REVIEW OF LITERATURE

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MULTIVARIATE ANALYSIS OF ACINETOBACTER SPECIES IN A TERTIARY CARE HOSPITAL.

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

APRIL 2017.

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CERTIFICATE

This is to certify that the enclosed work “Multivariate Analysis

of Acinetobacter species in a Tertiary Care Hospital” submitted by Dr. M. Banumathy 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 June 2015 to April 2016 under the guidance and supervision of Dr. N.Mythily, MD., Professor, Department of Microbiology and the conclusion reached in this study are her own.

Guide

Dr. N.MYTHILY, MD., Professor,

Department of Microbiology, Coimbatore Medical College, Coimbatore.

Dr. A.EDWIN JOE, MD., (F.M), B.L., Dr.A.DHANASEKARAN, MD., DCH.,

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. M. Banumathy solemnly declare that the dissertation entitled

“MULTIVARIATE ANALYSIS OF ACINETOBACTER SPECIES IN A TERTIARY CARE HOSPITAL” was done by me at Coimbatore Medical College Hospital, during the period from June 2015 to April 2016 under the guidance and supervision of Dr. N. Mythily, M.D., Professor, 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. M. Banumathy

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ACKNOWLEDGEMENT

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ACKNOWLEDGEMENT

I express my deep debt of gratitude to our respectful Dean Dr.

A. Edwin Joe MD., (F.M), B.L., for permitting me to do this study.

I wish to place my deep sense of gratitude and sincere thanks to Dr. A. Dhanasekaran DCH., 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 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.R.Senthilkumar and Dr. V.M. Theeba 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 Mr. R. Murugan and my daughter M.Preethika Rajshri 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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INTRODUCTION

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INTRODUCTION

Acinetobacter is an opportunistic bacterial pathogen primarily associated with Hospital-acquired infections. Multi-drug Resistant (MDR) Acinetobacter has increasingly become a formidable antigen in nosocomial and community acquired infections. So much so, that in recent years it has been designated as a red alert human pathogen generating alarm among the medical personnel . Antibiotic resistant bacteria currently imply an impending disaster worldwide and therefore

constitute a strong challenge when treating patients in hospital settings.¹ WHO has recently identified antimicrobial resistance as one of

the important causes of life threatening nosocomial infections among critically ill and immonocompromised individuals in treating human diseases. The most common and serious MDR pathogens have been encompassed within the acronym "ESKAPE" standing for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species.³

Carbapenems have emerged as the agent of choice for managing Acinetobacter species, as they are resistant to Aminoglycosides, Fluoroquinolones, Penicillins and third generation Cephalosporins. But increased use of these antibiotics have in turn resulted in the emergence of Carbapenem resistant strains². Emergence of such antibiotic resistant strains has made the infection untreatable.

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Hence characterization of the species and identification of antibiotic resistant strains is necessary for treatment and management of infections.

Acinetobacter species are encapsulated, aerobic, non-fermentative bacteria. They are ubiquitous - living in soil and water. They are able to live for longer periods in the environment both in wet and dry conditions.

Acinetobacter is an important nosocomial pathogen causing pneumonia and bacteraemia among patients admitted in the intensive care unit worldwide. It also accounts for 10% of community acquired pneumonias. Other human infections caused by Acinetobacter species include ventilator associated pneumonia, prosthetic valve endocarditis, endophthalmitis, meningitis following neurosurgical procedures, skin and wound infections, UTI and bacteraemia.

A.baumannii accounts for more than 80% of isolates causing human infections among all other species. It is also currently the third common isolate among cases of Gram-negative sepsis particularly in immunocompromised patients. Other species such as A. johnsonnii, A.lwoffii seem to be natural inhabitants of human skin and also commensals of the oropharynx and vagina.

Acinetobacter species are widely-distributed in nature. The organism`s ability to survive under a wide range of environmental conditions and to persist for extended periods of time on surfaces

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confers on it a survival advantage leading to frequent outbreaks of infection. In hospitals, they persist for months on clothing, bedclothes, bed rails, ventilators, and other environmental surface such as sinks and doorknobs.⁴

Risk factors specific for nosocomial infection include

 Prolonged hospital stay as in ICU

 Previous infection

 Treatment with broad spectrum antibiotics

 Indwelling central venous or urinary catheters

 Mechanical ventilation

 Breaches in infection control measures

 Introduction into hospital due to medical transfers from other hospital where organism is endemic.

One of the most striking features of Acinetobacter species is their capacity to produce multiple drug resistant mechanisms against major antibiotic classes. The drugs most effective in the early 80’s, were the common Cephalosporins, namely Ceftazidime and Cefotaxime, which were later replaced by the Carbapenem group of drugs.

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Due to increasing use of antibiotics there is selective pressure among the bacterial population leading to mutation of the genes which code for Carbapenemase - a beta-lactamase enzyme responsible for the emergence of antibiotic resistance in Acinetobacter. This has led to increased rate of secondary bacteraemia, greater duration of hospitalization and higher mortality rates.

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 carbapenem. Frequent co-existence of other antibiotic resistant genes complicates therapy and limits treatment options.

Class B type of beta-lactamase (Ambler Classification) is one of the carbapenamases , which has the ability to hydrolyse Penicillins, Cephalosporins, and Carbapenems. Accurate detection of carbapenemase production is essential for infection control and rational use of antibiotics⁵.

