Diagnosis of Infections Caused by Cocci PDF

PAGE 1 UNIVERSITÄTSMEDIZIN https://www.umfst.ro NEUMARKT A. M. https://edu.umch.de CAMPUS HAMBURG 2024 December Diagnosis of infections...

PAGE 1 UNIVERSITÄTSMEDIZIN https://www.umfst.ro NEUMARKT A. M. https://edu.umch.de CAMPUS HAMBURG 2024 December Diagnosis of infections caused by cocci: Staphylococcus spp., Streptococcus spp., Enterococcus spp., Neisseria spp., Haemophilus spp. PAGE 2  Staphylococcus spp. PAGE 3 Gram-positive cocci Streptococcus spp. Enterococcus spp. Genus Staphylococcus PAGE 4 General characteristics - non-fastidious - catalase positive - oxidase negative - aerobic, facultative anaerobic Gram-positive cocci Coagulase-positive staphylococci: S. aureus The classification of staphylococci is based on Coagulase-negative staphylococci (CNS): S.lugdunensis, S. free coagulase production schleiferi, S. hyicus, S. epidermidis, S. haemolyticus, S. saprophyticus, etc. Genus Staphylococcus PAGE 5 Specimens - Detection of colonization with S. aureus – screening for carriers medical personnel in units with a high risk of nosocomial infections patients undergoing surgical procedures (mainly orthopedics, thoracic surgery) epidemiological reasons - Appropriate specimens for screening: nasal swabs pharyngeal swabs skin swab rectal swab https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines- clinical-specimens.html https://blog.puritanmedproducts.com/nasopharyngeal- swab-vs-nasal-swab Genus Staphylococcus PAGE 6 Specimens Detection of staphylococci for diagnosis of infections - specimens are chosen according to the type of infection: - skin and wound infections, otitis, sinusitis, osteomyelitis, mastitis: pus (for S. aureus) - lower respiratory tract infections: sputum, tracheal, bronchial secretions (for S. aureus) - bloodstream infections: blood (for S. aureus, CNS) - intravascular catheter-associated infections: catheter tip (for S. aureus, CNS) - urinary tract infection: urine (for S. saprophyticus, S. aureus) - meningitis: cerebrospinal fluid (for CNS, S. aureus) Genus Staphylococcus PAGE 7 Direct examination of the specimen - macroscopical examination of the pathological product: visual examination of pus, usually creamy, yellowish - microscopical examination of the pathological product: direct smears from the specimen – Gram-positive cocci in clusters +/-leukocytes Genus Staphylococcus PAGE 8 Cultivation - non-fastidious organisms, usually grow easily on basic culture media, such as nutrient agar - blood agar - Chapman (Manitol Salt agar) medium - enrichment media: salted broth (7.5% NaCl) - the culture media are incubated at 37⁰C for 18-24 hours Genus Staphylococcus PAGE 9 Cultivation Blood agar Manitol salt agar Genus Staphylococcus PAGE 10 Identification - morphology and staining properties are studied on smears from pure cultures: - Gram-positive cocci in clusters - Colony morphology: - S colonies - a non-diffusible pigment may be present according to staphylococcal species: golden colonies in case of S. aureus Genus Staphylococcus PAGE 11 Identification Biochemical patterns: - catalase-positive - coagulase-positive: S. aureus; all other species are coagulase-negative (CNS) https://microbiologyinfo.com/coagulase-test-principal-procedure-types-interpretation-and-examples/ Genus Staphylococcus PAGE 12 Identification Biochemical patterns: - clumping factor positive: S. aureus and a few other CNS - mannitol fermentation S. aureus and some CNS species - beta-hemolysis: S. aureus, S. haemolyticus Other tests: - novobiocin resistance: Staphylococcus saprophyticus - presence of protein A in S. aureus – detection by latex agglutination https://www.thermofisher.com/order/catalog/product/R30859902 Genus Staphylococcus PAGE 13 Identification Biochemical patterns: - DN-ase production Genus Staphylococcus PAGE 14 Typing techniques Typing techniques are useful for comparing staphylococcal strains and establish their source during an epidemic. - phage typing: determines the susceptibility of S. aureus to different phages – classical typing method - molecular typing methods: pulsed-field gel electrophoresis (PFGE): macro-restriction fragments of the whole bacterial genome create the genetic fingerprint of the S. aureus; PFGE has high discriminatory power multi-locus sequence typing (MLST) – sequence-based typing method, which determines the allelic profile of 7 housekeeping genes; international databases are built based on MLST data Genus Staphylococcus PAGE 15 Antibiotic susceptibility testing (AST) - important resistance phenotypes to be tested: – methicillin-resistance (MR phenotype): resistance against all β-lactam antibiotics, including β-lactam/β-lactamase inhibitor combinations – inducible clindamycin resistance – D test (antagonism between erythromycin and clindamycin) Genus Streptococcus PAGE 16 General characteristics - aerobic, facultative anaerobic Gram-positive cocci; some species are obligate anaerobic - medically important streptococci prefer a CO2 enriched atmosphere - some of the species are fastidious catalase-negative oxidase negative Genus Streptococcus PAGE 17 Medically important species - S. pyogenes (Lancefield group A streptococcus – GAS) - S. agalactiae (Lancefield group B streptococcus – GBS) - Viridans group streptococci - S. pneumoniae (pneumococcus) - Lancefield groups are defined according to the C polysaccharide found in the cell wall of streptococci. https://commons.wikimedia.org/wiki/File:S trep_Classification.svg Genus Streptococcus PAGE 18 Specimens - S. pyogenes: - pharyngitis - throat swab - skin, wound infections: secretions - post-streptococcal diseases: blood for serology https://en.wikipedia.org/wiki/Streptococcal_pharyngitis https://en.wikipedia.org/wiki/Streptoco https://en.wikipedia.org/wiki/Erysipelas#/media/File:Facial_erysi ccal_pharyngitis#/media/File:Strep_thro pelas.jpg at2010.JPG Genus Streptococcus PAGE 19 Specimens - S. agalactiae: - screening for vaginal colonization in pregnant women: vaginal secretion (lower 1/3 of the vagina) and rectal swab - neonatal sepsis: blood - neonatal meningitis: cerebrospinal fluid https://en.wikipedia.org/wiki/Streptococcus_agalactiae#/ https://pixabay.com/ro/illustrations/fem media/File:Streptococcus_agalactiae_on_blood_agar.JPG eie-insarcinata-dr%c4%83gu%c5%a3- sarcina-5200901/ Genus Streptococcus PAGE 20 Specimens - Viridans group streptococci: present in the normal flora of the throat - endocarditis: blood - Streptococcus pneumoniae: - pneumonia: sputum, tracheal, bronchial lavage - meningitis: CSF - bloodstream infections: blood https://commons.wikimedia.org/wiki/File:Endocarditis_mitral._IMG_2895.jpg https://commons.wikimedia.org/wiki/File:X- ray_of_lobar_pneumonia.jpg Genus Streptococcus PAGE 21 Direct examination of the specimen - macroscopical examination of the pathological product: pneumococcal pneumonia - rusty sputum - microscopical examination of the pathological product: Gram-positive cocci in chains Genus Streptococcus PAGE 22 Direct examination of the specimen - microscopical examination of the pathological product: - Streptococcus pneumoniae: lance-shaped cocci in pairs, short chains Genus Streptococcus PAGE 23 Direct examination of the specimen - Antigen detection – rapid diagnostic methods - detection Streptococcus pyogenes antigens directly from throat swabs - used in combination with throat culture method, as false-negative results may occur - detection of Streptococcus pneumoniae antigens from urine in case of invasive pneumococcal disease or sputum in case of pneumonia http://www.bacteriainphotos.com/Latex_agglut ination_of_streptococci.html Genus Streptococcus PAGE 24 Cultivation - incubation: 35-37°C, 18-20 hours, 5% CO2 Genus Streptococcus PAGE 25 Identification - morphology (see direct examination - microscopy) - colony