Rita Cardines

Biofilm-growing intestinal anaerobic bacteria.

Sessile growth of anaerobic bacteria from the human intestinal tract has been poorly investigated, so far. We recently reported data on the close association existing between biliary stent clogging and polymicrobial biofilm development in its lumen. By exploiting the explanted stents as a rich source of anaerobic bacterial strains belonging to the genera Bacteroides, Clostridium, Fusobacterium, Finegoldia, Prevotella, and Veillonella, the present study focused on their ability to adhere, to grow in sessile mode and to form in vitro mono- or dual-species biofilms. Experiments on dual-species biofilm formation were planned on the basis of the anaerobic strains isolated from each clogged biliary stent, by selecting those in which a couple of anaerobic strains belonging to different species contributed to the polymicrobial biofilm development. Then, strains were investigated by field emission scanning electron microscopy and confocal laser scanning microscopy to reveal if they are able to grow as mono- and/or dual-species biofilms. As far as we know, this is the first report on the ability to adhere and form mono/dual-species biofilms exhibited by strains belonging to the species Bacteroides oralis, Clostridium difficile, Clostridium baratii, Clostridium fallax, Clostridium bifermentans, Finegoldia magna, and Fusobacterium necrophorum.

Microbial biofilms associated with biliary stent clogging.

Endoscopic stenting is a palliative approach for the treatment of diseases involving biliary obstruction. Its major limitation is represented by stent occlusion, followed by life-threatening cholangitis, often requiring stent removal and replacement. Although it has been suggested that microbial colonization of biliary stents could play a role in the clogging process, the so far available data, particularly on the role of anaerobic bacteria, are not enough for a comprehensive description of this phenomenon. Our study was focused on the analysis of 28 explanted biliary stents by culturing, denaturing gradient gel electrophoresis and scanning electron microscopy to identify all the aerobic/anaerobic bacteria and fungi involved in the colonization of devices and to verify the ability of isolated anaerobic bacterial strains to form a biofilm in order to better understand the mechanisms of stent clogging.

Invasive Type e Haemophilus influenzae Disease in Italy

We describe the first reported cases of invasive type e Haemophilus influenzae disease in Italy. All five cases occurred in adults. The isolates were susceptible to ampicillin and eight other antimicrobial agents. Molecular analysis showed two distinct type e strains circulating in Italy, both containing a single copy of the capsulation locus.

Presence of Multiple Copies of the Capsulation b Locus in Invasive Haemophilus influenzae Type b (Hib) Strains Isolated from Children with Hib Conjugate Vaccine Failure

Most invasive Haemophilus influenzae type b strains possess a duplication of the capsulation locus. Further amplification resulting in as many as 5 copies has been described. To verify whether amplification is involved in vaccine failure, the number of copies of the locus was determined by Southern blotting in 90 strains from children with true vaccine failure (TVF) between 1993 and 1999 and in 139 strains from unvaccinated children (50 collected between 1993 and 1999 and 89 collected between 1991 and 1992, before routine immunization was introduced). A significantly greater proportion of strains from TVFs contained multiple copies, compared with strains from control children (24% vs. 10%; P = .0379), which suggests that amplification of the capb locus may be a contributory factor in vaccine failure. The presence of multiple-copy strains was associated with disease other than meningitis.

Nontypeable Haemophilus influenzae meningitis in children : Phenotypic and genotypic characterization of isolates

