gram positive batera

7/29/2019 gram positive batera

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GRAM-POSITIVE BACTERIA

Universiti Malaysia KelantanFaculty of Veterinary Medicine

Bacteriology and Mycology (DVT2064)

25 November 2012

Lecture By: Dr. Erkihun Aklilu (Lecturer, MSc Molecular Biology)


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GRAM -POSITIV E BACTERIA

Staphylococcus

Streptococcus

Corynebacterium

Rhodococcus

Lecture By: Dr. Erkihun Aklilu (Lecturer, MSc Molecular Biology)


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The ge rm th eo ry of d isea se

Theo ry o f d isease accor din g t o Rob ert Koch(1843-19 10 ), a Germ an scien t ist

Kochs po st ula te s

A m icro -o rgan ism can be accep t ed as acau sat ive agen t o f an in fect io u s d isease o n ly if

t he fo llow ing cond it i ons are sat i sf ied .

25/ 11/ 2012 3Dr. Erkihu n Akli lu (Lecture r)


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1. The m icro -o rgan ism sho u ld be fo und in ever y caseo f t he d isease an d un der co nd it io ns w h ich exp lainthe patho logical changes and cl in i cal f eatu res.

2. It sho uld be possib le t o iso lat e t he causat ive agen tin p ure cu lt ure f ro m t he lesio n.

3. W hen su ch pu re cu lt u re is ino cu lat ed in t o

app ro pr iat e labo rat o r y an im al, t he lesion o f t hed isease shou ld be r ep r oduced .



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4. Th e m icro -o rgan ism sh o u ld b e fo u nd in every case o f t h ed ise ase an d u n d er co n d it io n s w h ich exp lain t h e p at h o lo gicalchanges and cl in i ca l featu res.

5 . It sh o u ld b e p o ssib le t o iso lat e t he cau sat ive agent in p u re

cu lt u r e f ro m t h e lesio n .

6 . W hen su ch p u re cu lt u re is in o cu lat ed int o app ro p r iat elab o rat o ry an im al, t h e lesio n o f t h e d isease sh o u ld b ereproduced.

7 . It sh ou ld b e po ssib le t o reiso lat e t h e b act er iu m in p urecu lt u re f rom t h e lesion p ro du ced in t h e exp er im ent alan imal .



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Coro llary to Koch s Post u late

Specif ic an t ibo dy t o t he bact er ium shou ld bedet ect ab le in t he serum d ur ing t he cou rse o f t he

disease.

Howeve r, t he re ar e except i ons t o Koch s post u l at e



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Exce pt ion s to Koch s post u late s

1. M any healt hy peop le and an im als car r y p at hogens bu td o n ot exh ib it sym p t o m s o f t h e d isease.

2 . Som e m icrobes are ver y d if f icu lt o r im p ossib le t o gro win vit ro (in t he labo rat o r y) in ar t if icial m ed ia. Eg.Treponem a pa ll idum

3. M any sp ecies are sp ecies sp ecif ic. Eg. Br u cell a a b o rt u s cau se ab or t io n in an im als b ut n o rep or t in h um an s.

4 . Cer t ain d iseases d evelop on ly w h en an op p o r t un ist icp at h o ge n in vad es an im m u n o co m p r o m i sed h o st .



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Gram -Po sit ive Bact er ia

Have t h ick o u t er m at r ix co m posed o f up t o 90% o fpept idoglycan layer

Th e cell w alls o f gram -p osit ive b act er ia are m ad e u po f t w ent y t im es as m uch m urein o r pept ido glycanthan gram -negat i ve bacte r ia.

