Systemic Bacteriology PDF

**MLS 536: Systemic Bacteriology** - Normal flora, pathogens sources of infection. - Laboratory diagnosis and identification of bacteria. - The enterobacteriaeceae, coliforms, gasteroenteritis, Salmonellosis, Shigellosis, Choleras, vibrios, Pseudomonas, Bacteriodes etc. - General pat...

**MLS 536: Systemic Bacteriology** - Normal flora, pathogens sources of infection. - Laboratory diagnosis and identification of bacteria. - The enterobacteriaeceae, coliforms, gasteroenteritis, Salmonellosis, Shigellosis, Choleras, vibrios, Pseudomonas, Bacteriodes etc. - General pathology, epidermilogy, features, diagnosis, control **ENTEROBACTERIACEAE** Enterobacteriaceae family contains a large number of genera that are biochemically and genetically related to one another. This group of organisms includes several that cause primary infections of the human gastrointestinal tract. Members of this family are major causes of opportunistic infection (including septicemia, pneumonia, and meningitis and urinary tract infections). Examples of genera that cause opportunistic infections are: Citrobacter, Enterobacter, Escherichia, Hafnia, Morganella, Providencia and Serratia. They are commonly found in the intestine of animals, hence got the name "enterobacteria" or "enteric bacteria". It is the most widely studied bacteria family. It contains several economically important and commensal bacteria; although some of them are pathogenic to humans, plants, and animals and can develop a fatal infection. The genera Escherichia, Klebsiella, Enterobacter, Serratia, and Citrobacter (collectively called the coliform bacilli) and Proteus include overt and opportunistic pathogens responsible for a wide range of infections. Many species are members of the normal intestinal flora. **Characteristics** Some of the common characteristics of Enterobacteria are: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. **Enterobacteriaceae Pathogenicity** A large number of species in the Enterobacteriaceae family are a part of the normal flora of the intestine of humans and animals.   However, they are frequently reported as a pathogen of plants, insects, and other animals including humans.  In animals, although they are normal gut flora, they cause gastrointestinal disorders like diarrhea, dysentery, mammary gland infection, hemorrhages, respiratory tract infections (RTIs), colonitis, etc. Enterobacteria are the major cause of [urinary tract infections (UTIs)](https://microbenotes.com/urinary-tract-infection-uti/) in humans. Besides, they cause respiratory tract infections (RTIs) (both lower and upper RTIs), bacteremia, septicemia, wounds and ulcers, gastrointestinal tract infections and disorders, systemic infections in internal organs, etc. Some species are well-known human pathogens, and even newer species in Enterobacteriaceae are emerging as human pathogens. Some most common human pathogens in this family are**:** *E. coli, Klebsiella spp., Salmonella spp., Shigella spp., Proteus spp., Morganella spp., Erwinia spp., Serratia marcescens, Citrobacter spp., Yersinia spp.* *E. coli, K. pneumoniae, *and*** **P. mirabilis* account for more than 80% of Enterobacteria species isolated from clinical samples. **Enterobacteriaceae ldentification** Like other bacteria, Enterobacteria can be identified by using either molecular diagnosis (PCR, DNA homology, 16s rRNA sequencing) system, biochemical tests, or antigen/antibody detection. The most commonly followed method used by microbiologists is a series of biochemical tests. Routinely followed biochemical tests are: - - - - - - - - **Enterobacteriaceae significances** ### **Positive significances** - - - ### **Negative significances** - - - **Antibiotics Resistance in Enterobactericeae** Enterobacteria are the most commonly encountered pathogenic bacteria in clinical cases and the environment. Hence, they are highly influenced by the intake of antibiotics for disease management or in agriculture and the environment. The scenario of antibiotic resistance among the members of Enterobacteriaceae is worst. Initially, beta-lactam antibiotics like derivatives of penicillin and the 1^st^ or 2^nd^ generation cephalosporins were considered effective against them. However, in the present situation due to the development of resistance against the Beta-Lactam antibiotics, the treatment option is very costly and limited. ESBL (extended-spectrum beta-lactamase), ABL (AmpC beta-lactamase), and other types of beta-lactamase-producing Enterobacteriaceae are responsible for several cases of human infection all around the world. Carbapenems also referred to as the last line of antibiotics, are also slowly losing their effectiveness due to the evolution of carbapenem's resistance Enterobacteria.  Carbapenems resistance genes are frequently encountered in several pathogenic species in the family. Drug resistance is increasing rapidly in *E. coli, Klebsiella spp., Salmonella spp., Shigella spp., *and* Citrobacter spp*. These resistance genes in these bacteria can be transferred horizontally among other members of Enterobacteriaceae, hence causing the rapid spread of resistance genes **COLIFORMS** **Coliform bacteria**  are defined as either [motile](https://en.wikipedia.org/wiki/Motile) or [non-motile](https://en.wikipedia.org/wiki/Non-motile) [Gram-negative](https://en.wikipedia.org/wiki/Gram-negative) non-[spore forming](https://en.wikipedia.org/wiki/Endospore) [bacilli](https://en.wikipedia.org/wiki/Bacillus_(shape)) that possess [β-galactosidase](https://en.wikipedia.org/wiki/Beta-galactosidase) to produce acids and gases under their optimal growth temperature of 35--37 °C. They can be aerobes or facultative aerobes, and are a commonly used [indicator](https://en.wikipedia.org/wiki/Indicator_bacteria) of low sanitary quality of foods, milk, and water. Coliforms can be found in the aquatic environment, in soil and on vegetation; they are universally present in large numbers in the feces of [warm-blooded](https://en.wikipedia.org/wiki/Warm-blooded) animals as they are known to inhabit the gastrointestinal system. While coliform bacteria are not normally causes of serious illness, they are easy to [culture](https://en.wikipedia.org/wiki/Microbiological_culture), and their presence is used to infer that other [pathogenic](https://en.wikipedia.org/wiki/Pathogen) organisms of fecal origin may be present in a sample, or that said sample is not safe to consume.  