Hindawi

International Journal of Bacteriology

Volume 2017, Article ID 4604789, 9 pages
https://doi.org/10.1155/2017/4604789

Research Article
Molecular Characterization of Salmonella from Human and
Animal Origins in Uganda

Atek Atwiine Kagirita,1 Andrew Baguma,1,2 Tonny Jimmy Owalla,3

Joel Bazira,2 and Samuel Majalija4
1
Department of Disease Surveillance and Outbreak, Uganda National Health Laboratories Services, Ministry of Health,
P.O. Box 7272, Kampala, Uganda
2
Department of Microbiology, Faculty of Medicine, Mbarara University of Science and Technology, P.O. Box 1410, Mbarara, Uganda
3
Division of Infectious Diseases, Med Biotech Laboratories, P.O. Box 9364, Kampala, Uganda
4
Department of Biosecurity, Ecosystem and Veterinary Public Health, College of Veterinary Medicine,
Animal Resources and Biosecurity, Makerere University, P.O. Box 7062, Kampala, Uganda

Correspondence should be addressed to Joel Bazira; jbazira@gmail.com

Received 18 December 2016; Revised 25 March 2017; Accepted 24 April 2017; Published 28 May 2017

Academic Editor: Kumar Venkitanarayanan

Copyright © 2017 Atek Atwiine Kagirita et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Sporadic Salmonella outbreaks with varying clinical presentations have been on the rise in various parts of Uganda. The sources
of outbreaks and factors underlying the different clinical manifestation are curtailed by paucity of information on Salmonella
genotypes and the associated virulence genes. This study reports molecular diversity of Salmonella enterica and their genetic
virulence profiles among human and animal isolates. Characterization was done using Kauffman-White classification scheme and
virulence genes analysis using multiplex PCR. Overall, 52% of the isolates belonged to serogroup D, 16% to serogroup E, 15% to poly
F, H-S, and 12% to serogroup B. Serogroups A, C1, and C2 each consisted of only one isolate representing 5%. Virulence genes located
on SPI-1 [spaN and sipB] and on SPI-2 [spiA] in addition to pagC and msgA were equally distributed in isolates obtained from all
sources. Plasmid encoded virulence gene spvB was found in <5% of isolates from both human epidemic and animal origins whereas
it occurred in 80% of clinical isolates. This study reveals that serogroup D is the predominant Salmonella serogroup in circulation
and it is widely shared among animals and humans and calls for joint and coordinated surveillance for one health implementation
in Uganda.

1. Introduction by Salmonella enterica serovar Typhi (typhoid fever) and

the second usually takes the form of a self-limiting food
Salmonella, subspecies enterica, is an important food-borne poisoning (gastroenteritis) and is caused by a large number of
pathogen responsible for disease in animals and humans. It nontyphoidal Salmonella serovars (NTS) resulting in a range
has been the leading cause of many outbreaks and infections of clinical syndromes including diarrheal disease [3] and
around the world and is considered as one of the major causes fever frequently occurs in developing countries and affects
of human gastroenteritis worldwide [1]. Animals have been an estimated 21.5 million persons annually, spreading mainly
implicated as important sources of Salmonella-contaminated from person to person. In contrast, nontyphoidal salmonel-
food products that are responsible for human salmonellosis losis is a worldwide disease of humans and animals and has
and in the United States approximately 40,000 cases of been long recognized as a public health problem responsible
salmonellosis are reported resulting in 600 deaths [1, 2]. for an estimated 1.4 million cases/year, with ∼16,000 hospi-
The clinical outcome of Salmonella infection in humans talizations and 600 deaths in developed countries [4, 5]. In
presents in two broad features; first, it manifests as a Sub-Saharan Africa, nontyphoidal Salmonella are emerging
serious systemic infection (enteric fever) caused mainly as a prominent cause of invasive disease in infants and young
2 International Journal of Bacteriology

