Biofilm Formation by Environmental Microbes Isolated from Hospitals in Karachi, Pakistan

Abstract

The purpose of this study was to isolate and identify the microbes from hospital environmental samples and to evaluate the potential of biofilm formation by isolated microbes. For this, 125 surface swabs of different environmental samples were taken from PNS SHIFA hospital, Karachi, Pakistan. Bacteria and fungi were isolated and identified by culture plate method. Trypticase soy broth (TBS) media was used for biofilm development by microbes in plastic tubes. Developed biofilm in tubes was visualized with crystal violet staining method and then biofilm forming potential was estimated by measuring the optical density through spectrophotometer. Total 202 microbes including 126(62.38%) bacteria and 76(37.62%) fungi were isolated and identified. Among environmental samples, hospital ward curtains and medical trays were highly contaminated with bacteria and fungi (with 26% each of total assemblage respectively). Staphylococcus aureus was in highest abundance followed by Candida albicans with 28.7% and 15.8% of total assemblage of isolation respectively. Moreover; Staphylococcus aureus followed by Candida albicans also found to have highest potential to form biofilm with 30.25% and 23.52% of total assemblage of biofilm formation respectively which clearly indicates that Staphylococcus aureus and Candida albicans may recognized as major agents of hospital acquired infection and can relate with enhanced potential of biofilm formation. Highest abundance and biofilm formation potential of bacteria and fungi in combination can also underline a direct extensive and striking interaction between prokaryotic and eukaryotic cells in biofilm.

Full text

American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS)
ISSN (Print) 2313-4410, ISSN (Online) 2313-4402
© Global Society of Scientific Research and Researchers
http://asrjetsjournal.org/
Biofilm Formation by Environmental Microbes Isolated
from Hospitals in Karachi, Pakistan
Aneela Shaheen a*, Rakhshanda Baqai b
aDepartment of Microbiology, University of Karachi, Karachi-75270, Pakistan
b Dadabhoy Institute of Higher Education, Main campus Karachi, Karachi, Pakistan
aEmail: aneelashaheen84@gmail.com
bEmail: r_baqai@yahoo.com
Abstract
The purpose of this study was to isolate and identify the microbes from hospital environmental samples and to
evaluate the potential of biofilm formation by isolated microbes. For this, 125 surface swabs of different
environmental samples were taken from PNS SHIFA hospital, Karachi, Pakistan. Bacteria and fungi were
isolated and identified by culture plate method. Trypticase soy broth (TBS) media was used for biofilm
development by microbes in plastic tubes. Developed biofilm in tubes was visualized with crystal violet staining
method and then biofilm forming potential was estimated by measuring the optical density through
spectrophotometer. Total 202 microbes including 126(62.38%) bacteria and 76(37.62%) fungi were isolated and
identified. Among environmental samples, hospital ward curtains and medical trays were highly contaminated
with bacteria and fungi (with 26% each of total assemblage respectively). Staphylococcus aureus was in highest
abundance followed by Candida albicans with 28.7% and 15.8% of total assemblage of isolation respectively.
Moreover; Staphylococcus aureus followed by Candida albicans also found to have highest potential to form
biofilm with 30.25% and 23.52% of total assemblage of biofilm formation respectively which clearly indicates
that Staphylococcus aureus and Candida albicans may recognized as major agents of hospital acquired infection
and can relate with enhanced potential of biofilm formation. Highest abundance and biofilm formation potential
of bacteria and fungi in combination can also underline a direct extensive and striking interaction between
prokaryotic and eukaryotic cells in biofilm.
Keywords: Biofilm; Bacteria; Fungi; Hospital acquired infection.
------------------------------------------------------------------------
* Corresponding author.
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1. Introduction
In natural environments, microorganisms occur mostly as surface attached cells rather than as planktonic or free
floating cells [1, 2, 3, 4]. In response to stressed conditions, microbes develop survival strategy for which they
tend to attach to available surfaces and form biofilms. Not all microbes attached to surfaces can form a biofilm.
