OPEN Emerging Microbes and Infections (2016) 5, e42; doi:10.1038/emi.2016.38
www.nature.com/emi
ORIGINAL ARTICLE
Predominance and high antibiotic resistance of the
emerging Clostridium difficile genotypes NAPCR1 and
NAP9 in a Costa Rican hospital over a 2-year period
without outbreaks
Diana López-Ureña, Carlos Quesada-Gómez, Mónica Montoya-Ramírez, María del Mar Gamboa-Coronado,
Teresita Somogyi, César Rodríguez and Evelyn Rodríguez-Cavallini
Clostridium difficile is the major causative agent of nosocomial antibiotic-associated diarrhea. In a 2009 outbreak of C. difficile-
associated diarrhea that was recorded in a major Costa Rican hospital, the hypervirulent NAP1 strain (45%) predominated
together with a local genotype variant (NAPCR1, 31%). Both strains were fluoroquinolone-resistant and the NAPCR1 genotype,
in addition, was resistant to clindamycin and rifampicin. We now report on the genotypes and antibiotic susceptibilities of 68
C. difficile isolates from a major Costa Rican hospital over a 2-year period without outbreaks. In contrast to our previous
findings, no NAP1 strains were detected, and for the first time in a Costa Rican hospital, a significant fraction of the isolates
were NAP9 strains (n=14, 21%). The local NAPCR1 genotype remained prevalent (n=18, 26%) and coexisted with 14 strains
(21%) of classic hospital NAP types (NAP2, NAP4, and NAP6), eight new genotypes (12%), four environmental strains classified
as NAP10 or NAP11 (6%), three strains without NAP designation (4%) and seven non-toxigenic strains (10%). All 68 strains
were resistant to ciprofloxacin, 88% were resistant to clindamycin and 50% were resistant to moxifloxacin and rifampicin.
Metronidazole and vancomycin susceptibilities were universal. The NAPCR1 and NAP9 strains, which have been associated with
more severe clinical infections, were more resistant to antibiotics than the other strains. Altogether, our results confirm that the
epidemiology of C. difficile infection is dynamic and that A−B+ strains from the NAP9 type are on the rise not only in the
developed world. Moreover, our results reveal that the local NAPCR1 strains still circulate in the country without causing
outbreaks but with equally high antibiotic-resistance rates and levels.
Emerging Microbes and Infections (2016) 5, e42; doi:10.1038/emi.2016.38; published online 11 May 2016
Keywords: antibiotic resistance; Costa Rica; emerging Clostridium difficile; NAPCR1; NAP9
INTRODUCTION remains controversial because, as Cartman et al.8 demonstrated, these
Clostridium difficile has become the leading cause of nosocomial deletions have little effect in significantly increasing toxin production.
diarrhea in adults.1 Clinical manifestations of C. difficile infections Furthermore, variations in the combined repetitive oligopeptide
(CDI) vary from asymptomatic to fulminant colitis, including domain of TcdB have been associated with increases in the virulence
pseudomembranous colitis (PMC) or antibiotic-associated diarrhea. of epidemic strains.9 A minority of toxigenic C. difficile strains also
There may be complications, such as toxic megacolon, colonic produce a third toxin, known as binary toxin Clostridium difficile toxin
perforation and a few extraintestinal manifestations.2 (CDT), that is encoded by cdtA and cdtB at the CdtLoc locus and
Most disease-causing isolates of C. difficile produce one or two is separated from the PaLoc.10 CDT is composed of an ADP-
toxins, i.e., TcdA and TcdB. These toxins enter intestinal epithelial ribosyltransferase that blocks actin polymerization and a binding
cells and glycosylate various families of cytoplasmic GTPases,3 which component that is involved in toxin delivery.11 Although it is believed
leads to actin depolymerization with the loss of internal cell that CDT affects the cytoskeleton and enhances the adhesion and
architecture, apoptosis, villus destruction and a mucosal inflammatory colonization of C. difficile,10 its role in CDI remains controversial.12
response.4,5 The genes encoding toxins A and B (tcdA and tcdB) are Various methods have been used to type C. difficile strains.
