- Volume 53, Issue 9, 2004

Pseudomonas aeruginosa produces multiple virulence factors and causes different types of infections. Previous clinical studies identified P. aeruginosa isolates that lack individual virulence factors. However, the impact of losing several virulence factors simultaneously on the in vivo virulence of P. aeruginosa is not completely understood. The P. aeruginosa cell-to-cell communication system, or quorum sensing (QS), controls the production of several virulence factors. Animal studies using constructed QS mutants indicated that loss of the QS system severely impacts the virulence of P. aeruginosa. In this study, we tried to determine if deficiency within the QS system compromises the ability of P. aeruginosa to establish infections in humans. We have identified five QS-deficient strains through screening 200 isolates from patients with urinary tract, lower respiratory tract and wound infections. These strains lacked LasB and LasA activities and produced either no or very low levels of the autoinducers N-(3-oxododecanoyl) homoserine lactone and N-butyryl homoserine lactone. PCR analysis revealed that three isolates contained all four QS genes (lasI, lasR, rhlI and rhlR) while two isolates lacked both the lasR and rhlR genes. We also examined the five isolates for other virulence factors. The isolates produced variable levels of exotoxin A and, with one exception, were deficient in pyocyanin production. One isolate produced the type III secretion system (TTSS) effector proteins ExoS and ExoT, two isolates produced ExoT only and two isolates produced no TTSS proteins. The isolates produced weak to moderate biofilms on abiotic surfaces. Analysis of the patients’ data revealed that two of the isolates represented a single strain that was isolated twice from the same patient within a 1 month interval. One QS-deficient clinical isolate (CI-1) lacked all tested virulence factors and produced a weak biofilm. These results suggest that naturally occurring QS-deficient strains of P. aeruginosa do occur and are capable of causing infections; and, that besides the known virulence factors, additional factors may contribute to the ability of certain strains such as CI-1 to establish an infection.

The immunogenicity and protective effect of a DNA vaccine encoding the heat-shock protein (Hsp) GroEL of Chlamydophila abortus AB7, an obligate intracellular bacterium that causes abortion in sheep, was evaluated in pregnant and non-pregnant mouse models. The C. abortus groEL gene was cloned by screening a genomic library constructed in λFIX II arms with a nucleic acid probe corresponding to the central portion of the groEL gene from C. abortus. Sequence analysis of a positive clone revealed an open reading frame of 1632 bp encoding a 544 amino acid polypeptide with a predicted molecular mass of 58 256 Da and highly similar to GroEL of Chlamydia trachomatis (93 %) and Chlamydophila pneumoniae (94 %). As observed in other sequenced chlamydial genomes, the groEL gene belongs to an operon comprising another gene encoding the Hsp GroES. OF1 outbred mice were immunized intramuscularly with plasmid DNA carrying the groEL gene three times at 3 week intervals and challenged 2 weeks after the last DNA injection. In pregnant mice, no reduction in abortion was observed and the DNA vaccination failed to reduce the bacterial infection in the placenta and spleen of mice. Nevertheless, partial protection of fetuses was obtained. Immunization of non-pregnant mice with the groEL gene resulted in a specific humoral response with the predominant IgG2a isotype, suggesting a Th1-type immune response. The anti-GroEL antibodies showed no neutralizing effect in vitro on C. abortus infectivity. Although the DNA vaccine induced a delayed-type hypersensitivity response, it failed to elicit an efficient cellular immune response since the mice were not protected against bacterial challenge.

Validation of a PCR test to detect hilA gene sequences of Salmonella spp. was performed in blood and faeces samples from typhoid fever and salmonellosis patients. Sensitivity (S), specificity (SP), positive predictive value (PPV) and negative predictive value (NPV) of the PCR in blood samples were performed by testing: 37 patients with clinical diagnosis of typhoid fever, 34 of them confirmed by isolation of S. Typhi from blood cultures; 35 patients infected with other pathogens corroborated by blood culture (Klebsiella pneumoniae, 9; Serratia marcescens, 5; Escherichia coli, 4; Pseudomonas aeruginosa, 9; Providencia alcalifaciens, 4 and Enterobacter cloacae, 4) and blood samples from 150 healthy volunteers. To evaluate S, SP, PPV and NPV of the PCR in faeces samples we studied: 34 patients with enteritis due Salmonella spp. (S. Typhimurium, 21; S. Enteritidis, 9; S. Choleraesuis, 3 and S. Agona, 1); faeces samples from 35 patients with enteric infection due to Shigella sonnei (8), Shigella flexneri (10), enteropathogenic E. coli (12), Aeromonas hydrophila (5) and faeces samples from 150 healthy volunteers. The S, SP, PPV and NPV of the PCR in blood samples were all 100 %. PCR detected three patients with clinical diagnosis of typhoid fever and negative blood cultures. In faeces samples, S was 97 %, SP 100 %, PPV 100 % and NPV 99 %. The lowest number of c.f.u. ml−1 detected by PCR in blood samples was 1×101 and in faeces samples 4×102.

