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Abstract
Present study aimed at the isolation and characterization of bacterial pathogens from stool
samples of diarrhoeal patients and evaluation of their antibacterial susceptibility pattern against
commonly used antibiotics. Samples were collected from Divisional Headquarter (DHQ) and
Allied Hospital, Faisalabad. Differential and selective media were used to isolate bacterial
pathogens which were identified through cultural characteristics, microscopy and biochemical
tests. Disc diffusion assay was done using Muller Hinton agar medium and Minimum Inhibitory
Concentration (MIC) was determined using broth dilution method against isolated pathogens.
According to the results, all 141 (100%) samples were positive for some bacteria. Frequently of
occurrence was; Bacillus cereus (65.95%), E. coli (48.51%), Salmonella typhi (27.65%),
Pseudomonas aeruginosa (8.51%) and Staphylococcus aureus (4.25%). Antibacterial activity of
used antibiotics i.e. Ofloxacin, Gentamicin, Amoxicillin, Cefotaxime against B. cereus was
33.447±3.429, 22.500±3.110, 23.277±3.059 and 12.617±2.889, against E. coli 11.701±2.082,
13.448±2.025, 3.382±1.985 and 0.886±0.179, against Salmonella typhi 25.00±6.130,
14.692±4.786, 7.30±3.72 and 0.1601±0.0256, against Pseudomonas aeruginosa 15.583±3.260,
15.167±2.552, 0.00±0.00, 0.00±0.00 and against Staphylococcus aureus 28.00±3.22,
12.333±2.160, 5.83±4.71 and 0.00±0.00, respectively. Pseudomonas aeruginosa showed
resistance against Amoxicillin and Cefotaxime whereas Staphylococcus aureus was found
resistant against Cefotaxime. Statistical analysis through one way ANOVA revealed that
Ofloxacin and Gentamicin had significant (p<0.05) activity against all isolates as compared to
the other antibiotics used in this study.
Keywords: Diarrhoea, Bacterial pathogens, Antibacterial susceptibility, Disc diffusion assay,
Minimum Inhibitory Concentration
1
Introduction
One billion people have poor excess to water and about 2.6 billion lack basic sanitation
facilities worldwide [1]. Ten to eleven million children die before the age of five in low and
middle income countries [2]. Among many other infectious diseases, diarrohea is an important
cause of morbidity and mortality in human. There has been much less progress in reducing
diarrohea over past decade and 21% mortality rate was estimated in the children of less than five
years of age throughout the world [3]. Approximately 2 million children die in low and middle
income countries only due to diarrohea [4]. It accounts for 12% of all deaths due to infectious
disease in the world. Approximately 90% of diarrhoeal cases in children are in low and middle
income countries [5]. Among different types of diarrhoea, acute watery diarrhoea is more
common and has morbidity rate of 80% and mortality rate of 50% in children [6]. Persistent
diarrhoea has the morbidity rate of only 10% but have increased risk of death due to
malnutrition. Globally, persistent diarrhoea has mortality rate of 35%, but Asia is proved to be
more responsible for more than half the diarrhoeal deaths [7].
Diarrhoea can be caused by a variety of microorganisms that may be viral, bacterial, and
protozoan. Most diarrhoea causing microbes have medium to low virulence. Shigella in few
numbers can cause symptomatic disease [8]. High concentration of Vibrio cholerae requires for
causing disease in healthy individual. The other bacterial etiological agents are Escherichia coli,
Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella and Shigella
serotypes. The dangerous form of the diarrhoea is cholera, caused by Vibrio cholerae. The fluid
loss in this condition if goes untreated leads to death [9]. Dysentery is the passing of stools with
blood. Common cause of the dysentery is Shigella spp. and amebiosis [10, 11]. To find
diarrhoeal causative agent from diarrhoea episodes is difficult even after under controlled
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conditions as pathogens are not found in half of the cases in modern hospitals [12]. The viruses
associated with acute gastroenteritis are Rotavirus, Norovirus, Enteric Adenovirus, Calicivirus
and Astrovirus and among these the Rotavirus is the most common cause of severe diarrhoea
[13]. Route of the parasite transmission is either through food or water. Parasites like Cyclospora
cayetanenisis, Cryptosporidium, Entamoeba histolytica and Giardia lamblia also cause diarrhoea
[14].
