PREVALENCE AND ANTIMICROBIAL SUSCEPTIBILITY OF GRAM-

NEGATIVE BACTERIA IN THE URINE OF PATIENTS ATTENDING AT

BIHS GENERAL HOSPITAL

M.S. THESIS

A Thesis Proposal submitted to the Department of Microbiology, Bangladesh

University of Health Sciences, in Partial fulfillment of the requirements for
the Degree of Master of Science in Microbiology.

SUBMITTED BY:

ABDIAZIZ MOHAMED YUSUF

MS in Microbiology

ID NO: O1202202002

Session: fall-20

Department of Microbiology

Bangladesh University of Health Sciences

August, 2021

TABLE OF CONTENTS

i
Abstract............................................................................................................................................v

CHAPTER ONE: INTRODUCTION..........................................................................................1

1.1 Background of the study...................................................................................................1

1.2 Rationale of the study.......................................................................................................3

1.3 Research Questions...........................................................................................................4

1.4 Research Objectives..........................................................................................................4

1.4.1 General Objectives.....................................................................................................4

1.4.2 Specific Objectives....................................................................................................4

1.5 Conceptual framework......................................................................................................5

1.6 List of variables.................................................................................................................5

1.7 Operational Definitions.....................................................................................................6

CHAPTER TWO: LITERATURE REVIEW.........................................................................7

2.1 UTI pathogenesis..............................................................................................................7

2.2 Entry of Bacteria into the Urinary Tract...........................................................................8

2.3 Routes of Bacterial Infection............................................................................................8

2.4 Symptoms of Urinary Tract Infection (UTI).....................................................................9

2.5 Gram Negative Bacteria that cause UTI...........................................................................9

2.5.1 Escherichia coli (E. coli)............................................................................................9

2.5.2 Proteus species.........................................................................................................10

2.5.3 Klebsiella species.....................................................................................................11

2.5.4 Pseudomonas aeroginosa.........................................................................................12

2.5.5 Serratia marcescens.................................................................................................13

2.5.6 Acinetobacter species..............................................................................................14

2.6 Laboratory Diagnosis of Urinary Tract Infection...........................................................15

2.6.1 Specimen Collection................................................................................................15

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 2.6.2 Specimen transportation..........................................................................................15

2.6.3 Specimen processing...............................................................................................16

2.6.4 Cultures and the laboratory diagnosis of UTIs........................................................17

2.7 Antimicrobial Susceptibility...........................................................................................18

2.7.1 History of Antibiotic and Antibiotic Resistance......................................................18

The Tide Turns, the Superbugs Arrive.......................................................................................19

2.8 Multiple Drug-Resistance (MDR), Extensively Drug-Resistant (XDR) and Pandrug-

Resistant (PDR)..........................................................................................................................21

2.9 Extended Spectrum Beta Lactamase (ESBL).................................................................22

2.10 AmpC beta-Lactamase....................................................................................................23

2.11 Carbapenemases..............................................................................................................24

2.12 Risk factors of Urinary Tract Infections.........................................................................25

2.12.1 Pregnancy................................................................................................................25

2.12.2 Socio Economic Status............................................................................................25

2.12.3 Anaemia...................................................................................................................25

2.12.4 Diabetes...................................................................................................................25

2.12.5 Sexual Activity........................................................................................................26

2.12.6 Catheterization.........................................................................................................26

2.12.7 Unhygienic Practices...............................................................................................26

CHAPTER THREE: MATERIAL AND METHODS.............................................................27

3.1 Study Design...................................................................................................................27

3.2 Study Area.......................................................................................................................27

3.3 Study Population.............................................................................................................27

3.4 Study Period....................................................................................................................27

3.5 Study Criteria..................................................................................................................27

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 3.5.1 Inclusion criteria......................................................................................................27

3.5.2 Exclusion Criteria....................................................................................................27

3.6 Sampling Technique.......................................................................................................27

3.7 Sample Size Determination.............................................................................................27

3.8 Data Collection...............................................................................................................28

3.8.1 Data collection instrument.......................................................................................28

3.8.2 Urine Sample Collection..........................................................................................28

3.8.3 Material and methods..............................................................................................29

3.9 Data analysis and interpretation......................................................................................30

3.10 Ethical consideration.......................................................................................................30

REFERENCES.......................................................................................................................32

ANNEXURE A: WORK PLAN............................................................................................36

Abstract
Urinary tract infection (UTI) is a common health problem in both community and nosocomial
settings. UTI is among one of the most common infections occurring particularly in women. As

iv
reported by the National Ambulatory Medical Care Survey, UTI alone is responsible for nearly
seven million patient visits in outpatient department (OPD) as well as up to one million visits in
hospital emergency department, resulting in about 100,000 hospitalizations. Nearly 50–60% of
all women suffer from an episode of UTI at least once in their lifetime. Antibiotic resistance is
increasing day by day. Many gram-negative bacteria show different multi drug resistance as they
contain AmpC beta-lactamase and ESBL enzyme in different age and sex groups. The aim of this
study is to estimate the prevalence of gram-negative organisms in the urinary tract among
patients attending at BIHS General Hospital and also to determine their antibiotic sensitivity
pattern to enable formulation of drugs for urinary tract infection in patients attending at BIHS
General Hospital. Descriptive cross-sectional study will be conducted in this study and samples
will be selected by using simple random sampling to select a sample of 80 specimens from
participants in the study. The data collected will be analyzed using Statistical Package for Social
Sciences (SPSS) version 22 where the researcher will be presented the results in the form of
tables and graph where applicable. The findings of this study will help the clinicians to known
pathogenic bacteria commonly responsible with UTI and susceptibility patterns in order to
choose the right empirical treatment.

v
 CHAPTER ONE: INTRODUCTION

1.1 Background of the study

A urinary tract infection (UTI) is an infection that affects part of the urinary tract. When it affects
the lower urinary tract, it is known as a bladder infection (cystitis) and when it affects the upper
urinary tract it is known as a kidney infection (pyelonephritis) 1

Urinary tract infection (UTI) is a common health problem in both community and nosocomial
settings. UTI is among one of the most common infections occurring particularly in women. As
reported by the National Ambulatory Medical Care Survey, UTI alone is responsible for nearly
seven million patient visits in outpatient department (OPD) as well as up to one million visits in
hospital emergency department, resulting in about 100,000 hospitalizations. Nearly 50–60% of
all women suffer from an episode of UTI at least once in their lifetime 2.

UTI is known to occur in all populations but has a particular impact on females of all ages and
males at two extremes of life, immuno-compromised patients and anyone with function or
structural abnormalities of the urinary and excretory system. UTIs are caused by both Gram-
negative and Gram-positive bacteria, as well as by certain fungi. The most common causative
agent for both uncomplicated and complicated UTIs is uropathogenic Escherichia coli (UPEC).
For the other agents involved in uncomplicated and complicated UTIs is followed in prevalence
by Klebsiella pneumonia, Staphylococcus saprophyticus, Enterococcus faecalis, group B
Streptococcus (GBS), Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and
Candida spp 3.

E coli cause 70-95% of both upper and lower UTIs. Various organisms are responsible for the
remainder of infections, including Staphylococcus saprophyticus, Proteus species, Neisseria
gonorrhoeae, Klebsiella species, Enterococcus faecalis, other Enterobacteriaceae, and Yeast.
Some species are more common in certain subgroups, such as Staphylococcus saprophyticus in
young women. However, Saprophyticus produces acute cystitis in older women and young
women and should not automatically have regarded as a contaminant in the urine culture of these
individuals 4.

