Clinical Bacteriology Lecture Notes (WEEK 1-4) PDF

CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 WEEK 1: GRAM NEGATIVE COCCI & FASTIDIOUS GRAM NEGATIVE BACILLI General Characteristics of Neisseria spp....

CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 WEEK 1: GRAM NEGATIVE COCCI & FASTIDIOUS GRAM NEGATIVE BACILLI General Characteristics of Neisseria spp. NEISSERIACEAE are: Plump cocci, coccobacilli, or rods Gram negative Nonmotile Strictly or preferentially aerobic Optimal temperature, 32C – 36C The family NEISSERIACEAE contains the 4 genus: NEISSERIA are: KINGELLA are: EIKENELLA are: SIMONSIELLA are: Cocci Bacilli Bacilli Bacilli Adjacent both sides Pairs and chains Catalase (-) Gliding motility flattened Cell division-one plane Oxidase (+) Normal huma oral biota Cell division-two planes Catalase (-) Large, flat multicellular Catalase (+) Oxidase (+) filaments Oxidase (+) Catalase (-) Oxidase (+) NEISSERIA SPECIES are: KINGELLA SPECIES are: EIKENELLA SPECIES are: Human isolates Kingella denitrificans Eikenella corrodens PATHOGENS: Kingella kingae Neisseria gonorrhoeae Kingella oralis Neisseria meningitidis Neisseria weaver (bacillus) – normal oral biota of dogs, wounds from dog bites MNEMONICS: Neisseria G,M,W NONPATHOGENS but may cause opportunistic infections in human hosts: Neisseria cinerea Neisseria elongata (bacillus) Neisseria flavescens Neisseria lactamica Neisseria mucosa Neisseria polysaccharrea Neisseria sicca Neisseria subflava (biovars flava, perflava, subflava) *Moraxella catarrhalis, although morphologically and biochemically similar to the Neisseria spp., is NOT a member of the family Neisseriaceae. Most Neisseria spp. is: ü Aerobic All Neisseria spp. is: ü Nonmotile ü Cytochrome oxidase ü Non-spore-forming ü Catalase positive ü Gram-negative EXCEPT: ü Diplococci ´ Neisseria bacilliformis EXCEPT : ´ Neisseria elongata subsp. ´ Neisseria weaver ´ Nitroreducens ´ Neisseria elongata ´ Neisseria bacilliformis Because they are catalase negative. Because they are rod-shaped. MNEMONICS: BEN MNEMONICS: WEB MORALES, MARIA MARIELLA 1 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 Many Neisseria spp are: CAPNOPHILIC - this means they require carbon dioxide to grow - they also need humid atmosphere - They can grow anaerobically if alternative electron acceptors (ex. nitrites) are available – oxidizing agents NATURAL HABITAT OF NEISERRIA spp: - Mucous membranes of respiratory and urogenital tracts - Usually found here but does not mean it is a normal flora. PRIMARY HUMAN PATHOGENS: - N. gonorrhoeae – significant to humans, will never be part of normal flora of the body. always pathogenic - N. meningitidis – significant to humans, can be found in respiratory tract of carriers - N. weaver – from animals PATHOGENIC NEISSERIA SPP. are: - Fastidious organisms – it needs a complex, strict, optimal type of nutritional requirement and is sensitive. - Sensitive to unfavorable environmental conditions – needs careful preservation and transport - Require iron for growth – these organisms compete with iron requirement of human body For N. weaver: - Commensal in the upper respiratory tract of dogs - Pathogenic In animals All other Neisseria spp. are considered opportunistic pathogens. (they can cause infection to immunocompromised) *Moraxella was formerly known as Neisseria. MORALES, MARIA MARIELLA 2 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 Pathogenic Neisseria spp. VIRULENCE FACTORS: FACTORS THAT CONTRIBUTE TO THEIR VIRULENCE: ü Receptors for human transferrin - Transferrin is type of protein that transports iron. - Decrease iron, increase transferrin ü Capsule (N. meningitidis) - This causes mucoid colony appearance in CAP. ü Pili (fimbriae) - This pili serves as an adherence to the surface of epithelial cells. Neisseria gonorrhoeae has 5 TYPES OF PILI: T1 – virulent form, containing presence of pili T2 – virulent form, containing presence of pili T3 – avirulent form, lacking the presence of pili (devoid) T4 – avirulent form, lacking the presence of pili (devoid) T5 – avirulent form, lacking the presence of pili (devoid) *During collection of swab N. gonorrhoeae, at the start of infection, colony appearance will contain T1 &T2 *On subculture, appearance will be T3-T5, pili are lost. ü Cell membrane proteins Cell membrane proteins are I, II, III and LOS What do you call the major outer membrane porin protein? - Protein I (por) They are coded by two genes: - por A - por B *N. MENINGITIDIS HAS: both por A and por B *N. GONORRHOEAE HAS: por B only What do you call the group of proteins that facilitate the adherence to phagocytic and epithelial cells? - Protein II (Opa) What do you call the modified protein that blocks the serum bactericidal (IgG) action against organism? - Protein III (Reduction modified protein RMP) ü Lipooligosaccharide (LOS) or endotoxin - LOS endotoxin increases virulence is a major in vivo virulence factor that mediates damage to body tissues and elicits inflammatory response. ü Immunoglobulin A (1gA) protease that cleaves IgA on mucosal surface Neisseria gonorrhoeae Human - only natural host, Agent of the sexually transmitted disease (gonorrhea) PRIMARY SITE OF INFECTION OF GONORRHEA: ü Urethra ü Endocervix ü Anal canal – anal sex ü Pharynx – oral sex ü Conjunctiva – newborn babies with mothers with gonorrhea (ophthalmia neonatorum) EPIDEMIOLOGY OF N. GONORRHOEAE Most commonly transmitted through sexual contact Asymptomatic carrier - primary reservoir - one of the first symptoms is release of yellowish discharge National reportable disease - Confirmed cases must be reported to state health laboratories (US) Highest rates of infection: - Men and women between the age of 20 and 24 - Gonorrhea is the second to Chlamydia trachomatis of confirmed sexually transmitted bacterial infection in the US. MORALES, MARIA MARIELLA 3 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 CLINICAL INFECTIONS: INCUBATION PERIOD of N. GONORRHOEAE? approx. 2 to 7 days *may be asymptomatic in 3rd day but can manifest in 4th day onwards GONORRHEA IS ACUTE PYOGENIC INFECTION OF THE? ü Urogenital tract ü Pharynx ü Conjunctiva of the eye INFECTIONS ARE LESS LIKELY IN? ü Disseminated infections – possible if N. gonorrhea is AHU strain ü Anal canal IN MEN: N. GONORRHOEAE IN MEN ARE: Transmitted only by intimate sexual contact Asymptomatic gonococcal infection is uncommon. 90% of infected men show symptoms of acute infection. 