Prospective study on the Microbial profile of Bacterial infections before and after Flap Reconstructive Surgery

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PROSPECTIVE STUDY ON THE MICROBIAL PROFILE OF BACTERIAL INFECTIONS BEFORE AND AFTER FLAP RECONSTRUCTIVE SURGERY.

Dissertation submitted in

Partial fulfillment of the Regulations required for the award of M.D. DEGREE

In

MICROBIOLOGY– BRANCH IV The Tamil Nadu

DR. M.G.R. MEDICAL UNIVERSITY Chennai

MAY 2018.

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CERTIFICATE

This is to certify that the enclosed work “Prospective study on the Microbial profile of Bacterial infections before and after Flap Reconstructive Surgery” submitted by Dr. P. Santhanalakshmi to The Tamilnadu Dr. MGR Medical University is based on bonafide cases studied and analysed by the candidate in the Department of Microbiology, Coimbatore Medical College Hospital during the period from July 2016 to June 2017 under the guidance and supervision of Dr. N.Mythily, MD., Professor & HOD, Department of Microbiology and the conclusion reached in this study are her own.

Guide

Dr. N.MYTHILY, MD., Professor & HOD,

Department of Microbiology, Coimbatore Medical College, Coimbatore.

Dr. B. ASOKAN. M.S., M.Ch., Dr., N.MYTHILY, MD.,

Dean, Professor & HOD,

Coimbatore Medical College and Hospital, Department of Microbiology,

Coimbatore – 14. Coimbatore Medical College,

Coimbatore – 14.

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DECLARATION

I, Dr. P. Santhanalakshmi solemnly declare that the dissertation entitled

“PROSPECTIVE STUDY ON THE MICROBIAL PROFILE OF BACTERIAL INFECTIONS BEFORE AND AFTER FLAP RECONSTRUCTIVE SURGERY” was done by me at Coimbatore Medical College Hospital, during the period from July 2016 to June 2017 under the guidance and supervision of Dr. N. Mythily, M.D., Professor & HOD, Department of Microbiology, Coimbatore Medical College, Coimbatore.

This dissertation is submitted to The Tamilnadu Dr. MGR Medical University towards the partial fulfilment of the requirement for the award of M.D. Degree (Branch – IV) in Microbiology.

I have not submitted this dissertation on my previous occasion to any University for the award of any degree.

Place:

Date :

Dr. P. Santhanalakshmi.

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ACKNOWLEDGEMENT

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ACKNOWLEDGEMENT

I express my deep debt of gratitude to our respectful Dean, Dr.B. Asokan, M.S., M.Ch., for permitting me to do this study.

I wish to place my deep sense of gratitude and sincere thanks to Dr. N. Mythily MD., Professor and Head of the Department of Microbiology, for the constant encouragement and timely advice given to me during the course of my post-graduation.

I express my deep sense of gratitude and indebtedness to Professor

& HOD, Dr. N. Mythily MD., for her constant guidance, valuable advice and inspiration throughout my study.

I sincerely place my thanks to Associate Professor Dr.P.Sankar,M.D., for his support and encouragement.

I express my sincere thanks to my Assistant Professors Dr.S.Deepa M.D., Dr.N.Bharathi Santhose M.D., Dr.B.Padmini M.D., Dr.C.Ashok Kumar MD., and Dr. R.Radhika MD., for their valuable suggestions.

My special thanks to my post graduate colleagues Dr.K.Sivaram, Dr.S.Viji and Dr. N.Vandarkuzhali and other post graduates in the department of Microbiology for their co-operation in completing my study.

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I would grossly fail in my duty, if I do not mention here of my subjects who have undergone the pain and discomfort of the investigations during this study.

I take this opportunity to thank all the technical staffs in the Department of Microbiology who gave me their kind co-operation throughout my study.

I affectionately thank my beloved husband Dr.M.Prasanna Kumar and my sons P.Sai Arjun and P.Sai Vijay who are giving their constant support throughout my entire post-graduation course without which this work would not have been successful.

I am thankful to God, who have been with me all throughout my way to reach the destination.

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CERTIFICATE - II

This is to certify that this dissertation work titled “Prospective study on the Microbial profile of Bacterial infections before and after Flap Reconstructive Surgery” of the candidate Dr. P. Santhanalakshmi with registration Number 201514251 for the award of Doctor of Medicine in the branch of Microbiology. I personally verified the urkund.com website for the purpose of plagiarism Check. I found that the uploaded thesis file contains from introduction to conclusion pages and result shows 6%

percentage of plagiarism in the dissertation.

Guide sign with Seal.

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INTRODUCTION

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INTRODUCTION

Flap Reconstructive surgery is a boon to patients with soft tissue defects due to various causes. The confounding factor which hinders healing is infection of the flaps which poses a challenge to the surgeon, as well as microbiologist.²⁴ Infection depends on the microbial environment, nature of the wound, as well as host factors. The spectrum of bacterial isolates and their antimicrobial susceptibility pattern aids the clinician in rational use of prophylactic antibiotics thereby improving the success rate of the reconstructed flaps.

Prevalence of soft tissue infections in reconstructive surgery depends upon the nature of the trauma and the class of wound. Deep fractures worsens the condition and can lead to chronic osteomyelitis, non-union, loss of function or limb loss.²⁰

Soft tissue defects include loss of skin, subcutaneous tissue, muscle with vascular supply, nerves, and associated segmental bone fractures.

Traumatic soft tissue damage compromises the system of motion, circulation or sensibility and therefore jeopardize functional rehabilitation.

Treatment options available for soft tissue coverage are Rotational muscle flaps, Fascio -cutaneous flaps, Adipo-fascial flaps, Negative pressure wound therapy, and Free tissue transfer.¹¹ Muscle flaps offer pliability and can eradicate dead space, and overcome residual bacterial infection in bone, improve blood flow and can provide a vascular recipient

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bed. Of these Rotational muscle flaps has now become the gold standard option for large complex defects.

Increase in the duration of infection and geographic area has a direct impact on the rate of infection. Various complications of the reconstructed wound are chronic infection, acute rejection of the flap, and limb loss.¹⁹ Chronic infection plays a major role among which, Trauma and Burns wound are the most common.

