EnterobacteriaceaeThe family Enterobacteriaceae inhabits the large bowel of man and animals, soil, water and decaying matter. These m.o. are gram negative bacilli, and are called “ enteric bacilli” or “ enterics”.These m.o. are responsible for the majority of nosocomial infections ( hospital-acquired ) as UTI , wound infections, pneumonia, meningitis and septicaemia.
Classification I. Edwards and Ewings: Tribe Genera 1. Escherichieae Escherichia Shigella 2. Edwaedsielleae Edwardsiella 3. Salmonelleae Salmonella 4. Citrobacteriaceae Citrobacter 5. Klebsielleae Klebsiella Enterobacter Hafnia Serratia Pantoea 6. Proteeae Proteus Morganella Providencia 7. Yersinieae Yersinia
Classification/cont…….:II. Practical Classification:Group I : Eschericheae Genus I : Escherichia Genus II : Edwardsiella Genus III : Citrobacter Genus IV : Salmonella Genus V : ShigellaGroup II : Klebsielleae Genus I : Klebsiella Genus II : Enterobacter Genus III : Hafnia Genus IV : Serratia
Group III : Proteeae Genus : ProteusGroup IV : Yersinieae Genus : YersiniaGroup V : Erwinieae Genus : ErwiniaMorphology:The enterics are small, gram negative, non-spore forming rods/sometimes coccobacilli. They could be motile with peritrichous flagellae as E.coli, Salmonella and Proteus, or non-motile as Klebsiella, Shigella and Yersinia. Also, these m.o. could be capsulated with well-defined capsule as Klebsiella, ill-defined loose coating “slime layer” as in E. coli, or non-capsulated as Proteus. Moreover, these m.o. have fimbriae (pili) for attachment to the host, other bacteria, or phage.
Cell Wall Ultrastructure: 1. Cytoplasmic membrane. 2. Murein (20%); for cellular rigidity. 3. Lipoprotein. 4. Lipid bilayer; phospholipids. 5. Lipopolysaccharides, proteins; for antiginicity and endotoxic portion. N.B. : 3, 4, & 5 = 80% of the cell wall.
Biochemical Properties:The enteric bacilli are facultative anaerobic or aerobic m.o. All these m.o. can ferment glucose with acid production, reduce nitrate to nitrite, and exhibit no cytochrome oxidase activity.They are divided into 2 groups according to their effect on lactose. The lactose fermenters (LF) are collectively called “coliforms” and include mainly Escherichia, Klebsiella, Citrobacter and Enterobacter. The non-lactose fermenters (NLF) include Salmonella, Shigella and proteus.Also, IMViC phenomena (I = Indol, M = Methyl Red, V = Voges Proskauer, C = Citrate Utilization); E.coli = ++--, Klebsiella = --++
Cutural Characteristics: I. Non-differential Non-selective Media: Examples: Blood agar, Brain-Heart Infusion. The various genera can NOT be distinguished on these media. The colonies appear as gray, white, opaque and watery. Klebsiella on blood agar shows large mucoid non-pigmented colonies. Proteus shows swarming phenomena (successive waves of translucent growth on the surface of agar). Pathogenic E.coli may produce beta haemolysis on blood agar.
II. Differential and/or Selective Media1. MacConkey’s Medium: Contains lactose, bile pigments and crystal violet (to prevent the growth of gram positive m.o. and fastidious gram negative m.o.) and neutral red as an indicator (change colour according to pH). The colonies of the lactose fermenter enterics appear rose pink in colour, while the non-lactose fermenter appear pale colonies.Lactose fermenter; Classical (18-24 hr): E.coli, Klebsiella ; Rapid (5 hr) : Enterobacter; Slow (weak ; 24-48hr): Citrobacter, Providencia, Serratia, Hafnia, Edwardsiella, Arizona and Erwinia. The latter m.o. are ONPG positive.Non- lactose fermenter: Shigella, Salmonella and Proteus. These m.o. are ONPG negative.
2. Eosin – Methylene Blue Media (EMB):This media contains two sugars; lactose , and sucrose, with eosin and methylene blue which inhibit the growth of gram positive and fastidious gram negative bacteria. The lactose fermenter m.o. appear as greenish - black colonies with metalic sheen, while the non-lactose fermenter colonies appear as non-pigmentd semi-translucent or colourless colonies.
3. Deoxyxholate or Deoxycholate- Citrate Agar (DCA):These media contain deoxycholate which prevent the growth of gram + & gram – fastidious m.o. These media favour the growth of Salmonella and Shigella.4. Shigella-Salmonella Agar (SS-Agar):This medium is a selective medium for Shigella and Salmonella. The latter m.o. appears black, while the former as pale (non-black) colonies.