part of a so-called pathogenicity locus (PaLoc), which also includes While pulsed-field gel electrophoresis (PFGE) is predominantly used
tcdR (a sigma factor that promotes the transcription of both of the in North America, ribotyping by PCR is most often used in Europe.
toxin genes), tcdE (a potential holin) and tcdC (a potential negative PFGE NAP1 strains correspond to ribotype 027 and harbor toxin A,
regulator of tcdA and tcdB).6 Although deletions in tcdC have been toxin B, CDT, a 18-bp mutation in tcdC and a point mutation
claimed to favor TcdA and TcdB hypersecretion,7 this conjecture in this gene at position 117. In contrast, most TcdA-negative and
Laboratory of Research in Anaerobic Bacteriology and Research Center in Tropical Diseases, University of Costa Rica, San José 11501-2060, Costa Rica
Correspondence: E Rodríguez-Cavallini
E-mail: evelynrodricava@gmail.com
Received 8 October 2015; revised 18 December 2015; accepted 7 January 2016
Antibiotic resistance of emerging C. difficile
D López-Ureña et al
2
TcdB-positive isolates belong to NAP9 and correspond to PCR C. difficile strain ATCC 700057 (tcdA−, tcdB−, tcdC−, cdtB−) were used
ribotype 017.13 as controls.
The effects, severity, complications, recurrence and even death rate For the PFGE typing, we obtained chromosomal SmaI macrores-
of CDI have increased since 2003 in accordance with the increased triction patterns with a published method19 and a CHEF-DRIII
isolation rates of hypervirulent strains, such as NAP1 and NAP9.14,15 variable angle system. Gel pictures were analyzed with BioNumerics
The NAP1 strains have been associated with higher sporulation v4.6 (Applied Maths, Austin, TX, USA) and compared with the
rates and greater resistance to antimicrobials, especially fluoro- databases of the National Microbiology Laboratory of Public Health
quinolones,14,16 whereas the NAP9 strains possess a TcdB that is Agency of Canada.
capable of exerting a variant cytopathic effect.17
During a C. difficile outbreak in a major Costa Rican hospital in Antibiotic susceptibility testing
2009, the hypervirulent NAP1 strain (45%) and the NAPCR1 strains The minimum inhibitory concentrations (MIC) for clindamycin,
(31%) were the predominant genotypes. Both types of strains were ciprofloxacin, moxifloxacin, rifampicin, metronidazole and vancomy-
resistant to ciprofloxacin, moxifloxacin and levofloxacin, and the cin were determined using E-test strips (AB bioMérieux, Askim,
NAPCR1 strains were also resistant to clindamycin and rifampicin. Sweden) and Brucella agar plates containing 5% blood, 1 μg/mL
NAP9 and the other seven classical nosocomial strains were present vitamin K and 5 μg/mL hemin according to established guidelines.22
but in minor proportions.18,19 Since this outbreak, the distribution of For susceptibility categorization, we used the resistance breakpoints
C. difficile genotypes in other Costa Rican hospitals has not been recommended by the CLSI;23 i.e., 8 μg/mL for clindamycin, cipro-
reported. To determine whether the NAP1 and NAPCR1 genotypes floxacin and moxifloxacin and 32 μg/mL for metronidazole. For
were dominant in a non-pediatric hospital over a two-year period rifampicin and vancomycin, we adopted the breakpoints recom-
without C. difficile outbreaks, 68 isolates from diarrheic patients were mended in the document M100-S21 for Staphylococcus aureus because
genotyped using PFGE and the antimicrobial susceptibilities of the no values have been defined for anaerobic bacteria; these values were
isolates were tested. This information contributes to an understanding 4 μg/mL for rifampicin and 16 μg/mL for vancomycin.