Establishment of biofilm from a planktonic culture through surface attachment is a continuum process.
Microorganisms attached to a substrate produce extracellular polymeric substances (EPS), exhibiting an altered
phenotype compared with corresponding planktonic cells, especially regarding growth, gene transcription,
protein production and intercellular interaction within biofilms [5, 6, 7]. It can be formed by different type of
micro-organisms, including bacteria, viruses, fungi and others can be form on almost any biological or
inanimate surface [8] and due to biofilm development, they are able to survive on dry surfaces for extended
periods [9].
In the hospital environment, surface like indwelling medical devices and prostheses, water lines and tubing on
endoscopes and on wounds [10, 11] microorganisms colonize on all abiotic and biotic surfaces available in form
of biofilms, making them as a source of infection for hospitalized individuals and create an important public
health problem [12, 13, 14]. Pathogen causing nosocomial infection may also be acquired from patients (own’s
skin, gut, respiratory flora) as well as hospital staff (surgical of clinical staff) or contact with surrounding
environmental sources [15, 16, 17]. That is why biofilm is considered to be involved in 65% of nosocomial
infection [18, 19, 20].
Neither dryness of microbes on to hospital surfaces has been studied nor attachment of bacteria to that surfaces
and formation of biofilms studied in detail. Vickery et al. [19], tried to study them in detailed and took the
several hospital surfaces samples after cleaning and bleach disinfection by cutting the materials out of the
hospital environment and they found the viable meticillin-resistant Staphylococcus aureus (MRSA) in biofilm on
three surface samples. Some other research findings have occurred that biofilm attachment may appears to be
loosened by some biocides but it is difficult to remove bacteria from biofilms through cleaning with disinfection
[21]. Furthermore, surface attached bacteria have reduced susceptibility to antibiotics and may also have
reduced susceptibility to biocide. Such findings are clear justification for failures of adequate biocides in
hospital disinfection, contributing to failures in hospital cleaning [22].
A biofilm changes the lifestyle of microbes in such a way that it directly enhances their virulence. Such surface
attached microbes have remarkable involvement in chronic bacterial infections and cannot easily be
exterminated by conventional therapy as biofilm can be up to 1000 folds more resistant to antibiotic treatment
than the same organism growing planktonically [1, 23, 24, 25]. Biofilm infections can be caused by a single
microbial species or by a mixture of bacterial or fungal species [26]. Infectious diseases caused by biofilms are
dental caries (caused by acidogenic Gram-positive cocci Streptococcus sp.), periodontitis (Gram-negative
anaerobic oral bacteria), otitis media, or middle ear infection (non-type able Haemophilus influenza), chronic
tonsillitis (by various species), cystic fibrosis pneumonia (Pseudomonas aeruginosa, Burkholderia cepacia),
endocarditis (streptococci, staphylococci), necrotizing fasciitis (Group A streptococci), musculoskeletal
infections (Gram-positive cocci), osteomyelitis (various species), biliary tract infection (enteric bacteria),
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infectious kidney stones (Gram-negative rods), bacterial prostatitis (Escherichia coli and other Gram-negative
bacteria). Organisms like P. aeruginosa, Staphylococci and E. coli are associated to such hospital acquired
infections that are caused by foreign body materials, such as contamination of implants, catheters, contact lenses
and prostheses [27, 28].
Among fungi, Candida species are known as most recognized nosocomial pathogen and associated with hospital
acquired infection with mortality rate of 35%. US National Nosocomial Infections Surveillance System rank
Candida species as the fourth most common cause of bloodstream infection, behind coagulase-negative
staphylococci, Staphylococcus aureus and enterococci [29]. Diseases that are associated with Candida species
biofilms: nosocomial pneumonias and urinary tract infections [30].
