Genetic polymorphisms identify in species/biovars of Brucella isolated in China between 1953 and 2013 by MLST.

BACKGROUND: Brucellosis incidence in China is divided into three stages: high incidence (1950s-1960s), decline (1970s-1980s), and re-emergence (1990s-2010s). At the re-emergence stage, Brucellosis incidence grew exponentially and spread to all 32 provinces. We describe the magnitude and the etiological distribution changes in mainland China by genotyping data and emphasize its recent reemergence. We also provide the genetic diversity and molecular epidemiological characteristics of Brucella. RESULTS: From a total of 206 Brucella isolates, 19 MLST genotypes (STs) were identified and 13 new STs(ST71-83)were found. MLST grouped the population into three clusters. B. melitensis, B. abortus and B. suis were grouped into cluster 1, 2 and 3 respectively. The predominant genotype in the first cluster by MLST, remained unchanged during the three stages. However, the proportion of genotypes in the three stages had changed. More isolates were clustered in ST8 at the re-emergence stage. STs71-74, which were not found in the two former stages, appeared at the re-emergence stage. CONCLUSIONS: The changing molecular epidemiology of brucellosis improve our understanding of apparent geographic expansion from the historically affected north of China to southern provinces in recent reemergence.

In vitro antimicrobial susceptibility testing of human Brucella melitensis isolates from Ulanqab of Inner Mongolia, China.

BACKGROUND: Brucellosis is an endemic disease in the Inner Mongolia Autonomous Region of China and Ulanqab exhibits the highest prevalence of brucellosis in this region. Due to the complex nature of Brucellosis, a cure for this disease has proven to be elusive. Furthermore, the reduced susceptibility of Brucella spp. to antimicrobial agents has been reported as a potential cause of therapeutic failure. However, detailed in vitro antimicrobial susceptibility patterns pertaining to Brucella isolates from this region have not yet been published. The aim of this study was to evaluate the antibiotic susceptibility profile of Brucella melitensis clinical isolates from Ulanqab, Inner Mongolia, China. METHODS: A total of 85 B. melitesis isolates were obtained from humans in Ulanqab of Inner Mongolia, China; the antimicrobial susceptibility of 85 clinical isolates to nine antibiotics was assessed using the E-test method according to the CLSI (Clinical and Laboratory Standards Institute) guidelines. RESULTS: All of the tested isolates were susceptible to minocycline, sparfloxacin, doxycycline, tetracycline, ciprofloxacin, gentamicin and levofloxacin. Resistance to rifampin and cotrimoxazole was observed in 1.0% (1/85) and 7.0% (6/85) of the isolates, respectively. However, rpoB gene mutations were not observed in single isolates exhibiting resistance to rifampin. CONCLUSIONS: We observed that B. melitensis isolates are susceptible to the majority of the tested antibiotics. Furthermore, minocycline and sparfloxacin exhibited extremely high bactericidal effects in relation to the B. melitensis isolates. The sensitivity of commonly used drugs for the treatment of brucellosis should be regularly monitored. To the best of our knowledge, this is the first report of rifampin and cotrimoxazole resistant isolates of B. melitensis in China. In summary, based on the findings from this study, we suggest that antibiotic administration and use should be rationalized to prevent future drug resistance.

Comparative genomic analysis between newly sequenced Brucella suis Vaccine Strain S2 and the Virulent Brucella suis Strain 1330.

