A Review Of Testing Methods And Medications Used In The Treatment Of Commonly Seen Bacterial And Fungal Conditions In Podiatry Jack Hutter DPM, C.ped, FACFAS, FPWCA, Diplomate, ABPFAS
Gram Stain - gram + or Morphology -coccus, bacillus, single, clusters, chains, encapsulation, spore forming Growth oxygen requirements -aerobic, anaerobic, intermediate, facultative, obligate Genetic information -RNA, DNA Coagulase testing -coagulase positive, coagulase negative Growth on culture medium Tests above are taken in totality when attempting to establish organism identity
Gram Stain the ability of the organism to take up stain - Crystal violet/safranin microscope slide test used to determine absence or presence of organism - Also provides information on organism morphology ( rods, cocci, single, chain, cluster, pleomorphic ) - Bacteria stain either purple ( gram positive ), or pink (gram negative ) - Gram positive bacteria can withstand drying out, gram negative thrive in a moist environment - Gram negative bacteria have multiple cell walls and withstand strong inhibitory chemicals (psuedomonas can grow in chlorinated water ) - A negative gram stain does not rule out infection as some organisms do not take up stain
Coagulase testing - presence of a protein produced by several microorganisms enabling the conversion of fibrinogen to fibrin - Used to differentiate between different types of Staphylococcus species - Coagulase negativity excludes Staphylococcus aureus ( staph aureus is coagulase positive ) - Other less frequently encountered Staphylcocci are also coagulase positive including Staphylococcus aureus anaerobius
Growth oxygen requirements - survival in the presence or absence of oxygen - Aerobic organisms thrive in a oxygen rich environment - Anaerobic organisms die if in an oxygen rich environment - Some organisms are intermediate in their oxygen requirements - Bacteria are also classified as facultative or obligate anaerobes - Facultative anaerobes are organisms that can make ATP by aerobic respiration if oxygen is present, but without oxygen are also capable of switching to fermentation for cell function - Obligate anaerobes die when oxygen is present
GROWTH ON CULTURE MEDIUM -SOURCES FOR OBTAINING THE SPECIMEN Drainage, whatever the source Aspirate Blood Draw Tissue biopsy Remember that the testing and culture identify the organism(s), whereas the sensitivity determines the appropriate antimicrobial choice
Drainage -Sterile swab and preservative medium contained in aerobic or anaerobic culturette -Swab drainage if possible, skin alone without drainage may produce a false result -Preliminary result in 24 hours usually reported along with gram stain, provide basic information about whether organisms are present and what type -Usually at least 48 hours for final result which identifies organism(s) AND provides antimicrobial sensitivity pattern (MIC) -Aseptic debridement of overlying necrotic tissue allows a more reliable culture, reduces contaminant -Amount of growth ( heavy, moderate, few ) on the culture medium helps in determining pathogen from contaminant -Be on guard for results reported strains suggest normal flora, as even Staph epidermidis can be a pathogen, request sensitivities, -Supply your lab with appropriate clinical information along with the specimen -Process the culture immediately, delay causes degradation
Aerobic, Anaerobic Culture Transfer
ASPIRATE Sterile prep of area to be aspirated Draw aspirate into a 3cc syringe Either submit aspirate within drawing syringe, capped off and without the needle, or deposit an appropriate amount of the aspirate aseptically onto the tip of a culturette, cap off and process immediately BLOOD DRAW Done in the case of suspected septicemia The best time to draw is just before or at the rise in oral temperature Three cultures are drawn, each every three hours for the most reliable results Preferably done before beginning antobiotics Most common bacteria to cause septicemia are gram (-) rods ( E. coli, enterobacter ), second is gram (+) cocci staph aureus
