Identifying the mechanism underlying treatment failure for Salmonella Paratyphi A infection using next-generation sequencing - a case report.

BACKGROUND: Salmonella is a notorious pathogen that causes gastroenteritis in humans and the emergence of resistance to third-generation cephalosporins and azithromycin have raised concern. There has been rare case of Salmonella Paratyphi A infection accompanied by spondylitis. Here, we report a case of initial antibiotic treatment failure in a Korean man with Salmonella Paratyphi A infection and conducted next-generation sequencing (NGS) to determine the cause of failure of initial treatment for Salmonella Paratyphi A infection. CASE PRESENTATION: A 70-year-old man was admitted to Chosun University Hospital with reported consistent low back pain with a history of having 5 days of chills and fever in another hospital a month ago. He was administered ceftriaxone (2 g daily) for 18 days including initial treatment to cover Salmonella enterica. The antimicrobial susceptibility test using MIC plate, found that the identified organism was resistant to ciprofloxacin and nalidixic acid. Moreover, the Salmonella Paratyphi A isolates were found to have an MIC > 16 mg/L for azithromycin, as he had resistance to both azithromycin and nalidixic acid, the treatment was switched to a combination of ciprofloxacin and cefotaxime. We carried out next-generation sequencing (NGS) to determine the cause of failure of initial treatment for Salmonella Paratyphi A infection. NGS showed that the amino acid substitution GyrA S83F and the expression of multiple RNA-family efflux pumps led to a high-level resistance to quinolone. No genes related to ceftriaxone resistance, such as CTX-M, CMY-2, or other extended-spectrum beta-lactamases were identified in Salmonella enterica Paratyphi A using NGS. The GyrA S83F mutation and the expression of multiple RNA-family efflux pumps may have contributed to the treatment failure of ceftriaxone, even though the MIC of the isolate to ceftriaxone was less than 1. CONCLUSION: This case involved a Salmonella Paratyphi A infection accompanied by spondylitis. To our knowledge, this is the first report to elucidate the mechanism underlying antimicrobial resistance using NGS.

Treatment Response in Enteric Fever in an Era of Increasing Antimicrobial Resistance: An Individual Patient Data Analysis of 2092 Participants Enrolled into 4 Randomized, Controlled Trials in Nepal.

BACKGROUND.: Enteric fever, caused by Salmonella Typhi and Salmonella Paratyphi A, is the leading cause of bacterial febrile disease in South Asia. METHODS.: Individual data from 2092 patients with enteric fever randomized into 4 trials in Kathmandu, Nepal, were pooled. All trials compared gatifloxacin with 1 of the following comparator drugs: cefixime, chloramphenicol, ofloxacin, or ceftriaxone. Treatment outcomes were evaluated according to antimicrobial if S. Typhi/Paratyphi were isolated from blood. We additionally investigated the impact of changing bacterial antimicrobial susceptibility on outcome. RESULTS.: Overall, 855 (41%) patients had either S. Typhi (n = 581, 28%) or S. Paratyphi A (n = 274, 13%) cultured from blood. There were 139 (6.6%) treatment failures with 1 death. Except for the last trial with ceftriaxone, the fluoroquinolone gatifloxacin was associated with equivalent or better fever clearance times and lower treatment failure rates in comparison to all other antimicrobials. However, we additionally found that the minimum inhibitory concentrations (MICs) against fluoroquinolones have risen significantly since 2005 and were associated with increasing fever clearance times. Notably, all organisms were susceptible to ceftriaxone throughout the study period (2005-2014), and the MICs against azithromycin declined, confirming the utility of these alternative drugs for enteric fever treatment. CONCLUSION.: The World Health Organization and local government health ministries in South Asia still recommend fluoroquinolones for enteric fever. This policy should change based on the evidence provided here. Rapid diagnostics are urgently required given the large numbers of suspected enteric fever patients with a negative culture.

Decreased ciprofloxacin susceptibility in Salmonella Typhi and Paratyphi infections in ill-returned travellers: the impact on clinical outcome and future treatment options.

The emergence of decreased ciprofloxacin susceptibility (DCS) in Salmonella enterica serovar Typhi and serovar Paratyphi A, B or C limits treatment options. We studied the impact of DCS isolates on the fate of travellers returning with enteric fever and possible alternative treatment options. We evaluated the clinical features, susceptibility data and efficacy of empirical treatment in patients with positive blood cultures of a DCS isolate compared to patients infected with a ciprofloxacin-susceptible (CS) isolate in the period from January 2002 to August 2008. In addition, the pharmacokinetic and pharmacodynamic parameters of ciprofloxacin, levofloxacin and gatifloxacin were determined to assess if increasing the dose would result in adequate unbound fraction of the drug 24-h area under the concentration-time curve/minimum inhibitory concentration (ƒAUC(0-24)/MIC) ratio. Patients with DCS more often returned from the Indian subcontinent and had a longer fever clearance time and length of hospital stay compared to patients in whom the initial empirical therapy was adequate. The mean ƒAUC(0-24)/MIC was 41.3 ± 18.8 in the patients with DCS and 585.4 ± 219 in patients with a CS isolate. For DCS isolates, the mean ƒAUC0-24/MIC for levofloxacin was 60.5 ± 28.7 and for gatifloxacin, it was 97.9 ± 28.0. Increasing the dose to an adequate ƒAUC(0-24)/MIC ratio will lead to conceivably toxic drug levels in 50% of the patients treated with ciprofloxacin. Emerging DCS isolates has led to the failure of empirical treatment in ill-returned travellers. We demonstrated that, in some cases, an adequate ƒAUC(0-24)/MIC ratio could be achieved by increasing the dose of ciprofloxacin or by the use of alternative fluoroquinolones.

