ENTEROBACTER AND PROTEOUS bacterial.pptx

bacteria

1. To Dr.A.K.Arora Professor Department of Vety.Microbiology By Rajwent singh L-2017-V-60-M KLEBSIELLA,PROTEUS, ENTEROBACTOR

2. KLEBSIELLA SPECIES  electron microscope image of  Klebsiella pneumoniae

3. gram-negative (pink) cell wall is composed of a thin peptidoglycan layer and an outer membrane. non-motile lactose fermenting rod-shape organism facultative anaerobe capsulated.

4. found in nose, mouth, skin, and intestinal tract cause disease pneumonia, bloodstream infections, wound infections, urinary tract infections, and meningitis.  ideal growth temperature 35° to 37°c.

5. List of species K. granulomatis K. oxytoca K. michiganensis K. pneumoniae  (type-species) K. p.  subsp.  ozaenae K. p.  subsp.  pneumoniae K. p.  subsp.  rhinoscleromatis K. quasipneumoniae K. q.  subsp.  quasipneumoniae K. q.  subsp.  similipneumoniae K. variicola

6.   K. variicola  is emerging pathogen of humans and animals and causes bovine mastitis. Bacterial bronchopneumonia caused by  Klebsiella  species in dogs. Dogs and cats have immunosuppresion results from drugs, malnutrition, stress, endocrine diseases, and   feline leukemia virus ,  feline immunodeficiency virus infection , and  canine distemper . Signs of bacterial pneumonia in dogs productive and soft cough, nasal discharge, and exercise intolerance.

7. PATHOGENESIS Klebsiella  infections spread through exposure to the bacteria via respiratory tract, which causes pneumonia, or the blood to cause an infection in the bloostream . Klebsiella  infections most well-known in hospitals spread through person-to-person contact by contaminated hands of surrounded people in hospitals. Klebsiella   spread very easily and rapidly, but not through the air.

8. VIRULENCE FACTORS 1. Capsular polysaccharide antigens The capsular polysaccharide antigens of K. pneumoniae can be classified into 77 serotypes. The K1 and K2 isolates generally more virulent than non-K1/K2 isolates in terms of lethality for a mouse when injected intraperitoneally .

9. Macrophages with the lectin or mannose receptor can recognize the capsular sugar sequences of mannose-alpha-2/3-mannose or L-rhamnose-alpha-2/3-L-rhamnose. The K1 or K2 antigens lack these mannose or rhamnose sequences, which can protect these isolates from lectinophagocytosis . K. pneumoniae with K1 or K2 capsule serotypes often hypermucoviscous than non-K1/K2 strains

10. Outer layer of the capsule of most K. pneumoniae consists of a network structure of fine fibers derived from capsular polysaccharide, known as the exopolysaccharide web. This structure encoded by capsular polysaccharide (cps) gene cluster on the chromosome and positively regulated by plasmid-mediated genes, including rmpA (regulator of mucoid phenotype gene A) , rmpA2 and magA ( mucoid associated gene A)

11. RmpA gene Capsule of K. pneumoniae encoded by the capsular polysaccharide (cps) gene cluster , which regulated by plasmid-born rmpA and rmpA2 genes to enhance extracapsular polysaccharide synthesis and produce hypermucoviscosity phenotype. MagA gene MagA encodes a 43 kD outer membrane protein. MagA gene located within an operon that specific to the serotype K1 capsular polysaccharide (cps) gene cluster . operon containing magA responsible for K1 capsular serotype

12. Using transposon mutagenesis to identify candidate virulence genes demonstrated magA -positive strains had a mucoviscous exopolysaccharide web, resisted phagocytosis , and produced liver abscesses.

13. Lipopolysaccharide K. pneumoniae serum resistance mainly conferred by the lipopolysaccharide O side chain, which can impede C1q or C3b from binding to the bacterial cell membrane, thereby protecting the organism from subsequent complement-mediated membrane damage and cell death. Klebsiella lipopolysaccharide may also contribute to virulence by other mechanisms. These include increasing lethality in pulmonary infection by enhancing the propensity for bacteremia , and acting as an endotoxin , by triggering cytokine pathways leading to the sepsis syndrome and septic shock.

