ENTEROBACTERIACEAE

             The predominant aerobic bacterial flora of the large intestine of human beings and animals is composed of non-sporing, non-acid fast, Gram negative bacilli. Members of this group are included in a complex family, Enterobacteriaceae. Some are capsulated, some not, some are motile and some non-motile. They are oxidase negative and catalase positive and reduce nitrates to nitrites, aerobes and facultative anaerobes, grow readily on ordinary media, MacConkey agar and ferment glucose in peptone water with the production of either acid or acid and gas. The property of lactose fermentation is of great importance for the isolation and identification of enterobacteria from clinical specimens.

1.  Escherichia

            Common species E. coli, which is a parasite, living only in human or animal intestine. In faeces, it remains viable only for some days. Detection of E. coli in drinking water is taken as evidence of recent pollution with human or animal faeces.

Morphology & Cultural Characteristics - It is a Gram negative, straight rod, measuring 1.3 X 0.4- 0.7 µm arranged singly or in pairs. It is motile by peritrichate flagella. Spores are not formed. Some strains possess capsules and fimbriae.

 It is an aerobe and a facultative anaerobe. The temperature range is 10-40°C. Good growth on ordinary media. Colonies are large, thick, grayish white, moist, smooth, opaque or partially translucent discs. Many pathogenic isolates have polysaccharide capsules, \ some occur in the mucoid form. Also pathogens are hemolytic on blood agar. On Mac Conkey’s medium, colonies are bright pink due to lactose fermentation. It can be inhibited by sodium selenite, sodium tetrathionate and brilliant green, 7% NaCl.

Biochemical reactions - The 4 biochemical tests are the indole, methyl red, Voges-proskauer and Citrate utilization tests, ie., IMViC. E. coli is indole and MR positive. VP and citrate negative. Gelatin is not liquefied, H₂S not formed and urea is not split. It ferments glucose, mannitol, maltose, lactose, sucrose with the production of acid and gas.

Antigenic Structure - E. coli has four antigens – the somatic antigen O, the capsular antigen K, flagellar antigen H and the fimbrial antigen F.

·         O antigens : heat stable, lipopolysaccharide antigens of cell wall, 173 different O antigens have been described.

·         K antigens : acidic polysaccharide, located in the envelope or microcapsule, 103 antigens are known.

·         H antigens : thermolabile, 75 antigens have been recognized.

·         F antigens : thermolabile proteins.

The K antigen located in the envelope or microcapsule encloses the O antigen and renders the strain inagglutinable by the O antiserum. It may also contribute to virulence factors in E. coli - surface Ag and toxins. Fimbriae also promote virulence, which is important in urinary tract infections. E. coli produce 2 kinds of toxins - hemolysins and enterotoxins. Hemolysins do not appear to be relevant, but enterotoxins are important in the pathogenesis of diarrhea. Three types of enterotoxins - heat labile toxin (LT), heat stable toxin (ST) and verotoxin (VT) (because of its toxic effect on vero cells, a cell line derived from African green monkey kidney cells).

On the basis of O antigens, E. coli is subdivided into a number of O groups, which are divided into subgroups on the basis of K antigens and again divided based on the H antigens. Thus a strain of  E. coli is recorded as O1111: K58: H12.

Resistance / Viability - E. coli  is excreted in faeces of man and animals in very large numbers and contaminate soil and water. It is killed by moist heat at 60⁰C usually within 30 minutes. In water it can be killed by 0.5-1 part per million chlorine.

Toxins –

1.      Enterotoxins : ETEC strains produce a heat labile toxin (LT) or a heat stable toxin (ST) or both, in which the production is under the genetic control of transmissible plasmids called ent plasmids.(a) Heat-Labile Toxin – a heat labile protein which is destroyed by heating at 65⁰C for 30 minutes. It is composed of one enzymatically active polypeptide A subunit and 5 identical subunits. The toxin binds to the epithelial cells of the small intestine, enters and activates adenylate cyclase, which results in the conversion of  ATP to cAMP, which initiates hypersecretion of electrolytes and fluids into the lumen resulting in watery diarrhoea.(b) Heat-Stable Toxin- a low molecular weight polypeptide and poorly immunogenic, not destroyed by heating at 100⁰C for 30 minutes. It activates guanylate cyclase, therefore increased production of  cGMP.

