**Meet the editor**

Dr. Shymaa Enany is an assistant professor of Microbiology and Immunology at the Suez Canal University, Egypt. She received her PhD degree from the School of Medical and Dental Sciences, Niigata University, Japan, and completed her postdoctoral work in collaboration with many laboratories in San Diego, California, USA, and in Niigata, Japan. She is an editorial board member

and a reviewer in many journals and scientific associations and has many publications in eminent journals as well as books. She has as an excellent experience in bacterial genomics and proteomics.

Contents

**Preface VII**

Shymaa Enany

**Treatment 57**

Gabriela Ursu

**Molecular Methods 73**

**Mammalian Cells 111**

Chapter 6 **Clostridium difficile in the ICU 129**

Fred Racine and Alex G. Therien

Tomás

Chapter 1 **Introductory Chapter: Clostridium difficile Infection Overview 1**

**Epidemiology, Antimicrobial Resistance and Treatment 5** Joana Isidro, Aristides L. Mendes, Mónica Serrano, Adriano O.

Laura Fernández-García, Lucia Blasco, María López and Maria

Luminiţa Smaranda Iancu, Andrei Florin Cârlan and Ramona

Mary Ann Cox, Lorraine D. Hernandez, Pulkit Gupta, Zuo Zhang,

William C. Sherman, Chris Lewis, Jong O. Lee and David N. Herndon

Chapter 5 **Assays for Measuring C. difficile Toxin Activity and Inhibition in**

Chapter 2 **Overview of Clostridium difficile Infection: Life Cycle,**

Chapter 3 **Clostridium difficile Infection: Pathogenesis, Diagnosis and**

Chapter 4 **Clostridium difficile Infection Diagnosis by Biological**

Henriques and Mónica Oleastro

## Contents

## **Preface XI**


Preface

tients, and residents.

have been accomplished.

*Clostridium difficile* , or you can call it as *C. difficile* or *C. diff*, is a bacterium living everywhere around us, in the air, water, and soil and in the feces of humans and animals, living normal‐ ly together with other microorganisms inside human alimentary canal in a balance number to each other. People can easily become infected with *C. difficile* if they touch contaminated clothing, sheets, or other objects and then touch their mouths. Also, when people take anti‐ biotics to knock out a certain bacterial infection, these antibiotics can trigger a disturbance in the bacterial balance inside their gut leading to overgrowth of *C. difficile* causing symptoms

*C. difficile* infections are most often spread in the healthcare facilities between workers, pa‐

Each year in the United States, almost a half million people get sick from *C. difficile*, and approximately 29,000 patients died within 30 days of its initial diagnosis. Nowadays, *C. diffi‐*

Therefore, we decided to write this book to discuss the numerous diagnosis methods and

This book consists of six review chapters. Each chapter starts with a brief introduction, in‐ cluding its aim, and then goes on to provide detailed information about current research relevant to the field. The first chapter is the introductory one that gives an overview of the *C. difficile* as an important pathogen to allow the reader to form a complete picture about this bacterium and its subsequent infection. Through the chapters within, the authors ex‐ plored *C. difficile* life cycle including growth, spore formation, and germination. They exam‐ ined *C. difficile* epidemiology and the different antimicrobial resistance patterns. Recent

We believe that our book is an excellent one for microbiologists, especially those who are interested in *C. difficile*. We hope you enjoy reading it. Finally, we would like to thank all the contributing authors without whose dedication and brilliant research, this project would not

Department of Microbiology and Immunology, Faculty of Pharmacy

**Dr. Shymaa Enany**

Ismailia, Egypt

Suez Canal University

the suitable treatment presented by international leaders in their respective fields.

developments in treatment and prevention of *C. difficile* are also reviewed here.

ranging from diarrhea to life-threatening inflammation of the colon.

*cile* infections have become more frequent, severe, and difficult to treat.