KIRBY-BAUER AGAR DIFFUSION TECHNIQUE 1. Isolate a bacterial colony from the original growth media 2.Allow the bacteria to grow in broth medium 3.Cover a Mueller-Hinton agar plate completely with the bacterial isolate 4.Place antibiotic impregnated sensitivity plates at intervals on the surface 5.Incubate 18 20 hours 6.Examine for clear areas around the individual discs which represent bacterial growth inhibition 7.Results are reported as resistant, intermediate or non-resistant 8. Anaerobes are cultured in appropriate oxygen free conditions
MINIMUM INHIBITORY CONCENTRATION More specific than simply reporting resistant or nonresistant Will determine the degree of sensitivity of a bacterial species to a particular antibiotic Gives the practitioner the ability to chose the most effective antibiotic for a particular organism The lower the number, the more sensitive the antibiotic and more susceptible the organism to it Also reports if the organism is resistant to the antibiotic
THE TEST PROTOCOL A pure culture of a single organism is grown on Mueller-Hinton broth The culture is standardized to 1,000,000 cells per milliliter The antibiotics to be tested are diluted a number of times A 1:1 mixture of the bacteria and the antibiotic to be tested is created in separate multiple dilution vials A series of inoculated vials is used for each tested antibiotic The inoculated vials with serially diluted antibiotic are incubated 18 hours After incubation, the series of dilution vials is observed for bacterial growth The last vial in the series that does not demonstrate growth corresponds with the minimum inhibitory concentration of the antibiotic Comparison of MIC values allows the practitioner to determine appropriate antibiotic coverage
ULCERATIVE WOUNDS Diabetic Venous Pressure TRAUMATIC WOUNDS Acute injury Chronic trauma (repetitive skin trauma, web space breakdown, fissures ) Post operative
ULCERATIVE WOUNDS Diabetic wounds -Perform bacterial aerobic ( and anaerobic if deep, sinus tract and chronic ), and fungal culture and sensitivity -gram stain is helpful for preliminary confirmation of infection ( reported as many vs. few of a particular type of organism ), but culture is necessary for ID of the organism -Debridement of necrotic, overlying tissue before culture provides a more reliable result
ULCERATIVE WOUNDS Venous wounds -Often chronic, with mixed bag of pathogens combined with contaminant -Anaerobes less frequent than aerobes unless extensive necrosis -Culture also for fungi, especially yeast, more common in elderly and immuno-compromised patients -Be aware of mixed diabetic and venous wound -Gram stain helps to determine if temporary antibiotic coverage is indicated, culture results will confirm the proper antibiotic and dosage
ULCERATIVE WOUNDS Pressure ulcer -Frequently chronic -Debilitated or wheelchair bound patient -Mixed pressure and diabetic etiologies not uncommon -Undermining, sinus tract, deep involvement, -Anaerobic and aerobic culture after debridement
TRAUMATIC WOUNDS Acute injury Less necrosis Unless deep puncture, aerobic culture primarily If infection, cultures likely to be skin structure flora staph, strep Chronic injury Repetitive trauma to skin Web space with maceration, heel fissures with dyshydrosis Culture mixed bag of organisms, not necessarily all pathogens Aerobic culture and sensitivity if drainage Skin specimen biopsy with culture if no drainage but suspect infection
SURGICAL WOUND Immediate post operative infection 24 to 72 hours post op Pain, rubor, swelling, gapping of incision, drainage Patient may be febrile, chills, night sweats if septic Remove sutures, express drainage, gram stain and aerobic C&S Start broad spectrum antibiotic ( penicillinase resistant, Augmentin or cephalosporin, clindamycin if penicillin allergy ) and watch for C&S results 48 hours Chronic post operative infection Dehiscence More tissue necrosis, deeper involvement Debridement, gram stain, aerobic and anaerobic culture and sensitivity Start broad spectrum antibiotic but may likely change pending culture and sensitivity results