morphology on blood agar plate: - small S colonies with beta-hemolysis = beta-hemolytic streptococci https://link.springer.com/referenceworkentry/ 10.1007/0-387-30744-3_3 Genus Streptococcus PAGE 26 Identification - susceptibility to BACITRACIN - is diagnostic for Streptococcus pyogenes Genus Streptococcus PAGE 27 Identification - positive CAMP test is diagnostic for Streptococcus agalactiae (a streak of S. aureus is inoculated perpendicular on the tested streptococcus streak on a blood agar plate - an arrow shaped enlargement of hemolysis appears where the two streaks meet) Genus Streptococcus PAGE 28 Identification - Streptococcus pneumoniae – alfa-hemolytic streptococci Genus Streptococcus PAGE 29 Identification - Susceptibility to OPTOCHIN - is diagnostic for Streptococcus pneumoniae Bile Optochin Esculine Viridans streptococci Resistant Resistant - Pneumococci Susceptible Susceptible - Genus Streptococcus PAGE 30 Identification - Susceptibility to OPTOCHIN - is diagnostic for Streptococcus pneumoniae Genus Streptococcus PAGE 31 Antibiotic susceptibility testing (AST) - β hemolytic streptococci are susceptible to penicillin; testing is not necessary for routine laboratories - in case of penicillin allergy, alternative antibiotics used to treat streptococcal infections (such as macrolides) are tested - AST is performed for other streptococci - medically important resistance phenotypes: Streptococcus pneumoniae with decreased susceptibility or resistant to penicillin (SPRP) Genus Streptococcus PAGE 32 Serology - serological tests are necessary to document recent streptococcal infections in case of suspected post- streptococcal complications - complications have immune-mediated pathogenesis, and they appear when the bacterium is already eliminated from the human body (approx. 3-4 weeks after a streptococcal infection) - Streptolysin O = SLO = Ag - induces the formation of anti-SLO antibodies = ASLO - ASLO titer peaks at about 4 to 5 weeks from the infection - useful marker of a recent streptococcal infection - an elevated ASLO titer may support but not definitely diagnose a post-streptococcal complication - anti-hyaluronidase, anti-deoxyribonuclease B, anti-streptokinase: more specific antibody tests – their clinical significance is the same as for ASLO Genus Enterococcus PAGE 33 General characteristics - non-fastidious - catalase negative - oxidase negative - aerobic, facultative anaerobic Gram-positive cocci Genus Enterococcus PAGE 34 Medically important species - Enterococcus faecalis, Enterococcus faecium - Former known as group D streptococci - enterococci are present in the digestive tract - they have low-grade pathogenicity - increasing role as nosocomial pathogens - enterococcal infections are difficult to treat! (natural resistance against several antibiotic classes) - frequent contaminants of poor quality, superficial specimens! The main sites of colonization in hospitalized patients are soft tissue wounds, ulcers and https://ridacom.com/en/products/view/1991 the gastrointestinal tract (GIT) Genus Enterococcus PAGE 35 Specimens - endocarditis, bloodstream infections: blood - urinary tract infections: urine - intra-abdominal infections: peritoneal fluid, bile - nosocomial infections – specimens according to location https://commons.wikimedia.org/wiki/File:Gallblad https://commons.wikimedia.org/wiki/File:Endocarditis_mitral._IMG_2895.jpg derAnatomy-en.svg Genus Enterococcus PAGE 36 Direct examination of the specimen - microscopical examination of the pathological product: - Gram-positive cocci in chains; cocci are usually large and oval +/- leukocytes Genus Enterococcus PAGE 37 Identification - morphology (see: Direct examination - microscopy) - colony morphology: - small, grayish S colonies, with or without α-hemolysis Genus Enterococcus PAGE 38 Identification - biochemical pattern: - catalase negative - growth on ABE culture medium (agar bile-esculin, contains 40% bile) as enterococci are resistant to bile - hydrolyze esculin in the ABE – black precipitate is formed Genus Enterococcus PAGE 39 Antibiotic susceptibility testing (AST) - natural resistance against many antibiotics - medically important resistance phenotypes: - vancomycin resistance (VRE – vancomycin- resistant enterococci) - high-level aminoglycoside resistance (HLAR): all enterococci have a low-level resistance against aminoglycosides, but aminoglycosides may be used in association with other antibiotics (lactams or vancomycin) unless high-level resistance is detected http://cdstest.net/manual/tables/plates/  saprophytic PAGE 40 non-fastidious ▪ Neisseria spp. pathogenic Neisseria meningitidis Gram-negative cocci fastidious Neisseria gonorrhoeae Haemophilus spp.  PAGE 41 Neisseria meningitidis ▪ Meningitis ▪ Disseminated infections ▪ Pneumonia Neisseria gonorrhoeae ▪ Gonorrhea ▪ Disseminated infections (spread to blood to skin or joints) ▪ Opthalmia neonatorum ▪ Asymptomatic infections! Genus Neisseria PAGE 42 Neisseria gonorrhoeae Specimens ▪ urethral, endocervical or prostatic secretions (genito- urinary infections) ▪ rectal swab – proctitis (anal infections) ▪ throat swab - pharyngitis ▪ conjunctival secretion - conjunctivitis ▪ in disseminated gonococcal disease: specimens according to localization Neisseria gonorrhoeae, Waterscan Genus Neisseria PAGE 43 Neisseria meningitidis Specimens ▪ nasopharyngeal swab (carriers) ▪ cerebrospinal fluid - meningitis ▪ blood - meningitis Neisseria meningitidis, Electron Microscopy, Waterscan Pathogenic Neisseria spp. are susceptible to drying, temperature changes → inoculate immediately or use transport media with charcoal. Genus Neisseria PAGE 44 Direct examination of the specimen - microscopy: Gram-negative cocci in diplo (kidney-shaped cocci) Neisseria gonorrhoeae, Gram stain, 100x Genus Neisseria PAGE 45 Direct examination of the specimen - microscopy: Gram-negative cocci in diplo (kidney-shaped cocci) Neisseria gonorrhoeae, Gram stain, 100x Genus Neisseria PAGE 46 Direct examination of the specimen - microscopy: Gram-negative cocci in diplo (kidney-shaped cocci) Neisseria gonorrhoeae, Giemsa stain, 100x Genus Neisseria PAGE 47 Direct examination of the specimen - rapid diagnosis of Neisseria meningitidis from cerebrospinal fluid or blood ▪ nucleic acid detection ▪ antigen detection by agglutination Genus Neisseria PAGE 48 Cultivation - specimen collection and transport conditions greatly influence the success of isolation - nutritionally exigent bacteria - special culture media are used: chocolate agar (unselective), chocolate agar supplemented with antibiotics (selective for Neisseria spp.) - incubation in CO2 enriched atmosphere for 48-72 hours (microaerophilic bacteria) Genus Neisseria PAGE 49 Identification - morphology: Gram-negative diplococci - N. gonorrhoeae is more fastidious that N. meningitidis and lacks the capsule Genus Neisseria PAGE 50 Identification - small S colonies (like pearls) - oxidase-positive – common property for all Neisseria spp. - species are identified according to sugar fermentation pattern Neisseria gonorrhoeae chocolate agar Antibiotic susceptibility testing  - meningitis Haemophilus influenzae - epiglottitis PAGE 51 - pneumonia - sinusitis, otitis - sepsis Haemophilus parainfluenzae - endocarditis - opportunistic infections Haemophilus spp. Haemophilus aegyptius - conjunctivitis (“pink eye”) Haemophilus ducreyi - chancroid (STD) other Haemophilus species Haemophilus influenzae PAGE 52 General characteristics - thin, microaerophilic Gram-negative rod that requires the presence of X and V factors for growth Haemophilus influenzae b type Photo Researchers Science Photo Library Haemophilus influenzae PAGE 53 Specimens - nasopharyngeal secretions - pus – otitis, sinusitis - sputum, other lower respiratory tract