Insect-pathogenic nematodes harboring Photorhabdus spp are used as biopesticides

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Insect-pathogenic nematodes harboring Photorhabdus spp are used as

biopesticides in a number of countries, including the United States

and Australia. Agricultural scientists are also attempting to develop

insect-resistant transgenic crops by using insecticidal toxin genes

derived from Photorhabdus spp.

 

 

 

 

 

 

Photorhabdus Species: Bioluminescent Bacteria as Emerging Human Pathogens?

 

John G. Gerrard,* Samantha McNevin,† David Alfredson,* Ross

Forgan-Smith,† and Neil Fraser‡

*Gold Coast Hospital, Southport, Queensland, Australia; †Queensland

Medical Laboratory, West End, Queensland, Australia; and ‡Harbour City

Family Practice, Gladstone, Queensland, Australia

 

Suggested citation for this article: Gerrard JG, McNevin S,

Alfredson D, Forgan-Smith R, Fraser N. Photorhabdus species:

bioluminescent bacteria as human pathogens? Emerg Infect Dis [serial

online] 2003 Feb [date cited]. Available from: URL:

http://www.cdc.gov/ncidod/EID/vol9no2/02-0222.htm

 

We report two Australian patients with soft tissue infections due

to Photorhabdus species. Recognized as important insect pathogens,

Photorhabdus spp. are bioluminescent gram-negative bacilli. Bacteria

belonging to the genus are emerging as a cause of both localized soft

tissue and disseminated infections in humans in the United States and

Australia. The source of infection in humans remains unknown.

 

Bioluminescence is the production of visible light by a chemical

reaction in a living organism. The property is rarely reported in the

clinical bacteriology laboratory because bacterial bioluminescence is

seen primarily in marine species. Photorhabdus spp (family:

Enterobacteriaceae) are the only terrestrial bacteria known to exhibit

this property (1). The classification within the genus is complex with

three currently recognized species: P. luminescens, P. temperata, and

P. asymbiotica (2). Several subspecies are recognized.

 

Photorhabdus spp. have been the subject of intensive study by

agricultural scientists because of the role these bacteria play in

controlling insects. Insects, like humans, are subject to infestation

by nematodes (3). Photorhabdus spp. inhabit the gut of some

insect-pathogenic nematodes (Heterorhabditis spp.), where they form a

symbiotic relationship. Nematode species of this type are able to

invade the larvae of susceptible insects and release Photorhabdus spp.

The bacteria proliferate and promote nematode reproduction by killing

the insect larvae.

 

Insect-pathogenic nematodes harboring Photorhabdus spp are used as

biopesticides in a number of countries, including the United States

and Australia. Agricultural scientists are also attempting to develop

insect-resistant transgenic crops by using insecticidal toxin genes

derived from Photorhabdus spp. (4).

 

Genes encoding homologues of insecticidal toxins from Photorhabdus spp

occur naturally within the genome of Yersinia pestis, the cause of

plague. Lateral transfer of genetic material between Photorhabdus and

Yersinia species is thought to have resulted from their common

association with insects as bacterial pathogens (5).

 

Human infection with Photorhabdus spp. has been described in two

previous publications—six cases from the United States (6) and four

cases from South Eastern Australia (Victoria and New South Wales) (1).

We report two additional recent human cases of Photorhabdus infection

from the Australian state of Queensland.

The Study

Patient 1

 

A 39-year-old male pest controller from Gladstone on a routine visit

to his general practitioner in April 2001 inquired about the recent

appearance of a red macule, 8 mm in diameter, on the medial aspect of

his right ankle. No specific treatment was given. When he was seen

again 18 days later, a painful, necrotic ulcer, about 12 mm in

diameter, had developed at the original site of the red spot. A

gram-negative organism later identified as Photorhabdus sp. was

isolated in pure growth from the exudate. The patient began a 10-day

course of oral cephalexin. When he was observed again 11 days later,

he exhibited a persistent discharge with surrounding cellulitis. He

was therefore prescribed a 10-day course of oral

amoxycillin-clavulanate. Three weeks later, the ulcer appeared to be

healing; after another 6 weeks, signs of infection had again

developed. A gram-negative organism was isolated from the exudate but

was not formally identified.

