Learn — Bacteriology

15 notes · 45 questions on the topic

Rod-shaped bacteria

Rod-shaped bacteria, or bacilli, are organisms whose length exceeds their breadth, as opposed to the spherical cocci and the spiral forms. Shape is the first thing a Gram film reports and, taken with the Gram reaction and the arrangement of the cells, it narrows the possibilities enough for treatment and isolation to begin hours before the culture is ready.

The Gram-positive rods that matter are Bacillus, which is aerobic and spore-forming (anthracis and cereus), Clostridium, which is anaerobic and spore-forming (tetani, perfringens, botulinum and difficile), Corynebacterium diphtheriae, which is club-shaped and arranged in Chinese-letter or V and L patterns with metachromatic granules on Albert stain, Listeria monocytogenes, and the acid-fast mycobacteria.
The Gram-negative rods are the larger group: the enteric organisms Escherichia coli, Klebsiella, Salmonella, Shigella, Proteus, Enterobacter and Serratia, the non-fermenters Pseudomonas aeruginosa and Acinetobacter, the curved Vibrio cholerae, the spiral Helicobacter pylori and Campylobacter, and the strict anaerobe Bacteroides fragilis.
Coccobacilli sit between the two shapes and are a favourite examination category: Haemophilus influenzae, Brucella, Bordetella pertussis, Acinetobacter and Pasteurella.
Arrangement is itself a clue: the bamboo-stick chains of Bacillus anthracis, the Chinese-letter arrangement of Corynebacterium, the box-car shape of Clostridium perfringens, the drumstick of Clostridium tetani with its terminal spore, and the comma of Vibrio cholerae.
Only two genera of medical importance form spores and both are Gram-positive rods, Bacillus and Clostridium, which is why killing spores rather than merely disinfecting is what defines sterilisation.
The Gram-negative rods carry lipopolysaccharide endotoxin in their outer membrane and are behind most hospital-acquired urinary, bloodstream and ventilator-associated infections, and behind the carbapenem-resistant isolates that require contact precautions.

Gram-positive organisms

A Gram-positive organism is one that retains the crystal violet-iodine complex after decolourisation and therefore appears purple, because its cell wall is a thick multi-layered peptidoglycan mesh with teichoic acid and no outer membrane. That single structural fact explains their behaviour: they are susceptible to penicillin, lysozyme and vancomycin, they act mainly through secreted exotoxins rather than endotoxin, and they survive drying on ward surfaces and linen.

The Gram stain in order: crystal violet for about a minute, Gram iodine as the mordant for about a minute, decolourisation with acetone or alcohol for a few seconds, and safranin or dilute carbol fuchsin as the counterstain; decolourisation is the critical step, and over-decolourising makes a Gram-positive organism read as Gram-negative.
Gram-positive cocci: Staphylococcus in grape-like clusters and catalase positive, with Staphylococcus aureus additionally coagulase positive; Streptococcus in chains or pairs and catalase negative; and Enterococcus, which grows in bile and in 6.5 percent salt.
Gram-positive rods: the spore formers Bacillus and Clostridium and the non-sporing Corynebacterium diphtheriae, Listeria monocytogenes and Nocardia; the mycobacteria have a Gram-positive type of wall but stain poorly and need the acid-fast stain instead.
Gram-positive organisms characteristically produce exotoxins, proteins with a specific action that can be converted into toxoid vaccines, such as tetanus, diphtheria and botulinum toxins, the toxic shock syndrome toxin and the staphylococcal enterotoxins; they carry no lipopolysaccharide endotoxin.
Antibiotic choice follows the wall: vancomycin cannot cross a Gram-negative outer membrane and therefore works only against Gram-positive organisms, and it is the drug for methicillin-resistant Staphylococcus aureus, while penicillin remains the drug for Streptococcus pyogenes, which has never become resistant to it.
Old cultures, prior antibiotics and heavy heat fixation make Gram-positive cells lose their colour, so a Gram film is made from a young culture and read alongside a known control on the same slide.

Endotoxin vs exotoxin

Exotoxins are proteins actively secreted by living bacteria, mostly Gram-positive, each with a specific target and a specific clinical syndrome, while endotoxin is the lipopolysaccharide of the Gram-negative outer membrane, released when the cell is lysed, whose lipid A component sets off a non-specific inflammatory cascade. The distinction decides treatment: an exotoxin can be neutralised with antitoxin and prevented with a toxoid vaccine, whereas endotoxin can be neither, and killing the organism can briefly make endotoxaemia worse.

