Identification of Proteus vulgaris from an Unknown SamplePraise Selah G. Dagoc

Mambajao, Camiguin

Abstract

Identification of microorganisms from unknown sample is a routine work for a Registered Medical Technologist assigned in the Microbiology section of the laboratory. For medical technology students, this activity serves as an important tool in exercising the students’ skills and knowledge in the processing and analysis of the various methods used in proper identification of isolates. In this activity, the student was able to identify Proteus vulgaris from an unknown sample with the use of scientific steps and procedures for proper identification of microorganisms in bacteriology.

Objectives

Through this activity, the student should be able to:

Practice skills on proper identification of microorganisms from sample.

Properly execute the steps in identification of microorganisms.

Know the background and diseases associated with the microorganism isolated and identified from the sample.

Describe the characteristics of the isolate. Discuss the infections caused by the isolate and

its treatment.

Introduction

Proteus vulgaris is one of the most commonly isolated members of Proteus sp., along with Proteus mirabilis. The genus Proteus is a member of a large gram-negative bacilli family, Enterobacteriaceae. Proteus organisms are known to be one of those to cause serious infections in humans, along with Escherichia, Klebsiella, Enterobacter, and Serratia species. (Struble, et al., 2009)

Proteus species are normal flora of the human intestinal tract, along with Escherichia coli and Klebsiella species, of which E. coli is the predominant resident. Proteus is also found in multiple

environmental habitats, including long-term care facilities and hospitals. In hospital settings, it is not unusual for gram-negative bacilli to colonize both the skin and oral mucosa of both patients and hospital personnel wherein infection primarily occurs. (Struble, et al., 2009)

Patients with recurrent infections, those with structural abnormalities of the urinary tract, those who have had urethral instrumentation, and those whose infections were acquired in the hospital have also been found to have an increased frequency of infection caused by Proteus and other organisms like Klebsiella, Enterobacter, Pseudomonas, Enterococci, Staphylococci. (Struble, et al., 2009)

P. vulgaris is an enteric bacterium, which means it is found in the intestinal tract (Deacon, J.); it is also found in the soil, contaminated water, or decomposing organic substances (University of Texas, 1995). While it is a pathogenic bacterium and has been shown to cause urinary tract infections, it is also part of the natural flora of the intestinal tract (Struble, et al., 2009); and because the microbe can live on the skin of some people, P. vulgaris is a common pathogen in hospital wound infections, especially in the immunosuppressed. (Struble, et al., 2009)

Conversely, Proteus vulgaris is easily isolated from individuals in long-term care facilities and hospitals and from patients with underlying diseases

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or compromised immune systems. (Struble, et al., 2009)

But, while P. vulgaris does contribute to the Proteus infections, it is not the most likely candidate for community-bourn disease; Proteus mirabilis causes a majority (90%) of those diseases (Struble, et al., 2009). However, P. vulgaris is the lead pathogen in causing hospital-bourn Proteus diseases. Unfortunately, it is not susceptible to ampicillins or cephalosporans. (University of Texas, 1995)

The reason the urinary tract is such a hospitable environment for the colonization of the microbe includes the microbes ability to degrade urea to ammonia with the enzyme urease. (Deacon, J., Struble, et al., 2009)

The bacterium is a gram-negative rod with flagella. As a gram-negative rod, it has an extracytoplasmic outer membrane. It creates an endotoxin, which can cause a deadly systemic inflammatory response in 20 to 50 per cent of its victims. (Struble, et al., 2009)

It has been shown that its optimal growth temperature was at 37 C. (Struble, et al., 2009) P. vulgaris is a chemoheterotroph, which means it uses carbon sources like glucose for energy and carbon (American Academy of Family Physicians, 2004); as a chemoheterotroph, it ferments glucose but not lactose or mannitol. (Clayton State University, 2004) However, because it is a facultative anaerobe, the glucose fermentation only occurs in anaerobic conditions; if placed in non-ideal, aerobic conditions, the microbe will use a variety of organic molecules to survive. (Deacon, J.)

When identifying the microbe, several tests can be used. It will test positive on the citrate test (Deacon, J.) and urease test (Struble, et al., 2009). Because it ferments glucose but not mannitol or lactose, it will only test positive in the glucose/carbohydrate utilization tests.