1
Another study conducted in Dhaka city was found that among the gram-negative bacilli, the
predominant isolate was E. coli 73.33% followed by other Bacilli such as Klebsiella spp. 16.67%
and Pseudomonas 6.67. On the other hand, the bacteria that cause urinary tract infections
typically enter the bladder via the urethra. However, infection may also occur via the blood
or lymph.It is believed that the bacteria are usually transmitted to the urethra from the bowel,
with females at greater risk due to their anatomy After gaining entry to the bladder, E. Coli are
able to attach to the bladder wall and form a biofilm that resists the body's immune response 5.

Antimicrobial resistance (AMR) occurs when microbes evolve mechanisms that protect them

from the effects of antimicrobials. Antibiotics are used for the control of bacterial infections in
human. UTI is treated often by broad-spectrum antibiotics, and treatment is started empirically
without performing culture and sensitivity. This inappropriate and non-judicious usage of
antibiotics has resulted in the development of worldwide antibiotic resistance in bacteria, leading
to the emergence of multi resistant strains of bacterial pathogens. According to a survey
conducted by the European Survey of Antibiotic Consumption, multidrug-resistant (MDR)
bacterial strains were accountable for a mortality rate nearly about 25,000 Europeans/year
usually due to complications of UTIs 6.

Hence, it is necessary to circumvent non-judicious use of antibiotics that lead to the emergence
of antimicrobial resistance and most appropriate antibiotics should be opted for first-choice
empiric treatment of UTI. The antimicrobial susceptibility pattern among bacteria varies from
country to country. The Infectious Diseases Society of America recommends that regional
surveillance should be conducted to monitor changes in susceptibility of uropathogens in specific
regions 7.

A study conducted in Qassim University affiliated hospitals was found in 92% (n = 82/89) of
samples, with most (80%) being resistant to at least two drugs. Antibiotic resistance was
commonly observed in Ampicillin (88.3%), Piperacillin (72.7%), Clindamycin (66.7%),
Amoxicillin/Clavulanic acid (66.2%), and Trimethoprim/Sulfamethoxazole (50%). The
commonly isolated microorganisms were Escherichia coli 24 (27%), Klebsiella pneumoniae 11

2
(12.4%), Proteus mirabilis 4 (4.5%), Pseudomonas aeruginosa 4 (4.5%), Enterobacter cloacae 5
(5.6%), Enterococcus faecalis 5 (5.6%), and Staphylococcus saprophyticus 3 (3.4%) 8.

UTI is a serious ailment in human due to increasing frequency, recurrence and difficulty in
eradication; it poses stiff challenge to the medical professionals. It is much more common in
women than in men, due to anatomical and physiological reasons; by virtue of its position
urinogenital tract is more vulnerable to bacterial infections caused by both internal and external
flora 9.
The population at risk of UTI includes new born (including the premature), mature girls, sexually
active females and elderly females. About 3% of all women in the United States visit a physician
10
at least once each year for UTIs, and at least 50% of women report at least one UTI in lifetime .
UTI can lead to renal scars and if undiagnosed leads to permanent renal damage causing
hypertension or end stage renal disease. The diagnosis of UTI is difficult in the neonatal period
because the signs and symptoms are no specific in this age group. The incidence in the neonates
is 0.01% - 1% and an also be as high as 10% in low birth weight and preterm babies 11.

1.2 Rationale of the study

Gram-negative bacteria are the most common pathogens associated with urinary tract infections
(UTI). The resistance of these gram-negative bacteria to various antibiotics is increasing with
time in world wide. Antimicrobial resistance is a worldwide problem, with severe implications
for developing countries, where a high infectious disease burden coexists with rapid emergence
and spread of antimicrobial resistance.
Among bacteria, Gram-negative organisms are most commonly isolated from urine samples of
with Escherichia coli (E. coli) accounting for 70% to 90% of infections
Klebsiella species, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter, and Serratia are
other Gram-negative bacteria isolated from pediatric patients. However, only 10% of the cases
are caused by Gram-positive bacteria and include Enterococcus, Staphylococcus,
and Streptococcus agalactiae. E. coli and Klebsiella spp. were also the most frequent pathogens
12
causing UTI reported by two distinct studies conducted in Turkey and Senegal, respectively .

3
The aim of this study was to determine the prevalence and antimicrobial susceptibility of gram-
negative bacteria in the urine of patient attending at BIHS General Hospital.

The findings of this study will help the clinicians to known pathogenic bacteria commonly
responsible with UTI and susceptibility patterns in order to choose the right empirical treatment.

1.3 Research Questions

 What is the prevalence of gram-negative organisms in the urinary tract among

patients attending at BIHS General Hospital?

 What are the antibiotic susceptibility patterns of gram-negative bacteria causing

UTI in patients attending at BIHS General Hospital?
1.4 Research Objectives

1.4.1 General Objectives

- The main objective of this study was to determine the prevalence and antimicrobial
susceptibility of gram-negative bacteria in the urine of patients attending at BIHS General
Hospital.

1.4.2 Specific Objectives

 To estimate the prevalence of gram-negative organisms in the urinary tract among

patients attending at BIHS General Hospital.

 To determine their antibiotic sensitivity pattern to enable formulation of drugs for

urinary tract infection in patients attending at BIHS General Hospital.

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1.5 Conceptual framework

Independent variables
Dependent variables

1.
Socio-demographic/Economic
2.
variables
3.
 Age
4.
 Gender
 Educational level
 Marital status Different isolated
Microorganisms
 Occupation status
 Income of the patients

Clinical variables

 Biochemical test
 Culture
 Gram stain
 Samples
 Antibiotic Disks

Figure 1: conceptual frame work

1.6 List of variables

1.6.1 Independent variables

1. Socio-demographic/Economic Variables

 Age
 Sex
 Educational level
 Religion
 Occupation

5
  Socioeconomic status
 income of patients
 Family size
 Living/Housing status

2. Clinical Variables
 Culture
 Biochemical tests
 Gram stain
 Samples
 Multiple drug resistance

1.6.2 Dependent variables

- Different isolated microorganisms

1.7 Operational Definitions

Prevalence:  is the proportion of a particular population found to be affected by a medical
condition (typically a disease or a risk factor such as smoking or seat-belt use) at a specific time.

Antibiotic susceptibility testing: is the measurement of the susceptibility of bacteria to

antibiotics.

Gram-negative bacteria: are bacteria that do not retain the crystal violet stain used in

the gram-staining method of bacterial differentiation.

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 CHAPTER TWO: LITERATURE REVIEW

2.1 UTI pathogenesis

Escherichia coli (E. coli) are the most common uropathogen of all forms of UTI and are
responsible for 80% of cases. The commonest urinary pathogen accounting for over 80% of
community-acquired infection is due to E. coli. However, other organisms gain a greater
foothold in patients with complicated UTI. Enterobacteriaceae and E. coli in particular are the
notorious pathogens causing infections by adhering to, invading, and replicating the umbrella
cells of the bladder epithelium 13

E. coli replication is facilitated by inflammation, leading to increased bacterial survival and

invasion to the deeper layers of the urothelium. Consequently, these urothelial cells become
reservoirs in which pathogens persist in a quiescent state becomes reservoirs and may be the
source of recurrent UTIs. In general practice, there are concerns that some common infections
are becoming increasingly difficult to treat and that complications due to antibiotic resistant
bacteria may take longer to resolve. The distribution of urinary pathogens in hospitalized patients
is differs with E. coli accounting for about 50% of infections. Enterococcus, Klebsiella,
Enterobacter, Citrobacter, Serratia, Pseudomonas aeruginosa, Providencia, and Staphylococcus
epidermidis account for most of the rest 14.

