Surveillance and outbreak reports
Clostri di u m di ffiCi le ri botypes 001, 017, an d 027
are assoCiate d with lethal C. di ffiCi le i n feCtion i n
hesse, Ge rmany
M Arvand (email@example.com)1, A M Hauri1, N H Zaiss2, W Witte2, G Bettge-Weller1
1. Hesse State Health Office, Centre for Health Protection, Dillenburg, Germany
2. Robert Koch Institute, Wernigerode, Germany
This article was published on 12 November 2009.
Citation style for this article: Arvand M, Hauri AM, Zaiss NH, Witte W, Bettge-Weller G. Clostridium difficile ribotypes 001, 017, and 027 are associated with lethal C.
difficile infection in Hesse, Germany. Euro Surveill. 2009;14(45):pii=19403. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19403
From January 2008 to April 2009, 72 cases of severe Clostridium
difficile infection were reported from 18 different districts in the
state of Hesse, Germany. A total of 41 C. difficile isolates from 41
patients were subjected to PCR ribotyping. PCR ribotype (RT) 027
was the most prevalent strain accounting for 24 of 41 (59%) of
typed isolates, followed by RT 001 (eight isolates, 20%), RT 017
and 042 (two isolates each), and RT 003, 066, 078, 081, and
RKI-034 (one isolate each). Eighteen patients had died within
30 days after admission. C. difficile was reported as underlying
cause of or contributing to death in 14 patients, indicating a case
fatality rate of 19%. The patients with lethal outcome attributable
to C. difficile were 59-89 years-old (median 78 years). Ribotyping
results were available for seven isolates associated with lethal
outcome, which were identified as RT 027 in three and as RT 001
and 017 in two cases each. Our data suggest that C. difficile RT
027 is prevalent in some hospitals in Hesse and that, in addition
to the possibly more virulent RT 027, other toxigenic C. difficile
strains like RT 001 and 017 are associated with lethal C. difficile
infections in this region.
Clostridium difficile infection (CDI) is a major cause of
morbidity and mortality from healthcare-associated infections
in economically developed countries. CDI is primarily linked
with hospital admission and prior antimicrobial treatment. The
symptoms can range from mild diarrhoea to serious manifestations
such as pseudomembranous colitis, toxic megacolon or perforation
of colon . In recent years, a hypervirulent strain, which has
been characterised by pulsed field gel-electrophoresis as North
American pulsed-field gel electrophoresis type 1 (NAP1) and by
PCR as ribotype (RT) 027, has emerged in North America, Canada,
and several European countries [2-6]. This strain has primarily
been described in association with hospital outbreaks but may also
cause community-acquired infection. RT 027 is characterised by
production of C. difficile toxins A and B and a third toxin (binary
toxin), deletions in the regulatory gene tcdC that potentially
allow increased toxin A and B production, and resistance to new
fluoroquinolones such as moxifloxacin [7,8].
In Germany, a hospital associated outbreak of the C. difficile
RT 027 strain was reported in 2007 from Rheinland-Palatina in
south-western Germany . Since then, RT 027 has sporadically
been isolated in other geographic regions of Germany . A recent
study found a high prevalence (55%) of C. difficile RT 001 in
patients with C. difficile-associated diarrhoea (CDAD) in southern
Germany . Isolates corresponding to RT 001 did not contain
the binary toxin genes cdtA and cdtB and displayed resistance to
moxifloxacin and erythromycin .
In December 2007, a requirement for mandatory notification
of severe CDI was introduced in Germany . According to this
requirement, severe CDI was defined as pseudomembranous colitis
confirmed by endoscopy or histology, or CDAD or toxic megacolon
with positive laboratory results for C. difficile associated with one
of the following conditions:
• readmission to the hospital because of recurrent CDI,
• admission to intensive care unit because of CDAD or its
• abdominal surgery because of toxic megacolon, perforation or
• death within 30 day after CDAD, with CDI as underlying cause
or contributing to death,
• detection of RT 027.