Likewise, when observing the plated colonies, it will be noticed on non-selective media a "swarming" behavior, where the microbe grows in waves

(Deacon, J.). The bacterium grows and stops in waves, creating what appears to be distinctive rings in the manner of tree rings. This feature is the result of the microbe’s flagella, which allow it to be extremely motile. The plate will also smell like burnt chocolate. (American Academy of Family Physicians, 2004)

These biochemical properties and physical observations helped in the indentification of the unknown, which turned out to be P. vulgaris.

Materials

To identify the bacteria from the unknown sample, the following materials were used:

Sheep Blood agar plate (SBAP) MacConkey Agar plate (MAC) Test tube containing a Tryptic Soy broth with

an unknown sample Test Battery for identification of gram negative

bacilli:- IMViC (Sulfide Indole Motility medium,

Methyl Red – Voges-Proskauer Medium, and Simmons Citrate Agar)

- Phenylalanine Deaminase Agar Slant- Triple Sugar Iron (TSI) Agar- Lysine Iron Agar (LIA)- Urea broth- OF-Carbohydrates (Mannitol, Sucrose,

Lactose???) Inoculating loop Inoculating needle Alcohol lamp Test tube rack Forceps Glass slides

Methods

As a part of the final activity for the bacteriology laboratory class, each bacteriology student was given a Tryptic Soy Broth containing an unknown

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isolate which the student must identify using the appropriate methods he/she has learned from class. This activity serves as an application of all the lessons that were taught and to test if the student understood and remembered the various tests that were mentioned in the laboratory and lecture classes in bacteriology. The author received the TSB tube number 8.

On the first day of the activity, the student inoculated sample using an inoculating loop from the TSB broth into the Sheep Blood Agar Plate (SBAP) and MacConkey plate (MAC) through the multiple interrupted streak method. After inoculation, SBAP was placed in a candle jar and incubated at 37C for 24 hours. Whereas, MAC was directly placed into the incubator and incubated at 37C for 24 hours.

The next day, SBAP and MAC were removed from the incubator and colony appearance from each of the plates was noted. Afterward, gram staining was performed to identify gram stain reaction of the isolate and guide in biochemical testing. A colony from SBAP was transferred onto a pre-heated (sterile) glass slide with the use of a sterile inoculating loop. Smearing was done by spreading the colony in a circular motion. The smear was then air-dried and heat-fixed before staining. Next, gram stain was applied following the appropriate order and time for each reagent: Crystal Violet (primary stain) – 1 minute, Gram’s Iodine (mordant) – 1 minute, 95% Ethyl Alcohol (decolorizer) – 30 seconds, and Safranin (counter stain) – 45 seconds. Upon drying of the stained smear, the slide was examined under the microscope set on oil immersion objective. Colonies observed were pink rods, indicating gram negative reaction, and were arranged in singles, pairs and clusters. Because of this finding, the student worker performed biochemical testing using the test battery for gram negative bacilli which consists of: IMViC (SIM, MR-VP & Citrate), Amino Acid Degradation tests (Phenylalanine & Lysine Iron Agar), Urea hydrolysis test, Triple Sugar Iron Agar, and Carbohydrate Fermentation Tests (Mannitol, etc..????). Sample was inoculated from MAC plate

into MR-VP and Urea using a sterile inoculating loop. The loop was used to get a part of the colony and aseptically transferred into each broth by tapping the loop inside the tubes. On the other hand, sterile inoculating needles were used to transfer samples from the plate (MAC) into the solid and semi-solid media. Transfer into the SIM medium was done by stabbing the medium until 3/4th to the bottom. Inoculation on citrate and phenylalanine was done by streaking the slant in a zigzag pattern using a sterile inoculating needle. On TSI, transfer was done using a sterile inoculating needle by stabbing the butt area and streaking the slant in a zigzag pattern. Stabbing was done twice in LIA after which the slant is streaked in a zigzag pattern only once. Full stabs were done on the OF tests. These tubes were then placed in an incubator and incubated at 37C for 24 hours.

On the following day, the test batteries were removed from the incubator and results observed. On SIM, 2-5 drops of paradimethylaminobenzaldehyde (PDAB) were added for Indole test. For MR-VP, 1-2 drops of methyl red were put into the MR tube, and 10 drops of 5% alpha-naphthol followed by 15-20 drops of 40% KOH were added to the VP tube. On phenylalanine slant, 4-5 drops of 10% aqueous ferric chloride was added.

Results

Swarming, circular, flat, undulate pink colonies were observed in the MacConkey Agar plate (figure 1.3) whereas swarming, flat, gray colonies were seen on the Sheep Blood Agar plate (figure 1.1 & figure 1.2). This colony characteristic illustrates a presumptive identification of a Proteus sp. giving the student a guide on what to expect.