2.      Haemolysin : bring about lysis of RBCs of some species,  E. coli  haemolytic strains obtain iron from the lysed erythrocytes of the host.

3.      Verotoxin : a phage encoded cytotoxin, cytotoxic to Vero cells, shiga-like toxins.

Pathogenicity - E. coli forms a part of normal intestinal flora of man and animals. It can cause four main types of syndromes : urinary tract infections, diarrhea, pyogenic infections and septicaemia.

            Urinary tract infection may be by urinary obstruction due to prostatic enlargement or pregnancy. The asymptomatic infection in pregnant women, if untreated will become symptomatic and causes hypertension which leads to prematurity or perinatal death of the foetus. UTIs(Urinary Tract Infections) are among the most common of all infections. It cause inflammation of the urethra ie., urethritis and inflammation of the bladder, ie., cystitis. Since infection easily spreads from the urethra to the bladder, most infections are urethrocystitis. Infectious agents (other than E. coli-Pseudomonas,Proteus,Klebsiella) reach the bladder of females faster than males because of the short urethra of females, and so women are affected more. A major cause of UTIs is incomplete emptying of the bladder during urination, \ retained urine serves as a reservoir for microbial growth, thereby infection. Pregnant ladies are more susceptible to infection because of the compressing of bladder by expanding uterus, decreased flow of urine through the ureters etc. and \there will be some amount of urine in the bladder always. While in men the prostate tends to enlarge with age and constrict the urethra. Some people do not visit the bathroom often enough, thus it is important for both females and males to empty the bladder frequently and completely. Infections usually begin in the lower urethra and ascend, to cause inflammation of the kidneys or pyelonephritis. Less often, infections begin in the kidneys and descend to urethra.

Laboratory Diagnosis- Normal urine is sterile, but during voiding may become contaminated with genital commensals. Therfore, clean-voided midstream urine is collected. The urine must be processed immediately as the contaminating bacteria readily multiply otherwise refrigerated. Microscopic examination of urine is done to detect the presence of increased number of polymorphs. The deposit of the centrifuged urine examined for the presence of pus cells, RBCs and bacteria. Presence of more than 3 pus cells per high field is suggestive of infection. The culture of urine can be prepared by streaking on Mac Conkey medium, Blood agar and the population estimated by TPC method.

UTI is treated with antibiotics - amoxicillin, sulfonamides. It can be prevented by good personal hygiene and frequent complete emptying of the bladder.

            Diarrhoea causing E. coli is divided into 5 different strains :

1.      Enteropathogenic E. coli (EPEC) : Associated with diarrhea in infants and children and is sporadic. It adhere to the mucosa of the upper small intestine intimately, cause disruption of  microvilli, do not ordinarily produce enterotoxins. Identified by plating on blood agar and Mac Conkey and serotyped.

2.      Enterotoxigenic E. coli (ETEC) (Traveller’s diarrhoea) : It is endemic and affect all age groups. It contain a plasmid that enables them to make an enterotoxin. They attach to the mucosa by attatchment pili or fimbriae.

3.      Enteroinvasive E. coli (EIEC) : It have a plasmid with a gene coding for a particular surface antigen, K antigen, which enables the strains to attach and invade mucosal cells. These strains are non-motile, non-lactose fermenter. Resembles shigellosis, occur in children, adults.

4.      Enterohemorrhage E. coli (EHEC) : causes bloody diarrhea. It invades vascular endothelial cells, occur in young children and elders. Sporadic food poisoning. The source of infection is contamination by human, animal faeces. Changing life styles and eating habits, with growing popularity of fast foods have led to a remarkable increase in EHEC food poisoning. Other than non vegetables, the source of infection can be salad vegetables, in which the bacteria found beneath the skin and deeper tissues. Washing & cooking would not remove the bacteria from such vegetables.

