BACILLACEAE

YUWONO, MD

• 1872, Ferdinand Cohn murid Robert Koch menemukan Bacillus subtilis • Genus Bacillus, family Bacillaceae• Ciri khas famili ini endospora• Ciri genus: Gram-positif, batang, aerobik atau fakultatif, membentuk endospora

• Ubiquitous in nature• Endospora resisten zat kimia dan fisik• Siklus pembentukan endospora• Produsen antibiotik• Spora dan kristal proteinnya toksik untuk beberapa serangga• Patogen Bacillus anthracis

Bacillus coagulans

Great diversity in physiology:• Mendegradasi hampir semua substrat tumbuhan dan hewan termasuk

selulosa, starch, pectin, protein, agar, hidrokarbon• Produksi antibiotik• Nitrifikasi, denitrifikasi, fiksasi nitrogen, facultative lithotrophy; autotrophy;

acidophily; alkaliphily; psychrophily; thermophily dan parasitism.

Spora merupakan strategi bertahan di tanah dimana banyak sekali bakteri. Spora juga dapat ditemukan di udara

Nutrisi dan Pertumbuhan • Hampir semua Bacillus versatile

chemoheterotrophs yaitu mampu respirasi menggunakan berbagai simple organic compounds (sugars, amino acids, organic acids).

• pada beberapa kasus kadang memfermentasi karbohidrat memproduksi glycerol dan butanediol.

• Bacillus megaterium memerlukan non organic growth factors

• Mayoritas mesofil optimum 30-45ºC,tapi ada juga yang termofilik pada 65ºC

Mucoid-type colonies of an encapsulated Bacillus species

Generation time di laboratorium 25 menitBacillus mudah diisolasi. Teknik sederhana dengan pasteurize a diluted soil sample pada 80ºC 15 menit plate pada nutrient agar dan inkubasi 37ºC 24 jam-beberapa hari. Setelah 24 jam ada koloni basilus yang khas: catalase-positive, Gram-positive, endospore-forming rods

Surface Structure of Bacillus Seperti permuakaan Gram-positif lainnya kompleks berhubungan dengan adherence, resistance dan tactical responses

Vegetative cell surface terdiri dari: capsule, proteinaceous surface layer (S-layer), several layers of peptidoglycan sheeting dan protein pada outer surface membran plasma

Surface of a Bacillus. Transmission E.M. C=Capsule; S=S-layer; P=Peptidoglycan

S-layers: Crystalline protein atau glycoprotein subunit yang disebut S-layers, fungsi belum diketahui –mungkin berhubungan dengan interaksi bakteri-metal, bermuatan negatif dan dapat mencegah autoaglutinasi pada B. stearothermophilus

Kapsul B. anthracis, B. subtilis, B. megaterium dan B. licheniformis: poly-D- or L-glutamic acid.  B. circulans, B. megaterium, B. mycoides dan B. pumilus: karbohidrat.Polisakarida beberapa Bacillus bereaksi silang dengan genus patogen lain seperti B. mycoides dengan Streptococcus pneumoniae type III; B. pumilus dengan Neisseria meningitidis group A; B.alveli dengan Haemophilus influenzae type B. Di bawah TEM beberapa kapsul polipeptida dan polisakarida tampak fibriler. Kapsul mudah dilihat dengan mikroskop cahaya. Media berkapsul bersifat mukoid atau slimy colony

Kapsul Bacillus anthracis FA stain (kiri), negative stain (india ink) (kanan)

Cell Walls Tidak bervariasi seperti pada Gram-positif umumnya. Dinding sel vegetatif terbuat dari meso-diaminopimelic acid (DAP) kecuali pada B. sphaericus, B. pasteurii dan B. globisporus dari lysine

Flagella Mayoritas Bacillus motil yaitu memiliki peritrichous flagella. Flagel tetap stabil hingga pH 11.

