The family Enterobacteriaceae (enterics) contains Gram (-) facultative rods that are common intestinal (colon) microbes most of which are either commensal or even mutualistic in the colon.
These are the most frequently isolated bacteria in the clinical lab coming from fecal samples infections of fecal origin. They also colonize mucous membranes outside the gut especially in hospitalized patients
In extraintestinal sites they are frequently associated with UTI lower respiratory tract and wound infections. In fact there is hardly an anatomic site they do not infect in some cases.
A few genera are not normal colon microbes - they are primary intestinal pathogens (a.k.a. enteropathogenic)
The enteropathogenic enterics cause GI conditions such as diarrhea enteritis dysentery and enteric fevers (e.g. typhoid and paratyphoid)
Most genera are easily grown and most look very similar making presumptive ID based upon morphology relatively difficult
cellular small short Gram (-) rods in singles and pairs
Proteus and Klebsiella are exceptions. Proteus (waves on the ocean) swarms on an agar surface and Klebsiella forms very mucoid colonies.
Another exception is Yersinia entercolitica because it grows better at room temp than at body temp. Y. entercolitica colonies are 1 mm or less in diameter after 24h incubation on blood or MacConkey agar at 35oC whereas typical enterics produce colonies 2-4 mm in diameter in these growth conditions
4 Preliminary grouping
Many carbohydrates fermented growth acid gas
Ferment glucose some to acid (MR)
some neutral (VP)
Some ferment lactose coliforms (E. coli)
Reduce nitrates to nitrites
Catalase positive - facultative
Motile some by polar monotrichous flagella and some by peritrichous flagella. Few are non-motile all Shigella some Klebsiella and some Salmonella()
5 Preliminary grouping
The most frequently isolated enterics have been categorized into subgroups or tribes that share similar characteristics. These groupings are not scientifically legitimate and are almost exclusively used for the enterics. Below NOT on test.
Escherichieae Escherichia and Shigella
Salmonelleae Salmonella Citrobacter
Klebsielleae Klebsiella Enterobacter Serratia and Hafnia
Proteeae Proteus Providencia Morganella
6 Cultural characteristics
As mentioned previously enterics are non-fastidious and bile-resistant but fairly antibiotic sensitive typically inhibited on media with colistin nalidixic acid polymyxin or cefaperazone
Most enteric selective media contain bile salts and/or dyes with bile-like properties (surfactants) that inhibit non-enteric bacteria
MacConkey agar is the most commonly used medium of this type. It contains bile salts and crystal violet. Eosin-Methylene Blue (EMB) is an alternative to MacConkey. EMB contains eosin and methylene blue. SS agar (Salmonella Shigella) is similar to MacConkey but also indicates sulfur reduction.
All of these media contain lactose and a pH indicator to differentiate lactose fermenters (yes or no and how much). Fecal coliforms such as E. coli produce acid from lactose fermentation resulting in darkened colonies on these media.
7 Cultural characteristics
Even though the name SS makes this medium sound ideal several literature reports state that some strains of Shigella fail to grow on it
XLD and Hektoen are selective enteric agars used for primary plating and subculture of enrichment broths and studies have shown that XLD and Hektoen are less likely to inhibit enteric species (produce higher yields than SS).
Like SS XLD and HE are also based on lactose fermentation and indicate sulfur reduction. Hydrogen sulfide production results in black colonies and on some media the agar becomes black as well.
The H2S test component of these media is particularly helpful because Salmonella Proteus Edwardsiella and an occasional Citrobacter produce hydrogen sulfide whereas most normal gut microbiota do not. Salmonella Edwardsiella are significant in this list as enteric pathogens
All Shigella species are H2S-negative
8 Cultural characteristics
Enrichment media have been used for enterics in the past. Using selenite broth allows Salmonella (resistant) which is often a distinct minority in the gut of carriers to quickly outgrow the other normal gut microbiota.
