Dataset Information

Reliable Identification of Environmental Pseudomonas Isolates Using the rpoD Gene.

ABSTRACT: 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.

SUBMITTER: Girard L

PROVIDER: S-EPMC7463772 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Reliable Identification of Environmental Pseudomonas Isolates Using the rpoD Gene.

Girard Léa L Lood Cédric C Rokni-Zadeh Hassan H van Noort Vera V Lavigne Rob R De Mot René R

Microorganisms 20200731 8

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 ide ...[more]

PMID: 32752051

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Project description: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 the 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-nucleotide (nt) region of the rpoD gene enabling taxonomic differentiation of Pseudomonas species in soil samples. The method was benchmarked on a 16-member Pseudomonas species mock community. All 16 species were correctly and semiquantitatively identified using rpoD gene amplicons, whereas 16S rRNA V3-V4 amplicon sequencing only correctly identified one species. We analyzed the Pseudomonas profiles in 13 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 16S rRNA gene abundance, of which each sampling site contained a unique Pseudomonas composition. Thirty culturable Pseudomonas strains were isolated from each grassland site and 10 from each agriculture site and identified by Sanger sequencing of the rpoD gene. In all cases, the rpoD amplicon approach identified more species than were found by cultivation, including hard-to-culture nonfluorescent pseudomonads, as well as more than were found by 16S rRNA V3-V4 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. IMPORTANCE A high-throughput sequencing-based method for profiling of Pseudomonas species in soil microbiomes was developed and identified more species than 16S rRNA gene sequencing or cultivation. Pseudomonas species are used as biocontrol organisms and plant growth-promoting agents, and the method will allow tracing of specific species of Pseudomonas as well as enable screening of environmental samples for further isolation and exploitation.

Project description:Background:Pseudomonas aeruginosa is ubiquitous, has intrinsic antibiotic resistance mechanisms, and is associated with serious hospital-associated infections. It has evolved from being a burn wound infection into a major nosocomial threat. In this study, we compared and correlated the antimicrobial resistance, virulence traits and clonal relatedness between clinical and fresh water environmental isolates of P. aeruginosa. Methods:219 P. aeruginosa isolates were studied: (a) 105 clinical isolates from 1977 to 1985 (n = 52) and 2015 (n = 53), and (b) 114 environmental isolates from different fresh water sources. All isolates were subjected to ERIC-PCR typing, antimicrobial susceptibility testing and virulence factor genes screening. Results:Clinical and environmental isolates of P. aeruginosa were genetically heterogenous, with only four clinical isolates showing 100% identical ERIC-PCR patterns to seven environmental isolates. Most of the clinical and environmental isolates were sensitive to almost all of the antipseudomonal drugs, except for ticarcillin/clavulanic acid. Increased resistant isolates was seen in 2015 compared to that of the archived isolates; four MDR strains were detected and all were retrieved in 2015. All clinical isolates retrieved from 1977 to 1985 were susceptible to ceftazidime and ciprofloxacin; but in comparison, the clinical isolates recovered in 2015 exhibited 9.4% resistance to ceftazidime and 5.7% to ciprofloxacin; a rise in resistance to imipenem (3.8% to 7.5%), piperacillin (9.6% to 11.3%) and amikacin (1.9% to 5.7%) and a slight drop in resistance rates to piperacillin/tazobactam (7.7% to 7.5%), ticarcillin/clavulanic acid (19.2% to 18.9%), meropenem (15.4% to 7.5%), doripenem (11.5% to 7.5%), gentamicin (7.7% to 7.5%) and netilmicin (7.7% to 7.5%). Environmental isolates were resistant to piperacillin/tazobactam (1.8%), ciprofloxacin (1.8%), piperacillin (4.4%) and carbapenems (doripenem 11.4%, meropenem 8.8% and imipenem 2.6%). Both clinical and environmental isolates showed high prevalence of virulence factor genes, but none were detected in 10 (9.5%) clinical and 18 (15.8%) environmental isolates. The exoT gene was not detected in any of the clinical isolates. Resistance to carbapenems (meropenem, doripenem and imipenem), β-lactamase inhibitors (ticarcillin/clavulanic acid and piperacillin/tazobactam), piperacillin, ceftazidime and ciprofloxacin was observed in some of the isolates without virulence factor genes. Five virulence-negative isolates were susceptible to all of the antimicrobials. Only one MDR strain harbored none of the virulence factor genes. Conclusion:Over a period of 30 years, a rise in antipseudomonal drug resistance particularly to ceftazidime and ciprofloxacin was observed in two hospitals in Malaysia. The occurrence of resistant environmental isolates from densely populated areas is relevant and gives rise to collective anxiety to the community at large.

Dataset Information

Reliable Identification of Environmental Pseudomonas Isolates Using the rpoD Gene.

Publications

Reliable Identification of Environmental <i>Pseudomonas</i> Isolates Using the <i>rpoD</i> Gene.

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