Biochemical Identification Of Gram-Negative Bacteria PDF

Biochemical Identification of Gram-Negative Bacteria **Miscellaneous Tests Citrate Utilization**\ The citrate test determines whether an organism can use sodium citrate as a sole carbon source. Simmons' citrate medium is frequently used to determine citrate utilization. In addition to citrate, the...

Biochemical Identification of Gram-Negative Bacteria **Miscellaneous Tests Citrate Utilization**\ The citrate test determines whether an organism can use sodium citrate as a sole carbon source. Simmons' citrate medium is frequently used to determine citrate utilization. In addition to citrate, the test medium contains ammonium salts as the sole nitrogen source. Bacteria able to use citrate will use the ammonium salts, releasing ammonia. The alkaline pH that results from use of the ammonium salts changes the pH indicator (bromthymol blue) in the medium from green to blue. It is important to use a light inoculum because dead organisms can be a source of carbon, producing a false-positive reaction Christensen's citrate medium is an alternative test medium. This medium incorporates phenol red (as the pH indicator) and organic nitrogen. At an alkaline pH, the indicator turns from yellow to pink.\ \ \ **DNase**\ DNA is a polynucleotide composed of repeating purine and pyrimidine mononucleotide monomeric units. Most bacterial DNases are endonucleases cleaving internal phosphodiester bonds resulting in smaller subunits. Extracellular DNase can be produced by numerous bacteria, such as Staphylococcus aureus and Serratia marcescens. DNase test medium usually contains 0.2% DNA. A heavy inoculum of bacteria is streaked onto the surface of the medium in a straight line; several organisms can be tested at once. The plate is incubated at 35°C for 18 to 24 hours, and then 1N HCl is added to the surface of the plate. Unhydrolyzed DNA is insoluble in HCl and forms a precipitate. Oligonucleotides formed from the action of DNase dissolve in the acid, forming a clear zone (halo) around the inoculum.\ \ **Gelatin Liquefaction** Gelatin is a protein derived from animal collagen. Numerous bacteria produce gelatinases---proteolytic enzymes that break down gelatin into amino acids. Gelatinase activity is detected by loss of gelling (liquefaction) of gelatin. Gelatinase activity is affected by several factors, including the size of the inoculum and incubation temperature. Some bacteria produce larger amounts of gelatinase at room temperature compared with 35°C. It may be necessary to incubate media several weeks to detect a positive reaction. Several methods are available for detecting gelatinase.\ \ **Indole Production**\ \ Indole is one of the degradation products of the amino acid tryptophan. Organisms that possess the enzyme tryptophanase are capable of deaminating tryptophan with the formation of the intermediate degradation products of indole, pyruvic acid, and ammonia. Bacteria are inoculated into tryptophan or peptone broth. Most commercial peptone broth contains enough tryptophan for a positive reaction; tryptophan can be added to a fnal concentration of 1%. After inoculation, the broth should be incubated at 35°C for 48 hours. After incubation, one of two methods can be used to detect indole. In the Ehrlich indole test, the indole is extracted from the broth culture by the addition of 1 mL of xylene. After the xylene is added, the tube is shaken well. After waiting a few minutes for the xylene to rise to the top, 0.5 mL of Ehrlich's reagent, containing para-dimethylaminobenzaldehyde (PDAB), is added. If indole is present, a red color develops after the addition of PDAB (Figure 9-9). Alternatively, Kovac's reagent, which also contains PDAB, can be used. This method does not use a xylene extraction. Approximately 5 drops of Kovac's reagent is added directly to the broth culture. The tube is shaken, and if indole is present, a red color develops. The Ehrlich method is more sensitive than Kovac's reagent and is preferred with nonfermentative bacteria. If indole-nitrate (trypticase nitrate) medium is used, the indole test can be performed from the same broth culture as a nitrate test. Before adding any reagents, the broth is divided in half, one aliquot for the indole test and the other for the nitrate test. A rapid indole test using p-dimethylaminocinnamaldehyde is available. Isolated bacterial colonies are smeared onto flter paper that has been moistened with p-dimethylaminocinnamaldehyde. The formation of a bluegreen color within 2 minutes is a positive reaction.