 

The patient was prescribed an additional 7-day course of oral

cephalexin. When he was observed 3 months later, the infection had

resolved. In his recent work as a pest controller, he had been

spraying chemical insecticides under houses and in foreign cargo

ships. He had never used insect pathogenic nematodes as a biopesticide.

Patient 2

 

A 78-year-old man from the Queensland Gold Coast sought treatment in

January 1999 with a 3-day history of a painful, swollen right foot.

The patient had a history of polymyalgia rheumatica for which he was

taking prednisone, 8 mg daily. In January 1999, after working barefoot

in the garden, the man noted intense pain in his right forefoot and a

very small amount of bloody discharge from the web space between his

fourth and fifth toes.

 

The next day he was seen by his general practitioner who treated him

with oral dicloxacillin. Two days later he was admitted to the

hospital with increasingly severe pain with extensive redness and

swelling extending to his right knee. He was noted to be afebrile with

a mild neutrophil leukocytosis. He was started on a regimen of

intravenous dicloxacillin and gentamicin.

 

Surgical debridement of the right foot was required on three occasions

during the first 8 days of his admission. Pus was collected for

culture on two of these occasions, and tissue was obtained during the

third. An organism identified as Photorhabdus sp. was isolated in pure

culture from each of these operative specimens. The same organism was

also isolated, together with Staphylococcus aureus, from a superficial

swab collected in the emergency department on presentation. No

bacterial growth was obtained from blood cultures collected on admission.

 

The patient was treated with intravenous gentamicin for 2 weeks and

ceftazidime for 1 week. He was discharged on a 6-week course of oral

ciprofloxacin. The foot remained healed on follow-up 3 months later.

 

Photorhabdus spp. can be isolated and identified to genus level by

using techniques available in most clinical bacteriology laboratories.

A total of five isolates from the two patients described in the

current report were examined in our laboratories with standard

techniques (one from patient 1 and four from patient 2). The

phenotypic characteristics that the isolates displayed were typical of

the genus.

Figure 1

Figure

 

 

Click to view enlarged image

 

Figure 1. Photorhabdus isolate from patient 2, growing on tryptic soy

agar containing 5% sheep blood, after 48 hours' incubation at 35°C...

 

 

 

 

Figure 2

 

Figure

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Figure 2. Photorhabdus isolate from patient 2 after 5 days' growth at

room temperature on sheep blood agar...

 

 

 

Figure 3

 

Figure

Click to view enlarged image

 

Figure 3. Australian and American clinical isolates of Photorhabdus.

 

Colonies were formed after 24–48 hours on tryptic soy agar containing

either 5% sheep or horse blood (bioMérieux, Baulkham Hills, Australia)

at both 35°C and at room temperature, with a tendency to " swarm "

(Figure 1). The isolates also grew on MacConkey agar. On sheep and

horse blood agar, a thin line of annular hemolysis was observed 4–12

mm from the colony edge. The hemolysis was more apparent when the

isolates were incubated at room temperature (Figure 2). The organisms

were motile, gram-negative, rod-shaped bacteria. They were

facultatively anaerobic, oxidase negative, and strongly catalase

positive. Other biochemical reactions were as described previously (1).

 

The defining characteristic was the presence of faint luminescence,

which could be clearly seen with the naked eye when the colonies were

examined under conditions of total darkness. It was critical to this

examination that the observer's eyes be allowed to adjust to the

darkness for 10 minutes.

 

Two commercially available automated bacterial identification systems

were used in our laboratories: MicroScan Walkaway (Dade Behring Inc.,

MicroScan Division, West Sacramento, CA) and bioMerieux Vitek

(bioMérieux; Hazelwood, MO). Photorhabdus spp. do not currently appear

on the databases of either of these systems, which leads to

misidentification (Table 1).

 

Photorhabdus spp. have been shown to form a heterogeneous group based

on DNA-DNA hybridization studies, 16S rDNA sequencing and polymerase

chain reaction ribotyping (2). A polyphasic approach is now applied to

classifying isolates within the genus, dividing it into three species

and several subspecies. The American clinical isolates described by

Farmer et al. (6) belong to a new species, Photorhabdus asymbiotica

(2). A specific epithet has not yet been assigned to the Australian

clinical isolates but they also may form a new species within the

genus (7).

