April 29, 2009
Identification of Unknown Indigenous Bacteria
Introduction
Many bacteria can be found in and on nearly all areas of the healthy human body. These bacteria
are referred to as normal flora and they for the most part live in commensal and mutualistic
relationships with humans, often providing benefits such as aiding in digestion and preventing
colonization by pathogenic bacteria (2). Normal flora can also become parasitic and pathogenic
given the necessary conditions, such as when one species is killed by antibiotics allowing
another to establish a superinfection. Different parts of the human body exhibit different types of
normal flora. The epidermis and nares are both commonly inhabited by Staphylococcus and
Corynebacterium species while Streptococcus, Neisseria, and other Gram-negative cocci and
bacilli are often found in the oropharynx (2).
The purpose of this experiment was to isolate, purify, and identify normal flora from the
hand, nares, and oropharynx.
Materials and Methods
Swabs were taken of the hand, nares, and oropharynx and inoculated onto trypticase soy (TSA)
and blood agar (BAP). Many bacteria were found growing on swabs from each plate. Wellisolated colonies were subcultured onto new TSA and BAP and then isolated and tested in an
effort to determine their identity. A colony of each isolated species was Gram-stained and
subsequently further tested.
Species 1 was streaked for isolation on MacConkey and Blood CNA in order to verify its
Gram stain characteristics. A catalase test was also performed, which was then followed by
CTA glucose, OF glucose, modified oxidase, and motility tests. Order and types of tests were
determined from information in Koneman's Color Atlas and Textbook of Diagnostic
Microbiology (3).
Species 2 was streaked on Mannitol Salts Agar (MSA) and DNase. A modified oxidase
test was next performed, as were tests for aerobic and anaerobic growth. CTA glucose and
sucrose, as well as a nitrate reduction test were performed. Order and types of tests were
determined from information in the Laboratory Applications in Microbiology: A Case Study
Approach and the Koneman's Color Atlas and Textbook of Diagnostic Microbiology (1,3).
Species 3 was streaked for isolation on MacConkey and Blood CNA in order to verify
its Gram stain characteristics. Modified oxidase, motility, nitrate reduction, and catalase tests
were performed next. A Triple Sugar Iron (TSI) test was conducted as well as indole and
MR/VP tests. Species 3 was then streaked for isolation on Hektoen Enteric (HE) and Xylose
Lysine Desoxycholate (XLD) agar for fermentation characteristics. Tests for the presence of
citrase and urease were conducted next. An Analytical Profile Index (API 20E) strip was utilized
as a means of efficiently conducting the necessary remaining tests, which included various
decarboxylase and carbohydrate fermentation tests, among others. Order and types of tests were
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determined from information in Koneman's Color Atlas and Textbook of Diagnostic
Microbiology (3).
Results
Table 1 Macroscopic observations of bacteria examined
Species 1
Species 2
Source
Hand
Nares
Colony Color
White
White
Colony Size
Medium
Medium
Colony Texture
Smooth
Smooth
Colony Shape
Circular, entire,
Circular, entire,
convex
raised
Observations
Opaque
Opaque
Species 3
Nares
White
Medium
Smooth
Circular, entire,
raised
Opaque
Colony morphology was determined by observing single colonies on TSA.
Table 2 Microscopic observations of bacteria examined
Species 1
Species 2
Gram Stain
Variable
Positive
Cell Size
1.0-1.5 µm diameter 0.7-1.0 µm diameter
Cell Shape
Cocci
Cocci
Arrangement
Pairs, chains, clusters Pairs, chains, tetrads,
clusters
Species 3
Variable
0.7-1.7 µm long
Coccobacilli
Pairs, chains, clusters
Microscopic observations made after Gram-staining and viewing under oil immersion at
1000X magnification. The Gram stain results of species 1 and 2 were ambiguous and so they
were consequently plated on CNA and MacConkey agar to determine whether they were Grampositive or negative (see Tables 3 and 5).
Figure 2 Gram stain of Species 2
magnified 1000X
Figure 1 Gram stain of Species 1
magnified 1000X
2
Figure 3 Gram stain of Species 3
magnified 1000X
Table 3 Results of tests performed on species 1
Growth on Blood CNA
Growth on MacConkey
Fermentation on MacConkey
Hemolysis on Blood Agar
Catalase
CTA Glucose Fermentation
OF Glucose (open tube/covered tube)
Modified Oxidase
Motility
Negative
Positive
Weak positive
Gamma
Positive
Negative
Negative/Negative
Negative
Negative
While species 1 grew on MacConkey agar, it unable to grow on blood CNA. Weak
lactose fermentation was present on MacConkey agar as indicated by light purple/pink colonies.
