Curved Bacilli

Gram Negative Curved Bacilli There are certain microorganisms that share this type of morphology which is sometimes is described as comma-shaped. The following m.o. are curved gram negative bacilli: 1. Vibrio. 2. Aeromonas. 3. Plesiomonas. 4. Campylobacter. 5. Helicobacter


Characteristics of Gram Negative Curved Bacilli: These m.o. are: 1. Gram negative curved bacilli. 2. Motile by a single polar flagellum. 3. Many live in fresh water, and some in sea water (called marine m.o.). 4. Some of these m.o. can cause diarrhoea in humans.


Family: Vibrionacea Genus : Vibrio Genus Vibrio is classified practically into 10 important species: 1. Vibrio cholerae : Causes epidemic and pandemic cholera. 2. Non-cholera Vibrios: Cause ear, wound, soft tissue, and extra-intestinal infections. The important species of this group of m.o. are: V. mimicus, V.vulnificus, V. hollisae*, V. fluvialis*, V. damsela*, V. furnissi*, V. alginolyticus, V. metschnikovii. = Group F Vibrio. 3. Vibrio parahaemolyticus: Causes gastroenteritis, and possibly extra-intestinal infections.


Vibrio cholerae: Pacini in 1854 obseved this microorganism, and Robet Koch in 1883 described it as a comma-shaped m.o. Vibrios have 2 important antigens; H (shared), and O (specific) which is lipopolysaccharides. There are at least 139 O - antigen groups. Vibrio cholerae of O group 1 (o1) and 139 (O139) cause the classical disease cholera, while the non-O1/ non-O139 V. cholerae cause cholera-like disease. Both O1 and O 139 V.cholerae can be of two biotypes: 1. Classical 2. El-Tor Then both biotypes can be subgrouped into serotypes according to 3 antigenic factors (A, B, and C) : Ogawa (A,B), Inaba (A,C), and Hikojima (A,B.C).

Vibrio cholerae (O-1 and O-139): These m.o. are highly aerobic, very actively motile (described as shooting stars or darting motility), and Pacini and Koch were able to detect these m.o. with the help of this type of motility. The best way to detect motility is by dark-field or contrast microscopy. After a prolonged culture, the m.o. become straight (similar to enteric bacilli). These m.o. can tolerate temprature of range 18-37.

Biochemical characteristics of V. cholerae: They ferment glucose, maltose, mannite, and sucrose with acid production only. These m.o. are respiratory (oxidase positive) and fermentative (catalase positive). They give positive cholera-red reaction on nitrate peptone medium by reducing nitrate to nitrite, and produce indol (+ve) on tryptophan medium. When the latter 2 tests are combined, red colour is also produced due to nitroso-indol when H2SO4 is added. The TSI shows A/A profile (yellow colour). The optimum pH for the growth of these m.o. is 7.0, but they can tolerate up to 8.5 - 9.0. They are very sensitive to acidic pH (less than 6), therefore, they are quite susceptible to gastric juice (a major barrier against V. cholerae). V. cholerae 0-139 is similar to El-Tor, but it differs in being capsulated.

Culture of V. cholerae:This m.o. can be cultivated on different media:1. Alkaline Peptone Water (APW; Sea Water):This medium is of pH 8.5 – 9.0 and used for the primary (first) isolation from clinical specimens. The m.o. grow forming a surface pellicle within 8 hours. Then, from this medium the m.o. can be sub- cultured on solid media (see below). Many m.o. can not tolerate such pH, butPseudomonas does.2. Thiosulfate Citrate Bile Salt Sucrose Agar (TCBS):The non-inoculated medium is olive green in colour, and contains an indicator called bromo-thymol blue.V.Cholerae is sucrose- fermenter, and due to acid production, the pH drops and colour of the medium and colonies become yellow. Aeromonas can produce similar chages in this medium.


Cont/…culture V. cholerae3. Tellurite Taurocholate Gelatin Agar (TTGA):On this medium the colonies show iridescence and are surrounded by cloudy zone of gelatin hydrolysis.4. Meat Extract Agar:On this medium the colonies appear translucent green to red bronzy in colour and in old cultures the colonies become opaque and rough.5. Taurocholate Pepton Broth:This medium can be used primary cultivation of the m.o., then sub-cultured on other media.6. MacConkey’s Agar:V.Cholerae colonies are of non-lactose fermenters.7. Blood Agar:El-Tor growth shows beta-haemolysis ( vesus the classical).In general, the colonies of V. cholerae are convex, smooth, granular.