It is notable that, in women, the colonization of the vaginal and periurethral mucous can precede
UTI, the infection can ascend, causing cystitis and, if not treated, pyelonephritis. Suprapubic
vesical puncture permits diagnosis confirmation of urinary tract infection, at any quantity of
identified colonies. However, as it is an invasive examination, it is not normally used. Thus, the
gold standard urine culture is considered for investigation of UTI. Aerobic non-fermenting gram-
negative bacilli (non-fermenters) are a heterogeneous group of organisms that are either
incapable of utilizing carbohydrates as a source of energy or degrade them via oxidative rather
than fermentative pathway 15.

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 2.2 Entry of Bacteria into the Urinary Tract

Most bacteria cause UTI, but the most common is E.coli which is responsible or about 80-85%
of UTI. Urinary tract infections are categorised as ascending or descending routes. Three years
later, Hooton Stamm (1997) stated that infections occurs when microorganism usually bacteria
from digestive tract cling to the opening of the urethra and begin to multiply. Four routes have
been purposed the ascending routes, from the urethra to bladder, then by the urether to kidney,
the haematogenous routes, with seeding of the urether to the kidney during the course of
bacteraemia; intestine to kidney by way of lymphatic and direct infections. These bacteria may
be present in the vaginal and rectal areas 16.

2.3 Routes of Bacterial Infection

It is known that infections from the ascending route affect mostly the kidney which emerges to
the urethra and peurethral tissues into the bladder and then enter into the urether finally into renal
pelvis, several factors can be dispose the urinary tract to infection. Any abnormality of the
urinary tract that obstructs the flow of urine sets the pace for infection to occur. Quoting Fogazzi
and his colleague a few bacteria that manage to invade the urinary defence and enter the bladder
can multiply to high levels during this time causes infection. An enlarged prostate gland can also
slow the flow of urine, thus raising the risk of infection. Another commonly known cause of UTI
is the use of diaphragm for contraception in which the diaphragm may press on the neck of the
bladders, preventing it from emulsifying completely and leaving a pool of stagnant urine for
bacteria may also enter when the diaghram is left for longer time than required. Catheters are
also known to be 24 tubes that is placed in the bladders to drain off urine when a patient is
unconscious or deeply ill from surgery. The route of the entry of infection is the connection that
is linked to the outside and this makes it easier for pathogens to reach the bladder. The normal
urinary tract is sterile but gets infected with normal flora by overcoming the natural defence of
the normal sterile urinary tract, thus acting as opportunistic pathogens 17.

8
 2.4 Symptoms of Urinary Tract Infection (UTI)

An individual might be infected without having on the symptoms showing up, while most have
the symptoms. Based on the records, the most common clinical symptoms associated with UTI
that brings medical attention are those referable to the urinary includes; Dysuria – its early
symptoms, may be burning or pain on the tip of the penis (for men), itching or in pain during
urination, discomfort in the lower abdomen and a frequent urge to urinate may arise. The clinical
preservation associated with acute pynerhitis is familiar and include recurrent regions and fever,
nausea and vomiting etc. the clinical signs associated with it is divided into two categories.
Those related to infection and they are related to degree and location of injury within the kidney,
consequently the infectious aspect of the disease may be minor. Although intermittent episodes
of full bi-non pyelone nephritis may occur, these are the exceptions. More common is
asymptomatic symptoms referable to lower urinary vague complaints or flank or abdominal
discomfort and intermittent low grade fever 18.

2.5 Gram Negative Bacteria that cause UTI

2.5.1 Escherichia coli (E. coli)

Escherichia coli are a rod-shaped, Gram-negative bacterium, and classified as a member of the

family Enterobacteriaceae within the Gamma proteobacteria class. Escherichia coli are among
one of the well-studied bacteria. Escherichia coli can grow rapidly under optimal growth
conditions, replicating in ~20 min. Many gene manipulation systems have been developed
using E. coli as the host bacterium, producing countless enzymes and other industrial products.
Genome sequence analysis of E. coli was first reported in 1997. Since then, more than
4800 E. coli genomes have been sequenced. The fast-growth characteristics of E. coli make it
suitable to study the evolution of micro-organisms and a long-term experimental evolution study
of E. coli involving more than 50 000 generations is ongoing . Escherichia coli includes not only
commensal strains but also pathogenic ones that cause a variety of human diseases—resulting in
more than 2 million deaths each year. There are six well-studied intestinal pathotypes
of E. coli, including Shiga toxin-producing E. coli (STEC), Enteropathogenic E. coli (EPEC),
Enterotoxigenic E.coli (ETEC), diffusely adherent E. coli and Enteroinvasive E. coli,

9
including Shigella strains. These strains are classified by virulence properties and pathogenicity
mechanisms causing gastrointestinal diseases such as diarrhoea 19.

Enterohaemorrhagic E. coli (EHEC) is one type of STEC that can cause severe enteric diseases,
such as haemolytic uraemic syndrome and haemorrhagic colitis leading to acute renal failure and
often death. Escherichia coli O157:H7, the most well-known serotype of EHEC, has caused
many outbreaks of water- and food-borne diseases in many countries. The incidence of non-
O157 STEC has been increasing in recent years, including those caused by serotypes O26, O45,
O103, O111, O121 and O145. Some E. coli strains can also cause extra-intestinal diseases, and
are called extra-intestinal pathogenic E. coli (ExPEC). The ExPEC, which were defined by
disease association, include uropathogenic E. coli, neonatal meningitis-associated E. coli and
sepsis-causing E. coli Pathogenic E. coli strains are implicated in many waterborne outbreaks,
and STEC and EPEC have been frequently reported to be responsible for waterborne outbreaks
worldwide. Pathogenic E. coli contamination of the environment may occur through manure and
other animal wastes, wastewaters from slaughterhouses and effluent from wastewater treatment
plants. Although extensive studies have been done on the clinical aspects of the
pathogenic E. coli strains, including mode of pathogenesis, diagnosis and sources their
prevalence in the environment has not been extensively examined in greater detail. This
emphasizes the need for additional studies to understand the ecology of these bacteria in
extraintestinal environments 20.

2.5.2 Proteus species

There are four species of proteus, of which three cause disease (Jensen, 1992), since it belongs to
the family of Enterobacteriaceae, general characteristics are applied on this genus. All strains are
motile. They may swarm on blood agar, producing concentric zones or event film. On
MacConkey agar, colonies are colorless, flat, often swarm slightly and are 2-3 mm in diameter
(this is specific to Proteus vulgaris and Proteus mirabilis). Other species do not swarm. They are
resistant to Polymxin B and Colistin. Proteus species can resemble non-motile salmonella
biochemically, and can agglutinate in polyvalent salmonella antisera. Proteus species do not
ferment lactose, but have shown to be capable lactose fermenters depending on the species in a
triple sugar iron (TSI) test. They are also oxidase negative but catalase and nitrate positive. Other

10
species tests are the urease (which is essential test to differentiate Proteus from Salmonella) and
phenylalanine deaminase tests. All strains are urease positive. They have been isolated from
human feaces, urine, abdominal, neck, groin, and hip wounds, infected conjunctiva, sacral
decubitus, and sputum membrane of the genus Proteus are widespread in the environment and
are found in the human gastrointestinal. The most common infection caused by Proteus spp is
urinary tract infections (UTIs). Proteus spp can be found to colonize the vaginal introitus prior to
onset of bactenruria. Therefore, like Escherichia coli, Proteus species causes urinary tract
infections by ascending from the rectum to the periurethra and bladder 21.