of CDI epidemiology worldwide and has the potential to guide local
prevention efforts and treatment strategies. RESULTS
No outbreaks were reported from October 2010 to August 2012 in the
MATERIALS AND METHODS hospital under study (Figure 1). Our genotyping procedure revealed
Isolates and bacteriological procedures that 28 isolates were positive for tcdA and tcdB, negative for cdtB and
This study included 68 C. difficile isolates that were obtained from the carried wild-type tcdC; these results were expected for classic hospital
diarrheal stools of non-pediatric patients who were admitted to a strains. Eighteen isolates exhibited the characteristic NAPCR1 pattern
major hospital in Costa Rica with 633 beds between October 2010 and (i.e., tcdA+, tcdB+, cdtB− and tcdC with a deletion), 14 exhibited the A
August 2012. All patients had been identified as having hospital- −B+ strain pattern (i.e., tcdA−, tcdB+, cdtB− and wild-type tcdC) and 1
acquired CDI according to the criteria from the Infectious Diseases isolate exhibited all 3 toxins and a deletion in tcdC. Seven isolates were
Society of America.20 Toxins A and B were detected in the stool non-toxigenic (Table 1).
samples by the hospital's clinical laboratory, and the samples with Although we observed a variety of genotypes (Figure 2 and Table 1),
positive results were inoculated onto cefoxitin–cycloserine fructose the local NAPCR1 genotype predominated (n= 18, 26%). The NAP9
agar plates (CCFA, Oxoid, Hampshire, UK). Yellow colonies on CCFA genotype was the second-most prevalent genotype (n= 14, 21%),
were cryopreserved at − 80 °C in brain–heart infusion broth with 20% followed by the 14 isolates (21%) from the other traditional hospital
glycerol and sent to the Laboratory of Research in Anaerobic pulsotypes of NAP2 (n= 2), NAP4 (n= 9), and NAP6 (n= 3).
Bacteriology at the University of Costa Rica for further analysis and Environment- or community-associated pulsotypes, such as NAP10
identification. There, the strains were subcultured in selective and NAP11, were observed (n= 4, 6%) as were new pulsotypes (n= 8,
C. difficile moxalactam norfloxacin medium (Oxoid) and later on 12%) and known pulsotypes with no NAP designations (100, 196 and
Brucella agar plates (BD Diagnostics, Franklin Lakes, NJ, USA) 178; n= 3, 4%). Interestingly, no NAP1 strains were detected.
supplemented with 5% lysed horse blood (Oxoid) and 1 μg/mL Antimicrobial susceptibility tests revealed that 88% of the isolates
vitamin K (Sigma-Aldrich, St. Louis, MO, USA) and (blood agar were resistant to clindamycin with very high MICs (4256 μg/mL,
vitamin K) under an atmosphere composed of 90% N2, 5% H2 and
5% CO2 in an anaerobic chamber (Bactron II; ShellLab, Cornelius,
OR, USA) at 37 °C for 48 h. The identities of the isolates were
phenotypically confirmed using selective media, the rapID 32A system
(bioMériuex, Marcy-l'Étoile, France) and chartreuse fluorescence on
blood agar vitamin K under long-wave ultraviolet light and genoty-
pically confirmed through PCR-based detection of the C. difficile
marker tpi and molecular typing by PFGE.21
Molecular typing
Genomic DNA from each strain was obtained from overnight cultures
in brain–heart infusion broth (Oxoid) using the InstaGene reagent
(Bio-Rad, Hercules, CA, USA). Fragments of tcdA, tcdB, cdtB and tcdC
were amplified by PCR using known primers and conditions.21
+ + + Figure 1 Epidemic curves for the stool samples that were positiveA NAP1/027 strain (tcdA , tcdB , 18 bp-deletion in tcdC, cdtB ), a for C. difficile (CD) toxins and were collected in a major hospital in Costa
NAP7 strain (tcdA+, tcdB+, tcdC deletion418 pb, cdtB+), an A−B+ Rica from 2010 to 2012. The cases were diagnosed based on clinical
strain (tcdA−, tcdB+, wild-type tcdC, cdtB−) and the non-toxigenic evidence and toxin detection.
