RAPID STR

Group 1 - IUP Class

GROUP 1
MEMBERS
 FIRST JOURNAL

ANTIBACTERIAL EFFECT OF GREEN TEA

AND POMEGRANATE PEEL EXTRACTS ON

STREPTOCOCCUS MUTANS OF
ORTHODONTIC TREATED PATIENTS
 INTRODUCTION
There are many different types of bacteria in dental caries, as
well as fungi, protozoa, and viruses. Streptococcus mutans is
the main bacteria causing dental caries, and this bacteria can
cause demineralization. Orthodontic treatment may lead to
changes in the oral environment, which may increase the
number of S. mutans in saliva. The pathogenesis of dental
caries has developed to a level where many antimicrobial and
anti- biotic agents are no longer effective. This has resulted in
resistance to many of these agents, which is beneficial for
both patients and practitioners. The plants include green tea
and pomegranate.
 MATERIALS AND METHODS
Sample Collection
Prior to orthodontic treatment:
Approx. 1ml of unstimulated whole saliva samples were obtained in the morning after
an overnight fast. Plaque samples were collected from the buccal and labial aspects
of the anterior teeth and the four first molars. Recorded as P1.

After placement of the orthodontic appliance:

Three months after placement of orthodontic appliance. 1ml of unstimulated whole
saliva samples and plaque samples were recollected from the same site and were
recorded as P2.
 MATERIALS AND METHODS
Inclusion and Exclusion

Inclusion Exclusion
Patients with permanent dentition, Smoking, pregnancy, poor
no clinical and/or radiographic general health, history of
manifestations of periodontal periodontal treatment, antibiotic
disease and no history of any administration.
smoking, no history of any
systemic illness and no antibi
 ISOLATION AND CULTIVATION

100 micro Liter Cultured in MS Incubated S. mutans

of each sample Agar (Good for anaerobically in collony is culture
S. mutans 37 C for 24 h in BHIB
growth)
IDENTIFICATION
Colonial shape and form
Gram staining
Microscopic examination
Catalase test
Remel RapID STR system and
ERIC (easy-to-use, reliable
electronic code compendium)
system electronic program.
 MICROBIOLOGICAL
PROCEDURE
100 gram of green tea and
pomegranate peel for each three
samples with two replicas (total six
samples)
Samples are subjected to different
dose of gamma irradiation (0, 5, and
10 kGy)
 SAMPLE EXTRACTION
COLD EXTRACTION METHOD (MACERATION)

20 mg of each samples were distilated by 200 ml of polar (aquades and

70% ethanol) and non-polar (70% hexane)
Let sit for 72 hours at 37°C with gentle shaking at 120 rpm
Filtered through double layers of muslin
Centrifuged at 6000 rpm for 10 min and finally filtered again through
Whatman filter paper no.1
The extracts were collected in Petri dishes, concentrated under low
pressure, and heated at 40°C untill evaporation.
 SAMPLE EXTRACTION
HOT EXTRACTION METHOD (DECOCTION)

20 g of green tea or pomegranate peels powder was mixed with

200 ml of sterile distilled water
Boiled at 70–80°C for half an hour using magnetic stirrer.
The mixtures were filtered by muslin cloths.
The filtrates were centrifuged for 10 min at 6000 rpm.
The extracts were collected in Petri dishes, concentrated under low
pressure, and heated at 40°C till evaporation.
RESULT & DISCUSSION
 ISOLATION, IDENTIFICATITON, AND
SELECTION OF THE BACTERIAL

ISOLATES
20 participants: each person collecting whole saliva and plaque
samples before (P1= 20 samples) and after orthodontic brackets
bonding for 3 months (P2= 20 samples)
10 Streptococcal isolates obtained from MS agar. This agar
promotes the growth of streptococci and suppresses other
bacterial species
Further purification by sub culturing on MS agar, selecting single
colonies
Identification: gram+staining, microscopic examination (cocci
arranged in chains), and catalase test (catalase negative)
S.mutans isolates enzymatic spectra were tested by Remel
RapID STR Panel
 EXTRACTION