children, although estimates of incidences have been carried biochemical identification of Salmonella were done using
out in isolation, giving no overall demographic picture [6]. standardized selective and identification media [14]. The
Notably, the severity of the infection and whether it identified Salmonella isolates were then used in subsequent
remains localized in the intestine or disseminates to the analysis.
bloodstream depend on the Salmonella serotype, immune
status of the host, and the virulence of the Salmonella isolate 2.3. Serogrouping of Salmonella Isolates. Using commercial
[7]. The recognition of the virulence in Salmonella as a monovalent and polyvalent Salmonella O antisera (Statens
major determinant of the outcome of human infection has Serum Institute, Denmark), serogrouping was carried out
been well appreciated [7], and among the various known by slide agglutination tests according to the manufacturer’s
virulence factors, studies indicated that Salmonella species instruction manual. Approximately 10–15 𝜇L of diluted anti-
which putatively possess virulence genes such as hilA and sera was delivered onto a glass slide. An equal volume of
invA are consistently associated with severe illness compared bacterial suspension was added onto the slide and mixed with
with those which lack such genes [8, 9]. a loop. The slide was rocked back and forth for 60 seconds
Recently, there have been increased frequencies of spo- while observing agglutination. An isolate was considered to
radic and epidemic Salmonella outbreaks associated with be positive if agglutination occurred. The Salmonella isolates
high prevalence of intestinal perforation in humans in were classified into O serogroups based on surface antigens
Uganda [10]. Although the common Salmonella serotypes [15]. Polyvalent group F, H-S were isolates that reacted
circulating in Uganda and their antibiograms had been fairly positive to polyvalent O Salmonella A-S group antigen but
studied [11, 12], information regarding the casual relationship negative to monovalent specific O antigen of serogroups A,
between the human-animal overlap of S. enterica serovars or B, C1, C2, D, E, and G.
their virulence profiles had remained scarce and subsequently
warranted an investigation. We hereby report the circulating 2.4. Antimicrobial Susceptibility Testing of Isolates. The antibi-
Salmonella serotypes and virulence-associated genes in sam- otic susceptibility profiling of the Salmonella isolates was
ples obtained from both humans and animals sources. determined by Kirby-Bauer disk diffusion method [16].
Seven different antibiotics were used in this study, namely,
2. Materials and Methods tetracycline (30 𝜇g), chloramphenicol (30 𝜇g), trimethoprim-
sulphamethoxazole (25 𝜇g), ciprofloxacin (5 𝜇g), nalidixic
2.1. Study Design, Population, and Sampling. The study acid (30 𝜇g), ampicillin (10 𝜇g), and ceftriaxone (30 𝜇g) (Mast
utilized archived Salmonella isolate cultures preserved in Group Ltd, Merseyside, UK). Antimicrobial susceptibility
20% glycerol (v/w) soft agar at −80∘ C from both human was done on Muller Hinton agar (Mast Group Ltd, Mersey-
and animal specimen obtained between 2007 and 2009. side, UK) and Escherichia coli ATCC 25329 control strain
The human isolates were obtained from patients diagnosed was used. The diameter of zonal clearance was measured
with gastroenteritis from three sources: Mulago National in millimeters and results were recorded as susceptible (S),
Referral Hospital, Tororo Hospital, and Kasese District. The intermediate (I), and resistant (R) according to CLSI, perfor-
human isolates obtained from stool samples or blood cultures mance standards for antimicrobial susceptibility testing [17].
of patients with sporadic gastroenteritis visiting Mulago
National Referral Hospital and Tororo Hospital from 2007 2.5. Molecular Analysis
to 2009 are hereafter referred to as sporadic human clinical
isolates. Additional Salmonella isolates from Kasese District 2.5.1. Total DNA Extraction and Purification. Genomic DNA
were collected during an epidemic in the same period, here from Salmonella isolates cultured in a sodium thioglycolate
referred to as human epidemic outbreaks isolates. The animal broth was extracted using the chloroform-isoamyl alcohol
isolates were collected from rectal faeces in slaughter cattle at method. Briefly, Cells were harvested by centrifugation at
the Kampala city abattoir and poultry anal swabs obtained 2400 g for 10 min in an IEC CL31R multispeed centrifuge
from different geographical places in Kampala city between (Thermo Scientific). The supernatant was discarded and
2007 and 2009 and archived at the Microbiology Labora- the pellet resuspended in 400 𝜇L Tris-EDTA (TE) buffer,
tory, College of Veterinary Medicine, Animal Resources and containing 0.01 M Tris-HCl pH 7.4, 0.001 M EDTA with gentle
Biosecurity, Makerere University. The study employed census agitation. Seventy microliters of 10% SDS (sodium dodecyl
method of sample size estimation. Due to the number of sulphate) (Fisher Scientific) was added to the suspension,
individuals targeted, the selection of Salmonella isolates was followed by 5 𝜇L of (10 mg/mL stock solution) proteinase
done using a nonprobability purposive method [13] of all K (Ambion). The samples were then incubated for 1 hr at
isolates collected from 2007 to 2009. A total of 69 Salmonella 65∘ C in hybridization oven (Biometra OV2, Anachem, UK).
isolates were used in this study, 48 were human isolates, and After incubation, 100 𝜇L of 5 M NaCl followed by 100 𝜇L
21 were animal isolates. CTAB/NaCl (prewarmed at 70∘ C) was added to the solutions.
The solution was gently inverted for 10 sec, incubated at 65∘ C
2.2. Recovery and Identification of Salmonella. Archived bac- for 20 min, and then cooled at room temperature for 5 min.
terial samples were thawed and inoculated into sodium Seven hundred and fifty microliters of chloroform-isoamyl
thioglycolate broth using sterile inoculating loop, incubated alcohol (24 : 1, Sigma-Aldrich) was added and centrifuged at
overnight at 37∘ C, growth detected by observing turbidity. 1300 ×g for 15 min. The supernatants were transferred to new
All further procedures like enrichment, subculturing, and tubes, treated with 5 𝜇L RNase A (5 mg/mL in RNase A buffer
International Journal of Bacteriology 3

Table 1: Distribution of Salmonella isolates obtained from different origins according to serogroups.

