Alexandria University Semester: Autumn Medical Research Institute Academic year: 2019-2020 Microbiology Department Time allowed: 3 hours Master degree in Molecular and Diagnostic Date: Saturday 15/1/2020 Microbiology Course Title: Medical Bacteriology Course Code: 1706701 Midterm exam. (Model answer) Total Marks: 60

Student Name: ______All questions to be answered

Question I: Choose the correct answer (30 marks)

A. MCQ: (0.5 mark each/ Total 22 marks) 1.All are true about diagnostic tests of Enteropathogenic E coli EXCEPT:

A. Shows localized adherence on HEp-2(LA) B. has positive fluorescent actin staining test C. lacks Stx expression D. shows PCR amplification using a variety of primers for detecting eae gene, EAF plasmid E. has a 60-MDa plasmid encoding hemolysin

2.All are true about Enterohemorrhagic E.coli EXCEPT:

A. causes Hemorrhagic colitis and Hemolytic Uremic Syndrome B. expresses Shiga C. causes A/E lesions on epithelial cells D. possesses a 60-MDa plasmid encoding hemolysin E. could be identified as sorbitol positive colonies on Sorbitol MacConkey agar that agglutinate in O157 antiserum

3. All are true about Klebsiella (K) EXCEPT:

A. It has 4 species B. K pneumoniae has 3 subspecies C. All Klebsiella are ONPG positive D. All Klebsiella possess a polysaccharide capsule protecting against phagocytosis, antimicrobial absorption and contributing to its virulence

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4.All are true about Hafnia alvei EXCEPT:

A. Genus Hafnia is composed of 1 species B. Hafnia alvei is a Gram-negative enteric and oropharyngeal bacillus C. It is usually is nonpathogenic D. H alvei may be a potential pathogen in patients with a chronic underlying illness E. A delayed positive lactose fermenting is a major characteristic of Hafnia

5.All are true about Proteus EXCEPT:

A. The genus Proteus consists of 4 species B. They are opportunistic pathogens C. They are non-lactose fermenters D. They are oxidase negative E. They can deaminate phenyl alanine F. They are mannose positive

6.All are true about Morganella morganii EXCEPT: They are

A. Urease negative B. PDA positive C. H2S negative D. Indole positive E. shows no swarming 7.All are true about Yersinia pestis EXCEPT:

A. Human Y. pestis infection takes three forms: pneumonic, septicemic, and bubonic plagues. B. Yersinia pestis is a Gram-negative rod-shaped bacterium with bipolar staining C. The transmission of Y. pestis is by rats D. humans are an accidental host E. The macrophages are unable to kill Y. pestis

8.All are true about Yersinia enterocolitica EXCEPT:

A. They are Gram-negative cocco-bacilli with bipolar staining, belonging to the family enterobacteriaceae B. They grow on ordinary media C. They have an optimal growth temperature of 25 ˚ -30˚ C D. They ferment lactose in Cefsulodin, Irgasan, Novobiocin Agar, producing an acid pH which gives the colonies their red color and the "bull's eye appearance E. Fecal samples suspected of containing Y enterocolitica are kept at 4˚C for 1-3 weeks with \ weekly subculture on CIN Agar

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9.All are true about serological classification of Salmonella EXCEPT:

A. Salmonella are divided into serogroups by means of somatic (O) antigens B. Each group has one specific O antigen, and several nonspecific (O) factors. C. O groups are now designated using the characteristic O factor .Letters are provisionally kept into brackets D. Each serogroup is subdivided into serotypes by means of flagellar (H) antigens E. Some strains in the same serotype carry an H-antigen specific for the serotype phase I antigen. designated with small letters F. Some strains carry H antigen which may be possessed by other Salmonella group Phase II and designated by Arabic numbers.

10. All are zoonotic pathogens EXCEPT:

A. Salmonella typhi B. Ecoli O157 C. Yersinia pestis D. Yersinia pseudotuberculosis

11. The most common clinical form of M.tuberculosis infection is A. Pulmonary tuberculosis B. Latent TB infection C. Extra Pulmonary tuberculosis

12. The TB infected individual becomes tuberculin-positive when:

A. T-cell activation of macrophages occurs B. Gamma interferon is liberated C. macrophages are capable of destroying M.tuberculosis D. All of the above

13. M.tuberculosis has slow growth rate because it takes ………… to proceed through one division cycle. This is due to …………………… .

