Epsilon Archive for Student Projects

Abstract

The
focus
of
this
project
was
to
characterize
the
occurrence
of
ESBL-­‐producing
bacteria
in
two
irrigation
water
ponds
in
Southern
Sweden.
Samples
were
taken
from
two
ponds
nearby
each
other,
from
which
analyses
were
made
based
on
levels
of
community
and
individual
isolates.
Community
samples
were
used
for
attempts
for
characterization
of
resistance
patterns
in
irrigation
water
using
the
Omnilog
PM-­‐plate
system.
The
growth
of
community
microbiota
in
the
presence
of
the
following
antibiotic
substances
was
assessed;
amikacin,
gentamicin,
kanamycin,
penicillin
G,
ampicillin,
piperacillin,
trimethoprim,
potassium
tellurite
and
ciprofloxacin.
Isolates
were
pure
cultured
using
semi-­‐selective
media:
LB,
Rainbow
Agar,
mEnterococcus,
0.1
TSA
and
VRBD.
A
total
of
177
isolates
were
isolated
randomly.
Each
isolate
was
identified
using
the
Omnilog
GENIII-­‐system
of
biochemical
characterization.
Further,
the
isolates
were
tested
on
Brilliance
ESBL-­‐agar
for
their
ability
to
grow.
Out
of
the
total
number
of
isolates,
30
were
selected,
preferably
Enterobacteriaceae
or
Pseudomonas
spp.,
based
on
their
high
similarity
to
library
strain
during
the
identification
in
the
Omnilog-­‐system.
These
were
further
characterized
using
Etest-­‐sticks.
Three
selected
strains
were
picked
for
further
analyses
using
exogenous
isolation
for
attempts
to
simulate
horizontal
gene
transfer,
and
Omnilog
PM-­‐panels
containing
various
antibiotics
of
interest.
Selections
of
samples
were
chosen
for
molecular
tests
targeting
plasmid-­‐borne
resistance
genes
CTX-­‐M1,
CTX-­‐M2
and
SHV.
The
method
for
assessing
community-­‐resistance
patterns
showed
ambiguous
data
that
was
difficult
to
interpret;
further
optimization
is
recommended.
For
all
tested
PM-­‐plates,
generally
greatest
differences
between
substances
were
found
among
wells
with
the
strongest
concentration
of
antibiotic
substance.
A
total
of
175
isolates
displayed
ability
to
grow
on
ESBL-­‐agar.
Etests
confirmed
ability
of
isolates
to
resist
many
β-­‐lactam
antibiotics
and
many
were
clinically
resistant
to
certain
substances.
No
plasmids
were
transferred
during
exogenous
isolation.
During
molecular
tests,
a
few
matches
were
found
for
the
CTX-­‐M1
gene.
Isolates
analyzed
with
Omnilog
PM-­‐plates
showed
resistance
to
most
compounds
tested.
The
isolates
showed
greatest
resistance
toward
penicillin
G,
ampicillin,
amikacin,
kanamycin
and
trimethoprim.
In
the
study,
it
was
established
that
resistance
among
bacteria
was
frequently
occurring
in
the
irrigation
water,
and
β-­‐lactamase
producing
bacteria
was
very
common.
Extended
studies
are
needed
to
assess
the
frequency-­‐
and
occurrence
of
horizontal
gene
transfer
(HGF)
in
this
environment.
Although
some
potential
corresponding
data
is
found,
it
is
at
this
point
not
possible
to
determine
whether
the
resistances
are
due
to
antibiotics
used
in
veterinary
medicine,
human
medicine
or
one
additional
reason
is
travel
and
trade.
in
irrigation
water
using
the
Omnilog
PM-­‐plate
system.
The
growth
of
community
microbiota
in
the
presence
of
the
following
antibiotic
substances
was
assessed;
amikacin,
gentamicin,
kanamycin,
penicillin
G,
ampicillin,
piperacillin,
trimethoprim,
potassium
tellurite
and
ciprofloxacin.
