Paola Mastrantonio

Molecular Analysis of the Pathogenicity Locus and Polymorphism in the Putative Negative Regulator of Toxin Production (TcdC) among Clostridium difficile Clinical Isolates

ABSTRACT The pathogenicity locus (PaLoc) of Clostridium difficile contains toxin A and B genes and three accessory genes, including tcdD and tcdC, which are supposed to code for the positive and negative regulators of toxin expression, respectively. Different studies have described variations in C. difficile toxin A and B genes, but little is known about C. difficile variants for the accessory genes. The PaLoc of several C. difficile clinical isolates was investigated by three different PCR methods with the aim to identify variant strains. Of the toxinogenic C. difficile strains examined, 25% showed variations. No correlation between C. difficile variant strains and key patient groups was found. Interestingly, all of these strains showed a variant tcdC gene. Three different tcdC alleles were identified, and one of these had a nonsense mutation which reduced the TcdC protein from 232 to 61 amino acids. It is possible that different TcdC variants affect toxin production differently, a hypothesis with important implications for the pathogenic potential of variant C. difficile strains.

Emergence of reduced susceptibility to metronidazole in Clostridium difficile

OBJECTIVES Antimicrobial treatment for Clostridium difficile infection (CDI) has typically been metronidazole, although reports have questioned the efficacy of this option. We screened recently isolated C. difficile (2005-06) for susceptibility to metronidazole and compared results for historic isolates (1995-2001). METHODS C. difficile ribotypes 001 (n = 86), 106 (n = 81) and 027 (n = 48) and isolates from the 10 other most prevalent ribotypes in Leeds (n = 57) were screened using spiral gradient endpoint analysis (SGE). C. difficile with metronidazole SGE MICs > or = 6 mg/L were analysed further by agar incorporation and Etest. Multiple-locus variable-number tandem-repeat analysis (MLVA) typing was performed for 28 C. difficile isolates. RESULTS No reduced metronidazole susceptibility was observed in C. difficile ribotypes 106 and 027 (geometric mean SGE MICs 1.11 and 0.90 mg/L, respectively). In contrast, 21 (24.4%) C. difficile ribotype 001 demonstrated reduced susceptibility to metronidazole (geometric mean SGE MICs 3.51 mg/L, P < 0.001). Variations in susceptibility were observed relating to the method and media, but increased metronidazole MICs were confirmed by an agar incorporation method. Geometric mean agar incorporation MICs for historic C. difficile ribotype 001 (n = 72) were 1.03 (range 0.25-2) mg/L compared with 5.94 (4-8) mg/L (P < 0.001) for recent isolates displaying reduced metronidazole susceptibility. MLVA typing revealed two clonal complexes of C. difficile with reduced susceptibility to metronidazole. CONCLUSIONS We have demonstrated the emergence of reduced susceptibility to metronidazole in 24.4% of the recent C. difficile ribotype 001 isolates from our institution. Our observations could have implications in the clinical setting due to the poor penetration of metronidazole into the colon.

Multidrug resistance in European Clostridium difficile clinical isolates

OBJECTIVES Multidrug resistance and antibiotic resistance mechanisms were investigated in 316 Clostridium difficile clinical isolates collected during the first European surveillance on C. difficile in 2005. METHODS MICs of eight different antibiotics were determined using Etest. Reserpine- and carbonyl cyanide m-chlorophenylhydrazone-sensitive efflux was tested using the agar dilution method. Molecular analysis of the resistance mechanisms was performed using PCR assays, PCR mapping and sequencing. RESULTS One hundred and forty-eight C. difficile strains were resistant to at least one antibiotic and 82 (55%) were multidrug resistant. In particular, 48% of these isolates were resistant to erythromycin, clindamycin, moxifloxacin and rifampicin. New genetic elements or determinants conferring resistance to erythromycin/clindamycin or tetracycline were identified. Even if most multiresistant strains carried an erm(B) gene, quite a few were erm(B) negative. In-depth analysis of the underlying mechanism in these isolates was carried out, including analysis of 23S rDNA and the ribosomal proteins L4 and L22. Interestingly, resistance to rifampicin was observed in multidrug-resistant strains in association with resistance to fluoroquinolones. Mutations in the rpo(B) and gyrA genes were identified as the cause of resistance to these antibiotics, respectively. CONCLUSIONS Characterization of multidrug-resistant C. difficile clinical isolates shows that antibiotic resistance is changing, involving new determinants and mechanisms and providing this pathogen with potential advantages over the co-resident gut flora. The present paper provides, for the first time, a comprehensive picture of the different characteristics of multidrug-resistant C. difficile strains in Europe in 2005 and represents an important source of data for future comparative European studies.

