Prevalence and Antimicrobial Resistance of Salmonella Isolated from Raw Milk Samples Collected from Kersa District, Jimma Zone, Southwest Ethiopia
Salmonella sp. is one of the most commonly reported foods borne disease
all over the world and developing countries at large. The cattle heath protection
is the basic for production of microbiologically safe and sufficient milk and
also preferable for consumption by human being. So that, antimicrobial resistant
Salmonella were the big threat to public health concern. The increasing
rate of antimicrobial resistance strains were main reason existing for aggravated
bacterial disease. Thus, this study was done to indicate the frequency of antimicrobial
resistance Salmonella isolates from rawcows milk in individual
farmers and dairy farms of Kersa district that is ready for consumption. A cross
sectional study was conducted by collecting rawmilk samples from dairy farms
and individual farmers. Isolation and identification was made by serological
and different traditional biochemical tests methods. The prevalence of Salmonella
spp. in raw milk of the study area was 20%. The isolated Salmonella spp.
were resistant to at least two or more antimicrobials which used in this study.
Among tested drugs Nalidixic acid (80%) was most highly resistant; however,
most susceptible to Ciprofloxacin (95%).So, the study was aimed to determine
prevalence of antimicrobial resistance bacteria and to make the concerned bodies
to take corrective measure.
January 02, 2013; Accepted: February 16, 2013;
Published: May 02, 2013
Salmonella species are a major pathogenic bacterium that causes salmonellosis
on human being and other organisms in the world (Mrema
et al., 2006). Sever cases can result in systemic infections and
even death. WHO (2007) and CLSI (2005)
reports that, Salmonella covers 88% the food borne infection. The Garment;
fecal wastes from infected animals, storage material and ways of handlings are
important sources of Salmonella contamination of the raw milk. According
to Bauer et al. (1996) anti microbial resistance
is currently the greatest challenge to the effective treatment of infections
globally. For instance, more than 80% of food poisoning bacteria such as Salmonella
are reported as antibiotic resistant to at least one type of antimicrobial
and more than 50% as resistant to two or more (Dabassa
and Bacha, 2012). The use antibiotics during animal production were the
main reason for the development of antimicrobial resistant Salmonella spp.
(IFT, 2003). Globally, the three main causes of antimicrobial
resistance have been identified as use of antimicrobial agents in agriculture,
over-prescribing by physicians and misuse by patients (Dabassa
and Bacha, 2012). Routine assessment of patterns of emerging antibiotic
resistant Salmonella strains is of principal importance because such
information channeled to physicians and veterinarians help to timely redirect
drug use so as to diminish the development and spread of resistance. The study
of prevalence and antimicrobial resistance salmonella in milk and milk
products were at a juvenile stage in Ethiopia. However, studies made elsewhere
indicated that milk and milk products are important sources of Salmonella
particularly among those raw milk consumers (Jay, 2000;
Olowe et al., 2007). Different studies indicated
that Salmonella were highly prevalent in Ethiopia both in veterinary
and public setups (Dabassa and Bacha, 2012) even if,
reports from apparently raw cows milk were limited. Thus, this study was
aimed to isolate and identify the antimicrobial resistance Salmonella
from raw cows milk of Kersa District, Southwest Ethiopia.
MATERIALS AND METHODS
Study area: The study was conducted in oromia regional state Jimma Zone,
Kersa Woreda, south western part of Ethiopia from December, 2010 to June, 2011.
Based on figures published by the Central Statistical Agency in 2005, the district
(woreda) has an estimated total population of 329,629, of whom 162,690 were
men and 166,939 women. Agriculture is the major source of economy and it includes
mainly the growing of coffee and cattle rearing. The altitude of this woreda
ranges from 1740 to 2660 above sea level.
Study population: In Kersa district there are many individual farmers
and Dairy farms of milk producers. The four dairy farms from which the study
was investigated have 7-25 lactating Holstein-Friesian cross-bred cows. The
raw milk produced by individual farmer from local lactating cows is consumed
by many farm families in their home whereas the dairy farm owners brought the
milk to local consumers , restaurants and cafeterias at Jimma town that is the
districts of towns of Jimma.
Sample collection: Preliminary visits were made on the distribution
of owners of dairy farms and households vending raw milk in the study area prior
to resuming the actual sample collection. Seven area namely, Ankaso, Serbo town,
Merewa, Bedabuna, Siphanawi, Seredo and Minko were selected purposively based
on their potential for production of milk. The target sampling populations were
defined as all households in the study area who owned milk cows. Of the total
population, some of the households were selected randomly from a list of farmers
registered as milk producers in their respective kebeles.
