This study investigated methods for detecting biofilm formation in Staphylococcal isolates from neonatal infections. Staphylococci were the most common cause of infection. Three phenotypic methods (tube method, microtiter plate method, Congo red agar) and a genotypic PCR method were used to detect biofilm formation. The tube method had the highest sensitivity and specificity compared to the PCR method. Strong biofilm formers showed higher resistance to vancomycin and meropenem. Both genotypic and phenotypic methods should be used to fully identify biofilm formation in Staphylococci.

1. COMPARATIVE STUDY ON DETECTION METHODS OF
STAPHYLOCOCCAL BIOFILM FORMATION IN
NEONATAL INFECTIONS
Submitted
by
Eman A. AbdAlrahman
Ass.lecturer of Microbiology & Immunology
AL-Azhar university- Asuit Faculty of Medicine
1

2. 2

3. MICROBIAL BIOFILM
Biofilms are highly organized communities of
microbes made up of one or more species
attached to biotic or abiotic solid surface and
often encased in an extracellular matrix
which composed of proteins ,polysaccharides
and nucleic acids ( Zarnowski et al., 2014).
3

4. STAGES OF BIOFILM
FORMATION.
Figure 1. Stages of biofilm formation
(Monroe, 2007). 4

5. MICROBIAL BIOFILM
 Biofilms have been found to be involved in 80%
of all microbial infections in the body. Infectious
processes in which biofilms have been
implicated are urinary tract infections, middle
ear infections and endocarditis (Rogers ,2008).
5

6. Biofilms can also be formed on the inert
surfaces of implanted devices such as
catheters, prosthetic cardiac valves and
intrauterine devices (Auler et al., 2009).
MICROBIAL BIOFILM
6

7. MICROBIAL BIOFILM
 Staphylococci are recognized as the most
frequent causes of neonatal nosocomial
infections, especially in premature newborns
or those hospitalized in NICU for along time as
they are submitted to invasive procedures and
antibiotic treatment (Schwab et al., 2007) .
7

8. MICROBIAL BIOFILM
 Staphylococci are also implicated as the most
common causes of biofilm-associated
infections. As they are frequent commensal
bacteria on the human skin and mucous
surfaces. So they can infect any medical device
that penetrates these surfaces ( Vuong &Otto 2002).
8

9. AIM
OF
THE
W
ORK
9

10. THE AIM OF THIS STUDY WAS
TO:-
 Investigate biofilm formation by phenotypic and
genotypic methods in Staphylococcal isolates
isolated from clinical specimens of newborns
suffering from neonatal sepsis.
 Compare the different methods for detection of
biofilm formation to reach the most appropriate
method.
10

11. CONT.
 Test antibiotic susceptibility of biofilm
forming staphylococci by minimum biofilm
inhibitory concentration (MBIC) and by
minimal inhibitory concentration (MIC).
11

12. Patients & Methods
12

13. PATIENTS AND METHODS
 This study was conducted on 130 neonates
with clinical signs and symptoms of sepsis
admitted to Alzhraa and Sayed Galal
NICUs from May 2012 to June 2013.
13

14. PATIENT AND METHODS
Inclusion criteria:
o Clinical suspicion of sepsis (e.g. poor reflexes,
lethargy, respiratory distress, bradycardia,
apnea, or bleeding
o And by laboratory investigations: C-reactive
protein, CBC and blood culture.)
14

15. PATIENT AND METHODS
Exclusion criteria:
o Neonates having major surgically
uncorrectable lethal anomalies.
o Preterm < 28 weeks.
o Birth weight < 750 gms.
15

16. PATIENT AND METHOD
 Blood cultures for 100 neonates and end
tracheal tube cultures for 30 neonates
with ventilator associated pneumonia
were done .
16

17. SAMPLE COLLECTION
1.Two ml of blood were
collected by vein puncture
under complete aseptic
condition and inoculated
immediately to blood
culture bottle.
17

18. 2.Tips of endotracheal tube were
aseptically cut by using a sterile
scalpel and immediately were
transferred to a sterile cup
containing trypticase soya broth as
a transport medium.
18