Antibody responses and persistence in the two years after immunization with two acellular vaccines and one whole-cell vaccine against pertussis

OBJECTIVE To evaluate the persistence of specific antibodies induced by primary immunization with three doses of two three-component acellular vaccines against pertussis with an observed efficacy of 84%, and one whole-cell vaccine with an observed efficacy of 36%. STUDY DESIGN Serum samples were collected from a subsample of 1572 children from the Italian double-blind, placebo-controlled, randomized trial of vaccines used in 15,601 children at three time points: before administration of the first dose of vaccine, and 1 month and approximately 15 months after administration of the third dose. Further evaluation included pooled cross-sectional analysis of serum specimens associated with episodes of cough (which were not laboratory confirmed as pertussis infection) occurring among the entire population enrolled in the trial. RESULTS With both acellular vaccines there was a fast and steep decrease in geometric mean antibody titers to pertussis toxin, filamentous hemagglutinin, and pertactin after vaccination. Mean titers were close to the limit of detection 15 months after primary immunization. The immunogenicity of the whole-cell study vaccine was poor 1 month after the third dose, and no antibody was detected in nearly all children 15 months after whole-cell vaccination. CONCLUSIONS Although the study acellular pertussis vaccines induced a strong primary specific antibody response in almost all recipients, the duration of the response was limited. Sustained high-level production of antibody to the antigens tested does not account for the observed efficacy of acellular pertussis vaccines.

Antigenic variants in Bordetella pertussis strains isolated from vaccinated and unvaccinated children

Bordetella pertussis shows polymorphism in two proteins, pertactin (Prn) and the pertussis toxin (PT) S1 subunit, which are important for immunity. A previous study has shown antigenic shifts in these proteins in the Dutch B. pertussis population, and it was suggested that these shifts were driven by vaccination. The recent Italian clinical trial provided the opportunity to compare the frequencies of Prn and PT S1 subunit variants in strains isolated from unvaccinated children, and from children vaccinated with two acellular and one whole-cell pertussis vaccine. Four Prn variants (Prn1, Prn2, Prn3 and Prn5) were found in the 129 strains analysed. Prn1, Prn2 and Prn3 have been described previously, whereas Prn5 is a novel variant. Prn1, Prn2, Prn3 and Prn5 were found in, respectively, 6, 41, 51 and 2% of the strains. The B. pertussis strains used to produce the vaccines administered in the clinical trial were found to produce Prn1, or a type which differed from Prn1 in one amino acid. The frequency of the Prn1 variant was found to be lowest in the strains isolated from vaccinated groups, suggesting that Prn1 strains are more affected by vaccine-induced immunity than Prn2 and Prn3 strains. Only one PT S1 type (S1A) was observed in the examined strains, which was distinct from the types produced by the vaccine strains (S1B and S1D). The S1A type also predominates in the Dutch B. pertussis population. The genetic relationship among B. pertussis strains analysed by IS1002-based DNA fingerprinting revealed that three fingerprint types predominate, representing more than 70% of the strains. Prn2 strains showed a greater variety of fingerprint types compared to Prn3, suggesting that Prn3 has emerged more recently. The results are discussed in the light of vaccine-driven evolution.