5.      Enteroaggregative E. coli (EAEC) : Associated with persistent diarrhea, produce a low molecular weight heat stable enterotoxin. It causes shortening of villi, hemorrhagic necrosis.

Laboratory Diagnosis – Faeces or rectal swabs is inoculated on Mac Conkey agar. Colonies tested by agglutination with polyvalent and monovalent O antisera.

           

            Pyogenic infections- E. coli is the most common cause of intra-abdominal infections.They also cause pyogenic infections in the perianal area and an important cause of neonatal meningitis. For the diagnosis pus, wound swab, blood, CSF etc. are processed.

Septicaemia- Blood stream invasion by E. coli may lead to fatal conditions like septic shock and Systemic Inflammatory Response Syndrome. For the diagnosis pus, wound swab, blood, CSF etc. are processed.

The strains show multiple drug resistance,so antibiotic sensitivity tesing of strains is important in treatment.

2.  Salmonella

            The genus Salmonella consists of bacilli that parastiise the intestines of a large number of vertebrate species and infect human beings, leading to enteric fever, gastroenteritis, septicaemia.

Morphology & Cultural Characteristics - Salmonella are gram negative rods,1-3 x 0.5 mm in size.They are motile with peritrichate flagella,gthe only non-motile type is S. gallinarum. They do not form capsules, spores , but  fimbriae.

They are aerobic and facultatively anaerobic, grows on simple media at pH 6-8, temperature 15-45°C. On nutrient agar or blood agar, colonies are large, 2-3 mm diameter, circular, low convex and smooth, translucent. On Mac Conkey, colourless colonies due to non-lactose fermentation. On bismuth sulphite medium, jet black colonies with a metallic sheen are formed due to H₂S production. The species which do not form  H₂S  produce green colonies. Tetrathionate broth, Selenite F Broth are used as enrichment media.

Biochemical Reactions - ferment glucose, maltose forming acid and gas. They are indole negative, MR positive, VP negative, Citrate positive. They need tryptophan as growth factor. Most salmonellae give a positive reaction for H₂S in triple sugar iron agar, fail to hydrolyse urea and deaminate phenylalanine.

Resistance -   The bacilli are killed by moist heat at 55°C for 1 hr, boiling, chlorination, pasteurization. In polluted water and soil,they survive for weeks and in ice for months. They are killed by 0.2% mercuric chloride or 5% phenol within 5 minutes.

Antigenic Structure - Salmonellae possess flagellar antigen H, somatic antigen O and surface antigen Vi. These antigens undergo variations.

·         H antigens : present on flagella, heat labile and alcohol labile, heating at 100⁰C for 30 minutes detaches all flagella.

·         O antigens :  heat stable polysaccharides form part of the cell wall LPS, withstand boiling for 2.5 hours, alcohol stable, hydrophilic.

·         Surface antigens : (a) Vi antigen – envelops the O antigen, heat labile, acidic polysaccharide, destroyed at 100⁰C for 1 hour. (b) M antigen- a loose extracellular polysaccharide slime, boiling for 2.5 hours destroys it. (c) F antigens – on fimbriae.

Pathogenicity - The most important species are S. typhi, S. paratyphi A, B and C which cause enteric/typhoid fever, S. choleraesuis which causes septicaemia and S. typhimurium / S .enteritidis which causes food poisoning/gastroenteritis.

Typhoid fever/Enteric fever : one of the most serious of the epidemic enteric infections, caused by S. typhi. The organisms enter the body through contaminated food or water and invade the mucosa of the upper small intestine. The bacteria attach themselves to the microvilli by means of adhesins on the bacterial surface From there they invade lymphoid tissues and are phagocytized by neutrophils and macrophages.They resist intracellular killing and multiply in the phagocytes, emerge and continue to to invade the blood stream, a transient bacteraemia. During this period the bacilli is seeded in the gall bladder, spleen, bone marrow, lymph nodes, lungs, kidneys, where further multiplication takes place. After multiplication in these organs, bacilli pass into the blood, causing a second and heavier bacteraemia. The organisms multiply in the gall bladder and are discharged continuously into the intestine, will become inflammed, especially Peyer’s patches, lymphoid tissues of ileum. They undergo necrosis and slough off, leaving behind characteristic typhoid ulcers .