Various model systems have been used to characterize the overall properties of biofilms. Mostly these model
systems were used to study bacterial biofilms. First and simplest model used to study biofilm was to grow the
adherent population on rough surfaces e.g. small dics cut from catheters. Then growth of adherent cells were
monitored quantitatively by a colourimetric assays [31]. One another method using similar principal has been
used to study the formation of biofilms on denture acrylic strips [32]. Now days, 96 well microtiter plates are
used for rapid processing of large numbers of samples even for antifungal agent susceptibility testing for
biofilms [33]. Microorganisms were grown in trypticase soy broth TSB) for biofilm formation [34]. The most
widely used method for biofilm formation in microtiter plates is the crystal violet (CV) staining method [35],
which only measured biofilm biomass at the bottom of the well. CV is a basic dye that stains both living and
dead cells in the extracellular matrix of biofilms by binding to negatively charged surface molecules and
polysaccharides [36]. Later, the method was modified for enhancing the efficiency and to quantify the biofilm
biomass in whole well by solubilization of dye by addition of acid [37, 38].
In this regard, we aimed to study our local hospital environment for availability of pathogenic bacteria and to the
best of our knowledge; no study has been carried out in Karachi hospital with reference to biofilm forming
potential as survival strategy. So we also aimed to develop a low cost in-vitro method that enables quantitation
of total amount of biofilm produced by microbes. For this purpose we isolate and identify the common bacterial
and fungal strains contaminating the hospital environment and to determine microbes potential to from biofilms.
For biofilm formation, a differential CV staining method was modified by replacing the microtiter plates with
sterile cheap plastic tubes, enabling the phenotypic biofilm formation in tubes.
2. Materials and methods
2.1 Sample collection
Total 125 surface swab samples were taken from different places of PNS SHIFA hospital located in Karachi,
Pakistan. All samples were taken with help of TRANSWAB (M40 Compliant, medical wire, UK) having amies
medium for transportation of aerobes and anaerobes. Out of 125 total samples, 40 samples were taken from
different medicine trays, 35 samples were taken from hand surface of ward boys and nurses, 30 samples were
taken from the patient′s bed linens and 20 samples were taken from hospital’s curtains surface. All swab
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samples were stored in ice box and transported to the laboratory within 2 hours.
2.2 Isolation and identification of microorganisms
Collected swab samples were subjected for culturing on bacterial cultural media: nutrient agar and fungal
cultural media: Sabroud dextrose agar (SDA) for propagation of bacteria and fungi. After inoculation, nutrient
agar plates were placed in inverted position and incubated at 37°C for 24 hours whereas SDA were placed
upward position and incubated at room temperature for one week. After incubation, bacterial colonies were
isolated and identified on the basis of bergey’s Manual of Determinative Bacteriology [39]. After one week,
isolated colonies of fungi from SDA plates were mounted by the use of 10% Potassium hydroxide (KOH) with
lactophenol cotton blue. All of isolated bacterial strains were then preserved and stocked in nutrient broth
containing 20% (vol/vol) glycerol and store at freezing temperature while fungal isolates were stored on SDA
slants.
2.3 Biofilm production by tube method and quantification with Crystal Violet staining
Trypticase soy broth (TSB) media in sterilized plastic tubes were used for production of biofilm. Under aseptic
conditions, 02 ml trypticase soy broth (TSB) media were inoculated with loopful of colonies from culture media
plates. TSB tubes were incubated at 37º C for 24 hours. After 24 hours, 2% glucose was added to each tube of
TSB and re-incubated at 37º C for next 24 hours. After incubation TSB growth medium was discarded from
tubes and washed three times with Phosphate buffer saline (PBS having 7.3 pH) under aseptic condition to
eliminate the unbound bacteria. To evaluate the formation of biofilm, remaining attached bacteria were fixed
with 2 ml of 99% methanol and tubes were left for 15 minutes. Then tubes were emptied and left to dry. To
visualize the biofilm production from microorganisms, cells attached to plastic tubes in form of biofilm were
stained with 0.2 ml of 2 % crystal violet for 5 minutes. Excess stain was rinsed off by placing tubes under
running tap water. Airs dried the tubes in inverted position and dye that adherent cells were solubilized with 1.5
ml of 33% glacial acetic acid. The optical density of each tube was determined at 570 nm wavelength by using
spectrophotometer to quantify the biofilm formation by microbes. The blank (negative control) was determined
for each tube by measuring the optical density of a tube filled with only PBS.