Individual cells of motile Bacillus species photographed on nutrient agar, 15,000X

  A.  B. subtilis;  B. B. polymyxa;  C.  B. laterosporis;  D. B. alveli.

Perbedaan endospora dan sel vegetatif Bacillus species

Property Vegetative cells Endospores

Surface coats

Typical Gram-positive murein cell wall polymer; crystalline S-layer

Thick spore coat, cortex, and unique peptidoglycan core wall; no S-layer

Microscopic appearance Nonrefractile Refractile

Calcium dipicolinic acid Absent Present in core

Cytoplasmic water activity High Very low

Enzymatic activity Present Absent

Macromolecular synthesis Present Absent

Heat resistance Low High

Resistance to chemicals and acids Low High

Radiation resistance Low High

Sensitivity to lysozymeSome sensitive; some resistant Resistant

Sensitivity to dyes and staining Sensitive Resistant

Formasi Spora

Genetik • Transforamsi pada Bacillus subtilis ditemukan pada1958• Generalized and specialized transduction juga terjadi pada B. subtilis• Genes affecting sporulation B. subtilis• Bacteriophages memediasi generalized transduction pada B subtilis, B.

cereus, B. megaterium, B. thuringiensis, B. anthracis dan B. stearothermophilus.

• Conjugative plasmids (fertility plasmids capable of bringing about their own transfer from one bacterium to another) juga terjadi pada bebrapa spesies

• Insecticidal delta toxin crystal protein B. thuringiensis dikode pada large plasmids. Plasmid ini dapat ditransfer antar spesies. Selain itu terbukti pula bahwatransmissible plasmids of Bacillus, a conjugative transposon (Tn925) berasal dari Enterococcus faecalis ke B. subtilis.

Ekologi Endospora resisten terhadap stres lingkungan. Primary habitat of the aerobic endospore-forming bacilli is the soil. Russian microbiologist Winogradsky menyebutnya "normal flora" of the soil.

Ecophysiological groups

Acidophiles: B. acidocaldarius, B. coagulans dan B. polymyxa. Alkaliphiles: B. alkalophilus dan B. pasteurii. Optimum pH 8 - 11 Halophiles: B. pantothenticus, B. pasteurii, beberapa tahan 10 % NaCl Psychrophiles atau psychrotrophs: B. globisporus, B. insolitus, B. marinus, B. macquariensis, B. megaterium, B. polymyxa, tumbuh pada 0C Thermophiles: B. acidocaldarius, B. schlegelii dan B. stearothermophilus, Acidophiles dan Lithoautotrophs pada kelompok ini tahan hingga 65C. Denitrifiers: B. azotoformans,  B. cereus, B. laterosporus, B. licheniformis, B. pasteurii, B. stearothermophilus (reduce NO3 to NO2). Nitrogen-fixers: B. macerans and B. polymyxa. B. macerans in soil and in decaying vegetable material. 

Pathogens of Insects: B. larvae, B. lentimorbis, and B. popilliae are invasive

pathogens. B. thuringiensis forms a parasporal crystal that is toxic to Lepidoptera.

B. larvae, B. lentimorbis and B. popilliae are a related cluster of Bacillus species,

being insect pathogens, with swollen sporangia, and typically catalase-negative.

Pathogens of Animals: B. anthracis, and B. cereus.  B. alvei, B. megaterium, B.

coagulans, B. laterosporus, B. subtilis, B. sphaericus, B. circulans, B. brevis, B.

licheniformis, B. macerans, B. pumilus, and B. thuringiensis have been isolated

from human infections.

Bacillus

• Classification– All are large Gram-positive bacilli– Are aerobic– Form endospores – Most are found in dust and soil– Bacillus anthracis is the major pathogen in the group

• Morphology and Cultural Characteristics (Bacillus anthracis)

Bacillus

– G+B with square cut ends– Forms endospores– Non-motile– Capsule is made of glutamic acid (a polypeptide)

The capsule is not found in cultured bacteria unless the bacteria are grown on bicarbonate containing media and in the presence of increased CO² concentrations. The organism has a capsule while inside the host which provides the conditions necessary for capsule production.

Gram stain of Bacillus sp.

Endospores of Bacillus sp,

Bacillus

– Grow well on ordinary lab media producing large granular colonies with a coarse texture.

