Gram-negative Rods

Swarming of Proteus spp

Proteus spp are Gram negative extremely, motile rods that are actively motile
at 25`C and weakly motile at 37`C. On moist agar P. vulgaris and P.
mirabilis tend to swarm, producing a bluish gray confluent surface growth.
Demonstrate this phenomenon by placing a drop from a culture of Proteus in
the centre of a blood agar plate and incubating the plate for 24 hours. Often
the swarming of Proteus must be suppressed to isolate other species of
bacteria in a culture. Proteus does not tend to swarm on deoxycholate agar.
MacConkey agar or EMB agar. Proteus will not swarm if the agar concentration
is increased to 7%. Demonstrate inhibition of swarming by plating Proteus on
deoxycholate, MacConkey agar, EMB agar and Blood agar (7% agar).

Presumtive Identification Of Pseudomonas Aeruginosa

As stated previously, Pseudomonas aeruginosa has been incriminated in 5-l5%
of all hospital acquired infections and thus is clinically important.
Pseudocel agar (BBL) is used to isolate selectively Pseudomonas aeruginosa
from other enterics and pseudomonads (the exception is Ps. fluorescens). The
agar contains inhibitor cetyltrimethylamine bromide (cetrimide). Streak a
mixture of E. coli and Ps. aeruginosa onto a plate of pseudocel agar,
incubate overnight and observe the colonies. Pseudocel allows the production
of fluorescein and thus the agar surrounding the agar should fluoresce under
ultraviolet light. Perform a Gram stain on the colonies and confirm that the
fluorescent colonies are oxidase positive with NTaxo discs. Pick a colony
and make a thick emulsion in saline. Drop this emulsion onto the disc which
will turn purple immediately if oxidase is present. E. coli is oxidase
negative and pseudomonads are oxidase positive.

Pigment formation by Pseudomonas

Pseudomonas aeruginosa has been implicated in 5-l5% of all hospital acquired
infections, where it may colonize burn sites wounds and the lower respiratory
tract with the formation of a characteristic blue or green pus. The unusual
colour is the result of pigments produced and released by Pseudomonas
aeruginosa. The fluorescein causes a green colour, while pyocyanin is blue.
Plate Ps. aeruginosa on to King's A and King's B media and incubate for 24 hr
at 37`C to demonstrate pigment production. The blue-green colour and a
grape-like odour from the colony is characteristic of Ps. aeruginosa.

Klebsiella Capsules

Members of the genus Klebsiella are Gram-negative, non-motile, encapsulated
rods. K. pneumoniae can cause enteritis in children, pneumonia. respiratory
tract infections septicemia, meningitis and urinary tract infections. K.
pneumoniae is particularly virulent because it possesses an extremely heavy
capsule. Plate K. pneumoniae onto blood agar, or T-soy agar or MacConkey
agar. It will give rise to large mucoid colonies that tend to coalesce. The
colony is so sticky that long strings of mucous can be pulled out of the
colony when it is touched with a loop. The capsule is easily visualized with
a capsule stain or by the quellung reaction with the appropriate
antiserum.cted from isolation plates.

The IMViC Reactions

The IMViC reactions are used to distinguish the coliform bacteria but they
may be applied to other microbes in the family enterobacteriacae. The letters
stand for the tests performed Indole, methyl red, Voges-Proskauer and citrate
(the "i" in IMViC is inserted for euphony).

The Indole Test

Bacteria deaminate tryptophan to indole and skatole, the two volatile
substances that give the characteristic foul odour to feces. Inoculate a tube
of peptone broth which contains tryptophan and incubate the culture for 48 hr
at 37`C. Test for the presence of indole by adding 4-5 drops of Kovacs
reagent. The paradimethylaminobenzaldehyde reacts with the indole to give a
red colour. The Enhrlich indole test is a more sensitive modification. The
indole is extracted by the addition of l ml of xylene to the 48 hr broth
cultures. Shake well and allow the solvent to rise to the surface. Carefully
pour 0.5 ml of Ehrlich's reagent (paradimethylaminobenzaldehyde) down the
side of the tube. If indole is present a red ring will appear just below the
solvent. Note. Since the indole is volatile and degradable, old cultures
cannot be used. Microbes break tryptophan down into indole, pyruvic acid and
ammonia in a single step by the use of the enzyme tryptophanase. Microbes
break tryptophan down into indole, pyruvic acid and ammonia in a single step
by use of the enzyme tryptophasase. In cultures that contain both protein and
carbohydrate (e.g. l% tryptone and l% glucose broth) rapid acid production
from the carbohydrate inhibits the formation or the activity of enzymes that
attack protein. The protein sparing action of carbohydrates indicates this
relationship. It is shown by the lack of indole formation from tryptone when
fermentable carbohydrate is available.

Methyl Red Test

The MR-VP broth (Clark and Luks medium) contains peptones, phosphate and
glucose. The methyl red test establishes if the microbe has hydrolyzed
glucose to an acid. The culture is incubated for 2-5 days at 37`C and a few
drops of methyl-red pH indicator are added. A distinct red colour is
regarded as a positive reaction and a yellow colour as a negative one. Methyl
red indicator is yellow at pH 6.2 or above; and red at pH 4.4 or below. Both
Escherchia coli and Aerobacter aerogenes hydrolyze glucose to acids. E. coli
produces formic, acetic, lactic and succinic acid (l.59 moles/Mole glucose)
and the final pH is 4.5 or lower. When A. aerogene<MU>s is grown on glucose
it produces less acid more ethanol and butanediol. The final pH is not
limiting and when the sugar is exhausted the peptone is attacked and the pH
rises to 6.2 which gives a negative methyl red test.

Voges-Proskauer Test

The Voges-Proskauer test detects the presence of acetoin or acetyl-
methyl-carbinol which is an intermediate in the formation of 2,3, butanediol.
Microbes are inoculated into MR-VP broth and the culture is incubated for 48
hr at 37`C. About l5 drops of -naphthol solution and l0 drops of 40% KOH
is added and the mixture shaken.

A positive reaction is the development of a red colour after l5-60 min. Under
alkaline conditions and in the presence of oxygen, acetyl-methyl- carbinol is
oxidized to diacetyl (Figure 5) which reacts with creatine to give a red
colour. Creatine is present in peptone.

Citrate Test

Microbes are inoculated onto Simmons citrate by making a single streak over
the surface of a slope. If after 2-3 days the slope is the original green
colour citrate is not utilized. If the slant has changed to a blue colour
and there is a streak of growth citrate is utilized as the sole carbon
source. Simmons citrate only contains salts, citric acid and bromothymol
blue pH indicator.

