Multiple-locus Variable-number Tandem Repeat Analysis Genotypes Research Articles

Multiple locus variable number tandem repeat analysis genotype polymorphism of Bacillus anthracis in China

Objective: To analyze the multiple locus variable number tandem repeat analysis (MLVA) genotype polymorphism of Bacillus (B.) anthracis and establish a MLVA genotype database of B. anthracis in China. Methods: B. anthracis strains isolated from different sources in China since 1947 were collected. Genotype identification was carried out using the MLVA15 genotyping protocol based on 15 variable number tandem repeat loci. The genotypes were uniformly numbered and named. The distribution characteristics of the MLVA genotypes of strains were analyzed. Software Bionumerics was used to construct clustering diagrams to analyze the genetic relationships. Results: The MLVA15 clustering analysis subdivided the isolates into 4 major groups and 91 genotypes, 54 of which were unique to China. The genotypes from MLVA15-CHN1 to MLVA15-CHN6 were widely distributed throughout China and in all eras, while other genotypes were restricted to certain regions or eras. Conclusions: This study established a MLVA genotype database of B. anthracis, which provides basis for the understanding of MLVA genetic polymorphisms and the control and molecular source tracing of the anthrax outbreaks in China.

MLVA and com1 genotyping of Coxiella burnetii in farmed ruminants in Great Britain

Coxiella burnetii, the causative agent of the zoonotic disease Q fever, has been shown to be endemic in Great Britain, but information on the prevailing genomic lineages or Genomic Groups (GGs) of Coxiella burnetii is limited. The aim of this study was to genotype C. burnetii isolates from infected farmed ruminants by Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) and identify their associated Genomic Group. A total of 51 Coxiella-containing abortion samples from farmed ruminants (sheep, goats, and cattle), which were collected in Great Britain during 2013–2018, were included in the study, 34 of which returned a C. burnetii MLVA genotype. All bovine samples (n = 18), 5/7 of the ovine samples, and 3/9 of the caprine samples belonged to an MLVA cluster which we could link to the MST20 genotype of GG III, whereas 6/9 of the caprine samples and 2/7 of the ovine samples belonged to MLVA clusters which we could link to the MST33 or MST32 genotypes of GG II (7 vs 1 sample(s), respectively). We also noted that the Coxiella-specific com1 gene contained unique mutations that could genomotype isolates, i.e. assign them to a Genomic Group. In conclusion, both goats and sheep in Great Britain (from 2014 onward) were found to carry the same MLVA genotypes (MST33-like; GG II) that were linked to a human Q fever outbreak in the Netherlands. This knowledge in combination with the usage of genotyping/genomotyping methods should prove useful in future surveillance programs and in the management of outbreaks.

Genotyping of Coxiella burnetii from Cattle by Multispacer Sequence Typing and Multiple Locus Variable Number of Tandem Repeat Analysis in the Republic of Korea

Genotyping of Coxiella burnetii using multispacer sequence typing (MST) and multiple locus variable number tandem repeat analysis (MLVA) was conducted from infected animals for the first time in the Republic of Korea. C. burnetii was detected by real-time PCR, and followed by MST and MLVA genotyping. The result showed that detected C. burnetii all had the same MLVA genotype, 6-13-2-7-9-10 for markers MS23-MS24-MS27-MS28-MS33-MS34, respectively, and genotype group 61 for MST. The same genotypes were previously identified in Poland. Importantly, this MLVA type was detected in humans in France, suggesting that the Korean strain can also potentially cause Q fever in humans. MST and MLVA were very useful tools for analyzing the molecular epidemiology of C. burnetii and helpful for interpreting the epidemiological relationship between isolates from domestic and international resources.

