Microbiology Review

Clinical and Diagnostic Sciences Tera Webb, MS, MLS(ASCP)CM

Antibiotic Targets and Mechanism of Action

TARGET

Cell Wall

DNA and RNA

Synthesis

Metabolic Pathways

Protein Synthesis

Cell Membrane

Staphylococcus

• Most common pathogen

• Media

• Microscopic

• Staphylococcus

aureus

• Staphylococcus

epidermidis

• Staphylococcus

saprophyticus

• Staphylococcus

lugdunersis

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Staphylococcus aureus

• Gram Stain • GPC in clusters

• Colony Morphology • Round, smooth, creamy, yellow to white colonies with beta- hemolysis

• Biochemical tests • Catalase positive • Coagulase positive

• Pathology • Wound & skin infections, toxic shock syndrome, food poisoning, pneumonia,

osteomyelitis, bacteremia, etc. • Virulence Factors

• Enterotoxins • Cytolytic toxins • Extracellular Enzymes • Protein A

Staphylococcus epidermidis

• Gram Stain

• GPC in clusters

• Colony Morphology

• Round, smooth, creamy, yellow to white colonies with no hemolysis

• Biochemical Tests

• Catalase positive

• Coagulase negative

• Pathology

• Normal skin flora, nosocomial infections, prosthetic valve endocarditis, catheters, septicemia

Other Coag-Negative Staphylococci

• Staphylococcus saprophyticus

• Add’l Biochemical: Novobiocin resistant

• Pathology: UTI’s in young, sexually active females

• Staphylococcus lugdunensis

• Add’l Biochemical: PYR positive

• Pathology: endocarditis

Staphylococcus - Antimicrobial Susceptibility

• Abnormal susceptibility patterns

• Beta-lactam resistance

• MRSA • Screening

• Treatment with vancomycin

• VISA and VRSA • Limitations to disk diffusion & automated methods

• Macrolide resistance • Resistance to clindamycin may not be obvious

• D test - Use erythromycin and clindamycin disks

• Inducible resistance is growth between disks but not on side of clindamycin disk

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Hemolysis

Streptococci and Enterococci are speciated based on hemolysin production and biochemical properties.

• α-Hemolysis

• RBCs are partially lysed, resulting in a greening of the area around the colony

• β-Hemolysis

• RBCs are completely lysed, resulting in a clear area around the colony

• γ-Hemolysis (nonhemolytic)

• RBCs are not lysed, so there is no change in agar color

Streptococcus

• Streptococcus pyogenes

• Streptococcus agalactiae

• Streptococcus pneumoniae

• Streptococcus bovis

• Viridians Streptococcus

• Lancefield groupings

• Brown’s classification

• Media

• Microscopic

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Streptococcus pyogenes

• Gram Stain • GPC in chains

• Colony Morphology • Small, white, transparent, smooth colony with

well-defined zone beta-hemolysis

• Biochemical Tests • Catalase negative • Strep Grouping “A”

• Pathology • Pharyngitis, scarlet fever, skin infections, toxic

shock syndrome, post-streptococcal sequelae • Virulence Factors

Streptococcus agalactiae • Gram Stain

• GPC in chains

• Colony Morphology • Small, greyish-white, transparent, smooth colony with small zone beta- hemolysis

• Biochemical Tests • Catalase negative • Strep Grouping “B” • Hippurate positive • CAMP positive

• Pathology • Neonatal sepsis • Neonatal meningitis

Streptococcus pneumoniae

• Gram Stain • GPC in pairs (lancet- shaped)

• Colony Morphology • Small, round, glistening, mucoid, dome -shaped colony with alpha - hemolysis

• Biochemical Tests • Catalase negative • Optochin (P) – Sensitive • Bile soluble

• Pathology • Pneumonia, sinusitis, otitis media, bacteremia, meningitis

Viridians Streptococcus

• Gram Stain

• GPC in chains

• Colony Morphology

• Small, round, colony with alpha -hemolysis

• Biochemical Tests

• Catalase negative

• Optochin (P) “R”

• Pathology

• Normal flora, opportunistic, subacute bacterial endocarditis

Streptococcus bovis

• Gram Stain • GPC in chains

• Colony Morphology • Small, greyish, smooth colony with gamma-hemolysis

• Biochemical Tests • Catalase negative • PYR negative • Bile Esculin positive • NaCl negative

• Pathology • Opportunistic

Enterococcus spp.

