1. Laboratory Methods for Diagnosis of Non-fermenting Gram-Negative Bacilli
Dr Mohammad Rahbar

2. General Characteristics of Non-fermenters
Nonfermenting gram-negative bacilli are grouped together because they fail to acidify oxidative-fermentative (OF) media overlaid with mineral oil or triple sugar iron agar (TSIA) butts .They prefer and grow much better in an aerobic environment ;some group members oxidize carbohydrates to derive energy for their metabolism ;they are referred to as oxidizers.

3. General Characteristics of Non-fomenters
Others do not break down carbohydrates at all and are inert or biochemically inactive; they are referred to as nonoxidizer or asaccharolytic .Additional characteristics can differentiate this group of nonfernenters from other gram-negative bacilli: motility ,pigmentation and their ability or lack of ability to grow on selective gram-negative media such as MacConkey agar.

4. General Characteristics of Non-fermenters
Most nonfermentative gram-negative bacilli are oxidase positive, a feature that differentiate them from the Enterobacteriaceae (except plesiomonas witch is oxidase positive.

5. General Characteristics of Non-fermenters
In general nonfermentative gram-negative bcilli are ubiquitous and found in most environments: in soil and water .on plants and decaying vegetation and in many foodstuffs. They prefer moist environment ,and in hospitals that can be isolated from nebulizers, dialysate,fluide saline and on catheters and other devices.

6. General Characteristics of Non-fermenters
Nonfermenters may withstand treatment with chlohexidine and quaternary ammonium compounds .They are rarely ,if ever part of the normal host flora but can easily colonize hospitalized patients, especially those who are immunocompromised .Nonfermentative gram-negative bacilli tend to be resistant to several Antimicrobial agents

7. TAXONOMY, BIOCHEMICAL CHARACTERISTICS, AND CLINICAL SIGNIFICANCE OF MEDICALL Y IMPORTANT GENERA OF NONFERMENTERS

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Unlike the Enterobacteriaceae the nonfermenting gram-negative
bacilli do not fit conveniently into a single family of well-characterized genera, and the correct taxonomic placement of many nonfermentative, gram-negative bacilli
(NFBs) remains unresolved.

9. Continue… Consequently, the study of nonfermenters
is often confusing for the beginning microbiologist. The major genera of nonfermenting, gram-negative bacilli
have been classified into at least 15 families.

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One approach to studying the nonfermenters is to group them on the basis of the presence or absence of motility and on the type of flagella present in strains that are motile.

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Organisms That Are Motile With Polar Flagella
Pseudomonads

12. Fluorescent Group.
The species within this group are all by the production of a water-soluble white to blue-green under long wavelength pyoverdin pigment that fluoresces (400-nm) ultraviolet light. Production of fluorescent pigments is particularly enhanced in media with a high
phosphate concentration.

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Although all three members of this group produce pyoverdin, only one species. P. aeruginosa ,produces the distinctive blue, water-soluble pigment pyocyanin

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Pseudomonas aeruginosa produces a characteristic appearance when grown on BAP. It appears as large gray colonies
with a spreading periphery and exhibits hemolysis. Colonies often have an alligator skin appearance and exhibit
a metallic sheen.

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Rapid identification of P. aeruginosa in culture can be made whenever the characteristics are observed: typical colony morphology production of diffusible pigments the presence of a fruity odor, and oxidase positivity .

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that produce a pungent, "rotten-potato" odor. There has been at least one report of a nosocomial outbreak caused by
strains of malodorous P. aeruginosa .

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Pseudomonas aeruginsa is the most frequently recovered from clinical specimens. p, aeruoginosa infection is especially prevalent among patients with burn wounds, cystic fibrosis, acute leukemia, organ transplants, and drug addiction.

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Infections commonly occur at any site where moisture tends to accumulate tracheostomies, indwelling catheters, burns, the external ear ("swimmer's ear"), and weeping cutaneous wounds. The exudation of bluish pus, with a grape-like odor from the production of pyocyanin, is characteristic.

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P aerugnosa also causes urinary tract and lower respiratory tract infections;
the latter can be severe and even life-threatening in immunocompromised hosts. The organism can also cause
devastating infections of the eye

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Pseudomonas keratitis. Infection of corneal ulcers, and endophthalmitis must be approached
as a medical emergency that can be fulminant and threaten permanent loss of vision. Individual cases of endocarditis,meningitis, brain abscess, and infections of bones and joints from hematogenous spread appear with regular
frequency in the literature.