P. mirabilis is by far the most common species identified in clinical specimens. P.mirabilis is a
common cause of both community-acquired and catheter-associated UTI, cystitis, pyelonephritis,
prostatitis, wound infections, and burn infections, and occasionally causes respiratory tract
infections, chronic supportive otitis media, eye infections (endophthalmitis), meningitis, and
meningo encephalitis. It is a common cause of bacteremia following catheter-associated UTI and
in rare cases has been reported to cause cellulitis, endocarditis, mastoiditis, empema, and
osteomyelitis. It has also been suggested that p. mirabilis could have a role in the etiology of
rheumatoid arthritis. P.vulgaris, previously considered biogroup 2, has been reported to cause
UTIs, wound infections, burn infections, bloodstream infections, and respiratory tract infections.
There has also been one case study of p. vulgaris causing bacteremia and brain abscesses, with
the suspected point of entry being the digestive tract 22.

2.5.3 Klebsiella species

Klebsiella species are a gram-negative bacillus that belongs to the family Enterobacteriaceae and
causes severe nosocomial infections, including pneumonia and primary blood stream infections.
Klebsiella pneumonia and Klebsiella a oxytoca are ubiquitous in nature and have two common
habitats; one is the environment, including surface area, soils, sewage, and plants and the other is
humans, including the skin, bowels, bladder, and respiratory tract. It can easily survive in
hospitals and is usually transmitted from patient to patient via the hands of health care personnel.
The identification and classification of Klebsiella spp are traditional based on morphological and
physiological properties of the bacteria. Morphological identification of typical and atypical

11
 23
colonies on selective culture is typically followed, in particular, by biochemical karag .
Klebsiella is an important nosocomial human pathogen that has the potential to cause severe
infections. K. pneumonia is gaining renewed interest because of emergence of multi drug
resistance due to ESBL production. Diabetes, alcoholism and previous surgeries were commonly
associated factors. Common infections caused by Klebsiella were found wound infections,
urinary tract infections and respiratory infections. Around a quarter of all Klebsiella isolates were
ESBL produces exhibiting multi drug resistance to commonly used antibiotics like
Cephalosporins. All isolates were sensitive to Carbapenems. The genus Klebsiella contains six
species and three subspecies. There are four species related to humans and they include k.
pneumonia subspecies pneumonia, ozaenae, and rhinoscleromatics; k.oxytoca; k. granulomatis
and variicola. The other two species, k.singaporensis and k. michiganensis have been isolated
from the soil and from a tooth brush holder respectively. The genus consist of over 77 capsular
antigens (k antigens), leading to different serogroups 13. These well-developed polysaccharides
capsules give the colonies their characteristic mucoid appearance. Klebsiella species are non-
motile, usually encapsulated rod-shaped bacteria, belonging to the family Enterobacteriaceae.
These bacteria produce lysine decarboxylase but not ornithine decarboxylase and are generally
positive in the voges-proskaur test as well as indole and urease tests. They are generally
facultatively anaerobic, and range from 0.3-1.0 mm in width and 0.6 mm in length 14. All strains
grow readily on ordinary media. On MacConkey agar, the colonies typically appear large,
mucoid, and red, with red pigment usually diffusing in to the surrounding agar, indicating
fermentation of lactose and acid production. They can cause bacteraemia and hepatic infections,
and have been isolated from a number of unusual infections, cholecystitis , crepitantmyonecrosis,
pyomyositis, necrotizing fasciitis, psoas muscle abscess, fascial space infections of the head and
neck, and septic arthritis, Klebsiella bacteria, including pneumonia; blood stream infections,
wound or surgical site infections, and meningitis. Healthy people usually do not get Klebsiella
infections however; people who are hospitalized and receiving treatment for other conditions
may be susceptible to these infections 23.

2.5.4 Pseudomonas aeroginosa

12
P. aeruginosa, a proteobacteria is a member of the Pseudomonadaceae family. Discovered in
1882 by the French bacteriologist and chemist Carle Gessard, this gram-negative bacteria
structure includes a .5-.8 µm by 1.5-3 µm rod shape utilizing one flagellum for mobilization.
Individualizing itself from most gram-negative bacteria, P. aeruginosa is positive in an oxidase
reaction. Moreover, P. aeruginosa is permanently unable to ferment lactose. P. aeruginosa is
found in water, plants, soil, and on the epidermis of animals. In nature, it is commonly found as a
plant on swimming through water or as a biofilm, clusters of bacteria sharing the same
phenotype and common chemical properties. Uniquely, P. aeruginosa can thrive and survive in a
variety of temperature and infrequent nutrition. The bacterium has been observed in previous
studies to grow in distilled water giving P. aeruginosa an advantage in adapting to changing
environments Being an opportunistic pathogen, P. aeruginosa requires a lack of immunity to
infect its host 24.

Moreover, this is the explanation as to why P. aeruginosa is such a sizeable nosocomial threat for
patients with ventilation machines, cancers, and burns. Colonization of P. aeruginosa in the
respiratory tract is associated with sepsis and death. Any patients that are immunosuppressed or
have experienced significant amounts of trauma are at risk for the colonization of an infection.
The mortality rate approximates 50% for said patients who obtain an infection. P.aeruginosa has
an acute ability to destroy human macrophages utilizing the production of exotoxin A to regress
the uptake of 3H-Thymidine by cells, thus giving P. aeruginosa its high toxicity. Hydrophilic
drugs like these β-lactams can pass through the complex outer membrane, so characteristic of P.
aeruginosa and other gram-negative bacteria, by way of small pores in said membrane. These are
referred to as porins and play an essential role in resistance to antimicrobials. The mechanisms
highlighted in this review are intended to express the acute ability of P. aeruginosa to develop
resistance to antibiotics. Therefore, stressing the importance of preventative measures and
developing new treatments for this bacterium. P. aeruginosa is a versatile pathogen that can
infect all tissues and systems of humans such as the urinary tract, respiratory tract, bone, joint,
soft tissue (burns) and dermatitis 25.

2.5.5 Serratia marcescens

13
Serratia marcescens is a species of rod-shaped, Gram-negative bacteria in the
family Yersiniaceae. It is a facultative anaerobe and an opportunistic pathogen. It was discovered
in 1819 by Bartolomeo Bizio in Padua, Italy. Marcescens is commonly involved in hospital-
acquired infections (HAIs), particularly catheter-associated bacteremia, urinary tract infections,
and wound infections, and is responsible for 1.4% of HAI cases in the United States. It is
commonly found in the respiratory and urinary tracts of hospitalized adults and in
the gastrointestinal systems of children. Due to its abundant presence in the environment, and its
preference for damp conditions, S. marcescens is commonly found growing in bathrooms
(especially on tile grout, shower corners, toilet water lines, and basins), where it manifests as a
pink, pink-orange, or orange discoloration and slimy film feeding off phosphorus-containing
materials or fatty substances such as soap and shampoo residue. S. marcescens is
a motile organism and can grow in temperatures ranging from 5–40 °C and in pH levels ranging
from 5 to 9. It is differentiated from other Gram-negative bacteria by its ability to
perform casein hydrolysis, which allows it to produce extracellular metalloproteinases which are
believed to function in cell-to-extracellular matrix interactions. Since this bacterium is a
facultative anaerobe, meaning that it can grow in either the presence of oxygen (aerobic) or in
the absence of oxygen (anaerobic), it is capable of nitrate reduction under anaerobic conditions.
Therefore, nitrate tests are positive since nitrate is generally used as the final electron acceptor
rather than oxygen. S. marcescens also exhibits tyrosine hydrolysis and citrate degradation 26.