• Gram Stain • GPC in chains

• Colony Morphology • Small, greyish, smooth colony with gamma-hemolysis

• Biochemical Tests • Catalase negative • PYR positive • Bile Esculin positive • NaCl positive

• Pathology • UTIs, Wounds, endocarditis, etc

Streptococcus -Antibiotic Susceptibility

• E. faecalis

• Usually susceptible to ampicillin and penicillin.

• Can acquire resistance to vancomycin.

• Occasionally produce beta- lactamase.

• E. faecium

• Often resistant to ampicillin and penicillin.

• Can acquire resistance to vancomycin.

• E. gallinarum and E. casseliflavus

• Have intrinsic low level vancomycin resistance

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Corynebacterium spp. aka Diphtheroids

• Gram Stain • Pleomorphic Gram positive rods • Often palisading • Can be club-shaped

• Colony Morphology • White, non-hemolytic, opaque colonies

• Biochemical Tests • Catalase positive • Nitrate variable • Glucose variable • Maltose variable • Sucrose variable • Urease variable

• Pathology • Considered normal skin flora or contaminant

Corynebacterium diphtheriae

• Gram Stain • Pleomorphic Gram positive rods • Often palisading • Can be club-shaped

• Colony Morphology • White, non-hemolytic, opaque colonies

• Biochemical Tests • Catalase positive • Nitrate positive • Glucose positive • Maltose positive • Sucrose negative • Urease variable

• Pathology • Produces diptheria toxin

Listeria monocytogenes

• Gram Stain • Small Gram positive rods (can be coccobacilli)

• Colony Morphology • Grayish white colony, small zone beta-hemolysis

• Biochemical tests • Catalase positive • Esculin hydrolysis positive • Hippurate hydrolysis positive • Motility • Wet prep = tumbling motility • 25°C semisolid media = inverted umbrella

• Pathology • Pregnant women: premature labor, spontaneous abortion • Neonates: sepsis, meningitis • Immunocompromised

Bacillus species

• Gram Stain • Large gram positive rods • Can see spores (if organism is stressed)

• Colony Morphology • “Ground glass appearance,” Large colonies,

rough edges, beta- hemolytic

• Biochemical tests • Catalase positive • Motile

• Pathology • Considered a contaminant, endophthalmitis,

food poisoning (B. cereus)

Bacillus anthracis

• Gram Stain • Large gram positive rods • Can see spores (if organism is stressed)

• Colony Morphology • “Ground glass appearance,” Large colonies, rough edges, NON- hemolytic

• Biochemical tests • Catalase positive • Non- motile

• Pathology • Causes anthrax

Lactobacillus sp.

• Gram Stain • Gram positive rods in chains

• Colony Morphology • Alpha hemolytic colonies

• Biochemical tests • Catalase negative

• Pathology • Normal female urogenital flora

Gardnerella vaginalis

• Gram Stain • Gram negative rod • Direct smear: Clue cells

• Colony Morphology • Tiny transparent colonies

• Biochemical tests • Catalase negative

• Pathology • Bacterial vaginitis

Differentiation of Major GNR Genera

• Utilization of Glucose

• Ferment “F”, oxidize “O” or inactive

• Oxidase

• Positive or negative

• Ability to grow on MacConkey agar

• Some GNRs that grow on BAP are unable to grow on MacConkey agar

Microbiology

Family: Enterobacteriaceae General Characteristics

• All ferment glucose • All reduce nitrate to nitrites • All grow on MAC • All are oxidase negative

