The sequences of the 16s rRNA gene of 40 strains of bacterial symbionts isolated from the nematodes Heterorhabditis spp. and seven bacterial symbionts of the nematodes Steinernema spp. which were isolated from different geographical areas, as well as the type strain ofXenorhabdus japonicus, were determined and compared to each other and to the sequences of several reference strains of members of the Enterobacteriaceae. Entomopathogenic bacteria that are symbiotic with nematodes in the genera Heterorhabditis and Steinemema have been known for many years (1,3,15,26), and originally these insectpathogenic bacteria were placed in the genus Xenorhabdus, with the luminous forms affiliated with the species Xenorhabdus luminescens and the nonluminous forms affiliated with several different species (Xenorhabdus nematophilus, Xenorhabdus beddingii, Xenorhabdus bovienii, Xenorhabdus japonicus, and Xenorhabdus poinarii). It was later proposed on the basis of physiological and genomic (DNA-DNA hybridization) analysis data that X. luminescens should be given genus status, and this taxon was designated Photorhabdus luminescens (6). All symbiotic P. luminescens strains to date have been isolated from nematodes belonging to the heterorhabditis group, while the Xenorhabdus species have been obtained from nematodes belonging to the genus Steinemema. Recently, several nonsymbiotic isolates of X. luminescens were described (13). These strains were obtained from human wounds, and while they share many properties with other X. luminescens strains, they are apparently not associated with nematodes. Although the genus Photorhabdus has been proposed (6), many of the details concerning the phylogeny and taxonomy of the genera Xenorhabdus and Photorhabdus have not yet been settled (23). The issues of phylogeny, taxonomy, and identification of strains take on importance for the following reasons: (i) these bacteria are economically important because of their ability to kill a variety of insect prey (9-11); (ii) these bacteria are often (but not always) symbiotically associated with nematode hosts, and the nature of the host-bacterium symbiosis has not yet been

Antibiotic poly-resistance (multidrug-, extreme-, and pan-drug resistance) is controlled by adaptive evolution. Darwinian and Lamarckian interpretations of resistance evolution are discussed. Arguments for, and against, pessimistic forecasts on a fatal “post-antibiotic era” are evaluated. In commensal niches, the appearance of a new antibiotic resistance often reduces fitness, but compensatory mutations may counteract this tendency. The appearance of new antibiotic resistance is frequently accompanied by a collateral sensitivity to other resistances. Organisms with an expanding open pan-genome, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae, can withstand an increased number of resistances by exploiting their evolutionary plasticity and disseminating clonally or poly-clonally. Multidrug-resistant pathogen clones can become predominant under antibiotic stress conditions but, under the influence of negative frequency-dependent selection, are prevented from rising to dominance in a population in a commensal niche. Antimicrobial peptides have a great potential to combat multidrug resistance, since antibiotic-resistant bacteria have shown a high frequency of collateral sensitivity to antimicrobial peptides. In addition, the mobility patterns of antibiotic resistance, and antimicrobial peptide resistance, genes are completely different. The integron trade in commensal niches is fortunately limited by the species-specificity of resistance genes. Hence, we theorize that the suggested post-antibiotic era has not yet come, and indeed might never come.

Xenorhabdus nematophilus is a symbiotic bacterium that inhabits the intestine of entomopathogenic nematodes. The bacterium-nematode symbiotic pair is pathogenic for larval-stage insects. The phase I cell type is the form of the bacterium normally associated with the nematode. A variant cell type, referred to as phase II, can form spontaneously under stationary-phase conditions. Phase II cells do not elaborate products normally associated with the phase I cell type. To better define phase variation in X. nematophilus, several strains (19061, AN6, F1, N2-4) of this bacterium were analyzed for new phenotypic traits. An analysis of pathogenicity in Manduca sexta larvae revealed that the phase II form of AN6 (AN6/II) was significantly less virulent than the phase I form (AN6/I). The variant form of N2-4 was also avirulent. On the other hand, F1/II and 19061/II were as virulent as the respective phase I cells. Strain 19061/II was found to be motile, and AN6/II regained motility when the bacteria were grown in low-osmolarity medium. In contrast, F1/II remained nonmotile. The phase II cells did not produce the outer membrane protein, OpnB, that is normally induced during the stationary phase. Both phase I and phase II cells were able to support nematode growth and development. These findings indicate that while certain phenotypic traits are common to all phase II cells, other characteristics, such as virulence and motility, are variable and can be influenced by environmental conditions.

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A relatively rapid and economic way of identifying and assigning nematodes to taxons, which had already been determined either by comparative sequence analysis of nuclear rDNA internal transcribed spacer (ITS) region or by other methods of molecular or conventional taxonomy, is provided. Molecular identification of entomopathogenic nematodes (EPN) can be upgraded by basing it on PhastSystem polyacrylamide gel electrophoresis (PAGE) analysis of restriction fragment length polymorphism (RFLP) patterns of polymerase chain reaction (PCR)‐amplified DNA derived from single nematodes of Steinernema or Heterorhabditis spp. Although analysis from single worms has previously been made on agarose gel, the resolution on PhastSystem PAGE gel is much higher. The DNA sequences selected for analysis were those constituting the internal transcribed spacer region between the 18S and 26S rDNA genes within the rRNA operon. RFLP analysis was carried out by gel electrophoresis on the PhastSystem (Pharmacia) as detailed elsewhere (Triga et al., Electrophoresis 1999, 20, 1272—1277). The downscaling from conventional agarose to PhastSystem gels resulted in pattern of DNA fragments differing from those obtained with agarose gel electrophoresis under conventional conditions by increasing the number of detected fragments. The approach supported previous species identifications and was able to identify several unclassified isolates, such as those from Hungary and Ireland, and provides a method for identification of previously unclassified strains. We confirmed that Heterorhabditis “Irish Type”, represented by two strains of different geographical origin, comprise a species different from H. megidis. We also confirmed that strain IS5 belongs to the species H. indicus rather than to H. bacteriophora, as had been suggested previously.

The DNA sequences constituting the internal transcribed spacer region, located between 18S and 26S rDNA genes within the rRNA operon, derived from single nematodes of two genera (Steinernema and Heterorhabditis) were amplified by polymerase chain reaction (PCR) and subjected to digestion by four restriction enzymes. The digests were analyzed by restriction fragment length polymorphism (RFLP) gel electrophoresis on the PhastSystem, using 7.5%T, 5%C(Bis) polyacrylamide. The downscaling from conventional agarose to PhastSystem gels permitted the analysis to be done on individual nematodes, rather than on mixed samples with average properties. The analysis time was reduced so as to allow for the electrophoretic separation on 200 samples/workday. The resulting patterns of DNA fragments differed from those obtained by agarose gel electrophoresis under conventional conditions by an increased number of detected fragments. The PhastSystem gel analysis provides the basis for taxonomical revisions.