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. 2010 Aug 4;48(10):3744–3749. doi: 10.1128/JCM.01171-10

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▿ †

D Bakker 1, J Corver 1, C Harmanus 1, A Goorhuis 1, E C Keessen 3, W N Fawley 2, M H Wilcox 2, E J Kuijper 1,*
PMCID: PMC2953124  PMID: 20686080

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.

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 isolation MLVA result (no. of tandem repeats for indicated locus)
A6Cdb B7Cd C6Cd E7Cd F3Cd G8Cd H9Cd
001 A 2008 NA 19 28 8 4 4 2
002 B 2008 NA 18 26 8 4 4 2
003 E 2008 NA 24 34 8 4 4 2
004 E 2008 NA 13 18 6 4 4 2
005 B 2008 NA 15 33 8 4 3 2
006 H 2008 NA 26 27 12 4 4 2
007 GP 2008 NA 17 49 7 4 1 2
008 I 2008 NA 16 32 8 4 4 2
009 J 2008 NA 22 30 8 4 6 2
010 A 2008 NA 16 26 5 4 6 2
011 M 2008 NA 25 25 8 4 4 2
012 T 2008 NA 18 29 5 4 0 2
013 U 2008 NA 21 39 5 4 6 2
014 GP 2008 NA 21 38 8 4 3 2
015 W 2008 NA 21 38 8 4 3 2
016 X Not supplied NA 19 39 5 4 4 2
017 GP 2008 NA 21 34 8 4 5 2
018 M 2008 NA 37 36 8 4 4 2
019 AA Not supplied NA 6 42 6 4 5 2
020 T Not supplied NA 21 32 8 4 3 2
021 E 2008 NA 19 24 5 4 4 2
022 GP 2008 NA 19 32 8 4 3 2
023 AD 2008 NA 19 32 8 4 3 2
024 E 2008 NA 19 38 5 4 5 2
025 E 2008 NA 37 36 8 4 4 2
026 GP 2008 NA 37 36 8 4 4 2
027 GP 2008 NA 37 36 8 4 4 2
028 M 2008 NA 19 38 5 4 5 2
029 AA Not supplied NA 21 39 5 4 6 2
030 AH 2008 NA 35 43 5 4 5 2
031 M 2008 NA 19 37 5 4 4 2
032 M 2008 NA 6 43 6 4 5 2
033 GP 2008 NA 21 41 5 4 6 2
034 AI 2008 NA 24 33 8 4 4 2
035 AJ 2008 NA 21 43 5 4 5 2
036 E 2008 NA 24 33 8 4 4 2
037 AJ 2008 NA 21 43 5 4 3 2
038 AI 2008 NA 21 39 8 4 3 2
039 AK 2008 NA 19 24 5 4 4 2
040 AI 2008 NA 24 33 8 4 4 2
041 AK 2008 NA 20 45 5 4 0 2
042 M 2008 NA 17 33 8 4 4 2
043 M 2008 NA 24 33 8 4 4 2
044 M 2008 NA 37 36 8 4 4 2
045 K 2008 NA 15 23 8 4 5 2
046 D Not supplied NA 18 26 8 4 4 2
047 G 2008 NA 19 32 8 4 8 2
048 G 2008 NA 28 34 8 4 4 2
049 C 2007 NA 20 33 8 4 3 2
050 G 2008 NA 19 24 5 4 4 2
051 AB 2007 NA 6 43 6 4 5 2
052 AC 2007 NA 15 43 7 4 5 2
053 D 2007 NA 6 42 6 4 5 2
054 F 2008 NA 19 39 5 4 5 2
055 Y 2007 NA 29 32 8 4 0 2
056 Y 2007 NA 37 36 8 4 4 2
057 AF 2008 NA 19 38 5 4 5 2
058 L 2007 NA 15 32 8 4 8 2
059 V 2007 NA 21 34 8 4 3 2
060 Z 2007 NA 20 37 5 4 4 2
061 AE 1953 NA 21 38 8 4 3 2
062 AL Not supplied NA 24 26 8 4 4 2
063 O 2007 NA 17 43 5 4 4 2
064 S 2008 NA 21 34 8 4 3 2
065 P Not supplied NA 35 38 8 4 3 2
066 Q 2008 NA 21 38 8 4 3 2
067 R 2008 NA 21 38 8 4 3 2
068 AL 2006 NA 26 38 12 2 4 8
069 AM 2007 NA 26 33 10 2 4 9
070 AM 2007 NA 26 33 11 2 4 9
071 AL 2007 NA 29 40 9 2 4 8
072 AN 2007 NA 24 41 9 2 4 8
073 AO 2007 NA 25 35 13 2 4 8
074 AP 2007 NA 20 20 12 2 4 8
075 AZ 2006 NA 26 34 11 2 4 8
076 AZ 2006 NA 21 34 12 2 4 9
077 AV 2007 NA 21 37 12 2 4 8
078 BB 2007 NA 31 35 11 2 4 8
079 BQ 2007 NA 21 30 9 2 4 8
