- Volume 50, Issue 5, 2001

To investigate whether adult-onset laryngeal papillomatosis induces serum antibodies to the human papillomavirus (HPV), 60 patients underwent a clinical examination, and HPV DNA from their laryngeal biopsy was assayed by PCR and HPV serology with virus-like particles as the antigen. Patients and controls (n = 53) showed no differences in their HPV 6 and 16 antibodies. Patients more often had HPV 11 antibodies, female patients more often than female controls or male patients. Of the female patients, 5 of 15 had a history of genital condylomas and, at the follow-up visit, 5 of 9 had cervical cytology consistent with genital HPV infection. The fact that HPV antibodies did not correlate with clinical features of the laryngeal disease or with HPV DNA detected in the larynx, suggests that HPV antibodies in female patients were induced by genital rather than laryngeal HPV infection. The high prevalence of abnormal Pap smears indicates that gynaecological examination of female adult-onset laryngeal papilloma patients is warranted.

The natural susceptibility of 221 Klebsiella strains to 71 antibiotics was examined. The strains were isolated from clinical specimens and the environment, and belonged to K. pneumoniae subsp. pneumoniae (n=40), K. pneumoniae subsp. ozaenae (37), K. pneumoniae subsp. rhinoscleromatis (10), K. oxytoca (44), K. planticola (40), K. ornithinolytica (25) and K. terrigena (25). MIC values were determined by a microdilution procedure in IsoSensitest broth according to the German standard (DIN). All Klebsiella spp. were naturally resistant or intermediate to amoxicillin, ticarcillin and to antibiotics to which other Enterobacteriaceae are also intrinsically resistant. Klebsiella spp. were naturally sensitive or intermediate to several penicillins, all tested cephalosporins, aminoglycosides, quinolones, tetracyclines, trimethoprim, co-trimoxazole, chloramphenicol and nitrofurantoin. K. pneumoniae subsp. ozaenae and subsp. rhinoscleromatis strains were generally more susceptible to antibiotics than strains of other Klebsiella taxa. K. pneumoniae subsp. rhinoscleromatis was the most susceptible taxon, being highly susceptible to cefuroxime, anti-folates and naturally intermediate to erythromycin and clarithromycin. K. pneumoniae subsp. ozaenae was most susceptible to glycopeptides. K. oxytoca and K. terrigena strains were least susceptible to cefazoline, cefoperazone and fosfomycin, respectively. The results of the present study describe a database of the natural antimicrobial susceptibility of Klebsiella spp., which can be used for the validation of antibiotic susceptibility results of these bacteria. MIC patterns to β-lactams indicate the expression of chromosomally encoded class A β-lactamases in all the species, including the subspecies of K. pneumoniae. Similar natural susceptibility patterns of K. planticola and K. ornithinolytica to all tested antibiotics support the status of K. ornithinolytica as a biovar of K. planticola.

Streptococcus intermedius, included in the ‘milleri group', is a commensal of the mouth and upper respiratory tract but it has often been associated with various pyogenic infections, such as endocarditis, pneumonia, abdominal or cerebral abscess, rarely with osteomyelitis, and exceptionally with muscular abscess. The first observed case of iliac osteomyelitis with gluteal muscle abscess caused by S. intermedius is reported. It is essential to recognise members of the ‘milleri group’ as possible agents of bone and muscle pyogenic infection because its management requires a timely diagnosis and prolonged antimicrobial treatment to achieve complete clinical and radiological recovery.

Chronic, idiopathic diffuse colitis is a well recognised clinical and pathological entity in captive rhesus monkeys. Six rhesus monkeys were diagnosed with clinically debilitating, chronic diarrhoea. Histologically, colonic tissues were characterised as chronic, moderate to severe colitis and typhlitis, with diffuse mononuclear inflammation of lamina propria, reactive lymphoid hyperplasia and multifocal micro-abscesses. Colonic tissues were cultured for Salmonella spp. and Shigella spp.; all results were negative. Samples were negative for Clostridium difficile A and B toxins, and special stains of colonic tissue for acid-fast bacteria were also negative. The six diarrhoeic monkeys tested gave negative results for serum IgG antibodies to herpes B virus, STLV, SRV and SIV. Colonic tissue from the six diarrhoeic and two clinically normal monkeys with histologically confirmed colitis from the same colony were also subjected to micro-aerobic culture. Micro-aerobic cultures from all eight monkeys incubated at 37°C and 42°C revealed pinpoint or spreading colonies on antibiotic-containing media. Bacteria were identified as gram-negative, oxidase positive and urease negative. Of the nine strains characterised biochemically, two separate biotypes (corresponding to different species by 16S rRNA analysis) were identified. One biotype (type 1), from non-diarrhoeic monkeys and the second biotype (type 2) from diarrhoeic animals with subclinical chronic colonic inflammation, differed by catalase activity, ability to reduce nitrate to nitrite and sensitivity to cephalothin. Complete 16S rRNA analysis of five of the nine strains characterised biochemically indicated that the organisms isolated were two novel Helicobacter spp. By electron microscopy, these novel helicobacters had spiral morphology with bipolar sheathed flagella. This is the first report describing the isolation of novel Helicobacter spp. from inflamed colons of rhesus monkeys. Studies are needed to determine whether these novel Helicobacter spp. play a causal role in the initiation and progression of chronic colitis in macaques. Further microbiological and histological analysis of this chronic idiopathic colitis syndrome in macaques may prove useful in understanding the aetiology and pathogenesis of inflammatory bowel disease in man.

