Providencia vermicola sp. nov., isolated from infective juveniles of the entomopathogenic nematode Steinernema thermophilum

The enterobacterial genus Providencia comprises five species that have been isolated from the colon and faeces of humans (Providencia alcalifaciens, Providencia rustigianii), wounds, urinary tract and respiratory tract of humans (Providencia stuartii), urinary tract of humans, poultry, faeces from reptiles and other environments (Providencia rettgeri) and from faeces of penguins (Providencia heimbachae) [summarized by Penner (1991)]. P. alcalifaciens and P. stuartii are known for their pathogenic potential, causing diarrhoea and bacteraemia, respectively. The description of several of these species was initially based upon DNA–DNA reassociation experiments, which indicated their previous misclassification in other genera or affiliation to other species (Brenner et al., 1978; Hickman-Brenner et al., 1983; Müller et al., 1986). The five species can be separated based on metabolic characteristics, such as acid production from some carbohydrates and several other standard tests (Müller et al., 1986).

The type strains of Providencia species (Table 1⇓) were obtained from the DSMZ. Isolation was performed as described by Akhurst (1980): a mixture of infective juveniles (IJs) of nematodes of Steinernema thermophilum belonging to different age groups, isolated from different larvae of the greater wax moth (Galleria mellonella), were surface sterilized in 0·1 % Hyamine 10×(methylbenzethonium chloride; Spectrum Chemicals & Laboratory Products) for 15–30 min. Nematodes were then washed three times in sterilized water in order to remove traces of Hyamine. The surface-sterilized IJs were suspended in Luria–Bertani (LB) broth or nutrient broth in a 1·5 ml microcentrifuge tube. Subsequently, the IJs were crushed manually by using a sterile tissue grinder. One loopful of the crushed suspension was then used to streak the indicator NBTA (5·0 g peptone, 3·0 g beef extract, 15 g agar, 0·025 g bromothymol blue, 0·04 g 2,3,5-triphenyl tetrazolium chloride; per litre of water) media plates. The plates were incubated for 24 h at 28 °C; individual colonies were purified by restreaking them onto fresh solid media. Isolates OP1^T, OP29 and VS3 were isolated independently from different nematode IJs at intervals of 2 weeks. These isolates were observed among other colony types, the strains of which were also isolated and await characterization (V. S. Somvanshi, E. Lang, S. Ganguly, J. Swiderski, A. K. Saxena and E. Stackebrandt, unpublished results).

Genomic DNA was extracted using the DNeasy tissue kit (Qiagen) following the manufacturer's instructions. The 16S rRNA gene was amplified as described by Rainey et al. (1996). PCR products were purified with the QIAquick PCR purification kit (Qiagen) and directly sequenced by using the CEQ Dye Terminator Cycle Sequencing kit. Products were separated on a CEQ 8000 Genetic Analysis System. The 16S rRNA gene sequences were aligned with corresponding sequences from the DSMZ database by using the ae2 editor (Maidak et al., 1997). Evolutionary distances were calculated by the method of Jukes & Cantor (1969). A distance analysis dendrogram was reconstructed by using the neighbour-joining algorithm and bootstrap values were determined based on 500 resamplings (Felsenstein, 1993). DNA was isolated using a French pressure cell (Thermo Spectronic) and purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA–DNA hybridization was carried out as described by De Ley et al. (1970), incorporating the modifications described by Huß et al. (1983), using a Cary 100 Bio UV/VIS-spectrophotometer (Varian) equipped with a Peltier-thermostatted 6×6 multicell changer and a temperature controller with in-situ temperature probe (Varian).

Automated ribotyping was carried out with the RiboPrinter microbial characterization system (Qualicon; DuPont). Riboprint analysis, using EcoRI, followed the methods described by Bruce (1996).