Sources of isolates
Animal Total
Serogroups Human clinical (𝑛 = 25) Human epidemic (𝑛 = 23)
(𝑛 = 21) 𝑛 = 69
Poly F, H-S 6 0 4 10
A 1 0 0 1
B 8 0 0 8
C1 1 0 0 1
C2 1 0 0 1
D 8 22 5 35
E 0 0 11 11
Untypable 0 1∗ 1∗ 2
All isolates were negative to monovalent O antisera G serogrouping. ∗ Untypable isolates.

containing 0.5 M NaCl, 0.01 M EDTA), and incubated at Table 2: Prevalence of Salmonella serogroups from different species
37∘ C for 30 min. DNA was precipitated with 500 𝜇L ice-cold of animals.
isopropanol containing 0.1 volume sodium acetate and mixed Chicken Cattle Pig Total
carefully by inverting the tubes. The pellets were incubated at Serogroup
𝑛 = 12 𝑛=7 𝑛=2 𝑁 = 21
−80∘ C for 30 min and centrifuged at 13000 ×g for 10 min. The
Poly F, H-S 4 0 0 4
supernatant was discarded and the pellets were washed twice
with 1 mL of cold 70% ethanol followed by centrifugation D 2 2 1 5
at 13000 ×g for 15 min. The pellets of DNA were dried at E 6 4 1 11
37∘ C for 30 min and reconstituted in 50 𝜇L elution buffer Nontypable 0 1 0 1
[Qiagen]. The quality of DNA was examined by running 4 𝜇L None of isolates in serogroups A, B, C1, and C2 were detected.
of the DNA sample on 0.85% agarose gel. The DNA was
subsequently stored at −20∘ C awaiting further analysis.
as judged by the molecular marker. These scores were entered
2.5.2. Multiplex PCR for Detection of Virulence Genes. In into a Microsoft Excel 2007 spreadsheet and then tabulated.
order to predict the virulence potential of Salmonella isolates
from different sources, the presence of 6 virulence genes
(spiA, pagC, msgA, sipB, spaN, and spvB) was screened as two
3. Results
runs; that is, run 1 consisted of 4 (spiA, pagC, msgA, sipB) and 3.1. General Serogroup Distribution Pattern of Salmonella
run 2 consisted of 2 (spaN and spvB) genes, respectively, using Isolates Obtained from Different Sources. The 69 Salmonella
multiplex PCR assays. isolates used in this study were obtained from three sources:
The forward and reverse primers used for detection of 25 (36%) from sporadic human clinical cases, 23 (34%)
virulence gene and the amplification conditions were adapted from human epidemic outbreaks, and 21 (30%) from animal
from Versalovic et al. [18]. Amplifications were performed in sources (Table 1). A total of 67 isolates were typable using
a 25 𝜇L reaction mixture comprising 2.5 𝜇L of template DNA, polyvalent O antisera for A-S antigens; the nontypable O
16.3 𝜇L of RNase free H2 O, 2.5 𝜇L of 10x PCR buffer, 3.0 𝜇L of antigen was expressed by 2 isolates which were recorded as
50 mM MgCl2 , 0.15 𝜇L of Taq 5 U/𝜇L [Thermo Fisher], 0.5 𝜇L NT as shown in Table 1. Further subtyping of 67 polyvalent A-
of 10 mM dNTPs mix (New England Biolabs), and 0.05 𝜇L S positive isolates segregated the isolates into serogroups A, B,
of 0.1 mM forward and reverse primers. Amplification was C1, C2, D, E, and G. The monovalent specific serotyping indi-
performed in a thermocycler (MJ Research, PTC 200 USA) cated that majority of isolates (52%) belonged to serogroup
under the following conditions: 1 cycle of initial denaturation D, serogroup E 16%, poly F, H-S 15%, and serogroup B with
at 95∘ C for 5 min, denaturation at 94∘ C for 30 sec, and 12% (Figure 1). The remaining serogroups A, C1, and C2 each
annealing for 1 min at 53∘ C for spvB, spiA, pagC, and msgA consisted of only one isolate representing 5% of the isolates
and at 56∘ C for sipB and spaN and 2 min at 72∘ C, with a (Table 1). Of the 10 isolates belonging to poly F, H-S, 6 (60%)
final cycle of 10 min at 72∘ C, followed by a hold at 4∘ C. were of human clinical origin and 40% were of animal origin.
The resulting PCR products were subjected to horizontal gel The animal isolates were obtained from pigs, 2 (9.7%), cattle,
electrophoresis in 2% agarose and visualized using BioDoc- 7 (33.3%), and chickens, 12 (57%), as in Table 2. All isolates
It Imaging (UVP, USA) and images retrieved from the belonging to serogroups A, B, C1, C2, and E were exclusively
computer in the data office. of human clinical and animal origins, respectively. Of the 35
isolates belonging to group D, the majority, 63%, were from
2.6. Data Analysis. Virulence gene results were analyzed human epidemic followed by 23% from human clinical cases
visually and an isolate was considered to contain the virulence and 14% from animal origins as shown in Table 1. Overall,
gene of interest if it produced an amplicon of the expected size the majority of the human isolates (30 of 48; 62%) belonged
4 International Journal of Bacteriology

Table 3: Antibiotic resistance profiles of Salmonella isolates according to serogroups.

