Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 1: Aerobic Gram-positive cocci

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1 Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Author: Mnr J.J. Gouws Licensed under a Creative Commons Attribution license. TABLE OF CONTENTS Introduction... 2 Table 1.1: Differentiation of the most common genera of Gram-positive cocci... 2 The MICROCOCCACEAE: Micrococcus species and Staphylococcus species... 2 Genus: Staphylococcus... 2 Table 1.2: Tests used to differentiate between Staphylococcus and Micrococcus... 3 Table 1.3: Diseases caused by the main pathogenic staphylococci in animals... 3 Identification of coagulase-negative staphylococci... 4 Table 1.4: Identification of coagulase-positive Staphylococci... 4 Table 1.5: Reactions of coagulase-positive staphylococci on purple agar with 1% maltose... 4 The STREPTOCCACEAE: Streptococcus and Enterococcus... 4 General differentiation of the streptococci... 5 Table 1.6: Biochemical reactions and other characteristics of staphylococci isolated from animals... 6 Table 1.7: Diseases caused by streptococci and enterococci... 8 Table 1.8: Biochemical reactions of important streptococci and enterococci from animals... 9 Table 1.9: Differentiation of streptococci, enterococci and group D streptococci Table 1.10: Differentiation of group C streptococci REFERENCES APPENDIX P a g e

2 Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria INTRODUCTION Gram-positive cocci represent a diverse group of bacteria belonging to several genera. They are grouped together because of their spherical to slightly oval shape and Gram-positive staining reactions. They do not form endospores. Motility is uncommon. Arrangement of the cells and occurrence of the enzyme catalase are important features used for separating genera. Another feature used for differentiating between genera is the ability to grow under anaerobic conditions. Grampositive cocci are divided into two families, namely the Micrococcaceae and the Streptococcaceae (Table 1.1). Gram-positive cocci are the bacteria, with the exception of the members of the family Enterobacteriaceae that are most frequently isolated from clinical specimens. Because these bacteria are widespread in nature and can be recovered as commensal inhabitants of the skin, mucous membranes and other body sites in animals, the interpretation of their recovery from patient specimens is difficult unless clinical manifestations of an infectious disease process are present. They cause a variety of diseases and therefore their isolation should be correlated with the clinical condition of the patient (Refer to Tables 1.2 and 1.7). Table 1.1: Differentiation of the most common genera of Gram-positive cocci Catalase reaction Strictly aerobic Facultative anaerobes Catalase positive Deinobacter Staphylococcus Deinococcus Stomatococcus (Micrococcaceae) Marinococcus Micrococcus Planococcus Catalase negative Aerococcus (Streptococcaceae) Enterococcus Gemella Lactococcus Leuconostoc Pediococcus Streptococcus THE MICROCOCCACEAE: MICROCOCCUS SPECIES AND STAPHYLOCOCCUS SPECIES Clinically significant species included in the family Micrococcaceae are the genera Staphylococcus, Micrococcus and Stomatococcus. Colonies of this group appear convex, entire and buttery with a white or yellow colour. They are all catalase positive (Stomatococcus is weakly positive). Grampositive cocci in clusters are seen in Gram stained smears. Micrococcus species are usually in tetrads (packets of 4). Table 1.2 includes parameters that differentiate between the two most commonly isolated genera Staphylococcus and Micrococcus. Genus: Staphylococcus For diagnostic purposes, the staphylococci are divided into two groups namely coagulase-positive and coagulase-negative species. The coagulase-positive staphylococci are generally considered to be 2 P a g e