Physiological and biochemical tests on the three isolates and the type strains of Providencia species were performed at 37 °C using API 20E, API 50CH strips (bioMérieux) and Biolog GN microplates. API 20E and API 50CH strips were used according to the manufacturer's instructions. Acid production from carbohydrates was tested on API 50CH strips with bioMérieux medium E. Biolog GN plates (Oxoid) were incubated for 24 h before readings were taken. Wells showing a photometric value above 25 or above 30 % of the highest value of an individual strain were scored as weak or positive, respectively. Conventional biochemical tests were performed according to standard methods (Smibert & Krieg, 1994). Cultural characteristics such as colony size, shape and colour were determined after 24 h incubation at 37 °C on trypticase soy agar (Merck) plates.

The almost complete 16S rRNA gene sequence of isolate OP1^T (1517 nt) was identical to that of strains OP29 and VS3. Highest sequence similarity values were found with members of the family Enterobacteriaceae, specifically with members of the genus Providencia (>98·1 %). The phylogenetically closest type strain was P. rettgeri DSM 4542^T (99·5 % 16S rRNA gene sequence similarity). Except for the branching of strain OP1^T and P. rettgeri DSM 4542^T (99 %), bootstrap values for the other branching points were low, indicating the tentative character of the topology of the tree (Fig. 1⇓).

Heterogeneity of the rrn operon, determined by the riboprinting method with the restriction enzyme EcoRI (Fig. 2⇓), indicated an identical riboprint pattern for the three isolates, thus confirming their high genomic similarity. This pattern was unique among those obtained for the type strains of Providencia species, which show <64 % 16S rRNA gene pattern similarity among themselves. Pattern similarity values for strains OP1^T, OP29 and VS3 and the type strains of recognized Providencia species are lower than 27 %. Riboprint similarities above 92 % are interpreted as indicating membership to the same ribogroup, i.e. to a taxon at the subspecific level (Anonymous, 1998). Similarity for the three new isolates was as high as 98 %. The presence of identical ribopatterns, together with identical 16S rRNA gene sequences and patterns of phenotypic characterization, for the three isolates indicates that they are descendants from the same clone recovered at different times from a mixture of IJs. These isolates are therefore considered to be members of the same species.

In order to investigate the physiological properties, Biolog GN and API tests were performed on strain OP1^T and the type strains of recognized Providencia species (Tables 1, 2 and 3⇑⇓⇓). All physiological characteristics of the new isolates were identical except for the utilization of four substrates for which a variable reaction was recorded (Table 3⇓).

Strain OP1^T is a Gram-negative, straight rod-shaped enterobacterium. It was positive for catalase and negative for oxidase, was able to ferment d-glucose and reduced nitrate. It produced acid from l-arabinose and 2-ketogluconate and utilized l-erythritol, d-glucosaminic acid and d-glucuronic acid, all of which were negative in the other Providencia species. The results for the physiochemical reactions for Providencia species that we obtained using the API and Biolog systems were in agreement with those obtained by traditional methods (Müller et al., 1986). Consistent with the description of other Providencia strains (Polster & Svobodova, 1964), strain OP1^T produced a brownish pigment and developed a characteristic smell on agar containing amino acids.

Previous DNA–DNA reassociation values generated under optimal hybridization conditions for the type strains of Providencia species ranged between 22 and 49 % (Hickman-Brenner et al., 1983; Müller et al., 1986). The corresponding 16S rRNA gene sequence similarity values are above 98 %. The level of DNA–DNA relatedness between strain OP1^T and the type strain of its closest phylogenetic relative, P. rettgeri DSM 4542^T, was 30 % (29 and 31 % similarity in two measurements using 2×SSC buffer at 66 °C).

The combination of phylogenetic, genomic and metabolic properties suggests that isolates OP29, VS3 and OP1^T should be included in the same species and that this represents a novel species within the genus Providencia. Several metabolic properties, determined by two independent API and Biolog test series, were found that are discriminatory and allow identification of the novel species. The name Providencia vermicola sp. nov. is proposed.