The most common types of beta-lactamases causing carbapenem resistance in Acinetobacter baumannii are the OXA-23, OXA-24, OXA- 40, OXA-58, and OXA-143 type serine beta-lactamases.

There is a significant increase in resistance pattern among the species of Acinetobacter in our hospital. So due to evolution of MDR, XDR and PDR strains rapid detection of these isolates is necessary for timely intervention to save the patient and initiate infection control measures.

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Phenotypic methods identify the carbapenemase production in general without any specification over the class of carbapenemase .Molecular characterization is the only available tool for discriminating different carbapenemase encoding genes among which Polymerase chain reaction is the most reliable method for identification of OXA and bla genes

The aim of this study is to determine the prevalence of MBL among carbapenem resistant strains of Acinetobacter species in our hospital and to investigate the presence of OXA type beta-lactamases in Meropenem- resistant Acinetobacter isolates using PCR. Hence this study may help in identification of Acinetobacter species , their resistance pattern and in the prevention and control of these hospital acquired infections thereby reducing mortality among neonatal and medical intensive care units.

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 last -line antimicrobials, thereby arresting the spread of antibiotic resistant strains so as to improve the clinical outcome among patients harbouring these organisms.

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AIM & OBJECTIVES

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AIM AND OBJECTIVES

AIM

The aim of the study is to isolate , identify the Acinetobacter species from various clinical samples and to demonstrate its antimicrobial resistance pattern with special emphasis on molecular characterization of Carbapenemase producing strains in our hospital.

OBJECTIVES

• To isolate and identify Acinetobacter species from all clinical specimens by conventional culture methods.

• To study the antibiotic susceptibility profile of Acinetobacter species by Disk diffusion - Kirby Bauer technique.

 Phenotypic identification of strains producing Extended spectrum β- lactamases and Carbapenemases among the isolates.

 Molecular characterization of the genes responsible for Carbapenemase production in Acinetobacter species by PCR.

• To formulate Antibiotic policies and proper infection control practices in various settings in our hospital in accordance with the prevalent multi-drug resistant strains.

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REVIEW OF LITERATURE

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REVIEW OF LITERATURE

HISTORY OF ACINETOBACTER:

1911: The Dutch microbiologist Beijerinck first isolated the organism from soil using minimal media enriched with calcium acetate and were named as Micrococcus calcoaceticus.⁶

1954: J. Brisou and A.R.Prevot created the genus Acinetobacter. The word ‘Acinetobacter’ is derived from Greek meaning non motile, bringing together Gram negative saprophytes which do not produce pigments.

1971: The Subcommittee on the Taxonomy of Moraxella and Allied Bacteria proposed that the genus Acinetobacter should include only the oxidase negative strains.

1980: The Approved Lists of Bacterial Names included two names, A,calcoaceticus for strains forming acid from sugars and A.lwoffii for non-acid-producing strains.

1984: Bergey’s Manual of Systematic Bacteriology classified Acinetobacter in the family Neisseriaceae, but more recently the molecular taxonomic studies have resulted in the reclassification of the organism in the new family of Moraxellaceae in 1991.

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1986: The genus was further subdivided into genomospecies, by Bouvet and Grimont based on DNA-DNA hydridization.⁷

1989: The number of genomospecies was increased to 17.

Currently there at least 25 genomospecies of Acinetobacter. Each genomospecies comprises a distinct DNA hybridization group and is given a numeric designation which has replaced previous names of species. 16 of these DNA homology groups can be differentiated by means of biochemical and growth tests, although only seven species have been named.

The organism based on 16s rRNA and rDNA- DNA hybridization assays, is classified in the super family II of the proteobacteria, Family Moraxellaceae, Genus Acinetobacter^8.

MORPHOLOGY:

The current definition of Acinetobacter comprises gram negative, non fermenting bacteria which are aerobic, non-fastidious, non motile, oxidase negative and catalase positive bacteria. It has a DNA G+C content of 39% to 47%.⁹ The organism is a short, plump, gram negative rod. It has a length of 1-1.5µm and a breadth of 1.5-2.5µm in the logarithmic phase of growth, but often becomes more coccoid in the stationary phase.⁹

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9 CLASSIFICATION :

Classification and typing of Acinetobacter is being essential for epidemiological purpose because of its importance in causing hospital acquired infection Currently only seven genomospecies have been named.

Genomospecies 1, 2, 3 and 13 are difficult to separate in the clinical lab and have been referred as Acinetobacter calcoaceticus - baumannii complex.

Genomospecies 1 A.calcoaceticus Genomospecies 2 A.baumannii Genomospecies 4 A.haemolyticus Genomospecies 5 A.junii

Genomospecies 7 A.johnsonii Genomospecies 8 A.lwoffii Genomospecies 12 A.radioresisten

Most of the remaining genomospecies are unnamed. A.baumannii, A.nosocomialis and A.pittii cause the majority of healthcare - associated infections. A.lwoffii and A.radioresisten colonize human skin and cause infection in immunocompromised hosts. A.calcoaceticus and A.johnsonii are prevalent in water and soil. A.baylyi is frequently isolated from sewage.¹⁰

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10 CULTURAL CHARACTERSTICS:

The organism is non-fastidious in its growth requirements and will grow freely on ordinary simple media without any enrichment. The members of this genus are aerobic and grow in a wide range of temperatures. The clinical isolates of Acinetobacter especially A.baumannii normally grow at 37°c, and some strains at 42°c. They grow on nutrient agar, Macconkey agar and 5% Sheep blood agar.

