| Abstract | Pripadnici roda Bartonella su Gram - negativne bacilarne bakterije, a danas je
poznato više od 40 vrsta, podvrsta i kandidata. Najznačajnija vrsta, Bartonella henselae,
glavni je uzročnik bolesti mačjeg ogreba (BMO) u ljudi. Primarni rezervoari bakterije
su mačke, asimptomatski nositelji uzročnika u svojim eritrocitima. Iako su protokoli za
dokazivanje bartonela različiti, uzgoj još uvijek predstavlja „zlatni standard”
dijagnostike. Ujedno predstavlja metodu dobivanja dostatne količine DNA za daljnje
genske analize, kao što je analiza sekvenci više gena (MLST). Zbog dugotrajnog rasta i
zahtjevnog umnažanja bartonela, uzgoj na hranjivim podlogama predstavlja
dijagnostički izazov.
Bartonele su proširene u cijelom svijetu i predstavljaju opasnost za zdravlje
ljudi. No kulturelna ili molekularna istraživanja vrsta u Hrvatskoj nisu do sada sustavno
provedena. Istovremeno serološka istraživanja i razne kliničke manifestacije ljudi
dokazuju prisutnost uzročnika u Hrvatskoj. Stoga je ovo prvo istraživanje takve vrste u
Hrvatskoj. B. henselae genski je heterogena i dijeli se na 37 sekvencijskih tipova (ST).
Osim istraživanja učestalosti i utvrđivanja optimalnog uzgojnog protokola, cilj ovog
rada je genski tipizirati izolate, steći uvid u raznolikost i rasprostranjenost genotipova te
doprinijeti poznavanju genetike samog uzročnika u jugoistočnoj Europi. Određivanje
sekvencijskih tipova izoliranih iz mačaka u Hrvatskoj značajno je, jer se oni razlikuju u
patogenosti i zoonotskom potencijalu.
Ukupno je pretraženo 279 mačaka. Metoda uzgoja uzoraka krvi 189 živih
mačaka s 13 lokacija u Republici Hrvatskoj izvedena je korištenjem pet čvrstih i dvije
bifazične hranjive podloge, a pretraženi su i lančanom reakcijom polimerazom.
Uzgojem na istim hranjivim podlogama analizirano je i 90 uzoraka krvi iz srca uginulih
mačaka. Za svaku životinju dostavljen je upitnik s anamnestičkim, kliničkim i
epizootiološkim podacima, s ciljem određivanja čimbenika rizika povezanih s
infekcijom mačaka. Za identifikaciju Bartonella sp. iz krvi i izolata konvencionalnim
PCR-om korištene su početnice za ciljne gene 16SrRNA i ITS (16S-23S rRNA).
Dokazivanje vrste B. henselae i njenih ST-ova provedeno je MLST metodom na osam
genskih lokusa iz izolata uzgojenih iz krvi 31 mačke. Aleli i ST-ovi određivani su korištenjem PubMLST baze podataka za bakteriju B. henselae i uspoređeni s
postojećima iz ostalih regija svijeta. Umnažanjem i sekvenciranjem 16SrRNA gena i
ITS regije dokazana je B. henselae i kod uginule mačke.
Bartonele su izdvojene iz 31 žive (16,4%) i jedne uginule (1,1%) mačke što daje
ukupnu učestalost infekcije od 11,5%. Istovremeno lančanom reakcijom polimerazom u
krvi 189 mačaka nije dokazan odsječak DNA Bartonella sp. Na svim korištenim
čvrstim hranjivim podlogama u primoizolaciji zabilježen je porast kolonija nakon četiri
do 56 dana inkubiranja. Najveća učestalost izdvajanja bakterije B. henselae utvrđena je
na BH (87,5%) i COL (82,4%) agaru. MLST analizom identificirano je 30 potpunih
alelnih profila iz kojih je proizašlo pet različitih sekvencijskih tipova. Najučestaliji ST5
izdvojen iz 17 (56,7%) mačaka, a dokazani su i ST6 (23,3%), ST1 (13,3%) i ST24
(3,3%). Usporedba alelnih profila rezultirala je jednim potpuno novim genotipom
(ST33), pripisanom jednom od tri klonalna kompleksa vrste B. henselae (CC2).