Emerging Microbes and Infections
Antibiotic resistance of emerging C. difficile
D López-Ureña et al
3
Table 2). Half of the isolates were resistant to moxifloxacin and group of clinical isolates from another hospital and confirmed the
rifampicin (MIC 432 μg/mL; Table 2). The MICs for metronidazole widespread distribution of these strains and their dominance even in
and vancomycin were rather low, and although all isolates were the absence of outbreaks. Moreover, the identification of NAPCR1
susceptible to both antibiotics, the MIC90 values were twice the MIC50 PFGE types reveals the ongoing evolution of this lineage and this
values (Table 2). species over a short time.
All isolates from the two most common genotypes (i.e., NAPCR1 The worldwide prevalence of clinically significant NAP9 strains
and NAP9) were resistant to clindamycin, moxifloxacin and rifampi- seems to be increasing,26 particularly in Asian countries.27 Our results
cin (Table 1). Among all of the clindamycin-resistant strains, only reinforce this view because this genotype was the second-most
those from the NAP and NAP9 genotypes exhibited MICs prevalent group. Many studies have found these strains inCR1
4256 μ 24,28g/mL. The remaining hospital, community and non- humans and in animals.29 NAP9 strains have been found once
18 27
toxigenic isolates and the strains from the new genotypes exhibited in Costa Rica and in Latin America, where they seem to be
low antibiotic-resistance levels (Table 1). gradually replacing other circulating genotypes. As observed in many
countries,30,31 our A−B+ strains were homogeneous, did not carry
DISCUSSION cdtAB, and harbored intact tcdC alleles. In contrast, in Australia, the
tcdA−, tcdB+We detected a predominance of C. difficile NAP and NAP9 strains strains are cdtB
+.13 Furthermore, because our NAP9
CR1
in the diarrheal stool samples of patients admitted to a hospital in strains were clindamycin-resistant, they may share a clonal origin with
which no C. difficile outbreaks had occurred during the period under the strains that caused epidemics in Canada, the Netherlands, Ireland
13
study. This knowledge is relevant from the clinical perspective because and Poland. We now know that these strains belong to the RT017
both genotypes have been associated with more severe cases of group (data not shown), but further studies are being performed to
CDI.19,24,25 confirm that their sequence type is indeed ST37 or ST86.
32
NAP strains have circulated in various Costa Rican hospitals Although NAP1 strains were previously isolated during a C. difficileCR1
since 2003 (López-Ureña D et al., 2003, unpublished data) and have outbreak at another Costa Rican hospital, we only found a single tcdA
+,
+ +
had major roles in the 2009 C. difficile outbreak in the San Juan de tcdB and cdtB strain with a tcdC deletion in this study. This strain
Dios Hospital.18 Here, we found NAP strains quite frequently in a did not give rise to the 001 macrorestriction pattern associated withCR1
NAP1 in our previous reports18,19 but rather exhibited a PFGE pattern
without a NAP designation (i.e., a 0196 macrorestriction pattern).
Other NAP strains coexisted including common inhabitants of
hospital environments, such as NAP2, NAP4 and NAP6 strains,33
and NAP10 and NAP11 strains with potential zoonotic or community
origins.34
Despite the marked increase in the recovery of clindamycin-
resistant anaerobic strains in Costa Rica in the last decade,35,36 this
antibiotic is still the first-choice antibiotic for infections by anaerobic
bacteria in Costa Rica and other geographic areas.37 The alarming
clindamycin resistance level of C. difficile observed in this study (88%)
is slightly lower than the level recorded during a 2009 outbreak at
another major hospital (97%)18 but is still much higher than the
values reported from other latitudes.38,39 Almost half of the strains, all
of which belonged to genotypes NAPCR1 and NAP9, had MICs
4256 μg/mL, whereas the remaining strains had MICs= 8 μg/mL.
