FOOD MICROBIOLOGY:

INFECTIONS OF THE GI
TRACT
BC5070 Infection Science 2
Dr Hamid Ghoddusi
h.ghoddusi@londonmet.ac.uk
 Factors affecting the growth of microorganisms in food

1. Intrinsic (properties of food)

 pH

 Moisture Content (Aw)

 Oxidation-reduction Potential (O2, …)

 Nutrient Content (C, N, F,…)

 Antimicrobial Constituents
 Biological Structures
 Factors affecting the growth of
microorganisms in food

2. Extrinsic Factors (properties of environment)

Temperature
Relative Humidity
Gases in the Environment
Presence of Other Microorganisms
FBD definition (WHO)

• ‘diseases, usually either infectious or toxic in

nature, caused by agents that enter the body
through the ingestion of food.’
• FBD are a serious and global problem
• Unsafe food causes >200 diseases:
• diarrhoea to cancers
• ~600 million (1 in10) fall ill annually
• ~420 000 die every year
• Children <5 (40%) deaths every year.
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Food Spoilage vs Food Poisoning

 Important to distinguish food poisoning from food

spoilage
 Poisoned food:
 the food looks, smells & tastes normal
 BUT when you eaten enough cause illness from the ingested
pathogens or toxins
 Spoiled food:
 not normally cause food poisoning because it is rejected by
the consumer before ingestion

5
Infections of the GI tract
• Caused by all 4 types of microorganism + worms
• Viruses- Norovirus (Winter vomiting bug), rotavirus
• Bacteria- Salmonella, Shigella, Campylobacter
• Protozoa- Giardia lamblia, Entamoeba histolytica,
Cryptosporidium parvum (Chloride resistant)
• Fungi- Candida albicans (e.g. in Immunocompromised)
• Worms
• Helminths (Worms), Strongyloides stercoralis
• Nematodes (round worms), Enterobius
• Cestodes (tape worms), Taenia solium
• Trematodes (flat worms) Fasciola hepatica
 Infection vs intoxication
• Infections • Intoxications
• Pathogens enters G.I. Tract and • Ingestion of a preformed
multiples toxin
• Bacteria may penetrate the • Sudden onset of symptoms
intestinal mucosa or may pass ( few hours )
to other systemic organs • Fever not always present
• Delay in appearance of
symptoms while pathogen
increases in number or invades
tissue
• Usually a fever
 Treatment of FBD

• Generally self limiting

• Recommended: rehydration therapy
• electrolyte salts to maximise absorption of
water
• Not recommended
• Antibiotics and anti-diarrhoeal drugs
• may encourage retention of organism in body
• do not reduce symptoms or limit illness
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Diagnostic
Stool samples
• Sent for analysis in cases of:
• Gastroenteritis (GE)
• Diarrhoea and vomiting (D&V)
• Food poisoning
• Tests performed:
• Culture for pathogens
• Microscopy for ova, cysts and
parasites (OCP)
• Electron microscopy for viral
particles
• ELISA for toxins, C. difficile,
• Multiplex PCR for stool
pathogens
Stools macroscopic appearance
• Formed stools may still
contain pathogens
• Semi-formed
• Liquid
• Note presence of blood and
mucous
• Cholera classic ‘rice-water’
stools
• Very watery with flecks of
food which look like grains
of rice
• Bristol Stool Chart
 Selective/Differential media
• Faeces has high content of normal microbial flora
• 10X more bacteria than cells in the body
• Selective components - Bile salts e.g. Na
Deoxycholate inhibit Gram positive organisms
• Differential components- pH indicator showing
which organisms ferment lactose producing acid
• In MacConkey:
• LF’s (lactose fermenters) bacteria will form Pink colonies
• NLF’s (Non lactose fermenters) – White colonies
• Salmonella and Shigella are NLF’s
• E. coli is LF
Name the major organisms that causes food-
borne diseases in the UK?

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 Campylobacter:prevalence
• a major cause of foodborne diarrhoeal illness in humans
• most common species: C. jejuni & C. coli
• the world most common bacteria causing gastroenteritis
• cause more cases of diarrhoea than foodborne Salmonella
(in developed & developing countries)
• socio-economically important:
• the high incidence of Campylobacter diarrhoea
• duration (long)
• possible sequelae

12
 Campylobacter:origin
• Origin: animal gut (cattle, sheep rodents, wild birds and
poultry) so a zoonotic pathogen
• Food vehicle: poultry, dairy products (notably
unpasteurised) and water
• frequently detected in foods derived from these animals

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 Salmonella
• Produce hydrogen sulphide (H2S) fish eye colonies on
DCA and SS agar
• Gram negative rods (Enterobacteriaceae)
• Oxidase negative
• Urease negative (Proteus urea positive)
• Identify using API 20E
• O and H antigen typing with specific antisera
• Over 2000 serovars in Kaufman White scheme

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 Salmonella species
• UK infections most commonly caused by:
• Salmonella Enteritidis
• Salmonella enterica Serotype Enteritidis

• Salmonella Typhimurium
• Salmonella enterica serovar Typhimurium

• Symptoms:
• diarrhoea, stomach cramps, vomiting and fever
• last for 1 to 2 days or may be prolonged, depending on host
factors, ingested dose, and strain characteristics

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 Salmonella
• Origin:
• Animal and poultry gut, survives in manure and the
environment Zoonotic pathogen
• Food vehicles:
• undercooked contaminated meat especially poultry, pork
and beef
• undercooked eggs and eggs containing dishes
• contaminated ready to eat fruit and vegetables (less)
• Cross-contamination: surfaces, kitchenware, towels and
other foods
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E. coli O 157 H7
• Entero-haemorrhagic E coli (EHEC)
• Haemolytic uraemic syndrome (HUS)
• Non sorbitol fermenter on SMA
• Gram negative rod ( Fam.
Enterobacteriacae)
• Oxidase negative, urease negative
• Identify using API 20E or Specific
antisera or latex agglutination
• Category 3 pathogen. Notifiable
disease
5. Verocytotoxigenic or enterohaemorrhagic E. coli
(VTEC or EHEC)

Serotype O:157:H7 most important type in human

infections. Causes outbreaks and sporadic cases.
Zoonotic pathogen. Toxic to Vero cells, a tissue culture
line derived from monkey kidney cells

Origin: animal gut (cattle, sheep)

Food vehicle: undercooked meat, dairy products (notably

unpasteurised) and water

Infective dose: can be very low, possibly 10 cells

PPAR: young and particularly the elderly

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19
 Listeria monocytogenes
• important foodborne pathogen because of nature of
illness:
• generally no gut symptoms
• 30% mortality
• min growth temp 2-4 oC (grow in refrigerated food)
• widespread occurrence
• ability to establish itself in the environment of food factories
• Origin: environment, including faeces of infected animals.
Can establish in food factories – dairies