Such pathogens include disease-causing [bacteria](https://en.wikipedia.org/wiki/Bacteria), [viruses](https://en.wikipedia.org/wiki/Viruses), or [protozoa](https://en.wikipedia.org/wiki/Protozoa) and many multicellular [parasites](https://en.wikipedia.org/wiki/Parasite). **GENUS: ESCHERICHIA** ***Escherichia coli*** It is the main species of medical important among the genus. **Characteristics:** *Escherichia* coli are gram negative motile, non sporing bacillus. Ferments lactose (grows on MaCconkey agar), Catalase positive and Oxidase negative. Some strains of *Escherichia* coli are capsulated. They live in the human gut and are usually harmless but some are pathogenic causing diarrhea and other symptoms as a result of ingestion of contaminated food or water. *Escherichia* coli is acquire by ingestion in the first few days after birth. **Clinical features:** Urinary tract infection- cystitis, pyelonephritis. Wound infection- appendicitis, peritonitis. Neonatal septicemia and meningitis. *E.coli* causes: 1\. acute diarrhea in infants and depending on the strain found in the gut, they can also cause diarrhea disease in adult. 2\. they cause urinary tract infection especially in pregnant women 3\. ocasionally they are associated with wound infection, septicemia, meningitis (especially in new born) 4\. they also cause otitis media The specimen for collection depends on the site of infection e.g urine in case of *Escherichia* coli, pus in case of wound infection, stool in case of diarrhea, blood in case of septiceamia, CSF in case of meningitis. **Epidemiology** *Escherichia coli* results in intestinal illness as well as infection outside of the intestine. Intestinal illness caused by *E. coli *is caused by one of five subtypes, and they are identified according to their O and H antigens. The O antigen is determined by a repeating polysaccharide chain present in the lipopolysaccharide (LPS) outer membrane, and the flagellum determines the H antigen. **Pathophysiology** 1\. **ETEC (Enterotoxigenic *E. coli*):** Causes watery (secretory) diarrhoea due to the production of plasmidmediated toxins (LT, ST) in infants and adults, particularly in developing countries. It is often referred to as traveller's diarrhoea. Many serogroups are involved. 2\. **EPEC (Enteropathogenic *E. coli*):** Causes vomiting, fever, and prolonged diarrhoea mainly in infants (less than 2 y). Due to bacteria adhering to epithelial cells, multiplying and causing lesions. Many serogroups are involved. EPEC is a major problem in developing countries. 3\. **EIEC (Enteroinvasive *E. coli*):** Causes dysentery (similar to shigellosis), fever and colitis, with blood, mucus, and many pus cells in faecal specimens. Due to bacteria invading and multiplying in epithelial cells. Many serogroups are involved. 4\. **EHEC (Enterohaemorrhagic *E. coli*):** Causes life-threatening haemorrhagic diarrhoea (colitis) in all ages, without pus cells, and often without fever. It can progress to haemolytic uraemic syndrome with renal failure. EHEC has been reported mainly from Europe and North America. Outbreaks have also occurred in refugee camps in Mozambique, Swaziland and Malawi. EHEC is due to cytotoxins damaging vascular endothelial cells, and is mainly associated with the serogroup 0157:H7. It is sometimes referred to as VTEC (verocytotoxin-producing E. coli, because it is toxic to vero monkey cells in culture). Infection occurs by ingesting contaminated meat products, unpasteurized milk and dairy products. 5\. **EaggEC (Enteroaggregative *E. coli*):** Causes chronic watery diarrhoea and vomiting, mainly in children. Due to the bacteria adhering to tissue cells often in stacks (aggregates). **NOTE:** Each type of *E. coli* except enterotoxigenic strains can cause inflammatory [enteritis](https://www.sciencedirect.com/topics/medicine-and-dentistry/enteritis). Both enterohemorrhagic and enteroaggregative *E. coli* pathotypes occur commonly in the US. **Laboratory diagnosis:** Specimen: Urine, pus, blood, stool, body fluid Smear: Gram-negative rods Culture: Lactose-fermenting mucoid colonies on MaCconkey agar and some strains are hemolytic on blood agar. **Biochemical reaction**: Produce indole from tryptophan containing peptone water. Reduce nitrate to nitrite. Mortility +V Citrate - Ve Urease -Ve Glucose A/G Lactose +Ve Eijkman test +V is the ability of *E.coli* to produce indole at 44^o^C by breaking tryptophan and been able to produced acid and gas from lactose fermentation at 44^o^C. *E.coli* has three types of surface antigen demonstrated by serotyping used in agglutination technique include 1\. Somatic antigens or with over160 serogroups designated as 01, 02, 03 etc. 2\. Capsular Ag. K Ag- designated 3\. Flagella Ag or (Hag with over 60 serogroup) and designated as H1, H2, H3 etc. Serology: For serotyping (Epidemiologic information) Treatment: Base on antibiotic sensitivity pattern **GENUS: KLEBSIELLA** **Characteristics:** *[Klebsiella](https://en.wikipedia.org/wiki/Klebsiella) *are non-motile, Gram-negative bacilli ranging from 1--2 μm in length. They are facultative anaerobes with a capsule composed of complex acid [polysaccharides](https://en.wikipedia.org/wiki/Polysaccharide) that allows them to withstand drying for several months. *Klebsiella* *pneumonia* is the most common *Klebsiella* species found in humans, the [gastrointestinal tracts](https://en.wikipedia.org/wiki/Gastrointestinal_tract) of animals, in sewage and in soil. On carbohydrate-rich media, *Klebsiella* colonies appear greyish-white in colour with a mucosal outer surface. The media used for selecting *Klebsiella* species in a mixed sample is an agar including ornithine, raffinose, and Koser citrate, where members of this genus will form yellow, wet-looking colonies**.