𝑁 (%) of resistant Salmonella serogroups against selected antibiotics

Antibiotics tested B C1 C2 D E F, H-S
(𝑛 = 8) (𝑛 = 1) (𝑛 = 1) (𝑛 = 35) (𝑛 = 11) (𝑛 = 10)
C (30 𝜇g) 6 (75) 0 0 07 (20) 0 5 (50)
SXT (25 𝜇g) 6 (75) 0 0 29 (83) 0 6 (60)
Amp (10 𝜇g) 8 (100) 1 (100) 1 (100) 34 (97) 7 (66) 8 (80)
T (30 𝜇g) 4 (50) 0 0 27 (77) 1 (9) 5 (50)

Poly F, H-S
[10, 15%]
Serogroup
Serogroup E
[11, 16%] A, C1, C2 [3, 5%]

Serogroup B
[8, 12%]

Serogroup D
[35, 52%]

Figure 1: General serogroup distribution pattern of Salmonella isolates obtained from different sources.

to serogroup D (Table 1). With respect to the source, 22 of (Cip), ceftriaxone (CRO), nalidixic acid (NA), chlorampheni-
23 (96%) isolates from human epidemic cases belonged to col (C), trimethoprim-sulphamethoxazole (SXT), ampicillin
group D (Table 1). While one isolate was nontypable, none (Amp), and tetracycline (T). All Salmonella serogroups and
of the Salmonella belonging to serogroups A, B, C1, C2, nontypable isolates showed no resistance to ciprofloxacin
and E were detected (Table 3). On the other hand, isolates and ceftriaxone while 12.5% resistance to nalidixic acid was
obtained from sporadic human infections, 35% (8 of 23), detected. Serogroup A isolate was resistant to all antibiotics
belonged to serogroup B or D, while 6 of 23 (26%) were assayed (Table 3).
poly F, H-S (Table 1). The remaining 3 isolates each belonged However, resistance pattern to the rest of the antibiotics
to serogroups A, C1, and C2, respectively. None of isolates varied widely among serogroups. Serogroup D showed the
in serogroup E or expressing a nontypable O antigen were highest variability from 20% to 97% resistance to chloram-
detected. phenicol, tetracycline, trimethoprim-sulphamethoxazole,
and ampicillin antibiotics. All the isolates of serogroups C1
3.2. Prevalence of Serogroups of Salmonella from Different and C2 were susceptible to all the antibiotics assayed
Species of Animals. The majority of the isolates, 11 of 21 except ampicillin. The resistance pattern for polyvalent
(52.4%), were of serogroup E, followed by 5 of 21 (24%) for group F, H-S ranged from 50% to 80% for tetracycline,
serogroup D, while 19% belonged to polyvalent F, H-S group trimethoprim-sulphamethoxazole, and ampicillin. Isolates
(Table 2). One isolate expressing a nontypable O antigen was belonging to serogroup B showed more than 50% resistance
detected. None of isolates in serogroups A, B, C1, C2, and G to all the above four antibiotics while isolates of group E
were detected. Of the 3 animal species from which specimens showed resistance to ampicillin and tetracycline. From this
were taken, chicken had all the three serogroups (E, D, and study, Salmonella isolates showed the highest resistance to
poly F, H-S) with 6 of 12 (50%) from serogroup E, 4 of 12 (33%) ampicillin followed by trimethoprim-sulphamethoxazole.
from serogroup D, and the rest from group F, H-S (Table 2). The most resistant serogroups were A and B and these were
Similarly, 4 out of 7 (57%) of isolates from cattle belonged predominantly found in human clinical specimens (Table 3).
to serogroup D. One isolate belonged to serogroups D and Over 60% of all the isolates were resistant to trimetho-
E each in the pig isolates (Table 2). prim-sulphamethoxazole, ampicillin, and tetracycline (Fig-
ure 2). The antibiotic susceptibility test revealed the pres-
3.3. Antimicrobial Resistance Profiling. To understand the ence of multiple drug resistance in both routine clinical
antimicrobial resistances profiles of the Salmonella under this and epidemic isolates. Furthermore, human clinical isolates
study, all 69 isolates were tested against seven antibiotics showed the highest variability in resistance patterns from
(Table 3). The antimicrobial drugs used were ciprofloxacin 60% to 100% for all antibiotics from tetracycline to ampicillin
International Journal of Bacteriology 5

Table 4: Distribution of virulence-associated genes among Salmonella isolates according to origins.

Number (%)
Virulence genes Clinical samples Human epidemic Cattle Pigs Poultry All samples
𝑛 = 25 𝑛 = 23 𝑛=7 𝑛=2 𝑛 = 12 𝑁 = 69
spvB 20 (80) 01 (4.3) 01 (14.3) 00 (0.0) 00 (0.0) 22 (31.9)
spiA 22 (88) 19 (82.6) 06 (85.7) 01 (50.0) 08 (66.7) 56 (81.0)
pagC 23 (92) 19 (82.6) 06 (85.7) 01 (50.0) 08 (66.7) 57 (82.6)
msgA 23 (92) 22 (95.6) 06 (85.7) 02 (100) 09 (75.0) 62 (89.9)
sipB 21 (84) 20 (87.0) 06 (85.7) 02 (100) 10 (83.3) 59 (85.5)
spaN 23 (92) 22 (95.6) 06 (85.7) 02 (100) 10 (83.3) 63 (91.3)

Table 5: Distribution of virulence-associated genes among predom-

T inant Salmonella serogroups.

