Relatedness of Human and Animal Clostridium difficile PCR Ribotype 078 Isolates Determined on the Basis of Multilocus Variable-Number Tandem-Repeat Analysis and Tetracycline Resistance

Totals of 102 and 56 type 078 strains of human and porcine origins, respectively, were available for this study. Table 1 lists the location of isolation and the year of isolation of each strain. All human type 078 strains were recovered from diarrheal patients. The “Leeds collection” (n = 67) consisted of 44 strains originating from an outbreak in Northern Ireland, 20 strains from other parts of the United Kingdom, and 3 strains originating from Ireland. The “Leiden collection” consisted of 35 strains from endemic cases in the years 2006 and 2007. The 56 porcine strains were collected from 11 Dutch pig farms in the years 2006, 2007, and 2009. All pig farms had the problem of persistent neonatal diarrhea. Forty-seven (84%) isolates were recovered from diarrheal piglets.

MLVA was adjusted for type 078 due to the lack of specific PCR products for the Clostridium difficile A6 (A6_Cd), B7_Cd, C6_Cd, and G8_Cd loci. Sequence analysis of 7 human type 078 strains and 8 porcine type 078 strains revealed multiple mismatches in the primer annealing sites for loci B7_Cd, C6_Cd, and G8_Cd and the absence of locus A6_Cd. We adjusted only the magnesium chloride concentration (4 mM) and annealing temperatures for the B7_Cd and G8_Cd loci (47°C) and also the C6_Cd locus (46°C). All MLVA PCRs were performed in a singleplex format. MLVA PCRs for the other loci and the analysis of the MLVA data were performed as previously described (22). The calculated variable number of tandem repeats (VNTR) of the adjusted MLVA was in complete concordance with the manually measured VNTR. The absence of the A6_Cd locus could theoretically result in less discriminative power of the MLVA. Therefore, we reanalyzed the MLVA (based on 7 loci) on previously typed ribotype 027 (n = 57) and 017 (n = 71) strains (7, 22). This reanalysis based on 6 loci resulted in equal numbers of genetically related clusters (GCs) and clonal complexes (CCs) (as based on 7 loci). Subsequently, we concluded that the optimized MLVA for type 078, based on 6 loci, is equally capable of discriminating between strains from various countries and origins.

All strains were tested for their susceptibility to tetracycline. The breakpoint for tetracycline was defined as a MIC of ≥8 mg/liter (2). Seventy-five of the 102 human strains and 15 of the 56 porcine strains were resistant to tetracycline. Tetracycline resistance was not found among randomly selected isolates of the 2 most common human types, types 001 (n = 10) and 014 (n = 10). There were significantly more human strains resistant to tetracycline than resistant porcine strains (P < 0.005; chi-square method). This difference could be explained by the fact that we included 44 outbreak strains. Thirty-eight (86%) of the outbreak strains were resistant to tetracycline, as opposed to 15 (65%) resistant strains originating from other parts of the United Kingdom and Ireland.

The origin of tetracycline resistance was investigated by detection of mobile elements, namely the Tn5397-like and Tn916-like transposons, as previously described (1). All tetracycline-resistant strains contained the Tn916-like transposon, harboring the tet(M) gene. Filter mating experiments demonstrated the transfer of the Tn916-like transposon from a donor strain to a recipient strain. We did not detect either of the transposons in tetracycline-susceptible strains. This observation suggests a high degree of relatedness of human and porcine isolates and is in agreement with recently published findings (1). However, we cannot exclude the possibility of horizontal transfer of the Tn916-like transposon, since this transposon is widely distributed in Gram-positive bacteria and additional data for tetracycline-resistant non-078-type strains are required (16, 17, 21). Recent publications show that tetracycline resistance is predominantly present in types 012, 017, 046, and 078 (2, 9, 15). A screening of randomly selected strains of these types demonstrated that tetracycline resistance in types 012 and 046 is conferred by the Tn5397-like transposon, whereas the tetracycline resistance in types 017 and 078 is conferred by the Tn916-like transposon.

Table 1 depicts the results of the MLVA of each strain per locus. A minimal spanning tree (MST) was constructed to determine the genetic relationships among strains as previously described (Fig. 1) (6). In total, 116 strains belonged to one of the GCs, defined by a summed tandem repeat difference (STRD) of ≤10. The largest GC (shown as green in Fig. 1) contained 103 strains, encompassing 47 porcine strains, 41 Leeds collection strains, and 15 Leiden collection strains. Fifty-five strains were susceptible to tetracycline, and 48 strains were resistant to tetracycline. The GC shown in yellow in Fig. 1 contained only strains which originated in the Netherlands, and these encompassed both tetracycline-susceptible and -resistant strains. The GC shown in blue in Fig. 1 contained only porcine strains susceptible to tetracycline which originated from one pig farm. The last GC (shown in purple in Fig. 1) contained only tetracycline-resistant strains from the Leiden collection. Sixteen of the 23 recognized CCs (defined by an STRD of ≤2) belonged to the largest GC, whereas the other 7 CCs were either single- or double-locus variants of the largest GC. Five CCs contained only porcine strains, and 13 CCs contained only human strains, of which 8 CCs were derived from the Leeds collection and 5 CCs were derived from the Leiden collection. The remaining 5 CCs contained both porcine and human strains of various countries. Two CCs contained human strains isolated in different locations from a specific region. Nine of the 23 CCs contained outbreak strains and strains from distinct settings. Interestingly, 12 CCs contained only strains resistant to tetracycline and 8 CCs contained both tetracycline-susceptible and -resistant strains. The remaining 3 CCs contained only tetracycline-susceptible strains. In total, 4 MLVA profiles that contained both human and porcine strains could be recognized. Overall, the MST could not differentiate between geographical origins or tetracycline phenotypes, irrespective of human or porcine origin.

The suggested high-level relatedness between human and porcine type 078 strains is in concordance with earlier publications based on MLVA, whole-genome analysis, and multilocus sequence typing (3, 8, 13, 20). The relatedness between human and porcine type 078 strains in this study could be an indication of a common source, as suggested in previous publications (3, 6, 20). However, the geographical locations of some related isolates are very distinct and cannot logically be explained by any direct epidemiological link. A possible common source of type 078 could be further supported by the observation that all tetracycline-resistant strains contain the mobile element Tn916-like transposon, which has also been described for tetracycline-resistant enterococci from human and porcine origins (1). Interspecies transmission or transmission through meat are suggested as sources of infection, but these are not yet established (10, 19, 23). However, data for a direct epidemiological link between human and porcine strains in this study are lacking. We also need to consider that the high-level relatedness between human and porcine type 078 strains could be the consequence of less natural variability in type 078 than in other types. A limitation of this study is the inclusion of porcine strains from only one country, while human strains were derived from 4 European countries. Further studies are needed to investigate the possible transmission routes between humans and animals.