3 Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria more pathogenic. The currently recognized species of coagulase-positive staphylococci are S. aureus, S. intermedius, S. pseudintermedius, S. aureus subsp. anaerobius, S.delphini, S. lutrae, S. schleiferi subsp. coagulans and S. hyicus. Coagulase-negative staphylococcal species (of which at least 20 have been described) may be identified by several biochemical characteristics. Its specific identification is usually only sought when it is isolated in almost pure culture, or are recovered from sites that are normally sterile such as joints or cerebrospinal fluid. Table 1.2: Tests used to differentiate between Staphylococcus and Micrococcus Test Staphylococcus Micrococcus Fermentation of glucose (anaerobic) + - Sensitivity to bacitracin - + Sensitivity to furazolidone (zone size > 10 mm) + - Sensitivity to lysostaphin + - Production of acid from glycerol in the presence of erythromycin (0,4 + - μg/ml) using peptone broth base Modified oxidase test - + Table 1.3: Diseases caused by the main pathogenic staphylococci in animals Species Host(s) Diseases Abscesses General Systemic infection Infections following surgery Cattle Mastitis Mastitis Sheep Tick pyaemia (lambs 2-5 weeks) Dermatitis Goats Mastitis Dermatitis S. aureus Botryomycosis of mammary glands Pigs Endometritis (necrotizing) Udder impetigo Horses Mastitis (acute) Botryomycosis (after castration) Exudative dermatitis Rabbits Abscesses Conjunctivitis Poultry Bumble foot Arthritis Dogs & cats Same as S.pseudintermedius S. aureus ss. anaerobius Sheep Caseous lymphadenitis S. pseudintermedius Dogs & cats Pyoderma Pustular dermatitis Pyometra Otitis externa Other infections e.g. osteomyelitis S. hyicus Pigs Greasy pig disease (exudative dermatitis) Septic polyarthritis S. schleiferi subsp. coagulans Dogs Otitis externa Identification of the coagulase-positive staphylococci is described in Tables 1.4, 1.5 and 1.6. Biochemical identification of all the staphylococci is described in Table 1.6. APISTAPH is a commercially available identification method for the staphylococci. 3 P a g e

4 Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Identification of coagulase-negative staphylococci Coagulase-negative staphylococci are not routinely identified to species level in a diagnostic laboratory. Certain tests must, however, be done to distinguish S. epidermidis which is coagulase- negative as well as S. pseudintermedius which may test coagulase-negative from the other coagulase-negative staphylococci which are regarded as commensal bacteria and are generally nonpathogenic. Table 1.4: Identification of coagulase-positive Staphylococci S. aureus S. pseudintermedius S. hyicus Polymyxin B sensitivity (300unit disk) Haemolysis on sheep blood agar Mannitol fermentation + + (delayed) - Maltose fermentation (Purple agar + 1% maltose) + ± - ± poor utilization Table 1.5: Reactions of coagulase-positive staphylococci on purple agar with 1% maltose Species Maltose fermentation Reactions on purple agar with 1% maltose S. aureus +++ Diffuse yellow colour around colonies. Rapid reaction within 24h incubation. S.pseudintermedius ± Little change in the medium. Slight yellowish zone under colonies and occasionally isolated colonies may have a yellowish tinge. S. hyicus - Diffuse deep purple (alkaline) zone around the colonies. THE STREPTOCCACEAE: STREPTOCOCCUS AND ENTEROCOCCUS The family Streptococcaceae includes the genera Streptococcus and Enterococcus. Inability to produce catalase differentiates them from the Micrococcaceae. Streptococci are Gram-positive cocci that occur in pairs or chains of varying lengths. Chain formation is usually more pronounced in broth medium. They are facultative anaerobes, catalase-negative, oxidase-negative and non-motile with the exception of some of the enterococci. The streptococci are fastidious and require the addition of blood or serum to media for growth. The streptococci are characterized by the growth of relatively small colonies on primary isolation media and are divided into -haemolytic, -haemolytic and nonhaemolytic ( -haemolytic) subgroups. The 4 P a g e -haemolytic streptococci can be differentiated biochemically and serologically. Streptoccus pneumoniae and the viridans group are -haemolytic and can be distinguished biochemically. The enterococci tolerate the bile salts in MacConkey agar and appear as small pin-point dark pink colonies. Enterococci grow in 6,5% NaCl. For identification of the Streptococcaceae refer to Tables 1.8, 1.9, and 1.10

