W. E. Peetermans

Quantification of Expression of Staphylococcus epidermidis Housekeeping Genes with Taqman Quantitative PCR during In Vitro Growth and under Different Conditions

The aims of the present study were (i) to develop and test a sensitive and reproducible method for the study of gene expression in staphylococci and (ii) to study the expression of five housekeeping genes which are involved in nucleic acid metabolism (gmk, guanylate kinase; the dihydrofolate reductase [DHFR] gene), glucose metabolism (tpi, triosephosphate isomerase), and protein metabolism (the 16S rRNA gene; hsp-60, heat-shock protein 60) during in vitro exponential and stationary growth. A modified method for instant mRNA isolation was combined with gene quantification via Taqman real-time quantitative PCR. The detection limit of our method was 10 copies of RNA. The average intersample variability was 16%. A 10-fold increase in the expression of the hsp-60 gene was induced by exposure to a 10 degrees C heat shock (37 to 47 degrees C) for 10 min. During in vitro growth, the expression of all five housekeeping genes showed rapid up-regulation after inoculation of the bacteria in brain heart infusion medum and started to decline during the mid-exponential-growth phase. Maximal gene expression was 110- to 300-fold higher than gene expression during stationary phase. This indicates that housekeeping metabolism is a very dynamic process that is extremely capable of adapting to different growth conditions. Expression of the 16S rRNA gene decreases significantly earlier than that of other housekeeping genes. This confirms earlier findings for Escherichia coli that a decline in bacterial ribosomal content (measured by 16S rRNA gene expression) precedes the decline in protein synthesis (measured by mRNA expression).

Polyclonal Staphylococcal Endocarditis Caused by Genetic Variability

Cultures of blood obtained from a patient with Staphylococcus epidermidis prosthetic valve endocarditis yielded 15 strains of S. epidermidis. Genome macrorestriction and amplified fragment-length polymorphism analyses of these strains showed that they belonged to 4 different, very closely related clones, suggesting that they were the result of genetic variability of an infecting strain during the infectious episode. In vivo experiments in a rat model for foreign body infections using 1 of the S. epidermidis strains from the patient showed genetic variability similar to that of the infecting strain. In the rat model, we also detected the simultaneous presence of different clones that were identical to those isolated from our patient, thus confirming the possibility of genetic variability. It is important to note that the 4 clones isolated from our patient presented with 2 different antibiograms. Therefore, in cases of foreign device-related infections due to coagulase-negative staphylococci, the possibility of polyclonal infection has to be taken into account, particularly as regards differences in antibiotic susceptibility.

Candidal Vertebral Osteomyelitis: Report of 6 Patients, and a Review

The incidence of deep-seated candidal infection is increasing, but candidal vertebral osteomyelitis is still rare. We describe 6 patients recently treated in our hospital. Conservative treatment failed in all. We reviewed the literature and identified 59 additional cases of candidal vertebral osteomyelitis. Candidemia was documented in 61.5% of them. The interval between the diagnosis of candidemia and the onset of symptoms of vertebral osteomyelitis varied widely, from days to >1 year. In patients without documented candidemia, there was a similar interval between the occurrence of risk factors for candidemia (present in 72% of the patients) and the onset of symptoms of vertebral osteomyelitis. Clinical, laboratory, and radiological findings are not specific for candidal spondylodiskitis. Final diagnosis is determined by means of culture of a biopsy specimen from the infected vertebra or disk. Treatment consisted of prolonged antifungal treatment, and it often included surgery. On the basis of our experience (for all 6 patients, initial conservative treatment with only antifungals failed), we recommend consideration of early surgical debridement in combination with prolonged antifungal therapy.

Zoonotic transmission of Cryptococcus neoformans from a magpie to an immunocompetent patient

Abstract.  Lagrou K, Van Eldere J, Keuleers S, Hagen F, Merckx R, Verhaegen J, Peetermans WE, Boekhout T (University Hospital Leuven, Leuven, Belgium; and Centraalbureau voor Schimmelcultures, CT Utrecht, the Netherlands) Zoonotic transmission of Cryptococcus neoformans from a magpie to an immunocompetent patient (Case report). J Intern Med 2005; 257: 385–388.

Saccharomyces fungemia complicating Saccharomyces boulardii treatment in a non-immunocompromised host.

