Content uploaded by Fazle Rabbi
Author content
All content in this area was uploaded by Fazle Rabbi on Mar 14, 2014
Content may be subject to copyright.
ORIGINALARTICLE
Stamford Journal of Microbiology, July 2011. Vol. 1, Issue 1
ISSN: 2074-5346
Fazle Alam Rabbi,1 Fazle Rabbi,1 T A Runun,1 Khadiza Zaman,1 M. Majibur Rahman,1
and Rashed Noor1‡
1Department of Microbiology, Stamford University Bangladesh
Received 12 February 2011/Accepted 24 April 2011
Various types of cooked food samples were collected from six different hospitals within the city of Dhaka
and they were analyzed for the presence (if any) of total aerobic count (TAC), total coliform count (TCC)
and total staphylococcal count (TSA) in order to determine the levels of contamination and to relate these
findings to the hygiene practice of the food handlers. According to Gulf standard for microbiological
criteria for foodstuff, all of the food samples exceeded the acceptable total aerobic count limit of 5 x 105
cfu/g while 4 out of 6 samples exceeded coliform count limit of 1x102 cfu/g. The total coliform counts were
found to be the highest in the fish (1.6 x 107 cfu/g) and egg (2.2 x 106 cfu/g) curry samples of hospital 1 and
hospital 4, respectively and were the lowest in the fish curry (2 x103 cfu/g) of hospital 4. Staphylococcus
aureus was found in all of the food samples with the highest occurrence (too numerous to count) in Dal
and Rice samples from hospitals 3 and 6, respectively. On Xylose Lysine Deoxycholate (XLD) agar, no
black centered colony but many colorless colonies were found which primarily indicated the absence of
Salmonella spp. in those samples. In order to identify pathogenic microorganisms from food samples, a
series of conventional biochemical tests were performed with 23 randomly selected isolates from
MacConkey, XLD, MSA agar plates. The isolates were presumptively identified as Escherichia coli,
Staphylococcus aureus, Shigella spp. and Pseudomonas spp. etc. The antibiotic susceptibility test was
performed with eleven selected isolates using six commonly prescribed antibiotics (ampicillin,
tetracyclines, ciprofloxacin, vancomycin, gentamicin and azithromycin). The results showed that six
isolates were resistant to vancomycin, two isolates were multidrug resistant and one isolate was
intermediately resistant to azithromycin. All the isolates were found to be sensitive to ciprofloxacin, and
gentamicin. Based on the data, it can be suggested that adequate hygiene practices are required after
cooking the foods and before serving them as they reconsider.
Hospital infections have generally been sourced
from endogenous and exogenous source for long time.
Endogenous infections arise from the patient himself
while he is in the hospital. Hospital patients, because
of their particular illness, become readily susceptible
to their own commensal flora and to the potentially
pathogenic microbes of their skin and mucous
membranes, i.e., the secondary and/or tertiary
infections.
Exogenous infections are derived from hospital
environment (1). Organisms from hospital
environment are generally virulent and naturally
resistant to the antibiotics (2). They represent the
selected microbial populations because most of the
less virulent and sensitive microbes are eliminated by
the antibiotic therapy in the hospital and by antibiotic
fall out in the hospital environment. Exogenous
infections may be transmitted by a variety of routes:
(a) air borne dusts and droplets, (b) direct contact with
carrier, (c) food utensils, etc. The other sources of
infection greatly count on the staff and visitors to the
hospital (3). So far reported, the organisms commonly
responsible for hospital infections could be
Staphylococcus aureus, Streptococci, Corynebacteria,
E. coli, Pseudomonas, Enterotoxigenic E. coli,
Shigella and Salmonella (1, 4, 5).
‡Corresponding Author. Mailing address: Dr. Rashed Noor, Dept. of
Microbiology, Stamford University Bangladesh, 51, Siddeswari Road, Dhaka,
Bangladesh. Phone: +88-02-8354577 (Ext-472), Fax: +88-02-8363698,
Email: noor.rashed@yahoo.com.
Hospital food is an essential as well as unavoidable
part of patient care. Good food can encourage patients
to eat well, giving them the nutrients they need to
recover from surgery or illness. Meals are offered to
each patient three times per day, according to a four-
week cycle menu. In addition to breakfast, lunch and
dinner, generally snacks are offered to each patient in
the afternon by the unit staff.