On Nutrient Agar the colonies are between 0.5-2mm in diameter, opaque to translucent, usually non - pigmented, white to cream colored colonies, smooth, circular , convex with entire edges and vary in consistency from butyrous to smooth and mucoid¹¹.

On Macconkey agar they produce non-lactose fermenting colonies . On 5% Sheep blood Agar at 37°C for 24 hours, colonies appear as smooth, opaque, greyish white, raised and creamy¹¹.

BIOCHEMICAL IDENTIFICATION:

Acinetobacter species are Oxidase negative (The oxidase provides a rapid test to distinguish them from Neisseria or Moraxella).

Acinetobacter can oxidize sugars but cannot ferment them. Rapid utilization of 10% glucose is seen with OF Medium. Acinetobacter sp.

are unable to reduce nitrate. Species differentiation is done on the basis of oxidation of glucose , gelatin liquefaction, hemolysis, growth at 37°C & 44°C and susceptibility to penicillin. A.baumannii is found to

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grow at 37°C and 44°C, whereas other strains do not do so. Haemolysis is seen only in A. haemolyticus.

PATHOGENESIS

The pathogenicity of A.baumannii depends on following factors 1. Its ability to adhere to surface utilizing pili.

2. To create biofilm on surfaces and human cells.

3. To survive in iron- limited environments within the host.

4. To acquire foreign genetic material to enhance survival.

5. To develop large number of antibiotic resistance mechanism.

Identification of Acinetobacter baumannii strains with monoclonal antibodies against the O antigens of their LPS by enzyme immunoassay and by western blot analysis was performed by Raphl et al¹².

VIRULENCE FACTORS:

Acinetobacter has several factors that contribute to its virulence.

One important factor is the outer membrane protein A (OmpA). This outer membrane protein binds to the host epithelial cells and mitochondria, after which it induces mitochondrial swelling . This is followed by the release of cytochrome c, a heme protein that leads to apoptosis¹³.

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The Omp A is a protein that is found on cell structure that causes resistance to complement and is responsible for biofilm formation.

Avril J.L et al , studied about the factors influencing the virulence of Acinetobacter. This virulence property can be subdivided into invasiveness, multiplication and spread within the host tissues and toxigenicity.

CATEGORY

CLASSES OF VIRULENCE FACTORS

(EXAMPLES)

EFFECTS IN HUMANS

Structural Aerobactin Fimbriae

Iron-repressible outer membrane Iron-uptake components

Lipopolysaccharide

Lipopolysaccharide(hydrophobic sugars in the O side chain) Outer membrane proteins Pili

Polysaccharide capsule

Increased virulence

Adhesion to epithelial cells Increased virulence

Survival in the bloodstream Pro-inflammatory response Adhesion to host cells Interference with cell permeability Adhesion to epithelial cells

Interference with

phagocytosis, survival in dry environment

Toxin Lipid A

Outer membrane protein A (Omp 38)

Toxicity, pyrogenicity Cytotoxicity

apoptosis cell death

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13 BIOFILMS

The biofilm produced by the organism plays a vital role in survival of the organism inside the host. The ability of the bacteria to form biofilm helps them to grow persistently in unfavorable conditions and environment. The factors that affect its ability to produce biofilm include nutritional availability and presence of pili and outer membrane protein. The initiation of the biofilm formation is by the pili and a protein called as biofilm associated protein (BAP).

The pili, once it gets attached to the surface starts the production of microcolonies that develops into a full biofilm structure.

The bacterial surface that contain the BAP helps in stabilization and maturation of the biofilm.The other proteins that contribute to the biofilm formation include phospholipases D and C.

CLINICAL MANIFESTATIONS OF ACINETOBACTER INFECTIONS:

Acinetobacter sp. (mainly A. baumannii ) have been implicated as the causative pathogen in nearly all types of nosocomial infection including blood stream infection, pneumonia, urinary tract infection, wound infection and meningitis. Pneumonia is the most common infection followed by urinary tract and soft tissue infections. Origin of nosocomial respiratory infections due to these organism have been traced to ventilators and bacteremia due to colonized intravenous catheters .

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K.K. Lahiri et al , studied about Acinetobacter species as

“Emerging nosocomial pathogens”. They are previously considered to be opportunistic pathogens and have been reported to cause a number of nosocomial infections in hospitalized patients like septicemia, pneumonia, wound sepsis, endocarditis and meningitis (2004).¹⁴

Pedro. B et al , showed incidence of Acinetobacter infection is more in patients with central venous lines and in patients on mechanical ventilation.¹⁵

COMMUNITY-ACQUIRED PNEUMONIA:

Chopade B.A. et al , isolated Acinetobacter sp. from upper respiratory tract of healthy humans. The colonization of bacteria on human mucosal surface is a pre-requisite for any infection. The attachment of bacteria to human tissue requires adhesion factors like lectins. The lectins are principally carbohydrate binding proteins. They are non-enzyme, non-immunoglobulin proteins that have at least one carbodydrate-binding domain.¹⁶

Community acquired pneumonia caused by A. baumannii has been found in tropical regions of Asia and Australia. It affects patients with impaired host defense mechanisms such as those with diabetes, renal failure, chronic alcohol abuse or underlying pulmonary disease. It is characterized by fulminant clinical course, secondary blood stream infections and a high mortality rate of 40 to 60%.