• Except Plesiomonas

• Majority motile • Gram-negative coccobacilli or rods

• Not very useful in identification other than ruling out other organisms

• Facultatively anaerobic • Culture morphology

• Large moist, gray colonies • Some mucoid

Lactose-Fermenters Escherichia coli

• Colony Morphology • Most beta-hemolytic

• Biochemical tests • Oxidase negative • Indole positive • Motile

• Pathology • Normal colon flora • Cause UTIs, septicemia, neonatal septicemia and meningitis and several GI tract diseases

• Traveller’s diarrhea (ETEC) • Dysentary (EIEC) • Diarrhea and Vomiting (EPEC) • Hemolytic colitis (EHEC)

Lactose-Fermenters Klebsiella

• Biochemical tests • Oxidase negative • Indole

• K. pneumoniae = negative • K. oxytoca = positive

• Urea positive • Citrate positive • Non-motile

• Polysaccharide capsule • Pathology

• UTIs, pneumonia

Lactose-Fermenters Enterobacter

• Biochemical tests

• Oxidase negative

• Indole negative

• Urea positive

• Citrate positive

• Motile

Slow-Lactose Fermenters Citrobacter

• Biochemical tests • Oxidase negative • Indole negative • H2S positive

• Motile • Pathology

• Opportunistic

Slow-Lactose Fermenters Serratia

• Colony Morphology • Colonies have a salmon red pigment

• Biochemical tests • Oxidase negative • Indole negative

• Motile • Pathology

• Opportunistic

Proteeae Phenylalanine deaminase & Urea positive

Proteus species • Colony Morphology

• Swarming motility

• Biochemical tests • Oxidase negative • Indole

• Proteus vulgaris = positive • Proteus miribilus = negative

• H2S positive

• Motile • Pathology

• Cause UTI’s • Opportunistic

Proteeae Phenylalanine deaminase & Urea positive

Providencia

• Biochemical tests

• Oxidase negative

• Indole positive

• Citrate positive

• Motile

Proteeae Phenylalanine deaminase & Urea positive

Morganella

• Biochemical tests

• Oxidase negative

• Indole negative

• Citrate positive

• Motile

Obligate Pathogens Salmonella

• Biochemical tests

• Oxidase negative

• Indole negative

• H2S positive

• Motile

• Pathology

• Diarrhea

• Septicemia

Obligate Pathogens Shigella

• Biochemical tests

• Oxidase negative

• Non-motile

• Pathology

• Diarrhea

Obligate Pathogens Yersinia

• Coccibacilli; bipolar staining

• Biochemical tests

• Oxidase negative

• Indole negative

• Motile

• Pathology

• Plague (Y. pestis)

• Diarrhea (Y. enterolitica)

Enterobactiaceae

Groupings

• Ferment Glucose

• Ferment Lactose

• Motile @ body temp

• Citrate

• H2S Producers

• Deaminate phenylalanine

• KIA: K/A no H2S

• KIA: A/A no H2S

• KIA: K/K no H2S

• KIA: K/A H2S

• KIA: A/A H2S

Differentation

• P. vulgaris vs. P. mirabilis

• Morganella vs. Providencia

• Enterobacter vs. Klebsiella

• K. pneumoniae vs. K. oxytoca

• E. coli vs. K. oxytoca

• E. aerogenes vs. E. cloacae

• Salmonella vs. Shigella

• Non-fermenting E.coli vs. Edwardsiella

• Providencia vs. Proteus

• Edwardsiella vs. Salmonella 37

Pseudomonas aeruginosa

• Gram Stain • Gram negative rod

• Colony Morphology • Beta-hemolytic; flat, spreading colony with fruity,

grape-like smell • Growth at 42oC

• Biochemical tests • Catalase positive • Oxidase positive • Oxidizes glucose

• Pathology • Pneumonia and wounds

Acinetobacter species

• Gram Stain • Gram Negative Rod

• Non-motile • Biochemical tests

• Catalase positive • Oxidase negative • Nitrate negative

• Acinetobacter baumannii • Glucose “O” • Grows at 42°C

• Acinetobacter lwoffii • Glucose Non-“F”, Non-“O” • No growth at 42°C

Stenotrophomonas maltophila

• Gram negative rod

• Oxidase negative

• Glucose “O”