10% are asymptomatic COMMON MANIFESTATION OF N. GONORRHOEAE IN MEN ü Acute urethritis – purulent discharge ARE: ü Dysuria – painful urination COMPLICATIONS OF N. GONORRHOEAE IN MEN ARE: ü Prostatitis ü Epididymitis ü Urethral stricture IN WOMEN: COMMON SITE OF INFECTION OF N. GONORRHOEAE IN endocervix WOMEN: SYMPTOMS OF N. GONORRHOEAE IN WOMEN ARE: ü Dysuria – painful urination ü Cervical discharge ü Lower abdominal pain *50% are asymptomatic COMPLICATIONS OF N. GONORRHOEAE IN WOMEN ARE: ü Pelvic Inflammatory Disease ü Sterility ü Ectopic pregnancy or ü Fitz-Hugh-Curtis Syndrome CLINICAL INFECTIONS IN OTHER SITES: BLOOD-BORNE DISSEMINATION OF N. GONORRHEA Less than 1% of all infections, resulting in purulent arthritis and OCCURS IN? rarely septicemia. Fever, intermittent bacteremia, and rash on the extremities may be present. Most disseminated gonococcal infections are attributed to from arginine, hypoxanthine, and uracil (AHU) strains the? affecting women SPS is NOT recommended anti-coagulant in blood culture sodium polyanethol sulfonate (SPS) inhibits N. gonorrhoeae media because? - Use gelatin to neutralize effects of SPS. GONOCOCCAL ARTHRITIS results to? ü Purulent arthritis ü Result of disseminated gonococcal bacteremia EXTRAGENITAL INFECTIONS – what diseases are outside Oropharyngeal infections – obtained from oral sex the urogenital tract? ü Pharyngitis – is the chief complaint in symptomatic Oropharyngeal infections. MORALES, MARIA MARIELLA 4 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 ANORECTAL INFECTIONS are? Most common in men who have sex with men 30% to 60% of women with genital infection can get anorectal infection. Rectal pain or bloody stools possible Most infections are asymptomatic or have nonspecific symptoms. WHAT IS THE DISEASE IN NEWBORNS ASSOCIATED WITH ü Ophthalmia neonatorum N. GONORRHEA? - Gonococcal eye infection occurs during vaginal delivery through infected birth canal. - If not treated immediately, newborn blindness can occur. - Infection is preventable with the application of eye drops at birth (erythromycin eye drops). - Every infant at birth, by law, must be treated. SPECIMEN COLLECTION AND TRANSPORT SPECIMENS COLLECTED FOR THE RECOVERY OF N. ü Rectal culture GONORRHOEAE are: - Insert swab 4 to 5 cm into the anal canal. ü Oral/pharyngeal ü Eye - aspiration ü Blood/joint fluids - aspiration *for penile discharge you should insert the swab in the male reproductive and then rotate, do not collect only on the outside discharge. When possible, notification of pending Neisseria sample should be made to the laboratory. What is the PREFFERED SWAB for specimen collection? ü Dacron or rayon Swabs that are NOT recommended? ´ Calcium alginate and cotton swabs are INHIBITORY to N. gonorrhoeae Direct plating of specimen to gonococcal-selective media gives optimal results. What are the TRANSPORT SYSTEM/MEDIA to be used? Transport systems are available that contain selective media and a CO, capnophilic atmosphere. ü JEMBEC, ü Gono-Pak, ü Transgrow Swab should be rolled in a? Specimen rolled in a "Z" pattern on media When direct plating is not possible, inoculated swabs ü Amies medium with charcoal, plated within 6 hours of should be placed in a transport system such as? collection. DIRECT MICROSCOPIC EXAMINATION: For Urogenital specimens, perform a: ü Perform Direct Gram Stain - this is the initial determination - check the morphology For Pharyngeal specimens: ´ Direct Gram stain is NOT recommended because commensal Neisseria spp. can be present, - Ex: N. meningitidis MORALES, MARIA MARIELLA 5 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 MICROSCOPIC MORPHOLOGY What is the MORPHOLOGY OF URETHRAL DISCHARGE OF ü Kidney bean-shaped gram-negative intracellular A SYMPTOMATIC MALE? diplococci - inside the neutrophil you will see diplococci (intracellular) ü When present, 89% likely to be gonococcal infection For Avirulent forms (lacking pili) are seen as: - T3, T4, T5 ü Extracellular gram-negative diplococci (outside) In women: ü 50% to 70% correlation of direct Gram stain with culture ü Less diagnostic because of similar vaginal flora ü Discharge in symptomatic women ü Gram stain may be helpful but culture is necessary for confirmation. ü Gram stain with more than five polymorphonuclear neutrophils per field with no bacteria suggests nongonococcal urethritis. - Chlamydia is the causative agent of nongonococcal urethritis. DIRECT GRAM-STAINED SMEARS: A. Male urethral discharge showing intracellular and B. Direct smear with more than five polymorphonuclear extracellular gram-negative diplococci diagnostic for N. neutrophils per field but not bacteria suggestive of gonorrhoeae. nongonococcal urethritis N. GONORRHOEAE CULTURE AND MEDIA INCUBATION CHARACTERISTICS: What is the general media culture used for N. gonorrhoeae? Chocolate Agar Plate (CAP) - this because iron is needed for growth - If blood agar plate will be used, heat it to lyse the RBC and release iron Chocolate agar supplemented with inhibitors for gram- Prevent overgrowth of normal flora positive, gram-negative, and yeast - selective media that makes only N. gonorrhoeae grow INOCULATED CULTURE MEDIA MUST BE? ü incubated at 35° C in 3% to 5% carbon dioxide (CO2). - Use CO2 incubator, generating pouch, or candle extinction jar - this is to maintain anaerobiosis because Neisseria is capnophilic MORALES, MARIA MARIELLA 6 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 SELECTIVE MEDIA FOR N. GONORRHOEAE & N. MENINGITIDIS Vancomycin: inhibits the growth of other gram-positive organisms Colistin: inhibits the growth of gram-negative organisms Nystatin: inhibits the growth or yeast Trimethoprim: inhibits the growth of swarming proteus spp. N. GONORRHOEAE COLONY MORPHOLOGY: Colony morphology on MODIFIED THAYER-MARTIN (MTM) ü Small, gray to tan AGAR ü Translucent, raised T1 and T2 colony types ü Small and raised ü Appear bright in reflected light T3 to T5 colony types ü Large and flat MORALES, MARIA MARIELLA 7 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 N. GONORRHOEAE MICROSCOPIC MORPHOLOGY: Gram stain must be performed on suspected isolates to verify appearance of gram-negative diplococci. Some gram-negative bacilli (rods) occasionally grow on gonococcal selective media (such as Kingella and Acinetobacter spp.). To differentiate some gram-negative rods from gram-negative The microscopic morphology of Neisseria gonorrhoeae remains diplococci, the organism can be streaked to a plate, and a 10- gram-negative diplococci (arrow) using the 10-unit penicillin disk unit penicillin disk can be added. After growth, the edge of (x1000) zone of inhibition is stained to visualize microscopic morphology - Neisseria can be also resistant to penicillin The gram-negative rod microscopic morphology of Kingella The elongated gram-negative rod microscopic morphology of spp. after the penicillin disk test (x1000) Acinetobacter app. after the penicillin disk test (x1000) N. GONORRHOEAE DEFINITIVE IDENTIFICATION: Other oxidase-positive gram-negative diplococci can grow on selective media from the same sites where N. gonorrhoeae is expected. Examples: N. cinerea, N. meningitidis, M. catarrhalis Further tests are required to differentiate these organisms. (Ex: Carbohydrate test) CHARACTERISTICS OF SIGNIFICANT SPECIES OF NEISSERIA, MORAXELLA, AND KINGELLA MORALES, MARIA MARIELLA 8 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 N. GONORRHOEAE ANTIMICROBIAL RESISTANCE: Plasmid-mediated penicillinase-producing N. gonorrhoeae - the organism acquires plasmid with B-Lactamase (PPNG) production Chromosome-mediated penicillin resistance (PenR) - these isolates were B-Lactamase negative - Resulting in alterations in penicillin binding proteins (PBPs) Plasmid-mediated tetracycline resistance (TRNG) Chromosomal-mediated resistance to