All clinical isolates of Salmonella enterica serovars Typhi and Paratyphi tested were capable of biofilm formation in vitro. The optimized conditions for maximum biofilm formation were adherence test medium incubated at 37°C and 150 rpm for 48 hours. Of the Salmonella Typhi isolates tested, 61% were very strong biofilm producers, 29% were strong producers, and 11% were moderate producers. For Salmonella Paratyphi isolates, 69% were very strong producers, 23% were strong producers, and 8% were moderate producers. The ability of these clinical isolates to form biofilms is concerning from a public health perspective as it could facilitate transmission to new hosts and cause disease.
Bacillus thuringiensis, an aerobic, Gram positive, spore forming bacterium produces unique proteinaceous crystalline parasporal inclusions during sporulation which have insecticidal properties. Besides being widely used as an insecticide in agriculture, Bt has been found to be useful in several fields like medicine, endoparasite control, bacteriocin production as well as enzyme production. Parasporin, a new category of bacterial parasporal protein capable of discriminately killing the cancer cells have been discovered. There are six classes of parasporins having different mode of action and cell specificities against cancer and tumor cells (Ohba et al., 2009).Bt proteins have also been used successfully to suppress the population levels of medically important Dipteran pests like mosquitoes by use of mosquitocidal strains that produce Cry proteins (Zhang et al., 2012) as well as potential therapeutic agent against protozoan disease Leishmaniases (El-Sadawy et al., 2008). Crystal proteins, like Cry5B from Bacillus thuringiensis are found to be safe to vertebrates and have been shown to have efficacy against intestinal hookworm parasites (Capello et al., 2006). Thus the multifarious applications of Bacillus thuringiensis have made it a microbe to reckon with and further study its genome for future developments.
Staphylococcus aureus is one of the most versatile nosocomial (i.e.
acquired in hospital) and dangerous human pathogen. In spite of the
introduction of antimicrobial agents and improvements in the
frequency and morbidity of staphylococcal diseases in the twentieth
century, staphylococci have persisted as an important hospital and
community pathogen. Thereafter, methicillin-resistant S. aureus
emerged as a major pathogen worldwide. A total of 38 positive clinical
isolates from various clinical samples received from different hospitals
of Dehradun included from March 2014 to August 2014. 38 samples
had bacterial growth, among these isolates 17(44.7%) were
Staphylococcus aureus. The present study was designed to investigate
antibiotic susceptibility pattern and the role of biofilm in isolates of various clinical
samples (Urine, Blood, Semen and Pus), by examining the ability of isolates to form biofilm
and produce signaling molecules and by developing a wound model, to relate laboratory
findings with in vivo activity by exploring the possibility of detecting biofilm markers in
dressings removed from chronic infections. The presence of biofilm was confirmed by
specialized microscopy techniques or by detecting biofilm markers. Various antibiotics had a
greater effect on viability when used at higher antibiotic concentrations (≥100 mg/L) and on
younger (6h) biofilms. The antibiotics used for antibiotic susceptibility testing were
Ofloxacin, Erythromycin, Amoxicillin, and Ciprofloxacin.
Should we be testing endoscopes? One life - Central Sterilising Club (csc) ...OneLife SA
The use of contaminated endoscopes can lead to patient-to-patient transmission of pathogens and infections. Studies evaluated cleanliness of patient-ready scopes after reprocessing and revealed between 10% and 30% of residual contamination.
Should therefore surveillance be standardized?
Effect of Agrobacterium Induced Necrosis, Antibiotic Induced Phytotoxicity an...Sandip Magdum
Agrobacterium tumefaciens infection and antibiotic wash are the critical steps of Agrobacterium mediated plant transformation procedure, most time responsible for lower transformation efficiency due to necrosis and phytotoxicity caused by biotic stress of Agrobacterium and abiotic stress by antibiotics respectively. Ammi majus Egyptian origin medicinal plant and Pearl millet cereal grain crop were studied for their stress responses to Agrobacterium mediated transformation (AMT). Agrobacterium strains LBA4404 (O.D.=0.6-0.8) and EHA105 (O.D.=0.2-0.4) were used for transformation experiments to infect calli of Ammi majus and embryogenic calli of Pearl millet respectively. Incase of antibiotic wash, Cefotaxime 500 mg L-1 was used for LBA4404 infected Ammi majus calli and Timentin 300 mg L-1 was used for EHA105 infected embryogenic calli of Pearl millet.
