Identification of natural compounds which inhibit biofilm formation in clinical isolates of Klebsiella pneumoniae

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Abstract
Klebsiella pneumoniae, an important opportunistic pathogen, exists as a biofilm in persistent infections and in-dwelling medical devices. With the objective of identifying natural compounds inhibiting biofilm formation in K. pneumoniae, 35 clinical isolates were screened,out of which 7 strong biofilm producers were identified. Six natural compounds were tested for their inhibitory effects on bacterial growth and biofilm formation by determining the minimum inhibitory concentration and minimum concentration for biofilm inhibition (MBIC) for each compound. The results show that reserpine followed by linoleic acid, were the most potent biofilm inhibitors. Reserpine, an efflux pump inhibitor was effective at biofilm inhibition at a concentration of 0.0156 mg/mL, 64-fold lower concentration than its MIC. Linoleic acid, an essential fatty acid was effective as a biofilm inhibitor at 0.0312 mg/mL, which is 32-fold lower than its MIC. Berberine, another plant derived antimicrobial, chitosan and eugenol had an MBIC value of 0.0635 mg/mL. Curcumin, a natural phenolic compound was effective at biofilm inhibition at a concentration of 0.25 mg/mL, which is 50 fold less than its MIC. Notably, the MIC and MBIC data on these 6 natural compounds was reproducible in all seven high biofilm forming isolates of K. pneumoniae. The present report is a comprehensive comparative analysis of the dose dependent inhibition of various natural compounds on biofilm formation in K. pneumoniae.
Indian Journal of Experimental Biology
Vol. 51, September 2013, pp. 764-772
Identification of natural compounds which inhibit biofilm formation in clinical
isolates of Klebsiella pneumoniae
H Magesh
a
, Arun Kumar
a
, Ayesha Alam
a
, Priyam
a
, Uma Sekar
b
, Venil N Sumantran
c
& Rama Vaidyanathan
a*
a
Department of Biotechnology, Dr. M.G.R.Educational and Research Institute,
E.V.R. Periyar Salai, Maduravoyal, Chennai 600 095, India
b
Department of Microbiology, Sri Ramachandra University, Porur, Chennai 600 116, India
c
Bhupat & Jyothi Mehta School of Biosciences, Indian Institute of Technology, Chennai 600 036, India
Received 14 November 2012; revised 18 June 2013
Klebsiella pneumoniae, an important opportunistic pathogen, exists as a biofilm in persistent infections and in-dwelling
medical devices. With the objective of identifying natural compounds inhibiting biofilm formation in K. pneumoniae,
35clinical isolates were screened,out of which 7 strong biofilm producers were identified. Six natural compounds were
tested for their inhibitory effects on bacterial growth and biofilm formation by determining the minimum inhibitory
concentration and minimum concentration for biofilm inhibition (MBIC) for each compound. The results show that
reserpine followed by linoleic acid, were the most potent biofilm inhibitors. Reserpine, an efflux pump inhibitor was
effective at biofilm inhibition at a concentration of 0.0156 mg/mL, 64-fold lower concentration than its MIC. Linoleic acid,
an essential fatty acid was effective as a biofilm inhibitor at 0.0312 mg/mL, which is 32-fold lower than its MIC. Berberine,
another plant derived antimicrobial, chitosan and eugenol had an MBIC value of 0.0635 mg/mL. Curcumin, a natural
phenolic compound was effective at biofilm inhibition at a concentration of 0.25 mg/mL, which is 50 fold less than its MIC.
Notably, the MIC and MBIC data on these 6 natural compounds was reproducible in all seven high biofilm forming isolates
of K. pneumoniae. The present report is a comprehensive comparative analysis of the dose dependent inhibition of various
natural compounds on biofilm formation in K. pneumoniae.
Keywords: Biofilm inhibition, Efflux pumps inhibitors, Klebsiella pneumoniae,Multi-drug resistance,
Minimum inhibitory concentration
Persistent infections are a global challenge for human
beings, claiming millions of lives every year and
demanding huge medical and social resources. One
common survival strategy employed by bacteria
pathogens is to form a biofilm, an amorphous and
dynamic structure that is not only resistant to
antibiotics, but also resistant to host immune
clearance
1
. Biofilm formation is a two-stage biological
process controlled by surface adhesins and cell-to-cell
communication pathways. Aggregated bacterial cells
protected and/or coated by extracellular matrix, are
insensitive to both nutritional stimulation and hostile
attacks. In the human body, biofilms may trigger
persistent infections with chronic inflammation.
