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.

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.

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.

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.

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 .

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 .

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

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.

Macrophage apoptosis associated with Salmonella enterica serovar Typhi plasmid.

The present study was undertaken to investigate the relationship between plasmid isolated from S. enterica serovar Typhi (pRST98) and macrophage apoptosis. pRST98 was transferred into an attenuated S. enterica serovar Typhimurium strain RIA to create a transconjugant pRST98/RIA. Standard S. enterica serovar Typhimurium virulence strain SR-11 was used as a positive control, and RIA as a negative one. Murine macrophage-like cell line (J774A.1) was used as an infectious cell model in vitro. In order to determine the inhibition and bactericidal effect of amikacin (AMK) to extracellular bacteria and the best optimization co-culture ratio between Salmonella and J774A.1, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of AMK to strains SR-11, pRST98/RIA and RIA and multiplicity of infection (MOI) were detected first, and then J774A.1 was infected by the above three serovar Typhimurium strains. Apoptosis of J774A.1 was examined with electron microscopy and flow cytometry after annexin-V/propidium iodide labeling at 0, 1, 3, 6, 12 and 24 h. Mitochondrial membrane potential was detected by JC-1 staining method. It was demonstrated that MIC of AMK to the three strains was 10µg/ml, MBC was 80µg/ml, and optimal MOI was 100:1. pRST98/RIA resulted in a higher apoptosis of J774A.1 than RIA, apoptotic features such as chromatin margination could be observed after 3 h, and death of J774A.1 cells was associated with the loss of mitochondrial membrane potential. These results indicated that pRST98 could enhance the virulence of its host bacteria, evidenced by increased macrophage apoptosis.

A Case of Multidrug-Resistant Salmonella enterica Serovar Typhi Treated with a Bench to Bedside Approach

We report a relapsed case of a 25 year-old man with multi-drug resistant Salmonella serovar Typhi (MDRST) bacteremia who had recently returned from travel in India. Due to unresponsiveness to ciprofloxacin and ceftriaxone, we examined the strain's resistance to quinolones and extended-spectrum beta-lactamases (ESBLs). The strain had a single gyrA mutation at codon 83 (Ser83Phe), which explains its decreased susceptibility to fluoroquinolone and resistance to nalidixic acid. In the screening tests of ESBLs, TEM-1 was positive, which is beta-lactamase but not ESBL. The patient was finally successfully treated with meropenem and aztreonam. In the presence of clinical unresponsiveness despite favorable sensitivity tests, further laboratory evaluations are needed, which should include studies of genes related to antibiotic resistance and ESBLs. In addition, further prospective trials should be done about the possible inclusion of antibiotics not yet mentioned in the current guidelines. With MDRST on the rise worldwide, the most optimal and effective line of antibiotic defense needs to be devised.

A Case of Typhoid Fever to Failed Ciprofloxacin, Infected in Korea / 감염과화학요법

Typhoid fever caused by Salmonella enterica serovar Typhi (S. typhi) and usually ciprofloxacin is first used for treatment. However, the incidence of fluoroquinolone resistance or reduced susceptibility in S. typhi has been increased in Asia over the past decade and there have been reports of failed treatment with ciprofloxacin. Recently, if typhoid fever does not improved with ciprofloxacin treatment, S. typhi with reduced susceptibility to ciprofloxacin should be considered. We experienced a case of nalidixic acid-resistant S. typhi infection that was refractory to treatment with ciprofloxacin in Korea. A 47-year-old woman presented with fever and headache for 14 days. Blood culture revealed the presence of S. typhi that was susceptible to ciprofloxacin. However, she remained feverish and new symptoms of abdominal pain and bloody diarrhea developed after 5 days treatment with ciprofloxacin and subsequent testing showed that isolate was resistant to nalidixic acid.

A Case of Typhoid Fever to Failed Ciprofloxacin, Infected in Korea / 감염과화학요법

Typhoid fever caused by Salmonella enterica serovar Typhi (S. typhi) and usually ciprofloxacin is first used for treatment. However, the incidence of fluoroquinolone resistance or reduced susceptibility in S. typhi has been increased in Asia over the past decade and there have been reports of failed treatment with ciprofloxacin. Recently, if typhoid fever does not improved with ciprofloxacin treatment, S. typhi with reduced susceptibility to ciprofloxacin should be considered. We experienced a case of nalidixic acid-resistant S. typhi infection that was refractory to treatment with ciprofloxacin in Korea. A 47-year-old woman presented with fever and headache for 14 days. Blood culture revealed the presence of S. typhi that was susceptible to ciprofloxacin. However, she remained feverish and new symptoms of abdominal pain and bloody diarrhea developed after 5 days treatment with ciprofloxacin and subsequent testing showed that isolate was resistant to nalidixic acid.