A total of 100 samples of raw cow milk were separately collected at different
occasions using random sampling technique. Individual raw cow milk samples were
collected aseptically in sterilized 300 mL screw caped bottles from individual
farmers in duplicate and that of dairy farms in triplicate, over a period of
6 months (December to June, 2011). The collected milk samples were transported
to Postgraduate and Research Laboratory of Biology Department, College of Natural
Sciences, Jimma University, using cold chains. After transportation samples
temporarily were kept under refrigerator at 4°C until processed for the
detection of Salmonella within 3 to 8 h of collection.
Isolation and identification of salmonella: The procedure has been used
for detection of Salmonella from milk was as per the ISO-6579: 2002 standard.
Milk sample was dispersed into suitable non-selective medium (buffered peptone
water). One militer of the pre-enrichment culture was transferred into selective
enrichment broth (10 mL Rappaport Vassiliadis soy peptone (RVS) and was incubated
at 41.5±0.5°C for 18-24 h. Subsequently; the enriched sample was
streaked onto each of the Brilliant Green Agar (BGA) and Xylose Lysine Deoxycholate
agar (XLD) and incubated at 37°C for 24 h. The presumptive Salmonella
colony on the XLD and BGA was selected and identified by using serologically
and a series of biochemical tests including reactions on Lysine Iron Agar (LIA),
Triple Sugar Iron agar (TSA), Urea agar, Simmon citrate agar and SIM medium.
Serological tests: The salmonella somatic (O) antigens of the
isolates were determined by slide agglutination test and flagillar (H) antigens
were also identified using a tube agglutination techniques described by (Standard
Microbiological Methods of the Member Companies of the Corn
Refiners Association (2007) and Ewing (1986).
Antimicrobial susceptibility test of salmonella isolates: The antimicrobial
susceptibility testing for Salmonella isolates were carried out following
the Kirby-Bauer disc diffusion method on Mueller-Hinton agar (Oxoid CM0337 Basingstoke,
England) as described in the Clinical and Laboratory Standards Institute (CLSI)
guidelines (NCCLS, 2002). The isolates were tested with
their respective concentration (in brackets) for the following antibiotics(all
from Oxoid);Chloramphenicol (30 μg), Gentamycin (10 μg), Streptomycin
(10 μg), Tetracyline (30 μg), Ciprofloxacillin (30 μg), Kanamycin
(30 μg), Nalidixic acid (30 μg) and Amikacin (30 μg) all from
Oxoid. A standardized suspension of the bacterial isolates was prepared and
adjusted to the 0.5 Mc Farland turbidity standard. Subsequently it was streaked
in to the Muller-Hinton Agar; the antibiotic discs were dispensed on the medium
and incubated at 35°C for 18 h, followed by measurement of zone of inhibition
manually. Finally, the isolates were classified as sensitive and resistant,
as described by Vlkova et al. (2006).The bacterial
characteristics were the main criteria used to select the antimicrobial agents.
Moreover, selection was also based on their mechanisms of action. Salmonella
ATCC 14028 were used as reference strains for quality control of the antibiotics
Frequency of isolation of salmonella: Twenty samples of the total
100 samples were positive for Salmonella isolates. Thus, prevalence
of Salmonella spp, in raw milk of the study area was 20%. With regards
to frequency distribution among selected sites Salmonella spp. were not
detected in 3 of the dairy farms and 2 kebeles. Comparatively high prevalence
of Salmonella was encountered in samples collected from Sipanawi and
serbo (35.71%, 5/14 each) followed by Ankeso (30%, 6/16). The frequency of isolation
of Salmonella in dairy farms (8.33%, 1/12) was not comparable to the
frequency from individual farms (Table 1).
Polyvalent Flagellar (H) and polyvalent somatic (O) tests were confirmed that
20% of the isolate were Salmonella (Table 1) In addition
to that, Salmonella spp. was also tested for the antibiotic susceptibility.
|| Frequency of Salmonella in raw cows milk
||Antibiotic susceptibility patterns of Salmonella isolates
in raw milk
Salmonella isolates were showed highly resistance to Nalidixic acid
(80%) followed by Tetracyline and Kanamycin (35% each) and Amikacin (30%), Gentamycin,
Chloramphenicol and Streptomycin (25% each) and Ciprofloxacin (5%) (Table
Total of 9 Multiple Drug Resistance (MDR) pattern were also observed. The highest
MDR noted was Chl/Te/Nal (15%, 3/20). The maximum MDR registered was resistance
to four antibiotics with the combination Kan/Chl/Nal/Amk being more frequent
(Table 3). In general, MDR to three and four antibiotics dominate
the resistance patterns (25%, 5/20 each).