## Preface

*Clostridium difficile* , or you can call it as *C. difficile* or *C. diff*, is a bacterium living everywhere around us, in the air, water, and soil and in the feces of humans and animals, living normal‐ ly together with other microorganisms inside human alimentary canal in a balance number to each other. People can easily become infected with *C. difficile* if they touch contaminated clothing, sheets, or other objects and then touch their mouths. Also, when people take anti‐ biotics to knock out a certain bacterial infection, these antibiotics can trigger a disturbance in the bacterial balance inside their gut leading to overgrowth of *C. difficile* causing symptoms ranging from diarrhea to life-threatening inflammation of the colon.

*C. difficile* infections are most often spread in the healthcare facilities between workers, pa‐ tients, and residents.

Each year in the United States, almost a half million people get sick from *C. difficile*, and approximately 29,000 patients died within 30 days of its initial diagnosis. Nowadays, *C. diffi‐ cile* infections have become more frequent, severe, and difficult to treat.

Therefore, we decided to write this book to discuss the numerous diagnosis methods and the suitable treatment presented by international leaders in their respective fields.

This book consists of six review chapters. Each chapter starts with a brief introduction, in‐ cluding its aim, and then goes on to provide detailed information about current research relevant to the field. The first chapter is the introductory one that gives an overview of the *C. difficile* as an important pathogen to allow the reader to form a complete picture about this bacterium and its subsequent infection. Through the chapters within, the authors ex‐ plored *C. difficile* life cycle including growth, spore formation, and germination. They exam‐ ined *C. difficile* epidemiology and the different antimicrobial resistance patterns. Recent developments in treatment and prevention of *C. difficile* are also reviewed here.

We believe that our book is an excellent one for microbiologists, especially those who are interested in *C. difficile*. We hope you enjoy reading it. Finally, we would like to thank all the contributing authors without whose dedication and brilliant research, this project would not have been accomplished.

> **Dr. Shymaa Enany** Department of Microbiology and Immunology, Faculty of Pharmacy Suez Canal University Ismailia, Egypt

**Chapter 1**

**Provisional chapter**

**Introductory Chapter:** *Clostridium difficile* **Infection**

*Clostridium difficile* (*C. difficile*) is an anaerobic, spore‐forming Gram‐positive bacillus that was first described in 1935 as part of the intestinal microflora in neonates, even so it was not identi‐ fied as a causative agent of human disease until 1978 [1]. The clinical presentation of *C. difficile* infection (CDI) could be asymptomatic, mild or moderate diarrhea and fulminant colitis [2, 3].

Center of Disease Control and Prevention showed an elevation in the incidence and the sever‐ ity of CDI [4]. More than 250,000 person need to be hospitalized due to CDI, and around 14,000 people die from it in the United States every year [5]. Among hospitalized patients, the incidence of CDI differs every year and from location to another. It has been elevating, to nearly 15 per 1000 hospital dismissal [6] and around 20 cases per 100,000 individual in the community [7]. *C. difficile* can only colonize the gut when the normal intestinal microflora is changed by the usage of antibiotics and that was proved by the 16S ribosomal RNA sequenc‐ ing [8]. Therefore, the antibiotics usage remains the most important risk factors for *C. difficile* infection. Many antibiotics are associated with the CDI such as ampicillin, amoxicillin, cephalosporins, clindamycin, fluoroquinolones, trimethoprim and sulfonamides [9]. Another risk factor for CDI is the age; the severity of the infection increases as the age increases [10]. Poor hand hygiene has also previously been shown to play a part in CDI transmission [11]. Hospitalization considers also a main risk factor as it brings together many CDI risk factors in one place such as the use of antibiotics, the spore contaminated media, inappropriate hand

**Introductory Chapter: Clostridium difficile Infection** 

DOI: 10.5772/intechopen.69983

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Overview**

**Overview**

Shymaa Enany

**1. Introduction**

**2. Epidemiology of CDI**

hygiene and the elderly patients [12].