BACKGROUND: Brucellosis is a bacterial disease caused by Brucella infection. In the late fifties, Brucella suis vaccine strain S2 with reduced virulence was obtained by serial transfer of a virulent B. suis biovar 1 strain in China. It has been widely used for vaccination in China since 1971. Until now, the mechanisms underlie virulence attenuation of S2 are still unknown. RESULTS: In this paper, the whole genome sequencing of S2 was carried out by Illumina Hiseq2000 sequencing method. We further performed the comparative genomic analysis to find out the differences between S2 and the virulent Brucella suis strain 1330. We found premature stops in outer membrane autotransporter omaA and eryD genes. Single mutations were found in phosphatidylcholine synthase, phosphorglucosamine mutase, pyruvate kinase and FliF, which have been reported to be related to the virulence of Brucella or other bacteria. Of the other different proteins between S2 and 1330, such as Omp2b, periplasmic sugar-binding protein, and oligopeptide ABC transporter, no definitive implications related to bacterial virulence were found, which await further investigation. CONCLUSIONS: The data presented here provided the rational basis for designing Brucella vaccines that could be used in other strains.

Rapid detection of Haemophilus influenzae and Haemophilus parainfluenzae in nasopharyngeal swabs by multiplex PCR.

OBJECTIVE: To establish multiplex PCR-based assays for detecting H.influenzae and H.parainfluenzae. And the PCR-based assays were applied to detect the carriage rates of H.influenzae and H.parainfluenzae in nasopharyngeal swab specimens which were collected from healthy children. METHODS: Multiplex primers for species-specific PCR were designed by using DNAstar soft based on the sequences of 16S rRNA genes from genus Haemophilus to detect H.influenzae and H.parainfluenzae. RESULTS: The sensitivity of the 16S rRNA PCR assay for detecting H.influenzae and H.parainfluenzae was 97.53% and 100% respectively, and the specificity was 95.89% and 96.63% respectively. Youden's Index on the ability to detect H.influenzae and H.parainfluenzae was 0.9342 and 0.9663 respectively. 666 nasopharyngeal swab specimens were collected from healthy children. The detection rates of H.influenzae and H.parainfluenzae were 14.11% and 16.07% respectively by using isolation and culture methods. The detection rates of H.influenzae and H.parainfluenzae were 43.54% and 57.96% respectively by 16S rRNA PCR assays. The carriage rates of serotypes a, b, c, d, e, f and non-typeable isolates were 0% (0/666), 0.15% (1/666), 1.20% (8/666), 0.15% (1/666), 1.20% (8/666), 1.80% (12/666), 95.50% (636/666) respectively. CONCLUSION: The multiplex PCR assays were very rapid, reliable and feasible methods for detection of H.influenzae and H.parainfluenzae in pharyngeal swab specimens which were compared to conventional isolation and culture methods. 95.5% of H.influenzae strains in healthy children were nontypeable. The encapsulated or typable strains were mainly three serotypes which was c, e, and f serotype.

A Flavobacterium lindanitolerans strain isolated from the ascites sample of a Chinese patient with EV71 virus infection.

A strain of Flavobacterium lindanitolerans isolated from a sick child's ascites was described. The 16S rRNA gene of the strain was 100% identical to that of Flavobacterium lindanitolerans which was first identified in India in 2008. It was first described that the isolate required X factor (Hemin) for growth in the optimal conditions of 37 °C with 5% CO(2). The isolate produced indole and H(2)S. It did not present hemolytic feature on blood agar.

Molecular epidemiology of Brucella abortus strains from cattle in Inner Mongolia, China.

Although the prevalence of brucellosis in Inner Mongolia Autonomous Region currently remains high, data available on the epidemiological of circulating Brucella abortus strains were limited. A total of 75 isolates obtained from cattle, sheep, and humans were analysed using both the classical method and multiple locus variable-number tandem repeat analysis (MLVA). There are at least three B. abortus biovars (1, 3 and 6) in this region, and B. abortus biovar 3 is the predominant one. Ten known MLVA-11 genotypes were identified, of which five genotypes (72, 75, 78, 82 and 210) were shared among strains from this study and others previously collected in two to seven different nations, suggesting that this population has multiple geographic origins. An MLVA-16 assay sorted the 75 B. abortus strains into two groups (I and II), 5 clusters (A-E) and 44 genotypes (GT1-44), with 26 unique genotypes represented by single isolates, indicating that these B. abortus brucellosis cases were not directly epidemiologically related. The remaining 18 shared genotypes (among a total of 47 isolates) were represented by two to eight isolates, suggesting that there were epidemiologically related pathogens from each shared genotype among the cases. Importantly, the cluster B1 branch including 22 cluster isolates with identical or similar genotypes confirmed the occurrence of a concentrated outbreak epidemic in the eastern region during 1988-1995. This work will contribute to better understanding of B. abortus brucellosis epidemiology in Inner Mongolia.

