Background
Clostridium difficile infection (CDI) is a major cause of healthcare-associated infections worldwide. While the hypervirulent ribotype 027 strain with binary toxin has led to outbreaks only in North America and Europe [
1,
2], toxin A-negative (A
-B
+)
C. difficile infections have been reported worldwide [
3‐
5]. The A
-B
+ strain infections occur in an epidemic or sporadic form worldwide, while A
-B
+ CDI is more frequent in East Asian countries [
4,
6‐
9].
Initially toxin A was considered to be the most important factor responsible for diarrheal disease [
5]; however, several reports have documented that A
-B
+ strains can lead to similar symptoms, from mild diarrhea to severe pseudomembranous colitis [
10,
11]. The risk factors and clinical characteristics of A
-B
+ CDI have not been clearly documented.
In this study, we investigated the presence of A-B+ strains in healthcare-associated CDI (HA-CDI) in Korea, compared the clinical characteristics of A-B+ CDI and A+B+ (tcdA-positive tcdB-positive) CDI, and analyzed the risk factors for acquisition of A-B+ CDI. We also assessed the susceptibilities to clindamycin and moxifloxacin of the A-B+ and A+B+ strains.
Results
During the study period, 138 C. difficile isolates were obtained from HA-CDI patients; 11 isolates (8.0%) were confirmed by multiplex PCR to have binary toxin genes, 22 (15.9%) were A-B+ strains, and 105 (76.1%) were A+B+ strains. Consequently, 22 patients with A-B+ strains and 105 with A+B+ strains were enrolled.
Comparison of the demographic and clinical characteristics of the A-B+ and A+B+ CDI groups
Demographic and clinical characteristics were compared in the 22 A
-B
+ patients and the 105 A
+B
+ patients (Table
1). There were no differences in age, sex, length of hospital stay, history of recent surgery or Charlson score between the two groups; however, more cases of chronic obstructive pulmonary disease (COPD) as an underlying disease were identified in the A
-B
+ group (
p = 0.049). No differences were found between the two groups with regard to factors associated with the CDI severity score. Using a definition of severe CDI adapted from Zar et al. [
18,
19], severe CDI occurred at a similar rate in the A
+B
+ and the A
-B
+ group (19% and 18.2% respectively,
p = 0.925). WBC counts and CRP levels were higher and the albumin level lower in the A
+B
+ CDI group, but these differences was not statistically significant. Clinical characteristics associated with CDI, such as stool frequency, mucoid stool, abdominal pain or tenderness, were not significantly different between the groups. The A
-B
+ group was 1.067 times more likely to develop pseudomembranous colitis than the A
+B
+ group, as measured by odds ratio.
Table 1
Demographic and clinical characteristics of patients infected with Clostridium difficile tcdA-positive tcdB-positive (A+B+) strains and with tcdA-negative tcdB-positive (A-B+) strains
Sex (female) | N (%) | 54 (51.4) | 8 (36.4) | 0.244 |
Age | Mean (SD) | 65.2 (15.10) | 58.5 (19.12) | 0.143 |
Length of hospital stay | Mean (SD) | 34.8 (44.41) | 24.3 (21.20) | 0.583 |
Charlson score | Mean (SD) | 3.17 (2.482) | 2.50 (2.345) | 0.172 |
Underlying disease |
COPD | N (%) | 8 (7.6) | 5 (22.7) | 0.049 |
CVA | N (%) | 28 (26.7) | 2 (9.1) | 0.100 |
Surgical procedure | N (%) | 28 (26.7) | 2 (9.1) | 0.100 |
Severity score | Mean (SD) | 0.96 (0.746) | 1.00 (0.873) | 0.961 |
Old age b
| N (%) | 67 (63.8) | 12 (54.5) | 0.472 |
Fever c
| N (%) | 10 (9.5) | 5 (22.7) | 0.137 |
Hypoalbuminemia d
| N (%) | 10 (9.5) | 1 (4.5) | 0.688 |
Leukocytosis e
| N (%) | 14 (13.3) | 4 (18.2) | 0.515 |
Severe CDI f
| N (%) | 20 (19.0) | 4 (18.2) | 0.925 |
Clinical findings |
Pain | N (%) | 25 (23.8) | 10 (45.5) | 0.063 |
Tenderness | N (%) | 36 (34.3) | 10 (45.5) | 0.338 |
Stool Fr ≥10/day | N (%) | 21 (20.4) | 2 (9.1) | 0.362 |
Mucoid stool | N (%) | 19 (18.1) | 8 (36.4) | 0.083 |
Laboratory findings |
WBC (cells/mm3) | Mean (SD) | 10892 (5527) | 10263 (7746) | 0.239 |
Albumin (mg/dL) | Mean (SD) | 3.1 (0.57) | 3.4 (0.53) | 0.073 |
CRP (mg/dL) | Mean (SD) | 7.40 (6.397) | 5.98 (6.194) | 0.139 |
Pseudomembrane g
| N (%) | 10/26 (38.5) | 2/5 (40.0) | |
Comparison of outcomes in the A-B+ and A+B+ CDI groups
Of the 127 patients with HA-CDI, 7 were discharged before diagnosis and 3 died from an underlying disease before they had begun treatment. In 32 patients, diarrhea improved or resolved without treatment, and 85 patients (16 A
-B
+ and 69 A
+B
+ CDI cases) completed treatment (16/22 vs. 69/105,
p = 0.623). Treatment outcomes were compared in the two groups. The treatment regimens (metronidazole or vancomycin) did not differ significantly (
p = 0.681). The rates of global cure, failure and mortality were respectively 62.3%, 5.8% and 10.1% in the A
+B
+ group and 75%, 12.5% and 12.5% in the A
-B
+ group (
p = 0.398, 0.315, and 0.675, respectively) (Table
2). One case of mortality attributable to CDI occurred in the A
+B
+ group. Additionally 15 of 69 patients (21.7%) in the A
+B
+ group and none in the A
-B
+ group (0/16) experienced CDI recurrence after completion of treatment; however, the difference in recurrence rate between the groups did not reach statistical significance (
p = 0.063). Clinical outcomes were not significantly different between the metronidazole and vancomycin treatment groups (measured in all patients,
p for trend = 0.597).