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Most cases of endocarditis require valve replacement because the infection is difficult to eradicate. P. aeruginosa dermatitis and otitis externa outbreaks associated with swimming-pool and hot-tub use are well described. The CDC reported at least 75 cases during six outbreaks occurring between 1997 and Sporadic P. aeruginosa infections following ear piercing have also been reported.

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P .aerugillosa produces several substances that are thought to enhance the colonization and infection of host tissue. These substances, together with a variety of virulence factors, including lipopolysaccharide (LPS), exotoxinA, leukocidin, extracellular slime, proteases, phospholipase,and several other enzymes (Box 7-5), make P. aerugillo.l'a the most clinically significant bacteria among the NFB.

23. Virulence Factors
An unusual mucoid morphotype of P. aeruginosa is frequently recovered from respiratory secretions of patients with cystic fibrosis who are chronically infected with P. aeruginosa The mucoid morphotype is due to the production of large amounts of a (called alginate)that surrounds the cell. The production of alginate is ultimately responsible for the poor prognosis and high mortality rates among patients with cystic fibrosis.

24. Virulence Factors of Pseudomonas aeruginosa
Alginate:
Capsular polysaccharide that allows
infecting bacteria to adhere to lung epithelial cell surfaces and form biofilms which, in turn, protect the bacteria from antibiotics and the body's immune
system

25. Virulence factors PIlli Surface appendages that allow adherence
of organism to GM-I ganglioside receptors on host epithelial cell surfaces
Neuraminidase
Removes sialic acid residues from GM-I
ganglioside receptors. Facilitating binding pili

26. Viurlence factors Exotoxin A :
Tissue destruction, inhibition of protein
synthesis; interrupts cell activity . Enterotoxin
Interrupts normal gastrointestinal
activity. leading to diarrhea

27. Virulence Factors Exoenzyme S: Inhibits protein synthesis
Phospholipase C:
Destroys cytoplasmic pulmonary surfactant; inactivates opsonins

28. Virulence Factors Elastase: Cleaves immunoglobulins and , disrupts
neutrophil activity
Leukocidin:
Inhibits neutrophil and lymphocyte function

29. Virulence Factors Pyocyanins:
Suppress other bacteria and disrupt ciliary activity; cause oxidative damage to tissues, particularly oxygenated tissues such as lung

30. Summary Key Tests for Identification P. aeruginosa
Minimum Requirements for Definitive Identification of P. aeruginosa
Identification based on all of the following:
I. Gram-negative rod
2. Oxidase-positive
3. Typical smell (fruity grape-like odor or corn tortilla)
4. Recognizable colony morphology
a. On blood or chocolate agar appear as large colonies with
metallic sheen, mucoid, rough. or pigmented (pyocyanin)
and often p-hemolytic

31. Summary Key Tests for Identification P. aeruginosa
b. On MacConkey, appear as lactose-negative with greenpigmentation, or metallic sheen
Limitations:
I. Rare Aeromonas isolates may resemble P. aerugirrosa
(lacking the typical smell) but will be spot indole-positive
(P. aeruginosq are indole-negative).
2. Some Burklto/der;a cepac;a isolates from patients with
cystic fibrosis may exhibit morphotypes that resemble P.aeruginosa.

32. Colonies of Pseudomonas aeruginosa typically display beta hemolysis, a metallic sheen, and blue or green pigment.

33. Pseudomonas aeruginosa (beta hemolysis and metallic sheen

34. Pseudomonas aeruginosa (beta hemolysis with transmitted light)

35. . Pseudomonas aeruginosa (beta hemolysis with transmitted light

36. FIG. 5. Pseudomonas aeruginosa (beta hemolysis and pigment with transmitted light

37. encapsulated strain of Pseudomonas aeruginosa recovered from a cystic fibrosis patient at 24 hours.

38. Same plate as FIG. 23 at 48 hours, this strain of Pseudomonas aeruginosa make abundant, mucoid capsular material.

40. Acinetobacter
The genus Acinetobacter ,now a member of the family Moraxellaceae ,cosist of 25 ِDNA homology groups or genomospeecies .Only 10 species have been officially named:the two species most commonly seen in clinical specimens are : A.baumannii and A. lwoffii

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Acinetobacter spp are unique in the environment in soil, water and foodstuffs in the hospital environment they have been associated with ventilator ,humidifies catheter and other devices. About 25% of adults carry the organism in their phrynx.If not harboring Acinetobacter spp ,already hospitalized patients may become easily colonized,

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As many as 45% of patents with a trachetomy may be colonized. When Acinetobacter spp isolated from urine, feces ,vaginal secretion ,and many different type of respiratory specimens, they are often considered insignificant colonizer or contaminants.

