Gram positive bacteria are a diverse group. The most clinically important gram ositive pathogens include Staphylococcus, Streptococcus and Entercocci. 

Gram-positive cocci

Enterococcus: are gram positive cocci to elongated cocci in single or short chains. The are catalase negative, facultatively anaerobic. The Enterococci family can be found as part of the normal flora of the gastrointesitnal 30 tracts of humans and other animals. Over the last 30 years, Enterococci have become major nosocomial pathogens. They can cause infections at a variety of sites, including the urinary tract, bloodstream, endocardium, abdomen and biliary tract, as well as burn wounds and indwelling devices. Among the 15 species of Enterococci, E faecalis and E faecium are the most commonly associated with clinical infection. 

–Enterococcus faecalis: causes mostly nosocomial or opportunistic urinary tract infections, wound infections and bacteremia in seriously ill elderly pateints.

–Enterococcus faecium: E. faecium has rapidly evolved as a worldwide nosocomial pathogen by successfully adapting to conditions in a nosocomial setting and acquiring resistance against glycopeptides. They have acquired a number of traits  making them successful in the hospital environment; such as an increase in antibiotic resistance genes and virulence genes enhancing biofilm formation and colonization

Micrococcus: are gram positive cocci in pairs, tetrads or clusters which are catalase positive, strictly aerobic and does not produce acid from glucose.

–Micrococcus luteus: is an opportunistic infection of immunocompromised patients.

Staphylococci (Staphylococcus): are  (so gram test can be used to identify) cocci which are most commonly found in clusters (another identification clue that distinguishes them from say Strept). 

Because stophylococci are carried on the skin surface and in the nasopharynx, shedding of the bacteria is common and is responsible for many hospital acquired infections. They are sususpectable to disinfectants and antiseptic solutions but are capable of survival on dry surfaces for long periods. Transfer to a susceptible individual can be either by direct contact or by means of fomits (contaminated clothing or bed lines). 

Treatment for staphylococcal infections include  (like cephalosporin) although plasmids encoding beta-lactamases are common. 

The only species of Staphylococci which produces the enzyme coagulase is S. aureus whereas all other species are coagulase negative (a distinguishing characteristic).

Group A streptococci are responsible for a wide variety of human diseases, the most common of which are nasopharyngitis and imeptigo. Nearly all clinical isolates have the antiphagocytic factor, M protein, on their surface. This virulence factor displays extreme antigenic diversity within its amino terminal region. It is these highly variable poritions of M proteins which form the basis of the serological type whchme which was formulated in the 1930s perior to knowledge of any structural detail. Group A streptococci can be divided into two major classes partly on the basis of their immunoreactivity with a pair of monoclonal antibodies directed to eptiopes which lie within the relatively conserved half of M proteins. Class I isolates are defined as those binding one or both monoclonal antiboides, whereas class II isolates do not bind either monoclonal antibody. In addition, the classes differ in their ability to exhibit opacity factor activity, and in several pathogenic properties of these organisms (US 5,556,944). 

–S. aureus: 

–S. epidermidis are circular, white, arranged in grape-like clusters, raised on sheep blood agar, coagulase negative (distinguish S. aureus above), non-hemolytic, catalase positive, facultative anaerobes. They are part of the human skin flora and can be found in mucous membranes. They are opportunisitc in that people with compromised immune systems are at risk for developing an infection. 

Streptoccoci are and may occur in pairs or as chains. They are catalase negative (unlike Staphylococcus) and most species are  . 4 different schemes are used for classifying these organisms: 1) clinical presentation (pyogenic, oral, enteric);2) serological properties (Lancefield groupings); 3) hemolytic patterns (complete B hemolysis, incomplete alpha hemolysis, and no gamma-hemolysis) and 4. biochemical properties.

–Streptococcus intermedius: is a Gram-positive bacteria that can cause brain and liver abscesses. It secretes a member of the cholesterol-dependent cytolysis (CDC) toxin family called Streptococcus intermedius intermedilysin (ILY). CDC play various roles in the pathogenesis of a wide variety of human and animal diseases caused by Gram positive bacterial. Human CD59 is a receptor for the cholesterol-dependent cytolysin intermedilysin (Giddings, Nature Structural & molecular biology, 2004). 

–Streptococcus pyogenes (S. pyogenes) 

 –Streptococcus agalactiae (Group B Streptococcus): cause newborn meningitis and pneumonia. Colonies are larger than Group A, and beta hemolysis zone is smaller. Group B Streptococci requires . They have an outermost capsule like Group A. They colonize the upper respiratory tract, lower GI tract, and vagina. Streptococcus agalactiae (Group B Streptococcus, GBS) is an important human pathogen that colonizes the urogenital and/or the lower gastro-intestinal tract of up to 40% of healthy women of reproductive age and is a leading cause of sepsis and meningitis in the neonates. GBS can also infect the elderly and immuno-compromised adults, and is responsible for mastitis in bovines. Like other Gram-positive bacteria, GBS can form biofilm-like three-dimensional structures that could enhance its ability to colonize and persist in the host. Biofilm formation by GBS has been investigated in vitro and appears tightly controlled by environmental conditions.See Mayo Clinic

–Streptococcus pneumoniae

–Viridan Streptococci are alpha and non-hemolytic streptococci. They are most often associated with dental caries. 

Gram + Bacilli

Bacillus: Bacillus species are  which may form chains. They are spore formers and some have capsules. They require aerobic growth and can be grown on blood agar. The pathogenic ones secrete exotoxins.