Effects of Agrobacterium infection, antibiotic and NaOCl washes on Agrobacterium removal and both explants physiological changes during transformation experimental procedures were studied. At the end of the experiments explants survival efficiency of Ammi majus and pearl millet were 8% and 5% respectively. Biotic and abiotic stress factors responsible for lower efficiency were investigated with various other factors and strategies were discussed which are need to be considered for higher transformation events and target tissue survival.
Bacillus thuringiensis, an aerobic, Gram positive, spore forming bacterium produces unique proteinaceous crystalline parasporal inclusions during sporulation which have insecticidal properties. Besides being widely used as an insecticide in agriculture, Bt has been found to be useful in several fields like medicine, endoparasite control, bacteriocin production as well as enzyme production. Parasporin, a new category of bacterial parasporal protein capable of discriminately killing the cancer cells have been discovered. There are six classes of parasporins having different mode of action and cell specificities against cancer and tumor cells (Ohba et al., 2009).Bt proteins have also been used successfully to suppress the population levels of medically important Dipteran pests like mosquitoes by use of mosquitocidal strains that produce Cry proteins (Zhang et al., 2012) as well as potential therapeutic agent against protozoan disease Leishmaniases (El-Sadawy et al., 2008). Crystal proteins, like Cry5B from Bacillus thuringiensis are found to be safe to vertebrates and have been shown to have efficacy against intestinal hookworm parasites (Capello et al., 2006). Thus the multifarious applications of Bacillus thuringiensis have made it a microbe to reckon with and further study its genome for future developments.
Staphylococcus aureus is one of the most versatile nosocomial (i.e.
acquired in hospital) and dangerous human pathogen. In spite of the
introduction of antimicrobial agents and improvements in the
frequency and morbidity of staphylococcal diseases in the twentieth
century, staphylococci have persisted as an important hospital and
community pathogen. Thereafter, methicillin-resistant S. aureus
emerged as a major pathogen worldwide. A total of 38 positive clinical
isolates from various clinical samples received from different hospitals
of Dehradun included from March 2014 to August 2014. 38 samples
had bacterial growth, among these isolates 17(44.7%) were
Staphylococcus aureus. The present study was designed to investigate
antibiotic susceptibility pattern and the role of biofilm in isolates of various clinical
samples (Urine, Blood, Semen and Pus), by examining the ability of isolates to form biofilm
and produce signaling molecules and by developing a wound model, to relate laboratory
findings with in vivo activity by exploring the possibility of detecting biofilm markers in
dressings removed from chronic infections. The presence of biofilm was confirmed by
specialized microscopy techniques or by detecting biofilm markers. Various antibiotics had a
greater effect on viability when used at higher antibiotic concentrations (≥100 mg/L) and on
younger (6h) biofilms. The antibiotics used for antibiotic susceptibility testing were
Ofloxacin, Erythromycin, Amoxicillin, and Ciprofloxacin.
Should we be testing endoscopes? One life - Central Sterilising Club (csc) ...OneLife SA
The use of contaminated endoscopes can lead to patient-to-patient transmission of pathogens and infections. Studies evaluated cleanliness of patient-ready scopes after reprocessing and revealed between 10% and 30% of residual contamination.
Should therefore surveillance be standardized?
Effect of Agrobacterium Induced Necrosis, Antibiotic Induced Phytotoxicity an...Sandip Magdum
Agrobacterium tumefaciens infection and antibiotic wash are the critical steps of Agrobacterium mediated plant transformation procedure, most time responsible for lower transformation efficiency due to necrosis and phytotoxicity caused by biotic stress of Agrobacterium and abiotic stress by antibiotics respectively. Ammi majus Egyptian origin medicinal plant and Pearl millet cereal grain crop were studied for their stress responses to Agrobacterium mediated transformation (AMT). Agrobacterium strains LBA4404 (O.D.=0.6-0.8) and EHA105 (O.D.=0.2-0.4) were used for transformation experiments to infect calli of Ammi majus and embryogenic calli of Pearl millet respectively. Incase of antibiotic wash, Cefotaxime 500 mg L-1 was used for LBA4404 infected Ammi majus calli and Timentin 300 mg L-1 was used for EHA105 infected embryogenic calli of Pearl millet.