14. Pili ( fimbriae ) Strains of K. pneumoniae express two morphologically and functionally distinct filaments: type 1 and the type 3 pili Type 1 pili heteropolymeric mannose-binding fibers produced by all members of the Enterobacteriaceae family, which mediate adherence to many types of epithelial cells like the bladder epithelium. The type 3 fimbrial adhesion protein ( MrkD adhesin ) plays a central role in the virulence of K. pneumoniae by attaching to host cells, such as of the urogenital , respiratory, and intestinal tracts . This can result in bacterial colonization, subsequent proliferation on the host mucosal surfaces, and clinical infection such as pyelonephritis or pneumonia.

15. Siderophores Iron an essential factor in the growth of Enterobacteriaceae such as Klebsiella . Because of the scarcity of iron in the microenvironment, K. pneumoniae and other bacteria have developed multiple mechanisms to enhance iron uptake. These include synthesis of iron chelators ( siderophores ), such as enterobactin (also called enterochelin ) and aerobactin , the aerobactin receptor to introduce exogenous aerobactin , and the kfu iron uptake system.

16. ISOLATION AND IDENTIFICAION 1.DIFFERENTIAL MEDIA: EMB agar – Large, slimy, irregular colonies ; pink centre and colourless periphery ; very mucoid . Mac conkey or Deoxycholate agar - Large, slimy, vicious confluent growth, red centred colonies 2.SELECTIVE MEDIA: Deoxycholate extract agar- inhibited or slight growth of mucoid , pink to red colonies.

17. K.Pneumoniae Macconkey agar.

18. 3.INHIBITED MEDIA: Bismuth sulfite agar : usually inhibited; occassional growth of small black, brown or green colonies. Brilliant green agar- inhibited or growth of mucoid yellow green colonies with yellow zone.

19. BIOCHEMICAL PROFILE ( K.pneumoniae ) Lactose fermentation : yes IMViC :- (--++) Indole production – negative reaction Methyl red test – negative reaction Voges-Proskauer – positive reaction Citrate utilisation - positive reaction H2S production in TSI agar - negative reaction Lysine decarboxylase - positive reaction Urease activity - positive reaction

20. Clinical Sign In animals,  Klebsiella  bacteria an important cause of metritis and infertility in horses, mastitis in bovines, hematogenous osteomyelitis originating in pulmonary lesions in cattle, and accumulation of pus in the chest ( pyothorax ) in horses. Bacterial bronchopneumonia or pneumonia caused by  Klebsiella  species a common lung disease, particularly in dogs. Affected dogs and cats usually have immunosuppresion which results from drugs, malnutrition, stress, endocrine diseases, and other infections, including feline leukemia virus, feline immunodeficiency virus infection, and canine distemper.

21. Signs of bacterial pneumonia in dogs include productive and soft cough, nasal discharge, and exercise intolerance. Cats rarely have cough. Klebsiella  species responsible for approximately 7% of urinary tract infections in dogs. Rhinoscleroma a chronic granulomatous infection that affects the upper respiratory tract from the nose down to the trachea.

22. MASTITS BY KLEBSIELLA One in four animals contracting clinical  Klebsiella   mastitis dies within days. Klebsiella   can stay silent inside the udder. Infection of the mammary gland with Klebsiella causes an immune response in which the bacteria are killed by white blood cells and endotoxin (LPS) released. This immune response and the resulting inflammation cause clinical symptoms. The endotoxins induce to severe changes in vascular permeability, and increase of somatic cells in mammary gland and milk, resulting in edema, depression, toxemia, and severe peracute or acute clinical signs of mastitis.

23. Mastitis in dairy cow

24. Klebsiella cases are approximately 1/3 mild, 1/3 moderate and 1/3 severe Klebsiella invade deeply into the udder tissue and damage the secretory capacity of the gland. Thus some Klebsiella infections become chronic, and affected cows suffer long-term decreased milk production.

25. clinical signs clinical signs in two weeks prior to lactation or in the first weeks of the dry period. Fever, agalactia , anorexia, depression, rumen static, and dehydration most common clinical signs of peracute mastitis in cattle. The mammary gland presents marked swelling and regions containing signs of congestion and necrosis. Mammary secretion watery to serous, containing small flakes and great increase of neutrophils . These high migrations of neutrophils into the affected quarter associated to severe leukopenia and neutropenia in bovine mastitis.