19. SAMPLE PROCESSING
1. Blood culture bottles and cups containing the
tips of endotracheal tubes were immediately
transferred to microbiological lab for over
night incubation under aerobic condition at
37°C.
19

20. SAMPLE PROCESSING
2. Sub cultures from blood cultures and
endotracheal tubes cultures were done on
blood agar, nutrient agar, macCkonkey’s
agar and sabroud dextrose agar which were
incubated at 37°C for 24h under aerobic
condition.
20

21. CONT.
 Subcultures from blood cultures
were done each other day and it was
discarded after two weeks.
21

22. ISOLATION AND IDENTIFICATION OF
STAPHYLOCOCCI FROM CLINICAL
SPECIMENS.
 Colony morphology.
o Gram staining.
o Catalase test .
o Oxidase test .
o Tube coagulase test .
o Mannite fermentation.
o Novobiocin sensetivity .
(Green Wood, 2012)
Novobiocin sensitivity
Mannitol salt agar
22

23. DETECTION OF BIOFILM
FORMATION
 Three phenotypic methods
• Tube method (TM).
• Microtiter plate method (MTP).
• Congo red agar method(CRA).
 Genotypic method.
• PCR was used to detect icaAD gene(coding for
biofilm formation). 23

24. (1) MICROTITER PLATE METHOD(MTP)
A single
colony of
the staph
isolate
from
nutrient
agar.
Resuspend in
2ml trypticase
soya broth
The tube
was
incubated
overnight
at 37°C
under
aerobic
conditions.
Dispense
200ul in
each well)
200
incubated
overnight at
37°C under
aerobic
conditions
for
fixation
for 5 min
then
washed
by tap
water.
200µl
Naac
etate
200 ul crystal
violet was
added to each
well for biofilm
staining for 30
minutes
washed 3 times with
taps water using
Pasteur pipette and
was left at room
temperature to air dry.
read using a
microtiter-plate
reader at 630nm.
24

25. )2(TUBE METHOD
A loopful of each
test organism
was inoculated
in 2 ml of
trypticase soy
broth with 1%
glucose in a
sterile falcon
tube
Incubate the tubes
at 37oC for 24 h
tubes were
decanted and
washed with
phosphate
buffer saline
(pH 7.3) and
dried.
Tubes were then stained with
crystal violet for 30 minute.
Excess stain was
washed with distilled
water. The tubes were
dried in inverted
position.
25

26. )3(CONGO RED AGAR METHOD
 Congo red agar plates were inoculated
staphylococcal isolates and incubated
aerobically for 24 to 48 hours at 37°C .
 Positive result was indicated by black
colonies.
 Non biofilm producers remained pink.
Congo red agar
26

27. )4(POLYMERASE CHAIN REACTION
(PCR) FOR DETECTION OF ICAAD
GENES
1. DNA was extracted manually by boiling.
(Eftekhar et al., 2009).
2. Amplification of icaAD were performed by
using specific primers (Qiagen).
3. PCR product electrophoresed in 2%
agarose gel containing 1μl/mL ethidium
bromide.
27

28.  The bands were visualized by ultraviolet
illumination (Foto/Phoresis I) and checked
for size against molecular weight markers
using 50 bp ladder (Qiagen) (Arciola et
al.,2001).
28

29. ANTIBIOTIC SUSCEPTIBILITY
TESTING FOR BIOFILM FORMING
ISOLATES.
Antimicrobial-susceptibility testing by:
A. Disk Diffusion) (CLSI 2012) .
B. MIC. Meropenem and Vancomycin
antibiotics were used as the tested
isolates were commonly sensitive to
them.
29

30.  MICs were determined by broth micro
dilution method using 96 wells micro titer
plate and results were interpreted
according to CLSI, guidelines (2012).
30