both penicillin and tetracycline (CMRNG) Chromosomal-mediated spectinomycin resistance Chromosomal-mediated fluoroquinolone resistance (QRNG) N. GONORRHOEAE TREATMENT: According to Centers for Disease Control and Prevention On 2007 CDC no longer recommended use of (CDC) fluoroquinolones due to resistance - Chromosomal-mediated fluoroquinolone resistance (QRNG) 2015 STD Treatment Guidelines: ü Ceftriaxone (cephalosporin) is currently recommended. Co-infection with Chlamydia trachomatis frequently occurs: ü Ceftriaxone plus azithromycin is recommended regiment Neisseria meningitidis GENERAL CHARACTERISTICS OF NEISSERIA MENINGITIDIS Neisseria meningitidis is only found on? Found only in humans Neisseria meningitidis may be characterized as? May be commensal or an invasive pathogen Neisseria meningitidis is an Important etiologic agent of? epidemic meningitis and meningococcemia Neisseria meningitidis is rarely associated with? Rarely associated with pneumonia, purulent arthritis, or endophthalmitis Where can you recover Neisseria meningitidis? Has been recovered from urogenital and rectal sites Neisseria meningitis causes meningitis because of? Because it is capsulated EPIDEMIOLOGY OF N. MENINGITIDIS Found on the mucosal surfaces ü Nasopharynx and oropharynx in approximately 30% of population Transmitted by close contact with respiratory droplet secretions from carrier to new host Only a few colonized hosts develop meningococcal disease. ü Highest incidence in infants and adolescents Populations at risk of invasive disease: ü Splenic patients – patients without spleen ü Individuals with complement deficiencies – they cannot fight infection ü Populations at risk of serious disease: o Smokers, o People exposed to smoke, o Individuals with concurrent upper respiratory tract viral infection Contributors to the spread of meningococcus: ü Crowded living conditions ü Military recruits ü College students Meningococcus disease is uncommon in developed nations. MORALES, MARIA MARIELLA 9 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 CLINICAL INFECTIONS OF N. MENINGITIDIS INCUBATION PERIOD OF N. meningitidis? ü Incubation period is 1 to 10 days by colonizing mucosa. ü A small number have bacteria enter bloodstream and potentially the central nervous system (CNS). - because of its capsule it can evade the action of phagocytic cells ü Leads to septicemia and/or meningitis CHARACTERIZATION OF MENINGITIS: ü Frontal headache ü stiff neck ü Confusion ü photophobia ü Nausea and vomiting ü Sometimes fever ü Purulent meningitis What are the 2 main diseases when N. meningitidis enters ü fulminant meningococcemia the bloodstream? ü or meningitis What do you call the hemorrhage in the adrenal glands Waterhouse-Friderichsen syndrome caused by N. meningitidis? MENINGOCOCCEMIA IN INFANTS AND HEMORRHAGE IN WATERHOUSE-FRIDERICHSEN SYNDROME Petechial skin rash associated with meningococcemia in an Hemorrhage (dark red areas) in the adrenal gland, infant. Waterhouse-Friderichsen syndrome. N. MENINGITIDIS SPECIMEN COLLECTION AND TRANSPORT: SPECIMENS OF CHOICE: ü Cerebrospinal fluid (CSF) ü Blood* ü Nasopharyngeal swabs or aspirates ü Joint fluids ü Less commonly sputum and urogenital sites SPS IS NOT RECOMMENDED FOR BLOOD CULTURE: *Because N. meningitides is inhibited by SPS, non-SPS-containing blood collection system must be used. N. MENINGITIDIS DIRECT MICROSCOPIC EXAMINATION On gram-stained smears from specimens such as CSF, intracellular and extracellular gram-negative diplococci Meningococci appear as? The highest yield of positive results of CSF gram stain is When specimens are centrifuged. obtained when? - Concentration of CSF can improve detection on direct exam. How many mL of CSF and it should be centrifuged at? At least 1 mL CSF should be centrifuged at x1000g for 10 minutes. - The sediment is used for direct Gram stain and culture What centrifuge is used for best results? Cytocentrifuge is recommended and increases the sensitivity for detection by concentrating small numbers of organisms 100- fold. MORALES, MARIA MARIELLA 10 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 DIRECT GRAM-STAINED SMEAR OF CSF: Direct gram-stained smear of CSF illustrating intracellular and extracellular gram-negative diplococci (arrows) of Neisseria meningitidis. N. MENINGITIDIS CULTURE AND INCUBATION: For isolation of N. meningitidis, it should be cultured on: sheep blood agar (SBA) and chocolate agar (CAP) - For specimens with normal flora, use selective media Cultures should be INCUBATED at? Incubate at 35°C at 3% to 5% CO2 - Neisseria are capnophilic Plates should be examined every? Examine plates daily for 72 hours. N. meningitidis isolates should use what BSL? Suspected isolates should be handled in biosafety level 2 (BSL-2) cabinet. COLONIES AFTER 48 HRS OF INCUBATION: Growth of Neisseria meningitidis after 48 hours on chocolate (CAP) agar (left) and sheep blood agar (SBA) (right). Of the Neisseria pathogens, only the meningococcus grows on SBA and CAP agar. IDENTIFICATION OF N. MENINGITIDIS: Identification is based on? ü colony morphology, ü microscopic morphology, ü and the oxidase test. Colonies usually grow within how many hours? Colonies usually grow within 18 to 24 hours. COLONY MORPHOLOGY: ü Medium-sized, gray, convex ü Encapsulated strains are mucoid ü Blood underneath colonies tend to have green tinge. MORALES, MARIA MARIELLA 11 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 What is the result of NEISSERIA IN OXIDASE AND Oxidase test - positive CATALASE TEST? Catalase test – positive DEFINITIVE DIAGNOSIS can be made via: ü carbohydrate methods (organism uses glucose and maltose). - N. gonorrhoeae can ferment: GLUCOSE ONLY - N. meningitidis can ferment: GLUCOSE and MALTOSE ü chromogenic enzyme tests. ü multitest assays. If testing identifies an isolate as N. meningitidis, serotyping should be done using? Molecular techniques | MALDI-TOF MS N. MENINGITIDIS TREATMENT: FOR CONFIRMED MENINGITIS: Penicillin FOR MENINGOCOCCEMIA: Third-generation cephalosporins FOR CLOSE CONTACTS OF PATIENTS: Chemoprophylaxis with rifampin or ciprofloxacin Use Azithromycin if ciprofloxacin is resistant. NOT RECOMMENDED: Routine susceptibility testing is not recommended. Chemoprophylaxis not recommended for asymptomatic carriers N. MENINGITIDIS VACCINE: Two quadrivalent polysaccharide-protein conjugated vaccines are available for some subtypes (only for A, C, Y, and W-135). Vaccinations are recommended for ü military recruits. ü splenic patients older than 2 years of age. ü laboratory scientists who work with N. meningitidis. ü other high-risk groups. Moraxella catarrhalis Moraxella catarrhalis belongs to the family: Family: MORAXELLACEAE Moraxellaceae has 3 genus: MORAXELLA ACINETOBACTER PSYCHROBACTER Moraxella catarrhalis is characterized as: Commensal only of the upper respiratory tract Carbohydrate fermentation of M. catarrhalis: ASACCHAROLYTIC: CANNOT ferment glucose, maltose, sucrose or lactose *Carbohydrate fermentation of Other Neisseria spp: N. subflava & N. sicca - positive to glucose, maltose, and sucrose N. lactamica - positive to glucose, maltose, and