Klebsiella pneumoniae is an important
opportunistic pathogen and is a common cause of
urinary tract infections, respiratory tract infections,
and septicemia, especially in immuno-compromised
individuals
2
. Factors that are implicated in the
virulence of K. pneumoniae strains include the capsular
serotype, lipopolysaccharide, iron-scavenging systems,
fimbrial and non-fimbrial adhesions. The ability of
bacteria to form a biofilm on host tissue surfaces is an
important step in the development of infection.
As a correlation between biofilm formation and
bacterial persistence has been established
3
, the
possibility of using drugs targeting biofilm formation
in combination with the current antibiotics is
emerging as a potential therapeutic approach for this
type of bacterial persistent infection. With an increase
in reports of multi drug resistant infections, and the
importance of biofilm formation resulting in
decreasing susceptibility to antibiotics, there is an
urgency to discover molecules targeting the inhibition
of biofilm formation of bacteria.The objective of the
present study was to identify natural compounds for
their potential to inhibit biofilm formation. Natural
products chitosan, eugenol, curcumin and linoleic acid
_________
*
Correspondent author
Telephone: 91 44 23782176
Fax: 91 44 2378 3165
E-mail: ramavaidy@gmail.com
MAGESH et al.: BIOFILM INHIBITION ACTIVITY OF NATURAL COMPOUNDS
765
which are known antibacterials
4-7
and efflux pump
inhibitors reserpine and berberine
8
were analyzed for
their potential to suppress biofilm formation in high
biofilm forming strains of K. pneumoniae. For each
compound, we quantitatively compared the potencies
for bacterial growth inhibition versus biofilm
inhibition. Such data would enable selection of natural
compounds which could synergize with established
anti-microbials, and increase their therapeutic efficacy.
With the objective of identifying natural
compounds that have biofilm inhibition activity, high
biofilm producing clinical isolates of multi drug
resistant (MDR) K. pneumoniae were first screened
and subsequently the effects of the 6 test compounds
were analyzed on biofilm formation. The analysis
showed a dose dependent biofilm inhibitory activity
for each of the 6 test compounds. The results from
this analysis will be useful in designing combinatorial
treatment of antibiotics and biofilm inhibitors.
Materials and Methods
Preparation of natural compoundsThe following
natural compounds were dissolved at a concentration
of 10 mg/mL in dimethylsulfoxide (DMSO,
Loba-Chemie, Pvt. Ltd. Mumbai, India), reserpine
(Sigma-Aldrich Co. LLC., USA), berberine
(Sigma-Aldrich Co. LLC., USA), ciprofloxacin
(Sigma-Aldrich Co. LLC., USA), eugenol (HiMedia
Laboratories Pvt. Ltd., Mumbai, India), linoleic acid
(Sisco Research Laboratories Mumbai Pvt. Ltd.),
chitosan (Axiogen Pvt. Ltd., India) and curcumin
(M/S. Agrihub Pvt. Ltd., Tuticorin, India).
Bacterial strains and antimicrobial sensitivity
testClinical isolates (35) of multidrug resistant
K. pneumoniae were collected from a tertiary care
hospital in Chennai during November 2009-February
2010. The isolates were numbered from 1 - 35. Based
on the source of the isolate, a prefix U for urine, B for
blood and S for sputum was given. The isolates were
tested for their antimicrobial sensitivity to ciprofloxacin
(5 µg disk
-1
, HiMedia Laboratories Pvt. Ltd., Mumbai,
India), cefotaxime (30 µg disk
-1
, HiMedia Laboratories
Pvt. Ltd., Mumbai, India), and amoxyclav (30 µg disk
-1
,
HiMedia Laboratories Pvt. Ltd., Mumbai, India) using
recommended guidelines. The isolates were scored for
their antimicrobial resistance according to the CLSI
guidelines
9
.
Determination of minimum inhibitory
concentration of natural compoundsThe MIC for
each of the natural compounds was determined using
the tissue culture plate method
10,11
. The bacterial
isolates were maintained in Brain heart infusion agar
(HiMedia Laboratories Pvt. Ltd. Mumbai, India) plate
and inoculated into 5 mL of Mueller Hinton broth
(HiMedia Laboratories Pvt. Ltd. Mumbai, India) and
incubated for 18 h at 37 °C in shaker. The overnight
culture was adjusted to 0.5 McFarland standards
[0.5 mL 1.17% (w/v) BaCl
2
× 2H
2
O + 99.5 mL 1%
(w/v) H
2
SO
4
]. Overnight culture (10 µL, corresponding
to 0.5× 10
6
cfu) was added to 100 µL of Mueller Hinton
broth and incubated overnight.
