- Volume 55, Issue 6, 2006

In a previous article, the authors reported that exposing wild-type enteropathogenic Escherichia coli (EPEC) to chemically synthesized N-acetyllactosamine glycosides covalently coupled to BSA (LacNAc–BSA) inhibited localized adherence (LA) by these organisms and also caused them to lose their bundle-forming pili (BFP), the filamentous surface appendages responsible for their LA phenotype. This effect has now been further investigated by screening a panel of LacNAc–BSA-related glycosides for their ability to inhibit EPEC LA, which revealed that LacNAc–BSA retained its status as the most effective inhibitor of EPEC LA. It was also shown that LacNAc–BSA did not cause the loss of BFP in an EPEC strain containing a non-polar mutation in the bfpF gene and, as a consequence, unable to retract its BFP. LacNAc–BSA also effectively inhibited LA of the bfpF mutant EPEC. Taken together, these observations suggest that, as well as triggering BfpF-mediated BFP retraction, LacNAc–BSA likely functions as a competitive inhibitor of EPEC binding to LacNAc-related receptors on host cells. Moreover, transmission electron microscopy revealed that LacNAc conjugated to gold nanoparticles bound specifically to BFP. This observation indicated that either the major BFP structural subunit (BfpA) itself or, possibly, an accessory protein co-assembled with BfpA into the BFP filaments, contains a LacNAc-specific EPEC adhesin. The results suggest a mechanism whereby the initial binding of EPEC to LacNAc-like receptors on host cells triggers BfpF-mediated BFP retraction. This could then expedite the intimate adherence phase of the multi-step EPEC colonization process by drawing the organisms closer to the host-cell plasma membrane.

The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa has been highly successful at colonizing cystic fibrosis (CF) patients throughout the UK, has replaced previously established strains in CF patients, has caused infections of non-CF parents of CF patients, and can cause greater morbidity in CF than other strains of P. aeruginosa. Using suppression subtractive hybridization (SSH) to identify strain-specific sequences, a diagnostic test for the LES based on PCR amplification of SSH sequence PS21 had previously been developed. In this study, the SSH sequence database of LES was substantially increased, using both extension of previous sequences and new rounds of subtraction. Of 92 SSH sequences identified as present in the LES but absent from strain PAO1, 25 were assessed for prevalence amongst a strain panel consisting mainly of LES and non-LES CF isolates. Preliminary analysis of genome sequence data indicated that all SSH sequences that were LES specific or found only rarely in other strains of P. aeruginosa were present on one of three contigs. All of the SSH sequences screened were either unstable amongst LES isolates or were not completely LES specific. Rare false positives were found with the PS21 test. The authors suggest that a second PCR assay designed to detect SSH sequence LESF9 can be used to confirm the identity of the most prevalent CF epidemic lineage in the UK.

The ability of Acanthamoeba to feed on Gram-negative bacteria, as well as to harbour potential pathogens, such as Legionella pneumophila, Coxiella burnetii, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes and Mycobacterium avium, suggest that both amoebae and bacteria are involved in complex interactions, which may play important roles in the environment and in human health. In this study, Acanthamoeba castellanii (a keratitis isolate belonging to the T4 genotype) was used and its interactions with Escherichia coli (strain K1, a cerebrospinal fluid isolate from a meningitis patient, O18 : K1 : H7, and a K-12 laboratory strain, HB101) were studied. The invasive K1 isolate exhibited a significantly higher association with A. castellanii than the non-invasive K-12 isolate. Similarly, K1 showed significantly increased invasion and/or uptake by A. castellanii in gentamicin protection assays than the non-invasive K-12. Using several mutants derived from K1, it was observed that outer-membrane protein A (OmpA) and LPS were crucial bacterial determinants responsible for E. coli K1 interactions with A. castellanii. Once inside the cell, E. coli K1 remained viable and multiplied within A. castellanii, while E. coli K-12 was killed. Again, OmpA and LPS were crucial for E. coli K1 intracellular survival in A. castellanii. In conclusion, these findings suggest that E. coli K1 interactions with A. castellanii are carefully regulated by the virulence of E. coli.