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 Listeria monocytogenes
• Food vehicle: soft cheese (pasteurised and non-
pasteurised milk) and pate
• Pregnant women advised to avoid
• Importance in soft cheese
• may be present in low numbers, but
• it can grow at <10oC
• in soft cheeses, the pH increases as the cheese ripens
• pH stops control of growth
• consequently these two hurdles controlling growth may not be
effective

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 Norovirus: the virus

Single-stranded RNA virus

Calciviridae family
Two human genera: noroviruses and sapoviruses
Six genogroups (I through VI)
I, II, and IV affect humans
35 genotypes
Hundreds of different strains

http://www.youtube.com/watch?v=UTr47Z4ZvGU
Norovirus: the virus

• Formerly called Norwalk agent or Norwalk

virus
• SRSV (Small, Round, Structured Virus)
• ~ 32 nm
• Need a living host to multiply
• Cannot multiply in food / water
• Origin: NA- these viruses need human host
 Norovirus: the virus
• very contagious and can cause gastroenteritis
diarrhoea and vomiting, fever, abdominal pain,
sometimes a headache or general pains
• important for very young children and the
elderly
• originally called winter vomiting disease,
Why?

24
Norovirus: the virus

• Can survive:
• 12 hours on a surface
• 12 days in contaminated fabric
• A study demonstrated survival for 61
days in well water
 How common is Norovirus?

• causes an estimated three million cases of diarrhoea and

vomiting each year
• estimated that it was responsible for around 380,000
cases of food poisoning in the UK (FSA, 2020)
• affects people of all ages

https://www.food.gov.uk/safety-hygiene/norovirus
 How does Norovirus spread?

• easily transmitted from one person to

another
• contact with an infected person
• consuming contaminated food or water
• contact with contaminated surfaces or objects
• likely to spread in enclosed areas
• a lot of contact
• e.g. schools, hospitals and nursing homes
 Sources of outbreaks

• Shellfish (including oysters, cockles)-filter –

feeders
• eaten raw or lightly cooked
• Outbreaks often associated with faecal contamination of growing
beds and unlicensed harvesting

•Infected food handlers

• especially of foods needing “hands-on” operations to be eaten without further
processing: sandwiches, prepared salads, pastry fillings - and peeled
grapes!

•Water and ice

• private water supplies (non-mains) where there is evidence of faecal
contamination
 4 C’s

• 1. Cleaning - wash hands properly and keep

them clean

• 2. Cross contamination - avoid cross

contamination

• 3. Cooking - cook food properly

• 4. Chilling - chill food properly

http://cookincastlewales.co.uk/sites/default/files/food_hygiene_4_c_activity_
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book.pdf
Sexually Transmitted
Infections: Viruses
BC5070: Infection Science 2
Transmission
• Def: the passing of a pathogen that causes a communicable disease from an
infected host individual or group to other individual or group, regardless of whether
the latter individual was previously infected
• Means of transmission
1. droplet contact (small particles suspended in air. Covid)
2. direct physical contact (touching infected person. Includes sexual contact)
3. indirect physical contact (touching contaminated surface. Includes soil)
4. airborne transmission (small dry and wet particles remain in air long time)
5. fecal-oral (unwashed hands, contaminated food)
6. indirect via vector
7. vertical
Sexual Transmission
• The mechanisms of transfer of a disease during sexual activity with
another person, including vaginal or anal sex or (less commonly)
through oral sex
• Either directly between surfaces in contact during intercourse (the
usual route for bacterial infections and those infections causing sores)
• Or from secretions (semen etc.) which carry infectious agents that get
into the partner's blood stream through tiny tears in the penis, vagina
or rectum (this is a more usual route for viruses)
• Anal sex is considerably more hazardous as damage occurs more
frequently
The Baltimore Classification
Parvoviridae Reoviridae
Herpesviridae Picornaviridae

Orthomyxoviridae

Retroviridae Hepadnaviridae
Examples of Viruses Transmitted Sexually

• hepadnavirus (HBV) causes hepatitis B

• herpes simplex (HSV) causes herpetic lesions of cervix and urethra

• papillomavirus (HPV) causes genital warts and cancer

• lentivirus (HIV) linked to AIDS

Hepatitis B Virus (HBV)
• Hepadnavirus group
• Has a circular DNA genome, replicates
through an RNA intermediate (needs
reverse transcriptase). Complex
replication process.
• Replication occurs in the liver but viral
proteins can spread to the blood
• Causes hepatitis type B a disease of
the liver
• 1/3 of the world population have
been infected (350 million chronic
carriers)
HBV Transmission
• Exposure to infectious blood or body fluids (semen or vaginal fluid)
• Perinatal infection is common in developing countries
• Other routes of transmission: healthcare work settings, transfusion,
dialysis, acupuncture, tattooing, sharing razors or toothbrushes

No evidence for more simple types of

transmission through saliva, tears, airborne,
direct contact etc.
HBV Disease
• Acute: when a person is first infected.
Self limiting when our immune
system succeeds in clearing the virus
• Acute: liver inflammation, vomiting,
jaundice
• Chronic: if the infection persist more
than 6 months. Long standing stages
• Chronic: cirrhosis, liver cancer
• Affects functions of the liver as
replication occurs in hepatocytes
• Host immune response causes both
viral clearance and liver damage
Treatment & Prevention
• 95% of acute infections do not require treatment as they are cleared
spontaneously
• Children and immunocompromised patients plus chronic infections
can require treatment
• Antiviral drugs: lamivudine, adefovir, tenofovir (RT -reverse transcriptase-
inhibitors), entecavir (base analog)
• Vaccine available – subunit (or acellular) vaccine. 3 doses provides
almost lifelong Ab immunity
Hepatitis Viruses Summary
• HAV: acute, notifiable, vaccine available
• HBV: very infectious. If chronic has high morbidity (liver cancer).
Treatments stop replication. Subunit vaccine available
• HCV: chronic infection. Difficult to treat. No vaccine. Transmission by
intravenous drug use
• HDV: delta. Unusual. Requires HBV for replication
• HEV: unusual in UK. Causes acute disease. High mortality in
immunocompromised and pregnant women
Herpes Simplex Virus (HSV)
• Group I: Herpesviridae
• dsDNA

• They are large DNA viruses with envelopes (membrane)

• They have the ability to establish latency and reactivate following
initial infection
HSV Diseases & Diagnosis
• Herpes, a friend for life.
• HSV 1 – Cold sores,
• HSV 2 – Genital Herpes
• Diagnosis:-
• Viral culture is time consuming
and labour intensive, therefore…
• Serology:
• ELISA for IgG and IgM antibodies
• IgM shows a primary infection
• IgG shows a prior infection
 Replication – latency
1) Virus infects epithelial cells and
enters sensory neurons, which are
non-dividing allowing only limited
replication
3

2) Virus travels along the neuron

axon and enters neuron cell body
2
where it establishes latency
(anterograde transport)
Only latency associated transcripts
expressed and these are required
to establish and maintain latency..

