Antimicrobial resistance and genomic characteristics of Salmonella enterica serovar Derby isolates from clinical patients and food sources in Hangzhou / 中华微生物学和免疫学杂志

Objective:To analyze the antimicrobial resistance and genomic characteristics of Salmonella enterica serovar Derby strains isolated from human and food sources in Hangzhou. Methods:A total of 60 Salmonella enterica serovar Derby strains isolated in Hangzhou during the period from 2015 to 2020 were subjected to antimicrobial susceptibility testing, pulsed field gel electrophoresis (PFGE) typing and whole-genome sequencing. Multilocus sequence typing (MLST), core genome multilocus sequence typing (cgMLST) and the identification of antimicrobial resistance genes were performed using the sequencing data. Phylogenetic tree based on the single nucleotide polymorphism (SNP) sites in the 60 genomes from Hangzhou and 379 genomes from public databases was constructed. Results:No significant difference was observed in the drug resistance rates between the clinical strains and food strains in Hangzhou. The multidrug resistance (MDR) rate was 76.7% (46/60). All of the 60 Salmonella Derby strains were positive for the antimicrobial resistance genes aac(6′)- Iaa and fosA7. The 60 strains were subtyped into 46 molecular types by PFGE and 53 molecular types by cgMLST(HC2). Except for one strain belonging to ST3220, the other Salmonella Derby strains were ST40. The phylogenetic analysis showed that some strains isolated in Hangzhou were close to the strains in Southeast Asia, suggesting the possibility of cross-border transmission of ST40 strains, with the main food sources being pork and fish; other strains were close to those circulating in Beijing, Guangzhou, Hubei, Chongqing and other provinces, suggesting the possibility of cross-province transmission of the strains, with the main food sources being pork, beef and chicken. Conclusions:The epidemic of Salmonella Derby in Hangzhou was mainly caused by the spread of ST40 strains and MDR was common. Clinical infections might be closely related to the consumption of pork, beef, chicken and fish. There was the possibility of cross-border transmission of Salmonella Derby between Hangzhou and Southeast Asia and cross-province transmission in China.

Extended-spectrum Beta-lactamases Encoding Genes among Salmonella Enterica serovar Typhi Isolates in Patients with Typhoid Fever from four Academic Medical Centers in Lagos, Nigeria

ABSTRACT Background: There is scarce information about the occurrence of extended-spectrum β-lactamases (ESBLs) in Salmonella enterica serovar Typhi (S. Typhi) from patients with typhoid fever. Objective: To study the antimicrobial resistance and ESBL encoding genes among S. Typhi isolates in aforesaid patients from Lagos, Nigeria. Methods: S. Typhi isolates were collected from blood samples of typhoid fever patients from 4 academic medical centers in Lagos, Nigeria. The identification of isolates and their antibiotic susceptibility testing were performed by standard bacteriological techniques and disc diffusion method, respectively. The production of ESBLs was investigated using combination disk test (CDT) and polymerase chain reaction (PCR). Results: A total of 27 S. Typhi isolates was collected. All isolates were susceptible to imipenem and nitrofurantoin. Fifteen (55.6%) isolates were multidrug-resistant (MDR). The CDT test showed 11 (40.7%) ESBL producer isolates. However, the PCR revealed a higher occurrence rate for ESBL producers (66.7%, n = 18/27). The ESBL genes were as follows: blaCTX-M (37.0%, n = 10/27), blaSHV (18.5%, n = 5/27), and blaTEM (44.4%, n = 12/27). All ESBL positive S. Typhi isolates were MDR. Conclusions: This study showed the emergence of ESBL-harboring S. Typhi in patients with typhoid fever from Nigeria.

A modified gentamicin protection assay for detecting invasive phenotype of Salmonella enterica serovar Enteritidis / 生物工程学报