Phylogenetic and Multiple-Locus Variable number tandem repeat analysis of Mycobacterium avium subsp. paratuberculosis isolates from Argentina

Paratuberculosis is a worldwide chronic enteric disease of ruminants, caused by Mycobacterium avium subsp. paratuberculosis (MAP). While MAP has been widely investigated all around the world, little is known about the different strains that circulate in each country. This study describes the genetic diversity of MAP isolates from different bovine and deer herds from Argentina, analyzed by Multiple-Locus Variable number tandem repeat Analysis (MLVA), as well as the phylogenetic relatedness between geographically distant isolates through Whole Genome Sequencing (WGS) and core-genome analysis. A total of 90 MAP isolates were analyzed. The results showed seven different MLVA genotypes, with almost 75% of them belonging to pattern INMV 1, described in all the herds studied. WGS results suggested the presence of a common INMV 1 strain circulating throughout the country. Our results allow confirming the coexistence of different strains in time and space and the mixed infections identified in some animals. These observations suggest the absence of animal monitoring prior to introduction to the herds and the need for a control program in the country. This study represents the first to report WGS of MAP strains in Argentina.

Molecular detection of Coxiella burnetii in small ruminants and genotyping of specimens collected from goats in Poland

BackgroundCoxiella burnetii is the etiological agent of Q fever, a zoonosis affecting many animal species including sheep and goats. The aims of this study were to evaluate the shedding of Coxiella burnetii in small ruminant herds and to identify the pathogen’s genotypes and sequence types (STs) using multiple-locus variable number tandem repeat analysis (MLVA) and multispacer sequence typing (MST) methods.ResultsOverall, 165 samples from 43 herds of goats and 9 flocks of sheep were collected including bulk tank milk (BTM), individual milk samples, vaginal swabs, tissue sections from stillborn kids, feces and placentas. These were tested by real-time PCR targeting the IS1111 element. C. burnetii infection was confirmed in 51.16% of the herds of goats and 22.2% of the flocks of sheep. Six out of nine samples originating from goats were successfully genotyped using the MLVA method. The presence was confirmed of two widely distributed MLVA genotypes (I and J) and genotype PL1 previously reported only in cattle. Only one sequence type (ST61) was identified; however, the majority of specimens represented partial STs and some of them may belong to ST61. Other partial STs could possibly be ST74.ConclusionThis study confirmed the relatively common occurrence of Coxiella burnetii in small ruminant herds in Poland. Interestingly, all genotyped samples represent cattle-associated MLVA genotypes.

Seroprevalence trend of human brucellosis and MLVA genotyping characteristics of Brucella melitensis in Shaanxi Province, China, during 2008-2020.

In this study, a total of 179,907 blood samples from populations with suspected Brucella spp. infections were collected between 2008 and 2020 and analyzed by the Rose Bengal plate test (RBPT) and serum agglutination test (SAT). Moreover, conventional biotyping, B. abortus-melitensis-ovis-suis polymerase chain reaction (AMOS-PCR), and multiple-locus variable-number tandem repeat analysis (MLVA) was applied to characterize the isolated strains. A total of 8103 (4.50%) samples were positive in RBPT, while 7705 (4.28%, 95% confidence interval (CI) 4.19-4.37) samples were positive in SAT. There was a significant difference in seroprevalence for human brucellosis over time, in different areas and different cities (districts) (χ2 = 2 = 32.23, 1984.14, and 3749.51, p < .05). The highest seropositivity (8.22% (4, 965/60393; 95% CI 8.00-8.44) was observed in Yulin City, which borders Inner Mongolia, Ningxia, and Gansu Province, China, regions that have a high incidence of human brucellosis. Moreover, 174 Brucella strains were obtained, including nine with B. melitensis bv. 1, 145 with B. melitensis bv. 3, and 20 with B. melitensis variants. After random selection, 132 B. melitensis were further genotyped using MLVA-16. The 132 strains were sorted into 100 MLVA-16 genotypes (GTs) (GT 1-100), 81 of which were single GTs represented by singular independent strains. The remaining 19 shared GTs involved 51 strains, and each GT included two to seven isolates from the Shaan northern and Guanzhong areas. These data indicated that although sporadic cases were a dominant epidemic characteristic of human brucellosis in this province, more than 38.6% (51/132) outbreaks were also found in the Shaan northern area and Guanzhong areas. The 47 shared MLVA-16 GTs were observed in strains (n = 71) from this study and strains (n = 337) from 19 other provinces of China. These data suggest that strains from the northern provinces are a potential source of human brucellosis cases in Shaanxi Province. It is urgent to strengthen the surveillance and control of the trade and transfer of infected sheep among regions.