After an incubation time of 6 to 12 hours selenite broth is subcultured to a selective agar greatly enhancing the likelihood of isolating Salmonella in carriers
Selenite broth may not be needed in acutely ill patients because Salmonella will likely be the predominate gut bacterium
Another enrichment broth medium GN is claimed by the manufacturer to enrich for both Salmonella and Shigella. To be effective GN must be subcultured after only 4 to 6 hours of incubation. Why
9 Cultural characteristics
Toxigenic E. coli strains have the same cultural characteristics as commensal strains of E. coli making the former difficult to detect
One exception is verotoxin (shiga) producing E. coli (VTEC) a.k.a enterohemorrhagic (EHEC) E. coli O157H7
The majority of these strains do not ferment the carbohydrate sorbitol whereas the normal strains usually do
Sorbitol MacConkey (SMAC) contains the same ingredients as regular MacConkey except that sorbitol replaces lactose
Commensal E. coli produce the same dark colonies on SMAC as on regular MacConkey but EHEC colonies are colorless
Colonies suggestive of these strains are usually further tested for the toxin with commercially available ELISA tests
Gram (-) rods that behave like enterics on Mac EMB etc should be screened with the oxidase test. All enterics are oxidase (-)
Few non-enterics resemble the enterics and are oxidase (-) and these are easily distinguished with further testing
Cultures with typical enteric morphology on Mac are oxidase (-) and are indole () presumptively ID as E. coli with a high probability. The last 2 tests must be run on cells from a medium devoid of pH indicators SBA colonies normally used.
Proteus has the distinctive swarming morphology. Over 95 of Proteus isolates from clinical specimens is Proteus mirabilis which is the only frequently isolated Proteus that is indole (-) making this an easy organism to ID.
P. penneri is also indole (-) however it is extremely rare in clinical specimens and can be differentiated from P. mirabilis based upon different antimicrobial sensitivity patterns
The occasionally isolated P. vulgaris is indole ().
Before the advent of semi-automated and automated ID systems certain enterics were presumptively identified by the small battery of biochemical tests indole methyl red Voges Proskauer and Citrate the so-called IMViC series
Some labs still use IMViC so questions about it may still appear on certification examinations. Below NOT on test unless
Oxidase (-) Gram (-) rods isolated from normally sterile sites such as blood and spinal fluid should be completely identified
Isolates that appear to be enterics and are oxidase (-) should be tested for glucose fermentation all enterics ferment glucose.
Kliglers iron agar (KIA) Triple Sugar Iron (TSI) agar O/F glucose glucose fermentation broth and other media can be used for this purpose. All are based upon a pH indicator color change via acid from glucose.
KIA and TSA the most commonly used media for this purpose in clinical labs are deep slants containing phenol red as a pH indicator. To inoculate you stab the butt and streak the slant.
The development of a yellow (acid) butt with a red (alkaline) slant after 12-24h of 35oC incubation indicates that the organism ferments glucose only (K/A is shorthand is for alkaline slant acid butt)
13 Test for Fermentation of Glucose Kliglers or Triple Sugar Iron (K/A) Stab inoculate with a needle and streak the slant Incubate 35oC 12-24h with loose cap Acid butt alkaline slant (K/A) indicates fermentation of glucose only 14 Test for Fermentation of Glucose Kliglers or Triple Sugar Iron (A/A) Stab inoculate with a needle and streak the slant Incubate 35oC 12-24h with loose cap Acid throughout indicates fermentation of glucose and lactose 15 Test for Fermentation of Glucose Kliglers or Triple Sugar Iron (K/K or NC) Stab inoculate with a needle and streak the slant Incubate 35oC 12-24h with loose cap No acid indicates non-fermenter Rules out enteric 16 Identification
Hugh and Leifsons (HL) oxidation/fermentation (O/F) medium contains a pH indicator (Bromthymol blue - BTB) peptone and a carbohydrate of your choice. BTB is green when neutral blue when alkaline and yellow when acid
Two tubes of HL glucose media are stabbed to the butt with a needle and 1 of the tubes is overlaid with sterile mineral oil to prevent O2 diffusion this is the F tube.