1 3)..and for the virus to be reactivated

during which the virus travels back to
epithelial cells and undergoes lytic
replication producing infectious virus
(retrograde transport, recurrent!!)
Treatment & Prevention for herpesvirus
• No vaccine available
• Treatment based on antiviral therapies
• Best known drugs are acyclovir and gancyclovir which can be applied
as creams or taken orally
Human Papilloma Virus (HPV)
• Small DNA virus of the papillomaviridae family
• Establish productive infections only in keratinocytes (skin) or mucous
membranes
• Most infections are subclinical but some become warts and
occasionally cancer
• More than 30-40 types of HPV are transmitted
Human Papilloma Virus (HPV)
• Papillomata are warts, HPV = wart virus
• Many different strains of the virus cause warts
• Some strains associated with cervical cancer
• Not tested for directly, clinical diagnosis
• Cervical smear to examine for abnormal cells
Human Papilloma Virus (HPV) strains
Disease HPV Type
Plantar warts (verruca) 1, 2, 4

Common warts (hands, fingers) 1, 2, 4, 26, 27, 29, 41, 57

Flat warts 3, 10, 27, 28, 41, 49

Genital warts 6, 11, 30, 40-45, 51, 54

Cervical cancer 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58

Precancerous changes 16, 18, 34, 39, 42, 55

Laryngeal papillomas 6, 11, 30

HPV Treatment & Prevention
• Prevention – Condoms (incomplete protection)
• Treatment Cryotherapy with liquid nitrogen
• HPV Vaccine to reduce cervical cancer
• In England, girls and boys aged 12 to 13 years are routinely offered the 1st
HPV vaccination when they're in school Year 8. The 2nd dose is offered 6 to
24 months after the 1st dose.
• Cervical cytology screening
 • HPV vaccines prevent
HPV Vaccine infection by certain types of
human papillomavirus.
• Available HPV vaccines
protect against either two, four,
or nine types of HPV.
• All HPV vaccines protect
against at least HPV types 16
and 18, which cause the
greatest risk of cervical cancer.
• HPV types 6 and 11 cause
around 90% of genital warts,
so using Gardasil 9 helps
protect girls and boys against
both cancer and genital warts
HIV & AIDS
• Human Immunodeficiency Virus causes
• Acquired Immunodeficiency Syndrome
• Follows 2-10 years after infection if untreated
• During this time there is gradual impairment of the immune system
• Death results not from HIV per se but from opportunistic infections
that take hold as the immune system can no longer control them
HIV Transmission
Why does HIV cause immunodeficiency?

• The target cell for HIV is the CD4+ T helper cell which
is infected and killed
• CD4+ T helper cells have an important role in the
immune response to viruses
• They help B cells produce anti-viral antibody
• They help CD8+ cytotoxic T cells lyse and destroy virus-
infected cells
HIV Life Cycle
Treatment
• Antiretroviral drugs (emtricitabine, tenofovir, darunavir, raltegravir).
• Valacyclovir for Herpesviruses
• Gancyclovir for CMV (cytomegalovirus)
• Trimethoprim sulfamethoxazole for Pneumocystis carinii
To assess effectiveness of treatment
• qPCR to assess the number of virus particles. This should fall with
treatment.

• CD4:CD8 lymphocyte ratio. The CD 4 count should improve with

treatment reducing the ratio. CD4 positive T helper cells are the cells
infected by the HIV virus.
Water and Environmental Microbiology
Dr Sarah Atchia
Microbial flora of aquatic environments
• Most environments contain large varieties of microbial species
• Most species are adapted for relatively nutrient poor environment
Factors Influencing Microbial Flora
• Temperature
• pH
• Nutrient content
• Salt content
• Extreme environments
Pollution
• Sewage contamination
• Litter
• Chemicals e.g. Fertilisers
• Point source of pollution
• Release of wastes into watercourses
• Nonpoint source of pollution
• Run off from fields etc
• Eutrophication (nutrient enrichment)
Waterborne Disease
• May cause up to 3.4 million deaths/year globally
• Pathogens may be transmitted via a variety of types of water
• Growth occurs in the intestines
• Microorganisms are shed in the faeces
• Infections are transmitted by the faecal-oral route
• Involves survival of acidic environment of the stomach
Viruses
• Enteroviruses e.g. poliovirus
• Hepatitis A
Bacteria
• Vibrio species
• Gram negative curved rods
• Vibrio cholerae
• Enterobacteriaceae species
• Enteric bacteria which are members of the intestinal microbiota of
humans and animals.
• Can be divided into coliforms and noncoliforms
• Found in water, soil and on vegetation
• Salmonella typhi
• Escherichia coli
Vibrio cholerae
• Gram-negative curved rod
causes cholera
• Endemic in India, Pakistan and
Central and South America
• Epidemics
• Pandemics
• 3-5 million affected and 100,000
deaths a year worldwide

Vibrio cholerae: Electronmicrograph x12,000

Cholera
• Ingestion of large inoculum (108 – 109 vibrios) causes disease
• Vibrios attach to small intestine
• Cholera produced by enterotoxin
• Results in ‘rice water’ diarrhoea and massive fluid loss
• Leads to severe dehydration
Cholera
• Treatment involves replacement of fluid and electrolytes
• Antibiotic therapy not normally recommended, but may increase
speed of bacterial elimination, e.g. Azithromycin, doxycycline,
Ciprofloxacin
• Vaccines for protection against some strains
• E.g. oral inactivated vaccine Dukoral
Salmonella typhi
• Gram-negative rod causes typhoid fever
• Up to 20 million cases and 150,000 deaths a year world wide
• Children, the elderly and travellers most at risk
• Most common in warmer months
• Ingestion of large inoculum (106 – 108 bacteria) causes disease
• Bacteria invade cells in small intestine
• Strains causing enteric fever pass through intestines and to other
organs
Typhoid Fever
• Enteric fever (typhoid fever)
• Bacteremic phase:
systemic illness (fever, headache, myalgias)
• Gastrointestinal phase:
diarrhoea, hepatosplenomegaly, rose spots
• Complications:
Intestinal haemorrhage, respiratory disease, encephalitis
Protozoa
• Cryptosporidium parvum
• Giardia lamblia
• Entamoeba histolytica
Cryptosporidium parvum
•Causative agent of cryptosporidiosis
•Cells attach to intestinal epithelium
•Life cycle includes production of oocysts
• Oocysts and cysts are highly resistant to
chlorine (Cryptosporidium up to x14
than Giardia)
• Protozoan parasites may infect wide
range of animal hosts