Bacillus is a very diverse genus with more than 200 species, and the identification and diagnosis of potential disease-causing Bacillus species from patient material by the clinical lab can be challenging. This is especially the case for members of the B. cereus family. Despite their different species names, all these bacteria are often considered to be members of one species based on their high content of shared genes. See Cote

–B. anthracis (anthrax):

–B. cereus: are rod shaped, beta hemolytic, facultative anaerobes and can produce endospores. Bacillus cereus is an endospore-forming, gram-positive bacterium that commonly resides in the soil. Because of its location, Bacillus cereus is usually found on a variety of foods that come into close contact with contaminated soil, and can cause two different types of food borne illnesses: emetic and diarrheal. They are endemic and soil dwelling. Consequently, food products of plant origin frequently contain their spores. There is a marked seasonal variation in the spore content of raw milk, with higher levels during the pasture period, when the cows tetes may contain their spores from soil. Dirty teats that are not cleansed before milking are an important contamination source, particularly during wet weather. Some strains cause food poisoning which occurs due to survival of the bacterial endospores when food is improperly cooked. They can also cause opportunistic infections. After the ingestion of such contaminated foods, Bacillus cereus may incubate inside the infected organism for up to six hours before showing any symptoms that the emetic form displays like nausea, vomiting and abdominal cramps. Strains that produce the diarrheal form of the disease grow in a variety of foods ranging from vegetables to even meat products. Patients experience profuse diarrhea with abdominal pain and cramps…which begin around eight to sixteen hours after ingestion of the contaminated food. B. cereus illness is related to many foods – beef, turkey, rice, beans, vegetables. Specifically, the diarrheal illness is often related to meats, milk, vegetables, and fish. The emetic-type illness is most often associated with rice products, but it has also been associated with other types of starchy products such as potato, pasta, and cheese products. See Medic8. 

Many strains of B. cereus are able to produce toxins and cause distinct types of food poisoning. Concerns over B. cereus contamination have increased because of the rapidly expanding number of chilled foods that may be pasteurized but may still contain viable spores. Spores from B. cereus can germinate and outgrow during storage, even at low temperatures. Major efforts to battle this increasing problem are focusing on determining the causes of the spore’s resistance and the mechanisms of germination. See Vries

While B. cereus is associated mainly with food poisoning, it is being increasingly reported to be a cause of serious and potentially fatal non-gastrointestinal-tract infections. The pathogenicity of B. cereus, whether intestinal or nonintestinal, is intimately associated with the production of tissue-destructive exoenzymes. Among these secreted toxins are four hemolysins, three distinct phospholipases, an emesis-inducing toxin, and proteases. The major hurdle in evaluating B. cereus when isolated from a clinical specimen is overcoming its stigma as an insignificant contaminant. Outside its notoriety in association with food poisoning and severe eye infections, this bacterium has been incriminated in a multitude of other clinical conditions such as anthrax-like progressive pneumonia, fulminant sepsis, and devastating central nervous system infections, particularly in immunosuppressed individuals, intravenous drug abusers, and neonates. See Botone

–B. clausii is a commonly used Bacillus spp. probiotic. Clinical data support its use for the treatment and prevention of gut barrier impairment. Small trials have investigated use in preterm neonates to prevent infection, treatment of nasal allergies and upper respiratory infections in children, and treatment of acute or chronic diarrhea, small-intestine bacterial overgrowth (SIBO), and adverse effects of Helicobacter pylori therapy in adults. See Drugs.com. Probiotic milk has been previously demonstrated to reduce the number of respiratory infections (RI) among children attending day care centres. See Marseglia

However caution should be exercised because Commensal/normal flora which are otherwise termed as ‘good bacteria’ are now causing infections in different group of patients, mostly immunocompromised individuals. Various host and environmental factors play a pivotal role in microbial transmigration from their normal habitat into the blood and other body sites. This has been reported for B. clausii. See Princess

–B. subtillis: is rod shaped, catalase positive, and can form a protective endospore. It is found in the soil and also considered as a normal human gut commensal. B.subtilis is non-pathogenic but can contaminate food and be considered an opportunistic pathogen among the immuno-compromised. They are used on seeds, vegetables, and plants as a fungicide because of their ability to produce antibiotics. B.subtilis inhabits the root system of the plant completing with disease causing organisms. Some B. subtilis strains are capable of producing toxins for insects. These strains are used by farms to protect their crops. See Microchem Laboratory

Clostridium (see outline)

Lactobacillus: are gram positive rods, sometimes in chains. They are catalase engative and have no endospores. They are isolated form a variety of foods (dairy, fish, grain, meat) and many are in normal flora (mouth, intestines, and vagina). There are no key pathogens in the clinial setting

Lysteria

–Listeria monocytogenes: is a ubiquitous, facultative intracellular, gram positive bacillus that causes listeriosis, an often fatal disease in newborns, pregnant women, and the immunocompromised. It is one of the most virulent food born pathogens. See CDC website

Mycobacteria: are obligate, . They are called “acid fast” in that they are very resistent to destaining after you stain them due to their complex cell wall lipids (mycolic acids). They are slow growing organisms and produce pigment depending on the presence or absence of light. Once inside a cell, mycobacteria are resistent to phagoccytosis. Some important types of mycobacteria are the following:

–Mycobacterium tuberculosis:

–M. leprae: occurs mostly in the tropics. Diagnosis is by way of a skin test reagent (lepromin), extracted from autoclaved tissue harvested from patients with leprosy. Therapy is with dapsone which has to be administered for years.

–M. avium is rare in immunocompetent people but a common opportunistic bacterial infection in patients. M. avium complex (MAC) is the most common cause for both disseminated Mycobacterium disease and death in patients with AIDS in the developed world (~25%-50% of adults and 10% of children with AIDS are infected). 

–M. kansasii is a pulmonary infection, resembling TB. It has a photoinducible pigment.

–M. marinum are acquired by contact with contaminated fresh water or salt water. For this reason, they are called “swimming pool granulomas.” It responds well to antibiotics.

–M. fortuitum are most commonly associated with disease following the introduction of the bacteria into deep subcutaneous tissues by trauma or intravenous catheter, contaminated wound dressing, etc. Infections with these organisms are increasing as more invasive procedures are performed on hospitalized patients. These organisms are faster growing than other mycobacteria.

Gram Positive Bacteria that Infects Non-Humans

Paenibacillus larvae is a Gram-positive, spore-forming bacterium that is the causative agent of American foul brood (AFB), the most devastating bacterial disease of the honeybee. P. larvae is antibiotic resistant, complicating treatment efforts. Bacteriophages that target P. larvae are rapidly emerging as a promising treatment. See Genon

Microbiologists have differentiated bacterial based on their biochemical diversity using various biochemical tests. Such tests often include differential medial which are artificial mixtures of chemicals and organic substrates intended to exploit different microbial abilities to perform in ways that might never actually be expressed in nautre. For example, even though an organisms may never greatly lower the pH of its natural environgment, the microbiologist can manipulate conditions in a lab such as by cultivating a microbe with medium spiked with a specific sugar or other substrate.