2.5.6 Acinetobacter species

Acinetobacters are short, plump, gram-negative (but sometimes difficult to destain) rods,
typically 1.0 to 1.5 by 1.5 to 2.5 mm in the logarithmic phase of growth but often becoming more
coccoid in the stationary phase. Pairing or clustering of cells often occurs. Gram stain variability,
as well as variations in cell size and arrangement, can often be observed within a single pure
culture. Acinetobacter spp. normally form smooth, sometimes mucoid, pale yellow to greyish-
white colonies on solid media, although some environmental strains that produce a diffusible
brown pigment have been described. The genus Acinetobacter is now defined as including
gramnegative (but sometimes difficult to destain) coccobacilli, with a DNA G1C content of 39 to
47 mol%, that are strictly aerobic, non-motile, catalase positive, and oxidase negative. Good
growth occurs on complex media between 20 and 308C without growth factor requirements,
14
while nitrates are reduced only rarely. Crucially, extracted DNA is able to transform mutant
strain BD413 trpE27 to the wild-type phenotype 27.

Nosocomial urinary tract infection is caused only infrequently by Acinetobacter spp. It occurs
most commonly in elderly debilitated patients, in patients confined to ICUs, and in patients with
permanent indwelling urinary catheters. Most patients (80%) tend to be men, perhaps reflecting
the higher prevalence of indwelling urinary catheters in this population as a result of prostatic
enlargement. It should, however, be noted that not every isolation of Acinetobacter spp. from the
urinary tract of patients with an indwelling urinary catheter can be correlated with actual
infection. Acinetobacters can be grown readily on common laboratory media such as nutrient
agar and tryptic soy agar, although defined media consisting of a mineral base containing
ammonium or nitrate salts and one or more carbon sources have been used for specific purposes
28
.

2.6 Laboratory Diagnosis of Urinary Tract Infection

2.6.1 Specimen Collection

Suprapubic aspiration is the best method to avoid contamination of specimens with bacteria in
the distal urethra. This collection method is used infrequently because it is not indicated
clinically (except in rare circumstances), it is invasive and uncomfortable, and it requires too
much time and too many resources to be practical. Collection of urine by use of a single catheter
(straight catheter technique) is the next-best technique for obtaining urine specimens with
minimal contamination, but, again, it is not indicated clinically for most patients because it is too
labor intensive and costly for routine use and it is invasive. It has added disadvantages, because
the process of inserting a catheter through the urethra can introduce bacteria into the bladder (and
thereby cause UTI), and rare complications have been reported. Most urine specimens are
obtained from adult patients via the clean-catch midstream technique. This technique has the
following advantages: it is neither invasive nor uncomfortable, it is simple and inexpensive, it
can be performed in almost any clinical setting, there is no risk of introducing bacteria into the
bladder by catheterization, and there is no risk of complications. Colony counts from urine

15
specimens collected by this method correlate reasonably well with those of specimens collected
via suprapubic aspiration or straight catheterization 29.

2.6.2 Specimen transportation

Several studies have demonstrated the adverse effect of delays in transportation or processing of
urine specimens on their quality. In each study, urine specimens were plated within 2 h after
collection and then were plated again after delays of up to 24 h; results were compared to
determine whether delays in plating resulted in an increase in colony counts. In each of the
studies, some of the cultures that were delayed showed increases in the number of colony
forming units (cfu) per mL to >10 5 cfu/mL, thereby leading to false-positive results. It should be
noted that these 3 studies were performed before the publication of current criteria for
interpreting quantitative urine cultures and that the effect on interpretation would have been even
greater if colony counts of 102 or 103 cfu/mL were used to define probable infection in specific
patients. On the basis of the results of these and other similar studies, it is currently
recommended that urine specimens be plated within 2 h after collection unless specimens have
been refrigerated or kept in a preservative 30.

2.6.3 Specimen processing

Routine urine cultures should be plated using calibrated loops for the semi quantitative method.
This method has the advantage of providing information regarding the number of cfu/mL, as
well as providing isolated colonies for identification and susceptibility testing. The types of
media used for routine cultures should be limited to blood agar and MacConkey's agar. For urine
specimens obtained from outpatients, it is not necessary to routinely inoculate a medium that is
selective for gram-positive bacteria, because nearly all UTIs in outpatients are caused by aerobic
and facultative gram-negative bacteria.

Even in patient populations in which Staphylococcus saprophyticus is a common cause of UTIs,

it is not necessary to use selective media. In contrast, urine specimens obtained from hospitalized
patients are likely to contain enterococci, which have emerged as the second most common cause
of nosocomial infections. Laboratories may want to consider inoculating urine specimens

16
obtained from hospitalized patients, or from patients in whom gram-positive bacterial infection is
suspected but not documented, to a medium that is selective for gram-positive cocci. A medium
such as phenylethyl alcohol suppresses the growth of swarming Proteus species and other gram-
negative bacilli that can overgrow gram-positive cocci in the specimen. Urine cultures should be
incubated overnight at 35°C–37°C in ambient air before being read. There is no added benefit to
incubating routine urine cultures for 48 h, provided that specimens are incubated for a full 24 h
and that urine specimens containing <104 uropathogens or specimens from patients with
suspected funguria are incubated for 48 h 31.

2.6.4 Cultures and the laboratory diagnosis of UTIs

Routine bacterial urine cultures: Urine culture may not be necessary as part of the evaluation
of outpatients with uncomplicated UTIs, However, urine cultures are necessary for outpatients
who have recurrent UTIs, experience treatment failures, or have complicated UTIs. Urine
cultures are also necessary for inpatients that develop UTIs. The bacterial culture remains an
important test in the diagnosis of UTI, not only because it helps to document infection, but also
because it is necessary for determination of the identity of the infecting microorganism(s) and for
antimicrobial susceptibility testing. This is particularly true because of the increased incidence of
antimicrobial resistance. The most commonly used criterion for defining significant bacteriuria is
the presence of ⩾105 cfu per milliliter of urine.

This criterion was established only for women with acute pyelonephritis or women who were
asymptomatic but had multiple urine cultures that yielded this number of bacteria; however, the
criterion is often applied to other patient populations. Most patients with UTIs, however, do not
fall into either category, and 30%–50% of patients with acute urethral syndrome will have
colony counts of <105 cfu/mL.

Catheterized patients (who may have low concentrations of bacteria that can progress to higher
concentrations) and many patients with infections of the lower urinary tract have colony counts
much lower than 105 cfu/Ml if the specimens are obtained via suprapubic aspirate or
catheterization. Accordingly, the most appropriate diagnostic criterion for urine culture

17
specimens obtained via suprapubic aspirate or catheterization is a bacterial concentration of
⩾102 cfu/mL 32.

Anaerobic bacteria urine cultures: The normal flora of the large intestine, vagina, and skin
contain large numbers of anaerobic bacteria. Because anaerobic bacteria cause UTIs only in rare
circumstances, however, recovery of anaerobic bacteria from urine by culture is of no clinical
relevance for most patients with UTIs. Urine cultures for anaerobic bacteria should be limited to
patients with anatomic abnormalities (e.g. Enterovesicular fistulae) that increase the likelihood of
infection with anaerobic bacteria.

2.7 Antimicrobial Susceptibility

2.7.1 History of Antibiotic and Antibiotic Resistance

1928. Alexander Fleming, a Scottish biologist, took the fight against infections to new level
when he identified penicillin, making this the year that the modern antibiotic era began.

1943. Penicillin was on it is way to mass production and was used heavily to treat allied troops
fighting in Europe during World War II. Considered a miracle drug, it was soon available to the
public, despite warning from Alexander Fleming that overuse could lead to mutant bacteria.
Because bacteria can transfer genes horizontally- from one bacterium to another immediately – it
is ability to share resistance was adding to a growing threat unheeded by many scientists and
doctors of the day.