From a regular blood agar plate species 1 was able to grow and was determined to be γhemolytic because there was no change in the agar surrounding the colonies. A positive catalase
test was indicated by the release of bubbles when hydrogen peroxide was added to a small
sample of organisms. Species 1 tested negative for fermentation of glucose in CTA because the
agar did not change from red/orange to yellow. The oxidative-fermentative glucose test yielded
no color change in either the open or closed tubes and therefore was negative for oxidative and
fermentative metabolism. A modified oxidase test was performed and was marked as negative
because the sample of organisms inoculated did not turn blue within a few seconds. Species 1
was negative for motility per a motility test in which the inoculum did not grow outward from
the stab culture.
Table 4 Results of tests performed on species 2
Catalse
Positive
3
Hemolysis on Blood Agar
Growth on MSA
Fermentation on MSA
DNase
Modified Oxidase
Aerobic Growth
Anaerobic Growth
CTA Sucrose Fermentation
CTA Maltose Fermentation
Nitrate Reduction
Arginine Dihydrolase
Gamma
Positive
Negative
Negative
Negative
Positive
Positive
Positive
Positive
Positive
Negative
Species 2 tested catalase-positive when bubbles were released after hydrogen peroxide
was added to a sample of it. Gamma-hemolysis was seen on blood agar as there was no change
in the agar surrounding the colonies. Species 2 was able to grow on MSA but was unable to
ferment mannitol as was apparent by red color of the colonies. A lack of clearing around
bacterial growth on a DNase plate indicated that species 2 was negative for DNase. The inability
of the organisms to turn blue after a few seconds into a modified oxidase test indicated a
negative result. Species 2 was able to grow in both aerobic and anaerobic settings. Maltose and
sucrose were fermented as indicated by a change in color of agar from red/orange to yellow. A
positive nitrate reduction test was seen as the color of a nitrate broth changed to red after the
addition of reagents A and B. Species 2 tested negative for arginine dihydrolase as was seen
when the broth in the both the control tube and the arginine tube turned yellow.
Table 5 Results of tests performed on species 3
Growth on Blood CNA
Negative
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Growth on MacConkey
Fermentation on MacConkey
Modified Oxidase
Motility
Nitrate Reduction
Catalase
Glucose Fermentation (TSI)
Lactose and/or Sucrose Fermentation (TSI)
Sulfur Production (TSI)
Gas Production (TSI)
Indole (on plate)
MR
VP
Growth on HE
Growth on XLD
Fermentation on XLD
Citrate
Urease
ONPG
Arginine Dihydrolase
Lysine Decarboxylase
Ornithine Decarboxylase
Tryptophan Deaminase
Gelatin Hydrolysis
Glucose Fermentation
Mannitol Fermentation
Inositol Fermentation
Sorbitol Fermentation
Rhamnose Fermentation
Sucrose Fermentation
Melibiose Fermentation
Amygdalin Fermentation
Arabinose Fermentation
Positive
Negative
Negative
Positive
Positive
Positive
Positive
Negative
Negative
Positive
Negative
Negative
Positive
Negative
Weak positive
Positive
Positive
Negative
Positive
Negative
Positive
Positive
Negative
Negative
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Species 3 did not grow on blood CNA but it did grow on MacConkey agar. Species 3
was unable to ferment lactose on MacConkey agar which was indicated by the absence of a
purple color in both the colonies and the agar. A negative oxidase test was seen after a sample of
organisms failed to turn blue after a few seconds into the test. By growing outward from its
point of inoculation in a stab culture, species 3 was found to be a motile organism. Species 3
was found to be capable of reducing nitrate to nitrate after the test broth turned red when
reagents A and B were added. Bubbles were released by a small sample of the organisms when
hydrogen peroxide was added to them, indicating the presence of catalase. A TSI test showed a
yellow butt with a red slant, indicating the ability of species 3 to ferment glucose but not lactose
or sucrose, respectively. The TSI slant showed no black precipitate, which meant that no
hydrogen sulfide was produced. Species 3 produces gas when it ferments carbohydrates, as was
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evident by the presence of air pockets in the TSI tube. An indole test was performed on a plate
with a small amount of bacteria that showed no color change upon the addition of Kovac’s
reagent. Species 3 tested negative for the MR test because it did not turn red upon the addition
of methyl red to a broth of the organisms. Species 3 did, however, test positive for the VP test by
means of turning red after the addition of α-napthol and potassium hydroxide to a bacterial broth.
No growth was observed on HE agar. A poor amount of growth on XLD agar had turned orange,
which indicated that species 3 was capable of fermenting xylose. Blue color on a citrate slant
indicated a positive result for the citrate test. A lack of color change on a urease slant was
indicative of a negative result. Species 3 was positive for ONPG per an API strip in which the
testing medium turned from clear to yellow. An arginine dihydrolase test was found to be
negative because no color change was observed while lysine and ornithine decarboxylase tests
turned red on the API strip and thus were positive results. A tryptophan deaminase test turned a
dark color on the API strip while the gelatin hydrolysis cupule remained clear, each indicative of
negative results. Glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin,
and arabinose all turned yellow on the API strip, indicating a positive result for the fermentation
of these carbohydrates.