Growth Requirements of Haemophilus

Members of the genus Haemophilus are small, Gram-negative, aerobic bacilli
which are non-sporulating, non-motile. They require enriched media with
either blood or blood products (e.g. Blood agar, Chocolate agar, Levinthol's
transparent agar and Filde's medium). Whole blood supplies the two factors
necessary for growth of H. influenza Lemin (Factor X) and NAD (Factor V).
Other species of the genus require either factor X, or V or both. H.
haemolyticus, H. parahaemolyticus and H. ducreyi cause beta hemolysis on
blood agar. Rabbit or horse blood agar is used for detection of hemolysis
because human and sheep blood agar often contain inhibitory compounds.

Determination of growth requirements of: Haemophilus species

l. Add saline (l ml) to the slant of Haemophilus and make an even cell
suspension.

2. Pour the suspension evenly over the surface of a tryptose agar plate and
allow the plate to dry. The tryptose agar alone will not support the growth
Haemophilus.

3. Place discs impregnated with Factor V, Factor X and both Factors on the
agar and incubate for 24 hr at 37`C in l0% CO2.

4. Observe the plate. Isolates requiring only Factor V will grow around the
Factor V and X,V disc. Those isolates requiring only Factor X will grow
around the Factor X and X,V discs. If both factors are needed growth occurs
only around the X,V disc.

Satellitism

H. influenza grows poorly in blood agar because it has an inadequate supply
of Factor V. The tiny colourless colonies are less than l mm in diameter.
The colonies are much larger on blood agar when they are growing near
colonies of S. aureus which synthesize extra NAD that diffuses into the
medium. The characteristic stimulation of growth around colonies of S.
aureus is called "satellitism" and it is used in clinical laboratories to
identify H. influenza. Demonstrate "satellitism" by smearing H. influenza
over the entire surface of a blood agar plate and then making a single streak
inoculation of S. aureus down the centre of the plate. Incubate at 37`C for
24 hr and observe.

Enrichment Media for Salmonella

Clinical specimens from patients with gastroenteritis, enteric fever, etc.,
may contain relatively few pathogens amid vast numbers of normal flora. The
normal flora would overgrow the pathogens if plated on enriched media. In
practice an enrichment media such as gram-negative broth, selenite broth or
tetrathionate broth are used. These enrichment media contain inhibitors that
will slow the growth of most microbes temporarily and allow the unrestricted
growth of Salmonella and Shigella. Use a Pasteur pipette and inoculate tubes
of GN broth, selinite broth and tetrathionate broth with a drop of an equal
mixture of E. coli and Salmonella. Incubate the broths overnight and then
streak a loopful of each broth onto MacConkey plates. There should be
relatively more colonies of Salmonella (grey colonies) than the E. coli (pink
colonies). The G-N broth (H. ajna broth) contains deoxycholate and citrate to
inhibit gram positive microbes and limiting amounts of mannitol to control
growth of Proteus species. As the name suggests Selenite broth contains
sodium selenite and E. coli is more susceptible to its toxicity than
Salmonella typhosa. E. coli is inhibited successfully for 8-l2 hours while
the Salmonella is unaffected. Proteus grows well in selenite broth. The bile
salts in tetrathionate broth inhibit Gram positive microbes and the iodine
inhibits most enterics.

Differential Media for Salmonella

Differential media characterize microbes by distinctive colonial appearances
in culture. Many agars contain a carbohydrate and an indicator dye in a
decolourized state. Fermentation of the carbohydrate to acid or aldehyde
will change the colour of the colony and the surrounding medium. The major
differential media for the identification of Salmonella and Shigella are
MacConkey, EMB and deoxycholate agar. The bile salts in deoxycholate agar
inhibit Gram positive microbes. Microbes that ferment lactose appear red as
opposed to grey if lactose is not utilized. The MacConkey agar contains all
the major ingredients of deoxycholate agar plus the inhibitor crystal-violet.
Colonies appear similar to those on deoxycholate agar. The lactose positive
colonies may be surrounded by a zone of precipitated bile because the acids
produced by the fermentation of lactose react with the bile salts and
subsequently absorb the neutral red. EMB in addition to lactose and sucrose
has eosin and methylene blue as an indicator to differentiate between lactose
fermentors and non-fermentors. Colonies of Salmonella are a transparent
amber colour or colourless while those of E. coli are blue-black and have a
metallic sheen.

Selective Media for Salmonella

Selective media are complex media that differentiate between organisms and
are very selective in their action towards certain microbes. Thus,
Salmone-la-Shigella agar (S-S) and Bismuth Sulfite eliminate the coliform
bacilli and Proteus spp with minor effects on Salmonella. Any coliform
colonies that do develop are differentiated readily from the salmonella on
the basis of colour and Brilliant green agar has the inhibitor Brilliant
green as well as lactose, saccharose and the pH indicator phenol red.
Salmonella and Shigella form pink to white opaque colonies surrounded by red
medium as does Proteus. The other enterics grown poorly but colonies that do
form are yellow-green in colour surrounded by a yellow-green zone. The
Bismuth sulfite agar is extremely selective for S. typhosa which form
characteristically black colonies when grown on this medium. A very heavy
inoculum is used since other coliforms are eliminated almost completely.
Hense small numbers of Salmonella can be detected quite easily, especially if
an enrichment broth is used first. A detailed description of the Bismuth
sulfite agar is given on pages l39-l44 of your Difco manual. The major
inhibitors in Salmonella-Shigella agar (SS) are bile salts and brilliant
green and as such is similar to the GN broth except that bile salts are a
crude form of sodium deoxycholate and contain several inhibitors. Salmonella
or Shigella form colourless colonies. E. coli and other lactose fermentors
that do grow form red colonies. Many of the coliforms will grow on XLD agar
which contains xylose, lysine and deoxycholate. Salmonella colonies are red
with a black center. Shigella is just red but some pseudomonods or P.
rettgeri give a falsely positive red. The other coliforms usually give rise
to yellow colonies. Use an inoculum containing equal numbers of Salmonella
and E. coli and streak a sample onto a plate of every agar described in this
section. Incubate and observe the difference in morphology and the
difference in the relative numbers of colonies of Salmonella and E. coli.