Molecular characterization of Tb, a new approach for an ancient Brucellaphage.

Tb (Tbilisi), the reference Brucellaphage strain, was classified as a member of the Podoviridae family with icosahedral capsids (57 +/- 2 nm diameter) and short tails (32 +/- 3 nm long). Brucellaphage DNA was double stranded and unmethylated; its molecular size was 34.5 kilobase pairs. Some sequences were found through RAPD analysis, TA cloning technology, and structural proteins were observed by using SDS-PAGE. Thus, the results have laid the foundation for the wider use of Brucellaphage's basic mechanisms and practical applications.

[Public health situation and needs assessment after Wenchuan earthquake in Anxian, Sichuan province].

OBJECTIVE: To explore the public health situation and needs in Anxian after Wenchuan earthquake so as to make an effective strategy for disease control and prevention. METHODS: 69 concentrated settlements with 100 residents were investigated. Probability proportion to size was adopted for sampling of 2200 residents from 687 scattered households (about 440 000 scattered residents). The content of this survey included drinking water, food hygiene, environment sanitation, planning immunity and medical health service, disease surveillance and so on. SPSS 16.0 was used for data analysis, and statistical interpretation was used to describe the results. RESULTS: 90.9% (31/66) resettled residents in Anxian lived in tents, 7.6% (5/66) lived in the movable-plate house, 93.3% (621/666) scattered households lived in tents and 71.9% (446/621) of them lived in tents which were built by residents themselves; the rate of drinking water disinfection in resettlement sites and scattered households were 97.1% (66/68) and 94.6% (650/687); 12.8% scattered residents had mouldy or food; 50% of resettlement sites raised animals; 43.6% (17/39) medical station didn't have bacterin inoculation service; 66.7% (10/15) lacked sufficient disinfection equipment; register rate was 50.0% (33/66) and report rate of symptoms and infectious diseases was 56.1% (37/66). CONCLUSION: There was still some risk of enteric and vector-borne diseases in Anxian, therefore, some tailored measures should be very important.

[Mental health assessment among scattered residents after Wenchuan earthquake in Anxian, Sichuan province].

OBJECTIVE: To explore the mental health status of residents scattered living in Anxian after Wenchuan earthquake so as to provide scientific basis for further mental health intervention. METHODS: A face to face interview was conducted among the scattered residents with designed questionnaire, which had three parts of the physical and emotional reaction, the relax methods and the social care and supports expected. Two-stage probability proportional to size (PPS) sample method was performed to sample 2184 from 0.44 million scattered residents in Anxian. On the basis of statistical description, mental health of different characteristics groups was compared. RESULTS: Three main symptoms of posttraumatic stress disorders in 2184 residents (11.23+/-3.44) were higher than the 103 fire victims in Hunan in 2003 (10.06+/-3.26), three factor scores of SCL-90 (5.76+/-1.74) were higher than normal in 1998 repair mode (n=23 891) (4.72+/-1.44), and the statistical difference was observed (t=10.77, P<0.05; t=706.04, P<0.05). Comparing the mental health of different groups, some significant differences were found by age, gender and education background. CONCLUSION: The earthquake disaster brought prevalent physical and emotional reaction. Elderly people, female, junior students need mental intervention immediately. Therefore, strengthen the mental education and assistance (especially in high risk groups) would be of more significance.

Whole-Genome Sequencing of a Brucella melitensis Strain (BMWS93) Isolated from a Bank Clerk and Exhibiting Complete Resistance to Rifampin.