Shymaa Enany

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69983

**Provisional chapter**

## **Introductory Chapter:** *Clostridium difficile* **Infection Overview Overview**

**Introductory Chapter: Clostridium difficile Infection** 

DOI: 10.5772/intechopen.69983

Shymaa Enany Shymaa Enany Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69983

## **1. Introduction**

*Clostridium difficile* (*C. difficile*) is an anaerobic, spore‐forming Gram‐positive bacillus that was first described in 1935 as part of the intestinal microflora in neonates, even so it was not identi‐ fied as a causative agent of human disease until 1978 [1]. The clinical presentation of *C. difficile* infection (CDI) could be asymptomatic, mild or moderate diarrhea and fulminant colitis [2, 3].

## **2. Epidemiology of CDI**

Center of Disease Control and Prevention showed an elevation in the incidence and the sever‐ ity of CDI [4]. More than 250,000 person need to be hospitalized due to CDI, and around 14,000 people die from it in the United States every year [5]. Among hospitalized patients, the incidence of CDI differs every year and from location to another. It has been elevating, to nearly 15 per 1000 hospital dismissal [6] and around 20 cases per 100,000 individual in the community [7]. *C. difficile* can only colonize the gut when the normal intestinal microflora is changed by the usage of antibiotics and that was proved by the 16S ribosomal RNA sequenc‐ ing [8]. Therefore, the antibiotics usage remains the most important risk factors for *C. difficile* infection. Many antibiotics are associated with the CDI such as ampicillin, amoxicillin, cephalosporins, clindamycin, fluoroquinolones, trimethoprim and sulfonamides [9]. Another risk factor for CDI is the age; the severity of the infection increases as the age increases [10]. Poor hand hygiene has also previously been shown to play a part in CDI transmission [11]. Hospitalization considers also a main risk factor as it brings together many CDI risk factors in one place such as the use of antibiotics, the spore contaminated media, inappropriate hand hygiene and the elderly patients [12].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **3.** *C. difficile* **virulence factors**

*C. difficile* has many virulence factors including toxins, sporulation, surface layer proteins and adherence. It produces many toxins such as the enterotoxin TcdA, the cytotoxin TcdB and the binary toxin CDT [12]. Theses toxins cause disruption of the actin cytoskeleton and tight junc‐ tion and cause a decrease in the transepithelial resistance, fluid accumulation and damage of the intestinal epithelium [13].

or vancomycin, and this regime is proved to be successful in 50% of patients [20]. Second recurrent infection can be treated with fidaxomicin which proved to prevent further episodes of *C. difficile* [21]. The fecal microbial transplantation is one of the bacterio‐therapy used to prevent CDI. It is referring to the infusion of fecal suspension from a healthy person to rein‐

Introductory Chapter: *Clostridium difficile* Infection Overview

http://dx.doi.org/10.5772/intechopen.69983

3

Since the CDI causes common and serious problems, many researchers have focused on improving the prevention and the treatment of CDI. In this book, we have focused on study‐ ing the pathogenesis and the virulence factors of *C. difficile* including toxins and trying to

Microbiology and Immunology Department, Faculty of Pharmacy, Suez Canal University,

[1] Bartlett JG. *Clostridium difficile*: History of its role as an enteric pathogen and the cur‐ rent state of knowledge about the organism. Clinical Infectious Diseases : An Official Publication of the Infectious Diseases Society of America. 1994;**18**(Suppl 4):S265‐S272 [2] Bartlett JG. Clinical practice. Antibiotic‐associated diarrhea. The New England Journal

[3] Kelly CP, LaMont JT. *Clostridium difficile*—More difficult than ever. The New England

[4] McDonald LC, Owings M, Jernigan DB. *Clostridium difficile* infection in patients dis‐ charged from US short‐stay hospitals, 1996‐2003. Emerging Infectious Diseases. 2006;**12**:

[5] Centers for Disease Control and Prevention. Antibiotic resistance threats in the United

[6] Steiner C, Barrett M, Weiss A.HCUP Projections: *Clostridium difficile* Hospitalizations 2001 to 2013. Rockville, MD: Agency for Healthcare Research and Quality; 2014

explore the different diagnostic tools and preventive therapeutic methods.