There are two pathophysiological mechanisms by which bacteria cause disease. Either
they produce toxins that affect the intestinal fluid or by the intrusion of the microbes. Cholera
toxin causes the fluid secretion. Transmission route of the bacteria is by the fecal-oral route by
means of unhygienic water, food, person to person interaction or by the direct contact. Diarrhoea
is transmitted by means of domestic storage containers. Protection against diarrhoeal agents also
relies on the microflora of the gastro-intestinal tract. Most of the pathogens are killed by the
hydrochloric acid. People having low level of gastric acid are more prone to contact diarrhoea
than others. Interrelated factors like family formation, sanitizing conditions, availability of clean
water and food, household environment, society trends, policies and individual behavior also
affect the rate of morbidity and mortality by the diarrhoea [15].
Antibiotics are not generally recommended to children with infectious causes [6]. But
antibiotics are given in case of having cholera and dysentery [16]. Zinc is used to reduce the
severity and duration of the disease and lower the incidence of diarrhoea in the following 2-3
months [17]. Now, it is recommended to use zinc as soon as possible for the treatment of
diarrhoea [18]. Most severe form is the persistent or chronic diarrhoea, for its treatment initially
correction of malnutrition is required [19]. Rehydration is essential by maintaining the
electrolytes. Symptomatic relief is the second priority [20]. In 1979, Oral rehydration therapy
3
(ORT) was introduced and became an important task for the CDD program (Control of
Diarrhoeal Disease). ORT contains sodium, a carbohydrate and water [21].
Keeping in view the importance of the disease and associated losses, the present study
was conducted to know the occurrence of the bacterial pathogens responsible for diarrhoea in
District Faisalabad Punjab Pakistan and to engender information regarding the effectiveness of
commonly used antibiotic against diarrhoea. It is hoped that the findings will be helpful to
establish an effective treatment and thus better control of diarrhoea in future.
Materials and Methods
Sample collection
A total of 141 stool samples of either sex and from different age groups were collected
from District Headquarter Hospital (DHQ) and Allied Hospital Faisalabad Punjab Pakistan.
Samples were obtained in sterile containers, properly labelled and transported in the Carry-Blair
Medium (Oxoid, UK) along with complete data sheet and under aseptic conditions to
Postgraduate Research Laboratory of Department of Microbiology, Govt College University
Faisalabad Pakistan [22].
Isolation and purification of bacterial pathogens
The stool samples were inoculated first on Nutrient agar (Scharlau, Spain) plates by
swabbing and incubated at 37°C for 24 h. For purification, the different colonies from initial
growth were streaked on MacConkey’s agar (Oxoid, UK) and Salmonella Shigella agar (Oxoid,
UK) plates and incubated at 37°C for 24 h [23].
Identification and biochemical characterization
Identification was done on the basis of cultural characteristics on different media and
microscopic examination of stained smears through Gram staining procedure. Biochemical
4
characterization was done using catalase, methyl red, indole production, Voges Proskauer,
coagulase, oxidase and triple iron suger tests [24, 25, 26].
Antibiotic susceptibility testing
a. Disc diffusion method
The agar disc diffusion method (Kirby-Bauer method) was done using Muller-Hinton agar
medium (Oxoid, UK). The antibacterial susceptibility of bacterial isolates was determined
against selected commonly used antibiotics including Ofloxacin, Gentamicin, Amoxicillin and
Cefotaxime. Turbidity of inoculum was adjusted to 0.5 McFarland and diameter of Zone of
inhibition were measured in mm [27, 28].
b. Minimum Inhibitory Concentration (MIC)
The MIC is the lowest concentration of the antibiotics which prevents visible growth of
microbes. It was measured by Micro-broth dilution method using 96-well microtitration plate.
Muller-Hinton broth (Oxoid, UK) was used with phenol red (0.01%) as an indicator [29].
Statistical analysis
The data was statistically analyzed by computing coefficient of variance and comparing
the mean with standard deviation and One-way Analysis of Variance (ANOVA) using the
Minitab 15.
Results
Sample distribution
Out of total 141 diarrhoeal stool samples collected, 100% pathogen response rate was
found and different bacteriological agents responsible for diarrhoea from the same specimen
were isolated. The patients were categorized into three age groups from infants to the older ones.
First group (G1), was from 1 day to 5 years (n=60) second group (G2) aged between 5-30 years
5
(n=45) and third group (G 3) from 30 years to onwards (n=36). The 57 (40.42%) samples were
from male patients and 84 (59.58%) were of females.
