Bacterial analysis master set

Microbes as forensic indicators in cases of suspicious death

1) Was death due to ongoing disease or a fatal infection resulting from a medical procedure?

> someone dies because of long term condition affecting them their whole life or infected with adverse pathogen

> infection in blood or fatal food poisoning? (usually pathogenic E. coli)

2) Diagnosis of fatal food poisoning.

3) Was death due to drug overdose or infection acquired through intravenous injection (e.g. anthrax contaminated heroin)?

4) Was death due to natural causes or a malicious act?

> use of bacteria in bioterrorism, very easy to grow in lab and some very pathogenic

Why are microbes so important

4lbs of bacteria in your gut (micro biome), that are responsible for regulating behaviour, cancer susceptibility and disease susceptibility. Help with digestion. Also super bugs such as MRSA (methyl resistant staph aureus) are very dangerous especially to immunocompromised individuals.

Essential for life on earth for it to exist but some are very pathogenic and cause ill health.

Microbes as forensic indicators

In order to use microbes as forensic indicators one must appreciate and understand:

1) What is the normal abundance and diversity of microbes on the body during life?

2) How does abundance and diversity of microbes on the body change after death?

What is the normal abundance and diversity of microbes on the body during life?

On going debate past ten years. Scientists used to believe number of bacterial cells compared to human cells outnumbers 10:1 (10x more bacterial cells). Quite impressive as we're made up of billions of cells.

After more research 5 years ago, it's 3:1 (3x more bacterial cells) on and in body

Now it's more 1:1 ratio

Ratio changes depending on the organ/part of human. Lower small intestine 10^11 vs large intestine 10^14 vs stomach 10^7 bacteria number. So there's more bacteria in parts of body than other. How will we use bacteria to determine how long someone's been dead if we're covered in bacteria.

Human microbiome project

Swabbed lots of human volunteers in every area possible (ears, toe web space, back etc.) and used molecular biology and compared to. To find all the different types of bacteria found on human body in different areas. Many phyla were found including actinobacteria, proteobacteria, Cyanobacteria, bacteroidetes etc.

Conclusions - first study that showed there's very specific bacteria found on different areas of your body. Further research now shows we each individually differ with the bacteria found on our bodies.

How do we identify bacteria

Microbiology or molecular biology techniques but only 1% of bacteria can be cultured on a nutrient agar plate.

Sequence 16S ribosomal RNA gene (16S rRNA) can be used to identify bacteria.

This gene is found in all prokaryotes (i.e. bacteria and archaea) but is distinct from 18S rRNA, found in eukaryotes.

Therefore can analyse bacterial gene separate from that of plants, fungi or animals.

Also, the 16S rRNA gene is only about 1.5 kilobases long and this makes it particularly easy and cheap to sequence.

Profile all 16SrRNA of bacteria found on dead body instead of 1%

Why can't standard microbiology techniques be used in forensics

Swab dead body take petrified dish half filled nutrient agar, wipe swab over then put in incubator 37°C overnight and have lots of bacteria growing everywhere. Only going to culture 1%, the other 99% of diverse bacteria found on dead body won't grow on nutrient agar. They have very specific growing environments (iron, calcium, other carbon source needed). Molecular approach does however take longer and more expensive.

How do the bacteria change when a human dies

• Forensic microbiology a.k.a. post mortem microbiology or microbiology of death

•Thanatomicrobiome (microorganisms found in the body and fluids after death)

> Do some increase, decrease, disappear?

•Can the thanatomicrobiome be used to determine time of death?

Can we use the Thanatomicrobiome to understand how long someone's been dead for

• Difficult to use as it's a new science

• Limited access to human cadavers

• Lots of research carried out on an animal model as easier than human being e.g. rats, pigs. Need ethical approval

• Many variables which affect microbiome which will grow on dead body - need to know about medical history of deceased, environmental conditions affect microbiome etc.

> human body is broken down over time on different soils with different microbes and chemistries which all affect the bacteria growing

• Correct interpretation of results

• CONTAMINATION!

> you're outside and bacteria are everywhere so how do you know the bacteria you have is largely from that dead body. Contamination also possible in lab.

The five main stages of decomposition

Five main stages of decomposition: fresh, bloat, active and advanced decay and skeletonization (Payne, 1965):

•Fresh: failure of immune system leads to proliferation of intestinal microbiome. Microbes migrate to other organs.

•Oxygen decreases and microbial population and diversity decreases.

•Bloat: tissue autolysis and nutrient release. Domination of anaerobic bacteria. Production and accumulation of gases due to fermentation.

•Active decay: internal pressure of gases causes the rupture of the skin. Aerobic conditions, domination of aerobic bacteria.

•Advanced decay: Slow depletion of nutrients, decrease in microbial activity.