The microbiology of acute and chronic sinusitis has been studied extensively. Establishing the concomitant distribution of the causative organisms in cases that involve multiple sinuses is of scientific and practical importance. This study evaluated the aerobic and anaerobic microbiology of acute and chronic sinusitis in patients with involvement of multiple sinuses. The 155 patients evaluated had sinusitis of either the maxillary, ethmoid or frontal sinuses (any combination) and had organisms recovered from two to four concomitantly infected sinuses. Similar aerobic, facultatively anaerobic and anaerobic organisms were recovered from all groups of patients. In patients who had organisms isolated from two sinuses and had acute sinusitis, 31 (56 %) of the 55 isolates were found only in a single sinus, and 24 (44 %) were recovered concomitantly from two sinuses. In those with chronic infection 31 (34 %) of the 91 isolates were recovered only from a single sinus, and 60 (66 %) were found concomitantly from two sinuses. Anaerobic bacteria were more often isolated concomitantly from two sinuses (50 of 70) than aerobic and facultatively anaerobic (ten of 21, P < 0.05). Similar findings were observed in patients who had organisms isolated from three or four sinuses. β-Lactamase-producing bacteria were more often isolated from patients with chronic infection (58–83 %) as compared to those with acute infections (32–43 %). These findings illustrate that there are differences in the distribution of organisms in single patients who suffer from infections in multiple sinuses and emphasize the importance of obtaining cultures from all infected sinuses.

Genetic variants of Clostridium difficile have been reported with increasing frequency, but their true incidence is unknown. C. difficile strains have been classified into variant toxinotypes according to variations in the pathogenicity locus encoding the major virulence factors, toxins A and B. Some strains produce an additional toxin, binary toxin CDT. This survey of clinical isolates (153) from patients in a single hospital set out to ascertain the distribution of variant toxinotypes and strains possessing binary toxin genes. A PCR-RFLP-based method of toxinotyping identified 123 (80.4 %) isolates as toxinotype 0, 13 (8.5 %) strains as non-toxigenic and 17 (11.1 %) as belonging to variant toxinotypes. Binary toxin genes were amplified by PCR in nine strains (5.8 %), all of which were variant toxinotypes. Toxin variants of C. difficile are pathogenic and commonly isolated and need to be considered when evaluating new diagnostic testing strategies for C. difficile disease.

Subinhibitory concentrations (sub-MICs) of antibiotics, although not able to kill bacteria, can modify their physico-chemical characteristics and the architecture of their outermost surface and may interfere with some bacterial functions. This study investigated the ability of sub-MIC piperacillin/tazobactam (P/T) to interfere with the bacterial virulence parameters of adhesiveness, cell-surface hydrophobicity, motility, biofilm formation and sensitivity to oxidative stress. Antimicrobial activity against five Pseudomonas aeruginosa clinical isolates, representative of clonal lineages of 96 strains of nosocomial origin, and six control strains (ATCC 27853, PAO1, AK1, MT1562, PT623, PAO1algC) was evaluated in vitro using the NCCLS microdilution method. The effects of sub-MIC on bacterial adhesion and biofilm formation were studied using a modified microtitre plate assay. The relative cell-surface hydrophobicity of P. aeruginosa strains was determined by measuring their ability to adhere to n-hexadecane. P. aeruginosa that had been exposed overnight to P/T and incubated with P/T in the plate were also screened for their ability to swim using flagella and to twitch and for their sensitivity to oxidative stress. The results obtained showed that the impact of sub-MIC P/T on bacterial characteristics was different for the various strains of P. aeruginosa. There was a change in bacterial morphology and hydrophobicity that could explain a significant decrease in adhesion values in all clinical isolates and controls tested, a decrease in biofilm formation, a significant increase in sensitivity to oxidative stress, a significant decrease in flagellum-mediated swimming and a decrease in type IV fimbriae-mediated twitching. The results obtained indicate that sub-MIC P/T interferes with the pathogenic potential of P. aeruginosa.

