APPLIED MICROBIOLOGY

1. E – Hospital associated infections

2. E - Emerging vs non emerging infections
3. E – Pyrexia of Unknown Origin
4. E – Zoonosis
5. SN – Normal flora of human body
Prebiotics vs Probiotics
Harmful effects of normal flora
Bacteriology of milk
6. SN – Diarrhea vs dysentery
7. SN – Bacteraemia and septicaemia
8. SN – Food poisoning
9. SN – Antibiogram
10.SN – Coagulase
11.SN – Significant bacteriuria
12.SN – Laboratory diagnosis of water & water borne diseases
13.SN – National Immunisation schedule
Biomedical waste management
14.SN – Urinary tract infections
15.SN – Sexually transmitted diseases
16.SN – Congenital infections
17.SN – Vector borne diseases
18.SN – Bacteriology of milk
19.SN – Faecal examination concentration methods
PCR
20.SN – Recombinant technology and implications
 ESSAY 1

WHAT ARE THE MAJOR TYPES OF HOSPITAL INFECTIONS?

EXPLAIN THEM IN DETAIL. LIST THE UNIVERSAL PRECAUTIONS
TAKEN TO PREVENT HOSPITAL ASSOCIATED INFECTIONS.

HOSPITAL ACQUIRED INFECTIONS

Infections that occur after 48 hours of admission / procedure in the hospital and up to 30 days
after procedure / discharge from the hospital.

TYPES OF HOSPITAL ASSOCIATED INFECTIONS:

The common types of HAI are:
1. Catheter – Related Blood stream infection – CRBSI, CLABSI
2. Ventilator associated Pneumonia
3. Catheter – Associated Urinary Tract Infections
4. Surgical site infection
5. Post – antibiotic Diarrhea
6. Others – Hepatitis, Tetanus

1. CATHETER-RELATED BLOODSTREAM INFECTION:

- Commonly caused by infected intravenous cannula

RISK FACTORS:

Device related:

● Central or Peripheral lines

● Type of Catheter
● Number of Lumens

Patient Related:

● Immunocompromised
● Total parenteral nutrition
 ● Loss of Skin Integrity

Caregiver Related: Poor Hand Hygiene

COMMON ORGANISMS:
● Intrinsic Contamination: Klebsiella, Enterobacter, Pseudomonas
● Extrinsic Contamination: CoNS, S. aureus
PREVENTION:
● Hand Hygiene before and after insertion
● Use Maximum sterile PPE
● Care of IV lines
● Skin preparation by Antiseptics

2. VENTILATOR- ASSOCIATED PNEUMONIA

Defined as the Pneumonia that occurs 48-72 hours after Endotracheal Ventilation.

RISK FACTOR:
Device Related – Nasogastric Tube
Ventilation Duration
Intervention Related – Stress Ulcer prophylaxis
Tracheostomy

COMMON ORGANISMS:
Generally caused by Multi-drug resistant strains of
● Acinetobacter baumannii
● Pseudomonas
● Klebsiella

PREVENTION:
● Daily Oral care with Chlorhexidine 2% solution
● Adherence to Hand Hygiene
● Elevation of the head of bed to 30-450
3. CATHETER- ASSOCIATED URINARY TRACT INFECTION

● Most Common HAI

● Caused due to the Presence of an Indwelling Catheter

RISK FACTORS:
Device Related: Long-Term Catheterization
Latex type of Catheter
Patient Related: Female Gender
Older Age
Diabetes Mellitus
Incomplete emptying of bladder

COMMON ORGANISMS:
● E. coli
● Proteus
● Enterococcus

PREVENTION:
● Catheter care
● Appropriate type of Catheter for long- term use
● Using Closed drainage system
● Using Sterile Items for Catheter Insertion

4. SURGICAL SITE INFECTION

● Infections that develop at the Surgical Site within 30 days of Surgery

● Can cause Morbidity and Mortality if left untreated.

RISK FACTORS:
Patient Related: Age>60 years
Malnutrition, Diabetes
Higher Wound Class
 Immunosuppression
Procedure Related: Improper surgical scab
Inadequate skin antisepsis
Prolonged Operative Time

COMMON ORGANISM:
● S. pyogenes
● MRSA
● E. coli

PREVENTION:
● Preoperative Bathing
● SAP must be provided
● Surgical hand disinfection
● Surgical site preparation

5. OTHER INFECTIONS

NEEDLESTICK AND SHARPS INJURIES

● Carries risk of infection by bloodborne pathogens like Hepatitis – B, C, D and HIV
● They can arise due to:
1. Device with needles
2. Syringes with Exposed Needles
3. Butterfly needles

6. ANTIBIOTIC – ASSOCIATED DIARRHEA

● Occurs due to prolonged uses of Antibiotics

● The Normal Gut Flora gets suppressed due to antibiotics

COMMON ORGANISMS: Clostridium difficile

 Reference: Ananthanarayan and Paniker’s Textbook of Microbiology E/11 Page No. 602

STANDARD / UNIVERSAL PRECAUTIONS OF HAIS:

● They are indicated while handling all Patients, Specimens and Sharps.
● Components include:
1. Hand Hygiene
2. Personal Protective Equipment (PPE)
3. Biomedical Waste
4. Spillage Cleaning
5. Disinfection of Patient care items
6. Environmental Cleaning
7. Sharp
8. Respiratory hygiene and cough etiquette

HAND HYGIENE:

- The Most important measure to avoid transmission of harmful microbes

TYPES:
● Hand Rub
 ● Hand Wash

5 MOMENTS FOR HAND HYGIENE:

1. Before touching a patient
2. Before Cleaning / Aseptic procedures
3. After Body Fluid Exposure
4. After Touching a Patient
5. After Touching Patient’s Surroundings.

PERSONAL PROTECTIVE EQUIPMENT (PPE):

● Used to Protect the Skin and Mucous membranes of HCWS from exposure to
Body fluids and from the HCW to the Patient.

TYPES:
1. Gloves
2. Should be worn during Anticipation of Contact
1. Should not be Reused
2. Same glove Shouldn’t be used on more than one patient.
3. Mask
4. Plastic apron / Gown
⮚ Should be worn to Protect Skin and Clothing during procedures
where contact of Body Fluid is anticipated
o Goggles / Face Shield
⮚ Used in Procedures that are likely to generate Sprays of Body Fluids.
5. Shoe Cover
6. Head Cover

BIOMEDICAL WASTE:

● All Biomedical Waste should be Segregated and Disposed appropriately.

DISINFECTION OF PATIENT CARE ITEMS:

● Ensure that all Patient Care Items such as Instruments, Devices, and Linens are
disinfected before reuse.

SPILLAGE CLEANING:

● Spillage of body fluids should be considered infectious and to be Cleaned at the

earliest.
● The area is cleaned with Hypochlorite.

SHARP:

● Safe use and Disposal of Sharp

● Sharp are disposed in Sharp Box which is a puncture – proof container

ENVIRONMENTAL CLEANING:

● Sterilization and Disinfection of Surface and floors in a hospital is important in

preventing Transmission of HAIs.

ESSAY 2

EMERGING AND RE-EMERGING INFECTIONS

EMERGING INFECTIONS:

● Infectious diseases
● Infections in humans have increased in the past 2 decades or threatens to increase in
near future
These include:

1. New infections, as a result of evolution

2. Known infections, spreading to new geographical populations
3. Previously unidentified infections
 Causes:

● Ecological disruption and human intrusion into new ecological system

● Climate change
● Urbanization and Industrialization
● International trade of goods and services
● Increased number of immunocompromised hosts

Ex: COVID-19 pandemic

RE-EMERGING INFECTIONS:

● Old infections
● Clinically silent, or reduced in numbers, and have re-emerged in the community

This can occur as a result of:

1. Antimicrobial resistance in agents

2. Breakdown in public health measures

Ex: Chikungunya re-emergence in 2005

Drug resistance and re-emergence:

▪ Occurs as a result of development of antimicrobial resistance

▪ Ex:
● Multidrug resistant tuberculosis (MDR-TB)
● Extensively drug resistant tuberculosis (EDR-TB)
● Vancomycin resistant enterococci (VRE)

MANAGEMENT OF EMERGING INFECTIOUS DISEASE:

- A proactive and planned approach to ensure the appropriate prevention and control of
the spread of disease.