5. Xylose- Lysine – Deoxycholate (XLD):This medium contains xylose, lactose, sucrose and lysine with phenol red as an indicator. Also, it contains sodium deoxycholate and sodium thiosulphate. On this medium Salmonella, Shigella and Proteus can grow. Both proteus and Salmonella produce H2S; so their colonies appear black in colour. However, Salmonella colonies are surrounded by pink halo due to lysine decarboxylation. Shigella have no black colour.
6. Hektoen Enteric Agar (HE): Used for the cultivation of enteric bacilli; the lactose fermenter colonies appera yellow, while the non-lactose fermenter appear faint green. III. Enrichment Broth: Examples: Selenite broth, G-N browth and tetrathionate broth. These media can be used for the enrichment of the growth of Salmonella as S. typhi or paratyphi.
Antigenic Structure: The antigenic structure identification is used for epidemiological, classification and taxonomic studies. There are: 1. Capsular antigens (K ;from Kapsel): These are polysaccharides. They stimulate IgM production and could be virulence factors as Vi of Salmonella. 2. Flagellar antigens (H ; from Hauch = Breath): These are protein antigens that stimulate IgG production, and are used for motility.They have 2 phases; phase I is specific, while phase II is non-specific (Shared). 3. Somatic antigens (O; from Ohne Hauch= without breath): These are lipopolysaccharides of the cell wall and are composed of 3 regions:
1. O- specific antigen; polymers of repeating oligosaccharides, and are used for serotyping of m.o. 2. Core polysaccharides; constant in a particular genus. 3. Lipid A moiety; which is the toxic portion (endotoxin).
Determinant of pathogenicity: 1. Endotoxins. 2. Enterotoxins: These are exotoxins that exert their activities on the small intestine causing transduction of fluid into the lumen. Many m.o. can produce them as enteropathogenic E. coli, Klebsiella pneumoniae, Enterobacter cloacae, Shigella dysenteriae, flexneri or sonnei. 3. Other factors: Invasiveness (penetration) as Shigella; Capsule as Klebsiella pneumoniae; or Vi antigen of Salmonella. Salmonella, Shigella and Yersinia are always pathogenic m.o. However, other m.o. in the intestine (stool) could part of normal flora.
Anti-Microbial Drugs against Enterics: 1. Broad spectrum penicillins as Ampicillin, or carbenicillin and the new ones as piperacillin. 2. Cephalosporins (see the generations). 3. Aminoglycosides. 4. Colistin and polymyxin B. 5. Chloramphenicol or tetracyclines. 6. Cotrimoxazole 7. Fluoroquinolones
Endotoxins:Endotoxins are generally heat stable molecules, and have M.W. Of 100 – 200 KD. They have the following effects on the body (endotoxic shock):1. Fever: Endotoxins can stimulate the liberation of IL-1 (endogenous pyrogen) which stimulates the thermoregulatory centre in the hypothalamus. They induce their action within 60-90 min.2. Leucopenia: In early stages the endotoxins induce leucopenia, followed by leucocytosis.
3. Hypotension: Endotoxins can cause arteriolar and venular vasocontriction ( causing chill), followed consequently by peripheral vascular dilatation, increased vascular permeability, decreased venous return, increased cardiac out put, stagnation in the microcirculation, peripheral vasoconstriction, then shock. This leads to impaired organ perfusion. 4. Impaired organ perfusion and acidosis: The vital organs (kidneys, heart, lungs and brain) become anoxic, and perform inadequatly. This results in acidosis.
5. Activation of C3 complement component: The alternative complement pathway is activated by endotoxins. (See Lect. On complement). 6. Disseminated intravascular coagulation (DIC): Endotoxins activate factor XII (Hageman)which is the first step of the intrinsic clotting system. Also, fibrinogen changes to fibrin, and plasminogen to plasmin. 7. Death: All of the above mentioned effects could lead to death. N.B.: Naloxone (opiate antagonist) can increase B.P. in endotoxic shock.
Genus : EscherichiaThere is a single important species to humans which is E. coli. It is the main m.o. present as normal flora in the large bowel (stool). These are opportunistic m.o.; outside the intestine, most of the enteric bacilli including E.coli become pathogenic. The important question when these m.o. are isolated from the stool “ Are they pathogenic?”. Example, E.coli when isolated from the stool in cases of diarrhoea.