26. Transmission Three weeks prior to calving, after a prolonged rainy period. The excess of rain and deficient management of organic material certainly increased humidity and fecal material in the environment, which probably primary sources of the microorganism to mammary gland. Fecal shedding of K. pneumonia and excess of organic material in the environment between milking would be associated with bovine mastitis .

27. TREATMENT Symptomatic treatment Fluid therapy Anti-inflammatory drugs Antibiotics (Third gen. c ephalosporins ( eg , cefotaxime , ceftriaxone ), carbapenems ( eg , imipenem / cilastatin ), aminoglycosides ( eg , gentamicin , amikacin ), and quinolon )

28. Prevention . SRP vaccine ( siderophore receptor and porin proteins) -induced antibodies bind and block transfer of iron and nutrients through bacterial cell wall pores, starving bacteria of needed nutrients, specifically iron. J5 bacterin Vaccine ( approximately 60 days and 28 days before the expected calving date) Pre- and post-milking teat disinfection and good milking hygiene is important to minimize transmission during milking. Apply fresh bedding Avoid the use of sawdust and recycled manure bedding.

29. Chronically infected cows should be segregated and milked last, and culled when possible.

30. PROTEUS SPECIES Proteus vulgaris electron micrograph

31. PROTEUS SPECIES They named after the Greek deity Proteus who had the ability to assume different shapes. They give a characteristic spreading growth (‘swarming’) on appropriate solid media, oxidatively deaminate amino acids, hydrolyze urea but fail to acidify mannose. Have five named species: Proteus mirabilis, Proteus vulgaris , Proteus myxofaciens , Proteus penneri , Proteus hauseri .

32. Proteus strains grow well on ordinary media, but most strains, particularly if grown at 22–30C, do not form discrete colonies on nutrient agar. they exhibit spreading growth (swarming) even on the surface of appropriate well-dried media.

33. Proteus cells readily destroyed by moist heat at 55C for 1 h and by common disinfectants such as phenolics or halogens.

34. Urinary track infections caused by  Proteus mirabilis frequent in dogs and cats.Infection with the organism can lead to formation of struvite stones in kidneys(dog and cat).  Proteus  species occasionally involved in  ear infections  in dogs and cats  Cause diarrhea in mink, lambs, calves, goats, and puppies.

35. PATHOGENICITY AND VIRULENCE FACTORS P. mirabilis  causes between 1-10% of all urinary tract infections, Urease –most important of all virulence factors,it hydrolyzes the urea in urine to ammonia and carbon dioxide The formation of ammonia also leads to alkalinization of the urine and at pH values above 8, calcium and magnesium ions precipitated in the form of struvite and carbonate-apatite crystals. Urease mediates virulence via the production of urinary stones. These stones can block urinary flow and cause tissue damage; they can also become quite large (> 1 cm 2 ) . The precipitated minerals may mix with bacteria adherent to a urinary catheter, forming a crystalline biofilm and eventually blocking urine flow through the catheter.

36.  A  ureC   urease mutant incapable of forming stones, and this has a direct impact on the ability of  P. mirabilis to cause UTI. Due to the prominent role of urease in  P. mirabilis  virulence, this enzyme an active target of investigation to identify clinically useful inhibitors. Since the ability of  P. mirabilis  to generate urinary stones and crystalline biofilms dependent upon alkaline pH, another approach to prevent catheter blockage to acidify the urine.

37. FLAGELLA Like many bacteria,  P. mirabilis  uses flagella to swim through liquids and toward chemical gradients P. mirabilis  has a short rod shape and typically possesses a few peritrichous flagella. All genes encoding flagellar components, including the class I flagellar master regulatory genes  flhDC  (PMI1671-72), are found within a single 54 kb locus in the chromosome (PMI1617-72). P. mirabilis  encodes two flagellins , FlaA (PMI1620) and FlaB (PMI1619) (also known as FliC1 and FliC2)  

38. FlaA the major flagellin . flaA   transcribed as a monocistronic message and  flaB  message is generally undetectable. Recombination between  flaA  and  flaB  can occur. This phenomenon discovered when  flaA  mutants were often found to revert to a motile phenotype; these revertants produced antigenically distinct flagella that the product of recombination resulting in a hybrid  flaAB  gene .

