Strain Differences

In some situations it is helpful to distinguish among different strains of a given species. This is particularly useful when only certain strains cause disease. For example, of the multitude of E. coli strains, relatively few have been shown to cause intestinal disease. This is because only a few strains have the virulence factors, such as toxin production, necessary to cause disease. Thus, finding

Chapter 10 Identification and Classification of Prokaryotes

PERSPECTIVE 10.1 Tracing the Source of an Outbreak of Foodborne Disease

In October 1996, physicians at the Children's Hospital and Medical Center in Seattle,Washington, noticed a slight increase in cases of children with severe bloody diarrhea. They quickly recognized that the children were infected with Escherichia coliO157:H7, a strain that can cause a fatal disease.Within days, the source of the outbreak was traced to contaminated unpasteurized apple juice, prompting the manufacturer to immediately recall that and related products from all stores in their seven-state distribution area.The prompt action by physicians, health officials and the juice manufacturer ended the outbreak and averted a potential widespread tragedy. ■ E. co/i O157:H7, p. 615

While the sequence of events that led to the recognition and cessation of the E. coli O157:H7 outbreak may sound quite simple, they are actually very complex. For one thing, most strains of E. coli are normal inhabitants of the Intestine and can be found In nearly any stool sample. How was this particular strain separated and distinguished from the hundreds of E. co/Z strains that do not cause diarrheal disease? It is also true that sporadic cases of E. co/i O157:H7 infections occur regularly, originating from unrelated sources. How was it recognized that these cases were connected?

To identify E. co/i O157:H7 in a stool specimen, the sample is plated onto a special agar medium designed to distinguish it from strains that typically inhabit the large intestine. One such medium is sorbitol-MacConkey, a modified version of MacConkey agar in which the lactose is replaced with the carbohydrate sorbitol. On this medium, most E. co/iO157:H7 isolates are colorless because they do not ferment sorbitol. In contrast, common strains of E. co/i ferment the carbohydrate, giving rise to pink colonies. Serology is then used to determine if the colorless E. co/i colonies are serotype O157; those that test positive are then generally tested to confirm they are serotype H7.

The next task is to determine whether or not two isolates of E. co/i O157:H7 originated from the same source. DNA is extracted and purified from each isolate and is then digested with restriction enzymes. Pulsed-field gel electrophoresis is generally used to compare the resulting restriction fragment length polymorphism (RFLP) patterns of the isolates.Those that have identical patterns are presumed to have originated from the same source.The patients from whom those isolates originated can then be questioned to determine their likely point of contact with the disease-causing organism. Culture methods are then used to try to isolate the organism from the suspected source. If that attempt is successful, the RFLP pattern of that isolate is then compared with those of the related cases.

E. coli in feces is of no significance, unless the strain is identified as one that causes disease. Detecting strain differences is also helpful in tracing the source of an outbreak (see Perspective 10.1).

The methods used to characterize different strains are summarized in table 10.5.

Biochemical Typing

Biochemical tests are used to identify various species of bacteria, but they can also be used to distinguish strains. If the biochemical variation is uncommon, it can be used for tracing the source of certain disease outbreaks. A strain that has a characteristic biochemical pattern is called a biovar or a biotype. For example, a biochemical variant of Vibrio cholerae called Eltor is increasingly being implicated in outbreaks of cholera around the world. Because this biovar can be readily distinguished, its spread can be traced. ■ Vibrio cholerae, p. 611

Serological Typing

Proteins and carbohydrates that vary among strains can be used to differentiate strains. For example, E. coli and other Gramnegative bacteria vary in the antigenic structure of certain parts of the lipopolysaccharide portion of the cell wall, the O antigen (see figure 11.15). The composition of the flagella, the H antigen, can also vary. The strain designation of E. coli O157:H7 refers to the structure of its lipopolysaccharide and its flagella. A strain that differs serologically from other strains is sometimes called a serovar or a serotype. ■ lipopolysaccharide, p. 59 ■ E. coli O157:H7, p. 615

Table 10.5 Summary of Methods Used to Characterize Different Strains

Method

Characteristics

Biochemical Typing

Biochemical tests are most commonly used to identify various species of bacteria, but in some cases they can be used to distinguish different strains. A strain that has a characteristic biochemical pattern is called a biovar or a biotype.