3. Results
Out of 125 different environmental samples from hospital, total 202 bacterial and fungal strains were isolated
and identified by culture plate method according to Bergey’s manual of determinative bacteriology [39] and
Practical Mycology: Manual for identification of fungi [40]. Bacteria were in abundance in different
environmental samples of hospital with 62.38 % of total isolation assemblage followed by fungi with 37.62 %.
Among environmental samples, hospital ward curtains and medical trays were highly contaminated with
bacteria and fungi (with 26 % each of total assemblage). The numbers of isolated bacteria and fungi in each set
of hospital environmental samples are shown in figure 1. which clearly indicating the highest number of
bacterial and fungal isolation in hospital ward curtains and medical trays set of samples.
Among isolated bacteria and fungi Staphylococcus aureus followed by Candida albicans were occurred
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frequently with 28.7% and 15.8% of total assemblage of isolation as shown in figure 2.
Figure 1: Comparative isolation of microbes from hospital different environmental samples
Figure 2: Total percentage composition of microbes isolates from environmental samples of hospital (%)
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Isolated bacteria and fungi were then tested for their ability to form biofilm in trypticase soy broth (TSB) media
as shown in figure 3 where enhanced growth is seen in some tubes and some showed less growth in TSB media.
Then biofilm forming potential was visualized by crystal violet staining method and indicated the clear picture
of positive potential of isolated strains to form strong and moderate, weak or no biofilm. Figure 4 shows the
clear picture of microbe’s potential for biofilm formation as indicated a purple color biofilm formation on
bottom and wall of tubes which were formed, washed, stained with crystal violet.
Figure 3: Growth of microbes in TSB media after 24 hours of incubation, promoting to form biofilm (right) and
comparative TSB media tubes after 48 hours of incubation with 2 % glucose, indicating enhanced growth and
no growth of microbe in TSB tube (left)
Figure 4: Biofilm formation by microbe having positive potential for biofilm formation (right) and clear tube
indicating microbe have no potential to form biofilm (Left) after both tubes stained with crystal violet.
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These biofilms were then solubilized and then quantify the biofilm forming potential by measuring the optical
density of crystals violet color on 570 mm wavelength to differentiate the microbes in strong, moderate, weak or
no biofilm forming microbes. It should be noted here that among isolated bacteria and fungi Staphylococcus
aureus followed by Candida albicans were also have strong potential to form biofilm with 30.25% and 23.52%
of total assemblage of isolated microbes to form biofilm as presented in figure 5. Together these results indicate
a direct interaction of such empowering potential of microbes to form biofilm with survival of microbes in
extreme condition such as hospitals etc.
Figure 5: Comparative percentage Potential of biofilm formation by microbes isolated from hospital
environmental samples (%)
4. Discussion
Increased prevalence in secondary infections (mostly by bacteria and fungi) in hospitalized patients throughout
the world enlightens the urgent need to assess the causes of hospital acquired infections. One of the main
highlighted finding by scientists worldwide is biofilm formation by surface attached bacteria. Although it is a
general public ideology that hospital environment is not a enriched providing media for microbial growth but It
was find out by the world wide scientists that microbes are likely to be attached on hospital dry surfaces and
they survive for unusually long periods (weeks to months) in developed biofilms that may take major role on the
prevalence of contaminations with pathogens and number of bacteria that are on hospital dry surfaces. This has
important implications, particularly for hospital outbreak investigation [41]. Coagulase negative
Staphylococcus, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Proteus mirabilis,
Pseudomonas species, Klebsiella species, and Enterococcus species are generally known to cause nosocomial
infections, and may be the common cause of colonization in indwelling medical devices even responsible for
biofilm production [27, 42, 43].