lactose except sucrose M. catarrhalis is Isolated only from? Isolated only from humans M. CATARRHALIS CLINICAL INFECTIONS Opportunistic pathogen and cause of URT infection in healthy children and the elderly Can cause RT infections in adults with chronic obstructive pulmonary disease (COPD) Predisposition may include advanced age, immunodeficiency, neutropenia and chronic debilitating diseases 3rd most common cause of acute otitis media and sinusitis in children MORALES, MARIA MARIELLA 12 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 IDENTIFICATION OF NEISSERIA SPECIES AND MORAXELLA CATARRHALIS Direct smear of an otitis media specimens showing On CHOC (CAP) growth of M.catarrhalis after 48 hours like intracellular gram-negative diplococci. "wagon wheel" appearance. - similar to Neisseria spp. MORAXELLA CATARRHALIS LABORATORY DIAGNOSIS SPECIMEN COLLECTION AND IDENTIFICATION SPECIMENS used for M. catarrhalis? Specimens from ü middle ear effusion, ü nasopharynx, ü sinus aspirates, ü sputum aspirates ü or bronchial aspirates What is the CULTURE MEDIA used for M. catarrhalis? Sheep Blood Agar (SBA) and Chocolate agar plate (CAP) What is the COLONY APPEARANCE on SBA and CAP? On SBA and Choc (CAP), appear as smooth, opaque, gray to white colonies (hockey puck) because it remains intact when pushed across the plate with a loop - Older colonies give a "wagon wheel" appearance What is the OPTIMAL GROWTH TEMPERATURE of M. Optimal growth temperature at 35° - 37° Celsius catarrhalis? M. catarrhalis OXIDASE AND CATALASE TEST result? Oxidase (+) and Catalase (+) Differentiate M. catarrhalis from Neisseria species by: (+) DNAse and butyrate esterase reactions (substrate is Tributyrin) Haemophilus and Other Fastidious Gram-Negative Bacilli HACEK Group and Similar Microorganisms HACEK group is from the family: Family: Pasteurellaceae It has 4 genera: HAEMOPHILUS ACTINOBACILLUS PASTEURELLA AGGREGATIBACTER General characteristics of Pasteurellaceae: ü gram negative, pleomorphic, coccoid shaped to rod- shaped cells that are nonmotile and facultatively anaerobic ü They form nitrates to nitrites and are oxidase (-) and catalase (+). ü Require special nutrients for isolation and identification General characteristics of HACEK: ü fastidious, pleomorphic (many shapes), gram negative bacilli HACEK group is composed of: ü Haemophilus ü Aggregatibacter ü Cardiobacterium ü Eikenella ü Kingella HACEK reside in: reside in the human oral cavity and can cause endocarditis. MORALES, MARIA MARIELLA 13 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 Other fastidious gram-negative bacilli include: ü Capnocytophaga, ü Brucella and ü Francisella Many members of the Genus Pasteurella, Brucella and zoonoses (animal diseases that are transmitted to humans Francisella causes: from the primary animal host) HAEMOPHILUS GENERAL CHARACTERISTICS OF HAEMOPHILUS: ü Gram negative, pleomorphic coccobacilli or rods in direct smears of clinical material to long filaments. ü Nonmotile and facultatively anaerobic ü Ferment Carbohydrates ü Reduce nitrates to nitrites ü Obligate parasites in the mucous membranes of humans and animals Haemophilus OXIDASE AND CATALASE test result? Oxidase (-) and catalase (+) 8 HAEMOPHILUS SPECIES ASSOCIATED WITH HUMANS: ü H. influenzae, ü H. parahaemolyticus, ü H. paraphroheamolyticus, ü H.parainfluenzae, ü H. haemolyticus, ü H.pittmaniae, ü H. aegvptius ü H. ducreyi (causes the STD chancroid) What are the MAJOR HAEMOPHILLUS PATHOGENIC ü H. influenzae, SPECIES? ü H. aegyptius and ü H. ducreyi – causative agent of soft chancre Haemophilus means? Derived from the Greek word which means "blood lover" What are the REQUIRED PERFORMED GROWTH FACTORS X factor PRESENT IN BLOOD? - hemin or hematin - X for “unknown” V factor - nicotinamide adenine dinucleotide (NAD) - V for “vitamin” or both What is the CULTURE MEDIA used for isolation of Clinical laboratories use Chocolate Agar Plate for the Haemophilus? recovery of Haemophilus spp from clinical specimens. Why? The lysing of red cells by heat in the preparation of CHOC (CAP) releases both the X and the V factors and inactivates NADases. What is the PHENOMENON THAT HELPS IN THE Satellitism: a phenomenon that helps in the recognition of RECOGNITION OF HAEMOPHILUS SPP? Haemophilus sp. that require V factor. This occurs when an organism such as Staphylococcus aureus, Streptococcus pneumonia or Neisseria species produces V factor as a by-product of metabolism Haemophilus influenzae satellitism around and between large, white, hemolytic staphylococci. The small, grey and glistening colony is H. influenza (arrow). Satellitism: The isolate obtains X factor from the SBA and V factor from one of these organisms. On SBA, tiny colonies are growing around the V factor-producing organisms. The picture shows H. influenzae satellitism around colonies of S.aureus. MORALES, MARIA MARIELLA 14 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 VIRULENCE FACTORS OF H. INLUENZAE 1. CAPSULE - Capsule plays a significant role in the serologic grouping of Haemophilus influenzae into 6 antigenically distinct types: (a,b,c,d,e,f) based on the differences in the capsular polysaccharide *Haemophilus influenzae can cause meningitis because it is capsulated WHAT IS THE LEADING CAUSE OF MENINGITIS IN In unvaccinated children, type b is the leading cause of UNVACCINATED CHILDREN? meningitis that occurred primarily in young children Has antiphagocytic and anticomplementary activity of the type b capsule contribute to its virulence and the pathogenesis of invasive disease. *In neisseria meningitis: Type y is the most common What is the STRAIN OF HAEMOPHILUS INFLUENZAE THAT Non-typable. H. influenzae (NTHi) - strains that not IS NON-CAPUSLATED? encapsulated. 2. IMMUNOGLOBULIN A (IgA) PROTEASES - the only member of the genus that produces lgA protease which can cleave secretory lgA present on human mucosal surfaces of the respiratory tract 3. ADHERENCE BY FIMBRIAE AND OTHER STRUCTURES - most NTHi strains are adherent to human epithelial cells and most serotype b are not adherent. - The lack of adherent capability of type b explain why type b cause systemic infections. - The NTHi strains however cause more localized infections such as acute conjunctivitis because of its adherent capability. 4. OUTER MEMBRANE PROTEINS AND - responsible for a specific activity such as invasiveness, LIPOPOLYSACCHARIDES (LPS) attachment and antiphagocytic function. - LPS has a paralyzing effect on the sweeping motion of ciliated respiratory epithelium. CLINICAL MANIFESTATION OF H. INFLUENZAE Clinical Manifestations of H. influenzae has 2 PATTERNS 1. Invasive disease caused by encapsulated strains OF DISEASE: ü bacteremia ü septicemia ü meningitis ü arthritis ü epiglottitis ü Tracheitis ü and pneumonia. 