080 AQ 2006 NA 23 30 9 2 4 8
081 BE 2006 NA 20 41 12 2 4 8
082 BF 2006 NA 22 31 10 2 4 8
083 BG 2006 NA 24 31 11 2 4 8
084 BE 2007 NA 31 36 7 2 4 8
085 BG 2006 NA 27 31 10 2 4 8
086 BG 2006 NA 23 33 9 2 4 8
087 BG 2006 NA 23 33 9 2 4 8
088 AR 2006 NA 23 30 11 2 4 8
089 AU 2007 NA 25 39 12 2 4 8
090 BA 2006 NA 23 25 11 2 4 8
091 BJ 2006 NA 26 32 10 2 4 8
092 AT 2007 NA 18 52 11 2 4 8
093 BI 2006 NA 27 34 12 2 4 8
094 AY 2007 NA 29 40 9 2 4 8
095 AX 2007 NA 29 40 9 2 4 8
096 AW 2007 NA 26 43 13 2 4 8
097 BC 2006 NA 19 28 10 2 4 8
098 BD 2007 NA 18 32 9 2 4 8
099 BP 2006 NA 23 41 12 2 4 8
100 BK 2007 NA 18 33 10 2 4 8
101 BK 2007 NA 18 33 10 2 4 8
102 AS 2005 NA 18 33 9 2 4 8
103 BL 2006 NA 21 37 9 2 4 8
104 BL 2006 NA 21 37 9 2 4 8
105 BM 2007 NA 23 36 11 2 4 8
106 BN 2007 NA 19 32 9 2 4 8
107 BN 2007 NA 19 32 9 2 4 8
108 BN 2007 NA 13 25 9 2 4 8
109 BN 2007 NA 18 34 4 2 4 8
110 BN 2007 NA 13 32 9 2 4 8
111 BN 2007 NA 18 33 9 2 4 8
112 BN 2007 NA 18 34 4 2 4 8
113 BO 2007 NA 21 37 9 2 4 8
114 BR 2009 NA 8 43 8 4 4 2
115 BR 2009 NA 28 43 8 4 4 2
116 BS 2009 NA 25 33 8 4 6 2
117 BS 2009 NA 21 32 8 4 3 2
118 BS 2009 NA 25 32 8 4 3 2
119 BS 2009 NA 25 32 8 4 3 2
120 BS 2009 NA 24 30 8 4 3 2
121 BS 2009 NA 23 31 8 4 3 2
122 BS 2009 NA 23 32 8 4 3 2
123 BS 2009 NA 24 30 8 4 3 2
124 BS 2009 NA 23 32 8 4 3 2
125 BT 2009 NA 20 45 8 4 4 2
126 BT 2009 NA 20 44 8 4 4 2
127 BT 2009 NA 16 38 8 4 4 2
128 BT 2009 NA 18 40 8 4 5 2
129 BU 2009 NA 21 34 8 4 4 2
130 BU 2009 NA 18 37 8 4 4 2
131 BU 2009 NA 15 35 8 4 4 2
132 BU 2009 NA 16 35 8 4 4 2
133 BU 2009 NA 16 35 8 4 4 2
134 BV 2009 NA 16 39 8 4 3 2
135 BV 2009 NA 15 38 8 4 3 2
136 BV 2009 NA 16 36 8 4 4 2
137 BV 2009 NA 28 38 8 4 5 2
138 BV 2009 NA 21 38 8 4 3 2
139 BV 2009 NA 21 38 8 4 5 2
140 BV 2009 NA 21 38 8 4 5 2
141 BV 2009 NA 15 38 8 4 4 2
142 BV 2009 NA 15 35 8 4 3 2
143 BV 2009 NA 16 38 8 4 5 2
144 BW 2009 NA 16 36 8 4 4 2
145 BW 2009 NA 17 33 8 4 4 2
146 BW 2009 NA 21 38 8 4 4 2
147 BW 2009 NA 20 37 8 4 4 2
148 BW 2009 NA 17 34 8 4 4 2
149 BW 2009 NA 23 34 8 4 4 2
150 BW 2009 NA 11 35 8 4 4 2
151 BX 2009 NA 8 35 8 4 1 2
152 BX 2009 NA 18 33 8 4 6 2
153 BX 2009 NA 18 33 8 4 4 2
154 BX 2009 NA 18 32 8 4 4 2
155 BX 2009 NA 20 33 8 4 3 2
156 BX 2009 NA 18 33 8 4 3 2
157 BX 2009 NA 8 35 8 4 6 2
158 BX 2009 NA 12 34 8 4 3 2
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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.

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.

FIG. 1.

FIG. 1.

Open in a new tab

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.

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.

Footnotes

▿

Published ahead of print on 4 August 2010.

†

The authors have paid a fee to allow immediate free access to this article.

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