The Hesse State Health Office (HSHO) receives notifications on
severe CDI from local health authorities of the state of Hesse, which
is located in western Germany and has approximately six million
inhabitants. Following the introduction of the federal notification
requirement, we initiated a pilot study to characterise C. difficile
isolates associated with severe CDI in Hesse by offering for free
a complete microbiological diagnostic service including culture,
toxin detection, antimicrobial resistance testing and ribotyping
to those healthcare facilities in Hesse that do not have access to
these analyses. In this report, we present the results of our study
during the first 16 months after introduction of these measures.
Patients and methods
From January 2008 to April 2009, 60 patients with notifiable
CDI were reported by local health authorities via electronic
notification system (SurvNet) to the HSHO. A total of 24
C. difficile isolates from 24 of these patients had been submitted
by the microbiological laboratories of the respective hospitals to a
national reference laboratory for C. difficile (Institute for Medical
Microbiology, University of Mainz, or Robert Koch Institute (RKI),
Wernigerode, Germany) for ribotyping. The ribotyping results of
these isolates were reported to HSHO along with the case reports
and corresponded in 23 of 24 cases to RT 027.
In addition, we received 22 stool samples from 17 patients
with severe CDI that were sent to the microbiological laboratory
of HSHO for detection and molecular typing of C. difficile during
the study period. Comparison of the electronic notification reports
with the data of these 17 patients revealed that 12 of them had
not been reported by the electronic notification system. These
cases were additionally enrolled in this study. The 17 patients
were hospitalised in 13 different hospitals. Seventeen isolates
(one isolate per patient) were forwarded to the national reference
laboratory at the RKI for PCR ribotyping.
C. difficile culture, toxin analysis, and antimicrobial susceptibility
Faecal culture for C. difficile was performed on C. difficile-
selective agar containing cycloserine, cefoxitin, and amphotericin
B (Bio Mérieux) under anaerobic conditions. Identification of
C. difficile was performed by routine microbiologic techniques and a
rapid confirmatory latex agglutination test for C. difficile (Microgen
Bioproducts). Twelve of 17 C. difficile isolates that were isolated in
the HSHO laboratories were tested for in vitro toxin production with
an ELISA detecting toxin A and/or B (Biopharm). Of the remaining
five cases, four had been tested positive for toxin A/B directly from
the stool specimen and were therefore considered to be toxin-
positive. One isolate was lost because of fungal contamination
and could not be used for ELISA or antimicrobial susceptibility
testing. Sixteen isolates were subjected to susceptibility testing for
erythromycin and moxifloxacin by E-test (AB-Biodisc).
PCR ribotyping was performed at the RKI according the protocol
of Bidet et al. , except that PCR Products were run on 1.5%
agarose gels in 1× TBE at 85 volts for 4 h. Through cooperation
with the reference laboratory for C. difficile at the Leiden
University Medical Centre in the Netherlands and the German
reference laboratory for gastrointestinal infections in Freiburg,
the RKI accumulated a reference strain collection of 76 different
C. difficile ribotypes, including 25 reference strains from the Cardiff
Anaerobe Reference Laboratory in Wales, United Kingdom .
PCR ribotypes that differed from reference patterns by at least one
band were assigned novel PCR ribotypes and marked with the prefix
RKI . Ribotyping at the University of Mainz was performed as
described by Brazier et al.  by using the 25 reference strains
from the Cardiff Anaerobe Reference Laboratory.
From January 2008 to April 2009, a total of 72 severe CDI
cases were reported to the HSHO by local health authorities or by
clinicians in Hesse (Figure 1).
Thirty-eight patients (53%) were male and 34 (47%) were
female. The patients´ age ranged from 30 to 94 years with a median
age of 80 years (Figure 2).
