Clostridium difficile epidemiology has changed in recent years, with the emergence of highly virulent types associated with severe infections, high rates of recurrences and mortality. Antibiotic resistance plays an important role in driving these epidemiological changes and the emergence of new types. While clindamycin resistance was driving historical endemic types, new types are associated with resistance to fluoroquinolones. Furthermore, resistance to multiple antibiotics is a common feature of the newly emergent strains and, in general, of many epidemic isolates. A reduced susceptibility to antibiotics used for C. difficile infection (CDI) treatment, in particular to metronidazole, has recently been described in several studies. Furthermore, an increased number of strains show resistance to rifamycins, used for the treatment of relapsing CDI. Several mechanisms of resistance have been identified in C. difficile, including acquisition of genetic elements and alterations of the antibiotic target sites. The C. difficile genome contains a plethora of mobile genetic elements, many of them involved in antibiotic resistance. Transfer of genetic elements among C. difficile strains or between C. difficile and other bacterial species can occur through different mechanisms that facilitate their spread. Investigations of the fitness cost in C. difficile indicate that both genetic elements and mutations in the molecular targets of antibiotics can be maintained regardless of the burden imposed on fitness, suggesting that resistances may persist in the C. difficile population also in absence of antibiotic selective pressure. The rapid evolution of antibiotic resistance and its composite nature complicate strategies in the treatment and prevention of CDI. The rapid identification of new phenotypic and genotypic traits, the implementation of effective antimicrobial stewardship and infection control programs, and the development of alternative therapies are needed to prevent and contain the spread of resistance and to ensure an efficacious therapy for CDI.

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 and antibiotic resistance mechanisms were investigated in 316 Clostridium difficile clinical isolates collected during the first European surveillance on C. difficile in 2005. 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. 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. 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.

In the last three decades, Clostridium difficile infection (CDI) has increased in incidence and severity in many countries worldwide. The increase in CDI incidence has been particularly apparent among surgical patients. Therefore, prevention of CDI and optimization of management in the surgical patient are paramount. An international multidisciplinary panel of experts from the World Society of Emergency Surgery (WSES) updated its guidelines for management of CDI in surgical patients according to the most recent available literature. The update includes recent changes introduced in the management of this infection.

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.

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 ≥32 μg 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.