Exotoxin: a heat-labile protein destroyed by moderate heat, highly antigenic, extremely potent in microgram or smaller amounts, with a specific pharmacological action, secreted by living cells and produced by Gram-positive organisms and by some Gram-negative ones.
Endotoxin: lipopolysaccharide in the Gram-negative outer membrane with lipid A as the toxic moiety, heat stable enough to survive autoclaving, weakly antigenic, far less potent, non-specific in its effects, and released only when the bacterial cell breaks up.
Only exotoxins can be converted by formaldehyde into toxoids, which is why there are vaccines against tetanus and diphtheria and none against endotoxin, and only exotoxins can be neutralised by ready-made antitoxin such as human tetanus immunoglobulin or diphtheria antitoxin.
The named exotoxins and their mechanisms: tetanospasmin blocks the release of the inhibitory transmitters glycine and GABA and causes spastic paralysis, botulinum toxin blocks acetylcholine release and causes descending flaccid paralysis, diphtheria toxin inhibits protein synthesis by ADP-ribosylating elongation factor 2, cholera toxin permanently activates adenylate cyclase so the gut pours out water and electrolyte, and the toxic shock syndrome toxin and the streptococcal pyrogenic exotoxins act as superantigens.
Endotoxin acts by making macrophages release tumour necrosis factor and interleukin-1 and by activating complement and the coagulation cascade, producing the fever, hypotension, disseminated intravascular coagulation and multi-organ failure of Gram-negative septic shock.
Endotoxin is a pyrogen that survives the autoclave, which is why intravenous fluids, giving sets and glassware must be pyrogen-free and are depyrogenated by prolonged dry heat rather than merely sterilised, and why a contaminated fluid can cause rigors even after every organism in it is dead.
The Limulus amoebocyte lysate test, which gels in the presence of minute amounts of lipopolysaccharide, is the standard laboratory assay for endotoxin in fluids and on devices.
Two exceptions the examination likes: the staphylococcal enterotoxin of food poisoning and the heat-stable enterotoxin of Escherichia coli are exotoxins that resist heat, which is why reheating the food does not make it safe.

Bacterial growth curve

The bacterial growth curve is the plot of the logarithm of the viable count against time when an organism is inoculated into a fixed volume of liquid medium, and it runs in four phases: lag, log or exponential, stationary and decline. It is not laboratory decoration, because cell-wall antibiotics work only on dividing organisms, spores and most toxins appear in the stationary phase, and the generation time explains why one culture is read next morning and another after six weeks.

Lag phase: the count does not rise but the cells enlarge and synthesise the enzymes needed for the new medium, and its length depends on the size and age of the inoculum, so a young actively growing inoculum shortens it.
Log or exponential phase: the cells divide at a constant generation time, the culture is most uniform and metabolically active, the organisms are at their most susceptible to antibiotics and physical agents, and this is the phase from which Gram films and biochemical tests are made.
Stationary phase: division is balanced by death as nutrients run out and toxic products accumulate, the viable count plateaus, and this is when spores, exotoxins, antibiotics and other secondary metabolites are formed and the cells become more resistant.
Decline or death phase: the viable count falls exponentially with autolysis, a few organisms survive much longer, and bizarre involution forms appear in old cultures.
Generation time is the examinable number: about 20 minutes for Escherichia coli, which is why an overnight plate can be read in the morning; 15 to 20 hours for Mycobacterium tuberculosis, which is why Lowenstein-Jensen culture takes 6 to 8 weeks; and around 30 hours for Treponema pallidum.
Total count, which includes dead cells and is made by counting chamber or turbidity, must be distinguished from viable count in colony-forming units by pour plate or the Miles and Misra method, because only the viable count says whether treatment or disinfection has worked.

Neisseria gonorrhoeae

Neisseria gonorrhoeae is a fastidious, oxidase-positive Gram-negative diplococcus with adjacent flattened sides, seen inside neutrophils in a smear of urethral pus, that infects the columnar and transitional epithelium of the genital tract, rectum, pharynx and conjunctiva. It matters because it is often silent in women until it has caused pelvic inflammatory disease and infertility, because it blinds newborns within days of birth, and because it has become resistant to almost every antibiotic ever used against it.