Human brucellosis has become the most severe public health problem in the Ulanqab region of Inner Mongolia, China. Brucella melitensis BMWS93 was obtained from a blood sample taken from a bank clerk in the Ulanqab region of Inner Mongolia, China, and antimicrobial susceptibility testing in vitro showed no zone of inhibition, which confirmed resistance to rifampin. Therefore, whole-genome sequencing of this isolate was performed to better understand the mechanism of this resistance.

Molecular Investigation of the Transmission Pattern of Brucella suis 3 From Inner Mongolia, China.

Brucellosis is an endemic disease in China affecting both humans and livestock. The aim of the present study was to analyze two Brucella strains isolated from sheep spleens from Ulanqab in Inner Mongolia, China using classical and molecular typing techniques. The two strains were identified as Brucella suis biovar 3 and were closely related to isolates previously obtained from two different hosts (human and swine) in Guangxi Province. Our results suggest that B. suis can be directly or indirectly transferred from swine to sheep, which act as reservoirs for B. suis infection and later transmitted to humans. Multiple locus variable-number tandem repeat analysis (MLVA) is a useful tool for tracing the geographical origin of brucellosis infections and elucidating its transmission patterns.

A case report of spontaneous abortion caused by Brucella melitensis biovar 3.

BACKGROUND: Brucellosis is a worldwide zoonotic disease caused by Brucella spp. Brucella invades the body through the skin mucosa, digestive tract, and respiratory tract. However, only a few studies on human spontaneous abortion attributable to Brucella have been reported. In this work, the patient living in Shanxi Province in China who had suffered a spontaneous abortion was underwent pathogen detection and Brucella melitensis biovar 3 was identified. CASE PRESENTATION: The patient in this study was 22 years old. On July 16, 2015, she was admitted to Shanxi Grand Hospital, Shanxi Province, China because of one day of vaginal bleeding and three days of abdominal distension accompanied by fever after five months of amenorrhea. A serum tube agglutination test for brucellosis and blood culture were positive. At the time of discharge, she was prescribed oral doxycycline (100 mg/dose, twice a day) and rifampicin (600 mg/dose, once daily) for 6 weeks as recommended by the World Health Organization (WHO). No recurrence was observed during the six months of follow-up after the cessation of antibiotic treatment. CONCLUSIONS: This is the first reported case of miscarriage resulting from Brucella melitensis biovar 3 isolated from a pregnant woman who was infected through unpasteurized milk in China. Brucellosis infection was overlooked in the Maternity Hospital because of physician unawareness. Early recognition and prompt treatment of brucellosis infection are crucial for a successful outcome in pregnancy.

[Comparative study on identity of B. ovis 019 strain by traditional methods and HOOF-prints technique].

B. ovis 019 strain was identified by traditional methods and HOOF-Prints technique. Software DNAMAN was used to analyze phylogenetic tree for B. ovis 019 and reference strains of B. abortus 2308, B. melitensis 16M, B. suis 1330 and B. ovis 63/290. The results showed that the similarity between B. ovis 019 and B. ovis 63/290 was higher than that between B. ovis 019 and the other reference strains, which was in accordance with the results by conventional bacteriological methods. HOOF-Prints technique would be a promising method for identifying Brucella species and even biovars.

MLVA Genotyping Characteristics of Human Brucella melitensis Isolated from Ulanqab of Inner Mongolia, China.