Serological Identification

The four major pathogens of the genus Salmonella. Salmonella typhi, S.
choleraseris, S. enteritidis and S. typhimusium, were isolated by l892.
Subsequent studies on the identification of Salmonella culminated in the
development of the antigenic schema of Kaufman and White which delineates
over l,000 specific antigenic types of Salmonella. Salmonella cause
bacteremia, enteric fevers and gastroenteritis. Transmission is by food,
water, human carriers and poultry. Serological identification helps locate
the source of the infection and prevents further spread. Serological
identification is based upon the detection of specific antigens on the
Salmonella. First the organism is placed into a "O" group depending upon the
somatic antigens present and identification is confirmed by antigenic
analysis of the flagellar "H" antigens. The somatic antigen (O = ohne Hauch)
is a heat stable polysaccharide associated with the cell wall. These are the
first antigens that are determined using the slide agglutination technique.
The flagellar antigen (H = Hauch) is a heat labile protein located in the
flagella. A tube agglutination technique is used to determine the H
serotype. S. typhosa and S. paratyphi C contain a third type of antigen
called the Vi (virulence) antigen, which is a heat-labile envelope antigen
that the O antigens. The virulence antigen is detected by passive
hemagglutination.

Slide Agglutination Test

Salonella types are characterized by the identification of both their somatic
"O" and flagellar "H" antigens. A thermolabile envelope antigen designated
Vi is usually found in freshly isolated strains of S. typhosa and S.
paratyphi C. This Vi antigen may block the O antigen and may be removed by
heat treatment. The O antigens are designated by Arabic numerals and the H
antigens by Arabic numerals and letters. The somatic antigens are separated
from the formula for flagellar antigens by a colon (:). The formulae of the
Salmonella strains are included in the following table. (Difco #0357). The
Kaufman-White Scheme is an ordered arrangement of Salmonella types into
groups based on a common somatic antigen. The groups are designated by Roman
Capital letters A-I. Compiled results show that 98% of isolates are
serologically in somatic groups A - E. These groups are identified by somatic
antigens as follows: A 2 B 4 C1 7 C2 8 D 9E1E2E3 3 Because most of the
Salmonella types isolated in this country fall within the serological groups
A - E any laboratory can presumtively identify their isolates by the use of
polyvalent O anti serum and six individual O antisera representing somatic
groups A - E. The additional use of six sera prepared against flagellar
antigens a,b,c,di and l-7 permits exact identification of the more important
serotypes causing human disease. An anti-V1 serum is also needed for
identification of S. typhosa and S. paratyphi C.

Slide Test for "O" Antigens

l. Inoculate a slant of T-Soy agar with Salmonella and incubate for l8-24
hours at 37`C.

2. Add 0.5-l.0 ml of saline to the slant and emulsify the growth until a
heavy smooth suspension is obtained.

3. Test to make sure that the culture does not autoagglutinate: by placing a
drop of culture suspension on a microscope slide and placing a drop of 0.2%
acriflavine in saline next to it. Gradually mix the two drops with the loop.
If the cells remain in homogeneous suspension the culture is considered
smooth. If the cells agglutinate, streak out the suspension on blood agar and
isolate another colony.

4. Take a 4" x 4" glass plate and draw 8 circles with a grease pencil. Label
the circles l-8. Place a drop of saline in the first circle and drop of poly
valent antiserum in the second. To each of the others add anti A, B, C,
etc., then add a loop full of the cell suspension to each drop and rock the
glass plate gently to mix the liquids. A positive reaction will be a rapid
and complete agglutination within a 30-60 second period after mixing the
bacteria with the antiserum. A negative reaction obtained with an unheated
suspension should be verified by boiling the suspension in a water bath for
30 minutes, cooling, and retesting. This method will remove any Vi antigen
that is masking the O antigens.

Tube test for H antigens

Tube Test for "H" Antigens Cultures to be tested for flagella antigens must
be actively motile.

l. Inoculate T-soy broth with the actively growing culture and incubate
overnight.

2. Dilute the broth culture with an equal volume (l0 cc) of 0.6% formol-
saline to prevent destruction of the Flagella.

3. Add 0.5 ml of the formalized broth to small test-tubes (l2 x 75 mm) using
l tube for each of the flagella sera to be tested.

4. Add 0.5 ml of the Flagellar sera to the tubes and incubate at 50`C for 2
hours. A positive result is complete agglutination of the cells and this
usually occurs within the first 30 minutes.

Passive Hemagglutination of Vi Ag

After fixation of polysaccharide or similar Ags on the surface of red blood
cells and the addition of homologous Antiserum, there occurs an agglutination
of the red blood cells. Because the red blood cells do not participate in
this reaction directly but are merely a marker of the reaction it is termed
"passive". The advantage of this reaction is that it permits detection of
the reaction of an antibody with a non-precipitating hapten by visualization
of the red blood cell agglutination. This method is very sensitive in the
detection of small amounts of hapten or Ag.

Coating with Vi Antigen

You will be provided with l Mg of Vi Ag. Coating of the rbc's with Vi Ag.

l. Wash l ml of a 5% suspension of sheep rbs's twice with phosphate-
buffered saline, pH 7.2.

2. Centrifuge to obtain a packed pellet of sheep rbc's and discard the
supernatant.

3. Dissolve 250 ug Vi Ag in l0 ml saline.

4. Add 50 ul of the packed sheep red blood cells to the Vi Ag (shake).

5. Incubate at 37`C with occasional shaking for 30 minutes.

6. Wash 3 times with phosphate buffered saline to remove excess Ag.

7. Suspend the coated sheep red blood cells in l0 ml of phosphate- buffered
saline (l:200).


Hemagglutination Test for Vi Antigen

l. Heat inactivate the antisera at 56`C for 30 minutes.

2. Add to each of l6 tubes 0.2 ml of phosphate buffered saline.

3. Add 0.2 ml of antiserum to the first tube and serially dilute the
antiserum.

4. Add 200 ul of Ag - coated sheep rbs to each tube. Shake vigorously.

5. Incubate at 37`C for 30 minutes.

6. Centrifuge at l000 rmp for l minute.

7. Read the agglutination grossly. The titer is the reciprocal of the
highest dilution which gives a visible agglutination.

Typhoid Mary

In l904, when an epidemic of typhoid broke out in Oyster Bay, New York, the
pattern of contamination put officials on the trail of Mary Mallon who had
been employed in the area as a cook. Nicknamed "Typhoid Mary", she managed to
elude the authorities until l907 when she was discovered working as a cook in
a Park Avenue home. Overtaken with considerable difficulty, she was rushed to
Willard Parker Hospital where she was found to be so loaded with typhoid
bacilli that some physicians referred to her as the human culture tube. Mary
- the first typhoid carrier identified in America - was committed, that year,
to North Brother Island in the New York Harbour. Despite a Supreme Court
appeal, she remained there until l9l0 when health officials released her on
her promise not to accept food handling employment. Four years later when
epidemics broke out in a New Jersey sanitarium and Sloane Maternity Hospital,
authorities feared that "Typhoid Mary" had broken her promise. Investigations
revealed that she had been employed in the kitchens of both institutions, but
she managed to leave before investigators arrived. She was finally captured
in a suburban New York home. Attributed with 5l original cases of typhoid and
3 deaths, "Typhoid Mary" was sent back to North Brother Island where she
remained in isolation until l938 when she died from the after-effects of a
stroke.