YIELDS

Control sample: un-irritated, treated sample: gamma irradiated (5 and

10 kGy) of pomegranate peels and green tea in water, hexane and
ethanol
Solvent selection is essential for obtaining extracts with higher yields.
Statistical analysis by ANOVA shows that there was a significant
difference between extract yields
Higher yields: 70% ethanol (10 kGy, irradiated), pomegranate peels and
green tea leaves
Reason: solvent affinity with the phytomolecules and the polarity of the
solvent. Ethanol and water are polar solvents, gave higher yields,
compared to nonpolar solvent
Gamma irradiation up to 10 kGy is an effective method for enhancing
extraction yields. This is due to degradation of non-soluble
components with high molecular weight
The irradiation effect is enhanced in ethanol solvent due to ability to
extract both polar and semi-polar compounds
ESTIMATION OF TOTAL PHENOL
CONTENT (TPC)

 ESTIMATION OF TOTAL PHENOL

CONTENT (TPC)
Since most phenolic compounds have polar properties that make them very soluble in polar solvents
(such as ethanol and water), using the nonpolar solvent hexane resulted in the least quantity of phenolic
compound extracts.

Irradiation exerts its effects as direct and indirect mechanisms.

direct mechanism: the increase in total phenols and flavonoids is due to the release of phenolic
compounds from glycosidic components and the
degradation of larger phenolic compounds into
smaller ones by gamma irradiation treatment.
indirect mechanism: the radiolysis of water results in the production of free radicals such as hydroxyl
radicals, hydroperoxide radicals, and hydrated electrons.

The irradiated green tea and pomegranate groups resulted in inhibition of S mutans adhesion to the tooth
surface. This could be attributed to the biological effect of gamma radiation mainly due to hydrolysis of
the water content of irradiated plants and formation of free radicals. Formed free radicals will react with
other components of plant cells and result in the accumulation of phenolic compounds thus increase its
antibacterial effect.

 Determination of Fractional Inhibitory

Concentration (FIC) and Fractional
Inhibitory Concentration Index (FICI) values

The interactive inhibition commonly measured by the checkerboard method,

which was used for the determi- nation of synergy between the antibiotics
and natural antimicrobials. Each combination (from 2MIC to 1/32 MIC) of
irradiated (10kGy) pomegranate peels and green tea leaves extracts was
tested. The most effective combination regimens were (6.25 green tea and
1.562 peels mg/ml), which showed a strong synergistic inter- action (FICI
value was 0.375) against S. mutans. Not all of the regimens exhibiting a
synergistic effect

 Adhesion of bacteria to the

cells

The effect of different concentrations of pomegranate peels and green

tea leaves irradiated (10kGy) ethanolic extracts on the adherence of S.
mutans to tooth surface was studied. The results showed that the
synergistic combinations concentration (SCC), and MBC of the tested
extracts were prevented the colonization and adherence of S. mutans to
the tooth surface as 1.0% chlorhexidine gluconate.

The bacterial adhesion to the cells can alter by the irreversible damage of
the bacterial cytoplasmic membrane by catechins.
 CONCLUSIONS
Gamma-irradiated (10kGy) ethanol extracts of green tea
and pomegranate peels have a high inhibition effect on
S. mutans bacteria and decrease its adherence to the
tooth surface. The efficacy of irradiated (10kGy)
pomegranate peels and green tea leaves extracts
combinations against S. mutans was significantly higher
compared to each one alone. The combinations of plant
extracts offer enhanced antimicrobial efficacy due to
the synergistic effects besides slowing the development
of resistance.