A. 20 hours - single copy of ribosome gene B. 20 days - require certain growth factor C. 20 hours - require certain growth factor D. 20 days - single copy of ribosome gene

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14. The QuantiFERON-TB IN Gold test (QFT-G) all are true EXCEPT:

A. Is an immunologic test used to aid in the diagnosis of latent tuberculosis infection and tuberculosis disease. B. A sample of whole blood is incubated with M.tuberculosis three specific antigens C. It measures the number of peripheral mononuclear cells releasing IFN-γ in the supernatant of the stimulated whole blood sample.

15. Sterility may be the long- term result of gonorrhea if inflammation and scarring take place in the A. Urethra B. Labia majora C. Rectum D. Fallopian tubes.

16. All the following are diseases caused by rickettsiae EXCEPT: A. Scrub typhus B. Rocky Mountain spotted fever C. Trench fever D. Tularemia

17. In addition to the urethra, the organism of gonorrhea may also infect the A. Muscles of the upper extremity B. Eyes C. Muscles of the lower extremity D. liver

18. The symptoms of chlamydia closely resemble those of A. Syphilis B. Granuloma inguinale C. Gonorrhea D. Cytomegalovirus infection

19. Swollen lymph nodes, also known as buboes, are characteristic signs of the infection A. Trichomoniasis B. Prostatitis C. Toxic shock syndrome D. Lymphogranuloma venereum

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20. An organism is isolated and several tests are run. Catalase negative,hydrogen sulphite positive, nonmotile, and non hemolytic. A. Listeria monocytogenes B. Erysipelothrix rhusiopathiae C. Strept. pyogenes D. Corynebacterium

21. The internalin on the surface of listeria binds with which of the following host epithelial cells A. lectin like molecule B. carbohydrates C. cadherin D. sugar

22. Organism resistant to degradative lysosomal enzymes includes A. Neisseria meningitidis B. Legionella pneumophila C. M.leprae D. Both b and c

23. There has been much speculation on the pathogenesis of group B streptococcal disease in the neonate. One of the most likely pathogenic mechanisms is A. Complement C5a, a potent chemoattractant, activates PMNs B. The streptococci are resistant to penicillin C. The alternative complement pathway is activated D. In the absence of specific , opsonization, phagocyte recognition, and killing do not proceed normally

24. A 70-year-old female patient was readmitted to a local hospital with fever and chills following cardiac surgery at a major teaching institution. Blood cultures were taken and a Gram-positive coccus grew from the blood cultures within 24 hours. Initial tests indicated that this isolate was resistant to penicillin. The most likely identification is A. Streptococcus pneumoniae B. Neisseria C. Group A streptococcus D. Enterococcus E. Group B streptococcus

25. An elderly man with chronic heart and lung disease develops Legionella pneumonia. By what route was the infection most likely to have been acquired? A. Respiratory route, from an infected person. B. Inhalation of aerosolized water from a source containing protozoa. C. Using snuff that contained spores. D. Inhalation of spores from moldy hay. E. Exposure to livestock or animal products.

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26. Listeria can invade the immune system by which of the following methods? A. markers on its cell wall B. high grade of motility to move cell to cell C. evading of the phagosome D. survival in the phagosome

27. All of the following is true about Non- Halophilic vibrios EXCEPT: A. It can grow in media containing more than 5-6% Nacl. B. produces yellow colonies (sucrose fermenter) on TCBS C. Eg: v. cholera: O1 +O 139: clssical cholera D. Non cholera vibrio: O2 - O138: diarrheal illness

28. All of the following aminoglycosides is used against Pseudomonas aeruginosa EXCEPT: A. Gentamicin B. Kanamycin C. Amikacin D. Tobramycin

29. All of the following cephalosporins is used against Pseudomonas aeruginosa EXCEPT: A. Ceftazidime B. Ceftriaxone C. Cefipime D. Cefobid

30. All of the following is true about Stenotrophomonas EXCEPT: A. Oxidase negative B. Citrate positive C. DNAase test negative D. Susceptible to trimethoprim

31. All of the following is true about Burkholderia EXCEPT: A. Oxidase negative B. Citrate positive C. DNAase test negative D. Susceptible to trimethoprim

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32. All of the following is true about stool Antigen test for H.pylori EXCEPT: A. Non-invasive test B. Has high sensitivity and specificity C. Can be used for monitoring of treatment D. Not affected by antibiotics intake