It was previously reported that two oligonucleotide primer sets (spn9802 and spn9828) for discriminating Streptococcus pneumoniae from pneumococcus-like oral streptococcal isolates using PCR had been developed. In this study, PCR amplification of the lytA, ply, spn9802 and spn9828 genes was used to identify presumptive S. pneumoniae. Two genetic groups were identified by analysing sputum samples from 28 patients with community-acquired pneumonia: the lytA-positive, ply-positive, spn9802-positive and spn9828-negative group, and the lytA-positive, ply-positive, spn9802-positive and spn9828-positive group. Isolates of the former group were resistant to optochin, while those of the latter group showed susceptibility to optochin. The lytA-positive, ply-positive, spn9802-negative and spn9828-negative isolates, and lytA-positive, ply-positive, spn9802-negative and spn9828-positive isolates, were not detected in sputum from patients with pneumonia. Subsequently, a total of 92 saliva samples from healthy individuals was screened by PCR using these primer sets. The lytA-positive, ply-positive, spn9802-positive and spn9828-negative group was identified more frequently in saliva from healthy children than in saliva from older healthy individuals and patients with pneumonia. The lytA-positive, ply-positive, spn9802-positive and spn9828-positive group was found frequently in saliva from healthy children, and in saliva and sputum from patients with pneumonia. This study demonstrates a rapid, optimal screening method for the genotypic identification of presumptive S. pneumoniae by PCR using four genes highly specific for S. pneumoniae.

Infection with human papillomavirus (HPV) is the main cause of cervical cancer, the principal cancer in women in most developing countries. Molecular epidemiologic evidence clearly indicates that certain types of HPV are the principal cause of invasive cervical cancer and cervical intraepithelial neoplasia. Comprehensive, high-throughput typing assays for HPV, however, are not currently available. By combining L1 consensus PCR and multiplex hybridization using a Luminex xMAP system-based suspension array, the authors developed a rapid high-throughput assay, the HPV DNA suspension array (HPV-SA), capable of simultaneously typing 26 HPVs, including 18 high-risk HPV genotypes and eight low-risk HPV genotypes. The performance of the HPV-SA applied to 26 synthetic oligonucleotide targets was evaluated. The HPV-SA system perfectly discriminated 18 high-risk HPV targets from eight low-risk HPV targets. To assess the clinical applicability of the assay, the HPV-SA was performed with 133 MY09/MY11 primer set-mediated PCR (MY-PCR)-positive clinical specimens; of the 133 samples, 121 were positive by HPV-SA. Both single and multiple types were easily identified. The authors believe that improvement of the assay may be useful for epidemiological studies, cancer-screening programmes, the monitoring of therapeutic interventions, and the evaluation of the efficacy of HPV vaccine trials.

Members of the Burkholderia cepacia complex are important bacterial pathogens in cystic fibrosis (CF) patients. The B. cepacia complex currently consists of nine genetic subgroups (genomovars) of different epidemiological relevance and possibly of different pathogenic potential in humans. In this study, a new approach was developed for the rapid identification of B. cepacia genomovar I, Burkholderia multivorans (genomovar II), Burkholderia cenocepacia (lineage III-A and III-B), Burkholderia stabilis (genomovar IV) and Burkholderia vietnamiensis (genomovar V), which cause the large majority of infections in CF patients. The method was based on the detection of differences in the recA gene sequence by using rapid-cycle PCR and genomovar-specific fluorescence resonance energy transfer (FRET) probes. The genomovar status of all 39 B. cepacia complex strains tested (genomovars I–V) was identified by melting-curve analysis. Each FRET probe produced a specific fluorescence signal only with the respective genomovar, and not with other B. cepacia complex strains and Burkholderia spp. The identification system was easy to handle and revealed B. cepacia complex genomovar I–V status from culture isolates within about 1 h.

In this study, the ease of selection of clarithromycin resistance was investigated in clarithromycin-susceptible Helicobacter pylori strains isolated from patients with H. pylori infection prior to the administration of triple-combination eradication therapy (clarithromycin plus amoxicillin plus a proton pump inhibitor). Clarithromycin-susceptible strains isolated from ten patients in whom the eradication therapy was successful and from six patients in whom the eradication therapy was unsuccessful were exposed serially to subinhibitory concentrations of clarithromycin. The number of transfers required for the MICs of the strains to increase by 8- and 32-fold were 6.6 and 7.2, respectively, in the successful eradication group, and as few as 2.4 and 1.5, respectively, in the unsuccessful eradication group. The number of transfers required for the A2142G or A2143G point mutation of the 23S rRNA gene to be detected in the strains were 5 and 8, respectively, for the strains in the successful eradication group, and 1 and 2, respectively, for the strains in the unsuccessful eradication group. These results suggest that patients in the unsuccessful eradication group were infected with strains of H. pylori that readily became resistant to clarithromycin on exposure to the drug.

Yersinia enterocolitica 4/O : 3 is the most frequent cause of sporadic human yersiniosis in Finland and Germany. To investigate the possible link between pigs and humans, 282 human and 534 porcine strains from Finland and Germany were characterized with PFGE using NotI, ApaI and XhoI enzymes. Most of the human strains (>80 %) were indistinguishable from the porcine strains in both countries and most of the genotypes (178/182) were different in Finland and Germany. The indistinguishable genotypes among human and porcine strains together with different genotypes in Finland and Germany indicate that pigs are an important source of sporadic yersiniosis in both countries.

