Bacteriology: Gram-Positive & Gram-Negative Bacteria

• Study of bacteria covers isolation, growth, cultural, morphological, biochemical, and antigenic characteristics.
• It includes epidemiology, pathogenesis, pathogenicity, diagnosis, prevention, and control of bacterial diseases.

Gram-Positive Bacteria

• Includes Staphylococcus, Streptococcus, Corynebacterium, Trueperella, Rhodococcus, Listeria, and Erysipelothrix.
• Also includes Bacillus, Mycobacterium, Clostridium, Actinomyces, Nocardia, Streptomyces, and Dermatophilus.

Gram-Negative Bacteria

• Includes Family Enterobacteriaceae like E.coli, Klebsiella, Salmonella, Yersinia, and Proteus.
• Includes Pseudomonas and Burkholderia.
• Includes Pasteurella and Mannheimia.
• Includes Actinobacillus and Haemophilus.
• Includes Brucella and Vibrio.
• Includes Campylobacter.
• Includes Bordetella and Moraxella.
• Gram-negative anaerobes include Bacteriodes, Dichlobacteria, and Fusobacterium.
• Leptospira and other Spirochaetes are included.
• Includes Mycoplasma, Coxiella, Neorickettsia, Ehrlichia, Anaplasma, and Rickettsia.
• Includes Chlamydia and Chlamydophila.
• Emerging, re-emerging, and transboundary bacterial pathogens are also a consideration.