Molecular and Genomic Analysis of Genes Encoding Surface-Anchored Proteins from Clostridium difficile

ABSTRACT The gene slpA, encoding the S-layer precursor protein in the virulent Clostridium difficile strains C253 and 79–685, was identified. The precursor protein carries a C-terminal highly conserved anchoring domain, similar to the one found in the Cwp66 adhesin (previously characterized in strain 79–685), an SLH domain, and a variable N-terminal domain mediating cell adherence. The genes encoding the S-layer precursor proteins and the Cwp66 adhesin are present in a genetic locus carrying 17 open reading frames, 11 of which encode a similar two-domain architecture, likely to include surface-anchored proteins.

Fluoroquinolone resistance in Clostridium difficile isolates from a prospective study of C. difficile infections in Europe.

The European Study Group on Clostridium difficile (ESGCD) conducted a prospective study in 2005 to monitor and characterize C. difficile strains circulating in European hospitals, collecting 411 isolates. Eighty-three of these isolates, showing resistance or intermediate resistance to moxifloxacin (MX), were selected for this study to assess susceptibility to other fluoroquinolones (FQs) and to analyse the gyr genes, encoding the DNA gyrase subunits GyrA and GyrB. Twenty MX-susceptible isolates from the surveillance study were included for comparison. Overall, one amino acid substitution in GyrA (Thr82 to Ile) and four different substitutions in GyrB (Ser416 to Ala, Asp426 to Asn, Asp426 to Val and Arg447 to Lys) were identified. A high level of resistance (MIC >or=32 microg ml(-1)) to MX, ciprofloxacin (CI), gatifloxacin (GA) and levofloxacin (LE) was found in 68 isolates showing the amino acid substitution Thr82 to Ile in GyrA, in eight isolates with the substitutions Thr82 to Ile in GyrA and Ser416 to Ala in GyrB, in two isolates showing the substitution Asp426 to Asn in GyrB and in one isolate with Asp426 to Val in GyrB. The remaining four isolates showed high MICs for CI and LE, but different MIC levels for MX and GA. In particular, intermediate levels of resistance to MX were shown by two isolates, one with the substitution Thr82 to Ile in GyrA, and one showing Asp426 to Asn in GyrB. The substitution Arg447 to Lys in GyrB was found in two strains resistant to MX, CI and LE but susceptible to GA. No substitutions in GyrA were found in the FQ-susceptible strains, whereas two strains showed the amino acid change Ser416 to Ala in GyrB. Thr82 to Ile was the most frequent amino acid change identified in the C. difficile isolates examined. In contrast to previous observations, 10% of the isolates showed this substitution in association with Ser416 to Ala in GyrB. The other amino acid changes found were characteristic of a few strains belonging to certain types and/or countries. Two new substitutions for C. difficile, Ser416 to Ala and Arg447 to Lys, were found in GyrB. Whereas the former does not seem to have a key role in resistance, since it was also detected in susceptible strains, the latter substitution occurred in the same position where other amino acid variations take place in resistant Escherichia coli and other C. difficile strains. A large number of C. difficile isolates now show an alarming pattern of resistance to the majority of FQs currently used in hospitals and outpatient settings, therefore judicious use of these antibiotics and continuous monitoring of in vitro resistance are necessary.

Cell-mediated immunity and antibody responses to Bordetella pertussis antigens in children with a history of pertussis infection and in recipients of an acellular pertussis vaccine

Cell-mediated immunity (CMI) and antibody responses to Bordetella pertussis antigens were assessed 4-6 years after primary infant immunization with diphtheria-tetanus tricomponent acellular pertussis (DTaP) or diphtheria-tetanus (DT) vaccine in a country with high endemicity of B. pertussis infection. CMI to the B. pertussis antigens (especially to the pertussis toxin [PT]) was more frequent and/or intense in DTaP than in DT recipients. No lymphoproliferation differences were found between those with and without a history of pertussis although the DT recipients produced very little interferon-gamma after antigen (particularly PT and filamentous hemagglutinin [FHA]) stimulation. In contrast, seropositivity to PT, but not to pertactin or FHA, was more frequent in DT recipients with history of pertussis than in all other subjects. Thus, years after disease or vaccination, CMI response to PT or circulating PT antibodies appears to be the main distinctive feature of pertussis-protected DTaP recipients or pertussis-affected DT recipients.

