Transcript E. coli - wendyachmmad

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• Escherichia coli, originally known as Bacterium coli commune, was
identified in 1885 by Theodor Escherich. E. coli is widely distributed in
the intestine of humans and warm-blooded animals and is the
predominant facultative anaerobe in the bowel and part of the
essential intestinal flora that maintains the physiology of the healthy
host .
• E. coli is a member of the family Enterobacteriaceae , which includes
many genera, including known pathogens such as Salmonella,
Shigella, and Yersinia.
• Most strains of E. coli are not regarded as pathogens, but they can be
opportunistic pathogens that cause infections in immunocompromised
hosts.
• If fecal coliform counts over 200 colonies/100 ml of water sample in
the river, there is a greater chance that pathogenic organisms are also
present. Such waters has a greater chance to cause diseases by
infecting through cuts in the skin, the nose, mouth, or the ears.
Diseases and illness such as typhoid fever, hepatitis, gastroenteritis,
dysentery, and ear infections can be borned by waters with high fecal
coliform counts. There are also pathogenic strains of E. coli that when
ingested, causes gastrointestinal illness in healthy humans
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In 1892, Shardinger proposed the use of E. coli as an indicator of fecal
contamination. This was based on the premise that the pathogens are
relatively scarce in water, making them difficult and time-consuming to
monitor directly but the E. coli is abundant in human and animal feces
and not usually found in other niches. And also. There are correlation
between fecal coliform counts and the probability of contracting a
disease from the water.
Furthermore, since E. coli could be easily detected by its ability to ferment
glucose (later changed to lactose), it was easier to isolate than known
gastrointestinal pathogens. Hence, the presence of E. coli in food or
water became accepted as indicative of recent fecal contamination and
the possible presence of frank pathogens.
Although the concept of using E. coli as an indirect indicator of health risk
was sound, it was complicated in practice, due to the presence of other
enteric bacteria like Citrobacter, Klebsiella and Enterobacter that can
also ferment lactose and are similar to E. coli in phenotypic
characteristics, so that they are not easily distinguished. As a result,
the term "coliform" was coined to describe this group of enteric
bacteria.
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Organisme Indikator
Syarat organisme indikator :
1. Keberadaannya dapat terdeteksi pada semua sampel yang
akan diperiksa,
2. Pertumbuhan dan jumlahnya memiliki korelasi negatif
langsung dengan kualitas sampel,
3. Dapat dideteksi dan dihitung jumlahnya dengan mudah dan
dalam waktu singkat, serta dapat dibedakan dengan jelas
dari organisme lain,
4. Memiliki resistensi yang tinggi terhadap lingkungan luar (di
luar habitat aslinya) agar dapat diisolasi.
(Jay 2001: 387 & 389)
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Coliform is not a taxonomic classification but rather a working
definition used to describe a group of Gram-negative, facultative
anaerobic rod-shaped bacteria that ferments lactose to produce acid
and gas within 48 h at 35°C.
•
In 1914, the U.S. Public Health Service adopted the enumeration
of coliforms as a more convenient standard of sanitary
significance.
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Although coliforms were easy to detect, their association with fecal •
contamination was questionable because some coliforms are found
naturally in environmental samples. This led to the introduction of
the fecal coliforms as an indicator of contamination.
Fecal coliform, first defined based on the works of Eijkman is a
subset of total coliforms that grows and ferments lactose at elevated
incubation temperature, hence also referred to as thermotolerant
coliforms. Fecal coliform analyses are done at 45.5°C for food
testing, except for water, shellfish and shellfish harvest water
analyses, which use 44.5°C
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The fecal coliform group consists mostly of E. coli but some other enterics
such as Klebsiella can also ferment lactose at these temperatures and
therefore, be considered as fecal coliforms. The inclusion of Klebsiella
spp. in the working definition of fecal coliforms diminished the
correlation of this group with fecal contamination. As a result, E. coli
has reemerged as an indicator, partly facilitated by the introduction of
newer methods that can rapidly identify E. coli.
Currently, all 3 groups are used as indicators but in different
applications.
Detection of coliforms is used as an indicator of sanitary quality of
water or as a general indicator of sanitary condition in the foodprocessing environment.
Fecal coliforms remain the standard indicator of choice for shellfish
and shellfish harvest waters; and
E. coli is used to indicate recent fecal contamination or unsanitary
processing.
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Almost all the methods used to detect E. coli, total coliforms or fecal
coliforms are enumeration methods that are based on lactose
fermentation.
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The most probable number test, also called the multiple tube fermentation
assay, is a statistical, multi-step assay consisting of presumptive, confirmed
and completed phases. It is an alternative to plate counts or membrane
techniques, especially for samples with higher turbidity. The MPN procedure
is a tube-dilution method using a nutrient-rich medium, which is less
sensitive to toxicity and supports the growth of environmentally-stressed
organisms. The MPN method detects and estimates the bacteria in water
samples (and can be applied to foods and soils) by the multiple
fermentation tube technique. The number of bacteria per 100 ml of sample
is estimated by the use of probability tables.
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In the assay, serial dilutions of a sample are inoculated into broth media.
Analysts score the number of gas positive (fermentation of lactose) tubes,
from which the other 2 phases of the assay are performed and then uses
the combinations of positive results to consult a statistical tables, to
estimate the number of organisms present.
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Typically only the first 2 phases are performed in coliform and fecal coliform
analysis, while all 3 phases are done for E. coli.
•
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The 3-tube MPN test is used for testing most foods. The 5-tube
MPN is used for water, shellfish and shellfish harvest water testing
and there is also a 10-tube MPN method that is used to test bottled
water or samples that are not expected to be highly contaminated.
