VPM: Veterinary Bacteriology and Mycology
Oct. 3-4th, 2012
LABORATORY 5A - ENTEROBACTERIACEAE
The Enterobacteriaceae is a large family of gram-negative bacilli. They grow readily on common culture media. Organisms can also be isolated and differentiated using a variety of selective/ differential enteric media such as MacConkey agar (MAC). On MAC,organisms that ferment lactose (LFs) grow as pink colonies while those that do not ferment lactose (NLFs) are colourless.
A.LAB EXERCISES
1. A swab of feces from a neonatal calf with diarrhea is provided. Use the swab to inoculate a BA and MAC plate. Label your plates for incubation overnight. Tomorrow you will examine your plates and refer to demonstration material to assist you with a presumptive identification.
B.DEMONSTRATIONS
1.Examine the following cultures growing on BA and MAC plates. Note the colour and colony type and record. Which are lactose fermenters?
Escherichia coli
Klebsiella pneumoniae
Proteus mirabilis
Salmonella
2.Observe and record the TSIagar reactions (see Lab Handbook, p. 10-11) provided.
E. coli
Salmonella
3.Examine E. coli, Klebsiella pneumoniae, and Proteus mirabilis inoculated into urea, indole and citrate tubed biochemical tests (see Laboratory Handbook, p. 7 and 10) and incubated for 24 hrs.
4.Examine the photomicrograph and prepared slide (under oil on microscope) of an India ink wet mount of K. pneumoniae for the presence of a capsule.
5.Examine the brochures on different types of vaccines and antibody supplements for animals to protect against enterotoxigenic E. coli(ETEC), Salmonella, and other enteric pathogens.
C.QUESTIONS AND DISCUSSIONS
1.Which media would you use to isolate E. coli from diarrheic feces? Explain? Which other diarrhea-causing bacteria will grow on the media you selected?
2.Describe the growth of P. mirabilis on BA and MAC agar. Can you differentiate it from Salmonellacolonies?
3.How do the TSI agar reactions of Salmonella differ from E. coli?
4.What specimens would you collect for bacteriological culture from a case of septicemia?
a.Live animal
b.Dead animal
5.Are members of Enterobacteriaceae generally susceptible or resistant to penicillin?
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6Why are New Delhi metallo-beta-lactamase 1 (NDM-1)-producing Enterobacteriaceae, such as E. coli, Klebsiella pneumoniae, and Enterobacter cloacae of such concern?
7.Why do oral antibiotics predispose to salmonellosis?
LABORATORY 5B
ENTEROBACTERIACEAE and other INTESTINAL INFECTIONS
A.THE GASTROINTESTINAL TRACT AS A MICROBIAL HABITAT
The normal microbial flora is a host-defence barrier, the result of a host-parasite balance which has evolved over the millennia. The flora is remarkably stable. Particular microorganisms are adapted to each site along the gastrointestinal tract and occupy the optimal niche available to them. It is difficult for exogenous bacteria to establish because (1) they may have to compete with existing flora for mucosal receptors; (2) they may be inhibited by metabolic by-products, especially fatty acids; (3) they have to compete with existing flora adapted to the fierce competition for nutrients within the intestine.
B.GASTROINTESTINAL MICROFLORA
The gastrointestinal microflora is highly complex. In the large bowel of humans there are about 500 species of bacteria at >108 bacteria per gram of content. The large bowel of animals contains 1011-12 bacteria per gram; if it were one order higher feces would be solid bacteria. These bacteria are largely anaerobic, outnumbering facultative anaerobes such as E. coli by 100 to 1. Many of these anaerobes are strict and most are nonpathogenic, lacking all virulence mechanisms. Many genera are found in the intestine, of which the most common are Bacteroides, Fusobacterium, Eubacterium, Peptostreptococcus and Bifidobacterium. These bacteria colonize in particular sites of the intestinal tract soon after birth, multiply to large numbers and remain at these numbers throughout life. There is evidence that a similarity exists between the antigenic determinants on the surface of mucosal intestinal epithelial cells and the indigenous microflora. These bacteria provide a stimulus for the development of the intestinal wall and of the immune system, as well as making major contributions to digestion in herbivores and preventing the establishment of certain pathogens. Perhaps one reason that diarrhea is so common in young animals is that indigenous flora has not become firmly established.
