Identification of Unknown Indigenous Bacteria

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 1

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 Species 3 Source Hand Nares Nares Colony Color White White White Colony Size Medium Medium Medium Colony Texture Smooth Smooth Smooth Colony Shape Circular, entire, convex Circular, entire, raised Circular, entire, raised Observations Opaque Opaque Opaque Colony morphology was determined by observing single colonies on TSA. Table 2 Microscopic observations of bacteria examined Species 1 Species 2 Species 3 Gram Stain Variable Variable Cell Size 1.0-1.5 µm diameter 0.7-1.0 µm diameter 0.7-1.7 µm long Cell Shape Cocci Cocci Coccobacilli Arrangement Pairs, chains, clusters Pairs, chains, tetrads, clusters 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 1 Gram stain of Species 1 magnified 1000X Figure 2 Gram stain of Species 2 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 Weak positive Gamma / 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 γ- hemolytic 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 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 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 4

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 Weak 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 5

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 6

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 7

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 8

*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 + OF Glucose - Acinetobacter baumannii Acinetobacter lwoffii Bordetella holmesii Bordetella parapetussis No Pigment Acinetobacter lwoffii Brown Pigment Bordetella holmesii Bordetella parapetussis Figure 5 Dichotomous key for Gram-positive cocci 9

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 + Nitrate Reduction - Staphylococcus warneri Staphylococcus saprophyticus Mannitol Fermentation + Mannitol Fermentation - Staphylococcus aureus Staphylococcus epidermidis Staphylococcus gallinarium Figure 6 Dichotomous key for Gram-negative rods/coccobacilli 10

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 + Urease - Enterobacter gergoviae Enterobacter aerogenes References 11

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. 12