Genetic and antimicrobial resistance profiles of non-O157 Shiga toxin-producing Escherichia coli from different sources in Egypt

BackgroundThe Shiga toxin-producing Escherichia coli (STEC) represented a great risk to public health. In this study, 60 STEC strains recovered from broiler and duck fecal samples, cow’s milk, cattle beef, human urine, and ear discharge were screened for 12 virulence genes, phenotypic and genotypic antimicrobial resistance, and multiple-locus variable-number tandem-repeat analysis (MLVA).ResultsThe majority of strains harbored Shiga toxin 1 (stx1) and stx1d, stx2 and stx2e, and ehxA genes, while a minority harbored stx2c subtype and eaeA. We identified 10 stx gene combinations; most of strains 31/60 (51.7%) exhibited four copies of stx genes, namely the stx1, stx1d, stx2, and stx2e, and the strains exhibited a high range of multiple antimicrobial resistance indices. The resistance genes blaCTX-M-1 and blaTEM were detected. For the oxytetracycline resistance genes, most of strains contained tetA, tetB, tetE, and tetG while the tetC was present at low frequency. MLVA genotyping resolved 26 unique genotypes; genotype 21 was highly prevalent. The six highly discriminatory loci DI = 0.9138 are suitable for the preliminary genotyping of STEC from animals and humans.ConclusionsThe STEC isolated from animals are virulent, resistant to antimicrobials, and genetically diverse, thus demands greater attention for the potential risk to human.

A Suggested Diagnostic Approach for Sporadic Anthrax in Cattle to Protect Public Health.

The repeated occurrence of anthrax in grazing animals should be a reminder of a widespread presence of Bacillus anthracis spores in the environment. Its rapid diagnosis is critical to protect public health. Here, we report a case of anthrax in cattle that was investigated using conventional and molecular methods. In 2015, six cows suddenly died within three days and the number of dead animals increased to a total of 12 within two weeks. At necropsy, anthrax was suspected. Therefore, spleen tissue samples were collected (from 6/12 animals) and laboratory tests (microscopy, cultivation, and real-time PCR) performed. The results of tissue staining for microscopy and cultivation were in congruence, while B. anthracis real-time PCR outperformed both. Spleen tissues from all six animals were real-time PCR-positive, while B. anthracis was successfully cultivated and detected by microscopy from the spleen of only three animals. Additionally, the ear tissue from another (1/12) cow tested positive by real-time PCR, supporting the suitability of ear clippings for molecular confirmation of B. anthracis. Genotyping of the isolates using multiple-locus variable-number tandem repeat analysis (MLVA) revealed a common source of infection as all three typed isolates had an indistinguishable MLVA genotype, which has not been observed previously in Europe. The results indicate that molecular testing should be selected as the first-line tool for confirming anthrax outbreaks in animals to ensure timely protection of public health.

Correlating Genotyping Data of Coxiella burnetii with Genomic Groups.

Coxiella burnetii is a zoonotic pathogen that resides in wild and domesticated animals across the globe and causes a febrile illness, Q fever, in humans. Several distinct genetic lineages or genomic groups have been shown to exist, with evidence for different virulence potential of these lineages. Multispacer Sequence Typing (MST) and Multiple-Locus Variable number tandem repeat Analysis (MLVA) are being used to genotype strains. However, it is unclear how these typing schemes correlate with each other or with the classification into different genomic groups. Here, we created extensive databases for published MLVA and MST genotypes of C. burnetii and analysed the associated metadata, revealing associations between animal host and human disease type. We established a new classification scheme that assigns both MST and MLVA genotypes to a genomic group and which revealed additional sub-lineages in two genomic groups. Finally, we report a novel, rapid genomotyping method for assigning an isolate into a genomic group based on the Cox51 spacer sequence. We conclude that by pooling and streamlining existing datasets, associations between genotype and clinical outcome or host source were identified, which in combination with our novel genomotyping method, should enable an estimation of the disease potential of new C. burnetii isolates.

Multidrug-Resistant Acinetobacter baumannii Genetic Characterization and Spread in Lithuania in 2014, 2016, and 2018.

