Letters to the Editor 181

negative. These were identified as E. rhusiopathiae by the API Coryne strip (Bio M6rieux). An echocardiogram re- vealed no vegetations, though trivial aortic regurgitation was noted on Doppler studies. The patient did not receive any antibiotic therapy and her fever settled within 24 h. A further five pairs of blood cultures were taken over the next two weeks; all were sterile. At outpatient review a month after discharge she was asymptomatic, one year later she remains in good health.

Erysipelothrix rhusiopathiae is a facultatively anaerobic, non spore-bearing, Gram positive bacillus which is widely distributed in the environment. 1 It causes infection in do- mestic animals, especially pigs. Human infection is an oc- cupational disease of butchers, abattoir workers, fish processors and veterinarians. 2 Typically, the fingers are infected after a puncture wound. This results in a localised cellulitis known as erysipeloid. Erysipelothrix rhusiopathiae bacteraemia is uncommon, but a recent review 3 reports 50 cases, of which 90% were in patients with endocarditis. This infection commonly affects the aortic valve and has a higher mortality than endocarditis due to other causes) Bacteraemia without endocarditis does occur. A case re- port and review of the literature from 1985-1991 de- scribes eight cases ofE. rhusiopathiae bacteraemia without endocarditis. 4 Seven of these had an underlying im- munosuppressive disorder (three with chronic liver dis- ease, four receiving corticosteroids). The authors conclude that E. rhusiopathiae bacteraemia without endocarditis is more common than previously thought and occurs mainly in the immunocompromised. Our patient was not im- munocompromised; her alcohol consumption was the only recognised risk factor for E. rhusiopathiae bac- teraemia. 3 Clinically, she had a resolving erysipeloid lesion of her finger and did not have endocarditis. The source of her infection may have been animal manure in her garden soil.

Bacteraemic E. rhusiopathiae infection may occur more commonly than reports suggest. It may be missed because blood cultures are not often taken from a patient like ours with a mild illness self-limiting illness. Furthermore, Gram positive rods cultured from blood are often dismissed as "diphtheroids" and not fully identified. Reports of bac- teraemia with this organism in immunocompromised patients may simply reflect the more thorough iden- tification of blood culture isolates from such patients. This case and those reported by Garcia-Restoy et al. 4 suggest that the association between E. rhusiopathiae bacteraemia and endocarditis is not as strong as previously reported.

Acknowledgement

We are grateful to Dr. A. D. Bryceson for permission to report details of ihis patient.

A. Fakoya, 1 R. P. Bendall, 1'2 D. R. Churchill, 1 J. E Doherty 1 and G. L. Ridgway 2

1 Hospital for Tropical Diseases, 4 St Pancras Way, London, and

2 Department of Clinical Microbiology, UCL Hospitals,

Grafton Way, London W C I E 6AU, U.K.

References

1 McClain JB. Erysipelothrix rhusiopathiae. In: Mandell GL, Douglas RG, Bennett JE Eds. Principles and Practice of Infectious Disease (3rd ed). New York: Churchill Livingstone, 1990; 1599-1600.

2 Reboli AC, Farrar WE. Erysipelothrix rhusiopathiae: an occupational pathogen. Clin Microbiol Rev 1989; 2: 354-359.

3 Gorby GL, Peacock JE Jr. Erysipelothrix rhusiopathiae endocarditis: mi- crobiologic, epidemiologic, and clinical features of an occupational disease. Rev Infect Dis 1.988; lO: 317-325.

4 Garcia-Restoy E, Espejo E, Bella F, Llebot J. Bacteremia due to Er- !tsipelothrix rhusiopathiae in immunocompromised hosts without en- docarditis. Rev Infect Dis 1991; 13:1252-1253.

Simultaneous Infection by a Sensitive and a Multiresistant Strain of Salmonella paratyphiA

Accepted for publication 7 July 1994

Sir,

Simultaneous infection by sensitive and resistant strains of Salmonella species have recently been reported in Journal of InfectionJ '2 I would like to report a case of infection with S. paratgphi A with two colony types showing different antimicrobial susceptibility patterns.

A 25-year-old male presented to the Emergency De- partment with an 8-day history of fever, headaches, night sweats, rigors, right upper quadrant pain and diarrhoea. He had been travelling for 7 months in Kenya, Zaire and Zimbabwe before spending 4 weeks in India, where he started to experience diarrhoea. He had not been treated with antibiotics. Physical examination was un- remarkable, apart from his temperature being 39 °C. He was mildly anaemic with a haemoglobin of 12.1 g/dl (reference range 13.5-18.0) and there was mild de- rangement of hepatocellular enzymes. White cell count was 6.3 x 109/1 (reference range 4.0-11.0 x 10 9) with a left shift. He had taken malaria prophylaxis and thick and thin films were negative for malaria parasites. A set of blood cultures were taken and he was discharged home for follow-up as an outpatient. The next day a blood culture bottle was growing Gram-negative rods and he was admitted for intravenous ceftriaxone, l g daily. Fur- ther blood cultures and faeces specimens were taken.