Oxidation-Fermentation (O-F) Test: is designed to differentiate bacteria on the basis of fermentative or oxidative metabolism of carbohydrates.

In oxidation pathways a carbohydrate is direclty oxidized to pyruvate and is further converted to CO2 and energy by way of the krebs cycle and the electron transport chain (ETC). An inorganic molecule such as oxygen is required to act as the final electron acceptor.

Fermentation also converts carbohydrates to pyruvate but uses it to produce one or more acids (as well as other compounds). As a consequence, fermenters identified by this test acidify O-F medium to a greater exent that do oxidizers.

The O-F test is used to differentiate bacteria based on their ability to oxidize or ferment specific sugars.

Triple Sugar Iron Agar (TSIA) / Kligler Iron Agar: TSIA is a rich medium designed to differentiate bacteria on the basis of glucose fermentation, lactose fermentation, sucrose fermentation and sulfur reduction. In addition to the three carbohydrates, it includes animal proteins as sources of carbon and nitrogen, and both ferrous sulfate and sodium theiosulfate as sources of oxidized sulfure. Phenol red is the pH indicator, and the iron in the ferrous sulfate is the hydrogen sulfide indicator.

The medium is prepared as a shallow agar slant with a deep butt, thereby providing both aerobic and anaerobic growth envirnoments. It is inoculated by a stab in the agar butt followed by a fishtail streak of the slant. When TSIA is inoculated with a glucose only fermenter, acid products lower the pH and turn the entire medium yellow within a few hours. As the glucose depletes, the organisms located in the aerobic region (slant) will begin to break down available amino acids, producing NH3 and raising the pH (18-24 hours). This only occurs in the slant due to the anaerobic conditions in the butt. Thus, a TSIA with a red slant and yellow butt after 24 hour incubation indicates that the bacteria ferments glucose but not lactose.

Organisms that are able to ferment glucose and lactose and/or sucrose also turn the medium yellow throughout. However, because the lactose and sucrose concentrations are 10 times higher than that of glucose, resulting in greater acid production, both slant and but will remain yellow after 24 hours.

Catalase test: The electron transport chains of aerobic and facultatively anaerobic bacterial are composed of molecules capable of accepting and donating electrons as conditions require. One carier molcule in the ETC called falvoprotein can bypass the next carrier in the chain and transfer electrons direclty to oxygen. This alternative pathway produces hydrogen peroxide (H2O2) and superoxide radical (O2-). Organisms that produce these toxins also produce enzymes capable of breaking them down. Superoxide dismutasecatalyzes conversion of superoxide radicals to hydrogen peroxide. Catalase converts hydrogen peroxide into water and gaseous oxygen.

Bacterial that produce catalase can be detected easily using hydrogen peroxide. When hydrogen peroxide is added to a catalase-postive culture, oxygen gas bubbles form. The test is commonly used to differentiate members of the catalase-positive Micrococcaceae from the catalase negative Streptococcaceae.

Oxidase Test: When glucose enters a cell, it is first split (oxidzied) in glycolysis where it is converted to two molecules of pyruvate and reduces two NAD (coenzyme) molecules to NADH (+H+). Then each of the pyruvate molecules is oxidized and converted to a two carbon molecule called acetyl-CoA and one molecule of CO2, which reduces another NAD to NADH. Then the Krebs cycle finishes the oxidation by producing two more molecules of CO2 (per acetyl-CoA) and reduces three more NADs and one FAD to FADH2. The cell is thus beocming full of reduced coenzymes. In order to continue oxidizing glucose, these coenzymes must be converted back to the oxidized state. This i the job of the electron transport chain.

Many aerobes, microarophiles, facultative anaerobes, and even some anaerobes have ETCs. The functions of the ETC are to transport electrons down a chain of molecules with increasingly positive reduction potentials to the terminal electron acceptor (1/2O2, NO32-, SO43-) and generate a protein motive force by pumping H+ out of the cell thus creating an ionic imbalance that will drive the production of ATP by way of membrane ATPases. The protons pumped out of the cell come from the hydrogen atoms whose electrons are being transferred down the chain. Some organisms use more than one type of ETC depending on the availability of oxygen or toher preferred terminal electron acceptor. E coli, for example, has two pathways for respiring aerobically and at least one for respiring anaerobically. Many bacteria have ETCs resembling mitochondria ETCs in eukaryotes. These chains contain a series of foru large enzymes broadly named Complexes I, II, III, and Iv, wach of which contains several molecules jointly able to transfer electrons and use the free energy released in the reactions. The last enzyme in the chain, Complex IV, is called cytochrome c oxidasebecause it makes the final electron transfer of the chain from cytochrome c, residing in the periplasm to oxygen inside the cell.

The oxidase test is designed to identify the presence of cytochrome c oxidase which has the ability to not only oxidize cytochrome c, but to catalyze the reduction of cyctochrome c by a chromogenic reducing agent called tetramethyl-p-phenylenediamine. Chromogenic reducing agents are chemicals that develop color as they become oxidized.

Tests Detecting Hydrolytic Enzymes: Reactions that use water to split complex molecules are called hydrolysis (or hydrolytic) reactions. The enzymes required for these reactions are called hydrolytic enzymes.

(i) Starch Hydrolysis: Starch is too large to pass through bacterail cell membranes and thus must be split into smaller fragments or individual glucose molecules. Organisms that produce and secrete the extracellular enzymes alpha-amylase and oligo-1,6-glucosidase are able to hydrloyze starch by breaking the glycoside linkages between sugar subunits. Starch agar is a medium of beef extract, soluble starch and agar. When organisms produce alpha-amylase and oligo-1,6-glucosidase they hydrolyze the starch in the area surrounding their growth. Because both starch and its sugar subunits are nearly invisible in the medium, iodine is added to detect the presence or absence of starch in the vicinity around the bacterail growth. Iodine reacts with starch and produces a blue or dark brown color.