Clostridium difficile Isolates Resistant to Fluoroquinolones in Italy: Emergence of PCR Ribotype 018

ABSTRACT Recent evidence strongly suggests an association between the use of fluoroquinolones and Clostridium difficile infection (CDI). Resistance to fluoroquinolones has been described not only in the hypervirulent strain 027, but also in other important PCR ribotypes circulating in hospital settings. In a European prospective study conducted in 2005, strains resistant to moxifloxacin represented 37.5% of C. difficile clinical isolates. In this study, we investigated a sample of 147 toxigenic C. difficile isolates, collected in Italy from 1985 to 2008, for the presence of mutations in gyr genes that conferred resistance to fluoroquinolones based on a LightCycler assay. Results were confirmed by the determination of MICs for moxifloxacin. Strains resistant to moxifloxacin were also investigated for resistance to three other fluoroquinolones and for a possible association between fluoroquinolone and macrolide-lincosamide-streptogramin B resistance. C. difficile isolates were typed by PCR ribotyping. In total, 50 clinical isolates showed substitutions in gyr genes and were resistant to fluoroquinolones. Ninety-six percent of the C. difficile resistant isolates showed the substitution Thr82-to-Ile in GyrA, as already observed in the majority of resistant strains worldwide. A significant increase of resistance (P < 0.001) was observed in the period 2002 to 2008 (56% resistant) compared to the period 1985 to 2001 (10% resistant). Coresistance with erythromycin and/or clindamycin was found in 96% (48/50) of the isolates analyzed and, interestingly, 84% of resistant strains were erm(B) negative. The majority of the fluoroquinolone-resistant isolates belonged to PCR ribotype 126 or 018. PCR ribotype 126 was the most frequently found from 2002 to 2005, whereas PCR ribotype 018 was predominant in 2007 and 2008 and still represents the majority of strains typed in our laboratory. Overall, the results demonstrate an increasing number of C. difficile strains resistant to fluoroquinolones in Italy and changes in the prevalence and type of C. difficile isolates resistant to fluoroquinolones circulating over time.

Bordetella pertussis-Infected Human Monocyte-Derived Dendritic Cells Undergo Maturation and Induce Th1 Polarization and Interleukin-23 Expression

ABSTRACT Bordetella pertussis, the causative agent of whooping cough, is internalized by several cell types, including epithelial cells, monocytes, and neutrophils. Although its ability to survive intracellularly is still debated, it has been proven that cell-mediated immunity (CMI) plays a pivotal role in protection. In this study we aimed to clarify the interaction of B. pertussis with human monocyte-derived dendritic cells (MDDC), evaluating the ability of the bacterium to enter MDDC, to survive intracellularly, to interfere with the maturation process and functional activities, and to influence the host immune responses. The results obtained showed that B. pertussis had a low capability to be internalized by—and to survive in—MDDC. Upon contact with the bacteria, immature MDDC were induced to undergo phenotypic maturation and acquired antigen-presenting-cell functions. Despite the high levels of interleukin-10 (IL-10) and the barely detectable levels of IL-12 induced by B. pertussis, the bacterium induced maturation of MDDC and T helper 1 (Th1) polarized effector cells. Gene expression analysis of the IL-12 cytokine family clearly demonstrated that B. pertussis induced high levels of the p40 and p19 subunits of IL-23 yet failed to induce the expression of the p35 subunit of IL-12. Overall our findings show that B. pertussis, even if it survives only briefly in MDDC, promotes the synthesis of IL-23, a newly discovered Th1 polarizing cytokine. A Th1-oriented immune response is thus allowed, relevant in the induction of an adequate CMI response, and typical of protection induced by natural infection or vaccination with whole-cell vaccines.