\ \ **Malonate Utilization**\ The malonate test determines whether the organism is capable of using sodium malonate as its sole carbon source. Malonate broth normally contains bromthymol blue as a pH indicator. Bacteria able to use malonate as a sole carbon source also use ammonium sulfate as a nitrogen source. A positive test results in increased alkalinity from utilization of the ammonium sulfate, changing the indicator from green to blue\ \ **Motility**\ Motility can be determined by microscopic examination of bacteria or by observing growth in a semisolid medium. Motility test media have agar concentrations of 0.4% or less to allow for the free spread of microorganisms. A single stab is made into the center of the medium. Best results are obtained if the stab is made as straight as possible. After incubation, movement away from the stab line or a hazy appearance throughout the medium indicates a motile organism. Incubation temperature is important. Some bacteria are motile only at room temperature, but this temperature may not be optimal for growth. It is suggested that two motility tubes be inoculated, one incubated at room temperature and the other at 35°C. Comparing inoculated with uninoculated tubes may help in interpreting results.\ \ **Nitrate and Nitrite Reduction**\ The nitrate reduction test determines whether an organism has the ability to reduce nitrate to nitrite and reduce nitrite further to nitrogen gas (N2). The organism is inoculated into a nutrient broth containing a nitrogen source. After 24 hours of incubation, N,N-dimethyl-α-naphthylamine and sulfanilic acid is added. A red color indicates the presence of nitrite. Nitrate reduction test reaction Nutrient broth with po0 1. % tassium nitrate Nitrate reductase Nitrite Sulfanilic acid → → → + N N, -Dimethyl- -naphthylamine Diazo red dye α → If no color develops, this may indicate that nitrate has not been reduced or that nitrate has been reduced further to N2, nitric oxide (NO), or nitrous oxide (N2O), which the reagents will not detect. Adding a small amount of zinc dust will help to determine whether the test has produced a true-negative result or whether the lack of color production was due to reduction beyond nitrite. Zinc dust reduces nitrate to nitrite. Therefore, development of a red color after the addition of zinc confrms a true-negative test result. Alternatively, a small glass tube, called a Durham tube, can be inserted into the broth upside down when the medium is aliquoted into test tubes. During incubation, if nitrogen gas is produced, it will be trapped in the inverted Durham tube.\ \ **Oxidase**\ The oxidase test determines the presence of the cytochrome oxidase system that oxidizes reduced cytochrome with molecular oxygen. The oxidase test is helpful in differentiating between the Enterobacteriaceae, which are oxidase-negative, and the pseudomonads, which are oxidase-positive. The oxidase test is also useful in identifying Neisseria spp., which are oxidase-positive. A modifed oxidase test is used to distinguish Staphylococcus from Micrococcus. Several methods for performing an oxidase test are available. Kovac's oxidase test uses a 0.5% or 1% aqueous solution of tetramethyl-ρ-phenylenediamine dihydrochloride. A drop of the reagent is added to flter paper, and a wooden applicator stick is used to rub a colony onto the moistened flter paper. The development of a lavender color within 10 to 15 seconds is a positive reaction. ρ-Aminodimethylaniline oxalate is less sensitive than tetramethyl-ρ-phenylenediamine dihydrochloride, but it is cheaper and more stable. Commercial forms of oxidase reagent are available in glass ampules and on filter paper disks.\ \ **Urease**\ The urease test determines whether a microorganism can hydrolyze urea, releasing a suffcient amount of ammonia to produce a color change by a pH indicator. Urease hydrolyzes urea to form ammonia, water, and CO2. Different formulations of urea agar are available, but Christensen's urea agar is generally preferred. The surface of the agar slant is inoculated but not stabbed. The medium contains phenol red as the pH indicator. The resulting alkaline pH from hydrolysis of urea is indicated by a bright pink color.\ \ **Lysine Iron Agar Slant**\ The lysine iron agar (LIA) test is a tubed agar slant. It contains the amino acid lysine, glucose, ferric ammonium citrate, and sodium thiosulfate. The pH indicator is bromcresol purple. LIA is used primarily to determine whether the bacteria decarboxylate or deaminate lysine (see Figure 9-6). H2S production is also detected in this medium. LIA is inoculated in the same manner as a TSI agar slant. LIA is most useful in conjunction with TSI in screening stool specimens for the presence of enteric pathogens, differentiating Salmonella spp. (lysine-positive) from Citrobacter spp. (lysine-negative). Decarboxylation occurs anaerobically only; the presence of a dark purple butt is positive for lysine decarboxylation. The production of H2S can mask the purple color in the butt of the tube. Because H2S production in LIA occurs only in an alkaline environment, a black precipitate indicating H2S is also a positive result for decarboxylation. LIA is also useful in differentiating Proteus, Morganella, and Providencia spp. from most other members of Enterobacteriaceae. This group of enteric deaminates (attacks the NH2 group instead of the carboxyl group) amino acids. In the LIA slant, deamination of lysine turns the original light purple color slant to a plum or reddish-purple color; the butt turns yellow because of glucose fermentation.