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Shigella spp. are a leading cause of human diarrheal disease worldwide, with Shigella flexneri being the most frequently isolated species in developing countries. This serogroup is presently classified into 19 serotypes worldwide. We report here a multicenter validation of a multiplex-PCR-based strategy previously developed by Q. Sun, R. Lan, Y. Wang, A. Zhao, et al. (J Clin Microbiol 49:3766–3770, 2011) for molecular serotyping of S. flexneri. This study was performed by seven international laboratories, with a panel of 71 strains (researchers were blind to their identity) as well as 279 strains collected from each laboratory’s own local culture collections. This collaborative work found a high extent of agreement among laboratories, calculated through interrater reliability (IRR) measures for the PCR test that proved its robustness. Agreement with the traditional method (serology) was also observed in all laboratories for 14 serotypes studied, while specific genetic events could be responsible for the discrepancies among methodologies in the other 5 serotypes, as determined by PCR product sequencing in most of the cases. This work provided an empirical framework that allowed the use of this molecular method to serotype S. flexneri and showed several advantages over the traditional method of serological typing. These advantages included overcoming the problem of availability of suitable antisera in testing laboratories as well as facilitating the analysis of multiple samples at the same time. The method is also less time-consuming for completion and easier to implement in routine laboratories. We recommend that this PCR be adopted, as it is a reliable diagnostic and characterization methodology that can be used globally for laboratory-based shigella surveillance.

Recently, strains of a novel Shigella flexneri serotype called 1d have been isolated from diarrheal patients in China. They are distinguished by the presence of a hitherto unknown combination of type O-factor I and group O-factor 7,8, both associated with lateral α-D-glucosyl groups on the basal linear O-polysaccharide. A serologically identical strain, 036_1d, has been constructed in the laboratory by sequential infection of serotype Y by serotype-converting bacteriophages SfX and SfI. In this work, using 1D and 2D (1)H and (13)C NMR spectroscopy, we established the following structure of the O-antigen of S. flexneri serotype 1d and demonstrated that the O-antigen of the 036_1d construct has the same structure: [structure: see text].

We report on the isolation of 5 Shigella flexneri strains displaying a novel serotype, 1d, that shares serologic features from both S. flexneri serotypes 1a and X. The 1d strains contained serotype-converting bacteriophages SfI and SfX in tandem on the chromosome. These strains were likely originated from serotype X strains through SfI infection.

The O-specific polysaccharide (O-antigen) of Escherichia coli O133 was obtained by mild acid hydrolysis of the lipopolysaccharide of E. coli O133. The structure of the hexasaccharide repeating unit of the polysaccharide was elucidated by (1)H and (13)C NMR spectroscopy, including a two-dimensional (1)H-(1)H ROESY experiment: Functions of genes in the O-antigen gene cluster were putatively identified by comparison with sequences in the available databases and, particularly, an encoded predicted multifunctional glycosyltransferase was assigned to three α-l-rhamnosidic linkages.

Surveys were carried out to better understand the tick vector ecology and genetic diversity of Huaiyangshan virus (HYSV) in both regions of endemicity and regions of nonendemicity. Haemaphysalis longicornis ticks were dominant in regions of endemicity, while Rhipicephalus microplus is more abundant in regions of nonendemicity. HYSV RNA was found in human and both tick species, with greater prevalence in H. longicornis and lesser prevalence in R. microplus . Phylogenetic analyses indicate that HYSV is a novel species of the genus Phlebovirus .