To the 96-well micro titer plate 150 µL of sterile
Mueller Hinton broth (HiMedia Laboratories Pvt. Ltd.
Mumbai, India) was added and two fold serial
dilutions of the natural compounds were made starting
with the first well by adding 50µL of the test
compound dissolved at a concentration of 4 mg/mL.
To each of the wells 10µL of the diluted culture
(0.5 McFarland standard) was added. This resulted in
the final concentration of the compound ranging from
2 mg/mL in the first well to 0.0078 mg/mL in the
9
th
well. In curcumin, higher concentrations were used
to determine its MIC. The tissue culture plate was
then incubated at 37 °C in stationary condition for
overnight. The growth of the bacterial culture was
measured at a wavelength of 595 nm with Bio-Rad
Model iMark Micro plate Absorbance Reader.
Twotypes of negative controls and one positive
controlwere used in each assay. The Vehicle control’
contained the solvent used for dissolving the test
compounds (10µL DMSO) and 100 µL of media in
10
th
well. The ‘Media control’ lacked bacteria and
plant compounds, and only contained media (100 µL
of MHB broth in the 11
th
well).The “untreated control”
lacked plant compounds, but contained growing
bacteria (positive control:10 µL bacterial culture and
100 µL of sterile MHB media in 12
th
well).
The MIC was defined as the minimum
concentration of the extract that did not allow any
visible growth or turbidity of the organism in broth.
MIC
90
refers to concentration of the test compound
required to prevent the growth of 90% of organisms
tested. For each compound the MIC was tested for the
nine isolates in duplicates. The concentration at which
all the isolates failed to grow is taken as MIC.
Screening of K. pneumoniae for biofilm formation
In this method, test strains were cultured on fresh brain
heart infusion agar (BHI) plate and inoculated in sterile
brain heart infusion broth and incubated overnight at
37 °C without shaking. The overnight culture was
diluted to 0.5 McFarland standard in fresh BHI medium.
INDIAN J EXP BIOL, SEPTEMBER 2013
766
The modified Tissue Culture Plate (TCP) method
was used for screening biofilm formation in
K. pneumoniae isolates
12,13
. In the TCP method, an
overnight culture of each isolate was adjusted to a
McFarland standard of 0.5. An aliquot of 10 µL of the
culture was added to 100 µL of the fresh BHI broth
and incubated overnight. After 24 h, the planktonic
cells were aspirated, and wells were washed with
phosphate buffer saline (PBS, pH 7.2) to remove free
floating bacteria. Biofilms which adhered to the wells
were fixed with 2% sodium acetate and stained with
0.1% crystal violet (0.1% w/v, aqueous solution,
HiMedia Laboratories Pvt. Ltd., Mumbai, India).
Excess stain was washed with deionized water and
plates were dried. The absorbance of stained adherent
bacteria (dried polysaccharides) were determined by
Bio-Rad Model iMark Micro plate Absorbance Reader
at 595 nm. To compensate for background absorbance,
OD values from sterile medium well were averaged
and subtracted from all test values. The experiment was
repeated twice. Each isolate was analyzed in triplicate.
Thirty five clinical isolates and a control strain
MTCC432 was screened by the TCP assay.
Quantitation of biofilm dataBiofilm forming
potential of all 35 test cultures could be quantitatively
compared as the incubation was started with the same
cell number for each of isolate. Further, free forming
(planktonic) cells did not contribute to biofilm formation
since they were removed at the start of the experiment.
Therefore, varying amounts of biofilm formation by
various isolates could be quantitated by comparing OD
values of stained adherent cells. Isolates which gave an
OD<0.120 were classified as non-adherent and weak
biofilm producers; O.D. values of 0.120 to 0.240 were
classified as moderately adherent and moderate biofilm
producers; O.D. value of > 0.240 was classified as
strongly adherent and high biofilm producers.