The clinical symptoms included diarrhoea (72 cases),
recurrent infection leading to hospital admission (19 cases),
pseudomembranous colitis (nine cases), sepsis (five cases), colitis
(two cases), and colon perforation, peritonitis and pancreatitis
(one case each). Twenty-three of the cases were reported because
F i g u r e 3
Assignment of C. difficile isolates collected from patients
with severe CDI to PCR ribotypes, Hesse, Germany (n=41)
001 003017 027 042066078081RKI-034
Number of cases
F i g u r e 2
Age distribution of patients with severe C. difficile infection
in Hesse, Germany (n=72)
Number of cases
F i g u r e 1
Cases of severe C. difficile infection reported from January
2008 to April 2009 in Hesse, Germany (n=72)
Number of cases
of detection of RT 027. The clinical outcome was disclosed in
60 cases (86%). The infection was lethal within 30 days after
diagnosis in 18 cases (25%). Infection by C. difficile was reported
as underlying cause of or contributing to death in 13 cases, and in
one case as the most probable cause of death. The patients with
lethal outcome that could be attributed to CDI were between 59
and 89 years-old, with a median age of 78 years.
PCR ribotypes, toxin production, antimicrobial susceptibility
Ribotyping results were available for 41 isolates obtained from
41 of the 72 patients with severe CDI. Twenty-four ribotyping results
were reported to our institution via electronic notification system,
while 17 isolates were isolated in the microbiological laboratory of
our institution and forwarded for ribotyping to the national reference
laboratory at the RKI. A total of 24 isolates were identified as RT
027, eight isolates as RT 001, two isolates each as RT 017 and
042, and one isolate each as RT 003, 066, 078 and 081. One
isolate could not be assigned to any known RT and was designated
as RKI-034 (Figure 3).
Production of toxin A and/or B was assessed in culture
supernatants of the 12 C. difficile isolates cultured in our institution
from patients with severe CDI. All isolates were tested positive for
toxin A and/or B production. Interestingly, direct toxin detection in
stool samples was negative in four of these 12 cases, confirming
the higher sensitivity of culture compared to direct toxin detection
in stool samples. Antimicrobial susceptibility results were available
for 16 isolates. Six of the eight RT 001 isolates were tested and
displayed resistance to moxifloxacin and erythromycin. Both RT
017 isolates, one of the two RT 042 isolates and the RT 078 isolate
were resistant to moxifloxacin. Six isolates were susceptible to
moxifloxacin. These results suggest that resistance to moxifloxacin
is not a specific marker for RT 027.
Characterisation of C. difficile isolates associated with lethal
Eighteen (25%) patients had died during the hospitalisation
period associated with severe CDI. Ribotyping results were available
for seven of the cases with lethal outcome and identified RT 027
in three cases and RT 001 and 017 in two cases each (Figure
3). The clinical symptoms, previous antimicrobial therapy, and
antimicrobial susceptibility results of these seven cases are
summarised in the Table 1.
In this study, we present the first results on surveillance of severe
CDI in the state of Hesse with approximately six million inhabitants.
A total of 72 cases of severe CDI were included in this study.
Sixty cases were reported through the federal notification system,
whereas 12 additional cases were enrolled because of our offer to
analyse samples from patients with severe CDI in our diagnostic
laboratory at no charge. Taking into account possible underreporting
and the restricted use of microbiological diagnostic tools such as
culture and ribotyping because of economic considerations, it can
be hypothesised that the real incidence of severe CDI might be
markedly higher in our region.
Sixty-nine (96%) of 72 patients included in this study were older
than 60 years. The median age was 80 years. We observed a high
rate (19%) of disease-related fatality in our study. Eleven of 14
patients with lethal outcome that was attributable to CDAD were
older than 70 years. This finding is in accordance with the results
of a recent study that identified advanced age (over 70 years) as
a significant risk factor for illness and death among patients with
CDAD . However, it can not be ruled out that the emergence
and circulation of epidemic and highly virulent C. difficile strain(s)
may have contributed to an increased case fatality rate in our study.