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15 HOSPITAL-ACQUIRED PNEUMONIA:

Ventilator associated pneumonia is the commonest infection caused by A. baumannii. Ventilator-associated pneumonia occurs after the patient has been intubated and on mechanical support for more than 48 hours. Longer period of stay in the hospital especially in patients with assisted mechanical ventilation along with prolonged use of broad spectrum antibiotics are found to be the risk factors that has been implicated in these cases.¹⁷

Buxton. AE et al , studied about the Nosocomial respiratory tract infection and its colonization with Acinetobacter calcoaceticus.

Retrospective studies demonstrated that the respiratory tract infection is associated with endotracheal intubation and continuous positive pressure ventilation .¹⁸

BLOODSTREAM INFECTIONS

Acinetobacter was the tenth most common pathogen, accounting for 1.3% of blood stream infections. Blood stream infection due to Acinetobacter tend to occur late during hospitalization, a mean duration of 26 days after hospital admission. Sources include intravascular catheters, pneumonia, urinary tract infection and wound infection. The mortality rate due to A.baumannii was found to be the third higher after P.aeruginosa and candida sp. Other Acinetobacter sp. have been also associated with catheter -related blood stream infections.¹⁹

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Vinodhkumar C.S et al , highlights Acinetobacter sp. As an important pathogen in neonatal blood stream infection. Septicemia due to Acinetobacter species, are common in babies with predisposing factors like intravascular catheterization, endotracheal intubation, parenteral nutrition, broad spectrum antibiotic therapy and artificial ventilation .²⁰

SKIN AND SOFT TISSUE INFECTIONS:

Acinetobacter causes about 2.1% of the ICU related skin and soft tissue infections .²¹

URINARY TRACT INFECTION:

Acinetobacter baumannii is one of the most common pathogen that causes urinary tract infection and is frequently isolated in catheter- associated infections and accounts for about 1.6% .²²

INFECTIONS IN BURNS PATIENTS

It is one of the common pathogen isolated from the burns patient, and is very difficult to eradicate.

MENINGITIS

Nosocomial meningitis is most commonly caused by gram negative pathogens which also includes Acinetobacter. It is often associated with patients who have undergone previous surgery usually neurological and with an external ventricular drain.

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17 MISCELLANEOUS INFECTION

Other manifestations include endocarditis involving prosthetic valves, endopthalmitis or keratitis following surgery and a single case of bloody diarrhea has also been reported.²³

Jann-Tay-Wang et al , studied about community acquired Acinetobacter baumannii bacteremia in 19 adult patients in Taiwan. They found that malignancy was the most frequent underlying disease. All 14 isolates obtained from adult patients were identified as A.baumannii by 16s ribosomal DNA sequencing and were found to be genetically distinct by pulsed- field-gel electrophoresis (2002).²⁴

EPIDEMIOLOGY OF ACINETOBACTER INFECTIONS:

Acinetobacter sp. are implicated in nosocomial infections and are increasingly difficult to treat as majority of the isolates are multidrug or pan drug resistant (MDR, XDR and PDR). The infections are being documented with increased frequency over the last few years all over the world . Recent studies by Andrew et al, have documented that hospital environments such as Intensive care units, can harbor carbapenem- resistant A.baumannii. Only after closing the affected ICU and thorough cleaning of the environment and equipment with hypochlorite and terminal disinfection, were the resistant strains eliminated.²⁵

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18 Natural Habitats:

Acinetobacter species are ubiquitous in soil, water and food stuffs in the hospital environment. The ability of Acinetobacter species to survive for weeks in the hospital environment leads to frequent nosocomial infections. Patient and staff movement within the health care facility leads to spread of infection . Essentially any moist or dry surface within a patient care area may become contaminated and responsible for transmission of infections.

These species have been associated with ventilators, sinks, faucets, humidifiers, hydrotherapy pools, curtains, pillows, bedrails, supply carts, infusion pumps, equipment control touch pads, catheters and other devices. Patients with both recent and remote history of infection are able to contaminate their surrounding environment. About 25% of adults carry the organisms on their skin and about 7% carry the organism in their pharynx¹⁰.

Smith P.W. et al studied, room humidifiers as a source of Acinetobacter infections. Twenty four patients contracted systemic infections with Acinetobacter sp. during a four month period. Unheated room humidifiers at the patient`s bedside were implicated as the source of infection. The outbreaks were terminated with the removal of the humidifiers.²⁶

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A study by Kappstein. I et al , showed aerators consisting of several wire meshes which served as reservoir of Acinetobacter species in causing outbreak of bacteremia in paediatric oncology patients.²⁷

RISK FACTORS FOR ACQUIRING ACINETOBACTER SPECIES INFECTION:

Risk factors for acquiring infections can be divided as community acquired and hospital acquired.⁷

RISK FACTORS FOR COMMUNITY ACQUIRED

ACINETOBACTER SPP. INFECTION

• Alcoholism

• Cigarette smoking

• Chronic lung disease

• Diabetes mellitus.⁷

The risk factors for nosocomial infection include length of hospital stay, previous surgery and infection, treatment with broad spectrum antibiotics, indwelling urinary catheters, admission to a burns unit or intensive care unit, parenteral nutrition, mechanical ventilation⁷ and probably colonisation of these bacteria in the patients themselves.