 SECOND JOURNAL

CHARACTERIZATION OF
ENTEROCOCCUS SPECIES ISOLATED
FROM MARINE RECREATIONAL WATERS
BY

 INTRODUCTION

Enterococcus levels in a water body can be used as a way to determine its quality.
Enterococcus spp. are Gram- positive, aerotolerant anaerobes and are present in high
numbers in the intestines of birds and mammals. Despite being common, not all
enterococci have a fecal link; several species are associated with plants. Currently,
Enterococci monitoring can help to subsidize the assessment of the potential presence
of pathogens in waters. This is widely used in microbiology and in the sanitary analysis of
waters. There are some biochemical systems that are limited in how reliable they are
when testing environmental samples. This is because they are laborious to set up and less
reliable when doing so. On the other hand, although 16S rRNA gene sequencing has been
considered the standard methodology for identifying most bacterial species, it is
expensive, time-consuming and laborious in screening microbes in a large number of
samples. This methodology relies on the analysis of the mass spectra of peptides and
small proteins from microbial cells, which are specific for each microbial species.
 MATERIALS & METHOD
1. Recreational water samples collection and isolation of

presumptive enterococci cells

Seawater samples were collected for regulatory monitoring purpose

Sampling was made at 1 m depth using a 500 mL sterile plastic bottles and cooled down (ice

packs) for transportation within 24 h.

For EPA Method 1600, 1–100 mL volumes of sample were membrane-filtered onto mEI and up to
five enterococci characteristic colonies per sample were randomly selected and subcultured

onto brain heart infusion (BHI)

After 18 to 24 h of incubation at 35 °C, cells were stored in 15% glycerol BHI at −80 °C
Enterococcus genus screen tests were performed as follow:
Gram stain
catalase test
growth and hydrolysis in bile esculin
growth at 45 °C
growth in BHI 6.5% NaCl.
 MATERIALS & METHOD
2. Identification of enterococci isolates by API® Strep20

system

The stored isolates were slightly thawed before being subcultured onto BHI agar for 24 hours at

35 °C and then identified using the API® Strep 20 System (2003)

Biomérieux's Analytical Profile Index WEB software was used to generate the identification of the

isolates
Strain identification (ID) to the species level was divided into four subgroups:
excellent species identification, ( ≥99.9%; )
very good species identification, ( ≥99.0%; )
good species identification,( ≥90.0% )
acceptable species identification, ( ≥80.0% )
 RESULT
The successful identification rates ranged from 92% (117 out of 127 isolates) for the API® 20 Strep system and
100% for the MALDI-TOF MS analysis (see Table 1).

Comparing the ability of both systems to identify the isolates strains at the genus level, the API® 20 Strep
system was able to identify 111 strains
as belonging to Enterococcus genus whereas MALDI-TOF MS analysis
identified 117 Enterococcus isolates (see Table 1).
 RESULT

Concordance rates were also analyzed for the identification of Enterococcus species. Among
the 111 Enterococcus genera. Strains, 64% (n=71) isolates were confirmed to belong to the same
Enterococcus species.

However, this method failed to achieve definitive species identification of the five phyla,
resulting in a clear distinction of Enterococcus phyla (Table 2).

 RESULT
It is likely that these isolates are E. faecium, since
this species shows more variability in the
biochemical identification tests The API® 20 Strep
system reached a low agreement rate (14%) when
compared to the gold standard methodology
(Table 3), especially among those isolates
belonging to the species like E. hirae (43%, n =
15/35) that were misidentified as E. durans.

Two isolates identified as E. hirae and E. faecium

by 16S rRNA gene sequencing were erroneously
identified by API® 20 Strep system and MALDI-TOF
MS (Table 3).

 CONCLUSION
MS are bacterial species frequently targeted for investigation due to their role in the etiology of
dental caries. Differentiation of S. mutans and S. sobrinus is an essential part of exploring the role of
these organisms in disease progression and the impact of the presence of either/both on a
subject’s caries experience. Of vital importance to the study of these organisms is an identification
protocol that allows us to distinguish between the two species in an easy, accurate, and timely
manner. While conducting a 5-year birth cohort study in a Northern Plains American Indian tribe,
the need for a more rapid procedure for isolating and identifying high volumes of MS was
recognized. We report here on the development of an accurate and rapid method for MS
identification. Accuracy, ease of use, material and time requirements for morphological
differentiation on selective agar, biochemical tests, and various combinations of PCR primers were
compared. The final protocol included preliminary identification based on colony morphology
followed by PCR confirmation of species identification using primers targeting regions of the
glucosyltransferase (gtf) genes of SM and SS. This method of isolation and identification was found
to be highly accurate, more rapid than the previous methodology used, and easily learned. It
resulted in more efficient use of both time and material resources.
 THIRD JOURNAL