Transmission is sexual and vertical during vaginal delivery, the incubation period is short at about 2 to 7 days, and while men present with dysuria and a profuse purulent urethral discharge, a large proportion of infected women have no symptoms at all.
A Gram-stained smear of urethral pus showing Gram-negative diplococci inside polymorphs is effectively diagnostic in a symptomatic man, but it is unreliable on cervical, rectal and pharyngeal specimens, where a nucleic acid amplification test is the test of choice.
Culture needs enriched selective medium such as chocolate agar, Thayer-Martin or modified New York City medium containing vancomycin, colistin and nystatin to suppress the other flora, incubated at 35 to 37 degrees Celsius in 5 to 10 percent carbon dioxide in a candle jar; the organism is extremely delicate, so the swab is plated at the bedside or sent in transport medium and is never refrigerated.
Biochemically Neisseria gonorrhoeae ferments glucose only while Neisseria meningitidis ferments both glucose and maltose, which is the classical distinguishing pair.
The complications are pelvic inflammatory disease with tubal infertility and ectopic pregnancy, epididymo-orchitis and urethral stricture in men, disseminated gonococcal infection with septic arthritis and pustular skin lesions, and ophthalmia neonatorum.
Treatment is a single intramuscular dose of ceftriaxone, given together with doxycycline or azithromycin unless chlamydial co-infection has been excluded; India's syndromic approach uses the colour-coded NACO kits, with the grey kit for urethral and cervical discharge containing single oral doses of cefixime and azithromycin, and the quinolones and penicillins are no longer used because resistance is widespread.
Every case is managed as a package: treat the index patient, treat all sexual partners, offer testing for syphilis, HIV and hepatitis B with consent and counselling, provide condoms with instruction, and keep the whole consultation confidential.

Salmonella typhi (typhoid)

Typhoid or enteric fever is a systemic infection by Salmonella enterica serotype Typhi, a motile non-lactose-fermenting Gram-negative bacillus that is swallowed in contaminated food or water, invades through the Peyer patches of the ileum and multiplies inside macrophages before entering the blood. It is not a diarrhoeal disease but a bacteraemia, which is why the diagnosis is made from blood in the first week and why the danger in the third week is perforation of an ulcerated Peyer patch.

Transmission is faecal-oral through contaminated water, food, milk and ice, and the reservoir is exclusively human: cases, convalescent carriers and chronic carriers who harbour the organism in the gall bladder and shed it for more than a year.
The incubation period is usually 7 to 14 days and the classical untreated course runs in weekly steps: step-ladder fever with relative bradycardia in the first week, rose spots, splenomegaly and toxaemia in the second, and complications in the third.
The diagnostic window decides the specimen: blood culture is the test of the first week and is positive in about 80 percent of cases then, stool and urine cultures become positive in the second and third weeks, and bone marrow culture is the most sensitive of all and stays positive even after antibiotics have been started.
The media are examinable: blood is inoculated into bile broth or brain-heart infusion, stool is enriched in selenite F broth, and the organism grows as pale non-lactose-fermenting colonies on MacConkey agar, as colonies with black centres on xylose lysine deoxycholate or deoxycholate citrate agar, and as jet-black colonies with a metallic sheen on Wilson-Blair bismuth sulphite agar.
The Widal test measures agglutinins to the O and H antigens, becomes positive only from the end of the first week, requires a rising titre in paired sera taken 7 to 10 days apart, and is unreliable in an endemic population and after vaccination, so a single high titre is not a diagnosis and it never replaces culture.
The third-week complications are the emergency: intestinal perforation and haemorrhage from the ulcerated Peyer patches, and less often myocarditis, encephalopathy and cholecystitis, so a patient whose abdomen suddenly becomes rigid after days of fever is perforating until proved otherwise.
Treatment is a third-generation cephalosporin such as ceftriaxone for severe illness and azithromycin for uncomplicated disease, because chloramphenicol, ampicillin, co-trimoxazole and the fluoroquinolones have lost ground to resistance, and extensively drug-resistant strains reported from the subcontinent leave little beyond azithromycin and the carbapenems.
Prevention is safe water, sanitation, food hygiene, hand washing and the exclusion of carriers from food handling, together with vaccination: the typhoid conjugate vaccine is given as a single dose from 6 months of age and the older Vi polysaccharide vaccine from 2 years.

Borrelia

Borrelia are large, loosely coiled, arthropod-borne spirochaetes that, unlike the other medically important spirochaetes, are thick enough to be seen on an ordinary Giemsa- or Leishman-stained blood film. Two syndromes matter: relapsing fever from Borrelia recurrentis and the tick-borne species, in which the organism changes its surface antigens to produce repeated waves of fever, and Lyme disease from the Borrelia burgdorferi complex, which begins as an expanding skin lesion and ends months later in the joints, heart and nervous system.