5 Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria General differentiation of the streptococci Haemolysis: The main types of haemolysis are: Beta ( )-haemolysis: a clear zone of haemolysis around the colony. Alpha ( )-haemolysis: a zone of greening or partial haemolysis. Gamma ( )-haemolysis: no haemolysis. Generally the -haemolytic streptococci tend to be the most pathogenic for animals. Lancefield grouping: A serological grouping method determined by the C-substance that is a group-specific cell wall polysaccharide antigen. 5 P a g e

6 Test S. aureus S. aureus subsp. anaerobius S. intermedius S. pseudintermedius S. hyicus S. delphini S.s. coagulanss S.s. schleiferi S. lugdunensis S. epidermidis S. caprae S. gallinarum S. arlettae S. lentus S. equorum S. simulans S saprophyticus S. capitis S. sacchrolytiocus S. warneri/pasteuri S. haemolyticus S. auricularis S. cohnii cohnii S. cohnii urealyticum S. xylosus S. carnosus S. felis S. piscifermentans Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Table 1.6: Biochemical reactions and other characteristics of staphylococci isolated from animals Colony >5mm d - d - d d + - s d Pigme nt produc tion Coagul ase d - - d + d - - d - - d d - d d d + d d Clumpi ng factor DNase Test Beta- Haem + + d d d d d - - d d d d - d d - - d - 6 P a g e

7 Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria olysis Phosp hatase Ureas e d d d d d d d - - d d d + + d d? (Manni tol salt agar) Mannit ol fermen tation + d w - d d d d + - d d - d d (purple agar + 1% maltos e) Trehal ose + + w d - + d + + d d w + d - d + d d d d d - d Xylose Sucros e Aescul in Hydrol d P a g e

8 Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria ysis Novobi ocin Polym yxin B S S S S S S S S R R R R S R R S S R R R S S S S S + = positive reaction; - = negative reaction; ± = variable reaction (90% or more weakly positive); d = 11-89% positive; ( ) = delayed reaction; S = sensitive; R = resistant; = information lacking; * = canine strains usually not pigmented Table 1.7: Diseases caused by streptococci and enterococci Lancefield group Species Host(s) Disease Humans Scarlet fever, septic sore throat, and rheumatic fever A S. pyogenes Cattle Mastitis (rare) Foals Lymphangitis Cattle and goats, sheep Chronic mastitis B S. agalactiae Humans and dogs Neonatal septicaemia Cats Kidney and uterine infections S. dysgalactiae Cattle Acute mastitis Lambs Polyarthritis S. equisimilis Horses Abscesses, endometritis and mastitis Pigs, cattle, birds, dogs Various suppurative conditions C S. equi subs. equi Horses Strangles, purpura haemorrhagica Horses Mastitis, abortion, secondary pneumonia, navel infection Cattle Metritis and mastitis S. equi subs Pigs Septicaemia and arthritis in 1-3 week old piglets zooepidemicus Poultry Septicaemia and vegetative endocarditis Lambs Pericarditis and pneumonia D Enterococcus faecalis Septicaemia in chickens, bovine mastitis, endocarditis in cattle and lambs, urinary infections in dogs E. faecium Many species (opportunistic) E. durans S equinis S. bovis Many species Opportunistic infections E S. porcinus Pigs Jowl abscesses and lymphadenitis G S. canis Carnivores Neonatal septicaemia, genital, skin and wound infections Cattle Occasional mastitis D S. suis Pigs Meningitis, arthritis, pneumonia and septicaemia Humans Meningitis and septicaemia 8 P a g e