Risk factors for primary Helicobacter pylori resistance in 186 children with gastroduodenal diseases (44 from villages/small towns and 130 from large towns/cities) in 2000–2003 were tested. Susceptibility was tested by a limited agar dilution method. Overall resistance rates to metronidazole, clarithromycin, tetracycline and both metronidazole and clarithromycin were 14.5, 11.9, 3.3 and 4.3 %, respectively. No amoxycillin resistance was observed. Tetracycline resistance was found in six children aged 7–18 years. Clarithromycin resistance was more common in children from small towns/villages (22.7 %) than in those from large towns/cities (8.5 %, P < 0.05). There were no significant differences (P > 0.05) in resistance rates between children from northern Bulgaria and those from southern regions. Resistance rates in duodenal ulcer patients and other children were, respectively, 10.5 and 15 % (P > 0.20) for metronidazole and 10.5 and 12 % (P > 0.20) for clarithromycin. No combined resistance to metronidazole and clarithromycin was found in 22 children aged 1–7 years and in 34 children living in northern Bulgaria. There were no significant associations of resistance with sex and age group (1–7- versus 8–18-year-old children) for all antibacterial agents tested. In conclusion, primary H. pylori resistance was absent (for metronidazole + clarithromycin) or low (4.5 % for clarithromycin) in children aged 1–7 years. Place of residence was associated with clarithromycin resistance rates.

The effects of imipenem and meropenem on the transcriptional expression of resistance-related genes oprD, oprM and oprN in Pseudomonas aeruginosa were studied by quantitative real-time PCR. Four strains were examined: the type strain PT5 (PAO1), its derivatives M7 and PT149, and a clinical isolate, PaKT3. The derivative M7 is a nalB mutant, overexpressing the MexAB–OprM pump, and the derivative PT149 is a nfxC-type mutant, overexpressing the MexEF–OprN pump while it is down-regulated for the OprD protein. After 18 h incubation in broth, the cultures were divided into three portions. Two were supplemented with antibiotics and the other was left antibiotic-free as the control. After a further 45 min incubation, total RNA was isolated from the strains by guanidine denaturation and acid-phenol/chloroform extraction. DNA-free total RNAs were immediately reverse-transcribed by MMuLV reverse transcriptase. Concentrations of mRNAs obtained by quantitative PCR were expressed relative to uninduced portions of the strains. The results showed that oprD was relatively stable against carbapenem antibiotics. oprM was induced significantly by imipenem in only one strain and oprN was induced by imipenem in most of the strains. The responses at the mRNA level found here were unexpected and suggested a chaotic, unpredictable regulatory mechanism.

The purpose of this study was to develop a simple procedure for cell lysis and DNA extraction for direct detection of Mycobacterium ulcerans in aquatic insects, gills and intestinal contents of fish, molluscs and human tissue samples using a nested PCR method specific for the insertion sequence IS2404. The simultaneous action of sodium N-lauroyl sarcosine, guanidinium isothiocyanate, chloroform and Tris-saturated phenol on mycobacteria, followed by a DNA purification method using mini-columns fitted with silica-cellulose membranes was successfully employed to extract DNA from cultured bacteria, environmental and human tissue samples. All specimens were collected from Buruli ulcer endemic regions. M. ulcerans DNA was detected in 11 of 57 aquatic insects, one of six molluscs and three of 15 fish, supporting the hypothesis that the fauna of major Buruli ulcer endemic foci in swampy terrain of tropical and subtropical regions can be a source of M. ulcerans infection.

An outbreak of infections due to a rare subspecies of Cryptococcus neoformans (var. gattii) was recognized on Vancouver Island (VI), British Columbia, in 2002, which had affected 59, mostly immunocompetent, individuals since 1999. The objectives of this study were to: (1) determine if the outbreak had spread to Vancouver and its surrounding communities and (2) review the epidemiological, clinical and pathological features of all cryptococcal infections in patients admitted to the Vancouver Hospital and Health Sciences Centre (VHHSC) over a 5 year period. VHHSC microbiology and pathology databases were searched for cryptococcal infections from 1 June 1997 to 31 December 2002. Hospital charts of all identified patients were reviewed. Available cryptococcal isolates and histopathological specimens were reviewed. Twenty-six cases of cryptococcosis were identified in both HIV-positive (n = 15) and HIV-negative (n = 11) patients. C. neoformans var. grubii was cultured from 13 patients, of whom 10 were HIV-positive. The outbreak strain, C. neoformans var. gattii, was detected in three patients; all had travelled to VI. C. neoformans var. neoformans was cultured from two patients, Cryptococcus laurentii was cultured from one, and seven patients had cryptococcosis based on histopathology alone, without cultures. The majority (10/15) of the HIV-positive patients developed systemic disease whilst HIV-negative patients (8/11) presented with pulmonary cryptococcosis. Lung biopsies revealed necrotizing and/or fibrosing granulomas, with cryptococcal cells in 5 of 10 specimens. Brain biopsies showed cryptococcal organisms within leptomeninges and deeper structures with minimal associated inflammation. This retrospective study demonstrated a sharp increase in the total number of C. neoformans infections in both immunocompromised and immunocompetent patients at the VHHSC in 2002. There was no evidence of spread of the outbreak strain to the Greater Vancouver area. This is the first correlation of clinical and investigational findings of cryptococcosis in a region in North America where C. neoformans varieties gattii and grubii are endemic.