Background Hemorrhagic fever-like illness caused by a novel Bunyavirus, Huaiyangshan virus (HYSV, also known as Severe Fever with Thrombocytopenia virus [SFTSV] and Fever, Thrombocytopenia and Leukopenia Syndrome [FTLS]), has recently been described in China. Methods Patients with laboratory-confirmed HYSV infection who were admitted to Union Hospital or Zhongnan Hospital between April 2010 and October 2010 were included in this study. Clinical and routine laboratory data were collected and blood, throat swab, urine, or feces were obtained when possible. Viral RNA was quantified by real-time reverse-transcriptase polymerase chain reaction. Blood levels of a range of cytokines, chemokines, and acute phase proteins were assayed. Results A total of 49 patients with hemorrhagic fever caused by HYSV were included; 8 (16.3%) patients died. A fatal outcome was associated with high viral RNA load in blood at admission, as well as higher serum liver transaminase levels, more pronounced coagulation disturbances (activated partial thromboplastin time, thrombin time), and higher levels of acute phase proteins (phospholipase A, fibrinogen, hepcidin), cytokines (interleukin [IL]-6, IL-10, interferon-γ), and chemokines (IL-8, monocyte chemotactic protein 1, macrophage inflammatory protein 1b). The levels of these host parameters correlated with viral RNA levels. Blood viral RNA levels gradually declined over 3-4 weeks after illness onset, accompanied by resolution of symptoms and laboratory abnormalities. Viral RNA was also detectable in throat, urine, and fecal specimens of a substantial proportion of patients, including all fatal cases assayed. CONCLUSIONS. Viral replication and host immune responses play an important role in determining the severity and clinical outcome in patients with infection by HYSV.

O-Antigens (O-specific polysaccharides) of Shigella flexneri, a primary cause of shigellosis, are distinguished by a wide diversity of chemical modifications following the oligosaccharide O-unit assembly. The present review is devoted to structural, serological, and genetic aspects of these modifications, including O-acetylation and phosphorylation with phosphoethanolamine that have been identified recently. The modifications confer the host with specific immunodeterminants (O-factors or O-antigen epitopes), which accounts for the antigenic diversity of S. flexneri considered as a virulence factor of the pathogen. Totally, 30 O-antigen variants have been recognized in these bacteria, the corresponding O-factors characterized using specific antibodies, and a significant extension of the serotyping scheme of S. flexneri on this basis is suggested. Multiple genes responsible for the O-antigen modifications and the resultant serotype conversions of S. flexneri have been identified. The genetic mechanisms of the O-antigen diversification by acquisition of mobile genetic elements, including prophages and plasmids, followed occasionally by gene mobilization and inactivation have been revealed. These findings further our understanding of the genetics and antigenicity of S. flexneri and assist control of shigellosis.

Shigella spp. are the causative agent of shigellosis with S higella flexneri serotype 2a being the most prevalent in developing countries. Epidemiological surveillance in China found that a new serotype of S. flexneri appeared in 2001 and replaced serotype 2a in 2003 as the most prevalent serotype in Henan Province. The new serotype also became the dominant serotype in 7 of the 10 other provinces under surveillance in China by 2007. The serotype was identified as a variant of serotype X. It differs from serotype X by agglutination to the monovalent anti-IV type antiserum and the group antigen-specific monoclonal antibody MASF IV-I. Genome sequencing of a serotype X variant isolate, 2002017, showed that it acquired a Shigella serotype conversion island, also as an SfX bacteriophage, containing gtr genes for type X-specific glucosylation. Multilocus sequence typing of 15 genes from 37 serotype X variant isolates and 69 isolates of eight other serotypes, 1a, 2a, 2b, 3a, 4a, 5b, X, and Y, found that all belong to a new sequence type (ST), ST91. Pulsed-field gel electrophoresis revealed 154 pulse types with 655 S. flexneri isolates analyzed and identified 57 serotype switching events. The data suggest that S. flexneri epidemics in China have been caused by a single epidemic clone, ST91, with frequent serotype switching to evade infection-induced immunity to serotypes to which the population was exposed previously. The clone has also acquired resistance to multiple antibiotics. These findings underscore the challenges to the current vaccine development and control strategies for shigellosis.