9 Acid from S. pyogeens S. agalactiae S dysgalactiae S. dysgalactiae subsp equisimilis S. equi subsp equi S. equi subs. zooepidemicus S. equi subs. ruminantorum Enterococcus faecalis S. ovis S. bovis S. equinis S. porcinus E. canis Enterococcus avium S. suis type 2 S. suis type 1 S. uberis S. pneumoniae S. delph S. iniae Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Ungroupable S. uberis Cattle Mastitis Humans and primates Pneumonia, septicaemia and meningitis S. pneumoniae Guinea-pigs and rat colonies Pneumonia (outbreaks can occur) Table 1.8: Biochemical reactions of important streptococci and enterococci from animals Lancefield Group A B C C C C C D D D E G Q D D Arabinose - Inulin (+) (+) Lactose v (+) (+) v Maltose + + (+)- + + Mannitol v v _ Raffinose (+) Ribose Salicin + (+) - (+) (+) v + Sorbitol v Sucrose Trehalose v v + (+) CAMP Aesculin hydrolysis v (+) - Growth in 6,5% NaCl (+) - - (+) - Haemolysis ( ) + = positive reaction; - = negative reaction; (+) = majority of strains positive; v = variable reactions; o = information not available 9 P a g e

10 Applied Veterinary Bacteriology and Mycology: Introduction Identification of aerobic and facultative anaerobic bacteria Table 1.9: Differentiation of streptococci, enterococci and group D streptococci Streptococci Enterococci Group D streptococci Growth on Mac Conkey Agar Growth at 45 C Growth in 6,5% NaCl Tolerance for 40% bile Table 1.10: Differentiation of group C streptococci Trehalose Sorbitol Lactose Maltose Salicin Ribose S. equi subsp. equi S. equi subsp. equi (atypical isolates) (+) - v S. equi subsp zooepidemicus (-) + + S. dysgalactiae subsp equisimilis + - v + (+) + S. dysgalactiae REFERENCES 1. Carter, G.R., Cole, J.R. jr. Diagnostic Procedures in Veterinary Bacteriology and Mycology. Fifth Edition. Academic Press, ISBN Quinn, P.J., Carter, M.E., Markey, B. and Carter, G.R. Clinical Veterinary Microbiology. Wolfe Publishers, ISBN Quin, P.J., Markey, B.K., Leonard, F.C., FitzPatrick, E.S., Fanning, S., Hartigan, P.J. Veterinary Microbiology and Microbial Disease. Second Edition, Wiley-Blackwell, ISBN APPENDIX 1 Media and Techniques ALKALINE PHOSPHATASE TEST Nutrient agar + 0,01% phenolphthalein diphosphate is incubated for hours at 37 C. The plate with colonies is held inverted over an open bottle of ammonia. Interpretation Positive - Colonies bright pink. Negative - Unchanged colonies. 10 P a g e

11 Applied Veterinary Bacteriology and Mycology: Introduction Identification of aerobic and facultative anaerobic bacteria Sodium hippurate test Sodium hippurate 10g Heart infusion broth 1 litre Sterilise at 115 C for 20 minutes. Reagent*: Ferric chloride 12,0g Concentrated HCl 2,5ml Distilled water 100,0ml The acid is diluted with 75ml of the distilled water and the ferric chloride is dissolved in this fluid by warming. The volume is adjusted to 100ml with distilled water. Inoculate 1 tube and incubate with 1 uninoculated control tube for 24 hours at 37 C. Centrifuge the tubes. Add 0,2ml reagent* to 0,8ml of the supernatant. Interpretation Positive reaction = Permanent precipitate. Negative reaction = No precipitate Sodium Chloride (6.5%) Tolerance Heart infusion broth 25 g NaCl 60g 1,6g bromocresol purple in 1 ml 100 ml 95% ethanol Dextrose Distilled water 1 g 1000 ml Dispense in screw-capped bottles or tubes and sterilize by autoclaving at 121 C for 15 minutes. Interpretation Positive: The indicator changes form purple to yellow and/or growth is observed. Negative: No change in indicator nor is growth observed. 11 P a g e