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Genus Staphylococcus Most common pathogenic species in the genus: Staphylococcus aureus – pyogenic wound infections, mastitis, metritis, arthritis, dermatitis, septicemia, omphalitis, urinary tract infections and other infections in different animal species. S.pseudintermedius – pyoderma, dermatitis...

Genus Staphylococcus Most common pathogenic species in the genus: Staphylococcus aureus – pyogenic wound infections, mastitis, metritis, arthritis, dermatitis, septicemia, omphalitis, urinary tract infections and other infections in different animal species. S.pseudintermedius – pyoderma, dermatitis, pyometra, endometritis, cystitis, otitis externa, conjuctivitis, airway infections, osteomyelitis, arthritis, pyogenic wound infections mostly in dogs and cats, less often in cows and horses. S.schleiferi s. coagulans – otitis externa in dogs. S.felis – otitis externa, cystitis, abscesses, conjunctivitis and wound infections in cats. S.epidermidis – pyogenic wound infections, dermatitis, abscesses in different animal species, mastitis in cows. S.hyicus – exudative epidermitis in piglets, polyarthritis in birds. Rare skin infections in horses and cows. S. haemolyticus – mastitis in cows. Samples: samples sent to the laboratory depend on the type of staphylococcal infection and the clinical signs. Since the inflammatory processes are mainly observed on the skin, usually exudate, secretions, discharge, pus etc. is taken from wounds, natural openings and other inflamed sites. In the case of clinical and subclinical mastitis, milk samples are sent to the laboratory. Examination of samples: the usual media used for inoculation are Nutrient Agar (NA), Blood agar (BA) – universal media, Baird Parker agar (BPA), Mannitol salt agar (MSA) – selective-differential media, and Columbia Naladixic Acid agar (CNA) – selective medium. To identify the genus and/or species a Gram stain is done after a pure culture is obtained. Then catalase, coagulase test and other biochemical tests are performed (DNase, mannitol and maltose fermentation, VP tests). In most cases, especially to identify coagulase negative species, it is necessary to perform complex biochemical tests. For this commercial identification systems (API Staph, Crystal Gram-Positive ID Kit, VITEK® 2 GP), MALDI-TOF mass spectrometry or molecular methods (PCR) are used. Microscopy: staphylococci are gram-positive, non-motile, non-spore forming cocci with a diameter of 0.5-1.5 μm. In stained smears staphylococci tend to be arranged in pairs, tetrads, or more often, grouped in irregular grape-like clusters. Note that a fresh culture is gram-positive, but older cultures may stain unevenly! Conditions for cultivation: staphylococci are classified as facultative anaerobes (except for S. aureus s. anaerobius – microaerophilic, and S. saccharolyticus – anaerobic) and are cultivated in aerobic conditions. Temperature range for growth is 6-48 ºC, but optimal growth temperature is 30-37 ºC. Staphylococci can live in and endure high NaCl concentrations (5-10%). Colony morphology: on NA staphylococci form pigmented, round colonies, 2-3 mm in diameter with smooth, shiny and curved surface. The consistency is usually butyrous. The characteristic colonies are usually seen in 24h. On BA coagulase positive staphylococci produce β-haemolysis. S. aureus and S. pseudintermedius can produce double haemolysis. Identification: during identification process staphylococci need to be differentiated from other G+ cocci (streptococci, enterococci, micrococci). To do this catalase test is performed, which is positive for staphylococci (enterococci, streptococci are catalase negative). Unlike for micrococci the modified oxidase test is negative for staphylococci and they are susceptible to furazolidone (antibiotic), but resistant to bacitracin (antibiotic). A great importance is given to colony morphology on selective-differential media – BPA and MSA. To identify coagulase positive staphylococci it is essential to use these tests – mannitol fermentation (positive for S.aureus (>90%), negative for S. pseudintermedius (11-89%)), DNase test (positive for S. aureus), VP test (positive for S. aureus (>90%), negative for S. pseudintermedius (>90%). For precise species identification (especially for coagulase negative staphylococci) it is recommended to use MALDI TOF MS or PCR. Genus Streptococcus Most common pathogenic species in the genus: Streptococcus pyogenes – mastitis in cows, lymphangitis in foals. S.agalactiae – chronic mastitis in ruminants, neonatal septicemia in dogs, kidney and uterine infections in cats. S.dysgalatiae – acute mastitis in cows, polyarthritis in lambs. S.equisimilis – abscesses, endometritis, abortion and mastitis in horses, various suppurative conditions in pigs, cattle, dogs and birds. S.equi s. equi – strangles, genital and suppurative conditions, mastitis and purpura haemorrhagica in horses. S.equi s.zooepidemicus – arthritis, mastitis, abortion, secondary pneumonia and navel infections in horses, mastitis and metritis in cattle, septicemia and arthritis in 1-3 week old piglets, septicemia and vegetative endocarditis in poultry, pericarditis and pneumonia in lambs. S. canis – neonatal septicemia, genital, skin and wound infections, otitis, mastitis, prostatis, abortion, conjunctivitis, lymphadenitis in dogs and cats. Occasional mastitis in cattle. S. suis – meningitis, arthritis, pneumonia, endocarditis and septicemia in pigs. S. uberis – mastitis in cattle. S. pneumoniae – pneumonia in horses, guinea pigs and rats. Pneumonia, septicemia and meningitis in primates. Samples: samples sent to the laboratory depend on the type of streptococcal infection and the clinical signs. In the case of clinical and subclinical mastitis, milk samples are sent to the laboratory. Usually exudate, secretions, discharge, pus etc. is taken from wounds, natural openings and other inflamed sites. Samples should be examined right away, because streptococci are sensitive to desiccation. If immediate examination is not possible, the pus, or other exudates obtained with a cotton swab, should be