Infections of the soft tissue defects is an important cause of mortality and morbidity and prevalence ranges from 20-60%.³³ Antibiotic use in the perioperative period has been shown to reduce the incidence of infection. Successful invasion, and proliferation by one or more species of micro-organisms anywhere within the body’s sterile tissues, sometimes results in pus formation. The rate of nosocomial infections are higher in patients with

1. Open wound defects,

2. Immuno-compromised status of the patient, 3. Prolonged hospitalisation and invasive procedures

Soft tissue cover is indicated for various defects like Trauma, stump wound in diabetic foot, III Degree burns, carcinoma, and connective tissue disorders. Early Soft tissue coverage diminishes the rate of infection, non-union, and subsequent amputation.⁴⁸ Indications for rotational muscle flap wound closure may include traumatic wounds, exposed bone with

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osteomyelitis, non healing wounds, surgical wound dehiscence recalcitrant to nonoperative therapies, fractures, and total arthroplasty.

In spite of the relevant use of peri operative antibiotic cover infection rate is high due to critical pathogens which causes multi drug resistant infections in pre-operative and post-operative wounds.

Bacterial infection is the usual cause of suppuration and includes Staphylococcus aureus, Pseudomonas aeruginosa, Proteus species, Escherichia coli, and Klebsiella species. Pyogenic infections are either polymicrobial or monomicrobial and endogenous or exogenous based on the type of wound the spectrum of isolates varies.

In Post traumatic wound infections - Staphylococcus aureus, Proteus species, Acinetobacter species, and Pseudomonas aeruginosa are the common organisms.

In Surgical site infections Staphylococcus aureus, followed by Pseudomonas aeruginosa, E.coli, Klebsiella pneumoniae, Proteus mirabilis, and Acinetobacter were the common organisms.⁴⁷

In Diabetic wound infection the commonest isolates were Pseudomonas aeruginosa, followed by Klebsiella, E.coli, Staphylococcus aureus, and Proteus species.

Burns and Diabetic wounds infections, also had a similar bacteriological profile as that of Diabetic wounds.⁴³

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Injudicious use of antibiotics have in turn resulted in the emergence of antibiotic resistant strains. Hence identification of the species and their resistant pattern is important in the treatment of such infections and survival of the flap.

The increase in the rates of antibiotic resistance is a major concern in both non-fermenting bacilli and members of the Enterobacteriaceae family. Antimicrobial resistance in addition hampers the effectiveness of treatment and thus the patient remain infectious for a long time thereby increasing the spread of resistant microorganisms to others.

In February 2017 WHO published a list of Antibiotic resistant

“Priority Pathogens” requiring newer antimicrobials, which is classified into critical, high, and medium priority pathogens.⁴⁴

Critical pathogens requiring newer antibiotics include Pseudomonas, Acinetobacter, Enterobacteriaceae (Proteus, Klebsiella, E.coli, Serratia).

These bacteria are resistant to number of antibiotics including Penicillin’s, III generation Cephalosporins & Carbapenems, which is the best available antibiotic for treating them.

LIST OF “WHO” PRIORITY PATHOGENS REQUIRING NEWER ANTIBIOTICS :

Priority I : Critical

1.Acinetobacter - carbapenem resistant.

2. Pseudomonas - carbapenem resistant.

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3. Enterobacteriaceae (Proteus, Klebsiella, E.coli, Serratia) - carbapenem resistant, ESBL producing.⁴⁴

The global spread of Carbapenemase - producing Gram-negative pathogens is of special concern in healthcare and community settings.

Carbapenemase confers resistance to most beta-lactam antibiotics including penicillin, cephalosporins and carbapenems. Carbapenems have emerged as the agent of choice for managing Enterobacteriaceae, as they are resistant to aminoglycosides, fluoroquinolones, Penicillin and third generation cephalosporins.⁴⁵

Nosocomial isolates of S. aureus, Enterococcus species, E.coli, Klebsiella, Pseudomonas, & Acinetobacter, of late have become multi drug resistant, extremely drug resistant and pan-drug resistant, producing beta-

lactamases including ESBL, MBL & amp-C. According to Ambler, β-Lactamases are classified into four types of which MBL, belongs to

class B.⁴⁶ Metallo-β-lactamases (MBL), has the ability to hydrolyse Penicillin’s, Cephalosporins, and Carbapenems. Proteus mirabilis, Klebsiella pneumoniae, and pseudomonas aeruginosa were the predominant MBL producers.

The Aim of the current study is to evaluate the impact of infection in reconstructed flaps caused by various organisms and their antibacterial susceptibility among the isolates.¹¹ This can be done by phenotypic as well as genotypic methods.

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Phenotypic methods such as combined disk diffusion test, double diffusion test, modified Hodge test and E strip test identify the Carbapenemase production in general without any specification over the class of Carbapenemase. To identify MDR, XDR, and PDR strains, Molecular characterization is the only available tool for differentiation of Carbapenemase encoding genes.⁸ The most sensitive method is PCR, used to identify the genes causing Carbapenemase production.

Hence this study helps in the characterization of various organisms, their resistance pattern and in the prevention and control of these hospital acquired infections thereby reducing mortality and morbidity among patients undergoing flap reconstructive surgery in Plastic surgery wards.

Early detection is critical, the benefits of which include timely execution of strict infection control practices, formulating an effective antibiotic policy to prevent the spread of these MBL producing strains, and treatment with alternative higher antimicrobial agents.

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AIM & OBJECTIVES

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AIMS AND OBJECTIVES

AIM:

The aim of the study is to isolate , identify the organisms from pus samples obtained from pre-operative and post-operative wounds in the department of plastic surgery and to demonstrate the antimicrobial resistance pattern with special emphasis on molecular characterization of the resistant strains prevalent in our hospital.

OBJECTIVES:

 To isolate and identify various organisms from pus samples by conventional culture methods.

 To study the antibiotic susceptibility profile of organisms by Disk diffusion - Kirby Bauer technique.

 Phenotypic identification of strains producing Beta lactamases among the isolates.

 Molecular characterization of the genes responsible for the resistant pattern by PCR.