Cryptosporidium oocysts: acid-fast stain

Microbiological Analysis
• Water cannot be examined for presence of all pathogens
• Waterborne disease is largely due to faecal pollution
• Therefore specific indicator organisms are utilised as markers of risk
Indicator Organisms
• Criteria required:
• Suitable for analysis of all water types
• Indicator must reflect presence of enteric pathogens
• Indicator should survive longer than enteric pathogens
• Indicator should not grow in contaminated water giving inflated counts
• Testing procedure should be specific for the indicator
• Testing procedure should be easy to perform
• Indicator should be harmless
• Indicator level must directly reflect the degree of faecal pollution
Coliforms
• Facultatively anaerobic
• Gram-negative
• Non-sporing
• Rod-shaped
• Ferment lactose and produce gas within 48 h at 35 oC
Coliforms
• Mostly of intestinal origin
• Members of Enterobacteriaceae and make up 10% of intestinal flora
• Slow die-out rate in comparison to other intestinal pathogens
• Contain Escherichia coli, Klebsiella pneumoniae Citrobacter (intestinal
inhabitant)
• Enterobacter aerogenes (non-intestinal inhabitant)
Most probable number method
• Principle: Probability of viable cells being present in a diluted
sample follows Poisson distribution

• Data are analysed using MPN tables

73
Membrane Filtration Technique
• Sample passed through a membrane filter
• Filter traps bacteria
• Filter transferred to media e.g. EMB
• Total or faecal coliforms may be counted
• Conditions may be varied to be more or less selective
• ‘Resuscitation’ step may be included for stressed microorganisms
 Detection of Protozoa
• Lack of correlation between presence of Cryptosporidium oocysts and
indicator organisms
• Can only be removed by filtration and sedimentation
• Large volumes of water (1-100 L) must be analysed due to low
infective dose
• Concentration of samples
• Membrane filtration
• Flocculation
Types of Water
• Drinking water
• Public supplies
• Private supplies
• Water containers
• Natural mineral water
• Recreational waters
• Bathing waters
Drinking Water
• In the UK water for human consumption is covered by the Drinking
Water Inspectorate
• Testing is required for total coliforms, faecal coliforms and total colony
counts
• Samples must be negative for coliforms and E.coli
• Samples tested for colony counts at 22 oC and 37 oC
• Continuous monitoring of ‘at risk’ water treatment works for
Cryptosporidium oocysts
• Requirement: <10 cryptosporidial oocysts /100L
Incidence of Waterborne Disease
• Low incidence of waterborne disease in developed countries
• Incidence of typhoid and cholera declined
• Chlorination major factor
Incidence of Waterborne Disease
• High incidence of waterborne disease still present in developing
countries
• Cryptosporidiosis may still be a threat in developed countries
• Outbreak in Milwaukee 1993
• 400,000 People infected, 85 fatalities
• Traced to municipal water supply
• Heavy rain lead to water supply contamination
• Outbreak in Lancashire 2015
• 300,000 homes affected
Sexually Transmitted
Infections
STI’s
BC5070 Infection Science 2
Dr M P Botey-Salo
STIs can be caused by the 4 Microorganism
Groups
• Bacterial STIs
• Neisseria gonorrhoae - Gonorrhoea
• Chlamydia trachomatis - Chlamydia
• Treponema pallidum – Syphilis
• Viral STIs
• (Covered in previous lecture)
• HIV, HSV (herpes simplex), HPV, HBV
• Protozoal
• Trichomonas vaginalis
• Fungal –
• Candida albicans, Thrush (not STI)
Neisseria gonorrheae
• Also known as “gonococcus” (GC)
• Diagnosis:
• In females from high vaginal swab (HVS) or endocervical swab. Or from purulent exudates
• In males from urethral swab or purulent exudates
• Gram staining from swabs
• Grow on chocolate and Thayer Martin selective agar, 370C, 10% CO2
• Forming grey watery colonies
• Has complex growth requirements
• Gram negative diplococci
• Oxidase positive
• Glucose positive
• Use specific GC antisera to ID serologically
• Combined GC – Chlamydia PCR
 GC pathology
• N. gonorrhoeae belongs to the genus Neisseria, of which N. gonorrhoeae and Neisseria meningitidis (also known as the meningococcus) a leading cause
of bacterial meningitis

• In 2012, the WHO reported 78 million cases of gonorrhea occurring worldwide between people ages 15–49
Gonorrhoea and syphilis at record levels in 2022 - GOV.UK (www.gov.uk)

• WHO surveillance of clinical strains of N. gonorrhoeae has identified strains that are resistant to most available antibiotics.

• N. gonorrhoeae mainly colonizes the genital mucosa, but it can also colonize the ocular, nasopharyngeal, and anal mucosa

• Pathology largely results from damage that is caused by the activation of innate immune responses at the sites of colonization as N. gonorrhoeae does
not express potent exotoxins.

• Complications from untreated, ascending, female genital tract infections can include pelvic inflammatory disease, infertility, and ectopic pregnancy.

• Maternal transmission to children during birth can also lead to neonatal blindness.

• Untreated N. gonorrhoeae infection can also lead to disseminated gonococcal infection (DGI), potentially giving rise to infectious arthritis and endocarditis

• Quillin & Seifert (2018)

 Symptoms
• Initially it was thought that female genital infections are mostly asymptomatic and male
infections are mostly symptomatic. However, there are many studies that show
asymptomatic infections are common in both genders

• This supposition that female infections are mostly asymptomatic and males are
symptomatic is mainly based on the fact that overt symptoms in males are easier to
diagnose, due to the purulent exudate from the penis and resultant painful urination.

• Clinical manifestations in women are more likely to go unnoticed, as inflammation does

not occur in the same niche as urination and thus is less likely to be painful.

• Moreover, symptoms of gonorrheal infection in women are more likely to be

nonspecific, as the vaginal discharge that is caused by neutrophil influx may be
mistaken for bacterial vaginosis, yeast infection, hormonal variation in vaginal
secretions, or normal variability in secretions
Transmission
• N. gonorrhoeae cannot survive outside the host. Is an
obligate human colonizer. Transmission between hosts
relies on sexual contacts
• N. gonorrhoeae attaches to sperm and is easily
transmitted through the ejaculates, as they contain a
high number of bacteria.
• The efficiency of transmission from women to their
partners is maintained is less apparent.
Gonococcal conjunctivitis (gonococcal ophthalmia
neonatorum)

• If Neisseria or Chlamydia
present in birth canal,
Neonatal Prophylaxis
the child can get infected •Erythromycin (0.5%) ophthalmic
ointment, or
•Tetracycline (1%) ophthalmic ointment
Costumbrado et al 2022
GC on Thayer Martin media
• Chocolate agar in one side (left),
chocolate agar with antibiotics in
the other (right).
• Antibiotics prevent normal flora
from growing
• Gonorrhea growing on the right
side
Treatment
• For uncomplicated gonorrhoea = a single 500mg IM dose of
ceftriaxone
• If also possible chlamydial infection suspected = doxycycline for 7 days

• In case of allergies, gentamicin can be used

St. Cyr S, Barbee L, Workowski KA, et al. Update to CDC’s Treatment Guidelines for Gonococcal Infection, 2020.
MMWR Morb Mortal Wkly Rep 2020;69:1911–1916. DOI: http://dx.doi.org/10.15585/mmwr.mm6950a6external icon
Chlamydia
• Most common STI
• May affect up to 1 in 10 sexually
active men and women
• There are over 100,000 cases
per year in UK
• Chlamydia is caused by the
bacterium Chlamydia Chlamydia in brown
trachomatis
• Gram negative bacteria that only
replicates in a host cell