Microscopic determination of biofilm formation
The test strain was cultured in brain heart infusion
broth. A sterile glass slide was kept in a sterile
petriplate and overlaid with 20 mLof test strain
inoculated in Brain Heart Infusion broth. After 24 and
48h of incubation, the slide was taken out aseptically
and washed with phosphate buffer saline (pH 7.2) to
remove free floating planktonic bacteria. The biofilm
was fixed with 2% sodium acetate and stained with
0.1% crystal violet stain, washed and air dried. The
slide was examined under Trinocular microscope at
100x oil immersion. Photomicrographs of adhered
bacterial biofilms were recorded.
Biofilm inhibition assayOnly those isolates of
K. pneumoniae which were classified as strong
biofilm producers were used. Test compounds were
dissolved in DMSO (10 mg/mL), and two fold
dilutions were made to result in a final concentration
ranging from 2-0.0078 mg/mL in the wells after the
addition of the freshly diluted brain heart infusion
broth culture containing 10
6
cfu of the strong biofilm
forming isolate per well. After incubation for 24 h at
37 ºC, the tissue culture plate was washed, fixed and
biofilms were stained and visualized as outlined
above. The inhibitory effect of the plant compound on
biofilm production was calculated by subtracting the
media control.The MBIC is the concentration of the
natural compound at which the biofilm formation was
reduced to an Absorbance 595<0.12 OD. Each assay
for MBIC determination was performed in triplicate.
Statistical analysisStatistical analyses were
performed with MS-Excel 2010. Data are shown as
mean ± SD unless otherwise stated (Figs 1 and 3). For
each bacteria, the biofilm formation assay was
performed in triplicates and the mean OD was taken
for the analysis. The data from a total of 35 bacteria
were considered for the test. The biofilm formation of
the bacteria was significantly different at OD>0.240
and the level of significance were tested by Sign test
as the data were not normally distributed
(Shapiro–Wilks statistics). The null hypothesis
H0: <0.240 against the alternative H1:=> 0.25 was
tested. Statistical significance was set at P< 0.05.
Results
Antimicrobial sensitivity testThe bacterial
isolates collected were highly resistant to antibiotics.
Out of 35 isolates, 31 were resistant to cefotaxime, a
third generation cephalosporin antibiotic, whereas
24 isolates were resistant to ciprofloxacin. All the
isolates were resistant to amoxyclav, a combination of
amoxicillin and clavulanic acid. In this study, we
focused on screening the biofilm forming potential of
all the 35 MDR isolates.
Classification of K. pneumoniae based on biofilm
formationThe 35MDR isolates of K. pneumoniae
were analysed for biofilm formation using the TCP
method and the results are shown in Fig. 1. Isolates
were classified by their biofilm forming potential.
Seven isolates were strong biofilm producers, 13 were
moderate producers, and 15 were poor biofilm
producers. K. pneumoniaeMTCC 423 which was not
part of this set of hospital isolates was included as an
MAGESH et al.: BIOFILM INHIBITION ACTIVITY OF NATURAL COMPOUNDS
767
internal assay control and found it to be a moderate
biofilm producer. Statistical analysis showed that the
seven strong isolates produced significantly greater
amounts of biofilms relative to the moderate and poor
biofilm producers. Based on intensity of biofilm
staining, these seven isolates of K. pneumoniae were
classified as strong biofilm producers. The magnitude
of biofilm formation in these 7 strains was
significantly greater than that produced by the poor
(biofilm producers (P=0.0019) (Fig. 1). Among these
seven strong biofilm producers, three (B15, B16 and
B24) were isolated from blood samples; two from
sputum samples (S13 and S20) and two were from
urinary samples (U5 and U25). The highest biofilm
producers were observed in the two urinary isolates
U5 and U25, which gave an OD of 0.47 and
0.5 respectively. Biofilm formation in the strong
biofilm producing strains was 4.5 fold greater than that
of the poor biofilm producers. The Fig. 1 also provides
a quantitative method for identifying and demarcating
biofilm producing versus non-biofilm producing
strains. Thus, biofilm producing strains when assayed
by the modified TCP method gave an OD value of
>0.120, whereas non-biofilm producing strains gave an
OD value of <0.120 by this assay.
Photomicroscopic analysis of the biofilm
producersTo visualize biofilm formation, one
representative isolate from the categories of strong,
moderate, and non-biofilm producers was analyzed by
the microscopic slide assay (Fig. 2). The biofilm
formation was clearly visible for the strong biofilm
producer U25, followed by the moderate biofilm
producer U6 at 24 h (Fig. 2A) and 48 h (Fig. 2B). The
strong biofilm producer (isolate U5) also showed
strong adherence to the slide (data not shown). The
non-biofilm producers, the isolate U23 did not show
biofilm formation even after 48 h. One of the
strongest biofilm producers, isolate U5, was chosen
for further studies for screening biofilm inhibitors.