33. Strict anaerobes lack following enzymes: A. Catalase B. Peroxidase C. Superoxide dismutase D. All the above

34. A middle-aged woman presented with fishy odour vaginal discharge shows clue cells in a wet preparation. She probably has: A. Bacterial vaginosis B. Chlamydia trachomatis C. Neisseria gonorrhoeae D. Trichomonas

35. Lemierre’s disease is a serious infection of the head and neck associated with which of the following anaerobes? A. Prevotella melaninogenica B. Bacteroides thetaiotamicron C. Porphyromonas gingivalis D. Fusobacterium necrophorum

36. Elek’s gel precipitation test is for A. Gonococcus B. Diphtheria C. Hemophillus D. Anthrax

37. To diagnose congenital syphilis: A. Non-Treponemal are detected at birth B. Treponemal antibodies are detected at birth C. Both treponemal and non-treponemal antibodies are detected after 6 months D. Treponemal or non-treponemal antibodies are detected monthly over 6 months

38. Erythema migrans is characteristic of: A. Weil’s disease B. Lyme disease C. Congenital syphilis D. Tertiary syphilis

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39. Follow-up of response to treatment in patients with syphilis can be performed by: A. Detection of Treponemal antibodies B. Detection of Non-treponemal antibodies C. Detection of Both treponemal and non-treponemal antibodies D. Molecular methods such as PCR

40. Nagler’s reaction is useful for the identification of: A. tetani B. C. D. Clostridium difficile

41. ………… is the causative agent of chancroid, one of the most common sexually transmitted disease and is characterized by painful lesions and genital ulcers. A. Neisseria gonorrhoeae B. Haemophilus haemolyticus C. Haemophilus ducreyi D. Treponema pallidum

42. Which encapsulated H. Influenzae serotype causes 95% of invasive disease (meaning the bacteria enters the bloodstream). A. Serotype A B. Serotype B C. Serotype C D. Serotype D

43. Brucellosis is also known as: A. Undulant fever, B. Mediterranean fever, C. Malta fever D. All of the above

44. are: A. Anaerobes B. They possess spores C. Gram positive D. Motile E. All of the above

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B. True or False (0.5 mark each/ Total 8 marks)

45. Y.enterocolitica and Y.pestis have a remarkable 97-100% homology (F) 46. Staphylococcus aureus is susceptible to lysis by the lysostaphin but is resistant to lysis by lysozyme. (T) 47. Staphylococcal cassette chromosome (SCC) carries the mec region encoding methicillin resistance. (T) 48. The Xpert MTB/RIF is an automated phenotypic test for detection of Mycobacterium tuberculosis (MTB) and its resistance to rifampin. F 49. The eyes of newborns are usually treated as a preventive measure to preclude gonococcal ophthalmia and chlamydial ophthalmia. (True) 50. Differentiation of N.gonorrhea and N. meningitides can be done by glucose fermentation. (False) 51. The antiphagocytic ability of E. rhusiopathiae likely results from production of antibodies (FALSE) 52. Listeria Phospholipase – is involved in the uptake of the bacterium into epithelial cells. (FALSE) 53. Legionnaires disease is commonly accompanied by extrapulmonary manifestations. (TRUE) 54. Wagatsuma agar contains low salt content, D-mannitol, blood group O. F 55. Conditions required for Campylobacter growth is reduced CO2 (5% CO2) ,02 (10%). F 56. Bacteroides are Gram positive coccobacilli or bacilli. (False) 57. Chancre is painless and can pass unnoticed. T 58. Syphilis can be transmitted from the mother to her fetus only during delivery. F 59. Pseudomembranous colitis is diagnosed by detection of one or both C difficile in stool and by endoscopic observation of pseudomembranes or microabscesses in patients who have diarrhea and have been given antibiotics. T

60. H influenzae does not grow on sheep blood agar except around colonies of staphylococci what is known as “satellite phenomenon”. T

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Question II: Answer the following questions. (30 marks)

