that conforms the family, together with the knowledge about their abundance and vast global distribution, it surprises that most of them have been described relatively recent to our days. Two notable exceptions are Rhodonostoc capsulatum (Molisch, Die purpurbakterien nach neuen untersuchungen, vols i–vii. G. Fischer, Jena, pp 1–95, 1907) and Micrococcus denitrificans Beijerinck and Minkman (Zentbl Bakteriol, Parasitenkd, Infektionskr Hyg. Abt II 25:30–63, 1910), early basonyms of Rhodobacter capsulatus and Paracoccus denitrificans, respectively. The fact that so many descriptions within this family are recent means that some studies have been concomitant and pose a challenge not only for pure taxonomic studies but also for interpreting other studies in which a rapidly evolving nomenclature had to be used anyway. The metabolic and ecological diversity of the group adds further complexity. In spite of all these difficulties, the picture is far from being a chaos and it can be considered an exciting and important bacterial group to study.
Rhodobacteraceae are, fundamentally, aquatic bacteria that frequently thrive in marine environments. They comprise mainly aerobic photo- and chemoheterotrophs but also purple non-sulfur bacteria which perform photosynthesis in anaerobic environments. They are deeply involved in sulfur and carbon biogeochemical cycling and symbiosis with aquatic micro- and macroorganisms.
One hundred genera are currently recognized as members of the family although the Stappia group, Ahrensia, Agaricicola, and Rhodothalassium do not belong, phylogenetically, to the family. The 90 other genera are distributed in 5 phylogenetic groups (the Rhodobacter, the Paracoccus, the Rhodovulum, the Amaricoccus, and the Roseobacter clades) that might be considered a family on its own.

The Family Rhodobacteraceae

This review provides a state-of-the-art description of the performance of Sanger cycle sequencing of the 16S rRNA gene for routine identification of bacteria in the clinical microbiology laboratory. A detailed description of the technology and current methodology is outlined with a major focus on proper data analyses and interpretation of sequences. The remainder of the article is focused on a comprehensive evaluation of the application of this method for identification of bacterial pathogens based on analyses of 16S multialignment sequences. In particular, the existing limitations of similarity within 16S for genus- and species-level differentiation of clinically relevant pathogens and the lack of sequence data currently available in public databases is highlighted. A multiyear experience is described of a large regional clinical microbiology service with direct 16S broad-range PCR followed by cycle sequencing for direct detection of pathogens in appropriate clinical samples. The ability of proteomics (matrix-assisted desorption ionization-time of flight) versus 16S sequencing for bacterial identification and genotyping is compared. Finally, the potential for whole-genome analysis by next-generation sequencing (NGS) to replace 16S sequencing for routine diagnostic use is presented for several applications, including the barriers that must be overcome to fully implement newer genomic methods in clinical microbiology. A future challenge for large clinical, reference, and research laboratories, as well as for industry, will be the translation of vast amounts of accrued NGS microbial data into convenient algorithm testing schemes for various applications (i.e., microbial identification, genotyping, and metagenomics and microbiome analyses) so that clinically relevant information can be reported to physicians in a format that is understood and actionable. These challenges will not be faced by clinical microbiologists alone but by every scientist involved in a domain where natural diversity of genes and gene sequences plays a critical role in disease, health, pathogenicity, epidemiology, and other aspects of life-forms. Overcoming these challenges will require global multidisciplinary efforts across fields that do not normally interact with the clinical arena to make vast amounts of sequencing data clinically interpretable and actionable at the bedside.