Hospitals are in general thought to be the most
hygienic place. However a poorly-run hospital can be
responsible for any sort of food-borne illnesses and
hence can serve as a reservoir of pathogens. The role of
foodstuffs, contaminated by potentially pathogenic
bacteria, has long been established as one of the most
common causes of gastroenteritis, but the control of
this condition remains a major public health problem in
all communities.
31
Bacteria such as Salmonella and E. coli can affect
any kitchen, regardless of its location, if the person
preparing the food is not careful. Most bacteria found
in food that leads to food poisoning are the result of
improper preparation. While one would think that a
hospital, among all places, would be especially mindful
of this threat, the individuals responsible for preparing
meals are not doctors themselves. Instead, chefs and
cooks run the kitchen, like anywhere else, and can
make the same mistakes that commercial cooks can
make. So there is every possibility of contamination
b
efore or after cooking of a food as well as during
serving. Possible sources of contamination may
Microbiological Quality Assessment of Foods collected from Different
Hospitals within Dhaka City
RABBI ET AL, 2011
account from washing water, insects and rodents,
contaminated hands or people having skin infection,
hair or hair products in food, unhygienic kitchen
environment, contaminated equipment, contaminated
air or dust, personal hygiene, lack of adequate
sanitation etc.
E. coli and S. aureus are amongst the most common
pathogens found on hands (6, 7). It is also revealed
that most hospital food handlers were carriers of S.
aureus. Food poisoning by Staphylococcus affects
hundreds of thousands of people each year (8).
In addition, the antimicrobial resistance of bacteria,
isolated from food and other sources, against
commonly used antibiotics has been increasing day by
day (9). Considering all of these, it is most essential to
maintain microbiological quality and safety of foods
in the hospitals.
On the basis of these background, present study was
designed to assess the microbiological quality of foods
collected from different hospitals within Dhaka City
by detecting the overall microbial load, identification
of the pathogenic microorganisms and evaluation of
the antimicrobial susceptibility tests of the identified
bacteria.
MATERIALS AND METHODS
The study was confined to Dhaka City. It was a randomized cross sectional
survey conducted in the city. A total of 12 food samples were collected from 6
different hospitals around Dhaka city.
About 200 gram of solid or semisolid samples were collected aseptically
using sterile container and then were placed in insulated box. Food samples
were then mixed with sterile saline solution and were homogenized with
stomacher for 5-10 mins and serial dilutions were made up to 10-5 dilution.
Bacteriological analysis were performed by standard method (10, 11). The
microbiological condition of safety and hygiene were assayed using the
methods recommended by ICMSF (10).
The quality of samples was assessed by determining aerobic plate count
(APC), total coliform count (TCC), total staphylococcal count (TSC).
Inoculation and incubation. Nutrient agar plate (NA), MacConkey agar
plate and Mannitol salt agar plate (MSA) were used to determine APC, TCC,
TSC respectively. Spread plate technique was used to inoculate the samples
on various culture media for enumeration of microbes. Six hours enrichment
in selenite broth was done before inoculating the sample to the Xylose lysine
deoxycholate agar (XLD) in order to determine the Salmonella and Shigella.
Bacterial count. Plate count was restricted to 30-300 colonies and plates
containing more than 300 colonies were designated as too numerous to count
(TNTC) and plates containing fewer than 30 colonies were designated as too
few to count (TFTC). The following formula was used for enumeration:
Number of cells per ml = number of colonies x dilution factor/ volume of
sample used =cfu/g
Identification of microorganisms. Identification of bacterial isolates was
carried out according to Bergey’s manual (12). For further confirmation
several biochemical tests were performed according to the manual of methods
for general Bacteriology by American Society of Microbiology(13) to identify
the bacteria.
Antibiotic Susceptibility test. Antibiotic susceptibility test of the identified
organisms was determined by Kirby-Bauer method using Mueller-Hinton agar
medium(14). Microbes to be tested were grown in Mueller-Hinton broth
medium until the turbidity was equal to 0.5 McFarland standard. Table 1
shows the antibiotics that were used in this experiment.
RESULTS
Total aerobic plate count (APC), total coliform
count (TCC) and total staphylococcal count is shown
in the Table 2.
