- Volume 49, Issue 9, 2000

Current methods for the detection of Cryptosporidium oocysts in water samples are both time-consuming and subject to variation in sensitivity. A genus-specific PCR assay was designed for the specific amplification of a 552-bp region of the 18S rRNA gene. Post-amplification endonuclease restriction generated unique digest patterns that enabled differentiation between the three species, C. muris, C. baileyi and C. parvum, the major human pathogen. Theoretical restriction profiles for other Cryptosporidium species were also predicted. The assay routinely detected 10 oocysts in 10-ml purified oocyst preparations, but sensitivity was found to be 103–104-fold lower in environmental water samples. The use of Chelex resin and an immunomagnetic separation procedure overcame this inhibition. This provided detection levels of 101–103 oocysts, depending on water turbidity. Rapid and sensitive pathogen detection methods are essential for the water industry. The results of this study demonstrate that PCR has the potential to improve current detection capabilities greatly by differentiating the major human pathogens from non-pathogenic species. This will greatly facilitate a closer examination of the epidemiology of this important pathogen.

The sensitivity of culture in BactecTM Plus Aerobic/F* culture vials of body fluids from adult patients at a university hospital was compared with that of conventional culture methods, including enrichment in Schaedler broth. Previous antibiotic therapy was recorded at the time of sampling. Analysis of culture results took account of the clinical significance of isolates and impact on therapy. Of 336 specimens evaluated, 81 (24%) yielded positive cultures, of which 50 cultures (15%) were considered to be clinically significant (yielding 71 isolates) and 31 (9%) were considered contaminated. Of the 71 pathogens, 16 (23%) were isolated in the Bactec system only, whereas 13 (18%) grew in conventional media only; 12 of the latter were strict anaerobes. Among clinically significant positive specimens, 19 (38%) were from patients receiving antibiotic therapy. In 27 cases (8% of all specimens and 54% of significantly positive cultures), the isolation of a pathogen led to modification of therapy. Overall, culture in the Bactec system showed higher sensitivity for the isolation of aerobic micro-organisms than Schaedler broth. Most of the difference was due to a better recovery of Streptococcaceae. Additional pathogens found only in resin-containing Bactec media led to 30% of all culture-influenced modifications of empirical therapy. These data confirm that culture of normally sterile body fluids frequently yields results that are useful for guiding therapy. Although more costly than standard enrichment broth, the resin-containing Bactec Plus Aerobic/F* vial can be advantageous for culture of aerobic pathogens from these specimens, particularly in patients receiving antibiotic therapy.

The bactericidal activity and mechanisms of action of the new fluoroquinolone gemifloxacin were investigated against the laboratory strains Escherichia coli KL16, Staphylococcus aureus E3T and Streptococcus pneumoniae C3LN4. Gemifloxacin was found to be highly bactericidal against these bacteria, producing a biphasic dose- response curve typical of the fluoroquinolones. This novel fluoroquinolone was more bactericidal than all other fluoroquinolones so far tested (ciprofloxacin, ofloxacin, enoxacin, lomefloxacin, levofloxacin, clinafloxacin, trovafloxacin, DV-7751 and sitafloxacin) against S. aureus and was more bactericidal than most other fluoroquinolones against E. coli or Str. pneumoniae. These data show gemifloxacin to be an improved member of the fluoroquinolone class of antibacterial agents.

The aerobic and anaerobic microbiology of intra-abdominal infections associated with diverticulitis was studied in 110 specimens from the peritoneal cavity after intestinal perforation and in 22 specimens from abdominal abscesses. Anaerobic bacteria only were isolated from 17 (15%) of the peritoneal specimens, aerobic bacteria only from 12 (11%) and mixed aerobic and anaerobic flora from 81 (74%). A total of 339 bacterial isolates was detected in peritoneal cultures (3.1 per specimen), comprising 155 aerobes (1.4 per specimen) and 184 anaerobes (1.7 per specimen). Anaerobic bacteria only were isolated in 4 (18%) abscesses, aerobes alone in one (5%) and mixed aerobic and anaerobic flora in 17 (77%). A total of 72 bacterial isolates (3.3 per specimen) was detected in abdominal abscesses – 35 aerobes (1.6 per specimen) and 37 aerobes (1.7 per specimen). The predominant aerobic and facultative bacteria in abdominal infections were Escherichia coli and Streptococcus spp. The most frequently isolated anaerobes were Bacteroides spp. (B. fragilis group), Peptostreptococcus, Clostridium and Fusobacterium spp.

Variants of large colony β-haemolytic Lancefield group A, C and G streptococci that are non-haemolytic or α-haemolytic on sheep blood agar have been detected in clinical specimens due to their enhanced haemolytic activity when grown on a new selective and differential blood agar medium containing colistin, nalidixic acid and pH 7.5-adjusted PIPES buffer (CNA-P). The large colony Lancefield group C and G isolates were identified as Streptococcus dysgalactiae subsp. equisimilis by API 20 Strep classification and 16S rDNA profiling. The haemolytic activity of these variants on various blood agar media, including CNA-P, was closely similar to that of known streptolysin S-defective mutants of S. pyogenes and was blocked by addition of cholesterol, a specific inhibitor of the streptolysin O family of haemolysins. As haemolysin variants could be detected in large numbers in cultures from patients with clinical symptoms of pharyngitis it is suggested that they may function as primary pathogens in such infections. The high frequency with which haemolysin variants were isolated from clinical specimens during a 3-month trial (3%, 13% and 10%, respectively, of group A, C and G streptococcal isolates) indicated that a substantial proportion of streptococcal infections may go undetected if only conventional sheep blood agar media are used in clinical laboratories for the detection of β-haemolytic streptococci. As haemolysin variants have been implicated in the development of serious streptococcal sequelae, further investigation of the full extent of their contribution to streptococcal disease is indicated.

The aim of this study was to establish bacterial profiles in gastric biopsy specimens from patients with Helicobacter pylori-associated gastritis by means of temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments. Specimens from eight patients with asymptomatic gastritis and five histologically normal controls revealed a Helicobacter-specific band in the TTGE profile with increased amounts of Helicobacter-specific DNA in the biopsies from most of the gastritis patients. DNA from other genera including Enterococcus, Pseudomonas, Streptococcus, Staphylococcus and Stomatococcus was also found in the stomach. In the absence of gastric inflammation, Helicobacter spp. appeared to be part of a complex, presumably indigenous microbial flora found in the biopsy specimens from the stomach.