Isolation, identification, and molecular characterization of strains of Photorhabdus luminescens from infected humans in Australia.
We describe the isolation of Photorhabdus (Xenorhabdus) luminescens from four Australian patients: two with multiple skin lesions, one with bacteremia only, and one with disseminated infection. One of the patients had multiple skin lesions following the bite of a spider, while the lesions in the other patient were possibly associated with a spider bite. The source of infection for the remaining two patients is unknown. As a member of the family Enterobacteriaceae, P. luminescens is unusual in that it fails to reduce nitrate and ferments only glucose and mannose. It gives negative reactions for lysine decarboxylase, arginine dihydrolase, and ornithine decarboxylase (Moeller). The species is motile, utilizes citrate, hydrolyzes urea, and usually produces a unique type of annular hemolysis on sheep blood agar plates incubated at 25 degrees C. A weak bioluminescence is the defining characteristic. P. luminescens is an insect pathogen and is symbiotically associated with entomopathogenic nematodes. Its isolation from human clinical specimens has been reported previously from the United States. Restriction fragment length polymorphism-PCR analysis of the 16S rRNA gene demonstrated a high level of similarity among the Australian clinical strains and significant differences between the Australian clinical strains and the U.S. clinical strains. However, numerical analyses of the data suggest that the two groups of clinical strains are more similar to each other than they are to the symbiotic strains found in nematodes. This is the first report of the isolation of P. luminescens from infected humans in Australia and the second report of the isolation of this species from infected humans worldwide. (+info)
Polyphasic classification of the genus Photorhabdus and proposal of new taxa: P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P. luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov. and P. asymbiotica sp. nov.
The taxonomic position of Photorhabdus strains was examined through the results of DNA relatedness (S1 nuclease method) studies associated with the determination of delta Tm, 16S rRNA phylogenetic inferences and phenotypic characterization, including morphological, auxanographic, biochemical and physiological properties. Three genomic species were delineated on a consensus assessment. One of these species corresponded to Photorhabdus luminescens, since strains were at least 50% related to the type strain of this species with delta Tm less than 7 degrees C. The two other species were novel genomic species II and III, which were less than 40% related to each other with delta Tm higher than 9 degrees C. A comparison of the complete 16S rDNA sequences of several representatives of genomic species II and genomic species III revealed that each of them formed a stable lineage independent of the cluster generated by P. luminescens strains. The genomic species differed in their maximum temperatures for growth. A correlation with the ecological origin of the bacterial samples was noticed. The heat-tolerant group I (maximum growth temperature 35-39 degrees C) corresponded to the symbionts of Heterorhabditis bacteriophora groups Brecon and HP88 and Heterorhabditis indica, nematodes living in warm and tropical countries, respectively. Group II (maximum growth temperature 33-35 degrees C) encompassed symbionts from Heterorhabditis megidis, Heterorhabditis zealandica and group NC1 of H. bacteriophora, nematodes isolated in temperate climates. Group III were bacteria isolated from human specimens. Two new species, Photorhabdus temperata sp. nov. (type strain CIP 105563T) and Photorhabdus asymbiotica sp. nov. (type strain ATCC 43950T), are proposed for genomic species II and III, respectively. Species I and II can be separated into sub-groups on the basis of high DNA-DNA relatedness (more than 80% DNA binding with delta Tm < 1.5 degrees C), 16S rDNA branching and phenotypic characters. Therefore, we propose that the two species P. luminescens and P. temperata should be subdivided into subspecies as follows: P. luminescens subsp. luminescens subsp. nov. (type strain ATCC 29999T), P. luminescens subsp. akhurstii subsp. nov. (type strain CIP 105564T), P. luminescens subsp. laumondii subsp. nov. (type strain CIP 105565T) and P. temperata subsp. temperata subsp. nov. (+info)
Occurrence of natural dixenic associations between the symbiont Photorhabdus luminescens and bacteria related to Ochrobactrum spp. in tropical entomopathogenic Heterorhabditis spp. (Nematoda, Rhabditida).