BACKGROUND Streptococcus suis emerged to cause an unusual outbreak of streptococcal toxic-shock-like syndrome (STSLS) in 2005. The mechanisms involved are unknown. METHODS Clinical, laboratory, and epidemiologic data on patients infected with culture-confirmed S. suis were analyzed. The strain involved in the outbreak, "epidemic" strain ST7, was compared with both a classical highly pathogenic strain, ST1, and an intermediately pathogenic strain, ST25, to determine both its capacity to induce cytokines in experimentally infected mice and its genomic difference. RESULTS Of 38 patients infected with culture-confirmed S. suis, 14 presented with STSLS. During the early phase of the disease, serum levels of interleukin (IL)-1beta, IL-6, IL-8, IL-12p70, interferon-gamma, and tumor necrosis factor-alpha were more elevated in patients with STSLS than in those with meningitis only. Serum levels of proinflammatory cytokines were significantly higher in mice infected with ST7 than in those infected with either ST1 or ST25. Genomic comparisons with ST25 showed that ST1 had acquired 132 genomic islands, including 5 pathogenicity islands, and that ST7, the epidemic strain, had acquired an additional 5 genomic islands. CONCLUSION Intermediately pathogenic strain ST25 has evolved to become highly pathogenic strain ST1, which, in turn, has more recently evolved to become epidemic strain ST7. ST7 has the ability to stimulate the production of massive amounts of proinflammatory cytokines, leading to STSLS.

Streptococcus suis sequence type (ST) 7 has been spreading throughout China. To determine events associated with its emergence, we tested 114 isolates. In all 106 ST7 strains responsible for human outbreaks and sporadic infections, the tetracycline-resistance gene, tetM, was detected on the conjugative transposon Tn916. Horizontal transmission of tetM is suspected.

An outbreak of Streptococcus suis serotype 2 emerged in the summer of 2005 in Sichuan Province, and sporadic infections occurred in 4 additional provinces of China. In total, 99 S. suis strains were isolated and analyzed in this study: 88 isolates from human patients and 11 from diseased pigs. We defined 98 of 99 isolates as pulse type I by using pulsed-field gel electrophoresis analysis of SmaI-digested chromosomal DNA. Furthermore, multilocus sequence typing classified 97 of 98 members of the pulse type I in the same sequence type (ST), ST-7. Isolates of ST-7 were more toxic to peripheral blood mononuclear cells than ST-1 strains. S. suis ST-7, the causative agent, was a single-locus variant of ST-1 with increased virulence. These findings strongly suggest that ST-7 is an emerging, highly virulent S. suis clone that caused the largest S. suis outbreak ever described.

Background O-antigen (O-polysaccharide) of the lipopolysaccharide is a highly variable cell component of the outer membrane in Shigella flexneri. It defines the serospecificity and plays an important role in the pathogenesis of shigellosis. There are two distinct O-antigen forms for the 19 serotypes of S. flexneri: one for serotypes 1–5, X, Y, 7 (and their subtypes), and the other for serotype 6. Although having different basal O-polysaccharide structures, the two forms share a common disaccharide fragment [→2)-α-l-RhapIII-(1 → 2)-α-l-RhapII]. In serotype 6 and some non-6 serotypes, RhaIII is O-acetylated at position either 3 or 4 (3/4-O-acetylation), conferring to the hosts a novel antigenic determinant named O-factor 9. An acyltransferase gene (oacB) responsible for this modification has been identified in serotypes 1a, 1b, 2a, 5a, and Y, but not in serotype 6. Results Using genetic, serological, and chemical approaches, another acyltransferase gene named oacC was demonstrated to be responsible for the 3/4-O-acetylation on RhaIII in the O-antigen of S. flexneri serotype 6. Inactivation of the oacC gene resulted in the loss of the 3/4-O-acetyltion, and the cloned oacC gene restored this modification upon transformation. In accordance with the similarity in the acceptor substrate structure and high sequence homology (72% identity) between oacC and oacB, oacC has the interchangeable function with the oacB gene in mediation of the 3/4-O-acetylation. The oacC gene is located in a prophage on the chromosome and presented in all 77 serotype 6 strains tested. Conclusions Identification and functional characterization of the O-acetyltransferase encoding gene, oacC, shows that it is involved in O-antigen modification by 3/4-O-acetylation on RhaIII specific to serotype 6. Electronic supplementary material The online version of this article (doi:10.1186/s12866-014-0266-7) contains supplementary material, which is available to authorized users.