Performance and Application of 16S rRNA Gene Cycle Sequencing for Routine Identification of Bacteria in the Clinical Microbiology Laboratory

Background The role of rodents in Leptospira epidemiology and transmission is well known worldwide. Rats are known to carry different pathogenic serovars of Leptospira spp. capable of causing disease in humans and animals. Wild rats (Rattus spp.), especially the Norway/brown rat (Rattus norvegicus) and the black rat (R. rattus), are the most important sources of Leptospira infection, as they are abundant in urban and peridomestic environments. In this study, we compiled and summarized available data in the literature on global prevalence of Leptospira exposure and infection in rats, as well as compared the global distribution of Leptospira spp. in rats with respect to prevalence, geographic location, method of detection, diversity of serogroups/serovars, and species of rat. Methods We conducted a thorough literature search using PubMed without restrictions on publication date as well as Google Scholar to manually search for other relevant articles. Abstracts were included if they described data pertaining to Leptospira spp. in rats (Rattus spp.) from any geographic region around the world, including reviews. The data extracted from the articles selected included the author(s), year of publication, geographic location, method(s) of detection used, species of rat(s), sample size, prevalence of Leptospira spp. (overall and within each rat species), and information on species, serogroups, and/or serovars of Leptospira spp. detected. Findings A thorough search on PubMed retrieved 303 titles. After screening the articles for duplicates and inclusion/exclusion criteria, as well as manual inclusion of relevant articles, 145 articles were included in this review. Leptospira prevalence in rats varied considerably based on geographic location, with some reporting zero prevalence in countries such as Madagascar, Tanzania, and the Faroe Islands, and others reporting as high as >80% prevalence in studies done in Brazil, India, and the Philippines. The top five countries that were reported based on number of articles include India (n = 13), Malaysia (n = 9), Brazil (n = 8), Thailand (n = 7), and France (n = 6). Methods of detecting or isolating Leptospira spp. also varied among studies. Studies among different Rattus species reported a higher Leptospira prevalence in R. norvegicus. The serovar Icterohaemorrhagiae was the most prevalent serovar reported in Rattus spp. worldwide. Additionally, this literature review provided evidence for Leptospira infection in laboratory rodent colonies within controlled environments, implicating the zoonotic potential to laboratory animal caretakers. Conclusions Reports on global distribution of Leptospira infection in rats varies widely, with considerably high prevalence reported in many countries. This literature review emphasizes the need for enhanced surveillance programs using standardized methods for assessing Leptospira exposure or infection in rats. This review also demonstrated several weaknesses to the current methods of reporting the prevalence of Leptospira spp. in rats worldwide. As such, this necessitates a call for standardized protocols for the testing and reporting of such studies, especially pertaining to the diagnostic methods used. A deeper understanding of the ecology and epidemiology of Leptospira spp. in rats in urban environments is warranted. It is also pertinent for rat control programs to be proposed in conjunction with increased efforts for public awareness and education regarding leptospirosis transmission and prevention.

/pdf/leptospira-infection-in-rats-a-literature-review-of-global-3v2g3rxqem.pdf

Leptospira infection in rats: A literature review of global prevalence and distribution.

Twelve strains of gram-negative, nonfermenting rods recovered mainly from septicemic patients were studied using conventional and molecular methods The phenotypic profiles of these strains most closely resembled Psychrobacter phenylpyruvicus They produced catalase, oxidase, urease, and H(2)S (lead acetate paper) but did not produce indole, reduce nitrate or nitrite, or hydrolyze gelatin or esculin No acid production was observed in a King's oxidation-fermentation base containing d-glucose, d-xylose, d-mannitol, sucrose, lactose, or maltose All strains were nonmotile and nonpigmented Most strains produced green discoloration on blood agar All strains grew at 25 degrees C and 35 degrees C and most grew on MacConkey agar They shared a common cellular fatty acid (CFA) profile characterized by large amounts (56% to 90%) of 18:1omega7c and the presence of 3-OH-10:0, 16:1omega7c, 16:0, and 19:0cycomega8c that overall was most similar to that of Rhodobacter species but was quite distinct from that of P phenylpyruvicus The MICs for most beta-lactams, fluoroquinolones, aminoglycosides, and carbapenems were low MICs for aztreonam and piperacillin were higher, with MICs for some strains of > 64 mg/liter and > 128 mg/liter, respectively Polyphasic analysis of these strains, including morphological, biochemical, CFA composition, DNA-DNA hybridization, 16S rRNA gene sequencing, and percent guanine-plus-cytosine (G+C) content analysis, demonstrated that these strains and Rhodobacter massiliensis represent a new genus, "Haematobacter" (proposed name), with the species H missouriensis (type strain H1892(T) = CCUG 52307(T) = CIP 109176(T)) and H massiliensis comb nov (type strain Framboise(T) = CCUG 47968(T) = CIP 107725(T)) and an unnamed genomospecies

Identification of “Haematobacter,” a New Genus of Aerobic Gram-Negative Rods Isolated from Clinical Specimens, and Reclassification of Rhodobacter massiliensis as “Haematobacter massiliensis comb. nov.”