Bacterial resistance to antimicrobial agents plays an important role in the treatment of bacterial infections in healthcare institutions. The spread of multidrug-resistant bacteria can occur during inter- and intra-hospital transmissions among patients and hospital personnel. For this reason, more studies must be conducted to understand how resistance occurs in bacteria and how it moves between hospitals by comparing data from different years and looking out for any patterns that might emerge. Multidrug-resistant (MDR) Acinetobacter spp. was studied at 14 healthcare institutions in Lithuania during 2014, 2016, and 2018 using samples from human bloodstream infections. In total, 194 isolates were collected and identified using MALDI-TOF and VITEK2 analyzers as Acinetobacter baumannii group bacteria. After that, the isolates were analyzed for the presence of different resistance genes (20 genes were analyzed) and characterized by using the Rep-PCR and MLVA (multiple-locus variable-number tandem repeat analysis) genotyping methods. The results of the study showed the relatedness of the different Acinetobacter spp. isolates and a possible circulation of resistance genes or profiles during the different years of the study. This study provides essential information, such as variability and diversity of resistance genes, genetic profiling, and clustering of isolates, to better understand the antimicrobial resistance patterns of Acinetobacter spp. These results can be used to strengthen the control of multidrug-resistant infections in healthcare institutions and to prevent potential outbreaks of this pathogen in the future.

Investigation of Genetic Relatedness of Brucella Strains in Countries Along the Silk Road.

In this study, MLVA (multiple-locus variable-number tandem repeat analysis) genotype data of Brucella strains from 11 countries along the Silk Road were downloaded from the MLVAbank. MLVA data of strains were applied to the constructed Minimum Spanning Tree to explore the species/biovars distribution, geographic origins, and genetic relationships of the strains analyzed. Moreover, whole-genome sequencing–single-nucleotide polymorphism (WGS-SNP) phylogenetic analysis of the genome of Brucella melitensis strains from GenBank was performed to discriminate the relatedness of strains further and investigate the transmission pattern of B. melitensis brucellosis. A total of 1,503 Brucella strains were analyzed in this study: 431 Brucella abortus strains (29.8%), 1,009 B. melitensis strains (65.7%), and 63 Brucella suis strains (4.5%). B. melitensis biovar 3 was the dominant species and was shown to be widespread in all of the examined regions, suggesting that the prevention and surveillance of the B. melitensis population are a main challenge in these countries. A wide host spectrum was observed for this Brucella population; many animal reservoirs are a potential reason for the continuous brucellosis circulation in these countries. Although the B. abortus strains from the examined regions had common geographic origins, only a few shared genotypes were observed in different countries. These data revealed that the majority of B. abortus strains were spreading within the national borders. However, the B. melitensis strains from Italy originated from a Western Mediterranean lineage; strains from the other 10 countries originated from Eastern Mediterranean lineage, and this lineage was shared by strains from three to nine different countries, suggesting that the introduction and reintroduction of the disease in the 10 countries might have occurred in the past. Furthermore, the most shared MLVA-16 genotypes were formed in the B. melitensis strains from China, Kazakhstan, and Turkey, suggesting that the introduction and trade in sheep and goats have occurred frequently in these countries. WGS-SNP analysis showed that the B. melitensis in this study originated from the Malta (Italy) region. According to their territorial affiliation between four clade strains from these countries in genotype B, the absence of a clear differentiation suggests that strains continuously expand and spread in countries along with Silk Road. Active exchange and trade of animals (sheep and goats) among these countries are reasonable explanations. B. suis strains from different nations showed unique geographic origins and epidemiological characteristics. Therefore, there is an urgent need for the control of transfer and trade of infected sheep (goats) in countries along the Silk Road, namely, the strengthening of the entry–exit quarantine of sheep and goats and improvements in the diagnosis of animal brucellosis.

Antimicrobial susceptibility and molecular characteristics of Mycoplasma pneumoniae isolates across different regions of China