Isolates
were
pure
cultured
using
semi-­‐selective
media:
LB,
Rainbow
Agar,
mEnterococcus,
0.1
TSA
and
VRBD.
A
total
of
177
isolates
were
isolated
randomly.
Each
isolate
was
identified
using
the
Omnilog
GENIII-­‐system
of
biochemical
characterization.
Further,
the
isolates
were
tested
on
Brilliance
ESBL-­‐agar
for
their
ability
to
grow.
Out
of
the
total
number
of
isolates,
30
were
selected,
preferably
Enterobacteriaceae
or
Pseudomonas
spp.,
based
on
their
high
similarity
to
library
strain
during
the
identification
in
the
Omnilog-­‐system.
These
were
further
characterized
using
Etest-­‐sticks.
Three
selected
strains
were
picked
for
further
analyses
using
exogenous
isolation
for
attempts
to
simulate
horizontal
gene
transfer,
and
Omnilog
PM-­‐panels
containing
various
antibiotics
of
interest.
Selections
of
samples
were
chosen
for
molecular
tests
targeting
plasmid-­‐borne
resistance
genes
CTX-­‐M1,
CTX-­‐M2
and
SHV.
The
method
for
assessing
community-­‐resistance
patterns
showed
ambiguous
data
that
was
difficult
to
interpret;
further
optimization
is
recommended.
For
all
tested
PM-­‐plates,
generally
greatest
differences
between
substances
were
found
among
wells
with
the
strongest
concentration
of
antibiotic
substance.
A
total
of
175
isolates
displayed
ability
to
grow
on
ESBL-­‐agar.
Etests
confirmed
ability
of
isolates
to
resist
many
β-­‐lactam
antibiotics
and
many
were
clinically
resistant
to
certain
substances.
No
plasmids
were
transferred
during
exogenous
isolation.
During
molecular
tests,
a
few
matches
were
found
for
the
CTX-­‐M1
gene.
Isolates
analyzed
with
Omnilog
PM-­‐plates
showed
resistance
to
most
compounds
tested.
The
isolates
showed
greatest
resistance
toward
penicillin
G,
ampicillin,
amikacin,
kanamycin
and
trimethoprim.
In
the
study,
it
was
established
that
resistance
among
bacteria
was
frequently
occurring
in
the
irrigation
water,
and
β-­‐lactamase
producing
bacteria
was
very
common.
Extended
studies
are
needed
to
assess
the
frequency-­‐
and
occurrence
of
horizontal
gene
transfer
(HGF)
in
this
environment.
Although
some
potential
corresponding
data
is
found,
it
is
at
this
point
not
possible
to
determine
whether
the
resistances
are
due
to
antibiotics
used
in
veterinary
medicine,
human
medicine
or
one
additional
reason
is
travel
and
trade.

Målet
med
detta
projekt
var
att
karaktärisera
förekomsten
av
ESBL-­‐producerande
bakterier
i
bevattningsvatten
i
södra
Sverige.
Prover
togs
från
två
närbelägna
bevattningsdammar,
från
vilka
analyser
gjordes
på
enskilda
isolat
och
direkta
prover.
De
prover
som
användes
utan
att
selektera
fram
enskilda
isolat
användes
för
att
testa-­‐
och
utvärdera
en
metod
för
karakterisering
av
resistensmönster
i
bevattningsvatten
med
hjälp
av
Omnilogs
system
med
PM-­‐plattor.
I
detta
test
bedömdes
tillväxten
av
mikroorganismerna
i
närvaro
av
följande
antibiotiska
ämnen:
amikacin,
gentamicin,
kanamycin,
penicillin
G,
ampicillin,
piperacillin,
trimetoprim,
kalium
tellurit
och
ciprofloxacin.
Ur
samma
prover
isolerade
enskilda
isolat
på
följande
medium:
LB,
Rainbow
Agar,
mEnterococcus,
0,1
TSA
och
VRBD.