• No growth at 42°C; Sometimes can develop a lavender-green to light purple pigment

Vibrio cholerae

• Three major groups:

• V. cholerae O1

• V. cholerae O129

• V. cholerae non-O1

• Virulence factors

• Cholera toxin—Causes mucosal cell hypersecretion of water

• Others—Enterotoxins (zonula occludens toxin [ZOT] and accessory cholera enterotoxin [ACE]); O1 and O139 antigens

• Spectrum of disease

• Cholera

• Profuse, watery diarrhea leading to dehydration (“rice-water” stool)

• Hypotension

• Nonepidemic diarrhea, associated with non-O1 group

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Aeromonas hydrophilia

• Gram negative rods

• Beta-hemolytic on BAP

• Grows on MAC

• Oxidase positive

• Glucose “F”

Campylobacter jejuni

• Curved Gram negative rod

• Grows at both 37°C & 42°C in 10% CO2 (microaerophilic)

• Oxidase positive

• Darting motility on wet prep

• Hippurate hydrolysis positive

Helicobacter pylori

• Curved Gram negative rod

• Urease positive

• Oxidase positive

Haemophilus species

• Gram negative coccobacilli • Fastidious

• Will not grow on sheep blood agar • Must use Chocolate agar • Requires CO2

• Porphyrin test • Delta aminolevulinic acid (ALA) is substrate • Determines if organism has enzyme required to produce hemin (X factor) • If positive, organism does not require X factor

Haemophilus species

• Gram negative coccobacilli • Fastidious • Porphyrin test • Pathology

• H. influenzae: Meningitis, epiglottitis, pneumonia, otitis media • H. aegyptius: conjunctivitis • H. ducreyi: STD - chancoid

Other Gram-negative Rods Eikenella corrodens

• Gram Stain

• Gram-negative rods

• Colony Morphology

• Nonhemolytic colonies

• Pathology

• Colonize the URT

HACEK Group General Characteristics

• Gram-negative bacilli • Require an increased CO2 (5%-10%) environment • Significant cause of endocarditis • Usual flora of the oral cavity • Thus are pathogens associated with human bite wounds causing septicemia and subacute

endocarditis • Opportunists in immunocompromised hosts

• H – Haemophilus spp. (Now Aggregatibacter aphrophilus) • A – Actinobacillus actinomycetemcomitans (Now Aggregatibacter actinomycetemcomitans) • C – Cardiobacterium hominis • E – Eikenella corrodens • K – Kingella spp.

Brucella species

• Gram Stain

• Gram-negative coccibacilli

• Zoonotic infection acquired from animals or animal products

• Most common species for human brucellosis

• B. mellitensis (most common isolate), B. abortus, B. canis, B. suis

• Pathology

• Brucellosis or undulant fever

Francisella species

• Gram Stain

• Gram-negative coccibacilli

• Facultative intracellular parasite

• Strict aerobe

• Pathology

• Zoonotic infection

• Causes tularemia

• Acute febrile, granulomatous disease with rapid onset and flulike symptoms

Legionella Species

• Intracellular parasite of cells • Enter and multiply within host cells • Particularly bronchoalveolar macrophages • Legionella pneumophila

• Legionnaires’ disease • Pontiac fever

• Sources • Present in environmental water sources • Air conditioning systems

• Detection • Culture: BCYE agar • Antigen in the urine: Sensitive • Direct fluorescent antibody staining • Serology