BackgroundIn China mainland, most Mycoplasma pneumoniae related studies are carried out in Beijing and Shanghai, while rare studies are performed in the other regions. In this study, we analyzed the molecular biology characteristics and antimicrobial susceptibility of clinical isolates of M. pneumoniae from 5 regions between January 2017 and December 2018.MethodsGenotyping was performed to 154 M. pneumoniae isolates from 5 cities using PCR and multiple-locus variable-number tandem repeat analysis (MLVA) method. Antimicrobial susceptibility test was performed to all the isolates against 4 antibiotics. Sequencing was performed to the amplification products of the 23S rRNA drug resistant gene.ResultsGenotype I was detected in 118 M. pneumoniae isolates (76.6%), and genotype II was identified in 36 isolates (23.4%). The majority (92.2%) of the MLVA genotypes were 4–5–7-2 and 3–5–6-2, which represented the genotype I and II, respectively. The total macrolide (ML) resistance rate was 79.7%. The minimum inhibitory concentration (MIC) of the erythromycin was in a range of 128- > 256 μg/ml, while that for the azithromycin was 2-32 μg/ml. There were mutations in the 23S rRNA in each ML resistance isolate. Jilin city showed the highest prevalence of genotype I (100%) and ML resistance rate (100%), while Jinan showed the lowest prevalence of genotype I (45.5%) and ML resistance rate (54.5%).ConclusionsA large variance was identified in the M. pneumoniae genotype and ML resistance among the 5 cities. The proportion of M. pneumoniae with a genotype II genotype (3–5–6-2) showed an increased trend.

Shedding and genetic diversity of Coxiella burnetii in Polish dairy cattle.

Q fever is a worldwide zoonotic disease reported in humans and many animal species including cattle. The aims of this study were to evaluate the prevalence of Coxiella (C.) burnetii shedding in Polish dairy cattle herds and to identify the pathogen’s genotypes and sequence types (STs) using multiple-locus variable number tandem repeat analysis (MLVA) and multispacer sequence typing (MST) methods. The presence of C. burnetii DNA was detected using a commercial real-time PCR kit, targeting the IS1111 element. Overall, 1,439 samples from 279 herds were tested including: 897 individual milk specimens, 101 bulk tank milk samples, 409 genital tract swabs and 32 placentas. Furthermore, 30 consumer milk samples, including 10 from vending machines and 77 dairy products were also analyzed. C. burnetii shedding was confirmed in 31.54% of tested cattle herds as well as in 69.16% of consumer milk and dairy products. Among real-time PCR–positive samples, 49 specimens obtained from 49 cattle herds and 8 samples of purchased dairy products were selected for genotyping. Overall, five previously known MLVA genotypes (I, J, BG, BE, and NM) and three new ones (proposed as PL1, PL2, and PL3) were identified. Two MST sequence types were recorded: ST16 and a novel sequence (ST61). The new genotypes and sequence types need further research particularly into their pathogenicity to humans.

Genotyping of pathogenic leptospira by Multiple Locus Variable-number Tandem Repeat Analysis (MLVA)

