Fluoroquinolone Resistance Mechanisms of Shigella flexneri Isolated in Bangladesh

Objective

To investigate the prevalence and mechanisms of fluoroquinolone resistance in Shigella species isolated in Bangladesh and to compare with similar strains isolated in China.

Methods

A total of 3789 Shigella isolates collected from Clinical Microbiology Laboratory of icddr,b, during 2004–2010 were analyzed for antibiotic susceptibility. Analysis of plasmids, plasmid-mediated quinolone-resistance genes, PFGE, and sequencing of genes of the quinolone-resistance-determining regions (QRDR) were conducted in representative strains isolated in Bangladesh and compared with strains isolated in Zhengding, China. In addition, the role of efflux-pump was studied by using the efflux-pump inhibitor carbonyl cyanide-m-chlorophenylhydrazone (CCCP).

Results

Resistance to ciprofloxacin in Shigella species increased from 0% in 2004 to 44% in 2010 and S. flexneri was the predominant species. Of Shigella spp, ciprofloxacin resistant (Cip^R) strains were mostly found among S. flexneri (8.3%), followed by S. sonnei (1.5%). Within S. flexneri (n = 2181), 14.5% were resistance to ciprofloxacin of which serotype 2a was predominant (96%). MIC of ciprofloxacin, norfloxacin, and ofloxacin were 6–32 mg/L, 8–32 mg/L, and 8–24 mg/L, respectively in S. flexneri 2a isolates. Sequencing of QRDR genes of resistant isolates showed double mutations in gyrA gene (Ser⁸³Leu, Asp⁸⁷Asn/Gly) and single mutation in parC gene (Ser⁸⁰Ile). A difference in amino acid substitution at position 87 was found between strains isolated in Bangladesh (Asp⁸⁷Asn) and China (Asp⁸⁷Gly) except for one. A novel mutation at position 211 (His→Tyr) in gyrA gene was detected only in the Bangladeshi strains. Susceptibility to ciprofloxacin was increased by the presence of CCCP indicating the involvement of energy dependent active efflux pumps. A single PFGE type was found in isolates from Bangladesh and China suggesting their genetic relatedness.

Conclusions

Emergence of fluoroquinolone resistance in Shigella undermines a major challenge in current treatment strategies which needs to be followed up by using empirical therapeutic strategies.     

痢疾杆菌染色体中噬菌体插入序列的分析

噬菌体作为细菌的病毒通过参与细菌的代谢过程完成自身的繁殖和复制.自80多年前噬菌体被发现以来,噬菌体因其遗传物质和结构组成十分简单并且与宿主细胞相互作用非常密切一直被作为研究生命基本规律的良好的模型系统.     

痢疾杆菌酸抗性系统相关基因缺失突变体的构建

依赖于谷氨酸脱羧酶的酸抗性系统对痢疾杆菌在宿主细胞内的生存和繁殖至关重要,hdeA、hdeB、yhiE和yhiF是其中几个重要的酸抗性基因。借助Red系统的重组功能,PCR扩增两翼与目的基因上下游同源的抗性基因片段,电击转化痢疾杆菌2457T,对筛选到的阳性转化子再导入编码FLP位点特异性重组酶的质粒pCP20以去除抗性基因。结果成功的敲除了hdeA、hdeB、yhiE和yhiF等4个酸抗性系统相关基因,为深入研究痢疾杆菌酸抗性基因的调控网络奠定了基础.     

