https://doi.org/10.52973/rcfcv-e34470
Received: 09/06/2024 Accepted: 23/07/2024 Published: 16/12/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34470
ABSTRACT
Brucellosis remains a critical zoonotic infection with profound
implications for public health across diverse regions, including
the Middle East, Asia, the Arabian Peninsula, the Mediterranean,
Africa, and South and Central American countries. This global threat
necessitates ongoing investigation and surveillance. Accordingly,
this study aimed to elucidate the presence and characteristics of
Brucella spp. isolated from patients in a province of eastern Türkiye. A
combination of conventional and molecular techniques was employed
to achieve comprehensive species and biovar determination. A total
of 189 human Brucella spp. strains isolated from blood cultures at
Bitlis State Hospital between 2010 and 2020 were included in the
study. Identication tests for the isolates comprised assessing
serum requirement for growth, oxidase and urease production, as
well as lysis testing with Tbilisi phage and R/C phage. Additional
conventional biotyping tests involved evaluating hydrogen sulde
(H
2
S) production, carbon dioxide (CO
2
) requirement for growth, and
growth in media containing thionin, basic fuchsin, and safranin.
Furthermore, agglutination with Brucella A and M type monospecic
antisera was performed. The isolates also underwent multiplex PCR,
specically the Bruce–Ladder PCR method, for biotyping. The results
demonstrated the predominance of Brucella melitensis strains in
human brucellosis cases, as identied by both conventional and
molecular methods. Specically, 185 isolates were classied as
B. melitensis biovar 3, with the remaining 5 isolates classied as
B. melitensis biovar 1. In conclusion, this distribution underscores
the signicant role of B. melitensis in the epidemiology of human
brucellosis in the region. The current study highlights the ecacy
of both conventional and molecular methods in Brucella spp.
identication, with particular emphasis on the Bruce–Ladder PCR
method’s superiority in terms of rapidity and compatibility with
traditional techniques. Continued research and surveillance efforts
are imperative to deepen our understanding of the epidemiology and
dynamics of this zoonotic disease.
Key words: Brucellosis; Brucella melitensis; multiplex polymerase
chain reaction
RESUMEN
La brucelosis es una infección zoonótica crítica con profundas
implicaciones para la salud pública en diversas regiones, incluido el
Medio Oriente, Asia, la Península Arábiga, el Mediterráneo, África y
países de América del Sur y Central. Esta amenaza global requiere
investigación y vigilancia continúa. En consecuencia, este estudio
tuvo como objetivo dilucidar la presencia y las características de las
especies de Brucella aisladas de pacientes en una provincia del este
de Turquía. Se empleó una combinación de técnicas convencionales
y moleculares para lograr una determinación integral de especies
y biovares. En el estudio se incluyeron un total de 189 cepas de
Brucella humana aisladas de hemocultivos en el Hospital Estatal
de Bitlis entre 2010 y 2020. Las pruebas de identicación para los
aislados comprendieron la evaluación de los requisitos de suero
para el crecimiento, la producción de oxidasa y ureasa, así como
pruebas de lisis con fagos Tbilisi y fagos R/C. Otras pruebas de
biotipado convencionales incluyeron la evaluación de la producción
de sulfuro de hidrógeno (H
2
S), los requisitos de dióxido de carbono
(CO
2
) para el crecimiento y el crecimiento en medios que contienen
colorantes tionina, fucsina básica y safranina O. Además, se realizó
aglutinación con antisueros monoespecícos tipo Brucella A y M.
Además, los aislados se sometieron a PCR múltiple, el método de
PCR Bruce–Ladder para biotipado. Los resultados demostraron el
predominio de cepas de Brucella melitensis en los casos de brucelosis
humana, identicadas tanto por métodos convencionales como
moleculares. Especícamente, 185 aislamientos se clasicaron como
B. melitensis biovar 3, y los 5 aislamientos restantes se clasicaron
como B. melitensis biovar 1. En conclusión, esta distribución subraya el
importante papel de B. melitensis en la epidemiología de la brucelosis
humana en la región. Este estudio destaca la ecacia de los métodos
convencionales y moleculares en la identicación de especies de
Brucella, haciendo hincapié en la superioridad del método Bruce–
Ladder PCR en términos de rapidez y compatibilidad con las técnicas
tradicionales. Es imprescindible realizar esfuerzos continuos de
investigación y vigilancia para profundizar nuestra comprensión de
la epidemiología y la dinámica de esta enfermedad zoonótica.