Outbreaks are frequently located in intensive care units and burns units involving patients on mechanical ventilation. Sources of

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transmission which have been identified include equipments such as resuscitator bags, mattresses, bedpans. ⁷

According to Maragakis.LL et al , patients on mechanical ventilation infected with resistant strains of Acinetobacter sp. have higher hospitalization costs, and longer ICU and hospital stay.²⁸

In a recent study by Prasanth et al, mechanical ventilation and a particular antimicrobial pattern, i.e. resistance to Ceftazidime, Cefotaxime, Amikacin, Ciprofloxacin, Olfoxacin contributed to increased mortality in hospitalized patients, infected with Acinetobacter sp.²⁹

ANTIMICROBIAL SUSCEPTIBILITY PATTERN FOR ACINETOBACTER

Acinetobacter species is inherently resistant to the β-lactam group of drugs. Till 1984-85, the drugs most effective against Acinetobacter infections were Ceftazidime (80%) and Imipenem (100%). Data available from the early 1990’s, showed an increased resistance to the Cephalosporin and Carbapenem group.

This trend continued, through the years and the latest data shows that the resistance to Ceftazidime is about 50-60% and to Imipenem in the range of 20%-30% , thus showing an upward trend.³⁰ Multidrug resistance is defined as resistance to more than three of the following five drug classes:

• Cephalosporins (Ceftazidime or Cefepime),

• Carbepenems (Imipenem or Meropenem),

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• Ampicillin-sulbactam,

• Fluoroquinolones (Ciprofloxacin or Levofloxacin)

• Aminoglycosides (Gentamicin, Tobramycin, or Amikacin).

MDR strains are those which are resistant to all three classes of antimicrobials such as Aminoglycosides, Fluoroquinolones and Cephalosporins. XDR strains are resistant to the above mentioned three classes of antimicrobials and carbapenems. PDR strains are those which are resistant to all antimicrobials mentioned above and also Colistin³¹. MECHANISMS OF RESISTANCE TO SELECTED ANTIBIOTICS:

A wide range of antibiotic resistance mechanisms have been described in Acinetobacter species. Antimicrobial resistance in Acinetobacter is both intrinsic and acquired. Intrinsic resistance is due to naturally occurring plasmid mediated genes such as OXA51.Acquired resistance may be due to either chromosomal or plasmid mediated β lactamases, DNA gyrase mutation or decreased outer membrane

permeability through porin loss and aminoglycoside inactivating enzymes.

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

b) Alteration in penicillin binding proteins,

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

The efflux pumps, beta lactamase or enzymes which cause hydrolysis of the beta lactam ring are classified by two systems:

I ) Ambler’s classification which is based on amino acid sequences

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

AMBLER’S CLASSIFICATION:

Class A –Penicillinases [TEM,SHV].

Class B-Metallo-β-lactamases [IMP,VIM]-These need zinc as a moiety for its functioning.

Class C- Cephalosporinases [AmpC].

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

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

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GROUP ENZYME MOL.CLASS

INHIBITED BY CLAVULANIC

ACID.

EXAMPLE

1 Cephalosporinases C No P99,MIP-1

2a Penicillinase A Yes S.aureus

2b Broad spectrum A Yes SHV-1

2be Extended spectrum

A Yes TEM-3

2br Inhibitor resistant A Diminished TEM-30

2c Carbenicillinase A Yes CARB-3

2d Cloxacillinase D or A Yes OXA-1

2e Cephalosporinase A Yes FEC-1

2f Carbapenemase A Yes IMC-1

3 Carbapenemase A No IMP-1

4 Penicillinase A No SAR-2

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24 CLASS A BETA-LACTAMASES:

The Beta-lactamase enzymes are both chromosomally as well as plasmid mediated. The AmpC Cephalosporinases are chromosomally mediated and they are found inherently in all A.baumannii strains. They have an upstream gene termed as ISAbal which regulates the over expression of this class A Beta-Lactamase.³²

Ambler class A group Extended spectrum beta lactamase (ESBL) have been described in these organisms, which includes VEB-1 from France, PER-1 from Turkey& Belgium, TEM-92, and TEM-116 from Italy and SHV-12 from China.

The most important enzymes described are the carbapenem hydrolysing enzymes, that include 2 main class of enzymes- Ambler class D OXA type (group 2d of the Bush classification), which are the serine oxacillinases and are the most widespread and also metallo beta lactamases from Ambler class B.

ESBL A.Baumannii which has CTX-M-2 has enhanced ability to hydrolyse cefotaxime and ceftriaxone which is epidemic in countries like Japan and Bolivia but the overall prevalence of CTX-M is low in the Acinetobacter species when compared to Enterobacteriaceae.³³

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25 CLASS B BETA-LACTAMASES

These Beta-lactams are able to hydrolyse betalactam as well as carbapenems with the exception of aztreonam. Class B beta-lactamases have a metallic iron in the active site which is zinc that acts as catalyst and it is absent in class A and D Carbapenemases. So they are called as Metallo-beta-lactamases. IMP-MBL are found in different parts of the world and they are mostly detected in the class one integron . Though IMP-MBL are not commonly found in Acinetobacter baumannii, several types have been described IMP-1, IMP-2, IMP-4,IMP-5,IMP-6 and IMP-11³⁴

In Italy, MBL isolated in pseudomonas aeruginosa encoded by Verona intergon has been reported as VIM-1. Acinetobacter baumannii which contains VIM-2 has been reported from Korea³⁴.

The description of Seoul imipenemase SIM-1, a novel MBL, has also been reported. SIM-1 belongs to the B1 sub class. It suggests that its gene cassette could have originated from ATCC 55044 superintegron of Pseudomonas alcaligenes³⁵.