** Main species of medical importance are: *K. pneumoniae***:** It is found as a commensal in the intestinal tract, and also found in moist environment in hospitals. It is an important nosocomial pathogen. It causes Pneumonia, Urinary tract infection, Septicaemia and meningitis (especially in neonates). Wound infection and peritonitis. ***K. rhinoscleromatis*:** It causes rhinoscleroma of nose and pharynx to extensive destruction of nasopharynx (hebra nose) ***K. ozaenae*:** It causes ozena manifesting with foul smelling nasal discharge leading to chronic atrophic rhinitis. *K. aerogenes,* both K. Edwardsii and K. Atlantae rarely cause respiratory infection including pneumoniae Laboratory diagnosis of klebsiella species: **Specimen**: Sputum, urine, pus, CSF, body fluid Smear: Gram-negative rods **Culture:** Large, mucoid, lactose-fermenting colonies on mac conkey agar, and shows stringy type growth when cultured in broth medium. **Serology:** Capsular polysaccharide serotyping More than 80 serotypes of *K. pneumoniae* recognized. **Treatment**: Based on sensitivity testing **Quellung reaction:** When pneumococci on a slide smear are mixed with a small amount of antiserum (serum with antibodies to the capsular antigens) and methylene blue, the capsule will appear to swell. This technique allows for rapid identification of this organism. **GENUS:** [***ENTEROBACTER***](https://en.wikipedia.org/wiki/Enterobacter) **Characteristics:** *[Enterobacter](https://en.wikipedia.org/wiki/Enterobacter)** ***are motile, flagellated bacilli known for causing infections such as [bacteremia](https://en.wikipedia.org/wiki/Bacteremia), respiratory tract infections, [urinary tract infections](https://en.wikipedia.org/wiki/Urinary_tract_infection), infections of areas where surgery occurred, and in extreme cases [meningitis](https://en.wikipedia.org/wiki/Meningitis), [sinusitis](https://en.wikipedia.org/wiki/Sinusitis) and [osteomyelitis](https://en.wikipedia.org/wiki/Osteomyelitis). To determine the presence of *Enterobacter* in a sample, they are first grown on MaCconkey agar to confirm they are [lactose](https://en.wikipedia.org/wiki/Lactose) fermenting. It is gram-negative lactose fermenting motile rods, and found as a commensal in the intestinal tract of humans and animals and moist environments. Medical important species is *Enterobacter aerogens*. It produces mucoid colony resembling klebsiella on MaCconkey agar. *Enterobacter aerogens* is associated with urinary tract infection, wound infection and septicaemia in immunocompromised and chronically deblitated patients. An indole test will differentiate Enterobacter from *Escherichia*, as Enterobacter are indole negative and Escherichia is positive. *Enterobacter* are distinguished from *Klebsiella* because of their differences in motility. **IDENTIFICATION TEST** They are gram negative bacilli Indole -- Negative Urease -- Negative H~2~S -- Negative Motility -- Negative Lactose -- positive Glucose -- positive (A/G) Mannitol -- positive Members of these genera include: *E.aerogenes, E.cloacae, E.liqufaciens, and E.sakazakii* **GENUS: [*CITROBACTER*](https://en.wikipedia.org/wiki/Citrobacter)** **Characteristics:** [*Citrobacter*](https://en.wikipedia.org/wiki/Citrobacter): are [peritrichous](https://en.wikipedia.org/wiki/Peritrichous) facultative anaerobic bacilli between 0.6--6μm in length. *Citrobacter* species inhabit intestinal flora without causing harm, but can lead to urinary tract infections, [bacteremia](https://en.wikipedia.org/wiki/Bacteremia), brain abscesses, [pneumonia](https://en.wikipedia.org/wiki/Pneumonia), intra-abdominal sepsis, meningitis, and joint infections if they are given the opportunity. Infections of a *Citrobacter* species have a mortality rate between 33--48%, with infants and [immunocompromised](https://en.wikipedia.org/wiki/Immunocompromised) individuals being more susceptible. **Medical important species** is *Citrobacter freundii*. *Citrobacter freundii* is associated with urinary tract infection, wound infection and septicaemia in immunocompromised and chronically debilitated patients. **IDENTIFICATION TEST** Indole -ve Urease -ve Citrate +ve Lactose +ve (Late lactose fermenter) Mortilty +ve Glucose +ve acid/gas **GENUS: SALMONELLA** Most isolates of salmonellae are motile It grows readily on simple media It never ferment sucrose or sucrose Form acid +/- acid from glucose or mannose **Epidemiology** The genus *Salmonella* contains over 2,000 sero-species and is one of the most important pathogens in the family *Enterobacteriaceae*. Salmonellosis is a worldwide disease of humans and animals. Animals are the main reservoir, and the disease is usually food borne, although it can be spread from person to person. Species of medical importance are: *S. typhi* *S. paratyphi* *S. enteritidis* Clinical features: 1\. Enteric fever It is caused by *S.typhi* and *S.paratyphi*, and transmitted by fecal-oral route via contaminated food and drinks. - Exposure to the O and H antigens stimulates the production of specific antibodies - Natural infection provides limited protection #### 2. Enteritis and Septicemia: - Surface antigens: play a role in attachment and intracellular survival - Invasiveness -  Penetrate mucus, adhere to and invade into epithelial layer ([enterocytes](https://science.umd.edu/classroom/bsci424/Definitions.htm#Enterocyte)) of terminal [small intestine](https://science.umd.edu/classroom/bsci424/Definitions.htm#SmallIntestine) ([ileum](https://science.umd.edu/classroom/bsci424/Definitions.htm#Ileum)) and further into subepithelial tissue - Bacterial cells are internalized in endocytic vacuoles in which the organisms multiply -  PMN\'s confine infection to gastrointestinal (GI) tract, but organisms may spread hematogenously (through blood, i.e., septicemia) to other body sites -  Inflammatory response mediates release of prostaglandins, stimulating cAMP and active fluid secretion with loose diarrheal stools - Epithelial destruction occurs during late stage of disease **Exotoxins** - Several *Salmonella* serotypes produce enterotoxins similar to both the heat-labile ([LT](https://science.umd.edu/classroom/bsci424/HostParasiteInteractions/Exotoxin.htm#HLT)) and heat-stable enterotoxins ([ST](https://science.umd.edu/classroom/bsci424/HostParasiteInteractions/Exotoxin.htm#ST))**, **but their effect has not been identified - A** **distinct cytotoxin is also produced and may be involved in invasion and cell destruction **Invasivenes** - Pass through intestinal epithelial cells in [ileocecal](https://science.umd.edu/classroom/bsci424/Definitions.htm#Ileocecal) region and infect the regional [lymphatic system](https://science.umd.edu/classroom/bsci424/Definitions.htm#LymphaticSystem), invade the bloodstream, and infect other parts of the [reticuloen dothelial system](https://science.umd.edu/classroom/bsci424/Definitions.htm#RES) - Organisms are phagocytosed by [macrophages](https://science.umd.edu/classroom/bsci424/Definitions.htm#Macrophage) and [monocytes](https://science.umd.edu/classroom/bsci424/Definitions.htm#Monocyte), but survive, multiply and are transported to the liver, [spleen](https://science.umd.edu/classroom/bsci424/Definitions.htm#Spleen), and bone marrow where they continue to replicate - During the second week, the organisms reenter the bloodstream and cause prolonged bacteremia; biliary tree and other organs are infected; gradually increasing and sustained fever most likely from endotoxemia - During the second to third week, the bacteria colonize the gall bladder and reinfect the intestinal tract with diarrheal symptoms and possible necrosis of the Peyer\'s patches - **Endotoxin**: could be responsible for the fever or the enteric lesions seen in typhoid fever Incubation period: 10-14 days Predisposing factors: - Reduced gastric acidity - Disrupted intestinal microbial flora - Compromised local intestinal immunity Both manifest with persistent fever, headache, malaise, chills, enlargement of liver and spleen, and skin rashes. Paratyphoid fever is milder than typhoid fever. **Complications:** Intestinal perforation Lower gastrointestinal bleeding Dissemination to different body organs including meninges and brain **Mortality rate:** Untreated cases: 10-15% Treated cases: \< 1% 2\. Bacteremia with focal lesions Causative agent: *S. choleraesuis* Manifests with blood stream invasion with focal lesions in lungs, bones and meninges Intestinal manifestation are often absent 3\. Gastroenteritis It is caused by *S. enteritidis* *S. typhimurium* Incubation period= 8-48 hrs It manifests with initial watery diarrhea, and later bloody mucoid diarrhea associated with crampy abdominal pain and tenesmus. Bacteremia is rare (2-3 % of cases). It usually resolves in 2-3 days. 1\. Blood, Bone marrow, stool, urine and serum for enteric fever. Blood -- 80% positive in the first week.. Stool- 70-80% positive in the second and third week..Urine- 20% positive in the third and fourth week..Serum for widal test- positive after the second week of illness. 2\. Stool for gastroenteritis. Gram reaction: Gram-negative rods Culture: Bacteriologic methods for salmonella isolation 1. Differential medium. For rapid isolation of lactose non-fermenters E.g.EMB agar MaCconkey agar Deoxycholate agar 2\. Selective medium. Favor growth of salmonella and shigella over other enterobacteriaceae E.g. SS agar Hekton Enteric agar, XLD agar Deoxycholate-Citrate agar 3\. Enrichment cultures. Inhibit replication of normal intestinal flora and permit replication of salmonella E.g. Selenite F broth, Tetrathionate broth, Non-lactose fermenting, H~2~S producing colonies in MaCconkey agar. Biochemical reaction: Generally produce gas and acid from carbohydrate; except *S. typhi* which does not produce gas. Serology: (widal test) a. Tube dilution agglutination test. b. Used to determine antibody titers in patients with unknown illness Method: Serial dilutions of unknown serum are tested against antigens from representative salmonella species. The highest diluted serum with positive result is taken as a tite Interpretation of result High or rising titer to O antigen (≥ 1:160) suggests active infection. High or rising titer to H antigen (≥ 1:160) suggests past infection or immunization. High titer to the Vi antigen occurs in some cases Causes of false positive Widal test \- Malaria infection \- Other acute febrile illness \- Poor quality reagent Causes of false negative widal test \- Specimen collected after antibiotic administration \- Specimen collected at early stage of diseases \- Technical errors a\. Slide agglutination test/ Kauffman-White system Used to identify unknown cultures with known sera Required: Salmonella O and H polyvalent antiserum Method:. Mix known sera with unknown culture on a slide.. Clumping occurs within a few seconds in positive result NB: Slide agglutination test is important for preliminary identification of culture Treatment: 1\. for cases Chloramphenicol Fluoroquinolones 3rd generation cephalosporins 2\. For carriers Ampicillin followed by cholecystectomy NB: Salmonellae persist in gall bladder in chronic carriers **Prevention and control** - Enteritis: Supportive therapy, dehydration prevention, balancing electrolytes; - Avoid antibiotics in gastroenteritis to prevent prolonged carrier state - Enteric fever or septicemia: ampicillin or chloramphenicol; resistant isolates occur - *S. typhi *carried in gall bladder; surgically remove and treat with ampicillin; 85% cure of carrier state - Observe water standards -  Cook and store foods properly - Treat carriers, special precautions with food handlers - Vaccines for typhoid fever - Sanity measures like hygienic food and drinks handling, and avoid carriers from food handling until properly treated - Provision of vaccine Injectable acetone-killed *S. typhi* suspensions Oral live, avirulent mutant strain of *S. typhi* in high endemic areas **GASTROENTERITIS (INFLAMATION S.INTESTINE ND STOMACH)** Gastroenteritis is the inflammation of the mucus membranes of the stomach and the S.intestine usually by microorganisms e.g. *S aureus, B cereus, Clostridum, E.coli, Shigella,* Salmonella*, vibrio,* Yesinia, Campylobacter*, Rota virus, Giardia lambia.* It is caused by *S. enteritis* *S. typhimurium* The etiologic agents maybe toxigenic, invasive or both. The infection is spread by facal-oral rout either directly or indirectly. The infection occurs as a result of a carrier, handling the food by preparation of the food in utensils infected by handling or washing with infected water or by flies perching on the food after feeding on expired faces Dietetic error may sometimes result in Gastroenteritis. **Food poisoning** It applied to illness acquired from eating food contaminated with chemical poisoning, pre-formed bacteria toxins or live bacteria or poisonous material vegetation e.g. Berries. Food poisoning results in vomiting with or without diarrhea. Condition enabling the growth of the bacteria and the production of enterotoxins in the contaminated food are pre-requisites for bacteria food poisoning (long time improper storage, wrong handling, and cold food e.t.c). Food borne infection may result from the infection of only a few pathogenic microorganisms. Gastro intestinal infections are very common especially in developing countries. Public health prevention, through fostering good hygiene and providing sanitary/ potable water and food supplies is of most important **GENUS: SHIGELLA** Shigella is a genus of the bacterial family Enterobacteriaceae. Shigellae are Gram-negative, non-motile, non-spore forming, rod-shaped bacteria, very closely related to *Escherichia coli.