Strategic planning should include:

1. Phase I (non-alert) is a routine, preparatory state

 2. Phase II (alert) is the detection, confirmation and declaration of changes
identified during non-alert conditions
3. Phase III (response) includes the ongoing assessment of information and the
planning and implementation of an appropriate response, which includes the
coordination and mobilization of resources to support intervention activities
4. Phase IV (follow-up) activities include re-evaluation

RECOMMENDATION:

● Strengthening epidemiological surveillance & laboratory capabilities and services.

Establishment of a rapid response team.
● Monitoring antimicrobial resistance.
● Establishment of international disease surveillance, networking and advocacy.
● Screening on International travels and trades.
● Networks of laboratories that link countries and regions need to be established.
● Strong national and regional public health systems.

Essay 3
PYREXIA OF UNKNOWN ORIGIN

Fever persisting for more than 2 weeks, with no clear diagnosis despite intelligent and
intensive investigation.

● Pyrexia of unknown origin (PUO) was classically defined as a temperature above

38.0°C on multiple occasions for more than 3 weeks, without diagnosis, despite initial
investigation in hospital for 1 week.
● The definition has been relaxed to allow for investigation over 3 days of inpatient
care, three outpatient visits or 1 week of intensive ambulatory investigation.
● Subsets of PUO are described as HIV-1 related, immune-deficient or nosocomial.
● Up to one-third of cases of PUO remain undiagnosed.
ETIOLOGY:
CLINICAL ASSESSMENT:
● Rare causes, such as periodic fever syndromes, should be considered in those with a
family history.
● Children and younger adults are more likely to have infectious causes – in particular,
viral infections.
● Older adults are more likely to have certain infectious and non-infectious causes.
● Detailed history and examination should be repeated at regular intervals to detect
emerging features (e.g., rashes, signs of infective endocarditis or features of
vasculitis).
● In men, the prostate should be considered as a potential source of infection.
● Clinicians should be alert to the possibility of factitious fever, in which high
temperature recordings are engineered by the patient.
INVESTIGATIONS:
● If initial investigation of fever is negative, further microbiological and
non-microbiological investigations should be considered.
● As with initial investigation of fever described above, the selection and prioritisation
of tests will be influenced by the geographical location of potential exposure to
pathogens.
● These will usually include: induced sputum or other specimens for mycobacterial
stains and culture
• serological tests, including an HIV test, and ferritin estimation
• imaging of the abdomen by ultrasonography or computed tomography (CT)
• echocardiography.
● Lesions identified on imaging should usually be biopsied in order to seek evidence of
relevant pathogens by culture, histopathology or nucleic acid detection.
● Particularly in patients who have received prior antimicrobials, 16S rRNA analysis
may aid diagnosis if a microorganism is not cultured.
● The chance of a successful diagnosis is greatest if procedures for obtaining and
transporting the correct samples in the appropriate media are carefully planned
between the clinical team, the radiologist or surgeon performing the procedure, and
the local microbiologist and histopathologist.
● Positron emission tomography (PET) scans – for diagnosis of vasculitis or help
selection of biopsy sites.
● Liver biopsy – to identify idiopathic granulomatous hepatitis – if there are
biochemical or radiological abnormalities.
● Bone marrow biopsies reveal haematological malignancy, myelodysplasia or
tuberculosis and for identifying brucellosis, typhoid fever or visceral leishmaniasis.
● Bone marrow – for culture and microscopy.
● Laparoscopy is occasionally undertaken with biopsy of abnormal tissues.
● Splenic aspiration – diagnostic test for suspected visceral leishmaniasis.
● Temporal artery biopsy should be considered in patients over the age of 50 years,
even in the absence of physical signs or a raised ESR.
PROGNOSIS:

- No cause is found in approximately 10% of PUO cases, but as long as there is no

significant weight loss or signs of another disease, the long-term mortality is low.
 ESSAY 4
ZOONOSIS
Any disease or infection that is naturally transmissible from vertebrate animals to humans.
Zoonoses may be associated with bacterial, viral, or parasitic, or due to fungal agents.

CLASSIFICATION (in terms of their reservoir hosts):

* Anthropozoonoses: Infections transmitted from animals to man.
* Zooanthroponoses: From man to animals
* Amphixenoses: In both man and animals transmitted in either direction.

COMMON ZOONOTIC INFECTIONS:

1. PLAGUE (YERSINIA PESTIS)
The tribe Yersinieae – genus Yersinia – 11species (3 are important human pathogens)
1) Yersinia pestis: It is the agent of plague
2) Y. pseudotuberculosis and Y. enterocolitica – both cause yersiniosis, a
self-limiting gastrointestinal illness

EPIDEMIOLOGICAL FACTORS:
* Reservoir: Wild rodents, such as gerbils (Tatera indica), field mice and the
bandicoot
*Source of infection- infected wild rodents, rat fleas and cases of pneumonic plague
*Vector:
1) Rat flea (common); species – Xenopsylla cheopis (most efficient) and X.
astia and X. brasiliensis
2) Human flea (Pulex irritans) – rare*

MODE OF TRANSMISSION:
- Bite of an infected rat flea (most common), human flea
- Direct contact with tissues of infected animal (rodents)
- Droplet inhalation (man to man)
 *Blocked flea: In a blood meal, the fleas suck blood containing bacilli from infected
rodents. In the gut of the flea, the bacilli multiply enormously and may block the
proventriculus-called as blocked flea.
*Extrinsic incubation period – Interval between the flea acquiring the infection
through blood meal and becoming blocked flea (2 weeks for Xenopsylla cheopis)
*Cheopis index – Average number of X. cheopis per rat Plague outbreak in places
having cheopis index > 1

VIRULENCE FACTORS OF Y. PESTIS:

Fraction 1 (F1) antigen: It is a capsular protein antigen, encoded by a plasmid (pFra);
principal virulence factor; acts by inhibiting phagocytosis. It is a highly antigenic -
Immunodiagnostic marker of infection.
Other virulence factors – Phospholipase D, surface proteases, pH 6 antigen,
lipopolysaccharide, type III secretion system, adhesins and siderophore.

HUMAN PLAGUE: CLINICAL TYPES

(1) Bubonic, (case-fatality ratio is nearly 30-60%)
(2) Pneumonic – Rare but highly infectious (C: F=30-100%)
(3) Septicaemic

BUBONIC PLAGUE – Most common type

Transmission – By the bite of an infected rat flea.
Incubation period – About 2-7 days
Symptoms – Fever, malaise, headache, and painful lymphadenitis
Buboes: Regional lymph nodes appear as tense, tender swellings called buboes; site- inguinal
(common), crural, (axillary & cervical in children), or submaxillary.

PNEUMONIC PLAGUE -
Transmission – Inhalation of bacilli in droplets expelled from contaminated persons or
animals.
Incubation period – About 1-3 days
Symptoms – Fever, headache, and respiratory symptoms (productive cough or haemoptysis,
dyspnea, and chest pain)
Agent of bioterrorism-aerosolized Y. pestis is a possible source of bioterrorism attack,

SEPTICAEMIC PLAGUE (BLACK DEATH)

Primary septicaemic plague – rare; Secondary septicaemic plague – common
Incubation period – about 2-7 days
Symptoms – haemorrhages in skin and mucosa → gangrene of the affected site

LABORATORY DIAGNOSIS OF PLAGUE:

SPECIMEN COLLECTION
• Bubonic plague – pus or fluid aspirated from buboes
• Pneumonic plague – sputum and blood
• Septicaemic plague – blood and splenic aspirate (post-mortem)

TRANSPORT MEDIUM (e.g., Cary-Blair medium)

DIRECT MICROSCOPY-
*Gram staining
*Wayson stain or methylene blue staining

CULTURE (Y. pestis is aerobic and facultatively anaerobic)

*Blood agar
*MacConkey agar

CULTURE SMEAR
Gram staining of culture smear reveals pleomorphism.

MOTILITY TESTING
Y. pestis-nonmotile both at 25°C and 37°C; other Yersinia species-motile at 25°C and
non-motile at 37°C.
IDENTIFICATION

AUTOMATED IDENTIFICATION SYSTEMS

*MALDI-TOF – can be used to differentiate three biotypes of Y. pestis

CONVENTIONAL BIOCHEMICAL TESTS

*ICUT tests:
Indole test (negative), citrate test (negative), urease test (negative) and TSI (triple
sugar iron agar) test shows alkaline/acid.

