diabetes research and clinical practice 105 (2014) 285–294
Contents available at ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Review
Clostridium difficile infection in diabetes Hui-Qi Qu a,*, Zhi-Dong Jiang b a
Human Genetics Center, The University of Texas School of Public Health, Houston, TX, USA Center for Infectious Diseases, Division of Epidemiology, Human Genetics and Environmental Sciences, The University of Texas School of Public Health, Houston, TX, USA b
article info
abstract
Article history:
Diabetes-related hospitalization and hospital utilization is a serious challenge to the health
Received 12 August 2013
care system, a situation which may be further aggravated by nosocomial Clostridium difficile
Received in revised form
(C. difficile) infection (CDI). Studies have demonstrated that diabetes increases the risk of
26 January 2014
recurrent CDI with OR (95% CI) 2.99 (1.88, 4.76). C. difficile is a gram-positive, spore-forming
Accepted 13 June 2014
anaerobic bacterium which is widely distributed in the environment. Up to 7% of healthy
Available online 21 June 2014
adults and up to 45% of infants may have asymptomatic intestinal carriage of C. difficile. A
Keywords:
based molecular typing methods are available for typing C. difficile isolates. C. difficile
Clostridium difficile
virulence evolved independently in the highly epidemic lineages, associated with the
large number of strains of C. difficile have been identified. A number of PCR or sequence-
Diabetes
expression of toxin genes and other virulence factors. This article briefly reviews recent
Host immunity
progresses in the bateriology of C. difficile and highlights the limited knowledge of potential
Gut microbiota
mechanisms for the increased risk of CDI in diabetes which warrants further research. # 2014 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2. 3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clostridium difficile infection and diabetes . . . . . Bateriology of Clostridium difficile . . . . . . . . . . . . 3.1. Molecular typing of C. difficile . . . . . . . . . 3.2. Molecular pathogenicity of C. difficile . . . 3.2.1. tcdA and tcdB . . . . . . . . . . . . . . . . 3.2.2. tcdR, tcdE, and tcdC in PaLoc . . . . 3.2.3. cdtA and cdtB . . . . . . . . . . . . . . . . 3.2.4. Other virulence factors . . . . . . . . Mechanistic study on the increased CDI risk in Acknowledgements . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
........ ........ ........ ........ ........ ........ ........ ........ ........ diabetes . ........ ........
. . . . . . . . . . . .
* Corresponding author. Tel.: +1 713 500 9950; fax: +1 713 500 0900. E-mail address: [email protected] (H.-Q. Qu). http://dx.doi.org/10.1016/j.diabres.2014.06.002 0168-8227/# 2014 Elsevier Ireland Ltd. All rights reserved.
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286 286 286 287 287 287 288 288 288 289 289 290
286
1.
diabetes research and clinical practice 105 (2014) 285–294
Introduction
Clostridium difficile (C. difficile) infection (CDI) is a major nosocomial infection [1]. The infection mostly occurs after the gut microbiota is disrupted by the administration of antibiotics, which impairs colonization resistance against C. difficile [2]. CDI accounts for approximately 15% to 25% of antibiotic-associated diarrhea [3]. In addition to antibiotic use, advanced age (>65 years old), impaired immune function, contact with CDI patients or their health care providers, and the use of acid-suppressive therapy, particularly proton pump inhibitors, are common factors increasing the risk of CDI [4–6]. The clinical symptoms include diarrhea, abdominal pain and fever. Sigmoidoscopic examination may reveal pseudomembranes colitis. In rare case, CDI may cause fulminant colitis in which patients may develop septic shock, toxic megacolon, and intestinal perforation [7,8]. Stool culture is the most sensitive test for laboratory diagnosis of CDI and essential for epidemiological studies, whereas it is not clinical practical because of its slow turnaround time [9,10]. Stool culture takes as long as 9 days for results, i.e. 2 to 5 days for culture, 2 days for enrichment, and 1 to 2 days for the isolate toxin cell culture cytotoxicity neutralization assay (CCNA) [10]. Instead, C. difficile glutamate dehydrogenase (GDH) test of stool specimens by enzyme immunoasssays (EIAs) followed by tests for toxins is recommended for CDI diagnosis in suspected patients [11]. The toxin testing by a direct stool CCNA requires only 1 to 2 days [10]. In cases that GDH testing is positive but toxin testing