(ii) Casein Hydrolysis Test: Casease is an enzyme that some bacteria produce to hydrolyze the milk protein casein. The presence of casease can be detected with milk agar. Casease positive organisms will secrete casease which will diffuse into the medium around the colonies and create a zone of clearing where the casein has been hydrolyzed.

Nitrate Reduction Test: Anaerobic respiration involves the reduction of (i.e., transfer of electrons to) an inorganic molecule other than oxygen. Nitrate reduction is one such example. Many Gram-negative bacteria (including most Enterobacteriaceae) contain the enzyme nitrate reductase and perform a single step reduction of nitrate to nitrite (NO3- to NO2).

Nitrate broth is an undefined medium of beef extract, peptone, and ptoassium nitrate (KNO3). An inverted Durham tube is placed in each broth to trap a portion of any gase produced. In contrast to many differential media, no color indicators are included. The color reactions obtained in nitrate broth take place as a result of reactions between metabolic products and reagents added after incubation.

Differential Tests for Enterobacteriaceae   See right hand panel

Motility Tests: See outline

Selective media can be used to  inhibit unwanted bacterial and thereby differentiate bacterial types. Media for isolating intestinal pathogens (MacConkey agar, Hektoen enteric (HE) agar) contain bile salts as a selective agent. Other agents that have selective properties are dyes, such as methylene blue and crysal violet and antimicrobila drugs. 

Selective media should be contrasted with differentail media which do not inhibit the growth of any particular microorganisms but are designed to siplay visible differences in how they grow. Differentiation shows up as variations in colony size or color (e.g., formations of gas bubbles and precipitates or media color changes). A single medium can be both selective and differential. MacConkey agar, for example is both slective and differentail due to its ability to suppress the growth of some organisms while producing a visual distinction among the ones that do grow. 

Selective Media for Isolation of Gram-negative Rods

The following types of media are useful for isolation and differentiation of Gram-negative organisms such as members of the family Enterobacteriaceae (gut bacteria).

MacConkey Agar: contains lactose, bile salts, neutral red and crystal violet. Bile salts and crystal violet inhibit growth of Gram-positve bacteria. Neutral red dye is a pH indicator that is colorless above a pH of 6.8 and red at a pH less than 6.5. Acid accumulating from lactose fermentation turns the dye red. Lactose fermenters turn a shade of red on MacConkey Agar, whereas lactose nonfermenters retain their normal color or the color of the medium. 

(1) Pour or no growth: organism is inhibited by crystal violet and/or bile so is Gram-positive.

(2) Good growth: organism is probably Gram-negative.

(3) Pink to red growth with or without bile precipitate: organism produces acid from lactose fermentation and is probably coliform

(4) Growth is colorless: organism does not ferment lactose is probably noncoliform. 

Eosin Methylene Blue (EMB) Agar: is commonly used for the test for the presence of coliforms in environmental samples. It is a complex (chemically undefined), selective, and differential medium. It contains peptone, lactose, sucrose and the dyes eosin Y and methylene blue. The dyes inhibit the growth of Gram-positive organisms and they react with vigorous lactose fermenters and (in the acidic environment) turn the growth dark purple or black. This dark growth is typical of Escherichia coliand is usually accompanied by a green metallic sheen. Other less aggressive lactose fermenters such as Enterobacter or Klebsiella species produce colonies that can range from pink to dark purple on the medium. 

(1) Poor growth or no growth: Organism is inhibited by eosin and methylene blue so is Gram positive.

(2) Good growth: organism is not inhibited by eosin and methylene blue os is gram-negative.

(3) Growth is pink and mucoid: organism ferments lactose with little acid production and is possible coliform.

(4) Dark purple to black, with or without green metallic sheen: organism ferments lactose and/or sucrose with acid production and is probable coliform.

(5) Growth is olorless (no pink, purple, or metallic sheen): organism does nto ferment lactose or sucrose and is probably noncoliform. 

 Other Types of Differential Media

HE Agar: differentiates Salmonella and Shigella from each other and form other enterics based on their ability to overcome the inhibitory effects of bile, reduce sulfur to H2S and ferment lactose, sucrose or salicin.

The test is based on the ability to ferment lactose, sucrose, or salicin and to reduce sulfur to hydrogen sulfide gas (H2S). Sodium thiosulfate is included as the source of oxidized sulfur. Ferric ammonium citrate is included as a source of oxidized iron to react with any sulfur that becomes reduced (H2S) to form the black precipitate ferrous sulfide (FeS). Bile salts are included to prevent or inhibit growth of Gram positive organisms. The bile salts also have a moderate inhibitory effect on enterics, so relatively high concentrations of animal tissue and yeast extract are included to offset this situation. Bromthymol blue and acid fuchsin dyes are added to indicate pH changes. Differentation is possible as a reslt of the various colors produced in the colonies and in the agar. 

Enterics that produce acid from fermentaiton will produce yellow to salmmon pink colonies. Neither Salmonella nor Shigella species ferment any of the sugars but do break down the animal tissue which raises the pH and gives the colonies a blue-green color. Salmonella species also reduce sulfur to H2S, so the solonies formed also contain FeS which makes them partially or completely black. 

XLD Agar: favors growth of Salmonella, Shigella or Providencia based on its ability to overcome the inhibitory effects of desoxycholate and differentiates them based on their ability to reduce sulfur to H2S, decarboxylate the amino acid lysine and ferment xylose or lactose.

Thioglycollate Broth: is designed to promote growth of a wide variety of fastidious microorganisms. Sodium thioglycollate in the media consumes oxygen and permits the growth of anaerobes. Oxygen removed during autoclaving will diffuse back into the meidum as the tubes cool. This produces a gradient of concentration from full aerobic at the top to anaerobic at the bottom. Thus fresh media will appear clear to straw colored with a pink region at the top. 

Experiment: Label 4 tubes having thioglycollate broth medium as 1. E coli (this is a facultative anaerobe) , B. subtilis (stribe aerobe so should see growth at top but nored because uses all the O2, 3. S. Aurues (facultative anaerobe (will see growth everywhere) and 4. control (expect to be just like the fresh medial with pink at top). 

Protocol Strategies:

(1) look at streaked colonies.