S. J. Microbiol.
TABLE 1. Potency of antibiotic discs used (Oxoid, UK)
Antibiotic use
d
Potency
(µg/disc)
Ampicillin (AMP) 15
Azithromycin (AZM) 15
Ciprofloxacin (CIP) 5
Gentamicin (CN) 10
Tetracycline (TE) 30
Vancomycin (VA) 30
From table 2 it is seen that all the food samples
exceeded the acceptable microbiological standard and
were highly contaminated. Except the rice samples, all
other samples contained more than 103 cfu/g coliforms
which is highly unacceptable. All samples were also
seriously contaminated by various species of
Staphylococci which were further confirmed through
biochemical tests and cultural characteristics.
TABLE 2. Aerobic plate count, total coliform count, total
Staphylococcal count in different types of food collected from
six different hospitals around Dhaka city.
Source Food type APC TCC TSC
(cfu/g) (cfu/g) (cfu/g)
H-1
Fish
7 x10
6
1.6 x10
7
1.7 x10
7
Rice TNTC Nil
4 x10
6
H-2
Dal
1.2 x10
7
7.2 x10
4
TNTC
H-3 Vegetable TNTC
4.3 x10
5
1.9 x10
6
H-4 Egg TNTC
2.2 x10
6
2.2 x10
5
Fish 9.8 x10
6
2 x10
3
1.7 x10
4
H-5
Fish
TNTC
1.3 x10
5
6.2 x10
5
H-6 Rice TNTC Nil TNTC
During the study we have isolated Staphylococcus
aureus, and S. epidermidis from the food samples.
Suspected colonies of Shigella sp (isolate no 4 and 26)
were isolated from fish and rice sample of hospital 1
and hospital 4, respectively. Suspected colonies of
Pseudomonas spp (Isolate no 13) were isolated from
vegetables collected from hospital 3. Table 3 shows
the cultural characteristics and the biochemical tests of
randomly selected 26 isolates.
32
S. J. Microbiol. Vol. 1, Issue 1
TABLE 3. Cultural characteristics and the biochemical tests of randomly selected twenty six (26) isolates collected from different hospital
33
Microbiological quality assessment of foods
Continued
S. J. Microbiol. Vol 1, Issue 1
34
Vol. 1, Issue 1 Microbiological quality assessment of foods
According to the above cultural and biochemical
tests, the following organisms were suspected. (Table
4)
TABLE 4. Suspected organisms isolated from the hospital food.
Isolate
Suspected
Isolate
Suspecte
d
No
Organism
No
Organism
1 S. aureus
14
S. aureus
2 S. epidermidis
15
S. epidermidis
3 E. coli
16
S. aureus
4 Shigella sp.
17
S. epidermidis
5 S. epidermidis
18
E. coli
6 S. aureus
19
E. coli
7 S. aureus
20
S. aureus
8 S. epidermidis
21
S. epidermidis
9 E. coli
22
E. coli
10
E. coli
23
Pseudomonas
sp
11
S. aureus
24
S. aureus
12
S. epidermidis
25
S. epidermidis
13
Pseudomonas
sp
26
Shigella
sp
.
Antibiotic susceptibility test. 11 randomly selected
bacterial isolates were tested for antibiotic
susceptibility by Kirby-Bauer method using 6
(ampicillin, tetracycline, ciprofloxacin, vancomycin,
gentamicin and azithromycin) commonly prescribed
antibiotics. The drug resistance pattern varied
considerably with different drugs. The highest
resistance was shown against vancomycin (6 out of 11
isolates). Moreover, 2 isolates were multidrug
resistant against ampicillin, tetracyclines in addition to
vancomycin and 1 isolate was intermediately resistant
to azithromycin. All the isolates were found to be
sensitive to ciprofloxacin, and gentamicin. Table 5
shows the antibiogram pattern of different isolates.
DISCUSSION
In the present study, we have isolated both indicator
and pathogenic microorganisms and unfortunately
neither of the food samples collected could meet the
microbiological standard in terms of aerobic plate
count (APC) or total Staphylococci count (TSC).
Although there is no available epidemiological data
about the risks of food-borne diseases resulting from
these food supply in Bangladesh, sparse information
about the risk of street-vended foods in other
developing countries has been published (15).
In case of the total coliform counts, the highest
occurrence was in fish (1.6 x107 cfu/g) and egg (2
x106 cfu/g) curry samples of hospitals-1 and hospital-
4, respectively and the lowest was in the fish curry (2
x103 cfu/g) of hospitals-4. Coliform count was nil in
the rice samples. The presence of total coliforms led
us to assume the presence of other harmful &
pathogenic microorganisms such as Salmonella spp.