\ \ **Motility-Indole-Ornithine Agar**\ Motility indole ornithine (MIO) agar is a semisolid agar medium used to detect motility and indole and ornithine decarboxylase production. MIO is useful in differentiating Klebsiella spp. from Enterobacter and Serratia spp. The medium is inoculated by making a straight stab down the center of the medium with an inoculating needle. Motility is shown by a clouding of the medium or spreading growth from the line of inoculation. Ornithine decarboxylation is indicated by a purple color throughout the medium. Because MIO is a semisolid medium, it does not have to be overlaid with mineral oil to provide anaerobic conditions. Indole production is detected by the addition of Kovac's reagent; a pink to red color is formed in the reagent area if the test result is positive. Ornithine decarboxylase and motility should be read first\ before the addition of Kovac's reagent.\ \ \ **Sulfide-Indole-Motility Agar**\ Sulfide indole motility (SIM) medium is a semisolid agar helpful in differentiating gram-negative bacteria in the Enterobacteriaceae. An inoculating needle is used to make a straight stab down the center of the medium. Cloudiness spreading from the inoculation line is positive for motility. The production of H2S is indicated by a black precipitate, and a pink to red color after the addition of Kovac's reagent is positive for indole. **Manual Multitest Systems\ Principles of Identification**\ Commercial identification systems fall into one of five categories or a combination thereof: (1) pH-based reactions, (2) enzyme-based reactions, (3) utilization of carbon sources, (4) visual detection of bacterial growth, or (5) detection of volatile or nonvolatile fatty acids by gas chromatography. Identification of bacteria and yeast can be facilitated by the use of automated or packaged kit systems by which organisms are identified with computer-assisted or computer-derived books of numeric codes. These numeric codes are generated based on the metabolic profiles of each organism. Each metabolic reaction, or phenotype, is translated into one of two responses: plus (+) for positive reactions and minus (−) for negative reactions. These plus-minus sequences are catalogued as binary numbers and stored in a computer database. Binary codes are computer converted into code profile numbers that represent the identifying phenotype of specific organisms. After metabolic profiles have been translated into numbers, a percent probability of correct identification is assigned based on the comparison of the unknown profile with known profiles within the database. As more organisms are included in the database, the genus and species designations and probabilities become more precise. With all the name changes that occur, it is sometimes difficult to maintain the current taxonomy in a database. For example, from the early 1960s to 2003, the number of genera of Enterobacteriaceae increased from 10 to 31, and the number of species increased from 24 to 130. All commercial suppliers of multicomponent biochemical test systems provide users with one or more of the following: a computer with profile number database, a computer-derived code book or compact disk, access to a Web site, or access to a telephone inquiry center to facilitate matching profile numbers with species.\ **\ Analytical Profile Index** The Analytical Profle Index (API; bioMérieux Vitek, Hazelwood, MO) was released in 1970. The system for identification of gram-negative fermentative bacteria (the family Enterobacteriaceae) is called the API 20E. This system has a series of 20 cupules attached to a plastic strip. Inside the cupules are lyophilized, pH-based substrates. A bacterial suspension made in saline is used to rehydrate the reagents in the cupules. The principles of the tests are the same or similar to the principles of tests performed in test tubes. Some of the cupules, such as those for amino acid deaminases and dehydrolase, require a mineral oil overlay. The strip is incubated 18 to 24 hours at 35°C, and reagents are added to some of the cupules. The last test used to determine the profile number is the oxidase test, which is not part of the API strip. Results are recorded, and a 7-digit code profile number is determined. After determining the code profile number, a database provided by the manufacturer is consulted, providing the most probable identification. If the identification is in question, nitrate reduction and motility are supplemental tests that can be used to obtain an additional digit for the profile number. The API system has been a standard product in many clinical microbiology laboratories and has remained unchanged. Accuracy for commonly isolated Enterobacteriaceae, such as Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis, has