(2) obtain 3 plaes, TP, EMB and MAC. Draw line down middle to separate the plate. Label one side “1” and the other “2”

(3) Using sterile techniques, touch one colony and single streak on side 1. Repeat for the other 2 plates.  Then choose a different colony and repeat on side 2.

Motility Test: The motility test uses a semisolid medium. Agar is typically 1.5% to 0.4% which is just enough to maintain its form while allowing movement of motile bacteria. A tetrazolium salt (TTc) can be added in the medium. TTC is used as an electron acceptor. When it gets reduced it is red and insoluble. A positive result for motility is indicated whne the red (reduced) TTC radiates outward from a central stabl. A negative result shows red only along the stab line.

Experiment: label tubeshaving the motility media (with tape) with 1. proteus vulgaris (should show + results), B. cerus (should show + results), S. Aureus ((should show – results) and a control. Stab the tubes 1/2-3/4 way. Incubate at 37C.

Wet Mount Preparation: is made by placing your specimen in a drop of water on a microscope slide and then covering it with a cover glass. Because no stain is used and most cells are transparent viewing is best done with as little illumination as possible. Viewing must be done quickly because of drying of your preparation. Motility is detected by independent darting of the cells. Be careful not to mistake motility with bacteria appearing to herd across the filed which is due to the water receding.

Hanging Drop Preparation: allows longer observation of your specimen because it does not dry out as quickly. A thin ring of petroleum jelly is applied around the well of a depression slide. A drop of water is then placed in the center of the cover glass and living microbes are then transferred into it. A depression microscope slide is next carefully placed over the cover glass in such a way that the drop is received into the depression and is undistrubed. The petroleum jelly will cause the cover glass to stick to the slide. The preparation can now be picked up inverted so the cover glass is on top and placed under a microscope. As with the wet mount viewing is best down with as little illumination as possible. Motility is observed when cells move over greater distances than simply the motility caused by Brownian motion created by colission with water molecules.

Generally

Enterobacteriaceae are the largest group of medically important bacteria which comprise a family of Gram negative rods that mostly inhabit the intestinal tract. The port of entry into the human body is usually orally.

Many enterics are harmless gut commensals or opportunists. However, some are pathogens (e.g., E coli which can cause bloody diarrhea, Klebsiella pneumoniae which can cause various types of pneumonia, Proteus mirabilis which can cause urinary tract infections, Salmonella tryphi which can cause typhoid fever, Shigella dysenteriae which can cause bacillary dysentery and Yersinia pestis which can cause the plague.

Each year, infections from major foodborn pathogens are responsible for an estimated 9.4 million illnesses, 56,000 hospitalization and 1,350 deaths in the U.S. To evaluate progress toward prevention of enteric infections in the US, the Foodborn Diseases Active Surveillance Network (FoodNet) conducts surveillance for laboratory-diagnosed infections causes by eith pathogens transmitted commonly through food at 10 U.S. sites. Campylobacter and Salmonella are the leading causes of bacterial enteric infections transmitted commonly by food. During 2022, FoodNet identified higher incidences of Shiga toxin-producing Escherichia coli, Yersinia, Vibrio and Cyclospora infections compared with 2016-2018. See CDC

 Detection and Isolation

Example: Frm a carbapenemas proudcing enterobacterales (CPE) surveillance specimen, asthe stool specimen was inoculated on CHROMID CARBA SMART agar. The next day, scant gray colonies grew on the OXA side of the plate. The organism was identified as E. coli by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectromety (Bruker Daltonics). owever, becasue the colonies were gray, not pink on the agar, and du to a known limitation that MALDI_TOF cannot distinguish E. coli from Sigella, serotyping uisng Shigella antisera and biochemical reactions were set up. Kligler ion agar was alkaline over acid with no H2S and produced litttle gas The organisms was negative for lysine, ornithin, indole and citrate. Motolity was negative, and the isolate showed agglutination with S. Flexneri group B antisera, types 1-6, and no agglutination with antisera for other Sigella groups. The identification was confirmed by the BD Phoenix system, which gave a 99% S. Flexneri identificaiton of the isolate. The isolate was forwarded to the Provincial laboratory and was serotyped as S. fleneri type 2a. Stool culture ordered as a reflex test after detection of Shigella species was multiplex PCR identified the organism when it grew as non-lactose fermenting colonies on MacConkey agar. DNA was extracted from an overnight culture of the bacterial isolate using the easyMag system (bioMerieux). Pufified DNA was prepared for sequencing using the Rapid Barcoding Sequencing Kit SQK-LSK114 (Oxford Nanopore Technologies, UK). Sequencing occurred on a GridION system using a R10.4.1 flow cell FLO-MIN114, Oxford Nanopore Technologies) with High-accuracy model basecalling with data captured over 26 hours. Data was evaluted with the MinKNOW software 23.04.5. A consensus genome was constructed using Flye 2.9. The consensus genome for botht he bacterial isoalte and plasmid were anlzyed thorugh the Reistance Gene Identificaiton tool within the Comprehensive Antibiotic Resistance Database. Hits demosntrating a greater than 95% identity and 95% lenght of reference sequence were included in analysis. The database identified 5 reistance genes present within the plasmid. The plasmid consensus sequence was visualized and annotated using Geneious Prime v2023.0.1 (Biomaters, US). “Detection of OXA-181 carbapenemase in Shigella flexneri”

1. Selective Media

MacConkey Agar: See outline

Eosin Methylene Blue Agar: See outline

Hektoen Enteric Agar: See outline

Major characteritiscs are the following:

• Growth on MacConkey agar: See differential media right hand column
• Gram negative rods
• Oxidase negative but most are catalase-positive
• acid production from glucose (with or without gas)
• Most reduce NO3 to NO2

2. Serological Classification

The serologic classification of Enterobacteriaceae is based on 3 major groups of antigens: somatic O LPS, capsular K antigens, and the flagellar H proteins. For example. “E. coli O157:H7” refers to the O antigen and flagellar protein.

One protocol might be to conduct whole-genome sequencing by extracting DNA using easyMag (bioMerieux), sequencing on a GridION system with a R10.4.1 flow cell (oxform nanopore Technologies) and analyzaing the data with MinKNOW 23.04.5 (oxform Nanopore Technologies) to construct a consensus genome using multiplex assay.