is negative, stool culture with isolate toxin testing is most useful and may detect additional 23% toxigenic C. difficile [10]. Stool culture followed by identification of a toxigenic isolate thus provides the standard against which other clinical test results should be compared, and the availability of an isolate also allows for strain typing and antimicrobial susceptibility testing [9,10]. Oral metronidazole or vancomycin are effective in most cases. However, 15–20% patients may have relapsing infection. Lipiarmycin (also known as clostomicin, tiacumicin, diffimicin, PAR-101, OPT-80, fidaxomicin, and Dificid), an 18membered macrocyclic-lactone antibiotic produced by Actinomycete species, is a novel antibiotic for CDI by inhibition of the switch region of bacterial RNA polymerase (RNAP) [12]. Lipiarmycin has high in vitro activity against C. difficile but low activity against the typical intestinal flora, and is barely absorbed systemically and achieves high stool concentrations [13]. Recurrence is less frequent with lipiarmycin than with vancomycin [14]. Fecal bacteriotherapy to restore disordered gut microbiota has been shown to be highly effective for recurrent CDI patients with disease resolution in 92% patients [15]. Fecal bacteriotherapy involves infusing intestinal microorganisms in a liquid suspension of stool from a healthy donor to restore the intestinal microbiota of a diseased individual [15,16]. To date, fecal bacteriotherapy still presents significant scientific and regulatory challenges. Although donor material is widely available, the complex in stool composition raises concerns about its safety and acceptability, and prevents the procedure from becoming a standard therapeutic option [17]. Further randomized placebo-controlled study on its efficacy and
safety are still underway [18]. Monoclonal antibodies against C. difficile toxins are also under development [19]. Endospores are pivotal to C. difficile transmission. C. difficile spores can survive up to 5 months in environment [20]. C. difficile spores are resistant to prolonged exposure to high temperatures and 70% ethanol, but are effectively inactivated by sporicidal detergents [21]. The proper practice of hand hygiene to prevent C. difficile transmission is to wash hands with soap and water to remove any spores because alcohol-based hand sanitizers cannot kill the spores [9]. CDI is linked to 14,000 deaths in the US each year (http:// www.cdc.gov/vitalsigns/hai/). Due to the emerging hypervirulent strains of C. difficile, this number may still increase [22]. CDI has formed a serious economic burden to the health care system. For the US health care system, the conservative estimation of direct cost for management of CDI is $3.4 billion per year [23].
2.
Clostridium difficile infection and diabetes
Diabetes-related hospitalization and hospital utilization is a serious challenge to health care system [24], a situation which may be further aggravated by nosocomial CDI. According to the American Diabetes Association, nearly 26 million children and adults in the United States have diabetes; another 79 million Americans have prediabetes and are at risk for developing type 2 diabetes. Recent estimates project that as many as 1 in 3 American adults will have diabetes in 2050 unless we take steps to stop diabetes [25]. Diabetes impairs host immunity, and increase the risk of a number of infectious diseases [26–28]. With the international pandemic of type 2 diabetes, it is becoming a major driving force of the epidemic of infectious diseases. The infectious diseases with increased risk in diabetes include CDI. Studies have demonstrated that diabetes increases the risk of recurrent CDI [29,30]. The combined effect of the two studies [29,30] has OR (95% CI) = 2.99(1.88, 4.76) for the increased risk of recurrent CDI in diabetes. In addition, studies showed that diabetes increases the risk of metronidazole treatment failure [31], and is independently associated with C. difficile positivity (CDP) status [32]. To understand the pathogenesis mechanisms of diabetes-increased risk of infectious diseases is a critical approach to gain knowledge for the control of these situations. Also diabetes can serve as a model to understand the interaction between host and pathogens, as demonstrated by our study on the molecular mechanisms of increased risk of tuberculosis in diabetes [27,28]. To discuss these issues, we reviewed recent progress of the molecular bateriology of C. difficile and highlight the limited knowledge of some possible mechanisms in the next sections.
3.