been reported to be 87.7% at 24 hours of incubation. For less commonly isolated Enterobacteriaceae, the accuracy is 78.7% at 24 hours. BioMérieux markets many multitest API systems, including systems for gram-positive cocci, nonfermentive gram-negative bacilli (20NE), and a rapid system for identifcation of the Enterobacteriaceae in 4 hours (RapID 20E). Other multitest systems include Crystal E/NF (BD Diagnostic Systems, Sparks, MD), RapID NF Plus (Remel, Lenexa, KS), Microbact (Oxoid, Ltd, Basingstoke, UK), Enterotube II (BD Diagnostic Systems), Uni-N/F-Tek plate (Remel), Micro-ID (Remel), and GN2 MicroPlate (Biolog, Inc, Hayward, CA). Siemens Healthcare Diagnostics (Tarrytown, NY) offers the MicroScan systems either semiautomated or fully automated. Biolog markets the MicroLog 1 and 2, manually read systems; the MicroLog MicroStation, a semiautomated system; and the OmniLog ID, a fully automated system. The Biolog database is one of the largest, with more than 500 species or taxa.\ \ **Rapid and Automated Identification Systems**\ Until the late 1970s, microbiologists relied on the growth and isolation of bacteria in broth culture and agar media. Once bacteria are cultured in vitro, their biochemical or metabolic characteristics can be used for identification. Isolation of the infectious agent from clinical samples typically requires 24 to 48 hours. Identification protocols often require another 24 hours of incubation in the presence of specifc biochemical substrates. Rapid diagnosis of infectious disease remains a major challenge for the clinical microbiology laboratory. The clinical outcome of rapid and accurate reporting of results should directly affect patient care in two ways: (1) early diagnosis and (2) subsequent selection of appropriate antimicrobial therapy. When these outcomes are achieved, the clinical microbiology laboratory will have a proactive, rather than retrospective, effect on patient management. Rapid reporting also becomes increasingly important in light of today's diagnostic challenges. Newly emerging pathogens, recognition of old pathogens in different clinical settings, world travel, increasing nosocomial infections, and the prevalence of multidrug-resistant organisms all contribute to the need for the design and development of new identification capabilities. Some of the major concepts and applications of rapid methods and automation currently available for the identification of clinically significant microorganisms are listed in the following sections. Although not exhaustive, the principles of most rapid identification technologies found in the clinical microbiology laboratory have been included. Other rapid identification methods are discussed in Chapters 10 and 11. Specific identification methods are discussed in greater detail in chapters in association with specific organism descriptions.\ \ **The Term Rapid**\ Direct microscopic examination of body fuids provides results within 15 to 30 minutes; these results are often valuable in patient management. For example, Gram stain results from a cerebrospinal fuid specimen along with blood and spinal fuid chemistry (glucose and protein) and hematology (complete blood count and differential) may be critical in establishing the cause of infectious meningitis. Chapter 7 discusses the microscopic examination of clinical samples. For decades, microbiologists relied on the ability of an organism to ferment sugars, degrade amino acids, and produce unique end products for identifcation purposes. Diagnosis of an infectious disease has been a complex, laborious, and frequently slow process. In the late 1950s and 1960s, traditional biochemical tests became miniaturized. Smaller test tubes and molded plastic vessels were introduced. These changes made testing more convenient but did not improve turnaround times in reporting results. In the 1970s, microbiologists began to rely on computerized databases so that numerous results could be considered simultaneously and the most statistically probable result could be regarded as the identification of the unknown organism. This development improved reliability of the results but still did not improve reporting turnaround times. In the mid to late 1970s, semiautomated instruments for identification and susceptibility testing gradually appeared. In many laboratory settings, these instruments shortened turnaround times, yielded greater precision, improved productivity, and provided accurate test results. The phrase rapid method encompasses a wide variety of procedures and techniques and has been loosely applied to any procedure affording results faster than the conventional method. Rapid methods exist for microscopy, biochemical identification, antigen detection, and antibody detection. Rapid is a relative term used to describe time and depends on the procedures being compared. For example, a 3-hour enzyme immunoassay method to detect Clostridium difficile toxin is