Patrizia Spigaglia

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.

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.

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.

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.

ErmB Determinants and Tn916-Like Elements in Clinical Isolates of Clostridium difficile

ABSTRACT Erythromycin and tetracycline resistance was analyzed in 37 Clostridium difficile clinical isolates. Strains of different clonal origins showed different erythromycin and tetracycline resistance determinants and different genetic arrangements of the elements. In strains of recent isolation, the presence of Tn916-like elements, never found before in C. difficile clinical isolates, has been demonstrated.

Immunomodulatory activities of surface-layer proteins obtained from epidemic and hypervirulent Clostridium difficile strains.

Surface-layer proteins (SLPs) have been detected in all Clostridium difficile strains and play a role in adhesion, although an involvement in the inflammatory process may also be supposed, as they cover the bacterial surface and are immunodominant antigens. The aim of this study was to evaluate the immunomodulatory properties of SLPs obtained from hypervirulent and epidemic (H/E) or non-H/E C. difficile strains, to try to determine whether they contribute to hypervirulence. SLPs were purified from H/E PCR ribotype 027 and 001 and non-H/E PCR ribotype 012 C. difficile strains, and the ability to modulate these properties was studied in human ex vivo models of monocytes and monocyte-derived dendritic cells (MDDCs). The results indicated that SLPs were able to induce immunomodulatory cytokines [interleukin (IL)-1β, IL-6 and IL-10] in monocytes. SLPs induced maturation of MDDCs, which acquired enhanced antigen-presenting activity, a crucial function of the mature stage. SLP-primed MDDCs expressed high levels of IL-10, an important regulatory cytokine. No significant differences were found in the activation induced in monocytes and MDDCs by SLP preparations from H/E and non-H/E strains. Overall, these findings show an important role for SLPs in modulation of the immune response to C. difficile. However, SLPs from H/E strains did not show a specific immunomodulatory pattern compared with SLPs from non-H/E strains, suggesting that SLPs are not involved in the increased severity of infection peculiar to H/E strains.

Analysis of metronidazole susceptibility in different Clostridium difficile PCR ribotypes

OBJECTIVES Susceptibility to metronidazole was investigated in 81 Clostridium difficile strains, belonging to nine different PCR ribotypes, by three different laboratory methods. METHODS MICs for 81 C. difficile clinical isolates were determined by Etest, the agar dilution method (ADM) and the agar incorporation method (AIM). Twenty selected strains were also subjected to subinhibitory concentrations of metronidazole and the MIC heterogeneity was analysed in colonies from each strain that showed increased values before and after exposure to the antibiotic, using ADM and AIM. RESULTS Overall, the MICs obtained by Etest were lower compared with those obtained by ADM and AIM, causing discrepancies in the categorization (as susceptible or having reduced susceptibility) of some strains. Reduced susceptibility to metronidazole was observed using both ADM and AIM, with higher MIC values by AIM in isolates belonging to PCR ribotypes 001 and 010. An increase in MICs after exposure to metronidazole was observed for strains belonging to these PCR ribotypes (by Etest and ADM, but not by AIM). In particular, MICs for colonies from strains belonging to either PCR ribotype 001 or 010 were less heterogeneous by AIM compared with by ADM, suggesting a better ability of AIM to detect strains with reduced susceptibility. CONCLUSIONS These results suggest that the presence of C. difficile subpopulations with reduced susceptibility to metronidazole in the human intestine may be one of the factors responsible for reduced antibiotic efficacy in vivo. The possibility that higher MICs may have often gone unnoticed underlines the importance of choosing the best method for MIC determination and the necessity to monitor C. difficile susceptibility to metronidazole.

Molecular Analysis of the gyrA and gyrB Quinolone Resistance-Determining Regions of Fluoroquinolone-Resistant Clostridium difficile Mutants Selected In Vitro

ABSTRACT Recent studies have suggested that exposure to fluoroquinolones represents a risk factor for the development of Clostridium difficile infections and that the acquisition of resistance to the newer fluoroquinolones is the major reason facilitating wide dissemination. In particular, moxifloxacin (MX) and levofloxacin (LE) have been recently associated with outbreaks caused by the C. difficile toxinotype III/PCR ribotype 027/pulsed-field gel electrophoresis type NAP1 strain. In this study, we evaluated the potential of MX and LE in the in vitro development of fluoroquinolone resistance mediated by GyrA and GyrB alterations. Resistant mutants were obtained from five C. difficile parent strains, susceptible to MX, LE, and gatifloxacin (GA) and belonging to different toxinotypes, by selection in the presence of increasing concentrations of MX and LE. Stable mutants showing substitutions in GyrA and/or GyrB were obtained from the parent strains after selection by both antibiotics. Mutants had MICs ranging from 8 to 128 μg/ml for MX, from 8 to 256 μg/ml for LE, and from 1.5 to ≥32 μg/ml for GA. The frequency of mutation ranged from 3.8 × 10−6 to 6.6 × 10−5 for MX and from 1.0 × 10−6 to 2.4 × 10−5 for LE. In total, six different substitutions in GyrA and five in GyrB were observed in this study. The majority of these substitutions has already been described for clinical isolates or has occurred at positions known to be involved in fluoroquinolone resistance. In particular, the substitution Thr82 to Ile in GyrA, the most common found in resistant C. difficile clinical isolates, was observed after selection with LE, whereas the substitution Asp426 to Val in GyrB, recently described in toxin A-negative/toxin B-positive epidemic strains, was observed after selection with MX. Interestingly, a reduced susceptibility to fluoroquinolones was observed in colonies isolated after the first and second steps of selection by both MX and LE, with no substitution in GyrA or GyrB. The results suggest a relevant role of fluoroquinolones in the emergence and selection of fluoroquinolone-resistant C. difficile strains also in vivo.