The t h ick ou t er m at r ix o f pept idoglycan , t eicho ic

acid , p o lysacch ar id es, an d o t her p ro tein s ser ve t heb act e ria in m an y asp ect s



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Gram -Posit ive Bact er ia

The Pept idoglycan Layer



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Gram -Posit ive Bact er iaThe Pept idoglycan Layer



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Fun ct ions of Bacte rial Ce ll W all

Provides shape t o t he b acter ium

Gives r ig idit y to t he o rganism

Protec ts th e bacter ia f rom env ironm ent

Provid es st aining charact er ist ics t o t he b act er iu m

Conta ins recep to r si tes fo r p hages/ com p lem ents

Site of act ion o f ant ibod y and col icin

Cont a ins to x ic com po nent s to ho st



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STAPHYLOCOCCUS

Fro m Greek,

Staphyle grap ean d

Kokkos granu le



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STAPHYLOCOCCUS

Characteristics Gram -posi t ive b act er ia Abou t 1 m in d iam e ter

Oxidase-negative

Catalase-posit ive Non spore- form ing

Non-mo t i l e

Facult at ive anaero bes

Sph er ical cells usually arr anged in grape -like clu st ers Single cocci , pairs, tet rads and chains are seen in l iqu idcu l tu res

25/ 11/ 2012 13Dr. Erkihu n Akli lu (Lecturer )


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STAPHYLOCOCCUS

Characteristics

Can readi ly grow in ord inary m edia under aerobic andm icroaeroph i l ic cond i t ion s

grow m ost rap id ly a t 37o

C but fo rm p igm ent best a t roomtem perature o f 20-25 oC

Colo nies in sol id m edia are roun d, sm oo t h, raised and gl isten ing.

Relat ively resistant to dr ying , heat , and 9 % NaCI, bu t readi ly

inhib i ted by 3 % hexachloro phen e



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STAPHYLOCOCCUS

Characteristics

Com m on comm ensa ls on sk in and m ucous mem brane

Often cause pyogenic in fect ion s

Path ogen ic spe cies of t en p ro du ce co agulase

Im po rt ant an im al path ogens include S. au reu s, S. int erm ediu s,S. hy icus

S. aureus and S. interm edius are coagu lase posit ive w hi le S.

hycus is coagulase var iab le Coagulase negat ive stap hylococcus are less v i ru lent bu t m ay

occasion al ly cause disease in anim als and m an



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STAPHYLOCOCCUS

Ant igen ic str uctu re:

Pept idoglycan( M ucope pt ide): Is im po rt ant in t he pat ho genesis of infect ion l ike el ici t ing

produc t ion o f cy tok ines and o pson ic an t ibod ies Chemo at t r actant f o r po l ym orphs

Act i vate com p lem en t

Teichoic acid : Po lymer o f g lycero l or r ib i to l pho sphate

Protein A : Im por tant in im m uno logic d iagnost i c test

(coagglu t inat ion test ) .

Capsule : Ant i -phagosi t i c prop er ty



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STAPHYLOCOCCUS

An t igen ic st ructu re: Enzymes

Catalase- Prod uced by staph ylococci Con vert s H20 2 in to H20 and 0 2

Coagulase Clots ox idated or c it ra ted p lasm a

Coagulase m ay depo si t f ibr in on t he sur face oforganism and a l ter ingest ion b y ph agocyt ic ce l ls

Clum ping fa ctor A sur face com po un d t hat is respo nsib le for adh erence

of th e organ ism to f i b r inogen and f i b r in



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STAPHYLOCOCCUS

An tigenic str uctu re: Enzymes

Hyaluronidase Spre ading facto r

Proteina ses a nd l ipa ses

Staphylokinase Fibrinolysin

- lactamase Pro vides resist ance of st aphylo coccus to - lactam ant ib io t i c

l ike penici l l in.

Dnase: Deoxyr ibon ucleot id ase Nuclease



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STAPHYLOCOCCUS

An t igen ic st ructu re:

Toxins

Exotoxins(, , , )

M ult ip le solub le heat -stab le, gut en zym e resist anttox ins

Cause st aph ylococcal fo od po ison ing

Toxic sho ck syn d ro m e t oxin (TSST)

Superant igen desquam at i ve tox in Produced by S.aureus

and Causes fever, sho ck, m ult iple-o rgan fai lur e an d skinrash.