Ford, M. (2019) medical Microbiology 3rd ed. Oxford University Press

Chlamydia trachomatis
• Causes:
• NSU Non-specific urethritis
• NGU Non-gonococcal urethritis
• Intracellular bacterium (does not grow on media)
• Usually asymptomatic
• Can cause
• Salpingitis (inflammation of fallopian tubes) and infertility
• Eye infection: Trachoma = irreversible blindness
• LGV Lymphogranuloma venereum
Treponema pallidum
• Causative organism of Syphilis
• Does not grow on artificial
media
• Spirochaete bacterium
Syphilis Stages
• Syphilis has 3 characteristic stages: primary, secondary and tertiary
(latent) disease
• Primary: genital lesion on mucosal surface (called chancre) followed
by a reddish rash
• Secondary: In some patients, the primary lesion may have healed.
This stage begins 3-6 months post infection (rash over body, or soles
of feet and hands). Generally resolves in few weeks.
• Tertiary: spontaneous mucocutaneous relapse (~ 12 months later).
Most common clinical conditions are neurological and cardiovascular
Syphilitic signs
Rash

Ulceration:
Desquamation chancre
Diagnosis & Treatment
• Difficult to diagnose due to different stages where lesions and rash
can disappear
Treatment:
-15th century: mercury
Also bismuth (less toxic) was used
- From 1943: penicillin is 1st line of treatment
- In case of penicillin allergies: deoxycycline and erythromycin
Screening for STI’s
• Culture for GC, fastidious, organism dies off rapidly at
low temperatures, false negative
• Best to culture directly from patient to agar by
nurse/physician, in the clinic and incubated
• Chlamydia does not grow on artificial media
• Need cell or animal culture
• ELISA for antibodies, low at acute stage
• What is the answer to better testing?
NAAT’s
• Nucleic acid amplification techniques, PCR.
• High sensitivity and specificity
• Primers to unique DNA sequences of organisms, this
gives high specificity
• Very low numbers of organisms required, this gives high
sensitivity
• No living organisms required
• Combined CG chlamydia PCR available
• This can be automated, Faster results, 4 hours
Problems with NAAT’s
• Technically demanding
• Risk of contamination, false positives
• Separate preparation and testing areas
• Test area-High levels of amplified DNA
• No antimicrobial sensitivity testing
Urinary Tract infections
(UTIs)
Dr Sarah Atchia
Urinary System
Urinary System
• Urine produced in kidneys passes through ureter into bladder and is
emptied via urethra.
• Infection may be produced when microorganisms ascend the urethra
into the bladder and then into the kidney.
• UTIs normally caused by microorganisms from gastrointestinal tract or
(rarely) via the bloodstream.
Normal Flora of Urinary Tract
• Urinary tract is normally sterile
• Prevention of infection ascending into bladder or
kidneys
• Flushing mechanism of urine from bladder
• Valve prevents reflux of urine from bladder into ureter
• Characteristics of urine
• low pH,
• high osmolarity
• High urea concentration
• IgA
Lower Urinary Tract Infections
• 95% of UTI are acute cystitis due to spread of
organisms up the urethra
• Symptoms
• Dysuria (pain when passing urine)
• Passing urine more frequently
• Suprapubic pain (lower abdomen)
• Urethral syndrome describes patients with UTI
symptoms but no significant bacteriuria.
Upper urinary tract infections

• Pyelonephritis -urinary tract infection which has ascended to the

kidney
• Symptoms
• Flank pain and fever
• Nausea and vomiting
Urinary Samples
• For urinary tract infection (UTI)
• Also cystitis (inflammation of the bladder)
• Mid stream urine (MSU)
• First urine passed washes away organisms from distal
urethra.
• Clean catch urine
• Sampling carried out in children.
• Catheter stream urine (CSU)
• Suprapubic aspirate (SPA)
• Sampling carried out in neonates and young children
Urinary Samples
• Samples should be kept at 4 oC for a maximum of 24 hours
• Dip-slide technique
• Uses agar-coated slides
• Boric acid may be used as a preservative
• Rant-Shepherd method
• Uses a microtitre tray and microscope
Urinalysis
• Appearance
• Microscopy
• Dipstick
• Culture
• Bacterial enumeration
• Antibiotic sensitivity test
Appearance
• Normally clear, straw coloured fluid
• Cloudy urine- ? WBC’s and bacteria
• Bloodstained- Haematuria
• Lysed blood- haemoglobinuria
• Purulent, green pus filled urine
Urine dipstick
• Leukocytes (WBC) indicate infection
• Nitrites (bacterial products) indicate bacteria
• Haemoglobin indicates haematuria
• Protein indicates infection, nephropathy
• Glucose indicates diabetes mellitus
• Ketones indicate fat breakdown
• pH, acidity or alkalinity. Normally acidic
• Specific gravity (SG) how concentrated
Microscopy
• Examine for white blood cells, pyuria
• Examine for red blood cells, haematuria
• Presence of bacteria, bacteriuria
• Casts showing tubular stasis/ damage
• Crystals indicating kidney stones, Ca oxalate
• Epithelial cells = poorly taken sample
• Presence of yeasts
• Parasites e.g. Schistosoma haematobium
Culture
Calibrated loop technique
• Use 1ml loop of urine
• 1 colony = 1000 CFU/ml
• 100 colonies = 105 CFU/ml (108 cfu/l)
• Significant bacteriuria is taken as greater than 100,000
(105) CFU/ml
• Sometimes lower counts may be significant.
• Cut off of pure growth of 104 organisms per ml.
Culture
• Cysteine lactose electrolyte deficient (CLED) agar
• Differentiates LF’s (yellow) from NLF’s (blue)
• Supports growth of Streptococcus pyogenes
• Electrolyte deficiency Inhibits swarming of Proteus species
• Chromogenic agar available
• Different colours for different organisms
LF’s and NLF’s on CLED
Potential pathogens
• Escherichia coli
• Proteus mirabilis
• Klebsiella species
• Pseudomonas aeruginosa
• Enterococcus faecalis
• Staphylococcus species
• Other Enterobacteriaceae
• Mycobacterium tuberculosis
Escherichia coli
• Most common cause of UTI
• Lactose fermenter (yellow cols) on CLED
• Gram negative rods (Enterobacteriacae)
• Oxidase negative, urease negative
• Identify using API 20E
• Perform antibiotic sensitivity testing
Escherichia coli
• Women are x15 more likely to acquire UTIs than men due to shorter
urethras.
• Some E.coli infections may spread to the kidneys from the bladder via
ureters.
• Acute pyelonephritis may develop involving fever, bacteriuria and
nausea
• E.coli 0157:H7 can cause fatal UTIs e.g. haemolytic uremic syndrome.
What is biotechnology?
• Def 1: Using scientific methods with organisms to
produce new products or new forms of organisms
• Def 2: Any technique that uses living organisms or
substances from those organisms to make or modify a
product, to improve plants or animals, or to develop
micro-organisms for specific uses
Genetic engineering
• Manipulation of genes is called genetic engineering or recombinant
DNA technology
• Genetic engineering involves taking one or more genes from a
location in one organism and either
• Transferring them to another organism
• Putting them back into the original organism in different combinations
The Central Dogma of Molecular Biology