Relapsing fever comes in two forms: epidemic louse-borne relapsing fever from Borrelia recurrentis, spread by the body louse in crowded, cold and unhygienic conditions and transmitted when a crushed louse is rubbed into broken skin, and endemic tick-borne relapsing fever from species such as Borrelia duttonii carried by soft ticks.
The relapses are caused by antigenic variation: each febrile wave ends when antibody clears the current surface type, and the next wave is a new antigenic variant, producing repeated crises of high fever separated by afebrile intervals.
Relapsing fever is one of the few bacterial infections diagnosed on a peripheral blood smear, taken at the height of the fever and stained by Giemsa or Wright or examined by dark ground; treatment is doxycycline, with erythromycin or penicillin in children and pregnancy, and delousing of the patient, the clothing and the contacts is part of the treatment.
Lyme disease is transmitted by Ixodes ticks and requires prolonged attachment, commonly quoted as around 36 to 48 hours, so prompt correct tick removal prevents it; it is not endemic across most of India, and the history is one of forest exposure or travel.
The stages of Lyme disease are erythema migrans, an expanding red ring with central clearing at the bite site within days to a month, then early disseminated disease with multiple skin lesions, facial palsy, lymphocytic meningitis and atrioventricular block, and finally late disease with an intermittent large-joint arthritis, typically of the knee.
Lyme serology is a two-tier test, an enzyme immunoassay followed by a confirmatory immunoblot, and it is negative in the first weeks, so a classical erythema migrans in an exposed person is treated on the clinical picture alone with doxycycline, or amoxicillin or cefuroxime in children under 8 years and in pregnancy.

Treponema pallidum (syphilis)

Syphilis is a chronic sexually transmitted infection caused by Treponema pallidum, a slender regularly coiled spirochaete that is too thin to be seen on a Gram film and cannot be grown on any artificial medium, so it is diagnosed by dark-ground microscopy and by serology. It matters most in pregnancy, where it crosses the placenta and causes stillbirth, prematurity and congenital syphilis, and where one correctly given injection of benzathine penicillin prevents all of it.

Transmission is by sexual contact, by transplacental passage to the fetus and rarely by transfusion; the organism dies quickly outside the body, so it does not spread by fomites.
Primary syphilis appears after an incubation period of about 9 to 90 days as a single painless indurated clean-based ulcer, the hard chancre, with painless rubbery regional lymphadenopathy, and it heals on its own, which is why patients do not attend.
Secondary syphilis follows weeks to months later with a symmetrical non-itchy maculopapular rash that characteristically involves the palms and soles, generalised lymphadenopathy, mucous patches, snail-track ulcers and the highly infectious moist condylomata lata; this is the most infectious stage.
After a latent stage, a proportion of untreated patients develop tertiary syphilis years later with gummas, cardiovascular syphilis with aortitis and aortic regurgitation, and neurosyphilis with tabes dorsalis, general paresis and the small irregular Argyll Robertson pupil that accommodates but does not react to light.
Serology comes in two families answering different questions: the non-treponemal tests VDRL and RPR are cheap, become positive a week or two after the chancre, and are used for screening and, because the titre falls with cure, for monitoring treatment; the treponemal tests TPHA, FTA-ABS and the rapid treponemal tests confirm the diagnosis but stay positive for life and cannot show whether treatment worked.
Two traps in the non-treponemal test: biological false positives in pregnancy, malaria, leprosy, tuberculosis, viral infections, systemic lupus erythematosus and the antiphospholipid syndrome, and the prozone phenomenon, in which a very high antibody level in secondary syphilis gives a falsely negative undiluted test that turns positive when the serum is diluted.
Treatment is benzathine penicillin G by deep intramuscular injection: a single dose of 2.4 million units for primary, secondary and early latent disease, and three weekly doses for late latent disease or disease of unknown duration, with intravenous aqueous crystalline penicillin for neurosyphilis. Penicillin is the only agent proven to treat the fetus, so a pregnant woman with a genuine penicillin allergy is desensitised and still given penicillin rather than an alternative.
Every pregnant woman is screened at her first antenatal visit, in India with the dual HIV-syphilis rapid test under the national programme, and screening is repeated later in pregnancy where risk is high, because congenital syphilis is preventable and its elimination is a programme target.

Vibrio cholerae

Vibrio cholerae is a comma-shaped, actively motile Gram-negative bacillus that is not invasive at all: it stays in the lumen of the small intestine and secretes cholera toxin, which locks adenylate cyclase on so that the enterocyte pumps out chloride and water. The result is the painless odourless rice-water stool that can strip an adult of many litres in a few hours, so cholera kills by dehydration and is cured by fluid rather than by antibiotics.