Antimicrobial Resistance of Shigella flexneri Serotype 1b Isolates in China

Shigella flexneri serotype 1b is among the most prominent serotypes in developing countries, followed by serotype 2a. However, only limited data is available on the global phenotypic and genotypic characteristics of S. flexneri 1b. In the present study, 40 S. flexneri 1b isolates from different regions of China were confirmed by serotyping and biochemical characterization. Antimicrobial susceptibility testing showed that 85% of these isolates were multidrug-resistant strains and antibiotic susceptibility profiles varied between geographical locations. Strains from Yunnan were far more resistant than those from Xinjiang, while only one strain from Shanghai was resistant to ceftazidime and aztreonam. Fifteen cephalosporin resistant isolates were identified in this study. ESBL genes (bla _SHV, bla _TEM, bla _OXA, and bla _CTX-M) and ampC genes (bla _MOX, bla _FOX, bla _MIR(ACT-1), bla _DHA, bla _CIT and bla _ACC) were subsequently detected among the 15 isolates. The results showed that these strains were positive only for bla _TEM, bla _OXA, bla _CTX-M, intI1, and intI2. Furthermore, pulsed-field gel electrophoresis (PFGE) analysis showed that the 40 isolates formed different profiles, and the PFGE patterns of Xinjiang isolates were distinct from Yunnan and Shanghai isolates by one obvious, large, missing band. In summary, similarities in resistance patterns were observed in strains with the same PFGE pattern. Overall, the results supported the need for more prudent selection and use of antibiotics in China. We suggest that antibiotic susceptibility testing should be performed at the start of an outbreak, and antibiotic use should be restricted to severe Shigella cases, based on resistance pattern variations observed in different regions. The data obtained in the current study might help to develop a strategy for the treatment of infections caused by S. flexneri 1b in China.     

Impact of either Elevated or Decreased Levels of Cytochrome bd Expression on Shigella flexneri Virulence

Shigella spp. are the major cause of bacillary dysentery worldwide. The pathogenic process involves bacterial invasion and lysis of the phagocytic vacuole, followed by replication and movement within the cell cytoplasm and, ultimately, spread directly into adjacent cells. This study demonstrates that S. flexneri cytochrome bd expression is necessary for normal intracellular survival and virulence. Cytochrome bd is one of two terminal oxidases in the bacterial respiratory chain that reduce molecular oxygen to water, utilizing intermediates shuttled through the electron transport chain. S. flexneri mutants that contain a disruption in the cydC locus, which leads to defective cytochrome bd expression, or in the riboflavin (ribE) or ubiquinol-8 (ubiH) biosynthetic pathway, which leads to elevated cytochrome bd expression, were evaluated in intracellular survival and virulence assays. The cydC mutant formed significantly smaller plaques, had significantly decreased intracellular survival, and had a 100-fold increase in lethal dose for mice compared with the wild type. The ribE and ubiH mutants each formed significantly larger plaques and had a 10-fold decrease in lethal dose for mice compared with the wild type. The data indicate that expression of cytochrome bd is required for S. flexneri intracellular survival and virulence.     

Factors Affecting Virulence of Shigella flexneri: Defective Methionine Synthesis in an Escherichia coli-Shigella Hybrid

An Escherichia coli-Shigella flexneri hybrid of intermediate virulence was studied to determine whether its shorter survival in host cells might be due to a metabolic defect. Investigation of its growth in minimal glucose medium showed that the hybrid, like its E. coli parent, had a longer lag phase and a slower growth rate than its virulent Shigella parent. Methionine was found to increase the growth rate of the hybrid. The Shigella parent of the hybrid can synthesize methionine normally, but the E. coli parent has a point mutation in its metA gene. Since it synthesizes enough methionine to grow slowly, it is postulated that the hybrid strain has a hybrid metA gene, that is, a gene composed partly of deoxyribonucleic acid (DNA) from E. coli, with the balance of the DNA from S. flexneri. Phage P1-mediated transduction, with the metA(-)E. coli parent as recipient and the Shigella parent as donor, yielded a few transductants that responded to methionine in the same way the hybrid did. Many more transductants of the hybrid type were produced when the hybrid strain was used as a donor. It is suggested that this poorly functioning gene acts synergistically with the hybrid strain's relaxed synthesis of ribonucleic acid to prevent its survival in the host.     