Occurrence of bacterial isolates
Single pathogen was detected in only 20 (14.18%) samples whereas combinations were
found in 121 (85.81%) samples. Bacillus cereus and E. coli were the most frequently detected
pathogens followed by the Salmonella typhi, Pseudomonas aeruginosa, and Staphylococcus
aureus. The percentage occurrence of isolated pathogens was; Bacillus cereus (43%), E. coli
(31%), Salmonella typhi (18%), Pseudomonas aeruginosa (5%) and Staphylococcus aureus (3%)
(Figure 1).
Identification of bacterial isolates
a. Colony characteristics
The growth of E. coli was observed as pink, shiny, smooth, round colonies with raised
surface and varying growth pattern on MacConkey’s agar. Bacillus Cereus has large, smooth,
pink colonies with mousy smell on MacConkey’s agar. Lactose non fermenter colonies on the
MacConkey’s agar and central black, small size colonies with smooth to rough in appearance on
the Salmonella-Shigella agar were identified as Salmonella spp. Pseudomonas aeruginosa was
found to be Non lactose fermenter with colourless, irregular and round colonies having sweet
odour on the MacConkey’s agar. Smooth, creamy and yellow colonies on the Staphylococcus
110 agar were observed for Staphylococcus aureus (Figure 2 a, b, c, d).
b. Microscopic examination
Gram’s staining was done to identify the bacterial isolates on the basis of their
morphology, arrangement, and staining characters. Different isolates showed difference in these
parameters which helped in their identification (Table 1 and Figure 3 a, b).
6
c. Biochemical characterization
Different biochemical tests were used to confirm the bacterial isolates including methyl
red, Voges Proskauer, catalase, indole production, coagulase and oxidase tests. The results are
shown in Table 2 and Figure 4 a, b.
Disc diffusion method
The diameter of Zones of inhibition were measured in mm. Bacillus cereus showed the
highest sensitivity against Ofloxacin with mean diameter of 33.447±SD and least sensitivity
against Cefotaxime as 12.617±SD. E. coli revealed highest sensitivity against Gentamicin with
13.448±SD mean diameter and it was found resistant against Cefotaxime. Salmonella typhi
exhibited the highest sensitivity against Gentamicin with diameter 13.448±SD whereas found
resistant against Cefotaxime. Pseudomonas aeruginosa displayed highest sensitivity against
Gentamicin with 15.583±SD mean diameter and indicated resistance against Cefotaxime and
Amoxicillin. Staphylococcus aureus presented highest sensitivity against Ofloxacin with the
28.00±SD mean diameter and found resistant against Cefotaxime (Table 3 and Figure 5 a, b, c,
d).
Minimum Inhibitory Concentration (MIC)
The MIC of different antibiotics was measured as the minimum concentration of the
antibiotic that inhibited the visible growth of microorganisms. Statistically, B. cereus was highly
sensitive against Metronidazole with the mean ± SD as (7.526±2.513) and E. coli against
Ciprofloxacin with mean ± SD (2.491±0.770). Salmonella typhi was highly sensitive against
Amoxicillin with the mean ± SD (2.362±0.792) wheras Pseudomonas aeruginosa showed
highest sensitivity against Ofloxacin with the mean ± SD (7.916±2.574). Staphylococcus aureus
was highly sensitive against Ciprofloxacin with the mean ± SD (2.340±0.854) (Table 4).
7
Discussion
The present study was performed to determine the type and occurrence of bacterial
pathogens involved in diarrhoeal cases from patients of both sex and different age groups in
District Faisalabad Punjab Pakistan. The antibacterial susceptibility pattern of isolated bacteria
was also studied against commonly used antibiotics. From the collected stool samples, bacterial
pathogens like Bacillus cereus, E. coli, Salmonella, Staphylococcus aureus and Pseudomonas
aeruginosa species were isolated and identified. Previous literature also reported the presence of
similar type of bacteria in the stool specimens of diarrhoeal patients [30, 31, 32, 33].
Isolates were identified on the basis of colony characteristics on different selective media.
Bacillus cereus showed pink colour colonies on the MacConkey’s agar due to the lactose
fermentation. It forms large colonies because of adapting a wide range of environmental
conditions [34, 35]. Salmonella typhi produced small black colour colonies on Salmonella
Shigella agar as it produces the hydrogen sulphide [36]. Pseudomonas aeruginosa did not
ferment lactose, so it appeared colourless on the MacConkey’s agar [37]. Staphylococcus aureus
showed yellow colour colonies on the blood agar media [38]. Isolates were identified by the
separation of the lactose fermenter and non-lactose fermenter bacteria using the novel approach
presented by Khan et al. [39].