Nine different C. difficile ribotypes were associated with severe
CDI in our study. Ribotypes 027 and 001 were the most prevalent
strains, while all other ribotypes were encountered only once or
twice. Twenty-four of 41 typed isolates (59%) were RT 027. Since
detection of RT 027 represents a case definition criterion for severe
CDI in Germany, the high proportion of RT 027 may at least partially
be attributed to a sampling bias. However, since the majority of RT
027 isolates were reported from a distinct district, a local outbreak
in a particular hospital in that region can not be excluded. Further
studies are required to evaluate this hypothesis. Taken together,
our data show unequivocally that C. difficile 027 has emerged and
is prevalent in Hesse.
Eight isolates (20%) were identified as RT 001 in this study.
The high prevalence of RT 001 in our study is in accordance with
Ta b l e
Clinical data of patients with lethal C. difficile infection for whom isolates were available for analysis and ribotyping (n=7)
Clinical symptoms Previous antimicrobial
Patient 1, 83, f 9 Mar 2008medicine CDAD, dialysis, hemi-colectomy, ceftriaxon, clarithromycin,
Patient 2, 62, f 20 Mar 2008medicineCDAD, colitis, peritonitisceftriaxon, vancomycin,
Patient 3, 86, m22 Jul 2008 medicinefracture, intracranial bleeding,
Patient 4, 83, m31 Jul 2008medicineurinary tract infection, CDAD,
Patient 5, 73, f9 Sept 2008 geriatrics cystitis, CDAD, readmissionlevofloxacin, vancomycin n.d.R027
Patient 6, 72, m 10 Oct 2008 urology gastroenteritis, CDADunknown, metronidazoleRR 017
Patient 7, 59, m11 Dec 2008medicine pseudomembranous colitis, sepsisclarithromycin, amoxicillin,
CDAD: Clostridium difficile-associated diarrhoea; n.d.: not defined; R: resistant; S: sensitive.
4 www.eurosurveillance.org Download full-text
the results of Borgmann et al. who found a high prevalence (55%)
of RT 001 in patients with CDAD in southern Germany in 2008
. Thus, RT 001 appears to be a common C. difficile genotype in
western and southern Germany. It is noteworthy that RT 001 used
to be the most prevalent strain associated with hospital outbreaks
in English hospitals in 2005, but its prevalence has declined to
7.8% of isolates in 2007-2008 . Future studies are necessary
to follow up the distribution of this ribotype in Germany.
One of the isolates in our study was identified as RT 078. An
increased prevalence of CDI due to this ribotype in the Netherlands
has been reported by Goorhuis et al. . In the latter study, CDI
due to both RT 078 and RT 027 presented with similar severity,
but CDI associated with RT 078 affected a younger population
and was more frequently community-associated. In our study, the
patient suffering from severe CDI due to RT 078 was 60 years-old
and therefore younger than the average. Our results indicate that
RT 078 is prevalent in hospitals in Hesse. They are in agreement
with the data by Rupnik et al.  who found RT 078 in 7.5%
of C. difficile isolates collected from hospitals in Göttingen and
the surrounding regions in the Lower Saxonia, Germany in 2006.
Ribotyping results were available for seven isolates associated
with lethal CDI; three isolates were identified as RT 027, and two
isolates each as RT 001 and 017. Our data suggest that, along
with the hypervirulent RT 027, other toxigenic C. difficile strains
such as RT 001 and 017 are associated with severe and lethal
CDI in Hesse. It is noteworthy that ribotyping results were not
available for half of the lethal cases of CDI in this study. Therefore,
it is possible that also other ribotypes may be involved in severe
CDI with lethal outcome. Our experience shows that offering the
possibility to submit samples from patients with severe CDI to
a specialised laboratory at no charge may help to collect more
In conclusion, the results presented here suggest that severe CDI
is prevalent among hospitalised patients in Hesse. Severe CDI was
associated with a high case fatality rate, especially in patients over
70 years of age. Nine different C. difficile ribotypes were associated
with severe CDI. Lethal infections were observed in association with
RT 001, 017, and 027. This study underlines the need for further
studies on molecular epidemiology of C. difficile.
We thank the staff of local health authorities in Hesse for excellent
cooperation. This project was supported by a grant of the Antibiotic
Resistance Surveillance (ARS) programme of the German Federal Ministry
of Health to WW.
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