Salmonella enterica serovar Enteritidis (SE) is one of the most important zoonotic pathogens that cause enteritis and systemic infection in animals and human. Understanding invasive capacities of SE isolates is of vital importance to elucidate pathogenesis of Salmonella infection. To improve the throughput capacity and repeatability of classical gentamicin protection assay (GPA), a modified PGA was developed by taking high-throughput advantage of 96-well cell plates and multichannel pipettes. In addition, drop plate technique rather than spread plate method was applied in the modified GPA protocol for bacterial enumeration. The modified GPA protocol was evaluated by phenotyping intracellular replication of a high virulent and a low virulent SE isolates, JL228 and LN248, in a phagocytic cell line RAW264.7. The protocol was then applied in invasive phenotype determination of 16 SE strains to non-phagocytes (HT-29) and the intracellular replication of 43 SE strains to phagocytes (RAW264.7). Significant lower intra-group and inter-group coefficient of variations of the modified GPA was observed, implying good repeatability and reproducibility over traditional protocol. Further, replication phenotypes were also correlated with those from direct observation by confocal microscopy. Collectively, the improved GPA protocol had advantages of high throughput capacity, good repeatability and reliability, it was also noticed that the protocol also represented a fast and labor-saving alternative scheme for the invasive phenotype determination of Salmonella Enteritidis, and providing reliable phenotype profiles for Salmonella-host interplay interpretation.

Role of methylation in regulating PhoP activity in Salmonella enterica serovar Typhimurium / 上海交通大学学报（医学版）

Objective :To investigate the correlation between methylation and the activity of transcriptional regulator PhoP in Salmonella enterica serovar Typhimurium (S. Typhimurium), and screen for the methyltransferase of PhoP. Methods :The methylation level of PhoP in S. Typhimurium under different culture conditions was determined by Western blotting. The transcription levels of phoP and the genes regulated by phoP were examined to screen for the methyltransferase of PhoP after overexpressing methyltransferase candidates predicted by bioinformatics. And then methyltransferase assay was verified in vitro. The transcription levels of phoP and its methyltransferase were determined by real-time PCR in high concentration of Mg2+ or weak acid conditions. Results :As the concentration of Mg2+increased in the medium, the methylation level of PhoP increased, and its methylation level decreased after S. Typhimurium was stimulated by weak acid. In the screening of 9 methyltransferases predicted by bioinformatics, overexpression of STM14_0023 reduced the transcription level of phoP and its downstream genes and the protein level of PhoP in vivo, but knockout of STM14_0023 had no effect on the expression of phoP and its downstream genes. STM14_0023 was capable of methylating PhoP in vitro and could also increase the methylation level of PhoP after overexpression of STM14_0023 in vivo. Under the condition of high concentration of Mg2+ when the expression of phoP was inhibited, the transcriptional level of STM14_0023 was reduced. Conclusion :STM14_0023 is the methyltransferase of PhoP, and methylation inhibits PhoP activity.

Screening of putative transcriptional regulators in Salmonella enterica serovar Typhimurium / 上海交通大学学报（医学版）

Objective: To uncover new transcriptional regulators by screening putative transcriptional regulators in Salmonella enterica serovar Typhimurium (S. Typhimurium) through genome informatics and molecular biology analyses. Methods: S. Typhimurium genome informatics was analyzed and 30 putative transcriptional regulators were screened out. All candidate genes were deleted by λ-Red system. The log-phase acid tolerance response (ATR) ability was compared between knock-out (KO) strains and wild type (WT) strain. Cell model was used to detect the invasion ability and intracellular ability of KO strains that showed differences in acid stress compared to WT strain. The transcription level of phoP was detected by realtime-PCR in the mutants involved in both ATR and cell infection. Results: All 30 deletion mutants were successfully constructed. ΔSTM14_0739, ΔSTM14_2717 and ΔSTM14_1646 showed increased log-phase ATR ability, while ATR abilities of ΔSTM14_4338, ΔSTM14_1965 and ΔSTM14_1878 decreased, compared with WT. ΔSTM14_0739 and ΔSTM14_2717 mutants showed weaker invasion ability in HeLa cells than WT, and ΔSTM14_1878 and ΔSTM14_2717 mutants showed stronger replication ability in RAW264.7 cells than WT. Realtime-PCR suggested STM14_2717 deletion had no effect on phoP transcription. Conclusion: This work discovers unknown transcriptional regulators, and provides clues for future research in S. Typhimurium pathogenesis.