Discussion
Species 1, which was isolated from the hand, was determined to be Acinetobacter lwoffii, a nonmotile, oxidase-negative, nonsaccharolytic, Gram-negative coccobacilli (3). Species 1 appeared
as Gram-variable cocci when viewed under 1000X magnification (see Figure 1), which is
consistent with A. lwoffii (3). In order to determine whether species 1 was Gram-positive or
negative, it was streaked on the selective MacConkey and blood CNA media. By being able to
grow on MacConkey and not CNA, species 1 was determined to be Gram-negative. Because
coccobacilli can sometimes appear as cocci, the possibility that species 1 was a coccobacilli had
to be considered. Species 1 was quickly differentiated from other Gram-negative species,
specifically the Enterobacteraceae, by its inability to ferment glucose (see Figure 4). The ability
of the isolate to grow on MacConkey agar was used as the next step in its identification, ruling
out Moraxella catarrhalis, several Neisseria species, and CDC Groups NO-1 and EO-5. At this
point it was apparent that species 1 was likely an Acinetobacter or Bordetella species, which was
confirmed by oxidase and motility tests. A negative oxidase test indicated an absence of the
enzyme cytochrome oxidase as an electron acceptor in the electron transport chain. A negative
motility test was consistent with an absence of flagella. Species 1 was negative for an oxidativefermentative glucose test, meaning that it could use glucose neither oxidatively or
fermentatively, eliminating Acinetobacter baumannii from the possibilities. Finally, the lack of
pigmentation of the colonies was used to differentiate species 1 from Bordetella holmesii and B.
parapertussis, leading to the identification of species 1 as A. lwoffii.
Many nonsaccharolytic Acinetobacter species are found on human skin. Acinetobacter
baumannii is the most common isolate, followed by A. lwoffii (3). As with many normal flora of
the skin, A. lwoffii has been known to cause nosocomial infections. The most common infections
caused by A. lwoffii are pneumonia, endocarditis, meningitis, peritonitis, wound infections, and
urinary tract infections (UTIs) (3). More than any other Acinetobacter species, A. lwoffii has
been associated with meningitis (3). Infections with Acinetobacter spp. tend to be problematic
due to antibiotic resistance, specifically to penicillin, ampicillin, cephalothin, and
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chloramphenicol, although A. lwoffii is the most sensitive of the genus (3). Infections may be
treated with a combination of an aminoglycoside and ticarcillin or piperacillin (3).
Species 2 was isolated from the nares and was identified as the non-motile, non-sporeforming, catalase-positive, Gram-positive cocci, Staphylococcus epidermidis (3). Upon the
determination of species 2 as Gram-positive cocci by Gram stain a catalase test was performed in
order to differentiate between Staphylococcaceae and Streptococcaceae (see Figures 2 and 5).
Species 2 tested positive for catalase, meaning that it was a Staphylococcaceae and that it could
break down toxic hydrogen peroxide into gas and water. The ability of Staphylococcaceae to
perform this reaction is important because they often encounter radical oxygen species due to the
fact that they live on the skin and in the nares. Catalase is also useful because it allows bacteria
to avoid being killed by toxic oxygen used by host immune cells. Species 2 was found to be able
to grow both aerobically and anaerobically, meaning that it was a facultative anaerobe. The
ability to ferment the carbohydrates sucrose and maltose was noted as well, meaning that species
2 could use other simple sugars during anaerobic fermentation. These characteristics were used
to eliminate several other Staphylococcus species from those being considered for the identity of
species 2. The isolate was next found to be able to reduce nitrate to nitrite in order to extract
oxygen, eliminating more Staphylococcus species from the possibilities of the identity of species
2. Finally the inability of species 2 to ferment mannitol allowed for the identification of species
2 as Staphylococcus epidermidis.
Staphylococcaceae are some the most frequently isolated bacteria from clinical
specimens with Staphylococcus epidermidis being the most commonly isolated of the genus (3).
Because S. epidermidis is normal flora, nearly all of the infections it causes are nosocomial (3).
These nosocomial infections include UTIs, surgical wound infections, and infections of
prosthetic devices, all of which seem to be enhanced by the ability of S. epidermidis to adhere to
surfaces such as catheters through use of cell-surface and extracellular macromolecules (3).
When treating infections with S. epidermidis, resistance to oxacillin and methicillin must be
considered (3). Decreased susceptibility to vancomycin has also recently developed in many
strains (3).