Morphology:Grom negative bacilli, motile, and some are capsulated (slime layer).Cultural characteristics:Faculatative anaerobes, grow on simple media. They are lactose fermenter; on media with lactose as MacConkey’s they produse rose pink colonies. See the other media (lecture 1).Biochemical activity:They ferment glucose, lactose, maltose, mannite, sucrose and salicin with production of acid and gas. The IMVic phenomena results are ++-- . They are urease – and do not produce H2S (vs Klebsiella)
Serological characters: 1. O (somatic) antigens : about 150. 2. K (capsular) antigens : about 90; of B,A & L subtypes. 3. H (flagellar) antigens : about 50. Serotyping is important to detect pathogenic strains. Enteropathogenic E.coli possess O antigens with numerical designations,e.g., 26, 55, 111, 119.....etc. The serotypes are written as O:K:H ,e.g., 111: 58 (B4): 2
Virulence factors: E.coli has several clearly identified components that contribute to its ability to cause disease: 1. Capsular (K) polysaccharides antigens which interfer with phagocytosis. 2. Pili (colonization factors) which enable the m.o. to adher to mucosal cells. 3. Endotoxin (see lecture 1). 4. Enterotoxins: They are genitically determined by a plasmid and are of 2 types:
1. Heat labile (LT): It is similar to the LT of V.cholerae . They are exotoxins that adhere to the small intestine. The Lt has M.W. Of 85000 Daltons and composed of 2 portions ( A and B). The A portion is the TOXIC portion with M.W. Of 26000 Daltons. The B portion is for attachment (binding). The toxic portion can stimulate cAMP of the small intestine epithelium which increases the intestinal permeability that results in fluid loss by excretion of water and Cl- and prevention of Na+ absorption and diarrhoea.
2. Heat stable (ST): It has M.W. Of 5000 Daltons, it is quiker in action than the LT (4 hours VS 10 hours), while its effect is weaker than that of LT (1/100). Moreover, ST stimulate cGMP. Diseases caused by E.coli: 1. Urinary tract infections: E. coli is the commenest cause of UTI. Certain strains known as uropathogenic E. coli colonize the vagina and periurethral region from where they ascend to the bladder or kidney causing cystitis or pyelonephritis. These strains possess pili with adhesive proteins that bind to specific receptors on the UT epithelium.
2. Neonatal meningitis: E.coli causes 40% of neonatal meningitis followed by group B streptococci. Such strains usually possess capsular antigen K1. 3. It causes sepsis, bacteraemia and endotoxic shock specially in immunoc- ompromised hosts. 4. It is an important cause of nosocomial infections. 5. Intestinal diseases: Different strains of E.coli can cause diarrhoea through different mechanisms:
A- Enterotoxigenic E. coli (ETEC): cause “traveller’s diarrhoea” and infantile diarrhoea. The diarrhoea is watery and ranges from mild to severe (cholera-like) and may be fatal. These m.o. Liberate both LT & St enterotoxins.B- Enteropathogenic E. coli (EPEC): Certain serotypes, e.g., O55, O111, cause outbreaks of neonatal diarrhoea in nurseries. They act mainly by adhering tightly to intestinal mucosa resulting in loss of microvilli and cupping of cells around the bacteria. Thus, preventing the normal functions of absorption and secretion.
C- Enteroinvasive E.coli (EIEC): These cause dysentery- like diarrhoea through invasion of intestinal epithelial cells.D- Enterohaemorrhagic E. coli (EHEC): These belong mainly to serotype O157:H7. They produce a toxin known as “verotoxin” (so called because it is toxic to Vero (monkey) cells in tissue culture). It is similar to shiga toxin (of Shigella). These strains are associated with outbreaks of haemorrhagic colitis, which is a severe form of diarrhoea associated with bloody discharge, which mainly follows ingestion of undercooked hamburger at fast-food restaurants. Some patients may end up with a life-threating complication called haemolytic- uraemic syndrome.
E. Enteroaggregative E. coli (EAggEC): These exhibit a specific pattern of aggregative adherence to the mucosa in patches. They are not invasive. They cause persistentdiarrhoea in children and in HIV patients. F. Diffusely adherent E coil (DAEC): Adhere to the epithelial cells in a diffuse pattern and carry a gene encoded surface fimbria. It is associated with both UTI and diarrhea.
Diagnosis: Specimens: urine, pus, stool, CSF ..etc. Direct examination & culture. Serotyping ELISA & Immunofluorescence ; to detect verotoxin & shiga toxin. PCR
Indicators of faecal pollution of water:There are some organisms that normally occur in the stools and if isolated from a water sample , this means that the water is contaminated with stools. These organisms are E. coli, Enterococcus faecalis and Cl. perfringens.Treatment:Mentioned in lecture 1.Prevention of traveller’s diarrhoea by daily use of bismuth subsalicylate, tetracycline and caution in food and drink. Early treatment of this condition is by the use of cotrimoxazole.