* Shigellosis is an infectious disease caused by various species of Shigella. People infected with Shigella develop diarrhea, fever, and stomach cramps starting a day or two after they are exposed to the bacterium. The diarrhea is often bloody. Shigellosis usually resolves in 5 to 7 days, but in some persons, especially young children and the elderly, the diarrhea can be so severe that the patient needs to be hospitalized. A severe infection with high fever may also be associated with seizures in children less than 2 years old. Some persons who are infected may have no symptoms at all, but may still transmit the Shigella bacteria to others. Shigella were discovered over 100 years ago by a Japanese microbiologist named Shiga, for whom the genus are named. There are four species of *Shigella: boydii, dysenteriae, flexneri, and sonnei. Shigella sonnei,* also known as \"Group D\" Shigella, accounts for over two-thirds of the shigellosis in the United States. *Shigella flexneri*, or \"group B \"Shigella, accounts for almost all of the rest. Other types of Shigella are rare in this country, although they are important causes of disease in the developing world. One type, *Shigella dysenteriae* type 1, causes deadly epidemics in many developing regions and nations. Pathogenesis and Clinical features: ### Pathogenesis Shigella infection is initiated by ingestion of shigellae (usually via fecal-oral contamination). An early symptom, diarrhea (possibly elicited by enterotoxins and/or cytotoxin), may occur as the organisms pass through the small intestine. The hallmarks of shigellosis are bacterial invasion of the colonic epithelium and inflammatory colitis. These are interdependent processes amplified by local release of cytokines and by the infiltration of inflammatory elements. Colitis in the recto sigmoid mucosa, with concomitant malabsorption, results in the characteristic sign of bacillary dysentery: scanty, unformed stools tinged with blood and mucus. Shigellosis occurs mostly at the extremes of life or in individual who are usually suffering from other diseases. Young children over 6months of age are frequently infected than adults. In fact, most shigella infection occurs in children 10years of age. These organisms are strictly human pathogens. i.e are not found in other animal spp. Except in higher primates. They are also not found in other organs of the body. Their activities are localized in the gut i.e localized infection. Incubation period is 1-2days and symptoms include sudden onset of abdominal pains, fever, and watery diarrhea colon involvement dysentery. **IDENTIFICATION TEST** Gram -ve bacilli Mortility -ve H2S -ve KCN -ve Citrate -- ve Glucose acid only Route of infection is fecal-oral route inoculum dose of organisms Pathogenicity determinant: Toxins: **Endotoxin:** irritate the bowel wall Exotoxin: **Enterotoxin** and neurotoxin *S. dysenritiae* type 1(shiga bacillus) produce heat labile exotoxin mediated diarrhea. Incubation period: 1-2 days It causes shigellosis (bacillary dysentery) characterized by sudden onset of bloody mucoid diarrhea, abdominal cramp, tenesmus, fever, generalized muscle ache and weakness. **Reiter\'s syndrome** Persons with diarrhea usually recover completely, although it may be several months before their bowel habits are entirely normal. About 3% of persons who are infected with *Shigella flexner*i may subsequently develop pains in their joints, irritation of the eyes, and painful urination. This condition is called Reiter\'s syndrome. It can last for months or years, and can lead to chronic arthritis which is difficult to treat. Reiter\'s syndrome is a late complication of S. Flexner infection, especially in persons with a certain genetic predisposition, namely HLA-B27. **Hemolytic Uremic Syndrome (HUS)** Hemolytic-uremic syndrome (HUS) can occur after *S. dysenteriae* type 1 infection. Convulsions may occur in children; the mechanism may be related to a rapid rate of temperature elevation or metabolic alterations, and is associated with the production of the Shiga toxin, which is discussed below. **Transmission** Shigellas are transmitted from an infected person to another who becomes infected. Shigella are present in the diarrheal stools of infected persons while they are sick and for a week or two afterwards. Most Shigella infections are the result of the bacterium passing from stools or soiled fingers of one person to the mouth of another person. This happens when basic hygiene and hand washing habits are inadequate. It is particularly likely to occur among toddlers who are not fully toilet-trained. Family members and playmates of such children are at high risk of becoming infected. The spread of Shigella from an infected person to other persons can be stopped by frequent and careful hand washing with soap, a practice that is important among all age groups. Part of the reason for the efficiency of transmission is because a very small inoculum (10 to 200 organisms) is sufficient to cause infection. As a result, spread can easily occur by the fecal-oral route and occurs in areas where hygiene is poor. Epidemics may be foodborne or waterborne. Shigella can also be transmitted by flies. Shigella infections may be acquired from eating food that has become contaminated by infected food handlers. Vegetables can become contaminated if they are harvested from a field with contaminated sewage or wherein infected field workers defecate. Flies can breed in infected feces and then contaminate food. Shigella infections can also be acquired by drinking or swimming in contaminated water. Water may become contaminated if sewage runs into it, or even if someone with shigellosis swims or bathes or, much less, defecates, in it. Complication: Dehydration Electrolyte and acid-base disturbance High prevalence: Poor sanitation, Poor personal hygiene, Polluted water supply, Young children are frequently affected. **Laboratory diagnosis**: Specimen: Stool, serum Gram reaction: Gram-negative non-motile rods. **Culture:** Non-lactose fermenting colonies on MaCconkey agar and SS agar. **Biochemical reaction:** It produces acid but not gas from carbohydrate. **Serology:** For sero grouping and serotyping. It is not used to diagnose shigella