placed in transport medium. Examination of samples: the usual media used for inoculation are NA, BA or Columbia Blood agar (CBA) – universal media, Chromogenic Streptococcus agar (CSA), Edward’s aesculin agar (EAA) – selective-differential media, and CNA or Streptococcus selective agar (SSA) – selective media. To identify the genus a Gram stain and catalase test is done after a pure culture is obtained. A great importance is given to colony morphology on selective-differential media. To identify the species, it is possible to do serological testing (Latex agglutination test) and to perform complex biochemical tests. For this commercial identification systems (API Strep, Crystal Gram-Positive ID Kit, VITEK® 2 GP), MALDI-TOF mass spectrometry or molecular methods (PCR) are used. Microscopy: streptococci are gram-positive, non-motile, non-spore forming round to oval cocci with a diameter of 0.5-1 μm. In stained smears streptococci tend to be arranged in shorter or longer chains (S. pneumoniae – has a pronounced arrangement only in pairs; S. agalactiae – tends to form the longest chains – up to 40 cocci). Note that a fresh culture is gram-positive, but older cultures may stain unevenly! Conditions for cultivation and colony morphology: inoculated media are incubated in aerobic conditions, in 37 ºC for 24-48h. Streptococci show better growth in reduced O2 conditions (aerotolerant anaerobes) and in the presence of 5% CO2. After 24h they form round, non-pigmented, smooth, convex, shiny colonies with the diameter of 0.5-1 mm. Colony consistency is usually butyrous, but can be mucoid in some species. Streptococci show weak growth on universal media (NA), but growth is optimal on BA and other enriched media. Depending on the group and the species streptococci may form α or β haemolysis on BA. Identification: during identification process streptococci need to be differentiated from other G+ cocci (staphylococci, enterococci, micrococci). To do this catalase test is performed, which is negative for streptococci. Carbohydrate fermentation in streptococci is variable and depends on the species. For this reason biochemical tests are not routinely used for identification of streptococci. S. uberis is capable of fermenting aesculin. CAMP factor production is seen with group B streptococci – S. agalactiae. One of the streptococcal classification systems used is the Lancefield grouping system, therefore to identify some streptococcal species, latex agglutination test can be used. For precise species identification it is recommended to use MALDI TOF MS or PCR. Genus Enterococcus Enterococci (enteric cocci) used to be classified as group D streptococci, but since 1984 they have been reclassified as a seperate genus Enterococcus. Most common pathogenic species in the genus: Enterococcus faecalis – is a part of normal microbiota in mammals and causes opportunistic infections such as colitis, enteritis and disbacteriosis in different animals. Septicaemia in chickens, bovine mastitis, endocarditis in cattle and lambs, urinary tract infections in dogs. Causes almost 90% of entercococcal diseases. E. faecium – is a part of normal microbiota in mammals and causes opportunistic infections such as septicemia in poultry, urinary tract infections, hepatic abscesses, meningitis and mastitis in cattle. E. durans, E. hirae, E.villorum – is a part of normal microbiota in mammals and is associated with diarrhea in puppies, kittens, and adult dogs and cats, foals, calves and piglets. Samples: wide range of samples, starting from discharge and exudate from natural openings and other inflamed sites, tissue samples and faeces to water samples. Examination of samples: the usual media used for inoculation are NA, BA or CBA – universal media, Enterococcus chromogenic agar (ECA), Aesculin bile azide agar (ABAA),Enterococcus selective agar (ESA or Slanetz Bartley agar (SBA)) – selective-differential media, or CNA – selective media. To identify the genus a Gram stain and catalase test is done after a pure culture is obtained. A great importance is given to colony morphology on selective-differential media. To confirm the genus, Latex agglutination test is performed (enterococci belong to group D in Lancefield classification). To identify a species, complex biochemical tests can be performed. For this commercial identification systems (API Strep, Crystal Gram-Positive ID Kit, VITEK® 2 GP), MALDI-TOF mass spectrometry or molecular methods (PCR) are used Microscopy: enterococci are gram-positive, non-spore forming cocci with a diameter of 0.6-2.0 μm. Most enterococci are non-motile, but some are motile (for example, E. gallinarum). In stained smears these cocci tend to be arranged singly, in pairs or in short chains. Enterococci from liquid media are more often arranged in short chains and visually it is hard to distinguish enterococci from streptococci. Slight polymorphism is characteristic of enterococci, meaning they can differ from one to another in size and form. Conditions for cultivation and colony morphology:enterococci are classified as facultative anaerobic, but show slightly better growth in reduced O2 conditions. Temperature range for growth is 10-40 ºC, but optimal growth temperature is 35-37 ºC. On universal media enterococci tend to grow well and produce round, greyish, cloudy and shiny colonies with a diameter of 1-2 mm. Even though a lot of enterococcal species do not produce haemolysis, some enterococci may form α or β haemolysis on BA. Identification: primary identification is based on catalase test – enterococci are negative, but occasionaly some strains are weakly positive. Enterococci are able to grow in 6.5% NaCl and in 40% bile concentration. To confirm the genus, Latex agglutination test is performed (enterococci belong to group D in Lancefield classification). A great importance is given to colony morphology on selective-differential media – ECA, ESA (SBA) and ABAA. For precise species identification it is recommended to use commercial identification systems, MALDI TOF MS or PCR. Enterococcus selective agar or Slanetz-Bartley agar showing red-brown to pink colonies of enterococci.