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.

Revised ciprofloxacin breakpoints for Salmonella Typhi: Its implications in India.

The rise of multidrug resistant strains of Salmonella Typhi in the last decade of the previous century led to the use of fl uoroquinolones as the drug of choice. However, over the past few years fl uoroquinolone resistance has been increasingly reported. In accordance with the revised Clinical and Laboratory Standards Institute (CLSI) breakpoints, only 3% of the isolates were susceptible to ciprofl oxacin in comparison to 95% as per the earlier guidelines when 488 isolates collected between 2010 and 2012 were re-interpreted. Interestingly, re-emergence of strains susceptible to chloramphenicol, ampicillin and cotrimoxazole is being seen. Amidst the changing susceptibility profi le, azithromycin remains a promising alternative.

Study of the role of efflux pump in ciprofloxacin resistance in Salmonella enterica serotype Typhi.

Purpose: There are increasing reports on failure of clinical response to ciprofl oxacin in typhoid fever despite the strain being sensitive to drug in in-vitro using standard guidelines and showing mutations in DNA gyrase. But this increased MIC and clinical failures with ciprofl oxacin are not always co-related with mutations presently identifi ed in gyrA and parC genes. This shows that there may be other mechanisms such as an active drug effl ux pump responsible as has been shown in other Enterobacteriaceae. This study was carried out to determine the role of effl ux pump in Salmonella Typhi isolates. Materials and Methods: Total 25 already characterized nalidixic acid sensitive and nalidixic acid resistant S. Typhi strains with different range of ciprofl oxacin MIC were included to study the role of effl ux pump in the presence of CCCP (effl ux pump inhibitor). For genotypic characterization, the entire acrR gene was sequenced to confi rm the presence of any mutation in the gene. Results: The MIC of ciprofl oxacin remained same in the presence and absence of CCCP in the studied strains and no signifi cant mutations were found in the acrR gene in any of the isolates studied. Conclusions: No role of effl ux pump in ciprofl oxacin resistance was found in strains studied. There is a need to explore further mechanism of ciprofl oxacin resistance in Salmonella Typhi.

Enteric Fever in Children at Dhulikhel Hospital.

Introduction: Typhoid fever is one of the most common public health problems in Nepal. It occurs in all parts of the world where water supplies and sanitation are sub-standard. In Dhulikhel hospital, this is one of the top acute febrile illnesses in inpatient department. The objectives of this study were to evaluate the clinical and laboratory parameters including culture and sensitivity, the response to therapy, and complications of enteric fever among child cases at Dhulikhel Hospital. Materials and Methods: This retrospective study was conducted at Dhulikhel Hospital, Kathmandu University Teaching Hospital from January 2009 to June 2011. Statistical analysis was done with SPSS. Results: There were total of 138 cases of enteric fever admitted. There were 73 (53%) male and 65 (47%) female. Eighty-one percent were above five years of age. The most common clinical presentation was fever (100%) followed by headache and G I symptoms. Hepatomegaly was the most common sign seen among the cases and was seen in 110cases (79.71%). Most of the patients had normal WBC count 100 (72.46%) Widal test was positive in 70 (50.72%) cases and blood culture was positive in 52(37.68%) cases. Nalidixic acid was found to be resistant in 26 (50%) cases. Complications were seen in only 7 (5%) enteric fever cases. Conclusion: Typhoid fever is predominant in school going children in Nepal with slight male predominance. Fever lasting over 3 days followed by headache and GI symptoms are the major presenting symptoms. In making the diagnosis, the isolation of bacteria from blood is the “gold standard”. Nalidixic acid resistant Salmonella typhi is on the increasing trend. Pneumonia was found to be the most common complication among all other complications seen in enteric cases. In Dhulikhel Hospital this is one of the top acute febrile illnesses in inpatient department.

Clinical Profile and Sensitivity Pattern of Salmonella Serotypes in Children: A Hospital Based Study.

Introduction: Enteric fever is a systemic infection caused by the bacteria, Salmonella enterica serovar Typhi (S.typhi) and Salmonella enterica serovara Paratyphi (S. paratyphi A, B and C). Most of the burden of the disease is limited to the developing world and the disease still has the issues like wide spectrum of clinical presentation and multidrug resistance. Objectives: This study was done to analyze the clinical profile and antibiotic sensitivity pattern in the cases of culture positive enteric fever. Methods: A prospective cross-sectional study was conducted in Civil Service Hospital from February 2010 to January 2011 in the paediatric population in the age group of 2 to 14 years. Children with Salmonella species isolated in blood culture were included in the study. Results: Out of the 40 children with culture positive enteric fever, male to female ratio was 1.3:1 with common age group between 11-14 years. S typhi was isolated in 25 cases while S. paratyphi in 15 cases. Clinical features of S. typhi and S. paratyphi were indistinguishable. Both S.typhi and S. paratyphi were found to be 100% sensitive to drugs like Ceftriaxone, Cefotaxime, Cefixime and Chloramphenicol. Sensitivity to Ofloxacin was 100% in S. paratyphi and 92% in S.typhi. Similarly sensitivity of Azithromycin was 92% and 93% for S.typhi and S. paratyphi respectively. Conclusion: Salmonella serotype is still 100 % sensitive to third generation cephalosporin. Some percentage of resistance is seen with Ofloxacin in S. typhi and with Azithromycin in both S.typhi and S. paratyphi.