CLASS C BETA-LACTAMASES:

In Acinetobacter baumannii, the bla gene that codes for class C Betalactamases, hydrolyse Penicillin and a narrow group of Extended Spectrum Cephalosporins. There are up to 28 variants of bla gene that are listed in the gene bank. Like many gram negative bacteria,

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Acinetobacter also has a chromosomally encoded class C beta-lactamase, AmpC gene. It could have been descended from a common beta- lactamase gene ancestry and Acinetobacter derived cephalosporinases called as ADC`s³⁶.

CLASS D BETA-LACTAMASES:

The OXA Beta-lactamases which belong to the class D produces Oxacillinases and some of the OXA have the ability to hydrolyze Extended Spectrum Cephalosporins. The most important feature of these OXA is that they can inactivate even Carbapenems. They were first described in a clinical isolate in Scotland ( OXA-23) even before the introduction of Carbapenems and named as ARI-1 (Acinetobacter resistant to imipenem). Since then it has been discovered in many parts of the world like England, Brazil, Singapore, Korea and China³⁶.

The first Oxacillinase to be described with Carbapenem hydrolyzing property was from Edinburgh, Scotland. It was named as ARI-1(for Acinetobacter resistant to Imipenem) and was found to be of plasmid origin.It was later renamed as bla. It was found to be of 273 amino acid length showing sequence homology to Ambler class D, Oxacillin-hydrolyzing enzymes.

Two highly conserved motifs, S-T-F-K and K-T-G (positions 79 to 82 and 216 to 218), believed to contribute to the function of the serine active site were also defined. A third motif, F-G-N at positions

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152 to 154, which differs from the corresponding motif in all other OXA enzymes by the presence of phenylalanine instead of tyrosine, was identified. This unique substitution was postulated to have significant biochemical effects and was possibly a factor in the evolution of carbapenem resistance in Acinetobacter³⁷.

An important factor has been implicated in carbapenem resistance in Acinetobacter baumannii which are associated with ISAbaI and ISAba3 These are naturally occurring plasmids that are found in them³⁷. These isolates have relatively high MIC for imipenem and Meropenem (i.e) >

32µg/ml. Minimum inhibitory concentration (MIC) using agar dilution method determines whether the bacteria is susceptible, intermediate and resistant. A lower Minimum inhibitory concentration indicates that less drug is required for inhibiting the growth of the organism. Hence drugs with lower MIC scores are more effective antimicrobial agents. Since MIC values are high in these plasmid mediated ISAbaI and ISAba3 , they contribute more to Carbapenem resistance. MIC scores are important in diagnostic microbiological laboratories to confirm resistance of microorganisms to an antimicrobial agents and also monitor the activity of newer antimicrobial agents. Clinicians use MIC scores to choose which antibiotics to administer to patients with specific infections and to identify an effective dose of the antibiotic. . Minimum inhibitory concentration (MIC) scores aid in improving outcomes for patients and prevent evolution of drug resistance mechanisms. Most of

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these Oxacillinases are chromosomally mediated. The Carbapenem- hydrolyzing Oxacillinases are thought to be chromosomally mediated enzymes and are given in table 1

OXA Carbapenemases in Acinetobacter baumannii Carbapenemase type

OXA Carbapenemases³⁷

Carbapenemase type OXA carbapenemases

Acquired/Chromosomal OXA-24, OXA-25, OXA-26, OXA-

40, OXA-58a

Plasmid OXA-23, OXA-58a

Naturally occurring OXA-51/69 like OXA-64, OXA-65, OXA-66, OXA- 68, OXA-70, OXA-71, OXA-78, OXA-79, OXA-80, OXA-82

An OXA-58 has been described both as chromosomal and as a plasmid mediated carbapenemase in A.baumannii. OXA-51/69-like Beta- lactamases are located chromosomally in Acinetobacter baumannii .It has been described in all parts of the world and are called as naturally occurring and their expression depends upon the presence of ISAbal. The OXA groups of Carbapenems are divided into eight sub groups³⁸.

• The first group has OXA- 23 which is also called as ARI-1 (Acientobacter resistant to imipenem) . They differ by 2-5 aimno

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acids from other enzymes. The enzymes that belong to this group are OXA -27 and OXA -49.

• Second group vary by 1-5 amino acids in their genetic sequence . They contain OXA -24, OXA- 25, OXA -26 and OXA-50.

• The third group mainly contains OXA- 51 like enzymes and the fourth group has only as OXA -58 like cluster which also includes OXA-64-66, OXA-68-71 and OXA-75-78. They diverge by 1-12 amino acids and are naturally occuring in Acientobacter baumannii

• The fifth group have a chromosomally encoded enzyme which is seen in Klebsiella Pneumoniae (OXA-55) and is derived by five substitution in Shewanella algae.

• The sixth group is derived from Shewanella onidensis and differ by 20 substitutions

• The seventh and eight branches of class D are represented by OXA-50 (enzyme of P. aeruginosa ) and the OXA-60 ( enzymes in Ralstonia picketti)

CHANGES IN OMPS AND PBPS:

In Acinetobacter baumannii, other non - enzymatic mechanisms such as change in outer membrane protein, alteration in efflux pump expression and affinity of penicillin binding proteins cause resistance to

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both beta-lactam and carbapenems. The resistance to imipenem and Meropenem has been associated with the loss of a 29-kD protein called as CarO⁴² which belongs to the OMP found only in Moraxellaceae.