Palabras clave: Brucelosis; Brucella melitensis; reacción en cadena
de la polimerasa multiplex
Molecular typing of Brucella species in human brucellosis cases from
Eastern Türkiye
Tipicación molecular de especies de Brucella en casos de brucelosis humana en el este de Turquía
İbrahim Halil Şahin
1
, Sevil Erdenliğ Gürbilek
2
, Nida Özcan
3
* , Ahmet Murat Saytekin
2
, Ayfer Güllü Yücetepe
2
1
Bitlis Eren University, Vocational School of Health Services, Department of Microbiology. Bitlis, Türkiye.
2
Harran University, Faculty of Veterinary Medicine, Department of Microbiology. Şanlıurfa, Türkiye.
3
Dicle University, Faculty of Medicine, Department of Medical Microbiology. Diyarbakır, Türkiye.
*Corresponding author: nida.ozcan@dicle.edu.tr
Biotyping of human Brucella species in Eastern Türkiye / Şahin et al. _______________________________________________________________
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INTRODUCTION
Brucellosis is one of the most common zoonotic infections all
over the world. It is transmitted to humans by direct contact with
infected animals or by ingestion of contaminated dairy products and
raw milk [1, 2]. Brucellosis causes mild u–like symptoms to serious
clinical conditions such as meningitis, and is often accompanied by
musculoskeletal system involvement [3]. As a result of successful
intervention measures, including vaccination, the incidence of
brucellosis has decreased in North America and Europe. On the other
hand, it continues to be an important zoonotic infection affecting public
health in the Middle East, Asia, Arabian Peninsula, Mediterranean,
Africa, South and Central American countries [4]. The incidence of
brucellosis in Turkey varies between regions, due to differences in
climatic conditions, animal husbandry practices, methods of processing
milk and dairy products, nutritional habits and socioeconomic status
[5]. In countries where brucellosis is endemic, including Turkey, human
brucellosis cases are signicantly underreported; thus, it is believed
that the incidence of brucellosis in the community is much higher
than reported. Therefore, there is a need for a system for mandatory
laboratory–based surveillance of the disease [6].
Blood culture is considered the gold standard in the diagnosis of
brucellosis. However, it necessitates a lengthy duration, biosafety
level 3 laboratory infrastructure, and experienced personnel, while
also posing the potential risk of contamination to laboratory personnel
during procedures [7, 8]. Molecular diagnostic tests are recommended
by researchers for rapid and accurate disease diagnosis in the
laboratory due to their high sensitivity, rapid results and safety from
contamination [9, 10, 11].
It was aimed to identify Brucella spp. isolated from patients in the
eastern region of Turkey, determine up to species and biovar levels
using conventional and multiplex polymerase chain reaction (PCR)
methods, and investigate the compatibility of these methods.
MATERIALS AND METHODS
Between 2010 and 2020, growth was detected in 1,701 of the blood
cultures taken from 7,964 patients at Bitlis State Hospital. Brucella
spp. grew in 189 (11.1%) of them. 103 of the patients were male and 85
were female, mean age of the patients was 27.3 years (0–75 years). In
the same period, brucellosis standart tube agglutination (STA) tests
were performed on 50,000 patients and was found to be positive at
a titer of 1/160 and above in 3,954 (7.2%) patients.
Among 189 patients with Brucella growth in culture, the standard
tube agglutination (STA) test was not performed in 3 patients. Of the
remaining patients, 7 tested negative, while 11 exhibited a titer of
1/80. In 151 patients, STA titers were 1/160 or higher, with the following
distribution: 17 patients at 1/160, 16 at 1/320, 26 at 1/640, 70 at 1/1280,
37 at 1/2560, and 1 patient at 1/5120. Of the patients, 167 presented to
the hospital with brucellosis–related complaints (such as fever, muscle
and bone pain), and 22 presented with non–specic complaints (such
as menstrual irregularity, anemia, gastroenteritis).