The epidemic serogroups are O1, with the classical and El Tor biotypes and the Ogawa, Inaba and Hikojima serotypes, and O139; El Tor is the strain of the current pandemic and is hardier, producing more carriers and more mild cases, which is what lets it spread so far.
Transmission is faecal-oral through contaminated water and food, and the incubation period is short, from a few hours to 5 days and commonly 1 to 2 days, which is why an outbreak appears to explode.
The stool is painless, effortless and voluminous, colourless with flecks of mucus like rice water, with no blood, no pus and no tenesmus, and there is little or no fever; the patient becomes hypovolaemic with sunken eyes, washerwoman's hands, muscle cramps, a feeble pulse and anuria while remaining fully conscious.
In the laboratory a hanging drop shows the darting or shooting-star motility, the enrichment and transport fluid is alkaline peptone water at about pH 8.6 because the organism thrives in alkali and is killed by acid, and the selective plate is thiosulphate citrate bile-salt sucrose agar, on which Vibrio cholerae ferments sucrose to give large yellow colonies; Cary-Blair is the transport medium when there is a delay.
Treatment is measured and aggressive rehydration: Ringer lactate is the fluid of choice for severe dehydration and low-osmolarity oral rehydration solution for the rest, and the WHO Plan C volume is 100 mL/kg, given to a patient of 1 year and over as 30 mL/kg in the first 30 minutes and the remaining 70 mL/kg over the next two and a half hours, with oral rehydration solution started as soon as the patient can drink.
An antibiotic is an adjunct only in moderate and severe cases: a single dose of doxycycline in adults, with azithromycin preferred in children and in pregnancy, shortens the diarrhoea, reduces the stool volume and shortens shedding, but it never substitutes for fluid.
Cholera is an outbreak disease and a reportable event under the International Health Regulations, so the first suspected case is notified the same day, a treatment centre with cholera cots and measured intake-output charting is set up, stools and vomitus are disinfected, the water supply is chlorinated, and the two-dose killed oral cholera vaccine is used alongside water, sanitation and hygiene measures.
Zinc for 14 days is given to children as in any acute diarrhoea and breastfeeding continues throughout, and food is restarted as soon as the patient can eat, because starving a patient with diarrhoea prolongs it.

Food poisoning — bacterial

Bacterial food poisoning is illness that follows eating food containing either a preformed bacterial toxin or a large number of live organisms, and it is distinguished from other diarrhoeal illness by the fact that several people who shared a meal fall ill together. The incubation period is the diagnostic instrument: a very short one means a toxin that was already in the food, while a longer one means the organism had to multiply or invade in the gut.

One to six hours with vomiting predominant and little or no fever means preformed toxin: Staphylococcus aureus enterotoxin from food handled by someone with a boil or septic finger, such as custards, cream, milk products and sandwiches, and the emetic form of Bacillus cereus from reheated fried rice.
Eight to sixteen hours with watery diarrhoea and cramps and little vomiting means Clostridium perfringens type A from meat and gravies cooked in bulk and kept warm, or the diarrhoeal form of Bacillus cereus.
Twelve hours to three days or more, with fever and sometimes blood, means the invasive organisms: Salmonella from eggs, poultry and milk, Shigella with its very small infecting dose and person-to-person spread, Campylobacter jejuni from undercooked poultry with Guillain-Barre syndrome as a late complication, and Vibrio parahaemolyticus from seafood.
Enterohaemorrhagic Escherichia coli O157:H7 causes bloody diarrhoea after about 3 to 4 days and may be followed by the haemolytic uraemic syndrome of haemolysis, thrombocytopenia and acute kidney injury, especially in children, so antibiotics and antimotility drugs are avoided in suspected cases because they increase that risk.
Botulism is the neurological food poisoning: Clostridium botulinum toxin from improperly processed canned, bottled or fermented food, 12 to 36 hours after the meal, causing a descending symmetrical flaccid paralysis that begins with blurred vision, diplopia, ptosis, dysarthria and dysphagia in an afebrile fully conscious patient, while infant botulism follows honey given in the first year of life.
The staphylococcal enterotoxin and the Bacillus cereus emetic toxin are heat stable, so reheating the food does not make it safe; this is the classic explanation for why the warmed-up hostel custard or picnic rice still causes illness.
Most food poisoning is treated with fluid and electrolyte replacement alone, antibiotics being reserved for defined situations such as invasive salmonellosis in the very young, the elderly or the immunocompromised and for shigellosis, and antimotility drugs are avoided in bloody or febrile diarrhoea.
The outbreak duties are examined as often as the organisms: refrigerate the suspected food and its containers for the laboratory, collect stool and vomitus from cases before antibiotics, make a line list with the time of the meal and the time of onset, draw the epidemic curve, examine and exclude food handlers with skin sepsis or diarrhoea, and notify the medical officer and the surveillance unit.