•Skeletonization: microbial profile of dry remains resembles soil microbiome.

HOW THE THANATOMICROBIOME CHANGES DURING DECOMPOSITION

How does your microbiome change during these stages?

1. Bacteria may all be in intestine and stomach. Will start migrating to different organs. Small intestine -> large intestine/stomach/lungs

2. Anaerobic bacteria will begin proliferating

3. During active decay, gases produced by microbes will rupture skin so aerobic conditions start again. Have fluctuating dynamics of conditions through stages

4. No nutrients so whole bacterial population will die off then especially when get to skeletonization

If these populations are changing through these 5 stages what's the best time to look at that

Study example looking at thanatomicrobiome using pigs

Used pigs in fresh water environment as couldn't get a hold of human bodies. One of first studies to look at thanatomicrobiome and see what bacteria were found, quantify them etc. lots of proteobacteria found (highly present in water and soil) huge drop at day 14 and 21 then body of pig completely taken over by other bacteria including tenericutes which take the lead in prevalence.

Study example looking at thanatomicrobiome using human bodies

30 human bodies or volunteers 18-90 years old. Looked at thanatomicrobiome changes in soil. Took bodies and put on soil in research facility and put cages over to protect from birds etc. swabbed bodies over time to see bacteria present.

Proteobacteria, acidobacteria and bacteroidetes proliferate up to date 5/6 then get more proteobacteria and firmicutes and outcompete the proteobacteria. Profiling the firmicutes over time could give reliable estimate of how long body has been dead. Therefore this group of bacteria could be one to concentrate on.

Done in US in 2/3 areas. But many differate climates and conditions around world so must do experiment elsewhere to see if results hold up. E.g. found in cold country then bacterial populations different

Study example looking at skeletalization stage of human bones

Could the bones be swabbed and profile bacteria found to estimate how long dead for? Used molecular biology to profile bacteria. A = partially skeletonised B = skeletonised C = dry remains D = soil. Firmicutes decreased as time goes on compared to how they increased in other study using flesh of bodies.

Bigger study going into more detail on bacteria found on human remains

Bigger study taking lots of human remains taking remain, swabbing and profiling bacteria the grouped bacteria into different clades on phylogenetic tree. Found a core set of microbes so went into more detail than firmicutes. Went into genera of bacteria. Phylogenetic tree showed core group of bacteria existing on thanatomicrobiome. Did in different climates, with people suffering with different health conditions. With people who died at different ages. No matter these differences still got this core micro biome. Should this be a blueprint or bacteria used when come across dead body, more research needs to be done but this a step in right direction.

MICROBES IN SALIVA AS FORENSIC INDICATORS

•Human bites nasty because saliva contains bacteria capable of causing a serious wound infection.

•These bacteria may be useful in identifying the person who inflicted the bite.

•Streptococci naturally present in mouths, is extremely diverse and their genomic profile appears to be characteristic for an individual.

•Volunteers bite themselves after which samples were taken over varying time periods.

•Live bacteria recovered from bite site for > 24 hours afterwards provided was relatively undisturbed, as well as from fabrics.

•Genomic profiles of the bacteria provided a unique identifier of the person responsible.

MICROBES AS FORENSIC INDICATORS- PROBLEMS

•Logistic problems: If you don't refrigerate a body within 24 hours this may affect thanatomicrobiome, as there is no immune system therefore bacteria from intestinal walls migrate into circulatory and lymphatic system (migration phenomena). Then have a mass of loads of bacteria if decomposes.

•Some organs are more sterile than the rest e.g. brain, liver, spleen or heart. Presence of bacteria could be due to migration. Must decide which organ best to look at.

• Problem: What if you find E. coli, Staphylococcus aureus, Candida albicans or Cryptococcus neoformans on dead body. Is this infection or contamination from skin? How do we understand what's a contaminant or not?

• An acceptable level of contamination is needed.

More issues when using microbes as forensic indicators

• Individual characteristics e.g. obesity. Must know individual medical records.

•Bacterial infection? Decay more rapid if person suffered from septicaemia or other bacterial infections. Did someone die from bacterial infection r other cause.

•Death due to open wound? More aerobic bacterial species as microbes enters through wound which may contaminate whole analysis. Excessive blood loss because the lack of fluid in the blood vessels makes it harder for bacteria to penetrate the body.

•What if someone is suffering from disturbed nutrient management e.g. anaemia or poisoned, struggled to digest food? The body will decompose slower due to unfavourable conditions.

The effect of soil on thanotomicrobiome

Microbial density varies enormously with the soil type but figures are generally in the region of 2x109 per gram in the top metre and 108 per gram at a depth of 1-8 metres.