Effects of Agrobacterium infection, antibiotic and NaOCl washes on Agrobacterium removal and both explants physiological changes during transformation experimental procedures were studied. At the end of the experiments explants survival efficiency of Ammi majus and pearl millet were 8% and 5% respectively. Biotic and abiotic stress factors responsible for lower efficiency were investigated with various other factors and strategies were discussed which are need to be considered for higher transformation events and target tissue survival.
Staphylococcus aureus is a most important bacterium that causes nosocomial infections and the etiologic
agent of a wide range of diseases related with major mortality and morbidity. Total 36 positive clinical
samples viz. urine, blood and pus collected from different patients were found to harbor Staphylococcus
aureus with a maximum isolation from pus samples i.e. 30 (83.33%) and minimum isolation from urine
samples i.e. 2 (5.56%). The degree of capacity to biofilm forming Staphylococcus aureus isolates to
different phenotypic analysis of biofilm formation by Congo red agar (CRA) Nil, Tube method (TM) 5
(13.89%), and Tissue culture plate method (TCP) 12 (33.33%). The application of silver and Selenium
nanoparticles as antimicrobials are gaining relevance in the medical field. Silver nanoparticles, due to
their unique properties, use in day-by-day many applications in human life. The major uses of silver
nanoparticles in the clinical and medical fields consist of investigative applications and curative
applications. Selenium metal is an essential micronutrient for human beings and animals. Selenium
nanoparticles showed the highest bactericidal and antimicrobial properties. Minimal biofilm eradication
concentrations (MBEC) were determined by 96-well microtitre plate. The antibacterial effects of silver and
selenium nanoparticles were evaluated with respect to growth, biofilm formation of Staphylococcus aureus
strains. Among the three biofilm forming Staphylococcus aureus strains showed OD450 i.e. 0.019, 0.039,
0.075 value ≤0.080 for AgNPs whereas SeNPs couldn’t showed any ≤ 0.080 value against biofilm forming
S. aureus strains. In case of MBEC test, AgNPs showed more affective against biofilm forming
Staphylococcus aureus strains compared of SeNPs. Finally we suggested that AgNPs showing best
antimicrobial activity against SeNPs.
Virulence Phenotype, Physicochemical Properties and Biofilm Formation of Pseu...IJERA Editor
Potable water piping has been demonstrated to serve as a reservoir for opportunistic pathogens bacteria such as Pseudomonas aeruginosa. In this report, we describe the characterization of P. aeruginosa strains isolated from water intended for human consumption by the presence of virulence factors. These strains expressed their suitability for adhesion and the formation of biofilms on polyethylene (PE). Also In this work, we were able to elucidate the factors intervening in adhesion and biofilm formation by showing the role of the substrate, the environment and bacteria. Strong correlation was observed between physicochemical properties especially the electron donor property and the surface percentage covered by cells. These results indicate that this property plays a crucial role in Pseudomonas aeruginosa adherence on the PE surface. In addition, if no relationship was found between the adhesion results and hydrophobicity, it means that this property was not involved in the adhesion process of Pseudomonas aeruginosa on the PE surface.
Evaluation of resistance profile of pseudomonas aeruginosa with reference to ...iosrjce
IOSR Journal of Dental and Medical Sciences is one of the speciality Journal in Dental Science and Medical Science published by International Organization of Scientific Research (IOSR). The Journal publishes papers of the highest scientific merit and widest possible scope work in all areas related to medical and dental science. The Journal welcome review articles, leading medical and clinical research articles, technical notes, case reports and others.
The effect of silver nanoparticles on Staphylococcus epidermidis biofilm biom...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Bacterial biofilm has been considered responsible for many deaths and high health costs worldwide. Their better protection against antibacterial agents compared to free living cells leads to poor treatment efficiency. Nanotechnology is promising approach to combat biofilm infections. The aim of the present study was to eradicate Staphylococcus epidermidis biofilm with silver nanoparticles (SNPs).
Materials and Methods:
SNPs were used at different concentrations (two fold dilutions) and incubation times (24, 48, 72 h). The crystal violet staining and pour plate assays were used to assess biofilm biomass and bacterial viability, respectively. The ability of SNPs on biofilm matrix eradication was assessed through optical density ratio (ODr). Positive control was defined as an ODr =1.0.