An O reaction is indicated by a yellow O tube and no change in the F tube (yellow in the O tube occurs due to CO2 from respiration reacting with water to make carbonic acid). An F reaction is indicated by yellow in both tubes these organisms are glucose fermentors such as enterics
Blue in either tube is an indication of amine accumulation from peptone utilization useful data for some Gram (-) non-fermenting (GNNF) organisms HL is better for GNNFs than KIA or TSI
17 Test for Utilization of Glucose Fermentation in Hugh and Leifsons test Stab inoculate with a needle Incubate 35oC 12-24h Acid below the oil indicates glucose fermentation No oil Oil 18 Test for Utilization of Glucose Oxidation in Hugh and Leifsons test Stab inoculate with a needle Incubate 35oC 12-24h Acid in top of open tube only indicates oxidation of glucose This rules out enterics No oil Oil 19 Test for Utilization of Glucose Nonfermenter Non-oxidizer (Inert) Stab inoculate with a needle Incubate 35oC 12-24h No oil No acid in either tubenonfermenter/nonoxidizer This rules out enterics Oil 20 Screening for Enteric Pathogens
Enteric isolates from stool specimens can be screened with certain media/tests to eliminate some possibilities. This makes the final ID easier but the down side is that it takes time.
Salmonella Shigella and Edwardsiella are enteric pathogens that produce clear and colorless colonies on enteric agars.
The Proteus group (Proteus Providencia and Morganella) and a few others are not enteric pathogens. However these may ( with exceptions) also produce colorless colonies on enteric agars
All colorless colonies growing on enteric agars could be subjected to a complete battery of tests but this would not be practical since over 90 of non-lactose fermenters isolated from feces are not the 3 enteric pathogens mentioned above.
The use of a few tests is effective for ruling out Salmonella Shigella before using an expensive automated or semi-automated product / approach.
21 Screening for Enteric Pathogens
KIA (or TSI) Lysine Iron Agar (LIA) and Christensens Urea agar are media used for screening enteric pathogens.
The fermentation/pH and the H2S reactions for the enteric bacteria as a group were discussed in previouss in this series.
Remember that Salmonella Shigella and Edwardsiella produce alkaline slants and acid butts in KIA or TSI and that Salmonella Edwardsiella are sulfur positive but Shigella is sulfur (-)
Proteus species and Citrobacter fruendii are usually also H2S positive but they are not enteric pathogens
Organisms that give any other reaction pattern on these media are not enteric pathogens
See KIA or TSI reaction patterns on following s
22 Screening for Enteric Pathogens Kliglers Reactions (K/A) Stab inoculate with a needle and streak the slant Incubate 35oC 12-24h with loose cap Shigella cannot rule out 23 Screening for Enteric Pathogens Kliglers Reactions (K/A) Stab inoculate with a needle and streak the slant Incubate 35oC 12-24h with loose cap Salmonella Edwardsiella cannot be ruled out 24 Screening for Enteric Pathogens Kliglers Reactions (A/A) Stab inoculate with a needle and streak the slant Incubate 35oC 12-24h with cap loose Not an enteric pathogen (typical of E.coli Klebsiella or Enterobacter) 25 Screening for Enteric Pathogens Kliglers Reactions (A/A) Stab inoculate with a needle and streak the slant Incubate 35oC 12-24h with cap loose Not an enteric pathogen (typical of most Citrobacter species) 26 Screening for Enteric Pathogens Kliglers Reactions (K/NC) Stab inoculate with a needle and streak the slant Incubate 35oC 12-24h with loose cap No acid in butt rules out all enterics including enteric pathogens typical of Pseudomonas and Acinetobacter 27 Screening for Enteric Pathogens
LIA is also a deep slant and is inoculated as is KIA TSI
LIA contains sulfur iron the amino acid lysine a small amount of glucose and the pH indicator Bromcresol Purple (BCP) which is purple if alkaline and yellow if acidic
The by-product of lysine deamination (occurs near the top - aerobic) reacts with the iron causing a color change from purple to red.