Background: With the decline in the incidence of invasive Haemophilus influenzae type b disease as result of routine immunization of infants, the potential emergence of nontypeable H. influenzae (NTHi) strains as important pathogens has been suggested. Methods: From June 1997 to July 2006, 9 cases of NTHi meningitis in children aged ≤60 months were detected. The 9 NTHi isolates were characterized. Antimicrobial susceptibility patterns were determined by E-test. The transpeptidase domain of penicillin binding protein 3 of a β-lactamase negative ampicillin-resistant strain was sequenced. Genetic relatedness among isolates was assessed by pulsed field gel electrophoresis and by multilocus sequence typing. The presence of HMW and Hia adhesins and hemagglutinating fimbriae was investigated by PCR and Western Blotting. Results: The 9 cases of NTHi meningitis did not occur in specific risk groups, except for one patient. Of the 9 NTHi isolates, 2 were β-lactamase producers and 1 showed the β-lactamase negative ampicillin-resistant phenotype. Sequencing of the penicillin binding protein 3 revealed novel amino acid substitutions. A high degree of genetic diversity among isolates was demonstrated by pulsed field gel electrophoresis. Multilocus sequence genotyping confirmed that the 9 NTHi isolates did not belong to related phylogenetic clusters. HMW adhesins were found in 2 isolates, and 5 strains possessed Hia. No hemagglutinating fimbriae were detected, even though 2 isolates contained hifA gene sequences. Conclusion: NTHi isolates from cases of meningitis in children are genetically diverse. Distribution of adhesins among the isolates we examined is unusual: most strains express Hia that generally occurs in a minority of strains in NTHi, suggesting that this adhesin may play a role in virulence mechanisms of NTHi causing meningitis.

First Characterization of Heterogeneous Resistance to Imipenem in Invasive Nontypeable Haemophilus influenzae Isolates

ABSTRACT This study describes the first two reported invasive nontypeable Haemophilus influenzae (NTHI) isolates (strains 183 and 184) with heterogeneous resistance to imipenem. For both isolates, Etest showed imipenem MICs of ≥32 μg/ml. When the two strains were examined by the quantitative method of population analysis, both strain populations were heterogeneously resistant to imipenem and contained subpopulations growing in the presence of up to 32 μg of imipenem/ml at frequencies of 1.7 × 10−5 and 1.5 × 10−7, respectively. By pulsed-field gel electrophoresis analysis, the two isolates appeared to be genetically closely related. The sequencing of the ftsI gene encoding penicillin-binding protein 3 (PBP 3) and comparison with the sequence of the imipenem-susceptible H. influenzae strain Rd identified a pattern of six amino acid substitutions shared between strains 183 and 184; an additional change was unique to strain 183. No relationship between mutations in the dacB gene encoding PBP 4 and imipenem resistance was found. The replacement of the ftsI gene in the imipenem-susceptible strain Rd (for which the MIC of imipenem is 0.38 to 1 μg/ml) with ftsI from strain 183 resulted in a transformant for which the MIC of imipenem ranged from 4 to 8 μg/ml as determined by Etest. The Rd/183 transformant population showed heterogeneous resistance to imipenem; it contained subpopulations growing in the presence of up to 32 μg of imipenem/ml at a frequency of 3.3 ×10−8. The presence of additional resistance mechanisms, such as the overexpression of the AcrAB efflux pump, was investigated and does not seem to be involved. These data indicate that the heterogeneous imipenem resistance phenotype of our NTHI clone depends largely on the PBP 3 amino acid substitutions. We speculated that bacterial regulatory networks may play a role in the control of the heterogeneous expression of the resistance phenotype.

Molecular typing and long-term comparison of Clostridium difficile strains by pulsed-field gel electrophoresis and PCR-ribotyping

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.

Variation in expression of HMW1 and HMW2 adhesins in invasive nontypeable Haemophilus influenzae isolates

BackgroundAmong surface antigens of nontypeable Haemophilus influenzae (NTHi), the HMW1 and HMW2 proteins are the major adhesins promoting colonization of the upper respiratory tract. Since they are potential vaccine candidates, knowledge concerning variation in HMW proteins expression among clinical isolates is of great interest. In this study, expression of hmw1A and hmw2A genes was evaluated by quantitative real-time reverse transcription-PCR in 3 NTHi invasive isolates (strains 56, 72, 91) and in the prototype strain 12. Number of 7-bp repeats within the hmwA promoters and presence of HMW proteins by Western blotting were also determined.ResultsResults showed that gene transcription varied not only among different isolates but also between the hmw1A and hmw2A genes from the same isolate. Compared to that found in prototype strain 12, up-regulation of the hmw1A gene expression was found in strain 56, down-regulation of both hmw1A and hmw2A genes transcripts was observed in strain 72 whereas the two hmwA genes appeared differentially expressed in strain 91 with the hmw1A transcript enhanced but the hmw2A transcript reduced.ConclusionIncreasing numbers of 7-bp repeats within the hmwA promoters generally correlated with decreased amounts of mRNA transcript, however additional control mechanisms contributing to modulation of hmw1A gene seem to be present.