Determination of minimum inhibitory
concentration of natural compoundsTo confirm the
antibacterial activity of the 6 natural compounds
(curcumin, eugenol, linoleic acid, chitosan, reserpine
and berberine), their minimal inhibitory concentration
was determined in 9 MDR isolates of K. pneumoniae
including the 7 high biofilm forming isolates. Table 1
shows the MIC of each test compound for 9 MDR
isolates. Eugenol was found to be the most potent
antimicrobial compound, followed by linoleic acid,
chitosan, reserpine, berberine and curcumin.
Fig. 1Biofilm formation of 35 MDR K. pneumoniae determined by using the Tissue Culture Plate assay after incubation for 24 h
at
37 ºC. Grid lines drawn at 0.120 OD and 0.240 OD demarcate the strong, moderate and non-
biofilm formers. Those isolates with
OD >0.240 are considered s
trong, biofilm formers; 0.120 to 0.240 OD medium biofilm formers, and <0.120 OD non biofilm former. The
magnitude of biofilm formation by the strong biofilm formers was significantly greater than that produced by the non-biofilm producers
(P=0.0019).
INDIAN J EXP BIOL, SEPTEMBER 2013
768
Determination of minimum concentration of natural
compounds required for biofilm inhibitionThe
modified TCP assay could quantitatively differentiate
between strong, moderate, and weak biofilm producers
with statistical significance (Fig. 1). These statistically
significant results validated the biofilm assay method.
Therefore, this assay was used to identify compounds
which could significantly inhibit biofilm formed by
strong biofilm producers. The lowest concentration of a
test compound required to inhibit biofilm formation by a
strong biofilm producer to the level of a non-biofilm
producer (OD<0.12), was determined. This
concentration was referred to as minimal biofilm
inhibitory concentration (MBIC) of the test compound.
Using the strong biofilm producing isolate U5,
clear and significant dose dependent inhibition of
biofilm formation by the six different test compounds
was demonstrated (Fig. 3). Reserpine was the most
potent biofilm inhibitor, since 0.0156 mg/mL was
sufficient to inhibit the biofilm formation completely
(Fig. 3). Next, linoleic acid showed an MBIC at
0.0312 mg/mL. Chitosan, a known biofilm inhibitor,
gave an MBIC value of 0.0625 mg/mL, as did berberine.
Eugenol, an essential oil found in clove and Ocimum
plants also gave an MBIC value of 0.0625 mg/mL.
Finally, curcumin showed an MBIC at 0.25mg/mL.
Effect of the determined MBIC concentration
against all the strong biofilm producersHaving
Fig. 2Photomicrograph of glass slides with adherent bacterial films stained with Crystal violet stain observed
under Trinocular
microscopeat 100X oil immersion. S Strong (Isolate U25), M Moderate (isolate U6) and N
Non/Weak biofilm producer
(Isolate U23) after 24 hrs (Panel A) and 48 hrs (Panel B).

Supplementary resources

October 2013
H Magesh · Arun Kumar · Ayesha Alam · Priyam · Rama Vaidyanathan
October 2013
H Magesh · Arun Kumar · Ayesha Alam · Priyam · Rama Vaidyanathan
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    Efflux pumps are widely implicated in antibiotic resistance because they can extrude the majority of clinically relevant antibiotics from within cells to the extracellular environment. However, there is increasing evidence from many studies to suggest that the pumps also play a role in biofilm formation. These studies have involved investigating the effects of efflux pump gene mutagenesis and efflux pump inhibitors on biofilm formation, and measuring the levels of efflux pump gene expression in biofilms. In particular, several key pathogenic species associated with increasing multidrug resistance, such as Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, have been investigated, whilst other studies have focused on Salmonella enterica serovar Typhimurium as a model organism and problematic pathogen. Studies have shown that efflux pumps, including AcrAB-TolC of E. coli, MexAB-OprM of P. aeruginosa, AdeFGH of A. baumannii and AcrD of S. enterica, play important roles in biofilm formation. The substrates for such pumps, and whether changes in their efflux activity affect biofilm formation directly or indirectly, remain to be determined. By understanding the roles that efflux pumps play in biofilm formation, novel therapeutic strategies can be developed to inhibit their function, to help disrupt biofilms and improve the treatment of infections. This review will discuss and evaluate the evidence for the roles of efflux pumps in biofilm formation and the potential approaches to overcome the increasing problem of biofilm-based infections.