Practical procedure of cultivation of V. cholerae:. 1. Direct stool examination: a. Motility: Dark field or contrast microscopy. B. Microscopical exam.: mucous, epith. cells, and large numbers of m.o., but no pus cells are seen. c. Fluorescent antibody staining.2. 2 ml of faeces + 20 ml APW pH 8.0 – 8.5 are incubated for 5 hours. At the same time, TCBS is heavily inoculated, and left for overnight.3. From the APW, TCBS and another APW are inoculated and left either for 5 hours or overnight.4. The colonies are subjected to further identification (see below):

Further Identification of V. cholerae: 1. String test: A drop of 0.5% Na-deoxycholate is mixed with a colony (?); after 60 seconds, with a loop there will a thread of tenacious nature. If positive, it means V. cholerae. 2. Polyvalent anti-sera (anti-O1, anti- O139): To confirm that the V. cholerae belongs to O-1 or O-139, agglutination test with specific polyvalent sera and emulsified colonies is carried out. 3. Biochemical tests including cholera red reaction. 4. Differentiation between classical & El-Tor Biotypes: See the following Table. 5. Serotyping: by specific antisera against A, B and A antigenic factors to determine Ogawa, Inaba & Hikojima.

Table: Differentiation between Classical and El-Tor biotypes of V. cholerae.

tests
classical
El-Tor
Voges-Proskauer
-
+
Chicken RBC Agglutination
-
+
Polymyxin-B sensitivity (50 U)
+
-
Group IV cholera- phage sensitivity
+
-
Bet – Haemolysis(On Blood Agar) -
+



Factors of Pathogenicity of V. cholerae: 1. Adherance and Motility: The actively motile m.o. can adhere to the intestinal wall (pathogenic), and vice versa for the non-motile (non-pathogenic). V.cholerae does not usually reach to the blood stream. 2. Enterotoxin: It is an important factor in the pathogenicity of the m.o. and has a M.W. of 84000 Daltons. It is composed of of 6 light chains of 8000 Daltons each, and one heavy chain of 28000 Daltons which has 2 portions: a. Toxic portion (A1): 98% protein, 1% lipid, and 1% CHO . b. Stabilizer portion (A2). The A1 portion increases the intracellular cAMP of the intestinal epithelium. The cAMP prevents reabsorption of Na (1 X), excretion of NaHCO3 (2 X),chloride and K (3-5 X). This leads to accumulation of water in large quantities (> 20 liters/day), acidosis, hypokalaemia, and death.

Cholera: Cholera is an acute infectious disease characterized by severe vomiting and watery diarrhoea (rice water stool) resulting in dehydration and collapse. The disease is endemic in the Indian subcontinent and used to occur in world wide epidemics. Infection occurs by oral route through contaminated food or drink and 10 trillions m.o. are required to cause cholera. Source of infection is a case or a carrier who excretes the organism in the stool. The incubation period is of 1-4 days. The organisms attach to the microvilli of the brush border of epithelial cells, where they multiply and liberate cholera enterotoxin which exerts the above mentioned effects. Convalescent carriers occur, but chronic carriers are rare. The stool contains mucous, epith. cells, large numbers of m.o. and no pus cells.

Diagnosis of cholera: A. First case in non-endemic area: Any comma-shaped motile m.o. detected in a stool sample should be thoroughly identified before giving a report as positive for cholera (see identification). B. Second case during an epidemic: In an established epidemic, cases can be diagnosed by direct stool examination mainly for motility, and immobilization by specific anti-O antisera. Then, continue the identification procedure. PCR assays have been developed for detection of cholera toxin genes. Finally, mortality from untreated cholera may reach up to 25% - 50%.