 To formulate antibiotics and proper infection control practices in our hospital and help the patients to overcome the infections caused by the prevalent multi-drug resistant strains.

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REVIEW OF LITERATURE

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REVIEW OF LITERATURE HISTORY:

Early Antiquity: Flap reconstruction had started its way back in 1000-800 BC, when Sushrutha of India described a regional flap for nose.

Since then the evolution of flap reconstruction showed enormous advancement especially after World War II. In 1400 AD, Because of inadequate measures to prevent and treat infections major extremity injuries frequently led to death by either exsanguination or eventual sepsis.

Antibiotic revolution: Infection was a major curse to those patients, and was considered as the “Dark Ages” till the discovery of sulphonamides. Later on Penicillin was discovered by Alexander Fleming in 1928, followed by streptomycin which dramatically reduced infection rates and increased survival from trauma reducing mortality.

Future-Evidence Based practice: Wound Healing as an outcome is also subjected to numerous uncontrolled variables including reconstructive timing, wound factors and patient factors. . Bacterial pathogens have developed many strategies of survival, which during their evolution have formed very sophisticated defence mechanisms. Pathogenic bacteria have worked out many different ways to overcome the host defence mechanism including virulence factors. Genotyping techniques have allowed the identification of cluster of genes which code for the virulence factors.

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9 FLAP RECONSTRUCTIVE SURGERY:

Flap reconstructive surgery, which includes skin and soft tissue coverage are the most common post operative complications even in hospitals with most modern facilities and standard protocols of preoperative preparation and antimicrobial prophylaxis.

DEFINITION:

Flap reconstructive surgery is a modern technique in plastic and reconstructive surgery where any type of tissue is lifted from a donor site and moved to recipient site with intact blood supply. They are classified under surgical site infections and are the third commonest nosocomial infections and account for approximately 10-40% of all health care associated infections(HAI).

CAUSES:

Soft tissue defects requiring Flap reconstructive surgery are;

1. Traumatic loss after Road traffic Accidents.

2. Residual stump infections in Diabetics, 3. III Degree burns(full thickness burns), 4. Carcinoma requiring total resection,

5. Connective tissue disorders(soft tissue excision).

Incidence of infections in soft tissue defects both pre-operatively and post-operatively depends on the nature of the wound, and the type of wound.

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10 Classification of wounds:

The Centres for Disease Control & Prevention (CDC) Atlanta, has formulated “Guidelines for Prevention of Surgical Site Infections”, and identified surgical wound classification.

1. Clean wound:

 Elective, primarily closed, no acute inflammation encountered.

 Entrance of respiratory, alimentary, genital, or uninfected urinary tracts are not encountered.

 No break in surgical technique.

2. Clean-Contaminated wound:

 Non elective cases that is otherwise clean.

 Controlled opening of normally colonized body cavity including Biliary tract, appendix, vagina and oropharynx.

 Minimal spillage or break in sterile technique.

3. Contaminated wound:

 Open, fresh, & Accidental wounds.

 Acute non purulent inflammation encountered.

 Major break in technique.

4. Dirty wound:

 Old traumatic wounds with retained, devitalized tissue.

 Wound with existing clinical infection.

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Based on the source of infection, they are classified as 1. Primary:

Present in the host and acquired from endogenous source.

2. Secondary:

They are present outside the body and are acquired from exogenous source such as Operation Theatre( inadequate air filtration, poor antisepsis) or the surgeon ( poor hand washing).

Wound can be classified according to severity as 1. Major:

Discharging significant quantity of pus with systemic signs.

2. Minor:

Discharging minimal quantity of pus with no systemic signs.

FACTORS AFECTING WOUND HEALING:

Certain conditions which diminish the efficacy of the immune response and delays wound healing are:

a. Ageing:

Diseases are more common in older age group since this will affect the circulation and oxygenation to the wound bed. Swift, in 2003 observed that the physiological process during ageing, makes the older patients more prone for poor wound healing.⁴⁹ Every phase of healing undergoes age

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related changes including delayed angiogenesis, decreased secretion of growth factors, impairment of macrophage function, delayed epithelisation, reduced turn over of collagen and remodelling. Skin elasticity is reduced and collagen replacement is also poor and this in turn affects wound healing. As immunity decreases with ageing they are more prone to infection.

b. Obesity:

Increased tension on wound edges due to Obesity results in wound dehiscence and also reduces micro perfusion thereby decreasing the oxygen availability to wound. Anaya and Dellinger et al., and De Mello, in 2002 observed that obesity is related to defective wound healing, due to impaired tissue perfusion.⁵⁰

c. Stress :

During stress normal cell mediated immunity is impaired and wound healing is delayed. Kiecott-Glaser at al., 1995 observed that physiological stress is associated with poor wound healing.⁵¹

d. Diabetes:

Studies conducted by Jorge Acosta in 2013 revealed that, vascular changes occurring in diabetic patients results in hypoxia, impaired angiogenesis and neovascularisation and dysfunction of fibroblasts leading to impaired wound healing.⁵²

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13 e. Dehydration :

Electrolyte imbalance that occurs during dehydration will affect cellular function and wound healing. So fluid resuscitation has to be done in post operative patients to prevent hypovolemia as studied by Posthauer, Mary Ellen RD in 2006.⁵³

f. Effective hand washing :

Transfer of pathogenic organisms through person or objects were prevented by simple hand washing technique.

g. Medication:

Hoffman et al., studied in 2004 that drugs such as anticoagulants, immune suppressants, anti-inflammatory and cytotoxic drugs, reduce wound healing due to its interference with cell division or clotting process.⁵⁴ h. Nutrition:

Studies conducted by Campos et al, in 2008 revealed that protein is essential for wound healing and it will be delayed in case of protein calorie malnutrition.⁵⁵

 Maintenance of blood sugar level is essential for wound healing to occur.

 Oxygen is an essential factor in the hydroxylation of amino acid proline and lysine during collagen synthesis.

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 For proper functioning of immune system minerals such as copper and zinc deficiency leads to defective formation of granulation tissue and affects cell multiplication, fibroplasias, collagen synthesis and epithelial covering of wounds.