Shigella flexneri O-antigen is an important and highly variable cell component presented on the outer leaflet of the outer membrane. Most Shigella flexneri bacteria share an O-antigen backbone composed of →2)-α-L-Rhap(III)-(1→2)-α-L-Rhap(II)-(1→3)-α-L-Rhap(I)-(1→3)-β-D-GlcpNAc-(1→ repeats, which can be modified by adding various chemical groups to different sugars, giving rise to diverse O-antigen structures and, correspondingly, to various serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on Rha(I) or/and Rha(III), and phosphorylation with phosphoethanolamine on Rha(II) or/and Rha(III). Recently, a new O-antigen modification, namely, O-acetylation at position 6 of N-acetylglucosamine (GlcNAc), has been identified in S. flexneri serotypes 2a, 3a, Y, and Yv. In this study, the genetic basis of the 6-O-acetylation of GlcNAc in S. flexneri was elucidated. An O-acyltransferase gene designated oacD was found to be responsible for this modification. The oacD gene is carried on serotype-converting bacteriophage SfII, which is integrated into the host chromosome by lysogeny to form a prophage responsible for the evolvement of serotype 2 of S. flexneri. The OacD-mediated 6-O-acetylation also occurs in some other S. flexneri serotypes that carry a cryptic SfII prophage with a dysfunctional gtr locus for type II glucosylation. The 6-O-acetylation on GlcNAc confers to the host a novel O-antigen epitope, provisionally named O-factor 10. These findings enhance our understanding of the mechanisms of the O-antigen variation and enable further studies to understand the contribution of the O-acetylation to the antigenicity and pathogenicity of S. flexneri.

Shigella flexneri is the major cause of shigellosis in developing countries. All serotypes except for serotype 6 share an O-antigen backbone composed of a →2)-α- l -Rha p III -(1→2)-α- l -Rha p II -(1→3)-α- l -Rha p I -(1→3)-β- d -Glc p NAc-(1→ tetrasaccharide repeat. It can be modified by the addition of a glucosyl group to one or more sugar residues and/or an O -acetyl group to Rha I and/or a phosphoethanolamine to Rha II and/or Rha III . These modifications give rise to type I-, IC-, II-, IV-, and V- and to group 6-, 7,8-, and MASF IV-1-specific antigenic determinants, which comprise the current serotyping scheme of S. flexneri . Recently, another O-antigen modification created by adding an O -acetyl group to Rha III at position 3 or 4 (3/4- O -acetylation) has been found in S. flexneri serotypes 1a, 1b, 2a, 5a, Y, and 6. A new O -acyltransferase gene named oacB has been shown to mediate the 3/4- O -acetylation in serotypes 1a, 1b, 2a, 5a, and Y but not in 6. In this work, we studied the distribution of the 3/4- O -acetylation in S. flexneri and the antigenicity that resulted from this modification. PCR screening of the oacB gene in clinical isolates of S. flexneri demonstrated that the oacB -mediated 3/4- O -acetylation is widespread in serotypes 1a, 1b, 2a, 5a, and Y. Serological analysis indicated that this modification confers the host with a novel antigenic determinant that is provisionally named group O factor 9. These findings enhance our understanding of the varieties of O-antigenic determinants related to O-antigen modification in S. flexneri and will assist epidemiological studies and vaccine development.

Shigella flexneri is the major cause of shigellosis in developing countries. A new S. flexneri serotype, Xv, appeared in 2000 and replaced serotype 2a as the most prevalent serotype in China. Serotype Xv is a variant of serotype X, with phosphoethanolamine modification of its O antigen mediated by a plasmid that contained the opt gene. Serotype Xv isolates belong to sequence type 91 (ST91). In this study, whole-genome sequencing of 59 S. flexneri isolates of 14 serotypes (serotypes 1 to 4, Y, Yv, X, and Xv) indicated that ST91 arose around 1993 by acquiring multidrug resistance (MDR) and spread across China within a decade. A comparative analysis of the chromosome and opt -carrying plasmid pSFXv_2 revealed independent origins of 3 serotype Xv clusters in China, with different divergence times. Using 18 cluster-dividing single-nucleotide polymorphisms (SNPs), SNP typing divided 380 isolates from 3 provinces (Henan, Gansu, and Anhui) into 5 SNP genotypes (SGs). One SG predominated in each province, but substantial interregional spread of SGs was also evident. These findings suggest that MDR is the key selective pressure for the emergence of the S. flexneri epidemic clone and that Shigella epidemics in China were caused by a combination of local expansion and interregional spread of serotype Xv.