Samples were collected from 128 symptomatic humans, 83 dogs, 49 mice, and 20 rats (Rattus rattus: 16; Rattus norvegicus: 4) in neighborhoods where human leptospirosis have been reported within the principal sea-port city of Colombia. Seroprevalences were assessed against 19 pathogenic, 1 intermediate pathogenic, and 1 saprophytic Leptospira serogroups. Pathogenic Leptospira were confirmed using conventional Leptospira-specific polymerase chain-reaction and pulsed-field gel electrophoresis analysis was used for serovar identification. Seroprevalences of 20.4%, 12.5%, 25.0%, 22.9%, and 12.4% were obtained against one to seven different serogroups in mice, R. rattus, R. norvegicus, dogs, and humans, respectively. The DNA was confirmed to be from pathogenic Leptospira by detecting the lipL32 gene in 12.5%, 3.7%, and 0.03% of the R. rattus, dog, and human samples, respectively. The first genetically typed Colombian isolate was obtained from a rat and identified as Leptospira interrogans serovar Icterohaemorrhagiae/Copenhageni.

/pdf/cross-sectional-study-of-leptospira-seroprevalence-in-humans-5a2h3nk77w.pdf

Cross-Sectional Study of Leptospira Seroprevalence in Humans, Rats, Mice, and Dogs in a Main Tropical Sea-Port City

Introduction: Leptospirosis is a bacterial disease transmitted directly or indirectly from animals to humans that may result in severe hemorrhagic, hepatic/renal and pulmonary disease. There are 20 known Leptospira species and hundreds of serovars, some of which belong to different species. It is essential to identify pathogenic Leptospira serovars and their potential reservoirs to prepare adequate control strategies. Objective: To characterize the Leptospira serovars isolated from rodents, dogs, pigs and water samplesin Colombia. Materials and methods: Leptospira organisms were isolated and cultured, and pathogenic strains were identified using a polymerase chain-reaction (PCR). Leptospira DNA and Salmonella Braenderup H9812 (molecular weight standard) DNA were cleaved using NotI and subjected to pulsed-field gel electrophoresis (PFGE). The PFGE patterns were analyzed based on bacterial strain-typing criteria and Dice coefficients (DCs) between these isolates and over 200 Leptospira organisms isolated from other parts of the world. Results: All of the isolates were pathogenic strains, and five were genetically characterized. The P275 (84% DC) and P282 (95% DC) pig isolates were related to the Leptospira interrogans Pomona serovar; the I15 (DC: 100%) rat isolate was identical to the Leptospira interrogans Icterohameorrhagiae or Copenhageni serovars, while the C67 (64% DC) dog and A42 (60% DC) water isolates were not related (< 73.7% DC) to any of the 200 reference serovars; the closest serovars were the Leptospira noguchii Nicaragua and Orleans serovars, respectively. Conclusion: This was the first molecular characterization of Colombian Leptospira spp isolates; these isolates will be used to develop a Colombian diagnostic panel. doi: http://dx.doi.org/10.7705/biomedica.v33i0.731

/pdf/molecular-serovar-characterization-of-leptospira-isolates-vn1go2mo95.pdf

Molecular serovar characterization of Leptospira isolates from animals and water in Colombia.

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Identification of “Haematobacter,” a New Genus of Aerobic Gram-Negative Rods Isolated from Clinical Specimens, and Reclassification of Rhodobacter massiliensis as “Haematobacter massiliensis comb. nov.”

Cross-Sectional Study of Leptospira Seroprevalence in Humans, Rats, Mice, and Dogs in a Main Tropical Sea-Port City

Molecular serovar characterization of Leptospira isolates from animals and water in Colombia.