Cell

Transcription DNA

mRNA
Translation Ribosome

Polypeptide
(protein)
How do we go from DNA (4 nucleotides) to a protein (enzyme) containing 20
different amino acids?
• An extremely complex and amazing enzyme called a ribosome reads messenger RNA, produced
from the DNA, and converts it into amino-acid chains
• To pick the right amino acids, a ribosome takes the nucleotides in sets of three (codon) to encode
for the 20 amino acids
• DNA consists of 4 different bases (A, G, C, T)
• 3 bases in a codon
• 4³ = 64 different codon combinations possible with a triplet codon of three nucleotides;
• 20 possible amino acids vs 64 codon?
• several different codons can encode for the same amino acid
• Also, there is a stop codon that marks the end of a gene
• At the beginning of the chain there is a section of bases that is called a promoter. A gene,
therefore, consists of a promoter, a set of codons for the amino acids in a specific enzyme, and a
stop codon
• That is all that a gene is
The genetic code is universal

• Since all living organisms…

• use the same DNA
• use the same code book
• read their genes the same way

The start codon is the first codon of a messenger RNA (mRNA) transcript
translated by a ribosome.

The start codon always codes for methionine in eukaryotes and a modified
Met (fMet) in prokaryotes.

The most common start codon is AUG

WHO ARE THE MAJOR PLAYERS IN BIOTECHNOLOGY?

• Biotech involves the use of all living forms. However, MO

(microorganisms) play the major role because:
• Have relatively simple structures
• Ease of mass growth/cultivation
• Speed of growth
• Reproduce quickly so they are useful for studies involving transfer of genetic
information
• Rapid growth in media (cheap substrates /wastes)
• Diversity of metabolites/potential products
• Ability to readily undergo genetic manipulation
• Large numbers of microbes can be used in an experiment to obtain
statistically reliable results at a reasonable cost
 Plasmid is an extra chromosomal
DNA a nucleic acid that DNA (capable of replicating
contains the genetic independently of the
instructions chromosomal DNA)

Plasmid,
Bacteria-Genetic

smaller,
circular
pieces of
DNA.
material

The DNA segments that carry DNA is found in cytoplasm (no

this genetic information are nucleus)
called genes
Plasmid usually encodes expendable
In Eukaryotes DNA is stored in nucleus functions, e.g., antibiotic resistance
rDNA technology
Plasmids are used to insert new genes into bacteria

cut DNA
gene we
want
like what?
…insulin transformed
…HGH
…lactase cut plasmid DNA bacteria

ligase

insert “gene we want”

recombinant into plasmid...
plasmid “glue” together
Molecular Tools for rDNA Technology

• Restriction enzymes
• Restriction EndoNuclease
• DNA cutting enzyme

• DNA Ligase
• DNA Sealing enzyme
Restriction Enzymes
• The ability to cut DNA predictably is due to the use of
restriction enzymes
• They were first identified in and isolated from the
bacteria that use them as a natural defense
mechanism to cut up the invading DNA of
bacteriophages – viruses that infect bacteria
• Restriction endonucleases: the
nucleases that cleave DNA at particular sites
by the recognition of specific sequences 5’….GAATTC.….3’
….CTTAAG….
e.g. EcoRI
To name a restriction endonuclease:

• e.g.

EcoRI
the 1st such
Escherichia coli R1 enzyme found
Genus species strain
digestion of a DNA fragment with endonuclease EcoRI

(The largest fragment)

(The smallest
fragment)

 Consider a linear DNA

molecule with 6 copies of
GAATTC:
it will be cut into 7
fragments which could be
separated in the gel
electrophoresis by size
What is DNA Ligase?
• DNA Ligase actively participates in cellular DNA repair processes.

• DNA Ligase joins (Seals) the DNA fragment by forming a

phospho-di-ester bond
 Purpose of recombinant technology
Cell containing gene
Bacterium
of interest
1 Gene inserted into
plasmid
Ex: gene that encodes insulin

Bacterial Plasmid
chromosome
Gene of
Recombinant interest
DNA of
DNA (plasmid) 2 chromosome
2 Plasmid put into
bacterial cell

Recombinant
bacterium

Fig. 20-2a
Fig. 20-2b

Recombinant
bacterium

3 Host cell grown in culture

to form a clone of cells
containing the “cloned”
gene of interest

Gene of Protein expressed

Interest by gene of interest

Copies of gene Protein harvested

4 Basic research and

Basic various applications Basic
research research
on gene on protein

Gene for pest Gene used to alter Protein dissolves Human growth hor-
resistance inserted bacteria for cleaning blood clots in heart mone treats stunted
into plants up toxic waste attack therapy growth
What PCR does?
• PCR amplifies DNA
• Makes lots and lots of copies of a few copies of DNA
• Can copy different lengths of DNA, doesn’t have to copy the whole length of a DNA
molecule
• One gene
• Several genes
• Lots of genes
• Artificial process which imitates natural DNA replication
• Why we need PCR?
• to "amplify" - copy - small segments of DNA because
• significant amounts of a sample of DNA are necessary for molecular and genetic
analyses
• http://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf
• PCR animation

http://www.sumanasinc.com/webcontent/animat
ions/content/pcr.html
Some topics related to medical
biotechnology (molecular medicine)
I. Human Genome Project
 its influence on medical biotechnology
II. Detecting infection organisms
 Improved diagnosis of disease
III. Detecting Genetic Diseases
 Earlier detection of genetic predispositions to disease
IV. Pharmacogenomics
 "custom drugs"
V. Gene therapy
 and control systems for drugs
Meningitis and CSF
Analysis

BC5072
Presenter: Assoc Prof Sheelagh Heugh
2023-24
Lecture content
• Good Laboratory Practice
• CSF characteristics
• Microorganisms that cause meningitis:
• Neisseria meningitidis
• Haemophilus influenziae
• Streptococcus pneumoniae
• Escherichia coli
• Streptococcus agalactiae
• Listeria monocytogenes
• Cryptococcus neoformans
GLP (Good Laboratory Practice) in Microbiology
• Ensure well taken samples by clinical staff
• Correct transport and storage conditions
• Patient sample and request form details should
match
• Quality systems in place, staff training
• Standard operating procedures (SOP’s)
• Aseptic techniques used. Sterile equipment.
• Controls for all procedures
 What is Meningitis?