EFFLUX PUMPS:

They are a simple and single mechanism that causes resistance to many classes of antibiotics. These pumps cause the efflux of compounds that are toxic to the bacterial cell which also includes antibiotics. The efflux pumps that are found in various species of bacteria belong to the small multidrug resistant superfamily, the major facilitator superfamily, the multiple and toxic compound extrusion superfamily and the resistance nodulation -cell division family.³⁹

The efflux pump that is found in Acinetobacter baumannii is called as the Ade ABC efflux pump . They belong to the family called as the resistance-nodulation-cell division family. These efflux pumps efficiently pump antibiotics like Fluoro-quinolones, Trimethoprim, Chloramphenicol, Erythromycin, Tetracyclines, Cefotaxime and Aminoglycosides out of the cell by over expression of this efflux pump. The expression of these pumps is managed by a two step regulator (adeR) and sensor (adeS) system.

This over expression is due to a single point mutation in the gene adeR adeS.⁴⁰

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The resistance to amino glycosides is mediated via modifying enzymes which have all been found to be located within the class I integrons.

The major class of enzymes found is acetyltransferases, nucleotidyltransferases and phosphotransferases. Also efflux pumps have been documented which throw out the antibiotic from within the cell.

The pumps which have been implicated in Acinetobacter sp. are AdeABC from the RND class and also AbeM from the MATE class.

The newest addition is the methylation of 16S r RNA from Korea and Japan which confers high level resistance to all amino glycosides.

With the use of PCR mapping, it showed that the genes that encode for aminoglycoside modifying enzymes are aphA1, aphA6, aacC1, aacC2, aacA4, aadA1, and aadB⁴¹ along with the production of enzymes.

QUINOLONES:

The resistance to quinolones is by modification in the structure of DNA-gyrase which leads to low affinity for the binding of the drug to the DNA complex enzyme. This modification of the structure is caused by mutation in the gene specifically in the region gyrA and parC.

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They also have efflux pump which includes RND type pump and MATE pump and AdeM pump. So far qnr gene which is a plasmid mediated quinolones resistance gene have not been reported in A.baumannii.

TETRACYCLINES:

Efflux pump and ribosomal protection causes resistance to Tetracyclines and their derivatives. The efflux pump are coded by tet(A)-tet(E). Till date tet(A)⁴² has coded only for resistance against Tetracylines but not against Minocyclines. Resistance to Tigecycline is conferred by AdeABC efflux pump in A.baumannii as these strains show threefold increase in MIC. These efflux pumps are coded by adeB gene and these strains are also resistant to Gentamicin, Levofloxacin, and Chloramphenicol.

CARBAPENEMS:

The resistance to Carbapenems is caused by the production of beta-lactamases. It is also contributed by reduced affinity to PBP's for Carbapenems, increased efflux of antibiotics and decreased permeability of the outer membrane. In Acinetobacter baumannii, the combined action of AmpC Beta-lactamase with weak Carbapenemase activity along with other mechanisms such as decreased production, and reduced affinity to PBP's and loss of OMP's contribute to resistance.

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Most of the OXA type carbapenemases cause only reduced susceptibility to Carbapenems . The clinical detection of the organism producing theses enzymes remains to be difficult.⁴³ The chromosomal location of the gene that encodes OXA and also increased selective pressure contribute to their spread.

POLYMYXINS AND COLISTIN :

Polymixin E and Polymixin B are used to treat the multidrug resistant Acientobacter baumanni infections. Polymyxin B resistance genes were reported by Urban et al in 2001 and were called as heteroresistant genes. The complication of these heteroresistant genes have to be studied from the patient outcome who are under treatment of polymyxin.

The modification of the lipopolysaccharide that is acylation, acidification and the presence of antigen that interferes with binding of the drug with the cell membrane could be the possible mechanism for the resistance of Polymyxin and colistin⁴⁴ .

When compared to Carbapenems the resistance to Polymyxin and Colistin is lower due to their infrequent use as they are reserve drugs used only for the treatment of carbapenem producing bacteria. But there are treatment failure reports from various parts of the world. About 28% of Colistin resistant Acinetobacter baumannii have been reported in Asia.

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Hetero-resistance: Among the Colistin resistant group of bacteria (Acinetobacter baumannii, K.pneumoniae and P.aeruginosa ), hetero resistance occurs due to mutations which lead to changes in bacterial cell membrane and outer membrane protein and cross resistance that occur between Colistin and Polymyxin⁴⁵. Of late Colistin resistance due to erm gene has also been documented in E.coli world over.

OTHER ANTIBIOTICS:

The resistance to trimethoprim-sulfamethoxazole and chloramphenicol have also been reported in multidrug resistant Acinetobacter baumannii strains. The genes encoding for the resistance of these drugs include dhfr & cat . The role of efflux pumps in the resistance against these antibiotics could also be another possibility⁴⁶.

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MECHANISM OF ANTIBIOTIC RESISTANCE IN ACINETOBACTER SPECIES⁵³

Mechanism Acinetobacter species

β-Lactamases AmpC cephalosporinase +

Inducible _

TEM +

SHV +

CTX-M +

PER +

VEB +

OXA +

IMP +

VIM +

SPM _

GIM _

PSE _

GES _

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Mechanism Acientobacter species

IBC _

OMP changes +

AMEs Adenylating +

Phosphorylating +

Acetylating +

Topisomerase mutations gryA +

parC +

Efflux pumps +

Mobile genetic elements +

Integrons +

Membrane changes and resistance to

polymyxin _ MANAGEMENT OF ACINETOBACTER INFECTIONS

Acinetobacter may colonize the skin, pharynx , GIT, urethra, conjunctiva and vagina, so care must be taken to rule out colonization and environmental contamination. The colonization does not require any special treatment but the identification of resistance to multiple antibiotics are of utmost importance.