Brucellosis is a serious public health problem in Ulanqab, which is a region located in the middle of the Inner Mongolia Autonomous Region adjacent to Shanxi and Hebei provinces. The disease is prevalent in both the latter provinces and Ulanqab with the highest prevalence of brucellosis occurring in Inner Mongolia. The MLVA-16 scheme is a genotyping tool for assessing genetic diversity and relationships among isolates. Moreover, this genotyping tool can also be applied to epidemiological trace-back investigations. This study reports the occurrence of at least two B. melitensis biovars (1 and 3) in Ulanqab, encompassing 22 and 94 isolates, respectively. B. melitensis biovar 3 was the predominant biovar in the area examined. Panel 1 (MLVA-8) identified three genotypes (42, 63, and 114), with genotype 42 (n = 101) representing 87% of the tested strains. MLVA-11 identified eight genotypes (116, 111, 297, 291, and 342-345) from 116 of the analyzed isolates. All of these isolates were identified as belonging to the East Mediterranean group. Genotype 116 (n = 94) was the predominant genotype and represented 81% of the isolates. The isolates pertaining to this genotype were distributed throughout most of Ulanqab and neighboring regions. The MLVA-16 scheme showed the presence of 69 genotypes, with 46 genotypes being represented by single isolates. This analysis revealed that Ulanqab brucellosis cases had epidemiologically unrelated and sporadic characteristics. The remaining 23 genotypes were shared (between a total of 70 isolates) with each genotype being represented by two to eight isolates. These data indicate that these cases were epidemiologically related. MLVA genotyping confirmed the occurrence of a multipoint outbreak epidemic and intrafamilial brucellosis. Extensive genotype-sharing events were observed among isolates from different regions of Ulanqab and from other provinces of China. These findings suggest either a lack of control of animal movement between different regions or the circulation of contaminated animal products in the market. Our study is the first comprehensive genotyping and genetic analysis of B. melitensis in Ulanqab. We believe that this study will help to improve the effectiveness of brucellosis control programs.

Multi-locus variable-number tandem repeat analysis of Chinese Brucella strains isolated from 1953 to 2013.

BACKGROUND: Brucellosis was a common human and livestock disease caused by Brucella strains, the category B priority pathogens by the US Center for Disease Control (CDC). Identified as a priority disease in human and livestock populations, the increasing incidence in recent years in China needs urgent control measures for this disease but the molecular background important for monitoring the epidemiology of Brucella strains at the national level is still lacking. METHODS: A total of 600 Brucella isolates collected during 60 years (from 1953 to 2013) in China were genotyped by multiple locus variable-number tandem repeat analysis (MLVA) and the variation degree of MLVA11 loci was calculated by the Hunter Gaston Diversity Index (HGDI) values. The charts and map were processed by Excel 2013, and cluster analysis and epidemiological distribution was performed using BioNumerics (version 5.1). RESULTS: The 600 representative Brucella isolates fell into 104 genotypes with 58 singleton genotypes by the MLVA11 assay, including B. melitensis biovars 2 and 3 (five main genotypes), B. abortus biovars 1 and 3 (two main genotypes), B. suis biovars 1 and 3 (three main genotypes), and B. canis (two main genotypes) respectively. While most B. suis biovar 1 and biovar 3 were respectively found in northern provinces and southern provinces, B. melitensis and B. abortus strains were dominant in China. Canine Brucellosis was only found in animals without any human cases reported. Eight Brucellosis epidemic peaks emerged during the 60 years between 1953 and 2013: 1955 - 1959, 1962 - 1969, 1971 - 1975, 1977 - 1983, 1985 - 1989, 1992 - 1997, 2000 - 2008 and 2010 - 2013 in China. CONCLUSIONS: Brucellosis has its unique molecular epidemiological patterns with specific spatial and temporal distribution according to MLVA. TRIAL REGISTRATION: IDOP-D-16-00101.

MLVA and MLST typing of Brucella from Qinghai, China.