– On BA may cause beta hemolysis.• Virulence factors

– Capsule helps organism to resist phagocytosis but antibodies are not protective.

– Exotoxin is very complex and is produced only when the bacteria is growing in animal tissues. Toxin production is mediated by a temperature sensitive plasmid. The toxin consists of three protein components (maximum toxicity occurs when all three components are present).

 

Bacillus sp. On BA

Bacillus• Protective antigen (PA) reacts with host cell tissue receptors

where it is proteolytically activated so that it binds LF and EF to allow entry of LF and EF into the host cells. Injection of PA into a host will result in the production of antibody against PA that provides short term immunity.

•  Lethal factor (LF) Both PA and LF are required for lethal activity.

•  Edema factor (EF) Both PA and EF are required for edema to occur. EF has adenylate cyclase activity and it becomes active when it enters eucaryotic cells. This results in an increase of cAMP and resulting edema.

Bacillus

• Clinical significance – Anthrax which is the disease caused by B. anthracis

is essentially a disease of animals who acquire the organism by ingestion or inhalation of spores. The spores are extremely resistant to adverse chemicals and physical environments. They may remain a source of infection in soil for 20-30 years.

–  Man acquires anthrax usually from contact with animal products; less commonly from working in an agricultural setting with infected animals.

Bacillus

– Man may acquire the organism through skin abrasions, by inhalation of spores, or by ingestion. The disease that develops depends upon the mode of transmission:• Pulmonary (Woolsorter‘s disease) Spores are inhaled and

germinate in the lungs where they multiply and spread to cause a fatal septicemia or meningitis. This is the most serious form of the disease.

•  Intestinal anthrax results from ingestion of spores.

 

Bacillus• Cutaneous anthrax when organism comes in through skin

abrasions; most common form; most damage comes from the invasion but the toxin may play a role; spores enter through a cut, germinate, and vegetative cells multiply; there is an acute host inflammatory response, but the capsule probably prevents phagocytosis; exotoxin release and invasive damage result in rapid development of a malignant pustule; occasionally, without treatment, the organism will disseminate to cause septicemia and death in a few days (rare because of B-lysin activity in the bloodstream that kills the bacteria); vascular injury with edema, hemorrhage, and thrombosis may occur; death results from respiratory failure and anoxia caused by actions of the toxin on the central nervous system (toxemic degeneration).

Anthrax Anthrax:primarily a disease of domesticated and wild animals terutama

herbivora.Manusia terkena karena konbtak dengan hewan-hewan tersebut: flesh, bones, hides, hair dan excrement. Paling sering cutaneous anthrax didapat lewat kujlit atau mukosa yang terluka,minor scratch atau abrasion

pada face or neck or arms terinokulasi spora dari tanah. Spora tumbuh menjadi vegetatif berupa gelatinous edema kemudian papule within 12-36

jam, kemudian cepat menjadi vesicle, pustule (malignant pustule) dan akhgirnya necrotic ulcer yang dapat menyebar menjadi septikemia.

Lymphatic swelling dalam 7 hari sebagai tanda septikemia umumnya fatal.

Anthrax

Inhalation anthrax (woolsorters' disease) karena menghirup spore-containing

dust where animal hair or hides are being handled. Dimulai dengan demam

tinggi dan nyeri dada dan perdarahan sistemik dan fatal.

Gastrointestinal anthrax analog dengan cutaneous anthrax karena tertelan

poorly cooked meat from infected animals. Gastrointestinal anthrax jarang tapi

kadang menjadi wabah

Patologi ditentukan virulence: antiphagoytic capsule, and powerful lethal toxin

ie. the anthrax toxin.

Anthrax

Anthraks kutaneus paling sering terjadi pada manusiaAnthraks pneunonik fatal jika terjadi

ESCHAR

B. Anthracis

B. Anthracis (kanan) dan B. cereus (kiri)

Bacillus cereus• Ditemukan di lingkungan seperti tanah, debu• Patogen oportunistik pada luka, abses paru dan LCS• Keracunan makanan menyebabkan diare, mual dan muntah terutama makanan yang dimasak kurang matang atau makanan yang disimpan/diawetkan

Koloni: “Medusa-head” morphology

Bacillus cereus food poisoning

Bacillus cereus causes two types of food-borne intoxications:1. Characterized by nausea and vomiting and abdominal cramps and has an

incubation period of 1 to 6 hours. It resembles Staphylococcus aureus food poisoning in its symptoms and incubation period. This is the "short-incubation" or emetic form of the disease.

