Research Article
01 October 2010

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

ABSTRACT

Totals of 102 and 56 Clostridium difficile type 078 strains of human and porcine origins, respectively, from four European countries were investigated by an optimized multilocus variable-number tandem-repeat analysis (MLVA) and for tetracycline susceptibility. Eighty-five percent of all isolates were genetically related, irrespective of human or porcine origin. Human strains were significantly more resistant to tetracycline than porcine strains. All tetracycline-resistant strains contained the Tn916-like transposon harboring the tet(M) gene. We conclude that strains from human and porcine origins are genetically related, irrespective of the country of origin. This may reflect a lack of diversity and/or common source.
Recently, we reported that Clostridium difficile PCR ribotype 078 (type 078) is an increasing cause of Clostridium difficile infections (CDI) in humans in the Netherlands, with disease severity similar to that of the hypervirulent type 027 (6). Also, the incidence of CDI in England caused by type 078 has increased (24). In addition, recent studies have demonstrated that type 078 is the predominant type in cattle and pigs (11, 18). In the Netherlands, we have noticed an overlap in the occurrence of human CDI cases caused by type 078 and the distribution of pig farms in the eastern part of the country (6). This suggests a possible link between human and porcine type 078 strains.
To investigate the relatedness between human and porcine type 078 strains, we applied a multilocus variable-number tandem-repeat analysis (MLVA) developed for C. difficile to a collection of type 078 isolates (6, 7, 22). This MLVA has been proven to be more discriminatory than other genotyping methods (5, 12). Since it has been suggested that the wide dissemination of Staphylococcus aureus sequence type 398 (ST398) in pigs and humans is associated with the frequent usage of tetracycline in pig farms, we also investigated susceptibility to tetracycline and the genetic origin of tetracycline resistance (4, 14, 25).

Clostridium difficile strains.

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.

Modification of MLVA.

MLVA was adjusted for type 078 due to the lack of specific PCR products for the Clostridium difficile A6 (A6Cd), B7Cd, C6Cd, and G8Cd 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 B7Cd, C6Cd, and G8Cd and the absence of locus A6Cd. We adjusted only the magnesium chloride concentration (4 mM) and annealing temperatures for the B7Cd and G8Cd loci (47°C) and also the C6Cd 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 A6Cd 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.

Tetracycline susceptibility.

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.

Application of optimized MLVA.

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.

Conclusions.