specimens - pneumonia - cerebrospinal fluid - meningitis - blood – sepsis - H. influenzae strains are susceptible to temperature changes – rapid transport is needed (do not refrigerate specimens in which the presence of Haemophilus spp. is suspected) Haemophilus influenzae PAGE 54 Direct examination of the specimen - microscopy: short and thin Gram-negative rods Image Source: Manitoba Health and Fine Art America Haemophilus influenzae PAGE 55 Direct examination of the specimen - rapid detection of H. influenzae b serotype antigens from CSF Haemophilus influenzae PAGE 56 Cultivation ₋ fastidious ₋ CO2 5-10% ₋ nutritionally exigent bacteria: they need the presence of NAD (V factor) and hemin (X factor) ₋ chocolate agar, chocolate agar with antibiotics (selective) Haemophilus influenzae PAGE 57 Identification - morphology (direct examination – microscopy) - colonies: ▪ S colonies (dew drops) - testing for X, V factor necessity Haemophilus influenzae PAGE 58 Identification - satelitism (Haemophilus influenzae may grow on blood agar plate around S. aureus colonies: S. aureus produces V factor and hemolysins that lyses red blood cells leading to the release of X factor) Haemophilus influenzae PAGE 59 Identification ₋ Ag detection: Haemophilus influenzae b serotype (according to capsular antigens) – Hib – the most virulent strains associated with disease (most Haemophilus spp. have a polysaccharide capsule => six antigenic serotypes) ₋ NAATs Haemophilus influenzae PAGE 60 Antibiotic susceptibility testing ₋ performed on special culture media (Haemophilus test medium) ₋ detection of beta-lactamase production may be sufficient PAGE 61  PAGE 1 UNIVERSITÄTSMEDIZIN https://www.umfst.ro https://edu.umch.de NEUMARKT A. M. CAMPUS HAMBURG 2024 November Enterobacterales, Non-fermentative Gram negative rods, Vibrio cholerae Gram-negative enteric rods PAGE 2 non-fastidious aerobic, facultative anaerobic bacteria. all species belonging to this family are glucose fermenters, catalase positive, oxidase negative Major pathogens: Salmonella spp., Shigella spp. and Yersinia spp. Other representatives of the family are members of the intestinal normal flora and are opportunistic pathogens. Escherichia coli PAGE 3 Specimens - Intestinal infections due to enteric pathotypes of E. coli: ▪ stool, bile - Extraintestinal infections: according to localization ▪ urine, pus, cerebrospinal fluid, blood, etc. Escherichia coli PAGE 4 Direct examination of the specimen - microscopy - Gram-negative rods with rounded ends Escherichia coli Electron Microscopy, University of California Escherichia coli PAGE 5 Escherichia coli PAGE 6 Cultivation - blood agar plate Escherichia coli PAGE 7 Cultivation - MacConkey agar Escherichia coli PAGE 8 Cultivation - Lactose agar Escherichia coli PAGE 9 Cultivation - Levine Medium Escherichia coli PAGE 10 Cultivation - Levine Medium Escherichia coli PAGE 11 Identification - morphology - colonies S – colonies ▪ on a blood-agar plate: some strains are β-hemolytic ▪ lactose-containing media: lactose positive colonies MacConkey: red colonies lactose agar: yellow colonies Escherichia coli PAGE 12 Escherichia coli PAGE 13 Biochemical patterns: ▪ SIM agar: o H2S negative o motility: + o indole positive ▪ TSI agar o glucose positive (+/- gas formation) o H2S negative o lactose, sucrose positive ▪ Simmons agar o no growth ▪ urea medium o urease negative Escherichia coli PAGE 14 Antigenic structure E. coli: ▪ agglutination reaction to identify specific pathotypes ▪ enterohemorrhagic E. coli (EHEC): O157:H7 Antibiotic susceptibility testing - important resistance phenotypes: ESBL (extended-spectrum β-lactamase) producer strains DOI: 10.14744/hnhj.2019.55706 Klebsiella genus PAGE 15 Medically important representatives ▪ K. pneumoniae ▪ K. oxytoca Specimens - urine - sputum - pus - cerebrospinal fluid - blood Klebsiella genus PAGE 16 Direct examination - microscopy Gram stain: Gram-negative cocobacilli in pairs Klebsiella genus PAGE 17 Direct examination - microscopy negative stain: capsule Klebsiella genus PAGE 18 Direct examination - microscopy Electron microscopy Klebsiella pneumoniae Electron Microscopy, CDC/ Janice Carr Klebsiella genus PAGE 19 Cultivation - blood agar plate Klebsiella genus PAGE 20 Cultivation - Lactose agar Klebsiella genus PAGE 21 Cultivation - MacConkey agar Klebsiella genus PAGE 22 Identification ▪ morphology ▪ colonies M colonies ▪ lactose-containing media: lactose-positive ▪ MacConkey: lactose positive (red) colonies ▪ lactose agar: lactose positive (yellow) colonies Klebsiella genus PAGE 23 Klebsiella genus PAGE 24 Biochemical pattern ▪ SIM agar o H2S negative o motility: negative o indole variable ▪ TSI agar o glucose positive o H2S negative o lactose, sucrose positive ▪ Simmons agar o growth ▪ urea agar o urease positive Klebsiella genus PAGE 25 Antigenic structure ▪ Quellung reaction – Identification of capsular antigens Antibiotic susceptibility testing Important resistance phenotypes ESBL (extended spectrum β-lactamase) production KPC (Klebsiella pneumoniae carbapenemase-producing) Proteus genus PAGE 26 Medically important species: P. vulgaris P. mirabilis P. penneri Specimens - urine - sputum - pus - cerebrospinal fluid - blood, etc. Proteus mirabilis Electron Microscopy, Visuals Unlimited/Corbis Proteus genus PAGE 27 Direct examination - microscopy - Gram-negative rods Proteus genus PAGE 28 Cultivation - blood agar plate Proteus genus PAGE 29 Cultivation - Lactose agar Proteus genus PAGE 30 Cultivation - MacConkey agar Proteus genus PAGE 31 Proteus genus PAGE 32 Identification - morphology (see microscopy) - colonies ▪ on culture media without selective agents (without bile salts): o swarming ▪ on culture media with selective agents: o S colonies ▪ lactose-containing media: lactose-negative colonies ▪ iron salts containing media: black centered colonies Proteus genus PAGE 33 Biochemical patterns ▪ SIM agar o H2S positive o motility: positive o indole variable ∙ P. mirabilis: indole negative ∙ P. vulgaris: indole positive ▪ TSI agar o glucose positive o H2S positive o lactose, sucrose negative ▪ Simmons agar o growth ▪ urea agar o urease positive Proteus genus PAGE 34 Antibiotic susceptibility testing – should be performed Shigella genus PAGE 35 Medically important species: Shigella dysenteriae Shigella flexneri Shigella boydii Shigella sonnei Shigella spp. cause dysenteria, Shigella sonnei, Electron Microscopy, Mediscan/Corbis Specimens - stool - food (source detection) Shigella genus PAGE 36 Direct examination - Visual examination of the stool: blood, pus and mucus are present - Microscopy: Gram-negative rods Shigella genus PAGE 37 Cultivation - MacConkey agar - SS agar https://microbiologyinfo.com/salmonella-shigella-ss-agar-composition- principle-uses-preparation-and-result-interpretation/ Shigella genus PAGE 38 Shigella genus PAGE 39 Identification - morphology - colonies ▪ S colonies, lactose negative - biochemical patterns ▪ SIM agar o H2S negative o motility negative o indole variable ▪ TSI agar o glucose positive o H2S negative o lactose, sucrose negative ▪ Simmons agar o no growth (green) ▪ urea agar o urea negative Shigella genus PAGE 40 Antigenic structure ▪ allows species identification (agglutination) - pathogenicity tests ▪ Serény test – eye infection in guinea pig ▪ tests on cell cultures. https://doi.org/10.1371/journal.pone.0100264 Antibiotic susceptibility testing Although may appear susceptible in vitro, strains belonging to Shigella genus are resistant to aminoglycosides, 1st and 2nd generation cephalosporins, therefore these antibiotics should neither be tested nor used in therapy. Salmonella genus PAGE 41 Medically important species ▪ Salmonella Typhi, Salmonella Paratyphi A, B – associated with typhoid fever ▪ Salmonella