 Vitamins A, and C plays a vital role in collagen synthesis. Reduced vitamin levels leads to less immunity and poor wound healing.

 Fats and Carbohydrates: Energy required for wound healing will be provided by carbohydrates and also by fats.

i. Oxygenation:

Hypoxemia results in poor wound healing. Rodriguez et al., 2008 observed that, for proper wound healing to occur, oxygen is essential, as it induces epithelisation, and provides immunity to patients.⁵⁶

j. Smoking :

Smoking impairs wound healing. Sorensen in 2012, observed that smoking causes delay in healing of wound due to its vasoconstrictive effects on blood vessels.⁵⁷

RISK FACTORS OF FLAP INFECTIONS⁵⁸ :

Multiple risk factors are involved in wound infections. Among these three major determinants are;

A. Bacterial factors B. Local wound factors C. Patient factors

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15 A. Bacterial factors :

1. Infection at remote site.

2. Recent hospitalization.

3. Long duration of procedure.

4. Different classes of wounds.

5. Antibiotics given previously.

6. Preoperative cleaning.

7. Bacterial number, virulence and antimicrobial resistance.

Bacterial factors causing infection are its virulence factors and bacterial load. The development of surgical site infection depends on the ability of the microorganisms to produce toxins and to resist phagocytosis and intracellular destruction

Studies conducted on traumatic wounds revealed that contamination of wounds with bacteria of >10⁵ organisms frequently causes infection.

Incidence of infection was more with longer preoperative stay, shaving done before surgery, increased duration of surgery and infection present at remote site.

B. Local wound factors:

1. Blood supply.

A good blood supply is an essential factor in the process of wound repair because it will provide oxygen and nutrients necessary for cellular and biochemical process of wound repair and it is also necessary for removal of wound metabolites.

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16 2. Surgical technique.⁴⁹

Good surgical technique includes gentle handling of tissue, maintaining haemostasis and avoiding dead space in the wound.

3. Haematoma and necrosis.

The necrotic tissue present in the wound causes tissue swelling and this will decrease the blood supply to the wound and the presence of these substances in the wound acts as a good culture media for bacterial growth thereby causing delayed wound healing.

4. Sutures.

Ideal suture should hold the tissue in opposition and cause only minimal tissue reaction and it should be quickly absorbed so that infection is not encouraged.

5. Drains.

Bacterial contamination of wound was more with the usage of drains, Lilani SP(10,) in his study observed that wound infection rate was 22.41% in cases where drains were used when compared to 3.03% in cases where drains were not used.⁵⁹

6. Foreign bodies.

It depends on the invasiveness of operation and surgical skills, as surgery breaks the barrier defense mechanism.

C. Patient related factors:

1. Age

2. Immuno-suppression.

3. Malnutrition.

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17 4. Steroids.

5. Malignancy.

6. Obesity.

7. Diabetes.

8. Smoking.

9. Perioperative transfusion.

Maintaining normothermia and delivering oxygen at Oxygen Saturation of 80% or higher during the operation and control of blood glucose level in the perioperative period can reduce surgical wound infections.

ETIOLOGY OF FLAP INFECTION^53,55

Multiple factors are involved in wound infections and the contribution of these factors varies in different types of surgeries.

The Source of infection can be 1. Exogenous or

2. Endogenous.

Organisms, when present on the body surface or hollow viscera can cause infection.

Spectrum of Bacteria:

Most of the Gram negative Bacilli in wound infections belong to Enterobacteriaceae family, are facultative anaerobes such as Escherichia, Proteus and Klebsiella.

Staphylococcus aureus is the most common Gram positive organism causing wound infections. Since development of antibiotic resistance is

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more common with Staphylococcus aureus such infections require treatment with Penicillinase resistant antibiotics. Initial extensive use of β-lactam antibiotics, has led to the emergence of MRSA strains.

Spectrum of Bacteria in various wounds:

Staphylococci is the predominant organism causing infections in patients undergoing surgery for clean wounds, as they are skin commensals present at the site of skin incisions, accounting for about 2-4% of incidence.⁴⁸

Infection in clean contaminated and contaminated surgeries are from microorganisms colonizing the respiratory, genital, gastrointestinal and urinary tract and incidence is about 10-20%

In Dirty wounds microorganisms already present in the traumatized area as in case of RTA, Burns, etc., leading to sepsis, where the rate of incidence of infection is about 40-60%.⁶⁰

Cross Infection

Moreover exposure of the wound to the operating room environment, during the surgical procedure leads to a high bacterial load.

The longer the duration of surgery the more the contamination of wound.

PATHOGENESIS:

Measures to decrease the infection rate are as follows:⁶² 1. Reduce the bacterial contamination

Includes Pre- operative and Intra- operative measures.

2. Improve the host immunity.

Pre- operative

Intra-operative & Post-operative measures.

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19 I. Reduce the Bacterial contamination A.PRE-OPERATIVE MEASURES:

 Avoid preoperative antibiotic use (excluding surgical prophylaxis)

 Minimize pre-operative hospitalization

 Hair removal using electric clippers or depilators.

 Ensure timely administration of antibiotics one hour prior to surgery & within 6 hrs postoperatively.

 Elimination of nasal carriage of staphylococcus aureus.

B. INTRA-OPERATIVE MEASURES:

 Preparation of skin with povidone iodine or chlorhexidine solution, 6-8 hrs prior to surgery.

 Follow strict aseptic techniques.

 Maintain high flow of filtered air.

 Reduce prophylactic antibiotics in prolonged surgeries

 Minimize flash sterilization of surgical instruments

 Minimize use of drains.

II. Measures to improve host immunity are:

A. PRE-OPERATIVE MEASURES:

 Resolve malnutrition or obesity.

 Eliminate high risk factors such as Cigarette smoking & Hyper - glycaemia.

B. INTRA-OPERATIVE AND POST-OPERATIVE MEASURES:

 Minimise dead space, devitalized tissue and haematoma.

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 Supplemental oxygen therapy and perioperative normothermia.

Maintenance of adequate hydration and nutrition during immediate post-operative period & recovery period.