O antigen (O polysaccharide) is an important and highly variable cell component present on the surface of cells which defines the serospecificity of Gram-negative bacteria. Most O antigens of Shigella flexneri , a cause of shigellosis, share a backbone composed of →2)-α- l -Rha p III -(1→2)-α- l -Rha p II -(1→3)-α- l -Rha p I -(1→3)-β- d -Glc p NAc-(1→ repeats, which can be modified by adding various substituents, giving rise to 19 serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on Rha I , and phosphorylation with phosphoethanolamine on Rha II or/and Rha III . Recently, two new O-antigen modifications, namely, O-acetylation at position 3 or 4 of Rha III and position 6 of GlcNAc, have been identified in several S. flexneri serotypes. In this work, the genetic basis for the 3/4-O-acetylation on Rha III was elucidated. Bioinformatic analysis of the genome of S. flexneri serotype 2a strain Sf301, which carries 3/4-O-acetylation on Rha III , revealed an O-acyltransferase gene designated oacB . Genetic studies combined with O-antigen structure analysis demonstrated that this gene is responsible for the 3/4-O-acetylation in serotypes 1a, 1b, 2a, 5a, and Y but not serotype 6, which has a different O-antigen backbone structure. The oacB gene is carried by a transposon-like structure located in the proA-adrA region on the chromosome, which represents a novel mechanism of mobilization of O-antigen modification factors in S. flexneri . These findings enhance our knowledge of S. flexneri O-antigen modifications and shed light on the origin of new O-antigen variants.

Spatial distribution of bacillary dysentery incidence was mapped at the district level in Wuhan, China. And a generalized additive time series model was used to examine the effect of daily weather factors on bacillary dysentery in the high-risk areas, after controlling for potential confounding factors. Central districts were found to be the high-risk areas. The time series analysis found an acute effect of meteorological factors on bacillary dysentery occurrence. A positive association was found for mean temperature (excess risk (ER) for 1°C increase being 0.94% (95% confidence interval (CI): 0.46% to 1.43% on the lag day 2), while a negative effect was observed for relative humidity and rainfall, the ER for 1% increase in relative humidity was −0.21% (95% CI: −0.34% to −0.08%) and the ER for 1 mm increase in rainfall was −0.23% (95% CI: −0.37% to −0.09%). This study suggests that bacillary dysentery prevention and control strategy should consider local weather variations.

Shigella flexneri is the major human pathogen causing shigellosis. O-antigens of all S. flexneri serotypes (except for serotype 6) share the →2)-α-l-Rhap(III)-(1 → 2)-α-l-Rhap(II)-(1 → 3)-α-l-Rhap(I)-(1 → 3)-β-d-GlcpNAc-(1→ basic O-unit, whereas differences between the serotypes are conferred by phage-encoded glucosylation and/or O-acetylation at various positions. Recently, in serotype X and 4a variants called Xv and 4av, respectively, O-antigen modification with phosphoethanolamine (PEtN) has been identified, which is encoded by a plasmid-borne gene (lpt-O) for a PEtN-transferase and confers the monoclonal antibody IV-1(MASF IV-1) determinant to the bacteria. In this study, we elucidated the O-antigen structure of serotype Yv, another MASF IV-1-positive novel variant of S. flexneri. The serotype Yv O-antigen has the same basic carbohydrate backbone structure as that of the "classical" serotype Y, but differs in the presence of PEtN at position 3 of Rha(III) (major) or both Rha(II) and Rha(III) (minor). This pattern is similar to that of serotype 4av, but different from the pattern of serotype Xv, which is characterized by major PEtN modification on Rha(II). In serotype Yv, mono- and bisphosphorylated O-units generate a block-copolymeric structure, the former being partially O-acetylated at position 6 of GlcNAc and the latter lacking O-acetylation. Functional analysis revealed a correlation between the serotype-specific PEtN modification pattern and the lpt-O variation in different serotypes: lpt-O(RII) in serotype Xv is better tuned for phosphorylation of Rha(II) and lpt-O(RIII) in serotypes Yv and 4av for phosphorylation of Rha(III). These data enhance our knowledge of S. flexneri serotype conversion mechanisms and help to understand the biosynthesis process of the new O-antigen variants.