Results:
The crystal violet assay indicated that the biofilm matrixes were intact at different concentrations of SNOs and incubation times. There were no significant differences between these parameters (P >0.05). Bacterial enumeration studies revealed that higher concentrations of SNPs were more effective in killing bacteria than lower ones. Although, longer incubation times led to enhancement of anti-biofilm activity of SNPs.
Conclusion:
The anti-biofilm activity of SNPs was concentration- and time-dependent. The results of this study highlighted that SNPs were effective against cell viability; however they were ineffective against biomass.
EWMA Conference-Ep442-MODELLING WOUND BIOFILMS IN A THERMO-REVERSIBLE MATRIX ...EWMA
Benjamin Taylor1, 2, David Williams1 & Jon Nosworthy3.
1Cardiff University School of Dentistry
2Knowledge Transfer Partnership Associate
3Advanced Medical Solutions plc
EWMA Conference-Ep442-MODELLING WOUND BIOFILMS IN A THERMO-REVERSIBLE MATRIX ...EWMAConference
MODELLING WOUND BIOFILMS IN A THERMO-REVERSIBLE MATRIX WITH FLORESCENT MARKERS
Benjamin Taylor1, David Williams2, Jon Nosworthy3
1Cardiff University/Advanced Medical Solutions (Cardiff/Winsford, United Kingdom);
2Cardiff University (Cardiff, United Kingdom);
3Advanced Medical Solutions (Winsford, United Kingdom).
1. Introduction
Salmonella spp. are enteric pathogens notable for their ability to
cause a range of diseases including gastroenteritis, septicaemia,
osteomyelitis, pneumonia, meningitis, and arthritis1. The food
borne pathogen Salmonella enterica and various members of
the familyEnterobacteriaceae areabletoformbiofilmondifferent
biotic and abiotic surfaces2-3. A bioûlm is a group of
microorganisms that attach to each other and to a biotic or abiotic
surface, resulting in stability and protection from environmental
factors mediated in part by a self-initiated exopolysaccharide
(EPS) matrix4. Biofilms can become a persistent source of
contamination5 with increased ability to colonize and survive in
a harsh condition6. The formation of biofilms involve multiple
processes including initial surface attachment, monolayer
formation, migration to form multilayered microcolonies,
production of extracellular matrix and biofilm maturation with a
three dimensional architecture7. Asmall number of bacterial cells
adhere to the surface, a process facilitated by bacterial motility.
Cells that attach irreversibly to the surface divide, forming
microcolonies, that produce extracellular polymeric substances
(EPS), primarily polysaccharides8. The EPS attaches the cells to
the surface and stabilizes the colonies. With time, attached
Original Article
Bangladesh J Microbiol, Volume 31, Number 1&2, June-December 2014, pp 35-39
bacteria from the biofilm detach and disperse in order to survive
and colonize new niches9.
Understanding the formation of biofilms is important for their
control. Biofilms are less susceptible to antimicrobials than are
planktonic cells10. Bacteria within a biofilm are more resistant to
environmental factors because of limited availability of key
nutrients5,11 andowingtotheextracellularmatrix8.Withinabiofilm
there is reduced diffusion, physiological changes due to reduced
growth rates and production of enzymes which degrade
antimicrobial substances8 leading to increased resistance. Biofilms
are a concern in the food industry as they can lead to illness,
disease outbreaks with subsequent economic losses12. In the
medical field, bacterial biofilms are worrying concerns because
they can occur on the surfaces of medical devices and on tissue
surfaces within compromised organs13. Biofilms grow similarly in
the environment and in industrial systems14. In the present study,
the ability of clinical isolates of Salmonella enterica serovarTyphi
and Paratyphi to form biofilm in vitro was investigated. To our
knowledgethisconstitutesthefirstworld-widereportofthebiofilm
forming ability by clinical Salmonella enterica serovar Paratyphi.
Previous reports on biofilm-forming S. enterica serovar Paratyphi
were based on reference bacteria rather than on clinical isolates.