1948. Transferring antibiotic use from medicine to agriculture also happened in a rather

accidental way. Robert Stokstad, an animal nutritionist, and Thomas Jukes, a biochemist, were
working at the company Lederle to develop an “animal protein factor” that could enhance
chicken growth and boost poultry profits. The researchers were initially experimenting with
vitamin B12, which was believed to boost animal growth, but eventually found that the cellular
remains of Streptomyces aureofaciens bacteria — from which tetracyclines are extracted —
contained a lot of the vitamin. As the Lederle labs had also discovered the first tetracycline, the
researchers had access to plenty of these bacterial remains. Chicks that received supplements
including Streptomyces auerofaciens grew 24 percent more than those receiving liver extract,

18
which also contained high levels of B12. The bacteria shells, which still contained traces of the
antibiotics, were more effective in making them grow (and much cheaper than liver extract).This
discovery jumpstarted the process of routinely injecting antibiotics into animals.

Meanwhile, resistant staph bacteria were growing in hospitals. According to Harvard Magazine ,
resistant staph infections in hospitals had risen from 14 percent in 1946 to 59 percent in 1948.

1952. By this time, doctors were somewhat aware of the possibility of antibiotic resistance, but
overall they remained hopeful and optimistic about the success of these new drugs, which could
defeat diseases that had long plagued humanity, like cholera and syphilis. A study on antibiotic
resistance published that year concluded that syphilis could be treated “without any indications
of an increased incidence of [resistant] infections, and this work gives grounds for hoping that
the widespread use of penicillin will equally not result in an increasing incidence of infections
resistant to penicillin.”

1950s–1970s: The next couple decades were considered the golden era of antibiotics, due to the
sheer number of new drugs that were being developed: streptomycin, to treat serious infections
like endocarditis and the plague; ampicillin, which treats respiratory tract infections and
meningitis; and dozens of others. In the excitement surrounding the success of these antibiotics,
they were made readily available to the public, like penicillin, ultimately paving the way for
resistance to develop more easily.

The Tide Turns, the Superbugs Arrive

1955. As Fleming had predicted, resistance to penicillin gradually built up due to the
accessibility of the drug. By 1955, many countries had attempted to slow this resistance by
limiting penicillin use to prescription only, but it was too little too late: many bacterial strains
had already defeated the antibiotic, including staphylococci.

1960. In an attempt to defeat penicillin-resistant strains, scientists developed methicillin, a

different antibiotic in the penicillin class that could work against resistance. But within a year,
bacterial strains developed resistance to methicillin too — eventually called MRSA , methicillin-
resistant Staphylococcus aureus, or S. aureus. Now, MRSA can resist most antibiotics, and

19
infections are common in hospitals — making it one of the biggest forerunners of multiple-drug
resistant (MDR) bacteria.

1976. At Tufts University, a physician and researcher named Stuart Levy became one of the first
to identify antibiotic resistance due to their use in animals, and to clamor for greater awareness
of the problem. He worked on a study that year that examined how small amounts of antibiotics
in animal feed could cause resistance in humans. On a farm in Massachusetts, Levy fed
tetracycline to chickens, and found that tetracycline-resistant bacteria began to populate the
chickens’ gut flora within a week. Despite this, rampant use of antibiotics likes vancomycin
(which had previously been considered a “last resort” drug) throughout the 1980’s led to an
epidemic of resistant strains on farms and among humans.

1990s. A stronger resistant strain of MRSA began sickening normal, healthy people in the 1990s.
This perhaps created a greater public awareness of the danger of antimicrobial resistance.

By 2002, up to 60 percent of S. aureus cases in hospitals were resistant to methicillin.

In 2005, over 100,000 Americans were stricken with MRSA infections and some 20,000 died,
more than the amount of people who were dying from HIV and tuberculosis combined,
according to Harvard Magazine.

2012. As more researchers began working on the impending antibiotic-resistant epidemic, they
had to tackle the classification of multidrug-resistant bacteria, which were multiplying by the
minute. In a 2012 study , a team of scientists proposed adding the terms extensively drug-
resistant (XDR) and pandrug-resistant (PDR) to multidrug-resistant (MDR) bacteria to better
help them classify and potentially defeat these superbugs. It was the first time that researchers
had a unified set of definitions for MDR bacteria to better understand them.

2013. After decades of certain researchers calling for action, the FDA finally implemented
a plan to phase out certain antibiotic use in animals. But the extent to which this plan is effective
at reducing the massive damage already done is difficult to identify.

20
2014. In response to major superbug outbreaks like Klebsiella pneumoniae (which causes
pneumonia and bloodstream infections in the hospital) and gonorrhea strains all over the world,
the World Health Organization (WHO) released a statement noting that “this serious threat is no
longer a prediction for the future, it is happening right now in every region of the world and has
the potential to affect anyone, of any age, in any country.”

2015. McDonald's announced that it would phase out all meat sources that contained antibiotics,
marking the first step of a major fast food company to heed the public health warnings and take
action.

Since the 70s, very few new antimicrobial agents have been discovered, and the only way for
researchers to combat resistant bacteria has been to modify already existing antibiotics. This has
led to a standstill of sorts between current antibiotics and the rapidly adjusting “superbugs.”

Today, from farms to hospitals to everyday workplaces, animals and humans are likely teeming
with different forms of resistant bacteria. In 2013, a Consumer Reports investigation showed that
over half of ground turkey meat sold in the U.S. contained strains of drug-resistant bacteria.
According to the CDC , some 2 million people in the U.S. are infected with drug-resistant bugs
every year, and 23,000 of them die from these infections. Those numbers are likely to get worse
in the coming decades, according to recent reports .

The danger of the situation is mainly in its complexity, Rustav Aminov writes in a
2010 report on antibiotic resistance: “It is not a single grand challenge; it is rather a complex
problem requiring concerted efforts of microbiologists, ecologists, health care specialists,
educationalists, policy makers, legislative bodies, agricultural and pharmaceutical industry
workers, and the public to deal with. In fact, this should be of everyone's concern, because, in the
end, there is always a probability for any of us at some stage to get infected with a pathogen that
is resistant to antibiotic treatment.

21
 2.8 Multiple Drug-Resistance (MDR), Extensively Drug-Resistant (XDR) and Pandrug-
Resistant (PDR)

Multiple drug resistance (MDR), multi drug resistance or multi resistance is antimicrobial shown
by a species of microorganism to multiple antimicrobial drugs. The types most threatening to
public health are MDR bacteria that resist multiple antibiotics. Recognizing different degree of
MDR, the terms extensively drug resistance (XDR) and pandrug-resistant (PDR) have been
introduced. Infections caused by multidrug-resistant (MDR) Gram-negative bacteria such as
Acinetobacterbaumannii and Klebsiellapneumoniae constitute an important issue for establishing
a correct and appropriate therapy in patients su‫و‬erLnJ
‫ٴ‬ from diseases deriving from these
microorganisms, both in Intensive Care Units (ICUs) and in non-ICU settings. Carbapenemase-
producing klebsiellapneumoniae (CP-Kp) is the most common microorganism involved in
nosocomial infections over the last years. Outbreaks due to KPC (K. pneumoniaecarbapenemase)
have been detected in many countries around the world; indeed these infections have become
endemic other than in Europe also in United States, Israel, and China. Various microorganisms
have survived for thousands of years by their ability to adapt to antimicrobial agents. They do so
via spontaneous mutation or by DNA transfer. This process enables some bacteria to oppose the
action of certain antibiotics, rendering the antibiotics infective E.coli is the most common MDR
organism. Recognizing different degrees of MDR in bacteria, the terms extensively drug-
resistant (XDR) and pandrug-resistant (PDR) have been introduced. Extensively drug-
resistant (XDR) is the non-susceptibility of one bacteria species to all antimicrobial agents
except in two or less antimicrobial categories. Within XDR, pandrug-resistant (PDR) is the
non-susceptibility of bacteria to all antimicrobial agents in all antimicrobial categories 33.