A. Write short account on the following: (2 marks each/ total 24 marks)

1.Give a short account on E.coli heat labile toxin

1. E . coli heat labile toxin is an oligomeric toxin of of ca. 86 kDa composed of one 28-kDa A subunit and five identical 11.5-kDa B subunits. 2. The B subunits are arranged in a ring and bind strongly to the ganglioside GM1. 3. The A subunit is responsible for the enzymatic activity of the toxin 4. The genes encoding LT (elt or etx) reside on plasmids 5. The cellular target of LTis adenylate cyclase 6. The A1 peptide has an ADP-ribosyltransferase activity and acts by transferring an ADP-ribosyl moiety from NAD to the alpha subunit of the GTP-binding protein, GS, which stimulates adenylate cyclase activity. 7. ADP-ribosylation of the GSa subunit results in adenylate cyclase being permanently activated, leading to increased levels of intracellular cyclic AMP (cAMP). 8. cyclic AMP works by activating protein kinase A (PKA, cAMP-dependent protein kinase). 9. cAMPdependent protein kinase (A kinase) is activated leading to supranormal phosphorylation of chloride channels 10. The net result is stimulation of Cl- secretion from secretory crypt cells and inhibition of NaCl absorption by villus tip cells. 11. The increased luminal ion content draws water passively through the paracellular pathway, resulting in osmotic diarrhea

2.Give a short account on the virulence plasmid of Shigella

• All virulent strains of Shigella possess a large 230kb plasmid that mediates its virulence properties. • This so-called the invasion plasmid has been shown to encode the genes for several aspects of Shigella virulence • It encodes genes for: a) the synthesis of type IIIsecretion apparatus (membrane excretion protein ,surface protein antigen) (mxi,spa) b) the production of invasion plasmid antigens (Ipas); c) the induction of endocytic uptake of the bacteria and disruption of endocytic vacuoles d) the intra- and intercellular spreading (ICSA) e) the regulation of plasmid-encoded virulence genes(vir)

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3. Give a short account on how the Salmonella Pathogenicity Island 1 (SPI1) effector promote bacterial invasion resulting in the engulfment of Salmonella by the host cell.

1. the bacterium Salmonella alters cell shape by activating and deactivating small GTPases.

2. The bacterial SopE (outer protein) E protein is delivered to the host cytoplasm where it binds to Rac and Cdc42. 3. SopE acts as a guanine-nucleotide-exchange factor to favour the GTP-bound, activated form of Rac and Cdc42. 4. Activated Rac and Cdc42 stimulates membrane ruffling, which results in bacterial uptake, and activates nuclear factors that affect gene transcription. 5. The bacterial SptP(Tyrosine-protein phosphatase) protein, also delivered to the host cytoplasm, binds preferentially to GTP-bound Rac and enhances its GTPase activity. 6. Cell shape is restored in the absence of active Rac

7. The concerted efforts of the SPI1 effectors SipA, SipC, SopE, SopE2, and SptP result in the engulfment of Salmonella by the host cell.

4. Enumerate Virulence Factors related to pathogenesis of M.tuberculosis

MTB has special mechanisms for cell entry bind to mannose receptors on macrophages directly via LAM or indirectly via certain complement receptors or Fc receptors. MTB can grow intracellularly. ie;evading the immune system ( antibodies and complement are ineffective). Once MTB is phagocytosed, it can inhibit phagosome-lysosome fusion MTB interferes with the toxic effects of reactive oxygen intermediates produced in the process of phagocytosis by two mechanisms: 1. Compounds including glycolipids, sulfatides and LAM down regulate the oxidative cytotoxic mechanism. 2. Macrophage uptake via complement receptors may bypass the activation of a respiratory burst Antigen 85 complex. bind fibronectin. These proteins may aid in walling off the bacteria from the immune system and may facilitate tubercle formation . Fibronectin is an extracellular matrix protein and is a for several extracellular pathogenic bacteria binding to mucosal surfaces. Slow generation time. the immune system may not readily recognize the bacteria or may not be triggered sufficiently to eliminate them. ( M. leprae , Treponema pallidum and Borrelia burgdorferi ). High lipid concentration in cell wall,

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5. List the main difference between epidemic typhus and endemic typhus. • Typhus is a rickettsial disease accompanied by high fever and a skin rash. • Epidemic typhus is caused by Rickettsia prowazekii which is transmitted by body louce. • Endemic typhus is caused by Rickettsia typhi which is transmitted by rate fleas. • The symptoms of endemic typhus are generally less severe than epidemic typhus.