In vitro Biofilm FormationAbility of Clinical Isolates of Salmonella enterica
SerovarsTyphi and Paratyphi
MaishaMalihaMorium1
, SunjuktaAhsan1*
, M Shahidul Kabir2
, Marufa ZerinAkhter1
andMFaridulIslam3
1Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh, 2Department of Microbiology, Stamford University Bangladesh, Dhaka
1217, Bangladesh, 3Department of Microbiology, Square Hospital, Panthapath, Dhaka 1205, Bangladesh
In the present study the ability of clinical isolates of Salmonella enterica serovars Typhi (n = 30) and Paratyphi
A (n = 11) to form biofilm on abiotic surface was investigated. All isolates were found capable of biofilm
formation in a microtitre plate assay. Upon optimization of biofilm formation by the test isolates, Adherence
test medium (ATM) was found to be the best medium for biofilm formation by both S. enterica serovars Typhi
and Paratyphi. Growth was optimized by incubation at 37°C in an orbital shaker set at 150 rpm for 48-72
hours. Biofilms were best detected when washed with PBS (1X), stained with crystal violet (1%) and
subsequently washed with acetone (33%). Optical density (OD) readings were better correlated with growth
at 570 nm when compared to 600 nm. Of the 28 Salmonella Typhi isolates, 17 (61%) were very strong biofilm
producers, 8 (29%) were strong biofilm producers and 3 (11%) were moderate biofilm producers. On the other
hand, out of 13 S. Paratyphi, 9 (69%) were very strong biofilm producers, 3 (23%) were strong biofilm formers
and 1 (8%) was a moderate biofilm producer. None of them were weak biofilm producers. The present study
raises concern from a public health point of view because the ability of the clinical isolates to form biofilm
would indicate their ability of being transmitted from abiotic surface to uninfected host giving rise to
disease.
Keywords: Biofilm, Salmonella enterica Typhi, Paratyphi, Planktonic
*Corresponding author:
Sunjukta Ahsan, Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh.
Tel: +880 (02) 9661920-73; E-mail: sunjukta@du.ac.bd
2. MaterialsandMethods
Bacterial isolates
Twenty-eight Salmonella enteric serovar Typhi and 13 S. enterica
serovar Paratyphi clinical isolates obtained from a hospital in
Dhaka City, Bangladesh were used in this study. The isolates
were confirmed by biochemical and serological tests.
Optimization of incubation conditions for maximum biofilm
formation
(i) Media optimization
In spite of the fact that one earlier report has suggested the use
of adherence test medium (ATM) for Salmonella enterica serovar
Typhi15 a number of media were tested for their ability to support
maximum biofilm formation by the test strains. This was deemed
necessary as strain variations may cause differences in
requirement. The media used in this study included tryptic soy
broth (TSB), Mueller-Hinton broth (MHB), Luria-Bertani broth
(LB), Luria-Bertani broth with 5 g/l D-glucose (LB + glucose) and
adherence test media (ATM).
(ii) Incubation condition optimization
In all conditions of optimization, incubation temperature of 37°C
was used. Both static and shaker incubators were used. In shaker,
two different shaking conditions, viz., 120 and 150 rpm were used.
Time of incubation was also varied and biofilm production was
observed after 24, 48 and 72 h incubation.
(iii) Biofilm staining and washing condition optimization
Non-adherentcellswerewashedwithtwodifferentwashingsolution,
viz., double distilled H2O and 1X PBS (phosphate buffer saline).
Biofilmwasstainedwithcrystalvioletattwodifferentconcentrations
of0.03and1%for30min.Todetachbiofilmfromwallofthetube,two
differentsolutionswereused,viz.,80:20=ethanol:acetoneand33%
aceticacid,whichwereappliedfor15min.
(iv) Wave length and absorbance optimization
In case of 80:20 = ethanol:acetone solution absorbance was taken
at 600 nm. In case of 33% acetic acid solution absorbance took at
570 nm.Absorbance was obtained after 24, 48 and 72 h incubation
in ELISAplate reader.
Bacteriological medium
Adherence test medium (ATM) with slight modifications was
adapted following optimization of biofilm formation and medium
was prepared as described earlier15. This medium contained 60
mM NaCl,20mMKCl,111mMglucoseand30mM NaHCO3.The
pH was adjusted to 8.4. To this a supplement containing NH4Cl
(20 mM), Na2HPO4 (40 mM), (NH4)H2PO4 (50 mM), CaCl2 (999
µM), MgCl2 (980 µM), FeCl3 (86 µM) and Na2SO4 (40 mM) was
added separately.