Genus : KlebsiellaKlebsiella are normal inhabitants of the intestine and respiratory tract (5% of people). They are saprophytes in soil and water. Some may cause disease in man. The main species are the following four:1. K. pneumoniae ( Friedlander’s bacillus).2. K. oxytoca.3. K. ozaenae.4. K. rhinoscleromatis.
Morphology:Gram negative, nonmotile and capsulated bacilli.Cultural characters:They give large pink colonies on MacConkey’s.The colonies are mucoid & moist due to the production of abundant extracellular slime.Biochemical activities:They ferment glucose, lactose, maltose, mannite, sucrose and salicin with production of acid and gas. The IMVic phenomena shows - - + + result; with exceptions: K. ozaenae is indol +, MR + and VP - , K. rhinoscleromatis is MR +, VP- and citrate - .K. oxytoca is indol + . Klebsiellae are urease +.
Diseases caused by Klebsiella: A. Klebsiella pneumoniae causes : 1. Lobar pneumonia with extensive haemorrhagic necrotizing consolidation of the lung specially in middle-aged or old patients. The patient produces thick, nonputrid and bloody sputum. 2. Urinary tract infections. 3. Wound infections. 4. Meningitis. 5. Nosocomial infections. 6. Bacteraemia; K. pneumoniae has replaced E. Coli as the leading cause of bacteraemia. 7. Tropical sprue (diarrhoea ); due to enterotoxins.
B. K. rhinoscleromatis causes rhinoscleroma which is a granulomatous lesion in the nose and throat. C. K. ozaenae is associated with atrophic rhinitis. D. K. oxytoca causes nosocomial infections. Treatment: Klebsiellae are resistant to ampicillin & carbenicillin, but sensitive to cephalosporins (vs Enterobacter).
Genus : Enterobacter These m.o. are less frequent than Klebsiellae. These m.o. can ferment lactose rapidly and are motile. The important species is E. aerogenes (Aerobacter) are usually motile, free living m.o. and can cause UTI and sepsis. Citrobacter and Serratia: They are found in soil, water and stool. They may cause UTI, wound and blood stream infections in hospitalized and immunocompromised patients, specially those under invasive procedures such as respiratory intubation, or I.V. & urinary catheters. Generally Serratia are pigment producers (rose ) as S. marcescens that produces intense red pigments and are late lactose fermenters. These m.o. are sentive to the 3 rd generation cephalosporins, but not aminoglycosides or penicillins.
Genus : Shigella Members of the genus Shigella cause bacillary dysentery ( bloody diarrhoea ) in man. Shigella is primarily human pathogen; it is only isolated from primates. They are divided into four groups ( that belong to four species) according to O antigens: 1. Group A : Sh. dysentery ; 13 serotypes. 2. Group B : Sh. flexneri ; 8 serptypes. 3. Group C : Sh. Boydii ; 18 serotypes. 4. Group D : Sh. Sonnei ; 1 serotype.
Morphology:Gram negative bacilli, non-motile, and noncapsulated.Cultural characters:They produce pale non-lactose fermenting colonies (NLF) on MacConkey’s medium and DCA (see lecture1)’ except Sh. Sonnei which late lactose fermenter m.o.Biochemical activities:Sh.dysentery ferments glucose only with production of acid only. Sh. boydii and Sh. sonnei ferment glucose and mannite with acid production. The IMViC results are V - - - (V = variable). Also, the features on the following 2 media:
1. Kligler Iron Agar (KIA). 2. Triple Sugar Agar (TSI). These 2 media are prepared in tubes or bottles, and are put in slops. The preparation has butt (buttom) and slant (top or surface). KIA contains glucose and lactose, while TSI contains glucose, lactose and sucrose. Both media contain iron and an indicator called phenol red. Both media at alkaline pH are red, but at acidic pH are yellow. The report should contains the colour of the slant and butt, gas production, and H2S production. Shigella : Alkaline slant (red), Acidic butt (yellow), no gas, no H2S ( K / A / G- / H2s- ). Salmonella : K / A / G+ / H2S + . (Gas : seen as bubbles; H2S : Black colour).