Escherichia coli

• E. coli was discovered by Theodor Escherich in 1885.
• It has a worldwide distribution and are found in water or as commensals.
• E. coli is a natural inhabitant of the large intestine and lower small intestine of all mammals.
• It is usually present in large numbers in carnivores and omnivores than in herbivores.
• E. coli can survive in fecal particles, dust, and water for weeks or months.
• Presence of E. coli in water samples indicates fecal pollution.
• E. coli are gram-negative, non-spore forming, and non-acid-fast.
• They are straight, rod-shaped bacteria occurring singly or in pairs.
• They are motile by peritrichous flagella.
• Some strains possess a polysaccharide capsule.
• Most strains possess fimbriae.
• E. coli are facultative anaerobes with optimal growth between 22 and 37°C.
• They grow on most simple bacteriological media.
• Most can grow on D-glucose as the sole carbon source, though some require amino acids or vitamins.
• E. coli are chemoautotrophs with both respiratory and fermentative metabolism.
• They form both acid and gas from glucose.
• On MacConkey agar, E. coli forms bright pink colonies indicating acid production from lactose fermentation.
• On Eosine Methylene Blue agar (EMB), E. coli gives a distinctive metallic sheen distinguishing it from other Enterobacteriaceae.
• E. coli ferments glucose, reduces nitrates, and is oxidase negative.
• IMViC test results for E. coli are Indole +ve, Methyl-red +ve, Voges-Proskauer -ve, and Citrate -ve.
• E. coli is lactose positive and doesn't produce H₂S.
• The reaction in triple sugar iron agar slant is Y/Y/H2S negative.
• E. coli can be subdivided according to their O, H, and K antigens.
• There are 164 O types, 103 K antigens, and 75 H antigens.
• An example is O157:H7.
• Fimbrial antigens include K88(F4), K99(F5), 987P(F6), and F41.
• Capsule, cell wall/endotoxin, fimbriae/pili, and intimin are virulence factors.
• Other virulence factors include enterotoxins (heat stable ST, heat labile LT), and verotoxins or shiga-like toxins.
• Additional virulence factors include CNF (CNF-1, CNF-2), haemolysins, siderophores, colicins, and biofilm production.
• Pathotypes of E. coli include: ETEC (Enterotoxigenic), EPEC (Enteropathogenic), EIEC (Enteroinvasive), AEEC (Attaching and Effacing), EHEC (Enterohaemorrhagic), and DAEC (Diffusely Adherent).
• Diseases caused by E. coli include Enteric colibacillosis (white scour), Joint ill/Naval ill-calves, and Colisepticemia.
• Other diseases include Oedema disease of pigs (VT2e toxin), Coliform mastitis, and Urogenital tract infection.
• Diseases also include Omphalitis (Mushy-yolk disease), Airsacculitis, Colibacillosis, Hjarre's disease, or Coligranuloma.
• E. coli is killed at 55°C for 1 hour and 60°C for 20 minutes.
• It is susceptible to phenol, QAC, cresol, and other disinfectants.
• Formaldehyde vapor destroys the organisms.
• E. coli remains in the environment (water, feed, dust) for weeks.
• Heat-resistant strains can survive at 60°C for 15 min or 55°C for 60 min.
• In Cattle (K99) E. coli causes white scour or colisepticaemia and Joint ill in Calves.
• It can cause Adult-scouring, weakness, prostration, and death within hours after initial symptoms, and Mastitis.
• In Horses E. coli causes New born foals-joint ill, naval ill or sleepy foal disease.
• In Pigs (K88, K99) E. coli causes Piglet diarrhea and edema disease.
• In Poultry E. coli causes Colibacillosis, airsacculitis, Hjarre's disease, or coligranuloma and yolk sac infection.
• In Dogs E. coli causes Fading puppy syndrome in new born puppies, pyometra, UTI infection.
• Specimens to collect for diagnosis are faeces for enteric disease, tissue specimens for septicaemic cases, mastitis milk, mid-stream urine and cervical swabs from suspected cases of pyometra or metritis.
• Other diagnostic methods include clinical signs, pathology, isolation, identification, and serotyping.
• Also includes detection of heat labile enterotoxin using mouse adrenal cells or Chinese hamster ovary cells, and Ligated ileal loops techniques.
• Additional methods include detection of heat stable enterotoxins or fimbrial antigens by immunological methods or PCR, and Slide agglutination tests for O and H antigens for serotype identification.
• Antibiotics are usually not effective since the infectious load is very high.
• Death can occur within 24 hours, leaving insufficient time for antibiotics to be effective.
• Fluid rehydration can be an important adjunct.
• The best plan for enteric diseases is prophylaxis and good management practices.
• Vaccines can be used, mostly based on immunity to pili.
• Passive immunization can be achieved by immunizing sows with E. coli K88 antigen during gestation.
• This results in anti K-88 antibodies in the colostrum and milk which when ingested by piglets prevent the adhesive capacity of K88 antigen.
• In cattle, the mother should not be moved to a new environment shortly before calving.
• Three types of vaccines are available: live E. coli K88 antigen (oral vaccine), killed E. coli bacteria with K88 antigen (bacterin), and bacteria-free K88 antigen (subunit vaccine).
• Vaccination of pregnant cows with purified E. coli K99 fimbrial or whole cell preparations, combined with rotavirus antigen, can be used to enhance colostral protection.
• The use of antibiotics may lead to resistant strains of E. coli.
• Oxytetracycline, chlortetracycline, streptomycin, ciprofloxacin, and enrofloxacin has been used to prevent.