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The susceptible strains have been treated with Beta-lactams.

Mostly the third generation Cephalosporins and extended spectrum of Penicillins have been used for the treatment. In case of severe infections, Penicillin with Beta-lactam inhibitors and Carbepenem in combination with aminoglycosides have been used⁴⁷.

GLOBAL EPIDEMIOLOGY:

Acinetobacter baumannii is now an emerging Carbapenem resistant bacteria worldwide. It has been shown that the percentage of Carbapenem resistant bacteria have increased over the last ten years in countries like North America, Europe and Latin America.

In a study by Zied et al, in North America after an out-break of ESBL producing Klebsiella, imipenem was significantly used, following which the infection due to Carbapenem resistant A.baumannii was reported in large number of hospitals in 1991 and 1992, after which numerous out breaks have been reported because of multi drug resistant A.baumannii⁴⁸.

In the United States, a study was conducted by Amy et al, on most commonly isolated gram-negative bacteria in culture ( 1 June 2009-31 May 2012), Acinetobacter baumannii was one among the most common isolates next only to Escherichia coli, Klebsiella pneumoniae and Enterobacter Aerogenes⁴⁹. In these 44.8% of the isolates were multidrug resistant isolates, 33.6% were Aminoglycoside resistant, 36.4%

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were Beta-lactamase resistant, and 44.1% were Carbapenem and Flouroquinolone Resistant⁴⁹.

In the Middle East, the most commonly isolated gram negative non fermentative organism was P.aeruginosa (72.9%), followed by A.baumannii (25.3%) and S.maltophila (1.8%) as reported in March 2012. It was found that, out of the total number of Acinetobacter baumannii isolated, more than 50% were resistant strains. About 76.9%

resistant to Amikacin, 77.8% to Gentamicin, 5.4% to Imipenem, 95.5%

to Aztreonam, 90.6% to Tetracycline, 95.9% to Chloramphenicol, and 13.2% resistant to Polymyxin-B⁵⁰.

EPIDEMIOLOGY OF CARBAPENEM RESISTANCE IN INDIA In India, a one year study by Naz et al , from January 2013 to December 2013 conducted in various tertiary care hospitals in Delhi and other parts of India revealed that large number of isolates were multi-drug resistant. Of the total number of isolates, 88.6% showed resistance to Cefotaxime, 88.9% to Amikacin, 80.5% to Gentamicin , 33% were resistant to Piperacillin Tazobactam and 16% to Imipenem.

Resistance to Meropenem was found to be 15% and was less than 2% for Colistin and Polymyxin B⁵¹.

In a study by Anne M et al, conducted in 2010 in AIIMS 17.32% of isolates were resistant to Imipenem. In vellore medical college, study conducted by Anitha et al showed carbapenem

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resistance as 96.6% . Study by Alexander .P et al in mumbai showed 29% resistance to Imipenem⁵².

GLOBAL EPIDEMIOLOGY OF CARBAPENEM RESISTANCE IN ACINETOBACTER BAUMANNII:

The exposure of Acinetobacter baumannii to various groups of drugs in the intensive care units, gradually has led to the prevalence of resistance to Carbapenems world wide. The relevant resistant genes in Acinetobacter baumannii coding for enzymes such as Cephalosporianase, Carbapenemase and Oxacillinases are described below⁵³.

Table 2 Genes of Carbapenemase production.

Enzyme Described in association with

IMP-1,IMP-2,IMP-4,IMP- 5,VIM-1,VIM-3,VIM-3,VIM-4

Class 1 integron

NDM -1,NDM-2 Chromosome

OXA -23,OXA-58, OXA- 40/OXA -24

Chromosome / plasmid mediated

The OXA 51-like enzyme cluster varies from other OXA type Carbapenemases as it is chromosomally located and also inherent in Acinetobacter baumannii but its activity depends on the presence of ISAba1 element. The most commonly found type of OXA

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carbapenemase is bla OXA-24, which is similar to OXA-23 enzyme.

The bla OXA-58 is a plasmid mediated gene and has been reported in many hospital outbreaks and found in association with ISAba2.

Combination therapy:

The combination therapy for the treatment of MDR/XDR/PDR isolates is attributed to the following reasons⁵⁴.

• To Delay / prevent the occurrence of resistance gene in the bacterial strain due to antibiotic therapy.

• To improve the clinical outcome for the patient by using synergistic effect.

• To increase the Empirical coverage provided by the spectrum of activity of different drugs used in combination therapy

A retrospective study in intensive care units by Beceiro et al, showed a decrease in mortality, in patients treated with combination therapy (29%) ( Beta-lactams along with Aminoglycosides / Macrolides / Flouroquinolones ) as compared to Beta-lactam Monotherapy which accounts for 30% of mortality⁵⁴.

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REVIEW OF LITERATURE

ACKNOWLEDGEMENT

CONTENTS

INTRODUCTION

AIM & OBJECTIVES

REVIEW OF LITERATURE

MATERIALS & METHODS