Similar findings were also noticed in present study as out of total 202 microbial isolates, 126(62.38%) bacteria
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and 76(37.62%) fungi were isolated and identified in hospital environmental surface samples. Staphylococcus
aureus was highly positive followed by Candida albicans with 28.7% and 15.8% of total assemblage of
isolation. Moreover; Staphylococcus aureus followed by Candida albicans also have a highest potential to form
biofilm with 30.25% and 23.52% of total assemblage of isolated microbes. These reports clearly indicate that
biofilm forming potential is the main survival strategy for these microbes in hospital stress environment and
may become a source of hospital acquired infections. Potential of microbes to form biofilm were monitored by
Tube method (TM). Although this method is slightly more laborious and slower than the traditional 96-well
microtiter plate staining procedure, but it is cost effective than other techniques that are used to determine the
spatial location of biofilms.
The samples collected for this study, some were in direct use and some that were in-direct use of hospitalized
patient. Among environmental samples, hospital ward curtains and medical trays were highly contaminated with
bacteria and fungi (with 26% each of total assemblage respectively). According to Curran et al [44] giving the
reason that considering the presence of staphylococci as normal skin flora, the microorganisms can reach
catheters through skin abrasions and catheter provides a favorable environment for bacteria to form biofilm.
Biofilms provide a mixed bacterial community where the horizontal transfer of resistance genes and extensive
interspecies interactions may take place in these communities. Some studies indicated that fungi can regulate the
antibacterial action and bacteria can control the activity of antifungal agents in these biofilms. Such supporting
interspecies interactions might explain the enhanced antimicrobial resistance of these mixed-species biofilms
and potential of microbes to survive for longer periods on hospital dry surfaces. Presence of C. albicans in a
biofilm increased the resistance of slime-negative staphylococci to vancomycin and Candida resistance to
fluconazole was enhanced in the presence of slime-producing staphylococci [26].
So there must be step forward to take necessary action to overcome microbial growth in hospital environment.
There should be proper use and selection of biocides and detergents with the best all round performance which
are able to control surface attached microbial growth on surfaces. Proper cleaning of all surfaces should be
occurred on regular basis. Further research is also required to evaluate the prevalence and composition of
biofilms in situ on hard and soft hospital surfaces. Vickery et al [17] giving more suggestion to overcome the
prevalence of microbes and their biofilm forming potential and develop in-vitro models that represents the
microbes and biofilm present in hospital environment, harnessing surface science to develop a hospital
environment that reduces the chance of biofilm formation such as microfibre or automated room disinfection
technology and surface modification.
5. Conclusion
Staphylococcus aureus and Candida albicans have highest level of survival strategy due to biofilm that is why
they may become major agents of hospital acquired infection and their emergence as important nosocomial
pathogens can relate with enhanced potential of biofilm formation. Highest abundance and biofilm formation
potential of bacteria and fungi in combination can also underline a direct extensive and striking interaction
between prokaryotic and eukaryotic cells in biofilm.
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6. Recommendations
As Tube method for biofilm formation is cheap and cost effective so it can be recommended as a general
screening method for detection of biofilm producing bacteria in laboratories. There are many limitations in
study which need to be covered for full understanding of the prevalence and composition of biofilm developed
by microbes. Followings are some recommendations, which should be studied in future;
• To understand the hydrophobic characteristics of microbe for biomaterial, adhesion assay should also be
performed. Findings can be helpful for selections of biomaterial uses generally in hospital. This may limits
the spread of microbes from such sources and decrease the cause of secondary infections.
• Further research is also required to evaluate the genotypic characteristics of biofilm forming microbes, to
suppress the major genes responsible for biofilm formation.
• To develop the in-vitro model that fully represents the microbes and biofilm relationship presents in
hospital environments.
• Moreover, antibiotic susceptibility of isolated microbes should be performed which will be useful for the
best selection of disinfectant.
• Selection and use of the best disinfectant that have highest efficiency performance to kills every type of
microbes on small concentration basis.
Acknowledgements
The research work and finding described in this paper were from research project in Master degree Programme.
We are grateful to Department of Microbiology, University of Karachi for research support and PNS SHIFA
hospital, Karachi, for samplings support.
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