infection **Treatment:** Ciprofloxacin, Cotrimaxazole suppress acute clinical attacks of dysentery.Shorten the duration of symptoms. **Prevention and control**: Sanitary control of water, food and milk, sewage disposal and fly control Antibiotic treatment of infected individuals 2\. GENUS: PSEUDOMONAS **General characteristics:** Gram-negative motile aerobic rods having very simple growth requirement. It can be found in water, soil, sewage, vegetation, human and animal intestine.. Species of medical importance: *P. aeruginosa* *P. pseudomallei* *Pseudomonas aeruginosa*. Found in human and animal intestine, water, soil and moist environment in hospitals.. Primarily a nosocomial pathogens..Invasive and toxigenic, produces infections in patients with abnormal host defenses Antigenic characteristic:.Pili: Adhere to epithelial cells.Exopolysaccharide: Anti-phagocytic property/ inhibit pulmonary clearance.Lipopolysaccharide: Endotoxic effect. Enzymes.Elastases: Digests protein (elastin, collagen, IgG).Proteases.Hemolysins.Phospholipases C (heat labile): Degrade cytoplasmic membrane components.. Exotoxin A: Cytotoxic by blocking protein synthesis Clinical features: Pathogenic only when introduced into areas devoid of normal defenses e.g. breached mucus membrane or skin, use of IV line or urinary catheterization, neutropenia of any cause. Urinary tract infection \- Chronic, complicated Urinary tract infection and associated with indwelling catheter.. Wound infection of burn sites, pressure sores and ulcers..Septicaemia- "Ecthyma gangrenosum" skin lesion (haemorrhagic skin necrosis). Otitis external \- Malignant external ear infection in poorly treated diabetic patients.. Pneumonia- Infection of the lung in patients with cystic fibrosis.. Eye infection- Secondary to trauma or surgery **Laboratory diagnosis:** Specimen: pus, urine, sputum, blood, eye swabs, surface swabs Smear: Gram-negative rods Culture:.Obligate aerobe grows readily on all routine media over wide range of temperature (5-42 OC).. Bluish-green pigmented large colonies with characteristic "fruity" odor on culture media.. Biochemical reaction:. Oxidase positive. Catalase positive. Citrate positive. Indole negative. Produce acid from carbohydrate by oxidation, not by fermentation NB: identification of the bacteria is based on colony morphology, oxidase-positivity, characteristic pigment production and growth at 42 ^O^ c Treatment: Ticarcillin or piperacillin and aminoglicosides, Aztreonam, Imipenem Ceftazidime, Cefoperazone, Flouroquinolones **Prevention and control:** Special attention to sinks, water baths, showers and hot tubs Polyvalent vaccine to high risk groups. **GENUS: VIBRIOS** - Actively motile, gram-negative curved rods. - Species of medical importance: - Vibrio cholerae-01 - Vibrio cholerae **Characteristics:**. Found in fresh water, shellfish and other sea food.. Man is the major reservoir of V. cholerae-01, which causes epidemic cholera.. Readily killed by heat and drying; dies in polluted water but may survive in clean stagnant water, especially in alkaline, or sea water for 1-2 weeks. **Clinical features:** Route of infection is fecal-oral route. After ingestion of the V.cholerae-01, the bacteria adheres to the intestinal wall without invasion then produces an exotoxin causing excessive fluid secretion and diminished fluid absorption resulting in diarrhea (rice water stool) which is characterized by passage of voluminous watery diarrhea containing vibrio, epithelial cells and mucus; and result in severe dehydration. **Cholera** **Vibrio cholerae** Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrheal disease caused by the bacterium Vibrio cholerae. Transmission to humans is by water or food. The natural reservoir of the organism is not known. It was long assumed to be humans, but some evidence suggests that it is the aquatic environment. V. cholera produces cholera toxin, the model for enterotoxins, whose action on the mucosal epithelium is responsible for the characteristic diarrhea of the disease cholera. In its extreme manifestation, cholera is one of the most rapidly fatal illnesses known. A healthy person may become hypotensive within an hour of the onset of symptoms and may die within 2-3 hours if no treatment is provided. More commonly, the disease progresses from the first liquid stool to shock in 4-12 hours, with death following in 18 hours to several days. The clinical description of cholera begins with sudden onset of massive diarrhea. The patient may lose gallons of protein-free fluid and associated electrolytes, bicarbonates and ions within a day or two. This results from the activity of the cholera enterotoxin which activates the adenylate cyclase enzyme in the intestinal cells, converting them into pumps which extract water and electrolytes from blood and tissues and pump it into the lumen of the intestine. This loss of fluid leads to dehydration, anuria, acidosis and shock. The watery diarrhea is speckled with flakes of mucus and epithelial cells (\"rice-water stool\") and contains enormous numbers of vibrio. The loss of potassium ions may result in cardiac complications and circulatory failure. Untreated cholera frequently results in high (50-60%) mortality rates. **Treatment** of cholera involves the rapid intravenous replacement of the lost fluid and ions. Following this replacement, administration of isotonic maintenance solution should continue until the diarrhea ceases. If glucose is added to the maintenance solution it may be administered orally, thereby eliminating the need for sterility and iv\. administration. By this simple treatment regimen, patients on the brink of death seem to be miraculously cured and the mortality rate of cholera can be reduced more than ten-fold. Most antibiotics and chemotherapeutic agents have no value in cholera therapy, although a few (e.g. tetracyclines) may shorten the duration of diarrhea and reduce fluid loss. **History and spread of epidemic cholera** Cholera has smoldered in an endemic fashion on the Indian subcontinent for centuries. There are references to deaths due to dehydrating diarrhea dating back to Hippocrates and Sanskrit writings. There are several characteristics of the El Tor strain that confer upon it a high degree of \"epidemic virulence\" allowing it to spread across the world as previous strains have done. First, the ratio of cases to carriers is much less than in cholera due to classic