Improved method for rapid and

accurate isolation and identification
of

Streptococcus mutans and

Streptococcus sobrinus from human
plaque samples
 INTRODUCTION
A subgroup of viridans streptococci, the mutans streptococci group, includes both Streptococcus
mutans and Streptococcus sobrinus as primary etiological agents of dental caries. This information
means that there's a high chance that the two species of sobrinus are being investigated a lot, and
it's important to have an accurate identification protocol in place so that it is certain to be dealing
with the right species. There are several differences between S. mutans and S. sobrinus, which can
make it difficult to identify which variety is responsible for a particular infection. There are various
methods currently used for identification of S.mutans. Methods include: Morphological
differentiation on selective agars, Biochemical tests looking at sugar fermentation profiles, and PCR
identification. There are multiple options, but each has its own advantages and disadvantages.
Given the high volumes of plaque samples being analyzed, a rapid and accurate identification
system is needed that works consistently with clinical isolates. The RapID STR System is a method
that uses common and chromogenic substrates to identify Streptococci and other organisms that
have been isolated from human clinical specimens and are of medical importance. When used in a
diagnostic workflow, it isolates Streptococcus species or other closely related microorganisms
cultured on agar to help clinicians with treatment options for patients suspected of having bacterial
infections.
 MATERIALS
A total of 239 mothers who were pregnant or who had just given birth were recruited from a
Northern Plains American Indian Tribe. All onsite research team members were Native and were
under the guidance of a study director who was a senior dental hygienist in the Tribe. Approval was
obtained from the Aberdeen Area IRB, University of Iowa IRB, and the Tribal Research Review Board.
 METHODS
1. Collection and processing of plaque samples.

Samples were collected and processed. Briefly, trained and calibrated dental hygienists
collected whole mouth plaque samples from Native American Indian children and their
mothers (or primary caregivers) at 8 time points from the child’s birth to age 36 months by
swabbing all smooth surfaces of the teeth (oral mucosa and tongues of children prior to
tooth eruption)

2. Isolation and Preliminary Identification of Mutans streptococci

Ten well isolated, presumed MS colonies were selected from the MSKB plates and streaked
onto both sides of a Mutans streptococci identification (MS ID) plate (1/2 colony per side).
The MS ID plate is a two compartment I-plate containing Strep Mannitol Agar with bacitracin
(SMAB) and Modified SB20 agar (SB20M). The pH was adjusted to 7.6 before autoclaving.
Plates were incubated for 48 hours at 37°C, 5% CO2 and preliminary ID was recorded.
 METHODS
3. Rapid DNA extraction and PCR.
During the 48 hour incubation of the MS ID plates (at 24 hours or when sufficient colony growth was
observed), plates were removed briefly from the incubator and a single colony from each MS ID plate
was streaked onto Tryptic Soy Broth agar with 0.5% yeast extract (TSB-YE) to be used for rapid DNA
extraction and freezer stocks of each isolate. TSB-YE plates were streaked from the SMAB side of the MS
ID plates due to more rapid growth and more butyrous colonies. When sufficient growth was obtained,
a rapid DNA extraction method was used to obtain DNA from all MS isolates.