31. Over night
culture of
staph on
nutrient
agar.
Adjust to 0.5
McFarland
with trypticase
soya broth
The tube
was
incubated
overnight
at 37°C
under
aerobic
conditions.
Dispense
75ul in each
well) incubated
overnight at
37°C under
aerobic
conditions
200µl
Naac
etate
washed 3 times with
taps water using
Pasteur pipette and
was left at room
temperature to air dry.
read using a
microtiter-plate
reader at 630nm.
Leave it to
dry In
inverted
position
under
aseptic
condition
Volumes of 100 μl of
appropriate two-fold
dilutions of the
respective antimicrobial
agents in Mueller–
Hinton broth were
transferred into the dried
wells with established
biofilms.
The microtiter plates
were incubated for
18–20 hours at 37 ºC
Under
aseptic
condition
C- Determination of minimum biofilm inhibitory
concentration MBIC)(Cernohorská and Votava 2008).
31

32. RESULTS
32

33. RESULTS
Table(1):Gender distribution in the studied
group.
Gender No. of infants %
Male 83 63.8%
Female 47 36.2%
Total 130 100%
33

34. 34

35. Fig.3 Gestational age distribution in
the studied group.
35

36. 36

37. TABLE(2)TYPE OF MICROBIAL GROWTH IN BLOOD
AND ENDOTRACHEAL TUBE CULTURES.
Type of growth Blood and endo tracheal
tube cultures
No. %
Mono-microbial growth 95 73%
Poly microbial growth
(two isolates)
15 11.5%
No growth 20 15.3%
Total 130 100 37

38. Fig.4 Type of organisms isolated from blood cultures.
38

39. Fig.5 Types of organisms isolated from ETT cultures.
39

40. D
c
A
B
Fig.6 Detection of biofilm formation
by microtiter plate method ,A:strong,B: weak C: non biofilm
former D:negative control. 40

41. Biofilm former Non biofilm former Total
No. % No. %
50
28 56 22 44
Table(3).Detection of staphylococcal biofilm
formation by micro titer plate method.
41

42. Fig.7. Detection of biofilm formation of Staphylococcal isolates
by tube method. A: Negative control, B: Weak, C: Moderate, D:
strong biofilm former.
B C
A
DB C
A
42

43. Biofilm former Non biofilm former Total
No. % No. %
50
34 68% 16 %32
Table (4) Results of biofilm formation of Staphylococcal
isolates by tube method.
43

44. A
B
FIG. 8. Detection of biofilm formation of Staphylococcal
isolates by Congo red agar method A: biofilm former, B: Non
biofilm former. 44

45. Biofilm former Non biofilm former Total
No. % No. %
5044 88 6 12
.
Table (5) Results of biofilm formation of Staphylococcal
isolates by Congo red agar method.
45

46. Table(6).Results of detection of icaAD (genes coding
biofilm formation in staphylococci) by PCR.
PCR
(ica A)
(50)
PCR
)ica D(
)50(
+ve. -ve +ve. -ve
No % No % No % No %
28 56 22 44 28 56 22 44
46

47. Fig. (9): Agarose gel electrophoresis analysis of the
PCR amplification products of icaA.
Lane 1: DNA molecular weight markers (50bp)
Lane 2,3:are examples of positive isolates for icaA
gene;
Lane 4: examples of negative isolates for icaA gene
Lane5: positive control,
Lane 6: negative control (DNA template absent).
1 2 3 4 5 6
47

48. Fig. (10): Agrose gel electrophoresis analysis of the
PCR amplification products of icaD
Lane 1: DNA molecular weight markers (50bp)
Lane 2: negative control (DNA template absent).
Lane3: positive control.
Lane 4,6,7,8:are examples of positive isolates for icaD
gene;
1 2 3 4 5 6 7 8
48

49. Fig.11 Results of detection biofilm formation by
staphylococcal isolates by the tube (TM), microtiter plate
(MTP) and Congo red agar (CRA) methods.
49