Amp Number (%) of isolates per serogroup positive for

Virulence the different virulence genes
SXT genes F, H-S
D (𝑛 = 35) E (𝑛 = 11)
(𝑛 = 10)
C spvB 8 (22.8) 1 (9.1) 4 (40)
spiA 29 (82.9) 10 (90.9) 6 (60)
CRO
pagC 30 (85.7) 10 (90.9) 6 (60)
NA msgA 34 (97.1) 10 (90.9) 7 (70)
sipB 30 (85.7) 11 (100) 6 (60)
Cip spaN 34 (97.1) 11 (100) 7 (70)

0 20 40 60 80 100 120

Animal n = 21 Figures 4(a) and 4(b). Overall, the most prevalent virulence-
Epidemic n = 23 associated genes in human epidemic cases were msgA and
Clinical n = 25 spaN while spiA, sipB, and pagC were present in about 80%
Figure 2: A bar graph showing resistance patterns of Salmonella iso- of the isolates (Table 4, Figures 3(b) and 4(b)). Multiplex
lates according to origins. NA = nalidixic acid; Cip = ciprofloxacin; PCR assays for the amplification of (spvB, spiA, pagC, msgA,
CRO = ceftriaxone; C = chloramphenicol; SXT = trimethoprim- sipB, and spaN) genes were carried out in Salmonella isolates
sulphamethoxazole; Amp = ampicillin; T = tetracycline. from the animal samples (Figure 5). Isolates of animal origin
exhibited the same virulence-associated gene pattern as those
of human origin (Table 4 and Figure 5). Isolates from cattle
(Figure 2). Isolates obtained from human epidemic cases (>85%) possessed spaN, msgA, sipB, pagC, and spiA and only
registered >95% resistance to 3 antibiotics (trimethoprim- one had spvB (Table 4). Strikingly, SpvB was not found in
sulphamethoxazole, tetracycline, and ampicillin) and a rel- isolates from either pig or poultry. Overall, there appeared
atively low resistance to chloramphenicol at 9% compared to be no major differences in the virulence-associated gene
to human clinical cases isolates. On the other hand, animal profiles of animal (poultry, cattle, and pig) and outbreak
Salmonella isolates showed minimal resistance of less than sources, or any differences among the multiple isolates of
10% to trimethoprim-sulphamethoxazole and tetracycline human origin (Table 4).
but a higher resistance of 67% to ampicillin (Figure 2). All
isolates were susceptible to ceftriaxone and ciprofloxacin. 3.5. Carriage of Virulence Genes by Salmonella Isolates from
One isolate of clinical origin showed resistance to nalidixic Different Serogroups. The distribution of the 6 virulence-
acid. associated genes in Salmonella is summarized as shown in
Table 5. All the virulence genes assayed except spvB were
3.4. Distribution of Virulence Genes among Salmonella Isolates present in all the isolates belonging to serogroups A, B,
of Different Origins. As shown in Figures 3(a) and 3(b) C1, and C2 and in more than 80% of isolates belonging to
and Table 4, majority of Salmonella isolates carried spiA, serogroups D and E. spvB was present in 7 (87.5%) of the
pagC, and msgA genes regardless of origin. In contrast, spvB isolates in serogroup B while being found in all the isolates
was only detected in 1 out of 21 (5%) isolates from the belonging to serogroups A and C1. Conversely, none of the
epidemic and in 80% of the sporadic cases (Table 4). Further isolates in serogroup C2 possessed spvB and only 9 (25.7%)
amplification of two virulence genes (spaN and sipB) was isolates belonging to serogroup D had this virulence gene.
carried out and the resultant banding pattern is shown in spvB was found in 9.7% and 40% of isolates belonging to
6 International Journal of Bacteriology

1000

(bp)
717 bp (spvB)
500 550 bp (spiA)
400 454 bp (pagC)
200
189 bp (msgA)
100
(a)

1000
717 bp (spvB)
(bp)

500 550 bp (spiA)

400 454 bp (pagC)

200 189 bp (msgA)

100

(b)

Figure 3: Multiplex PCR results for spvB, sipA, pagC, and msgA genes in human Salmonella isolates. (a) M, 100 bp ladder; P, positive control;
N, negative control; lanes 2–17, human clinical isolates. (b) Lane M, 100 bp ladder; P, positive control; N, negative control; lanes 18–21, human
clinical isolates from sporadic cases; lanes 22–34, Salmonella isolates from human epidemic cases.
(bp)

1000 875 bp (sipB)

500 504 bp (spaN)

100

(a)
(bp)

1000 875 bp (sipB)

500 504 bp (spaN)

100

(b)

Figure 4: Multiplex PCR results for sipB and spaN genes in human Salmonella isolates. (a) Lane M, 100 bp ladder; P, positive control; N,
negative control; lanes 2–11, human clinical isolates. (b) Lane M, 100 bp ladder; P, positive control; N, negative control; lanes 26–35, Salmonella
isolates from human epidemic outbreaks.
International Journal of Bacteriology 7

1000
717 bp (spvB)
(bp) 500 550 bp (spiA)
400 454 bp (pagC)

200
189 bp (msgA)
100

Figure 5: Multiplex PCR results for spvB, sipA, pagC, and msgA genes in animal Salmonella isolates. Lane M, 100 bp ladder; P, positive control;
N, negative control; lanes 39–49, samples.