43. Acinetobacter baumannii
A. baumannii is the second most frequent nonfermenter encountered in clinical laboratories, but with only about one tenth the frequency of P. aerugi1losa. The following are the characteristics by which a presumptive identification can be made.

44. Clinical Infections
Acinetobacter spp are opportunistic accounting 1% to 3% of all nosocomial infections< they are second only to P.aeruginosa in frequency of isolation of all nonfermenters in the clinical microbiology laboratory.

45. Disease in particular with A.baumannii
UTI
Pneumonia, Tracheobronchitis,or both
Endocarditis with up 25% mortality
Meningitides
Septicemia Truman infections, Burn infections,
Eye infections.
A.lwoffii is much less virulent

46. Laboratory Diagnosis Appear as cocci or coccobacilli on Gram stain .
Grow well on MacConkey agar (colonies may have slightly pinkish tint ,a helpful characteristic when present
Exhibit rapid utilization of glucose, with production of acid
Are non- motile
Are penicilin resistant

47. Lab Diagnosis
The initial clue is the observation of tiny diplococci on Gram stains prepared directly from clinical materials. When Gram stains are prepared from agar or broth cultures, the cells may appear larger and more like coccobacilli

48. Lab Diagnosis
Acinetobacter species are not pigmented when grown on blood agar, a helpful characteristic in differentiating them from certain other nonfermenters, such as occasional oxidase-negative, nonmotile strains of Burkholderia cepacia.

49. Lab Diagnosis
However, colonies growing on Mac- Conkey agar may produce a faint pink tint or a deeper cornflower blue when observed on eosin methylene blue agar Resistance to penicillin helps distinguish
A. baumannii from the highly penicillin-sensitive Moraxel/ a species, which also usually appear as coccobacilli on Gram stain.

50. Lab Diagnosis
Most strains of Moraxel/a species are also cytochrome oxidase-positive. A. lwoffii is nonsaccharolytic and can be differentiated from A. baumannii because it produces
no acid when grown in media that contain carbohydrates.

51. Summary for Diagnoses of Acinetobacter Spp
Obligate Aerobe
Nonmotile
Oxidase Negative
Nonhemolytic
Saccharolytic: acidifies most OF carbohydrates ,including glucose and xylose.
Produce acid from lactose

52. Summary for Diagnoses of Acinetobacter Spp
Grows well on MacConkey agar
Resistant to penicillin

53. Although Acinetobacter baumanii is incapable of fermentation, its very strong lactose oxidation leads to weakly acid/purple colonies on MacConkey agar

54. Stenotrophomonas ma/tophi/ia
S. maltophilia is the third most frequently encountered nonfermenter in clinical laboratories. Before 1983 it was a member of genus Pseudomonas. It was later classified as a member of the plant pathogen Xanthomonas .Following DNA homology andsequencing analysis it was classified as a member of Stenotrophomonas

55. Continue… Isolates are ubiquitous in the envir-
ointment ,being found in water, sewage, and plant materials ; they are very common to the hospital environment ,where they can be found contaminating blood –drawing equipment ,disinfectant,tranducer, and other equipment.

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Clinically ,when S.maltophilia is isolated from clinical specimens ,it is initially regarded as saprophyte or colonize . Although not considered part of normal flora ,S.maltophilia cab quickly colonize the reparatory tracts hospitalized patients ,in particular those exposed to antimicrobial agents to which S. maltophilia may be inherently resistant

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These antimicrobial include cephalosporins ,penicillins, carbapenems .and aminoglycosides. With increased use of agents to which it is innately resistant ,there have been more reports of disease attributed to this organism. Reported disease include endocarditis, especially in a setting of prior intravenous drug abuse or heart surgery ,wound infections, including cellulitis and ecthyma gangrenosum,bactermia and rarely meningitides.

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With rare exceptions ,infections have occurred in a nosocomial setting .S.maltophilia is rarely associated with lower respiratory tract infections, although it has been isolated from 6.4 to 10.2 of patients with CF. Pseudoinfections have also occurred a result contaminated collection tubes or cups. (e.g Blood collection tubes)

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The single most important risk factor in affected individuals was the presence of a venous chatter. Most patients with bactermia responded well to therapy unless they had concomitant pneumonia or shock.