1.Mouth - The dominant bacteria are strict anaerobes including spirochetes, aerobic and anaerobic Actinomyces, and streptococci. Streptococci are important in the development of dental caries; spirochetes, Actinomyces viscosus, and Porphyromonas asaccharolytica (formerly named Bacteroides asaccharolyticus) are important in the development of periodontal disease.
2.Stomach - The acid pH of the stomach prevents the establishment of a bacterial flora in most animals. Many bacteria from the saliva are destroyed in the stomach.
3.Small Intestine - The upper small intestine has few bacteria present but numbers increase to about 106-7/gram of content in the lower ileum. In the upper intestine, low pH and bile salts limit bacterial growth and peristaltic movements sweep bacteria down the tract. The flora of the lower small intestine consists of the anaerobes described above as well as facultative anaerobes such as nonpathogenic E. coliand fecal enterococci and also oxygen-tolerant Clostridium species.
4.Large Intestine - The colon and caecum have a massive bacterial flora, predominantly anaerobic, with a highly complex ecology. The fatty acid products (acetic, butyric) of anaerobic fermentation coupled with low pH and Eh (oxidation-reduction potential) are toxic to members of the Enterobacteriaceae, such as Salmonella. Removing the anaerobic flora in mice by antibiotics can reduce the infective dose of Salmonella from 106 to 1 bacterium.
C.DISRUPTION OF THE INTESTINAL FLORA
The protective role of intestinal microflora can be compromised by the following:
1.In neonatal animals where the flora has not been fully established.
2.By treatment with oral antibiotics which select for resistant bacteria and for yeasts. Examples are: Clostridium difficile colitis in humans or rabbits; Clostridium spiroforme in rabbits selected for by clindamycin; and selection of Salmonella and Clostridium species in horses by tetracyclines.
3.From stress can result in detectable changes in the large bowel flora.
4.By a change in diet that may produce changes in the large bowel flora.
D.PATHOGENIC MECHANISMS IN INTESTINAL DISEASE
Most bacterial pathogens go through a two-stage process to initiate intestinal disease, firstly, by attaching to the target cell, and secondly, by producing a toxin or in some other way damaging enterocytes and/ or invading the intestinal mucosa. Attachment is particularly important in the small intestine in order to overcome the washout effect of peristalsis (examples - E. coli, V. cholerae, C. perfringens). Gastrointestinal pathogens cause disease in the small intestine, perhaps because there is less bacterial competition in this site. It has been noted that postweaning diarrhea in pigs, caused by enterotoxigenic E. coli (ETEC) is a multifactorial disease. The presence ofETEC alone is not always sufficient to cause disease. The major stress of weaning is critical. Even though piglets are already colonised with ETEC before weaning, clinical disease occurs only after weaning. (Goswami PS et al. Preliminary investigations of the distribution of Escherichia coli O149 in sows, piglets and their environment. Can J Vet Res 2011;75:57-60). Edema disease in pigs caused by Shiga toxin- producing E. coli (STEC) is also associated with weaning, stress, such as transport, or a switch to a high-protein diet.(Gyles CL, Prescott JF, Songer JG, Thoen CO. Editors. Pathogenesis of Bacterial Infections in Animals 2010. 4th Edition. Wiley –Blackwell. ISBN 978-0-8138-1237-3)
E.LABORATORY EXAMINATIONS
1.Direct Examination
In general direct examination of fecal material is not a useful diagnostic procedure due to the high number of different types of bacteria present in the large bowel. Many will mimic the shape of pathogens. Examples include: nonpathogenic spirochetes in swine which are similar to Brachyspira hyodysenteriae;and the large number of gram-negative rods which are morphologically similar to Salmonella and pathogenic E. coli etc. Exceptions include: clumps of acid-fast Mycobacterium avium subsp. paratuberculosis (Johne’s disease); gentle jejunum mucosal scrapings of neonates with E. coli diarrhea will show a 10:1 ratio of bacteria to epithelial cells. Insuspect clostridial enterotoxemia in dogs (due to either C. perfringens or C.difficile),large gram-positive bacilli seen in predominant numbers on a direct Gram-stained smear of feces can be helpful in making a presumptive diagnosis.