Only the Proteus group (Proteus species Morganella morganii and Providencia species) cause this reaction. This is important because the Proteus group mimics enteric pathogens
Lysine decarboxylation (occurs where O2 is low) forms alkaline amines. Enteric bacteria growing in the butt will ferment the glucose but lysine decarboxylation neutralizes the acid from fermentation and turns the butt purple. Presencf of growth distinguishes this from NC. Salmonella is lysine decarbox ().
A yellow butt reaction is typical of Shigella species
Keep in mind that Proteus Salmonella and Citrobacter produce the black color via sulfur reduction as well as the above results
28 Screening for Enteric Pathogens Lysine Iron Agar (K/K) Lysine decarb H2S Stab inoculate with a needle and streak the slant Incubate 35oC 12-24h with loose cap Typical reaction Salmonella species 29 Screening for Enteric Pathogens Lysine Iron Agar (Red/A) Lysine deam H2S Stab inoculate with a needle and streak the slant Incubate 35oC 12-24h with loose cap Typical reaction of Proteus 30 Screening for Enteric Pathogens Lysine Iron Agar (K/A) NC on slant acid butt H2S Stab inoculate with a needle and streak the slant Incubate 35oC 12-24h with loose cap Typical reaction of some Citrobacter species 31 Screening for Enteric Pathogens Lysine Iron Agar (K/A) Stab inoculate with a needle and streak the slant Incubate 35oC 12-24h with loose cap Typical reaction of Shigella and Some E. coli 32 Screening for Enteric Pathogens
Christensens urea agar (another deep slant) contains glucose a pH buffer urea and phenolphthalein as a pH indicator.
Urease produced by non-enteric pathogens growing on this medium changes the urea to ammonia
Depending on the amount of ammonia produced the phenolphthalein color change ranges from a very pale pink to darker pink or a deep fuschia
Salmonella and Shigella grow but do not produce urease the medium remains pale
A positive urease rules out Salmonella and Shigella
The Proteus group (Proteus Morganella Providencia) are urease positive
33 (No Transcript) 34 Screening for Enteric Pathogens Christensens Urea Agar (Urease ) Streak the slant Incubate 35oC 12-24h with loose cap Typical reaction of Salmonella and Shigella species (as well as E. coli ) 35 Screening for Enteric Pathogens Christensens Urea Agar (Urease weakly ) Streak the slant Incubate 35oC 12-24h with loose cap Typical reaction of Citrobacter Serratia and Klebsiella species rules Salmonella and Shigella 36 Screening for Enteric Pathogens Christensens Urea Agar (Urease strongly ) Streak the slant Incubate 35oC 12-24h with loose cap Typical reaction of Proteus species Rules out Salmonella and Shigella 37 Summary KIA and LIA Screen for enteric pathogens KIA Reactions X on test LIA React. R/A K/K K/K K/A K/A K/A- Prot. d Sal. a Sal. a Citr. d K/K Pseud. c d K/A- Prot. d Sal. a Edwr a. Edwr. a . Sal. a b Sal. a b K/A-- Morg. d Prov. d Sal. a Pleis. c Aerom. a Shig. a K/A Prot. d Sal. a Sal. a Sal. a b Citr. d Citr. d A/A- E. coli d Kleb. d E. coli d Kleb. d Enter. d A/A-- Serrat. d a Indicates enteric pathogen Sal. and Shig. should be serotyped b Indicates unusual reaction cOxidase positive d Not enteric pathogen 38 Serotyping
Isolates presumptively identified as Salmonella or Shigella should be confirmed by serotyping.
Isolates identified as Edwardsiella are not usually serotyped but are sent to a reference lab as this is an enteric pathogen
Enteropathogenic E. coli (diarrhea etc) can also be serotyped
Enterics have three types of antigens which can be detected by agglutination tests O (somatic) K or Vi and H antigens.
Somatic (of the body or of the body wall) or O antigens are heat stable and are part of the Gram (-) bacterial cell wall
K or Vi antigens are heat labile and if present represent the bacterial capsule. These are the most external antigens and therefore can mask the presence of O antigens (ie. will not show up on agglutination test). K is for the German Kapsule and Vi is derived from the word envelope.