Bateriology of Clostridium difficile
C. difficile is a gram-positive, spore-forming anaerobic bacterium. It was first isolated in meconium samples of normal newborn infants as a component of the normal intestinal flora by Hall and O’Toole [33]. Since the 1970s, C. difficile was recognized as the cause of antibiotic-associated pseudomembranous colitis [34–37]. C. difficile is widely distributed in the
diabetes research and clinical practice 105 (2014) 285–294
environment, e.g. soil, river and sea water, and won’t cause disease normally [38]. Up to 7% healthy adults [39] and up to 45% infants [40] may have asymptomatic intestinal carriage of C. difficile. The C. difficile genome consists of a circular chromosome of 4,290,252 bp encoding 3,776 predicted coding sequences (CDS), and a plasmid of 7881 bp encoding 11 CDSs [41]. For the encoded genes, 19.7% genes are conserved across different C. difficile strains [42].
3.1.
Molecular typing of C. difficile
To date, a large number of strains of C. difficile have been identified. A number of PCR or sequence-based molecular typing methods are available for typing C. difficile isolates, e.g. restriction endonuclease analysis (REA), pulsed-field gel electrophoresis (PFGE), PCR-ribotyping, multilocus variablenumber tandem-repeat analysis (MLVA), amplified fragment length polymorphism (AFLP), surface layer protein A gene sequence typing (slpAST), and multilocus sequence typing (MLST) [43,44]. Three of these methods have been most commonly used to type the strains of C. difficile, i.e. REA, PFGE, and PCR ribotyping [44]. REA and PFGE are restriction enzyme based methods. In the REA typing, the chromosomal DNA of C. difficile is usually digested with HindIII restriction enzyme, which has numerous restriction sites in the C. difficile genome [45]. Because of producing numerous DNA fragments after HindIII digestion, the banding pattern of REA electrophoresis is complicated to read, which limits the clinical application of REA. In the PFGE typing, the chromosomal DNA of C. difficile is usually digested with SmaI restriction enzyme, which gives 7– 15 restriction fragments ranging from 10 to 1100 kbp [46]. Compared with conventional electrophoresis which only separate DNA fragments
Contents available at ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Review
Clostridium difficile infection in diabetes Hui-Qi Qu a,*, Zhi-Dong Jiang b a
Human Genetics Center, The University of Texas School of Public Health, Houston, TX, USA Center for Infectious Diseases, Division of Epidemiology, Human Genetics and Environmental Sciences, The University of Texas School of Public Health, Houston, TX, USA b
article info
abstract
Article history:
Diabetes-related hospitalization and hospital utilization is a serious challenge to the health
Received 12 August 2013
care system, a situation which may be further aggravated by nosocomial Clostridium difficile
Received in revised form
(C. difficile) infection (CDI). Studies have demonstrated that diabetes increases the risk of
26 January 2014
recurrent CDI with OR (95% CI) 2.99 (1.88, 4.76). C. difficile is a gram-positive, spore-forming
Accepted 13 June 2014
anaerobic bacterium which is widely distributed in the environment. Up to 7% of healthy
Available online 21 June 2014
adults and up to 45% of infants may have asymptomatic intestinal carriage of C. difficile. A
Keywords:
based molecular typing methods are available for typing C. difficile isolates. C. difficile
Clostridium difficile
virulence evolved independently in the highly epidemic lineages, associated with the
large number of strains of C. difficile have been identified. A number of PCR or sequence-
Diabetes
expression of toxin genes and other virulence factors. This article briefly reviews recent
Host immunity
progresses in the bateriology of C. difficile and highlights the limited knowledge of potential
Gut microbiota
mechanisms for the increased risk of CDI in diabetes which warrants further research. # 2014 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2. 3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clostridium difficile infection and diabetes . . . . . Bateriology of Clostridium difficile . . . . . . . . . . . . 3.1. Molecular typing of C. difficile . . . . . . . . . 3.2. Molecular pathogenicity of C. difficile . . . 3.2.1. tcdA and tcdB . . . . . . . . . . . . . . . . 3.2.2. tcdR, tcdE, and tcdC in PaLoc . . . . 3.2.3. cdtA and cdtB . . . . . . . . . . . . . . . . 3.2.4. Other virulence factors . . . . . . . . Mechanistic study on the increased CDI risk in Acknowledgements . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
........ ........ ........ ........ ........ ........ ........ ........ ........ diabetes . ........ ........