Enteritidis, Typhimurium, etc. – acute gastroenteritis Salmonella typhimurium Salmonella typhimurium Electron Microscopy CDC/ Bette Jensen Electron Microscopy Rocky Mountain Laboratories, NIH Salmonella genus PAGE 42 Specimens ▪ typhoid fever blood, intestinal secretions (vomitus or duodenal fluid), stool samples are positive in 85-90% of patients within one week of disease onset; 20-30% later during the disease the highest sensitivity of detection: bone marrow aspirate urine culture – may be positive (timing unpredictable) ▪ detection of carriage - stool ▪ gastroenteritis – stool ▪ disseminated infection – blood Salmonella genus PAGE 43 Direct examination - microscopy - Gram-negative rods Salmonella genus PAGE 44 Cultivation - enrichment media Salmonella genus PAGE 45 Cultivation - Lactose agar Salmonella genus PAGE 46 Cultivation - MacConkey - SS Salmonella genus PAGE 47 Salmonella genus PAGE 48 Identification - colonies morphology ▪ MacConkey: – lactose-negative (transparent) S colonies ▪ SS - lactose-negative (transparent) S colonies, black center - biochemical patterns ▪ SIM agar o H2S positive o motility: positive o indole negative ▪ TSI agar o glucose positive o H2S positive o lactose, sucrose negative ▪ Simmons agar o growth ▪ Urea agar o urease negative Salmonella genus PAGE 49 Antigenic structure ▪ agglutination reactions (O, H, Vi antigens) ▪ based on O antigen serogroups are established ▪ serotypes are determined according to H antigens ▪ Vi antigens are surface antigens present in S. typhi, paratyphi serovars and may mask O antigens Salmonella genus PAGE 50 Serology Serology is applied for the diagnosis of typhoid fever: - Widal reaction – a classic diagnostic method used in developing countries to detect antibodies against O and H antigens based on quantitative agglutination test – questionable value Antibiotic susceptibility testing - although may appear susceptible in vitro, strains belonging to Salmonella genus are resistant to aminoglycosides, 1st and 2nd generation cephalosporins, therefore these antibiotics should neither be tested nor used in therapy Yersinia enterocolitica PAGE 51 Specimens - gastroenteritis – stool sample - mesenteric adenitis, mesenteric ileitis, and acute pseudoappendicitis – Yersinia enterocolitica lymph node tissue Visuals Unlimited/Corbis - sepsis – blood Direct examination – microscopy - Gram-negative rods Cultivation - MacConkey - Lactose agar - CIN agar (selective culture medium for Yersinia enterocolitica) Yersinia enterocolitica PAGE 52 Yersinia enterocolitica PAGE 53 Motility - (37°C) Motility + (30°C) Yersinia enterocolitica PAGE 54 Identification ▪ morphology ▪ colonies Mac Conkey: small, transparent lactose-negative S colonies CIN agar: bull’s eye colonies (red centered) ▪ biochemical patterns ⮚ SIM agar o H2S negative o motility - negative at 37° C - positive at 30° C o indole negative ⮚ TSI agar o glucose positive o H2S negative o lactose negative o ferments sucrose Simmons agar o no growth ⮚ Urea agar o positive Yersinia enterocolitica PAGE 55 Antigenic structure O3 and O9 are the most frequently encountered serogroups in Europe Antibiotic susceptibility testing - antibiogram should be performed for Yersinia spp. isolates  Colony morphology Citrate Glucose Lactose H2S Urease Motility Indole SS BAP MC LA utilization PAGE 56 E. coli - S L+ L+, S + + - - - + + colonies colonies Klebsiella - S L+ L+, M + + - + + - - pneumoniae colonies colonies Klebsiella oxytoca - S L+ L+, M + + - + + - + colonies colonies Proteus mirabilis L-, black swarmi L- L- + - + + + + - centre ng swarming Proteus vulgaris L-, black swarmi L- L- + - + + + + + centre ng Salmonella spp. L-, black S L- L- + - + - + + - centre colonies Shigella spp. L- S L- L- + - - - - - v colonies Non-fermentative Gram-negative bacteria PAGE 57 Pseudomonas aeruginosa PAGE 58 ▪ opportunistic pathogen associated with severe infections in immunocompromised hosts and hospitalized patients ▪ obligate aerobic ▪ glucose non-fermentative ▪ oxidase positive ▪ Gram-negative rod ▪ catalase positive Pseudomonas aeruginosa PAGE 59 Specimens - pus (surgical, burnt wound infections, necrotic skin lesions) - sputum, tracheal aspirates, bronchial aspirates - urine - blood - cerebrospinal fluid - conjunctival secretion - in case of hospital epidemics – environmental specimens Pseudomonas aeruginosa PAGE 60 Direct examination ▪ Visual examination purulent secretions may be greenish and have a characteristic odor ▪ Microscopy Gram-negative bacilli with flagella Pseudomonas aeruginosa CDC/ Janice Haney Carr Pseudomonas aeruginosa PAGE 61 Cultivation Non-fastidious organism, has very simple nutritional requirements ▪ Basic media ▪ Blood agar plate ▪ MacConkey agar ▪ Cetrimide agar (P. aeruginosa is resistant to cetrimide, a toxic substance that inhibits the growth of other bacteria) Pseudomonas aeruginosa PAGE 62 Identification - morphology (see direct examination – microscopy) - colony morphology ▪ Liquid media – pellicle, pigmented ring beneath the pellicle ▪ Solid media – shiny S colonies, lactose – negative colonies on lactose-containing media ▪ Blood agar plate – beta hemolysis Pseudomonas aeruginosa PAGE 63 Biochemical pattern ▪ oxidase positive, glucose non-fermenter, lactose negative ▪ pigment production – 2 soluble pigments o pyocyanin – blue – specific for P. aeruginosa, produced on special media with low iron content o pyoverdin - fluorescent yellow-green Pseudomonas aeruginosa PAGE 64 Biochemical pattern ▪ strong aroma (linden tree or lime tree flower-like) Typing ▪ epidemiological applications ▪ phage typing, molecular typing Pseudomonas aeruginosa PAGE 65 Antibiotic susceptibility testing - Intrinsic resistance against many antimicrobials - Acquired resistance loss of outer membrane proteins leads to decreased permeability or efflux pumps frequently affects carbapenem, fluoroquinolone activity beta-lactamases MDR strains – multidrug-resistant strains Acinetobacter genus PAGE 66 ubiquitous bacteria, which may colonize mucosal surfaces, especially in hospitalized patients under broad-spectrum antibiotic treatment opportunistic pathogens with high nosocomial potential https://doi-org.am.e-nformation.ro/10.1038/nrmicro1789 Acinetobacter genus PAGE 67 Medically important species: Acinetobacter baumannii Acinetobacter lwofii Acinetobacter calcoaceticus Specimens - sputum, other lower respiratory tract specimens - pus - urine - blood - CSF Acinetobacter genus PAGE 68 non-fastidious aerobic glucose non-fermentative oxidase negative Gram-negative coccobacilli Acinetobacter genus PAGE 69 Direct examination - microscopy: - pleomorphic bacteria (variable shape), short, coccoid bacilli in pairs, that resemble Neisseria spp. Acinetobacter baumannii CDC/ Janice Haney Carr Acinetobacter genus PAGE 70 Cultivation - basic media - blood agar plate - MacConkey agar Acinetobacter genus PAGE 71 Identification - morphology: pleomorphic bacteria (variable shape) - colony morphology: S colonies, grey Acinetobacter genus PAGE 72 Biochemical patterns glucose non-fermentative oxidase negative lactose negative Simmons positive A. baumannii – growth at 44°C Antibiotic susceptibility testing - Resistance to many antibiotic classes (natural and acquired) - MDR strains Vibrio cholerae PAGE 73 Vibrio cholerae is aerobic, oxidase positive, glucose fermenter Gram-negative curved rod, found in aquatic environments. Serogroups O1 and O139 are the causative agents of cholera. Specimens - Stool, rectal swab - Water, food samples Direct examination - Visual examination of the stool samples - watery stool with flakes of mucus and epithelial cells ("rice-water stool") Vibrio cholerae PAGE 74 Direct examination - Microscopy ▪ Gram stain – Gram-negative, thin, curved, comma-shaped bacillus with one polar flagellum ▪ Wet mount