• Infection of the fluid in the spinal cord and the

fluid that surrounds the brain
• Viral, Bacterial, Parasitic, Fungal
• Etiology is important because of the seriousness
of the illness and the treatment needed
Types of meningitis
• Bacterial (aka septic meningitis)
• Can be deadly. Requires immediate medical attention
• Caused by Neisseria meningitidis, Group B Streptococcus, Strep pneumoniae, L. monocytogenes, E.coli

• Viral (aka aseptic meningitis)

• Serious but usually less severe than bacterial meningitis
• Caused by Enteroviruses, herpesvirus, mumps, measles and flu viruses

• Fungal
• Rare. Infection by inhalation of fungal spores
• Immunocompromised people most at risk
• Cryptococcus neoformans (lives in environment. All over the world)

• Protozoal
• Parasitic meningitis less common than viral or bacterial
• Plasmodium, trypanosomes
• Amoebic meningitis. Rare but devastating. Naegleria fowleri
Signs and symptoms of meningitis
About the CSF (khasawneh et al, 2018) (Bothwell et al, 2019)

• CSF is a clear, proteinaceous fluid that exists in the

surrounding spaces of mammalian central nervous
systems (CNS)
• In the average adult human, there is roughly 150 mL of
CSF circulating at any given moment.
• CSF protects the brain and spinal cord by cushioning
them from mechanical injury
• Transports nutrients and hormones
• Plays a role in protein clearance within CNS, but the
mechanism is unclear
• How it is secreted, its flow and reabsorption/drainage is
debated
About the CSF (khasawneh et al, 2018) (Bothwell et al, 2019)
• CSF forms at a rate of
about 0.3–0.4 mL/min.
• The original theory of CSF
production views 75% of all
CSF being produced by the
choroid plexus epithelium,
while the remaining quarter
being produced by other
CNS structures
• In balanced physiological
conditions, the rate of CSF
formation must be equal to
the rate of absorption.
Absorption by arachnoid villi
CSF sample

• Under normal conditions CSF should be sterile

• CSF sample is taken in cases of suspected meningitis and other conditions
• Sample obtained by lumbar puncture (LP) (= spinal tap)
• Obtained in an aseptic manner
• As meningitis is a medical emergency
• Samples should be processed urgently
• Preliminary results reported when ready
• Any growth should be considered significant
• Gram staining of CSF possible to start treatment

Universal bottles UV sterilised

Tests done on CSF
• Macroscopic appearance
• Should be clear if no infection
• Microscopy for white and red blood cells
• Cell Differential on white blood cells
• Gram stain for bacteria
• Culture on appropriate culture media
• Antibiotic sensitivity test on any organisms
• India ink preparation for Cryptococcus neoformans
• CSF glucose and protein estimation
• Glucose should be ~ as blood. Protein is normally low
• In case of bacterial infection, Glucose low, prot high
• Latex antigen testing (Commercial kit)
Macroscopic appearance
• Should be clear and colourless
• Turbidity may indicate presence of white blood cells
(WBC) and red blood cells (RBC)
• If sample is bloodstained may be due to a poor
sample or subarachnoid haemorrhage
• Xanthochromic or yellow? bilirubin
• Spiders web clot suggests TB

Seehusen et al 2003
Potential pathogens of blood stream
• Neisseria meningitidis (Meningococcus)
• Haemophilus influenzae
• Streptococcus pneumoniae (Pneumococcus)
• Escherichia coli (Neonatal)
• Streptococcus agalactiae BHS group B
• Listeria monocytogenes (Soft cheeses)
• Cryptococcus neoformans (Fungus)
Neisseria meningitidis (aka
meningococcus)
• Grows on chocolate agar (5% CO2)
• Gram negative diplococci
• Oxidase positive
• Ferments maltose and glucose (MG)
• API NH (Neisseria, Haemophilus)
available
• 13 antigenic strains identified but
groups A, B, C, Y, W135 cause most
cases of disease. Serotyping
identification
• Notifiable disease
Pathogenicity of Neisseria meningitidis
• Virulence factors
• Capsulated (resistant to lytic enzymes)
• IgA protease (breaks down IgA antibodies). Prevents
opsonization (complement inactivation)
• Lipopolysaccharide (LPS) in the cell wall
• Activates coagulation leading to DIC (Disseminated Intravascular
Coagulation)
• Coagulation disseminates around the body using up coagulation
factors and platelets (Consumption coagulopathy)
• Low Platelets (thrombocytopenia) and low clotting factors lead to
bleeding into the tissues = Non blanching petechial rash
• Ischaemia, gangrene (amputation)
• Multiple organ failure
Meningococcal vaccine

Against groups A, C, Y and W-135 Against type B

Novartis
Licence granted by European
Commission on 22nd Jan 2013
Joint committee on vaccination and
immunisation to decide if
incorporated into the current
vaccine schedule
1800 cases a year
Haemophilus influenzae
• Growth on chocolate agar only (5%
CO2)
• Gram negative rods
• X and V factor dependent
• X factor = haematin, V factor = NAD
(they require heme and NAD for
growth)
• API NH (Neisseria, Haemophilus)
• Satellitism around Staph aureus on
blood agar
• Causes meningitis (not flu)
Neisseria looks similar, grey colonies: Gram staining next
Pathogenicity
• Capsulated
• IgA protease
• Excessive inflammatory response resulting in more severe
neurological sequalae
• May spread from ear infection (otitis media)
• May occur after head trauma
Haemophilus influenzae type B vaccine (Hib
Vaccine)
• Given routinely in UK since 1992
• Hib can cause meningitis, sepsis,
pneumonia, epiglottitis
• In UK Babies have 3 separate
doses of Hib vaccine – at 8,
12 and 16 weeks of age – as part
of the combined 6-in-1 vaccine
• A booster dose is also offered
when a child is 1 year old
Streptococcus pneumoniae
• Growth on Blood agar 24-
48 hours
• Alpha haemolysis (green,
partial haemolysis)
• Gram positive diplococci
• Catalase negative (Staph is
catalase positive)
• Optochin sensitive
• Bile soluble
• API Streptococcus
• Capsulated
Vaccines
Prevenar:
Pneumovax 23
against 23 pneumococcal variants For infants below 5 y.o.
For over 5 years old 90% cover of variants
Beta Lactams: target peptidoglycan layer
• Penicillins
• Cephalosporins
• Based on the beta lactam ring

Peptidoglycan
Cell wall peptidoglycan layer
Beta-lactam antibiotics
• Penicillins
• Ampicillin, Amoxicillin, Piperacillin, Flucloxacillin (Oxacillin, cloxacillin)
• Bind to penicillin binding proteins (PBP’s)
• PBP’s are peptidoglycan synthesising enzymes. When antibiotic binds into PBPs it halts cell
wall synthesis and bacteria become susceptible to osmotic changes