 

Antimicrobial sensitivity was assessed by using broth microdilution.

The isolates were sensitive to a broad range of antimicrobial agents

with activity against gram-negative bacteria including ciprofloxacin,

gentamicin, tetracycline, ceftriaxone, and amoxycillin-clavulanate.

Isolates from both patients were resistant to cephalothin and ampicillin.

Conclusions

 

Publication of information about these two cases brings to a total of

12 the number of human infections with Photorhabdus spp. documented in

the medical literature (Table 2 and Figure 3). The clinical picture

described in the 12 cases has generally been one of localized or more

commonly multifocal skin/soft tissue infection. Such infection has had

a tendency to relapse. The disseminated distribution of skin/soft

tissue infection in several cases suggests hematogenous spread.

Bacteremia was documented in 4/12 case-patients. Cough was documented

in two of the bacteremic case-patients. In one of these, isolates of a

Photorhabdus sp. were obtained from sputum as well as from blood and

skin/soft tissue.

 

Given the very limited clinical experience, making definitive

recommendations about treatment is not possible. Antimicrobial therapy

should be guided by in vitro sensitivities. The tendency for

Photorhabdus infection to relapse suggests that prolonged therapy for

a period of weeks would be prudent, perhaps with an oral fluoroquinolone.

 

Photorhabdus spp. are not human commensals. The patients apparently

acquired the pathogen from an unidentified source in the terrestrial

environment. This hypothesis is supported by the observations that at

least 4/6 of the Australian patients were engaged in outdoor

activities around the time of acquisition and that the initial site of

infection was on the lower limbs in more than half of Australian and

American case-patients.

 

Photorhabdus spp. have never been shown to live freely in soil,

although they will survive in soil under laboratory conditions (8).

Photorhabdus spp. have only been isolated naturally from two

nonclinical sources: insect-pathogenic nematodes (Heterorhabditis spp)

and the insects they parasitize (beetles, moths, and the like). It

seems likely therefore that Photorhabdus spp are transmitted to humans

by a terrestrial invertebrate (nematode or arthropod), but that vector

has not yet been identified.

 

Dr. Gerrard is Director of Infectious Diseases at the Gold Coast

Hospital and a clinical senior lecturer at the University of

Queensland, Australia. His research interests include clinical and

laboratory aspects of emerging bacterial pathogens.

 

References

 

1. Peel MM, Alfredson DA, Gerrard JG, Davis JM, Robson JM,

McDougall RJ, et al. Isolation, identification, and molecular

characterization of strains of Photorhabdus luminescens from infected

humans in Australia. J Clin Microbiol 1999;37:3647–53.

2. Fischer-Le Saux M, Viallard V, Brunel B, Normand P, Boemare NE.

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. Int J Syst Bacteriol

1999;49:1645–56.

3. Boemare N, Givaudan A, Brehelin M, Laumond C. Symbiosis and

pathogenicity of nematode-bacterium complexes. Symbiosis 1997;22:21–45.

4. ffrench-Constant RH, Bowen DJ. Novel insecticidal toxins from

nematode-symbiotic bacteria. Cell Mol Life Sci 2000; 57:828–33.

5. Parkhill J, Wren BW, Thomson NR, Titball RW, Holden MT, Prentice

MB, et al. Genome sequence of Yersinia pestis, the causative agent of

plague. Nature 2001;413:523–7.

6. Farmer JJ, Jorgensen JH, Grimont PAD, Ackhurst RJ, Poinar GO,

Ageron E, et al. Xenorhabdus luminescens (DNA Hybridization Group 5)

from human clinical specimens. J Clin Microbiol 1989;27:1594–1600.

7. Akhurst R, Smith K. Regulation and safety. In: Gaugler R,

editor. Entomopathogenic nematology. New York: CABI Publishing; 2002.

p. 311–32.

8. Bleakley BH, Chen X. Survival of insect pathogenic and human

clinical isolates of Photorhabdus luminescens in previously sterile

soil. Can J Microbiol 1999;45: 273–8.

 

 

Table 1. Misidentification of Photorhabdus isolates from patients 1

and 2 by commercially available bacterial identification systems