Područje, dob i po prvi puta prisutnosti crijevnih parazita utvrđeni su kao
čimbenici rizika za infekciju mačaka bakterijom B. henselae (p<0,05). Mačke starosti
≤12 mjeseci najučestalije su bile inficirane, a uočen je trend smanjivanja prevalencije s
porastom dobi mačaka. Statistički značajna povezanost s infekcijom utvrđena je u
mačaka s primorskih područja u odnosu na mačke s kontinenta, kao i u mačaka
uzorkovanih u razdoblju od siječnja do travnja u odnosu na ostatak godine.
Multivarijabilnom analizom prisutnost crijevnih parazita utvrđena je kao čimbenik
najsnažnije povezan s bakterijemijom mačaka. Metoda uzgoja korištenjem različitih
hranjivih podloga pokazala se uspješnom u izdvajanju većeg broja izolata. Dokaz
zoonotskog genotipa ST5 kod najvećeg broja mačaka predstvalja javnozdravstveni
značaj, osobito jer se u nekoliko mačaka dovodi u vezu s oboljenjima ljudi od BMO-a. |
| Abstract (english) | Introduction. Bacteria of the genus Bartonella sp. are small, short,
pleomorphic, Gram-negative coccobacilli (0.5 by 1 to 2 µm), and today almost 40
species are known, along with numerous unnamed or Candidatus species. Bartonella
henselae is the most important species of zoonotic significance, the principal etiologic
agent of cat scratch disease (CSD) in humans, most commonly manifested by localized
lymphadenopathy with or without bacteremia.
The main reservoirs of bacteria B. henselae are cats, and the causative agent is
most often transmitted to humans by contamination of the scratch site with feces of the
cat flea (Ctenocephalides felis), which contains viable multiplied pathogens. Cats are
chronic asymptomatic carriers of bacteria B. henselae, which infects their erythrocytes,
making isolation from the cat’s blood on blood agar plates more successful than from
other animals and humans.
Epizootiological studies have shown a prevalence of Bartonella infection in
cats of 10-30% by culture from cat’s blood. Although protocols for proving Bartonella
are different, isolation by cultue on agar plates is still considered the "gold standard" of
diagnosis. It is also a method by which sufficient amounts of DNA can be obtained for
further genetic analysis of bacteria B. henselae, such as analysis of multiple gene loci.
Due to the slow growth and fastidious cultivation, isolation on culture media is a
diagnostic challenge.
In spite of Bartonella are widespread throughout the world and pose a threat to
human health, research on species / isolates in Croatia has not been carried out
systematically so far. Serological studies and various clinical manifestations of people
has proven the presence of pathogens in Croatia. To date, no studies by culture and / or
molecular investigation of bacteria B. henselae in cats, or genotyping methods of
species / isolates have been performed, so this is the first study of its kind in Croatia.
B. henselae isolates show a considerable genetic heterogeneity and bacteria is
divided into sequence types (ST), which today are most often determined by multilocus sequence typing (MLST) analysis. Prior to the start of this study, 32 different STs were
known, and today there are a total of 37 enrolled in the PubMLST database
(https://pubmlst.org/organisms/bartonella-henselae). Beside to study the prevalence of
infection and optimal culture procedures, the aim of this paper is to perform B.
henselae-genotyping, gain insight into the diversity and distribution of sequence types
and contribute to knowledge of the genetics of the bacteria B. henselae in Southeast
Europe. Determining the sequence types isolated from cats in Croatia is important,
because they differ in virulence and zoonotic potential.