AEROBIC BACTERIA GRAM POSITIVE COCCI Streptococcus Facultative anaerobe Colony appearance describes three basic forms, alpha, beta, gamma -Alpha - strep viridans, causes incomplete hemolysis surrounding the colony -Beta causes complete hemolysis, the highest incidence of pathogenicity -Gamma no hemolysis, enterococcus group Lancefield classification of beta hemolytic strep ( group A and Group D ) based on the presence of a particular carbohydrate produced by antibodies
AEROBIC BACTERIA GRAM POSITIVE COCCI Staphylococcus -Gram (+), occur in clusters -Facultative anaerobe -Normal skin flora -Three groups depending upon colony appearance -Staph epidermidis ( coagulase negative, white colonies ) -Staph aureus ( coagulase positive, yellow colonies ) -Staph citreus ( pale green, rarely seen )
METHICILLIN RESISTANT STAPHLOCOCCUS AUREUS MRSA, AKA multidrug resistant staph aureus or oxacillin resistant staph aureus All the characterictics of Staph aureus, also resistant to cephalosporins Emerging as a serious pathogen with resistance to many antibiotics First reported in 1961 in UK, 1981 US, 2004 in Canada, world wide and worsening 2.4 % of healthy children in US are carriers, same percentage in adults Studies show that poor hygiene habits with health care professionals is a principal barrier to spread of MRSA
METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS Colonizes in the anterior nares and respiratory tract Usually purulent, can cause sepsis and shock Facultative anaerobe8 Two basic forms, community acquired and hospital acquired Community form is more virulent, can lead to necrotizing fascitis, septic arthritis, osteomyelitis Risk factors include immuno-compromised state, diabetes mellitus, IV drug use, quinolone antibiotic usage, children, elderly, healthcare facility workers, confined spaces with large populations, locker rooms
MRSA DIAGNOSIS Colony growth on Mueller-Hinton agar shows resistance to oxacillin disc Real time PCR, quantitative PCR for rapid detection Latex agglutination to detect PBP2a protein, a penicillin binding protein that gives Staph. aureus its oxacillin resistance MRSA TREATMENT Prevention through active patient screening with anterior nares cultures ( hospitals, nursing homes, long term care facilities ) (also schools, workplaces since community form is more virulent? Controversial ) Isolation of MRSA carrying patient/resident Decolonization with chlorhexidine or Phisohex full body scrub, and Bactroban 2% ointment in nostrils bid for one week Aseptic pecautions in patient care Terminal cleaning of treatment room Negative anterior nares cultures three separate times to confirm decolonization
MRSA TREATMENT NON MEDICATIONS Restricted use of certain antibiotics that promote colonization, cephalosporins and especially fluoroquinolones Essential oil effusion (cinnamon, lemon myrtle, melissa, mountain savory all inhibit MRSA, lemon grass and tea tree oil kill MRSA Cannibinoids Maggot therapy extremely effective (fly larvae debride and sterilize a wound ) MRSA TREATMENT CURRENT MEDICATONS The drug of choice for both community and hospital based MRSA is vancomycin Teichoplanin is a structural congener of vancomycin, but has a longer ½ life Administer through a PICC line ( peripherally inserted central catheter ) Newer drugs Linezolid and Daptomycin are also effective but have a greater activity spectrum A new strain VISA ( vancomycin intermediate resistant Staph aureus ) must be treated with Linezoid, Daptomycin, Synercid, or Tigecycline,
MRSA TREATMENT THE FUTURE New even more resistant strains are continuously evolving, requiring novel treatments that can circumvent the resistance mechanism Many drugs are in phase II and phase III trials, ( especially Aurograb, Nemonoxacin ) Phage therapy has been 95% efficient in mice Platensimycin
AEROBIC BACTERIA GRAM POSITIVE COCCI Enterococcus -Entercoccus faecalis -Rods appearing as pairs or chains -Facultative anaerobe GRAM POSITIVE RODS Mycobacteria ulcerans -Gram stain negative -Long, slender rods -Obligate anaerobe