Factors of pathogenicity: 1. Invasiveness: Shigella has the ability to penetrate the epithelium of the terminal ileum and colon. They multiply inside these cells and cause local inflammation, cell death, and sloughing. This could result in bloody diarrhoea, and the stool contains pus cells and RBC (vs Salmonella). 2. Enterotoxins: They are liberated by Shigellae of groups A, B and C. These toxins are similar to the LT enterotoxins of E. coli . Also, Sh. dysentery serotype 1 (Sh. Shiga) produce an exotoxin (shiga toxin) that acts on the gut as an enterotoxin causing diarrhoea similar to that caused by E. coli verotoxin, and on the CNS as a neurotoxin causing meningism and coma which account for the severity and fatal outcome of this infection ( mortality rate = 80-90%). 3. Endotoxins: They are lipopolysaccharides of the m.o. and they lead to irritation of the bowel wall.
Pathogenesis:Shigellae are quite pathogenic m.o. ; only 200 bacilli can cause bacillary dysentery. Shigella transmitted from person to person. The four F’s- fingers, flies, food, and faeces- are the principal factors in tramsmission. Food and water borne outbreaks occur in nurseries and mental hospitals where faecal-oral trtansmission is likely to occur.Infection is limited to the GIT without blood invasion (blood culture – ve). The toxin acts early producing voluminous non-bloody diarrhoea which is followed by invasion of the mucosa and wall of the large intestine and terminal ileum leading to necrosis, superficial ulcers, pseudomembrane formation and bleeding. Sh. flexneri is most common in Iraq.The I.P. Is 1 – 4 days. There is abdominal pain, diarrhoea, tenesmus and fever. Stools contain blood, mucous and pus. Sh. dysentery causes the severest local and systemic manifestations. Sh. sonnei causes mild disease. Recovery occurs spontaneously in most cases. However, in children ( 2/3 of cases ) and elderly, dehydration, acidosis and even death may occur. Very few remain as chronic carriers.
Diagnosis:1. Macroscopic examination of stools to detect mucous and blood.2. Microscopic examination reveals pus and RBCs.3. Stool samples are inoculated on MacConkey’s, DCA, and selenite broth.4. Subcultures are made on MacConkey’s and DCA after 48 hours. Non-lactose (pale) colonies are identified by: morphology, biochemical reactions and serologically by agglutination with specific antisera.
Prevention: 1. Non- specific prophylaxis by adequate sanitation. 2. Prevention of shigellosis is dependent on interruption of faecal-oral transmission by proper sewage disposal, chlorination of water, and personal hygiene (hand washing by food handlers, hot air blowers or washbussin taps operated by foot ..etc.). 3. Oral vaccine may alter the immune status of the intestine by increasing the humoral immunity. 4. Isolation of patients until being culture negative. 5. Carriers are not allowed to handle food or drink.
Treatment: 1. Restoration of the fluid and electrolytes balance in severe cases. 2. Antibiotics: Could decrease the severity and mortality of shigellosis , but prolong the course of bacilli excretion. a. Ampicillin or tetracycline. b. Cotrimoxazole. C. Fluoroquinolone (ciprofloxacin) is the drug of choice in severe cases. 3. Usually shigellosis is mild and self-limiting condition.
Genus : Salmonella Genus Salmonella is complex both serologically and biochemically. They infect both humans and animals, and can invade the extraintestinal tissues. Classification of Salmonella is also complex. The common two classification are Kauffmann- white (gives status of genus and species to the m.o.), and Ewing (gives the status of serotypes to the m.o.). However, Salmonellae are recently classified according to DNA- DNA hybridization studies into 7 groups. Nearly all of the Salmonella serotypes that infect humans are in group I .
Group I Salmonellae: This group includes > 1400 serotypes; the most important of which are: 1. Salmonella Typhi, Paratyphi A, B and C: Cause typhoid and paratyphoid fevers (enteric fevers). 2. Salmonella Typhimurium and Salmonella Enteritidis : Cause salmonella food poisoning or enterocolitis. 3. Salmonella Choleraesuis : Causes septicaemia with metastatic abscesses.
Morphology:Gram negative, motile, non- capsulated bacilli.Cultural characters:Facultative anaerobes. They grow on simple media. On MacConkey’s and DCA, the produce pale NLF colonies. Also, on bismuth sulphate agar (BSA) Salmonella typhi shows black colonies with metallic sheen (due to H2S production). On SS agar the colonies appear black in colour (see lecture 1; othe media).
Biochemical activities: They are non lactose fermenters, non sucrose fermenters and urease negative. They ferment glucose, maltose, and mannite with production of acid only in case of S. Typhi, and acid and gas in case of S. Paratyphi. Most of them produce H2S. The IMViC = - +- + Serological characters: They possess O and H (phase I & II) antigens. Salmonella is divided into serogroups according to the O antigen, and into serotypes according to the specific H antigens. Some may possess a capsular Vi or virulence antigen which frequently interfers with agglutination of some salmonella by O antisera,e.g., S. Typhi, and prevent phagocytosis & I.C. destruction.