To investigate if temperature affects the interaction of Haemophilus ducreyi with human epithelial cells, nine strains were used to evaluate the adhesion kinetics of the organism at 33°C and 37°C. The effect of the free toxin on the epithelial cells at those temperatures was also assessed. The cyto-adherence kinetics of H. ducreyi to the epithelial cells was significantly greater at 33°C (10 times more) than at 37°C in all seven clinical isolates tested. There was a significant difference in cell-associated H. ducreyi at 33°C as compared with 37°C. Control strains showed similar adhesion properties at both temperatures. However, the virulent strain CIP542 adhered in larger amounts than the avirulent strain A77. Electron microscopy revealed that there was more tissue necrosis at the lower than the higher temperature. The effect of the free toxin was the same at each temperature. However, strain A77 had significantly lower toxicity than strain CIP542 and the clinical isolates. These results suggest that H. ducreyi displays a temperature-dependent interaction with human epithelial cells, and this feature may play a role in the virulence of the organism in vivo. While the overall toxic effect of viable bacteria depends on the metabolic activity of the bacteria and is, therefore, higher at 33°C than at 37°C with the same initial inoculum, the effect of the extracted toxin at molecular level with fixed concentrations is a temperature-independent event.

A real-time PCR assay based on the TaqMan ® technology was developed for the detection of Bordetella pertussis and B. parapertussis in clinical samples. The assay was evaluated with 182 specimens from 153 patients with and without symptoms of pertussis. The analytical sensitivity ranged from 0.1 to 10 cfu for B. pertussis and B. parapertussis, respectively, and diagnostic sensitivity was 94.1% when culture was used as a reference. No sample from a patient without symptoms of pertussis was positive in PCR. Twenty-four of 28 patients who were negative by culture and positive by PCR assay met the CDC clinical case definition for pertussis; the remaining four patients had paroxysms of shorter duration. Intra- and inter-assay variation were <5% and results were available within 4 h.

Thirty-two related and 68 unrelated isolates of Clostridium difficile, isolated in different Italian hospitals since 1987, were analysed by PFGE and PCR-ribotyping to investigate their genetic relatedness. The isolates were classified into 28 groups by PFGE and 20 ribotypes by PCR-ribotyping. A single clone of C. difficile was recognised as the cause of three geographically and chronologically distant outbreaks. The correlation between PFGE and PCR-ribotyping results was good, with agreement for 77 (84%) of the 92 isolates typed by both methods. However, among sporadic isolates the discriminatory power of PFGE was more evident. Eight isolates that were untypable by PFGE could be analysed by PCR-ribotyping. The dendrograms generated showed that the genetic relatedness of the C. difficile isolates obtained by both techniques was comparable. The majority of the isolates in recent years appeared to be genetically unrelated to isolates from past infections. However, two clonal groups identified in all time periods had a common origin and this seems to indicate that they share some advantageous biological characteristics. The constant monitoring of C. difficile epidemiology will allow acquisition of further important data on this nosocomial pathogen.

A DNA fragment, isolated from a genomic DNA mini-library of Candida parapsilosis group I reference strain ATCC 22019, was sequenced and characterised. The fragment was first probed by Southern blotting against a pool of DNA from several yeasts. The hybridisation tests revealed that the probe was specific for strain ATCC 22019 and 49 (90.74%) of 54 C. parapsilosis clinical and soil isolates that were attributed to C. parapsilosis group I by the electrophoretic images of their restriction fragment length polymorphisms (RFLPs) and electrophoretic karyotype (EK). The remaining five negative isolates, according to the same criteria, were attributed to group II (one isolate) and III (four isolates). Two primers were selected from the probe DNA sequence and a PCR-based test was developed for specifically detecting C. parapsilosis group I isolates, which represent the majority of the common clinical isolates. The PCR assay confirmed the Southern hybridisation results. This PCR assay could be a simple and reproducible tool for the rapid, sensitive and species-specific identification of C. parapsilosis major group I isolates.

Serotype G6 and G8 rotaviruses are rarely found in man and may have originated in animals. Human serotype G6 and G8 rotaviruses, isolated from hospitalised children at various locations in Australia, were characterised. Deduced amino acid sequences of the major neutralising antigen, V7, showed significant identity to the cognate proteins of prototype human and bovine G6 and G8 viruses, respectively, and the strains reacted with G6 and G8 serotype-specific neutralising monoclonal antibodies, respectively, in an enzyme immunoassay. The VP4 type was determined as P[14] for all strains tested. Phylogenetic analysis of these and other human and bovine VP7 sequences suggested that a single inter-species transmission event, possibly from cattle, may have led to the emergence of G6 viruses in man. In contrast, the exchange of genes between human and bovine G8 viruses may have occurred on more than one occasion, or these genes may have originated in a different host.