Briefly, a 1 microlitre loop of bacterial cells was suspended in 140 μl TE buffer and placed in a
thermal cycler at 87°C for 5 minutes. Immediately after heating, samples were vortexed and
then centrifuged (4°C, 10 minutes, 10,000xg). Supernatant was placed in fresh tubes for use in ID
PCR. Following DNA extraction, freezer stock of each isolate was made and stored at -80°C.
 RESULTS

1. PRELIMINARY IDENTIFICATION OF MUTANS STREPTOCOCCI

In order to confirm that we saw morphological differences between the two species
similar to what was described by Saravia et al. (19), 37 assorted S. mutans and S. sobrinus
isolates (previously identified via fermentation profile) from 4 subjects were streaked onto
ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 6 SB20M agar. After 48 hours incubation
(37°C, 5% CO2), laboratory staff determined ID based on colony morphology observed on
the plates. All S. mutans isolates were successfully identified and 14/16 S. sobrinus were
correctly identified. In testing the MS ID plates, there were four typical combinations of
colony morphologies observed (Figure 1). On the SMAB agar, there were subtle, but
consistent differences in colony morphology between S. mutans and S. sobrinus. The
differences between the two species were more distinct on the SB20M agar. The accuracy
of the preliminary ID plates was 96.9% (649/670) across all isolates that were identified
through this method
 RESULTS

2. PCR identification of Mutans streptococci

Initial attempts at confirmation of species identification via PCR utilized the gtf D (SM) and gtf T (SS) primers
(Figure 2). Isolates (previously identified by fermentation profile) from 9 subjects (4 mothers, 5 children), all with
both S. mutans and S. sobrinus colonization, were selected to test the primers. Use of these primers resulted in a
7.4% (4/54) and 61.5% (32/52) false negative rate for S. mutans and S. sobrinus respectively. The second set of
primers tested, gtf B (SM) and gtf I (SS), were tested on isolates from 5 subjects (1 caregiver, 4 children) and
resulted in no false negative reactions for S. mutans (0/30) and 40% (6/15) false negatives for S. sobrinus isolates.
A subset of S. sobrinus isolates (n=20) that had produced multiple false negative reactions with the first two sets
of primers were then used to test two additional primer combinations, gtf B (SM)/gtf I-IN (SS), and the dex gene
targeted primers. Both primer sets were successful in identifying all S. sobrinus isolates. Examples of gtf B (SM)/gtf
I (SS), gtf B (SM)/gtf I-IN (SS), and dex PCR products can be found in Figure 3. The gtf B/gtf I-IN primer set was
selected for confirming the MS ID plate preliminary identification (Figure 4). Of the 670 isolates identified through
this protocol, 99.9% (669/670) were positively identified as either S. mutans or S. sobrinus using this primer set
 DISCUSSION

Distinguishing S. mutans and S. sobrinus is easily done once morphological

combinations are found and documented
error in identification become extremely rare
 gtf primers

The one previously used: produced an unacceptably high rate of false

negative PCR
Two sets of primer; gtf B (SM) and GTF I (SS)
resulted in positive identification of all S.mutans on the first round , and is
successful rate of subrinus identification on the second round

Selection of colonies & Fermentation

test

Through PCR confirmation within 4-5 days

Fewer agars are required as supposed to a series of 6
DNA are ready for AP-PCR (arbitrarily primed PCR) reactions as soon as the
ID PCR results were confirmed.
Second modified extraction for S.Sobrinus resulted in a more consistent
AP-PCR
Sequence-based methods are heavily utilized in the current study of
microorganisms found in the human microbiome. Metagenomic analysis offers
a method for quickly evaluating the microbiological make-up of a particular
sample.
It is crucial to keep these culture-based processes streamlined and
optimized, especially for labs lacking 16s rDNA sequencing capability for
isolate identification. The method used in this paper demonstrates how easily
S. mutans and S. sobrinus isolates from human plaque samples can be
distinguished, and it also gives us pristine cultures for next analysis.
 CONCLUSION
MALDI-TOF MS has been extensively used for
identification of microorganisms recovered from
clinical specimens. In this study, it has shown to
be a powerful tool for characterizing and
distinguishing environmental isolates. Results
corroborate with many authors showing that the
technique can largely contribute with taxonomic
and epidemiological studies. It also has the
potential to become an important tool for
bacterial source tracking method.
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