Journal Reading Stase Media dan Sterilisasi:

Comparison of direct plating and broth enrichment

culture methods for detection of potential bacterial
pathogens in respiratory secretions
Ravinder Kaur,* Jareth Wischmeyer, Matthew Morris and Michael E. Pichichero
Maria Silvia Merry
NIM 18/437866/PKU/17829

PROGRAM PENDIDIKAN DOKTER SPESIALIS I

MIKROBIOLOGI KLINIK
FK KMK UGM
JULI 2019
Presentasi Kasus Stase Media dan Sterilisasi 1
Presentasi Kasus Stase Media dan Sterilisasi 2
INTRODUCTION
• Colonization of the nasopharynx (NP) by potential respiratory
bacterial pathogens is frequent in early childhood and is the first step
in pathogenesis of invasive diseases and respiratory tract infections
such as pneumonia, sinusitis and acute otitis media
• The predominant respiratory bacterial pathogens that colonize the NP
of children are Streptococcus pneumoniae (Spn), non-typeable
Haemophilus influenzae (Hflu) and Moraxella catarrhalis (Mcat),
Other normal nasopharyngeal bacterial flora, Staphylococcus aureus
(SA), and alpha-haemolytic Streptococcus (AHS) are also common.

Presentasi Kasus Stase Media dan Sterilisasi 3

Presentasi Kasus Stase Media dan Sterilisasi 4
OBJECTIVES
• Compared the recovery of potential respiratory bacterial pathogens
and normal flora from nasopharyngeal specimens collected from
children during health and at the onset of acute otitis media (AOM)
by selective direct-plating and overnight broth-enrichment.
• Surveys of species present in the NP during different stages of
pathogenesis

Presentasi Kasus Stase Media dan Sterilisasi 5

 METHODS: Study Populations
• Children at the age of 6 months and prospectively followed until 30–36
months of age during 2006–2016.
• Children were healthy, full term birth, no craniofacial anomalies and no
known immune deficits.
• Received all doses of pneumococcal conjugate vaccine (PCV) according to
the U.S. schedule; either PCV7 or PCV13 depending on the date of their
enrollment, along with other routine childhood vaccinations.

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 METHODS : NP sample collections and processing
1 ml of sterile
Sample PBS
Collection into
and each nare and collecting
Processing
the solution in a50–100
2–3 h later, sterile container
μl of the sample was
transferred onto BD Tryptic Soy with 5% sheep
blood agar and BD Tryptic Soy chocolate agar
Plates
plates and were incubated
dilution streakedovernight at 37
to promote C, 5%
single
CO2.
colony An additional 100 μl of NW were
growth
transferred into 2 ml of BacT/Alert SA culture
media broth and incubated overnight at 37 C,
5% CO2 Presentasi Kasus Stase Media dan Sterilisasi 7
 METHODS : MEF sample collections and processing
collected
Sample byCollection
tympanocentesis, 8% tetracaine solution was
and Processing
instilled into the external auditory canal after placement of an
otowick

after ∼15 min a 20-gauge 3·5″ spinal needle attached to a 3

cc syringe was used to puncture the tympanic membrane and
collect MEF

were processed in the same manner as NW samples when

the children developed AOM to evaluate the impact of direct
culture versus the broth-enrichment of MEF
Presentasi Kasus Stase Media dan Sterilisasi 8
 METHODS : Identification
SpnSample
was identified using colony morphology,
Collection and a-haemolysis, P-disc sensitivity test.
Processing
Serotypes of Spn were determined by latex agglutination (Pneumotest-Latex, Statens
Serum Institute, Copenhagen, Denmark) according to the manufacturer’s instructions.
Quellung reactions were used to identify the serotype subgroup

Identification of Spn from AHS was conducted by the implementation of the P-

disc. Hflu identification was based on colony morphology, growth requirement for
hemin and nicotinamideadenine dinucleotide using Haemophilus ID Quad plates