182 Letters to the Editor

Table 1. Minimum inhibitory concentrations of antimicrobials

MIC (mg/1)

ampicillin tetracycline chloramphenicol ceftriaxone

resistant morphotype >256 >256 >256 0.25 susceptible morphotype 3 1.5 4 0.2 5

Three sets of blood cultures grew Gram-negative rods. All cultures produced large and small colony forms on blood agar. Biochemical (API 20E, Bio-M6rieux) and serological (Wellcome Diagnostics) identification were consistent with Salmonella paratyphi A for both colony types. The API 20E profile number was 0104552, very good identification as S. paratyphi A; biochemical tests performed at the reference enteric laboratory for both colony types were in agreement with this identification:

HaS production citrate utilisation lysine decarboxylase xylose fermentation dulcitol fermentation sorbitol fermentation

negative negative negative negative positive positive

Antimicrobial susceptibility testing using NCCLS agar dilution and modified Kirby-Bauer disc methods showed a markedly increased resistance of the small colony type. It was resistant to ampicillin, tetracycline, chlor- amphenicol, sulphamethoxazole/trimethoprim and strep- tomycin. The large colony type was sensitive to these antimicrobials and both morphotypes were sensitive to ceftriaxone, ciprofloxacin, imipenem and gentamicin. Minimum inhibitory concentrations (MICs) for the fol- lowing antimicrobials were determined for both morpho- types (Table 1).

The first faecal specimen cultured before admission cultured no pathogens but showed numerous Blastocystis hominis in the wet preparation. The second specimen taken one day after antimicrobial treatment grew the more resistant small colony S. paratyphi A only. Three days later a faecal specimen showed no pathogens.

The patient was treated for 4 days with ceftriaxone, ciprofioxacin 500mg twice daily was given for 2 weeks and the Blastocystis was treated with metronidazole 400mg 8 hourly for 7 days. Post-treatment faecal speci- mens have not grown S. paratyphi A and the patient remains well.

Prolonged incubation at 35°C of the large colony susceptible type of S. paratyphi A on Mueller-Hinton Agar with discs of ampicillin, chloramphenicol and sulpha- methoxazole/trimethoprim using a lawn inoculum

showed the emergence of resistant small colonies within the zones of inhibition around the discs.

There have been reports of a similar phenomenon occurring in Salmonella typhi isolated from patients with a history of recent travel in India. 1'2 In these reports the changes in resistance and morphology have been associated with a 120MDa plasmid. Gutmann et al) have reported associated morphology and resistance changes induced in the laboratory in a S. paratyphi A from a case of endocarditis and attributed this to changes in outer membrane components of the resistant mutant.

Using the methods described by Tenover et al. , 4 I detected plasmids of identical length in both of the morphotypes isolated from this case; the resistant morph- otype did not contain any additional plasmids. Phage typing performed by the Microbiological Diagnostic Unit of the University of Melbourne (Australian Salmonella Reference Laboratory) has shown the susceptible large colony type to be S. paratyphi A phage type 1. The resistant small colony type was susceptible to phages but did not conform to a recognised pattern (termed RDNC - reacts but does not conform). Both colony types have been forwarded to the Laboratory of Enteric Pathogens, Central Public Health Laboratory, London. Phage type conversion associated with acquisition of drug resistance plasmids in Salmonella species has been previously de- scribed. 5'6 Threlfall and Chart also described strains of S. enteritidis that spontaneously mutated irreversibly from smooth to rough strains with an associated change of phage type. 6

These findings support the proposal that multiple anti- biotic resistance in S. paratyphi A can be due to outer membrane changes and may not necessarily be plasmid- mediated as has been reported in other Salmonella species. The reports of Yaneza et al. 1 and Simpson et al. 2 suggested simultaneous infections with two strains of S. typhi, the resistant strain being selected out while on antimicrobial therapy. Gutmann et al. described spontaneous mutants of S. paratyphi A that appeared when the isolate was grown on ampicillin-containing media; 3 there was no mention of plasmids being involved with the acquisition of resistance. The strains I have described were isolated from multiple blood cultures before any antibiotic therapy was commenced and the different colony morphotypes

Letters to the Editor 183

were evident on the primary inoculation media, not under antibiotic pressure. This would suggest that the patient's S. paratyphi A strain underwent spontaneous mutation which exhibited increased antimicrobial re- sistance, perhaps by changes in outer membrane proteins, and not necessarily conferred by plasmid acquisition.

Michael Leung Microbiology Department,

Fremantle Hospital, Alma St, Fremantle,

Western Australia 6014, Australia

References 1 Yaneza A, Kumari P. Enteric fever simultaneously caused by a

sensitive and a multiresistant strain of Salmonella tBphi [letter]. ] Infect 1993; 27: 102-103.