examined in darkfield: motile bacteria - Rapid diagnosis - Detection of bacterial antigens (O1, O139) from stool samples by ELISA Vibrio cholerae PAGE 75 Cultivation - Non-fastidious organisms, which grow on alkaline pH - Alkaline peptone water - Blood agar - Special media (e.g. BSA - bile, salt, agar, TCBS – thiosulphate, citrate, bile salts, sucrose) TCBS agar Vibrio cholerae PAGE 76 Identification - Morphology (see direct methods) - Colony morphology ▪ Solid media – S colonies, watery appearance Biochemical pattern Oxidase positive Glucose fermentative Sucrose positive Antigenic structure antigen-antibody reactions to identify serogroups (O1, O139) ▪ Automated/Semiautomated Identification Systems ▪ Molecular typing Suspected strains are referred to reference laboratories for confirmation. PAGE 77  PAGE 1 UNIVERSITÄTSMEDIZIN https://www.umfst.ro NEUMARKT A. M. https://edu.umch.de CAMPUS HAMBURG 2024 December Diagnosis of infections caused by Moraxella spp. Diagnosis of syphilis. Diagnosis of tuberculosis. Moraxella genus PAGE 2 Moraxella catarrhalis – Pulmonary infections in patients with chronic pulmonary diseases – Sinusitis, otitis Specimens – sputum – other lower respiratory tract specimens - pneumonia Moraxella genus PAGE 3 Direct examination of the specimen - microscopy: Gram negative diplococci Cultivation - most non-selective media https://www.grepmed.com/images/11761/culture-catarrhalis-moraxella-microscopy-microbiology Moraxella genus PAGE 4 Identification morphology (direct examination – microscopy) oxidase positive Strictly aerobic bacteria Biochemical tests DN-ase positive Mass spectrometry Ag detection NAATs https://microbeonline.com/moraxella-catarrhalis/ Treponema pallidum PAGE 5 Treponema pallidum is the causative agent of syphilis, a sexually transmitted disease. Treponema pallidum PAGE 6 Specimens - exudates from lesions of primary and secondary syphilis (a clear, serous liquid must be obtained, free of erythrocytes). The area should first be cleaned with a sterile gauze, moistened in saline solution and the sample should be put on a glass slide - tissue or aspirates from lymph nodes – should be placed in 10% buffered formalin - a small section of the umbilical cord – for congenital syphilis - CSF - neurosyphilis - serum for serology (the current standard of diagnosis) For PCR, the sample should be collected on a sterile swab and then placed in a cryotube with a nucleic acid transport medium or a universal transport medium Treponema pallidum PAGE 7 Direct examination dark-field microscopy – wet mount direct fluorescent antibody test direct tests for T. pallidum in tissue samples nucleic acid amplification tests (PCR) Treponema pallidum PAGE 8 Dark-field microscopy – wet mount ▪ motile spirochetes with large, regular spirals, sizes: 6-16 /0,25μm (corkscrew spirals) ▪ can be performed only when the lesions are moist and the examination may be carried out immediately after collection Treponema pallidum PAGE 9 Direct fluorescent antibody test ▪ the most specific test when lesions are present ▪ detects antigens Treponema pallidum - serology PAGE 10 Nontreponemal serological tests (non-specific tests)/ screening tests - detect non-specific antibodies, with the help of non-specific antigens (such as cardiolipin) - a positive result indicates that the patient might have syphilis, but for a certain diagnostic a confirmatory test should be performed Treponemal serological tests (specific tests)/ confirmatory tests - detect antibodies formed against specific protein antigens, using specific treponemal antigens (proteins) Treponema pallidum PAGE 11 Direct tests for T. pallidum in tissue samples ▪ fluorescent antibody test in combination with a histological staining ▪ silver stain Treponema pallidum PAGE 12 Nucleic acid amplification tests (PCR) Cultivation T. pallidum does not grow on solid culture media - cell cultures (research) - inoculation in rabbit testicles (research, maintaining stock strains, obtaining treponemal antigens for diagnostic tests) Treponema pallidum PAGE 13 Nontreponemal serological tests (non-specific tests)/ screening tests ▪ VDRL (Venereal Disease Research Laboratory) – flocculation tests (precipitation in liquid medium) ▪ RPR (Rapid Plasma Reagin Test) - agglutination Characteristics Disadvantages tests become positive 1-4 weeks after the low specificity appearance of the primary lesion (chancre) rapid, simple, non-expensive the possibility of false-positive reactions due to cross-reactions recommended to monitor the course of disease positive reactions during screening must be after treatment (titers decrease with cure) confirmed with specific treponemal tests not accurate in detecting late syphilis Treponema pallidum PAGE 14 Treponemal serological tests (specific tests)/ confirmatory tests ▪ FTA – Abs (Fluorescent Treponemal Antibody Absorption Test) ▪ TPHA (Treponema pallidum hemagglutination assay) ▪ ELISA Characteristics Disadvantages remain positive for years, antibody titers expensive and time-consuming do not correlate with disease course (treponemal tests are not suitable for monitoring disease evolution) used to diagnose patients with late syphilis when nontreponemal tests are negative Treponema pallidum PAGE 15 Diagnostic algorithms for syphilis syphilis (past of present) Treponemal serological tests Non-treponemal tests (VDRL or RPR) negative result negative result Treponema pallidum PAGE 16 Diagnostic algorithms for syphilis The reverse sequence syphilis syphilis (past or present) Non-treponemal tests (VDRL or RPR) negative result Automated treponemal serological tests syphilis (past or Treponemal present) serological tests negative result Mycobacterium tuberculosis PAGE 17 strictly aerobic rod-shaped bacterium that cannot be stained by Gram due to the high lipid content of the cell wall the causative agent of tuberculosis Mycobacterium tuberculosis CDC/ Dr. Ray Butler; Janice Carr Mycobacterium tuberculosis PAGE 18 Specimens - pulmonary tuberculosis – sputum, other secretions of the lower respiratory tract, gastric secretion in case of infants - renal tuberculosis – urine (whole quantity voided in the morning) - genital tuberculosis – vaginal secretions, sperm, prostatic secretions - joint, bone tuberculosis – joint fluid, pus - lymph node tuberculosis – lymph node aspirate, pus - meningitis – CSF Mycobacterium tuberculosis PAGE 19 Direct examination Microscopical examinations - Ziehl-Neelsen stain (acid-fast staining) – long, thin bacilli, slightly curved, stained red (acid resistant), other elements are stained in blue - Primary stain: carbofuchsin (with heat) - Decolorizing agent: acid-alcohol - Counterstain: methylene blue - Kinyoun stain: acid fast stain (cold stain) - Auramine stain/ Auramine-rhodamine flurochrome staine (more sensitive) Mycobacterium tuberculosis PAGE 20 Direct examination Acid-fast stain: positive for: - Mycobacterium spp. - Nocardia spp. - Legionella micdadei - Rhodococcus spp. NAATs - PCR (polymerase chain reaction); - Automated DNA sequencing Mass spectrometry – MALDI-TOF MS Mycobacterium tuberculosis PAGE 21 Cultivation - Löwenstein-Jensen agar – egg-based selective medium - 37°C, up to 60-90 days Mycobacterium tuberculosis PAGE 22 Identification - morphology ▪ „serpentine cords”: bacilli form bundles in presence of cord factor - a toxic glycolipid found as a surface component of tubercle bacilli that is responsible for virulence and serpentine growth Mycobacterium tuberculosis PAGE 23 Identification Cultivation ▪ growth after 2-3 weeks of incubation ▪ R colonies, very dry, resembling breadcrumbs or cauliflower Mycobacterium tuberculosis PAGE 24 Antibiotic susceptibility testing - the usual disk diffusion method is not applicable - susceptibility can be determined on Löwenstein-Jensen agar with