Samples and quality control isolates
Blood samples were incubated at 37°C for 10 days (d) in automated
blood culture system (Biomerieux, BacT/ALERT®, France) . Positive
blood culture bottles were subcultured onto 5% sheep blood agar
plates and incubated at 37°C for 5 d. Following Gram staining, Brucella–
suspected colonies underwent catalase, oxidase, and urease tests.
Bacterial colonies displaying the morphology of small Gram negative
cocoid rods with positive catalase, oxidase, and urease tests were
identied as Brucella spp. and were stored at -80°C freezer (Ildam,
ILD–DF–720, Türkiye) until biotyping analysis.
Conventional biotyping of Brucella spp.
TSA (Tryptic soy agar) (Oxoid, United Kingdom) was used as the
basal medium for conventional identication and biotyping processes.
Initially, grown cultures were assessed for purity and colonial
morphology. Smooth and rough isolates were differentiated by checking
their colonial morphology using a stereomicroscope (Olympus, SZX10,
Japan) and were tested for agglutination using 0.1% neutral acriavin
(Sigma, Australia). Any agglutination observed rendered the strain
untypeable. Tests conducted to identify the species of the isolates
included assessment of serum requirement for growth, oxidase
and urease production, as well as lysis testing with Tbilisi phage at
routine test dilution (RTD) and 104–fold RTD and R/C phage at RTD. For
biotyping, further tests were conducted, including checking for the
production of hydrogen sulde (H
2
S), carbon dioxide (CO
2
) requirement
for growth, growth in media containing thionine, basic fuchsin, and
safranin O dyes. Agglutination with Brucella A and M type monospecic
antisera was also investigated. To distinguish between eld strains and
vaccine strains, growth on media containing penicillin, streptomycin,
thionine blue, and erythritol was tested [12, 13, 14].
Molecular typing of Brucella spp. by multiplex PCR (Bruce–ladder)
This assay was performed according to Anne Mayer–Scholl protocol
[11, 15]. To extract the bacterial genomic Deoxyribonucleic Acid (DNA),
a loopful of bacterial colonies was retrieved from the medium and
suspended in 200 μL of sterile distilled water. Boiling method was
used for DNA extraction. The amounts of isolated DNA were measured
(ThermoScientic, NanoDrop ND–1000, USA) and 50 – 150 ng was
used for each reaction tube. The test was conducted using a 25 µL
reaction mixture comprising 2× Qiagen Multiplex Master Mix (Qiagen,
Germany), 2 µM of each primer from a combination of nine primer
sets, and 1µl of template DNA. Amplications were astablished with
the sample denaturation step (95°C, 15 min), followed by 25 cycles of
template denaturation (94°C, 30 s), primer annealing (58°C, 90 s), and
primer extension (72°C, 180 s) steps. Following the nal cycle, samples
were further incubated at 72°C for 10 min. ( Rotor Gene, Qiagen,
Germany). The amplied products were subsequently separated
via electrophoresis (Orange, GRUN24H, India) on 1.5% agarose gels.
As quality control isolates, following strains were used: Brucella
melitensis 16M (ATCC 23456), the reference strain for B. melitensis
biovar (bv) 1; B. melitensis 63/9 (ATCC 23457), the reference strain for
B. melitensis bv 2; and B. melitensis Ether (ATCC 23458), the reference
strain for B. melitensis bv 3.