Shigella flexneri is a leading cause of bacterial dysentery in developing countries. Among the 15 known serotypes, four (1b, 3a, 3b and 4b) contain a group 6 epitope due to an acetyl group connected to the O-2 position of rhamnose III on the tetrasaccharide structure of the lipopolysaccharide. O-acetyltransferase encoded by a bacteriophage, Sf6, mediates the acetylation reaction. We found that the oac gene in serotype 1b strains was very different from that in serotypes 3a, 3b and 4b strains and is herein after referred to as oac 1b which shares with oac 88%-89% identity at the DNA level and 85% identity at the protein level. Considering that S. flexneri strains of serotypes 1-5 share a recent common ancestry, the divergent oac 1b is more likely to have been obtained from outside S. flexneri than to have undergone rapid divergence from the oac gene in the other serotypes (3a, 3b and 4b) within S. flexneri. The cloned oac 1b gene was found to perform the same acetylation function as oac. Analysis of the genomic regions flanking oac 1b showed that it was present in a prophage on the chromosome and the organizational structure is different from that of phage Sf6. Additionally, phage conversion assay showed that serotype 1b cannot be generated by infecting serotype 1a strains with Sf6. We conclude that oac 1b was carried by a non-Sf6 phage and is uniquely present in serotype 1b.

Shigella flexneri is the major pathogen causing bacillary dysentery in developing countries. S. flexneri is divided into at least 16 serotypes based on the combination of antigenic determinants present in the O-antigen. All the serotypes (except for serotype 6) share a basic O-unit containing one N-acetyl-d-glucosamine and three l-rhamnose residues, whereas differences between the serotypes are conferred by phage-encoded glucosylation and/or O-acetylation. Serotype Xv is a newly emerged and the most prevalent serotype in China, which can agglutinate with both MASF IV-1 and 7,8 monoclonal antibodies. The factor responsible for the presence of MASF IV-1 (E1037) epitope has not yet been identified. In this study, we analyzed the LPS structure of serotype Xv strains and found that the MASF IV-1 positive phenotype depends on an O-antigen modification with a phosphoethanolamine (PEtN) group attached at position 3 of one of the rhamnose residues. A plasmid carried gene, lpt-O (LPS phosphoethanolamine transferase for O–antigen), mediates the addition of PEtN for serotype Xv and other MASF IV-1 positive strains. These findings reveal a novel serotype conversion mechanism in S. flexneri and show the necessity of further extension of the serotype classification scheme recognizing the MASF IV-1 positive strains as distinctive subtypes.

Background Shigella flexneri is the major pathogen causing bacillary dysentery. Fifteen serotypes have been recognized up to now. The genesis of new S. flexneri serotypes is commonly mediated by serotype-converting bacteriophages. Untypeable or novel serotypes from natural infections had been reported worldwide but have not been generated in laboratory. Results A new S. flexneri serotype-serotype 1 d was generated when a S. flexneri serotype Y strain (native LPS) was sequentially infected with 2 serotype-converting bacteriophages, SfX first and then SfI. The new serotype 1 d strain agglutinated with both serotype X-specific anti-7;8 grouping serum and serotype 1a-specific anti- I typing serum, and differed from subserotypes 1a, 1b and 1c. Twenty four S. flexneri clinical isolates of serotype X were all converted to serotype 1 d by infection with phage SfI. PCR and sequencing revealed that SfI and SfX were integrated in tandem into the proA-yaiC region of the host chromosome. Conclusions These findings suggest a new S. flexneri serotype could be created in nature. Such a conversion may be constrained by susceptibility of a strain to infection by a given serotype-converting bacteriophage. This finding has significant implications in the emergence of new S. flexneri serotypes in nature.