2. Localized infection caused by the contiguous spread of NTHi strains - occurs within or close proximity to the respiratory tract. ü conjunctivitis, ü sinusitis ü and otitis media with effusion (middle ear infections ) MENINGITIS Before Hib (Haemophilus influenzae type b) vaccine - children between the ages of 3 months and 6 years were caused by serotype b. Symptoms include headache, stiff neck, other meningeal signs preceded by mild respiratory disease Bloodstream invasion and bacteremia spread follow colonization, invasion and replication of the bacteria in the respiratory mucous membranes. MORALES, MARIA MARIELLA 15 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 EPIGLOTTITIS Manifestations include rapid onset, acute inflammation and intense edema of the epiglottis causing complete airway obstruction which may require an emergency tracheostomy. To avoid causing further possible damage, the area is not swabbed for culture but is empirically treated based on signs and symptoms. Peak incidence occurs in children between 2 and 4 years old The most important aspect of treatment is maintenance of a secure airway BACTERIAL TRACHEITIS Serious life-threatening disease in young children Can arise after an acute, viral respiratory infection Initial presentation includes a mild to moderate illness for 2 to 7 days which may rapidly progress Use of broad-spectrum antimicrobial agents during the acute stages is important because thick secretions can occlude the trachea The disease must be differentiated from epiglottitis HAEMOPHILUS AEGYPTIUS Haemophilus aegyptius is also known as? Koch-Weeks bacillus Haemophilus aegyptius is observed in? Observed in conjunctivitis exudates from Egyptians by Koch in 1883 - Associated with an acute, contagious conjunctivitis commonly referred to as the "pink eye conjunctivitis” Haemophilus influenza biogroup aegyptius Can cause conjunctivitis primarily in pediatric populations In 1984, a severe systemic disease known as Brazilian purpuric fever (BPF) was caused by a clone of this bacteria. BPF is characterized by recurrent or concurrent conjunctivitis, high fever, vomiting, petechial, purpural rash, septicemia, shock and vascular collapse. HAEMOPHILUS PARAINFLUENZAE H. parainfluenzae is found on? Found in the oral cavity H. parainfluenzae incidence: Very low incidence of pathogenicity H. parainfluenzae is RARELY implicated as causative agent Rarely implicated as a causative agent of endocarditis of? If implicated, it has an insidious onset (progressing disease without marked symptoms); first symptoms appear approximately 1 month after a routine dental procedure What is the PRIMARY SITE OF INFECTION OF H. The mitral valve is the primary site of infection PARAINFLUENZAE? HAEMOPHILUS PARAHAEMOLYTICUS H. parahaemolyticus can cause? May be a cause of pharyngitis in the absence of other pathogens MORALES, MARIA MARIELLA 16 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 HAEMOPHILUS DUCREYI Haemophilus ducreyi causes the disease? Causative agent of chancroid (soft chancre), a sexually transmitted genital ulcer disease (GUD) Infects mucosal epithelium, genital and non-genital skin, and regional lymph nodes Chancroid facilitates the transmission of other STDs All patients who have GUD should also be tested for HIV along with syphilis and herpesvirus Nonindurated, painful lesion with irregular edge develops on the genitalia or perianal areas INCUBATION PERIOD of H. ducreyi? Incubation period is from 4 to 14 days Causative agent of SOFT CHANCRE? Haemophilus ducreyi Causative agent of HARD CHANCRE? Treponema pallidum (syphilis) MOST COMMON SITE OF INFECTION OF H. DUCREYI? Most common sites of infection are on the penis, the labia or within the vagina Clinical manifestations of H. ducreyi? Most infected patients have suppurative (pus-forming) enlarged, draining, inguinal lymph nodes (buboes) Men are symptomatic with inguinal tenderness and genital lesions while women are asymptomatic Lesions of chancroid penis, showing a draining bubo (arrow) in the adjacent groin area. Chancroid is caused by Haemophilus ducreyi. LABORATORY DIAGNOSIS OF HAEMOPHILUS SPECIES SPECIMEN PROCESSING AND ISOLATION Specimens for Haemophillus species may include: ü Blood ü CSF ü Middle ear exudate ü Joint fluids ü Upper and lower respiratory tract specimens -bronchial wash is recommended ü Conjunctival swabs ü Vaginal swabs ü Abscess drainage MORALES, MARIA MARIELLA 17 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 LABORATORY DIAGNOSIS (HAEMOPHILUS SPECIES) H. influenzae. Nasal and nasopharyngeal swabs have no clinical value in evaluation for respiratory tract infections caused by H. influenzae. H. ducreyi Genital specimens submitted for H.ducreyi, genital sites must be cleaned with sterile gauze moistened with sterile saline. Next, a swab premoistened with sterile phosphate buffered saline should be used to collect material from the base of the ulcer. Alternatively, pus can be aspirated from buboes if they are present ü Prompt transportation and processing are vital for isolation ü Direct plating on selective media at the bedside is preferred instead of transport media CULTURE MEDIA USED for isolation of Haemophilus Chocolate agar plate (CAP) is the commonly used medium species? Culture media should be incubated at? incubated between 33 to 37 degrees Celsius with 5 to 10% CO2 What is the excellent medium for the isolation of CAP supplemented with Bacitracin (300 mg/L) HAEMOPHILUS SPECIES FROM RESPIRATORY SPECIMENS? Specimens submitted for H.ducreyi and H.aegyptius must be plated to a special media. SPECIAL MEDIA FOR H. AEQYPTIUS: enriched CHOC (CAP) supplemented with 1% IsoVitaleX or Vitox SPECIAL MEDIA FOR H. DUCREYI: enriched CHOC(CAP) or Nairobi biplate (GC agar base) with 2% bovine hemoglobin and 5% fetal calf serum on one half and Mueller Hinton agar with 5% chocolatized horse blood on the other). Both sides contain 3 mg /L of vancomycin. COLONY MORPHOLOGY Colonies of H. influenzae on (CAP): ü appear translucent, tannish, moist, smooth and convex with a distinct "mousy or bleach like" odor. Colonies of H. parainfluenzae on (CAP): ü appear as tannish and drier with a medium to large size compared with H. Influenzae. Colonies of H. ducreyi on (CAP): ü appear as small, flat, smooth, nonmucoid, transparent to opaque colonies or appear as tan or yellow. ü They produce "clumpy" nonhomogeneous appearance when suspended in saline. MICROSCOPIC MORPHOLOGY Haemophilus spp. appear as? Small, gram-negative coccobacilli Because the organism is small and pleomorphic it often stains a faint pink which resembles the amorphous serous material (serum-like or proteinaceous background material) in Gram stains Capsules of H. influenzae: Capsules of H. influenzae may be observed in Gram-stained smears as clear, nonstaining areas (halos) surrounding the organisms in purulent secretions MORALES, MARIA MARIELLA 18 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 Direct smear of Haemophilus influenzae in CSF in a case of meningitis. Note the intracellular and Gram stain of Haemophilus influenzae colony. Bacilli extracellular, gram-negative coccobacilli (arrow) are slight more elongated MICROSCOPIC MORPHOLOGY What stain can help in gram-staining of Haemophilus? Because of the low specificity and sensitivity of Gram stains, acridine orange or methylene blue stain may help in detecting Haemophilus. Gram stains of H. ducrevi colonies: from genital lesions may show pale staining gram