2. Manifested primarily by abdominal cramps and diarrhea with an incubation period of 8 to 16 hours. Diarrhea may be a small volume or profuse and watery. This type is referred to as the "long-incubation" or diarrheal form of the disease, and it resembles more food poisoning caused by Clostridium perfringens. In either type, the illness usually lasts less than 24 hours after onset.

The short-incubation form is caused by a preformed heat-stable enterotoxin. The mechanism and site of action of this toxin are unknown. The long-incubation form is mediated by a heat-labile enterotoxin which activates intestinal adenylate cyclase and causes intestinal fluid secretion.

Bacillus subtilis dan spesies lain• Ditemukan di lingkungan• Patogen oportunistik

Bacillus subtilis dan spesies lain• Ditemukan di lingkungan• Patogen oportunistik

GRAM POSITIVE BATANG NON SPORAGRAM POSITIVE BATANG NON SPORA

CASECASE

The patient was a 2,150 gram female neonate

Born at 32 weeks gestation. She had poor vital signs and she was

intubated at birth because of poor respiratory effort.

The mother presented at the time of delivery with complaints of lower

abdominal pains with a temperature of 39oC and a white blood cell

count of 25,000/mm3.

The mother was given ampicillin at the time of delivery. She

recovered.

Blood cultures were taken from the infant soon after birth. These

grew Gram-positive coccobacilli that produced beta hemolysis on

sheep blood agar.

Lumbar punctures were attempted, but they were too traumatic and

non-revealing.

Listeria monocytogenesGram positif, batang pendek bulat (kokobasiler)Fakultatif anaerob, b-hemolytic on sheep blood agarTumbuh pada suhu 4ºC. Katalase positifCommonly associated with dairy products

Listeria monocytogenesGram positif, batang pendek bulat (kokobasiler)Fakultatif anaerob, b-hemolytic on sheep blood agarTumbuh pada suhu 4ºC. Katalase positifCommonly associated with dairy products

b-hemolysis

Gejala klinis• Meningoencephalitis: Most common in neonates• Low grade septicemia during pregnancy• Granulomatosis infantiseptica-intrauterine Early onset syndrome- before delivery characterized by septicemia, disseminated abscesses and granulomas

Late onset syndrome- meningitis 2-3 weeks after birth (MUST BE differentiated from Group B Streptococcal meningitis)

Bacillus

• Antibiotic susceptibility and treatment– Penicillin or tetracycline– A short-term PA vaccine is available for industrial

workers and others at high risk.• Other Bacillus species

– Bacillus subtilis, and occasional other species may occasionally cause opportunistic infections.

– Bacillus cereus is a major cause of enterotoxin food poisoning; the toxin is protein in nature and can be destroyed by heating; food poisoning occurs after ingestion of pre-formed toxin; vomiting occurs 1-5 hours after ingestion 

Bacillus

– B. cereus is also an opportunistic pathogen that has been cultured from cases of septicemia, endocarditis, meningitis, wound infections, pneumonia, and fulminant eye infections; in addition to the enterotoxin that bacteria may produce, a dermonecrotic and a lethal toxin, hemolysins, lecithinase, proteases, and nucleases may be involved in its pathogenesis; clindamycin with or without gentamycin may be used for treatment infections

Basil gram (+) berspora

I.Aerob a.Bacillus cereus b.B.subtilis c.B.anthracis

II.Anaerob a.Clostridium tetani b.Cl.perfringens (welchii) c.Cl.botulinum d.Cl.difficile

Bacillus anthracis

Menyebabkan peny.anthrax (zoonosis). Infeksi terjadi pd manusia melalui spora.1.kulit : berupa papulavesikula pustula maligna ulkus nekrotik. kalau menjalar keseluruh tubuh septikemia.2.gasterointestinal :melalui makanan (daging hewan)3.pneumonia.per inhalasi paru paru secara alamiah : wool’sorter disease. dapat dibuat senjata biologis (WMD) bersifat sangat fatal (mematikan)

Sifat sifat B.anthracis batang besar,ujung persegi,1 x 3-4 um, tersusun spt rantai panjang (mirip bambu) – berspora sentral- nonmotil.