ObjectiveTo introduce the method of molecular genotyping (MLVA) to determine the genotype of field isolates of leptospira.IntroductionLeptospirosis (ictherohemoglobinuria, Leptospirosis biliousness) is a natural focal and zoonotic infectious disease dangerous for humans and farm animals. It is important to identify specific leptospira strains isolated from rodents or sick and suspicious animals by the serotype or genotype. In comparison with serotyping using micro agglutination test (MAT), molecular genotyping makes it possible to accurately identify a specific pathogen strain. The genetic classification now becomes more significant than the phenotypic classification.MethodsSpecific oligonucleotide primers, which flank fragments of the genome locus of pathogenic leptospira varies in terms of the number of tandem repeats VNTR-4, -7, -10 specific for L.interrogans, L.kirschneri, and L.borgpetersenii were used. The amplification products were detected using agar gel electrophoresis with the following identification of the fragment length with a molecular weight marker and comparison with the collection of VNTR profiles of the strains described in the literature.ResultsIt was established that the method of leptospira molecular genotyping by determining the number of variable tandem repeats of a locus (VNTR-variable number tandem repeats analysis) is suitable for molecular epizootology studies in Ukraine. The advantages of the method are the simplicity of performance and availability for diagnostic and research laboratories in Ukraine compared to other pathogen genome sequencing based genotyping methods, in particular Multilocus sequence typing (MLST) or Multispacer Sequence Typing (MST), which require complex equipment and operating conditions. The reference strain of Leptospira M20 serotype Copengageni serogroup Icterohaemorrhagiae from the NAAS IVM collection of was studied and its VNTR profile was identified with the genotype of the strain Fiocruz L1-130 that is described in the literature as a serotype of Copengageni serogroup Icterohaemorrhagiae. The genotype of the leptospira field isolate obtained from a rat in Lviv Oblast of Ukraine was specified and its identity was established in the aforementioned genotype. The obtained data support the prospects of using MLVA genotyping method to study the distribution of different genotypes of leptospira. The research will continue to study the specificities of molecular epizootology of leptospirosis in Ukraine.ConclusionsThe method of leptospira molecular genotyping by multilocus analysis of the number of variable tandem repeats has been tested in the Leptospirosis Research Laboratory in collaboration with the Museum of Microorganisms at the National Academy of Sciences, the Ukraine Institute of Veterinary Medicine, the ELISA and PCR Research Laboratory, and the Bila Tserkva National Agrarian University. The genotype of the reference strain has been correlated with its serological profile; identification of the genotype of the field isolate pathogenic leptospira has been completed. The tested method is planned to be implemented in surveillance and control over leptospirosis spreading in Ukraine, and aimed to help in development and improvement of leptospirosis vaccine formulations. Additionally, method of Multiple-Locus Variable number tandem repeat Analysis will be used for molecular epidemiology research in Ukraine.ReferencesSalaün L, Mérien F, Gurianova S, Baranton G, Picardeau M. Application of multilocus variable-number tandem-repeat analysis for molecular typing of the agent of leptospirosis. J Clin Microbiol. 2006;44(11):3954-3962. doi:10.1128/JCM.00336-06.Caimi K, Repetto SA, Varni V, Ruybal P. Infection , Genetics and Evolution Leptospira species molecular epidemiology in the genomic era. Infect Genet Evol. 2017;54(July):478-485. doi:10.1016/j.meegid.2017.08.013.Ayral F, Zilber AL, Bicout DJ, Kodjo A, Artois M, Djelouadji Z. Distribution of leptospira interrogans by multispacer sequence typing in urban Norway rats (Rattus norvegicus): A survey in France in 2011-2013. PLoS One. 2015;10(10):1-14. doi:10.1371/journal.pone.0139604.

Relationship between macrolide resistance mutations in Mycoplasma pneumoniae and its genotype

Objective To analyze the relationship between macrolide resistance mutations in Mycoplasma pneumoniae (Mp) and its genotype by multiple-locus variable-number tandem-repeat analysis (MLVA). Methods One hundred and forty-three Mp-positive specimens were collected in Beijing (54 collected at the Affiliated Children′s Hospital of the Capital Institute of Pediatrics), the United States (59 collected at four different geographical locations: Kansas City, Missouri; Seattle, Washington; New York, New York; Chicago, Illinois) and Australia (30 provided by the diagnostic laboratory at the Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Sydney). Nested PCR was used to detect mutations in 23S rRNA. A capillary electrophoresis-based single tube multiplex PCR (mPCR-CE) was used to analyze the MLVA types of Mp in those samples. Results A2063G mutation was identified in 57 specimens including 49 from Beijing, seven from the United States and one from Australia. The 143 Mp-positive specimens were typed into 10 distinct MLVA types. Fifty-four specimens collected in Beijing belonged to four MLVA types, which were M4-5-7-2 (44/54, 81.5%), M3-5-6-2 (7/54, 13.0%), M4-5-6-2 (2/54, 3.70%) and M4-5-5-2 (1/54, 1.85%). Fifty-nine specimens collected in the United States belonged to six MLVA types including M4-5-7-2 (27/59, 45.8%), M3-5-6-2 (18/59, 30.5%), M3-6-6-2 (11/59, 18.6%), M3-5-6-1 (1/59, 1.69%), M4-5-7-3 (1/59, 1.69%) and M5-5-7-2 (1/59, 1.69%). Thirty specimens of Mp from Australia were grouped to five types with M3-5-6-2 (12/30, 40.0%) and M4-5-7-2 (10/30, 33.3%) and M3-5-7-2 (5/30, 16.7%) being the predominant types. Macrolide resistance mutations were detected in 57 out of 143 specimens (49 from Beijing, seven from the United States and one from Sydney) and 50 of them were MLVA type of M4-5-7-2. Conclusion The MLVA type of M4-5-7-2 is associated with macrolide resistance in Mp. Key words: Mycoplasma pneumoniae; Macrolide resistance; 23S rRNA; MLVA genotype

Genome Plasticity and Polymorphisms in Critical Genes Correlate with Increased Virulence of Dutch Outbreak-Related Coxiella burnetii Strains.