Shigella flexneri is the major Shigella species that causes diarrheal disease in developing countries. It is further subdivided into 15 serotypes based on O-antigen structure. Serotyping of S. flexneri is important for epidemiological purposes. In this study, we developed a multiplex PCR assay targeting the O-antigen synthesis gene wzx and the O-antigen modification genes gtrI , gtrIC , gtrII , oac , gtrIV , gtrV , and gtrX for molecular serotyping of S. flexneri. The multiplex PCR assay contained eight sets of specific PCRs in a single tube and can identify 14 of the 15 serotypes (the exception being serotype Xv) of S. flexneri recognized thus far. A nearly perfect concordance (97.8%) between multiplex PCR assay and slide agglutination was observed when 358 S. flexneri strains of various serotypes were analyzed, except that 8 strains were carrying additional cryptic and/or defective serotype-specific genes. The multiplex PCR assay provides a rapid and specific method for the serotype identification of S. flexneri .

From April to July in 2009 and 2010, unexplained severe hemorrhagic fever-like illnesses occurred in farmers from the Huaiyangshan mountains range. Clinical specimens (blood, urine, feces, and throat swabs) from suspected patients were obtained and stored. Mosquitoes and ticks in affected regions were collected. Virus was isolated from 2 patients and characterized by whole genome sequencing. Virus detection in additional patients and arthropods was done by virus-specific reverse transcription (RT) PCR. Clinical and epidemiological data of RT-PCR confirmed patients were analyzed. An unknown virus was isolated from blood of two patients and from Haemaphysalis ticks collected from dogs. Whole genome sequence analysis identified the virus as a novel member of the family Bunyaviridae, most closely related to the viruses of the genus Phlebovirus within which it forms a separate lineage. Subsequently, infection was confirmed by RT-PCR in 33 of 58 suspected patients. The illness in these patients was characterized by fever, severe malaise, nausea, vomiting, and diarrhea. Prominent laboratory findings included low white cell- and platelet counts, coagulation disturbances, and elevation of liver enzymes. Hemorrhagic complications were observed in 3 cases, 5 (15%) patients died. A novel tick-borne Bunyavirus causing life-threatening hemorrhagic fever in humans has emerged in the Huaiyangshan mountain areas of China. Further studies are needed to determine the epidemiology, geographic distribution and vertebrate animal ecology of this virus

We constructed a food-grade secretion expression vector and used it for reporter protein expression in live delivery vehicle L lactis MBP71. The p32 fragment, which containing the stronger p32 promoter, was amplified by polymerase chain reaction (PCR) with the plasmid pMG36e as template. After being purified, the p32 fragment was ligated with SPusp45 fragment amplified from genomic DNA of L lactis MG1363. The fusion fragment p32-SPusp45 was inserted into the food-grade vector pSH91 to construct a secretion expression vector, pSQ. The coding sequence of NucA (nucA) was also amplified from Staphylococcus aureus chromosome and inserted into pSQ under the control of p32 promoter to construct a recombinant plasmid pSQ-nucA. Nuclease plate activity assay and zymograme assay demonstrate that NucA was secretion expressed from L lactis harboring the recombinant plasmid pSQ- nucA, and the quantity of NucA secreted into supernamant was about ten times more than which in cell lysate. Results also indicate that expression efficiency of L lactis/pSQ-nucA was higher than that of L lactis/pSQZ-nucA, constructed by us earlier.