Immunity against cholera: The immunity against cholera could be achieved by: 1. Gastric juice: It is a major barrier against infection by V. cholerae. 2. Secretory IgA: Intestinal secretory IgA prevents the attachment of m.o. to the intestinal wall. 3. Vibriocidal IgG: Anti-toxic Ab, presents in the serum. 4. Solid immunity can stay for 3 years after infection. 5. Immunoprophylaxis: a. Heat killed vaccine: Given in 2 subcutaneous doses. It stimulate anti-bacterial and not anti-toxic antibodies. Therefore, its role in protection is questionable. b. Live oral vaccine: Protects against cholera enterotoxin, and is prepared by recombinant DNA tech. c. O-139 : New vaccine using the Bengal strain (O139)

Treatment and control of cholera: 1. Intravenous fluid and electrolytes replacement: This is the most important part of the therapy. 2. Oral anti-microbial drugs: Have a secondary role in the treatment. Tetracyclines are the most effective drugs. 3. Control: The main control measures are: a. Community and personal hygiene. b. Isolation of cases and proper disposal of sewage. c. Chemoprophylaxis by using tetracyclines for exposed persons. d. Immunoprophylaxis; see above (immunity).


Vibrio parahaemolyticusThese are gram negative, motile, curved, and non-sucrose bacilli. They are halophilic (salt –loving) in tolerating 2-11 % NaCl depending on their biotypes. These are marine m.o., and cause diarrhoea in some countries with sea water as Japan. There are reports that these m.o. are detected in south Iraq (Basrah). The diarrhoea is usually a self limiting condition and may be associated with constitutional symptoms as nasea, vomiting and fever. However, some times broad spectrum anti-microbial may be required as co-trimoxazole.

Aeromonas: These m.o. are gram negative, comma-shaped, motile, and sucrose- fermenter bacilli. Some species cause diarrhoea, wound infection, UTI, cellulitis, osteomylitis and septicaemia. They are NLF bacilli. A. Hydrophila (water-lover) may cause human diseases, and can be recovered from sink taps, drain pipes, and water faucets. Plesiomonas: These m.o. have similar morphology to Aeromonas, but are non-sucrose fermenter. They may cause diarrhoea, neonatal menigitis, cellulitis, septic arthritis, and acute cholycystitis. P. shigelloides is an important species that may cause human diseases and is LF m.o.. Treatment for Aeromonas & Plesiomonas: Anti-microbial agents effective against gram negative m.o. or broad spectrum drugs

Family : Campylobacter In this family there are two genera: 1. Genus: Campylobacter; with 18 species. 2. Genus: Acrobacter; with 4 species. Genus: Campylobacter These microorganisms have long been known as animal pathogens including poultry, dogs, cats, sheep and cattle. There are two important species that can cause human infection: 1. C. jejuni. 2. C. coli. Transmission of these m.o. is usually via contaminated food (as chicken) , milk or water, while person to person transmission plays a minor role .

Morphology:Small gram negative rods comma-shaped or S-shaped. Motility is of darting type with cork screw-like movement. These m.o. have a single polar flagellum.Cultural characteristics:These microorganisms are microaerophilic (requires 5% O2, 10% CO2, and 85% N2) which is provided by Gas-Generating Kit. Also these m.o. are thermophilic (tolerate temperature up to 42-43 C which is useful characteristic in their isolation and differentiation.The important media used for cultivation is:1. Skirrow’s medium: Contains blood agar and anti-microbials including vancomycin, polymyxin B, and trimethoprim to make it selective for the m.o.2. Campy-BAP: Similar to (1) but with cephalothin.


Colonies of Campylobacter: The colonies are colourless, rounded and convex. For culturing plates are put at 37 C and others at 42 C. Also, other media containing charcoal, or using filtration can be used. Biochemical Characteristics: Campylobacter are oxidase positive, catalase positive, H2S producers, and can reduce nitrate to nitrite. Also, these m.o. are urease negative (versus Helicobacter).

Pathogenesis and Clinical Picture: The m.o. are acquired by oral route by food and water contaminated with animal faeces or after direct contact with animals (as puppies with diarrhoea). The m.o. are quite sensitive to gastric juice and at least 10000 m.o. are required to breach this barrier. The I.P. is 1-6 days. In the intestine, they multiply and invade the intestinal wall (similar to- but less than Shigella). The m.o. liberate exotoxin and enterotoxin (V. cholerae- ike) causing damage to the intestinal tissue. This results in an acute onset of grossly bloody diarrhoea which is associated with cramps of abdominal pain. The stool contains R.B.C. and leucocytes (pus cells). Occasionally, the m.o. invade to the blood stream and giving picture of enteric fevers.