MICROBIAL AGENTS IN WOUNDS BEFORE AND AFTER FLAP RECONSTRUCTION:

Bacterial infection is the usual cause of suppuration and the most common organisms include Proteus species, Klebsiella species, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Acinetobacter species.

Infections of the post traumatic wound cultures revealed Proteus species, Acinetobacter species, Staphylococcus aureus and Pseudomonas aeruginosa.

Surgical site infections reveals Proteus species, Staphylococcus aureus, followed by Pseudomonas aeruginosa, E.coli, Klebsiella pneumoniae, Acinetobacter baumannii.⁵⁹

Infections of diabetic wounds showed the most common isolate being Pseudomonas aeruginosa, followed by Proteus species, Klebsiella, E.coli, and Staphylococcus aureus.⁵²

Burns wound infections often reveal Pseudomonas aeruginosa, to be the most common organism, after which Proteus species, Klebsiella pneumoniae, and Staphylococcus aureus were isolated.⁶¹

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Microbes isolated in the study prior to surgery and following surgery, did not show a marked difference. Proteus being the most common isolate, could be a potential pathogen causing wound infections.

Among the total isolates, Proteus constituted about 55% and Klebsiella 22%, E.coli was 15%, Pseudomonas -3.5% and Staphylococcus aureus &

Acinetobacter were of equal prevalence (i.e) 1%.

PROTEUS:⁶³

The first description of Proteus bacilli as putrefying bacteria was made by Gustav Hauser in 1885. Proteus was named after Greek God, which means Pleomorphic due to its constant change in the shape and size. They typically appear as bacilli and measure about 1-3µm in length and 0.4-0.6µm in diameter but also as cocco-bacilli singly or in chains, and young forms can be filamentous, arranged discretely.

NATURAL HABITAT:

SAPROPHYTES:

Micro organisms belonging to Proteus species are widely distributed in the natural environment. They can be found in polluted water and in soil and manure where they play an important role in decomposing organic matter. Proteus rods also exhibit proteolytic activity under aerobic and facultative anaerobic conditions. The oxidative deamination of amino acids and the ability to hydrolyse urea to ammonia and carbon dioxide are the most representative biochemical properties of these bacteria.

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22 COMMENSALS:

Besides the saprophytic mode of life in the natural environment they are also commensals in the intestines of humans & moist areas of the skin. Proteus bacilli under favourable conditions are able to cause pathological events such as nosocomial infections in intensive care units who are often immunocompromised.

OPPORTUNISTIC PATHOGENS:

Proteus bacilli are now treated as well known opportunistic pathogens that cause infections in humans. The genus Proteus belongs to the family Enterobacteriaceae. The most characteristic feature which distinguishes Proteus rods from other members of this family is the swarming phenomenon. According to Bergey’s Manual Proteus consists of five species: P. mirabilis, P. vulgaris, P. penneri, P. hauseri and P.

myxofaciens and three unnamed genomospecies.

Proteus species are opportunistic pathogens, causing urinary tract, wound and soft tissue infections and septicemia. Proteus mirabilis and Proteus vulgaris are the most common species of which Proteus mirabilis accounts for 90% of all Proteus infections. P. vulgaris is isolated from immuno-compromised patients or those on long-term antibiotic regimen.

Other Proteus species P. penneri and P.myxo faciens are rarely encountered in clinical specimens. They are all oxidase negative, actively motile, non spore forming, non capsulated, and are recognised by their ability to cause disease. The Proteus species which colonize the intestinal

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tract, differ from those found in the wounds in their ability to carry genes encoding antibiotic resistance.

VIRULENCE FACTORS:

Various virulence factors have been attributed to the pathogenicity of Proteus infections. Apart from antigens, fimbriae, and flagella chemical structure also plays an important role in the pathogenicity.

NAMING OF “H” AND “O” ANTIGEN:⁶⁴

In general, the terms H and O antigens are used to denote the flagellar and somatic antigens of any organism respectively. However the naming of these antigens were linked historically to the properties of Proteus.

H antigen is named from the ability of flagellated strains of Proteus to grow on agar as a thin film resembling the film of breath on a glass(

Greek word ‘Hauch’ means film of breath).

O antigen does not show the thin film, while strains carrying the somatic antigen grow on media( ‘Ohne Hauch’ means without film of breath).

S.No VIRULENCE FACTORS PROPERTIES

1 Fimbriae Adhesion

2 Flagella Swarming growth

3 Urease Renal stones

4 LPS Endotoxin

5 O and capsular polysaccharides Biofilm formation

6 Hemolysins Cytotoxicity

7 Agglutinins Cell aggregation

8 Proteases Ab degradation

9 Deaminases Iron acquisition

10 Zinc and phosphate transport system

Utilization

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24 FIMBRIAE:⁶⁶

Bacterial adherence to epithelial surfaces is thought to be one of the most important virulence factor in pathogenesis. Ultrastructural studies of Proteus have shown two types of fimbriae-thick(7nm diameter) and thin(4 nm diameter). The first type also known as type IV fimbriae was found to be mannose resistant and was named Proteus like fimbriae (MR/P). The second type is the type III fimbriae, which are mannose resistant Klebsiella like fimbriae(MR/K). The MR/P fimbriae were isolated from Sareneva et al study, and it was a 21 KDa protein. The MR/P fimbriae are strongly immunogenic and induce specific antibodies in chronic infected patients. MR/K fimbriae are heam agglutinins. It mainly binds to tissues and forms biofilms.

FLAGELLA:⁶⁷

The presence of flagella on the surface of pathogenic and opportunistic bacteria has been thought to facilitate the colonization and dissemination from the initial site. Proteus bacilli are dimorphic bacteria.

When grown in liquid media, these cells display swimming behaviour and have a distinct morphology i.e., they are motile with peritrichous flagella (6-10 flagella per cell) and 1-2 µm in diameter. When grown in solid media, they undergo morphogenesis and swarm over the surface & are non- septate, multi nucleated swarmer cells of 20-80 µm in diameter.