biotypes (1: 30-100 for El Tor vs. 1: 2 - 4 for \"classic\" biotypes). Second, the duration of carriage after infection is longer for the El Tor strain than the classic strains. Third, the El Tor strain survives for longer periods in the extra intestinal environment. Between1969 and 1974, El Tor replaced the classic strains in the heartland of endemic cholera, the Ganges River Delta of India. **Antigenic Variation and LPS Structure in Vibrio cholera** Antigenic variation plays an important role in the epidemiology and virulence of cholera. The emergence of the Bengal strain, mentioned above, is an example. The flagellar antigens of V. cholera are shared with many water vibrios and therefore are of no use in distinguishing strains causing epidemic cholera. O antigens, however, do distinguish strains of V. cholera into 139 known serotypes. Almost all of these strains of V. cholera are non-virulent. Until the emergence of the Bengal strain (which is \"non-O1\") a single serotype, designated O1, has been responsible for epidemic cholera. However, there are three distinct O1 biotypes, named Ogawa,Inaba and Hikojima, and each biotype may display the \"classical\" or El Tor phenotype. The Bengal strain (O139) is a new serological strain with a unique O-antigen which partly explains the lack of residual immunity. Antigenic Determinants of Vibrio cholerae Ogawa A, B ---------- --------- Inaba A, C Hikojima A, B, C **Laboratory diagnosis:** **Specimen:** Stool flecks **Smear**: Gram-negative motile curved rods Motility of vibrios is best seen using dark-field microscopy. Presumptive diagnosis: Inactivation of vibrios in a wet preparation after adding vibrio antiserum. Culture: 1\. TCBS (Thiosulphate citrate bile salt sucrose agar) media Selective media for primary isolation of V.cholerae.. Observe for large yellow sucrose-fermenting colonies after 18-24 hours of incubation. 2\. Alkaline peptone water: Enrichment media for V.cholerae-01 Growth on and just below the surface of peptone water within 4-6 hours at room temperature as well as 37 o c. Biochemical Reaction:.Oxidase - positive.. Ferment sucrose and maltose (acid; no gas)..Do not ferments L-arabinose. Treatment: Sensitive to tetracycline and chloramphenicol. Fluid and electrolyte replacement are the first line of management for cholera. **Pseudomonas and Pseudomonas-like Bacteria** The genus comprises a relatively large and important group of Gram-negative bacteria. Members of the genus are found abundantly as free-living organisms in soils, fresh water and marine environments, and in many other natural habitats. They may also be found in associations with plants and animals as normal flora or as agents of disease. For the purposes of this article, the term \"pseudomonad\" refers to a bacterium with ecophysiological properties similar to members of the genus Pseudomonas. Some of these bacteria were formerly in the genus Pseudomonas but have been moved to other genera, families, or orders among the alpha Proteobacteria because of their phylogenetic distance from Pseudomonas. Morphologically, members of the genus Pseudomonas (as well as most other pseudomonads) may be described as Gram-negative, non-spore forming, straight or slightly curved rods. They are typically motile by means of one or more polar flagella. These basic morphological characteristics, however, are common to many families of bacteria and so are of little value in the positive identification or diagnosis of a member of the genus Pseudomonas. Generally, common to all constituent species of the genus Pseudomonas are certain physiological properties such as chemoorganotrophic nutrition, aerobic metabolism, absence of fermentation, absence of photosynthesis, in ability to fix nitrogen, and capacity for growth at the expense of a large variety of organic substrates. **GENUS PSEUDOMONAS** **General characteristics of the genus Pseudomonas** Gram-negative Rod-shaped, 0.5-0.8 um x 1-3 um Strictly aerobic; the only anaerobic activities may be denitrification and arginine degradation to ornithine Motile by polar flagella; some strains also produce lateral flagella Oxidative, chemoorganotrophic metabolism Catalase-positive Usually oxidase-positive No organic growth factors are required Diffusible and/or insoluble pigments may be produced GC content of the DNA: 58-68 mol % *P. aeruginosa* has attracted the most attention from general and clinical microbiologists, geneticists, and biochemists. The list of materials from which this species can be isolated is almost endless; so that from a practical point of view, one can assume that the bacterium is present everywhere. Most strains of the species can be easily identified by a number of phenotypic characteristics never found in the same combination in other species. Most important among these are production of pigments, including pyacyanin, the ability to denitrify, and the ability to grow at 41°C. *P. aeruginosa* most strains of the species can be easily identified by a number of phenotypic characteristics never found in the same combination in other species. Most important among these are production of pigments, including pyocyanin, the ability to denitrify, and the ability to grow at 41°C. The classification of pseudomonas is based now on ribosomal Rrna and DNA homology and common cultural characteristics. The medically important Pseudomonas are: +-----------------------+-----------------------+-----------------------+ | S/N | rRNA homology group | Genus and species | | | and sub group | | +=======================+=======================+=======================+ | 1 | Fluorescent group | *P.aeruginosa* | | | | | | | | *P. flourescens* | | | | | | | | *P.putida* | +-----------------------+-----------------------+-----------------------+ | | Non- Fluorescent sp. | *P. stutzeri,* | | | | | | | | *P. mendocina* | +-----------------------+-----------------------+-----------------------+ | ii | | *P. cepacia,* | | | | | | | | *P. pseudomallei,* | | | | | | | | *P. mallei,* | | | | | | | | *P. pickettii* | +-----------------------+-----------------------+-----------------------+ | III | | Comamonas, | | | | | | | | acidovorans, | +-----------------------+-----------------------+-----------------------+ **IDENTIFICATION TEST** Gram- negative rod/bacilli Oxidase +ve Catalase + ve Gelatin Liquefaction +ve KCN +ve Growth on 0.4% cadmium sulphate agar +ve Growth at cetrimide agar +ve Indole -ve