In 2003 to 2004, the first five VIM-2 metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa (MPPA) isolates with an In4-like integron, In461 (aadB-blaVIM-2-aadA6), on conjugative plasmids were identified in three hospitals in Poland. In 2005 to 2015, MPPA expanded much in the country, and as many as 80 isolates in a collection of 454 MPPA (∼18%) had In461, one of the two most common MBL-encoding integrons. The organisms occurred in 49 hospitals in 33 cities of 11/16 main administrative regions. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) classified them into 55 pulsotypes and 35 sequence types (STs), respectively, revealing their remarkable genetic diversity overall, with only a few small clonal clusters. S1 nuclease/hybridization assays and mating of 63 representative isolates showed that ∼85% of these had large In461-carrying plasmids, ∼350 to 550 kb, usually self-transmitting with high efficiency (∼10-1 to 10-2 per donor cell). The plasmids from 19 isolates were sequenced and subjected to structural and single-nucleotide-polymorphism (SNP)-based phylogenetic analysis. These formed a subgroup within a family of IncP-2-type megaplasmids, observed worldwide in pseudomonads from various environments and conferring resistance/tolerance to multiple stress factors, including antibiotics. Their microdiversity in Poland arose mainly from acquisition of different accessory fragments, as well as new resistance genes and multiplication of these. Short-read sequence and/or PCR mapping confirmed the In461-carrying plasmids in the remaining isolates to be the IncP-2 types. The study demonstrated a large-scale epidemic spread of multidrug resistance plasmids in P. aeruginosa populations, creating an epidemiological threat. It contributes to the knowledge on IncP-2 types, which are interesting research objects in resistance epidemiology, environmental microbiology, and biotechnology.
- MeSH
- Anti-Bacterial Agents pharmacology MeSH
- Bacterial Proteins MeSH
- beta-Lactamases genetics metabolism MeSH
- Epidemics * MeSH
- Phylogeny MeSH
- Cross Infection * epidemiology MeSH
- Integrons genetics MeSH
- Humans MeSH
- Multilocus Sequence Typing MeSH
- Hospitals MeSH
- Pseudomonas Infections * drug therapy epidemiology MeSH
- Pseudomonas aeruginosa genetics metabolism MeSH
- Electrophoresis, Gel, Pulsed-Field MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Poland MeSH
We studied the diversity of Limnohabitans using reverse line blot hybridization with Limnohabitans lineage-specific probes in the freshwater canyon-shaped Římov reservoir (Czech Republic). To examine the succession of distinct lineages, we performed (i) a study of an intensive spring sampling program at the lacustrine part of the Římov reservoir (from ice melt through a phytoplankton peak to the clear-water phase), and (ii) a seasonal study (April to November) when the occurrence of distinct Limnohabitans lineages was related to the inherent longitudinal heterogeneity of the reservoir. Significant spatiotemporal changes in the compositions of distinct Limnohabitans lineages allowed for the identification of "generalists" that were always present throughout the whole season as well as "specialists" that appeared in the reservoir only for limited periods of time or irregularly. Our results indicate that some phytoplankton groups, such as cryptophytes or cyanobacteria, and zooplankton composition were the major factors modulating the distribution and dynamics of distinct Limnohabitans lineages. The highest Limnohabitans diversity was observed during the spring algal bloom, whereas the lowest was during the summer cyanobacterial bloom. The microdiversity also markedly increased upstream in the reservoir, being highest at the inflow, and thus likely reflecting strong influences of the watershed.IMPORTANCE The genus Limnohabitans is a typical freshwater bacterioplankton and is believed to play a significant role in inland freshwater habitats. This work is unique in detecting and tracing different closely related lineages of this bacterial genus in its natural conditions using the semiquantitative reverse line blot hybridization method and in discovering the factors influencing the microdiversity, subtype alternations, and seasonality.
- MeSH
- Comamonadaceae classification genetics isolation & purification MeSH
- Cryptophyta growth & development MeSH
- Ecosystem MeSH
- Eutrophication MeSH
- Phytoplankton growth & development MeSH
- Seasons MeSH
- Cyanobacteria growth & development MeSH
- Fresh Water microbiology MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
Among abundant freshwater Betaproteobacteria, only few groups are considered to be of central ecological importance. One of them is the well-studied genus Limnohabitans and mainly its R-BT subcluster, investigated previously mainly by fluorescence in situ hybridization methods. We designed, based on sequences from a large Limnohabitans culture collection, 18 RLBH (Reverse Line Blot Hybridization) probes specific for different groups within the genus Limnohabitans by targeting diagnostic sequences on their 16 S-23 S rRNA ITS regions. The developed probes covered in sum 92% of the available isolates. This set of probes was applied to environmental DNA originating from 161 different European standing freshwater habitats to reveal the microdiversity (intra-genus) patterns of the Limnohabitans genus along a pH gradient. Investigated habitats differed in various physicochemical parameters, and represented a very broad range of standing freshwater habitats. The Limnohabitans microdiversity, assessed as number of RLBH-defined groups detected, increased significantly along the gradient of rising pH of habitats. 14 out of 18 probes returned detection signals that allowed predictions on the distribution of distinct Limnohabitans groups. Most probe-defined Limnohabitans groups showed preferences for alkaline habitats, one for acidic, and some seemed to lack preferences. Complete niche-separation was indicated for some of the probe-targeted groups. Moreover, bimodal distributions observed for some groups of Limnohabitans, suggested further niche separation between genotypes within the same probe-defined group. Statistical analyses suggested that different environmental parameters such as pH, conductivity, oxygen and altitude influenced the distribution of distinct groups. The results of our study do not support the hypothesis that the wide ecological distribution of Limnohabitans bacteria in standing freshwater habitats results from generalist adaptations of these bacteria. Instead, our observations suggest that the genus Limnohabitans, as well as its R-BT subgroup, represent ecologically heterogeneous taxa, which underwent pronounced ecological diversification.
- MeSH
- RNA, Bacterial genetics MeSH
- Biodiversity * MeSH
- Comamonadaceae physiology MeSH
- Adaptation, Physiological physiology MeSH
- In Situ Hybridization, Fluorescence MeSH
- Hydrogen-Ion Concentration MeSH
- Water Microbiology * MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Fresh Water microbiology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