negative coccobacilli arranged singly or in groups (clusters) referred to as "school of fish" or "railroad tracks" LABORATORY IDENTIFICATION The first clue that an isolate may belong to genus Haemophilus species: ü Growth of Gram negative coccobacilli on CHOC(CAP) ü No growth on SBA and MacConkey agar -because RBCs are still not lysed on SBA and MacConeky Agar ü Testing for X and V factors ü Traditional Biochemicals ü Hemolysis on Media containing rabbit or horse RBC ü Oxidase ü Catalase Several manual and automated commercial ID systems can be used to identify and biotype Haemophilus species. X FACTOR AND V FACTOR REQUIREMENT: Testing for X and V factor requirements using impregnated strips or disks is the traditional approach for identification of Haemophilus species. MORALES, MARIA MARIELLA 19 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 THE HAEMOPHILUS QUAD PLATE The Haemophilus Quad Plate contains 4 zones: 1. Media with X factor only 2. Media with V factor only 3. Media with X and V factors 4. Media with X and V factors + horse RBC Haemophilus haemolyticus is generally beta hemolytic on horse blood Haemophilus influenza is negative. PORPHYRIN TEST Porphyrin test is used for? Alternative method for differentiating the heme-producing species of Haemophilus Porphyrin test is performed in? Performed in agar, in broth or on a disk What is the PRINCIPLE of Porphyrin test? Principle: based on the ability of the organism to convert the substrate delta aminolevulinic acid (ALA) into porphyrins or porphobilinogen which are intermediates in the synthesis of X factor. Porphobilinogen is detected by the addition of p- dimethylaminobenzaldehyde (Kovac's reagent). Positive result of Porphyrin test? red color forms in the lower aqueous phase if porphobilinogen is present. Porphyrins can be detected using? UV light with a wavelength of about 360 nm (Wood's lamp) Porphyrins fluoresce appears as? reddish orange under UV light. Other test: Biochemical test: Carbohydrate fermentation Tests can help further differentiate Haemophilus species MORALES, MARIA MARIELLA 20 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 TREATMENT Treatment for H. influenzae The current recommended treatment caused by H.influenza is cefotaxime or ceftriaxone. Chloramphenicol + ampicillin is also effective Alternative treatment Alternatives are trimethoprim-sulfamethoxazole, imipenem, and ciprofloxacin There is an increased resistance to Ampicillin and should not be used alone for initial therapy CHROMOGENIC CEPHALOSPORIN TEST A disk is impregnated with benzy|penicillin and bromcresol purple (pH indicator). Moistened with 1 or 2 drops of sterile distilled water. Using a sterile loop, colonies are smeared to the test strip. If the beta lactam ring of the benzy|penicillin is broken down by beta lactamase, penicilloic acid is formed, which cause a decrease in pH; this decrease in pH is shown by a color change from purple (negative) to yellow (postive) on the strip within 5 to 10 minutes. Only testing for Beta lactamase activity for assess ampicillin and amoxicillin efficacy is necessary. Non-life-threatening infection of H. influenzae Non-life-threatening infection of H. influenzae is treated with amoxicillin - clavulanate, an oral 2nd generation or 3rd generation cephalosporin or trimethoprim-sulfamethoxazole For H. ducreyi: For H. ducreyi, azithromycin, ceftriaxone, ciprofloxacin, or erythromycin is recommended. Haemophilus spp have increased resistance to? Increased resistance to ampicillin by Haemophilus species due to Beta lactamase or to altered penicillin -binding proteins In the US, approximately 25% of NTHi isolates are Beta lactamase positive. Rapid tests to detect Beta lactamase production? ü chromogenic cephalosporin test (Cefinase) ü Acidometric tests. (+) microorganism is resistant to ampicillin and amoxicillin HACEK Group HACEK is an acronym representing each genus in the group namely: 1. H- aemophilus species (H.paraphrophilus ) 2. A - ggregatibacter actinomycetemcomitans (formerly Actinobacillus actinomycetemcomitans and A. aphrophilus) 3. C- ardiobacterium hominis 4. E- ikenella corrodens 5. K- ingella species Members of this group of Gram-negative bacilli are fastidious needing an increased CO2 Dysgonic (slower or poorer growing) Predilection to heart valves attachment, usually damaged or prosthetic Important cause of endocarditis, the lesion (referred to as vegetation) is composed of fibrin, platelets, PMNs, monocytes and microorganisms. Include both the fermentative and the nonfermentive GNB All members are normal biota of the oral cavity which can be introduced to the bloodstream and resultant infections All members are opportunists which require a compromised host Risk factors for infective (bacterial) endocarditis include tooth extraction, his of endocarditis, gingival surgery , heart valve surgery and mitral valve prolapse MORALES, MARIA MARIELLA 21 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 AGGREGATIBACTER APHROPHILUS What is the most prevalent species in the HACEK group Aggregatibacter aphrophilus involved in endocarditis? Aggregatibacter aphrophilus is found in? Found in dental plaque and gingival scrapings Signs and symptoms: Signs and symptoms include ü fever, ü heart murmur, ü congestive heart failure ü embolism. *H. aprhophilus and H. paraphrophilus are re-classified into Aggregatibacter aphrophilus. AGGREGATIBACTER ACTINOMYCETEMCOMITANS Aggregatibacter actinomycetemcomitans appear as? Small bacilli to coccoid gram-negative bacilli which are nonmotile Distinctive "star shape with 4 to 6 points" in the center of the colonies What are the 6 serotypes of Aggregatibacter Divided into 6 serotypes (a,b,c,d,e,f) actinomycetemcomitans? Specimens of Aggregatibacter actinomycetemcomitans is Isolated from blood, lung tissue, abscesses of the mouth isolated from? and brain, and sinuses Aggregatibacter actinomycetemcomitans is the causative Causative agent of subacute bacterial endocarditis with an agent of? insidious and protracted presentation Aggregatibacter actinomycetemcomitans is the etiologic Etiologic pathogen in the development of periodontitis pathogen of? Major virulence factors: collagenase and a leukotoxin which is toxic to PMNs and monocytes How many hours for isolation? Isolates may require more than 24 hours for visible growth MORALES, MARIA MARIELLA 22 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 CARDIOBACTERIUM HOMINIS What are the important species of Cardiobacterium? C.hominis C. valvarum C. hominis appear as? pleomorphic, nonmotile, fastidious gram negative bacillus Organisms tend to form rosettes, swellings, long filaments, or sticklike structures in yeast extract Cardiobacterium hominis Normal microbiota of the: nose, mouth and throat as well as the GIT Clinical manifestation: Usual clinical manifestation is endocarditis, with very large vegetations and no demonstrable fever Infects the aortic valve more frequently than the other HACEK organisms Cardiobacterium hominis grow slowly on what culture Grow slowly on SBA and (CAP) media? What culture media should not be used in C. hominis? Do not grow