Streptococcus

Streptococci are catalase-negative Gram-positive cocci that divide in one plane and so appear in pairs and chains, and they are classified first by their haemolysis on blood agar (beta is complete, alpha is partial with green discolouration, gamma is none) and then by the Lancefield group of their cell wall carbohydrate. They matter more than any other genus in an Indian ward because Streptococcus pyogenes causes the sore throat and the skin sepsis that lead on to acute rheumatic fever, rheumatic heart disease and acute glomerulonephritis.

Group A Streptococcus pyogenes is beta-haemolytic, bacitracin sensitive and PYR positive, and causes pharyngitis, scarlet fever, impetigo, erysipelas, cellulitis, necrotising fasciitis and streptococcal toxic shock syndrome; its M protein is the main virulence factor and the basis of type-specific immunity.
The two non-suppurative sequelae are examined every year: acute rheumatic fever follows throat infection only, after a latent period of about 2 to 3 weeks, and is diagnosed on the revised Jones criteria, while acute post-streptococcal glomerulonephritis follows either throat or skin infection, after about 1 to 2 weeks from the throat and 3 to 6 weeks from the skin, and presents with smoky urine, puffiness and hypertension.
Group B Streptococcus agalactiae is CAMP-test and hippurate positive and colonises the vagina; it is the leading cause of early-onset neonatal sepsis, meningitis and pneumonia, which is why a colonised woman is given intravenous penicillin or ampicillin in labour, ideally at least 4 hours before delivery.
Among the alpha-haemolytic organisms, Streptococcus pneumoniae is optochin sensitive and bile soluble, lancet-shaped, capsulated and arranged in pairs, and causes pneumonia, meningitis and otitis media, while the viridans streptococci are optochin resistant and bile insoluble, live in the mouth and cause subacute infective endocarditis on damaged valves after dental work.
Enterococcus, formerly group D, grows on bile-esculin and in 6.5 percent sodium chloride, causes urinary, biliary and endocardial infection, and is intrinsically resistant to many drugs; vancomycin-resistant Enterococcus is a contact-precautions organism in any hospital.
Streptococcus pyogenes has never developed resistance to penicillin, so penicillin V or amoxicillin for a full 10 days, or a single intramuscular dose of benzathine penicillin, is the treatment for streptococcal pharyngitis, and the reason for treating a self-limiting sore throat at all is to prevent rheumatic fever.
Once acute rheumatic fever has occurred, secondary prophylaxis with intramuscular benzathine penicillin G every 3 weeks continues for years, and keeping the recall register and giving the injection on time is a nursing responsibility, because a missed injection is how rheumatic heart disease progresses.

Clostridium perfringens (gas gangrene)

Clostridium perfringens is a large box-car shaped, non-motile, anaerobic Gram-positive bacillus with a subterminal spore that lives in soil and in the human and animal gut. In a deep wound with dead muscle and a poor blood supply it multiplies and releases alpha toxin, a lecithinase that destroys cell membranes, producing clostridial myonecrosis or gas gangrene, which spreads through muscle within hours and is a surgical emergency rather than an antibiotic one.

The setting is a wound with devitalised tissue and low oxygen: road traffic and crush injuries contaminated with soil, farm injuries, compound fractures, deep burns and septic abortion, and in hospital an amputation stump or an operation on the bowel.
The alpha toxin is a lecithinase, also called phospholipase C, demonstrated by the Nagler reaction on egg yolk agar in which the opacity is abolished on the half of the plate spread with specific antitoxin; the organism also gives a double zone of haemolysis on blood agar, stormy clot fermentation in litmus milk, and very rapid growth in Robertson cooked meat medium.
The clinical picture is severe pain out of proportion to the appearance of the wound, tense oedema, a thin foul brownish discharge, crepitus from gas in the tissues, skin that turns bronze and then dusky and blistered, and a tachycardic toxic patient who is often strikingly alert; gas is visible on a plain X-ray.
The Gram film is quick and characteristic: large Gram-positive box-car bacilli with very few pus cells, because the toxin destroys the neutrophils, and spores are usually not seen in tissue.
Treatment is urgent wide surgical debridement or amputation, which is what saves the patient, supported by high-dose intravenous penicillin G with clindamycin, clindamycin being added because it suppresses toxin production, along with fluid resuscitation and correction of anaemia; hyperbaric oxygen is an adjunct where available and must never delay surgery.
The same species causes food poisoning through its enterotoxin from bulk-cooked meat kept warm, with diarrhoea and cramps 8 to 16 hours after the meal, and it also causes necrotising enteritis, so the wound disease and the food poisoning are different toxins of one organism.
Prevention is surgical rather than pharmacological: prompt thorough wound toilet with removal of all dead tissue and foreign material, leaving contaminated wounds open rather than closing them primarily, and giving tetanus prophylaxis at the same time, because the same wounds admit Clostridium tetani.