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STAPHYLOCOCCUS

An tigenic str uctu re: Toxins

Exfoliative toxin Epiderm oly t ic superant igen Causes general ized d esquam atio n of t he skin (stap hylo coccal

scalded skin syndr om e). Epiderm olytic toxin A:

Chrom osom al gene pro duct and heat stab le

Epiderm olytic toxin B: Plam id m ed iated and heat lab i le

Leukocidin: S aureus to xin w hich k i lls W BCs by for m ing po res and

incresing cat ion perm eab i li t y



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STAPHYLOCOCCUSD isea ses in an im als

Exudat ive ep iderm it is in piglet s (greasy-pig diseases): S.hyicus

Tick pyaem ia of lam bs : S. au reu s

Bovine staph ylococcal m ast i t is: S. au reu s Bot ryo m ycosis (Scirrh ou s cor d) (ho rse, pigs, catt le) : S.

aureus

W oun d in fect ion (mo st an imals) : S. aureu s, S. hy icu s, S.in termidus

M ast i t is: S. au reu s, S. hyicus, S. int erm ediu s

Bum ble foo t , omp ha li t is in pou l t r y : S. au reu s Pyoderm a, o t i t i s externa, cyst i t i s, endom etr i t i s in do gs: S.

in termedius



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STAPHYLOCOCCUS

Lab orat ory Diagno sis

Specimen: Su r face sw abs, pus, b loo d , sput um ,cerebrosp inal f lu id , exuda tes, m i lk

Smear : Gram posit ive co cci in clust ers, singly o r in

pairs. Culture: Grow w ell aero b ically and in a CO2 enr iched

o rd inary m ed ia at an o pt im al t em perat ure o f 35oC-37oC.

M edia: N o n -e nr ich ed m e dia (N u t r ie nt agar, b lo o d agar ) Selective m edium: m an nit o l salt agar (co nt ain s 7-10%

NaCl)



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STAPHYLOCOCCUS

Lab orat ory Diagno sis

Colony M orph ology

Colour : usua ll y w h i te , opaque and up t o 4m m indiameter .

Colon ies of bo v ine and h um an st ra ins of S. aureus aregolden yel low .

Sapr ophyt ic st ap hylococcusm ay be p igment ed

St aphylo coccus m ay pro du ce haem olysis on sheep / oxblo o d agar. Type s of haem o lysis (, , , )

S. aureus and S. in term edius prod uce doub le zones of na r row com p lete and w ide incom plete haem olysis onbloo d agar

S. hyicus is non -haem olyt ic25/ 11/ 2012 23Dr. Erkihu n Akli lu (Lecture r)


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25/ 11/ 2012 Dr. Erk ihu n Akli lu (Lect u rer ) 24

S. aureus on BA

S. aureus on M SA

M RSA on O RSABS. aureus on Gram -sta in ing


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STAPHYLOCOCCUS

Lab orat o ry Diagno sis

Bioche m ica l Test s



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STAPHYLOCOCCUS

Lab orat o ry Diagno sis

Bioche m ica l Test s



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STAPHYLOCOCCUS

Treatment

Pen icil l in sen sit ive st aphy lo coccipen icil l in ,ampici l l in

Pen icil l in resist an t st aphy lo coccicloxacill in,Nafci l l in

M et hici l l in resist ant st aph ylo cicci Vancom ycin



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STREPTOCOCCI

Ge ne ral Cha racte rist ics

N on-m o t i le , non -spo r u lat i ng, gr am - posit i ve f acu lt at i veanaerobes

Spher ical o r oval ce lls char act e r ist i call y f o r m ing pai rs o rch ain s d u r in g gro w t h

Oxidase negat ive

Catalase-negative

Grow w ell o n o rd inar y so lid m ed ia en r iched w it hb lo o d , se ru m o r glu co se .

M o st st rept ococci grow in so lid m ed ia as d isco idcolonies

Capsu lar st rep t oco ccal st rains give r ise t o m u co idcolonies



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STREPTOCOCCI

General Characteristics

St rept o cocci are f ragile and suscept ib le t odesiccation

W idely d ist r ibu t ed in nat ure and are fo und in u pperresp irato r y t ract , gast ro in t est inal t ract andge nit o u r in ar y t r act as n o r m a l m icr o bial f lo r a

Th ey ar e h et e ro ge ne ou s gr o up o f b act e ria



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STREPTOCOCCI

Classification

Cla ssifica t ion of St rep t occi ba sed on :