Mcat testing grey-white hemispheric colonies of waxy surface were identified

using hockey puck test and catarrhalis disc test. SA isolates were identified based
on colony morphology, clear b-haemolysis on blood agar plates, catalase positive
test and coagulase positive test.
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 METHODS : Data Analysis
NWSample Collection
direct-plating and NWand Processing culture results were
broth-enrichment
compared during health and at the onset of AOM illness; MEF and
MEF broth-enrichment culture results were also compared

chi-square test in GraphPad Prism version 6.1 for windows. P-

values of <0.05 were considered significant.

plots of bacterial species interactions in broth were prepared

using the ggplot2 package in R version 3.1.1

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RESULT: subject characteristic
Research Subject Antibiotic Usage in Healthy Group

18%
24%, 24%

Healthy No AB
AOM AB

76%, 76%
82%

Antibiotic Usage Antibiotic Usage in Sick Group

22%

No AB 43% No AB
AB 57% AB

78%

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RESULT: NW direct-plating and broth analysis

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RESULT: MEF bacterial recovery after broth-enrichment
In MEF samples broth-enrichment
resulted in increased isolation of
Spn (+10.4 %, P<0.001), Hflu (+4.4
%, P=0.39) and Mcat (+13.5 %,
<0.001) (Table 1). SA (44.4 %) and
AHS (15.9 %) were also more likely
to be isolated after
brothenrichment.
This data indicates that broth-
enrichment has value to improve
potential respiratory bacterial
pathogens’ identification in MEF.
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RESULT: In vitro bacterial–bacterial competition with
the inocula present naturally in NP