different antituberculosis agents included Mycobacterium tuberculosis PAGE 25 Indirect diagnosis: skin test (Mantoux test, tuberculin test) – based on delayed hypersensitivity reaction ▪ PPD (purified protein derivate) is intracutaneously injected in the forearm ▪ the test is read within 48-72 hour ▪ positive reaction: erythema, swelling and induration (raised and hard) with a diameter of 10 mm or greater – recent or past infection with M. tuberculosis Mycobacterium tuberculosis PAGE 26 Indirect diagnosis: skin test (Mantoux test, tuberculin test) – based on delayed hypersensitivity reaction ▪ vaccination with BCG => positive skin test ▪ false-positive test: infection with other mycobacteria ▪ false-negative reaction: in case of an impaired cellular immune response (e.g. AIDS patients) Mycobacterium tuberculosis PAGE 27 Indirect diagnosis - Serology - IGRAs (INF-γ release assays) - Quantiferon-TB Gold assay - T.SPOT.TB - Measure the cell-mediated immune response in whole blood samples - Not approved for persons younger than 17 years old or for immunosuppressed patients - Not affected by BCG vaccination; no cross-reactivity with other Mycobacterium spp. - Cannot distinguish between active and latent infections PAGE 28  PAGE 1 UNIVERSITÄTSMEDIZIN NEUMARKT A. M. https://edu.umch.de www.umfst.ro CAMPUS HAMBURG 2024 December Mycology Definitions PAGE 2 Fungi are eukaryotic microorganisms, uni- or pluricelular, heterotrophic, that have chitin in their cellular wall. https://biologydictionary.net/prokaryotes-vs-eukaryotes/ https://commons.m.wikimedia.org/wiki/File:Fungi_(26748490534).jpg Fungi classification PAGE 3 1. Yeasts – unicelullar microorganisms, round or elongated, which multiply mostly by budding. The bud is called blastospore or blastoconidia and can further divide into new individuals, forming a liniar group called pseudohyphae. https://chromoscience.com/fungal-infections-of-the-reproductive-system/ Thompson, Carlisle and Kadosh, 2011 Fungi classification PAGE 4 2. Molds – pluricellular microorganisms, with thallus formed out of tubular shaped filaments, with or without septa, called hyphae; hyphae are usually divided and https://biologydictionary.net/septate-vs-non-septate-hyphae/ together they form the mycelium. Cogulet, Antoine. “Caractérisation des mécanismes naturels https://bioisnotdifficult.blogspot.com/2020/04/kingdom-fungi-in-detail.html de vieillissement de lambris de bâtiments en bois.” (2018). Fungi classification PAGE 5 Mold in the Cold, Yeast in the Heat 3. Dimorphic – a very special category of fungi, which apear as yeast in the tissues of the infected organisms or at 37°C in vitro and as moulds when they are cultured at room temperature or at 30°C, on usual culture media. https://labpedia.net/fungus-fungal-infections-diagnosis-of-fungal-infection/ https://www.sciencedirect.com/topics/immunology-and-microbiology/eurotiomycetes Yeasts PAGE 6 Classification Ascomycete (Candida, Geotrichum, Saccharomyces) Basidiomycete (Cryptococcus, Malassezia, Rhodotorula, Trichosporon) Pneumocystis https://fungaljungle.weebly.com/types-of-fungus.html Yeasts PAGE 7 - Unicellular fungi that have a vegetative multiplication (asexuate) by budding or physion ubiquitous aerobic or facultative anaerobic microorganisms optimal cultivation temperature: 25-30°C, the pathogenic ones tolerate the temperature of 37°C Habitat: - plants, insects, animals, humans (skin, mucous membranes, digestive tract), soil, water, air Some yeasts can take two forms, depending on how they are multiplied: Anamorphic Teleomorphic (asexual multiplication) (sexual multiplication) Yeasts https://freesvg.org/1497804422 PAGE 8 Direct microscopic examination - with 20% KOH solution - with 20% KOH solution + fluorescent substance (calcofluor white) - Gram stained smear - yeast cells (approx. 3-5 mm), Gram-positive stained pseudohyphae - Indian ink - to highlight the capsule - May Grunwald Giemsa stained smear - for the detection of intracellular yeasts (Histoplasma spp.) - with 3% methylene blue - with adhesive tape, colored with methylene blue in lactophenol blue During the microscopic examination, the following are observed: the presence, shape and dimensions of the elements that are yeast characteristic: blastoconidia, pseudohyphae, hyphae (filaments), arthrospores. Genus Candida PAGE 9 Species ▪ Candida albicans ▪ non-albicans: C. krusei, C. glabrata,. C. tropicalis, C. parapsilosis, C. guillermondii, C. famata, etc. ❖ members of normal flora ❖ proliferate in the presence of risk factors: immunosuppression antibiotics pregnancy diabetes presence of intravascular catheters, intravenous drug use https://pixabay.com/illustrations/pregnant- woman-cute-pregnancy-5200901/ Genus Candida PAGE 10 Candida infections ▪ superficial infections - skin, nail, mucous membranes (vaginitis, esophagitis, oral candidiasis) ▪ deep infections - sepsis, endocarditis, abscesses in various organs, dissemination in neutropenic patients, shunt infection, pericarditis ▪ congenital candidiasis - systemic candidiasis in newborn with low birth weight https://www.voicedoctor.net/media/structural-injury- inflammation-fungal/candida/fungal-laryngitis-flovent https://commons.wikimedia.org/wiki/File:Human_tong ue_infected_with_oral_candidiasis.jpg Genus Candida PAGE 11 Morphology - studied on: wet mount, Gram staining, Giemsa staining ▪ blastospores big cells (3-8 μ), spherical or ovalar, Gram positive replicate by budding (burgeoning) https://www.researchgate.net/publication/309032798_Evaluation_de_la_formation_des_biofilms _de_Candida_sp_isolees_des_dispositifs_medicaux_au_CHU_de_Sidi_Bel_Abbes ▪ pseudomycelium https://en.wikipedia.org/wiki/Chlamyd ospore#/media/File:C_albicans_en.jpg elongated cells, still attached after division ▪ chlamydospore spherical cell with double contour larger than blastospores Genus Candida PAGE 12 Species Candida albicans Candida glabrata Candida krusei Genus Candida PAGE 13 Cultivation Sabouraud medium - supplemented with chloramphenicol, enable the development of yeasts and inhibit the multiplication of bacterial contaminants Genus Candida PAGE 14 Cultivation CandiSelect: - chromogenic media - incubation 30-37°C, 24-48 hours https://www.flickr.com/photos/7cius/32121601184 https://basicmedicalkey.com/13-commercial-methods-for- identification-and-susceptibility-testing-of-fungi/ Genus Candida PAGE 15 Identification ▪ sugar fermentation ▪ temperature of growth: C. albicans grow at 45°C most species grow at 37-40°C ▪ urease negative – differentiation from other yeasts Genus Candida PAGE 16 Identification Germ tube test – positive for C. albicans and dubliniensis ▪ prepare a 0.5 McFarland suspension in saline solution ▪ 0.5 ml of suspension is added to equal volume of human or horse blood serum → incubation in a water bath (36°C for 2 hours) → wet mount ▪ germination is observed as short filaments that emerge from blastospores (only in C. albicans, C. dubliniensis) https://microbiologyinfo.com/germ-tube-test-principle-procedure-results-interpretation-and-limitations/ Genus Candida PAGE 17 Identification Chlamydospore formation ▪ only for C. albicans and dubliniensis ▪ only if grown on special media (cornmeal agar or Tween 80) Genus Candida PAGE 18 Identification Automated methods of identification: - Vitek 2 Compact https://www.biomerieux-asean.com/file/369 https://de.bimedis.com/biomerieux-vitek-2-compact-60-m274395  Identification Genus Candida https://biobase-jn.en.alibaba.com/product/60837540251- 804530812/BIOBASE_Cheap_Price_Laboratory_and_Medica l_PCR_Machine_Thermal_Cycler_Real_Time_PCR_System.h tml Molecular techniques - identification based on 18S ribosomal DNA sequencing, identification by PCR PAGE 19 Genus Candida PAGE 20 Antifungal susceptibility testing not recommended as routine indicated in case