Gram negative (pink) rods (figure 2.1 & figure 2.2) were seen on the gram stain of a colony from SBAP. These colonies were found to be arranged in singles and clusters.

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Table 1-1. Biochemical Test ResultsSIM

MR VP Cit Urea LIA Phenyl TSIOF-Carbohydrates

Sulfide Indole Motility SUC MAL MAN

+ + - + - + + - (K/A) ++ (A/A

with H2S)ferm. ferm. ferm.

For the biochemical testing, SIM showed a nonmotile reaction demonstrated by the lack of a brushlike appearance. With the addition of 2-5 drops of paradimethylaminobenzaldehyde (PDAB), red color developed on the surface indicating a positive reaction for Indole. Sulfide reaction was also observed as demonstrated by the blackening of the medium (See figure 3.1 in appendix). For MR-VP (figure 3.2), 1-2 drops of methyl red were put into the MR tube and 10 drops of 5% alpha-naphthol followed by 15-20 drops of 40% KOH were added to the VP tube. A positive reaction (red color) was seen in the MR tube whereas VP tube exhibited a negative reaction (no change in color). Citrate tube (figure 3.3) showed a positive reaction as evident in the prussian blue color of the agar slant. The isolate was also found to be positive for urea hydrolysis (figure 3.4) as shown in the development of a red-violet color in the urea tube. In the LIA butt/slant (figure 3.6), a K/A reaction or negative lysine decarboxylation was observed (purple slant/yellow butt). H2S reaction was seen in TSI butt/slant (figure 3.5) as demonstrated in the blackening of the medium (A/A H2S). Gas production was not seen in TSI. For phenylalanine deaminase test, 4-5 drops of 10% aqueous ferric chloride was added to the slant after which a green color developed indicating a positive result. Fermentation (yellow color) was seen on OF-Sucrose and OF-Maltose whereas OF-Mannitol showed an inert reaction evident in the presence of a blue color near the surface of the butt. A summary of the biochemical test results is shown in table 1.

Discussion

In addition to these methods, Ultrasonography of the kidneys or a CT scan could also be considered as part of a workup for Proteus infection of the urinary tract that does not resolve quickly with antimicrobial therapy. Calices and/or perinephric abscesses should be excluded. (Struble, et al., 2009)

For treatment, the recommended empirical treatment includes an oral quinolone for 3 days or trimethoprim/sulfamethoxazole (TMP/SMZ) for 3 days for uncomplicated UTIs in women on an outpatient basis. Acute uncomplicated pyelonephritis in women can be treated with oral quinolones for 7-14 days, single-dose ceftriaxone or gentamicin followed by TMP/SMZ, or an oral cephalosporin or quinolone for 14 days as outpatient therapy. For hospitalized patients, therapy consists of parenteral (or oral once the oral route is available) ceftriaxone, quinolone, gentamicin (plus ampicillin), or aztreonam until defervescence. Then, an oral quinolone, cephalosporin, or TMP/SMZ for 14 days may be added to complete treatment. Complicated UTIs in men and women can be treated with a 10- to 21-day course of oral therapy (in the same manner as for hospitalized patients) as long as the follow-up is adequate. (Struble, et al., 2009)

However, serious and occasionally fatal hypersensitivity (ie, anaphylactoid) reactions have occurred in patients receiving antibiotics. These reactions are more likely to occur in persons with a history of sensitivity to multiple allergens. Cross-sensitivity between penicillins and cephalosporins has occurred. If a reaction occurs, discontinue the implicated drug unless the condition is life threatening and amenable only to therapy with that antibiotic. Serious anaphylactoid reactions require

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immediate emergency treatment with epinephrine. Oxygen, intravenous steroids, and airway management, including intubation, should also be used as indicated. (Struble, et al., 2009)

P. vulgaris, along with P. penneri, is resistant to ampicillin and first-generation cephalosporins. Activation of an inducible chromosomal beta-lactamase occurs in up to 30% of these strains. Imipenem, fourth-generation cephalosporins, aminoglycosides, TMP/SMZ, and quinolones have excellent activity (90%-100%). (Struble, et al., 2009)