Incubation period is 8-15 days. During the first week, the patient suffers from headache, malaise and fever due to an endotoxin. During the second week, the patient’s condition worsens as the organisms invade many tissues, including the intestinal mucosa and are excreted in the stools. The typhoid bacilli multiply in bile, organisms from the gall bladder reinfect the intestinal mucosa and lymphoid tissues such as Peyer’s patches. Characteristic “rose spots” often appear on the trunk and abdomen for a few days. Abdominal distention and tenderness and enlargement of the spleen occurs, but diarrhoea is absent. Leucocytes decrease in number. Some patients suffer internal hemorrhage, perforation of the bowel and pneumonia..By the fourth week, symptoms subside and immunity develops.

Laboratory Diagnosis – can be done by the isolation and identification of the causative agent from patient’s blood, faeces, urine, bone marrow etc., demonstration of circulating antigen, antibodies in the serum.

            Demonstration of antibodies in the patients serum is done by Widal test, an agglutination test which detects the presence of agglutinins against O and H antigens. ELISA, radioimmunoassay, counter immunoelectrophoresis also can be done.

Prophylaxis & Treatment – Best methods to control the enteric fever are proper sewage disposal, safe water supply, handling food hygienically, periodic examination to ascertain that the food handlers are not carriers. About 1-5% of persons recovering from typhoid fever continue to excrete the organisms in faeces.(temporary carriers and chronic carriers). For the treatment of chronic carriers chemotherapy with ampicillin, amoxycillin. Cell-mediated immunity provides protection against future infection. Chloramphenicol is the antibiotic of choice

            A live,attenuated oral typhoid vaccine stimulates cell-mediated, humoral immunity, secretes IgA which helps in prevention of mucosal invasion. Booster dose every 3 years.

            The source of infection is a patient ar a carrier.Patients who continue to shed typhoid bacilli in faeces for 3 week to 3 months after clinical cure are called convalescent carriers.Those who shed more than 3 month,but less than a year is called temporary carriers,those who shed over a year are chronic carriers.

            Salmonellosis(Gastroentritis): caused by S. enteritidis. It is found in the gastrointestinal tracts of many animals including poultry, wild birds, rodents. So it is a zoonotic disease. ie, the source of infection from animal products. Human infection results from the ingestion of contaminated food, such as poultry, meat, milk and milk products. The chicken eggs can become infected if the hens laying them are infected. They can enter through the shell if eggs are left on contaminated chicken feed or feces and grow inside. Also salmonellae have been traced to contaminated water and food contaminated by carriers. Salads, other unooked vegetables cause infection if contaminated through manure. Foods will be contaminated by droppings of rats, lizards etc.

Symptoms of salmonellosis include abdominal pain, fever and diarrhoea with blood and mucus. They appear 8-48 hrs after ingestion of organisms and are associated with the organisms invading the mucosa of small and large intestines. Fever due to endotoxins. It lasts 1-4 days.

Antibiotics  not  used,  because  it  increase  the  faecal  shedding  of   bacilli,   for   serious   cases it   is   recommended.   Prevention   is   by   maintenance   of   sanitary   water   and   food   supplies  and eradication of organisms from carriers.

3.  Proteus

            The members have a unique ability to oxidatively deaminate amino acids to the corresponding keto acids and ammonia. It is tested by growing the organism in a medium containing phenylalanine, from which phenylpyruvic acid is formed (PPA test). They are widely distributed in nature as saprophytes, found in decomposing animal matter, in sewage, in manured soil and in animal, human faeces. They are frequently present on moist areas of skin. They are responsible for urinary and septic infections.