Species 3 was also isolated from the nares and was identified as Enterobacter aerogenes,
a member of the normal flora of the human digestive tract that are characterized by heavy gas
production, motility, the ability to ferment many different carbohydrates, and that they are
ornithine-positive. While it is strange that normal flora of the intestine were found in the nares, a
possible explanation could be that they were inhaled and trapped in the nostrils after they were
made airborne by the flushing of a toilet. Upon Gram staining and viewing under 1000X
magnification, species 3 appeared as Gram-variable coccobacilli (see Figure 3). This is not a
characteristic of E. aerogenes, though, and was likely the result of an error in the staining
process. In order to determine the true staining characteristics of species 3, MacConkey and
CNA plates were utilized. Growth on MacConkey and lack of growth on CNA indicated that
species 3 was Gram-negative. An oxidase test was performed in order to differentiate from the
enteric and non-enteric Gram-negative coccobacilli, with a negative result indicating that species
3 did not have oxidase in its electron transport chain and therefore was enteric (see Figure 6).
Species 3 was next differentiated from the Klebsielleae for being motile and being able to
decarboxylate ornithine to produce amines (3). A negative MR test, which meant that strong
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acids were not produced from glucose fermentation, eliminated various other enterics from the
possibilities. By not being able to decarboxylate lysine, E. cloacae and others were ruled out
next. Finally, the inability of species 3 to produce urease to break down urea into ammonia and
carbon dioxide allowed for its identification as E. aerogenes.
Enterobacter aerogenes and E. cloacae are the most commonly clinically isolated
enterics. Most strains of E. aerogenes can ferment lactose, but strangely enough the strain
isolated in this experiment was unable to do so despite being able to ferment numerous other
carbohydrates (see Table 5) (3). Enterobacter aerogenes can cause opportunistic infections such
as UTIs, respiratory infections, and wound infections (3). Considering this, it is fortunate that
the bacteria were trapped in the hairs and mucous of the anterior nasal passage or else a
respiratory infection may have developed in the person from which the bacteria were cultured.
Figure 4 Dichotomous key for Gram-negative cocci/coccobacilli
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*There are few, if any bacteria that fit these categories
Glucose Fermentation +
Glucose Fermentation -
Other Gram-negative
cocci/coccobacilli
Growth on MacConkey +
Growth on MacConkey Moraxella catarrhalis Neisseria elongata
Neisseria weaveri
Neisseria flavescens
CDC Group EO-5
CDC Group NO-1
Oxidase +*
Oxidase -
Motility +*
Motility -
OF Glucose +
Acinetobacter baumannii
No Pigment
Acinetobacter lwoffii
Figure 5 Dichotomous key for Gram-positive cocci
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OF Glucose Acinetobacter lwoffii
Bordetella holmesii
Bordetella parapetussis
Brown Pigment
Bordetella holmesii
Bordetella parapetussis
Catalase +
Catalase Streptococcaceae
Anaerobic Growth/Sucrose Fermentation -
Anaerobic Growth/Sucrose Fermentation +
Staphylococcus cohnii
Staphylococcus carnosus
Staphylococcus sacchrolyticus
Staphylococcus caprae
Maltose Fermentation +
Maltose Fermentation Staphylococcus intermedius
Staphylococcus simulans
Staphylococcus capitis
Nitrate Reduction +
Mannitol Fermentation +
Staphylococcus aureus
Staphylococcus gallinarium
Nitrate Reduction Staphylococcus warneri
Staphylococcus saprophyticus
Mannitol Fermentation Staphylococcus epidermidis
Figure 6 Dichotomous key for Gram-negative rods/coccobacilli
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Oxidase +
Oxidase -
Gram-negative non-enteric coccobacilli
Ornithine Decarboxylase + / Motility +
Ornithine Decarboxylase - / Motility Klebsiella spp.
MR +
MR -
Various other Enterobacteraceae
Lysine Decarboxylase +
Lysine Decarboxylase Enterobacter aminogenus
Enterobacter canerogenus
Enterobacter cloacae
Enterobacter kobei
Enterobacter sakazakii
Urease +
Enterobacter gergoviae
Urease Enterobacter aerogenes
References
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1) Chess, B., ed. Laboratory Applications in Microbiology: A Case Study Approach. Boston,
MA: McGraw, 2009.
2) Todar, Kenneth. "The Normal Bacterial Flora of Humans." Todar's Online Textbook of
Bacteriology. 2008. 24 Apr. 2009
<http://www.textbookofbacteriology.net/normalflora.html>.
3) Winn, Washington, Jr., et al. Koneman's Color Atlas and Textbook of Diagnostic
Microbiology. 6th ed. Baltimore: Lippincott Williams & Wilkins, 2006.
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