Salmonella

• Salmonella are Gram-negative, non-capsulated, non-spore forming, short rods.
• They are motile by peritrichous flagella, except Salmonella Gallinarum and Salmonella Pullorum.
• Type-1 and Type-2 fimbriae are present.
• Strains of S. Gallinarum and S. Pullorum form type 2 fimbriae and are non-adhesive.
• Salmonella enterica is subdivided into six subspecies: Enterica (I), Salamae (II), Arizonae (IIIa), Diarizonae (IIIb), Indica (IV), and Houtenae (V).
• The genus Enterica is the largest and most important, containing all the species that commonly cause human and animal infections.
• Members of this subgenus are given a name similar to Salmonella enterica subsp. enterica serovar Typhimurium.
• Serotyping is based on the Kauffmann–White scheme, depending on the identification by O and H antigens of the strain and agglutination with capsular Vi antigen.
• The flagellar antigens of salmonellae occur in 1 or 2 phases.
• Phase 1 antigen is either specific for a species or shared by a few species only, termed as specific phase.
• Phase 2 antigens are widely shared and hence termed as nonspecific or group phase.
• Phase 1 antigens are designated as a, b, c…Z and after Z as Z1, Z2, Z3…etc.
• Phase 2 is designated as 1,2…etc.
• Selective enriched media for salmonellae includes tetrathionate broth, selenite broth, and rappaport vasiliadis medium.
• Host-adapted serotypes from pigs and poultry are more fastidious than others.
• Rappaport is highly suitable for Salmonella that does not tolerate selenite or tetrathionate broth.
• Other mediums include MacConkey agar, Brilliant green agar, XLD, and Salmonella Shigella (SS) agar.
• The majority of salmonellae, except some strains of S. Arizonae, are non-lactose fermenters and produce pale or colorless colonies on MacConkey agar.
• Most salmonellae give an alkaline reaction in brilliant green agar.
• On XLD medium, they produce H₂S and have red colonies with a black center (Black center with red skirt).
• Salmonella and Shigella agar produce colorless colonies with black center.
• The typical reaction for salmonellae in TSI (triple sugar iron) agar is an alkaline slant (red), acid butt (yellow) and superimposed H₂S (black) production (R/Y/H₂S+).
• The test for lysine decarboxylation is positive.
• Salmonella gives IMViC test  -,+,-,+.
• They ferment maltose, mannitol, mannose and glucose and produce acid and gas. 
• They do not ferment lactose, sucrose and salicin.
•  Urease negative. Most salmonellae produce H₂S except S.Choleraesuis and S.Paratyphi A.
•  Salmonella Pullorum ferments glucose and rhamnose while S.Gallinarum ferments dulcitiol and maltose.
• The bacilli are destroyed at 55°C in one hour or at 60°C in 15 minutes.
• Boiling or chlorination of water and pasteurization of milk destroys the bacilli.
• Cultures may be viable for years if prevented from drying.
• Salmonella is killed within 5 minutes by mercuric chloride (1:500) or 5% phenol.
• Salmonella virulence factors include defensive and offensive factors, Salmonella pathogenicity islands, and type III secretion systems.
• Salmonella pathogenicity island-1 is associated with intestinal invasion and onset of diarrhea.
• Salmonella pathogenicity island-2 is associated with intracellular survival and systemic infection.
• Infections caused by salmonella include enteric salmonellosis, septicaemic salmonellosis, and salmonellosis in poultry.
• Salmonellosis in poultry includes pullorum disease and fowl typhoid.
• Salmonella Dublin and Salmonella Typhimurium causes Enteritis and septicaemia, meningitis in calves, abortion, osteomyelitis, joint ill, and terminal dry gangrene in calves.
• Salmonella Choleresuis, Salmonella Typhisuis and Salmonella Typhimurium causes Outbreaks clinically similar to swine fever, Chronic enteritis in young pigs and Enteritis or septicaemia.
• Salmonella Abortusovis, Salmonella Typhimurium and Salmonella Brandenburg causes Abortion, Enteritis or septicaemia and.
• Salmonella Abortus equi and Salmonella Typhimurium causes Abortion in mares and Enteritis or septicaemia.
• Salmonella Pullorum, Salmonella Gallinarum. Salmonella Arizonae, Salmonella Enteritidis, Salmonella Typhimurium causes Pullorum disease (bacillary white diarrhea) in chicks and Fowl typhoid in all ages.
• Salmonella Typhi, Salmonella Paratyphi( Salmonella paratyphi A), Salmonella Schottmuelleri( Salmonella paratyphi B), Salmonella Hirschfeldri( Salmonella paratyphi C) and Salmonella Enteritidis causes Human Typhoid fever, Paratyphoid fever, Food poisoning.
• Used to diagnose salmonella is clinical samples including blood, diarrhoeic stool, PM samples, and serum.
• Other methods include whole blood agglutination test, tube agglutination test (widal test).
• A positive test shows Agglutinations in dilutions at a titre of 1 in 40.
• Isolation and identification via Tetrathionate broth or Selenite broth are useful.
• Serotyping and molecular detection can be done.
• For control and prevention, live vaccines give rise to solid immunity is used in horses, cattle, sheep and goat but the animal may remain as carrier throughout its life.
• Killed vaccines are safe but can stimulate only a temporary resistance.
• For cattle attenuated, live culture of S. Dublin vaccine has been developed.
• In poultry either killed or live attenuated vaccine stimulates good immune response.
• Treatment include different chemotherapeutic agents like Chloromycetin, Terramycin, neomycin, furazolidone and sulfasuxidine.
• Majority of serotypes are potential pathogens for both man and animals.
• Route of transmission is through milk, meat and.
• Salmonella food poisoning will result in symptoms of severe gastroenteritis.