Clostridium tetani

Clostridium tetani is a slender motile obligate anaerobic Gram-positive bacillus whose terminal round spore gives it the classical drumstick appearance and whose spores survive for years in soil, dust and animal faeces. It does not invade the body: it stays in the wound and releases tetanospasmin, which travels up the nerve and blocks the release of the inhibitory transmitters glycine and GABA, so the muscles lose their brake and go into the sustained spasm of tetanus.

The portal of entry is any wound with dead tissue and low oxygen, such as a puncture wound from a nail or thorn, a burn, an unclean umbilical stump, an unsafe abortion, a chronic ear discharge or an injection with a contaminated needle, and in a proportion of cases no wound is ever found.
The incubation period is usually 3 to 21 days and commonly about 8 days, and the prognosis worsens the shorter it is; the period of onset, the interval between the first symptom and the first generalised spasm, is the better guide, and under 48 hours is a bad sign.
The clinical picture is trismus or lockjaw first, then risus sardonicus from facial spasm, neck stiffness, board-like abdominal rigidity, opisthotonus and painful generalised spasms triggered by light, noise and touch in a patient who remains fully conscious, with autonomic instability giving labile blood pressure, tachycardia and sweating.
Neonatal tetanus follows contamination of the umbilical stump, presents typically between the third and tenth day of life with a baby who stops sucking and cries continuously before the spasms begin, and is prevented by maternal immunisation and the three cleans: clean hands, a clean delivery surface and a clean cord cut and stump.
The diagnosis is clinical, because the organism is grown in only a minority of cases; it swarms on blood agar and grows in Robertson cooked meat medium, and no laboratory test should ever delay treatment.
Treatment has four arms: neutralise the unbound toxin with human tetanus immunoglobulin, or equine antitoxin where that is all there is; remove the source by wound debridement; kill the organism with metronidazole, which is preferred to penicillin because penicillin is itself a GABA antagonist; and control the spasms with benzodiazepines, adding magnesium sulphate, neuromuscular blockade and ventilation in severe cases.
Nursing is the treatment in tetanus: a quiet dimly lit single room, minimal clustered handling with care given after sedation, suction and intubation equipment at the bedside, airway protection, nasogastric or intravenous nutrition for a high calorie requirement, pressure-area and eye care, and continuous monitoring for the autonomic storms.
Prevention is immunisation, and having tetanus does not immunise, so the patient must be given the full toxoid course during convalescence; India uses Td in place of plain tetanus toxoid in the Universal Immunisation Programme at 10 and 16 years and in pregnancy, and wound management combines a toxoid booster with human tetanus immunoglobulin for a dirty wound in anyone incompletely immunised or of unknown status.

Escherichia coli

Escherichia coli is a Gram-negative, non-sporing, motile rod of the family Enterobacteriaceae that lives harmlessly in the large intestine of every healthy person and is the standard indicator organism for faecal contamination of water. The same species becomes a pathogen when it carries virulence genes: it is the leading cause of urinary tract infection, a major cause of neonatal septicaemia and meningitis, and the agent of several distinct diarrhoeal syndromes, and hospital strains now commonly carry extended-spectrum beta-lactamase or carbapenemase resistance.

