Antibiotic resistome of Salmonella typhi: molecular determinants for the emergence of drug resistance / 医学前沿

Resistome is a cluster of microbial genes encoding proteins with necessary functions to resist the action of antibiotics. Resistome governs essential and separate biological functions to develop resistance against antibiotics. The widespread clinical and nonclinical uses of antibiotics over the years have combined to select antibiotic-resistant determinants and develop resistome in bacteria. At present, the emergence of drug resistance because of resistome is a significant problem faced by clinicians for the treatment of Salmonella infection. Antibiotic resistome is a dynamic and ever-expanding component in Salmonella. The foundation of resistome in Salmonella is laid long before; therefore, the antibiotic resistome of Salmonella is reviewed, discussed, and summarized. We have searched the literature using PubMed, MEDLINE, and Google Scholar with related key terms (resistome, Salmonella, antibiotics, drug resistance) and prepared this review. In this review, we summarize the status of resistance against antibiotics in S. typhi, highlight the seminal work in the resistome of S. typhi and the genes involved in the antibiotic resistance, and discuss the various methods to identify S. typhi resistome for the proactive identification of this infection and quick diagnosis of the disease.

Differential roles for pathogenicity islands SPI-13 and SPI-8 in the interaction of Salmonella Enteritidis and Salmonella Typhi with murine and human macrophages

BACKGROUND: Salmonella pathogenicity island (SPI)-13 is conserved in many serovars of S. enterica, including S. Enteritidis, S. Typhimurium and S. Gallinarum. However, it is absent in typhoid serovars such as S. Typhi and Paratyphi A, which carry SPI-8 at the same genomic location. Because the interaction with macrophages is a critical step in Salmonella pathogenicity, in this study we investigated the role played by SPI-13 and SPI-8 in the interaction of S. Enteritidis and S. Typhi with cultured murine (RAW264.7) and human (THP-1) macrophages. RESULTS: Our results showed that SPI-13 was required for internalization of S. Enteritidis in murine but not human macrophages. On the other hand, SPI-8 was not required for the interaction of S. Typhi with human or murine macrophages. Of note, the presence of an intact copy of SPI-13 in a S. Typhi mutant carrying a deletion of SPI-8 did not improve its ability to be internalized by, or survive in human or murine macrophages. CONCLUSIONS: Altogether, our results point out to different roles for SPI-13 and SPI-8 during Salmonella infection. While SPI-13 contributes to the interaction of S. Enteritidis with murine macrophages, SPI-8 is not required in the interaction of S. Typhi with murine or human macrophages. We hypothesized that typhoid serovars have lost SPI-13 and maintained SPI-8 to improve their fitness during another phase of human infection.

stg fimbrial operon from S. Typhi STH2370 contributes to association and cell disruption of epithelial and macrophage-like cells

BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi) stg operon, encoding a chaperone/usher fimbria (CU), contributes to an increased adherence to human epithelial cells. However, one report suggests that the presence of the Stg fimbria impairs the monocyte-bacteria association, as deduced by the lower level of invasion to macrophage-like cells observed when the stg fimbrial cluster was overexpressed. Nevertheless, since other CU fimbrial structures increase the entry of S. Typhi into macrophages, and considering that transcriptomic analyses revealed that stg operon is indeed expressed in macrophages, we reassessed the role of the stg operon in the interaction between S. Typhi strain STH2370 and human cells, including macrophage-like cells and mononuclear cells directly taken from human peripheral blood. RESULTS: We compared S. Typhi STH2370 WT, a Chilean clinical strain, and the S. Typhi STH2370 Astg mutant with respect to association and invasion using epithelial and macrophage-like cells. We observed that deletion of stg operon reduced the association and invasion of S. Typhi, in both cellular types. The presence of the cloned stg operon restored the WT phenotype in all the cases. Moreover, we compared Salmonella enterica sv. Typhimurium 14028s (S. Typhimurium, a serovar lacking stg operon) and S. Typhimurium heterologously expressing S. Typhi stg. We found that the latter presents an increased cell disruption of polarized epithelial cells and an increased association in both epithelial and macrophage-like cells. CONCLUSIONS: S. Typhi stg operon encodes a functional adhesin that participates in the interaction bacteria-eukary-otic cells, including epithelial cells and macrophages-like cells. The phenotypes associated to stg operon include increased association and consequent invasion in bacteria-eukaryotic cells, and cell disruption.