Phenotype characterization of 11 181 invasive Neisseria meningitidis isolates collected in Brazil from 1990 to 2001 was performed. Based on laboratory data, there were 7436 (67 %) serogroup B isolates, 3391 (30 %) C, 236 W135, 51 Y, four 29E, three X, one Z, and 59 of unknown serogroup. Phenotype B : 4,7 : P1.19,15 (54 %) remained the most common during the whole of the 12-year period. Two waves were observed within the serogroup C population: the most frequent phenotype C : 2b : P1.3 (47 %) was replaced after 1998 by non-typable isolates (C : NT : NST) (16 %).

Mycoplasma pneumoniae is increasingly recognized as a common and important pathogen in community settings, and is responsible for various pulmonary and extrapulmonary conditions in the normal population. However, the seroepidemiology of acute M. pneumoniae infection in HIV-infected individuals is still unclear worldwide. This study examined the seroprevalence of antibodies to M. pneumoniae in HIV-infected patients admitted with respiratory complaints at a tertiary AIDS care centre in Chennai, India. A commercial gelatin microparticle agglutination test (Serodia-Myco II, Fujirebio) was used for the determination of antibodies against M. pneumoniae in acute serum specimens. Of the 200 HIV-infected patients with underlying pulmonary conditions tested, 34 (17 % positivity; 95 % CI 12–23 %) had antibodies specific to M. pneumoniae, while among the 40 patients with no underlying pulmonary symptoms, five (12.5 % positivity; 95 % CI 4–27 %) had evidence of anti-M. pneumoniae antibody. This shows that the incidence of M. pneumoniae seropositivity is greater in patients with underlying pulmonary complaints. Most positive titres were found in the age group 28–37 years in the symptomatic and symptom-free groups (64.7 and 60 %, respectively). The positive titres ranged from 40 to >20 480. High titres (⩾320) were found in 10 out of the 39 patients (25.6 %). This seroprevalence study reports a 16.2 % prevalence of M. pneumoniae infections in HIV-infected patients by a particle agglutination test.

Three cases of symptomatic toxoplasmic lymphadenitis, together with a serologic profile of recent infection, are described, for which quantitative real-time PCR (LightCycler PCR) targeting different parasite genes was designed, in order to quantify Toxoplasma gondii DNA in acute and follow-up blood specimens. Similar parasite gene kinetics and DNA concentrations were observed in the patients studied. However, the profile of each target gene investigated was different. While the level of B1 DNA remained elevated for the entire time of observation, irrespective of clinical and serologic resolution, the SAG-1 gene was detected at the end of acute symptomatic disease, overlapping with a strong anti-T. gondii IgA antibody response, and persisting for over 3 months after infection and clinical recovery. With respect to the two bradyzoite genes investigated (SAG-4 and MAG-1), levels peaked during the symptomatic phase, but did not fall until 2 or 3 months of follow up. The real-time PCR assay with new alternative targets to the B1 gene may have potential for monitoring the clinical outcome of disease and for providing molecular information regarding the actual state of infection.

Group B Streptococcus (GBS) is an important pathogen responsible for a variety of diseases in newborns and the elderly. A clinical GBS isolate is considered nontypable (NT) when serological methods fail to identify it as one of nine known GBS serotypes. Eight clinical isolates (designated A1–A4, B1–B4) showed PFGE profiles similar to that of a GBS serotype V strain expressing R1, R4 surface proteins. These unique isolates were further characterized by immunologic and genetic methods. Rabbit sera to isolates A1 and A2 reacted weakly with concentrated HCl extracts of A1–A4 isolates, but not with those of B1–B4 isolates. In addition, a type V capsular polysaccharide (CPS) inhibition ELISA revealed that cell wall extracts from isolates A1–A4, but not from B1–B4, expressed low but measurable amounts of type V CPS. Molecular serotyping with PCR analysis showed that all eight isolates contained a type V-specific CPS gene (cpsO) and harboured the gene encoding the surface protein Alp3. Multilocus sequence typing identified isolate A1 as belonging to a new sequence type (ST) designated ST-173, whereas the other seven isolates keyed to ST-1. Sequencing of the 18 genes (17 736 bp) in the cps locus showed that each NT isolate harboured one to three unique polymorphisms, and also identified an IS1381 element in cpsE of the B4 isolate. Collectively, genetic and immunologic analyses revealed that these NT isolates expressing R1, R4 proteins have a genetic profile consistent with that of type V, an emergent, antigenically diverse and increasingly prevalent GBS serotype.