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.
FIG. 1.
FIG. 1. Minimum spanning tree analysis of 158 C. difficile type 078 isolates by MLVA. Each circle represents either one unique isolate or more isolates that have identical MLVA types. The numbers between the circles represent the summed tandem-repeat difference (STRD) between MLVA types. Thick lines represent single-locus variants, thin lines represent double-locus variants, and the interrupted lines represent triple-locus variants between MLVA types. Clonal clusters are defined by an STRD of ≤2, and genetically related clusters are defined by an STRD of ≤10. Porcine (n = 56) isolates and the various human isolates are shown in different colors and fonts as shown in the key (on the right side of the figure). The numbers represent isolates from different geographical locations. The Leeds collection consists of isolates from Northern Ireland (1 to 44), Yorkshire and Humber (45 to 53), southwest England (54 to 57), northwest England (58 to 62), eastern England (63 and 64), and Ireland (65 to 67). The Leiden collection consists of isolates from North Holland (68 to 76), South Holland (77 to 79), Utrecht (80 to 87), Gelderland (88 to 91), Brabant (92 and 93), Groningen (94 and 95), Friesland (96), Flevoland (97), Limburg (98), Overijssel (99 to 101), and Belgium (102). Porcine strains are numbers 103 to 158.
TABLE 1.
TABLE 1. For each strain, the results of the optimized MLVA for each of the 7 loci (A6Cd, B7Cd, C6Cd, G8Cd, E7Cd, F3Cd, and H9Cd)a
MST isolate no.Location of isolation (deidentified)Yr of isolationMLVA result (no. of tandem repeats for indicated locus)      
   A6CdbB7CdC6CdE7CdF3CdG8CdH9Cd
001A2008NA19288442
002B2008NA18268442
003E2008NA24348442
004E2008NA13186442
005B2008NA15338432
006H2008NA262712442
007GP2008NA17497412
008I2008NA16328442
009J2008NA22308462
010A2008NA16265462
011M2008NA25258442
012T2008NA18295402
013U2008NA21395462
014GP2008NA21388432
015W2008NA21388432
016XNot suppliedNA19395442
017GP2008NA21348452
018M2008NA37368442
019AANot suppliedNA6426452
020TNot suppliedNA21328432
021E2008NA19245442
022GP2008NA19328432
023AD2008NA19328432
024E2008NA19385452
025E2008NA37368442
026GP2008NA37368442
027GP2008NA37368442
028M2008NA19385452
029AANot suppliedNA21395462
030AH2008NA35435452
031M2008NA19375442
032M2008NA6436452
033GP2008NA21415462
034AI2008NA24338442
035AJ2008NA21435452
036E2008NA24338442
037AJ2008NA21435432
038AI2008NA21398432
039AK2008NA19245442
040AI2008NA24338442
041AK2008NA20455402
042M2008NA17338442
043M2008NA24338442
044M2008NA37368442
045K2008NA15238452
046DNot suppliedNA18268442
047G2008NA19328482
048G2008NA28348442
049C2007NA20338432
050G2008NA19245442
051AB2007NA6436452
052AC2007NA15437452
053D2007NA6426452
054F2008NA19395452
055Y2007NA29328402
056Y2007NA37368442
057AF2008NA19385452
058L2007NA15328482
059V2007NA21348432
060Z2007NA20375442
061AE1953NA21388432
062ALNot suppliedNA24268442
063O2007NA17435442
064S2008NA21348432
065PNot suppliedNA35388432
066Q2008NA21388432
067R2008NA21388432
068AL2006NA263812248
069AM2007NA263310249
070AM2007NA263311249
071AL2007NA29409248
072AN2007NA24419248
073AO2007NA253513248
074AP2007NA202012248
075AZ2006NA263411248
076AZ2006NA213412249
077AV2007NA213712248
078BB2007NA313511248
079BQ2007NA21309248
080AQ2006NA23309248
081BE2006NA204112248
082BF2006NA223110248
083BG2006NA243111248
084BE2007NA31367248
085BG2006NA273110248
086BG2006NA23339248
087BG2006NA23339248
088AR2006NA233011248
089AU2007NA253912248
090BA2006NA232511248
091BJ2006NA263210248
092AT2007NA185211248
093BI2006NA273412248
094AY2007NA29409248
095AX2007NA29409248
096AW2007NA264313248
097BC2006NA192810248
098BD2007NA18329248
099BP2006NA234112248
100BK2007NA183310248
101BK2007NA183310248
102AS2005NA18339248
103BL2006NA21379248
104BL2006NA21379248
105BM2007NA233611248
106BN2007NA19329248
107BN2007NA19329248
108BN2007NA13259248
109BN2007NA18344248
110BN2007NA13329248
111BN2007NA18339248
112BN2007NA18344248
113BO2007NA21379248
114BR2009NA8438442
115BR2009NA28438442
116BS2009NA25338462
117BS2009NA21328432
118BS2009NA25328432
119BS2009NA25328432
120BS2009NA24308432
121BS2009NA23318432
122BS2009NA23328432
123BS2009NA24308432
124BS2009NA23328432
125BT2009NA20458442
126BT2009NA20448442
127BT2009NA16388442
128BT2009NA18408452
129BU2009NA21348442
130BU2009NA18378442
131BU2009NA15358442
132BU2009NA16358442
133BU2009NA16358442
134BV2009NA16398432
135BV2009NA15388432
136BV2009NA16368442
137BV2009NA28388452
138BV2009NA21388432
139BV2009NA21388452
140BV2009NA21388452
141BV2009NA15388442
142BV2009NA15358432
143BV2009NA16388452
144BW2009NA16368442
145BW2009NA17338442
146BW2009NA21388442
147BW2009NA20378442
148BW2009NA17348442
149BW2009NA23348442
150BW2009NA11358442
151BX2009NA8358412
152BX2009NA18338462
153BX2009NA18338442
154BX2009NA18328442
155BX2009NA20338432
156BX2009NA18338432
157BX2009NA8358462
158BX2009NA12348432
a
All strains have an isolate number which corresponds to the number used in the MST (Fig. 1). Geographical locations correspond as follows: Northern Ireland, 1 to 44; Yorkshire and Humber, 45 to 53; southwest England, 54 to 57; northwest England, 58 to 62; eastern England, 63 and 64; Ireland, 65 to 67; North Holland, 68 to 76; South Holland, 77 to 79; Utrecht, 80 to 87; Gelderland, 88 to 91; Brabant, 92 and 93; Groningen, 94 and 95; Friesland, 96; Flevoland, 97; Limburg, 98; Overijssel, 99 to 101; Belgium, 102. Porcine strains are numbered 103 to 158. For each strain, a deidentified location (institute or pig farm) of isolation, year of isolation, and MLVA results for each locus are described.
b
NA, not applicable.

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cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 48Number 10October 2010
Pages: 3744 - 3749
PubMed: 20686080

History

Received: 9 June 2010
Revision received: 6 July 2010
Accepted: 29 July 2010
Published online: 1 October 2010

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D. Bakker
Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands
J. Corver
Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands
C. Harmanus
Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands
A. Goorhuis
Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands
E. C. Keessen
Institute for Risk Assessment Sciences, Veterinary Public Health Division, University of Utrecht, Utrecht, Netherlands
W. N. Fawley
Department of Microbiology, Leeds Teaching Hospitals and University of Leeds, Leeds, United Kingdom
M. H. Wilcox
Department of Microbiology, Leeds Teaching Hospitals and University of Leeds, Leeds, United Kingdom
E. J. Kuijper [email protected]
Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands

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