TREATMENT: “antibiotics”
Streptomycin has been the choice of treatment for plague(past)-given for 10 days.
Gentamicin is superior to streptomycin and currently recommended.
Levofloxacin, doxycycline, and chloramphenicol are also effective.

PREVENTION:
*Control of cases by early diagnosis, Isolation precaution
*Control of fleas by use of effective insecticides, control of rodents.
*Vaccine:
• Formalin killed vaccine (Sokhey's modification of original Haffkine vaccine) given
subcutaneously, two doses 4 weeks apart and a booster after 6 months.
• Live attenuated vaccine based on strain EV76

TULARAEMIA
CAUSATIVE AGENT – FRANCISELLA TULARENSIS
Primarily a plague-like disease of rodents and other small animals
F. tularensis - An agent of bioterrorism.

PREVALENCE:
F. tularensis – Four subspecies: Tularensis (most common & most virulent), holarctica,
novicida and mediasiatica.
CLINICAL MANIFESTATIONS:
* Ulceroglandular tularemia: most common form (75-85% of total cases) - ulcerative lesion
at the site of inoculation, with regional lymphadenopathy
Other forms-Pulmonary, oropharyngeal, oculoglandular form and typhoid-like illness
* Complications -Suppurated lymph nodes, acute kidney injury, hepatitis, rhabdomyolysis,
empyema, pericarditis, meningitis, osteomyelitis, and endocarditis.

LABORATORY DIAGNOSIS:
• Culture: special media-BCG agar (blood cysteine glucose agar)
• Specimen: Ulcer scrapings, and lymph node biopsy
• Species identification from colonies –
-By conventional biochemical tests or by automated identification systems such as VITEK.
- Antibody detection is the mainstay of diagnosis-Agglutination tests, ELISA, PCR.

TREATMENT:
Gentamicin – the drug of choice; given for 7-10 days. Doxycycline or ciprofloxacin –
alternatives.

3. BITE WOUND INFECTIONS

Bites and scratches from animals and humans allow the inoculation of microorganisms that
are commonly found in the animal's oral cavity, nose, or nail by lodging on the wound
surface.
* Can breach skin barrier and penetrate deeper causing osteomyelitis and septic arthritis.
* Invasion of lymphatics and blood cause bacteraemia, meningitis, brain abscess, and
endocarditis.
• Rarely, infection of the cutaneous nerves carries organisms to CNS (e.g., rabies causing
encephalitis).

DOG BITES
Accounts for 80% of all animal-bite wounds; infection- 15-20%
* Age/gender: children>adults, and males>females
*Site: often upper extremity, except for children <4 years -in head and neck region
*Microbiology:
- Aerobes - B-haemolytic streptococci, Pasteurella species, Staphylococcus, Eikenella
corrodens, and Capnocytophaga canimorsus.
- Anaerobes -Actinomyces, Fusobacterium, Prevotella, and Porphyromonas species
- Organisms causing systemic diseases: Rabies and tetanus.

CAT BITES
Cat bites and scratches → infection (>50% of cases) - higher risk of causing penetrating
injury-septic arthritis and osteomyelitis
*Victims -women>men
*Microbiology: Pasteurella multocida, Bartonella henselae (Causes cat-scratch disease),
Tularemia (Francisella tularensis), S. aureus, Anaerobes. Organisms causing systemic
disease – rabies (rare) and tetanus.

HUMAN BITES
Human bites may take place during fights, domestic abuse, sexual activity, or healthcare
workers caring for patients.
Infection-less → (10-15% of the time)
*Types of human bites: two types
- Occlusional injuries: inflicted by actual biting
- Clenched-fist injuries (more common): Occurs during fights.
* Microbiology:
- Aerobes – viridans streptococci, S. aureus, Eikenella corrodens (common in clenched-fist
injury), and Haemophilus influenzae
- Anaerobes – Fusobacterium nucleatum and Prevotella, Porphyromonas, and Pepto
streptococcus species
- Hospitalized patients → Enterobacteriaceae, non-fermenters in addition

OTHER ANIMAL BITES

* Rat bite infections, Snakebites, Bites of Old-World monkeys (Macaca)&seals
LABORATORY DIAGNOSIS FOR BITE WOUND INFECTIONS:
Specimen:
- Purulent exudate aspirated from depth of wound.
- Wound swab (most common)

AGENTS CAUSING BITE-WOUND INFECTIONS

- Pasteurellosis: (P. multocida – common species)

CLINICAL FEATURES: In humans, P. multocida is the most common cause of wound

infections after dog or cat bites.

SYMPTOMS: Affected area > red, swollen and painful with regional lymphadenopathy and
bacteraemia (severe cases) > osteomyelitis or endocarditis or meningitis.

LABORATORY DIAGNOSIS: Automated methods – MALDI-TOF or VITEK

TREATMENT: Penicillin G or amoxicillin-clavulanate is the drug of choice.

Capnocytophaga Infection Species:

C. ochracea, C. gingivalis and C. sputigena – In human mouth, cause periodontal diseases,
and sepsis / meningitis in immunocompromised hosts.
C. canimorsus and C. cynodegmi – In dogs.

LABORATORY DIAGNOSIS: Appearance - fusiform or filamentous gram-ve coccobacilli

- Automated methods such as MALDI TOF or VITEK.

TREATMENT: As they produce B-lactamases, ß lactam inhibitor combo i.e.: ampicillin

sulbactam is used as the drug of choice.
 4. RAT-BITE FEVER

Characterized by septic fever, petechial rashes, and painful polyarthritis with frequent
relapses. This is known as Haverhill fever or epidemic arthritic erythema.
It is caused by:
(1) Streptobacillus moniliformis
(2) Spirillum minus.

TRANSMISSION:
- By contact with rodents carrying these bacteria and Consumption of contaminated food or
water.
Streptobacillus moniliformis:
Gram-negative, highly pleomorphic nonmotile bacilli, which is frequently arranged in chains
and tangled filaments with bulbous swellings.
It has a tendency to form an L-form.

Spirillum minus: (known as Sudoku) -They are rigid, spirally coiled motile bacilli It doesn't
grow in artificial media.

TREATMENT:
Penicillin is the treatment of choice.
IMPORTANT ZOONOTIC INFECTIONS AFFECTING HUMAN
BEINGS & THEIR SOURCES
 Essay 5

NORMAL MICROBIAL FLORA OF HUMAN BODY AND

BACTERIOLOGY OF WATER, AIR AND FOOD

A. Explain the normal flora of the human body and their role?
ROLE OF BACTERIA:

1. MOUTH
● Oral bacteria produce certain vitamin c and cofactor like vitamin K, Biotin,
Riboflavin
● Prevention of colonization by exogenous pathogens.
● Help in maturation of the host immune system.
● Digestible enzymes like salivary amylase.

2. NASOPHARYNX:
● It can be considered the natural habitat of the common pathogenic bacteria which
causes infection of the nose, throat and bronchi.
● After insulin therapy, they may be predominant flora.

3. GIT:
● Produce vit B12, Vit K
● Control growth of harmful bacteria.
● Breakdown poison in large intestine.
● Bacteria produces enzyme that digest carbohydrate in plant cell wall

4. FGT –VAGINA:
● Normal vaginal flora is predominantly aerobic.
● Normal vaginal flora is dominated by lactobacilli.
● Lactic acid helps to maintain a normal vaginal pH of 3.8 to 4.2
● Acidic environment and other host immune factors inhibit the overgrowth of bacteria
● Some lactobacilli also produce H2O2, a potent microbicide.

5. SKIN:
● Skin is the organ of the human body that protects from the pathogens from the
environment and retards the loss of excessive water.
● Its other functions are insulation, temperature regulation sensation and synthesis of
vitamin D
 B. What is the difference between prebiotics and probiotics?

PREBIOTICS PROBIOTICS

● Prebiotics are defined as a ● Probiotics are referred to as live active

non-digestible special form of fibre microorganisms (bacteria) that when
or carbohydrate. administered in adequate amount will
have beneficial effect to its host.