Identification of Gram-neg Rods

Accurate identification of a gram-negative rod is one of the more difficult
tasks facing a bacteriologist because of the extreme heterogeneity of the
group. The Gram-negative aerobic rods may be divided arbitrarily into three
major sections; fermentative rods, non-fermentative (oxidative) rods and
coccobacilli. The fermentative rods mainly belong to the family
enterobacteriacae which is composed of 5 tribes; Eschericheae,
Edwardsielleae, Salmonelleae, Klebsielleae and Proteae. The five tribes
possess ll genera. In addition, the genera, Vibrio and Yersinia, which are
not enterobacteriacae often are considered with the fermentative rods. The
non-fermentors or oxidizers are the pseudomonods which are polar flagellates
and the genera, Pasteurella, are all short gram negative coccobacilli. For
convenience the characteristics of all these microbes are summarized in a
series of tables.

Enterobacteriacae

The family Enterobacteriacae comprises numerous inter-related genera all of
which are microscopically indistinguishable, 3 to 5 mu by 0.5 mu. relatively
straight rods with rounded ends, Gram-negative, variously motile, some are
capsulate, none possess spores. All members of the family ferment glucose,
with or without gas production. Their natural habitat is the intestinal
tract of man and animals; some, e.g., Escherichia coli, are part of the
normal flora whilst others, e.g., Shigellae and Salmonellae are pathogenic
for man. Not all of the genera of this family are considered.

Escherichia coli

Microscopy Strains have the general characteristics mentioned above; those
which are motile possess peritrichous flagella. Cultural appearances Grows
readily on all ordinary media and is aerobic and facultatively anaerobic.
Colonies are 2 to 4 mm. in diameter after l8 to 24 hours at 37`C, opaque and
convex with an entire edge. On MacConkey's medium colonies are rose-pink on
account of lactose fermentation; grow poorly if at all on
desoxycholate-citrate (D.C.A.) with small dense pink colonies. Biochemical
activities. The practical importance of these tests lies in differentiating
E. coli strains which are constantly in human faeces from the microscopically
similar common pathogenic species of Salmonellae and Shigellae. More than
95% of E. col<MU>i strains ferment lactose promptly and with gas production
so that confusion with Shigellae strains rarely occurs. Virtually all strains
produce indole so that this feature alone prevents wrong identification as a
member of the genus Salmonella which never produces indole; the absence of
urease activity separates E. coli from members of the genus Proteus at an
elementary level. As in the identification of any species within the
Enterobacteriacae the final decision rests on serological differentiation.
Serological Characters. This genus has been subjected to extensive antigenic
analysis and more than l40 somatic (O) antigens have been identified and 49
flagellar (H) antigens are known. Routine serotyping is not undertaken
except when strains are implicated in gastroenteritis; these enteropathogenic
strains possess K antigens - present in capsules or micro-capsules. Three
types of K antigen, L, A and B, can be differentiated by their stability in
various physical tests. Almost all enteropathogenic possess the B type of
antigen. Animal inoculation. IS NOT UNDERTAKEN IN DIAGNOSTIC BACTERIOLOGY but many strains are associated with natural disease in domestic animals and poultry.

Salmonellae

There are more than 7000 serotypes within the genus Salmonella; four of
these, Salmonella typhi, S. paratyphi A, S. paratyphi B, and S. paratyphi C
give rise to the enteric fevers and are parasitic only in the human
intestine. The other serotypes occur widely as parasites of mammals and
birds as well as of man and the human infection associated with them is in
the nature of acute gastroenteritis or bacterial food-poisoning. Microscopy
Identical with E. coli but with the exception of S. pullorum and S.
gallinarum, all types are motile and possess peritrichous flagella. Cultural
appearances Aerobic and facultatively anaerobic; have a wide temperature
range and like all enerobacteria grow readily on all ordinary media. Colonies
are 2 to 4 mm in diameter after l8 hours to 24 hours of incubation at 37`C
and on agar appear as greyish-white dome-shaped discs with an entire edge. On
MacConkey's and D.C.A. media colonies are similar in size and shape to those
on agar and are pale since lactose is not fermented. Biochemical activities
There is a profusion of biochemical tests but for normal diagnostic purposes
it is sufficient to determine that the organism ferments glucose, dulcitol,
and mannitol with gas production (N.B. - S. Typhi is anaerogenic), does not
ferment lactose or sucrose and does not produce indole. On this evidence and
the demonstration if motility further identification is by serological
analysis. Serological characters All motile salmonellae possess two main
antigens. The 'O' or somatic antigens are group antigens that are noted in
the table. Identification of these with group-specific antisera allows the
unknown organism to be allocated to one or other of the groups. The 'H' or
flagellar antigens of a single species may occur in either or both of two
phases; phase l antigen being shared by only a few other species whereas
phase 2 is shared by many; these phases are referred to respectively as
specific and non- specific phases. A few salmonellae possess a third antigen
occurring on the surface and designated the Vi antigen; when present it masks
Bacteriophage typing Certain salmonellae serotypes can be subdivided into
phage types for epidemiologic purposes. Animal inoculations Species vary in
their virulence for various laboratory animals; inoculation is of no
importance in diagnostic laboratory practice.

Shigellae

Members of this genus are the causative organisms of acute bacillary
dysentery which is world wide in distribution and endemic in many highly
developed countries. With the exception of certain simians man is the only
host. Microscopy Identical with E. col<MU>i but members of the genus
Shigell<MU>a are never ever motile or capsulate. Cultural appearances Similar
to E. coli but give pale (colourless) colonies on MacConkey's and D.C.A.
medium since with the exception of Shigella sonnei, they do not ferment
lactose. Shigellae grow abundantly on D.C.A. in comparison to E. coli.
Biochemical activities Group A strains are mannitol non-fermenting; members
of groups B and C must be differentiated by serological methods but it should
be noted that Sh. flesneri, serotype six strains are capable of biochemical
subdivision and that certain of these are exceptional in producing gas (in
small volumes). Group D strains are unique in fermenting lactose if
incubated beyond l8 to 24 hours and thus give pink colonies on MacConkey and
D.C.A. media. Serological Characters Each of the groups is serologically
distinctive; l0 serotypes are recognized with Group A and there is no
significant intra-group relationship. Group B comprises six serotypes, all
of which possess a common group antigen and each possess a type specific
component. Sh. flexneri types l, 2 and 4 can be divided each into two
subtypes on the presence or absence of minor group antigens. Group C contains
l5 serotypes which are serologically distinct from Group B strains in lacking
the flex group antigen. There are intra-group relationships so that for
identification of the serotype of a Sh. boydi<MU>i strain it is necessary to
use absorbed antisera. Group D strains are a single serological entity.
Shigella dysenteriae serotype l (Sh. shiga) produces a potent neurotoxin.
Animal inoculation Of no value in the diagnostic identification of Shigella.