H2S -ve VP -ve Strictly aerobic **Pathogenesis of *P. aeruginosa*** *P. aeruginosa* has long been known as an opportunistic pathogen, especially dreaded in the hospital environment. Early reports pointing to infection with this organism described blue pus associated with wound infections. *P. aeruginosa* has been isolated from wounds in almost all locations in the human or animal body, as well from purulent infections of the urinary and respiratory tracts. *P. aeruginosa* associated with pneumonia, enteritis, vaginitis, mastitis, and endometritis in animals is abundantly recorded in the literature. Since the tissue invasiveness of the organism is very limited, *P. aeruginosa* usually uses accidental ports of entry (burns, wounds, intravenous and urinary catheterization, surgical procedures, etc.) to gain access to its compromised host. Several extracellular products (proteases, elastase, etc.) help the invasion and dissemination of *P. aeruginosa*. Most isolates of the species produce exotoxin A, which is induced under the conditions of iron limitation that characterize many animal tissues. The target of this toxin is one of the elongation factors in translation during protein synthesis. Strains incapable of producing exotoxin A have reduced virulence. ***P.maltophilia (Strenotrophomonas maltophilia)*** These characteristics are similar to *P. aeruginosa* except that they are oxidase negative. They are widely distributed in the environment and they are hospital acquired. They are lysine decarboxylate positive. ***P. fluorescens and P. putida*** These species were described a few years after the description of *P. aeruginosa*. In human and veterinary medicine, while *P. aeruginosa* has historically been the most significant pathogenic species, it is now becoming evident that other species of the genus may be serious opportunistic pathogens. This includes the fluorescent pseudomonads, herein described, and certain non-fluorescent species which will be discussed below. Both *P. fluorescens* and its close relative, *P. putida*, have a natural history similar to *P. aeruginosa*. Two phenotypic characteristics of *P. fluorescens* that distinguish it from *P. putida* are its ability to grow at 4°C, and its ability to hydrolyze gelatin. These characteristics help explain its frequent involvement in spoilage of refrigerated food, in particular chicken and processed meats. If it\'s fluorescent, get rid of it! ***B. mallei*** (**Burkhoideria**) is the etiologic agent of glanders, a serious disease of equines, and *B. pseudomallei* causes a related disease. Glanders can be transmitted from equines to animals of other groups and also to humans. The disease can start in the respiratory tract or as an ulcerative process, rapidly spreading to the lymph nodes with fatal consequences. The organism is not found living freely in Nature, and normally passes from animal to animal. **Genus: Bacteroides** Bacteroides spp are non-spore forming gram-negative bacilli that are part of the human resident flora. Microbiologically, they are distinguished from other genera by growth in 20% bile. At present, the *Bacteroides* *fragilis* group consists of ten species: *B. fragilis* (the most frequent isolate), *B. distasonis*, *B. thetaiotaomicron*, *B. vulgatus*, *B. ovatus*, *B. eggerrthii*, *B. merdae*, *B. stercoris*, *B. uniformis*, and *B. caccae*. Bacteroides, the predominant genus in the human intestine, are important in numerous metabolic activities and may provide some level of protection from invasive pathogens. All 10 species are usually isolated from the colon, although infections caused by or associated with them can include virtually any organ. Isolation and identification of *Bacteroides* spp pose a hurdle to many clinical laboratories; most only identify the genus. Other laboratories may identify *B. fragilis* species, and lump all others into the *B. fragilis* group. Further, if more than three anaerobic organisms are isolated in a clinical specimen, many laboratories do not perform further identification. Those laboratories that do identify *Bacteroides* frequently do not perform susceptibility testing. Lack of identification and susceptibility testing of anaerobes can be attributed, in part, to its expense and the lack of timeliness in providing relevant information to the clinician. Thus, choice of therapy for infections that may involve *Bacteroides* is empiric. Characteristics of *Bacteroides* -------------------------------- - - - - - - - - Classification of *Bacteroides* ------------------------------- - ### **1. **Bacteroides fragilis group: - - - - - - - - - - - ### **2. **Bacteroides melaninogenicus group - - - **Epidermiology** *Bacteroides fragilis* are endogenous organisms of the GI tract. Spread of strains among patients is not known. Thus, infections due to this organism are most likely caused by endogenous strains. **Laboratory diagnosis** *Bacteroides fragilis* may be isolated as a single agent, such as in blood cultures, or more typically from mixed infections. The organism is aerotolerant, but requires an anaerobic environment to propagate. Simple identification from blood cultures includes Gram stain and growth on blood agar and *Bacteroides*-bile-esculin (BBE) agar for isolation and presumptive identification of *Bacteroides fragilis* group (as well as *Bilophila wadsworthia*). *B. fragilis* will appear as dark colonies with brown-black halos on BBE agar due to the hydrolysis of esculin. *B. fragilis* can be further presumptively identified by resistance to kanamycin, vancomycin and colistin, using a disk test, and will grow in 20% bile, produce catalase (most strains), and is variably indole positive. Many laboratories will confirm the identification using a rapid identification kit or individual fermentation reactions. ### **Biochemical tests for ***fragilis* - - - - - - - - - **Pathogenesis** Multiple virulence factors have been implicated in the pathogenesis of this organism. They include the capsular polysaccharide (which inhibits opsonophagocytosis and promotes abscess formation), pili and fimbriae (promotes adherence), and production of a number of different enzymes (hyaluronidase, hemolysin, peroxidase, collagenase, protease, heparinase, and neuraminidase). In addition, superoxide dismutase and catalase also considered virulence factors. These enzymes defend *B. fragilis* against oxygen radicals and increase aerotolerance