Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
155
SEROLOGICAL, MOLECULAR CHARACTERIZED AND PLASMID
MEDIATED ANTIBIOTICS RESISTANT PATTERNS OF SALMONELLA
SPP. FROM MILK AND OTHER SOURCES
Marwan M. Mohammed, Mohammed H. Khudor
Department of Microbiology, College of Veterinary Medicine, University of .Basrah ,Basrah,
Iraq.
ABSTRACT
This study was carried out for detection of Salmonella isolates from 278 different samples
(direct milk 50 samples, indirect milk 50 samples, feces 50 samples, teat swabs 50 samples ,
hand milker swabs 28 and 50 stool samples ) in Basrah during the period between 20 September
2015 to 5 January 2016. The results revealed that the incidence rate of Salmonella isolates in
samples was 6.1% by using API system, serotyping and PCR technique. Serological methods
revealed that high percentage of Salmonella serotype was Salmonella typhimurium 29.5%. The
highest resistance of Salmonella spp. isolates were found against chloramphenicol and rifampin
(100%). Whereas the lowest resistance was against ciprofloxacin (0.0%). Using plasmid curing
by temperature method showed that 41.1% of total Salmonella isolates were related plasmid
antimicrobial resistance.
INTRODUCTION
Salmonella serotypes remain a potential threat to human and animal health. Infection with
Salmonella may not lead to fatal disease but rather it may remain localized in the gastrointestinal
tract resulting in gastroenteritis or may take a septicemia form that can affect several
organ systems. Infected food animals that do not develop salmonellosis and those that recover
from the disease may become carriers of Salmonella and serve as sources of infection to humans
and animals. Generally, milk considered as nearly perfect food that it contains the essential
nutrients required by the body. However, it is a could be a vehicle for bringing people into
contact with potential microbial, in the developing countries where production of milk and milk
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
156
product takes place under poor hygienic, sanitary and Agricultural practices the safety of dairy
products with respect to food borne diseases is a major issue (1).
However, in some cases the diarrhea may be so severe that the patient becomes dangerously
dehydrated. In severe cases, the Salmonella infection may spread from the intestines to the blood
stream, and then to other body sites, and can cause death. The elderly, infants, and those with
impaired immune systems are more likely to develop severe illness. Some people afflicted with
salmonellosis later experience reactive arthritis, which can have long-lasting, disabling effects
(2). Many of the resistance genes on R plasmids are carried on transposons that can move from a
plasmid to the chromosome, from one plasmid to another, or from the chromosome to a plasmid.
Thus, if one organism has two different plasmids, an antibiotic-resistance gene can move from
one to the other (2).
MATERIALS AND METHODS
The present work was undertaken to isolate and identify Salmonella isolates apparently
depend on their cultural morphological, biochemical characterization, serological and molecular
detection, also plasmid curing method was used to determinate the role of plasmid in
antimicrobials resistance .
A total (287) samples were collected between 20 September 2015 to 5 January 2016 (Direct
milk 50 samples, indirect milk 50 samples, feces 50 samples, teat swabs 50 samples , hand
milkers swabs 28 and 50 stool samples ) in Basrah governorate.
The presence of Salmonella in samples were detected using non-selective enrichment medium
Peptone Bufferd Water (PBW) and incubated at 37ºc for 24 hours then using selective
enrichment medium selenite F broth, incubated at 37ºc for 24 hours,then subculture on
Salmonella -shighella agar (SSA) and Xylose Lysine Dexycholate Agar (XLD) ,incubation at
37ºc for 24 hours (3). The suspected Salmonella were transferred to Triple Sugar Iron (TSI) agar
by stabbing and streaking, incubated at 37°C for 24 hour, also transferred to urea medium tubes,
incubated at 37°C for 24 hour, one large colony inoculated into 5 ml 0.85% NaCl solution to
inoculate the API 20E strip according to the API 20E miniaturized identification system
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
157
(Biomerieux, France) for Salmonella Spp. serotyping was done at the Institute of Public Health,
Baghdad,Iraq.
For PCR assay, Salmonella isolates had been grown in 5 ml of Luria-Betani broth over night
at 37 ᵒC (4), then bacterial DNA extracted according to manufacture of bacterial extraction kit
(Genaid , Korea). The primers used for the detection of 16srRNA gene of Salmonella.(5).