Morphology & Cultural characteristics -They are Gram negative coccobacilli, 1-3mm long and 0.6 mm wide. In young cultures, most of them are long, curved and filamentous. They may be arranged singly, in pairs or in short chains. They are actively motile, also possess more than one type of fimbriae.

            They can grow on ordinary media like nutrient agar and culture emits characteristic putrefactive, fishy odour. P. mirabilis and P. vulgaris possess the ability to swarm on solid media. Swarming can be inhibited by increasing the concentration of agar in the medium 2-6% or by the addition of sodium azide or boric azide. Swarming does not occur on Mac Conkey agar on which smooth, colourless colonies are formed. Dienes phenomenon – When two identical strains of Proteus are inoculated at different points of the same culture plate, the resulting swarms of growth coalesce without signs of demarcation. While two different strains of Proteus species are inoculated, the spreading films of growth fail to coalesce and remain separated by a narrow but easily visible furrow. It has been used to determine the identity or non identity of variuos strains of  Proteus.

Biochemical reactions – All species of  Proteus produce acid from glucose and no acid from lactose, mannose etc. They reduces nitrates. Two important species-Proteus mirabilis, important urinary and nosocomial pathogen and Proteus vulgaris, found less commonly in human infections. Former is indole negative, latter positive. Both are urease positive.

 Antigenic structure – Motile strains possess O (somatic) and H (flagellar) antigens. O antigens are resistant to heat at 100⁰C, to ethanol and to dilute HCl whereas H antigens are destroyed by these treatments. O antigen has alkali-stable and alkali-labile fractions. The alkali stable portions of nonmotile strains OX19, OX2, OXK is shared by some rickettsiae, and this is the basis of Weil- Felix reaction for the diagnosis of some rickettsial diseases.

Pathogenicity – The genus Proteus is found in soil, water and faecally contaminated materials. Proteus mirabilis is the causative agent of urinary tract and wound infections. The urinary tract infections commonly involves the young boys and elderly people of both sexes often with diabetes or structural abnormalities of the urinary tract. Proteus vulgaris is more commonly recovered from infected sites in immunosuppressed hosts, particularly those receiving pro;onged regiments of antibiotics.

            UTI caused by Proteus tends to be more serious than E. coli as Proteus has a predilection for the upper urinary tract. It produces urease which liberates ammonia from urea. Ammonia inactivates complement, damages renal epithelium and makes the urine alkaline, the alkaline condition leads to the precipitation of phosphates in the urinary tract. It may even lead to hyperammonaemic encephalopathy and coma.

            Proteus may cause wound infections, bed sores, osteomyelitis and in neonates it may cause infection of the umbilical stump which often leads to bacteraemia and meningitis.

Laboratory Diagnosis – Culturing of the specimen on Mac Conkey agar or Deoxycholate agar. The isolate is identified by its morphological and biochemical tests and agglutination reactions.

Proteus bacilli are resistant to many antibiotics, like polymyxin B and colistin. Most strains are susceptible to aminoglycosides but they may acquire plasmids coding for resistance to aminoglycosides, trimethoprim and other antibiotics to which they are normally sensitive.

4.  Shigella

           

Morphology & Cultural characteristics - Shigella are short, Gram negative rods, 1-3 X 0.5mm in size. They are non-motile, non-sporing, non-capsulated. Fimbriae present. They are aerobes and facultative anaerobes, growing with in a temperature range 10-40°C, pH 7.4. Grow on ordinary media. Colonies are small, circular, convex, smooth and translucent. On Mac Conkey agar, colonies are coloulrless due to the absence of lactose fermentation. Deoxycholate citrate agar is a useful selective medium, smaller colonies without black centres. In XLD colonies are red and without black centres (salmonellae shows dark centres). Salmonella – Shigella (SS) agar is a highly selective medium for the isolation of Salmonella and Shigella. The high bile salt concentration and sodium citrate inhibit all gram positive bacteria, coliforms and many gram negative bacteria, also distinguishes between H₂S producers.