OUTER MEMBRANE PROTEINS(OMP) :⁶⁵

In general, OMP possess immunogenic properties and mitogenic activity for B cells. Further, OM lipoproteins and their synthetic analogy

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function as adjuvants which activates macrophages to produce tumour necrosis factor(TNF). The OMP of Proteus contains three major proteins, among which OMP A is a peptidoglycan associated matrix protein.

Three important properties of Outer membrane proteins : 1. OMP A mediates penetration of hydrophilic molecules

2. OMP A is a mitogen for B cells and displays strong antigenic activity.

3. OMP A inhibits oxygen radicals & enhances interleukin synthesis.

LIPOPOLYSACCHARIDE (O-antigen, endotoxin):⁶⁸

Proteus is antigenically heterogenous, principally because of structural differences of its host specific polysaccharide chain of LPS (O antigen) & H antigen. The serological classification scheme of Kauffman and Perch includes 49 O antigens and 19 H antigens. The common structural feature of Proteus O antigen is Uronic acid which plays an immunodominant role. O specific polysaccharides from P. vulgaris OX 19 and OX 2 and P. mirabilis OX K cross react with antibodies from patients of Rickettsia infections used in the diagnostic Weil-Felix test. This strongly suggest that the antigen common is LPS.

S.No Core Type LPS

1 I P. mirabilis O 28, O 23, O32. P. vulgaris O39

2 II P. mirabilis 1959

3 III P. mirabilis O28

4 IV P. mirabilis O38

5 V P. vulgaris O23

6 VI P. mirabilis O 11,O 13, O30, O31, O35, O36

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Biologically , LPS or endotoxin pathogenic factors of gram negative bacteria causes fever hypotension DIC and shock. Endotoxin can be released from cell surfaces of bacteria during their multiplication, lysis and death. LPS is a bio active molecule and acts through its biological centre (lipid A component) on various cell types including macrophages and monocytes. LPS binds with receptor in blood and activates CD 14 on macrophages and produces active lipids (Prostaglandins, Thromboxane A2 and platelet activating factor), oxygen free radicals (O2, H2O2 and NO ) and peptide mediators ( TNF α, IL-1, IL-6, IL-8, and IL – 10) which produces detrimental effects.

UREASE:⁶⁹

Urease catalyses the hydrolysis of urea to ammonia and carbon di oxide and increases urine pH, and precipitates Mg²⁺ and Ca²⁺ forming struvite stones. This forms a glycocalyx which facilitates the adhesion of bacteria to the cell surface. Bacteria can divide in the glycocalyx to form isolated clumps or micro colonies as a continuous biofilm. This enzyme is also a factor contributing to the pathogenicity of many bacteria including Proteus.

Mc lean et al, formed an etiological concept of stone formation as,

1. P.mirabilis, ascends the urinary tract, colonizes uroepithelium and facilitates bacterial adhesion.

2. Urease production elevates urine pH, protecting uro pathogens from antibiotics.

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3. Encrusted subdividing bacteria, produces glycocalyx with fossilized bacterial colony.

PROTEASE:⁶³

IgA, a secretory protein used to transport the antibody molecule is a predominant Immunoglobulin in mucus secretion. It is reported that different Proteus species produces different IgA proteases; the most characteristic feature of IgA protease is cleavage of IgA and IgG, as well as gelatin, casein. Allison et al, have demonstrated the differentiation of Proteus into multinucleate and hyper flagellate swarmer cells that increases virulence. One metalloprotease is Zap A which is extracellular, and digests immunoglobulins IgA1 and IgA 2.

HEMOLYSINS:⁷⁰

The history of investigation of Proteus haemolytic activity starts from 20^th century when Taylor observed lysis of erythrocytes in young broth culture of Proteus. Hughes et al described in 1997 that some strains produce haemolysis in blood agar plates. There are two types of haemolysins: intra cellular and cell bound haemolysins, which are calcium dependent or independent. Synthesis of haemolysins is associated with cytotoxic activity.

SIDEROPHORES:⁷¹

Virtually all bacteria need iron as an important nutritive compound.

It is indispensable for growth and metabolism. In the presence of iron deficiency, bacteria produce iron chelators called as siderophores, which bind iron and transport it to the bacterial cells by suitable receptor proteins

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and appropriate transport mechanisms. Synthesis of siderophores is under the control of chromosomal or plasmid genes. α-Hydroxy Iso-valeric acid is the siderophore produced by Proteus group. Eukaryotic proteins like transferrin and lactoferrin have high siderophore activity, and render prokaryotes iron deficient. The siderophore activity of α-keto acids, with deamination of amino acids by these bacteria was studied by Drechsel et al, which form stable iron chelators in the absence of siderophores. The possibility of iron uptake in the course of particular colonization and in the consequence is considered an important factor of pathogenicity of these bacilli.⁷¹

PATHOGENESIS OF PROTEUS IN INFECTIONS:

Proteus species possess an extra cytoplasmic outer membrane, a feature shared with other gram negative bacteria. In addition, the outer membrane contains a Lipid bilayer, Lipoproteins, Polysaccharides, and Lipopolysaccharides. Infection depends on the interaction between the infecting organism and the host defence mechanism. Various components of the membrane interplay with the host to determine virulence. Inoculum size is important and has a positive correlation with the risk of infection.

The first step in the infectious process is adherence of the microbe to the host tissue. Fimbriae facilitate adherence and thus enhance the capacity of the organism to produce disease. Proteus contains fimbriae(pili) which are tiny projections on the surface of the bacterium.

Pili enable organisms to attach to the selected host tissue sites. The presence of the fimbriae has been demonstrated to be important for

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attachment. Proteus induces apoptosis and epithelial cell desquamation by secreting Interleukin-6 and Interleukin-8. Additionally, they elaborate cytolytic haemolysins that lyse red blood cells and release iron, a bacterial growth factor.

Proteus multiplies in the necrotic, devitalised tissues leading to wound infections & produces Endotoxin which enters the blood stream from wounds, & induces sepsis resulting in SIRS(systemic inflammatory response syndrome) leading to high mortality rate.

CLINICAL MANIFESTATIONS OF PROTEUS INFECTIONS:⁶³ Proteus species are part of normal flora of the human gastro intestinal tract. Colonization of Genito urinary tract is commonly known to cause Urinary tract infection, Urolithiasis & and at times Prostatitis.