Material and methods. A total of 279 cats were searched. 189 blood samples
of live cats collected in 20 veterinary clinics from 13 different locations in the Republic
of Croatia were analyzed. Samples were inoculated on five types of solid and two types
of biphasic culture media, previously tested using ATCC strains of bacteria B. henselae
(49882) and B. clarridgeiae (700095), and analysed in parallel by polymerase chain
reaction (PCR). Any colony growth indicating species of the genus Bartonella was
screened by PCR, and isolates were stored in pure culture by freezing at –80 °C, in
broths with added glycerol, or in a commercial bacterial storage system. In addition to
samples of live cats, 90 samples of blood from the hearts of dead cats were analyzed,
tested only by culture. Each sample was accompanied by a questionnaire which contains
anamnestic, clinical and epizootiological data on animals, in order to determine the risk
factors associated with infection of cats with bacteria of the genus Bartonella sp.
Five solid culture media were used, Columbia agar with 5% sheep blood
(COL), Brain heart agar with 5% rabbit blood (BH), Chocolate agar with 10% sheep
blood (ČOK), Tryptic soy agar with 5% sheep blood (TSA) and Esculin blood agar with
5% sheep blood (EKA). In addition, two biphasic media were used, made by a
combination of liquid and solid culture media: Tryptic soy broth with Tryptic soy agar
(TSA+TSB) and Brucella broth with Brain heart agar (BH+BB). To identify Bartonella
sp. from blood and isolates by conventional PCR, primers for the target genes 16SrRNA
and ITS (16S-23S rRNA) were used.
Detection of B. henselae species and sequence types determination was
performed by multi locus sequence typing (MLST) at eight gene loci (16SrRNA, batR,
ftsZ, gltA, groEL, nlpD, ribC and rpoB) from isolates obtained by culture of blood samples of 31 cats. Nucleotide sequence determination was performed at the
commercial company Macrogen Inc. (Amsterdam, The Netherlands). The obtained
sequences were processed using the computer program BioNumercis, which compared
the nucleotide sequences of both directions, in order to obtain a unique nucleotide
sequence.
Alleles and STs were determined using the MLST online database
(https://pubmlst.org/organisms/bartonella-henselae) for bacteria B. henselae and were
assigned an eight-digit numerical code, consisting of a combination of alleles at each
locus. Alleles were compared with each other on the basis of categorical coefficient and
UPGMA, and presented in the form of dendrograms and MST (minimum spanning
tree). The obtained STs with this research were compared with the existing STs from
different countries and regions of the world.
In the statistical processing of epizootiological data, we used the computer
program STATA 13.1. Univariate and multivariante logistic regression was used to
analyze the statistical correlation of epizootiological data from the questionnaires and to
determine risk factors associated with B. henselae infection. Independent variables
statistically significant in univariate analysis (p-value <0,05) were included in
multivariable model and further tested to evaluate if remained associatied with feline
Bartonella bacteremia.
Results. Out of a total of 279 cats searched, Bartonella spp. were isolated
from 31 live and one dead cat. Determined prevalece was 16,4% (CI 11,074 – 21,730)
(total frequency of isolation was 11,5%, in live cats 16,4% and in dead 1,1%). On all
types of solid culture media used in the primoisolation a growth of colonies was
recorded, and the typical colonies for species B. henselae (hard, dry, difficult to break,
and firmly embedded to the surface) were observed after four to 56 days of incubation.
B. henselae was identified by amplification and sequencing of the 16SrRNA gene and
the ITS region. By analyzing the nucleotide sequences of eight gene loci of all isolates
uing the MLST method, five different sequence types (ST) of bacteria B. henselae were
identified. Out of 31 infected live cats, a total of 30 complete MLST allelic profiles
were obtained, and one was incomplete and was not assigned a sequence type with
certainty.