BACKGROUND: The Qinghai-Tibet Plateau (QTP) of China is an extensive pastoral and semi-pastoral area, and because of poverty and bad hygiene conditions, Brucella is highly prevalent in this region. In order to adequately prevent this disease in the QTP region it is important to determine the identity of Brucella species that caused the infection. METHODS: A total of 65 Brucella isolates were obtained from human, livestock and wild animals in Qinghai, a Chinese province in east of the QTP. Two molecular typing methods, MLVA (multi-locus variable-number tandem-repeat analysis) and MLST (multi locus sequence typing) were used to identify the species and genotypes of these isolates. FINDINGS: Both MLVA and MLST typing methods classified the 65 isolates into three species, B. melitensis, B. abortus and B. suis, which included 60, 4 and 1 isolates respectively. The MLVA method uniquely detected 34 (Bm01 ~ Bm34), 3 (Ba01 ~ Ba03), and 1 (Bs01) MLVA-16 genotypes for B. melitensis, B. abortus and B. suis, respectively. However, none of these genotypes exactly matched any of the genotypes in the Brucella2012 MLVA database. The MLST method identified five known ST types: ST7 and ST8 (B. melitensis), ST2 and ST5 (B. abortus), and ST14 (B. suis). We also detected a strain with a mutant type (3-2-3-2-?-5-3-8-2) of ST8 (3-2-3-2-1-5-3-8-2). Extensive genotype-sharing events could be observed among isolates from different host species. CONCLUSIONS: There were at least three Brucella (B. melitensis, B. abortus and B. suis) species in Qinghai, of which B. melitensis was the predominant species in the area examined. The Brucella population in Qinghai was very different from other regions of the world, possibly owing to the unique geographical characteristics such as extremely high altitude in QTP. There were extensive genotype-sharing events between isolates obtained from humans and other animals. Yaks, sheep and blue sheep were important zoonotic reservoirs of brucellosis causing species found in humans.

Typing Discrepancy Between Phenotypic and Molecular Characterization Revealing an Emerging Biovar 9 Variant of Smooth Phage-Resistant B. abortus Strain 8416 in China.

A newly isolated smooth colony morphology phage-resistant strain 8416 isolated from a 45-year-old cattle farm cleaner with clinical features of brucellosis in China was reported. The most unusual phenotype was its resistance to two Brucella phages Tbilisi and Weybridge, but sensitive to Berkeley 2, a pattern similar to that of Brucella melitensis biovar 1. VITEK 2 biochemical identification system found that both strain 8416 and B. melitensis strains shared positive ILATk, but negative in other B. abortus strains. However, routine biochemical and phenotypic characteristics of strain 8416 were most similar to that of B. abortus biovar 9 except CO2 requirement. In addition, multiple PCR molecular typing assays including AMOS-PCR, B. abortus special PCR (B-ab PCR) and a novel sub-biovar typing PCR, indicated that strain 8416 may belong to either biovar 3b or 9 of B. abortus. Surprisingly, further MLVA typing results showed that strain 8416 was most closely related to B. abortus biovar 3 in the Brucella MLVA database, primarily differing in 4 out of 16 screened loci. Therefore, due to the unusual discrepancy between phenotypic (biochemical reactions and particular phage lysis profile) and molecular typing characteristics, strain 8416 could not be exactly classified to any of the existing B. abortus biovars and might be a new variant of B. abortus biovar 9. The present study also indicates that the present phage typing scheme for Brucella sp. is subject to variation and the routine Brucella biovar typing needs further studies.

MLVA typing and antibiotic susceptibility of Brucella human isolates from Liaoning, China.

We have characterized clinical isolates of Brucella melitensis from Liaoning, China, using multiple-locus variable-number repeat analysis of 16 loci (MLVA-16) and evaluated the antimicrobial susceptibilities in vitro. All 31 isolates shared the panel 1 genotype 42 profile suggesting that they belong to the 'East Mediterranean' group. In vitro antibiotic susceptibility was determined by the broth microdilution testing. All isolates were sensitive to tested antibiotics (rifampin, doxycycline, ciprofloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, ceftriaxone, streptomycin and trimethoprim-sulfamethoxazole) in addition to azithromycin and clarithromycin. Partial nucleotide sequences of the 23SrRNA gene were analyzed for all isolates and single nucleotide polymorphism alterations of 2632 T/C were found. The MLVA-16 assay can be applied to the long-term surveillance and investigation of strain relatedness in regions of endemicity.