Klebsiella

The majority of types within this genus are saprophytic, e.g. in water
supplies. Other strains occur commensally in the intestinal tract in a
minority of healthy people. The most common sites in which Klebsiella
strains fulfill a pathogenic role in man is the urinary tract and the
respiratory tract. Microscopy Similar to the other members of the
Enterobacteriacae but are non- motile. Capsulate both in the tissues and on
'in vitro' cultivation. Cultural Appearances Colonies are large, high-convex
and mucoid on account of abundant production of extracellular slime; colonies
tend to coalesce. On MacConkey's medium the majority of strains give pink
colonies due to lactose fermentation. Biochemical activities Strain variation
is so great that it defies summary treatment in any case their characteristic
colonies allow easy differentiation from other enterobacteria. The majority
of strains are urease-producers but are much slower and less intense in this
regard than Proteus strains; their lack of motility and non-spreading growth
on ordinary further differentiated them from proteus. Serological characters
In addition to somatic antigens, strains also possess K (capsular) and M
(mucoid) antigens; in any one strain the K and M antigens are identical. The
presence of K and M antigens masks the O antigen so that capsular antisera
are sued to characterize strains. Thus, the genus is divided into 72 defined
serotypes by means of capsule-swelling reactions similar to the technique
employed in typing pneumococi. Animal inoculation Is not employed for
diagnostic purposes.

Proteus

Members of this genus are widely distributed in nature and are also found in
the faeces of animals and man. They occur as pathogens in wounds bedsores
and urinary tract infections; infection may be exogenous or endogenous in
origin. Microscopy Similar to other enterobacteria but very pleomorphic.
Motile. Cultural appearances Not in the least fastidious in regard to
temperature, atmosphere or nutritional requirements. On nutrient agar these
organisms rarely grow as isolated colonies but swarm in successive waves over
the surface; swarming is inhibited on MacConkey's medium or by increasing the
agar content of blood agar to 4%. Biochemical activities Four biochemical
types can be recognized (see chart). Lactose is not fermented so that on
MacConkey or D.C.A. media, pale colonies are noted. Differentiation from the
popular pale pathogens isolated on such media is effected by testing for
urease activity. Proteus species decompose urea rapidly with the liberation
of ammonia; Shigellae and Salmonellae do not produce urease. Serological
characters Pr. vulgaris and Pr. mirabilis have been subjected to serological
analysis and ll9 serotypes can be recognized on the basis of O and H
antigens, analogous with serotyping of Salmonellae. Certain types of Proteus
have antigens in common with the rickettsiae and this is the basis of the
Weil-felix reaction in the sero-diagnosis of the typhus fevers. Animal
inoculation Such methods are not employed for diagnostic purposes.

Vibrio

The genus Vibrio comprises members pathogenic to man, others pathogenic for
animals or insects and many which are commensal or saprophytic particularly
in water. !TOPIC Vibrio cholerae Popularly known as the comma bacillus and
the causative organism of Asiatic cholera. Microscopy When freshly isolated
they appear as definitely curved rods with rounded ends, 3 mu by 0.5 mu.
Often lose their curved appearance on artificial cultivation. Gram-negative.
Motile with a single terminal flagellum. Non-capsulate and non-sporing.
Cultural appearances Aerobic. Wide temperature range. Optimum 37`C. Grows
on ordinary media but sensitive to acid pH/ growth favoured by alkaline media
and the optimum reaction is about pH 8.2. On agar the colonies are not
distinctive, 2 to 3 mm in size after l8-24 hours at 37`C, low conves with an
entire edge, whiteish and translucent. Older colonies develop a light euchre
tint. Monsur's and Aronson's media are commonly employed for selective
cultivation; by inoculating a tube of peptone water with a flake of mucus
from the stool and incubating for only 6 to 8 hours vibrios, if present, can
be harvested from the surface in almost pure culture. Biochemical abilities
V. cholerae ferments glucose, sucrose, mannitol and maltose without gas
production but does not utilize lactose or dulcitol. It gives a positive
Cholera-red reaction when growing in peptone water due to the production of
indole and nitrites. This can be tested by adding for drops of sulphuric acid
to a 72 hour culture. V. cholerae is non-haemolytic when l ml of a 2-day
broth culture is added to l ml of a 5% suspension of sheep red cells (Greig
test). Serological characters There are three recognized serotypes of V.
cholerae - 'Inaba', 'Ogawa' and 'Hikojima' - all of which possess a common
'H' antigen but distinctive 'O' components. Animal inoculation No laboratory
animal is readily susceptible to the organism.

Vibrios are short, curved gram-negative rods which are oxidase positive and
motile by a single polar flagellum. Some species are members of the normal
flora, particularly the anaerobic vibrios of the oral flora. There are two
main pathogenic species, Vibrio cholerae, the cause of Asian cholera, and
Vibrio fetus which causes abortion in cattle and rarely infects man. Cholera
is a severe form of epidemic diarrhea, presently limited to Asia where it is
a major public health problem. The disease is characterized by a profuse
outpouring of intestinal fluid ("rice water" stools) causing rapid fluid
depletion. Pathologically there is a local acute enteritis without invasion
of the blood or lymph nodes. The spread is via water of food contaminated by
patients or carriers. There is no prolonged carrier state as in typhoid and
most patients stop excreting organisms within a few days and almost all by
three weeks.

Yersinia

The Yersinia (formerly Pasteurella) are short gram-negative rods that show
bipolar staining that causes them to resemble safety pins. Y. pestis which
causes plague is the most dangerous pathogen of this group although Y.
-pseudotuberculosis and Y. enterocolitia can infect man from animals.
Serological characters Virulent strains Y. pestis produces an Fl antigen in
the outer cell envelope. This antigen which is produced at 37`C but not at
30`C is largely responsible for stimulating antibacterial immunity. Mixed
cultures can be grown on media containing anti serum to Fl. Exposure of
colonies to chloroform vapours releases the Fl antigen and a ring of
precipitate develops around the colony. Phage specific for Y. pestis type Y.
pseudotuberculosis and those specific for Y. pseudotuberculosis may lyse
Salmonella or Shigella indicating a close antigenic relationship among the
gram-negative bacilli. Microscopy Short ovoid bacilli, l.5 mu by 0.7 mu,
Gram-negative, non motile.