It is a Gram-negative bacillus, non-acid-fast and non-sporing, motile by peritrichous flagella, and many pathogenic strains are capsulated; it is typed by its O (somatic), H (flagellar) and K (capsular) antigens, which is how the serotype O157:H7 gets its name.
On MacConkey agar it ferments lactose and gives flat pink colonies, on blood agar some strains are haemolytic, and on eosin methylene blue agar it gives the classical green metallic sheen; biochemically it is indole positive, methyl red positive, Voges-Proskauer negative and citrate negative.
Uropathogenic strains cause most community-acquired urinary tract infection; a clean-catch midstream specimen growing 100,000 or more colony-forming units per millilitre of a single organism is significant bacteriuria by the Kass count, and the specimen must reach the laboratory within about 2 hours or be refrigerated at 4 degrees Celsius, because a sample left on the counter grows contaminants and reads as infection.
The diarrhoeagenic pathotypes are examined as a set: enterotoxigenic (heat-labile and heat-stable toxins, travellers and childhood watery diarrhoea), enteropathogenic (infantile diarrhoea with the attaching and effacing lesion), enteroinvasive (dysentery resembling Shigella), enteroaggregative (persistent diarrhoea) and enterohaemorrhagic or Shiga-toxin producing, of which O157:H7 is the classic strain.
Enterohaemorrhagic E. coli is looked for on sorbitol MacConkey agar, on which O157:H7 does not ferment sorbitol and gives pale colourless colonies, with Shiga toxin detection or toxin gene amplification to confirm; the incubation period is usually 3 to 4 days, the infecting dose is very small, and the source is undercooked minced beef, unpasteurised milk, contaminated water or salad, with easy person-to-person spread.
Haemolytic uraemic syndrome follows about 5 to 10 days after the diarrhoea starts, chiefly in young children and the elderly, with the triad of microangiopathic haemolytic anaemia, thrombocytopenia and acute kidney injury; antibiotics and anti-motility drugs increase that risk and are withheld in suspected Shiga-toxin infection.
Neonatal meningitis and septicaemia are caused mainly by K1 capsulated strains, and E. coli is also a leading cause of hospital-acquired urinary tract infection, intra-abdominal sepsis and catheter-associated bloodstream infection.
Infection control is standard precautions for most patients, with contact precautions and dedicated equipment for an incontinent or diapered patient with diarrhoea and for any extended-spectrum beta-lactamase or carbapenem-resistant isolate; hand hygiene with soap and running water is used when hands are soiled, and culture plates and other laboratory waste are autoclaved at 121 degrees Celsius for a 15-minute holding time before they go into the yellow bag under the Biomedical Waste Management Rules 2016.

Mycoplasma

Mycoplasmas are the smallest free-living bacteria; they have no cell wall at all, their limiting membrane contains sterols, and they are therefore pleomorphic, invisible on a Gram stain and completely resistant to every cell-wall-active antibiotic such as the penicillins, cephalosporins and vancomycin. Mycoplasma pneumoniae is the important human pathogen of the group: it causes atypical or walking pneumonia in school-age children and young adults living close together, and it is diagnosed on the clinical picture with a nucleic acid test or serology rather than by culture.

They lack a cell wall, so they cannot be seen with a Gram stain and are demonstrated with Giemsa staining or dark-ground illumination; being plastic and filterable, they pass through filters that hold back ordinary bacteria.
The culture medium of choice is PPLO (pleuropneumonia-like organism) medium enriched with serum and yeast extract; growth is very slow, taking one to three weeks, so culture is a reference procedure and never guides ward treatment; typical mycoplasma colonies have a fried-egg appearance under low power, while Mycoplasma pneumoniae tends to form granular mulberry-like colonies.
The diagnostic test of choice is a nucleic acid amplification test on a throat swab or nasopharyngeal aspirate, which is positive early in the illness; serology needs specific IgM, which appears about a week after symptoms begin, or a fourfold rise between paired sera taken 2 to 3 weeks apart, and cold agglutinins rise in about half of cases but are not specific.
The incubation period is long, about 2 to 3 weeks, so cases in a hostel or barracks appear in waves over weeks rather than all at once; transmission is by respiratory droplets and close prolonged contact in households, hostels, boarding schools and military units, and the cough can persist for a month or more.
The clinical picture is fever, headache, malaise and a persistent dry cough with strikingly few chest signs, and a chest radiograph that looks far worse than the patient; bullous myringitis is the classical association, and extrapulmonary features include haemolysis from cold agglutinins, the rash and mucositis syndrome, Guillain-Barre syndrome, encephalitis and arthralgia.
Treatment is a macrolide such as azithromycin, which is preferred in children, or doxycycline or a respiratory fluoroquinolone in adolescents and adults; amoxicillin and ceftriaxone are useless because the organism has no cell wall for them to act on, and failure to improve on a beta-lactam is itself a clue.
Infection control is droplet precautions with a surgical mask for anyone working within a metre of the patient, along with cough etiquette, hand hygiene and cohorting in an institutional outbreak; having no cell wall, mycoplasmas are fragile outside the body and are destroyed by drying, by 70 percent alcohol and by routine autoclaving at 121 degrees Celsius with a 15-minute holding time.
Mycoplasma hominis and Ureaplasma urealyticum are the genital species, associated with non-gonococcal urethritis, pelvic inflammatory disease, chorioamnionitis and post-partum fever.

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