Yersinia

• Important species include Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis.
• Yersinia are non-lactose fermenters.
• All are motile except Y. pestis.
• They characteristically demonstrate bipolar staining in Giemsa staining.
• Wild and domestic animals may act as reservoir of Y. enterocolitica and Y. pseudotuberculosis.
• Pig is the natural reservoir of Y. enterocolitica serotype O3 biotype 4, which is an important pathogen in human.
• Xenopsylla cheopis (oriental rat flee) is a reservoir.
• Yersinia survive for long periods in cool wet conditions.
• Serotyping and biotyping methods are used for identifying pathogenic Yersiniae.
• Y. pseudotuberculosis has ten serotypes, I, II and III which are pathogenic.
• Y. enterocolitica has 5 biotypes and 50 serotypes.
• Serotype O:9 shares common antigens with Brucells species and it may induce false positive reactions in Brucella agglutination test.
• Cold enrichment is needed for recovery of Yersinia from feaces.
• Yersinia can be cultured in MacConkeys and Blood agar plate
• Yersinia has invasin and antiphagocytic protein for virulence.
• Additional virulence factors of Y. pestis included capsule, plasminogen activator and endotoxin.
• Pathogenic Yersiniae are facultative intracellular organisms.
• enterocolitica and Y. pseudotuberculosis is an enteric lesion characterized by growth in lymphoid tissue and it produces Mesenteric lymphnodes.
• Y. pestis causes generalized infection by having Anti phagocytic protein capsule and Endotoxin.
• Yersinia enterocolitica effects Pigs, other domestic animals, wild life and Humans to cause Subclinical enteric infection and gastro enterocolitis, it effects the Ewes resulting in Sporadic abortion.
• Yersinia pseudotuberculosis effects Farm deer, sheep, goats, cattle, buffalo, pigs and Humans to cause enteritis mesenteric lymphadenitis and enterocolitis mesenteric lymphadenitis, Effects Cattle, sheep goat to cause Sporadic abortion and Guinea pig and other lab, Animals to cause Focal Hepatic necrosis ,septicemia.

Yersinia Diseases

• Enteric yersiniosis is enteritis characterized by profuse watery diarrhea, times blood stained, mucosal ulceration and focal necrotic foci.
• Septicaemic yersiniosis creates pin point necrotic foci developed in the liver.
• Pseudotuberculosis in laboratory animals leads to diarrhea and gradual weight loss to emaciation and death with necrotic foci.
• Feline plague results from cat develop infection by ingestion of infected rodent causing bubonic, Pneumonic and septicaemic plague.
• Diagnostic methods includes history examination of clinical signs and symptoms as wel as post mortem lesions, demonstration of the organism in direct blood smear by giemsa, grams stain and FAT
• Further diagnostic methods: Isolation either via Cold enrichment, Blood agar, MLA, Endo agar or Serotyping or PCR based detection.
• Treatment diagnostic consists of History, clinical signs and.
• Symptoms Isolation, Serotyping treated with Formalin killed vaccine (Y. pseudotuberculosis) or Chloramphenicol or tetracyclines.

Other Enterobacteriaceae

• Shigella: Four species (S. boydii, S. dysenteriae, S. flexneri and S. sonnei) are etiological agents of diarrhoea and dysentery in man and subhuman primates. They are Gram -ve, non-motile, rod shaped organisms found in the intestinal flora of animals. Additionally Shigella are facultatively anaerobic and grows on blood agar & MacConkey agar at 37°C without haemolysis. It produces an enterotoxin acting on the epithelial lining of the terminal illeum and large intestine, producing bloody mucus stained stool.