● The powder from prebiotics can ● Probiotics are more fragile. They are
survive heat, cold, acid and even vulnerable to heat and stomach acid.
time. They may also kill over time

● Prebiotics perform their role by ● Probiotics fight the harmful bacterial

nourishing the bacteria that lives in species present in the gut.
the intestines.

● Prebiotics have been argued to be ● There are various stains of probiotics

beneficial in irritable bowel bacteria which are proven to be effective
syndrome, inflammatory bowel in the relief of irritable bowel syndrome
disease, colon polyps, cancer and and bowel infections symptoms.
even leaky guts.

● Serve as food for friendly bacteria ● Bacteria or yeast

within the gut.
 ● Available as food supplements and ● Available as food supplements and in
naturally occurring in certain foods certain food containing live cultures
such as chicory root, Jerusalem such as yoghurt, coconut.
artichoke, onion, leek, garlic, carrots
and dandelion.

C. What are the harmful effects that can be brought in by the

normal flora of the human body?

- There exists a delicate balance between the human body and the microbial flora that
live symbiotically with the host or as commensals.
- Any factor affecting this balance results in adverse effects.

Major factor responsible for altering the balance and causing infections are:

● Prolonged antibiotics therapy, immunosuppressive agents and immunodeficiency

diseases.
● Alteration of pH (as in the genitourinary tract in the elderly and during
pregnancy)
● Increased virulence of the commensals, production of toxins and change in
microbial flora.

HARMFUL EFFECTS:

❖ May be agents of the disease:

● Members of normal flora may cause various endogenous diseases.
 ● When the host immunity is lowered, the transient flora may invade and
produce disease. e.g., gram negative organism (E. coli) colonizing the
respiratory tract can cause pneumonia.
● If they enter the wrong site or tissue (e.g., Blood, sterile body cavities)- then
even the resistant flora can produce disease. e.g., E. coli which is a resident
flora of the intestine may cause urinary tract infection if entered into the
urinary tract.
❖ Transfer to susceptible hosts:
● Some pathogens of humans that are members of the normal flora for one host
can produce disease if transferred to the other host.
● E.g., The pathogen that colonizes the upper respiratory tract (such as
meningococcus, pneumococcus etc.) can produce disease in the susceptible
hosts.
❖ Bacterial synergism:
● Bacterial vitamins and growth factors produced by members of the normal
flora may promote the growth of the potential pathogens.
❖ Contribute to the drug resistance of pathogens:
● Some members of normal flora produce enzyme such as beta lactamases
which destroy the beta lactam antibiotics
● Thus, indirectly contributing to the drug resistance of pathogens that are
otherwise susceptible to the drug.
❖ Competition for host nutrients:
● Bacteria in GIT absorb some of the host’s nutrients for their survival.

DISEASES PRODUCED BY NORMAL ANATOMICAL SITE FROM WHICH THE

FLORA FLORA IS TRANSFERRED

❖ Urogenital infections including UTI ❖ Intestinal flora such as Escherichia coli,

Klebsiella, Proteus.

❖ Endocarditis ❖ Oral flora (Viridans streptococci)

❖ Dental caries and periodontal disease ❖ Oral flora (Streptococcus mutans)

❖ Peritonitis, abdominal infection ❖ Intestinal flora

❖ Pneumonia ❖ Transient respiratory flora

❖ Septicaemia ❖ From any site

D. Explain the bacteriology and the bacteriological examination

of milk?

BACTERIOLOGY:

● It is the branch of biology that deals with the study of minute organisms
called bacteria (singular bacterium).
● A branch of microbiology dealing with the identification, study, cultivation of
bacteria and with their application in medicine, agriculture, industry and
biotechnology.

BACTERIA:

● It is a single celled, often parasite microorganism without distinct nuclei or organized

cell structures. Various species are responsible for decay, fermentation, nitrogen
fixation and many plant and animal diseases.

They are:

● Prokaryotes
● Single celled organisms
● Size microscopic
● E. coli is 1.3 µm wide x1.0 µm long. 625 E. coli to make 1 inch.
BACTERIOLOGICAL EXAMINATION OF MILK:

- Routine bacteriological examination of milk consists of the following

● Viable method
● Test for coliform bacilli
● Methylene blue reduction test
● Phosphatase test
● Turbidity test

VIABLE COUNT:

Method:

● This is estimated by performing plate counts with serial dilution of milk sample.
● Raw milk always contains bacteria, varying in number from about 500 to several
million per ml.

Significance:

● The plate count gives a rough and direct assessment of the viable bacteria in the
milk
● It is easily explainable to the producer and gives a fair idea of the improvement
or deterioration in the condition of production.

TEST FOR COLIFORM BACILLI:

Method:

● This is performed by inoculating varying dilutions of milk into MacConkey’s

fluid medium
● And nothing about the production of acid and gas after incubation.

Significance:

● Contamination with coliforms comes mainly from dust, dirty utensils and daily
workers.
 ● This method is a useful indicator of faecal contamination and also contamination
by dust or unclean utensils.

METHYLENE BLUE REDUCTION TEST:

Method:

● This is the simple substitute for the viable count.

● It depends on the reduction of methylene blue by bacteria in milk when
incubated at 37ºC in complete darkness.

Significance:

● The rate of reduction is related to the degree of bacterial contamination.

● Raw milk is considered satisfactory if it fails to reduce the dye in 30 mins.
● The resazurin test is similar, except that the dye resazurin, on reduction, passes
through a series of colour changes- from blue to pink to colourless. This colour
change is noted after incubation with the milk for 10 mins.

PHOSPHATASE TEST:

Method:

● This is a check whether milk has been pasteurised.

● The enzyme phosphatase, which is normally present in milk, is inactivated if
pasteurization has been performed properly.

Significance:

● Residual phosphatase activity indicates that pasteurization has been inadequate.

● This test, if positive after proper pasteurization of milk, shows contamination after
pasteurization.
TURBIDITY TEST:

● This is a check on the sterilization of milk.

● If milk has been boiled or heated to the temperature prescribed for sterilization, all
heat – coagulable protein are precipitated
● If ammonium sulphate is then added to the milk, filtered and boiled for 5 mins, no
turbidity results.
● This test distinguishes between pasteurized and sterilized milk.
 SHORT NOTES 6

DIFFERENTIATE DIARRHEA AND DYSENTERY

DIARRHEA DYSENTERY

● Diarrhea is presented as watery stool ● Dysentery is presented as a mucoid stool that

with no blood and mucus. may be accompanied by blood.

● The patient may or may not be ● The patient usually complains of cramps and
accompanied by cramps or pain pain in the lower abdominal area.

● Fever is less common in diarrhea ● Fever is more common in dysentery

● Diarrhea is a disease that affect the ● Dysentery is a disease that affect the colon.
small bowel.

● Diarrhea infection is located and ● Dysentery not only upper epithelial cells and
targets only intestinal lumen and upper targeted but colon ulceration also results.
epithelial cells.

● There is no cell death in diarrhea and ● When a person get dysentery, the upper
the infection is only caused because of epithelial cells are attacked and destroyed by
the release of some toxins by the the pathogen or disease-causing agent.
infecting agent.

● The antimicrobial that are used to treat ● Treatment for dysentery can eradicate the
diarrhea do not eradicate the toxin left pathogen that is causing the infection and stop
behind. the inflammation.
● The effects of diarrhea are not that ● Dysentery can cause a lot of complications. If
serious, apart from a risk of left untreated.
dehydration.

● Diarrhea is mostly viral. E. coli can ● Dysentery is mostly bacterial E. coli, Shigella
also cause watery diarrhea. and Salmonella are the most common causative
organisms.

● Diarrhea does not need antibiotics. ● Dysentery almost always requires antibiotic
Oral rehydration solution or treatment. Intravenous antibiotics may be
intravenous fluid therapy may be used. needed in severely ill children.
 SHORT NOTE 7:

BACTEREMIA AND SEPTICEMIA

BACTEREMIA:
● The transient presence of Bacteria in the Bloodstream without Multiplication.
TYPES:
o TRANSIENT BACTEREMIA:
- May occur Spontaneously or with Minor events
- May lead to Septicaemia
- Normally cured by the Host immune mechanisms

o CONTINUOUS BACTEREMIA:
- Microbes are released into blood at a Fairly Constant Rate.
- Occurs in
● Septic Shock
● Endocarditis
● Early stages of: Enteric Fever and Brucellosis

o INTERMITTENT BACTEREMIA:
- Microbes are released Intermittently into blood.
- Occurs in
● Sequestered focus of infection (e.g., Undrained Abscess)
● Early Course of - Meningitis, Pneumonia, Septic Arthritis.