Table 2
Comparison of clinical outcomes in patients infected with tcdA-positive tcdB-positive (A+B+) strains of Clostridium difficile and those infected with tcdA-negative tcdB-positive (A-B+) strains
Global cure | 43 (62.3) | 12 (75.0) | 0.398 |
Failure | 4 (5.8) | 2 (12.5) | 0.315 |
Death | 7 (10.1) | 2 (12.5) | 0.675 |
Recurrence | 15 (21.7) | 0 (0.0) | 0.063 |
Total | 69 (100.0) | 16 (100.0) | |
Comparison of previous medication history in the A-B+ and A+B+ groups
Medication histories of the patients were obtained by retrospective review of their medical records. After excluding 5 cases of HA-CDI due to incomplete medical records, 21 cases of A
-B
+ CDI and 101 cases of A
+B
+ CDI were included in the analysis (Table
3).
Table 3
Previous medication in patients infected with tcdA-positive tcdB-positive (A+B+) strains of Clostridium difficile and with tcdA-negative tcdB-positive (A-B+) strains
Medication |
PPI | N (%) | 35 (34.7) | 6 (28.6) | 0.800 |
H2 blockers | N (%) | 49 (48.5) | 8 (38.1) | 0.474 |
Probiotics | N (%) | 49 (48.5) | 13 (61.9) | 0.339 |
Steroids | N (%) | 37 (36.6) | 7 (33.3) | 1.000 |
Chemotherapy | N (%) | 12 (11.9) | 3 (14.3) | 0.722 |
Antibiotics |
All-DDD | Mean (SD) | 25.4 (18.91) | 27.0 (21.83) | 0.989 |
Days of antibiotic use | Mean (SD) | 18.7 (12.25) | 16.6 (12.23) | 0.317 |
Antibiotics use |
Fluoroquinolones | N (%) | 46 (45.5) | 14 (66.7) | 0.096 |
2nd Quinolones | N (%) | 22 (21.8) | 8 (38.1) | 0.161 |
3rd Quinolones | N (%) | 34 (33.7) | 9 (42.9) | 0.458 |
Clindamycin | N (%) | 17 (16.8) | 11 (52.4) | 0.001 |
ESC | N (%) | 57 (56.4) | 11 (52.4) | 0.811 |
BL/BLI | N (%) | 36 (35.6) | 6 (28.6) | 0.620 |
Carbapenem | N (%) | 10 (9.9) | 2 (9.5) | 1.000 |
Glycopeptides | N (%) | 20 (19.8) | 2 (9.5) | 0.360 |
Metronidazole | N (%) | 29 (28.7) | 3 (14.3) | 0.275 |
Antibiotics dose | | | | |
Fluoroquinolones | Mean (SD) | 5.606 (9.2145) | 7.138 (10.0394) | 0.202 |
2nd Quinolones | Mean (SD) | 1.858 (5.1504) | 2.210 (4.7322) | 0.168 |
3rd Quinolones | Mean (SD) | 3.737 (7.2372) | 4.929 (9.3117) | 0.484 |
Clindamycin | Mean (SD) | 1.111 (3.1081) | 4.302 (5.1853) | <0.0001 |
ESC | Mean (SD) | 5.260 (7.7728) | 3.595 (4.5188) | 0.611 |
BL/BLI | Mean (SD) | 3.825 (9.2440) | 3.536 (8.2533) | 0.611 |
Carbapenem | Mean (SD) | 0.636 (3.3368) | 0.524 (1.7210) | 0.995 |
Glycopeptides | Mean (SD) | 1.255 (3.4253) | 0.548 (1.9359) | 0.270 |
Metronidazole | Mean (SD) | 2.850 (6.9772) | 1.397 (3.7901) | 0.194 |
Previous use of proton pump inhibitors, H2 blockers, probiotics, steroids or chemotherapy was not significantly different between the groups. The most commonly prescribed antibiotics in the A+B+ CDI group were extended-spectrum cephalosporins (ESCs) (56.4%) followed by fluoroquinolones (45.5%) and beta-lactam/beta-lactamase inhibitors (35.6%), while in the A-B+ group they were fluoroquinolones (66.7%), clindamycin (52.4%) or ESCs (52.4%). Calculating the amount of antibiotics as DDD showed that, in the A+B+ group, the order of antibiotics by quantity was similar to the order by frequency of use. In the A-B+ group, antibiotics consumed in largest quantities were fluoroquinolones, clindamycin and ESCs. Finally, a comparison of previous use and amount of antibiotics in the two groups showed that both the use and amount of clindamycin were significantly higher in the A-B+ group (p = 0.001 and p < 0.0001, respectively).