Shigella flexneri is one of the major etiologic causes of shigellosis in Guizhou Province, China. However, the genetic characteristics of circulating isolates are unknown. Phenotypic and molecular profiles of 60 S. flexneri isolates recovered in Guizhou between 1972 to 1982 and 2008 to 2010 were determined. Nine serotypes (1a, 2a, 3a, 1b, 2b, X, Y, 4av and Yv) were identified. Multi-locus sequence typing differentiated the isolates into 20 sequence types (STs); 18 were novel. Four STs, ST 129, ST 100, ST 126 and ST 18, were most abundant, accounting for 65% of the isolates. Thirty-nine NotI-pulsed field gel electrophoresis patterns (pulsotypes, PTs) were observed; eight PTs were represented by more than one isolate with six isolates sharing the PT 13 profile. Multi-locus variable-nucleotide tandem-repeat analysis recognized 44 different types (MTs); seven MTs were represented by more than one isolate and MT 1 was most commonly encountered. Correlation between genetic relationships and serotypes was observed among the isolates studied; the majority of isolates belonging to the same serotype from different years clustered together based on the molecular data. These clustered isolates were also from similar geographical origins. These results enhance our understanding of genetic relationships between S. flexneri in Guizhou Province and can be used to help understand the changing etiology of shigellosis in China.

Background The gene for New Delhi metallo-β-lactamase 1 (NDM-1) has been reported to be transmitted via plasmids which are easily transferable and capable of wide distribution. We report the isolation of two NDM-1 producing strains and possible in vivo transfer of blaNDM-1 in a patient. Methods Clinical samples were collected for bacterial culture and antibiotic susceptibility testing from a patient during a 34-day hospitalization. The presence of blaNDM-1 was detected by PCR and sequencing. Plasmids of interest were sequenced. Medical records were reviewed for evidence of association between the administration of antibiotics and the acquisition of the NDM-1 resistance. Results A NDM-1 positive Raoultella planticola was isolated from blood on the ninth day of hospitalization without administration of any carbapenem antibiotics and a NDM-1 positive Escherichia coli was isolated from feces on the 29th day of hospitalization and eight days after imipenem administration. The blaNDM-1 was carried by a 280 kb plasmid pRpNDM1-1 in R. planticola and a 58 kb plasmid pEcNDM1-4 in E. coli. The two plasmids shared a 4812 bp NDM-1-ISCR1 element which was found to be excisable from the plasmid as a free form and transferrable in vitro to a NDM-1 negative plasmid from E. coli. Conclusion blaNDM-1 was embedded in an ISCR1 complex class 1 integron as a novel 4812 bp NDM-1-ISCR1 element. The element was found to be able to self excise to become a free form, which may provide a new vehicle for NDM-1 dissemination. This mechanism could greatly accelerate the spread of NDM-1 mediated broad spectrum β-lactam resistance.

Shigella flexneri is the major cause of bacterial shigellosis in developing countries. S. flexneri is divided into at least 19 serotypes, the majority of which are modifications of the same basic O-antigen by glucosylation and/or O-acetylation of its sugar residues by phage encoded serotype-converting genes. Recently, a plasmid encoded phosphoethanolamine (PEtN) modification of the O-antigen has been reported, which is responsible for the presence of the MASF IV-1 determinant and results in conversion of traditional serotypes X, 4a and Y to novel serotypes Xv, 4av and Yv, respectively. In this study, we characterized 19 serotype Yv strains isolated in China. A variant of the O-antigen phosphoethanolamine transferase gene opt (formerly called lpt-O) carried by a pSFxv_2-like plasmid was found in serotype Yv strains, which specifies the phosphorylation pattern on the O-antigen of this serotype. For the majority of the O-antigen units, the PEtN modification occurs on Rha(III), while for a minority, modifications occur on both Rha(II) and Rha(III). Serotype-specific gene detection and PFGE analysis suggested that these serotype Yv isolates were originated from serotypes Y, Xv and 2a by acquisition of an opt-carrying plasmid and/or inactivation of serotype-specific gene gtrII or gtrX. These data, combined with those of serotypes Xv and 4av reported earlier, demonstrate that the plasmid-encoded PEtN modification is an important serotype conversion mechanism in S. flexneri, in addition to glucosylation and O-acetylation.