SEPTICEMIA:
❖ Refers to the Presence of Microbial Antigens and Endo / Exotoxins in the
Bloodstream.
❖ Sepsis – Response the Host mounts against these products.
❖ There is Active Bacterial Multiplication.
ETIOLOGY:

GRAM – POSITIVE COCCI

Staphylococci

Beta-haemolytic streptococci

Enterococci

Pneumococci

GRAM – NEGATIVE COCCI

Meningococci

GRAM – POSITIVE BACILLI

Bacillus anthracis

Listeria

GRAM – NEGATIVE BACILLI

E. coli

Klebsiella

PATHOGENESIS:

Bacteria may enter bloodstream:

– From an infective focus with the help of phagocytic cells carrying microbes
into capillaries or the lymphatic system.
– From breakages of blood vessels adjacent to the skin or mucosal surfaces.
– By introduction of contaminated material directly into the vascular system
SIGNS AND SYMPTOMS:

● Hypothermia / Fever
● Chills
● Hyperventilation
● Subsequent Respiratory Alkalosis

COMPLICATIONS:

▪ Septic
▪ Endotoxic
▪ Septic shock
▪ Acute renal failure
▪ Shock may lead to multiple organ failure (e.g., heart, lungs, liver, kidneys)
 SHORT NOTE 8:
FOOD POISONING

Food poisoning refers to an illness acquired through consumption of food or drink

contaminated either with microorganisms or their toxins.

CAUSES OF FOOD POISONING:

1-6 HOURS INCUBATION PERIOD:

Organisms Symptoms Common food sources

Staphylococcus aureus Vomiting, diarrhoea, Ham, poultry, salad, dairy

abdominal cramps products, pastries
due to Enterotoxin -
preformed heat stable toxin

Bacillus cereus (emetic) Vomiting, diarrhoea, Chinese fried rice

abdominal cramps
due to heat stable preformed
emetic toxin

8 - 16 HOURS INCUBATION PERIOD:

Organisms Symptoms Common food sources

Clostridium perfringens Abdominal cramps, diarrhoea Beef, poultry, legumes,

gravies

Bacillus cereus (diarrheal) Abdominal cramps, diarrhoea Meats, vegetables, dried

beans, cereals

> 16 HOURS INCUBATION PERIOD:

Organisms Symptoms Common food sources

Vibrio cholerae Watery diarrhoea Shellfish, water

Enterotoxigenic E. coli Watery diarrhoea Salads, cheese, meat

Enterohemorrhagic E. coli Bloody diarrhoea Ground beef, milk

Non typhoidal salmonella Inflammatory diarrhoea Meat, eggs, milk, juice

Shigella species Dysentery Potato, egg, salad, lettuce

Vibrio parahaemolyticus Dysentery Raw or undercooked shellfish

particularly oysters

Campylobacter jejuni Inflammatory diarrhoea Poultry, raw milk

Clostridium botulinum Flaccid paralysis diplopia Homemade improperly

dysphagia canned foot and honey in
infants

Listeria monocytogenes Fever and myalgia in Soft cheeses, raw sprouts,

pregnant women meats

Norovirus (virus) Watery diarrhoea, vomiting, Salads, fresh fruits, shellfish

abdominal cramps

Cyclospora (parasite) Watery diarrhoea, abdominal Raw fruits or vegetables

cramps herbs

Mycotoxicosis (fungus) Depends on type of fungal Nuts, maize, wheat, cereals

toxin.
e.g.: aflatoxin causes
hepatoma

TREATMENT:

Type of food poisoning Treatment

Staphylococcal food poisoning Supportive by correcting fluid and electrolyte

 imbalance

Bacillus cereus food poisoning Clindamycin, erythromycin, vancomycin,

tetracycline (resistant to penicillin as it
produces beta lactamase)

Shigellosis Fluoroquinolone like ciprofloxacin

Cholera Azithromycin

Food Botulism Heptavalent botulism equine serum antitoxin,

antibiotics like penicillin or metronidazole
 SHORT NOTE 9:
ANTIBIOGRAM

Overall profile of antimicrobial susceptibility testing results of a specific microorganism to a

battery of antimicrobial agents

ANTIBIOGRAM TYPING:
It classifies the organism into different groups based on their resistance pattern to different
antimicrobials.

HOSPITAL ANTIBIOGRAM :
Periodic summary of antimicrobial susceptibilities of local bacterial isolates submitted to the
hospital's clinical microbiology laboratory.
● It is the responsibility of the department of Microbiology to construct a hospital
antibiogram and share it with clinicians.

WHY DO WE NEED ANTIBIOGRAM?

● Antimicrobial-resistant bacterial infections are a challenging problem in the hospital
setting.
● Infections caused by resistant- and multidrug-resistant (MDR) bacteria not only
increase morbidity and mortality, but also increase overall healthcare costs, primarily
by prolonging hospital length of stay.

PROCEDURE:

1→Sample collection (E.g. - Blood, Pus, Urine, Sputum)

2→Isolation & Identification of microbes

3→Sample cultured and antibiotic disks added

4→After 24-48hours, Zones of inhibition (Clearings) are measured

5→Tables framed-labelling of microbes as Susceptible, Dose-dependent, Intermediate or
Resistant.

USES:
● Guides the clinicians in selecting the best empirical antimicrobial treatment-as
Inappropriate antimicrobial selection also has the potential to increase the risk for
resistance development.
● Useful tool for detecting and monitoring trends in antimicrobial resistance within the
hospital.
● Used to compare susceptibility rates across institutions.
● Since antimicrobial susceptibility testing is routinely done in any hospital, this typing
system provides the first clue to a microbiologist about outbreaks occurring in a
hospital.
● Evaluate the efficacy of a new antibiotics.
● Study the epidemiology of resistance.
 SHORT NOTE 10:
COAGULASE TEST

Biochemical test used to differentiate Staphylococcus aureus (positive) which produce the
enzyme coagulase, from S. epidermidis and S. saprophyticus (negative) which do not
produce coagulase i.e., Coagulase Negative Staphylococcus (CONS).

TYPES OF COAGULASE TEST:

A. Tube / Free coagulase test
B. Slide / Bound coagulase test

A. TUBE OR FREE COAGULASE TEST

Tube coagulase test detects the free coagulase which is an extracellular product of
Staphylococcus aureus.
PROCEDURE:
● 0.1 ml of a young broth culture or agar culture suspension of the isolate is
added to 0.5 ml heparinized or oxalated human or rabbit plasma.
● The tubes are incubated in the water bath at 37 degree Celsius for 3 to 6 hours
to demonstrate clotting.
PRINCIPLE:
1. Activation of plasma coagulase reacting factor (CRF) found in rabbit
plasma.
2. CRF reacts with coagulase.
 3. Formation of coagulase CRF complex.
4. Complex reacts with fibrinogen and activates it.
5. Fibrin clot is produced which is a positive test.

B. SLIDE OR BOUND COAGULASE TEST:

Slide coagulase tests detect the bound coagulase called the clumping factor.
PROCEDURE:
A drop of human or rabbit plasma is added to an emulsion of an isolate in a drop of
saline and mixed.
PRINCIPLE:
1. Clumping factor is bound to the bacterial cell wall.
2. It reacts directly with fibrinogen of human or rabbit plasma.
3. It results in the alteration of fibrinogen.
4. Precipitates on the staphylococcal cell.
5. Clumping of cocci occurs which is a positive test.
 SHORT NOTES 11:
SIGNIFICANT BACTERIURIA

● Bacteriological diagnosis of UTI is done by demonstrating significant bacteriuria

using semi-quantitative cultures developed by Kass.

FACT:
Normal urine is sterile but during voiding may become contaminated with
genital commensals i.e., normal urethral flora. The counts in the contaminated
urine would be lower than that in the case of UTI.