Haemophilus influenzae in children with cystic fibrosis: antimicrobial susceptibility, molecular epidemiology, distribution of adhesins and biofilm formation.

Haemophilus influenzae commonly infects the respiratory tract of patients with cystic fibrosis (CF), early in childhood. In this investigation, 79 H. influenzae isolates were recovered from the respiratory secretions of 64 CF patients (median age: 5 years) included in a 5-year follow-up study. Fifteen of the 64 patients contributed two or more H. influenzae isolates overtime. Serotyping, antibiotic susceptibility testing, genotyping, detection of both hmwA and hia adhesin genes and hypermutable strains was carried out. Biofilm formation ability was investigated. Most strains (72/79, 91.2%) were nonencapsulated or nontypeable (NTHi). Resistance to ampicillin (13.9%) and imipenem (17.7%) was the most detected. Few isolates (2.5%) exhibited the hypermutable phenotype. The NTHi strains showed 55 different genotypes, but 19 clusters of closely related strains were identified. Nine clusters included strains that cross-colonised several patients over a long-time period (mean: 3.7 years). Most patients with sequential isolates harboured strains genetically unrelated, but persistent colonisation with the same clone was observed in 37.5% of patients. Over 45% of NTHi strains contained hmwA-related sequences, 26.3%, hia, 8.3% both hmwA and hia, while 19.4% lacked both. A significant association was found between occurrence of an adhesive gene (irrespective of which) and both persistence (P<0.0001) and long-term cross-colonisation (P<0.0001). Mean biofilm level formed by the persistent strains was found significantly increased compared to non-persistent ones (P<0.0001). Hia-positive strains produced significantly more biofilm than hmwA-carrying strains (P<0.01). Although a high turnover of NTHi strains in FC patients was observed, distinct clones with increased capacity of persistence or cross-colonisation occurred.

Conservation and Diversity of HMW1 and HMW2 Adhesin Binding Domains among Invasive Nontypeable Haemophilus influenzae Isolates

ABSTRACT The pathogenesis of nontypeable Haemophilus influenzae (NTHi) begins with adhesion to the rhinopharyngeal mucosa. In almost 80% of NTHi clinical isolates, the HMW proteins are the major adhesins. The prototype HMW1 and HMW2 proteins, identified in NTHi strain 12, exhibit different binding specificities. The two binding domains have been localized in regions of maximal sequence dissimilarity (40% identity, 58% similarity). Two areas within these binding domains have been found essential for full level adhesive activity (designated the core-binding domains). To investigate the conservation and diversity of the HMW1 and HMW2 core-binding domains among isolates, PCR and DNA sequencing were used. First, we separately amplified the hmw1A-like and hmw2A-like structural genes in nine invasive NTHi isolates, discovering two new hmwA alleles, whose sequences are herein reported. Then, the hmw1A-like and hmw2A-like PCR products were used as the template in nested PCR to produce amplicons encompassing the encoding sequences of the two core-binding domains. In-depth sequence analysis was then performed among sequences of each group, with the support of specific computer programs. Overall, extensive sequence diversity among isolates was highlighted. However, similarity plots showed patterns consisting of peaks of relatively high similarity alternating with strongly divergent regions. The phylogenetic tree clearly indicated the HMW1-like and HMW2-like core-binding domain sequences as two clusters. Distinct sets of conserved amino acid motifs were identified within each group of sequences using the MEME/MOTIFSEARCH tool. Since HMW adhesins could represent candidates for future vaccines, identification of specific patterns of conserved motifs in otherwise highly variable regions is of great interest.