39. Fimbriae and adherence ability - Bacterial adhesion to epithelial surfaces thought to be one of the most important virulence factors playing a significant role in the initiation of UTI. Bacterial uropathogens exist mostly in the intestinal tract, from where they colonize the periurethral region and then ascend into the bladder, causing symptomatic or asymptomatic bacteriuria . Adhesion of bacterial cells to uroepithelial cells very important in this process in infections caused by Proteus spp.

40. The bacterial adhesion capacity most frequently associated with the presence of fimbriae on bacterial cells. Fimbriae indeed responsible for the attachment of Proteus bacilli to uroepithelial cells.. By electron microscopy, it possible to see bacteria with fimbriae bound to renal pelvic mucosa.

41. The bacteria with large numbers of fimbriae readily ingested by polymorphonuclear cell monolayers , whereas the bacilli with small numbers of fimbriae resistant to phagocytosis . Proteus strains have two types of fimbriae —thick (approximately 7 nm in filament diameter) and thin (4 nm in diameter). The first type, also known as type IV fimbriae , found to be mannose resistant and named Proteus-like fimbriae (MR/ P).

42. The second type the type III fimbriae , which mannose-resistant Klebsiella -like fimbriae (MR/K). These types of fimbriae associated with their ability to hemagglutinate untreated (MR/P) or tannic acid-treated (MR/K) erythrocytes from several animal species. MR/P fimbriae are more important for the adherence of bacteria to epithelial cells than MR/K hemagglutinins .

43. Flagella and swarming motility. Presence of flagella on the surface of pathogenic and opportunistic bacteria has been thought to facilitate the colonization and dissemination from the initial site. When transferred to a solid medium, Proteus bacilli undergo morphogenesis to swarmer cells and swarm over the surface of solid medium

44. This kind of growth of Proteus rods on solidified nutrient medium termed the swarming phenomenon. Swarming as a form of bacterial translocation across the solid surface of artificial or natural media characteristic not only for Proteus spp. but also for the gram-negative bacteria Vibrio spp.

45. The mechanism of overproduction of flagella by swarmer cells and the significance of this phenomenon in pathogenicity also closer to being understood. the synthesis of swimmer and swarmer cell flagella encoded by the genes flaA , flaB , flaC , and flaD . flaA and flaB tandemly linked and nearly identical copies of flagellin -determining genes.

46. Flagellin strongly immunogenic, it can be assumed that at least part of the immunoresponse of the host during the infection directed against this antigen. Thus, the possible changes in flagellin antigenicity may enable bacteria to escape the immunoresponse of the infected microorganisms.

47. Outer membrane proteins. OM proteins (OMP) possess immunogenic properties and mitogenic activity for B cells. OM lipoproteins and their synthetic analogs function as adjuvants and can also activate macrophages to produce tumor necrosis factor (TNF). The OM of P. mirabilis contains three major proteins of 39.0, 36.0, and 17.0 kDa . The 39-kDa protein identified as the OmpA protein , and the 36-kDa protein appeared to be a peptidoglycan -associated matrix protein.

48. OmpA effective as an immunomodulator of the immune response to LPS, greatly enhancing the level of O-specific IgG . 39-kDa( OmpA ) protein inhibits oxygen radicals, as well as interleukin-1 (IL-1) production, and enhances TNF (tumor necrosis factor)secretion by LPS-stimulated macrophages.

49. Lipopolysaccharide (O-antigen, endotoxin ) Proteus antigenically heterogeneous , principally because of structural differences of its O-specific polysaccharide chain of LPS (O antigen), as well as its H antigen. The serological classification(Kauffman and Perch)- 49 P. mirabilis and P. vulgaris O serogroups and 19 serologically distinct H antigens . O-specific polysaccharides from P. vulgaris OX19 and OX2, and P. mirabilis OXK, since strains belonging to these serogroups cross-react with antibodies from patients with different rickettsial infections and commonly used in the diagnostic Weil-Felix test .

50. Six types of core regions have been identified so far in Proteus LPS I - P. mirabilis O28, O23, O32, P. vulgaris O39 II - P. mirabilis 19,59 III - P. mirabilis O28 IV- P. mirabilis O38 V- P. vulgaris O23 VI- P. mirabilis O11, O13, O30, O31, O35, O36.