Enteric bacilli AEROBIC BACTERIA GRAM NEGATIVE RODS -Escherichia coli, enterobacter, klebsiella, proteus -May be normal flora in the GI tract, pathogenic if contamination of other location -Salmonella, Shigella not normal GI flora, enter through the environment Psuedomonas -P. Auergeuosa -Obligate anaerobe -May live in the GI tract, contaminate wounds especially if host is immunocompromised -Aquatic environment -Encapsulated, motile -Greenish color in clinical setting
ANAEROBIC BACTERIA Clostridium,Perfringens( Welchii ), tetani, botulinum -Gram positive rod -Obligate anaerobe -Normally in GI tract, 20% of the time on the skin -Gas gangrene, tetanus, botulism Bacteroides -Gram negative rod -Obligate anaerobe -GI tract, mouth, female GU tract -Abscess, gangrene, wound contaminant Streptococcus -Peptostreptococcus -Gram positive cocci, single or in chains -Often mixed aerobic/anaerobic infection, deep wounds, necrotizing fascitis -Extremely drug resistant
Assess liver and kidney function prior to antibiotic therapy Primary infection caused mostly by two skin structure bacteria, staphylococcus and streptococcus Secondary infection by contamination from non-skin structure organisms is precipitated by loss of skin integrity Immuno-compromised patients are at increased infection risk Antibiotics for treatment of skin infections are either bacteriocidal or bacteriostatic Each particular antibiotic acts by inhibiting a particular cell function or altering cell anatomy Antibiotic resistance often develops with prolonged antibiotic use Occasionally infections are due to a combination of anaerobic and aerobic organisms, and one must chose the antibiotic ( or combination of antibiotics ) with the greatest efficacy
Some bacteria produce enzymes called beta-lactamases, which provide resistance to antibiotics containing a beta-lactam ring such as the penicillins This occurs by breaking down the lactam ring in the molecular structure of the antibiotic Penicillinase is a specific type of beta-lactamase, which dictates the need for a penicillinase resistant antibiotic such as methacillin, oxacillin, dicloxicillin Ceplalosporins are generally resistant to betalactamase Clavulonic acid will inhibit penicillinase, and is added to a cillin to eliminate the beta-lactamase inactivation ( Augmentin )
GENERALLY RESISTANCE TESTING WILL CONFIRM THE APPROPRIATE ANTIBIOTIC Clostridium difficale -Overgrowth of human flora in the GI tract from over use of antibiotics - Treatment -D/C predisposing antibiotics -Fluids -Internal medicine/infectious disease consult -Penicillin G, Metronidazole Clostridium tetani -Puncture wound -Spores in soil -Gas gangrene -Surgical emergency, hyperbaric oxygen, PCN G, Doxycycline
Enterococcus faecalis -GI tract -Opportunistic contaminant of a wound or surgical site -Treatment -Medical management -Penicillin + Vancomycin, newer agents Quinupristin and Dalfopristin Escherichia coli -Normal GI flora -Opportunistic wound contaminant -Treatment -Medical management -Co-trimazole, Fluoroquinolone, Cephalosporin + Gentamycin combination
Psuedomonas Auerginosa -Water borne organism -Infects Damaged tissue, surgical sites -Bone, joint, skin, soft tissue are all susceptible -Treatment - Topical acetic acid, Aminogyycocide + beta-lactam, or Fluoroquinolone, -Local wound care Staphylococcus aureus -Human mucosal flora -Wound and surgical contaminant -MRSA potential -Treatment -Oxacillin, Cephalosporin, Augmentin, -Local wound care -Surgical invention if indicated
Staphylococcus Epidermidis -Normal human mucosal flora -Wound contaminant, prosthesis infection -Resistant to many antibiotics -Often reported as normal flora, if cultures show moderate or heavy growth, consider to be a pathogen and order sensitivities -Treatment -Clindamycin, Vancomycin in severe infections -Wound care -Surgical intervention if indicated Streptococcus Agalactiae, Pyogenes,Beta Group A and Group D -Human mucosal flora -Opportunistic wound contaminant with infection -Treatment -Penicillin G, Ampicillin, Aminoglycocide if severe infection -Local wound care Peptostreptococcus and related gram (+) anaerobic bacteria -Deep, chronic wound -Often mixed infection -Treatment -Penicillin G, Cephalosporins, Clindamycin, Vancomycin, or newer quinolone ( not cipro ) -Local wound care -Surgical debridement