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. During 2007–2010 the largest Q fever outbreak ever reported occurred in The Netherlands. It is anticipated that strains from this outbreak demonstrated an increased zoonotic potential as more than 40,000 individuals were assumed to be infected. The acquisition of novel genetic factors by these C. burnetii outbreak strains, such as virulence-related genes, has frequently been proposed and discussed, but is not proved yet. In the present study, the whole genome sequence of several Dutch strains (CbNL01 and CbNL12 genotypes), a few additionally selected strains from different geographical locations and publicly available genome sequences were used for a comparative bioinformatics approach. The study focuses on the identification of specific genetic differences in the outbreak related CbNL01 strains compared to other C. burnetii strains. In this approach we investigated the phylogenetic relationship and genomic aspects of virulence and host-specificity. Phylogenetic clustering of whole genome sequences showed a genotype-specific clustering that correlated with the clustering observed using Multiple Locus Variable-number Tandem Repeat Analysis (MLVA). Ortholog analysis on predicted genes and single nucleotide polymorphism (SNP) analysis of complete genome sequences demonstrated the presence of genotype-specific gene contents and SNP variations in C. burnetii strains. It also demonstrated that the currently used MLVA genotyping methods are highly discriminatory for the investigated outbreak strains. In the fully reconstructed genome sequence of the Dutch outbreak NL3262 strain of the CbNL01 genotype, a relatively large number of transposon-linked genes were identified as compared to the other published complete genome sequences of C. burnetii. Additionally, large numbers of SNPs in its membrane proteins and predicted virulence-associated genes were identified in all Dutch outbreak strains compared to the NM reference strain and other strains of the CbNL12 genotype. The presence of large numbers of transposable elements and mutated genes, thereof most likely resulted in high level of genome rearrangements and genotype-specific pathogenicity of outbreak strains. Thus, the epidemic potential of Dutch outbreak strains could be linked to increased genome plasticity and mutations in critical genes involved in virulence and the evasion of the host immune system.

Temperature-Dependent Growth Modeling of Environmental and Clinical Legionella pneumophila Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Genotypes.

Legionella pneumophila causes waterborne infections resulting in severe pneumonia. High-resolution genotyping of L. pneumophila isolates can be achieved by multiple-locus variable-number tandem-repeat analysis (MLVA). Recently, we found that different MLVA genotypes of L. pneumophila dominated different sites in a small drinking-water network, with a genotype-related temperature and abundance regime. The present study focuses on understanding the temperature-dependent growth kinetics of the genotypes that dominated the water network. Our aim was to model mathematically the influence of temperature on the growth kinetics of different environmental and clinical L. pneumophila genotypes and to compare it with the influence of their ecological niches. Environmental strains showed a distinct temperature preference, with significant differences among the growth kinetics of the three studied genotypes (Gt4, Gt6, and Gt15). Gt4 strains exhibited superior growth at lower temperatures (25 and 30°C), while Gt15 strains appeared to be best adapted to relatively higher temperatures (42 and 45°C). The temperature-dependent growth traits of the environmental genotypes were consistent with their distribution and temperature preferences in the water network. Clinical isolates exhibited significantly higher growth rates and reached higher maximal cell densities at 37°C and 42°C than the environmental strains. Further research on the growth preferences of L. pneumophila clinical and environmental genotypes will result in a better understanding of their ecological niches in drinking-water systems as well as in the human body.IMPORTANCELegionella pneumophila is a waterborne pathogen that threatens humans in developed countries. The bacteria inhabit natural and man-made freshwater environments. Here we demonstrate that different environmental L. pneumophila genotypes have different temperature-dependent growth kinetics. Moreover, Legionella strains that belong to the same species but were isolated from environmental and clinical sources possess adaptations for growth at different temperatures. These growth preferences may influence the bacterial colonization at specific ecological niches within the drinking-water network. Adaptations for growth at human body temperatures may facilitate the abilities of some L. pneumophila strains to infect and cause illness in humans. Our findings may be used as a tool to improve Legionella monitoring in drinking-water networks. Risk assessment models for predicting the risk of legionellosis should take into account not only Legionella concentrations but also the temperature-dependent growth kinetics of the isolates.