Address all correspondence to: shymaa21@yahoo.com

of Medicine. 2002;**346**:334‐339

Journal of Medicine. 2008;**359**:1932‐1940

States; 2013. Accessed December 15, 2015

stating the gut microbiota of the recipient.

**6. Conclusion**

**Author details**

Shymaa Enany

**References**

409‐415

Egypt

## **4. Diagnosis of CDI**

Diagnosis of *C. difficile* is easily done in the laboratory and usually performed for the patients suffering from diarrhea. Currently, CDI is diagnosed by several available diagnostic tests such as enzyme immunoassay (EIA), EIA for *C. difficile* glutamate dehydrogenase (GDH) or by DNA‐based tests which recognize the genes of *C. difficile* toxin in the stool sample. Additional diagnostic tests are available like toxigenic cultures and cell culture neutralization assays [14].

Stool culture for *C. difficile* requires anaerobic culture and is not widely available [9]. Radiography suggestive of CDI includes polypoid mucosal thickening, haustral fold thicken‐ ing or gaseous distention of the colon; however, radiographic features are not sensitive and not CDI specific [15]. Another diagnostic method is the endoscopy which is rarely required, but it may be helpful in case of doubt of CDI from the clinical signs with all the laboratory tests showed negative results or in patients with inflammatory bowel disease [16].

## **5. Prevention and treatment of CDI**

Since there is no effective vaccine for the CDI control, prevention of the CDI has been a demand and it has focused on barrier methods and environmental hygiene in a trial for pro‐ hibiting *C. difficile* spores and for the reduction of the CDI risk factors: isolation of CDI patient in private room, gowns and gloves usage, hand hygiene and the use of sporicidal solution for rooms [12]. Moreover, altering the antibiotic prescribing could be a good way for prevent‐ ing CDI spreading, since the possibilities of some antibiotics to stimuli CDI are smaller than others. Furthermore, the use of probiotics to prevent CDI could be a safe method. Probiotics usually formed of live microorganisms which give a lot of health benefits to the patient. These microorganisms work through direct activity against *C. difficile* through the inhibition of the bacterial adherence, the modification of the response of the host and the induction of produc‐ tion of specific IgA antitoxin [17, 18].

The treatment of CDI has not shown a big variation. For the acute infections, metronida‐ zole and oral vancomycin has been the mainstay of treatment since 1970. Fidaxomicin was approved in 2011 by the Food and Drug Administration for CDI treatment [19]. Treatment of the first recurrent CDI infection is recommended with a repeat course of either metronidazole or vancomycin, and this regime is proved to be successful in 50% of patients [20]. Second recurrent infection can be treated with fidaxomicin which proved to prevent further episodes of *C. difficile* [21]. The fecal microbial transplantation is one of the bacterio‐therapy used to prevent CDI. It is referring to the infusion of fecal suspension from a healthy person to rein‐ stating the gut microbiota of the recipient.

## **6. Conclusion**

**3.** *C. difficile* **virulence factors**

2 Clostridium Difficile - A Comprehensive Overview Clostridium Difficile - A Comprehensive Overview

the intestinal epithelium [13].

**5. Prevention and treatment of CDI**

tion of specific IgA antitoxin [17, 18].

**4. Diagnosis of CDI**

*C. difficile* has many virulence factors including toxins, sporulation, surface layer proteins and adherence. It produces many toxins such as the enterotoxin TcdA, the cytotoxin TcdB and the binary toxin CDT [12]. Theses toxins cause disruption of the actin cytoskeleton and tight junc‐ tion and cause a decrease in the transepithelial resistance, fluid accumulation and damage of

Diagnosis of *C. difficile* is easily done in the laboratory and usually performed for the patients suffering from diarrhea. Currently, CDI is diagnosed by several available diagnostic tests such as enzyme immunoassay (EIA), EIA for *C. difficile* glutamate dehydrogenase (GDH) or by DNA‐based tests which recognize the genes of *C. difficile* toxin in the stool sample. Additional diagnostic tests are available like toxigenic cultures and cell culture neutralization assays [14]. Stool culture for *C. difficile* requires anaerobic culture and is not widely available [9]. Radiography suggestive of CDI includes polypoid mucosal thickening, haustral fold thicken‐ ing or gaseous distention of the colon; however, radiographic features are not sensitive and not CDI specific [15]. Another diagnostic method is the endoscopy which is rarely required, but it may be helpful in case of doubt of CDI from the clinical signs with all the laboratory

tests showed negative results or in patients with inflammatory bowel disease [16].