Rapid detection of Salmonella Typhi by loop-mediated isothermal amplification (LAMP) method

An in-house loop-mediated isothermal amplification (LAMP) reaction was established and evaluated for sensitivity and specificity in detecting the presence of Salmonella Typhi (S. Typhi) isolates from Kelantan, Malaysia. Three sets of primers consisting of two outer and 4 inner were designed based on locus STBHUCCB_38510 of chaperone PapD of S. Typhi genes. The reaction was optimised using genomic DNA of S. Typhi ATCC7251 as the template. The products were visualised directly by colour changes of the reaction. Positive results were indicated by green fluorescence and negative by orange colour. The test was further evaluated for specificity, sensitivity and application on field samples. The results were compared with those obtained by gold standard culture method and Polymerase Chain Reaction (PCR). This method was highly specific and -10 times more sensitive in detecting S. Typhi compared to the optimised conventional polymerase chain reaction (PCR) method.

Investigation of an outbreak of Salmonella Typhi in Battalgazi district, Malatya, Turkey / Investigação de um surto de Salmonella Typhi no distrito de Battalgazi, Malatya, Turquia

Salmonella Typhi infections are important public health problems for the developing countries. In this study we investigated the molecular epidemiology of a suspected well-water borne S. Typhi outbreak occurred in a district of Malatya-Turkey. This outbreak affected 10 patients in two days. Arbitrary primed polymerase chain reaction (AP-PCR) based typing showed two clones, one had seven, and the other had three strains, supporting outbreak speculation. By adding chlorine to wells by local municipal authority, the outbreak ended within a very short time (about ten days).

As infecções por Salmonella Typhi são problemas importantes de saúde pública em países em desenvolvimento. Neste estudo, investigamos a epidemiologia molecular de surto de Salmonella Typhi, supostamente causado por água de poço, ocorrido no distrito de Battalgazi, Malatya, Turquia. Este surto afetou 10 pessoas em dois dias. A tipagem por AP-PCR (arbitrary primed polimerase chain reaction) indicou dois clones, um com sete isolados e outro com três isolados. Com a adição de cloro aos poços pelas autoridades locais, o surto terminou rapidamente (em dez dias).

Variabilidade genética de amostras de Salmonella Typhi isoladas de surto e de casos esporádicos ocorridos em Belém, Brasil / Genetic variability of Salmonella Typhi samples isolated from outbreaks and sporadic cases of typhoid fever in Belém, Brazil

INTRODUÇÃO: Salmonella Typhi é o agente da febre tifóide (doença caracterizada por febre, cefaléia, mialgia, artralgia, diarréia ou constipação), cujo quadro pode se complicar e levar o paciente a óbito. No Brasil, a febre tifóide é endêmica nas regiões Norte e Nordeste, com surtos ocorridos nos meses de intenso calor. OBJETIVO: Analisar e comparar a variabilidade genética de S. Typhi isoladas de surto e casos esporádicos de febre tifóide ocorridos em determinado período na cidade de Belém (PA). MATERIAL E MÉTODOS: Foram analisadas 20 amostras de S. Typhi: 10 isoladas de um surto ocorrido no bairro do Guamá, Belém, entre os meses de dezembro/2005 e março/2006, e 10 de casos esporádicos ocorridos em diferentes localidades da mesma cidade e no mesmo período do surto. A caracterização genética foi realizada pela análise do perfil de macrorrestrição obtido pela enzima XbaI e definido por eletroforese em gel de campo pulsado (PFGE). RESULTADOS: A análise de XbaI-PFGE das amostras estudadas demonstrou uma similaridade genética de 83 por cento a 100 por cento. CONCLUSÃO: Este estudo pôde demonstrar a relação clonal das amostras S. Typhi causadoras de surto e de casos esporádicos de febre tifóide ocorridos na cidade de Belém no período de dezembro/2005 a março/2006.

BACKGROUND: Salmonella Typhi is the causative agent of typhoid fever, illness characterized by fever, migraine, myalgia, arthralgia, diarrhea or constipation, which may have complications and cause death. In Brazil, the typhoid fever is endemic in the Northern and Northeastern regions, with outbreaks occurring in scorching months. OBJECTIVE: To analyse and compare the genetic variability of S. Typhi strains isolated from outbreaks and sporadic cases of typhoid fever occurred in the city of Belém (PA) between December 2005 and March 2006. MATERIAL AND METHODS: Twenty samples of S. Typhi were analyzed: 10 of them were isolated from an outbreak occurred in Guamá neighborhood in Belém, between December 2005 and March 2006, and the other 10 were isolated from sporadic cases in different neighborhoods of the same city in the same outbreak period. The genetic characterization was performed by macrorestriction analysis of genomic DNA with XbaI enzyme defined by pulsed-field gel electrophoresis (PFGE). RESULTS: The Xbal-PFGE analysis of the studied samples revealed a genetic similarity of 83 percent to 100 percent. CONCLUSION: This study demonstrated the clonal relation between the S. Typhi samples from the outbreak and from the sporadic cases of typhoid fever occurred in the city of Belém between December 2005 and March 2006.