on MacConkey agar Incubated at? Incubation with 5% CO2 (either aerobic or anaerobic) EIKENELLA CORRODENS General characteristics of Eikenella corrodens: Fastidious, gram negative coccobacilli, grow best under increased CO2 Nonmotile, oxidase positive, assaccharolytic COLONY/AGAR APPEARANCE? About 45% of the isolates "pit the agar" make a depression or corrode the surface of the agar Chlorine bleach like odor from the agar surface What culture media should not be used Eikenella Do not grow on MacConkey or EMB agar corrodens? Eikenella corrodens is resistant to? Resistant to clindamycin and narrow spectrum cephalosporins Clinical manifestations of Eikenella corrodens: Infections occur as a result of trauma, especially after human bites or fights "clenched fist wounds" or after the skin has been broken by human teeth Poor dental hygiene or oral surgery Cause of adult periodontitis, meningitis empyema, pneumonia, osteomyelitis, arthritis and post operative tissue infections In drug addicts, implicated in cellulitis as a result of direct inoculation of the organisms in the skin after oral contamination of needle paraphernalia (licking the needle clean) MORALES, MARIA MARIELLA 23 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 Predilection for attachment to heart valves and cause endocarditis KINGELLA General charctaresitics of Kingella: coccobacillary to short bacilli Nutritionally fastidious ü oxidase positive, ü catalase negative ü fermenters of glucose and other sugars but without gas Genus of Kingella consists of: consists of 4 species: ü K. kingae, ü K. denitrificans, ü K. oralis, and ü K. potus Catalase test differentiates them from Neisseria and Moraxella species (catalase positive) Capnocytophaga Belongs to the family Flavobacteriaceae Associated with septicemia notably in patients with neutropenia and not as commonly involved in endocarditis Fastidious, facultatively anaerobic GNB which requires increased CO2 Thin and often fusiform' or pointed ends resembling Fusobacterium species Produce gliding motility on solid surfaces colonies are often adherent and produce a yellow-orange pigment CARBOHYDRATE FERMENTATION: Ferment sucrose glucose, maltose and lactose TREATMENT: Susceptible to imipenem, erythromycin clindamycin, tetracycline, chloramphenicol quinolones and Beta lactams Resistant to Aminoglycosides MORALES, MARIA MARIELLA 24 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 Pasteurella Pasteurellosis = Zoonotic infection, a disease that humans acquire from exposure to infected animals or products made from infected animals Cutaneous infections resulting from animal bites is the most common clinical presentation which can result in inflammation and exudate production Pasteurella multocida is the most frequently isolated species which consists of 5 serogroups (A, B, D,E and F) defined-by capsular antigens Exhibits bipolar staining (safety pin appearance) All Pasteurella Species grow on SBA and CHOC (CAP) producing grayish colonies. No growth on MacConkey Brucella Important zoonotic disease Considered as category B select biological agents by CDC (easy to disseminate and cause moderate morbidity but low mortality Undulant fever, Mediterranean, Crimean and Malta fevers Brucellosis exposed to animals and animal products (vegetarians, hunters) and laboratory workers acquired through aerosol, percutaneous and oral routes of exposure Three clinical stages of brucellosis: acute, subchronic and chronic 1. Acute infection = nonspecific (fever, malaise, headache, anorexia, myalgia and back pain); within 1 week of exposure 2. Subchronic or undulant form = occurs within a year of exposure characterized by undulating fevers (normal temperature in the morning followed by high temperatures in the afternoon and evening), arthritis and epididymoorchitis which is inflammation of the epididymis and testis. 3. Chronic form = manifests 1 year after exposure with symptoms such as depression, arthritis and chronic fatigue syndrome 4 SPECIES OF BRUCELLA: ü B. melitensis, B.suis, B.abortus and B.canis General characteristics of Brucella: ü Small gram negative , aerobic, nonmotile, nonencapsulated bacteria, non-spore-formers and may appear as coccobacilli or bacilli. ü On SBA, they appear as smooth, raised and translucent colonies ü They grow on SBA and CHOC(CAP) within 18 hours ü Oxidase negative and catalase positive ü Urease positive within 2 hrs ü Should be handled under BSL-3 conditions because of the aerosol mode of transmission within a biological safety cabinet) Francisella Francisella tularensis is implicated in most human infections Tularemia = zoonotic disease (rabbit fever, deerfly fever, lemming fever and water rat trappers’ disease) o MOT (ingestion, inhalation, arthropod bite by ticks, biting flies and contact with infected tissue) o The most common clinical presentation is ulceroglandular which an ulcer forms at the site of inoculation and followed by an enlargement of the regional lymph nodes o Tularemia can also occur in pneumonic (contracted by inhalation route) glandular, oropharyngeal, oculoglandular and typhoidal forms. General characteristics of Francisella: Francisella species appear as small, nonmotile, nonsporeforming gram negative bacilli and are strictly aerobic Francisella species are fastidious requiring cysteine, cvstine, or biosulfate for growth MORALES, MARIA MARIELLA 25 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 WEEK 2: ENTEROBACTERIACEAE Key Charactersitics of the Family Enterobacteriaceae Members of the family Enterobacteriaceae are: ü gram negative ü non-spore forming ü facultatively anaerobic bacilli ü On gram stain, they can appear as coccobacilli or as straight rods. ü They are gram-negative bacilli and coccobacilli ü They all ferment glucose They do NOT produce cytochrome oxidase EXCEPT FOR: Plesiomonas They reduce nitrate to nitrite EXCEPT FOR: Photorhabdus And Xenorhabdus. They are motile at body temperatures EXCEPT FOR: Klebsiella, Shigella, And Yersinia. None has remarkable colony morphology on laboratory media. Klebsiella, Proteus, And Some Enterobacter Isolates, They appear large, moist, and gray on sheep blood agar (SBA), chocolate (CHOC) agar, and most nonselective media EXCEPT FOR: CLASSIFICATION Members of the family are also subcategorized into numerous tribes based on biochemical characteristics. The use of tribes in classifying the members in this family was proposed by Ewing in 1963 In classifying species into tribes, Ewing grouped bacterial species with similar biochemical characteristics. Within the tribes, organisms are classified further into genera and species. ENTEROBACTERIACEAE: VIRULENCE AND ANTIGENIC FACTORS The virulence of the Enterobacteriaceae genera is affected ü adhere, by many factors, such as the ability to: ü colonize, ü produce toxins, ü and invade tissue. What are the type of antigens that can be used in the O antigen / somatic antigen identification of different serologic groups? H antigen / flagellar antigen K antigen / capsular antigen What is O antigen/somatic antigen? this is a heat-stable antigen located on the cell wall. What is H antigen/flagellar