Polymerase chain reaction assays were carried out in 25 μI reaction volume, and the PCR
amplification conditions performed with a thermal cycler were precise to each single primer set
depending on their reference procedure, as shown in table 1.
Antibiotics susceptibility testing
The disc diffusion susceptibility test gives early indication of whether an organism is sensitive,
intermediate or resistant to a specific (12) antibiotics, based on the zone of inhibition around the
disc (6). table 2.
Plasmid curing
Physical agent such as elevated growth temperature is commonly used in plasmid curing then
used same antibiotics discs that used previously on Salmonella isolates dispensed onto the
surface of muller-Hinton agar plate. Then compared the resistance/ sensitive behavior after
curing procedure (7).
RESULTS
The results of this study were showed that the overall identification rate of Salmonella spp.
isolates according to conventional biochemical tests was 27/278 (9.7%), according to each of
API 20 E system, serological methods and molecular method were 17/278 (6.1%).
Serological methods revealed 17 serotypes as: Salmonella typhimurium 5 (29.5%).
Salmonella munchen 4 (23.5%). Salmonella kentucky 3 (17.6 %). ,while other isolates like
Salmonella enteritidis, Salmonella livingstones, Salmonella braenderup, Salmonella ohio and
Salmonella hato were 1 (5.8%) for each ,table 3.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
158
Seventeen isolates of Salmonella spp. which were identified by API 20 E system and
serological methods were subjected to DNA extraction and PCR assay for detection for 16s
rRNA(550bp).Positive results were seen in 17(100 %) of isolates subjected to PCR assay (figure
1).
The results of 17 isolates of Salmonella spp. were tested for their antimicrobial susceptibility
against 12 antimicrobials agents were showed that the highest resistance of Salmonella against
chloramphenicol, vancomycin, lincomycin and rifampin (100%). whereas the lowest resistance
was against ciprofloxacin (0.0%). Statistical analysis showed that there were high significant
differences (P<0.01) between antimicrobial agents ( table 4, figure 2).
.
Plasmid curing by temperature method showed that seven (41.1%) of total Salmonella isolates
were losing their ability to resistance ampicillin, amoxicillin, azithromycin, streptomycin,
ceftriaxone and chloramphenico (table 5,figure 3).
DISCUSSION
Salmonella infection in cattle continues to be a significant problem in intensive production
systems. It caused substantial economic loss both though mortality, carcasses condemnation, and
poor growth after clinical disease and in directly from animal carriage lead to cause of human
salmonellosis which is a major food borne infection in man (8).
The results of this study were showed that the total number of Salmonella isolated from milk
was 12% these results in line with Karshima et al.,(9) in west of India. Results of this study were
also showed that the total number of Salmonella isolates form fecal samples was 6%.these results
agreement with Zelalem et al.,(10).Total number of Salmonella isolated form teat swab samples
were (2%) these result agree with Gedawy et al., (8) The agreement and the difference in the
results may be due to the difference in the living condition, like housing conditions, feeding
habits, types of feed given for the cattle relied on vaccination and treatment procedures (8).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
159
The study showed that the total number of Salmonella isolated form stool samples were (6%)
these result agree with AL –Taie (11) in Bayblon and Mezal et al.,(12) . Results might be
explained by the recovery of adults animals from infection with the certain bacteria also the
human might be the carrier form unhealthy animal to healthy one.
Results of comparison of three different methods (API 20 E, serotyping and PCR) clarified
that there was great similarity in the results rate between API 20E and PCR assay (85.2%), these
results were in agreement with Jawad and Al-Hmadani (13).
By using disc diffusion method, 17 isolates of Salmonella spp. were submitted for their
antimicrobial susceptibility toward 12 antimicrobials. Most isolates show high resistance to
rifampin (100%) , vancomycin (100%), chloramphenicol (100%) and lincomycin (100%).while
most isolates show resistance to nalidixic acid 82%,trimthropin- sulphamethoxide 47%,
ampicillin 58.8% , amoxicillin 58.8% , ceftriaxone 17%, streptomycin 17%, azithromycin 17%
while showed no resistance percentage to ciprofloxacin. These results were in agreement with
the results of Al-Maliki (14) in Basrah and Harakeh et al., (15). There had been a major factor in
the antibiotic resistance between bacteria spp. (16).Many scientists reported that the original
cause of acquired resistance is using of antibiotics in cattle for different purposes such as growth
promotion, or prophylaxes, therapeutics (17).