Resistance - They are killed by moist heat at 56°C in  1 hr, 1% phenol in 30 minutes. In stools that are allowed to become acid through growth of  coliforms or other bacteria they tend to die within a few hours, but can survive for several days in faeces kept nonacid in buffered glycerol solution or preserved at 4⁰C.

Biochemical reactions - They are MR positive and negative for VP reaction, citrate utilisation, urease and H₂S  and reduce nitrates to nitrites. Catalase is produced. Glucose fermented with acid and no gas. S. sonnei is a late lactose fermenter.

Antigenic structure – All Shigella possess O antigens, and certain may possess K antigens. The K antigens if present interfere with the detection of the O antigen during serologic grouping. The K antigen is heat-labile and may be removed by boiling the organism in a cell suspension.

Toxins –

·         Endotoxin : All shigellae release an endotoxin after autolysis. It is thermostable lipopolysaccharide of the cell wall. It has irritating action on the intestinal  wall which causes diarrhoea and subsequent ulcers.

·         Exotoxin : It is a heat labile protein and acts as enterotoxin and neurotoxin. As enterotoxin it acts on the intestinal mucosa causing accumulation of the fluid in the lumen and as neurotoxin, it damages endothelial cells of small blood vessels of the central nervous system which results in neurological complications like polyneuritis, coma and meningism.

·         Some strains of S. dysenteriae produces a cytotoxin which is active on Vero cells and is known as Verotoxin (VT).

Pathogenicity - Shigellae cause bacillary dysentery. It is an acute diarrhoeal disease characterized in the more severe infections by the presence of blood and mucus in the stools. Humans appear to be the only natural hosts for shigellae. It spreads rapidly in overcrowded conditions with poor sanitation. Contaminated food, fingers, flies, faeces, and fomites spread the pathogens. Playing, bathing, washing clothes in contaminated water play significant roles in transmitting Shigella. Infection may also spread via flies, as flies contaminate food mainly by carrying infective material on their feet. Children aged 1-10 are more susceptible to Shigella. The ingestion of 10-100 organisms can be sufficient to cause infection, relatively unaffected by gastric acid or bile.

Once contaminated food or water ingested, Shigella survive the acidity of the stomach, pass through the small intestine and attach to large intestine and multiply in the gut lumen. Many bacteria adhere to the epithelial cells of the gut mucosa and induce these cells to ingest them. They then multiply within the epithelial cells, inflammatory reaction develops within the capillary thrombosis, leading to necrosis of patches of epithelium, which slough off, leaving behind superficial ulcers. The cellular response is mainly by PMNLs which can be seen on microscopic examination of stool, together with red cells and sloughed epithelium.

 After an incubation period of 1-4 days, abdominal cramps, fever, profuse diarrhoea with blood and mucus suddenly appear. In the most serious cases, diarrhoea cause dangerous protein deficiencies called kwashiorkor and vitamin B₁₂ deficiencies and also loss of electrolytes, all these causes neurological damage. Other than the endotoxin which elicits fever, S. dysentriae produces Shiga toxin, which acts as neurotoxin, which causes convolutions and coma. They cause ulceration and bleeding of the intestinal lining and sometimes deeper intestinal layers. Symptoms persist 2-7 days, cause severe dehydration and fluid, electrolytic imbalance.

Laboratory diagnosis - Diagnosis made by isolating bacillus from faeces. They are sensitive to acids in faeces, so they are maintained in a buffered transport medium. They are inoculated into Deoxycholate agar plates, colonies, biochemical studies.

Treatment - Restoring fluid and electrolytes by oral rehydration salt solution is essential for recovery. Intravenous replacement of fluids and electrolytes plus antibiotic therapy is necessary in the very young, the aged or debilitated and in severe cases of shigellosis. Antibiotics ampicillin, tetracycline, nalidixic acid are used. Nalidixic acid inhibits DNA synthesis, especially in gram negative pathogens. Oral vaccines now available.

Control consists of improving personal and environmental sanitation.