Capsulate in tissues or when freshly isolated. Non-sporing.
Characteristically shows bipolar staining. Pleomorphic on prolonged
cultivation or sub-culture. Cultural appearances Aerobic and facultatively
anaerobic. Optimum temperature = 30`C.

Grows on ordinary nutrient agar but more rapidly if blood or serum is added.
Colonies are small (l mm) greyish and semitransparent but become larger and
irregular in outline on continued cultivation.

Biochemical activities

There is some variation in strains within the species.

Haemophilus

Members of this genus are strictly parasitic in man and other animals and are
deeply viable outside their respective hosts. Haemophilus influenza is the
commonest member of the genus pathogenic to man with Haemophilus ducreyi less
commonly found.

Haemophilus influenzae

This organism is frequently found in the healthy human throat and is also
associated with infection of the respiratory tract. It is the cause of a
small proportion of cases of acute pyogenic meningitis in young children.
Microscopy Characteristically small coccobacilli l.5 mu by 0.5 mu.
Gram-negative, non-motile, capsulate in young cultures, non-sporing. Often
presents as very long filaments, particularly in cerebrospinal fluid from
cases of Haemophilus meningitis and also in old laboratory cultures. Cultural
appearances Aerobic and demands blood containing media for growth; blood agar
or better, chocolate blood agar, provides the X(Haematin) and
V(diphosphopyridine) factors required by H. influenzae. On these media,
colonies are minute (l-2 mm) and transparent; the organism grows in symbiosis
with staphylococci which produce V factor. Hence in the vicinity of
staphylococci, colonies of H. influenzae are larger in size - satellitism.
Biochemical activities Powers of fermenting carbohydrates are feeble and
irregular. Serological characters Smooth strains possess capsular
polysaccharide antigens; six types (a to f) can be recognized by a capsule
swelling reaction analogous to that employed in typing pneumococci. Animal
inoculation Not pathogenic for laboratory animals.

Haemophilus aegypticus

This species is for all practical purposes identical with H. influenzae and
associated with an acute and readily communicable type of conjunctivits.

Haemophilus ducreyi

The causative organism of a venerally transmitted infection - chancroid or
soft sore. In films made of the lesion or material aspirated from the
swollen regional lymph glands, Gram-negative, coccobacilli are noted and some
may be present intracellulary. Difficult to grow although requiring only the
X factor and colonies resemble those of H. influenzae; agglutination tests
with a specific anti- serum confirm the identity.

Bordetella

This generic title commemorates the isolation by Bordet of the whooping cough
bacillus, Bordetella pertusis; this organism along with two closely related
species Bord. parapertusis and Bord. bronchiseptica was originally classified
with Haemophilus but since they require neither X nor V factors for growth
they receive separate status.

Bordetella pertusis

Microscopy Bears a close resemblance to H. influenzae but is less
pleomorphic. Cultural appearances Complex enriched media such as
Bordet-Gengou's potato-blood-glycerol- agar are required for primary
isolation; even then, two to three days incubation at 37`C. are required
before colonies can be recognized. These are small, dome-shaped and highly
refractile. Biochemical activities No reliable fermentative properties.
Serological characters Freshly isolated strains are serologically homogeneous
- phase l organisms. Animal inoculation Of no practical value diagnotically.

Bordetella parapertusis

This organism has been isolated from a whooping-cough-like disease. Similar
in many respects to Bord. pertusis but produces darkening of underlying blood
in Bordet-Gengou medium. Unlike Bord. pertusis it produces urease. Strains
are serologically homogeneous and although there are antigenic components in
common with Bord. pertusis, parapertusis strains can be specifically
agglutinated with an absorbed antiserum.

Bordetella bronchiseptica

Rarely associated with man, but causes brochopneumonia in rodents and has
been isolated from dogs suffering from distemper. Morphologically it differs
from the other members of the genus in possessing peritrichous flagella.
Grows readily on ordinary media without blood and colonies are very similar
to those of Bord. pertusis. Urease is produced. Serologically related to
other members of the genus but capable of differentiation by employing
absorbed antiserum.

Pasteurella tularensis

The causative organism of tularemia in wild rodents and transmissible to man
by fleas and ticks, eg. as an occupational hazard in persons handling
rabbits. Originally reported from Tulare County, California. Microscopy Much
smaller (0.7 mu by 0.2 mu) than Y. pestis but otherwise similar and shows
bipolar staining. Cultural appearances Aerobe. Will not grow unless complex
media are provided, eg. blood agar containing added cystine and glucose.
Optimum temperature 37`C. Colonies similar to those of Y. pestis and often
showing alph-haemolysis. Biochemical activities Feebly fermentative without
gas production in various sugars. Serological inoculation Most rodents can be
infected experimentally and the organism is recoverable from heart-blood,
liver and spleen. The other members of the genus Pasteurella are
non-pathogenic to man.

Brucellae

All members of this genus are strictly parasitic on man or animals and are
characteristically intracellular. Brucellosis in the human subject is
characteristically associated with drinking unpasturized goat's milk
(Brucella melitensis) or cow's milk (Brucella abortus). In either case and
also with Brucella suis (affecting pigs) there is also an occupational hazard
to farm workers, abbattoir personnel and veterinary surgeons. Laboratory
workers may become infected in handling cultures.

Brucella Melitensis

Microscopy Varies in shape from coccal forms (0.5 mu) to small bacilli (l mu
0.5 mu) Gram-negative. Non-motile, Capsulate. Non-sporing. Cultural
appearances Aerobe. Grows on ordinary media but a more rapid and growth is
obtained by cultivation on liver-infusion agar. Colonies are low convex,
with an entire edge, transparent and approximately l mm in diameter; on
continued incubation they increase in size and become brownish. Biochemical
abilities In ordinary sugar mediano fermentation is observable but if a
pepton- free, buffered medium containing the relevant substrate is heavily
inoculated very consistent patterns are obtained. Serological characters Br.
melitensis is very closely related to the other members of the genus since
all possess two similar antigens; however, one of these latter is dominant in
Br. melitensis, thus it is possible to prepare an absorbed agglutinating
antiserum for this species. Animal inoculation Guinea pigs inoculated
intramuscularly or subcutaneously with Br. melitensis suffer a chronic
illness which is rarely fatal and differs from that caused by inoculation
with other Brucellae.

Brucella abortus: Brucella suis

These are closely similar to Br. melitensis in most biological
characteristics.