serogroups E and polyvalent F, H-S, respectively. All the 11 divergent serogroups (A, B, C1, and C2) not found in samples
isolates in serogroup E had sipB and spaN whereas 10 had from animal origin with exception of poly F, H-S, suggesting
msgA, pagC, and spiA. About 60% of isolates in polyvalent that the potential source of clinical infection could have been
F, H-S had spiA, pagC, and sipB while 7 (70%) had msgA and from the environment, soil, and water. However, the role of
spaN with 4 (40%) having spvB. Interestingly, none of the 2 animals as potential source of these pathogens could not be
nontypable isolates had any of the six virulence genes assayed ruled out since a small sample size was used. There is need for
in this study. a larger sample size in future studies to determine whether
such serogroups reported in this study may be infecting
4. Discussion animals in Uganda.
In terms of the distribution of drug resistance among
This study sought to characterize Salmonella enterica isolates Salmonella isolates, high variability of patterns with respect
from both animal and humans samples as well as detecting to serogroups and sources is confirmed. Although animals are
the associated virulence genes. Phenotyping showed a high known to be the main reservoir of Salmonella and a common
variation in serogroups among isolates obtained from all source of human contamination [1, 2], it is interesting to
the sources. Fifty-two percent of all the isolates belonged note that the frequency of antibiotic resistance to tetracy-
to serogroup D, 16% to serogroup E, 12% to serogroup B, cline, ampicillin, and trimethoprim-sulphamethoxazole in
and 15% to poly F, H-S while serogroups A, C1, and C2 this study was higher in isolates of human origin than in
each consisted of only one isolate. Not surprisingly, all the animal and/or poultry isolates. This observation conflicts
serogroups identified in this study belong to Salmonella with reports by other authors [23, 24] and extricates the
enterica I subspecies which is responsible for most of human antibiotic use in animal feeds as a potential source of
salmonellosis [19]. There was a remarkable variation in resistance induction to human infections. Plausible expla-
distribution of serogroups according to isolate sources. Of nations could be as follows: (i) selective pressure existing
the 35 isolates belonging to serogroup D, majority, 63%, in hospital environments that strongly contribute to the
were from human epidemic cases, 23% from routine clinical increasing resistance of strains isolated in such environments;
cases, and 14% from animal origins. This indicates that there (ii) irrational use of antibiotics through either easy access
is wide sharing of isolates between animals and humans, self-medication, poor prescription, or incomplete dosage in
incriminating animals partly as potential source of clinical the general population. The same reasons could also shed
salmonellosis. Previous studies have highlighted the signif- light on the observed resistance to chloramphenicol and
icance of zoonotic salmonellosis in human infections [20]. nalidixic acid exhibited by the clinical isolates collected from
It was observed that Salmonella isolates collected from the Kampala versus epidemic samples from Kasese District and
human epidemic used in this study were serotyped as S. Typhi further corroborated findings by Mahero et al., 2013 [24].
[10]. The fact that S. Typhi is not animal adapted [21] implies Although these isolates belonged to a different serogroup
that the animal isolates that shared serogroup D could be from those causing human epidemics, this poses a big
of different serotype. This is because the antigenic coat D is threat to public health interventions and calls for effective
shared by diverse Salmonella strains belonging to multiple surveillance program to ascertain its magnitude.
serotypes. Salmonella isolates incriminated in human epidemic out-
Conversely, isolates belonging to serogroup E were exclu- break, clinical cases, and Salmonella isolated from clinically
sively of animal origin and this result conforms to existing healthy animals were assayed for their possession of certain
literature showing that all serotypes in serogroup E are animal putative virulence genes in an effort to identify factors that
restricted [22]. One striking observation is that most of are important to the virulence of this organism. Of particular
the human clinical salmonellosis was caused by isolates of interest in this study were genes involved in intracellular
8 International Journal of Bacteriology