Totalt
isolerades
177
stammar
vilka
plockades
baserat
på
utseende
på
media,
där
heterogenitet
i
insamlat
material
var
målet.
Varje
isolat
identifierades
med
användning
av
Omnilog
GENIII-­‐system
av
biokemisk
karakterisering.
Vidare
var
isolaten
testades
på
Brilliance
ESBL-­‐agar
för
deras
förmåga
att
växa
genom
att
producera
betalaktamaser.
Av
det
totala
antalet
isolat
var
valdes
30
stycken,
företrädesvis
Enterobacteriaceae
eller
Pseudomonas
spp.,
baserat
på
likhet
med
modellorganismer
under
identifieringen
i
Omnilog-­‐systemet.
Dessa
isolat
karaktäriserades
ytterligare
med
Etest-­‐stickor.
Utifrån
dessa
tester
valdes
tre
stammar
för
ytterligare
analyser
där
försök
gjordes
att
överföra
plasmider
i
en
ansats
att
simulera
horisontell
genöverföring.
Isolaten
testades
också
med
Omnilog
PM-­‐paneler
med
olika
antibiotika
av
intresse.
Ett
antal
isolat
valdes
även
för
molekylära
tester
med
PCR
där
man
sökte
efter
de
plasmidburna
resistensgenerna
CTX-­‐M1,
CTX-­‐M2
och
SHV.
Data
från
tester
på
den
totala
mikrofloran
var
svår
att
tolka;
för
att
utveckla
metoden
till
ett
tillförlitligt
verktyg
krävs
vidare
optimering.
För
alla
test
med
PM-­‐plattor
(total
microbiota
och
enskilda
isolat)
visades
största
skillnader
mellan
olika
antibiotika
i
brunnar
med
starkast
koncentration.
Totalt
uppvisade
175
isolat
förmåga
att
växa
på
ESBL-­‐agar.
E-­‐tester
bekräftade
resistens
hos
isolat
mot
β-­‐laktam-­‐antibiotika.
Många
av
dessa
var
kliniskt
resistenta.
Inga
plasmider
överfördes
under
exogen
isolering.
Under
molekylära
tester
har
några
träffar
för
CTX-­‐M1
genen
hos
de
utvalda
isolaten,
men
inte
för
de
andra
generna.
Isolaten
som
analyserades
med
Omnilog
PM-­‐plattor
visade
resistens
mot
de
flesta
testade
substanserna.
Isolaten
visade
störst
resistens
mot
penicillin
G,
ampicillin,
amikacin,
kanamycin
och
trimetoprim.
I
studien
konstaterades
att
resistens
bland
bakterier
var
vanligt
i
bevattningsvattnet
och
att
bakterier
resistenta
mot
β-­‐laktam-­‐antibiotika
var
mycket
vanligt.
Vidare
studier
behövs
för
att
bedöma
frekvens-­‐och
förekomsten
av
horisontell
genöverföring
(HGF)
i
denna
miljö.
Även
angående
utredning
av
orsak
till
uppkomsten
av
resistens
krävs
mer
undersökningar.
Data
som
erhållits
skulle
kunna
visar
att
resistens
finns
mot
substanser
som
är
vanliga
inom
veterinärmedicin
(t
ex
penicillin
G).
Dock
är
dessa
substanser
även
vanliga
inom
humanmedicin
vilket
gör
att
det
inte
går
att
dra
någon
slutsats
angående
resistensens
ursprung
utifrån
data
i
denna
studie.
Spridning
av
bakterier
med
ESBL-­‐resistens
har
även
visats
ske
via
människor
som
exponeras-­‐
och
blir
bärare
av
resistenta
bakterier
via
exempelvis
resor.
Studier
visar
också
att
resistenta
bakterier
sprids
via
transport-­‐
och
handel
med
varor
och
djur.