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2.Culture
The main problem is to isolate specific bacterial pathogens against a background of a very large number of non-pathogenic organisms. Generally, this is not a difficulty with E. coli because enteropathogenic types (such as ETEC and STEC) will predominate in disease. For Salmonella, however, we generally require a broth enrichment process. One example is Rappaport broth medium (see Laboratory Handbook, p. 9) which is incubated for 24 hours before subculturing to another selective medium - Modified Semi-solid Rappaport-Vassiliadis Medium (MSRV), (see Laboratory Handbook, p. 9). Otherwise it would not be possible to recover 106Salmonella/gram of feces against a background of possibly 108 normal flora E. coli/gram. For Campylobacter jejuni (one of the curved, gram-negative gastrointesintestinal pathogens) we use a medium with various antibiotics, such as Modified Preston (Campy) agar (see Laboratory Handbook, p. 7) for selective isolation. Selective blood agar containing the antibiotic spectinomycin, incubated anaerobically at 42oC is used to isolate Brachyspira hyodysenteriae.
3.Biopsy and Histology
Bacteria can sometimes be demonstrated in histological preparations of intestine; e.g. Lawsonia intracellularis in proliferative adenomatosis of swine and horses (less frequently), Brachyspira hyodysenteriae in swine dysentery, Clostridium perfringens in necrotic enteritis, E. coli in E. coli diarrhea.
F.LABORATORY EXERCISES
Examine your BA and MAC plates inoculated yesterday with fecal-swabs from neonatal calves with diarrhea.
Is there a predominant colony? If so what are the colony features? What is the Gram-stain and microscopic morphology like?
Can you make a presumptive diagnosis? (see Question 4 below and the demonstration on virotyping)
G.DEMONSTRATIONS
1.Enrichment isolation of Salmonella from feces of an adult horse with diarrhea using Rappaport broth and MSRV medium. MSRV is a soft semi-solid Salmonella selective agar which allows motile Salmonella bacteria to swim across it. It will fall apart if you move the plate around too much or turn upside down.
PLEASE HANDLE THE MSRV PLATE CAREFULLY
AND DO NOT TURN UPSIDE DOWN!
2.Examine a selective Campylobacter agar that was inoculated with feces from a puppy with a moderate, mucoid diarrhea and incubated microaerophilically (reduced oxygen) for 48 hours. This colony grew at 42oC but not at 25oC. It is also, catalase, oxidase, H2S and hippurate positive.
Examine the gram-stain prepared from this culture. Note - these spiral/curved (“seagull-shaped”) pathogens are thin (0.2-0.5 μm) and stain very lightly with safranin. They can be challenging to see.
Colony character:
Gram morphology:
Identity? Campylobacter ______
Significance of this organism?
3.A silver-stained section from the ileum of a pig with proliferative intestinal adenomatosis. Locate the darkly stained, thin, curved bacteria making up microcolonies within the apical cytoplasm of crypt epithelial cells. What is the most likely candidate for this pathogen?
4.Examine the E. coli virotyping PCR testing information sheet by Gallant Custom Laboratories Inc., which is based on E. coli virotyping developed by the E. coli Laboratory (EcL) at the Université de Montréal, Quebec. Virotyping, which detects virulence genes of pathogenic E. coli using PCR methodology, is being used to replace serotyping ofE. coli isolates and can also be also performed following overnight enrichment of fecal samples.
H.QUESTIONS:
1.What serovars of Salmonella are common pathogens in animals? Can you name the top two Salmonella serovars in the world?
2.Why is Salmonella such a ubiquitous pathogen of animals? What factors predispose to infection?
3.Why is diarrhea so common in young animals?
4.How would you differentiate pathogenic E. coli from non-pathogenic E. coli after you have cultured a diarrheic fecal sample?
5.Will Salmonella grow at 42°C?
6.What specimens would you submit from calves with suspected E. coli diarrhea?
a.Live animal:
b.Dead animal:
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