SeeGene Allplex GI-EB gastrointestinal multiplex assay

3. Antibody Susceptibility Testing:

NG Biotech CARBA-5 Assay 

Comprehensive Antibiotic Resistance Database

Specific Types of Enterobacteriaceae

Some members of this family like E coli and K. pneumonia are part of the normal bowel flora that can cause opportunistic infections whereas others like Salmonella, Shigella and Yersinia are always associated with disease. Virulence factors include –endotoxins, –capsules, –antigenic phase variation and –antimicrobial resistance.

Escherichia coli

E. coli are part of the normal flora. E coli and other “coliforms” are commonly found only in the bowel. (coliforms are used as a public health indicator of fecal contamination of water). They are facultative anaerobe, gram-negative rods and lactose positive fermentators. Most infections are endogenous (come from the gut and get into other parts of the body that they should not be in). Enterohemorrhagic E. coli (EHEC) causes a Shiga-like toxin which causes bloody diarrhea. Enterotoxigenic E. coli (ETEC) causes traveler’s diarrhea and produces heat labile and heat stable enterotoxins similar to cholera which causes essential metabolites to flow out of the cell rather than into it. Together with group B streptococci, they are the most common cause of neonatal meningitis. They are also the most common gram negative organisms in septic patients. Virulence factors of pathogens include –adhesins, –exotoxins, –alpha hemolysins (which disrupt plasma membranes) and siderophores (which bind necessary ion). Escherichia coli: are gram-negative, facultative anaerobic, rod shaped bacteria which are often found in the lower intestinal tract of warm blooded animals as part of the natural flora. Some serotypes can cause serious food poisening in humans. The fecal-oral route is the major route through which pathogenic strains cause disease. It is one of the most widely studied prokaryotic organisms and has an important role in recombinant DNA.  E coli can be distinguished from other coliforms (rod-shaped gram-negative non-spore forming bacteria which can ferment lactose and commonly used to indicate sanitary quality of foods and water) by the ability to ferment lactose at 44C in the fecal coliform test.  See WHO

Enterobacter aerogenes:

Enterobacter aerogenes is a gram negative oxidase negative, catalase positive, citrate positive, indole negative, rod-shaped bacterium. They are found in the gastroinestinal tract and are opportunistic bacteria.

Klebsiella pneumoniae 

K. pneumoniae, a member of the Enterobacteriaceae family, has long been recognized as a formidable human pathogen associated with a wide range of infections, including urinary tract infections, pneumonia, bloodstream infections, and surgical site infections. The clinical significance of K. pneumoniae is underscored by its ability to exploit an array of virulence factors that facilitate adherence, colonization, and evasion of the host immune response. These virulence factors include capsule polysaccharides, lipopolysaccharides, fimbriae, and siderophores. Notably, the polysaccharide capsule confers resistance to phagocytosis and complements bacterial virulence by facilitating biofilm formation and mediating adherence to host tissues.

Klebsiella pneumoniae usually infects immunosuppressed individuals and cause a bloody sputum. They have an anti-phagocytic capsule. Carbapenem-resistant Enterobacteriaceae (CRE) are considered an urgent threat in the United States because they are associated with high morbidity and mortality, limited treatment options, and potential for rapid spread among patients. Carbapenemases, enzymes that confer resistance to the carbapenem class of antibiotics, are believed to contribute to increasing transmission and regional spread of CRE because the genes encoding these enzymes can reside on mobile plasmids and can be transferred among bacterial species. Klebsiella pneumoniae carbapenemase (KPC) is the most common carbapenemase seen in the United States, but isolates with the New Delhi metallo-β-lactamase (NDM) are emerging. Known risk factors for carbapenemase-producing CRE, including NDM, include health care exposures such as hospitalization outside the United States, recent overnight admissions to short-stay and long-term acute care hospitals, residence in long-term care facilities, surgical procedures, and having indwelling devices. Community-associated CRE lack these health care exposures and are rare in the United States. During 2014–2016, NDM-producing CRE were isolated from patients in Colorado without known health care risk factors. See CDC

Salmonella

Salmonella enterica are lactose negative (unlike E coli which are lactose +). A large infectious dose (106) is necessary. S. enterica is ubiquitous in animals. The source of most infections is from contaminated food products. Exposure to Salmonella bacteria can occur through food, drinking water, animal contact, environmental sources such as soil and water, and infected persons.

Some Salmonella serotypes are highly host-specific. For example, S. enterica serovar Dublin is primarily associated with infections in cattle and sheep, whereas serovar Gallinarum is almost exclusively associated with infections in poultry. In contrast, other serotypes, such as serovars Enteritidis and Typhimurium, are associated with infection of a wider range of human and animal hosts. See Attribution of Salmonella enterica to Food Sources by Using Whole-Genome Sequencing Data

Nontyphoidal Salmonella infections place a large burden on public health; an estimated 79 million cases of foodborne nontyphoidal Salmonella infection occurred in 2010.

Salmonella enterica serovar Infantis>  presents an ever-increasing threat to public health because of its spread throughout many countries and association with high levels of antimicrobial resistance (AMR).  Salmonella enterica subspecies enterica serovar Infantis is becoming an increasingly prevalent serovar globally. A 167% increase in human infections was observed in the United States during 2001–2016. nfantis is the predominant serovar isolated from broiler flocks and broiler meat, accounting for 56.7% of Salmonella isolates from broiler meat in 2018. Higher levels have been observed in Japan, at 72.2% of isolates from ground chicken, and levels of 84% were seen in broilers in Ecuador.

Salmonella typhi are an obligate pathogen of humans (do not spread from species to species) and cause enteric (typhoid) fever. They may form a carrier state (typhoid Mary) and invade macrophages. The infectious dose is small (10) compared to S. enterica.

Shigella

Shigella is primarily a pediatric disease although infections in male homosexuals are also observed. Transmission is by the fecal-oral route. Because as few as 200 bacilli can establish disease, Shigella bacteria are responsible for shigellosis. shigellosis spreads rapidly in communities where sanitary standards are low. A shiga like toxin is produced by S. dysenteriae causing a bloody diarrhea much as with EHEC. Shigella invades M cells and replicates in the cytoplasm. It goes from cell to cell using the actin machinery of the infected cell. In the US, most Shigella is already resistant to antibiotics such as ampicillin and trimethoprim/sulfamethoxazole. Woldwide, resistance to Cipro is also on the rise.