Bordetella Species

• Major pathogens • Bordetella pertussis • Bordetella parapertussis • Bordetella bronchiseptica

• Highly contagious disease • Exposure and incubation period of 1-2 weeks • Initially cold or flulike symptoms, sneezing, runny nose • Catarrhal phase 1-2 weeks (highly contagious)

• Paroxysmal phase: Sudden severe repetitious coughing

• Convalescent phase • Begin recovery about 4 weeks after initial symptoms

• Decrease in bouts of coughing but may take weeks or months for complete resolution • Pathology

• Causes ____________________

• B. bronchiseptica - Opportunistic pathogen involved in pneumonia and wound infections

Pathogenic Neisseria

• Gram Stain • Gram negative diplococci • Kidney bean shaped

• Colony Morphology • On modified Thayer- Martin (MTM) agar: • Small, gray, translucent, raised

• Biochemical tests • Catalase positive • Oxidase positive • Carbohydrate utilization ->

• Pathology • N. gonorrhea: Cervicitis, urethritis, conjunctivitis • N. meningitidis: meningococcemia

• Antimicrobials • Penicillin is drug of choice

• Resistance observed- Perform beta-lactamase

Organism Glucose Maltose Lactose Sucrose

Neisseria gonorrhoeae Positive Negative Negative Negative

Neisseria meningitidis Positive Positive Negative Negative

Neisseria lactamica Positive Positive Positive Negative

Nonpathogenic Neisseria Positive Positive Negative Positive

Moraxella catarrhalis Negative Negative Negative Negative

Clostridium perfringens

• Anaerobic organism • Gram Stain

• Large gram positive rods • Blunt, rounded ends (“boxcar” shaped) • Can see spores (if organism is stressed)

• Colony Morphology • Double zone of beta-hemolysis

• Biochemical tests • Catalase negative

• Pathology • Gas gangrene, food poisoning

Other Clostridium

• Clostridium difficile

• Clostridium tetani

• Clostridium botulium

B. fragilis group

• Main group of anaerobes isolated

• Consists 13 species

• Most common – B. fragilis, B. thetaiotamicron

• Natural habitat

• Predominant NF colon

• Also found oropharynx, female genital tract

• Clinical significance

• Mainly infections below diaphragm (intraabdominal, liver abscesses, other)

• GS- pale-staining neg rod, rounded ends, vacuoles, pleomorphic

• Colonies- white-gray, translucent or semi-opaque, NH

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Lipid-Mycolic Acid Walls Mycobacterium

Acid –Fast Stains • Ziehl-Neelson

• Kinyoun

Media • Lowenstein-Jensen

• Middlebrook Medium

• Mycobacterium culture bottles

Specimens • Respiratory

• Urine

• Blood & bone marrow

• Tissue & body fluids

Pathogens

• M. tuberculosis

• M. bovis

• M. ulcerans

• M. avium

• M. kansasii

• M. xenopi

• M. lepreae

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DNA Viruses

• Adenoviridae

• Mostly respiratory illness

• May cause gastroenteritis, conjunctivitis, cystitis, rashes

• Hepadnaviridae

• Hepatitis B

• Herpesviridae • Herpes simplex 1 and 2 • Varicella-zoster

• Papillomaviridae • Warts • Possibly cervical carcinomas

DNA Viruses

• Parvoviridae • Only one causes human disease: B19

• Others cause disease in animals

• Poxviridae • Largest viruses

• Causative agent of smallpox

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RNA Viruses

• Orthomyxoviridae

• Include influenza A, B, and C viruses

• Type A—most virulent in humans

• Coronaviridae • Respiratory and enteric disease

• Picornaviridae

• Important pathogens, include Enterovirus, Rhinovirus, Hepatovirus, Aphthovirus, Parechovirus, Erbovirus, Kobuvirus, Teschovirus