Biofilm assay
The ability of the bacterial strains to form bioûlms in polystyrene
(PS) microtitre plates was evaluated by using the method
described elsewhere16 with some modifications. Colonies from
an overnight plate was grown in adherence test medium (ATM) at
37°Cinanorbitalshaker(120rpm)toobtainaMacFarlandstandard
of 0.5 equivalents of cells. From this, 200 µl of bacterial suspension
was transferred into a well of a sterile 96-well PS microtitre plate
and the plate was then incubated for 48 hours at 37°C, 120 rpm to
allowbiofilmformation.Planktonicbacteriawereremovedandeach
well was washed thrice with phosphate buffered saline (dissolving
8gofNaCl,0.2gofKCl,1.44gofNa2HPO4,0.24gofKH2PO4 in1
l distilled H2O and pH adjusted to 7.4) to remove loosely attached
cells. Subsequently, 200 µl of 1% (wt/vol) crystal violet solution
(Sigma-Aldrich/ Life Science Chemilab SA,Athens, Greece) was
added into each well, and the plate was then incubated for 30 min
at room temperature.After being washed three times with 200 µl of
PBS to remove excess stain, the crystal violet was solubilised in
200 µl of 33% acetate solution. Dye absorbance at 570 nm was
measured using a microtitre plate reader (Sunrise, Tecan,
Männedorf, Switzerland). Each experiment (isolate) was done in
triplicate wells. Salmonella enteric serovar Typhimurium ATCC
14028 was used as a positive control for biofilm formation and
sterileATM were used as negative controls. When the absorbance
values of the crystal violet bound to the sample bacteria was shown
at least twice that of the control it was considered as positive
result for bioûlm formation. Based on the binding of crystal violet
by the biofilm forming bacteria the isolates were categorized as
very strong (VS) biofilm producers, strong (S) biofilm producers
and moderate (M) biofilm producers using a modified convention
described earlier17.
Results
Optimized incubation conditions for biofilm formation
Following optimization, best biofilm production was obtained in
ATM medium at 37°C in 150 rpm shaker after an incubation time
of 48 h.Among the two washing solutions used, 1X PBS was the
better washing solution. For staining of biofilm, 1% crystal violet
worked better than the lower concentration of 0.03% when used
for 30 min. As detachment solution, 33% acetic acid solution
worked better than ethanol:acetone = 80:20. Absorbance was
taken at 570 nm after 48 h of incubation. Figure 1 depicts the
OD600 values when using the optimized conditions for biofilm
formation. The results are mean of four readings and are expressed
as mean ± 1 standard deviation.
Biofilm formation by the S. enterica Typhi and Paratyphi isolates
All isolates of S. enterica Typhi and Paratyphi were tested in
triplicate were found to be capable of forming biofilm to different
extents. The degree of crystal violet retention is an indication of
the number of bacteria in the biofilm which bind to crystal violet.
Hence, the absorbance at 570 nm correlates positively with the
number of biofilm producer. The cut-off OD was taken as two
standard deviations above the mean value of negative control.
Figure 2 represents the mean OD values of the crystal violet
retained by the Salmonella Typhi isolates and the controls and
Figure 3 depicts that retained by S. enterica Paratyphi.
Morium et. al
36
3. 0
0.5
1
1.5
2
2.5
3
ODat570nm
Figure 1. Biofilm formation under optimized condition (ATM
medium incubated at 37°C in an orbital shaker adjusted to 150
rpm and incubated for 48-72 hours. Biofilms were washed with
PBS, 1X, stained with crystal violet, 1% and washed with
acetone, 33%). All experiments were carried out four times.
Results are mean OD570 ± SD.
Figure 2. Absorbance of crystal violet OD570 by clinical isolates
of Salmonella enterica serovar Typhi biofilms in modified ATM.
Results are mean of three readings ± 2 SD.
Figure 3. Absorbance of crystal violet OD570 by clinical isolates of Salmonella enterica serovar Paratyphi biofilms in modified
ATM. Results are mean of three readings ± 2 SD.