These findings indicate that only certain lineages acquire highly
Figure 2 Pulse field gel electrophoresis results of representative C. difficile efficient mechanisms of resistance to clindamycin.
genotypes that were isolated between October 2010 and August 2012 from Fluoroquinolone resistance is increasing in epidemic strains of
a hospital without a history of outbreaks during the period under study. C. difficile primarily due to the emergence of chromosomal mutations
Table 1 Antibiotic resistances of various Clostridium difficile genotypes recovered between October 2010 and August 2012 at a hospital
without a history of outbreaks during the period under study
Resistance (% isolates)
Genotype Number of isolates(%) Clindamycin Ciprofloxacin Moxifloxacin Rifampicin Metronidazole Vancomycin
NAPCR1/012 18 (26%) 100 100 100 100 0 0
NAP9/017 14 (21%) 100 100 100 100 0 0
NAP2, NAP4, NAP6 14 (21%) 93 100 7 0 0 0
NAP10, NAP11 4 (6%) 75 100 0 0 0 0
New genotypes 8 (12%) 88 100 12 8 0 0
No NAP designationa 3 (4%) 25 100 25 25 0 0
Non-toxigenic 7 (10%) 71 100 0 0 0 0
aIncludes SmaI patterns 100, 196 and 178.
Emerging Microbes and Infections
Antibiotic resistance of emerging C. difficile
D López-Ureña et al
4
Table 2 MICs and resistances of 68 isolates of Clostridium difficile persistence in the hospital environments and dominance over other
recovered between October 2010 and August 2012 from a Costa genotypes. Constant characterization of circulating C. difficile isolates
Rican hospital without a history of outbreaks during the period in terms of their population structures and antibiotic resistances not
under study only improves our understanding of the epidemiology of CDI but also
guides sanitary authorities and physicians in efforts to reduce the
MIC range MIC50 MIC90 Resistance burden associated with this emerging pathogen.
Antibiotic (μg/mL) (μg/mL) (μg/mL) (% isolates)
Clindamycin 1.5 4256 256 256 88 ACKNOWLEDGEMENTS–
Cipro oxacin 432 432 432 100 We thank Dr Michael R Mulvey, Dr George Golding and Tim Du (the Nationalfl
Moxi oxacin 0.75 432 432 432 50 Microbiology Laboratory, Winnipeg, MB, Canada) for access to the NAP typesfl –
Rifampicin o0.002 432 432 432 50 database and technical assistance with the PFGE and ribotyping analyses.–
Metronidazole 0.064 1.5 0.4 1 0 Pablo Vargas and Robin Cárdenas (the Laboratory of Research in Anaerobic–
Bacteriology, University of Costa Rica, San José, Costa Rica) are acknowledged
Vancomycin 0.38–4.0 1 2 0
for their technical assistance. This work was supported by the National Rector’s
Abbreviation: minimum inhibitory concentration, MIC. Council, Costa Rica (CONARE) and the Vice-Rectory for Research of the
University of Costa Rica through grants VI-803-B1-654 and VI-803-B3-003.
in DNA gyrase genes.38–40 As described in 2010, all strains from this
study were resistant to ciprofloxacin with MICs ⩾ 32 μg/mL.18
Moreover, half of the isolates were resistant to moxifloxacin. All of
1 Slimings C, Riley T. Antibiotics and hospital-acquired Clostridium difficile infection:
the NAPCR1 and NAP9 strains were resistant to both quinolones. Update of systematic review and meta-analysis. J Antimicrob Chemother 2014; 69:
Older fluoroquinolones, such as ciprofloxacin, exhibited moderate or 881–891.
poor activity against C. difficile, and the third- and fourth-generation 2 Shivashankar R, Khanna S, Kammer PP et al. Clinical factors associated with
development of severe-complicated Clostridium difficile infection. Clin Gastroenterol
fluoroquinolones, such as moxifloxacin, were effective against these Hepatol 2013; 11: 1466–1471.
bacteria. However, recent studies indicate that the rates of resistance to 3 Aktories K. Bacterial protein toxins that modify host regulatory GTPases. Nat Rev
moxi oxacin in C. difficile have increased dramatically in different Microbiol 2011; 9: 487–498.fl
4 Kim H, Kokkotou E, Na X et al. Clostridium difficile toxin A-induced colonocyte
countries.41–43 apoptosis involves p53-dependent p21(WAF1/CIP1) induction via p38 mitogen-
Resistance to rifampicin is not unusual in C. difficile (6%–39%), activated protein kinase. Gastroenterology 2005; 129: 1875–1888.
