Aerobic Gram-Positive Bacilli Part II Genital Culture Unit Please click audio icon to hear Carol’s narration Aerobic Gram-Positive Bacilli Part II Genital Culture Unit Hi I’m Carol Larson, your guide thru this tutorial about the aerobic gram-positive bacilli – part 2. During student lab, you received Part 1 of this lecture. This lesson will be of value to you as you rotate thru your Wound Culture Rotations and your Genital Culture Rotation. You will find an audio icon on each screen of this presentation. Please click on the icon to hear my narration for that specific screen. You may also follow along with the lecture handout in your student lab notes entitled “Miscellaneous Gram Positive Bacilli” starting on page 7. Division of Medical Technology Carol Larson MSEd, MT(ASCP)

Differentiation of Major GPR Genera Click icon for audio Differentiation of Major GPR Genera Gram stain Morphology Arrangement Formation of spores Catalase reaction Remember from the first Aerobic GPR lecture you had in student lab, that the aerobic gram-positive bacilli are differentiated by gram stain morphology and arrangement, formation of spores, and the catalase reaction.

GPR Discussed in Student Lab Click icon for audio GPR Discussed in Student Lab Spore-forming GPR, catalase positive Bacillus species Bacillus anthracis Bacillus cereus Nonspore-forming GPR, catalase positive Listeria monocytogenes Corynebacterium species (diphtheroids) Corynebacterium diphtheriae Corynebacterium jekeium Organisms that we looked at in student lab included the spore-forming GPRs that are catalase positive including the Bacillus species. We also looked at the nonspore forming, catalase positive GPRs including Listeria and Corynebacterium. Be sure to review the information relating to these organisms as you go thru your clinical rotations.

GPR Discussed in This Lecture Click icon for audio GPR Discussed in This Lecture Nonspore-forming GPR, catalase negative Gardnerella species Erysipelothrix species Lactobacillus species Branching Nocardioform GPR Nocardia species Streptomyces species For this lesson, we will now turn our attention to the nonspore forming catalase negative GPRs and the branching GPRs. The nonspore forming catalase negative GPRs include Gardnerella, Erysipelothrix and Lactobacillus. The branching GPRs include Nocardia and Streptomyces.

Gardnerella vaginalis Gram Stain GPR, pleomorphic, Non-spore forming, Catalase negative Click icon for audio Gardnerella vaginalis Gram Stain Pleomorphic gram-variable bacilli Non-sporulating Specimen gram stain: Clue cells Lets start with Gardnerella vaginalis, a pleomorphic, non-spore forming, gram-variable rod. Gardnerella decolorizes very quickly and appears gram-variable. On specimen gram stains, especially female vaginal specimens, we will often times see clue cells. These are epithelial cells covered with tiny bacilli especially around the edge of the cell. This picture shows a squamous epithelial cell covered with bacteria. When these are seen, it is a presumptive identification for Gardnerella.

Gardnerella vaginalis Colony Morphology GPR, pleomorphic, Non-spore forming, Catalase negative Click icon for audio Gardnerella vaginalis Colony Morphology Does not grow on sheep blood agar Human Blood Agar (V-agar, HBT-agar) Small colonies with diffuse zone of beta-hemolysis Gardnerella vaginalis does not grow on standard sheep blood agar, but it will grow on human blood agar such as V agar and Human Blood Bilayer Tween agar. It is beta-hemolytic on human blood agar and the colonies are very small. There is a nice picture on page 345 in your textbook.

Gardnerella vaginalis Identification GPR, pleomorphic, Non-spore forming, Catalase negative Click icon for audio Gardnerella vaginalis Identification Catalase negative Oxidase negative Sodium hippurate usually positive SPS sensitive Gardnerella vaginalis is catalase negative, oxidase negative, and usually Sodium hippurate positive. It is also sensitive to sodium polyanetholsulfonate (SPS). This is significant in that most blood culture broths used today contain SPS as an anticoagulant. If sepsis with Gardnerella is suspected, it is important to use blood culture broth that is SPS-free and supplemented with gelatin.

Gardnerella vaginalis Clinical Significance GPR, pleomorphic, Non-spore forming, Catalase negative Click icon for audio Gardnerella vaginalis Clinical Significance Normal vaginal flora Bacterial vaginosis Polymicrobic infection with Mobiluncus and Bacteroides Diagnosis Homogeneous, gray discharge Clue cells Amine or fishy odor when add 10% KOH Culture not necessary for diagnosis Gardnerella vaginalis is usually considered normal flora, but is also associated with bacterial vaginosis. This infection is usually a polymicrobic infection with Mobiluncus & Bacteroides. To diagnose this disease, we examine the vaginal specimen for the presence of a homogeneous, gray discharge, clue cells on the Gram stain or wet mount, and an amine or fishy odor when 1 drop of 10% KOH is added to the discharge on a slide. A culture is not recommended for the diagnosis as the other items provide a classic presentation of bacterial vaginosis.