Seven isolates (41.1%) from 17 isolates showed alteration in antibiotics resistance after
plasmid curing procedure, 28% of cured isolates loss their ability of resistance to ampicillin and
amoxicillin while 42% of cured isolates showed sensitive to azithromycin, chloramphenicol,
ceftriaxone, these results agree with Mirmomeni et al., (18) in Iran. Curing by elevated
temperature is an efficient curing agent. This may be due to the fact that the enzymes of DNA
replication become more affected by high temperature which it involves changing the shape
(folding of the polypeptide) of the enzyme responsible for DNA replication of plasmids, though
it could be that the change makes these enzymes inactive at this temperature (19).
Table (1) PCR primers, PCR conditions and references
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
160
Primer
Name
Nucleotide sequence
(5' to 3')
Size
(pb)
PCR conditions
References
16s rRNA
F: GCAACG CGA AGA ACC
TTA CC
R: GGT TAC CTT GTT ACG
ACT T
550
94°C for 5 min, 35 cycles of
94°C for 1 min, 50°C for 45 sec
and 72°C for, 72°C for 10 min
(White et
al., 2002)
Table (2): Types of antibiotics and their concentrations
No Antibiotics Code Concentration
1 Ampicillin AM 25 mcg
2 Amoxicillin AX 25 mcg
3 Azithromycin AZM 15 mcg
4 Ceftriaxone CRO 10 mcg
5 Chloramphenicol C 10 mcg
6 Ciprofloxacin CIP 10 mcg
7 Lincomycin L 10 mcg
8 Rifampin RA 5 mcg
9 Streptomycin S 10 mcg
10 Trimthropin
Sulphamethoxide
SXT 25 mcg
11 Nalidixic acid NA 30 mcg
12 Vancomycin VA 10 mcg
Table (3): Serotypes of Salmonella isolates with their percentage.
Serotype Number Percentage %
Salmonella Typhimurium 5 29.5
Salmonella Munchen 4 23.5
Salmonella Enteritidis 1 5.8
Salmonella Livingstones 1 5.8
Salmonella Braenderup 1 5.8
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
161
Table (4): Degree of susceptibility of Salmonella isolates against 12 antimicrobial agents.
Antimicrobial Code Resistance
%
Sensitivity
%
intermediate
%
Rifampin RA 100 %
(17/17)
Zero
(0/ 17)
Zero
(0/ 17)
Nalidixic acid NA 82 %
(11/17)
17.6 %
(3/ 17 )
17.6 %
(3/ 17 )
Trimthropin
Sulphamethoxide
SXT 47.0 %
(8/17)
52.9%
(9/17)
Zero
(0/ 17)
Chloramphenicol C 100 % Zero Zero
Salmonella Ohio 1 5.8
Salmonella Kentucky 3 17.6
Salmonella Hato 1 5.8
Total 17 100
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
162
(17/17) (0/ 17) (0/ 17)
Azithromycin AZM 17 %
(3/17)
82 %
(11/17)
17.6 %
(3/17)
Streptomycin S 17%
(3/17)
83%
(12/17)
11.7%
(2/17)
Vancomycin VA 100 %
(17/17)
0%
(0/ 17)
0%
(0/ 17)
Lincomycin L 100 %
(17/17)
Zero
(0/ 17)
Zero
(0/ 17)
Ceftriaxone CRO 17 %
(3/17)
83%
(14/17)
zero%
(0/17)
Ciprofloxacin
CIP 0%
(0/17)
100 %
(17/17)
zero %
(0/17)
Ampicillin AM 58.8%
(9/17)
29.4%
(5/17)
5.8%
(1/17)
Amoxicillin AX 58.8%
(10/17)
29.4%
(5/17)
Zero %
(0/17)
Figure (2): Antimicrobials susceptibility test for Salmonella isolates.
Table (5) Antimicrobial resistance of Salmonella serotypes before and after plasmid curing.