2 Simpson J, Threlfall EJ, Derrick G, Mellersh A. An unusual case of multiple enteric infections [letter]. l Infect 1993; 27: 103-105.

3 Gul.mann L, Billot-Klein D, Williamson R et al. Mutation of Salmonella paratyphi A conferring cross-resistance to several groups of antibiotics by :tecreased permeability and loss of invasiveness. Antimicrob Agents Chcmother 1988; 32: 195-201.

4 Ten over FC, Phillips K, Carlson LG. Plasmid fingerprinting of Gram- negative organisms. In: Isenberg ttD (Editor-in-Chief), Clinical Micro- biology Procedures Handbook. Washington D.C. American Society for Microbiology. 1992. 10.4.1-10.4.4:.

5 Frost JA, Ward LR, Rowe B. Acquisition of a drug resistance plasmid converts Salmonella enteritidis phage type 4 to phage type 24. Epi- demiol Infect 1989; 103: 243-248.

6 Threlfall EJ, Chart H. Interrelationships between strains of Salmonella enteritidds. Epidemiol Infect 1993; 111: 1-8.

Diagnostic and Therapeutic Problems due to Cat Scratch Disease

Accepted for publication 22 July 1994 Sir,

The aetiology of cat scratch disease (CSD) is still dispu- ted. 1'2 Afipia felis and Rochalimaea henselae are both sus- pected to be causative agents. I'2 In very few CSD cases, the aetiologic agent has been determined. Recently, we identified by 16S ribosomal RNA sequence analysis R. henselae in a childhood case of CSD.

A 6-year-old boy sustained voluminous inguinal adenopathies and developed fever to 38°C to 39°C, asthenia and headache. His remaining examination did not reveal any other abnormality. He had not been bitten or scratched by a cat in recent history, but he had rose thorn scratches and possessed a cat. Blood examination revealed 9.5 x 109 leukocytes/1 (50% neutrophils, 25% lymphocytes), rising to 11.1 × 109 leukocytes/1 (54% neutrophils, 37% lymphocytes), a

C-reactive protein level of 50mg/l, rising to 57rag/1. An hyperalpha2-globulinemia and hypergamma- globulinemia were also found. Chest X-ray was normal. Blood cultures were negative, acute and convalescent serologic tests were negative for antibodies directed against Cytomegalovirus, HIV, Epstein-Barr virus, My- coplasma sp., Rickettsia rickettsii, Coxiella burnetii, Brucella sp., Chlamydia, Toxoplasma gondii, and Streptococcus sp. The patient received a l O-day course of oral tri- methoprim-sulphamethoxazole (6 and 30mg/kg/day), then a lO-day course of oral oxacillin ( lO0mg/kg/ day), but no clinical improvement was noted. At this time, we saw the patient and performed aspiration of suppurating nodes. The histopathology of these lymph nodes was characterised by granulomas and non- specific inflammatory infiltrates. As CSD was suspected, examination of node fluid was performed after Gram stain and Wharthin-Starry silver impregnation stain. No bacteria were detected. Node fluid was cultured on various culture media: 5% fresh sheep blood agar, trypticase soy agar, chocolate blood agar incubated at 30°C and 35°C in either 5% CO~ environment or air. After 3 weeks, the culture plates were discarded since no colonies were found. Node fluid was also intraperitoneally (2 ml) injected to three athymic (conge- nitically deficient in T-lymphocytes) BALB/c mice. It was hypothesised that CSD aetiologic agents which are also associated with various clinical symptoms in immunodeficient patients, 2 may lead to chronic infection in T-lymphocyte deficient mice. After 2 weeks, mice were sacrificed and spleen and liver suspensions were plated onto appropriated culture media. No colonies were found after a 3-week incubation.

However, the patient reciprocal serum R. henselae antibody detection was positive at a 1/50 dilution by indirect immunofluorescent-antibody test to whole bacteria (cutoff 1/25), but negative to Afipia felis by the same method. No IgM was detected to R. henselae or A. felis after absorption of IgG. DNA was extracted from node fluid using SDS-proteinase K digestion. It was purified with phenolchloroform extraction and ethanol precipitation. The 16S rRNA gene were PCR amplified with primer specific to 16S rRNA gene of alpha subgroup of Proteabacteria. The PCR product was cloned in M13 phage and sequenced with Hot-Tub polymerase (Amersham International, Amersham, Eng- land). The variable parts of 16S rRNA gene sequenced were compared to previously reported sequences avail- able in Genbank, and R. henselae was subsequently identified as the causative agent of this CSD case. The child was orally given rifampicin (lOmg/kg/day) and became afebrile within 1 week. However, since ad- enopathies and the biological inflammatory serum signs