Limmatech Biologics is currently evaluating the sfety and immunogenicity of a Shigella vaccine candidate in a Phase I/II clinical trail in Kenyan children. The Sigella vaccine is tetravelnt in that it incorproates four anitgens. It covers up to 85 of diseases produced by Shigella.

Fhigella flexnerii: infection leads to shigellosis, an acute gastrointestina disease. Shigellosis affects socioeconomically disadvantaged and densely populated communiteis that have unsafe water, poor sanitation, and poor hygeine. (Dhabaan, “Detection of OXA-181 Carbapenemase in Sigella flexneri Emerging Infectious Diseases, 30(5), 2024).

Shigella sonnei: causes about 500k cases of diarhea in the US each year and is becoming increasingly resistant to antibiotics such as ciprofloxacin (Cipro). Cipro is often presecribed for travelers visiting other countries who develop diarhea while travelling. However, this may also be contributing to the bacterial resistance. Washing hands with soap and water, choosing hot foods and drinking only from sealed containers are protection measures.

Treatment:

The emergence of multidrug-resistant Shigella strains is a concerning trend. Multidrug-resistant strains resist multiple first line oral antimicrobials (i.e., ampicillin, trimethoprim/sulfamethoxazole, and ciprofloxacin). The situaiton is further complicated by enzyme-mediated beta-lactam resistance in Shigella bacteria, further impacting empiric therapy and making the isolates extensively drug resistant. Alhtough extesnively drug resistant isolates have remained susceptible to carbapenem therapy, carbapenem reistance in Shigells spp. thorugh imipenemase-type metallo-beta-lactamase, New Delhi metallo-beta-Lactamase, and Verona integron-encoded metallo-beta-lacamase has been reported. (Dhabaan, “Detection of OXA-181 Carbapenemase in Sigella flexneri Emerging Infectious Diseases, 30(5), 2024).

Yersinia 

Yersinia consists of several species. The Yersinia genus encompasses 2 enteropathogenic species, Y. enterocolitica and Y. pseudotuberculosis. Those bacteria are the cause of foodborne infections that range from mild enteritis, especially in children, to systemic infections in the elderly or patients with underlying disorders. See Genomic Characterization of Yersinia enterocolitica Isolates, Costa Rica

Y. pestis is the species that causes bubonic plague and was so devastating in the 14 century. This “black death” killed 25 million in Europe. It is transmitted via fleas and the reservoir is rodents, dogs and rabbits. It is a facultative intracellular bacterium and a zoonaotic

There are three clinical types of plague. 1) the first is bubonic (infected lymph nodes) which causes high fever and painful bubo (inflammatory swelling of lymph node). 2) The Second is pneumonic is the deadliest (almost 100% fatality rate) and transmissible by aerosol. 3) The third is septicemic (blood-born organisms).

Serratia: 

This genus comprises gram-negative rods c . 0.9–2 μm long and 0.5–0.8 μm in diameter, and is part of the family Enterobacteriaceae . They consists of the following recognized species: Serratia entomophila , S. ficaria , S. fonticola , S. grimesii , S. liquefaciens , S. marcescens , S. odorifera , S. plymuthica , S. proteamaculans , S. quinivorans , S. rubidaea and S. ureilytica. Most Serratia spp. are motile by peritrichous flagella and are facultative anaerobic, chemoorganotrophic bacteria with both a respiratory and a fermentative type of metabolism. Being ubiquitous inhabitants of soil, water and plant surfaces, Serratia spp. are commonly associated with raw food materials and cause spoilage of various foods. In addition, they are capable of colonizing a wide variety of surfaces in the digestive tracts of rodents, insects, fish and humans

S. marcescens is implicated in a wide range of serious infections including pneumoni. S. marcescens infection has been attributed to many different sources. Outbreaks of infection have been traced to medical equipment including nebulisers. See Antimicrobes

Specific Types found in Non-humans

Edwardsiellosis, caused by Edwardsiella tarda, has been reported worldwide in economically important fish species, including Japanese eel (Anguilla japonica), red sea bream (Pagrus major), yellowtail (Seriola quinqueradiata), channel catfish (Ictalurus punctatus), and turbot (Scophthalmus maximus).  Edwardsiella tarda is one of the serious fish pathogens, infecting both cultured and wild fish species. Research on edwardsiellosis has revealed that E. tarda has a broad host range and geographic distribution, and contains important virulence factors that enhance bacterial survival and pathogenesis in hosts. Although recent progress in edwardsiellosis research has enabled the development of numerous, highly effective vaccine candidates, these efforts have not been translated into a commercialized vaccine. see Park

Bacteria are classified according to 1) morphology 2) gram stain as well as 3) growth requirements.

(1) Morphology See right hand column

(2) Gram stain: is the most important tool for bacterial identification. It is a differential stain in which a decolorization step occurs between the application of two basic stains. The primary stain is crystal violet. Iodine is added as a mordant to enhance crystal violet staining by forming a crysal violet-iodine complex. Declolorization follows and is the most critical step in the procedure. Gram negative cells are declorozed by the solution whereas Gram positive cells are not. Gram negative cells can thereafter be colorized by the counterstain safranin. At the end, Gram positive cells appear purpose and gram negative cells appear reddish pink. 

(3) Growth Requirements

Although all bacteria have minimum nutrient requirements for growth which are 1) a carbon and nitrogen sources, 2) energy source, 3) water and 4) various ions, a great deal of diversity exists within the prokaryotic kingdom in specific growth requirements. For example, some bacteria are unable to grow in the presence of oxygen and are are referred to as obligate anaerobes whereas others require the presence of oxygen and are called obligate aerobes. The vast majority of bacterial grow in either the presence or the absence of oxygen and are called facultative. One may also classify bacteria based on their sources of energy and carbon. Those that derive energy from the oxidation of metals and get carbon from CO2 are referred to as chemotrophs. Those that derive energy from light and carbon from CO2 are referred to as phototrophs. Most pathogenic bacteria derive their energy and carbon from organic sources and are referred to as organotrophs.