. . . . . . . . . . . .
* Corresponding author. Tel.: +1 713 500 9950; fax: +1 713 500 0900. E-mail address: [email protected] (H.-Q. Qu). http://dx.doi.org/10.1016/j.diabres.2014.06.002 0168-8227/# 2014 Elsevier Ireland Ltd. All rights reserved.
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286 286 286 287 287 287 288 288 288 289 289 290
286
1.
diabetes research and clinical practice 105 (2014) 285–294
Introduction
Clostridium difficile (C. difficile) infection (CDI) is a major nosocomial infection [1]. The infection mostly occurs after the gut microbiota is disrupted by the administration of antibiotics, which impairs colonization resistance against C. difficile [2]. CDI accounts for approximately 15% to 25% of antibiotic-associated diarrhea [3]. In addition to antibiotic use, advanced age (>65 years old), impaired immune function, contact with CDI patients or their health care providers, and the use of acid-suppressive therapy, particularly proton pump inhibitors, are common factors increasing the risk of CDI [4–6]. The clinical symptoms include diarrhea, abdominal pain and fever. Sigmoidoscopic examination may reveal pseudomembranes colitis. In rare case, CDI may cause fulminant colitis in which patients may develop septic shock, toxic megacolon, and intestinal perforation [7,8]. Stool culture is the most sensitive test for laboratory diagnosis of CDI and essential for epidemiological studies, whereas it is not clinical practical because of its slow turnaround time [9,10]. Stool culture takes as long as 9 days for results, i.e. 2 to 5 days for culture, 2 days for enrichment, and 1 to 2 days for the isolate toxin cell culture cytotoxicity neutralization assay (CCNA) [10]. Instead, C. difficile glutamate dehydrogenase (GDH) test of stool specimens by enzyme immunoasssays (EIAs) followed by tests for toxins is recommended for CDI diagnosis in suspected patients [11]. The toxin testing by a direct stool CCNA requires only 1 to 2 days [10]. In cases that GDH testing is positive but toxin testing is negative, stool culture with isolate toxin testing is most useful and may detect additional 23% toxigenic C. difficile [10]. Stool culture followed by identification of a toxigenic isolate thus provides the standard against which other clinical test results should be compared, and the availability of an isolate also allows for strain typing and antimicrobial susceptibility testing [9,10]. Oral metronidazole or vancomycin are effective in most cases. However, 15–20% patients may have relapsing infection. Lipiarmycin (also known as clostomicin, tiacumicin, diffimicin, PAR-101, OPT-80, fidaxomicin, and Dificid), an 18membered macrocyclic-lactone antibiotic produced by Actinomycete species, is a novel antibiotic for CDI by inhibition of the switch region of bacterial RNA polymerase (RNAP) [12]. Lipiarmycin has high in vitro activity against C. difficile but low activity against the typical intestinal flora, and is barely absorbed systemically and achieves high stool concentrations [13]. Recurrence is less frequent with lipiarmycin than with vancomycin [14]. Fecal bacteriotherapy to restore disordered gut microbiota has been shown to be highly effective for recurrent CDI patients with disease resolution in 92% patients [15]. Fecal bacteriotherapy involves infusing intestinal microorganisms in a liquid suspension of stool from a healthy donor to restore the intestinal microbiota of a diseased individual [15,16]. To date, fecal bacteriotherapy still presents significant scientific and regulatory challenges. Although donor material is widely available, the complex in stool composition raises concerns about its safety and acceptability, and prevents the procedure from becoming a standard therapeutic option [17]. Further randomized placebo-controlled study on its efficacy and
safety are still underway [18]. Monoclonal antibodies against C. difficile toxins are also under development [19]. Endospores are pivotal to C. difficile transmission. C. difficile spores can survive up to 5 months in environment [20]. C. difficile spores are resistant to prolonged exposure to high temperatures and 70% ethanol, but are effectively inactivated by sporicidal detergents [21]. The proper practice of hand hygiene to prevent C. difficile transmission is to wash hands with soap and water to remove any spores because alcohol-based hand sanitizers cannot kill the spores [9]. CDI is linked to 14,000 deaths in the US each year (http:// www.cdc.gov/vitalsigns/hai/). Due to the emerging hypervirulent strains of C. difficile, this number may still increase [22]. CDI has formed a serious economic burden to the health care system. For the US health care system, the conservative estimation of direct cost for management of CDI is $3.4 billion per year [23].