1 . Co lony m or pho logy and hem o lyt i c r eact i on onbloo d agar

2. Sero log ic speci f ici ty o f t he cel l w all grou p speci f icsubstan ce and ot her cellw all capsular ant igens

3. Biochem ical react ion s and resistance to physica l

and chem ical facto rs4 . Eco logic f eat u r es



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STREPTOCOCCI

Classification

He m olyt ic react ion of st rep tococci:

H e m olysis App earance D esignat ion Exam ple

Co m plet e Clear, co lo u r less, sharp lydef in ed zo n e Bet a() S. pyogenes

Par t ial Green ish d isco lo rat io n(reduced h em o glo b in ) Alpha()

Viridansstreptococci

No ne No ch ange Gamma() Enterococci



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STREPTOCOCCIClassification

La ncefie ld gro up ing of st rep to cocci

St rep to co cci p ro du ce gro up sp ecif ic car bo hyd rat es(Ccarbohyd rates) iden t i f ied using group speci f ic an t i se rum.

It is designat ed A-H and K-V. The clin ically im p o r t an tst rep tococci are grouped unde r A ,B,C,D,F and G.

The m ain species and group s of m edical im po rt ance are: S. p yo gene s... Lancef ield gro up A S. agalact iae.. Lan cef ield gro up B Ent ero cocciLancef ield gro up D



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STREPTOCOCCI

Ant ige nic st ruct ure :

Grou p-spe cific cell w a ll a nt igen

St rep t o co ccal cell w all carb ohyd rat e is t h e b asis fo r

se ro lo gic gro u p in g o f st r ep t o co cci (Lan ce fie ld gro u p s A-H ,K-U)

M protein They are fou nd in h ai r- like pro ject ions of t he st rep to coccal

sur face and deter m ine v i ru lence M ajor v i ru lent facto r for group A st rept ococci

Ther e are tw o m ajor st ru ctu ral c lasses of M pro t ein(class I

& class II)25/ 11/ 2012 33Dr. Erkihu n Akli lu (Lecture r)


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STREPTOCOCCI

Ant ige nic st ruct ure :

M ore than e igh ty sero types o f M pro te in o f group Astreptococci

T sub sta nce:

Acid and heat lab i le un l ike M prot e in , and h as no re lat ion

to v i ru lence o f st rept ococci .



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STREPTOCOCCI

To xin s a n d e nzy m e s p ro d uce d b y St r ep t oco ccu s pyogenes(Gro up A -hem olyt ic stre pt ococci)

S. Pyogenes The m ost p ath ogen ic m em ber o f t he genus

It i s p resent as a com m ensa l in th e nasoph arynx in avar iab le pro po r t ion o f h ealt hy ind iv idu als.

Toxins a nd enzym es

St rep t okin ase: Fibr ino lysin

An act ive prot eo ly t ic enzym e w hich lyses f ibr in bycata ly t ic conversion o f p lasm inogen to p lasm in

For IV t reatm ent o f pu lm onary edem a and ar te r ia l andvenou s th rom bosis



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STREPTOCOCCI



Streptodornase: St rep t ococcal d eoxyr ibo nu clease

M ixtu res of st r ept ok inase and st rept od orn ase are used

in enzym at i c debr idem ent

NADase: N icot inam ide adenine d inuc leot idase

Hyaluronidase: Spr eadin g facto r

I t degrades t he grou nd sub stan ce of con nect ive t issue(hyaluro nic acid)



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STREPTOCOCCI



Hemolysins:

Tw o t ypes :St rept o lysin O and St rept o lysin S

Erythrogenic toxin: Pyrogenic exot ox ins

It is respo nsib le for t he ery t hem atous rash inscarlet feve r.



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STREPTOCOCCI

Path ogen ic Stre pt ococci

Bovine st rep to cocca l m a stit is S. aga lact ia e

S. dysga lact iae S. ub er is

Ent erococcus faecal is

S. pyo gen es

S. zoo epid em iccos



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STREPTOCOCCI

Pathogenic Streptococci

St ran gles in ho rses: S. equ i

Abscess and ot her sup pu rat ive con di t ions and

sept icaem ia in m any species of anim als: S. pyogenes: hu m an s

S. can is: dog s

S. su is: pigs

S. equisim i l is : hor ses



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STREPTOCOCCI

Diagnosis Hist o ry, cl in ical sign s an d p at h o logy m ay b e in d icat ive o f

st r ept ococcal in fect ion

Sam p les ar e co llect ed an d cu lt u red p ro m p t ly: st rep t oco ccalare h igh ly su sce p t ib le t o d e siccat i o n . Sam p le s in clu d e p u s an dexudates