Factors of pathogenicity: 1. Invasiveness: Salmonellae has the ability to penetrate the intestinal epithelium and reach to the lymphatics, then to the blood causing bacteraemia and septicaemia, and to many body organs. 2. Intracellular survival inside the phagocytic cells (mainly due to Vi antigen). 3. Endotoxins (see lecture 1). 4. Enterotoxins similar to the LT and ST enterotoxins of E. coli.
Diseases caused by Salmonella: Generally,Salmonella can be transmitted from animals to man, except S. Typhi and Paratyphi which are primarily human pathogens. Salmonellae are transmitted to man via poultry products, meat contaminated during slaughtering, or contaminated eggs.The diseases are of 3 categories: 1. Enterocolitis (food poisoning). 2. Enteric fevers. 3. Septicaemia with metastatic abscesses.
Salmonella enterocolitis (infective food poisoning, Gastroenteritis):Many members of salmonella are responsible S. Typhimurium and S. Enteritidis. These organisms are common pathogens of animals or poultry. The organism is transmitted from improperly cooked meat of infected animals, eggs of infected birds or from food contaminated with rat excereta.The disease is characterized by nausea, vomiting, abdominal discomfort, diarrhoea and slight fever.The condition occurs after multiplication of the organism in the intestine (i.e., it is an infection and not due to a toxin). Therefore the I.P. is 12 – 48 hours to allow for such multiplication. The m.o. are isolated from the stool ( blood culture is negative). Recovery follows within one week. It is self-limited and does not require medical treatment. Antibiotics are indicated only for neonates or persons with chronic diseases who are at risk of septicaemia and disseminated abscesses.
Septicaemia with metastatic abscesses: It is mainly caused by S. Choleraesuis. It occurs in patients with underlying chronic disease such as sickle cell anaemia or cancer or a child with enterocolitis. Bacteraemia results in the seeding of many organs, with osteomyelitis, pneumonia, and meningitis as the common sequelae. Metastatic abscesses frequently occur on top of damaged tissues ,e.g., infarcts and aortic aneurysms. Blood cultures are positive.
Enteric Fevers: Enteric fevers are caused by S.typhi and paratyphi A, B, and rarely C. The source of infection is the stool or urine of cases or carriers. The organism enters the body by the oral route in contaminated food or drinks, e.g., raw vegetables, fruits, drinks, or milk products. After ingestion, the organism multiplies in the intestine, then passes through the lymphatics to the blood streamcausing bacteraemia that persists for 1 week. It the disseminates to the kidney and is excreted in urine, and to the liver and is excreted in bile to reach the intestine again but in huge numbers. It may rarely reach the periosteum, lungs, or meninges. Manifestations appear after an incubation period of 2 weeks in the form of 3 satges:
Stages of enteric fevers: 1. First week:The m.o. (about 1000) enters the body, to the intestine, then lymphatics, then blood. The patient have malaise, headache, constipation, bradycardia, myalgia, backache, and fever. The fevers is usually low grade, but gradually increases in a step-ladder pattern. During this stage blood culture is positive (+ve), stool, bone marrow & urine cultures are negative (-ve), widal test is negative (-ve).
Second week: During this stage the m.o. are phagocytosed and multiply intra- cellularly. After rupture of these cells they invade various parenchymatous organs (kidney, liver, billiary ducts, spleen, meninges, bone marrow, or bone...etc.).The patient have high fever, ill and toxic. On examination the patient may have hepatosplenomegally, jaundice, and rose spots on the abdomen. Also, the patient have leucopenia then leucocytosis.Blood culture starts +ve then becomes –ve, but B.M., urine & stool Cutures are opposit (starst –ve then become +ve), and widal test is +ve.
Third week:The m.o. pass through the billiary ducts to the intestine in huge numbers, invade the Peyer’s patches, cause local lymphatic hyperplasia, and may lead to local ulceration and perforation of the intestinal wall, then death.During this stage the patient have diarrhoea and other systemic manifestations ( may be as pneumonia or meningitis ). At the end of the week the patient may look better and the fever may subside.Stool & urine cultures are (+ve), blood & B.M. culture are (-ve), and widal test is +ve).