Treatment of Campylobacter:Replacement of fluid and electrolytes is an essential step in the management.The m.o. are sensitive to erythromycin and nalidixic acid.Recovery occurs usually in one week.Complications:Gullian –barre syndrome ( acute demyelinating peripheral neuropathy with ascending flaccid paralysis) is an important complication. The pathogenesis involves gangliosides like –epitopes in the Campylobacter LPS that cross react with reactive targets in peripheral nerve tissue as well as host susceptibility factors.

Genus: Helicobacter There are many species of this new genus (proposed in 1989), and only a single species is important to humans which is H. pylori. The latter m.o. is now a well-known cause of gastritis, duodenal and gastric ulcer and is linked to gastric carcinoma and MALT (Mucosal Associated Lymphoid Tumours). Helicobacters are very similar to Campylobacters (even previously they were in the same genus), but are different in being urease positive, while Campylobacter are urease negative. Campylobacters are motile with one polar flagellum, while H. pylori have multiple polar flagella.

Morphology, Culture and Biochemical Tests:H. pylori are gram negative m.o., spiral in shape, actively motile by multiple flagellae. H. pylori can grow on the same media mentioned for Campylobacter. It is better to supplement the media with horse serum. They best grow at 37 C, and require 3-6 days of incubation.The m.o. are catalase positive, oxidase positive and urease positive. The latter character can be used for:1. Rapid urease test: In which gastric biopsy material is placed onto a medium containing urea with a colour indicator. If H. pylori is present, the urease splits the urea and results in shift of pH leading to colour change (from yellow to red). 2. Urease Breath Test: In which 14 C –labelled urea is ingested by the patient. If the m.o. is present, the urease activity generates radiolabelled CO2 that can be detected in patient’s breach (by detector).

Pathogenesis of H. pylori:These m.o. attaches to the mucous- secreting cells deep in the gastric mucosa away from the acidity of the stomach. The production of ammonia from urea by the organism’s urease, coupled with an inflammatory response leads to damage of the mucosa. Loss of the protective mucous coating, predisposes to gastritis and peptic ulcer. The ammonia also neutralizes gastric acidity, allowing the organism to survive.It is worth to mention that the m.o. prefer pH of 6-7, but sensitive to gastric juice.H. pylori is responsible for 85% of peptic ulcers. However, 80- 90 % of people in developing counties carry this m.o., while in developed counties the rate is 20- 40%. The main factors of pathogenicity are the active motility, proteases (damage mucosa) & urease.

Diagnosis of H. pylori Infection:1. Gastric Biopsy specimens: Minced in saline and examined: A. Smears stained with Gram’s or special stains which show the curved or spiral organism. B. Culture (see above).2. Detection of H. pylori Antibodies in patient’s serum: These antibodies are of 3 classes (IgM, IgG, and IgA). IgM is the first one to appear followed by IgG and IgA. ELISA can detect such antibodies. The detection of these antibodies does not differentiate between current/active and old/inactivate infections. However, these antibodies can be used for follow up of treatment of patients.3. Detection of H. pylori Antigens in the stool (ELISA).4. Rapid UreaseTest (see above).5. Urease Breath Test (see above).

Treatment of H. pylori infection:Peptic ulcers and gastritis could be treated by combination of the following drugs:Triple Therapy: Metronidazole + clarithromycin or amoxicillin or tetracycline + bismuth subcitrate (pepto-bismol).This treatment results in eradication of H. pylori in 70 – 95 % of patients and decreases recurrence rate of peptic ulcers.Immunity:The antibodies could stay for a long time after recovery. These antibodies have no active role in bactericidal effect against H. pylori.

Genus : YersiniaThese microorganisms are primarily animal pathogens and belong to the family Enterobacteriaceae. The most important species clinically are:1. Y. pestis : Causes plague in man.2. Y. enterocolitica : Causes enterocolitis and3. Y. pseudotuberculosis : mesenteric adenitis.Yersinia pestis:Rats and rodents are the natural host of this m.o. which is transmitted to man by the rat-flea Xenopsylla cheopis. Morphology:Gram negative short ovoid, non-motile bacilli. In tissue it forms a capsule-like outer envelop. When stained by Wayson stain, Geimsa or mythylene blue it shows marked bipolar staining “ safety pin appearance).