survival, adhesion, and invasiveness [25, 26]. The genes of Health Sciences, Makerere University, for laboratory assis-
include spaN, sipB, and spiA which are associated with type tance and technical support.
III secretory system [27–29]. The msgA, spvB, and pagC
genes encode products that are associated with cell invasion,
survival within the cell, and adhesin or pili production [30–
References
32]. [1] E. Scallan, F. J. Angulo, M. Kirk et al., “The global burden
Genes located on SPI-1 (spaN and sipB), and on SPI-2 of nontyphoidal Salmonella gastroenteritis,” Clinical Infectious
(spiA) along with pagC and msgA were found in 100% of Diseases, vol. 50, no. 6, pp. 882–889, 2010.
isolates belonging to serogroups A, C1, C2, and B and in more [2] CDC, “Salmonellosis associated with pet turtles-Wisconsin and
than 80% in isolates in serogroups D and E, suggesting a Wyoming,” Morbidity and Mortality Weekly Report (MMWR),
ubiquity of these genes in Salmonella serogroups. Similarly, vol. 54, no. 9, pp. 223–226, 2005.
these genes were present in over 80% of isolates obtained [3] S. I. Miller and D. A. Pegues, “Salmonella species, including
from either human clinical, epidemic, or animal origins Salmonella typhi,” in Principles and Practice of Infectious Dis-
suggesting their wide distribution among Salmonella isolates ease, G. L. Madell, J. E. Bennett, and R. Dolin, Eds., Churchill
regardless of their host of origin. A recent study assaying >50 Livingstone, New York, NY, USA, 5th edition, 2000.
genes also did not find major differences in the virulence- [4] G. K. Adak, S. M. Long, and S. J. O’Brien, “Trends in indigenous
associated gene profiles of the poultry (egg houses and farm) foodborne disease and deaths, England and Wales: 1992 to
and outbreak strains, nor any differences among the multiple 2000,” Gut, vol. 51, no. 6, pp. 832–841, 2002.
isolates of serovars [33]. Similarly, another study, which [5] A. C. Voetsch, T. J. Van Gilder, F. J. Angulo et al., “Food-
included 17 genes, did not find any significant difference Net estimate of the burden of illness caused by nontyphoidal
between sick and clinically healthy birds [34]. Salmonella infections in the United States,” Clinical Infectious
In contrast to these highly prevalent genes, spvB gene Diseases, vol. 38, no. 3, pp. S127-S134, 2004.
was found in <5% and 15% of isolates from human epidemic [6] S. M. Tennant, S. Diallo, H. Levy et al., “Identification by
and animal origins, respectively, whereas it occurred in about PCR of non-typhoidal Salmonella enterica serovars associated
80% of clinical isolates. In addition, this gene was present with invasive infections among febrile patients in Mali,” PLoS
in >80% of isolates belonging to serogroups A, B, and C1 Neglected Tropical Diseases, vol. 4, no. 3, article e621, 2010.
and in <23% of isolate in serogroups D, E, and poly F, H- [7] E. W. Hook, “Salmonellosis: certain factors influencing the
S. However, not all isolates of a plasmid bearing serogroups interaction of Salmonella and the human host,” Bulletin of the
contain this virulence plasmid, which could explain why spvB New York Academy of Medicine, vol. 37, no. 7, pp. 499–512, 1961.
was found in such a low proportion of isolates compared [8] M. L. Lesnick, N. E. Reiner, J. Fierer, and D. G. Guiney, “The
with chromosomal genes. Overall, the strong similarities in Salmonella spvB virulence gene encodes an enzyme that ADP-
virulence genotypes between isolates obtained from different ribosylates actin and destabilizes the cytoskeleton of eukaryotic
cells,” Molecular Microbiology, vol. 39, no. 6, pp. 1464–1470, 2001.
origins might indicate that the Salmonella of animal and
human clinical and epidemic cases are capable of causing [9] S. M. F. Castagna, M. Muller, M. Macagnan, C. R. Rodenbusch,
salmonellosis under conditions conducive to illness and that C. W. Canal, and M. Cardoso, “Detection of Salmonella sp.
from porcine origin: a comparison between a PCR method
virulence genotyping, at least with genes studied here, might
and standard microbiological techniques,” Brazilian Journal of
have marginal influence in enhancing severe infection. This Microbiology, vol. 36, no. 4, pp. 373–377, 2005.
study also reveals that serogroup D is the predominant
[10] K. P. Neil, S. V. Sodha, L. Lukwago et al., “A large out-
Salmonella serogroup in circulation and it is widely shared
break of typhoid fever associated with a high rate of intesti-
among animals and humans and calls for a joint and coordi- nal perforation—Kasese district, Uganda, 2008-2009,” Clinical
nated surveillance for one health implementation in Uganda. Infectious Diseases, vol. 54, no. 8, pp. 1091–1099, 2012.
To the best of our knowledge, the present study is the first
[11] K. Ikwap, J. Erume, D. O. Owiny et al., “Salmonella species
comprehensive study attempting to provide insight into the in piglets and weaners from Uganda: prevalence, antimicrobial
genetic diversity of Salmonella and its associated virulence resistance and herd-level risk factors,” Preventive Veterinary
genes circulating in both humans and animals in Uganda. Medicine, vol. 115, pp. 39–47, 2014.
[12] J. A. Afema, D. K. Byarugaba, D. H. Shah, E. Atukwase, M.
Conflicts of Interest Nambi, and W. M. Sischo, “Potential sources and transmis-
sion of Salmonella and antimicrobial resistance in Kampala,
There are no conflicts of interest concerning this article. Uganda,” PLoS ONE, vol. 11, no. 3, Article ID e0152130, 2016.
[13] M. Dolores and C. Tongco, “Purposive sampling as a tool for
Acknowledgments informant selection,” Ethnobotany Research & Applications, vol.
5, pp. 147–158, 2007.
The study was approved by Makerere University College of [14] World Health Organisation, “Manual for the laboratory identifi-
Health Science, School of Biomedical Sciences, Research and cation and antimicrobial testing of bacterial pathogens of public
Ethical Review Committee. The authors acknowledge the health importance in developing world,” WHO/CDS/CSR/
Microbiology Laboratory, College of Veterinary Medicine, RMD/2003, 2003.
Animal Resources and Biosecurity, Makerere University, for [15] G. Kauffman, “Kauffman White Scheme. WHO. Pd 172, 1, rev.
the permission to use archived animal isolates. They are 1,” Acta Pathologica et Microbiologica Scandinavica Section B-
grateful to the staff of Molecular Biology Laboratory, College Microbiology, vol. 61, p. 385, 1974.
International Journal of Bacteriology 9