Widal test: This test is to detect antibodies against H and O antigens of the Salmonellae causing enteric fevers. The anti-O starts on the 8th. & 9th. day of infection, but the anti-H appears on the 11th. & 12th. day.The anti-O agglutinins appear early and disappear faster than the anti-H antibodies. The anti-O is more important in the diagnosis than the anti-H antibodies. The significant antibody titre for both agglutinins is 1/160 or more. Vaccinated people may have anti-H antibodies only. In the first week of enteric fevers the test is negative. Cross reaction with other m.o. may occur as Yersinia, other species of Salmonellae, Brucella, certain E. Coli, or HBV...etc. Anti-Vi agglutinins appear in the sera of carriers. Rising titre in 10 days interval suggest active enteric fevers. Recently developed 2-ME test for widal reaction can differentiate between active-recent and inactive-old infections of enteric fevers.
Diagnosis of carriers: Stool & urine culture, & detection of Vi-agglutinins. Prophylaxis: 1. Sanitary measures. 2. Carriers must not be allowed to work as food handlers. 3. Vaccines; three types are available: A.Heat killed (TAB); SC. 2 doses of 1 month interval. B. Oral typhoid vaccine of avirulent mutant of S. Typhi. It give 90-95% prtection. C. Vi capsular polysaccharide of S. Typhi, I.M.
Treatment: The drug of choice is ciprofloxacin. Other common drugs are chloramphenicol, ampicillin, and co-trimoxazole. Mutiple drug resistance is aproblem, and antibiotic sensitivity is required. Third generation cephalosporin (ceftriazone) is used for resistant organisms. Ampicillin and ciprofloxacin are recommended for chronic carriers. If not useful, cholycystectomy can be done for carriers. About 10% of patients may relapce, 3% pass into chronic carriers mainly in females (male: female ratio 1:3).
ProteeaeThe Tribe Proteeae have the ability to deaminate phenyl alanine and produce pyruvic acid which can be detected by addition of 10% ferric chloride. This will lead to the production of green colour. This test is called Guthrie’s test.The Tribe Proteeae have 3 genera:1. Proteus.2. Providencia.3. Morganella
Proteus The genus proteus have 4 species: 1. P. vulgaris. 2. P.mirabilis. 3. P. penneri. 4. P. myxofaciens. Proteus species are found in soil and water, and are normal inhabitants of the intestine in man. They cause infections only when they leave the intestine mainly by the first 2 species. They cause UTI, otitis media, wound infections, pneumonia and bacteraemia. Infection may be hospital or community acquired. Proteus infections are resistant to antibiotics and sensitivity T. should be done.
Morphology: Gram negative bacilli, very pleomorphic and highly motile.Cultural chracteristics: Fucultative anaerobes. Due to their high motility, they give colonies which swarm (swarming phenomenon) as successive waves of trunslucent growth over the surface of adars (as blood, nutrient). On MacConkey’s agar, the m.o. Appear as NLF growth, with fishy bad odour.Biochemical Activities: They are urease +ve (differentiate it from Salmonella and Shigella). They produce H2S which blackens the butt of TSI agar. The IMVic phenomena is V+-V. P. vulgaris is indol +ve ornithine decarboxylase –ve, but P. mirabilis is opposit (- & + ve respectively). Hydrlysis of urea produces ammonia & CO2. Ammonia can increase the pH of urine (alkalinization) and then precipitate Ca & PO4 leading to stone formation. Therefore, acidification of urine is required during treatment of UTI by proteus.
Serological chracters: Certain types of Proteus (OX19, OXK, OX2) share antigens with Rickettsiae. Hence suspensions of these m.o. are used in sero-diagnosis of rickettsial infections (e.g., typhus) in the Weil Felix agglutination test. The test is less frequently used as other more specific tests are available. Treatment: Most strains are sensitive to aminoglycosides and cotrimoxazole. Cephalosporines or fluroquinolones are used for resistant strains.
Morganella:The species M. morganii is of medical importance since it can cause UTI, diarrhoea in children,or wound infection. This m.o. is ornithine decarb- oxylase +ve, citrate –ve and H2S –ve. M morganii it is divided into two species according to trehalose fermentation. Those that can ferment trehalose are related to M.morganii subspp sibonii, while those that cannot are named as M. mrganii subspp. morganii.
Providencia: P. alcalifaciens, P.stuartti, P. rettegeri, and the newly described species prustiganni are capable to deaminase phenylalanine and P. rettegeri can hydrolyze urea. The species P. rettgeri and stuartti are important in causing nosocomial infections, diarrhoea and wound infections.