COUNT:
● Greater than or equal to 10⁵ CFU/ml = SIGNIFICANT indicates infection.
● Between 10⁴ to 10⁵ CFU/ml = DOUBTFUL significance, must be clinically
correlated.
● Low count of less than 10⁴ CFU/ml = CONTAMINATION of urine during voiding
with commensal bacteria.

CONDITIONS WHERE LOW COUNTS CAN BE SIGNIFICANT:

● Patient on antibiotic or on diuretic treatment.
● Infection with some gram-positive organisms like Staphylococcus aureus.
● Pyelonephritis and acute urethral syndrome.
● Sample taken by suprapubic aspiration.
● In catheterized patients:
If the patient is symptomatic then a count of greater than or equal to 10³ CFU/ ml
is considered significant.
 SHORT NOTES 12:

A. LABORATORY ASSESSMENT INVOLVED IN THE

ESTIMATION OF PORTABILITY OF DRINKING
WATER

LABORATORY TESTING OF DRINKING WATER

MULTIPLE TUBE METHOD:

● Most common method

● Involves mixing specific amount of water to multiple tubes containing culture
medium

PROCEDURE:

● Most hospital water supplies are unpolluted

● Testing of unpolluted water is as follows
1. 50ml of water is added to one tube of 50ml of culture
2. 10ml of water is added to 5 tubes of 10ml of culture
3. After 24-48hrs of incubation,
I. Medium turns yellow to purple
i. Due to lactose fermentation
ii. Indicates presence of coliform bacteria
II. Medium becomes turbid
III. Gas is collected in Durham’s tube
4. Number of tubes turning purple is matched against McCardy
statistical table to determine MPN of coliform count per 100ml of
water
5. Depending upon MPN/100ml, the quality of water can be
interpreted
DIFFERENTIAL COLIFORM TEST (EIJKMAN TEST):

● Positive multiple tube test does not always indicate faecal contamination, as coliform
bacteria are naturally present
● This test is done to detect faecal E. coli
● Positive tubes from multiple tube test are sub cultured on lactose culture

RESULT:
1. Brilliant green broth is seen
2. Detection of lactose fermentation with acid and gas production at 44℃
3. Positive indole test at 44℃

MEMBRANE FILTRATION METHOD:

PROCEDURE:

1. Filtration of known amount of water through cellulose membrane of pore size

0.2µm-0.45µm
2. Bacteria retained on surface on membrane is cultured and incubated
3. Yellow colonies of coliform are obtained which can be counted to CFU/100ml of
water

ADVANTAGES:

1. Checking dialysis water

2. Testing a large amount of water

DISADVANTAGES:

1. Not suitable for turbid water

2. Expensive than multiple tube water
PRESENCE ABSENCE METHOD – MANJAS METHOD:

● Qualitative method
● Detects the presence or absence of an organism
● H2S coated strips used to detect Salmonella in water
ADVANTAGES:
1. Monitoring good quality drinking water
2. In outbreak situations where urgent reports are needed

B. WATER BORNE DISEASES

Hospital water may serve as a reservoir for waterborne pathogens.

Microbial contamination of water in hospital settings are of 2 types:

1. Enteric pathogens:
● Common in communities surrounding health care settings
● Due to faecal contamination of drinking water
● Transmitted by ingestion of contaminated water
● Cause diarrheal outbreaks and extraintestinal diseases

Ex:

i. Bacteria: E. coli, vibrio cholerae

ii. Viruses: Hepatitis A and E viruses, polioviruses
iii. Parasites: Entamoeba histolytica, Cyclospora

2. Common hospital pathogens:

● Includes multidrug resistant gram-negative bacilli, non-tuberculous
mycobacteria, etc.
● Commonly present in hospital environment, can contaminate hospital water
reservoirs such as potable water, dialysis water, ice and ice machines
● Transmitted by ingestion, aspiration, etc.
● Outbreaks caused by these microorganisms are a serious threat to high-risk
patients who are critically ill or immunocompromised.

- Several methods have been employed for detection and elimination of

microbial contamination of water.
 Short Notes 13:

A. What are routine and individual vaccines? Explain the

National Immunisation Schedule.

ROUTINE VACCINES
❖ They have been developed based on the prevalence of Infectious Diseases, their
Public Health Importance, availability, cost- benefit factors and Logistics.
❖ In India, the Expanded Programme on Immunisation (EPI) and the Universal
Immunisation Programme (UIP) provide Routine Vaccinations to provide protection
against VPDs for much of the target population.
❖ E.g.: BCG, OPV, Hep B birth dose given at Birth.

INDIVIDUAL VACCINES
❖ These are supplemented by Individual initiative.
❖ These vaccines may be omitted under National programmes due to Economic
Limitations.
❖ E.g.
● Varicella vaccine
● Typhoid vaccine

NATIONAL IMMUNISATION SCHEDULE

❖ Immunisation is one of the Most Logical and Cost-effective strategies of any
country for the Prevention of Childhood Sickness and Disabilities.
❖ The National Immunisation Schedule is recommended by the Ministry of Health,
Government of India.
❖ It includes those vaccines that are given free of cost to all children of our country.
Reference: Ananthanarayan and Paniker’s Textbook of Microbiology E/11
B. HOW DO WE SEGREGATE DIFFERENT BIOMEDICAL
WASTES?
 Short note 14:

URINARY TRACT INFECTION (UTI)

Disease caused by microbial invasion of the urinary tract that extends from the renal cortex of
the kidney to the urethral meatus.

CLASSIFICATION:
Two types – based on the anatomical sites involved

Two Types-based on the source of infection

- Health care associated example CAUTI (catheter associated UTI) – most common
- Community-acquired

PREDISPOSING FACTORS:
● Gender: Female > Male
● Age: Incidence of UTI increases with age
● Pregnancy: Anatomical and hormonal changes > asymptomatic bacteriuria.
● Structural and functional abnormality of urinary tract
1)Structural obstruction- urethral structure, renal and ureteric stones, prostatic
hypertrophy
2)Functional Obstruction-neurogenic bladder.
● Bacterial virulence – Pili, Fimbriae; Vesicoureteral reflux; Genetic factors.

ETIOLOGY:
Escherichia coli (uropathogenic coli) – common cause of UTI – 70 % of total cases.
PATHOGENESIS:
By two routes – ascending and descending routes.

ASCENDING ROUTE DESCENDING ROUTE

Most common route: Invasion of renal parenchyma through

Enteric endogenous bacteria enter the urinary hematogenous seeding of pathogens as the
tract by Intercourse or catheterization. spread of bacteraemia.

Gram negative bacilli – E. coli Gram positive bacilli – Mycobacterium

tuberculosis

Fungus – Candida albicans Salmonella

Gram positive cocci – Enterococcus species, Gram positive cocci - Staphylococcus aureus
staphylococcus saprophyticus

Parasites – Schistosoma haematobium, Leptospira

Trichomonas vaginalis

CLINICAL MANIFESTATIONS:
▪ Lower UTI: Asymptomatic bacteriuria, cystitis, urethritis, acute urethral syndrome
▪ Upper UTI: Pyelonephritis, ureteritis, perinephric abscess, renal abscess, renal
tuberculosis
▪ Immunological sequela: Post-streptococcal glomerulonephritis (PSGN)
 SHORT NOTES 15:

CAUSES AND PREVENTION OF SEXUALLY

TRANSMITTED DISEASES

Sexually transmitted diseases are a group of communicable diseases transmitted by sexual

contact.

CAUSES OF STDs:

1. Agents causing local manifestations: (genital tract infections)

● Lesions common in both sexes: Genital ulcers, urethritis
● Female genital tract infections: Vulvovaginitis, cervicitis
● Male genital tract infections: Prostatitis, Epididymis
2. Agents causing systemic manifestations: without producing local manifestations
● Do not primarily affect the genital system
● HIV, Hepatitis B

PREVENTION OF STDs:

▪ Education about safe sex practices

▪ Implementation of healthy sex practices
▪ Prophylactic use of barrier contraceptives
▪ Tracing and treatment of contacts
▪ Periodic screening of high-risk groups
 SHORT NOTES 16:

CONGENITAL INFECTIONS

→ Vertical transmission refers to the spread of infections from mother-to-baby.