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There is also a solid medium plating method for coliforms that uses
Violet Red Bile Agar, which contains neutral red pH indicator, so that
lactose fermentation results in formation of pink colonies. There are
also membrane filtration tests for coliform and fecal coliform that
measure aldehyde formation due to fermentation of lactose. This
chapter also includes variations of above tests that use fluorogenic
substrates to detect E. coli, special tests for shellfish analysis, a brief
consideration of bottled water testing and a method for testing large
volumes of citrus juices for presence of E. coli in conjunction with
the Juice HACCP rule.
•
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Sampling Procedures
It is best to use glass bottles but certain types of plastic bottle can be
used. Add 4-5 drops of sodium thiosulfate solution (100gr/lt) to each
clean sample bottle if the sample contains any residual chlorine.
Remove the stopper or cap just before sampling and avoid touching the
inside of the cap. If sampling by hand, use gloves and hold the bottle
near its base. Plunge it (opening downward) below the water surface,
then turn the bottle underwater into the current and away from you.
Avoid sampling the water surface because the surface film often contains
greater numbers of fecal coliform bacteria than is representative of
the river. Also, avoid sampling the sediments for the same reason,
unless this is intended. The same general sampling procedures apply
when using the extended rod sampler. When collecting samples,
leave some space in the sample container (an inch or so) to allow
mixing of the sample before-pipetting.
It is a good idea to collect several samples room any single location on
the river to minimize the variability that comes with sampling for
bacteria. Ideally, all samples should be tested within one hour of
collection. If this is not possible, the sample bottles should be placed
in ice and tested within six hour
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Uji Koliform dengan medium standar MTF
(Gaudy & Gaudy 1981)
Sampel
I. UJI PENDUGA
5 ml
0.5 ml
Asam +
Gas
0.05 ml
Inkubasi
24--48 jam
35° C
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LBG
5ml
LBT
5ml
LBT
5ml
Positif
koliform
II. Uji Penguat
1 ose
Inkubasi
24--48 jam
35° C
Gas
LB positif
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BGLB
Positif
koliform
III. Uji Pelengkap
Koloni hijau metalik:
positif E.coli
koloni merah:
positif koliform
pengecatan
Gram
Streak
24 jam
Streak
Inkubasi
24 jam
NA
35° C
BGLB
positif
EA
1 ose
Asam + gas
24--48 jam
LB
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• The presumptive tests are designed to grow the target bacteria. The
media used in the confirmed tests are designed to validate the
growth of target bacteria in the presumptive test.
• Confirmed test conditions are usually more stringent than
presumptive conditions. Thus, the presumptive test provides a
preliminary estimate of bacterial density based on enrichment in
minimally restrictive tube media. The results of this test are never
used without further analysis; the MPN must be carried through the
Confirmed Test for valid results.
• The MPN per 100 ml is calculated from the MPN table based upon
the Confirmed Test results. This value is based on the number of
positive and negative results observed when five 10 ml, five 1.0 ml
and five 0.1 ml volumes of sample are tested in
confirmed/completed tests. For example, if 3 of 5 tubes in the first
series (10 ml) were positive, 2 of 5 were positive in the second
series (1.0 ml) and 1 of 5 positive in the third series (0.1 ml) for
confirmed tests, then the pattern is read as 3-2-1. Referring to the
MPN index, 3-2-1 implies that the most probable number of bacteria
is 17/100 ml. However, the actual range may be between 7 and 40
(95% confidence).
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Substrat kromogenik-fluorogenik
(Manafi 1991; Manafi 1996; Jay 2001)
Contoh substrat kromogenik untuk deteksi koliform:
o-nitrophenyl-β-D-galactopyranoside (ONPG) dan 5-bromo-4-chloro3-indolyl--D-galactopyranoside (XGAL).
Contoh substrat fluorogenik untuk deteksi Escherichia coli:
4-methylumbelliferyl--D-glucoronide (MUG).
+ sample
- Kromogenik Perubahan warna medium
Medium
± 24 jam
- Fluorogenik  Perpendaran medium
(bila dipapar UV)
Hidrolisis substrat oleh enzim spesifik
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Fluorocult LMX broth
Mengandung: Triptosa, triptofan, natrium klorida, sorbitol, K2HPO4,
KH2PO4, garam sodium lauryl sulfat , 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (XGAL),
4-methylumbelliferyl --Dglucoronide (MUG), dan
1-isopropyl--D-1thiogalactopyranoside (IPTG).
IPTG (Inducer)
XGAL (Substrat kromogenik)
-galaktosidase
Aglycone bebas
Koliform
Cat indigo
Warna biru kehijauan
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MUG  Substrat fluorogenik
-glukuronidase
4-MU
Escherichia coli
Sinar UV
berpendar
Triptofan  asam amino
triptofanase
Escherichia coli
Indol
Reagen kovac
cincin merah
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Uji Koliform pada Fluorocult LMX broth
5 ml
0.5 ml
Warna
biru
kehijauan
0.05 ml
Inkubasi
24--48 jam
35° C
Fluorocult
LMX broth
ganda
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Fluorocult
LMX broth
tunggal
Fluorocult
LMX broth
tunggal
Positif
koliform
berpendar
E.coli
Fluorocult LMX
Positif koliform
Terbentuk cincin merah
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Pengecatan Gram
Cuci
Fiksasi
NA dari
fluorocult
LMX broth
Cuci
Akuades
steril
Cuci
Fiksasi
NA dari
Endo
agar
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Cuci
Akuades
steril
Amati
Uji Indol
Inkubasi
24--48 jam
35° C
NA dari
Endo agar
Tripton 1%
Terbentuk cincin merah
Positif E. coli.
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