5.  Klebsiella

Morphology & cultural characteristics - It is isolated from fatal cases of pneumonia. Gram negative, non-sporing, non-motile, 1-2 mm long and 0.5-0.8 mm wide with parallel or bulging sides and slightly pointed or rounded ends. They occur either in end to end pairs or are arranged singly. Freshly isolated strains possess a well defined polysaccharide capsule. It is produced in media rich in carbohydrates. Some extracellular polysaccharide is also secreted from the bacteria are loose soluble slime, accumulation of which gives mucoid appearance. They are non-flagellated, but most strains possess fimbriae of one or more of 3 types.

They grow well on ordinary media in a temperature range of 12-43⁰C with optimum growth at 37⁰C. on Mac Conkey agar, the colonies typically appear large, mucoid and pink.

Biochemical reactions - It ferments glucose, lactose, sucrose, mannitol with the production of acid and abundant gas. It is IMViC, - - + + . It forms urease. It is aerobic, seen in human intestine.

Pathogenicity - It causes pneumonia, an inflammation of lung tissue. Klebsiella initiate the disease process by colonizing the upper tract and then entering the lower respiratory tract during a deep breath or suppressed cough, or by large amounts of mucus. Once it enters the lower respiratory tract, attaches by means of pili and multiplies.

Pneumonia are classified by site of infection as lobar and bronchial. Lobar pneumonia affects one or more of the 5 major lobes of the lungs. Fibrin deposits are characteristic of lobar pneumonia. When they solidify, they cause consolidation or blockage of air spaces. Inflammation of the pleural membranes causing painful breathing follows. This is caused by K. pneumoniae, while bronchial pneumonia affects bronchi, caused by pneumococci (S. pneumoniae).

Pneumonia is transmitted by respiratory droplets and by carriers.The infected person suffers violent chills and high fever, chest pain, cough and sputum containing blood, mucus follow. Klebsiella causes an extremely severe pneumonia that can lead to chronic ulcerative lesions in the lungs and extensive destruction of lung tissue.

It is also an important cause of nosocomial infections. It causes pneumonia, urinary infection, pyogenic infections, septicemia.

Treatment – Clinical isolates of  Klebsiella contain R plasmids and are usually resistant to ampicillin, amoxycillin, cephalosporins.

 6.  Yersinia

Morphology & Cultural characteristics - It is a short, plump, ovoid, gram negative bacillus, about 1.5 X 0.7 mm in size, with rounded ends and convex sides, arranged singly. It shows “safety-pin” appearance on bipolar staining with methylene blue, ends densely stained and the central area clear. It shows pleomorphism. It is surrounded by a slime layer, non-motile, non-sporing, non-acid fast.

It is aerobic and facultatively anaerobic, pH 5-9.6, temperature 2-45°C. On nutrient agar, colonies are small, delicate, transparent discs, becomes opaque on continued incubation. Colonies on blood agar are dark brown due to the absorption of the blood pigment. Colourless colonies on Mac Conkey agar. In broth, flocculent growth at the bottom and along the sides of the tube. If grown in a flask of broth with oil or ghee on top, a characteristic growth occurs which hangs down into the broth from the surface (stalactite growth).

Biochemical reactions-  IMViC is - + - -, catalase positive, oxidase and urease negative. It is nitrate reduction positive and gelatin liquefaction negative. It ferments glucose, mannitol and maltose  with the production of acid but no gas.

Susceptibility - It is easily destroyed by heat at 55⁰C and 0.5% phenol within 15 minutes. Exposure to sunlight for 3-4 hours is also lethal, sensitive to drying and chemical disinfectants. It remains viable for long periods in cold, moist environments. On sealed agar slopes and in frozen tissues, it remains viable for years.

Antigenic Structure -

·         A heat-labile protein envelope antigen known as fraction – 1 or F1. it is formed in vivo at 37⁰C. it inhibits phagocytosis.

·         The V and W proteins are also formed by virulent strains of Y. pestis  at 37⁰C at low calcium concentrations. V antigen is a protein of molecular weight 90,000 and W antigen is an acidic lipoprotein of molecular weight 145,000. Their production is determined by a plasmid.