Several reports have documented the prevalence and distribution of Salmonella
in bulk tank (Sandgren et al., 2008). Evidence
(Hitoshi, 2006; Mahami et al.,
2011; Bauer et al., 1996) indicates that Salmonella
spp are agents for the cause of mastitis in dairy animals and may have contaminated
milk from the udder of infected animals and also reside in the intestinal tract
where they cause gastro-enteritis in animals and may have occurred in milk as
a result of faecal contamination. In the present study the prevalence of Salmonella
spp in raw milk was found 20%.The isolation rate of Salmonella in
this study was related to reports from Gaborone, Botswana 20% (Aaku
et al., 2004). However, it was higher than a study conducted by (Dabassa
and Bacha, 2012), who reported a prevalence of 7.6 and 13.63%, respectively.
Studies made on Salmonella isolation from raw milk and foodborne illness
associated with the consumption of Salmonella contaminated raw milk had
not been clearly documented so far in Ethiopia and Jimma zone in particular.
As a consequence of the high antimicrobial use in dairy farms and individual
cows, bacterial contaminants carried by milk and milk products often show high
levels of antimicrobial resistance (Sandgren et al.,
2008). Salmonella resistant for at least to two or more antimicrobials
which were observed in this study (70%) was lower than 83.3% conducted in Ethiopia
(Dabassa and Bacha, 2012) and elsewhere in the world
(Berge et al., 2004) (75%).
These change as results of increasing rate of wrong way-utilization of antibiotics
in the dairy farms which maintaining resistance genes in bacteria (Aaku
et al., 2004). As a result it should be of concern as it raises food
safety and ethical issues. In the present study, Salmonella isolates
were most susceptible to Ciprofloxacin (95%). This result was similar with the
result reported by (Hawkey, 2008; Sandgren
et al., 2008) from Nigeria.
In addition, the data from (Dabassa and Bacha, 2012)
has indicated that, the effectiveness of such drugs like ciprofloxacin as the
results of the drug were mostly not used for animal treatments. So that, the
result of this study indicated that resistance of Salmonella isolates
to those antibioticslike, Nalidixic acid (80%), Chloramphenicol, Gentamycin
and streptomycin (25% each), kanamycin and tetracycline (35% each) and Amikacin
(30%). However, Salmonella resistance to Tetracycline and Kanamycin (35%each)
and Gentamycin (25%) were found higher in the present study as compared to finding
of (Dabassa and Bacha, 2012; Mrema
et al., 2006) who found that 33.3% and 12%, respectively.
In the current study Nalidixic acid showed a least efficacy against Salmonella
isolates. In addition, the resistance to Nalidixic acid is consistent with the
prevalence of 89-92% reported from Kenya (Lakshmi et
al., 2006). Antimicrobial-resistant salmonella in rawmilk may
be able to colonize the gut if consumed by humans, thus making infections difficult
to treat. Evidence (Mahami et al., 2011; Akoachere
et al., 2009) indicates that the global rise of antimicrobial resistance
is mainly due to the exposure of this bacteria in human and veterinary medicine
and indiscriminate use of drug for the treatment of both human and animal disease
caused by Salmonella sp.
Examining the prevalence and drug resistance pattern of Salmonella from
raw milk in dairy farms and individual farmers is the best mechanism to plan
methods of reducing the ways of transmission of Salmonella between humans
and cattle. Likewise, it imperative in fighting the development of drug resistant
strains of Salmonella. The result obtained in this study (80%) is significantly
high to the widely observed food borne salmonellosis in the area. Moreover,
medium proportion (35-25%) of Salmonella isolates were resistance to
two or more of the antimicrobials drugs. This condition creates big problems
on human medical treatment. This finding showed that additional exploration
is necessary on the prevalence and antimicrobial resistance pattern of Salmonella,
which is food borne pathogen. In line with the experiential evidences, which
indicate the uncontrolled use of antimicrobials for animal and public health
treatments were the crucial reason for high rate of antimicrobial resistant
Forall necessary support to the successful completion of this research, Jimma
University, Department of biology members were gratefully acknowledged. We honestly
thank our friends who involved in critical reviewing and helpful comments for
this work. Dairy farm owners and other individual farmers who allowed taking
the milk sample for this research were also acknowledged.
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