Isolate
Serotype
Antimicrobial resistance
before curing
Antimicrobial resistance
after curing
A5 Salmonella Munchen RA,NA,SXT,C,VA,L,
AM,AX
RA, NA, SXT, C, VA, L.
A7 Salmonella Enteritidis RA,NA,C,S,AZM,VA,L,
CRO
RA, NA, C, VA, L.
A6 Salmonella Hato RA,NA,C,S,AZM,VA,L,
CRO
RA, NA, VA, L.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
163
الخصائص المصلیة ، والجزیئیة وطرز البلازمیدات فی مقاومة المضادات الحیاتیة فی عزلات السالمونیلا
المعزولة فی الحلیب ومصادر اخرى .
مروان میثم محمد ، محمد حسن خضر
فرع ألإحیاء المھجریة ، کلیة الطب البیطری ، جامعة البصرة ، البصرة ، العراق.
الخلاصة
تم جمع 278 عینة ( 50 عینة من الحلیب المباشر ، 50 عینة من الحلیب الغیر مباشر، 50 عینة براز الحیوانی ، 50 عینة
مسحھ من حلمات الثدی ، 28 عینة من أیادی الحلابین ، 50 عینة براز ) فی محافظة البصرة للفترة بین 20 ایلول 2015 لغایة
. 5 من کانون الثانی 2016
ان السالمونیلا (PCR, serotyping, API 20E) بینت الدراسة باستخدام طرق التشخیص الاکثر حساسیة و خصوصیة
متواجدة بنسبة 6.1 % فی عینات الدراسة اعلاه . کشفت طریقة التنمیط المصلی للسالمونیلا أن النمط المصلی
ھو الاکثر تواجدا بین الانماط المصلیة للسالمونیلا ( 29.5 ٪) . أظھرت عزلات السالمونیلا Salmonella typhimurium
مقاومة للمضادات الحیاتیة الکلورامفینیکول وریفامبین بنسبة ( 100 ٪)، فی حین لم تظھر ای مقاومة تذکر تجاه المضاد الحیاتی
٪ المعتمدة على الحرارة لتحدید دور البلازمیدات فی المقاومة تبین ان نسبة 41.1 curing سیبروفلوکساسین .أستخدام طریقة
من إجمالی عزلات السالمونیلا قد فقدت مقاومتھا للمضادات الحیاتیة مما یؤکد دور البلازمیدات المھم فی مقاومة مضادات
المیکروبات لعزلات السالمونیلا قید الدراسة .
REFERENCES
A4 Salmonella Kentucky RA,NA,SXT,C,VA,L,
AM, AX.
RA, NA, SXT, C, VA, L.
M6 Salmonella Munchen RA,NA,C,S,AZM,VA,L,
CRO
RA, NA, C, VA, L.
M12 Salmonella Braenderup RA,NA,SXT,C.VA.L RA,NA,SXT,C.VA.L
S21 Salmonella Typhimurium RA,NA,C,VA,L,AM,AX RA,NA, VA,L,AM,AX
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
164
1-Jordan D (2007). Antimicrobial resistance in animals and impacts on food safety and public
health. Infections, 28(4): 163-164
2-Menghistu, H. T.; Rathore, R.; Dhama, K. and Agarwal, R. K. (2011). Isolation,
identification and polymerase chain reaction (PCR) detection of Salmonella species from field
materials of poultry origin. International Journal of Microbiological Research. 2(2):135-142.
3-Huehn, S.; La Ragione R.; Anjum, M.; Saunders, M.; Woodward, M.; Bunge, C.
Helmuth, R.;Hauser, E.; Guerra, B.; Beutlich. J.; Brisabois, A.; Peters, T. ; Svensson, L.;
Madajczak, G.; Litrup, E.; Imre, A.; Herrera-Leon, S.,; Mevius, D.; Newell, DG.; Malorny,
B. (2010). Virulotyping and antimicrobial resistance typing of Salmonella enterica serovars
relevant to human health in Europe. Foodborne Pathogens Disease. (7): 523-535.