51. LPS endotoxins , cause a broad spectrum of pathophysiological effects such as fever, hypotension, disseminated intravascular coagulation, and lethal shock. LPS as a bacterial surface antigen recognized by specific antibodies produced by the host defense system. LPS from the S form of pathogenic bacteria contributes to their resistance against bactericidal action of serum and intracellular killing by phagocytes.

52. The possibility of development of local nephritis in dogs demonstrated after direct injection of lipid A into the renal pelvis. A dministration of lipid A results in an immunopathophysiological process in which complement activated by lipid A–anti-lipid A complexes causes inflammation.

53. The characteristic feature which distinguishes the Proteus core region from E. coli and Salmonella cores presence of D- galacturonic acid.

54. Capsule antigens P. mirabilis O6 and O57 and P. vulgaris O19 could synthesize a capsule antigen structure identical to the O-specific chain of their LPS . A cidic character of Proteus CPS, due to the presence of uronic acids, pyruvic acid, or phosphate groups, enabled them to bind metal cations (e.g., Mg21) via electrostatic interactions and it helps to form struvite in UTI.

55. Hemolysins - Hemolytic strains much more lethal for mice than nonhemolytic one. HpmA and HlyA cytotoxic for a wide variety of cell types and together with urease they play an important role in cell invasion and internalization. Hemolysin and urease expressed at higher levels in P. mirabilis than in P. vulgaris

56. ISOLATION AND IDENTIFICAION 1.DIFFERENTIAL MEDIA: EMB agar– Small, colourless, discrete colonies or “swarming” spreading growth (foul odour). Mac C onkey or Deoxycholate agar- Small, colourless, discrete colonies; occassional spreading growth. 2.SELECTIVE MEDIA: Deoxycholate extract agar- Small, discrete colourless colonies.

57. 3.INHIBITED MEDIA: Bismuth sulfite agar: Flat or slightly raised green colonies Brilliant green agar- inhibited or red colonies with intense red zone.

58. BIOCHEMICAL PROFILE Lactose fermentation : No IMViC :- Indole production – P.vulgaris +; P.mirabilis − Methyl red test – positive reaction Voges-Proskauer – reaction varies with individual species Citrate utilisation - reaction varies with individual species H2S production in TSI agar - positive reaction Lysine decarboxylase - negative reaction Urease activity - positive reaction

59. CLINICAL SIGNS Proteus mirabilis   commonly associated with recurrent urinary tract infections in dogs thus providing easily detectable clinical signs ( haematuria )   dysuria (straining to urinate) A burning sensation when urinating Passing frequent, small amounts of urine Urine that appears cloudy Urine that appears red, bright pink or cola-colored — a sign of blood in the urine Strong-smelling urine

60.  Intermittent pain. Other symptoms include nausea and vomiting, restlessness, sweating, and blood or a stone or a piece of a stone in the urine 14% of dogs may experience urinary tract infection during their lifetime. 9 to 32% UTI from P.mirabilis .

61. TREATMENT   Struvite stones / crystals form when the animal’s urine PH becomes neutral or alkaline (PH 7 or greater) and urine concentrated. Three primary treatment strategies for struvite bladder stones: 1) feeding a special diet to dissolve the stone(s), 2) non-surgical removal by urohydropropulsion and 3) surgical removal. FIRST-  These diets typically restricted in protein, phosphorus and magnesium and formulated to promote formation of acidic urine (with a pH less than 6.5).

62. x-rays should be performed approximately every four to six weeks during treatment. Non-surgical removal - If the bladder stones very small it may be possible to pass a special catheter into the bladder and then flush the stones out , called urohydropropulsion Surgical removal - Surgery indicated in dogs that have a large number of stones in their bladder

63. Symptomatic treatment Oral quinolone , cephalosporin, TMP/SULPHA drug for 14 days may be added to complete treatment.

64. ENTEROBACTER SPECIES Enterobacter aerogenes

65. ENTEROBACTER SPECIES They metabolically active organisms with most strains able to ferment glucose, usually with gas production. Their habitat soil and water, but they occasionally found in the animal bowel flora . They all reduce nitrate to nitrite but have negative reactions for indole production, H2S production, hydrolysis of gelatin , or production of lipase, DNase , or oxidase .