Serological Typing

Proteins and carbohydrates that vary among strains can be used to differentiate strains. A strain that has a characteristic serological type is called a serovar or a serotype.

Genomic Typing

Molecular methods can be used to detect restriction fragment length polymorphisms (RFLPs).

Pulsed-field gel electrophoresis

The genomic DNA is digested with an enzyme that cuts the chromosome into 10 to 20 large fragments.These are then separated using a special modification of gel electrophoresis.The pattern of sizes can be determined directly by looking at the stained gel.

Ribotyping

The genomic DNA is digested into many small fragments.These are then separated by gel electrophoresis and transferred to a membrane, which is then probed with labeled rDNA, resulting in a distinct pattern of bands.

Phage Typing

Strains of a given species sometimes differ in their susceptibility to various types of bacteriophage.

Antibiogram

Antibiotic susceptibility patterns can be used to characterize strains.

Genomic Typing

Molecular methods can be used to detect genomic variations that characterize certain strains. In some cases, these differences include genes that encode toxins or other proteins related to disease. For example, E. coli O157:H7 owes its virulence, in part, to the production of a toxin that is not encoded by most other strains of E. coli, which are normal intestinal inhabitants. The toxin gene can be detected using a probe that consists of a specific nucleotide sequence unique to that gene. Probes can also be used to detect genes that encode resistance to the antibiotic penicillin. Infections caused by organisms that have this gene must be treated with an alternative antimicrobial medication.

Subtle differences in DNA sequences can be used to distinguish among strains that are phenotypically identical. This is particularly important in tracing epidemics of foodborne illness in which a single source of contaminated food is shipped to several states. The ability to link geographically distant cases can enable public health officials to trace the source of the epidemic, leading to the recall of the implicated product and preventing further cases of disease. One method of doing this is to compare the pattern of fragment sizes produced when the same restriction enzyme is used to digest DNA from each organism. When the lengths of the restriction fragments vary among organisms, the fragments are said to be polymorphic. The different patterns of fragment sizes obtained by digesting DNA with restriction enzymes are called restriction fragment length polymorphisms (RFLPs) (see figure 9.9). Two isolates of the same species that have different RFLPs are considered different strains. Two isolates that have an identical RFLP may be the same strain. It cannot be concluded with absolute certainty, however, that they are indeed the same strain. ■ restriction enzymes, pp. 219,231 ■ restriction fragment length polymorphisms, p. 226

Common methods used to look for restriction fragment length polymorphisms include:

■ Pulsed-field gel electrophoresis This method uses a restriction enzyme that cuts infrequently along with a special type of gel electrophoresis that can separate very large fragments. Pulsed-field gel electrophoresis is used to separate these fragments, which are generally over 100,000 base pairs in length. The resulting bands can be visualized by staining the gel with ethidium bromide (figure 10.10).

■ Ribotyping This method uses a restriction enzyme that cuts genomic DNA into many small fragments. Southern blot hybridization is then done using a probe that hybridizes to only those fragments that have sequences encoding ribosomal RNA (see figure 9.8). Because bacteria generally have several rRNA genes, the probe hybridizes to several different restriction fragments, the pattern of which varies among strains (figure 10.11). ■ Southern blot hybridization, pp. 225, 236

To facilitate the tracking of foodborne disease outbreaks, the Centers for Disease Control recently established the National Molecular Subtyping Network for Foodborne

Figure 10.10 Detecting Restriction Fragment Length Polymorphisms (RFLPs) Using Pulsed-Field Gel Electrophoresis Genomic DNA is digested with a restriction enzyme that cuts infrequently; the resulting fragments are then separated by pulsed-field gel electrophoresis.