What are the key identification characteristics to determine if Gardnerella vaginalis is the cause of bacterial vaginosis? Examine vaginal specimen for presence of a homogeneous, gray discharge, clue cells on the Gram stain, and an amine or fishy odor when adding 10% KOH. A culture is not recommended, but if done, G. vaginalis grows on V-agar/HBT-agar and it beta hemolytic.

Lactobacillus species Gram Stain GPR, pleomorphic, Non-spore forming, Catalase negative Click icon for audio Lactobacillus species Gram Stain Two types Long slender GPR in chains Short GPCB Non-sporulating Lactobacillus species also can have two forms on the gram stain. They can be long slender gram positive rods in chains, or short coccobacilli. The are also non-sporulating. It is often difficult to differentiate the short coccobacilli of Lactobacillus from streptococci, so a gram stain of growth just outside the zone of inhibition surrounding a 10-U penicillin disk placed on a BAP inoculated with a lawn of the organism should show long bacilli rather than coccoid forms if it is Lactobacillus.

Lactobacillus species Colony Morphology GPR, pleomorphic, Non-spore forming, Catalase negative Click icon for audio Lactobacillus species Colony Morphology Grows on BAP Multiple colony morphologies Nonhemolytic or alpha hemolytic Microaerophilic Lactobacillus is microaerophilic and must be incubated in 5-10% CO2. They will also grow anaerobically. On sheep blood agar, Lactobacillus may have multiple colony morphologies ranging from pinpoint, alpha-hemolytic colonies resembling streptococci to rough, gray colonies.

Lactobacillus species Identification GPR, pleomorphic, Non-spore forming, Catalase negative Click icon for audio Lactobacillus species Identification Catalase negative Sucrose “F” Vancomycin “R” To identify Lactobacillus, the catalase is negative, it ferments sucrose, and is resistant to vancomycin. To differentiate from streptococci, as mentioned earlier, a gram stain of organism growing around a penicillin disk can be of assistance.

Lactobacillus species Clinical Significance GPR, pleomorphic, Non-spore forming, Catalase negative Click icon for audio Lactobacillus species Clinical Significance Normal flora Mouth GI tract Female vaginal tract Rarely pathogenic Endocarditis Meningitis Lactobacillus is considered normal flora of the mouth, gastrointestinal tract, and female genital tract. It is rarely considered pathogenic. It has been implicated in rare cases of endocarditis and meningitis.

What is the clinical significance of Lactobacillus species? It is considered normal flora of the mouth, gastrointestinal tract, and female genital tract

Review Nonspore-forming GPR, catalase negative Gardnerella species Click icon for audio Review Nonspore-forming GPR, catalase negative Gardnerella species Erysipelothrix species Lactobacillus species Thus far, we have learned about the non-spore forming, catalase negative GPRs. Gardnerella is most associated with bacterial vaginitis and is presumptively identified by the presence of clue cells, gram-variable rods, and beta-hemolysis on human blood agar. Erysipelothrix is the only GPR that is H2S positive and shows a test-tube brush appearance in the motility tube. It can cause erysipelas. Lactobacillus is considered normal flora and is generally identified by gram stain, colony morphology and catalase. All three of these organisms are catalase negative. Next, lets turn our attention to the branching nocardioform GPRs that include Nocardia and Streptomyces.

Nonspore-forming GPR, catalase negative Click icon for audio Aerobic GPR Summary Nonspore-forming GPR, catalase negative Gardnerella species Erysipelothrix species Lactobacillus species Branching Nocardioform GPR Nocardia species Streptomyces species In this lesson, I’ve taken a look at the aerobic gram-positive bacilli that are of two groups: nonspore-forming GPRs that are catalase negative, and the branching nocardioform GPRs. I addressed the key gram stain, colony morphology and biochemical tests used to identify these organisms. Though rarely causing disease, each one of these organisms when given the opportunity can be pathogenic. If you have additional questions relating to these organisms, your textbook provides a nice discussion on each one.

Who am I? Gardnerella vaginalis HBT media shows beta-hemolysis Gram Stain Causes Bacterial Vaginitis Gardnerella vaginalis