44,45 5 Chumbler NM, Farrow MA, Lapierre LA et al. Clostridium difficile toxin B causesespecially in multidrug-resistant strains. The reported MIC values epithelial cell necrosis through an autoprocessing-independent mechanism.
of ⩽ 0.002 μg/mL for susceptible strains and 432 μg/mL for resistant PLoS Pathog 2012; 8: e1003072.
strains largely match our findings.44,46 Up to 50% of our strains 6 Dingle KE, Elliott B, Robinson E et al. Evolutionary history of the Clostridium difficile
pathogenicity locus. Genome Biol Evol 2014; 6: 36–52.
were rifampicin-resistant, particularly those from the NAPCR1 and 7 Dupuy B, Govind R, Antunes A et al. Clostridium difficile toxin synthesis is negatively
NAP9 genotypes, which were also resistant to clindamycin, ciproflox- regulated by TcdC. J Med Microbiol 2008; 57: 685–689.
acin and moxi oxacin. Interestingly, all of the isolates that were 8 Cartman ST, Kelly ML, Heeg D et al. Precise manipulation of the Clostridium difficilefl
chromosome reveals a lack of association between the tcdc genotype and toxin
resistant to moxifloxacin were also rifampicin-resistant, and this production. Appl Environ Microbiol 2012; 78: 4683–4690.
association should be explored further. 9 Lanis JM, Heinlen LD, James JA et al. Clostridium difficile 027/BI/NAP1 encodes a
hypertoxic and antigenically variable form of TcdB. PLoS Pathog 2013; 9: e1003523.
The antibiotic-resistance levels of the less abundant genotypes were 10 Gerding DN, Johnson S, Rupnik M et al. Clostridium difficile binary toxin CDT:
rather low, but they supported the previously reported increased mechanism, epidemiology, and potential clinical importance. Gut Microbes 2014; 5:
resistance to fluoroquinolone of non-epidemic C. difficile strains40 15–27.
11 Schwan C, Stecher B, Tzivelekidis T et al. Clostridium difficile toxin CDT induces
and the ciprofloxacin resistance that is present in virtually all strains of formation of microtubule-based protrusions and increases adherence of bacteria.
C. difficile. PLoS Pathog 2009; 5: e1000626.
All of the isolates were susceptible to metronidazole and vancomy- 12 Kuehne SA, Collery MM, Kelly ML et al. Importance of toxin A, toxin B, and CDT in
virulence of an epidemic Clostridium difficile strain. J Infect Dis 2014; 209: 83–86.
cin. In agreement with the known metronidazole MIC of C. difficile,20 13 Elliott B, Squire MM, Thean S et al. New types of toxin A-negative, toxin B-positive
our MICs were invariably o2.0 μg/mL. However, a gradual change in strains among clinical isolates of Clostridium difficile in Australia. J Med Microbiol
2011; 60: 1108–1111.
the pattern of sensitivity to this antibiotic might be occurring within 14 Merrigan M, Venugopal A, Mallozzi M et al. Human hypervirulent Clostridium
our strains because the MIC90 for metronidazole was twice the MIC50. difficile strains exhibit increased sporulation as well as robust toxin production.
A similar situation might be occurring in terms of their vancomycin J Bacteriol 2010; 192: 4904–4911.
15 Kim J, Pai H, Seo M-R et al. Clinical and microbiologic characteristics of tcdA-negative
susceptibility because 12 strains (18%), six of which were classified as variant Clostridium difficile infections. BMC Infect Dis 2012; 12: 109.
NAPCR1, exhibited reduced vancomycin susceptibility with MICs 16 Razavi B, Apisarnthanarak A, Mundy LM. Clostridium difficile: emergence of hyperviru-
⩾ 2 μg/mL. These data strongly support the presence of continuous lence and fluoroquinolone resistance. Infection 2007; 35: 300–307.