Cultural characteristics: Facultative anaerobe. Optimum temp. 30 C. It grows on nutrient agar, however, better growth on blood agar. Pathogenicity: The m.o. is highly pathogenic to laboratory animals as rats or mice. When injected SC the animals die within few days. The m.o. have pathogenic factors as LPS, F1 antigen, V-W antigens and endotoxins; F1 & V-W are anti-phagocytic. Plague: Plague is a disease of wild rodents and is transmitted to man by bite of infected fleas. The m.o. multiplies in the draining L.N. (tender) causing bubonic plague. From there, it can spread to the blood causing septicaemic plague, then to the lungs causing pneumonic plague which can spread from person to person by droplets.


Cont/…plagueThe patient have painful lymphadenopathy specially in the groins and axillae. Also, the patient may have nausea, vomiting, diarrhoea, DIC, or meningitis. Mortality rate of plague is 50%, but from pneumonia is almost 100%.Diagnosis:1. Detection and isolation of the m.o. from aspirate of L.N., sputum or blood. 2. Serological diagnosis by ELISA or agglutination tests for rising antibody titers.Treatment:Streptomycin is the drug of choice. Tetracycline is an alternative. They may be used in combination.Prophylaxis:Anti-rat anti-flea measures, prevention of rats coming in ships, prophylactic tetracyclines & killed vaccine.

Y. Enterocolica & pseudotubrerculosis: They cause enterocolitis and mesenteric adenitis even some times suspected as appendicitis. They are transmitted to humans by contamination of food with excreta of domestic animals as dogs, cats and cattle. Morphology and culture: Similar to Y. pestis, and for culture the m.o. can tolerate temprature 4-30 C. Therefore, to isolate the m.o. culture at 4 C (cold enrichment) for 2-4 weeks of stool specimens to make selective for Yersinae (other m.o. can not tolerate as enteric bacilli). The m.o. are catalase +, urease +, oxidase -, motile at low temperature and not at 37 C.


Clinical picture Y. enterocolitica and pseudotuberculosis: The I.P. is 5-10 days. Both m.o. are important causes of diarrhoea (watery to bloody) in countries with cold climate and pork consumption, but not in Iraq (1-2%). Early symptoms are fever, abdominal pain, and diaarhoea.At times, the abdominal pain is severe and localized in the right lower quadrant, suggesting appendicitis. Later, arthralgia, arthritis or erythema nodosum (immunological reaction), meningitis and sepsis may develop. Treatment: Aminoglycosides as nitilmicin & gentamycin, 3rd generation cephalosporins, or ciprofloxacin.

Francisella tularensis & tularaemia This m.o. is widely found in animal reservoirs and is transmissible to humans by biting arthropods, direct contact with animals, inhalation of aerosols, or ingestion of contaminated food or water. The m.o. is small, gram negative, pleomorphic rod. Growth does not occur in most ordinary media, but small colonies appear in 1-3 days aerobically at 37 C on: 1. Glucose cysteine blood agar. 2. Glucose blood agar. When m.o. enter through skin abrasions, an ulcerating papule develops, and regional L.Ns. enlarge, but when inhaled pneumonitis occurs, and oculoglandular tularaemia may develop via the conjunctiva. Treatment: Streptomycin, or gentamicin therapy for 10 days. Tetracyclines may be equally effective. Ceftriazone is not effective.


Pasteurella These m.o. are primarily animal pathogens, but the can produce human diseases. They are gram negative, non-motile aerobic or facultative anaerobic cocco-bacilli with bipolar appearance on stained smears. They can grow on ordinary media at 37 C. They are oxidase +, and catalase +. The important species are: P. multocida, P. haemolytica, P. pneumotropica, and P. ureae. P. Mutocida is the most common m.o. in cat or dog bites. The other m.o. may cause man infections as wound, respiratory or suppurative. The common presentation is animal bite followed by an acute onset of redness, swelling and pain. Regional lymphadenopathy is variable and fever is often low- grade. Sometimes bacteraemia or chronic RTI may be see. Treatment: Aminoglycosides or tetracyclines.