Nature of the soil affects the rate of decay :

1) directly through chemical actions and

2) indirectly through its effect on the abundance and activity of soil organisms.

> Heavy clay soils are poorly aerated = low oxygen levels = reduces microbial activity = reduced rate of decay.

> Very acid soil reduces microbial activity but the low pH dissolves soft tissues and bone.

> High soil calcium content reduces chemical dissolution of bones but will not prevent microbial decomposition.

The effect of the environment on the thanatomicrobiome

During decay pH becomes more acidic owing to release of acids during autolysis and products of bacterial fermentation.

Body becomes anaerobic because all oxygen used up by bacteria and circulation system ceased to operate.

Consequently, majority of bacteria found in a dead body are anaerobic and spore forming, such as Clostridium perfringens.

Too hot/ too dry = microbial decay delayed and cannot be used to determine time since death.

Frozen = decomposition tends to occur from the outside to centre upon thawing.

MICROBES AND DRUG RESIDUES 1: DEGRADATION

•Microbes often metabolise drugs and degrade drug residues.

•Speed and extent of breakdown depends upon drug and environmental circumstances.

Example:

Nitrobenzodiazepines (e.g. Nitrazepam) degraded to amino compounds by Streptococcus and therefore difficult to detect if death was due to an overdose.

> suspected overdose but after drug analysis found drug content to be low, bacteria can be very good at breaking down chemical compounds including drugs

Example:

alcohol levels can rise following death as a consequence of microbial metabolism. Enhanced by high blood sugar levels at time of death.

> Microbes may cause an increase in drug concentrations. May ferment alcohol so levels are higher than what they are, feeding off sugars and creating lots of ethanol, end up with false alcohol poisoning cause of death

Case study of increased intoxication due to bacteria fermenting glucose to ethanol

14 month year old baby had high levels of alcohol which made prosecutors question whether the child was a victim of abuse having access to drink alcohol or parents gave. Child had huge glucose infusion prior to death. A bacteria was found and fermented glucose which produced ethanol.

Other residues that microbes can produce: GHB

- Gamma-hydroxybutyric acid (GHB) is naturally present at a very low level, thought to increase in body when die.

•This level increases enormously when GHB used as recreational or therapeutic drug - or for criminal purposes.

•GHB = class B now as used for malicious purposes to normally spike drinks

•low doses = 'upper' by clubbers = 'liquid ecstasy',

•high doses = confusion/ coma = spike drinks in 'date rape'.

•After GHB is ingested, there is an initial increase in concentration, then levels decline within a few hours

•After death [GHB] increases but not as high as levels immediately after taking the chemical as a drug.

•Therefore of forensic interest to know the extent to which levels of GHB found in a body might be ascribed to natural causes as opposed to misuse.

•Pseudomonas aeruginosa unique ability to produce GHB but not enough to account for all of the natural increase so other bacteria and/or physiological processes may contribute to the rise.

Gonorrhoea

Caused by bacterium Neisseria gonorrhoea and almost always sexually transmitted. Very prevalent. People have studied for long time and created very good database to determine what strain someone has.

Forensic need

a)Identify presence: culture/ DNA typing

b)Link/ absence of link between two individuals to establish whether sexual contact

Tracing the source of gonorrhoea infections

Doctors need genetic markers to study epidemiology of N. gonorrhoeae.

These could also prove useful in a forensic context.

However, unless strain involved was rare, it would be difficult to establish strong proof of association between two adults.

In absence of medical records it would also be difficult to establish the direction of transmission. This is because N. gonorrhoeae is a common infection among sexually promiscuous adults (or those who have a sexually promiscuous partner).

Therefore, if two adults share same locally circulating strain of N. gonorrhoea it is only weak circumstantial evidence of transmission from one to the other.

However, different in cases of gonorrhoea in children below the age of sexual consent, since N. gonorrhoeae is transmitted almost exclusively through sexual intercourse.

Identifying source of gonorrhoea

Identifying source of gonorrhoea

Genetic markers (porB and tbpBb):

a)Exhibits variation sufficient to exclude unlinked infected people.

b)Variation is easily identified

c) Does not arise too rapidly or too slowly to facilitate linkage between infected people.

Exclude non-infected people. Any cases of gonorrhoea in abuse cases can be compared to any match in the database.

Gonorrhoea and epidemiology

Allows to look at epidemiology of gonorrhoea. 2012-2017, saw more cases of gonorrhoea. Different strains of gonorrhoea can be found across UK. Gonorrhoea is evolving resistance. Old antibiotics are no longer prescribed and new ones are needed. Phylogenetic tree shows how closes related different strains of gonorrhoea are across world.