Pembiakan :koloni bulat,transparan,hemolisis(-),mencairkan gelatin.Kapsul terdiri dari asam glutamat, sedangkan badan berupa protein dan polisakarida.

Menghasilkan toksin :1. A protein2. Faktor edema3. Faktor lethal

Diagnosis laboratorium

1. Bahan pemeriksaan : pus, darah,sputum, jr biopsi 2. Pengecatan : basil gram (+),bentuk rantai.3. Pembiakan pd agar darah.4. Tes Ascoli : terbentuk cincin presipitat apabila serum imun + ekstrak jaringan terinfeksi.

Terapi : penisilinPencegahan : vaksin anthraxEpidemiologi : spora tanah hewan (domba) manusia

Kontrol :

1. Bangkai hewan dibakar.2. Produk tercemar di autoklaf 3. Gunakan sarung tangan,masker,baju pelindung, goggle, dll.4. Vaksinasi hewan ternak.

Clostridium tetani

Dijumpai di seluruh dunia : tanah,kotoran hewan,dll.Basil gr(+), anaerob, spora terminal(+), motil.Menghasilkan eksotoksin yg bekerja pd CNS.spasme ,hiperrefleksi, kejang ok tetanospasmin.

Kuman tidak invasif : masuk melalui luka yg dalam/kotor.(bayi :umbilikus, luka tusuk, lubang gigi, dll.)Spora vegetatif eksotoksin ke axon/darah CNS.Gejala : trismus (lockjaw),risus sardonicus.

Pencegahan : DPT,ATS profilaksis,booster TT.

Pengobatan : wound cleansing,penisilin,ATS & TT.

Pemeriksaan laboratorium

Spesimen : pus ,dibiakkan pd anaerobic jar .Tampak koloni kecil spt jala halus & berjalinan.Pada media cair (chopped cooked meat) : tumbuh pada dasar tabung.

Pemeriksaan lab jarang diminta !

Kalau secara klinis (+) gejala tetanus :Bayi : tetanus neonatorum, orang dewasa (luka kotor) langsung MRS terapi .

Clostridium botulinum

Penyebaran di seluruh dunia (tanah,kotoran hewan)Menghasilkan 7 jenis toksin, A s/d G.A, B, dan E : peny.pd.manusia (keracunan makanan)C : limber neck (unggas)D : botulismus pd peternak.

Penyakit timbul pasca mengkonsumsi makanan yang diawetkan : kalengan, makanan yg diasap,divakum.Pd bayi : madu sids (sudden infant death syndrome)

Toksin rusak jika dipanaskan 100º C / 20 menit

Gejala penyakit :18-96 jam pasca makan makanan yg tercemar flaccid paralysis berupa gangguan penglihatan, tak dpt menelan, kesulitan bicara, paralisis bulbar progresif, tanpa demam.

Kematian akibat paralisis otot pernafasan dan jantung.

Clostridium perfringens

Spora masuk melalui luka dalam/kotor (fraktur terbuka)Menyebabkan gas gangrene (clostridial myonecrosis) ok alfa toksin (lecithinase) yg menghidrolisis membran sel.

Gejala : nyeri hebat, menjalar pembentukan gas pd jr.subkutan & otot .Kalau diraba ber krepitasi.

Terapi : oksigen hiperbarik dan amputasi.

Clostridium difficile

Menyebabkan kolitis pseudomembranosa terutama pd anak.

Diare th clindamycin flora normal matiBerkembang Cl.diff toksin kolitis ps dapat berakibat fatal.

Terapi : vancomycin

Lab : mencari eksotoksin dr tinja pasien.

THANK YOU