In our study, Salmonella spp count was nil. However
considering the low sample size together with the
negative data, it is not claimed that hospital food are
free from Salmonella spp rather it does indicate that
the prevalence of Salmonella in hospital food is very
low. Besides, many colourless colonies were found on
XLD agar plate that were later presumptively
identified as Pseudomonas spp. and Shigella spp
On MSA agar media, huge Staphylococcal counts
were found in all the samples. Besides, two types of
colonies were found- yellow & white. The organisms
giving yellow colonies were assumed as S. aureus and
the organisms giving white colonies were assumed as
S. epidermidis. The result indicated that the food
samples contaminated with Staphylococci resulted
from the poor food handling practices.
Data on risk factors for foodborne diseases indicate
that the majority of outbreaks result from
inappropriate food handling practices (16). There are
also various reports that environmental conditions
offward
TABLE 5. Antibiogram pattern of different isolates
Isolate
Suspected
Antibiogram
Isolate
Suspected Antibiogram
no.
Organism
Resistant
Intermedi
Sensitive(S) no. Organism
Resistant
Intermedi
Sensitive(S)
(R)
ate (I)
(R)
ate (I)
03
E. coli - AZM AMP,VA,CIP, 20 S. aureus -
-
AMP,CIP,CN,
CN, TE
AZM,VA, TE
07
S. aureus - - AMP,VA,CIP, 21 S. epidermidis
VA
-
AMP,CIP,CN,
CN, TE,AZM
TE,AZM
12
S.
- - AMP,VA,CIP, 22 E. coli
TE,AMP,
-
CIP,CN, AZM
epidermidis
CN, TE,AZM
VA
13
Pseudomon VA - AMP,CIP,CN, 23
P
seudomonas
sp
VA
-
AMP,CIP,CN,
as
sp.
TE,AZM, TE,AZM
17
S.
- - AMP,VA,CIP, 26
Shigella
sp
.
VA
-
AMP,CIP,CN,
epidermidis
TE,CN, AZM
TE,AZM
19
E. coli
TE,AMP,
-
CIP,CN, AZM
VA
Note: *AMP (Ampicillin), AZM (Azithromycin) CIP (Ciprofloxacin), CN (Gentamicin), TE (Tetracycline), VA (Vancomycine).
35
RABBI ET AL, 2011 S. J. Microbiol.
possess significant effects on the risk of
microbiological contamination (17). Practices of
inadequate hygienic measures, mishandling,
improper storage, inadequate cooking and above all,
the unhygienic condition of the cooking premises are
responsible for food-borne outbreaks (18-20). Also,
the implication of food handler in food-borne disease
was observed in different studies by Hall and Hauser
(21). Food handlers play an important role in food
safety and in the occurrence of food poisoning
because they may introduce pathogens into food
during production, processing, distribution and/or
preparation (22). In our study, we have observed that
the personal hygiene status of the hospitals was not
satisfactory. The storage condition together with the
kitchen environment was also very poor in every
hospital. Most of the servers and cooks were also
illiterate and had a very poor knowledge about
personal hygiene and good manufacturing practice
(GMP).
One of the major reasons of morbidity & mortality
associated with gastrointestinal infections is the
antimicrobial resistance of the available drugs. In our
study, we have randomly selected 11 bacterial isolates
to determine the resistance against the 6 commonly
prescribed antibiotics (ampicillin, tetracyclines,
ciprofloxacin, vancomycin, gentamicin and
azithromycin). Except ciprofloxacin and gentamicin,
resistance was observed against the other four drugs.
The reasons for antimicrobial resistance include
inappropriate use of readily available antibiotics,
reuse of antibiotics, poor implementation of infection
control measures, improper disposal of hospital waste
and lack of education (3, 23-25).
Finally, it can be said that most of the hospital foods
are contaminated with huge load of microorganisms.
Presence of coliforms & other pathogenic bacteria
indicates poor hygienic features of the foods. Patients
might be taking contaminated foods with or without
their knowledge. This contamination might occurred
due to that most of the hospital workers are
uneducated & they are not at all concerned regarding
the good hygienic practice that reduce a considerable
number of pathogens from food samples. In hospitals,
where a high proportion of the patients might be
expected to react more severely to the ingestion of
bacterial-contaminated food, it is especially important
that the food prepared should be 'bacteriologically
clean' and that in the place where it is handled and
processed the standards of kitchen hygiene should be
high. It is, therefore, essential for people who handle
the foods to be properly trained on safe food handling
under special care by the concerned enterprises and
the governmental authorities. Lack of knowledge in
food safety by the owners and servers of the
restaurants promote the food contamination process
unconsciously.