of invasive infections, infections refractory to treatment determination of minimum inhibitory concentration by E-tests or microdilution https://doi.org/10.3389/fcimb.2021.720609 Genus Candida PAGE 21 Antifungal susceptibility testing Genus Cryptococcus PAGE 22 Species · Cryptococcus neoformans · Cryptococcus gattii Genus Cryptococcus PAGE 23 Morphology Microscopic examination - mature yeast cells (asexuate, anamorphic) are round and can present multiple buds (”Mickey Mouse” head) - pseudomycelia are absent - negative staining: can be used to show the presence of the capsule - teleomorphic yeast cells present hyphae with strangulations which form basidiospores https://microbenotes.com/cryptococcus-neoformans/ https://de.wikipedia.org/wiki/Cryptococcus_neoformans Genus Cryptococcus PAGE 24 Cultivation · on Sabouraud agar medium · incubation at 37°C, 48-72 hours Genus Geotrichum PAGE 25 Species Geotrichum candidum colonizes the intestinal tract may cause bronchopulmonary infections (pulmonary geotrichosis) infections/colonization of the oral and vaginal mucosa or of the skin rarely - sepsis, traumatic wound infections Genus Geotrichum PAGE 26 Morphology hyphae (7-12 μ wide) with branches (2-4 μ wide) the ends of the branches fragment, resulting in arthroconidia of rectangular shape (5-17 μ length/4-6 μ width) Genus Geotrichum PAGE 27 Cultivation - Sabouraud agar medium - incubation at 30-34°C, 24-48 hours - does not grow at 40°C Genus Rhodotorula PAGE 28 Species Rhodotorula mucilaginosa (rubra) present in air, soil, water colonizer of catheters or medical equipment (dialysis) fungal peritonitis in people with ambulatory peritoneal dialysis fungemia, endocarditis, meningitis in people with venous or immunocompromised catheters Genus Rhodotorula PAGE 29 Morphology unipolar budding cells, 2.5-5 μ pseudomycelium https://doi.org/10.1016/j.femsyr.2004.05.002 DOI: 10.5772/19235 Genus Rhodotorula PAGE 30 Cultivation - Sabouraud agar medium - incubation at 30-34°C, 24-48 hours - pigmented, coral pink-red "S" type colonies, sometimes mucous or rough Genus Malassezia PAGE 31 https://commons.wikimedia.org/wiki/File: Species Tinea_versicolor1.jpg Malassezia furfur Malassezia globosa Malassezia furfur https://commons.wikimedia.org/wiki/File:Pytriasis it can be part of the skin flora and the ear canal in hyperhydrotic people pityriasis versicolor (tinea versicolor) – excessive dandruff seborrheic hyperkeratosis is rare _versicolor.jpg blepharitis, neonatal pustulosis, folliculitis in immunocompromised seborrheic dermatitis, disseminated infection (lung) fungemia in people receiving lipid supportive therapy Globose malaisea most common causative agent of pityriasis versicolor Genus Malassezia PAGE 32 Morphology Malassezia furfur: unipolar budding cells; buds are almost the same size as the cell of origin Malassezia globosa: unipolar budding cells; buds are smaller in size compared to the cell of origin Genus Pneumocystis PAGE 33 Species Pneumocystis jirovecii - a common cause of pneumonia in HIV/AIDS infected patients - epidemics in institutionalized people (orphanages, in conditions of malnutrition or overpopulation) - it causes a mild infection in immunocompetent individuals https://www.cell.com/trends/microbiology/fulltext/S0966-842X(20)30077-9 Genus Pneumocystis PAGE 34 Cultivation can’t be grown routinely in the laboratory axenic culture (on germ-free animals) or on cell cultures https://en.wikipedia.org/wiki/Pneumocystis_jirovecii#/media/File:Pneumocystis_ji rovecii_AO_AL.jpg https://en.wikipedia.org/wiki/Pneumocystis_jirovecii#/media/File:Pneumocystis_carinii_01.jpg Molds PAGE 35 Classification Non-septate fungi (inferior molds) PAGE 36 = Zygomycetes Caracterized by: - macroscopically: fluffy, wool-like aspect of the colonies, white-grey colour, fast and invasive growth - microscopically: non-septated mycelia , made out of hyaline hyphae with irregular diameters Non-septate fungi (inferior molds) PAGE 37 Morphology Non-septate fungi (inferior molds) PAGE 38 Samples ▪ skin cells (raclate, biopsy, scotch-tape) – frequent face skin infections ▪ bone, hair, nails ▪ respiratory secretions in case of respiratory infections ▪ CSF in rhino-cerebral zygomycosis DOI: 10.1056/NEJM199508313330906 Rhizopus PAGE 39 - part of the Mucorales family - produces infections in immunocompromised patients - deep skin and organ infections Absidia PAGE 40 - part of the Mucorales family - produces infections in immunocompromised patients - deep skin and organ infections Mucor PAGE 41 - part of the Mucorales family - produces infections in immunocompromised patients - deep skin and organ infections Non-septate fungi (inferior molds) PAGE 42 Cultivation Potato-dextrose-agar medium (PDA) Sabouraud agar medium incubation at 32⁰C https://sciencelab.co.ke/products/potato-dextrose-agar Septate fungi (superior molds) PAGE 43 Hyaline = non-pigmented hyphae Aspergillus: A. fumigatus, A. flavus, A. niger, A. terreus Penicillium: over 200 species: P. notatum, P. commune, etc. Pathogenic for humans: P. marneffei Fusarium: over 100 species; Pathogenic: F. oxysporum, F. solani Acremonium: over 100 species Septate fungi (superior molds) PAGE 44 Cultivation Potato-dextrose-agar medium (PDA) Sabouraud agar medium with chloramphenicol Czapek medium – selective for Aspergillus spp. ▪ incubation at 30°C for superficial samples ▪ incubation at 37°C for deep site infections https://sciencelab.co.ke/products/potato-dextrose-agar https://www.sglab.com/product/czapek-dox-agar-90mm-plate/ Septate fungi (superior molds) PAGE 45 Identification Microscopy o form, length of hyphae o number of branches, presence of roots, type of spore-forming structures Culture characters size, shape, color of the colonies fluffy, wooly colonies flat colonies Growth time ▪ 24-48 h for inferior molds ▪ 24h-5 days for superior molds PCR methods Septate fungi (superior molds) PAGE 46 Aspergillus spp - microscopy - straight conidiophores - apical vesicle: uniseriate – just phialides biseriate – phialides + metula http://thunderhouse4-yuri.blogspot.com/2012/01/aspergillus-fumigatus.html - the conidia produced by the phialids can be arranged in a https://quizlet.com/492555410/fungi-flash-cards/ columnar or radial shape Septate fungi (superior molds) PAGE 47 Aspergillus niger Septate fungi (superior molds) PAGE 48 Aspergillus flavus Septate fungi (superior molds) PAGE 49 Penicillium spp - microscopy - straight or branched conidiophores - terminal phialides - conidia produced by the phialids DOI: 10.4103/0019-5278.165331 Septate fungi (superior molds) PAGE 50 Penicillium spp. Septate fungi (superior molds) PAGE 51 Fusarium spp. - microscopy - hyaline septate hyphae - conidiophores - phialides - conidia - macroconidia - microconidia Septate fungi (superior molds) https://commons.wikimedia.org/wiki/File:Fusarium_convolutans_(10.3897-mycokeys.34.25974)_Figure_5.jpg PAGE 52 Fusarium spp. https://commons.wikimedia.org/wiki/File:Fusarium_sp..jpg DOI: 10.5897/AJMR11.1081 Septate fungi (superior molds) PAGE 53 Antifungal susceptibility testing Not as routine Difficult, require special methods and reagents Dermatophytes PAGE 54 potential pathogens of humans and animals characteristic morphology - the presence of 2 types of conidia: ▪ microconidia (single cell) ▪ macroconidia (multiple cells) https://commons.wikimedia.org/wiki/File:Macroconidia_Microsporum_canis.JPG Dermatophytes PAGE 55 asexual forms (anamorphic): Epidermophyton, Microsporum, Trichophyton sexual forms: Arthroderma dermatophytes have the ability to break down keratin => superficial skin infections https://thebiologynotes.com/dermatophytosis/ Dermatophytes PAGE 56 Identification Direct microscopic examination samples taken are deposited on the glass slide and suspended in a drop of clarifying liquid (lactophenol or 1% KOH solution which digests the keratin and allows the visualization of the filaments) after 10-15 minutes cover with a cover slip examine under a microscope with a 20x objective the detection of dermatophytes is improved if fluorochromes (e.g. chalcofluor) are used DOI:10.3823/264 Dermatophytes PAGE 57 Identification Cultivation Sabouraud agar medium supplemented with chloramphenicol and cycloheximide the first culture is carried out in Petri dishes incubate at 25-30°C for approx. 