Apart from infections in Wounds & Burns, they also cause Abscesses.

They also play a role in causing severe infections like Sepsis, SIRS, Nosocomial pneumonia, Endocarditis, etc.

MANAGEMENT OF PROTEUS INFECTIONS:⁷²

Proteus species are intrinsically resistant to Nitrofurantoin, Tetracycline & Polymyxin B, due to various mechanisms.

Different classes of antibiotics were tested on Proteus species, for studying its antimicrobial sensitivity pattern. Results of the study revealed that Imipenem(IMP) was the most effective antibiotic with 100%

sensitivity followed by Amikacin(AK) with 81% sensitivity.

In a study conducted by Saleh A. Bahaswan et al(14), among 49.74%

of Total isolates, Proteus had an incidence of 20.33%. Its sensitivity to

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Imipenem was 91%, amikacin 61%, cefoxitin 48%, and piperacillin 44%.

Results also showed Proteus species was resistant to Cotrimoxazole, Ampicillin, and cefipime.⁵

Proteus species is sensitive to all major classes of antibiotics including Penicillins, cephalosporins, carbapenems, and intrinsically resistant to Nitrofurantoin, Tetracyclines and Polymyxin B.

Ampicillin :

Ampicillin is the prime member in the Penicillin group of β-lactam antibiotics, which is bactericidal against gram negative bacteria. Ampicillin is an irreversible inhibitor of enzyme transpeptidase, which is needed for cell wall synthesis of bacteria, and thus bactericidal.

Trimethoprim and sulfamethoxazole :

Blocks 2 consecutive steps in the biosynthesis of nucleic acids and proteins essential to many bacteria. SMZ inhibits bacterial synthesis of dihydrofolate, by competing with PABA. TMP serves as a competitive inhibitor of dihydrofolate reductase (DHFR), inhibiting the synthesis of tetrahydrofolate, the active form of folate. In vitro studies indicate that bacterial resistance develops more slowly with TMP/SMZ combination .

The spectrum of antibacterial activity of TMP/SMX includes, E coli, Klebsiella and Enterobacter species, Morganella morganii, P mirabilis, and P vulgaris.

Ciprofloxacin :

Ciprofloxacin is a broad spectrum antimicrobial carboxy fluoroquinolone. Ciprofloxacin inhibits bacterial topoisomerase II and IV

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(DNA gyrase), enzymes required for DNA replication, transcription, repair and recombination, thereby inhibiting cell division. It has in vitro activity against a wide range of gram-negative and gram-positive microorganisms.

Ceftriaxone :

A Third-generation cephalosporin, and β-lactam antibiotic with broad-spectrum especially gram-negative activity. The Bactericidal activity of ceftriaxone is by binding to penicillin-binding proteins (transamidases), thereby inhibiting cell wall synthesis. Peptidoglycan cross linking activity is irreversibly inhibited. It is highly stable in presence of beta-lactamases, like penicillinase and cephalosporinase, of both gram-negative and gram- positive bacteria.

Amoxycillin :

Amoxicillin is an extended spectrum, Penicillinase susceptible, semi- synthetic aminopenicillin. The Presence of amino group helps in the easy penetration of bacterial cell wall, after which it binds to PBP 1A, prevents its attachment and Inhibits the cell wall synthesis. Hence it has bactericidal activity against both gram-positive and gram-negative organisms.

Gentamicin :

Bactericidal aminoglycoside antibiotic that irreversibly binds to 30s ribosome inhibiting protein synthesis in susceptible microorganisms. Active against E. coli, Proteus species, Pseudomonas aeruginosa, Klebsiella, Enterobacter, and Serratia.

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Its a combination of extended spectrum penicillin antibiotic(

piperacillin) & β- lactamase inhibitor (tazobactam). Like all β-lactam antibiotic, Piperacillin exerts bactericidal activity by combining with PBPs and inhibiting cell wall synthesis. Active against various gram-positive and gram-negative aerobic and anaerobic bacteria. Tazobactam is an irreversible inhibitor of bacterial β-lactamases.

Tazobactam protects piperacillin against Richmond and Sykes II, III, IV & V beta lactamases; Staphylococcal penicillinase; and extended spectrum beta lactamases. However tazobactam has only species specific activity against class I chromosomally mediated beta lactamases and is particularly useful for treatment of mixed infections and presumptive therapy prior to the identification of the causative organisms.

Imipenem and Meropenem :

These “Last Line” β-lactam Carbapenem antibiotics have broadest spectrum of activity against gram-positive and gram-negative aerobes and anaerobes, and are used in treating mixed infections.

Being larger molecules, they enter Gram negative bacteria through Outer Membrane Porin channels. It permanently acylates the Penicillin Binding Proteins and inhibits the peptide cross linkages thereby inhibiting the cell wall synthesis, with continued autolysis.

Certain bacteria which are inherently resistant to most beta-lactam antibiotics, may also develop resistance to these Carbapenems rapidly

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during treatment. So periodic susceptibility testing is required when clinically indicated.

BETA-LACTAMASES:

Introduction

Even before Penicillin was used in medical practice, the first ever Beta-lactamases were identified in E. Coli. and remain the most important contributing factor for Antibiotic resistance in gram negative bacteria. ²⁶ Key Dates in Beta-lactamase emergence :

Year Enzyme Organism Place

1944 Pencillinase S. aureus -

1963 Tem – 1 E. Coli Athens

1974 SHV-1 E. Coli Switzerland

1978 OXA-10 P. aeruginosa -

1988 Metallo-beta-

lactamase

P. aeruginosa Japan

1991 OXA-14 P. aeruginosa Turkey

1991 PER-1 P .aeruginosa,

S. typhimurium

Turkey

BETA-LACTAMS:

The resistance to beta-lactams in the bacteria is caused by either one or a combination of factors like;

a) Hydrolysis by beta-lactamases,

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b) Alteration in penicillin binding proteins,

c) Change in the structure and number of the porin proteins of efflux pump that causes decrease in concentration of antibiotics in the bacteria.