Sir: Saccharomyces boulardii (SB) is used for the prevention and treatment of Clostridium difficile-related diarrhoea and in one study it was given successfully for the prevention of enteral feeding-associated diarrhoea in the ICU [1]. There are conflicting, but mostly positive, results about the efficacy of SB for the prevention of antibiotic-associated diarrhoea. Treatment with SB is generally thought to be safe as it only colonises mucosal surfaces. We report the case of a patient who developed fungemia with Saccharomyces cerevisiae (SC) during treatment with SB for enteral feeding-associated diarrhoea. A 74-year-old male patient was hospitalised with hemiplegia due to a subarachnoidal haematoma and underwent neurosurgery. During his stay in the ICU he received enteral nutrition through a nasogastric tube. Major diarrhoea developed (5±10 liquid stools a day), repeated testing for Clostridium difficile and other enteropathogens was negative. The problem persisted after enteral nutrition was stopped and treatment with SB (Perenterol, Biodiphar, Brussels) was started at two capsules (50 mg each) 6 times a day. After several days of treatment he developed sepsis with Klebsiella oxytoca and SC (2 of 3 aerobe blood culture bottles were positive). Catheter-tip culture of the only (intravenous) catheter the patient had remained sterile. Antibiotic treatment and fluconazole 200 mg i. v.b. i. d. were started. On clinical examination the patient had a severely distended abdomen. A sigmoidoscopy showed severe inflammation of the mucosal surface without pseudomembranes and pathological findings were unrevealing (aspecific inflammation). Subsequent blood cultures remained negative. The patient died 4 weeks later. Autopsy showed diffuse mucosal inflammation of the colon and a perforation of the sigmoid with faecal peritonitis. Biopsies of the colonic mucosa revealed multiple ulcerations, some of them causing perforation. The aetiology of the colitis remained uncertain. Yeast infection could not be demonstrated at necropsy. To the best of our knowledge, this is the second report of SC fungemia in a non-immunocompromised host receiving SB treatment for diarrhoea [2]. We think the fungemia was caused by translocation through the intestinal wall because this patient had macroscopic and microscopic colitis as an obvious portal of entry. We were unable to show with certainty that the strain of the patient was the same as the one administered, because the blood cultures were no longer available for genotypic differentiation. In most cases of SB fungemia in ICU, intravascular catheters were considered the probable portal of entry. Transmission via hands that were contaminated while manipulating Saccharomyces capsules for administration via nasogastric tubes (and subsequent contamination of the catheter) was the likely explanation. Transmural migration of Saccharomyces in critically ill patients, however, remains a concern. This case illustrates that there should be concern about the safety of SB in patients with active colitis although it is uncertain if the patients death was related to the fungemia. Colonic ulceration might predispose to translocation of SB through the intestinal wall even in the non-immunocompromised host. It has to be mentioned that although our patient was, generally speaking, nonimmunocompromised, a critically ill patient can probably be considered to be immunocompromised [3].

Three cases of destructive native valve endocarditis caused by Staphylococcus lugdunensis

Described here are three cases of acute native valve endocarditis due to the coagulase-negative pathogen Staphylococcus lugdunensis with serious complications. Two of the three patients died despite optimal antibiotic therapy and cardiovascular surgery. These cases demonstrate the aggressive nature of S. lugdunensis and emphasize the importance of identifying coagulase-negative staphylococci to the species level and not considering the isolation of S. lugdunensis from normally sterile body fluids as contamination. On the contrary, when this organism is found in patients with endocarditis, early surgery should be considered. The possibility that this organism could be misidentified as S. aureus because of ‘autocoagulation’ and that commercial identification systems may misidentify it as S. haemolyticus, S. hominis or S. warneri should also be remembered.

Increase in Penicillin Resistance Rates in Belgium due to Clonal Spread of a Penicillin-Resistant 23F Streptococcus pneumoniae Strain

Abstract In 1994 a sudden increase in penicillin resistance was observed in Belgium among invasive pneumococci. To determine whether this increase was due to clonal spread of a resistant strain or to de novo acquisition of penicillin resistance, pneumococci of capsular types 23F, 19, 14, 9, and 6, isolated in 1993 and 1994, were analyzed by capsular serotyping and DNA macrorestriction analysis, resolved by pulsed-field gel electrophoresis. Furthermore, pneumococcal isolates from northern France, a region with a high prevalence of penicillin resistance, and from southern Belgium, a region with a low but increasing prevalence of penicillin resistance, were analyzed. The rate of resistance of invasive pneumococci to penicillin increased from 2.3% in 1993 to 7.6% in 1994. Pneumococcal serotype 23F represented 26.7% of the penicillin-resistant isolates in 1993 and 40.4% in 1994, while the prevalence of serotype 23F decreased from 10.9% in 1993 to 8.8% in 1994. In 1994 up to 35.8% of serotype 23F isolates were penicillin resistant. The Belgian penicillin-resistant 23F isolates from 1994 were genetically closely related to the French 23F penicillin-resistant isolates and, as clones were clearly distinct from the other serotypes as well as from the penicillin-susceptible 23F isolates. These data demonstrate the important contribution of the clonal spread of a penicillin-resistant pneumococcal strain in the overall increase of penicillin resistance in our country.