REFERENCES
1. McFee, R. B. 2009. Nosocomial or hospital-acquired infections: an
overview. Dis. Mon. 55(7):422-38.
2. Gould, I. M. 2008.Antibiotic policies to control hospital-acquired
infection. J Antimicrob Chemother. 61(4):763-5.
3. Choudhury, M. R. 2005. Hospital Infections. In Modern Medical
Microbiology, (5th ed.), Vol. 43.
4. National Nosocomial Infections Surveillance (NNIS) System
Report, data summary from January 1992 through June 2003, issued
August 2003. Am J Infect Control, 2003. 31(8):481-98.
5. National Nosocomial Infections Surveillance (NNIS) System
Report, data summary from January 1992 through June 2004, issued
October 2004. Am J Infect Control 2004. 32(8):470-85.
6. Shojoei, H., J. Shooshtaripoor, and M. Amiri. 2006. Efficacy of
Simple Hand-Washing in Reduction of Microbial Hand Contamination
of Iranian Food Handlers. Food Research International, 2006. 39:525 –
529.
7. Oteri, T., and E. Ekanem. 1989. Food hygiene behavioural among
hospital food handlers. Public health 103:153 -159.
8. Hazariwala, A, et al. 2002. Distribution of Staphylococcal enterotoxin
genes among Staphylococcus aureus isolates from poultry and human
with invasive Staphylococcal disease. Avian Diseases 46:132 -136.
9. Vikas, G., et al. 2000. Sensitivity pattern of Salmonella serotypes.
BJID 6:281-282.
10. ICMSF. 1978. Sampling for Microbiological Analysis: Principles and
Specific Applications. In Microorganisms in Food, 2nd ed. Chapman &
Hall, London.
11. Thatcher, F. S., and C. DS. 1968. Microorganisms in Food: Their
Significance and Methods of Enumeration. University of Toronto Press,
Toronto.
12. Senath, P. H. A. 1986. In Bergey’s Manual of Systematic
Bacteriology. W. Wilkins, Editor.
13. Gerhardt, P. 1913. General Bacteriology. American Society for
Microbiology.
14. Barry, A., and C. Thornsberry. 1986. Susceptibility tests: Diffusion
test procedures. In Lannette, E. H., et al. (ed.), Manual of Clinical
Microbiology. ASM press, Washington, DC.
15. Sami, Z. and A. Bari, 1986. Food hygiene with reference to public
health. Viable bacterial counts of ready to eat foods served in
Rawalpindi Islamabad. J Pak Med Assoc 36(12): 304-8.
16. Jones, T. F., and F. Angulo. 2006. Eating in Restaurants: a risk factor
for food borne disease. Clinical Infection diseases 43:1324 - 1328.
17. FAO/WHO. 1974. Food-borne disease: Methods of sampling and
examination in surveillance programmes. 543, W.H.O. (WHO), Editor:
Geneva.
18. Bean N. H., et al. 1996. Surveillance of foodborne-disease outbreaks –
United States, 1988-1992. Morb. Mortal. Wkly. Rep 45(5):1-55.
19. Paulson, D. 1994. A comparative evaluation of different hand
cleansers. Dairy Food Environ Sanit 14:524-528.
20. Restaino, L., and C. Wind. 1990. Antimicrobial effectiveness of hand
washing for food establishments. Dairy Food Environ Sanit 10:136-
141.
21. Hall, H. and G. Hauser. 1966. Examination of faeces from food
handlers Salmonella, Shigella, enteropathogenic Escherichia coli and
Clostridium perfringens. Appl Microbiol. 14: 928-933.
22. Green, L., et al. 2005. Food service workers self reported food
preparation practices: an EHS Net study. International journal of
hygiene environmental health 67:27-35.
23. Hassan, M. M., et al. 2008. Pattern of medical waste management:
existing scenario in Dhaka City, Bangladesh. BMC Public Health 8:36.
24. Kapil, A. 2005. The challenge of antibiotic resistance: need to
contemplate. Indian J. Med. Res. 121(2):83-91.
25. Mulu, A., et al. 2006. Pattern and multiple drug resistance of bacterial
pathogens isolated from wound infection at University of Gondar
Teaching Hospital, Northwest Ethiopia. Ethiop. Med. J. 44(2):125-31.
36