Case study of sexual abuse and gonorrhoea

Child with gonorrhoea sampled from Bahia, strain cultured and DMA sequenced to look at genetic markers. Police also swabbed suspects underwear. Found two strains that were identical and this was concrete evidence child was abused by suspect.

Anthrax (Bacillus anthracis)

Humans + herbivorous mammals

Naturally occurs in wild animals

People can accidentally inhale spores and die as result.

Can survive years in soil.

Found around world. In life cycle once eaten all nutrients of thing reproducing on it produces spores which are extremely resistant and indestructible, produces unlimited amount

Clinical manifestations

Ways it can infect you:

Cutaneously (through skin): most common though now rare

Gastrointestinal: through eating infected meat

Inhalation: very rare naturally, most likely via bioterror

Pathogenicity of anthrax

Pathogenicity depends on toxin. Inside has a plasmid with very pathogenic toxins which causes cell death very quickly.

Genes coding for toxin +

full genomic sequence

known for several strain

= aids diagnosis

Ability to engineers for > pathogenicity + > resistance to antibiotics = good bio-warfare potential

Deliberate spread of anthrax, weaponisation of anthrax. Cases

Case 1: Aum Shrinikyo cult, Japan

• Released aerosol containing anthrax

spores in city in Japan

• No deaths as stolen from lab and used strain developed for preparing anthrax vaccine = harmless

• Started cult of couple hundred thousand people. Grew up anthrax and made into aerosol and let it spread from building top in Tokyo.

Case 2: America anthrax letters.

• Anthrax spores enclosed in letters, breathed in

• 5 people died, several more very ill

• Only case to date + no repeat

The Anthrax Letters: Handwriting, envelope + postal evidence

Addressed by hand.

Contained handwritten notes indicating the nature of their contents, threats to America and Israel and ended with 'Allah is great'.

Crudely written + spelling mistakes.

Intention = cast suspicions onto a Muslim

extremist organisation.

Looked into more deeply, found sent from New Jersey. Swabbed post boxes looking for anthrax, found one post box positive for it.

How was the anthrax put into the letters?

Difficult to put anthrax in letter from liquid suspension by drying out to spore stage and rub onto letter without breathing in. Assumed it must be a scientist using a sterile hood.

Analysis of the anthrax

Used molecular analysis and found it to be an Ames strain that was isolated decades ago in sick Texas cow. It was subsequently used for vaccine development. Then must've come from medical research laboratory?

Traced back to the US medical research institute

Was the anthrax weaponised

Was it mixed with an additive such as silica to put in aerosol to allow it to be spread? If weaponised then points to an Islamic move. If not then points to a lone wolf move. Still unknown if weaponised or not.

How old were the anthrax spores

Look at carbon 14 signatures, their signature just remains the same. Found produced in last 2 years so have they just been sat in the research facility in freezer?

Where were the anthrax spores produced

Water was from the north eastern states and

Theories on who was behind the anthrax attacks

Someone qualified with access to facilities?

Less qualified person with access to standard microbiology laboratory?

Bruce Irvins was suspected but he killed himself due to blame pointing at him. He was a highly trained insider with access to specialist equipment and facilities

Summary of anthrax attack

1)The anthrax belonged to the Ames strain

2) Don't whether it was weaponized.

3)Was produced shortly before the letters were sent.

4)Did not originate from an old stockpile.

5)Was most probably grown in the USA.

What is microbial forensics?

The use of microorganisms as evidence in criminal investigations.

What is the human microbiome?

The collection of microorganisms living in the human body.

What is the thanatomicrobiome?

The microbial community that develops after death.

What happens to bacteria after death?

Gut bacteria spread throughout the body when the immune system stops functioning.

Which organs can be colonised by bacteria after death?

Brain, liver, spleen and heart.

What is bacterial translocation?

The movement of bacteria from the gut to other organs after death.

What is the microbial clock?

Predictable bacterial succession patterns used to estimate time since death.

What can bacterial analysis reveal in forensic investigations?

Time since death, infections, disease and possible contamination.

How many microorganisms (e.g. bacteria) would you expect to find in a spoonful of soil?

10 billion

Which technique offers the highest resolution and is the most frequently used approach for bacterial and fungal analysis?

Next generation sequencing (NGS)

Which gene region is targeted for forensic sequencing of fungal and eukaryotic communities? 

18S rRNA

How do the symptoms of Anthrax poisoning typically manifest when used as an agent of biological warfare?

Inhalation

Which factors affect an individual’s microbiome?

Age, Diet, Health, Personal Habits, Geography

What is the necrobiome? 

community of species associated with decomposing remains

During which decomposition stage might you expect to find sulphate reducing bacteria around a corpse?

Bloat

Bacteria affect the metabolic rate of different substances in the body after death. When is the most reliable time to assess blood alcohol levels during a post-mortem?

3 days after death