Sam p les can b e p laced in t r an sp o rt m e d iu m

St ain ed sm e ar o f clin ical sam p les m ay r eveal gram - po sit ive

co cci in ch ain s25/ 11/ 2012 40Dr. Erkihu n Akli lu (Lecture r)


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STREPTOCOCCI

Diagnosis Sam p les sho u ld b e cu lt ured o n b lo od agar and M acCo n key

agar

In cu b at e agar p lat e s ae ro b ically at 3 7oC fo r 24 -48 h ou rs

St rep to co ccal co lo nies ar e sm a ll, t ran slu cen t an d so m e m ayb e m u co id

St rep tococci a re d i ff eren t iated based on :

Ty p e o f h ae m o l ysis

Lancef ie ld group ing

Biochem ical test ing25/ 11/ 2012 41Dr. Erkihu n Akli lu (Lecture r)


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STREPTOCOCCI

DiagnosisA. Types of haem olysis on blood agar

Bet a-haem o lysis: co m p let e haem o lysis o f clearzones ar ound co lon ies

Alpha-haem o lysis: par t i al, incom p lete haem o lysi s,gr een ish o r h azy zo n es ar o un d co lo n ie s

Gam m a-h ae m o lysis: n o o b se rvab le ch an ges in t h eb lo o d agar ar o un d co lo n ies



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STREPTOCOCCI

DiagnosisB. Lancef ie ld grouping: A sero logical m et hod o f

classif icat io n b ased o n t h e gr ou p-sp ecif ic C su bst an ceLancef ield group ing. Inc ludes:

Ring specifica t ion t est Extract C-sub stance by ac id or heat f ro m th e Streptococcus spp

The extract (ant igen) is layered o ver ant isera of d i f ferentspeci f ici t ies in narro w tu bes p laced in p last ic ine on s l ide

A pos it ive react ion is ind ica ted by t he fo rm at ion o f a w h i te r ing o fprec ip i ta te close to t he in te r face o f the t w o f lu ids w i th in 30m i n u te s



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STREPTOCOCCI

Diagnosis

B . Lancefield grouping: A sero lo gical m e th od o f classif icat io nbased on the group-speci f ic C substance Lance f ie ld group ing.Includes:

La tex a gglut ina t ion test : latex-coat ed group -speci f icant ibo dies are com m ercial ly avai lable for t he t est .

Ant igen is ext rac ted enzym at ica l ly f rom th e st rept ococcus spp

M ix ant iserum and ant igen t ogether on a sl ide

Posit ive react io n is indicated by agglut inat io n25/ 11/ 2012 44Dr. Erkihu n Akli lu (Lecture r)


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STREPTOCOCCI

Diagnosis

C. Biochem ical te st s: oxidase, cat alase, sugar fer m ent at io nt est s. Bioch em ical test s are avai lable com m ercial ly for r apidident i f i cat ion o f st rept ococc i

Spe cies Trehalo se Sorb it o l Lact ose M alto se

S. eq u i - - - +

S. zo o ep id em icu s - + + (+)-

S. equ isim ilis + - V +



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STREPTOCOCCI

Diagnosis

Different iat ion o f stre pt ococci associated w ithmastit is:

S. pyogenes: Bacit r acin sensit ive (al l gro up A)

S. aga lact iae: CAM P test posit ive w i th S. aur eus andCorynebacter ium pseudotu bercu losis (a l l group BStreptococci)



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STREPTOCOCCI

Diagnosis

Different iat ion o f stre pt ococci associated w ithmastit is:

S. uberis: Aescul in hydro lysis (black bro w n zones ofd isco lourat ion around dark co louredcolon ies on Edw ardsm e d i u m ) .