A study of clinical profile of multidrug resistant typhoid fever in children.

Background: Salmonella typhi infection remains a serious problem in developing country. It has been estimated that approximately 12.5 million cases of typhoid fever occurs annually in the developing countries with 7.7 million cases in Asia alone. The disease is predominantly a disease of school age children and young adults and is reported to be a milder in infants and young children. Methods: A total of 150 children with clinical and/or laboratory diagnosis of typhoid fever admitted to the tertiary care level government hospital attached with the Government medical college in Pediatrics ward were reviewed during the months of JUNE to MAY for demographic data such as age, sex, clinical features, result of laboratory tests and antibiotic sensitivity in vivo. Results: There were 88 male and 62 female patients, from the age group ranging from 2 years to 12 years. Predominant symptoms were fever, abdominal pain , vomiting and headache. Hepatomegaly was almost twice as frequent as spleenomegaly. Common clinical signs of typhoid fever in adults such as relative bradycardia and rose spots were seldom documented in children. Fever, Toxic look, coated tongue and hepato-spleenomegaly were common clinical signs of clinical presentation in children. The positivity rate of WIDAL test and Blood Culture was 71.33% and 5.33% respectively. Hepatitis, Bronchitis and Encephalopathy were commonly observed complications of Multidrug resistant typhoid fever in this study. Antibiotics sensitivity in vivo revealed resistance rates of 78.12% for Ampicillin, 84.2% for Trimethoprim-Sulfamethoxazole (Co-trimoxazole), 19.5% for Ciprofloxacin, 14.28% for Ofloxacin and 20% for Cefotaxime. Conclusions : No resistance was detected against Cefixime and Ceftriaxone. Except the two patients died during the period of observation of this clinical study, all paediatrics patients survive from their illness completely.

Novel restriction enzyme SSiI for the detection of mutation in GyrA gene of Salmonella enterica serovar Typhi.

Aim: Enteric fever is an ongoing problem in the developing nations. Resistance and reduced susceptibility to ciprofloxacin narrows the therapeutic options in enteric fever. The present study was carried out with the objective of determining molecular basis of resistance to fluoroquinolone among the clinical isolates of Salmonella enterica serovar Typhi from different parts of India. Materials and Methods: A total of 60 S.Typhi clinical isolates were subjected to antimicrobial susceptibility testing and determination of minimum inhibitory concentration (MIC) to ciprofloxacin and nalidixic acid. Polymerase chain reaction (PCR) for GyrA gene followed by restriction fragment length polymorphism (RFLP) with restriction enzyme (RE) SSiI was performed to detect mutation at position Ser83. Further confirmation of mutation was done by nucleotide sequencing of GyrA gene. Results: Isolates showed 100% sensitivity to first-line drugs ampicillin, chloramphenicol, and cotrimoxazole. Twelve of the 60 isolates (18%) were susceptible to nalidixic acid (NASST) and the remaining 48 (82%) were resistant to nalidixic acid (NARST). Of these 48 NARST strains, 46 (97.5%) had reduced susceptibility to ciprofloxacin (MIC 0.25-1.0 μg/mL), whereas 2 strains (2.75%) were resistant to ciprofloxacin (MIC 4.0 μg/mL). In RFLP analysis, all the NASST strains showed 3 fragments, whereas all the NARST strains showed 2 fragments due to the loss of 1 restriction site as a result of mutation. All the NARST strains with reduced susceptibility to ciprofloxacin (n = 46) had a single mutation in gyrA gene (Ser 83→Tyr or Ser 83→Phe), whereas double mutations (Ser 83→Phe and Asp 87→Asn) were found in each of the 2 ciprofloxacin-resistant strains. None of the NASST strains (n = 12) revealed any mutation. Conclusion: Our study exemplifies the correlation between nalidixic acid screening test, MIC values, and the detection of mutation in GyrA gene by PCR-RFLP with a novel RE SSiI.This was further confirmed by nucleotide sequencing.