[16] J. C. Sherris and M. Turck, “Antibiotic susceptibility testing by a virulence of Salmonella typhimurium in mice,” Infection and
standardized single disk method,” American Journal of Clinical Immunity, vol. 66, no. 6, pp. 2803–2808, 1998.
Pathology, vol. 45, no. 4, pp. 493–496, 1966. [32] E. Haghjoo and J. E. Galan, “Salmonella typhi encodes a
[17] Clinical and Laboratory Standard Institute (CLSI), “Perfor- functional cytolethal distending toxin that is delivered into host
mance standards for antimicrobial susceptibility testing; 20th cells by a bacterial internalization pathway,” Proceedings of the
informational supplement (June 2010, update),” CLSI Docu- National Academy of Sciences, vol. 101, pp. 4614–4619, 2004.
ment M100-S20-U, Clinical and Laboratory Standard Institute [33] W. Zou, S. F. Al-Khaldi, W. S. Branham et al., “Microarray
(CLSI), Wayne, Pa, USA, 2010. analysis of virulence gene profiles in Salmonella serovars from
[18] J. Versalovic, M. Schneider, F. J. de Bruijn, and J. R. Lupski, food/food animal environment,” Journal of Infection in Develop-
“Genomic fingerprinting of bacteria using repetitive sequence ing Countries, vol. 5, no. 2, pp. 94–105, 2011.
based PCR (rep-PCR),” Methods in Molecular Cell Biology, vol. [34] A. S. Jerod, M. L. Catherine, and K. N. Lisa, “Virulence
5, pp. 25–40, 1994. genotyping of Salmonella spp. with multiplex PCR,” Avian
[19] F. W. Brenner, R. G. Villar, F. J. Angulo, R. Tauxe, and B. Diseases, vol. 50, pp. 77–81, 2006.
Swaminathan, “Salmonella nomenclature,” Journal of Clinical
Microbiology, vol. 38, pp. 2465–2467, 2000.
[20] S. D. Alcaine, Y. Soyer, L. D. Warnick et al., “Multilocus
sequence typing supports the hypothesis that cow- and human-
associated Salmonella isolates represent distinct and overlap-
ping populations,” Applied and Environmental Microbiology,
vol. 72, no. 12, pp. 7575–7585, 2006.
[21] B. J. Morrow, J. E. Graham, and R. Curtiss, “Genomic sub-
tractive hybridization and selective capture of transcribed
sequences identify a novel Salmonella typhimurium fimbrial
operon and putative transcriptional regulator that are absent
from the Salmonella typhi genome,” Infection and Immunity, vol.
67, no. 10, pp. 5106–5116, 1999.
[22] CDC, Salmonella Surveillance: Annual Summary, US Depart-
ment of Health and Human Services, Atlanta, Ga, USA, 2006.
[23] C. Bacci, E. Boni, I. Alpigiani, E. Lanzoni, S. Bonardi, and
F. Brindani, “Phenotypic and genotypic features of antibiotic
resistance in Salmonella enterica isolated from chicken meat
and chicken and quail carcasses,” International Journal of Food
Microbiology, vol. 160, no. 1, pp. 16–23, 2012.
[24] M. Mahero, D. K. Byarugaba, D. K. Doetkott, S. Olet, and M.
L. Khaitsa, “Antimicrobial resistance and presence of class 1
integrons in Salmonella serovars isolated from clinical cases of
animals and humans in North Dakota and Uganda,” Clinical
Microbiology, vol. 2, article 128, 2013.
[25] L. K. Nolan, R. E. Wooley, J. Brown, and J. B. Payeur, “Com-
parison of phenotypic characteristics of Salmonella spp isolated
from healthy and ill (infected) chickens,” American Journal of
Veterinary Research, vol. 52, no. 9, pp. 1512–1517, 1991.
[26] T. J. Kottom, L. K. Nolan, and J. Brown, “Invasion of Caco-2
cells by Salmonella Typhimurium (Copenhagen) isolates from
healthy and sick chickens,” Avian Diseases, vol. 39, no. 4, pp.
867–872, 1995.
[27] H. Ochman, F. C. Soncini, F. Solomon, and E. A. Groisman,
“Identification of a pathogenicity island required for Salmonella
survival in host cells,” Proceedings of the National Academy of
Sciences, vol. 93, pp. 7800–7804, 1996.
[28] M. Suarez and H. Russmann, “Molecular mechanisms of
Salmonella invasion: the type III secretion system of the
pathogenicity island 1,” Internatl Microbiol, vol. 1, pp. 197–204,
1998.
[29] S. R. Waterman and D. W. Holden, “Functions and effectors of
the Salmonella pathogenicity island 2 type III secretion system,”
Cell Microbiol, vol. 5, pp. 501–511, 2003.
[30] E. A. Groisman and H. Ochman, “How Salmonella became a
pathogen,” Trends in Microbiology, vol. 5, no. 9, pp. 343–349,
1997.
[31] A. W. M. Van der Velden, A. J. Baumler, R. M. Tsolis, and
F. Heffron, “Multiple fimbrial adhesins are required for full
 International Journal of

Peptides

Advances in
BioMed
Research International
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Stem Cells
International
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Virolog y
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
International Journal of
Genomics
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014

Journal of
Nucleic Acids

=RRORJ\
International Journal of

Hindawi Publishing Corporation Hindawi Publishing Corporation

http://www.hindawi.com Volume 201 http://www.hindawi.com Volume 2014

Submit your manuscripts at

https://www.hindawi.com

Journal of The Scientific

Signal Transduction
Hindawi Publishing Corporation
World Journal
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014

Genetics Anatomy International Journal of Biochemistry Advances in

Research International
Hindawi Publishing Corporation
Research International
Hindawi Publishing Corporation
Microbiology
Hindawi Publishing Corporation
Research International
Hindawi Publishing Corporation
Bioinformatics
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014

Enzyme International Journal of Molecular Biology Journal of

Archaea
Hindawi Publishing Corporation
Research
Hindawi Publishing Corporation
Evolutionary Biology
Hindawi Publishing Corporation
International
Hindawi Publishing Corporation
Marine Biology
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014