→ Routes of infection:
● transplacental route (congenital infection)
● during delivery
● after delivery
→ A congenital infection is an infection that crosses the placenta to infect the foetus.
→ They often lead to defects in foetal development or even death.
→ TORCH is an acronym used for some common congenital infections.
● Toxoplasmosis
● Other infections
■ congenital syphilis
■ hepatitis B,
■ Coxsackie virus
■ Epstein-Barr virus
■ Varicella-Zoster virus
■ Plasmodium falciparum
■ Human Parvovirus
● Rubella
● Cytomegalovirus (CMV)
● Herpes simplex virus

PERINATAL INFECTIONS (DURING DELIVERY)

→ Infections occur while the baby moves through an infected birth canal.
→ Usually caused by the agents of STD s.
→ Also include the infections transmitted through contamination with faecal matter
during delivery.
→ Common examples of agents causing perinatal infections include:
● CMV
 ● Neisseria gonorrhoeae
● Chlamydia species
● Herpes simplex virus
● Human papillomavirus (genital warts)
● Group B streptococci

POSTNATAL INFECTIONS (AFTER DELIVERY)

● These infections spread from mother to baby following delivery, usually
during breastfeeding.
● Causative organisms
■ CMV
■ HIV
■ Group B streptococci.

CONGENITAL TOXOPLASMOSIS:
→ Toxoplasma is the most common parasite to be teratogenic.
→ Incidence – 1 per 1000 live births.
→ Causes encephalitis in HIV-infected individuals
→ Important cause of repeated abortion and infertility.

TRANSMISSION:
→ Mother acquiring Toxoplasma infection in pregnancy is usually asymptomatic.
→ Transplacental transmission of T. gondii from mother to-foetus can occur at any time
during the pregnancy.
→ Tachyzoites are the infective form

GESTATIONAL AGE:
→ The main factor that influences the foetal outcome
→ As the gestation proceeds, the chance of transmission of infection increases but the
severity of the infection declines
CLINICAL MANIFESTATIONS:
→ The classical triad:
● Chorioretinitis
● Hydrocephalus
● Intracranial calcifications

→ Other manifestations:
● Stillbirth
● Psychomotor disturbance
● Microcephaly

→ Ocular involvement:
● Eyes are involved later in life (2nd 3rd decade) when the cysts ruptures
● Causes bilateral chorioretinitis leading to profound visual impairment
● Other manifestations
▪ Blurred Vision
▪ Scotoma
▪ Photophobia
▪ Strabismus
▪ Glaucoma
● If ocular involvement occurs without a history of congenital infection, it is
mostly unilateral.

LABORATORY DIAGNOSIS:

ANTENATAL DIAGNOSIS:
→ Acute infection – Ultrasonography of foetus should be done at 20-24 weeks of
gestation and repeated every 2-4 weeks for detecting the lesions of congenital
infection

→ PCR and/or isolation: Amniotic fluid sample is collected, centrifuged and the pellet is
subjected to PCR and/or isolation in mouse or tissue culture If either or both found
positive, then antenatal T diagnosis is confirmed
 → If both negative: Warrant’s evaluation of the neonate to rule out any remote
possibility of infection.

POSTNATAL DIAGNOSIS:

▪ Isolation of the parasite at the time of delivery must be attempted from amniotic
fluid, placenta and cord leukocyte

▪ IgM and IgG: New-born and maternal sera are subjected to detect IgG
(Sabin-Feldman dye test, IFA or ELISA) and IgM (ELISA or IFA)

▪ IgG titre of 21,000 in neonate: Indicates possible T diagnosis which should be

confirmed by IgM testing
▪ IgM titre of neonate 21:4 after 2 weeks of age indicates probable diagnosis and guides
the clinicians to initiate the treatment to the neonate.
▪ Other tests IgA detection (neonatal and maternal blood)
o IgA appears to be more sensitive than IgM for the diagnosis. IgA antibodies
usually disappear within 10 days of birth, hence persistence of IgA beyond 10
days confirms the postnatal infection
o PCR in neonatal and maternal blood detecting specific genes of T. gondii also
confirms the diagnosis
o IgE detection (neonatal and maternal blood)
 Short Notes 17:
ROLE OF VECTORS IN TRANSMISSION OF INFECTIOUS
DISEASES

VECTOR-BORNE INFECTIONS:
- Vectors are living organisms that can transmit infectious pathogens
(parasites, viruses and bacteria) between humans, or from animals to
humans.

TYPES OF TRANSMISSION:
MECHANICAL TRANSMISSION:
The disease agent does not replicate or develop in/on the in vector; it is simply transported by
the vector (e.g., housefly) from one animal or environment to man.

BIOLOGICAL TRANSMISSION:

Steps:

1→Pathogen (through blood meal)

2→Enters vector

3→Replicates and /develops inside vector

4→Regurgitated /injected into susceptible animal

TYPES OF BIOLOGICAL TRANSMISSION:

TYPE DEVELOPMENT EXAMPLE

•Propagative Pathogen only multiplies Yersinia pestis in rat fleas

 inside the vector

• Cyclodevelopmental Pathogen develops into the Wuchereria bancrofti in

next stage without mosquitoes
multiplying

• Cyclopropagative Pathogen multiplies and also Plasmodium species in

enters to next developmental mosquito
stage

• Salivaria (anterior station) ● Pathogen multiplies Trypanosoma brucei in Tsetse

and reaches the fly
salivary glands of the
vector
● Infection by
regurgitating/ biting

• Stercoraria (posterior ● Pathogen multiplies Trypanosoma cruzi in

station) and reaches the Reduviid bug
rectum of the vector
● Infective forms are
released through
faeces

• Transovarian transmission Parent vector passes the Rickettsia rickettsii in ticks

pathogen to off springs and
the latter spreads infection to
Susceptible hosts
 Short Notes 18:
BACTERIOLOGY OF MILK
TYPES OF BACTERIA IN MILK

i) Acid-forming bacteria
i. Lactic streptococci
ii. S. lactis
iii. Enterococcus faecalis
iv. Lactobacilli
● Ferment the lactose in milk, producing acids, mainly lactic acids, which lead
to the formation of a smooth, gelatinous curd.

ii) Alkali-forming bacteria

i. Alcaligenes spp.,
ii. Aerobic spore bearers
iii. Achromobacter species.
● These render the milk alkaline.

iii) Gas-forming bacteria

i. Coliform bacilli
ii. C. perfringens
iii. C. butyricum.
● They produce acid and gas-a smooth, gelatinous curd riddled with gas bubbles
is formed.
● Coliform bacilli are responsible for the ropiness in milk.

iv) Proteolytic bacteria

i. Bacillus subtilis
ii. Bacillus cereus,
iii. Proteus vulgaris,
iv. Staphylococci
v. Micrococci
 v) Inert bacteria
● Produce no visible change in milk.
i. Cocci of the udder,
ii. Members of the Achromobacter group
iii. Pathogenic organisms in milk.

vi) Human milk

i. S. epidermidis
ii. S. mitis
iii. Gaffkya tetragena
iv. S. aureus

MILK BORNE DISEASES

Milk may be contaminated with

● Streptobacillus moniliformis from the nasal secretions of with rats
● Campylobacter jejuni from animal faeces.
● Yersinia enterocolitica

STERILISATION OF MILK

1. Pasteurisation
▪ In this method, all vegetative pathogens are killed by heating milk to 63-66°C
and maintaining it at this temperature for 30 minutes (the holder method) or by
heating milk to a temperature of 71°C and holding it at that temperature for at
least 15 seconds.
▪ The second step of this method is the rapid cooling of milk to 10°C or less.
▪ The phosphatase test is used to check the adequacy of pasteurisation.

2. Boiling
 ▪ The commonly-used household method of heating milk at or around boiling
point (which destroys all but the most resistant spores) is adequate for short
term purposes.
▪ The efficacy of such treatment is tested by the turbidity test.

3. Ultraheat treatment
▪ In this method, milk is heated to 132°C for one second under specified
conditions.

BACTERIOLOGICAL EXAMINATION OF MILK

1. Viable count
a. This is estimated by performing plate counts with serial dilutions of the milk
sample.
b. Raw milk always contains bacteria, varying in number from about 500 to
several million per ml.
c. The plate count gives a rough and direct assessment of the viable bacteria in a
sample of milk.
d. It is easily explainable to the producer and gives a fair idea of the
improvement or deterioration in the conditions of production.