Enterobacter sakazakii (Database #9008)

Enterobacter sakazakii (Database #9009)

Courtesy of Qualicon

Figure 10.11 Ribotyping Genomic DNA is digested with a restriction enzyme that cuts frequently. Southern blot hybridization is then done using a probe that hybridizes only to those fragments that have sequences encoding ribosomal RNA. Because bacteria generally have several rRNA genes, the probe hybridizes to several different restriction fragments, the pattern of which varies among strains.

Enterobacter sakazakii (Database #9008)

Enterobacter sakazakii (Database #9009)

Courtesy of Qualicon

Figure 10.11 Ribotyping Genomic DNA is digested with a restriction enzyme that cuts frequently. Southern blot hybridization is then done using a probe that hybridizes only to those fragments that have sequences encoding ribosomal RNA. Because bacteria generally have several rRNA genes, the probe hybridizes to several different restriction fragments, the pattern of which varies among strains.

Disease Surveillance (PulseNet), which catalogues the RFLPs of certain pathogenic organisms. Laboratories from around the country can submit RFLP patterns to a computer database and quickly receive information about other isolates showing the same patterns. Using this database, multistate foodborne disease outbreaks can more readily be recognized and traced.

Phage Typing

Strains of a given species sometimes differ in their susceptibility to various types of bacteriophages. A bacteriophage, or phage, is a virus that infects and multiplies within bacteria and usually lyses the infected cells; each type of phage has a limited host range. Lysis of the infected cell releases more phage, which in turn infect neighboring cells. The susceptibility of an organism to a particular type of phage can be readily demonstrated in the laboratory. First, a culture of the test organism is inoculated into melted, cooled nutrient agar and poured onto the surface of an agar plate, thus creating a uniform layer of cells. Then drops of different types of bacteriophage are

258 Chapter 10 Identification and Classification of Prokaryotes

Inoculum of staphylococcus to be typed

Agar medium

258 Chapter 10 Identification and Classification of Prokaryotes

Inoculum of staphylococcus to be typed

Agar medium

Petri dish

(a) An inoculum of S. aureus is spread over the surface of agar medium.

(a) An inoculum of S. aureus is spread over the surface of agar medium.

(b) 31 different bacteriophage suspensions are deposited in a fixed pattern.

(c) After incubation, different patterns of lysis are seen with different strains of S. aureus.

Figure 10.12 Phage typing carefully placed on the surface of the agar. During incubation, the bacteria multiply, forming a visible haze of cells. A clear area will form at each spot where bacteriophage was added, however, if the organism is susceptible to the type of phage. The patterns of clearing indicate the susceptibility of the test organism to different phages and it is these patterns that are compared to determine strain differences (figure 10.12). Bacteriophage typing has now largely been replaced by molecular methods that detect genomic differences, but it is still a useful tool for laboratories that lack sophisticated equipment. ■ bacteriophage, p. 323 ■ host range, p. 361

Bacteriophage Typing

Figure 10.13 An Antibiogram In this example, 12 different antimicrobial drugs incorporated in paper discs have been placed on two plates containing different cultures of Staphylococcus aureus. Clear areas represent zones of inhibited growth.The different patterns of clearing indicate that these are two different strains of S. aureus.

(c) After incubation, different patterns of lysis are seen with different strains of S. aureus.

Figure 10.12 Phage typing

Figure 10.13 An Antibiogram In this example, 12 different antimicrobial drugs incorporated in paper discs have been placed on two plates containing different cultures of Staphylococcus aureus. Clear areas represent zones of inhibited growth.The different patterns of clearing indicate that these are two different strains of S. aureus.

Antibiograms

Antibiotic susceptibility patterns, or antibiograms, can also be used to distinguish among different strains. Again, this method has now largely been replaced by molecular techniques. To determine the antibiogram, a culture is uniformly inoculated onto the surface of a nutritional agar medium. Paper discs, each of which has been impregnated with a given antibiotic, are then placed on the surface of the agar. During incubation, the organism will multiply to form a visible film of cells. A clear area, indicating lack of growth, will form around each antibiotic disc that inhibits the organism. Different strains will have different patterns of clearing (figure 10.13). ■ antibiotic, p. 508

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Responses

  • Iggi
    How would you differentiate the causitice strain from other strains of the same apecies?
    3 years ago

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