IMPLEMENTATION OF PRETRAVEL ADVICE : GOOD FOR MALARIA, BAD FOR DIARRHOEA

Abstract Pretravel immunisations and health advice can substantially reduce the incidence of travel-related diseases. The aim of this study was to evaluate the implementation of pretravel advice among a homogenous group of students, who received similar written information on vaccination requirements and health advice. They were referred to the travel clinic (50 %) or a general practitioner (50 %) for vaccination, counselling and prescriptions. Eighty-four out of 110 students (76 %) returned the questionnaire. Insect repellent was used by all and only 10 used the repellent for less than 75 % of the time spent in malaria endemic areas. Malaria chemoprophylaxis was taken by all but one : chloroquine plus proquanil by 12 and mefloquine by 71. Reported compliance with the dosing regimen was optimal in 64 students, 9 missed one dose and 10 stopped too early. Side effects due to antimalarials were reported by 25 (30 %). Diarrhoea during travel occurred in 43 students (51 %). Loperamide was used by 34 students with diarrhoea (79 %), but only 2 out of 27 students with moderate to severe diarrhoea used the recommended self-treatment with a fluoroquinolone antibiotic. In conclusion, the recommendations of malaria prophylaxis were well implemented by most travellers despite a high incidence of self-reported side effects to antimalarials. The incidence of traveller’s diarrhoea was high and the recommendation for early self-treatment of moderate to severe diarrhoea with a fluoroquinolone antibiotic in combination with loperamide was not put into practice.

A cluster of airport malaria in Belgium in 1995

In Europe 64 cases of airport malaria have been registered between 1969 and 1996, most of them in France, Switzerland and Belgium. In the summer of 1995 six cases of airport malaria occurred at the International airport of Brussels, Belgium. Of the six patients three were airport employees, three were occasional visitors. One patient died, the diagnosis was made by PCR amplification and DNA sequencing after exhumation. Two different species of Plasmodium were detected, and infections occurred on at least two different floors of the airport. An inquiry revealed that the cabin of airplanes is correctly sprayed, according to WHO recommendations, but that the inside of the hand luggage, the cargo hold, the animal compartment, the wheel bays and container flights remain possible shelters for infected mosquitoes. In a case of fever of unknown origin, airport malaria should be considered in the differential diagnosis, especially during hot summers, and when thrombocytopenia is present. Additional antimosquito measures should be generalised, encompassing highly exposed personnel, container content and handling buildings, animal cages, wheel bays, and the boundary between the sorting and the reception of luggage.

Staphylococcus aureus adherence to nasal epithelial cells in a physiological in vitro model

SummaryNasal carriage of Staphylococcus aureus represents a risk factor for subsequent invasive infections and interpatient transmission of strains. No physiological in vitro model of nasal epithelial cells is available to study both patient- and bacteria-related characteristics and their interaction, leading to adherence and colonization. Starting with tissues from human nasal polyps, a confluent, squamous, nonkeratinized epithelium in collagen-coated 96-well microtiter plates was obtained after 14 d. This in vitro cell-layer was characterized histologically, ultrastructurally, and immunohistochemically and showed features that were indistinguishable from those observed in the squamous nonkeratinized epithelium found in the posterior part of the vestibulum nasi. Adherence experiments were performed with four different 3H-thymidine-labeled Staphylococcus aureus strains. The effect of bacterial inoculum size, temperature of incubation, and incubation medium were studied. The adherence results were found to be reproducible, reliable and sensitive, allowing detection of small quantitative differences in adherence between the Staphylococcus aureus strains. There was no significant difference in adherence at 23° C and 37° C, nor between the incubation medium M199 and phosphate-buffered saline. Plastic adherence could be reduced and standardized with use of siliconized tips and a constant bacterial inoculum volume of 100 µl/well. This physiological and reliable in vitro cell-culture model offers a unique opportunity to study Staphylococcus aureus adherence to squamous, nonkeratinized nasal epithelial cells and both patient and bacterial characteristics involved in this interaction.