An Imported Case of Typhoid Fever Refractory to Ciprofloxacin Treatment / 감염과화학요법

Salmonella enterica serovar Typhi infection is widely prevalent in developing countries; its treatment has been complicated by the emergence of resistance to antimicrobial agents. Fluoroquinolones are orally administered antimicrobials effective against typhoid fever, including that caused by the multidrug-resistant S. Typhi. They are relatively inexpensive and more convenient to administer com pared to third-generation cephalosporins; hence, they constitute the drugs of choice for the treatment of typhoid fever in developing countries. In Asian countries, however, resistance to nalidixic acid-a prototype of quinolone antibiotics-diminishes the value of fluoroquinolones with regard to the treatment of typhoid fever. We experienced a case of nalidixic acid-resistant S. Typhi infection imported from Pakistan that was clinically refractory to ciprofloxacin treatment. A 31-year-old male presented with a fever of 14 days' duration after returning from his native Pakistan. Oral ciprofloxacin was empirically administered for three days without any beneficial effect. His illness, however, improved after the administration of ceftriaxone for three days. Blood culture revealed the presence of S. Typhi that was resistant to nalidixic acid (minimal inhibitory concentration > or =32 microgram/mL) but susceptible to ciprofloxacin (minimal inhibitory concentration=1.0 microgram/mL) in vitro.

An Imported Case of Typhoid Fever Refractory to Ciprofloxacin Treatment / 감염과화학요법

Salmonella enterica serovar Typhi infection is widely prevalent in developing countries; its treatment has been complicated by the emergence of resistance to antimicrobial agents. Fluoroquinolones are orally administered antimicrobials effective against typhoid fever, including that caused by the multidrug-resistant S. Typhi. They are relatively inexpensive and more convenient to administer com pared to third-generation cephalosporins; hence, they constitute the drugs of choice for the treatment of typhoid fever in developing countries. In Asian countries, however, resistance to nalidixic acid-a prototype of quinolone antibiotics-diminishes the value of fluoroquinolones with regard to the treatment of typhoid fever. We experienced a case of nalidixic acid-resistant S. Typhi infection imported from Pakistan that was clinically refractory to ciprofloxacin treatment. A 31-year-old male presented with a fever of 14 days' duration after returning from his native Pakistan. Oral ciprofloxacin was empirically administered for three days without any beneficial effect. His illness, however, improved after the administration of ceftriaxone for three days. Blood culture revealed the presence of S. Typhi that was resistant to nalidixic acid (minimal inhibitory concentration > or =32 microgram/mL) but susceptible to ciprofloxacin (minimal inhibitory concentration=1.0 microgram/mL) in vitro.

Genomic Characteristics and Identification of Salmonella enterica serovars Typhi and Paratyphi A Using Multiplex PCR / 대한임상미생물학회지

BACKGROUND: Salmonella enterica serovars often have a broad host range and cause some gastrointestinal and systemic diseases. The diagnosis of typhoid fever or paratyphoid fever is made by ordinary culture methods and biochemical tests. However, a more rapid and alternative method of diagnosing these diseases is in need since the classical diagnostic method requires several days for a result. Some researchers have already reported serovar Typhi detection methods with PCR using the fliC-d gene and the Vi capsular antigen gene. METHODS: Thirty-six Salmonella strains isolated at Pusan National University Hospital from 1997 to 2004 were used for a rapid identification of S. enterica serovars Typhi and Paratyphi A with multiplex PCR that uses the O (rfbE, rfbS), H (fliC-d, fliC-a), and Vi (viaB) antigen genes. To further characterize these Salmonella strains, we used PCR to detect genes (invA and enterotoxin) for proposed virulence factors and performed antimicrobial susceptibility testing, serotyping and pulsed-field gel electrophoresis for epidemiological characteristics. RESULTS: Most strains were resistant to ampicillin. By PCR, tyv, prt, fliC-d and viaB genes were detected in serovar Typhi, whereas only fliC-a and prt genes were found in serovar Paratyphi A. In addition, invA and enterotoxin genes were detected in both strains. CONCLUSION: This method enabled us to identify and differentiate serovars Typhi and Paratyphi A by only a single PCR assay. That is, clinically important human pathogens were more rapidly and specifically detected and identified with multiplex PCR.

A case of Salmonella enterica serovar Typhi endocarditis with multiple splenic infarctions / 대한내과학회지

Infective endocarditis is a very rare cardiac manifestation of salmonella infection, and splenic infarction is a rare noncardiac complication. We describe a case of Salmonella enterica serovar Typhi bacteremia which was complicated by infective endocarditis with multiple splenic infarctions in a previously healthy 47-year-old female. She didn't have any history of foreign travel. The isolate of Salmonella enterica serovar Typhi was susceptible to cephalosporins, aminoglycosides, quinolones but resistant to ampicillin. After 3 weeks of intravenous and oral therapy with ciprofloxacin, follow up transthoracic and transesophageal echocardiography showed no vegetation. In addition, follow up abdominal CT showed decreased size of splenic infarctions. The patient was treated with 2 weeks of intravenous and 4 weeks of oral ciprofloxacin, and was cured without sequelae or relapse for 6 months follow-up.