Diagnosis of typhoid fever by polymerase chain reaction.

OBJECTIVE: To determine the efficacy of nested polymerase chain reaction (PCR) in detecting Salmonella typhi gene sequences in blood and urine specimens and to determine the cut-off titer of Widal test using PCR as gold standard test for diagnosis of typhoid fever. METHODS: Study included 71 children between the ages of 8 months and 14 years; 52 of them were suspected cases of typhoid fever, 11 were febrile non-typhoid controls and 8 were apparently healthy children. Nested PCR in Blood and Urine, Blood culture, Widal test and Urine culture were done and their results analyzed. RESULTS: Among suspected typhoid cases, PCR in blood and urine had positivity of 82.7% each. Blood culture, Widal test (at cut off titer TO and/or TH > 1:160) and urine culture had positivity of 26.9%, 50% and 3.8% respectively. In one case, urine PCR was positive and blood PCR was negative. Similarly, in another case, PCR in blood was positive however urine tested negative. Considering PCR as gold standard, the antibody cut off titer was evaluated. A cut-off titer of TO > 1:80 and/or TH > 1:160 had sensitivity and specificity of 72.7% and 84.2%, while the respective figures were 50% and 89.5% when the cut-off titer was TO and/or TH > 1:160. CONCLUSION: The sensitivity, specificity, positive and negative predictive values, likelihood ratios were same for PCR based detection of S. typhi in blood and urine samples. Nested PCR had higher efficacy in detecting typhoid fever than Widal test, blood and urine cultures. A cut off titer of TO > 1:80 and/or TH > 1:160 was found to have better diagnostic value in this region.

Salmonella enterica serovar typhi: molecular analysis of strains with decreased susceptibility and resistant to ciprofloxacin in india from 2001-2003

Chromosomally-mediated reduced susceptibility to ciprofloxacin narrows the therapeutic options in enteric fever. We made a molecular comparison of clinical isolates of fluoroquinolone-resistant strains of Salmonella enterica serotype Typhi from January 2001 to May 2003; 178 isolates were subjected to antimicrobial susceptibility testing by the Kirby-Bauer method of disk diffusion, and agar dilution was used to determine the minimum inhibitory concentration (MIC) to ciprofloxacin. Nalidixic-acid resistant strains (NARST) were observed in 51 percent of the isolates, of which 98.9 percent had decreased susceptibility (MIC>0.125-1mug/mL) to ciprofloxacin. A single strain (4 mug/mL) was resistant to ciprofloxacin and double mutations were found in the gyrA gene (76 Asp->Asn, 44 leu->Ileu). Among seven NARST strains with reduced susceptibility, a single mutation was found in five strains, one of which had 76 Asp->Asn and two each had mutations at 87 Asp->Asn and 72 Phe->Tyr, respectively); no mutations could be detected in two isolates. Routine antimicrobial surveillance, coupled with molecular analysis of fluoroquinolone resistance, is crucial for revision of enteric fever therapeutics.

Identification and sequencing of Salmonella enterica serotype typhi isolates obtained from patients with perforation and non-perforation typhoid fever.

We describe the characterization of Salmonella enterica serovar Typhi, isolated from the blood of patients with perforation and non-perforation typhoid fever, by a combination of conventional microbiological tests, 16S rRNA gene sequencing, and flagellin gene and CDP-tyvelose epimerase (rfbE) gene sequencing. The 16S rRNA gene sequencing showed that there were four base mutations from perforation samples and only three from non-perforation samples. These findings indicated that the isolates were a strain of Salmonella enterica. The flagellin gene sequences from the two groups were 100% identical to that of the H1-d flagellin gene of serovar Typhi. Sequences of the rfbE from both groups were also 100% identical.

A prospective study of phage types & biotypes of Salmonella enterica serotype Typhi isolated from hospitalized children in Kolkata, India.