17 Chaves-Olarte E, Freer E, Parra A et al. R-Ras glucosylation and transient RhoA
monitoring programs both in clinics and the community. activation determine the cytopathic effect produced by toxin B variants from toxin
In this study, a local genotype of C. difficile was the most prevalent A-negative strains of Clostridium difficile. J Biol Chem 2003; 278: 7956–7963.
18 Quesada-Gómez C, Rodríguez C, Gamboa-Coronado MDM et al. Emergence of
strain in a set of 68 isolates that were recovered in a hospital over a Clostridium difficile NAP1 in Latin America. J Clin Microbiol 2010; 48: 669–670.
2-year period without outbreaks. This genotype has previously been 19 Quesada-Gómez C, López-Ureña D, Acuña-Amador L et al. Emergence of an outbreak-
found to be dominant in another hospital during an outbreak. The associated Clostridium difficile variant with increased virulence. J Clin Microbiol 2015;
53: 1216–1226.
second-most prevalent genotype was NAP9, which is in line with its 20 Cohen SH, Gerding DN, Johnson S et al. Clinical practice guidelines for Clostridium
increased prevalence in the USA,31 Europe,24,31 Asia25 and Australia.13 difficile infection in adults: 2010 update by the society for healthcare epidemiology of
The NAP and NAP9 genotypes exhibited high levels of antibiotic America (SHEA) and the infectious diseases society of America (IDSA). Infect ControlCR1 Hosp Epidemiol 2010; 31: 431–455.
resistance, which reflects the use of antibiotics to which the strains 21 Spigaglia P, Mastrantonio P. Comparative analysis of Clostridium difficile clinical
have developed resistance and the association between CDIs and isolates belonging to different genetic lineages and time periods. J Med Microbiol 2004;
53: 1129–1136.
increased antibiotic use. The high levels of resistance to several 22 Letournel-Glomaud C, Houssaye S, Milhaiha L et al. E-test antibiotics susceptibility of
antibiotics among the predominant genotypes could favor their strict anaerobic bacteria. Anaerobe 2003; 9: 281–284.
Emerging Microbes and Infections
Antibiotic resistance of emerging C. difficile
D López-Ureña et al
5
23 Clinical and Laboratory Standards Institute. Methods for Antimicrobial Susceptibility 36 Quesada-Gómez C, Rodríguez-Cavallini E, Rodríguez C. Scarce detection of mobile erm
Testing of Anaerobic Bacteria, Approved Standard, CLSI document M11-A8, CLSI: genes associated with tetQ in Bacteroides and Parabacteroides from Costa Rica.
Wayne, PA, USA; 2012. Available at http://shop.clsi.org/site/Sample_pdf/M11A8_ Anaerobe 2013; 21: 18–21.
sample.pdf. 37 Rashid M-U, Weintraub A, Nord CE. Development of antimicrobial resistance in the
24 Drudy D, Harnedy N, Fanning S et al. Emergence and control of fluoroquinolone- normal anaerobic microbiota during one year after administration of clindamycin or
resistant, toxin A–negative, toxin B–positive Clostridium difficile. Infect Control 2007; ciprofloxacin. Anaerobe 2015; 31: 72–77.
28: 932–940. 38 Huang H, Weintraub A, Fang H et al. Antimicrobial resistance in Clostridium difficile.
25 Du P, Cao B, Wang J et al. Sequence variation in tcdA and tcdB of Clostridium difficile: Int J Antimicrob Agents 2009; 34: 516–522.
ST37 with truncated tcdA is a potential epidemic strain in China. J Clin Microbiol 39 Tickler IA, Goering RV, Whitmore JD et al. Strain types and antimicrobial resistance
2014; 52: 3264–3270. patterns of Clostridium difficile isolates from the United States, 2011 to 2013.
26 Collins D, Hawkey P, Riley T. Epidemiology of Clostridium difficile infection in Asia. Antimicrob Agents Chemother 2014; 58: 4214–4218.