antigen? this is a heat-labile antigen found on the surface of flagella, structures responsible for motility. What is K antigen/capsular antigen this is a heat-labile polysaccharide found only in certain encapsulated species. Examples are the k1 antigen of E. Coli and the VI antigen of Salmonella Enterica subsp. Enterica serotype typhi. MORALES, MARIA MARIELLA 26 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 ENTEROBACTERIACEAE: CLINICAL SIGNIFICANCE The Enterobacteriaceae, with few exceptions, share a Salmonella, Shigella, And Yersinia, they can be resident common niche; they reside in the gastrointestinal (GI) tract. microbiota if confined to their natural environment. EXCEPT FOR: Enterobacteriaceae are responsible for what type of are responsible for a large number of opportunistic infections infection? when introduced into inappropriate body sites. Members of the family Enterobacteriaceae may be divided into Opportunistic pathogens TWO BROAD CATEGORIES BASED ON THEIR CLINICAL Primary pathogens. INFECTIONS: What is Opportunistic pathogen and their example? Often a part of the usual intestinal microbiota of both humans and animals. Outside their normal body sites, these organisms can produce serious extraintestinal, opportunistic infections Ex: E. Coli - one of the best-studied members of the Enterobacteriaceae, is a member of the normal bowel biota but can cause urinary tract infections (UTIs), septicemia, wound infections in healthy individuals, and meningitis in neonate What is Primary Pathogens and their example? Considered true pathogens; that is, they are not present as commensal biota in the GI tract of humans. These organisms produce infections resulting from ingestion of contaminated food or water or from other sources. Ex: Yersinia Spp, S. Enterica, Shigella Spp Mnemonics: YES MORALES, MARIA MARIELLA 27  CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 Opportunistic Members Of The Family Enterobacteriaceae And Associated Infections ENTEROBACTERIACEAE: ESCHERICHIA COLI Escherichia coli was first described by? Escherich In 1885, E. coli was INITIALLY considered a harmless member of the colon resident biota. It is now recognized as an important human pathogen, ü UTIs associated with a wide range of clinical syndromes including: ü Diarrheal disease ü central nervous system infections Most strains of E. Coli are: ü motile ü generally possess adhesive fimbriae and sex pili and ü O, H, and K antigens. Why is Escherichia and Shigella spp. are grouped together E. Coli O groups cross react with Shigella spp. which also in the tribe Escherichia? has O antigen What Enterobacteriaceae is so commonly isolated from E. coli (Escherichia coli) colon biota that is used as a primary marker of fecal contamination in water quality testing? What is the distinct morphology of E.coli on MAC? it appears lactose-positive (pink colony) with surrounding area of precipitated salts What is the distinct morphology of E.coli on EMB? green metallic sheen Escherichia spp. TSI H2S UREASE I M V C LDC ODC ADH PLD E. coli A/A - - + + - - + v v - A. Typical dry, lactose-positive Escherichia coli growing on B. Mucoid colonies of E. coli growing on MAC agar. MacConkey (MAC) agar. Note the pink precipitate surrounding the individual colonies. C. Non–lactose-fermenting E. coli compared with typical D. Green metallic sheen on EMB lactose-fermenting E. coli on MAC agar MORALES, MARIA MARIELLA 28 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 E. COLI IS ASSOCIATED WITH THE FOLLOWING PROPERTIES: E. coli ferments: GLUCOSE, LACTOSE, TREHALOSE, XYLOSE E. coli produce: INDOLE FROM TRYPTOPHAN E. coli ferments glucose by mixed acid pathway producing: METHYL RED (+) VOGES-PROSKAUER (-) E.coli DO NOT produce: H2S, DNASE, UREASE, or PHENYLALANINE DEAMINASE E. coli CAN NOT use: CITRATE AS A SOLE CARBON SOURCE Uropathogenic Escherichia coli What is the 1st common cause of UTIs in humans? E. coli What is the 2nd common cause of meningitis in humans? E. coli *What is the 1st common cause of meningitis in humans? - Streptococcus agalactiae E. coli strains that cause UTIs usually originate in the: large intestine. E. coli strain that cause acute pyelonephritis in have been shown to be the dominant resident e. Coli in the immunocompetent hosts: colon. What is the PRIMARY VIRULENCE FACTOR OF PILI – which allow uropathogenic strains to adhere to epithelial UROPATHOGENIC STRAINS OF E.COLI? cells and not be washed out with urine flow. What are the OTHER VIRULENCE FACTORS OF Cytolysins - characterized as hemolysins, can kill immune UROPATHOGENIC E.COLI? effector cells and inhibit phagocytosis and chemotaxis WBC Aerobactin - allows the bacterial cell to chelate iron; free iron is generally unavailable within the host for use by bacteria. Gastrointestinal Escherichia coli E. Coli may cause several different GI syndromes. Based on virulence factors, clinical manifestation, epidemiology, and different O and H serotypes, THERE ARE FIVE MAJOR CATEGORIES OF DIARRHEOGENIC E. COLI: Enterotoxigenic Escherichia coli (ETEC) Enteroinvasive Escherichia coli (EIEC) Enteropathogenic Escherichia coli (EPEC) Enterohemorrhagic Escherichia coli (EHEC) Enteroadherent: o Diffusely Adherent Escherichia coli (DAEC) o Enteroaggregative Escherichia coli (EAEC) MORALES, MARIA MARIELLA 29 CLINICAL BACTERIOLOGY (LECTURE) 1ST TERM – FINALS – AY 2024-2025 ENTEROTOXIGENIC ESCHERICHIA COLI (ETEC) ETEC STRAINS are associated with: Diarrhea of adults and especially children in tropical and subtropical climates, especially in developing countries. ETEC DIARRHEA is the most common cause of a diarrheal MONTEZUMA REVENGE, TURISTA OR TRAVELER’S disease sometimes referred to as? DIARRHEA ETEC INFECTION Is spread commonly via consumption Contaminated food or water, mostly poor hygiene and poor food of? handling. What HIGH INFECTIVE DOSE is necessary to initiate 𝟏𝟎𝟔 𝒕𝒐 𝟏𝟎𝟏𝟎 ORGANISMS disease in an immunocompetent host? Individuals with ____ are with high risk factor of ETEC: Achlorhydria - deficiency of hydrochloric acid within the stomach ETEC infection is characterized by: ü rice watery stool diarrhea, ü abdominal cramps, ü sometimes nausea, ü usually with no vomiting or fever; ü usually self-limiting. DIAGNOSIS OF ETEC INFECTION is made primarily by the characteristic symptoms and the isolation of solely lactose-fermenting organisms on differential media. Once the ETEC strains are established, they produce what ü Heat labile toxin (LT) toxin in the small intestine? - similar in action and amino acid sequence to cholera toxin form Vibrio cholerae. LT consists of two fragments: A portion – enzymatically active portion B moiety – binding portion This results in watery diarrhea similar to cholera. ENTEROPATHOGENIC ESCHERICHIA COLI (EPEC) EPEC has been known to be cause of: infantile diarrhea (EPEC:0111) EPEC is characterized as: Non-invasive, produces NO toxin, nosocomial Diarrheal outbreaks caused by EPEC have occurred in: hospital nurseries and daycare centers, but cases in adults are rarely seen. (nosocomial) IDENTIFIED TO CAUSE INTESTINAL INFECTIONS. EPEC O serogroups EPEC INFECTION is charac