4-Saverino, D.;McDermott, J.; Ferrari, D.; Terrile, M.; and Piatti, G. (2008). Identification
of Salmonella enteric serovar typhi DNA fragments with transcriptional activity under different
growth conditions. The Open Infectious Diseases Journal. 2: 32-38.
5-White, P.; Meglli, K.; Collins, D.; and Gormely, E. (2002). The prevalence and PCR
detection of Salmonella contamination in raw poultry. Veterinary Microbial journal. 89: 53-60
6-Bauer, A.W.; Kirby W.M.; Sherris J.C. and Turk A. (1966). Antibiotic susceptibility
testing by standardized single disk method- American J.Clin. Pathol. 45: 493-6.
7-Carlton B. C.; Brown B. J. (1981). Manual of Methods for General Bacteriology
.Washington, DC: American Society for Microbiology.(2): 222–242. Ed.
8-Callaway, T.; Keen. J.; Edrington T.S.; Baumgard L.H.; Spicer L.;Fonda E.S.; Griswold,
K.E. Overton T,; VanAmburgh M.E; Anderson. R.C.; Genovese K.; Poole. T.; Harvey R.;
Nisbet, D.(2005).Fecal prevalence and diversity of Salmonella species in lactating dairy cattle in
four states. Dairy Science journal. 88: 3603.
9-Karshima, N.S.; Pam, V.A.; Bata, S.I.;Dung, P.A.; Paman, N.D. (2013) Isolation of
Salmonella species from milk and locally processed milk products traded for human
consumption and associated risk factors in . Anim. Prod. Adv. (3): 69-74.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
165
10-Zelalem A.l; Nigatu K.; Zufan S.; Haile A.; Alehegne W.; Tesfu K.(2011). Prevalence and
antimicrobial resistance of Salmonella isolated from lactating cows and in contact humans in
dairy farms of Addis Ababa: a cross sectional study. BMC Infectious Diseases. 11: 222.
11-AL-Taie, H. I. (2009). Prevalence rate of Salmonella in Babylon province. Iraqi Journal of
Veterinary Medicine. 33:2.
12-Mezal, E. H.; Hanan, Z. K.; Saleh M. B. (2016). Identification, antimicrobial resistance of
Salmonella enteritidis isolates from children's diarrhea in Dhi Qar governorate during 2015.
European pharmaceutical journal medical research. 3(6): 01-6.
13-Jawad, A. A. and Al-Hamadani, A. H. (2011). Detection of fimA and fimC genes of
Salmonella isolates by using Polymerase Chain Reaction. Journal of Basrah Researches
(Sciences). 37(4).
14-Al-Maliki, G. M.; Mohamad, E. T.; AL-Abresm, A. N. and AL-Omairi, M. S. (2012).
Prevalence of Salmonella Enteritidis in aquatic bird's eggs (Anas platyrhncha) from farmer's
houses in Basrah marshes, Iraq. J .of Thi _Qar Univ. for Agri. Researches. 1 (2): 1-8.
15-Harakeh, S.; Yassine, H.; Gharios, M.; Barbourc, E.; El-Fadeld, S. H. M.; Toufeilib, I.
and Tannous, R. (2005). Isolation, molecular characterization and antimicrobial resistance
patterns of Salmonella and Escherichia coli isolates from meat-based fast food in Lebanon.
Science of the Total Environment. 341:33– 44.
16-Akbarmehr, J.; Salehi, T. Z. and Nikbakht, B. G. H. (2010). Identification of Salmonella
isolated from poultry by MPCR technique and evaluation of their hsp groEL gene diversity
based on the PCR-RFLP analysis. African Microbiology Research journal. 4(15).
17- Shah, A. H.; and Korejo, N. A. (2005). Antimicrobial Resistance profile of Salmonella
serovars isolated from chicken meat. J. Vet. Anim. Sci. 2: 40-46.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
166
18-Mirmomeni, M. H.; Hossienzadeh. C.;Ghazaey, S.(2007). Molecular study of Salmonella
Enteritidis poultry sample by PCR, plasmid curing and protein pattern analysis. Pakistan journal
of biological sciences. 10(10): 1562- 1570.
19-Kheder, A.K. (2002). Studies on antibiotic resistance by plasmid of Pseudomonas
aeruginosa. Ph.D. thesis .College of Education. University of
(11):856-861.