H antigens if present are also heat labile and are part of the bacterial flagella
Since K and H antigens are heat labile they can be removed by boiling the culture for 15 min.
After boiling cultures are tested for somatic antigens. Somatic antigen is the primary antigen used to group (serological groups or serotypes) Salmonella Shigella isolates via antigenic relatedness
95 of human clinically significant Salmonella belong to the somatic groups A through G
S. typhi belongs to group D and S. paratyphi to group C
Reference labs serotype these isolates using K and H antigens as well for confirmation. Most S. typhi strains and a few paratyphoid biotypes also posses the K antigen.
Three types of antigens possessed by enterics
H 41 Serotyping
Shigella species are divided into four somatic groups A through D each corresponding to a different species S. dysenteriae (A) S. flexneri (B) S. boydii (C) and S. sonnei (D)
Any Shigella species may also possess K antigens but since they are nonmotile they never posses H antigens
Enteropathogenic E. coli are relatively hard to detect serologically but most can be serotyped similarly to Salmonella and Shigella
E. coli O157H7 is a well known serotype but there are others
Serotyping each E. coli isolated from feces is impractical
Detecting them by culture is a problem because most toxigenic E. coli have the same cultural characteristics as indigenous colon E. coli but we did discuss a few ways to do this earlier - SMAC
Some labs make available to the physician serological procedures that test for the various toxins produced by E. coli directly from the stool specimen
The LGH Micro lab evaluated the following approach
For 1 year a direct ELISA test for the verotoxin (shiga) was run on every stool received for enteric pathogen culture only three were found to be positive in the 12-month period
Since each test cost about 10 the procedure was deemed not to be cost effective
The current policy of the LGH Micro lab is to culture all stools on SMAC and test only colorless colonies for the Shiga toxin with an ELISA procedure
This is not optimum because some EHEC strains have been shown to be sorbitol positive. Additionally this procedure does not deal with other enteropathogenic E. coli serotypes
Until research finds cost effective ways of detecting all EHEC and other enteropathogenic E. coli the LGH method will have to suffice
43 Clinical Significance
Enterics that cause primary intestinal infections include typhoidal and non-typhoidal Salmonella species (S. enteritidis) toxigenic strains of E. coli Shigella species and Yersinia entercolitica
Enterics that do not cause intestinal infections can be associated with several extraintestinal infections as discussed previously - includes UTIs LRT infections GI tract systemic etc
90 of modern non-intestinal enteric infections are caused by only three species E. coli K. pneumoniae and P. mirabilis
See following table for summary of common disease associations
44 Enterics and Common Infections They Produce Bacterial Species Diseases E. coli UTI septicemia neonatal sepsis diarrheal syndromes Shigella Diarrhea dysentery Edwardsiella Diarrhea wound infections enteric fever Citrobacter Oppor tunistic and nosocomial infections (wounds UTI) s 44-47 not on test -------- 45 Enterics and Common Infections They Produce Bacterial Species Diseases Klebsiella UTI pneumonia septicemia wounds Enterobacter Opportunistic and nosocomial infections (UTI and wounds) Serratia Opportunistic and nosocomial infections (woundUTI sepsis) Proteus UTI wound infection sepsis 46 Enterics and Common Infections They Produce Bacterial Species Diseases Providencia and Opportunistic and nosocomial Morganella infections (UTI wound sepsis) Yersinia pestis Bubonic and pneumonic plague Yersinia entercolitica Diarrhea mesenteric lymph- adenitis (mimicking appendicitis) Salmonella Diarrhea enteric fever septicemia 47 Intestinal Illness caused by E. coli Virulence Factor not known for sure heat labile and stable enterotoxin direct penetration spreadingfactors Shiga-like toxin verotoxin strongly adhere to mucosa
All Gram-negative bacteria (not only pathogens) possess lipopolysaccharide (LPS) in the outer membrane of their cell envelope (membrane(s) wall)
During an infection the envelope can be degraded by phagocytic cells or due to antibiotic therapy. LPS can also be introduced in injectable drugs IV solution etc.