Genus: Citrobacter: Previously classified with tribe salmonelleae, but it is now is reclassified into tribe citrobacteriaceae. It involves11 spp. Most hydrolyze urea slowly and ferment lactose giving colonies similar to E. coli. All grow on simmon's citrate medium and give positive reaction to MR. Citrobacter freundii is isolated from diarrheal stool culture and it is associated with hospital acquired infection (UTI, pneumonia, intra-abdominal abscess) because 80% of Citrobacter freundii produce H2Sand 50% fail to ferment lactose,therefore, it is mistaken with Salomenlla and is differentiated by urea hydrolysis.
New members of Enterobacteriaceae:Kluyvera: This genus is made up of 3 closely related species K. ascorbata, K. cryocrescens, K.geogiana. They have found in respiratory, urine and blood. Produce a blue violet pigment usually on non blood containing media. All species resemble E. coli colonies growing on MacConkey’s agar, but it is citrate positive. K. ascorbata does not ferment D-glucose at 5 C, while K. cryocrescens ferment glucose at this temperature.
Buttiauxella: It is consist from 7 species isolated from water only B. agrestis & B. noackiae has been isolated from human specimens. It is biochemically similar to Citerobacter and kluyvera, however, DNA hybridization differentiate it . B.agrestis biochemical activity of IMViC phenomena is: -+-+.
The Family: Pseudomonaceae There is an important genus in this family which Pseudomonas. These m.o. are oxidase +ve (opposit to Enterobacteriaceae). Also, these m.o. are non-fermantative for CHO, but oxidative; so they require O2. These m.o. are aerobic, gram negative, motile, and NLF bacilli. They are found in soil, sewage and water, some are commensals in the intestine, others cause UTI, wound infection, and otitis media. It causes severe infection in hospitalized and compromised hosts.
Species of Pseudomonas: The most important species are: 1. P. aeruginosa (the most important one). 2. P. fluorescens. 3. P. mallei. 4. P. putida 5. P. pseudomallei. 6. P. stutzeri. 7. P. maltophilia
Pseudomonas aeruginosa: This m.o. Is the most important species. It is very important cause of nosocomial infections (responsible for 10-20% of these infections). It is quite resistant to many anti-microbial agents (has R factor). Morphology: Gram negative, motile m.o. It has a single polar flagellum , has a slime layer around it, and can attach to mucous membranes and surfaces by pili which resist phagocytosis.
Biochemical and cultural characteristics:P.aeruginosa can grow on all media mentioned for Enterobacteriaceae as nutrient, blood, brain-heart infusion, and MacConkey’s agars. They apopear as NLF and have the ability to tolerate alkaline pH (e.g 8.5), therefore, can overgrow on cultures of V. cholerae. The optimum temp. is 37 C, but P. aeruginosa can tolerate 42 C. Also, on blood agar P. aeruginosa can produce beta-haemolysis. This m.o. can produce the following pigments:
1. Pyocyanin: Blue. 2. Pyoverdin : Green or yellowish-green. 3. Pyorubin: Red. 4. Pyomelanin: Black or dark brown. The most common are 1 & 2. The growth and clinical specimens of this m.o. usually appear green or greenish-blue. The pigments are part of the pathogenicity of this m.o. The colonies appear NLF, smooth, flat, rounded or with irregular edges, and with very characteristic odour (sweetish aromatic, or grape-like).
Oxidative-Fermantative Test (OF): Two tubes of semisolid media(have low concentration agar, high CHO content, and an indicator called bromothymol blue) are used. They are inoculated with the m.o.; one is covered by mineral oil and the other is left opened. After 24-48 hours the covered tube remains green, while the opened becomes yellow. Also, in this test, the motility could be seen, which have tree-like appearance mainly toward the top of the tube (aerobic m.o.).
Pathogenesis & factors of pathogenicity: 1. Enzymes: two proteases that cause skin haemorrhage and corneal destruction. 2. Exotoxins: A, B, & C. 3. Haemolysins : two types. 4. Pigments: could affect O2 uptake by tissues and cells including leucocytes. 5. Enterotoxins: result in diarrhoea. 6. Pili : attachment & resist phagocytosis.
Clinical Infections: 1. Local: Burns, wound infection, corneal ulceration, UTI, pneumonia (has martality rate of 50%), bacterial endocarditis, and gastroenteritis. 2. General: Bacteraemia, & septicaemia that has martality rate of 80%. Treatment: The main problem is the antimicrobial resistance of P. Aeruginosa. Many strains are sensitive to carbenicillin, new penicillins, carbapenems, new fluoroquinolones. Antibiogram sensitivity test is required. In emergency cases gentamicin and carbenicillin are useful as in cases of burns, pneumonia or septicaemia. Prevension: Pseudogen vaccine, hypergammaglobulin can be used.