6. Mention Legionella type IV secretion system. • The type IV secretion system, (dot/icm) • It is encoded by a set of dot (defective in organelle tracking) and icm (intracellular multiplication) genes. • is required by L. pneumophila to insert pores into the host plasma membrane, a process that could disturb phagosome maturation. • The Dot/Icm complex must act during phagocytosis to distract phagosome maturation to establish a protective-replication vacuole

7. Mention L.monocytogenes clinical diseases.

8. Urease enzyme is key factor for survival of H.pylori. Discuss this statement. • essential in colonization of gastric mucosa

• converts urea into bicarbonate (to CO2) and ammonia • Neutralize the local acid environment • Localized tissue damage as it is a strong immunogen which stimulates the immune response. • Cytotoxic to the gastric epith. Cells

9. Urea breath test is a non invasive method for H.pylori diagnosis. Discuss this test. • is a safe, easy, and accurate test for the presence of H. pylori in the stomach. • The breath test relies on the ability of H. pylori to break down the naturally occurring chemical, urea, into carbon dioxide which is absorbed from the stomach and eliminated from the body in the breath.

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• Ten to 20 minutes after swallowing a capsule containing a minute amount of radioactive urea, a breath sample is collected and analyzed for radioactive carbon dioxide • The presence of radioactive carbon dioxide in the breath (a positive test) means that there is active infection. • The test becomes negative (there is no radioactive carbon dioxide in the breath) shortly after eradication of the bacterium from the stomach with antibiotics.

10. Give a short note on including structure/type, mode of action, and factors affecting its production • Diphtheria toxin is a very potent consisting of 353 amino acids. heat-labile, single- chain, three domain polypeptide (62 kDa) The lethal dose for susceptible individuals is approximately 0.1 µg/kg body weight • Mode of action of diphtheria toxin: causes death of eukaryotic cells by inhibiting cellular protein synthesis by ribosylation of adenosine diphosphate leading to inactivation of elongation factor • it is composed of a single polypeptide chain containing two fragments, A and B, connected by a bond, and both are required for intoxication of tissue culture cells. • The fragment A possesses the active site of TD and it is responsible for the enzymatic activity, while the fragment B is responsible for the fixation of the toxin with receptors in the host cells • Two factors to produces diphtheria toxin and cause infection: Low extracellular concentrations of iron Presence of a lysogenic prophage in the bacterial chromosome.

11. Write short notes on the secondary stage of syphilis. • Approximately 6 weeks after the sore first appears, the second stage of the disease begins, due to dissemination of the organisms after reaching a sufficient number . • The most common symptom during this stage is a rash, spreading from the palms and soles of the feet towards the trunk . This is associated with generalized lymphadenopathy. • Other symptoms may occur such as tiredness, fever, sore throat, headaches, hoarseness, loss of appetite . Aseptic meningitis may also occur. • Alopecia and condylomas may occur later • (Condylomata lata is a cutaneous condition characterized by wart-like lesions on the genitals ). • The patient is very infectious at this stage. • These signs and symptoms will last 2-6 weeks and generally disappear in the absence of adequate treatment. • After healing of the lesions, the person enters a latent stage during which there are no signs or symptoms of the disease.

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12. Discuss toxins.

• The vegetative cells of C tetani produce the plasmid-encoded toxin tetanospasmin (150 kDa) that is cleaved by a bacterial protease into two peptides (50 and 100 kDa) linked by a disulfide bond. • The larger peptide initially binds to receptors on the presynaptic membranes of motor neurons. • It then migrates by the retrograde system to the cell bodies of these neurons to the spinal cord and brainstem. • The toxin diffuses to terminals of inhibitory cells, including both glycinergic and γ-aminobutyric acid (GABA)–secreting neurons from the brainstem. • The smaller peptide degrades , a protein required for docking of vesicles on the presynaptic membrane. Release of the inhibitory and GABA is blocked, and the motor neurons are not inhibited. • Hyperreflexia, muscle spasms, and spastic paralysis result. • Extremely small amounts of toxin can be lethal for humans.

i. which causes lysis of RBCs ii. Tetanospasmin is and essential pathogenic product

Tetanospasmin is toxic to humans and various animals when injected parenterally, but it is not toxic by the oral route

Tetanospasmin which causes increasing excitability of spinal cord neurons and muscle spasm

Complete: (2 marks each/ Total 6 marks)

1. Regarding the diagnosis of M.tuberculosis , Direct Z-N staining is more rapid but less sensitive because it requires M.tuberculosis concentration of 5-10x 103/ml sputum sample compared to culture on Lowenstein-Jensen medium which requires 10-100 viable bacilli/ ml.

2. Legionellae prevent bactericidal activity of the phagocyte by several mechanism such as inhibition of the oxidative burst, reduction in phagosome acidification, blocking of phagosome maturation and changes in organelle trafficking.

3. V.cholera strains comprise both serotypes O1 and O139.

GOOD LUCK

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