Advances on antimicrobial resistance research of Salmonella enterica Serovar Indiana / 中华预防医学杂志

Salmonella enterica Serovar Indiana is a common serotype of Salmonella isolated from food especially from poultry meat. Recently it demonstrated a raising tendency of infection cases and isolate numbers with high antimicrobial resistant rate against many common antimicrobials, including quinolones and cephalosporin which were regarded as the first line drug for the treatment of Salmonella infections, and this kind of Salmonella serotype was always carrying complex resistance mechanisms and also a variety of mobile elements, all of these features made the very clinical infections caused by Salmonella hard to treat and brought great difficulties and risks. Here, we review the prevalence of Samonella Indiana on national and international view, and we also anticipate the research progress on antimicrobial drug classes, multi drug resistance, co-resistance and resistance mechanism. We discuss the resistant genotypes, phenotypes, mechanism and transmission of Salmonella Indiana strains isolated from different origins. By introducing the resistance of Salmonella Indiana, we want to attract people's attention to this bacteria and its hazard, and offer some idea to evaluate and treat infections in clinical.

Identification of a toxin coding fragment in pBSSB1, a linear plasmid from Salmonella enterica serovar Typhi that can stabilize a multicopy plasmid

Objective: To identify the region conferring stability to pBSSB2 (a linear plasmid, pBSSB1, containing a kanamycin cassette), which is unique to Indonesian isolates of Salmonella enterica serovar Typhi. Methods: The open reading frame (ORF) 009 was identified as a toxin coding gene in the plasmid through introduction of translational termination codons in the ORF. Results: The stability function was located in a fragment that spanned nucleotides 5 766 to 6 828 in the linear plasmid genetic map. Ectopic expression of ORF009 in pBAD18 vector indicated ORF009 codes for a toxin. This fragment could stabilize plasmid pUC18 previously destabilized through mutation of the pcnB (plasmid copy number control) gene that codes for polyA polymerase. Majority of the cells expressing ORF009 were non-viable according to phase contrast microscopy. Conclusions: This study demonstrated that a linear plasmid fragment that carries a gene encoding a toxin possibly conferred stability to the parent plasmid. It was able to stabilize a multicopy plasmid of Escherichia coli.

Identification of a toxin coding fragment in pBSSB1, a linear plasmid from Salmonella enterica serovar Typhi that can stabilize a multicopy plasmid

Objective: To identify the region conferring stability to pBSSB2 (a linear plasmid, pBSSB1, containing a kanamycin cassette), which is unique to Indonesian isolates of Salmonella enterica serovar Typhi. Methods: The open reading frame (ORF) 009 was identified as a toxin coding gene in the plasmid through introduction of translational termination codons in the ORF. Results: The stability function was located in a fragment that spanned nucleotides 5766 to 6828 in the linear plasmid genetic map. Ectopic expression of ORF009 in pBAD18 vector indicated ORF009 codes for a toxin. This fragment could stabilize plasmid pUC18 previously destabilized through mutation of the pcnB (plasmid copy number control) gene that codes for polyA polymerase. Majority of the cells expressing ORF009 were non-viable according to phase contrast microscopy. Conclusions: This study demonstrated that a linear plasmid fragment that carries a gene encoding a toxin possibly conferred stability to the parent plasmid. It was able to stabilize a multicopy plasmid of Escherichia coli.

In vitro efficacy of several disinfectants against Salmonella enterica serovar Enteritidis and Escherichia coli strains from poultry / Eficácia de vários desinfetantes in vitro contra cepas de Salmonella entérica serovar Enteritidis e Escherichia coli aviária

ABSTRACT The efficacy of 28 individual or blended disinfectants against avian Salmonella enterica serovar Enteritidis and Escherichia coli strains was determined. An in vitro test in the presence and absence of serum as source of organic material was conducted. Povidone-iodine (releasing 1% available iodine), 1% potassium permanganate, 70% ethanol, 2% chlorhexidine digluconate and three commercial formulations based on quaternary ammonium compounds + formaldehyde or cresol derivates were the most effective against all strains tested and reduced bacterial counts by more than 106 times (6-log10) regardless of the presence of organic matter. These commercial compounds as well as ethanol and chlorhexidine among the individual substances tested might be helpful in the adoption of environmental control measures against these two enterobacteria in poultry industry.

RESUMO: A eficácia de 28 desinfetantes individuais ou combinados sobre cepas de Salmonella enterica serovar Enteritidis e Escherichia coli foi determinada. Um teste in vitro em presença e ausência de soro como fonte de matéria orgânica foi realizado. Iodopovidona (contendo 1% de iodo ativo), permanganato de potássio a 1%, etanol a 70%, digliconato de clorexidina e três formulações comerciais, baseadas em compostos de amônia quaternária + formaldeído ou em derivados de cresóis, foram mais eficazes contra as cepas bacterianas testadas, reduzindo em mais 106 vezes (6-log) a contagem bacteriana, independente da presença de matéria orgânica. Esses compostos comerciais, bem como o etanol e a clorexidina entre as substâncias químicas individuais avaliadas, podem ser úteis para a implementação de medidas de controle ambiental contra estas duas enterobacterias de importância para a indústria aviária.

Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α

A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.

Deletion of Salmonella enterica serovar typhimurium sipC gene

Objective: To construct a novel plasmid as Salmonella enterica serovar typhimurium (S. typhimurium) sipC gene knockouts candidate. Methods: In this research, 50 upstream and 30 downstream regions of S. typhimurium sipC gene and kanamycin gene were PCR amplified. Each of these DNA fragment was cloned into pGEM T-easy vector. The construct was confirmed by PCR and restriction digest. Results: PCR amplified 320, 206 and 835 bp DNA fragments were subcloned into pET-32 vector resulting with a plasmid called pET-32-sipC up-kan-sip C down. Conclusions: The new plasmid (pET-32-sipC up-kan-sip C down) is useful for genetic engineering and for future manipulation of S. typhimurium sipC gene.

Regulation of motility of Salmonella enterica serovar Typhi by QseBC / 军事医学

Objective To study the impact of QseBC on the motility of Salmonella enterica serovar Typhi ( S.Typhi ) . Methods The motility of wild-type ( WT) and null mutants (ΔqseB and ΔqseC) at mid-log phase was investigated by swimming assay.Quantitative RT-PCR was carried out to calculate the transcriptional variation of flhD and qseB among WT,ΔqseB andΔqseC.QseB overexpressing strain was constructed to compare its motility and flhD expression with the wild-type control.Results The result of motility assay showed that the motility of ΔqseB was similar to that of the WT strain , while the motility of ΔqseC was much lower than that of WT .qRT-PCR revealed that compared with WT , the expression of flhD was significantly decreased in ΔqseC while the expression of qseB was increased considerably .The motility of QseB overex-pressing strain was lower .Conclusion The expression of flhD may be regulated by QseBC which has an effect on the motil-ity of S.typhi, and the overexpression of QseB may inhibit the motility .

Deletion of Salmonella enterica serovar typhimurium sipC gene

Objective: To construct a novel plasmid as Salmonella enterica serovar typhimurium (S. typhimurium) sipC gene knockouts candidate. Methods: In this research, 5' upstream and 3' downstream regions of S. typhimurium sipC gene and kanamycin gene were PCR amplified. Each of these DNA fragment was cloned into pGEM T-easy vector. The construct was confirmed by PCR and restriction digest. Results: PCR amplified 320, 206 and 835 bp DNA fragments were subcloned into pET-32 vector resulting with a plasmid called pET-32-sipC up-kan- sip C down. Conclusions: The new plasmid (pET-32-sipC up-kan- sip C down) is useful for genetic engineering and for future manipulation of S. typhimurium sipC gene.

Antibacterial Activity of Phenolic Compounds Derived from Ginkgo biloba Sarcotestas against Food-Borne Pathogens.

Aims: We investigated the antibacterial activity of three groups of phenolic compounds obtained from the chloroform (CHCl3) extract of the fleshy seed coat (sarcotestas) of Ginkgo biloba. Study Design: An experimental study. Methodology: Inhibition of microbial growth was measured by an agar diffusion method and susceptibility tests were performed by the broth microdilution method. Bactericidal effect of Ginkgo biloba compound 5-7 against Salmonella enterica serovar Typhimurium was assessed by time-kill assay. Results: Ginkgo biloba compounds 5-7 and 8-10 showed high antimicrobial activity against Gram-positive and Gram-negative bacteria, including several food-borne pathogens. In particular, compounds 5-7 and 8-10, containing phenolic acids and bilobols, respectively, were highly effective against Salmonella enteric serovar Typhimurium, Listeria monocytogenes, Listeria innocua, Streptococcus pyogenes, Escherichia coli, and Shigella dysenteriae. On the opposite, compounds 1-4, containing cardanols, showed little antibacterial activity. Compounds 5-7 exerted a bactericidal and bacteriolytic effect on Salmonella enteric serovar Typhimurium with a Minimal Inhibitory Concentration (MIC) and a Minimal Bactericidal Concentration (MBC) of 8.3 μg ml–1. Conclusion: The results of this study indicate that phenolic compounds derived from Ginkgo biloba sarcotestas, because of their strong inhibitory characteristics towards food pathogens, can be considered ideal candidates for possible application in food microbiology due to their natural origins.