66. Their strains may be somewhat mucoid , but the amount of extracellular material formed usually not great. Motility has historically been an important distinguishing character between Enterobacter and Klebsiella . Growth at 37C usually good, many Enterobacter strains give atypical biochemical reactions unless tested at a lower temperature.

67. E.aerogenes resembles Klebsiella in its wide range of fermentative activity and in its ability to form gas from cellobiose , glycerol, and inositol , but it does not form gas from starch.

68. HABITAT AND PATHOGENICITY The normal habitat of Enterobacter believed to be soil and water, but the organism occasionally found in the feces and the respiratory tract of animal. Most infections of the urinary tract. It can cause bacteremia and meningitis .

69. Shlaes (1993) described Enterobacter bacteremia conducted in several patients. The results indicated that : (1) Emergence of resistance more frequent during treatment with third-generation cephalosporins than with other antibiotics. (2) The addition of an aminoglycoside to the cephalosporin did not prevent emergence of resistance.

70. (3) previous treatment with cephalosporins associated with bacteremia caused by multiresistant Enterobacter spp. (4) Infection with multiresistant Enterobacter spp.associated with a higher mortality than infection with susceptible strains.

71. Conclusion : avoidance of third-generation cephalosporins for surgical prophylaxis and therapy in patients in whom Enterobacter infections suspected or proven should lower the prevalence of Enterobacter bacteremias and mortality and prevent the emergence of multiresistance .

72. ISOLATION AND IDENTIFICAION 1.DIFFERENTIAL MEDIA: EMB agar – Large, shiny, mucoid colonies; pink centre and colourless periphery; no sheen. Mac conkey or Deoxycholate agar- Large, shiny, mucoid colonies; red centre and colourless periphery. 2.SELECTIVE MEDIA: Deoxycholate extract agar- inhibited, or slight growth of pink to red colonies.

73. Enterobacter aerogenes in Macconkey agar

74. 3.INHIBITED MEDIA: Bismuth sulfite agar : usually inhibited; occassional growth of small black, brown or green colonies. Brilliant green agar- inhibited or growth of mucoid yellow green colonies with yellow zone.

75. BIOCHEMICAL PROFILE ( E.aerogenes ) Lactose fermentation : Yes IMViC :- (--++) Indole production – negative reaction Methyl red test – negative reaction Voges-Proskauer – positive reaction Citrate utilisation - positive reaction H2S production in TSI agar - negative reaction Lysine decarboxylase - positive reaction Urease activity - negative reaction

76. Enterobacter Disease In Dogs. Enterobacter spp a  normally found in the skin and gastrointestinal microflora of dogs. Species which are pathogenic to dogs include: Enterobacter cloacae. Enterobacter hormaechei . A ssociated with pancreaticobiliary duct infections, nosocomial infections such as cystitis associated with urinary catheterization and post-operative empyema .

77. Zoonotic infections in humans have been attributed to these bacteria, which are normal residents of the canine oropharynx .

78. TRANSMISSION  Transmission through direct or indirect contact of mucosal surfaces with infectious agent (e.g. bacteria can transfer from contaminated feed and water) or, in the case of endogenous flora, through transfer to adjacent, susceptible. Enterobacter can also be spread through the fecal-oral route.

79. TREATMENT Enterobacter spp. multidrug resistent.can present therapeutic challenge. Treatment must be based on culture and sensitivity result. Empiric therapy may include amoxicillin/ clavulanate or chloramphenicol with severe infections. Multi drug therapy required due to the high level of resistance .

80. REFERENCES Topley and Wilson's Microbiology www.microbewiki.com Veterinary microbiology and microbial diseases by Quinn and Carter second edition (2012) The potential of selected Australian medicinal plants with anti-Proteus activity for the treatment and prevention of rheumatoid arthritis  (2015) Significance and Roles of Proteus spp. Bacteria in Natural Environments. Dominika Drzewiecka Microb Ecol. 2016;   Zachary D  et al  (2011) Cutaneous mucormycosis complicating a polymicrobial wound infection following a dog bite.  Case Rep Infect Dis   2011 :348046