Fragments of the lipid A portion of the LPS function as endotoxin in the body. Compared to exotoxins endotoxin is heat stable relatively (generally) mild in its affect and less specific in its affect.
Endotoxin stimulates phagocytic cells to release interleukin-1 (IL-1 formerly known as endogenous pyrogen). Il-1 travels to the hypothalamus via the blood circulation triggering an increase in body temperature (fever)
49 Virulence Factors
Il-1 also stimulates phagocytes to release another cytokine tumor necrosis factor (TNF) or cachectin (ku-kek-tin)
This substance binds to tissues in the body causing damage. One example is TNF causing damage to blood capillaries causing them to become leaky. Severe cases are sufficiently acute to result in septic shock. A massive dose of endotoxin would be required for such as this it is very rare.
Endotoxin can cause GI pathology which is usually mild
Endotoxin can also cause diffuse intravascular coagulation
Typhoid fever is a prime example of a disease in which endotoxin causes a significant amount of the pathology
50 Virulence Factors a few examplesX on test
Klebsiella pneumoniae large capsule that interferes with phagocytosis and aids in alveolar attachment
Shigella Shiga-like toxin a very potent enterotoxin
enteropathogenic E. coli verotoxin surface protein for adherence to intestinal mucosa
Proteus Urease produces a large quantity of ammonia in the kidney during upper UTI which is toxic to renal parenchyma
Salmonella direct invasion of mucosa anti-phagocytosis enterotoxigenic those that produce enteric fever invade the blood stream
Most E. coli involved in community acquired UTI are susceptible to many antibiotics. Most hospital strains become resistant to a myriad of antibiotics
K. pneumonia is almost always resistant to ampicillin
Enterobacter are resistant to first generation cephalosporins
Proteus mirabilis is intrinsically resistant to nitrofurantoin and tetracycline both frequently used for treating UTI
Providencia Morganella and Serratia are multi-resistant - often plasmid mediated and can easily be passed to other enterics via conjugation
Enteric isolates thought to be causing an infection are almost always tested in vitro with a large battery of antimicrobial agents
52 Intestinal Diseases Caused by Non Enterics
There are several bacteria that cause intestinal infections other than the enterics. Some grow on the enteric media used for primary isolation from clinical specimen (ex. MacConkey agar) and they often produce colonies indistinguishable from enterics
The Vibrio group (Vibrio Aeromonas and Pleisiomonas) is one of these and must be differentiated from the enterics
Members of the Vibrio group are oxidase positive a fact allowing quick differentiation from enterics
Cells from a sweep of a primary isolation plate to collect cells from each representative colony can be tested for oxidase. If the test is oxidase positive each colony is them tested individually to find the positive colony and additional testing can be conducted.
PowerShow.com is a leading presentation/slideshow sharing website. Whether your application is business, how-to, education, medicine, school, church, sales, marketing, online training or just for fun, PowerShow.com is a great resource. And, best of all, most of its cool features are free and easy to use.
You can use PowerShow.com to find and download example online PowerPoint ppt presentations on just about any topic you can imagine so you can learn how to improve your own slides and presentations for free. Or use it to find and download high-quality how-to PowerPoint ppt presentations with illustrated or animated slides that will teach you how to do something new, also for free. Or use it to upload your own PowerPoint slides so you can share them with your teachers, class, students, bosses, employees, customers, potential investors or the world. Or use it to create really cool photo slideshows - with 2D and 3D transitions, animation, and your choice of music - that you can share with your Facebook friends or Google+ circles. That's all free as well!
For a small fee you can get the industry's best online privacy or publicly promote your presentations and slide shows with top rankings. But aside from that it's free. We'll even convert your presentations and slide shows into the universal Flash format with all their original multimedia glory, including animation, 2D and 3D transition effects, embedded music or other audio, or even video embedded in slides. All for free. Most of the presentations and slideshows on PowerShow.com are free to view, many are even free to download. (You can choose whether to allow people to download your original PowerPoint presentations and photo slideshows for a fee or free or not at all.) Check out PowerShow.com today - for FREE. There is truly something for everyone!