2.9 Extended Spectrum Beta Lactamase (ESBL)

ESBL stands for extended spectrum beta Lactamase. An enzyme (chemical) which some
bacteria like Escherichia Coli and Klebsiella pneumonia produce (coliforms). Since 21st century,
it is thought that the emergence of extended-spectrum β-Lactamase (ESBL-) producing bacteria
may present an increasing risk of transmission of resistant strains in humans and animals. They is
a worrying global public health issue as infections caused by such enzyme-producing organisms
are associated with a higher morbidity and mortality and greater fiscal burden. The problem is

22
clearly severe in developing countries where studies on this subject, drug availability, and its
appropriate use were limited and resistance rate was high. ESBL-producing organisms are
capable of hydrolyzing penicillin, broad-spectrum cephalosporins, and monobactams, but they
do not affect the cephamycins or carbapenems, and their activity is inhibited by clavulanic acid.
In addition, ESBL-producing organisms are frequently exhibiting resistance to other
antimicrobial classes such as fluoroquinolones, aminoglycosides, and trimethoprim-
sulfamethoxazole due to associated resistance mechanisms, which may be either chromosomally
or plasmid-encoded .

The widespread use of third-generation cephalosporin was believed to be the major cause of
mutations in these enzymes that leads to the emergence of plasmid-encoded ESBLs. These
ESBLs were transferred between bacteria by plasmids, which were in turn spread by clonal
distribution between hospitals and countries through patient mobility. The presence of ESBLs
complicates antibiotic selection, especially in patients with serious infections, such as
bacteremia. The reason for this is that ESBL-producing bacteria, including those originating in
the community, are often multiresistant to various antibiotics; an interesting feature of isolates
that produce CTX-M (CTX stands for cefotaximases and M for Munich) is the coresistance to
the fluoroquinolones. Type CTX-M ESBLs have been described as an enzyme preferentially
hydrolyzing cefotaxime over ceftazidime and also hydrolyzing cefepime with high efficiency.

The spread and the burden of ESBL-producing bacteria are greater in developing countries.
Findings of a recent review showed that pooled prevalence of healthcare-associated infections in
resource-limited settings (15.5%) was twice the average prevalence in Europe (7.1%). Some
plausible reasons for this difference include the following conditions that are prevalent in low-
income countries: crowded hospitals, more extensive self-treatment and use of non-prescription
antimicrobials, poorer hygiene in general and particularly in hospitals, and less effective
infection control 34.

2.10 AmpC beta-Lactamase

AmpC β-lactamase preferentially hydrolyzes narrow-, broad-, and expanded-spectrum

cephalosporins and cephamycins and resists inhibition by clavulanate, sulbactam, and

23
tazobactam. Many gram-negative bacilli produce a chromosomally mediated AmpC which, when
hyper produced, may cause resistance to penicillins, aztreonam, cephamycins, and narrow-,
broad-, and expanded-spectrum cephalosporins. Because several terminologies are in use, for
simplicity, transmissible AmpC β-lactamase are referred to here as plasmid-mediated AmpC β-
lactamase. These enzymes have been detected in some isolates of Klebsiella spp,
Salmonella spp., C. freundii, E. aerogenes, P. mirabilis, and E. coli and are typically associated
with multidrug resistance. The most commonly encountered plasmid-mediated AmpC β-
lactamase belongs to the CMY, FOX, and DHA families. Accurate prevalence data are scarce
due to lack of testing, but they appear to be less common than ESBLs.

Laboratories should be able to detect AmpC β-lactamase because they have been associated with
false cephalosporin susceptibility and also to recognize isolates for which there is the potential to
falsely report isolates as ESBL negative. It is unnecessary to detect AmpC production in
organisms that produce an inducible chromosomal AmpC β-lactamase because the organism
identification is indicative of AmpC production; i.e., 100% of E. cloacae, E. aerogenes, C.
freundii, S. marcescens, Providencia sp., Morganella morganii, Hafnia alvei, Aeromonas sp.,
and P. aeruginosa isolates can be assumed to be AmpC producers. These organisms have the
potential to readily mutate to develop resistance during therapy with β-lactam antibiotics other
than the Carbapenems, penems, or zwitterionic (sometimes referred to as fourth-generation)
cephalosporins (e.g., cefepime) 35.

2.11 Carbapenemases

Carbapenemases are diverse enzymes that vary in the ability to hydrolyze carbapenems and other
β-lactams. Detection is a crucial infection control issue because (i) they are often associated with
extensive, sometimes total, antibiotic resistance and (ii) more-resistant organisms such as strains
of Pseudomonas and Acinetobacter spp. that have acquired a carbapenemase can be vectors
responsible for carbapenemase transmission to members of the family Enterobacteriaceae in
which the resistance mechanism is not recognized. The major concern is with transmissible and
not chromosomal carbapenemases. The transmissible enzymes can be acquired unpredictably by
important pathogens 3such as P. aeruginosa, A. baumannii, and members of the
family Enterobacteriaceae. The chromosomal enzymes occur predictably in less common

24
pathogens such as S. maltophilia, Aeromonas spp., Chryseobacterium spp., and others.
Carbapenemases belong to molecular classes A, B, and D. The class A enzymes (Bush group 2f)
are inhibited to various degrees by clavulanate and usually hydrolyze penicillins or
cephalosporins more efficiently than carbapenems.

For this reason, some, such as KPC enzymes, lack potent carbapenemase activity and may be
considered ESBLs that also hydrolyze carbapenems. Class A carbapenemases include the KPC,
IMI, and SME families, NMC-A, and some GES enzymes. They are most commonly produced
by members of the family Enterobacteriaceae but have been recently detected in isolates of P.
aeruginosa in Colombia and Puerto Rico and A. baumannii in Puerto Rico (41 Robledo,). Class
B enzymes (Bush group 3) are metallo-β-lactamases (MBLs) which typically hydrolyze
carbapenems efficiently but not aztreonam and resist currently available β-lactamase inhibitors
but are inhibited by chelating agents such as EDTA. The most important include the VIM and
IMP families and SPM-1, which have been detected in strains of P. aeruginosa, members of the
family Enterobacteriaceae, and A. baumannii. Most class D carbapenemases hydrolyze
carbapenems weakly and are inhibited poorly by clavulanate. They belong to the OXA family
and are most commonly produced by Acinetobacter spp. but have also been reported in some P.
aeruginosa, K. pneumoniae, and E. coli strains 36.

2.12 Risk factors of Urinary Tract Infections

2.12.1 Pregnancy

Stenqvist et al found that the frequency of bacteriuria increases by about 1% during pregnancy.
He also confirmed that the risk of acquiring bacteriuria increases with the duration of pregnancy
from 0.8% of bacteriuric women in the 12th gestational week to 2% at the end of pregnancy but
Kutley et al stated that the prevalence of asymptomatic bacteriuria in pregnancy varies from 4-
7% (range 2-11%) and it is similar to that observed in non-pregnant women 37.

2.12.2 Socio Economic Status

Prevalence is higher among lower socio economic classes. Kiningham et al., 1993 reported that
low socio- economic status, sickle cell trait, diabetes mellitus and grand multi parity predispose

25
to urinary tract infection, and each is associated with two-fold increase in the rate of bacteriuria
38
.