The most common genotype was ST5, isolated from 17 (56,7%) cats, and of
the other sequence types ST6 was detected in seven (23,3%) cats, ST1 in four (13,3%)
and ST24 in one (13,3%). The genotype of one cat was completely new due to a new
combination of alleles, and was deposited into an online database
(https://pubmlst.org/organisms/bartonella-henselae) as a sequence type ST33. Based on
the determined combination of alleles, MLST analysis assigned a numerical code 2-3-3-
1-2-1-1-2, and phylogenetic analysis determined affiliation to one of the three
previously described CCs of B. henselae species, clonal complex 2 (CC2).
The best efficiency of isolation of B. henselae – bacteria on seven types of
culture media, comparing the growth rate (days of incubation) and the isolation rate
(share of isolates obtained from infected blood samples on specific media), in the
primary isolation was on BH agar (87.5%, 21/24 isolates) and COL agar (82.4%, 14/17
isolates). Cat’s isolates with varying level of bacteremia were grown on both culture
media, on BH agar with a range of 16 to 1960 CFU / mL, and on COL agar from 40 to
3050 CFU / mL. Slightly lower isolation rate was observed on EKA (80,0%) and ČOK
(77,3%) agar and on biphasic medium TSA+TSB (80,0%). The advantage of a certain
culture medium in the isolation of a particular sequence type of B. henselae has not been
observed. B. henselae species was identified from an isolate grown on ČOK agar plate
from a blood sample of a dead cat by PCR and sequencing. By examining the blood of
live cats by PCR, we were unable to amplify the DNA fragment of Bartonella.
By processing epizootiological data from the live cat by survey questionnaires,
a statistically significant association (p <0,05) was found between the infection of cats
with B. henselae and the area, age of cats and and first-time findings of intestinal
parasites. The highest prevalence of infected animals was found in the cities of Zabok
(66,7%, 2/3), Rijeka (50,0%, 5/10), Pula (40,0%, 8/20) and Jastrebarsko (30,4 %, 7/23),
while in four out of the 13 locations (Osijek, Split, Vukovar and Velika Gorica) there
were no cats infected with Bartonella. Cats ≤12 months of age were the most frequently
infected (25,0%), and a trend of declining prevalence with increasing age of cats was
observed. In the coastal area, cats were significantly more frequently infected than on
the mainland, as were cats sampled between January and April compared to the rest of
the year. Multivariate analysis revealed the strongest association between feline
bacteremia and the presence of intestinal parasites.
Conclusion. The prevalence of cat infection with bacteria B. henselae
obtained on culture media (16,4%) is within the range found in similar studies in cats in
Europe and the rest of the world, and it is the third most common infection in the
domestic cat group. A slightly higher rate of isolation was observed in cats with respect
to lifestyle (going out, lack of owners). It was found that coastal cats were more
frequently infected with B. henselae than continental. Such a finding could be
associated with an average warmer climate of coastal locations, which is more
conducive to the life of cat fleas, the main vectors in Bartonella transmission. Infection
with intestinal parasites and age up to one year are risk factors that are significantly
associated with infection of cats with B. henselae. On the other hand, factors such as
sampling in the summer months, antibiotic administration, flea control treatments, and
age above one year are possible causes of differences in prevalence between sites.
The identification of five different sequence types from isolates of 30 cats
confirmed the genetic diversity of the bacterium B. henselae in the territory of the
Republic of Croatia. For the first time in the Republic of Croatia and for the first time
globally from the cat’s blood has been proven the ST33 genotype. It was not observed
association of other genotypes with particular location because they were equally
distributed. The culture method has proven successful for detecting B. henselae bacteria
from the blood of cats. Combined use of several different culture media, with extended
incubation time up to 60 days, are recommended for the isolation of bacteria B. henselae
by culture from the blood of the cats. The zoonotic genotype ST5 was isolated from
three cats in households with CSD patients suggesting that it is a common and virulent
zoonotic genotype. |