Methods used to detect bacteria

• Isolation and identification in Culture is the “gold standard for identification of bacteria. Types of culture media used include 

  • 1) agar plates, 

  • 2) liquid media (broth) which is used in blood culture bottles, 

  • 3) selective and differential media: See right hand panel. 

• Direct detection 

• serology which is evidence of a specific immune response to the bacterium. The problem with this approach is that it is retrospective. IgM is the first Ig to appear usually about 7-8 days after infection and indicates a recent infection. IgG appears later. 

Tools for Bacterial Detection

• genomic analysis of isolates can be down according to the following strategies:

  • 1) the presence of certain plasmids 

  • 2) PCR: Caroll (J. Clin. Microbiol. May 2000, 38(5), 1753-1757 disclsoed a nested PCR protocol for the detection of and discrimination between 14 species of gram positive and negative bacterial. First round PCR was with pan bacterial oligonucleotide primers based on conserved sequences of the 16S ribosomal gene, followed by a gram negative specific PCR which resulted in a 985 bp amplifcaiton product and a multiplex PCR which resulted in two PCR products, a 1,025 bp amplicon (all bacteria) and a 355 bp amplicon (gram positive bacteria only).  

  • 3) nucleic acid probes.

Medically Important Gram –

Morphology (Shell Shape) and Arrangement

Bacterial cells are much smaller than eukaryotic cells and come in a variety of morphologies (shapes) and arrangements. Determining cell morphology is an important first step in identifying a bacterial species. Bacteria are classified into 3 different shapes: 

(1) spheres (cocci, singular=coccus):

(2) rods (bacilli, singular=bacillus):

(3) spiral cells (spirilla,singular=spirillum):

Variations of these shapes include slightly curved rods (vibrios), short rods (coccobacilli) and flexible spirals (spirochetes). 

Cell arrangement, determined by the number of planes in which division occurs is also useful in identifying bacterial. Spirillas rarely are seen as anything other than single cells, but cocci and bacilli do form multicellular associations. 

The coccus is better suited for a dry environment than a bacillus because a sphere has less surface area and thus less moisture will be lost by osmosis in a dry environment. In a moist environment, the higher surface area to volume ratio of a rod shaped bacterium will allow greater efficiency in transferring water and solutes into and out of the cell, making it possible for a rod shaped cell to have a higher metabolic rate.

Bacteria have contributed significantly to the rise in infectious diase. Bacteria as a class include a huge number of organisms, generally classified by biochemical characteristics (i.e., Gram stain characteristics) or morphological characteristics (e.g., bacilli cocci).

Bacteria are classified taxonomically as to genera and species. Many bacteria are harmless to humans while others cause disease only opportunistically. A subset of bacteria are highly pathogenic.

Differences between Bacterial and Eukaryotics

Bacterial are single cell prokaryotes, which have the following differences with the eukaryotic cell:

(1) lacks a true membrane bound nucleus. Instead, the prokaryotic DNA molecule is found within the cytoplasm of the cell in a discrete area known as the nucleotid. Because there is no physical separation between transcription and translation as with eukaryotes, the two processes are “coupled” (occur simultaneously).

(2) surrounded by a cell wall (everything external to the cytoplasmic membrane) composed of peptidoglycan which interfers with phagocytosis, is mitogenic (stimulates mitosis of lymphocytes) and has pyrogenic activity (induces fever). The B-lactam antibiotics (penicillins and cephalosporins) inhibit peptidoglycan synthesis. Cell envelope architecture differs between gram +/- bacteria.

Plants, fungi and most protists also have cell walls but with a chemical structure different form that of peptidoglycan. 

(3) the cytoplasm is surrounded by a cytoplasmic membrane. (this is true with both gram + and -)

(4) in some cases enclosed in a capsule. The presence of a capsule is associated with virulence because it interferes with phagocytosis. Most capsules consist of repeating sequences of 2-3 sugars. This capsule encases an endospore which is an extremely resistent structure (contains calcium dipicolinate) that encases the genome. When conditions become favorable signals turn on genes that allows the spore to germinate.

The resistance of endospores to antibiotics is of immense concern. The endospore’s most external layer, the exosporium, is composed primarily of proteins, polysacharides and lipids. Inward from the exosporium, the spore coat is composed primarily of proteins, which envelops the peptidoglycan cortex. Interior to the corext, the highly lipid nature of the inner membrane renders the spore core impermeable to several chemical compounds. These protective aleyrs work collectively to passively dehydrate the spore core and maintain a state of dormany. Upon encountering favorable enironmental conditions, suficial sensory mechanisms of variable sportes recognize specific chemical signals, known as germinants and initiate metabilic casades that resultin the germination and outgrowth of the spore to its vegetative form. 

spore-forming bacterial species reported as being pathogen to humans, livestock and insects below to the general Bacillus and Clostridium. 

(5) Various structures like flagella (long helical) and pili (straight and smaller) may protrude from the cell. The presence of pili is a characteristic most common among pathogenic bacteria of the mucosal surfaces like neisseria gonorrhoeae which are able to adhere to genital tract mucosal surfaces.

(6) smaller than eukaryotic cells. Bacteria are about 1 micron whereas eukaryotes are typically greater than 5 microns. The genomes of bacteria are also smaller (2-5 million base pairs which encode about 4k genes)

(7) some bacterial (particularly gram –) contain additional DNA molecules called “plasmids” which replicate independently of the chromosomes and which often code for virulence factors. They are commonly exchanged among related bacteria via conjugation and transduction and occasionally by transformation.

(8) Bacteria reproduce by a process called binary fission rather than mitosis. Since there is only one DNA molecule, a spindle apparatus and other components of the mitotic cycle are unnecessary.

(9) there are no other organelles in their cytosol except ribosomes.

Bacteria are extremely complex at the molecular level, composed of thousands of proteins, large quantities of nucleic acids and a great many types of small organic molecules. However, the host is oblivious to the vast majority of these molecules. Only a handful of them (e.g., LPS, lipopeptides, lipoteichoic acid, flagellin and unmethylated DNA) incite an innate immuen response.