2.12.3 Anaemia

Bacteriuria in pregnancy was associated with maternal anemia ; also found that bacteriuria in
pregnancy was associated with maternal anemia. But Fatima et al 2006 reported that there is no
any association between bacteriuria and anemia 39.

2.12.4 Diabetes

Systematic review and meta-analysis of published data since 1966 to evaluate whether
asymptomatic bacteriuria (ASB) is more common in patients with diabetes than among control
subjects. Out of twenty-two studies which fullfilled the inclusion criteria, they found that ASB
was present in 439 of 3,579 (12.2%) patients with diabetes and in 121 of 2,702 (4.5%) healthy
control subjects. ASB was more common both in patients with type - diabetes and type 2
diabetes than in control subjects. The prevalence of ASB was higher in both women and men as
well as in children and adolescents with diabetes than in healthy control subjects. They
concluded that the prevalence of ASB is higher in all patients with diabetes compared with
control subjects 40.

2.12.5 Sexual Activity

Buckley et al, 1978 found that 30% of women have at least one log increase in bacteria in the
bladder immediately following sexual intercourse. Women who have been sexually active within
the past month are six-times more likely to 2000) and woman with a new sexual partner also has
an increased risk of infection 41.

2.12.6 Catheterization

Cope et al., 2009 reviewed all urine culture results at a veteran’s affairs medical center during a
3-month period. 280 episodes of culture proven catheter-associated bacteriuria occurred during 3
months of study. Out of 280, 164 cases were asymptomatic.

26
 2.12.7 Unhygienic Practices

Estimates of the prevalence of methods of management vary greatly across contexts but studies
report widespread use of unsanitary absorbents, and inadequate washing and drying of reused
absorbents across Africa, South East Asia and the Middle East. Studies in Africa have found use
of sanitary pads as low as 18% amongst Tanzanian women with the remainder using cloth or
toilet paper. Studies of Nigerian schoolgirls have found between 31% and 56% using toilet tissue
or cloth to absorb their menstrual blood as oppose to menstrual pads. Studies in India have found
between 43% and 88% of girls washing and reusing cotton cloth rather than using disposable
pads 42.

CHAPTER THREE: MATERIAL AND METHODS

3.1 Study Design

Descriptive cross-sectional study was conducted in this study.

3.2 Study Area

This study was conducted at BIHS General Hospital, Dhaka, Bangladesh, and rest of the
work was done in the department of Microbiology of BUHS.

3.3 Study Population

The study population was involved UTI symptomatic patients attending at BIHS General
Hospital.

3.4 Study Period

This study was carried out in the period between J to December 2021.

3.5 Study Criteria

3.5.1 Inclusion criteria

 Patients attending BIHS General Hospital, Dhaka, Bangladesh

3.5.2 Exclusion Criteria

27
  patients who are not willing to participate in this study
3.6 Sampling Technique

Sampling technique of this study was simple random sampling. Simple random sampling
was used to select a sample without bias from the accessible population

3.7 Sample Size Determination

The sample size was calculated by using Cochran’s formula:

N = (Z2 x p x q) /d2

Were:

N = sample size

p = Estimated proportion of an attribute that is present in the population (50 percentage)

q = (1-p) = 1 - 0.5 = 0.5

d = acceptable error 5% (here 10% of p) = 0.05

Z = 1.96 (at 95% confidence interval)

So, N = (1.96)2x 0.5 x 0.5/ (0.05)2

= 3.84 x 0.5 x 0.5 / (0.05)2

= 0.96 / 0.0025

= 384

Though statistically acceptable size is 384 but due to time and financial constraint, the study was
covered a total of 80 samples.

3.8 Data Collection

Semi-structured questionnaire that had translated to English and Bangla language was
prepared and designed in order to obtain clinical and socio demographic information from
participating patients and clinician to satisfy the objectives of the study

28
3.8.1 Data collection instrument

Data collection sheet, Laboratory reports, and direct contact in patient (if needed) was used.

3.8.2 Urine Sample Collection

On the urine sample container, Name, Age, Sex, Room number, and State of origin
including the date and time of collection was mentioned. Only patients who had not been
on antibiotic preceding one week of sample collection were enrolled. About 15- 20ml of
clean catch mid-stream urine samples were collected after clear instructions on collections
including cleaning the genitalia before voiding of urine. Urine samples were collected
using sterile wide mouth containers.

3.8.3 Material and methods

Methods:
Methods and tests were used this study is as follows:
1. Urine Analysis Test
1. Physical Examination
Physical examination of urine is to detect color, odor, clarity, volume, and specific gravity
of each sample.
2. Microscopically Examination
Microscopic examination entails the detection of crystals, cells, casts, and microorganisms.
2. Culture
DAY ONE
 Sample was collected and keep in a record book
 Sample was inoculated to the appropriate Agar (Media) and then incubates at 37°C for 18
to 24 hrs.

DAY TWO

 Inoculated media was checked for any growth.

 If any growth occurred, a colonies characteristic was noted.
 Gram stain was done from the growth colonies and their result was recorded.

29
  Biochemical test was done from the growth organism grown and their result was
recorded.
 Antibiotic sensitivity was done by using Muller-Hinton Agar.

DAY THREE

 Result of antibiotic sensitivity was noted.

 Result of remaining Biochemical test was reported
 All findings were entered in record book.

3. Antimicrobial Susceptibility Test

Antimicrobial sensitivity was tested for each isolated organism using the disk diffusion
method of Kirby-Baurer as described by the Clinical Laboratory Standard Institute (2004)
43.

A disc of blotting paper was impregnated with a known volume and appropriate
concentration of an antimicrobial and it was placed on a plate of Muller-Hinton agar
uniformly inoculated with test organism. The zone of inhibition was measured and
compared with table.

4. Biochemical Tests
Common Biochemical tests that were used in this study are include Oxidase test, Motility
test, Methyl red test, Indole test, Urease test and Citrate Utilization test.
5. Gram Stain Technique
Gram stain was done as per standard method 44.
Materials:

Media Agars: Blood Agar, MacConkey agar, CLED, Brain heart Infusion agar, Triple Sugar
Iron, citrate agar, MIU media.

Biochemical reagents: oxidase test reagents etc.

Antibiotic Disks: Cefoxitin(30µg) Cotrimoxazole (25µg) Ceftazidime(30µg), Meropenem

(10µg), Cefotaxime(30µg), Doxycycline(30µg), Ampicillin(10µg), Amoxicillin-clavulanate

30
(20µg), Chloramphenicol(30µg), Gentamycin(10µg), Nitrofurantoin(100µg),Tetracycline(30µg),
Ciprofloxacin(5µg), Colistin, Aztreonam(10µg), and Cephradine (5µg).

Other equipment: common laboratory equipment and materials.

3.9 Data analysis and interpretation

Data editing, clearing and analysis was done by statistical package for social science SPSS and
result was presented in the form of tables and graph where applicable.

3.10 Ethical consideration

 In this study, in order to maintain the ethical issue of research, the researcher sought
approval to conduct this study from the BUHS university ethics and research committee.
 Permission was sought from the BIH Hospital manager to take samples from their
victims and also will tell them that the information was used only for academic purpose
and the research was kept the solitude, confidentiality and secrecy of patients
Information.
 The researcher promised that this research was conducted in a fully ethical manner and
all copyrights were reserved.

31
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ANNEXURE A: WORK PLAN

Activities Year 2021

Jan Feb Mar Apr May Jun Jul Aug sep Oct Nov Dec

Development of
Research title

Development of
protocol and
protocol
presentation

Ethical clearance

Literature
review gathering

Data collection

Data entry and

36
Analysis

Thesis defense
and submission

37