Bacteria naturally associated with the symbiont Photorhabdus luminescens subsp. akhurstii were isolated from the entomopathogenic nematode Heterorhabditis indica. Bacterial isolates distinct from P. luminescens subsp. akhurstii were obtained from 33% of the samples. Fourteen bacterial isolates, from nematodes collected from three different Caribbean islands, were characterized by conventional phenotypic tests, restriction fragment length polymorphism and sequence analyses of PCR-amplified 16S rRNA genes (16S rDNAs). Isolates were grouped into three genotypes, each one being associated with one Caribbean island. Phenotypic characteristics and 16S rDNA analysis showed that the Photorhabdus-associated bacteria were closely related to Ochrobactrum anthropi for the group from Guadeloupe, and to Ochrobactrum intermedium for the two groups from the Dominican Republic and Puerto Rico. No pathogenicity of the Ochrobactrum spp. to the insects Galleria mellonella and Spodoptera littoralis (Lepidoptera) was detected. Since Ochrobactrum spp. are considered as human opportunist pathogens, the mass production of entomopathogenic nematodes for biological control requires strict vigilance. (+info)
Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct.
Strains of Staphylococcus aureus were transformed with plasmid DNA containing a Photorhabdus luminescens lux operon (luxABCDE) that was genetically modified to be functional in both gram-positive and gram-negative bacteria. S. aureus cells containing this novel lux construct, downstream of an appropriate promoter sequence, are highly bioluminescent, allowing the detection of fewer than 100 CFU in vitro (direct detection of exponentially dividing cells in liquid culture). Furthermore, these bacteria produce light stably at 37 degrees C and do not require exogenous aldehyde substrate, thus allowing S. aureus infections in living animals to be monitored by bioluminescence. Two strains of S. aureus 8325-4 that produce high levels of constitutive bioluminescence were injected into the thigh muscles of mice, and the animals were then either treated with the antibiotic amoxicillin or left untreated. Bioluminescence from bacteria present in the thighs of the mice was monitored in vivo over a period of 24 h. The effectiveness of the antibiotic in the treated animals could be measured by a decrease in the light signal. At 8 h, the infection in both groups of treated animals had begun to clear, as judged by a decrease in bioluminescence, and by 24 h no light signal could be detected. In contrast, both groups of untreated mice had strong bioluminescent signals at 24 h. Quantification of CFU from bacteria extracted from the thigh muscles of the mice correlated well with the bioluminescence data. This paper shows for the first time that bioluminescence offers a method for monitoring S. aureus infections in vivo that is sensitive and noninvasive and requires fewer animals than conventional methodologies. (+info)
A genomic sample sequence of the entomopathogenic bacterium Photorhabdus luminescens W14: potential implications for virulence.
Photorhabdus luminescens is a pathogenic bacterium that lives in the guts of insect-pathogenic nematodes. After invasion of an insect host by a nematode, bacteria are released from the nematode gut and help kill the insect, in which both the bacteria and the nematodes subsequently replicate. However, the bacterial virulence factors associated with this "symbiosis of pathogens" remain largely obscure. In order to identify genes encoding potential virulence factors, we performed approximately 2,000 random sequencing reads from a P. luminescens W14 genomic library. We then compared the sequences obtained to sequences in existing gene databases and to the Escherichia coli K-12 genome sequence. Here we describe the different classes of potential virulence factors found. These factors include genes that putatively encode Tc insecticidal toxin complexes, Rtx-like toxins, proteases and lipases, colicin and pyocins, and various antibiotics. They also include a diverse array of secretion (e.g., type III), iron uptake, and lipopolysaccharide production systems. We speculate on the potential functions of each of these gene classes in insect infection and also examine the extent to which the invertebrate pathogen P. luminescens shares potential antivertebrate virulence factors. The implications for understanding both the biology of this insect pathogen and links between the evolution of vertebrate virulence factors and the evolution of invertebrate virulence factors are discussed. (+info)
Plasmid-located pathogenicity determinants of Serratia entomophila, the causal agent of amber disease of grass grub, show similarity to the insecticidal toxins of Photorhabdus luminescens.