In addition, the use of chlorhexidine and triclosan in closed urinary catheterization systems and drug-impregnated catheters reduce the incidence of Proteus UTI in patients with long-term indwelling urinary catheters.3,4 While the use of these types of catheters for Proteus UTIs is helpful in containing the migration of Proteus in experimental models, this practice is not widespread, as other, more common, uropathogens are resistant to the drugs used in these systems. (Struble, et al., 2009)

A vaccine derived from purified mannose-resistant Proteus -like (MR/P) fimbriae proteins has been proven to prevent infection in mouse models and is under clinical research, but it is not available commercially. (Struble, et al., 2009)

In presence of struvite renal calculus associated with Proteus infection, surgery must be done to remove it. (Struble, et al., 2009)

Most nonurologic infections result in abscesses. Radical surgical debridement is the cornerstone of successful therapy. Amputation may be necessary if skin or muscle necrosis of an extremity is the presenting infection, but tissue recovery is often better than expected. Broad-spectrum antimicrobial therapy is started empirically and is modified by the results of smears and cultures. Mortality and morbidity rates are high, even with adequate treatment. (Struble, et al., 2009)

The discovery of stones requires an evaluation by a physician knowledgeable in the short- and long-

term management of stones, typically a urologist or nephrologist. (Struble, et al., 2009)

Summary and Conclusion

Based on the different tests performed, the student concludes that the organism isolated was Proteus vulgaris, an organism commonly associated with urinary tract infections. Moreover, infections caused by P. vulgaris are treatable with antimicrobial drugs and, in the case of renal stones, surgery.

References

Struble, K., et al. (2009). "Proteus Infections." E-medicine.com. Retrieved on March 14, 2010 from http://www.emedicine.com/med/ byname/proteus-infections.htm.

Deacon, Jim. The Microbial World: Proteus vulgaris and clinical diagnostics. Institute of Cell and Molecular Biology, University of Edinburgh. Retrieved on March 16, 2010 from http://helios.bto.ed.ac.uk/bto/microbes/ roteus.htm.

AAFP.com. Bacterial Identification. American Academy of Family Physicians 2004. Retrieved on March 16, 2010 from http://www.aafp.org/x2215.xml.

University of Texas, Houston (1995). Proteus. Retrieved on March 16, 2010 from http://medic.med.uth.tmc.edu/path/00001517.htm.

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Appendices

Appendix 1. Culture Plates

Figure 1.1 P.vulgaris colonies on Sheep Blood Agar plate. Colonies show a swarming, gray-colored appearance

Figure 1.2 A closer look on the colonies in SBAP will show the flat, undulate characteristic of the P. vulgaris colonies.

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Figure 1.3 P.vulgaris colonies on MacConkey Agar show the characteristic pink swarming colonies.

Appendix 2 Gram Staining

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Figure 2.1 Gram staining of the isolate shows gram negative (pink) rods, arranged in singles, pairs and clusters.

Figure 2.2 Gram negative rods of Proteus vulgaris.

Appendix 3 Biochemical Testing

Figure 3.1SIM MediumIsolate of P. vulgaris shows a positive reaction for Sulfide (darkening of the medium), Indole (reddish/pinkish surface upon addition

of Kovac’s reagent), and nonmotile (lack of brushlike growth).

Figure 3.2MR-VP MediumMR (left) shows a positive reaction – red color upon addition of methyl red.VP (right) showed a negative reaction –

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no change in color after adding 5% alpha naphthol and 40% KOH

Figure 3.3 CitrateP. vulgaris exhibits a positive reaction on Simmon’s Citrate medium as shown in the prussian blue color of the agar.

Figure 3.4 UreaP. vulgaris shows a positive reaction on urea hydrolysis test as demonstrated by the red-violet color of the broth.

Figure 3.5 TSITriple Sugar Iron tubes with P. vulgaris shows a positive reaction for H2S, evident in the blackening of the slant, along with the yellow color on the butt and part of the slant. The reaction on TSI is A/A with H2S.

Figure 3.6 LIAP. vulgaris shows a negative (K/A) reaction on Lysine Iron Agar as demonstrated by the purple slant and

yellow butt.

Figure 3.7 PhenylalanineP. vulgaris is positive on phenylalanine slant, evident in the presence of a

green color on the slant after the addition of 10% aqueous ferric chloride.

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Figure 3.8 OF-Carbohydrate TestsFermentation is observed in OF-Sucrose (left) and OF-Maltose (center) as seen in the yellow color of these two tubes. Whereas, reaction in OF-Mannitol (right) is inert in P. vulgaris isolate as shown in the blue color near the

surface of the tube.

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