Categorization of the isolates on the basis of strength of biofilm
formation
The isolates were classified as follows: non-producing, weak,
moderate, and strong-producing, based on the following optical
density (OD) average values: OD (Isolate) d” OD (Control) =
Non-biofilm-producer; OD (Control) d” OD (Isolate) d” 2 OD
(Control)=Weakbiofilmproducer;2OD(Control)d”OD(Isolate)
d” 4 OD (Control) = Moderate biofilm producer; 4 OD (Control)
d” OD (Isolate) = Strong biofilm producer. We modified these
interpretive criteria by adding yet another category, which we
described as 8 OD (Control) d” OD (Isolate) =Very strong biofilm
producer. In this case, the category ‘strong biofilm producer’
wasdescribedas4OD(Control)d”OD(Isolate)d”8OD(Control)
= Strong biofilm producer. According to this categorization, of
the 28 S. enterica Typhi isolates, 17 (61%) were very strong (VS)
biofilm producers, 8 (29%) were strong (S) biofilm producers and
3 (11%) were moderate (M) biofilm producers (Table 1). On the
other hand, 9 (69%) of the S. enterica Paratyphi were very strong
(VS) biofilm producers, 3 (23%) were strong (S) biofilm formers
and 1 (8%) was a moderate (M) biofilm producer.
Biofilm Formation by Clinical Salmonella
37
4. Microscopy of a sample biofilm
A sample of the biofilm was observed under the light microscope
(100x) following crystal violet staining. The micrograph indicated
clumps of biofilm bacteria (Figure 4).As negative control, blank
medium was used instead of bacterial culture. The micrograph
which reflected a blank view has not been shown here.
formation for both S. enterica Typhi and Paratyphi was observed
inATM medium at 37°C under vigorous aeration (150 rpm). This
finding is similar to that of Raza et al.15 who modified ATM
medium slightly for optimum biofilm formation by S. enterica
Typhi only.
There are several reports that describe the ability of Salmonella
to form biofilms on abiotic surfaces outside the host, such as
stainless steel19, plastic20, rubber21, glass2, cement22, marble and
granite14.All these surfaces are commonly encountered in farms,
slaughter houses, food industries and kitchens which raise the
risk for public health. It is strongly believed that the ability of
Salmonella to form biofilms on inanimate surfaces contributes
to its survival and persistence in non-host environments and its
transmission to new hosts. However, adhesion by Salmonella is
strain-dependent and probably influenced by surface structures,
such as cell wall and membrane proteins, fimbriae, flagella and
polysaccharides19,23-24 has reported on the ability of reference
S. enterica Typhi and Paratyphi isolates to form biofilm in
microtitre plate.
Conclusion
The significance of the present study lies in the fact that all
isolates were clinical in origin and most of them were very strong
biofilm producers. This raises the chance of formation of biofilms
by clinical Salmonella enterica serovars Typhi and Paratyphi on
abiotic surfaces, a condition which is of public health significance
since attached bacteria on commonly used plastic surfaces can
aid transmission to uninfected hosts and gives rise to disease.
Persistence of such biofilm bacteria on abiotic surfaces may form
the basis of future studies.
References
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Choleraesuis: Epidemiology, pathogenesis, clinical disease, and
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2. Prouty A and Gunn J. 2003. Comparative analysis of Salmonella
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Table 1. Categorization of the Salmonella enterica Typhi and S. Paratyphi isolates in terms of biofilm forming ability
Isolate Biofilm category OD control/OD sample
Salmonella Typhi isolates (n = 28)
S. Typhimurium VS 8 OD (Control) d” OD (Isolate)
S8,13,14,24,25,27,30,38,40,42,51,52,54,55,56,57,58 VS 8 OD (Control) d” OD (Isolate)
S4, 9, 20, 32, 34, 35, 45 and 49 S 4 OD (Control) d” OD (Isolate) d” 8 OD (Control)
S2,22,23 M 2 OD (Control) d” OD (Isolate) d” 4 OD (control)
Salmonella Paratyphi isolates (n = 13)
S1,10,12,26,28,29,36,37,50 VS 8 OD (Control) d” OD (Isolate)
S11,18,21 S 4 OD (Control) d” OD (Isolate) d” 8 OD (Control)
S19 M 2 OD (Control) d” OD (Isolate) d” 4 OD (Control)
VS = Very strong; S = Strong; M = Moderate.
Discussion
Salmonellae are recognized worldwide as major zoonotic
pathogens for both humans and animals. Most microorganisms
persist in a biofilm ecosystem and not as free-floating organisms.
The ability of Salmonella to attach to food surfaces was the first
published report on food-borne bacterial biofilms18.
In the present study, all investigated S. enterica Typhi and
Paratyphi isolates were found to be capable of forming biofilms.
Different culture media, incubation conditions solutions were
used for biofilm washing and detachment. The best biofilm
Figure 4. Light micrograph of a representative biofilm forming
Salmonella enterica serovar Typhi.
Morium et. al
38
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