Identification and Molecular Analysis of Antibiotic-Resistant Gram-negative

Bacteria Found in Three Freshwater Creeks Located in Bacoor City, Cavite

Juwan Howard R. Barlomento, Joanalaine A. Orsos, Angelica T. Saul

OVERVIEW

Majority of antibiotics used for human, plants and animals are excreted into the
environment as intact or decomposed form via various pathways, including wastewater effluent
discharge, runoff from land to which agricultural or human waste has been applied, and leaching
(Zhang et al., 2009). Antibiotic residues in the environment impose selective pressure on
bacterial populations, which results prevalence of resistant bacteria even at sub-inhibitory low
concentrations. Other pollutants are also known as selective agents (Stepanauskas et al., 2005,
2006). Additionally, the raw wastewater contaminated by antibiotics released into aquatic
environments often carries human and animal pathogenic bacteria, in addition to commensal
bacteria, and many of these organisms harbor antibiotic-resistance genes. Therefore, water
constitutes a way of dissemination of not only antibiotic-resistant bacteria, but also the resistance
genes, which genetically change in natural bacterial ecosystems (Baquero et al., 2008;
Rosenblatt-Farrell, 2009). The heavy and unregulated use of antibiotics along with the discharge
of untreated wastewater into the aquatic environments might cause significant contamination of
both antibiotic residues and ARB. Thus, it is of utmost importance that creeks and other bodies
of water located near houses and residential areas be kept clean to prevent antibiotic-resistant
bacteria from proliferating and contaminating the environment.

STATEMENT OF THE PROBLEM

SCOPE AND LIMITATION

Freshwater samples from the creeks will be collected only once a month. Samples
will only be taken from the three creeks within the inclusive barangays of Bacoor City. No other
locations outside the area of the city will be used. Water samples will only be collected during
the dry season to avoid possible contamination of the samples by rainwater. No other treatments
or drugs will be applied to the bacteria that will be obtained. Only Gram-negative bacteria will
be cultured and tested in this study. All tests will be conducted using the original nutrient agar
stock culture slants to avoid the spontaneous loss of antibiotic resistance sometimes associated
with frequent sub-culturing. Only isolates that grew on the Mueller-Hinton agar control plates
will be used in enumerations of MAR bacteria.

METHODOLOGY:

Selection of sampling sites

Three locations in Bacoor City will be used in this study. The water quality of the
portion of the creek near Floraville Subdivision, Barangay Panapaan I is murky, muddy and
stagnant. The creek runs beside a mall, school, carwash and a variety of stores, all of which
potentially dump their wastes directly into the creek. The second creek along Barangay Habay II
is also murky and muddy; however, the water is flowing. The murky creek flows beside
establishments, stores and houses. Solid waste like plastics can be found on both creeks. The
third creek is along Barangay Panapaan IV, murky and muddy flowing water. The creek is along
a residential area.

Sample collection

For the stagnant creek(s), three equal-sized (2 liter) samples of freshwater from
varying depths (surface, midzone and bottom) will be collected from a single location and the
samples from each depth will be sub-sampled. All samples will be aseptically collected in sterile
2-liter propylene storage bottles and will be transported to the laboratory in in ice-filled coolers
(Bissonnette et al., 1994). All samples will be processed within 6 hours from their collection.
Temperature and pH measurements will also be made at the time of sampling (Dalsgaard et al.,
2001).

Bacterial culturing

Serial tenfold dilutions of the creek samples will be prepared in physiological saline
and 0.1 ml aliquots will be streak-plated onto MacConkey agar (MA) (Merck, Copenhagen,
Denmark). (Dalsgaard et al., 2001).
Microbial Analysis and Antibiotic Sensitivity Testing

Antibiotic resistance will be assayed using a modified Kirby-Bauer disk-diffusion method

(Bauer et al., 1966). Cells will be grown at 30°C in Mueller-Hinton broth, and the resulting
cultures will be swabbed onto Mueller-Hinton agar (MHA) to achieve a lawn of confluent
bacterial growth, and antibiotic disks of standard concentration will be placed on each plate.
Plates will be incubated at 30°C for 24 hours. Organisms are classified as sensitive or resistant to
an antibiotic according to the diameter of the inhibition zone surrounding each antibiotic disk.
Organisms considered to be of intermediate resistance will be scored as sensitive. Prevalence of
resistant bacteria will be calculated as the numbers of bacteria growing on media containing
antibiotics divided by the numbers of bacteria growing on media without antibiotics.
(Bissonnette et al., 1995).

Purification and storage of isolates

Each isolate to be identified will be picked from a plate containing one of the
antibiotics to which it was resistant and was inoculated into tryptic soy broth (Difco) containing
0.3% yeast extract (Difco). After 24 hours of incubation at 35°C, a loopful of this culture is
streaked onto tryptic soy agar (TSA) (Difco) containing 0.3% yeast extract, and the plate is again
incubated for 24 hours at 35°C. A single colony from this plate is then used for identification.
(Armstrong et al, 1981).

Identification

Isolates are subjected to verification procedures (APHA, 1989) in order to categorize

the respective strains as coliforms or non-coliforms. Isolates will be inoculated into Sulfide-
Indole-Motility (SIM) and Oxidative/Fermentative (OF) basal medium containing 1% glucose to
determine motility and oxidative/fermentative metabolism, respectively. The colonies will be
subjected to catalase, Gram stain reactions, and morphological tests. Those isolates exhibiting a
fermentative metabolism will be identified by API 20E (Analytab Products, Inc.), whereas
isolates exhibiting oxidative metabolism will be identified using API Rapid NFT (Analytab
Products, Inc.). (Bissonnette et al, 1995). Isolates are placed into genera or groups on the basis of
cell morphology, colonial morphology, Gram stain, catalase and oxidase reactions, motility,
urease and indole tests, and glucose fermentation and oxidation. (Armstrong et al, 1981).
Plate counting

Standard plate counts of total heterotrophic bacteria will be performed on tryptone soy
agar (TSA) (Oxoid, Ballerup, Denmark). TSA plates will be incubated for 48 hours at 30oC
before bacteriological counts are performed. Colonies are enumerated with the aid of a
stereoscopic microscope (35x). The number of colony-forming units (CFU), colony size, and
pigmentation will be recorded from the plate most representative of the mean colony count, and
isolates will be sub-cultured for pure culture isolation. The prevalence of resistant bacteria is
calculated as the numbers of bacteria growing on media containing antibiotics divided by the
numbers of bacteria growing on media without antibiotics. (Dalsgaard et al., 2001)