Risk factors for A-B+ strain acquisition
We performed a multivariate logistic regression analysis to identify the risk factors for acquisition of
tcdA-negative strains. Age, sex, chronic obstructive pulmonary disease as an underlying disease, albumin level, and quinolone and clindamycin use were included in the analysis. Previous use of clindamycin was found to be a significant risk factor for A
-B
+
C. difficile infection (OR = 4.738, 95% CI 1.481–15.157,
p = 0.009). Age also had a statistically significant effect (OR = 0.966, 95% CI 0.935–0.998,
p = 0.038) (Table
4).
Table 4
Multivariate analysis of risk factors for tcdA-negative tcdB-positive (A-B+) Clostridium difficile infections
Age | 0.966 | 0.935 | 0.998 | 0.038 |
Sex | 1.436 | 0.482 | 4.273 | 0.516 |
Albumin | 2.584 | 0.901 | 7.412 | 0.077 |
COPD | 2.269 | 0.462 | 11.153 | 0.313 |
Fluoroquinolone use | 1.808 | 0.529 | 6.180 | 0.345 |
Clindamycin use | 4.738 | 1.481 | 15.157 | 0.009 |
Microbiologic studies
Toxinotyping was performed on the 138 isolates of C. difficile; 104 of the 105 A+B+ isolates (99.0%) belonged to toxinotype 0, and one isolate to toxinotype I; all 22 isolates of the A-B+ strain were toxinotype VIII; 8 of 11 binary toxin-producing isolates were toxinotype IV and 3 isolates, toxinotype III.
Clindamycin and moxifloxacin susceptibility testing was performed on only 123 isolates (21 A-B+ isolates and 102 A+B+ isolates) because subculture failed in the case of 4 isolates.
The A-B+ strains had significantly higher resistance rates to clindamycin and moxifloxacin than the A+B+ strains (p = 0.006 and 0.004, respectively). In the A-B+ group, the resistance rates to clindamycin and moxifloxacin were 100% (MIC range 16 6– > 256 mg/L) and 95.2% (MIC range 2–32 mg/L), respectively, whereas in the A+B+ group they were 69.6% and 63.7%, respectively, with wide ranges of MIC (MIC range 1.5– > 256 mg/L and <0.25–128 mg/L, respectively). Resistance to moxifloxacin was correlated with resistance to clindamycin (P < 0.0001). The moxifloxacin resistance rate among clindamycin-resistant A+B+ isolates was 88.7% (63/71) compared with 6.5% (2/31) among clindamycin-susceptible A+B+ isolates. By the same token, the clindamycin resistance rate for the moxifloxacin-resistant A+B + isolates was 96.9% (63/65), but only 21.6% (8/37) for the moxifloxacin-susceptible A+B+ isolates.
ErmB gene-specific PCR revealed that 17 of the 21 A-B+ isolates (81.0%) were ermB-positive. All 17 of these isolates were resistant to clindamycin, and 16 (94.1%) showed high level resistance (MIC > 256 mg/L). Among the 102 A+B+ isolates, there were 49 (48.0%) ermB-positive isolates, 37 of which (75.5%) were highly resistant to clindamycin.