2. Test for coliform bacilli

Indicator of
a. faecal contamination
b. contamination by dust
c. unclean utensils.

3. Methylene blue reduction test

a. Substitute for the viable count.

 b. It depends on the reduction of methylene blue by bacteria in milk when
incubated at 37°C in complete darkness.
c. The rate of reduction is related to the degree of bacterial contamination.
d. Raw milk is considered satisfactory if it fails to reduce the dye in 30 minutes.
e. The resazurin test is similar, except that the dye resazurin, on reduction, passes
through a series of colour changes from blue to pink to colourless.
f. The colour change is noted after incubation with the milk for 10 minutes.

4. Phosphatase test

a. This is a check on whether milk has been pasteurised.

b. The enzyme phosphatase, which is normally present in milk, is inactivated if
pasteurisation has been performed properly.

EXAMINATION FOR SPECIFIC PATHOGENS:

▪ Tubercle bacillus
o The milk is centrifuged at 3,000 rpm for 30 minutes and the sediment
inoculated into two guinea pigs.

▪ Brucella
o Isolation of brucella may be attempted by inoculating cream heavily on serum
dextrose agar or by injecting a centrifuged deposit of the milk sample
intramuscularly into guinea pigs.
o The animals are sacrificed after six weeks and the serum tested for agglutinins
and the spleen inoculated in culture media.
o Brucellosis in animals can also be detected by demonstrating the antibodies in
milk by the milk-ring or the whey agglutination tests.
 SHORT NOTES 19:
A. WHAT ARE THE CONCENTRATION METHODS FOR
FAECAL EXAMINATION?

Stool analysis:

It is a series of tests done on a stool (faeces) sample for the differential diagnosis of certain
diseases of the digestive system and include infection (such as from parasites, virus or
bacteria), poor nutrient absorption or cancer.

CLINICAL SIGNIFICANCE OF STOOL ANALYSIS:

● Diagnosis of digestive system infectious diseases: Bacteria, parasites, virus and

fungi.
● Diagnosis of pancreas disorder (inflammation); which is associated with
malabsorption of nutrients.
● Primary screening test for some type of digestive system malignancy such as:
Colon cancer.
● Primary screening test for peptic ulcer disease and some types of anaemia.

STOOL EXAMINATION DONE IN:

● Patients with abdominal pain

● Patients with diarrhoea
● Patient with anaemia
CONCENTRATION TECHNIQUES FOR FAECAL EXAMINATION:

CONCENTRATION TECHNIQUE:

● These methods are used when ova or parasites are not found in direct saline
preparation.
● But their presence is highly suspected or symptoms persist. ova of certain parasites
are scanty. E.g., Schistosoma, Taenia.
a) Flotation technique:
This method uses the high specific gravity of a solution to float the lighter ova and
cyst. They can be improved by centrifugation.
ADVANTAGE:
 ● Easy to perform
DISADVANTAGE:
● Delay in examination can result in distortion
● Larvae and some fluke eggs do not concentrate.
● Frequent checking of specific gravity.
b) Sedimentation technique:
● Use solution of lower specific gravity than the parasitic organisms (formalin
ethyl acetate technique)
● Recommended for general diagnostic laboratories due to easy to perform and
less prone to technical error.
E.g., Formalin ether sedimentation technique
● It is the recommended concentration procedures
● Most types of worm eggs (roundworm, tapeworms, schistosomes, fluke eggs),
larvae and protozoan cysts may be recovered by this method.
ADVANTAGES:
● Speed: One sample can be processed in 5 minutes.
● Broad spectrum: It will recover most ova, cyst, and larvae.
● The morphology of most parasites is retained for easy identification.

DISADVANTAGES:

● Requires several pieces of apparatus which does not make it an easy.

● The preparation contains some debris.
● Ether is flammable. Formalin is an irritant.
● Hymenolepis nana and Fasciola spp. Do not concentrate well.
 B. EXPLAIN THE LABORATORY USE OF PCR AND TYPES
IN MICROBIOLOGICAL DIAGNOSIS

- Technique in molecular biology used to amplify a single or few copies of a piece of DNA
to generate millions of copies of DNA. It was developed by Kary B Mullis.

It involves 3 basic steps:

1) DNA extraction from the organism.

2) Amplification of extracted DNA
● Denaturation at 95ºC
● Priming annealing (55ºC)
● Extension of the primer (72ºC)

3) Gel electrophoresis of amplified products.

LABORATORY APPLICATIONS OF PCR:

● It is an indispensable technique used in medical diagnostics and research

laboratories.
● Subcloning DNA targets using PCR
● PCR mediated in vitro mutagenesis
● Amplification of differentially expressed gene sequences
● Differential display reverse transcription PCR.

APPLICATIONS OF PCR:

● More sensitive: It can amplify very few copies of a specific DNA, so it is more
sensitive.
● More specific: Use of primers targeting specific DNA sequences of the organism
makes the PCR assays highly specific.
● PCR can be done to amplify the DNA of the organism
1) Either directly from the sample or
2) To confirm the organism grown in culture.
 ● PCR can also detect the organism that are highly fastidious or non-cultivated by
conventional culture methods
● PCR can be used to detect the genes in the organism responsible for drug resistance
(Eg mec A gene detection in Staphylococcus aureus)
● Detects genetic disease such as sickle cell anaemia, phenylketonuria, muscular
dystrophy.

DISADVANTAGES OF PCR:

● Qualitative, not quantitative

● Viability – cannot differentiate viable or non-viable.
● False positive amplification
● False negative

TYPES OF PCR:

● Long PCR
● Nested PCR
● Inverse PCR
● Quantitative PCR
● Hot start PCR

1) Long PCR:
It is used to amplify DNA over the entire length up to 25kb of genomic DNA
segments cloned.

2) Nested PCR:
Involve two consecutive PCR reactions of 25 cycles. The first PCR uses primers
external to the sequence of interest. The second PCR uses the product of the first PCR
in conjunction with one or more nested primers to amplify the sequence within the
region flanked by the initial set of primers.

3) Inverse PCR:
Used to amplify DNA of unknown sequence that is adjacent to known DNA
sequence.
4) Quantitative PCR:
Product amplification with respect to time, which is compared with standard DNA.
5) Hot start PCR:
Used to optimize the yield of the desired amplified product in PCR and
simultaneously to suppress nonspecific amplification.
 SHORT NOTE 20:
IMMUNOPROPHYLAXIS

▪ Vaccine is an immunobiological preparation that provides specific protection against a

given disease.
▪ Following vaccine administration, the immunogen (active ingredient of the vaccine)
stimulates the immune system of the body to produce active immunity in the form of
protective antibody and/or immunocompetent T cell response.

DNA VACCINE:

▪ DNA vaccines are experimental at present, have many advantages such as cost
effectiveness and mounting a stronger and wider range of immune response.
▪ The small pieces of DNA containing genes from the pathogenic microorganism are
injected into the host.
▪ The gene of interest gets integrated with the host cell genome and starts transcribing
the proteins against which the host mounts an immune response.
▪ Several vaccine trials are going on based on DNA vaccines.

EDIBLE VACCINE:

▪ New concept- introduced recently

▪ The gene encoding the orally active antigenic protein is isolated from the pathogen
and is transferred to suitable plant bacteria, which are then used to infect a
transgenic plant (e.g., banana, potato, etc.)
▪ The plants infected by the bacteria then start producing the antigen of interest on a
large scale.
▪ The appropriate plant parts having the antigen may be fed raw to animals or humans
to bring about immunization
▪ The advantages of the edible vaccines are:
● Low cost
 ● Ability to produce in large scale
● Administered orally
● Induce local immunity
● Heat stable

APPLICATIONS:

→ The edible vaccines are still under experimental stage; some formulations available
include
● Transgenic potatoes and tomatoes against diarrhoea
● Edible banana against Norwalk virus
REFERENCES

Ananthanarayan and Paniker’s Textbook of Microbiology

▪ Tenth Edition
▪ Eleventh Edition

Essentials of Medical Microbiology, Apurba Sastry

▪ First Edition
▪ Second Edition
▪ Third Edition

Review of Microbiology and Immunology, Apurba Sastry, Sixth Edition

Complete Microbiology for MBBS, CP Baveja Seventh Edition