S. pneum onia e: Capsule sw el l ing t est , bi le solub i l i ty,Optorchin-sensi t iv i ty,



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CORYNEBACTERIUM

Sm all Gram -posi t i ve p leom orp hic (coccoid, club and rodform s) bacter ia

St ained sm ear r eveals cel ls in p al isades of paral lel and angu lar

clusters resem blin g Chin ese lett ers

Non -m ot i le facu l ta t ive anaerobes

Catalase-positive, oxidase-negative

Fast id iou s, requ i re enr ichm ent for grow th

Cause pyo genic infect ion

M ost pat ho genic species are ho st speci f ic

Typ e species: C. dipt heriae, causes dipht heria in chi ldren25/ 11/ 2012 48Dr. Erkihu n Akli lu (Lecture r)


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CORYNEBACTERIUM

Diseases

Corynebacte r ium bov is

Ho st (cat t le): sub clinical m ast i t is

C. kut scher i

Ho st ( labor ato ry ro de nt s): sup erf ic ial absceses, causespu ru lent foc i in l iver, lun gs and lym ph no des

C. pseudot uberculosis (non-ni t r at e-reducing biot ype)

Host (Sheep an d goats): caseou s lym ph aden i t is

C. pseudot uberculosis (ni t ra te-reducing biot ype)

Ho st (ho rses, catt le): ulcerat ive lym ph agit is, abscesses



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CORYNEBACTERIUM

Diseases

C. renale ( type I )

Catt le: cyst i t is, p yelonep hr i t is

Sheep and goats: u lcerat ive (enzoo t ic) balano po st hi t is

C. pi losum (renale t ype I I) Catt le: cyst i t is, p yelonep hr i t is

C. cyst i t ides (renale t ype II I)

Catt le: severe cyst i t is, rarely pyelo ne p hr i t is

C. ulcer an s

Catt le: m ast i t is



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CORYNEBACTERIUM

Diagnosis

Specim en: p us, exud ates, t issue, sam ple, m id-st ream ur ine

Direct m icroscop y of Gram -st ained sm ear m ay revealco rynefo rm bacte r i a

Inoculate sam ple on t o b lood agar, select ive m edia (M cLeod sb lood agar, Loef f ler s m edium ) cont a in ing pot assium te l lur i te ,and M acConkey agar

Incubate aerobica lly at 370C for 24 t o 48 h ou rs

Iden t i f ica t ion : no grow th on M acConkey agar



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CORYNEBACTERIUM

Diagnosis

Colon ia l Cha ra cte rist ics:

C. bovis: a l ipophi l ic bacter ium . Sm al l w hi te, dry, non-haemoly t i c co lon ies

C. kut scheri : w hit ish colonies, occasiona l ly haem olyt ic C. pseudo t uber culosis: sm al l w hit ish colonies, surr oun ded by a

narrow zone of com plete haem olysis ev ident a f ter 72 ho urs ofincub at ion. Colonies becom e d ry, crum bly and cream -

co lou red w ith age

M e m b e rs o f C. renale group produce sm al l, non-haem oly t ic

colonies af t er 24ho urs incub at ion. Produ ce p igm ent a f t er 48ho urs of incubat ion



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CORYNEBACTERIUM

Diagnosis

Biochem ical te sts

Ni t rate reduc t ion : C. pseudot uberculosis biot ype

Al l path ogenic coryn ebacter ia are urease po si t ive except C.

bovis

Enh anced h aem olysis by C. pseudot uberculosis w heninoculated across a streak of Rhado coccus equi



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CORYNEBACTERIUM

Diagnosis



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RHODOCOCCUS

Gram -posit ive aerob ic bacter ia

No n-m ot i le cat alase-posit ive, oxidase-negat ive

W eakly acid fast

Grow s on n on -enr iched m edia Rod or coccibaci llus in shap e

Pro du ces pigm ent s, colo nies are pin k

I t fo rm s capsule. Pro du ces large, m oist ,

v iscid / m uco id co lon ies Im po r t ant spp . R. equ i



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RHODOCOCCUS

Diseases

Foals (1 - 4 m ont hs of age): sup pu rat ivebron chopn eum on ia and pu lm on ary abscessat ion

Horse: superficial abscessation

Pigs, Cat t le: m i ld cervical lym ph adeno pat hy

Cats: sub cutan eou s abscesses, m ediast inal granu lom as



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gram positive batera

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