Since there is no effective vaccine for the CDI control, prevention of the CDI has been a demand and it has focused on barrier methods and environmental hygiene in a trial for pro‐ hibiting *C. difficile* spores and for the reduction of the CDI risk factors: isolation of CDI patient in private room, gowns and gloves usage, hand hygiene and the use of sporicidal solution for rooms [12]. Moreover, altering the antibiotic prescribing could be a good way for prevent‐ ing CDI spreading, since the possibilities of some antibiotics to stimuli CDI are smaller than others. Furthermore, the use of probiotics to prevent CDI could be a safe method. Probiotics usually formed of live microorganisms which give a lot of health benefits to the patient. These microorganisms work through direct activity against *C. difficile* through the inhibition of the bacterial adherence, the modification of the response of the host and the induction of produc‐

The treatment of CDI has not shown a big variation. For the acute infections, metronida‐ zole and oral vancomycin has been the mainstay of treatment since 1970. Fidaxomicin was approved in 2011 by the Food and Drug Administration for CDI treatment [19]. Treatment of the first recurrent CDI infection is recommended with a repeat course of either metronidazole Since the CDI causes common and serious problems, many researchers have focused on improving the prevention and the treatment of CDI. In this book, we have focused on study‐ ing the pathogenesis and the virulence factors of *C. difficile* including toxins and trying to explore the different diagnostic tools and preventive therapeutic methods.

## **Author details**

#### Shymaa Enany

Address all correspondence to: shymaa21@yahoo.com

Microbiology and Immunology Department, Faculty of Pharmacy, Suez Canal University, Egypt

## **References**


[7] Chitnis AS, Holzbauer SM, Belflower RM, Winston LG, Bamberg WM, Lyons C, et al. Epidemiology of community‐associated *Clostridium difficile* infection, 2009 through 2011. JAMA Internal Medicine. 2013;**173**:1359‐1367

**Chapter 2**

**Overview of** *Clostridium difficile* **Infection: Life Cycle,**

Joana Isidro, Aristides L. Mendes, Mónica Serrano,

Adriano O. Henriques and Mónica Oleastro

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69053

prevention of *C. difficile* infection.

**persistence and dissemination vehicles**

**1.1.** *Clostridium difficile* **biology**

**Abstract**

**Epidemiology, Antimicrobial Resistance and Treatment**

The use of antimicrobial agents and acquired resistances explains in part the emergence and spreading of epidemic strains of *Clostridium difficile*. Continued use of antimicrobial therapy still represents an acute danger in triggering the emergence and spreading of new resistant and multiresistant strains including against first-line antibiotics. We examine the pathway of peptidoglycan synthesis in this organism and associated resistances, as well as resistance to other classes of antibiotics. The life cycle of *C. difficile* involves growth, spore formation and germination. Spores endow the organism with a formidable capacity of persistence in the environment and in the host, resistance, dissemination and infectious potential. Highly resistant spores produced by antibiotic-resistant/multiresistant strains may be one of the most serious challenges we face in what concerns the containment of *C. difficile*. Finally, we review recent developments in the treatment and

**Keywords:** *Clostridium difficile* infection, antibiotics, epidemiology, drug resistance,

**1.** *Clostridium difficile* **life cycle: antibiotic-resistant spores as infectious,** 

The human gut is the home of a community of as many as 1000 species of commensal, beneficial and pathogenic microorganisms. Recent studies suggest that at least half of the bacterial

> © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

spores, β-lactam antibiotics, fidaxomycin, *Clostridium scindens*