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    • Ranitaa Roy
      Ranitaa Roy
    • Monalisa Tiwari
    • Gianfranco Donelli
      Gianfranco Donelli
    • Vishvanath Tiwari
      Vishvanath Tiwari
    Biofilm refers to the complex, sessile communities of microbes found either attached to a surface or buried firmly in an extracellular matrix as aggregates. The biofilm matrix surrounding bacteria makes them tolerant to harsh conditions and resistant to antibacterial treatments. Moreover, the biofilms are responsible for causing a broad range of chronic diseases and due to the emergence of antibiotic resistance in bacteria it has really become difficult to treat them with efficacy. Furthermore, the antibiotics available till date are ineffective for treating these biofilm related infections due to their higher values of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), which may result in in-vivo toxicity. Hence, it is critically important to design or screen anti-biofilm molecules that can effectively minimize and eradicate biofilm related infections. In the present review, we have highlighted the mechanism of biofilm formation with reference to different models and various methods used for biofilm detection. A major focus has been put on various anti-biofilm molecules discovered or tested till date which may include herbal active compounds, chelating agents, peptide antibiotics, lantibiotics and synthetic chemical compounds along with their structures, mechanism of action and their respective MICs, MBCs, minimum biofilm inhibitory concentrations (MBICs) as well as the half maximal inhibitory concentration (IC50) values available in the literature so far. Different mode of action of anti biofilm molecules addressed here are inhibition via interference in the quorum sensing pathways, adhesion mechanism, disruption of extracellular DNA, protein, lipopolysaccharides, exopolysaccharides and secondary messengers involved in various signaling pathways. From this study, we conclude that the molecules considered here might be used to treat biofilm-associated infections after significant structural modifications, thereby investigating its effective delivery in the host. It should also be ensured that minimum effective concentration of these molecules must be capable of eradicating biofilm infections with maximum potency without posing any adverse side effects on the host.
  • Article
    • Sergio Gutiérrez
    • Alfredo Morán
    • Honorina Martínez-Blanco
    • Leandro B Rodríguez-Aparicio
      Leandro B Rodríguez-Aparicio
    The effect of generally recognised as safe (GRAS) plant metabolites in regulating the growth of human pathogenic and probiotic bacteria and in the formation of biofilm was investigated. Thymol, carvacrol and eugenol showed the strongest antibacterial action against both pathogenic and probiotic microorganisms, at a subinhibitory concentration (SIC) of ?50??g?ml(-1). Genistein, hydroquinone, p-hydroxybenzoic acid and resveratrol also showed antibacterial effects but at a wide concentration range (SIC?=?50-1000??g?ml(-1)). Catechin, gallic acid, protocatechuic acid and cranberry extracts were the most biologically compatible molecules (SIC???1000??g?ml(-1)). Regarding the effect on biofilm, it was observed that thymol, carvacrol and eugenol showed antibiofilm activity against all potential pathogenic bacteria tested whilst specifically enhancing probiotic aggregation. Catechin, genistein and cranberry extracts did not inhibit the pathogenic aggregation but they stimulated probiotic biofilm formation, whilst gallic acid, protocateuchic acid, hydroquinone, p-hydroxybenzoic acid and resveratrol did not show opposite effect on biofilm formation between pathogenic and probiotic microorganisms. These results indicate that an appropriate combination of GRAS plant metabolites, which have traditionally been used as dietary constituents due to their health-promoting characteristics, can also be extremely useful in the regulation of bacterial proliferation in the intestinal microbiota. Hence, it is suggested to apply these natural GRAS molecules as dietary supplements in the food industry in order to promote probiotic viability and to prevent or reduce colonisation or proliferation of intestinal pathogens.