RESULTS AND DISCUSSION
All 189 isolates grew in media containing thionine, basic fuchsin
and none of the isolates producted H
2
S and required CO
2
for growth,
therefore all isolates were identied as B. melitensis at the species
level. All isolates tested negative for Tbilisi and R/C phage lysis, and the
majority (184) were identied as bv 3 eld strain due to agglutination
with type A and M–type antisera. Agglutination was detected with only
M–type antisera among 5 isolates (isolates number 11, 35, 64, 151, and
178), thus they were identied as B. melitensis bv 1 eld strain. Using
multiplex polymerase chain reaction (m–PCR), molecular identication
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
152 bp
450 bp
587 bp
794 bp
1071 bp
1682 bp
Figure 1: Multiplex PCR agarose gel image of the isolates. Lane 1: 2000 bp Mid Range Ladder (Qiagen),
Lanes 2-16 and Lane 18: Isolates tested in the study, Lane 17: Negative Control (Water), Lane 19: Positive
Control-1 (Brucella melitensis 16M), Lane 20: Positive Control-2 (Brucella melitensis 63/9), Lane 21: Positive
Control-3 (Brucella melitensis Ether)
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of the isolates was conducted at the species level (FIG. 1). It was
observed that the results were in perfect concordance with those
obtained through conventional culture–based methods.
The prevalence of brucellosis varies across countries, with
the Mediterranean region being the most affected. Türkiye is
one of the countries where brucellosis is endemic, particularly in
the eastern Anatolia region. Bitlis is a city located in the eastern
Anatolia region of Turkey and its main source of income is animal
husbandry. Determining the species and subtypes of Brucella, as
well as distinguishing between wild and vaccine strains, is crucial for
detecting and controlling the source of the disease [16].
This study is the first biotyping study conducted with human
brucellosis cases in eastern Turkey. There are limited studies on
biotyping human isolates in Turkey. The current study was conducted
using both traditional and molecular methods, revealing that the
Brucella spp. isolated from patients in the region were predominantly
B. melitensis, with the majority of isolates (185 out of 189) being bv 3,
while 5 isolates were bv 1. The current study was found to be compatible
with other studies conducted in Central Anatolia region of Türkiye.
Bodur et al. [17] biotyped Brucella isolates obtained from human blood
and cerebrospinal uid samples in Ankara using conventional methods,
which were the same as in current study. In total, 41 isolates were
identied as B. melitensis at the species level, with 39 isolates typed
as bv 3 and 2 isolates typed as bv 1 [17]. Similarly, in their 2004 study
conducted in Ankara, Simsek et al. biotyped Brucella spp. isolated
from human blood using conventional methods and identied 65 of
the isolates as B. melitensis bv 3 and 5 of the isolates as B. melitensis
bv 1 [18]. In the study by Bolca et al. [19], in which they typed 26 human
Brucella spp. isolates in 3 provinces from the Marmara and Central
Anatolia regions of Türkiye, 22 isolates were identied as B. melitensis
bv 3 and 4 isolates were identied as B. melitensis bv 1 [19].
The current study was also compatible with Karagul et al.'s [20]
study of biovar distribution of livestock Brucella spp. isolates in
Türkiye. A total of 5,203 Brucella spp. eld livestock isolates from
different regions of Türkiye were tested by conventional methods.
In the period between 2010 and 2015, B. abortus bv 3 was found to be
the most common cause of brucellosis in cattle, while B. melitensis
bv 3 was the most common cause in sheep and goats. The study
examined the percentage of biovars in different regions. The results
showed that in Eastern Anatolia, the detection percentages of biovars
were as follows: 94.68% for B. abortus bv 3, 3.52% for B. melitensis
bv 3, 1.67% for B. abortus bv 1, and 0.06% for B. melitensis bv 1 [20].
Ica et al. [21] typed Brucella spp. isolated from human blood
(50 samples) and animal abortion (17 cattle, 12 sheep) materials in
Kayseri, a province in the Central Anatolia region of Türkiye, using both
conventional methods and the Enhanced AMOS–ERY PCR method. The
study revealed that all Brucella spp. isolated from cattle were typed
as B. abortus bv 3b, while those isolated from sheep and humans
were identied as B. melitensis bv 3 using both conventional and
molecular methods .
In a study conducted in Iran, the eastern neighbor of Türkiye, all
206 human isolates isolated in 2013 were identied as B. melitensis
bv 1 using conventional methods [22]. In another study conducted in
Iran in the same year, Mirnejad et al. [23] investigated the detection
and typing of Brucella spp. from blood samples using the PCR–RFLP
method. DNA belonging to the Brucella genus was detected by PCR
in 52 out of a total of 160 blood samples. Biotype determination was
performed using PCR–RFLP in 25 of the positive samples, with 14
samples (56%) identied as B. melitensis bv1, and the remaining
isolates (44%) characterized as B. abortus biotypes (bv 3, 5, 6 and 9).