Pseudomonas

Pseudomonas occurs widely in soil, water, sewage, etc., it is commensal in
the human intestine in small numbers and is also found, usually in
association with other bacteria, in infected wounds or burns and urinary
tract infections. Microscopy Identical to Proteus species but flagella are
few in number and polar in origin. Cultural appearances Aerobic. Wide
temperature range. Growth on nutrient agar gives colonies 2-4 mm in dia.
which are convex and with an entire edge.

Two striking characteristics are the sweet, must odour and the green
colouration which diffuses into the medium. Biochemical activities Of the
sugars commonly employed in diagnostic laboratories only glucose if fermented
and without gas production. Strains are encountered which do not produce the
pigments characteristic of the majority: such cultures can be classified as
Ps. pyocyanea if they give a positive oxidase reaction. Very few other gram
negative bacilli are responsive to the oxidase reagent and these in any case
react more slowly (more than 2 min) than Ps. pyocyanea which responds within
30 seconds. Serological characteristics Strains possess somatic and flagellar
antigens but no valid serotyping scheme has been introduced. Animal
inoculation No practical application.

Pseudomonas aeruginosa is a gram-negative polar flagellate rod which is found
in the environment and in the normal bowel flora. It produces a
characteristic blue-green pigment (pyocyanin) which diffuses into the medium
and may cause blue pus from lesions. Due to its virulence and resistance to
antibiotics, this organism has become an increasing cause of severe infection
in hospitals, particularly among debilitated patients and patients with
extensive burns. Treatment of systemic infections with Pseudomonas
aeruginosa is very difficult. Carbeniciliin, a new semi-synthetic penicillin
of expanded spectrum and gentamicin have promising activity against this
organism. Pseudomonas pseudomallei causes a severe, toxic form of pneumonitis
and systemic infection known as melioidosis which has been seen in Viet Nam.
Although this organism resembles other pseudomonads bacteriologically, its
antibiotic susceptibility pattern is quite different as it is resistant to
polymyxin, carbenicillin, and gentamicin.

Infection with Enterobacteriaceae and Similar Gram-negative Organisms This
group of organisms may cause a wide variety of infections both inside and
outside the gastrointestinal tract. Of the infections outside the GI tract,
urinary tract infections are by far the most common, but it should be
remembered that these organisms have been the cause of infections, in
virtually every organ system, including meningitis, pneumonia, and wound
infections. Hospital infections with E. coli, Klebsiella-Enterobacter,
Proteus, Mima-Herellea and Flavobacterium have increased in the last few
years relative to staphylococcal infections. This has been associated with:
l. Greater susceptibility of some groups of patients: eg. those treated with
cancer chemotherapeutics, immunosuppressives, etc. 2. Greater use of complex
respirator equipment. With better decontamination this is less important now
as a source. 3. Wide use of chemotherapeutics with activity against the
normal flora opening the way to superinfection. 4. The spread of R factor
resistance among the Enterobacteriaceae. A particularly devastating
manifestation of gram-negative infections is produced when they enter the
blood stream in "gram-negative sepsis". This is a clinical syndrome which
begins with a shaking chill and fever and may be rapidly followed within l-2
hours by hypotension and sometimes irreversible shock. This is due to the
release of endotoxin by the bacteria in the blood. The nature and effects of
endotoxin differ in a number of important features from those of the
exotoxins produced by C. diphtheriae and the toxigenic Clostridia and will be
discussed below. Bacterial endotoxin is an integral part of the cell walls of
gram- negative organisms being liberated in solution or suspension only by
methods which completely disrupt the cell. Chemically, they are the complex
lipopolysaccharide of the cell wall and are heat stable. Although injection
of endotoxin will induce antibody formation, in most cases it has not been
possible to produce an anti-endotoxin serum which will neutralize its in vivo
effects. Microgram amounts injected I.V. may have striking biologic effects
(eg. fever, leukopenia, hypotension), but quantitatively endotoxins are less
toxic than exotoxins. Finally, the effect of endotoxins is not specific for
the species tending to be the same for all enteric gram-negative rods. In
contrast, exotoxins in general are proteins, which are usually toxoidable and
are neutralized by specific antitoxin. They are relatively heat labile,
diffuse away from the bacterial cell, and have specific pharmacologic effects
characteristic for the species, depending on their cellular substrate. In
addition to infections produced by enteric gram-negative rods outside their
normal habitat in the GI tract, there are species which can produce disease
in or related to the bowel. These are members of the Salmonella and Shigella
Groups which are NOT NORMAL inhabitants of the GI tract. In addition
specific serotypes of E. coli which may be carried by adults without ill
effects cause severe diarrhea in infants. Members of the genus Vibrio, which
are not Enterobacteriaceae cause cholera, a severe diarrheal disease.

Enteric Fever

This may be produced by a number of species of Salmonella, the most prominent
of which is S. typhosa (typhi) -- "typhoid fever". Others include S.
paratyphi. These organisms are strict human pathogens. The infection is
through the ingestion of food or water which has been contaminated with the
organisms, usually by an unknowing carrier. Even small numbers are highly
infective. After ingestion the organisms multiply in the small intestine and
enter the intestinal lymphatics. They then travel via the thoracic duct into
the blood stream and are disseminated into many organs, including the kidneys
and intestines. At this point, after an incubation period of 7-l4 days,
blood cultures are positive and the patient experiences malaise, headache and
the gradual onset of a fever which increases each day, reaching a plateau
varying from l02`- l05`F each day. The organisms multiply in the R.E. system
generally and in the lymphoid tissue of the bowel producing hyperplasia and
necrosis of the lymphoid Peyer's patches. By the second or third week of
illness they are being excreted in the stool and sometimes in the urine.
Abdominal tenderness and distension and splenomegaly are prominent, and the
leukocyte count shifts from normal to a neutrophilic leukopenia. Diarrhea,
however, if it occurs, is late in the course. A characteristic rash referred
to as "rose spots" appears in the second to third week in about 30% of
patients. The typical disease (untreated) lasts 3-5 weeks ending with
gradual lysis of the fever over a period of days. The major complications of
the disease are gastointestinal hemorrhage and bowel perforation with
peritonitis. Following recovery a small percentage of patients become
carriers of the organism - sometimes for life. The organism is usually
located in the gall bladder or biliary passages. It is important that these
people are excluded from jobs involving food handling. Immunity following
infection is not solid but a killed whole bacterial vaccine is available and
is used in groups with a high risk of exposure (eg. overseas, military). It
does not give complete protection. The laboratory diagnosis is by isolation
of the organism from the blood (lst-2nd week) or sometimes from the urine.
Stool cultures must be plated on the proper indicator and selective media.
Agglutinating antibodies for the 'O' and 'H' antigens appear during the
second and third week of infection and are useful in diagnosis. Patients
with active typhoid typically show an 'O' titer which is much higher than the
'H' titer. A high 'H' with a low 'O' titer suggests either previous
vaccination or past infection. High titers to the "Vi" antigen suggest the
carrier state. Chloramphenicol is the drug of choice for typhoid fever but
ampicillin is also effective. Cholecystectomy may be necessary to eliminate
the carrier state as the organisms may be harboured in the gall bladder and
not respond to antibiotic treatment.