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    • Panan Pattiyathanee
    • Ratha-Korn Vilaichone
      Ratha-Korn Vilaichone
    • Nuntaree Chaichanawongsaroj
      Nuntaree Chaichanawongsaroj
    Helicobacter pylori is a leading etiologic agent causing peptic ulcer and gastric cancer. The alternative lifestyle as a biofilm facilitates H. pylori to survive in adverse environments. Here, we investigated effect of curcumin on H. pylori biofilm formation both qualitatively by pellicle assay and quantitatively by crystal violet staining. Three-dimensional structure of biofilm was imaged by scanning electron microscopy. The effect of curcumin on H. pylori adherence to HEp-2 cells was also investigated. Sub-inhibitory concentrations of curcumin inhibited the biofilm in dose dependent manner. However, H. pylori could restore ability to form biofilm during extended time of incubation. Scanning electron microscopy revealed less amorphous extracellular polymeric matrix, slow of morphological conversion to coccoid form with cell damage after curcumin treatment. Curcumin significantly decreased the ability of H. pylori to adhere to the HEp-2 cells. Our findings demonstrated advantages of curcumin to inhibit biofilm formation by H. pylori, making it as a potential complimentary medicine for curing of H. pylori-biofilm related infections.
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    • Nathaniel C Cady
      Nathaniel C Cady
    • Kurt Mckean
      Kurt Mckean
    • Jason Behnke
    • Rabi Ann Musah
      Rabi Ann Musah
    Using a microplate-based screening assay, the effects on Pseudomonas aeruginosa PAO1 biofilm formation of several S-substituted cysteine sulfoxides and their corresponding disulfide derivatives were evaluated. From our library of compounds, S-phenyl-L-cysteine sulfoxide and its breakdown product, diphenyl disulfide, significantly reduced the amount of biofilm formation by P. aeruginosa at levels equivalent to the active concentration of 4-nitropyridine-N-oxide (NPO) (1 mM). Unlike NPO, which is an established inhibitor of bacterial biofilms, our active compounds did not reduce planktonic cell growth and only affected biofilm formation. When used in a Drosophila-based infection model, both S-phenyl-L-cysteine sulfoxide and diphenyl disulfide significantly reduced the P. aeruginosa recovered 18 h post infection (relative to the control), and were non-lethal to the fly hosts. The possibility that the observed biofilm inhibitory effects were related to quorum sensing inhibition (QSI) was investigated using Escherichia coli-based reporters expressing P. aeruginosa lasR or rhIR response proteins, as well as an endogenous P. aeruginosa reporter from the lasI/lasR QS system. Inhibition of quorum sensing by S-phenyl-L-cysteine sulfoxide was observed in all of the reporter systems tested, whereas diphenyl disulfide did not exhibit QSI in either of the E. coli reporters, and showed very limited inhibition in the P. aeruginosa reporter. Since both compounds inhibit biofilm formation but do not show similar QSI activity, it is concluded that they may be functioning by different pathways. The hypothesis that biofilm inhibition by the two active compounds discovered in this work occurs through QSI is discussed.
  • Book
    • Clinical and Laboratory Standards Institute
  • Article
    Full-text available
    • Meng-Chuan Wu
    • Tzu-Lung Lin
    • Pei-Fang Hsieh
      Pei-Fang Hsieh
    • Jin-Town Wang
    Community-acquired pyogenic liver abscess (PLA) complicated with meningitis and endophthalmitis caused by Klebsiella pneumoniae is an emerging infectious disease. To investigate the mechanisms and effects of biofilm formation of K. pneumoniae causing PLA, microtiter plate assays were used to determine the levels of biofilm formed by K. pneumoniae clinical isolates and to screen for biofilm-altered mutants from a transposon mutant library of a K. pneumoniae PLA-associated strain. The biofilm formation of K. pneumoniae was examined by microtiter plate assay. Higher levels of biofilm formation were demonstrated by K. pneumoniae strains associated with PLA. A total of 23 biofilm-decreased mutants and 4 biofilm-increased mutants were identified. Among these mutants, a biofilm-decreased treC mutant displayed less mucoviscosity and produced less capsular polysaccharide (CPS), whereas a biofilm-increased sugE mutant displayed higher mucoviscosity and produced more CPS. The biofilm phenotypes of treC and sugE mutants also were confirmed by glass slide culture. Deletion of treC, which encodes trehalose-6-phosphate hydrolase, impaired bacterial trehalose utilization. Addition of glucose to the culture medium restored the capsule production and biofilm formation in the treC mutant. Transcriptional profile analysis suggested that the increase of CPS production in ΔsugE may reflect elevated cps gene expression (upregulated through rmpA) in combination with increased treC expression. In vivo competition assays demonstrated that the treC mutant strain was attenuated in competitiveness during intragastric infection in mice. Genes important for biofilm formation by K. pneumoniae PLA strain were identified using an in vitro assay. Among the identified genes, treC and sugE affect biofilm formation by modulating CPS production. The importance of treC in gastrointestinal tract colonization suggests that biofilm formation contributes to the establishment and persistence of K. pneumoniae infection.