50. Method Ica
+ve
Ica
-ve
Sensitivity specificity Positive
predictive
value
Negative
predictiv
e value
P
value
Microtiter
plate)+(
15 13
57.7% 40.9% 53.6% 45%
0.684
(NS)
Microtiter
plate (-)
11 9
Congo red
agar)+(
27 15
96.4% 25% 64.3% 83.3%
0.029
(HS)
Congo red
agar (-)
1 5
Tube
method
(+)
27 6
96.4% 70% 81.8% 93.3%
<0.01
(HS)
Tube
method (-)
1 14
Table (7) Statistical evaluation of the phenotypic
biofilm detection methods compared to detection of
icaAD genes in Staphylococcal isolates.
50

51. Fig. 12. Antibiotics resistance pattern of biofilm forming S. aureus
by disc diffusion method.
51

52. Fig. 12.Antibiotics resistance pattern of biofilm forming
S. epidermidis.
52

53.  Table.(8) Antibiotics resistance pattern in
biofilm forming staphylococci according to
the intensity of biofilm formation:
Antibiotics
Strong biofilm
forming
staphylococci
(3)
Moderate
biofilm forming
staphylococci
(6)
weak biofilm
forming
staphylococci
(19)
P value
Vancomycin 3 1 0 0.000
Meropenem 3 4 0 0.000
Cefazoline 3 6 9 0.082
Gentamycin 3 6 8 0.082
Erythromycin 3 6 8 0.082
Ceftrixone 3 6 17 0.771
53

54.  A statistically significant difference
between antibiotic susceptibility of
planktonic populations(performed by
MIC) and biofilm populations of the same
organism(performed by MBIC) was
detected in this study. As p value was
<0.05
54

55. CONCLUSION AND
RECOMMENDATIONS
55

56.  Staphylococci were identified in this study as
the most common cause of neonatal sepsis in
Sayed Glal and Alzahraa NICUs as 38.5% of
cases had staphylococcal infection.
 Also it was found that about 70% of
Staphylococcal isolates were biofilm former,
biofilm formation is considered as a one of the
most important virulence of Staphylococci. 56

57.  Comparing the phenotypic biofilm
detection methods with genotypic method
(PCR).TM was the most better tool for
screening of biofilm formation, as it has
both high specificity and sensitivity .While
CRA, although it is easier, but it has the
least specificity so it not recommended for
screening of biofilm formation. 57

58.  This study have confirmed previous data
presented by other authors that the molecular
presence of icaAD genes in the bacterial genome is
associated with the ability to form biofilms, but the
absence of these genes does not exclude this
phenomenon phenotypically.
 Therefore, it seems appropriate to use both
genotypic and phenotypic methods to improve the
identification of biofilm formation by staphylococci
58

59.  On the other hand, the biofilm-forming
ability of some strains in the absence of
icaAD gene highlights the importance of
further genetic investigations of ica
independent biofilm formation
mechanisms.
59

60.  Also it was found that Initial empirical
treatment with ampicillin and gentamicin
used routinely in NISUs is inadequate as
resistence to ampicillin and gentamycin in
this study was100% and 61% respectively.
Vancomycin and meropenem are still the
most effective antimicrobial agents against
staphylococcal isolates in NICUs. So it is
advised to consider them in the treatment of
resistant strains.
60

61.  Also it was found that strong biofilm
formers had complete resistance to all
tested antibiotics including vancomycin
and meropenem when tested by disc
diffusion and MIC methods( in planktonic
form ). Suggesting that strong biofilm
formers are more virulent than weak or
non biofilm formers even in their
planktonic form . 61

62.  A statistically significant difference
between antibiotic susceptibility of
planktonic populations and biofilm
populations of the same organism was
detected in this study.
 For this purpose detection of biofilm
formation and antibiotic susceptibility
pattern of bacteria in its biofilm form by
using MBIC should be considered in
routine diagnosis of bacterial infection.
62

63.  Current antibiotics have classically been
developed to treat infections involving
planktonic bacterial populations in acute
infection settings and are typically
ineffective in the eradication of bacteria in
biofilm- associated, persistent infections.
So many researches are needed to find
more specific antimicrobial agents and
ideal device surfaces that would help the
fight against biofilm formation.
63

64. 64