rapid compared with a 48-hour cell culture cytotoxicity assay. The fluorescent quenching--based oxygen sensor detection of Mycobacterium tuberculosis within 2 weeks with the BACTEC 9000 MB (BD Diagnostic Systems) is rapid compared with 6 weeks required to grow the organism on agar slants. Microscopic procedures using common stains and fluorescent antibody to detect specific organisms are rapid methods; so are some conventional procedures used for initial differentiation or presumptive identification of certain groups of organisms. These procedures have been modified to provide immediate results that may lead to presumptive identification. Rapid identification of clinical isolates often involves commercially packaged identification kits or fully automated instruments. These manufactured kits are usually miniaturized test systems that employ chromogenic or fluorogenic substrates to assess preformed enzymes. Chromogenic substrates are colorless; when cleaved by a microbial enzyme, a colored compound is produced. Fluorogenic substrates are nonfluorescent until cleaved by microbial enzymes. Reaction endpoints may be reached after 2 to 6 hours of incubation, although some may require an overnight incubation. The capabilities of these kits and systems vary widely. Certain systems may still require manual reading by the laboratory scientist, whereas others may use mechanized reading through spectrophotometry, numeric coding, and computerized databases. Some of these instruments also incubate, read, and interpret the enzymatic test results.\ \ **Rapid Biochemical Tests Performed on Isolated Colonies**\ Table 9-12 summarizes the principles, modes of action, and applications of certain established manual procedures for quick presumptive differentiation between groups of organisms or presumptive identification to bacterial species. Often more than one test must be performed for a presumptive identification. These tests may also provide direction about additional tests needed for definitive identification. Further discussion of these and other similar rapid biochemical methods is included in subsequent chapters as they apply to identification of specific organisms.\ \ **Identification Systems Relying on Carbohydrate Utilization or Chromogenic Substrates**\ Rapid tests for detection of end products resulting from carbohydrate metabolism or enzymatic tests using chromogenic substrates produce reaction endpoints in minutes to hours. Plastic cupules, reaction chambers, or filter paper strips contain desiccated or dehydrated reagents or substrates. In general, a suspension of bacterial cells or a loopful of an isolated colony is added to the system or rubbed off to a reaction area. A positive reaction is measured by enzymatic activity and color change. Multitest kits using conventional carbohydrate metabolism take advantage of one test inoculum distributed to multiple reaction sites to yield more than one result. To obtain more rapid results, conventional methods have been modified by decreasing the test substrate medium volume and increasing the concentration of bacteria in the inoculum. Methods based on enzyme substrates have certain advantages over conventional methods. Because enzymatic methods involve preformed enzymes, they do not require multiplication of the organism (growth independent). Endpoints are reached in minutes to a few hours. The tests are very sensitive for the presence of the enzyme, although not always specific for genus and species identification; however, sensitivity depends on the concentration and stability of the substrate, the enzyme, and the age of the inoculum. Several rapid modifications of conventional methods for bacterial and yeast identification are listed in Table 9-3. Remel markets a series of Rapid panels that can provide identification in about 4 hours (Figure 9-12).\ \ \ **Automated Identification Systems**\ Several automated identifcation systems are currently available; most of these systems use turbidity, colorimetry, or fuorescent assay principles. Panels of freeze-dried or lyophilized reagents are provided in microtiter trays or sealed cards. Fully automated systems incubate and read the reactions, and computer software interprets the results and provides the identifcation. An advantage of automated systems includes an interface to laboratory information systems, leading to decreased turnaround times for reporting of results. Other advantages include statistical prediction of correct identifcation, increased data acquisition and epidemiologic analysis, and automated standardization of identifcation profles that can reduce analytical errors. If these systems are used in conjunction with automated susceptibility testing, these data may be linked to the pharmacy for patient management applications. Theoretically, early reporting of results can shorten length of hospital stay, augment therapeutic management of appropriate antimicrobial agents, and thus decrease hospital costs.\ \