60. Important Reactions for Diagnosis of S.maltophilia
Yellow –tan pigment on tryptycase Soy agar.
Lavender-green pigment on sheep blood agar
Growth at 42C: positive
Oxidase =negative
Catalase :positive
Oxidize glucose in OF medium weakly positive
Oxidize maltose in OF medium strongly positive.

61. Important Reactions for Diagnosis of S.maltophilia
Pyoverdin=negative
ONPG = positive
DNASe = positive
Nitrate not reduced to nitrogen gas
Lysin decarboxylase=slovely positve
Arginine dehydrolase :negative
Ornithine decarboxylase : negative

62. Continue…. Esculin hydrolysis :positive Gelatin hydrlysis: positive
Susceptibility to SXT: positive
Susceptibility to colisitin:positive

63. Important Reactions for Diagnosis of S.maltophilia
The antibiotic susceptibility pattern can also be a clue to the identification of S. maltophilia, which is typically resistant
to most antibiotics, , butis susceptible to trimethoprim- and colistin.

64. Figure 8 Stenotrophomonas maltophilia on EMB

65. . Same plate as FIG. 19 at 48 hours, Stenotrophomonas has distinct non-lactose fermenting colonies. The indicator has turned an alkaline tan color

66. Gram Stain of S.maltophilia

67. Burkholderia B.cepacia
Burkholderia (Pseudomonas ) cepacia is a complex of nine distinct genomic species (geneomvars) that has in the past been called P.multivorans,P.kngiiand and EQ-1.Clinically B.cepacia is a lowgrade ,nosocomial pathogen that has most often been associate with pneumonia inpatients with CF or chronic granulomatous disease( CGD).

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It has been reported to cause endocarditis ( especially in drug addicts) pneumonia ,UTIs ,osteomylitis , dermatitis, and other wound infections resulting from use of contaminated water. It has been isolated from irrigation fluids, anesthetics ,nebulizers, detergents, and disinfections.

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Research supports the association of B,cepacia and increased severity of disease and death in patients with CF and CGD.In the United State about 3%of CF population is infected with B.cepacia ,but rats up to 30%in some adult CF patients populations have been repotted. Outside these population morbidity and mortality rates remain low and consideration needs to be given to the possibility of contamination rather than infection when isolated.

70. Laboratory Diagnosis
The organism grow well on most laboratory media but may also viability on Sheep blood agar in 3 to 4 days without appropriate transfers .
B.cepacia grow on MacConkey agar, but selective media containing antimicrobial to reduce the growth of P.aeruginosa ,as well as other gram-negative bacilli ,are available to increase the recovery of B.cepacia.

71. Lab Diagnosis
These media include PC( pseudomonas cepacia), OFPBL (oxidative fermentative base ,polymyxinB, bacitracin ,lactose )and BCSA ( B.cepacia selective agar). Studies have suggested that BCSA is most effective in reducing overgrowth while maintaining good recovery of B.cepacia.

72. Lab diagnosis
B.cepacia complex often produce a week oxidase reaction. Nearly all strains oxidize glucose, and many will oxidize maltose, lactose and manitol.
Most strains are LDC positive
Most strains are ONPG positive
Most strains are ODC negative.

73. Lab diagnosis Nitrate positive
Are motile by means of polar tuftsof flagella
They do not fluoresce like P .aeruginosa, but they can produce a nonfluorescing yellow or green pigment that may diffuse into media.

74. Lab Diagnosis
Colonies of B.cepacia are nonwrinkled ,and this may be used to differentiate isolates from P.stutzeri ,which also produce a yellow pigment.

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B.cepacia is usually susceptible to chloramphenicol, cetazidim, piperacillin, and SXT, but resistant to most other agents. Susceptibility to the carbapenems is variable. resistance can develop quite rapidly. The CLSI recommended that if disk diffusion method of susceptibility testing ,then only ceftazidime ,meropenem, minocycline and SXT should be reported.

76. . Same plate as FIG. 17 at 48 hours, Burkholderia cepacia displays small non-lactose fermenting colonies. Some strains appear somewhat purple due to strong lactose oxidation

77. Oxidation-fermentation Hugh-Leifson - Uninoculated

78. OF glucose oxidative metabolism

79. OFglucose fermentative metabolism (Enlarged view)

80. Amino acid decarboxylase

81. Figure 7 Nitrate reduction test (Labeled view)

82. Phenol red broth