Induction of deletion mutation on ompR gene of Salmonella enterica serovar Typhi isolates from asymptomatic typhoid carriers to evolve attenuated strains for vaccine development

Objective: To develop attenuated strains of Salmonella enterica serovar Typhi (. S. typhi) for the candidate vaccine by osmolar stress. Methods: S. typhi SS3 and SS5 strains were isolated from asymptomatic typhoid carriers in Namakkal, Tamil Nadu, India. Both strains were grown in LB (Luria Bertani) medium supplemented with various concentration of NaCl (0.1-0.7M) respectively. The effect of osmolar stress was determined at molecular level by PCR using MGR 06 and MGR 07 primers corresponding to ompR with chromosomal DNA of S. typhi SS3 and SS5 strains. Attenuation by osmolar stress results in deletion mutation of the S. typhi strains was determined by agglutination assays, precipitation method, SDS PAGE analysis and by animal models. Results: The 799 bp amplified ompR gene product from wild type S. typhi SS3 and SS5 illustrate the presence of virulent gene. Interestingly, there was only a 282 bp amplified product from S. typhi SS3 and SS5 grown in the presence of 0.5, 0.6 and 0.7 M NaCl. This illustrates the occurrence of deletion mutation in ompR gene at high concentration of NaCl. Furthermore, both the wild-type and mutant S. typhi outer membrane SDS-PAGE profile reveals the differences in the expression of ompF, ompC and ompA proteins. In mice, wild type and mutant strains lethal dose (LD

Comparative evaluation of multilocus variable-number tandem-repeat analysis and pulsed-field gel electrophoresis for genotyping Salmonella enterica serovar Typhi isolates / 中华微生物学和免疫学杂志

Objective To evaluate the capability of multilocus variable-number tandem-repeat ( VNTR) analysis ( MLVA) and pulsed-field gel electrophoresis ( PFGE) for genotyping Salmonella enterica serovar Typhi (S.Typhi) isolates.Methods Five polymorphic VNTRs (SAL02,SAL11,SAL16,SAL20, and TR4699 ) that were observed in S.Typhi strains from previous studies were selected to establish MLVA . Twenty-one epidemiologically unrelated S.Typhi strains that were isolated from Shenzhen ,China from 2002 to 2007 were genotyped by the established MLVA , and the results were compared with those by PFGE . Results The Simpson′s index of diversity ( D value ) for all five different VNTRs ranged from 0.838 to 0.943 .A total of 19 MLVA profiles and 19 PFGE profiles were found , respectively .The D value for both MLVA and PFGE were 0.99 and the test results from two analyses were identical .Conclusion The five polymorphic VNTRs analysis could be used as an alternative typing scheme for epidemiologic investigation of S.Typhi infection .

A low-pH medium in vitro or the environment within a macrophage decreases the transcriptional levels of fimA, fimZ and lrp in Salmonella enterica serovar Typhimurium.

Many Salmonella Typhimurium isolates produce type 1 fimbriae and exhibit fimbrial phase variation in vitro. Static broth culture favours the production of fimbriae, while solid agar medium inhibits the generation of these appendages. Little information is available regarding whether S. Typhimurium continues to produce type 1 fimbriae during in vivo growth. We used a type 1 fimbrial phase-variable strain S. Typhimurium LB5010 and its derivatives to infect RAW 264.7 macrophages. Following entry into macrophages, S. Typhimurium LB5010 gradually decreased the transcript levels of fimbrial subunit gene fimA, positive regulatory gene fimZ, and global regulatory gene lrp. A similar decrease in transcript levels was detected by RT-PCRwhen the pH of static brothmediumwas shifted frompH 7 to amore acidic pH 4. A fimA-deleted strain continued to multiply within macrophages as did the parental strain. An lrp deletion strain was unimpaired for in vitro growth at pH 7 or pH 4, while a strain harboring an lrp-containing plasmid exhibited impaired in vitro growth at pH 4. We propose that acidic medium, which resembles one aspect of the intracellular environment in a macrophage, inhibits type 1 fimbrial production by down-regulation of the expression of lrp, fimZ and fimA.