Isolates were confirmed on the basis of the results of biochemical tests. Bacillus cereus
was confirmed in the ninety three (93) samples as Gram positive rods with MR-VP positive.
Because of the accumulation of acids, pH of the medium drops due to which MR changes from
yellow to red colour and a tinge of red colour in VP medium due to the production of non-acidic
products [25, 26]. E. coli was identified in the sixty seven (67) samples with MR positive and VP
negative as the addition of acids causing the pH of the medium falls due to which MR altered
8
from yellow to red colour and a shade of red colour in VP medium because of the production of
non-acidic products. The indole production test was also positive as E. coli has the capability to
generate indole from tryptophan by using an enzyme tryptophanase and was identified in
presence of Kovac’s reagent [40]. Salmonella typhi was confirmed in the thirty nine (39) samples
because of the indole negative and smooth, small and colourless colonies due to lack of H2S
production on the Salmonella Shigella agar. Pseudomonas aeruginosa was identified in the
twelve (12) stool samples due to the oxidase positive test indicating the presence of cytochrome
enzyme. Staphylococcus aureus was confirmed in only six (06) samples showing catalase and
coagulase test positive due to the presence of catalase enzyme that produce bubbles on reacting
with the Hydrogen peroxide (H2O2) and releasing the products as water (H2O) and molecular
oxygen (O2) [25].
The percentage occurrence of Bacillus cereus was the highest (65.95%) among all the
isolates. Al-Khatib et al. [41] also reported similar results. Second most frequent isolated
pathogen in all age groups was E. coli (48.51%). The reason for this may be the improper
sanitization of utensils and unhygienic conditions in this region of the world. Third most
frequently isolated pathogen was the Salmonella (27.65%) which was prevalent in G3 group and
less common in G1 and G2. Frequency of occurrence of different bacterial pathogens in this
work was almost similar to the work of Manikandan and Amsath [42] with approximately 10%
variations.
In this study, commercially available and commonly used four antibiotics (Ofloxacin,
Gentamicin, Amoxicillin and Cefotaxime) were selected for antibacterial susceptibility testing.
Ofloxacin and Gentamicin are broad-spectrum antibiotics that inhibit the protein synthesis [43,
44] wheras Amoxicillin and Cefotaxime inhibit peptidoglycan of the both Gram positive and
9
Gram negative cells [45, 46]. The overall results of disc diffusion assay revealed that all isolated
pathogens showed statistically significant (p<0.05) sensitivity against Ofloxacin and Gentamicin
wheras they were resistant to Amoxicillin and Cefotaxime.
The B. cereus exhibited partial resistance against Amoxicillin and Cefotaxime, wheras it
was sensitive to Gentamicin and Ofloxacin. The result coincides with the work of Turnbull [47]
and Kiyomizu et al. [48]. E. coli showed resistance against Amoxicillin and Cefotaxime and
sensitivity against Gentamicin and Ofloxacin. Salmonella presented sensitivity against Ofloxacin
and resistance against Amoxicillin. A study conducted in the Bangladesh also revealed similar
results [49]. Staphylococcus aureus showed sensitivity against Ofloxacin and resistance against
Cefotaxime. Pseudomonas aeruginosa exhibited sensitivity against Ofloxacin and Gentamicin,
whereas it was resistant against Amoxicillin and Cefotaxime.
Conclusion
Based on the overall result of present study, it was concluded that Bacillus cereus was the
most frequently isolated organism from stool samples of diarrhoeal patients of both sex and all
age groups followed by E. coli, Salmonella typhi, Pseudomonas aeruginosa and Staphylococcus
aureus, respectively in District Faisalabad of Punjab Pakistan. The overall percentage of
occurrence of diarrhoeal cases was more in the children and females than in males. Ofloxacin
and Ciprofloxacin were found to be the most effective antibiotics for the treatment of diarrhoea
as compared to the Amoxicillin and Metronidazole used in this study.
Acknowledgement
The authors are highly thankful to Higher Education Commission (HEC) Islamabad
Pakistan for providing funds under IPFP start up grant for the successful execution of this study.
10
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