• Large family, smallest RNA viruses

• Hepeviridae

• Paramyxoviridae

• Filoviridae & Bunyaviridae

• Hemorrhagic Fevers 60

RNA Viruses

• Rhabdoviridae • Infect plants, insects, fish, birds, mammals, including humans

• Rabies virus belongs to this group

• Reoviridae • Rotavirus: gastroenteritis—common in children— accounts for approximately 50% of all cases of

diarrhea in children requiring hospitalization

• Retroviridae • Oncovirinae, Lentivirinae, Spumavirinae

• Example: HIV

• Togaviridae • Alphaviruses, Rubivirus—rubella virus

• Flaviviruses • Yellow fever, West Nile virus

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Fungal Characteristics

• Fungal colonies are vegetative • Generally grow as filamentous, multicellular organisms • They form filaments called hyphae or unicellular organisms • Mycelium is a collective mass of hyphae • Differ from plants—do not contain chlorophyll • Do not use photosynthesis • Have cell wall composed of strong flexible polysaccharide called chitin • Some synthesize toxins—ergot alkaloids, psychotropic agents, aflatoxins

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• Routine Media

• Sabouraud Dextrose Agar (SDA)

• Selective for dermatophytes

• Mycosel or Mycobiotic

• Selective and differential for yeasts

• CHROMagar Candida

• Niger seed

• Cornmeal agar

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Primary Culture Media

Direct Microscopic Exam

• Should be done on all specimens received for fungal culture

• Purpose

• Preliminary report

• Clues to identification

• Methods

• Wet Prep/KOH prep

• India Ink

• Calcofluor white

• Other stains

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Criteria for ID of Molds

• Growth Rate • Rapid, intermediate, slow growers

• Incubation conditions

• Colony Morphology • Hyaline or dematiaceous

• Pigment, texture, topography, etc

• Microscopic Morphology (natural growth state) • Hyphae

• Type of Sporulation

• Mold to yeast conversion

• Cycloheximide resistance

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Classification of Fungi

• Zygomycota

• Multinucleate molds, most are saprobes

• Nonseptate fungi

• Mucor, Absidia, Rhizopus, Rhizomucor

• Dermatophytes

• Filamentous fungi

• Trichophyton, Epidermophyton, Microsporum

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Classification of Fungi

• Dimorphic Fungi • Temperature triggers change

• Exist in yeast and mold form

• Blastomyces, Histoplasma, Coccidoides, and Sporothrix

• Yeast • single-celled fungi

• Reproduce by budding

• Candida albicans

• Cryptococcus neoformans

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Protozoans

• Defined by three common characteristics

• They are eukaryotes

• They are single-celled

• They lack a cell wall

• Majority are motile due to cilia, flagella, and/or pseudopodia

• Two distinctive forms

• Trophozoites

• Cysts

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Protozoan Parasites

• Amebas

Move by pseudopods

• Entamoeba histolytica

• Entamoeba coli

• Acanthamoeba

• Naegleria fowleri

Protozoan Parasites

• Flagellates

• Simple

• Giardia

• Trichomonas

• Several life-cycle forms

• Typanosmona cruzi

• Typanosoma brucei

• Leishmania

Protozoan Parasites

• Ciliates

• Balantidium coli

• Apicomplexa

Intracellular

• Plasmodium

• Toxoplasma

• Pneumocystis

• Cryptosporidium

Helminths

• Often found in blood, feces, and urine

• Must be distinguished from microbes

• Many parasitic helminths spend much of their life cycle in mammalian host

• Most of the ones affecting humans belong to flatworms and roundworms

• Adult animals usually large enough to be seen with the naked eye

• Eggs and larvae—microscopic

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Helminth Parasites

• Trematodes a.k.a. flukes

• Schistosoma

• Cestodes a.k.a. tapeworms

• Taenia saginata

• Taenia solium

• Hymenolepis nana

Helminth Parasites

• Nematodes a.k.a. roundworms

• Necator

• Ascaris

• Enterobius

• Loa Loa

• Filiariasis

• Wuchereria

• Brugia