• Citrobacter is a that Citrobacter freundi forms part of normal intestinal flora of domestic animals and are also found in soil and water. It is a Gram negative, facultatively anaerobic, motile, rod shaped organisms, which grows on blood agar and MacConkey agar at 37°C producing no haemolysis. In domestic animals, Citrobacter are opportunistic pathogens in a variety of extra intestinal infections.

• Edwardsiella tarda has a worldwide distribution and has been isolated from all classes of vertebrates, fish, amphibia, reptiles, birds and mammals.They are Gram negative, motile rods and facultatively anaerobic and grow on blood agar. However it grows on MacConkey agar at 37°C. It's non-haemolytic and lactose negative.E. tarda has been implicated as the cause of diarrhoea, wound infections and sepsis in animals and humans.

• Enterobacter clocae and E.agglomerans are the most commonly isolated species inhabits mainly the environment, but they inhabits the digestive tract of animals. They're Gram negative, motile rod shaped organisms, facultatively anaerobic. Additionally this species grow on blood agar and MacConkey agar at 37°C and are opportunistic pathogens and causes abortion and infection of genital tract in horses, abortion and mastitis in cattle and agalactia syndrome in pigs.

• Klebsiella also known as Friedlanders bacillus are species that occurs naturally in soil and water and normal intestinal flora of domestic animals. They are non-motile, encapsulated rods arranged singly, in pairs or short chains however the strains show a non haemolytic reaction on blood agar and appear lactose positive colonies on MacConkey agar. Klebsiella pneumoniae has O antigen (11 types) and K antigen (70 types). Klebsiella causes inflammation of the genital mucosa and abortion. Additionally, causes generalized infections of foals plus mastitis, generalized infections and enteritis of calves. In pigs and it causes atrophic rhinitis while, in poultry causes air sac and yolk sac infections also, however K. pneumoniae and K. oxytoca are the most commonly isolated species from domestic animals.

• Moragnella also known as M. morganii  is that normal intestinal flora however the species can also exists with in domesticated animals. It is gram-negative bacteria that is a motile rod organism with a facultatively anaerobic configuration. Grows on blood agar and MacConkey agar at 37° C. When the species finds an opportunity to invade its host it can cause multiple of effects one of those being an Urinary Tract infections.

• Proteus mirabilis and P.vulgaris which are normal intestinal flora that domesticated animals are host with however they can also be found located in the water of polluted areas and soil. In laboratory, the organism expresses its self as a gram negative rod shaped organism known for creating a pleomorphism pattern when observed through microscope. All strains produced both capsule and fimbriae this aids as the facilitator as these strains are also motile allowing the organism with a peritrichous flagella with characteristics of swarming. However, when they are not producing spores under specific settings, at a optimum temp 37 Celsius are know to produce acids and that is used in laboratories when testing for phenylpuruvic acid due to the colonies positive phenotype. Is mostly well know to be the culprit of endometritis, gastroenteritis and arthritis. It can also be the case of a severe diarrhoea condition to be present with piglet a urinary infections within dogs and cats as well as otits externa and ultimately the patient is put in a conditions of sepsis.

• Providencia rettgeri and P.stuartii are both found to be the norm within domesticated animals in their intestinal flora. Both will presents themselves as gram rods with motility by peritrichous flagella which will allow the a facultative anaerobic effect producing results on blood agar & MacConkey at 37° C. However both of those cultures will not produce hemolysins or be lactose negative. P.rettgeri is frequently isolated in pure culture from urinary infections.

• Serratia liquefaciens and S. marcescens are the organisms that make the intestinal flora more dynamic. However, those species are mainly located in soil and water that livestock can access as well. Both of them presents as Gram negative, motility rods when observed through a Microscope. Serratia will also demonstrates as an anaerobic organism if cultured properly. Through blood test they demonstrates a colorful colonies expressing either white, pink, or red. However, S. marcescens produces a deep red pigment plus express as lactose a positive colonies this organism also can be the reason to conjunctivitis, pneumonia, as well as Septicaemia’s can be isolated from endometriosis and are cases of abortion and mastitis in cattle.