Septicemic Salmonellosis

Salmonella may also produce an acute illness with abrupt onset and early
invasion of the blood stream with septic spiking fever. The organisms do not
localize in the bowel and stool cultures are negative. Wide dissemination of
the organisms may result in local abscesses, osteomyelitis, endocarditis,
etc. Diagnosis is by culture of blood or of a local lesion. Although S.
choleraesuis is the prototype of this type of infection, it may be caused by
many other species of Salmonella.

Gastroenteritis

Gastroenteritis, or "food poisoning", is presently the most common form of
Salmonella infection in the U.S. The disease has a short incubation period
(8-48 hours) following ingestion of food contaminated with the organisms
which was not cooked well enough to kill them. The infecting dose to healthy
individuals is high, and the responsible foodstuff has often been held at
temperatures suitable for bacterial multiplication. The illness is manifest
by nausea, vomiting and diarrhea and lasts 2-5 days. The organisms usually
originate from animal sources, the most common being poultry and poultry
products which may have been contaminated during processing. The source may
also be from human carriers, especially food handlers. The diagnosis is by
culturing the stool and the suspect food if available. Agglutination
reactions are not helpful and blood cultures are not positive. S.
typhimurium is the most common species in this group.

Shigellosis

Bacillary dysentery or Shigellosis is a disease characterized by acute
inflammation of the wall of the large intestine and terminal ileum leading to
necrosis of the mucous membrane, superficial ulceration, bleeding, and
formation of a "pseudomembrane" on the ulcerated area. The pseudomembrane is
composed of fibrin, leukocytes, cell debris, a necrotic mucous membrane, and
bacteria. Clinically, the incubation period is short (l-4 days) and the
onset sudden with abdominal pain, cramps, diarrhea and fever. The stools are
liquid, and after the first few movements contain mucus and blood. Their
passage is accompanied by much straining antenesmus (rectal spasms).
Spontaneous recovery generally occurs in a few days but small children
sometimes succumb to dehydration and acidosis. Dysentery may be produced by
any member of the genus Shigella (see previous notes). The disease produced
by S. dysenteriae (group A) (Shiga's bacillus) is particularly severe due, at
least in part, to the production of a neurotoxin. S. dysenteriae is
essentially limited to tropical countries. This is a disease of poor
sanitation. Transmission from man to man is via vehicles such as "food,
fingers, feces, and flies". Control is by isolation of cases and attention to
disinfection of excrement and proper sewage disposal. There is little
immunity to reinfection and no effective vaccine. Most patients do not
excrete the organisms for more than a week after the acute attack, but a
small percentage become persistent carriers. The carrier state is much more
common with S. dysenteriae than with the other groups although excretion
beyond one year is infrequent. The laboratory diagnosis is by culture of the
organisms from the stool on differential and selective media. Since the
disease is localized to the GI tract, blood cultures are rarely positive.
Serologic tests are not helpful. The most important therapy is fluid
replacement. Antibiotics, particularly ampicillin, shorten the period of
diarrhea and the excretion of Shigella.

EPEC Enteropathogenic E. Coli

The difficulties of establishing a relationship between strains of E. coli
and gastrointestinal disease are obvious, but certain strains of this
organism have been shown to produce a severe, epidemic diarrhea in infants.
These strains differ from the non-enteropathogenic strains only in their
antigenic makeup. There are about eleven recognized serotypes. In the
laboratory they are distinguished from other E. coli strains by agglutination
tests from colonies with antisera prepared against their 'O' somatic and
surface antigens. A direct fluorescent antibody technique is also useful in
laboratory diagnosis. These strains produce a severe form of neo-natal
diarrhea which may spread rapidly in a newborn nursery. Because of the
severity of the illness and its epidemic potential, the emphasis should be on
rapid diagnosis by culture of stool specimens or FAM, and prompt isolation
procedures. As with most diarrheal diseases, the most important mode of
treatment if fluid replacement although antibiotics, particularly neomycin,
are usually effective. Organisms of Other Genera

Vibrios are short, curved gram-negative rods which are oxidase positive and
motile by a single polar flagellum. Some species are members of the normal
flora, particularly the anaerobic vibrios of the oral flora. There are two
main pathogenic species, Vibrio cholerae, the cause of Asian cholera, and
Vibrio fetus which causes abortion in cattle and rarely infects man. Cholera
is a severe form of epidemic diarrhea, presently limited to Asia where it is
a major public health problem. The disease is characterized by a profuse
outpouring of intestinal fluid ("rice water" stools) causing rapid fluid
depletion. Pathologically there is a local acute enteritis without invasion
of the blood or lymph nodes. The spread is via water of food contaminated by
patients or carriers. There is no prolonged carrier state as in typhoid and
most patients stop excreting organisms within a few days and almost all by
three weeks.

Pseudomonas aeruginosa is a gram-negative polar flagellate rod which is found
in the environment and in the normal bowel flora. It produces a
characteristic blue-green pigment (pyocyanin) which diffuses into the medium
and may cause blue pus from lesions. Due to its virulence and resistance to
antibiotics, this organism has become an increasing cause of severe infection
in hospitals, particularly among debilitated patients and patients with
extensive burns. Treatment of systemic infections with Pseudomonas
aeruginosa is very difficult. Carbeniciliin, a new semi-synthetic penicillin
of expanded spectrum and gentamicin have promising activity against this
organism. Pseudomonas pseudomallei causes a severe, toxic form of pneumonitis
and systemic infection known as melioidosis which has been seen in Viet Nam.
Although this organism resembles other pseudomonads bacteriologically, its
antibiotic susceptibility pattern is quite different as it is resistant to
polymyxin, carbenicillin, and gentamicin.

Mima, Herellea

This is a taxonomically ill-defined group of gram-negative rods which are
non-motile and on Gram smear are small and often coccobacillary. Because they
are found in the genitourinary tract and are a rare cause of meningitis, they
have been confused with Neisseria on direct smears. They are resistant to
the pencillins.

Flavobacterium

These are free-living organisms found in water faucet aerators, sink traps,
etc. They are gram-negative rods which form yellow pigmented colonies.
Optimum growth temperature below 37`. Have caused disease in the compromised
host; eg. bacteremia after heart valve implantation, meningitis in the
newborn. They are derived from contaminated environment.