Genotyping of Brucella melitensis strains from dromedary camels (Camelus dromedarius) from the United Arab Emirates with multiple-locus variable-number tandem repeat analysis

Camel brucellosis is a widespread zoonotic disease in camel-rearing countries caused by Brucella melitensis and Brucella abortus. The aim of this study was the first genetic analysis of B. melitensis strains isolated from dromedary camels (Camelus dromedarius) using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA 16 and its MLVA 8 and MLVA11 subsets were used to determine the genotypes of 15 B. melitensis isolates from dromedary camels (11 strains) and other host species (4 strains) from the United Arab Emirates and the results were then compared to B. melitensis MLVA genotypes from other parts of the world. Five, including two novel genotypes were identified with MLVA 8. MLVA 16 further discriminated these five genotypes to ten variants. The eleven camel isolates clustered into four main genetic groups within the East-Mediterranean and African clades and this clustering correlated with the geographic origin of the hosts (United Arab Emirates, Kingdom of Saudi Arabia and Sudan) and the date of their isolation. The camel strains were also genetically related to strains isolated from wild and domestic ruminants from their close habitat or from other parts of the world. Although limited number of strains were analysed, based on our data imported animals from foreign countries, local small ruminants and wildlife species are hypothesized to be the main sources of camel brucellosis in the United Arab Emirates. MLVA was successfully applied to determine the epidemiological links between the different camel B. melitensis infections in the United Arab Emirates and it can be a beneficial tool in future disease control programs.

A Familial Cluster of Human Brucellosis Attributable to Contact with Imported Infected Goats in Shuyang, Jiangsu Province, China, 2013.

Brucellosis remains a serious public health issue in developing countries, including China. On August 8, 2013, four cases of brucellosis from one extended family were reported at Shuyang County, Jiangsu Province, China. Active case finding was performed to identify the source and the risk factors of the infection and to prevent additional cases. Multiple-locus variable number tandem repeat analysis (MLVA) was used for molecular subtyping analysis. Six people from two extended families met the case definition for brucellosis infection; four were blood culture positive for Brucella melitensis biotype 3. Four additional family members were found seropositive by using a serological test. Isolates from the four patients were indistinguishable by MLVA profiling, displaying a unique type for Jiangsu Province. Field epidemiological data combined with MLVA genotyping supported a common source of the isolates from the different patients. We recommend stronger reinforcement measures for animal quarantine practices, enhanced cooperation with veterinary service organizations, and implementation of measures that strengthen public education on brucellosis to prevent further human outbreaks in Jiangsu Province.

Identification of novel Coxiella burnetii genotypes from Ethiopian ticks.

Background Coxiella burnetii, the etiologic agent of Q fever, is a highly infectious zoonotic bacterium. Genetic information about the strains of this worldwide distributed agent circulating on the African continent is limited. The aim of the present study was the genetic characterization of C. burnetii DNA samples detected in ticks collected from Ethiopian cattle and their comparison with other genotypes found previously in other parts of the world.Methodology/Principal FindingsA total of 296 tick samples were screened by real-time PCR targeting the IS1111 region of C. burnetii genome and from the 32 positive samples, 8 cases with sufficient C. burnetii DNA load (Amblyomma cohaerens, n = 6; A. variegatum, n = 2) were characterized by multispacer sequence typing (MST) and multiple-locus variable-number tandem repeat analysis (MLVA). One novel sequence type (ST), the proposed ST52, was identified by MST. The MLVA-6 discriminated the proposed ST52 into two newly identified MLVA genotypes: type 24 or AH was detected in both Amblyomma species while type 26 or AI was found only in A. cohaerens.Conclusions/SignificanceBoth the MST and MLVA genotypes of the present work are closely related to previously described genotypes found primarily in cattle samples from different parts of the globe. This finding is congruent with the source hosts of the analyzed Ethiopian ticks, as these were also collected from cattle. The present study provides genotype information of C. burnetii from this seldom studied East-African region as well as further evidence for the presumed host-specific adaptation of this agent.