Discussion
The prevalence of toxin A-negative/toxin B-positive (A
-B
+) strains among CDI isolates varies depending on the country. In most of Europe and in North America, the prevalence of A
-B
+ strains has been reported to be only 0.2–8% [
6,
24]; however, such strains are thought to have caused several outbreaks in those regions. For example, in Poland the prevalence of A
-B
+ strains has increased from 11% to 45% [
25] since there was an outbreak. By contrast, Japan has reported a high prevalence of A
-B
+ strains; the peak incidence was 39% in 2000 [
10], and the incidence decreased to 12.7% in 2005–2008 [
8]. A report from Shanghai revealed that A
-B
+ strains were responsible for a third of all CDI cases [
4]. In Korea, the frequency of A
-B
+ strains was reported to be 4.9% in 1998 [
26], and it then increased to 50.9% in 2004–2005 [
7,
11]. In our study, the frequency of A
-B
+ strains among the HA-CDI patients in 2009 was 15.9%. Even given the expected fluctuations in time and space, such a high frequency of A
-B
+ strains over the East Asian region is interesting. Possible explanations could be similar profiles of antibiotic use, and spread of the pathogen through common foods, as well as the heavy traffic of people across the three countries.
The role of toxin A and toxin B in the pathogenecity of CDI has been debated. Although early studies suggested that toxigenic strains of
C. difficile always produce both toxin A and toxin B [
5], one study showed that toxin B is the key virulence factor [
27]. However, a recent study figured out that
C. difficile producing either one or both toxins showed cytotoxic activity in vitro that translated directly into virulence in vivo [
28]. Several studies of the clinical characteristics of A
-B
+ CDI [
8,
29‐
31] have shown that A
-B
+ strains cause a wide spectrum of diseases from asymptomatic colonization to life-threatening colitis [
31], and that there is no significant difference between the clinical manifestations and outcomes of CDI caused by A
-B
+ and A
+B
+ strains [
10]. However, another study reported more cases of pseudomembranous colitis on endoscopy in A
-B
+ patients than in A
+B
+ patients (70% vs. 40%,
p = 0.0016) [
11]. Furthermore, severe CDI caused by PCR ribotype 017 was responsible for 5% of cases in Germany, and all these cases had lethal outcomes [
30]. In another report, the mortality during a PCR ribotype 017 CDI outbreak attributable to that ribotype was similar to that attributable to PCR ribotype 027 (7.0% vs. 6.5%), and higher than that attributable to other types (7.0% vs. 1.6%) [
32]. Conversely, in our study there was no significant difference in the clinical characteristics, the laboratory findings or the incidence of pseudomembranous colitis between the study groups, and there were no deaths attributable to A
-B
+ CDI. The clinical outcomes also did not differ in the two groups, although there was no A
-B
+ CDI recurrence. In previous reports, the recurrence rate of A
-B
+ CDI varied from 9 to 35.7% and was similar to that of A
+B
+ CDI [
3,
29]. The reason that we observed no recurrence and no attributable death could be simply that the number of A
-B
+ cases in this non-epidemic setting was too low. Alternatively it could be because the A
-B
+ CDI developed in younger patients with lower Charlson scores, or because the shorter hospital stays among the A
-B
+ group prevented re-infection (however that effect was not statistically significant). Another possibility is that A
-B
+ CDI recurs less frequently than A
+B
+ CDI. To investigate this, a further study with a larger number of cases would be necessary.
In order to confirm that clindamycin use is an important risk factor for A
-B
+ CDI in a clinical setting, we compared the antimicrobial susceptibilities of the A
-B
+ and A
+B
+ strains. As expected [
33], all 21 A
-B
+ strains were resistant to clindamycin. High level resistance to clindamycin (MIC > 256 mg/L) in
C. difficile is usually linked to the
ermB gene, encoding resistance to macrolide-lincosamide-streptogramin B (MLS
B) [
31]. In this study, the percentage of
ermB-positive strains was 53.7% among all
C. difficile isolates and 81.0% among the A
-B
+ isolates. Among the A
-B
+ strains, 4 of the 21 isolates were
ermB-negative, and all of these were highly resistant to clindamycin. Clindamycin resistance in these strains could also be induced by other mechanisms such as other
erm genes, efflux mechanisms, or nucleotide substitutions in other genes [
34]. Further study of these resistance mechanisms would be desirable.
In previous studies, moxifloxacin resistance was closely associated with clindamycin resistance (95% CI 68–97%) [
34,
35]. It was reported that, of isolates resistant to fluoroquinolones, 98% were resistant to either erythromycin or clindamycin, although fluoroquinolone resistance in the absence of MLS
B resistance was rare [
35]. Similarly, in our study, resistance to moxifloxacin was highly correlated with clindamycin resistance (
p < 0.0001). An efflux pump may be responsible for the co-resistance to the MLS
B drugs and fluoroquinolone.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
J Kim contributed to protocol preparation, data collection and analysis, as well as manuscript preparation. H Pai provided the funding for this study and made a major contribution to the interpretation of data and appraisal of the manuscript. M Seo participated in the microbiologic studies, and J O Kang contributed to the microbiologic studies, discussion of results and revision of the manuscript. All authors read and approved the final manuscript.