Salmonella enterica serovar Typhi plasmid pR ST98 -mediated inhibition of autophagy promotes bacterial survival in infected fibroblasts.

pR ST98 is a chimeric plasmid isolated from Salmonella enterica serovar typhi (S. typhi) and mediates both drug-resistance and virulence of S. typhi. Autophagy has been recently reported as an important component of the innate immune response against intracellular pathogen. In this study, we investigated the effect of pR ST98 on cellular autophagy, apoptosis and bacterial survival in infected fibroblasts. S. typhi strain ST 8 carrying pR ST98 , Salmonella typhimurium strain SR-11 carrying a 100 Kb virulent plasmid, and avirulent S. typhi strain ST 10 without plasmid were tested in this experiment. Results showed that embryonic fibroblasts infected with ST 8 containing pR ST98 had decreased autophagy accompanied by increased bacterial survival and apoptosis. Further study showed that autophagy inducer rapamycin reversed pR ST98 -mediated inhibition of autophagy and reduced apoptosis in infected fibroblasts. Our data indicate that pR ST98 can inhibit autophagy, thus facilitating S. typhi survival and promoting apoptosis of host cells. This study contributes to understanding the underlying mechanism of pR ST98 -mediated virulence in S. typhi.

Molecular Characterisation of Salmonella enterica Serovar Typhi Isolated from Typhoidial Humans

Aims: Salmonella enterica serovar Typhi is the major causative agent for typhoidial fever around the globe among human population reported till date. Present research work was carried out for detection and molecular characterisation of Salmonella enterica serovar Typhi isolated from humans with Typhoidial fever by biochemical, phenotypical and virulence gene based polymerase chain reaction (PCR) techniques. The isolated strains were also investigated for antibiotic susceptibility patterns as a control measure. Methodology and Results: A total of 16 clinical samples were collected from the same numbers of patients (7 males and 9 females) from Coimbatore, Erode and Salem districts of Tamil Nadu and were processed via broth enrichment methods for isolation and identification of the causative agent S. enterica serovar Typhi. Microbiological and biochemical investigations revealed the presence of S. Typhi from 16 samples. The biotyping of the isolates showed that all the isolates belonged to biotype IV. The PCR analysis confirmed the presence of invA (Invasion gene, 244bp), tyv (Tyvelose epimerase gene, 615 bp), fliC-d (Phage-1 flagellin gene for d-antigen, 750 bp) and viaB (Vi antigen gene, 439bp) in all 16 clinical samples. The antibiotic susceptibility test that was carried out among the isolates against 12 antimicrobial agents, showed 100 % resistance to only ampicillin and 100 % sensitivity to carbenicillin, chloramphenicol, clindamycin, gentamycin, kanamycin and tetracycline. Conclusion, significance and impact of study: This study confirmed the association of virulent strains of S. enterica serovar Typhi from Typhoidial fever among human population and suggested that PCR based diagnostic could be very useful for the rapid detection of S. Typhi isolates. Present study emphasized the use of antibiotic like chloramphenicol or in combination with other antibiotics for the effective control of S. Typhi.

Expression of fljB: z66 on a linear plasmid of Salmonella enterica serovar typhi is dependent on FliA and FlhDC and regulated by OmpR

Salmonella enterica serovar Typhi z66-positive strains have two different flagellin genes, fliC:d/j and fljB:z66, located on the chromosome and on a linear plasmid, respectively. To investigate the mechanism underlying the expressional regulation of fljB:z66, gene deletion mutants of the regulators FliA, FlhDC, and OmpR were constructed in this study. The expression levels of fliC and fljB:z66 were analyzed by qRT-PCR in the wild-type strain and mutants at high and low osmolarity. The results show that the expression levels of both fljB:z66 and fliC were greatly reduced in fliA and flhDC mutants under both high and low osmotic conditions. In the ompR mutant, the expression levels of fljB:z66, fliC, fliA, and flhD were increased at low osmotic conditions. SDS-PAGE and western blotting analysis of the secreted proteins revealed that the FljB:z66 was almost absent in the fliA and flhDC mutants at both high and low osmolarity. In the wild-type strain, the fljB:z66 was more highly expressed under high-osmolarity conditions than under low-osmolarity conditions. However, this difference in expression disappeared in the ompR mutant. Translational expression assay of FljB:z66 showed that the FljB:z66 expression was decreased in ompR mutant at both low and high osmolarity. These results suggest that the expression of fljB:z66 in S. enterica serovar Typhi is dependent on FliA and FlihDC, and OmpR can regulate the expression and secretion of FljB:z66 in different osmolarity.