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.

Analysis of pulsotypes of salmonella typhi isolates and their clinical profiles in typhoid fever patients.

A study of genotyping (pulsotyping) of Salmonella typhi (S. typhi) isolates using pulse-field gel electrophoresis (PFGE) methods was performed to examine their genetic diversity, and relationship between genetic characteristics and clinical outcomes. Sixty-six S. typhi isolates obtained from sporadic hospitalized typhoid fever cases were used in this study. Four isolates were found identical and the dendogram constructed showed 33 pulsotypes in which 13 of them can be divided into 30 subtypes. Diversity among them were high as shown by the Dice coefficients that ranged from 0.486 to 1.000. Cluster analysis showed 2 main clusters with 65% degree of similarity, suggested that they were not originated from one clone. Further, at 90% degree of similarity, 9 clusters containing at least 3 isolates were determined to explore any possible existence of relationship between genetic profile and particular clinical outcomes. Clinical manifestations ranged from mild to severe were in fact distributed diversely among these clusters. Although the clinical data obtained were incomplete, 2 out of 4 patients infected by the S. typhi belonged to cluster 1 showed an elevation of total bilirubin, whereas it was not found in 19 other patients distributed in other 8 clusters. Even though specific clinical manifestations were apparently not found to relate with particular clusters of genotypes, S. typhi isolates grouped in cluster 1 seemed to show trophism to hepatobiliary system.

Minimal inhibitory concentration (M.I.C) of antibiotics to S.typhi strains isolated at Daklak from 1996-1998

For the first time in Daklak, S. typhi strains were isolated from cases of suspected typhoid fever. MIC was investigated on 50 strains of S. typhi in Daklak from 1996-1998. The results were as following: All these isolated S.typhi strains were fully sensitive to common antibiotics (1g/ml for Tetracycline, Trimethoprim/sulfamethoxazole and 4 g/ml for Ampicillin, Nalidixic acid and the third generation cephalosporin (Ceftriaxone M.I.C=0.125 g/ml) and the fluoquinolones (Ofloxacin M.I.C =0.25 g/ml), especially to 16% of Chloramphenicol-resistant isolates (with M.I.C = 64 g/ml). This is a great difference to compare with S.typhi strains isolated in Northern and Southern in Vietnam. Concerning the plasmid profile analysis, from 50 strains of S.typhi, the results showed that: All these strains did not harbour any R-plasmid. 43 strains harboured a plasmid lower than 70 Md. 7 strains did not harbour any kind of plasmids.

Evaluation of thiol broth for the culture of Salmonella typhi and other bacteria from blood

Thiol broth is known to neutralize various antimicrobial agents. Positivity of growth of various species of bacteria from blood in thiol broth was reported as similar to that in tryptic soy broth (TSB). As blood cultures are often used for the diagnosis of typhoid fever, and as patients may receive antimicrobial therapy before blood culture, the positivity and rapidity of growth of Salmonella typhi in thiol broth were compared to those in TSB. Routine blood culture samples from Yonsei Medical Center patients were inoculated in 50-ml amounts of TSB and thiol broth. The media were prepared from dehydrated products and did not contain CO2, but TSB contained 0.025% sodium polyanethol sulfonate (SPS). Growth of S. paratyphi-A, Klebsiella pneumoniae, Enterobacter sp., Serratia marcescens and alpha-hemolytic Streptococcus were similar in both media. However, greater positivity and shorter incubation time for macroscopic detection were noted in TSB with S. typhi, Escherichia coli and Staphylococcus aureus. It is concluded that thiol broth is inferior to TSB plus SPS for the culture of S. typhi from blood.

STUDIES ON THE CHANGES OF ANTIBIOTIC RESISTANCE OF SALMONELLA AND THEIR THERAPEUTIC IMPLICATIONS IN CHONGQING DISTRICT / 重庆医科大学学报

This paper reports the changes in susceptibility of Salmonella spp. to 32 antimicrobial agents from 1959 to 1988. The clinical observation on the treatment of typhoid fever caused by drug-sensitive and resistant strains of S. typhi arc also reported. Resistant Salmonella began to appear from the year of 1986. The in vitro studies indicated that drug-resistant S. typhi were resistant to commonly anti-typhoid agents such as chloramphenicol, SMZ/TMP, ampicillin and other drugs, but were very sensitive to new quinolones and the third generation cephalosporins. The .therapeutic effects correlate well with the in vitro sensitivity tests. Typhoids caused by drug-sensitive strains responded well when treated with chloramphenicol, SMZ/TMP or ampicillin, while those caused by resistant strains only responded well to norfloxacin or its combi-nation with other drugs