Both studies indicated that B. melitensis bv 1 exhibited the highest
prevalence in Iran [22, 24] .
In a study conducted in China in 2015, Brucella spp. isolates obtained
from human samples in Shanxi Province between 2009 and 2011 were
typed using traditional methods and conrmed by abortus–melitensis–
ovis–suis (AMOS)–PCR method. All 81 tested Brucella strains were
identied as B. melitensis bv 3 through conventional biotyping [24].
The majority of isolates in our study were also typed as bv 3.
Biotyping of human Brucella species in Eastern Türkiye / Şahin et al. _______________________________________________________________
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Lucero et al. [3]. analyzed Brucella strains isolated from humans
and animals from Latin American countries between 1968 and 2006.
Their study covered two different periods; the rst period included
isolates between 1968 and 1991 (half of the isolates were human and
half were animal isolates), and the second period included human
isolates from Argentina between 1994 and 2006. In the rst period, the
main Brucella strain in Argentina was B. suis, while B. melitensis strains
were dominant in Mexico and Peru. In Argentina, B. suis isolates
were dominant in the rst years of the study, and subsequently B.
melitensis human isolates increased. In the second period, covering
the years 1994–2006, of the 367 human isolates in Argentina, 145 were
B. melitensis, 144 were B.suis, 75 were B.abortus, and three were
B.canis. Biotyping of the isolates was as follows; Of the total 145 B.
melitensis isolates, 135 (93.1%) were bv 1, 7 were bv1a (4.8%), 2 were
bv 3 (1.4%) and 1 isolate was (0.7%) detected as bv 2a. The majority
of the 75 B. abortus isolates (86%) were detected as bv1 [25].
In a review of childhood brucellosis cases by Mantur et al. [25]. in
India, it was reported the majority of the isolates (number 43) were
identied as B. melitensis bv 1 and 1 isolate was typed as B. melitensis
bv 3 through microbiological, epidemiological and clinical evaluations.
The current research employed the Bruce–ladder PCR technique, a
multiplex PCR approach, for molecular typing of Brucella isolates.
The main advantage of using the Bruce–ladder PCR method over
previously used multiplex PCR assays is its ability to distinguish all
Brucella species and vaccine strains in a single test. The Bruce–ladder
PCR stands out in comparison to AMOS PCR, as it can identify DNA
from various Brucella strains, including Brucella strains from marine
mammals, B. abortus biovars (bv 3, 5, 6, 7, 9), B. suis biovars (bv 2–5),
B. neotomae and B. canis strains. Additional advantages include
the rapid yield of the test, the simplicity of sample preparation, and
reduced risks of contamination. As a result, the Bruce–ladder PCR
method is gaining recognition as an ecient way to identify Brucella
strains in both animal and human sources. Furthermore, it can be
used in any regular microbiology laboratory worldwide, rather than
being limited to specialized facilities [26].
CONCLUSIONS
An investigation was carried out on 189 Brucella spp. isolates
obtained from clinical cases in the Eastern Anatolia region of Türkiye.
In our study, 184 out of 189 isolates were identied as B. melitensis bv
3 eld strain, while the remaining 5 were identied as the B. melitensis
bv 1 eld strain. Traditional techniques were employed alongside
Multiplex PCR Bruce–Ladder for typing, and it was observed that the
Bruce–Ladder PCR method yielded results more rapidly compared to
conventional microbiological standard tests. Additionally, there was
100% agreement between the two methods, with a kappa value of 1.
ACKNOWLEDGEMENT
We extend our gratitude to the Pendik Veterinary Control and
Research Institute for providing monospecic serum and phages,
and to the Harran University Veterinary Faculty for providing standard
bacterial isolates. This study received no funding.
Conicts of interest
The authors declare no conicts of interest.
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