Serratia entomophila and Serratia proteamaculans cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 115-kb plasmid, pADAP, identified in S. entomophila is required for disease causation and, when introduced into Escherichia coli, enables that organism to cause amber disease. A 23-kb fragment of pADAP that conferred disease-causing ability on E. coli and a pADAP-cured strain of S. entomophila was isolated. Using insertion mutagenesis, the pathogenicity determinants were mapped to a 17-kb region of the clone. Sequence analysis of the 17-kb region showed that the predicted products of three of the open reading frames (sepA, sepB, and sepC) showed significant sequence similarity to components of the insecticidal toxin produced by the bacterium Photorhabdus luminescens. Transposon insertions in sepA, sepB, or sepC completely abolished both gut clearance and cessation of feeding on the 23-kb clone; when recombined back into pADAP, they abolished gut clearance but not cessation of feeding. These results suggest that SepA, SepB, and SepC together are sufficient for amber disease causation by S. entomophila and that another locus also able to exert a cessation-of-feeding effect is encoded elsewhere on pADAP. (+info)
A new broad-spectrum protease inhibitor from the entomopathogenic bacterium Photorhabdus luminescens.
A new protease inhibitor was purified to apparent homogeneity from a culture medium of Photorhabdus luminescens by ammonium sulfate precipitation and preparative isoelectric focusing followed by affinity chromatography. Ph. luminescens, a bacterium symbiotically associated with the insect-parasitic nematode Heterorhabditis bacteriophora, exists in two morphologically distinguishable phases (primary and secondary). It appears that only the secondary-phase bacterium produces this protease inhibitor. The protease inhibitor has an M:(r) of approximately 12000 as determined by SDS-PAGE. Its activity is stable over a pH range of 3.5-11 and at temperatures below 50 degrees C. The N-terminal 16 amino acids of the protease inhibitor were determined as STGIVTFKND(X)GEDIV and have a very high sequence homology with the N-terminal region of an endogenous inhibitor (IA-1) from the fruiting bodies of an edible mushroom, Pleurotus ostreatus. The purified protease inhibitor inactivated the homologous protease with an almost 1:1 stoichiometry. It also inhibited proteases from a related insect-nematode-symbiotic bacterium, Xenorhabdus nematophila. Interestingly, when present at a molar ratio of 5 to 1, this new protease inhibitor completely inactivated the activity of both trypsin and elastase. The activity of proteinase A and cathepsin G was partially inhibited by this bacterial protease inhibitor, but it had no effect on chymotrypsin, subtilisin, thermolysin and cathepsins B and D. The newly isolated protease inhibitor from the secondary-phase bacteria and its specific inhibition of its own protease provides an explanation as to why previous investigators failed to detect the presence of protease activity in the secondary-phase bacteria. The functional implications of the protease inhibitor are also discussed in relation to the physiology of nematode-symbiotic bacteria. (+info)
A genomic approach to gene fusion technology.
Gene expression profiling provides powerful analyses of transcriptional responses to cellular perturbation. In contrast to DNA array-based methods, reporter gene technology has been underused for this application. Here we describe a genomewide, genome-registered collection of Escherichia coli bioluminescent reporter gene fusions. DNA sequences from plasmid-borne, random fusions of E. coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed precise mapping of each fusion. The utility of this collection covering about 30% of the transcriptional units was tested by analyzing individual fusions representative of heat shock, SOS, OxyR, SoxRS, and cya/crp stress-responsive regulons. Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit. Thus, the collection mirrors E. coli's transcriptional wiring diagram. This genomewide collection of gene fusions provides an independent test of results from other gene expression analyses. Accordingly, a DNA microarray-based analysis of mitomycin C-treated E. coli indicated elevated expression of expected and unanticipated genes. Selected luxCDABE fusions corresponding to these up-regulated genes were used to confirm or contradict the DNA microarray results. The power of partnering gene fusion and DNA microarray technology to discover promoters and define operons was demonstrated when data from both suggested that a cluster of 20 genes encoding production of type I extracellular polysaccharide in E. coli form a single operon. (+info)