• Cephalosporins
• Cephalosporins are grouped together based on the type of bacteria that they’re most effective
against. These groups are referred to as generations. There are five generations of
cephalosporins
Penicillin Resistance (Bacteria defends
itself)
• Bacteria produce Beta lactamases
• Enzymes which denature the antibiotic
• Hydrolysis of the beta lactam ring
• Altered penicillin binding proteins (PBP2)
• Antibiotics unable to bind, become ineffective
• Extended spectrum beta lactamases (ESBL)
• Mutations of beta lactamases
• More broad spectrum with a larger range of activity
affecting more antibiotics
• Resistance to a wider range of penicillins
• Amp C ampicillin resistance gene
Beta Lactamase Inhibitors (we fight back)
• Clavulanic acid (clavulanate)
• Co-amoxiclav – combination of amoxicillin and clavulanic acid. Also
known as Augmentin
• Tazobactam – Combination of piperacillin and tazobactam. Also
known as Tazocin
• Sulbactam – Combination of amoxicillin and sulbactam. Also known as
Unasyn
Mechanisms of Bacterial Resistance
• Transfer of resistance genes
(conjugation)
• Altered target proteins
• Mutations alter protein
structure
• Altered permeability (efflux
pumps)
• Actively remove
antibiotics from the cell
• Inactivating enzymes
• Denature antibiotic
molecules
Infection Control
• There is a Cross infection team implementing good
practice. Composed of:
• Cross infection nurses monitoring infection rates
• Clinical microbiologist directing judicious use of antibiotics
• Biomedical scientist identifying organisms and reporting
antibiotic sensitivity patterns
• They supervise Hospital guidelines and practices
• Sterile equipment and aseptic techniques
• Antiseptics and disinfectants
 Microbiological Laboratory
Management and Quality Control
Dr Mohamed Ahmed
2023
What is Management?
• Organising workload of samples and processes to maximise efficiency
and minimise errors.
• Economy and value for money
• Quality management
• NEQAS: National External Quality Assessment Service. A UK-based global
enterprise that provides external quality-assessment services, enabling medical and
veterinary laboratories to fulfil quality goals and facilitate optimal patient care.
Why is it important to have a quality management
system in place?

• To ensure the laboratory and staff are complying with current regulations to ensure the results being
produced in the laboratory are accurate, precise and delivered in a timely manner. To minimise
errors and to spot them when they happen.
Many Aspects of Management
• Staff: training, qualifications, CPD (continuous
professional development), rotas, roles
• Equipment: analysers, centrifuges, supplies (reagents,
etc)
• Departments: Micro, blood, histology
• Procedures: SOP’s (Standard Operational Procedures),
Contingency planning
• Estates & Buildings: access, flow, utilities, safety,
environment (AC)
• Legal requirements: UKAS, MHRA, HSE, NEQAS
• Budget management: all this has to be paid for!
Staffing and Working hours
• Sufficient numbers to cover workload
• Shift system or on call roster. Working time directive
• Suitable qualifications and experience
• Induction process for familiarisation
• Training and competence assessment
• Files maintained by training officer
• Familiarisation with SOP’s
• CPD, Specialist portfolio, Higher specialist Diploma
Equipment
• Autoanalysers, incubators, safety cabinets…
• Suitable environment, Temp, humidity,
• Power supply, UPS (uninterrupted power supply). Back up
generators
• Periodic maintenance, daily, weekly, monthly
• Maintenance contract with manufacturers
• Calibration according to manufacturers
• Run and monitor controls daily. Look for trends
• Participate in NEQAS.
Reagents and consumables
• Good stock control system
• All supplies in date, long shelf life
• Need to ensure you don’t run out. Must always be
able to provide the service.
• Get balance right, don’t overstock.
• Correct storage environment, fridges, freezers
• Temperature monitoring, chart recorders
Procedures and Processes
• Standard operating procedures (SOP’s)
• Based on current best practice, methods etc
• Ensure everyone is doing the same thing
• Strict document control procedures. Why?
• Complete, signed documentation history.
• Any changes, all previous versions removed
• Don’t want some using old procedure
• Everyone doing the same thing
Estates
• Ensure building is fit for purpose
• Utilities, correct gas and electricity standards
• Fire alarm systems in place and equipment
• Restricted access to authorised personnel
• Correct health and safety signage, Superlab!
• All cupboards numbered and contents list
• Correct storage for flammable, corrosive etc
• All gases carefully controlled
Budgeting and Finance
• Limited budget. Need to get value for money.
• Staff salaries are a major cost
• Relationships with pathology companies
• Analyser with reagent rental
• MALDI-TOF £100K for analyser. Large up front cost but save money on
staff and reagents.
Internal Quality control
• Use of samples with known value
• Treated and run as routine clinical samples
• Used to ensure correct results are produced
• Run controls as per laboratory guidelines
• Some once a day, others with every test
• Control every new batch of reagents
• New methods need to be validated against the
current method – run in parallel
Action on control failure
• Repeat the controls ensuring in date, not expired
• Repeat using fresh controls
• Calibrate the analyser and repeat controls
• Change the reagent and repeat the controls
• Assess where the problem lies – root cause analysis
• Take corrective action – change reagent, fresh control,
review storage, procedures,
• Preventive action e.g. temperature monitoring,
• Check lot number and expiry date
NEQAS
• National External Quality Assessment Scheme
• Simulated samples sent to all registered laboratories
• Samples tested like routine specimens
• Lab results sent back to NEQAS
• Data collated and report sent out
• Can see individual performance
• Comparison with other laboratories
Evasion mechanisms. How
components of the immune
system are subverted
Evasion mechanisms. How Complement is
subverted.
• Examples:
• Salmonella produces an O-antigen capsule (lipopolysaccharide O). This capsule protects the
bacteria against MAC (membrane attack complex)
• Salmonella and E.coli can shed C5-C9. C5-C9 are the complement proteins that lead to the formation of the pore of
the MAC. C5-C9 bind to the capsule, but if capsule is shed, the pore of MAC cannot be formed

• E.coli and Neisseria bind Factor H & I to dissociate C3bB (so the alternative complement pathway
cannot proceed)

• Antigenic mimicry: Schistosoma binds host DAF (Decay accelerating factor) to

prevent deposition of C3b. (Hides own antigens from our immune system)
• DAF = is a protein in our cells that removes C3b so our own cells DO NOT get attacked by our
immune system.
• Individuals with genetic mutation that affects DAF will have a Complement system attacking
own cells.
• Bacteria do not have DAF.
Protein A Staphylococcus aureus
• Staph aureus has a capsule and
produces protein A.
• Protein A binds to Fc part of
antibody so
• The antibody is wrong way
around
• No response is elicited
Sabotage of phagocytosis via the capsule
• A big capsule will protect bacteria as will
“hide” the antigens on their membrane
• Bacterial capsules mainly found in Gram-
negative bacteria such as:
• E. coli
• Klebsiella pneumoniae
• Haemophilus influenzae
• Pseudomonas aeruginosa
• And in some Gram-positive bacteria:
• Bacillus megaterium
• Streptococcus pyogenes (hyaluronic acid
capsule)
• Strep pneumoniae
• Strep agalactiae