Acid tolerance of Shigella sonnei and Shigella flexneri

AIMS: Determination of the behaviour of Shigella sonnei and Sh. flexneri under acid conditions. METHODS AND RESULTS: The growth and survival of Shigella spp. (9 isolates) in acidified Brain Heart Infusion (BHI) (pH 5.0-3.25 with pH intervals of 0.25) was determined after 6, 24 and 30 h incubation at 37 degrees C. Subsequently, survival of shigellae was studied in apple juice and tomato juice stored at 7 degrees C and 22 degrees C for up to 14 days and in strawberries and a fresh fruit salad, kept at 4 degrees C for 4 and 48 h. CONCLUSIONS: The minimum pH for growth in acidified BHI for Sh. flexneri and Sh. sonnei was, respectively, pH 4.75 and pH 4.50. Survival in fruit juices and fresh fruits depended upon their pH, the type of strain and the incubation temperature. Shigella spp. Survived for up to 14 days in tomato juice and apple juice stored at 7 degrees C. The shortest survival time (2-8 d) was observed in apple juice at 22 degrees C. Sh. sonnei but not Sh. flexneri was recovered after 48 h from strawberries and fruit salad kept at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Acid foods, especially if kept at refrigeration temperatures, support survival of Shigella spp. and may cause Shigella food poisoning.     

Complete genome sequence of Shigella flexneri 5b and comparison with Shigella flexneri 2a

Background

On most common microarray platforms many genes are represented by multiple probes. Although this is quite common no one has systematically explored the concordance between probes mapped to the same gene.

Results

Here we present an analysis of all the cases of multiple probe sets measuring the same gene on the Affymetrix U133a GeneChip and found that although in the majority of cases both measurements tend to agree there are a significant number of cases in which the two measurements differ from each other. In these cases the measurements can not be simply averaged but rather should be handled individually.

Conclusion

Our analysis allows us to provide a comprehensive list of the correlation between all pairs of probe sets that are mapped to the same gene and thus allows microarray users to sort out the cases that deserve further analysis. Comparison between the set of highly correlated pairs and the set of pairs that tend to differ from each other reveals potential factors that may affect it.     

The pho regulon of Shigella flexneri

Growth of Escherichia coli K-12 in low-phosphate conditions results in the induction of the synthesis of many proteins, including the outer membrane porin PhoE, alkaline phosphatase, and the Pst system for the transport of phosphate (P_i). This response is controlled by a two-component regulatory system of which PhoB and PhoR are the response-regulator and the sensor/kinase, respectively. When Shigella flexneri was starved for P_i, neither PhoE nor alkaline phosphatase was produced. However, induction of the synthesis of the PstS protein was observed, indicating that S. flexneri contains a functional PhoB/PhoR regulatory system. Consistent with this notion, the introduction of the B. coli phoA gene in S. flexneri resulted in the induction of alkaline phosphatase synthesis under phosphate limitation. However, introduction of phoE on a plasmid did not lead to the expression of PhoE protein, indicating that S. flexneri PhoB does not recognize the phoE promoter region. The phoB gene was cloned and sequenced and in the deduced amino acid sequence two deviations from that of E. coli PhoB were detected. Site-directed mutagenesis revealed that one of these deviations, i.e. Leu-172, which is Arg in E. coli PhoB, is responsible for the lack of expression of the PhoE protein in S. flexneri.     

Use of the coagglutination reaction for identifying Shigella flexneri

The data on the use of the coagglutination test for the identification of Sh. flexneri are presented. The influence of different methods for the treatment of antisera on their heterologous reactions was studied and the advantage of coagglutinating reagents was revealed. 9 variants of coagglutinating preparations were obtained; their use made it possible to reduce the consumption of antisera and in some cases avoid their absorption. The problem of Sh. flexneri classification is discussed.     

Virulence plasmids of Shigella bacteria

The contemporary data on the genetical control of invasion process in dysentery causative agent coded by the virulence plasmids of the bacteria are presented. The examples of plasmid genes expression regulated by the genes-regulators located on the chromosome are presented. The mechanism for virulence genes regulation is discussed.     

弗氏志贺菌磷酸化蛋白的检测

目的:检测弗氏志贺菌全菌蛋白中被磷酸化修饰的蛋白。方法:制取弗氏2a志贺菌2457T野生株全菌磷酸化蛋白样品时加入磷酸化酶抑制剂,随后对样品进行双向电泳,以抗磷酸丝氨酸/苏氨酸/酪氨酸抗体为免疫探针,通过Western印迹找到被磷酸化的蛋白,并进行胶内酶解及MALDI-TOF质谱分析。结果与结论:共检测到13个磷酸化蛋白,其中9个为代谢途径中的酶。