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Discussion
• Broth-enrichment of MEF
samples was shown to be of
value.
• Competition between species
during broth-enrichment
resulted in normal flora out-
competing bacterial respiratory
pathogens with reproducible
patterns.
Presentasi Kasus Stase Media dan Sterilisasi
• A significant reduction in the
number of bacterial respiratory
pathogens isolated from NW
occurred with enrichment
during both healthy and AOM
visits.
• This suggests that broth-
enrichment is disadvantageous
because it reduces recovery of
the target isolates.
15
Discussion
• Broth-enrichment of MEF
samples was shown to be of
value.
• Competition between species
during broth-enrichment
resulted in normal flora out-
competing bacterial respiratory
pathogens with reproducible
patterns.
Presentasi Kasus Stase Media dan Sterilisasi
• A significant reduction in the
number of bacterial respiratory
pathogens isolated from NW
occurred with enrichment
during both healthy and AOM
visits.
• This suggests that broth-
enrichment is disadvantageous
because it reduces recovery of
the target isolates.
16
Discussion
• The increased number of
bacterial respiratory pathogens
recovered from MEF samples
after broth-enrichment was
significant and since the middle
ear is typically non-culturable or
home to only species of bacterial
respiratory pathogen during
AOM the enrichment might be
endorsed.
Presentasi Kasus Stase Media dan Sterilisasi
• Spn and Hflu are frequently found
to co-colonize the NP. It has been
shown that Hflu out-competes Spn
for survival through signalling of
nucleotide-binding oligomerization
domain-1 (Nod1) to facilitate host
clearance of Spn [21], but when
grown in broth, we found that Spn
predominates over Hflu.
17
Discussion
• The increased number of
bacterial respiratory pathogens
recovered from MEF samples
after broth-enrichment was
significant and since the middle
ear is typically non-culturable or
home to only species of bacterial
respiratory pathogen during
AOM the enrichment might be
endorsed.
Presentasi Kasus Stase Media dan Sterilisasi
• Spn and Hflu are frequently found
to co-colonize the NP. It has been
shown that Hflu out-competes Spn
for survival through signalling of
nucleotide-binding oligomerization
domain-1 (Nod1) to facilitate host
clearance of Spn [21], but when
grown in broth, we found that Spn
predominates over Hflu.
18
Discussion
• It has been shown that Spn and
SA are usually recovered from
different sites in the respiratory
tract (posterior NP for Spn and
anterior nares for SA)
• Our low rates of SA recovery
might be due to our sampling
process
Presentasi Kasus Stase Media dan Sterilisasi
• SA seems to be quite a unique
pathogen in terms of its relative
strength when competing with
other bacteria organisms in
broth. While we only isolated SA
in 94 out of 3442 visits (3 %), SA
out-competed respiratory
bacterial pathogens in ~75% of
samplings.
19
Discussion
• It has been shown that Spn and
SA are usually recovered from
different sites in the respiratory
tract (posterior NP for Spn and
anterior nares for SA)
• Our low rates of SA recovery
might be due to our sampling
process
Presentasi Kasus Stase Media dan Sterilisasi
• SA seems to be quite a unique
pathogen in terms of its relative
strength when competing with
other bacteria organisms in
broth. While we only isolated SA
in 94 out of 3442 visits (3 %), SA
out-competed respiratory
bacterial pathogens in ~75% of
samplings.
20
Discussion
• Previous studies have shown that SA
is susceptible to hydrogen peroxide
produced by other bacteria (most
notably Spn, S. viridans and other
Streptococcus species); however in
broth when SA was paired with Spn
(nine cases) or AHS (25 cases) in all
but one instance SA predominated.
This may suggest that broth
enrichment overcomes the effect of
production of H2O2.
Presentasi Kasus Stase Media dan Sterilisasi
• Hflu and Mcat did not compete
strongly with one another during
broth-enrichment. However,
AHS tended to predominate over
all otopathogens after broth-
enrichment, especially when
samples were taken at healthy
visits.
• This suggests that the broth we
used for enrichment was
selective for non-pathogenic
normal flora.
21
Discussion
• Previous studies have shown that SA
is susceptible to hydrogen peroxide
produced by other bacteria (most
notably Spn, S. viridans and other
Streptococcus species) [24, 25];
however in broth when SA was
paired with Spn (nine cases) or AHS
(25 cases) in all but one instance SA
predominated. This may suggest that
broth enrichment overcomes the
effect of production of H2O2.
Presentasi Kasus Stase Media dan Sterilisasi
• Hflu and Mcat did not compete
strongly with one another during
broth-enrichment. However,
AHS tended to predominate over
all otopathogens after broth-
enrichment, especially when
samples were taken at healthy
visits.
• This suggests that the broth we
used for enrichment was
selective for non-pathogenic
normal flora.
22
Conclusion
• We recommend using direct-plating • In contrast, we recommend
of NP samples for most broth-enrichment of MEF
circumstances when the laboratory samples because the process
is asked to identify potential results in significantly improved
bacterial respiratory pathogens. recovery of bacterial respiratory
Only if it is essential to identify every pathogens with marginal
possible potential bacterial additional technical time
respiratory pathogen would broth- because other flora are
enrichment be of value, accepting frequently present in the
that the technical time and effort inocula.
would be material.

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Additional
Point

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Miscellanous

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Critical Appraisal

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Paper Source: Journal of Medical Microbiology

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Critical Appraisal for Observational Prospective Study
I. Are the result valid?
NO CA QUESTION ANSWER BASED ON PAPER

1 Was the cohort representative of the population? Was YES, LARGE NUMBER OF PARTICIPANTS, WITH
everyone included who should have been? SPECIFIC INCLUSION CRITERIA

2 Were the tools validated, where applicable? YES, ACCORDING TO CLSI

3 Were the subjects and/or the outcome assessor blinded to CAN BE TOLD SO
exposure?

4 Have all important confounding factors been identified and YES BY INCLUSION AND EXCLUSION CRITERIA
accounted for in the design and/or analysis?

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Critical Appraisal for Observational Prospective Study
II. What are the result?
NO CA QUESTION ANSWER BASED ON PAPER

1 What are the result of this study? option to use direct plating or broth enrichment
2 How precise are the results? precise since they used large numbers of samples
3 Do you believe the results? yes

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Critical Appraisal for Observational Prospective Study
III. Are the result help locally?
NO CA QUESTION ANSWER BASED ON PAPER

1 Can the result be applied in local yes

population?
2 Do the result of this study fit with yes
the other evidence?
3 What are the implication of this giving suggestion to ude certain method in laboratory
study for practice?

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Question & Answer Session

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