Antimicrob Resist Infect Control 2013; 2: 21. 40 Spigaglia P, Barbanti F, Louie T et al. Molecular analysis of the gyrA and gyrB
27 Goorhuis A, Legaria MC, van den Berg RJ et al. Application of multiple-locus quinolone resistance-determining regions of fluoroquinolone-resistant Clostridium
variable-number tandem-repeat analysis to determine clonal spread of toxin A-negative difficile mutants selected in vitro. Antimicrob Agents Chemother 2009; 53:
Clostridium difficile in a general hospital in Buenos Aires, Argentina. Clin Microbiol 2463–2468.
Infect 2009; 15: 1080–1086. 41 Barbut F, Mastrantonio P, Delmée M et al. Prospective study of Clostridium difficile
28 Alfa MJ, Kabani A, Lyerly D et al. Characterization of a toxin A-negative, toxin infections in Europe with phenotypic and genotypic characterisation of the isolates. Clin
B-positive strain of Clostridium difficile responsible for a nosocomial outbreak of Microbiol Infect 2007; 13: 1048–1057.
Clostridium difficile-associated diarrhea. J Clin Microbiol 2000; 38: 2706–2714. 42 Bourgault A-M, Lamothe F, Loo VG et al. In vitro susceptibility of Clostridium
29 Thakur S, Sandfoss M, Kennedy-Stoskopf S et al. Detection of Clostridium difficile difficile clinical isolates from a multi-institutional outbreak in Southern
and Salmonella in feral swine population in North Carolina. J Wildl Dis 2011; 47: Québec, Canada. Antimicrob Agents Chemother 2006; 50: 3473–3475.
774–776. 43 Huang H, Wu S, Wang M et al. Clostridium difficile infections in a Shanghai hospital:
30 Johnson S, Sambol SP, Brazier JS et al. International typing study of toxin antimicrobial resistance, toxin profiles and ribotypes. Int J Antimicrob Agents 2009; 33:
A-negative, toxin B-positive Clostridium difficile variants. J Clin Microbiol 2003; 41: 339–342.
1543–1547. 44 Curry SR, Marsh JW, Shutt KA et al. High frequency of rifampin resistance identified in
31 Van den Berg RJ, Claas ECJ, Oyib DH et al. Characterization of toxin A-negative, toxin an epidemic Clostridium difficile clone from a large teaching hospital. Clin Infect Dis
B-positive Clostridium difficile isolates from outbreaks in different countries by 2009; 48: 425–429.
amplified fragment length polymorphism and PCR ribotyping. J Clin Microbiol 2004; 45 Spigaglia P, Barbanti F, Mastrantonio P. Multidrug resistance in European Clostridium
42: 1035–1041. difficile clinical isolates. J Antimicrob Chemother 2011; 66: 2227–2234.
32 Griffiths D, Fawley W, Kachrimanidou M et al. Multilocus sequence typing of 46 O’Connor JR, Galang MA, Sambol SP et al. Rifampin and rifaximin resistance in clinical
Clostridium difficile. J Clin Microbiol 2010; 48: 770–778. isolates of Clostridium difficile. Antimicrob Agents Chemother 2008; 52: 2813–2817.
33 Martin H, Willey B, Low DE et al. Characterization of Clostridium difficile strains
isolated from patients in Ontario, Canada, from 2004 to 2006. J Clin Microbiol 2008;
46: 2999–3004. This work is licensed under a Creative Commons Attribution 4.0
34 Lessa FC. Community-associated Clostridium difficile infection: how real is it? Anaerobe International License. The images or other third party material in this
2013; 24: 121–123. article are included in the article’s Creative Commons license, unless indicated otherwise
35 Molina J, Barrantes G, Quesada-Gómez C et al. Phenotypic and genotypic characteriza- in the credit line; if thematerial is not includedunder theCreativeCommons license, users
tion of multidrug-resistant Bacteroides, Parabacteroides spp., and Pseudoflavonifractor will need toobtainpermission fromthe licenseholder to reproduce thematerial.Toviewa
from a Costa Rican hospital. Microb Drug Resist 2014; 20: 478–484. copy of this license, visit http://creativecommons.org/licenses/by/4.0/
Emerging Microbes and Infections