All of these factors cause hydrolysis of the beta lactam ring and are classified into two systems:

I. Ambler’s classification- Molecular classification, which is based on amino acid sequences

II. Bush- Jacoby- Medeiros classification, which is a functional classification.

AMBLER’S CLASSIFICATION:⁷³ Class A – Penicillinases [TEM,SHV].

Class B - Metallo-β-lactamases [IMP,VIM]

These need zinc as a moiety for its functioning.

Class C - Cephalosporinases [Amp C].

Class D - Oxacillinases [OXA-23,OXA-58].

Classes A, C, D have a serine moiety at the active site.

METALLOBETALACTAMSASE (MBL):⁷⁴

The first MBL was reported in the 1960s from Bacillus cereus and thereafter eighteen MBLs are observed in various Gram- Negative bacteria.

These MBLs production is mostly chromosome Encoded which did not cause an obvious threat of spreading to other Bacteria. However the

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first plasmid mediated MBL was isolated in 1991 from Pseudomonas aeruginosa and was reported from Japan Which was named as IMP- 1 while VIM -1, another type of Acquired metallo-beta-lactamase, was first reported in 1999 from Italy.

Characteristics of metallo-beta-lactamase (MBL):

1. Metallo-beta-lactamase requires zinc for their catalytic activity.

2. Their activity is inhibited by metal chelators such as EDTA and THIOMERSAL compounds.

3. They hydrolyse all beta-lactam antibiotics including Penicillin, Cephalosporins and Carbapenems with exception of Aztreonam which is a Monobactam.

4. MBL producing strains are not susceptible to serine beta- lactamase inhibitors such as clavulunate.²⁷

Classification of MBL

I. According to Ambler Molecular classification – MBL belongs to class B. on the basis of their sequences, & class B is again divided into 3 subgroups B1, B2, & B3.⁷³

1.Class B1 :These enzymes posses the zinc coordinating residues of One cysteine and three Histidine and also will include the transferable MBLs.

2.Class B2:These enzymes will posses an Asparaginase at the first Position instead of Histidine in the principal zinc - binding motif. They are derived from the Serratia fonticola enzyme SFH –1 and Aeromonas species.

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3.Class B3: MBL L1 is the sole occupant of this class, functionally represented as a tetramer .²⁸

II. According to Bush-Jacoby- Medeiros (functional) Classification :⁷⁵ MBL belong to group 3.

On the basis of Imipenem and other beta-lactam antibiotic hydrolysing capacity, Group – 3 is again divided into 3 subgroups which include,

a. Group 3a - broad spectrum activity.

b. Group 3b - preferential activity towards Carbapenem.

c. Group 3c – hydrolyse Carbapenem poorly, as compared to other beta-lactam antibiotics²⁹.

3. MBL gene: According to location : a. Chromosomally encoded MBLs

b. Plasmid coded Transferable MBLs (a part of Integron).

Transferable MBL is divided into 4 groups on molecular basis as i). IMP (Imipenemase) Types

ii). VIM ( Verona Imipenemase ) Types iii). GIM (German Imipenemase) Types iv). SPM (Sao Paulo Imipenemase ) Types³⁰. TRANSFERABLE MBLs:⁷⁶

Genetic Apparatus of Transferable MBLs

Genes encoding IMP, VIM type as well as GIM –1 are found as gene cassettes in class 1 integrons³¹. The VIM and IMP type of MBLs are the most common^32,33 & IMP MBL genes are also found on class 3

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integrons. Integrons are capable of procuring cassettes via a site - specific recombination event between two DNA sites, one in the integron and one in the gene cassette.

Integrons consist of three regions:

1. The 5’ conserved region, consists of the integrase Gene, its adjacent recombination site, and a promoter, which facilitates Expression of the procured gene cassettes in the variable region

2. The 3’ conserved region, consists of a partially deleted qac gene, confers resistance to antiseptics and sulphonamides.

3. Variable region.

Gene cassettes are small pieces of circular DNA, comprising a single gene together with a recombination site termed a 59- base element, approximately 1 kb in size. Bla-VIM genes from some European countries have been found with a truncated 59 base element and the gene cassettes are likely to be “ fused”.

While gene cassettes carrying aminoglycoside and beta-lactam resistant genes can freely move from one integron to another, they cannot move from one organism to another. The assistance of other genetic elements such as plasmids and Transposons are necessary. The majority of MBL genes are found usually on plasmids.²⁸

BIOCHEMISTRY OF MBLs:⁷⁷

MBLs and serine beta-lactamase cleaves the amide bond of the beta-lactam ring and thus mediate resistance to beta-lactams. However, the way in which the two groups of enzymes achieve this varies

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considerably. MBLs possess a distinct set of amino acids that define the finite architecture of the active site which coordinates the Zinc ions. The zinc ions in turn usually coordinate two water molecules necessary for hydrolysis. The principle zinc binding motif is histidine-X-histidine-X aspartic acid (HXHXD), and while most MBLs accommodate two zinc Ions in their active site, the class B2 enzymes possess just a single Zinc ion.

It appears that most MBLs have a flexible loop that facilitates binding and hydrolysis of the beta-lactam substrates. MBLs possess a wide active site and accommodates most beta-lactam substrates, facilitating wide spectrum of activity. They are also impervious to the impeding effects of serine inhibitors such as clavulanic acid and sulbactam that are often treated as poor substrates. Interestingly, none of the MBLs hydrolyse aztreonam particularly well, and it has been speculated that it could be considered a therapeutic MBL inhibitor.

Risk factors for MBL colonization³⁶.

1. Prolonged hospital stay especially in ICU.

2. Irrational use of multiple antibiotics.

3. Patients on multiple invasive devices.

4. Presence of focal or generalized infection.

MECHANISM OF CARBAPENEM RESISTANCE:⁷⁹

Carbapenemase producing Enterobacteriaceae are gram negative bacteria that are nearly resistant to the carbapenem class of antibiotics, considered “the drug of last resort” for such infections. They have been

Prospective study on the Microbial profile of Bacterial infections before and after Flap Reconstructive Surgery

ACKNOWLEDGEMENT

CONTENTS

INTRODUCTION

AIM & OBJECTIVES

REVIEW OF LITERATURE