BACKGROUND & OBJECTIVES: Kolkata and its suburbs in eastern India faced an epidemic of typhoid fever in 1990. A prospective, hospital and laboratory based study over a period of 12 yr (1990-2001), on the phage typing and biotyping pattern of Salmonella enterica serotype Typhi was carried out, to see if there has been a change. METHODS: A total of 338 S. enterica serotype Typhi isolates from 1491 blood samples were phage typed and biotyped. The mean age of isolation was calculated. RESULTS: The age distribution of subjects (neonates to 12 yr) has been analysed. Of the 338 (22.7%) isolates obtained, eight different S. enterica serotype Typhi phage types were detected. Biotype I (95.8%) was more prevalent as compared to biotype II (4.1%). Phage type E1 was the commonest phage type in Kolkata and its suburbs. INTERPRETATION & CONCLUSION: The mean age at isolation was found to be 6.7 +/- 3.3 yr. Biotype I was predominant and it was of interest that all strains of phage type E1 belonged to biotype I.

Characterization of a phase 1-d epitope on Salmonella typhi flagellin and its role in the serodiagnosis of typhoid fever.

A monoclonal antibody (MAb) directed against Salmonella typhi 52 kDa flagellin protein has been previously produced by our group. In this study, we have demonstrated that the epitope specific to the MAb is unique to phase 1-d. To map the epitope, plasmids encoding different regions of S. typhi flagellin gene were constructed. Analysis of protein produced from each recombinant plasmid indicated that the epitope specific to the MAb resided within amino acids 171-303 (region IV) of S. typhi flagellin protein. The recombinant region IV flagellin was used to develop an ELISA for the detection of IgM antibody to S. typhi in serum. In the hemoculture-positive typhoid group, the developed ELISA was positive in 77 of 92 cases. In patients with non-typhoidal Salmonella, gram-positive and gram-negative bacteria or dengue virus, the ELISA was negative in all 78 cases. Two from 116 healthy control subjects had positive reactions with the assay. The calculated sensitivity, specificity, positive and negative predictive values of the test were 83.7%, 99.0%, 97.5% and 92.8%, respectively. With such high validity together with the requirement of only a single serum specimen and one day for performing the test, the developed ELISA should become a valuable diagnostic test for typhoid fever.

Pulsed-field gel electrophoresis as an epidemiologic tool in the investigation of laboratory acquired Salmonella typhi infection.

Strains of Salmonella typhi implicated in two separate cases of laboratory acquired infection from patients and the medical laboratory technologists who processed the patients' samples were analysed by pulsed-field gel electrophoresis. Although all four isolates were of bacteriophage type E1, PFGE was able to demonstrate that the strains responsible for the two laboratory acquired cases were not genetically related. The PFGE patterns of the isolates from the MLTs were found to be identical to those of the corresponding patients after digestion with restriction enzyme AvrII. This provided genetic as well as epidemiological evidence for the source of the laboratory acquired infections.

Mini-Mu technology in Salmonella typhi: isolation of stable MudJ operon fusions by cis complementation

This paper describes a method to achieve stable MudJ insertions in the Salmonella typhi Ty2 chromosome. The method is a modification of the genetic complementation system described previously for Salmonella typhimurium, which consists in placing the defective transposon (MudJ) near the transposase genes of a helper Mu phage on a single DNA fragment. This fragment is then introduced into a new bacterial host by means of P22 transduction. We constructed a S. typhi strain which carries MudJ and the Mu helper phage in the chromosome. This strain was induced to lytic growth and the lysate was used to infect S. typhi Ty2. The frequency of mutation was 2.0 x 10(-6) mutants per recipient bacterium. Superinfection with the Mu helper phage was about 1 percent. To determine the number of MudJ insertions, several mutants were subjected to Southern blot analysis. From a total of 25 mutants analyzed, only 4 contained more than one insertion. Our procedure compares well with the method described previously for the S. typhimurium-P22 system and can be applied to other Mu sensitive bacteria

Molecular cloning and expression of Salmonella typhi flagellin: characterization of 52 kDa specific antigen of S. typhi.

We previously reported monoclonal antibodies (MAbs) specific to S. typhi 52 kDa antigen which do not cross react with related protein antigens from 11 bacteria causing enteric fever and enteric fever-like illness. Using the combination of these specific MAbs and recombinant DNA technology, expression plasmids containing the antigen gene producing substantial amount of the S. typhi protein antigen have been established. Plasmid pSKM-T7 containing the specific 52 kDa antigen gene was cloned and the antigen expressed was detectable by immunoblotting using specific mAbs. The complete nucleotide sequence of this gene was compared with other bacterial sequences and found to be highly homologous with the flagellin gene H1-d of S. muenchen except in the hypervariable region in the central portion. The specific 52 kDa antigen of S. typhi detected by our MAbs is thus a flagellin.