4 weeks examine de culture media at least 2 times per week Dermatophytes PAGE 58 Identification in vitro hair perforation test - highlighting the presence of “perforating organs” of dermatophytes urease test nutritional needs test (vitamins or amino acids) cultivation on rice grains - differentiation of some species of microsporum thermotolerance test, thermostimulation (some species tolerate the temperature of 37°C, others are stimulated and others do not grow at all at this temperature) fluorescence test: examination of hair with uv light - in some dermatophyte infections fluorescence occurs Dermatophytes PAGE 59 Epidermophyton spp. E. flocossum - elongated macroconidia - mace appearance - sometimes arranged in a bouquet, contain 2-5 cells (lodges) - no microconidia Pathogenicity: tinea pedis (sometimes epidemic - gyms, swimming pools, shared showers) Dermatophytes PAGE 60 Trichophyton spp. T. ajelloi, T. mentagrophytes, T. rubrum, T. schonleinii, T. soudanese, T. terrestre, T. tonsuras, T. violaceum macro- and microconidia Dimorphic fungi PAGE 61 filamentous form in vitro and on culture media incubated at 30°C yeast form in vivo and on blood culture media highly pathogenic to humans and animals but not transmitted from human to human may affect both immunosuppressed and immunocompetent individuals contamination: aerogenous or transcutaneous imported infections DOI:10.1093/femsre/fuv035 Dimorphic fungi PAGE 62 Species Blastomyces dermatitidis Coccioides immitis Histoplasma capsulatum Paracoccidioides brasiliensis https://de.wikipedia.org/wiki/Paracoccidioides_brasiliensis https://universe84a.com/blastomyces-dermatitidis-general/ Practical work PAGE 63 1. Perform a wet mount (yeasts) 2. Perform a Gram stained smear (yeasts) 3. Microscopic examination of moulds: prepare an adhesive tape + lactophenol blue mount and examine it PAGE 64  PAGE 1 UNIVERSITÄTSMEDIZIN NEUMARKT A. M. https://edu.umch.de www.umfst.ro CAMPUS HAMBURG 2024 December Diagnosis of infections caused by Bacillus spp. Diagnosis of infections caused by anaerobic bacteria  PAGE 2 Pathogenic ▪ B. antharacis ▪ B. cereus Bacillus Non-pathogenic (Anthracoids) B. subtilis Bacillus anthracis PAGE 3 Disease: anthrax ▪ Cutaneous Infection ▪ Inhalational Infection (the woolsorters’ disease) ▪ Gastrointestinal Infection ▪ Injectional anthrax Complications: meningitis (5%) Potential infectious agents of bioterrorism (Category A – the greatest public health threat; easily transmitted and highly infectious)! Bacillus anthracis PAGE 4 Specimens - cutaneous anthrax - secretions from the lesion (vesicular fluid) - gastrointestinal anthrax - blood, feces - inhalation anthrax (woolsorters’ disease) - blood - sepsis - blood Meningitis - CSF Retrospective diagnosis – organ fragments Bacillus anthracis PAGE 5 Microscopy ▪ Gram stain Gram-positive rods with squared ends, specific arrangement in chains (bamboo) central spore Bacillus anthracis PAGE 6 Bacillus mycoides Bacillus antharacis, Gram 100x Microscopie în contrast de fază Sursa: Rutgers, The State University of New Jersey Bacillus anthracis PAGE 7 Cultivation - non-fastidious bacterium nutrient broth nutrient agar blood agar Bacillus anthracis PAGE 8 Identification morphology colonies ▪ R colonies - elevated central part, irregular, sometimes lobated margins ▪ M colonies – when plates are incubated in CO2 enriched atmosphere (capsule production) no hemolysis on blood agar plate Bacillus anthracis PAGE 9 Identification catalase - positive non-motile grows in high salt concentrations (7%) and low pH  Bacillus anthracis PAGE 10 Differential diagnosis (differentiation from other species: B. cereus, B. thuringiensis) ▪ B. anthracis is susceptible to penicillin and gamma phage (lytic bacteriophage) Retrospective diagnosis (veterinary medicine – confirmation of anthrax in animals) - Ascoli reaction – precipitation reaction in liquid - detects antigens from infected tissues  Bacillus cereus PAGE 11 Diseases Food poisoning: - diarrheal (ingestion of meat, poultry, pastas, vegetables) - emetic (fried rice) Ocular Disease Other Conditions: sepsis, meningitis, infections associated with central venous catheters, etc. To confirm food poisoning: - quantitative culture (more than 105 B. cereus cells per gram of food are present and the absence of other pathogens) B. cereus is resistant to penicillin. Diagnosis of infections caused by anaerobic bacteria PAGE 12 Endogenous anaerobic bacteria Gram-positive cocci: Peptostreptococcus, Peptococcus Gram-negative cocci: Veillonella are present in the Gram-positive bacilli: Lactobacillus, normal flora: oral cavity, Propionibacterium, Actinomyces, Eubacterium, digestive tract, vagina Bifidobacterium Gram-negative rods: Bacteroides spp., Prevotella, Porphyromonas, Fusobacterium, etc. Exogenous anaerobic bacteria Spore-forming Gram-positive bacilli – Clostridium spp. Diagnosis of infections caused by anaerobic bacteria PAGE 13 Clues for suspecting anaerobic infections, characteristics of anaerobic infections: deep abscesses (brain, liver, etc.) fowl smell presence of foreign bodies, necrotic tissue, poor blood supply, tumors, metabolic disorders contamination with anaerobic flora during surgery (abdominal, female genital area, oral cavity) usually mixed infections – several species are involved, including facultative anaerobic species Diagnosis of infections caused by anaerobic bacteria PAGE 14 Adequate specimens for detection anaerobic bacteria ▪ deep specimens (from bites, wounds contaminated with soil, crashed tissue) ▪ aspirated pus from abscesses ▪ gingival, perioral abscesses ▪ pus aspirated from paranasal sinuses ▪ lower respiratory tract specimens collected during bronchoalveolar lavage ▪ ulcers (“diabetic foot”, decubitus) ▪ tissue collected during surgery ▪ urine collected by suprapubic aspiration ▪ blood Diagnosis of infections caused by anaerobic bacteria PAGE 15 Inadequate specimens for detection of anaerobic infections superficial gingival secretions throat swab, nasopharyngeal swab sputum stool (exception: C. difficile) urine vaginal/cervical secretion superficial lesions Diagnosis of infections caused by anaerobic bacteria PAGE 16 Protection of specimens ▪ special containers (devoid of air) ▪ transport media Specimens should be kept at room temperature (do not refrigerate)!! Processing in 30 minutes – in case of small quantities (0,5 ml) in larger volume (over 1 ml) bacteria remain viable for 1-2 hours in the presence of pus in transport media viability is prolonged for a few days Diagnosis of infections caused by anaerobic bacteria PAGE 17 Microscopy: - describe morphology - note: Gram staining is frequently unreliable in case of anaerobic bacteria Diagnosis of infections caused by anaerobic bacteria PAGE 18 Cultivation - special media for anaerobic bacteria liquid media: thyoglicolate – used for enrichment solid media: anaerobic blood agar plate (Columbia, Schaedler, etc.) selective media for anaerobic bacteria (PEA- Phenyl-ethyl alcohol, BBE – Bacteroides bile-esculin, KVLB – kanamycin-vancomycin laked blood, EYA – egg-yolk agar, etc.) Diagnosis of infections caused by anaerobic bacteria PAGE 19 Incubation: in anaerobiosis classic methods (pyrogallol, Fortner method - historical) anaerobic jar – with oxygen reducing agents anaerobic chamber D