HUMAN FUNGAL INFECTIONS Internal fungal infection, potentially systemic -Sporotrichosis,Blastomycosis,Histoplasmosis Opportunistic fungal infections -Antibiotics which kill bacteria alter the normal flora, providing a medium for fungal invasion -Compromised host with reduced response to fungal infection ( AIDS, steroid therapy, methotrexate and similar drugs used to treat autoimmune disorders ) Skin and toenail fungal infection -What Podiatrists see most of the time -12% of all Americans have toenail fungus -Runs in families due to inherited tendencies -Rare in children unless one or both parents are infected -Accounts for 50% of all toenail abnormalities
THREE PRINCIPAL TYPES OF FUNGAL ORGANISMS AFFECTING SKIN AND/OR TOENAILS Dermatophytes -Aerobic -Trichophyton rubrum most common, T.mentagryphytes species ( no longer considered a human pathogen, now refers to mouse ringworm ), T. violaceum, T. interdigitale, T. tonsurans, T. verrucosum (cattle ringworm ), Epidermphyton floccosum, Microsporum gypseum Non dermatophyte molds -Aerobic -Opportunistic -Seen in patients over 60 y/o -Scopulariopsis, Alternaria, Penicillium,Aspergillus Yeast -Aerobic -Opportunistic -Primarily candida species
MAKING THE DIAGNOSIS Dermatophytes -KOH smear demonstrates single hyphae and combined branches of hyphae called mycelia -Culture with DTM Saboraud s agar or Mueller Hinton agar, shows diagnostic color change and colony growth Non dermatophyte molds -KOH shows no presence of hyphae -Culture as with dermatophyte, shows diagnostic colony growth, but no color change in Saboraud s agar Yeast -Can be polymorphic with KOH demonstrating psuedohyphae formed from budding yeast cells, spores on psuedomycelia -Gram stain + -DTM or Mueller Hinton culture shows creamy white, putty-like colony, no color change in medium
DTM results
TINEA PEDIS AKA athlete s foot -Trichophyton rubrum most common -Acute form at web spaces, moist areas -Clinically erythema, pruritis, maceration -Moccasin distribution when tinea unguim is also present Diagnosis -Hyphae seen on KOH prep rule out psoriasis, eczema -Skin culture (DTM scraping ) may confirm organism Treatment -Avoid exposure to fungal organisms with appropriate footwear and foot hygiene -OTC antifungal cream, ointment or gel for acute cases -Antifungal powder to reduce moisture and control reoccurrence -Moisture absorbing, wicking socks, shoes that breathe -More severe cases may require a short course of oral terbinifine -Often there is concurrent tinea unguim requiring treatment with ciclopirox and/or oral terbifine or iatroconazole
TINEA VERSICOLOR AKA pityriasis versicolor -Yeast organism, Malassezia globosa, m. fufur -Normal skin flora, becomes pathogenic under warm, humid conditions -Presents clinically as erythematous, ovoid macular area with fine scaling Diagnosis -KOH shows psuedohyphae -Culture shows yeast cells Treatment -Topically,nizoral, ciclopirox, ketoconazole, miconazole -Orally, fluconazole, ketoconazole ((Diflucan, preferred over ketoconazole, terbinifine, iatroconazole ) CANDIDIASIS Candida - Also a yeast infection, superficially in skin, web spaces (candidal intertrigo ) -Normal skin flora, controlled by the immune system and bacteria that occupy the same niche - Become pathogenic iatrogenically through bacterial antibiosis Diagnosis - KOH prep displays psuedohyphae and budding yeast cells -Yeast colonies on culture ( DTM creamy color, creamy in consistency) Treatment -Usually topical for skin, miconazole, nystatin, clotrimazole, - Oral fluconazole ( Diflucan ) in severe cases
ONYCHOMYCOSIS FOUR BASIC CLINICAL PRESENTATIONS 1. Distal subungual - Most common presentation, usually dermatophyte Trichophyton rubrum 2. White superficial 10% of cases -Superficial white islands on surface of toenail 3. Proximal subungual -Toenail is penetrated through the proximal nail fold -More frequently seen in immuno-compromised patients 4. Candidal -Opportunistic infection -develops secondary to trauma to toenail