·         Pesticin, fibrinolysin, coagulase, whose production is regulated by a single plasmid.

Pathogenicity - Y. pestis  is the causative agent of plague. Plague is a zoonotic disease, ie., the disease is spread by infected rodents, especially rats which transmit the organisms by animal to animal contact and also to humans by flea bites. It is a natural pathogen of rodents.

The flea, Xenopsylla cheopsis, which is a carrier, suffers from plague infection. When it bites a diseased rat, it sucks about 0.5 ml of blood per feed which contains about 5,000 to 50,000 bacilli. The plague organisms ingested from a sick rat multiply and block the flea’s digestive tract until blood meal, its food cannot pass through it. In flea it takes about 3 weeks (extrinsic incubation period). The flea gets hungrier, bites more ferociously and infects new victims as it dispenses plague organisms with each bite. Eventually flea dies infecting humans.

As infected rats die of plague, their body temperature drops, their blood coagulates, hungry fleas jumps to near by sources of warmth and liquid blood. The new host also a rat,  but in crowded rat-infested areas, or when contact is made with the carcass of a plague infected animal, the next host can be a human.

In man plague occurs in 3 forms : Bubonic, Septicaemic and Pneumonic.

·         Bubonic Plague – After an incubation period of 2-5 days, the lymph nodes draining the site of entry of bacillus become infected. As the plague bacillus usually enters through the bite of infected flea on the legs, the lymph nodes will enlarges known as buboes, ie., enlarged gland in groin and armpit. Patient develops fever, chills, nausea and malaise.

·         Septicaemic plague – The presence of bacteria in the blood denotes septicaemic plague. Massive involvement of blood vessels occurs resulting in haemorrhages in the skin and mucosa, the disease is also known as Black Death.

·         Pneumonic Plague – Clumps of bacteria may become entrapped in the lungs causing pneumonic plague. It can be transmitted from man to man by droplet infection. Patient develops fever, and cough, first the sputum is mucoid and later blood-tinged which is loaded with bacteria. Also severe chest pain, difficult and rapid breathing, cyanosis and circulatory failure.

Laboratory diagnosis – By demonstrating Y. pestis in stained films from buboes,sputum, blood cultures in septecaemic plague. Plague can be diagnosed by fluorescent antibody technique, complement fixation tests.

Prophylaxis – can be carried out by :

·         Control of fleas and rodents, by keeping houses and surroundings free from dirt and debris in which fleas can survive.

·         Spraying insecticides inside the rodent burrows and houses to kill the fleas.

·         After the fleas have been killed, kill the rat with rat-poison, otherwise the fleas will jump from the carcass of rats to man.

It is treated with streptomycin, tetracycline. Recovery confers life time immunity. Two types of vaccines : killed and live attenuated.

Other members of Enterobacteriaceae are citrobacter,enterobacter and serratia.

Citrobacter:  utilize citrate, grow in KCN medium, produce H₂S, ferment lactose. It is a normal intestinal inhabitant. It may cause infections of urinary tract, gall bladder, middle ear, meninges.

Enterobacter: Motile,capsulated, lactose fermenting, IMViC is - - + +. Normally found in faeces, sewage, soil and watery. They may be responsible for hospital infections.

Serratia : Forms a pink, red pigment. It is pleomorphic. It is a saprophyte found in water, soil, food. Nosocomial, associated with meningitis, septicimia, respiratory infections.

Pasteurella

            P. multocida  is a non motile, gram negative bacillus, resembles Yersinia, but oxidase positive, indole positive, non fermenters of lactose.

It is transmitted by cat or dog bites and scratches.It is part of the normal flora of many wild and domestic animal’s mouth,nasopharynx and gastrointestinal tracts.Infections are reddened usually localized in the soft tissue adjacent to the bite,often appear within 24 hours.

Virulence factors include production of an endotoxin and capsule that helps prevent phagocytosis.Penicillin is the drug of choice.