2.
Clostridium difficile infection and diabetes
Diabetes-related hospitalization and hospital utilization is a serious challenge to health care system [24], a situation which may be further aggravated by nosocomial CDI. According to the American Diabetes Association, nearly 26 million children and adults in the United States have diabetes; another 79 million Americans have prediabetes and are at risk for developing type 2 diabetes. Recent estimates project that as many as 1 in 3 American adults will have diabetes in 2050 unless we take steps to stop diabetes [25]. Diabetes impairs host immunity, and increase the risk of a number of infectious diseases [26–28]. With the international pandemic of type 2 diabetes, it is becoming a major driving force of the epidemic of infectious diseases. The infectious diseases with increased risk in diabetes include CDI. Studies have demonstrated that diabetes increases the risk of recurrent CDI [29,30]. The combined effect of the two studies [29,30] has OR (95% CI) = 2.99(1.88, 4.76) for the increased risk of recurrent CDI in diabetes. In addition, studies showed that diabetes increases the risk of metronidazole treatment failure [31], and is independently associated with C. difficile positivity (CDP) status [32]. To understand the pathogenesis mechanisms of diabetes-increased risk of infectious diseases is a critical approach to gain knowledge for the control of these situations. Also diabetes can serve as a model to understand the interaction between host and pathogens, as demonstrated by our study on the molecular mechanisms of increased risk of tuberculosis in diabetes [27,28]. To discuss these issues, we reviewed recent progress of the molecular bateriology of C. difficile and highlight the limited knowledge of some possible mechanisms in the next sections.
3.
Bateriology of Clostridium difficile
C. difficile is a gram-positive, spore-forming anaerobic bacterium. It was first isolated in meconium samples of normal newborn infants as a component of the normal intestinal flora by Hall and O’Toole [33]. Since the 1970s, C. difficile was recognized as the cause of antibiotic-associated pseudomembranous colitis [34–37]. C. difficile is widely distributed in the
diabetes research and clinical practice 105 (2014) 285–294
environment, e.g. soil, river and sea water, and won’t cause disease normally [38]. Up to 7% healthy adults [39] and up to 45% infants [40] may have asymptomatic intestinal carriage of C. difficile. The C. difficile genome consists of a circular chromosome of 4,290,252 bp encoding 3,776 predicted coding sequences (CDS), and a plasmid of 7881 bp encoding 11 CDSs [41]. For the encoded genes, 19.7% genes are conserved across different C. difficile strains [42].
3.1.
Molecular typing of C. difficile
To date, a large number of strains of C. difficile have been identified. A number of PCR or sequence-based molecular typing methods are available for typing C. difficile isolates, e.g. restriction endonuclease analysis (REA), pulsed-field gel electrophoresis (PFGE), PCR-ribotyping, multilocus variablenumber tandem-repeat analysis (MLVA), amplified fragment length polymorphism (AFLP), surface layer protein A gene sequence typing (slpAST), and multilocus sequence typing (MLST) [43,44]. Three of these methods have been most commonly used to type the strains of C. difficile, i.e. REA, PFGE, and PCR ribotyping [44]. REA and PFGE are restriction enzyme based methods. In the REA typing, the chromosomal DNA of C. difficile is usually digested with HindIII restriction enzyme, which has numerous restriction sites in the C. difficile genome [45]. Because of producing numerous DNA fragments after HindIII digestion, the banding pattern of REA electrophoresis is complicated to read, which limits the clinical application of REA. In the PFGE typing, the chromosomal DNA of C. difficile is usually digested with SmaI restriction enzyme, which gives 7– 15 restriction fragments ranging from 10 to 1100 kbp [46]. Compared with conventional electrophoresis which only separate DNA fragments