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    Full-text available
    • Ross P Carlson
      Ross P Carlson
    • Reed L Taffs
    • William M Davison
    • Philip S Stewart
    Surfaces coated with the naturally-occurring polysaccharide chitosan (partially deacetylated poly N-acetyl glucosamine) resisted biofilm formation by bacteria and yeast. Reductions in biofilm viable cell numbers ranging from 95% to 99.9997% were demonstrated for Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans on chitosan-coated surfaces over a 54-h experiment in comparison to controls. For instance, chitosan-coated surfaces reduced S. epidermidis surface-associated growth more than 5.5 (10)log units (99.9997%) compared to a control surface. As a comparison, coatings containing a combination of the antibiotics minocycline and rifampin reduced S. epidermidis growth by 3.9 (10)log units (99.99%) and coatings containing the antiseptic chlorhexidine did not significantly reduce S. epidermidis surface associated growth as compared to controls. The chitosan effects were confirmed with microscopy. Using time-lapse fluorescence microscopy and fluorescent-dye-loaded S. epidermidis, the permeabilization of these cells was observed as they alighted on chitosan-coated surfaces. This suggests chitosan disrupts cell membranes as microbes settle on the surface. Chitosan offers a flexible, biocompatible platform for designing coatings to protect surfaces from infection.
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    Full-text available
    • Li Chen
    • Yu-Mei Wen
      Yu-Mei Wen
    Bacterial biofilms can be viewed as a specific type of persistent bacterial infection. After initial invasion, microbes can attach to living and non-living surfaces, such as prosthetics and indwelling medical devices, and form a biofilm composed of extracellular polysaccharides, proteins, and other components. In hosts, biofilm formation may trigger drug resistance and inflammation, resulting in persistent infections. The clinical aspects of biofilm formation and leading strategies for biofilm inhibitors will be discussed in this mini-review.
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    • Victor Kuete
      Victor Kuete
    • Justin Kamga
      Justin Kamga
    • Louis P. Sandjo
      Louis P. Sandjo
    • Bonaventure T Ngadjui
      Bonaventure T Ngadjui
    Many plants of the family Moraceae are used in the treatment of infectious diseases. Ficus polita Vahl., an edible plant belonging to this family is used traditionally in case of dyspepsia, infectious diseases, abdominal pains and diarrhea. The present work was designed to assess the antimicrobial activity of the methanol extract from the roots of F. polita (FPR), as well as that of its fractions (FPR1-5) and two of the eight isolated compounds, namely euphol-3-O-cinnamate (1) and (E)-3,5,4'-trihydroxy-stilbene-3,5-O-β-D-diglucopyranoside (8). The liquid microdilution assay was used in the determination of the minimal inhibitory concentration (MIC) and the minimal microbicidal concentration (MMC), against seven bacterial and one fungal species. The results of the MIC determination showed that the crude extract, fractions FPR1, FPR2 and compound 8 were able to prevent the growth of the eight tested microorganisms. Other samples showed selective activity. The lowest MIC value of 64 μg/ml for the crude extract was recorded on 50% of the studied microbial species. The corresponding value for fractions of 32 μg/ml was obtained on Salmonella typhi, Escherichia coli and Candida albicans ATCC strains. The MIC values recorded with compound 8 on the resistant Pseudomonas aeruginosa PA01 strain was equal to that of chloramphenicol used as reference antibiotic. The obtained results highlighted the interesting antimicrobial potency of F. polita as well as that of compound 8, and provided scientific basis for the traditional use of this taxon in the treatment of microbial infections.
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    • Stefan Schwarz
      Stefan Schwarz
    • Peter Silley
      Peter Silley
    • S. Simjee
    • Wim Gaastra
      Wim Gaastra
    The accurate performance of antimicrobial susceptibility testing of bacteria from animal sources and the correct presentation of the results is a complex matter. A review of the published literature revealed a number of recurring errors with regard to methodology, quality control, appropriate interpretive criteria, and calculation of MIC(50) and MIC(90) values. Although more subjective, there is also no consensus regarding the definition of multiresistance. This Editorial is intended to provide guidance to authors on how to avoid these frequently detected shortcomings.
  • Article
    • Stefan Schwarz
      Stefan Schwarz
    • Peter Silley
      Peter Silley
    • S. Simjee
    • Wim Gaastra
      Wim Gaastra