IT S NOT ALWAYS A FUNGUS TOENAIL CHANGES THAT MAY REVEAL A SYSTEMIC CONDITION White toenail liver disease Half pink, half white toenail kidney disease Toenail is light reddish heart disease Yellow and thickened, reduced growth rate lung disease Pale nail bed anemia Yellowish toenail, slight blush at eponychium diabetes mellitus Thickened, hardened, brittle toenail peripheral vascular disease Thick, brownish yellow, friable, much like a mycotic nail psoriasis, lichen planus
MAKING THE DIAGNOSIS Involved toenail is thick, discolored, friable, lytic in appearance Single or multiple nails Partial or full nail plate involvement Look for concurrent skin involvement Tests include KOH, Periodic Acid-Schiff stain, culture on DTM or Mueller-Hinton agar MIC values have been established for oral antifungals against, dermatophytes, molds, yeasts KOH shows hyphae, mycelia ( dermatophyte ), none ( non dermatophyte ),psuedohyphae, psuedomycelia, yeast cells (yeast ) Periodic acid-schiff stain used if culture is inconclusive Culture shows characteristic colony growth and color changes as with skin culture -dermatophyte - white, fluffy colony, cherry red color change in agar ) -non-dermatophyte grayish, fufy colony, no color change in agar -yeast creamy white, putty- like, no color change in agar
CURRENT TRENDS IN TREATMENT Topical OTC, Ciclopirox, Amorolfine Systemic Terbinifine, Iatraconazole, Fluconazole Combined topical and systemic Toenail avulsion combined with short course of systemic Onychoplasty Infrared laser
QUANTIFYING TREATMENT SUCCESS Clinical cure Toenail physically appears improved, less discoloration, thickness, more attached to toenail bed Mycologic cure Toenail culture is negative Complete cure The preferred result, physical appearance is normal and toenail culture is negative One should perform cultures prior to and at the end of treatment
OBSERVATIONS ON TREATMENT SUCCESS Topical treatment should be reserved for mild cases Complete nail involvement infers matrix involvement, must be treated more aggressively than with topical alone Amorolfine 5% lacquer has shown complete cure rates of 38-46%, more effective than Ciclopirox, and only needs to be applied twice per week Amorolfine is only available in Europe Topical combined with systemic is more effective than systemic alone Systemic antifungals create a reservoir effect due a high affinity to the fat cells under the nail bed and matrix, arresting fungal growth in the nail bed and matrix MIC values show terbinifine is the most effective oral antifungal against dermatophytes, fluconazole is most effective against yeast
TREATMENT SUCCESS OBSERVATIONS CONTINUED Monitoring for liver function is necessary with SGOT, SGPT or liver panel as systemic antifungals can cause liver damage Systemic antifungals can also can cardiac abnormalities and heart failure Pulse dosing is more effective with iatroconazole, daily dosing is more effective for terbinifine Terbinifine shows complete cure rates around 60-70%, better than iatroconazole High reoccurrance rate is in part due to never reaching a mycologic cure, and cultures should be done at the end of the reservoir to confirm a mycologic cure Frequent aggressive nail debridement and removal of lacquer film from topical agent using alcohol or nail polish remover once per week improves treatment outcomes
THE FUTURE Improved topical medications ( topical terbinifine ) Amorolfine approval for use in Canada and USA New oral agents ( Posaconazole ) Nd:YAG infrared lasers very promising, showing at least 80% effectiveness -Fungicidal, will not harm surrounding tissues -Pinpointe ( FDA approved ) - Synergy Plus ( FDA approval anticipated ) -Cutera Genesis Plus ( FDA approval anticipated ) -Noveon laser ( dual infrared laser ) Ontario Osteopathic and Alternative Medicine Association LAFT, laser based method of treatment
Bacterial and fungal infections are conditions frequently seen in Podiatry practice Knowledge about the indications for performing specific laboratory tests is necessary in the appropriate treatment of these conditions Sensitivity testing is of critical importance in antibiotic selection Proper interpretation of test results allows for successful pairing of the infective organism with the appropriate antibiotic treatment regime
Thank you!