In vitro susceptibility of typhoidal Salmonellae against newer antimicrobial agents: a search for alternate treatment options.

OBJECTIVES: To determine the minimum inhibitory concentrations (MICs) of ceftriaxone, azithromycin, pefloxacin, cefipime and imipenem for Salmonella Typhi (S. Typhi) and Paratyphi. METHODS: One hundred and fifty four isolates of Salmonella Typhi and S. Paratyphi A, B and C growing in blood culture were selected. MICs of ceftriaxone, azithromycin, pefloxacin, cefipime and imipenem were performed by agar dilution method as recommended by clinical laboratory standard institutes. RESULTS: MIC90 of azithromycin and pefloxacin was 8 microg/ml, cefipime was 0.06 microg/ml and imipenem was 0.5 microg/ml. None of the strains were found to be resistant to ceftriaxone but 3 isolates showed higher MIC value of 2 microg/ml. CONCLUSION: Azithromycin appears a suitable alternate for the treatment of typhoid in the community. Imipenem and cefipime are good options in complicated cases to be treated in hospital settings. Pefloxacin cannot be used as MICs are higher. Presence of isolates with higher MIC of ceftriaxone is serious and stresses upon continuous laboratory surveillance to guide clinicians appropriately.

Analysis of Salmonella enterica serotype paratyphi A gene expression in the blood of bacteremic patients in Bangladesh.

BACKGROUND: Salmonella enterica serotype Paratyphi A is a human-restricted cause of paratyphoid fever, accounting for up to a fifth of all cases of enteric fever in Asia. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we applied an RNA analysis method, Selective Capture of Transcribed Sequences (SCOTS), and cDNA hybridization-microarray technology to identify S. Paratyphi A transcripts expressed by bacteria in the blood of three patients in Bangladesh. In total, we detected 1,798 S. Paratyphi A mRNAs expressed in the blood of infected humans (43.9% of the ORFeome). Of these, we identified 868 in at least two patients, and 315 in all three patients. S. Paratyphi A transcripts identified in at least two patients encode proteins involved in energy metabolism, nutrient and iron acquisition, vitamin biosynthesis, stress responses, oxidative stress resistance, and pathogenesis. A number of detected transcripts are expressed from PhoP and SlyA-regulated genes associated with intra-macrophage survival, genes contained within Salmonella Pathogenicity Islands (SPIs) 1-4, 6, 10, 13, and 16, as well as RpoS-regulated genes. The largest category of identified transcripts is that of encoding proteins with unknown function. When comparing levels of bacterial mRNA using in vivo samples collected from infected patients to samples from in vitro grown organisms, we found significant differences for 347, 391, and 456 S. Paratyphi A transcripts in each of three individual patients (approximately 9.7% of the ORFeome). Of these, expression of 194 transcripts (4.7% of ORFs) was concordant in two or more patients, and 41 in all patients. Genes encoding these transcripts are contained within SPI-1, 3, 6 and 10, PhoP-regulated genes, involved in energy metabolism, nutrient acquisition, drug resistance, or uncharacterized genes. Using quantitative RT-PCR, we confirmed increased gene expression in vivo for a subset of these genes. CONCLUSION/SIGNIFICANCE: To our knowledge, we describe the first microarray-based transcriptional analysis of a pathogen in the blood of naturally infected humans.

Ciprofloxacin treatment failure in a case of typhoid fever caused by Salmonella enterica serotype Paratyphi A with reduced susceptibility to ciprofloxacin.

This report describes a case of ciprofloxacin treatment failure in a patient with enteric fever caused by Salmonella enterica serotype Paratyphi A. The organism was isolated from a blood culture from a patient who was treated with oral ciprofloxacin (500 mg every 12 h) for 13 days. The organism showed reduced susceptibility to ciprofloxacin (MIC 0.75 microg ml-1) and was resistant to nalidixic acid. The patient was then placed on intravenous ceftriaxone (1 g every 12 h) and responded within 3 days. The patient was discharged after 9 days on ceftriaxone with no relapse on follow-up. This case adds to the increasing incidence of treatment failures with ciprofloxacin in typhoid fever caused by typhoid salmonellae with reduced susceptibility to ciprofloxacin. It also highlights the inadequacy of current laboratory methods for fluoroquinolone susceptibility testing in adequately predicting in vivo activity of ciprofloxacin against typhoid salmonellae and supports calls for new guidelines for fluoroquinolone susceptibility testing of these organisms.

Suboptimal clinical response to ciprofloxacin in patients with enteric fever due to Salmonella spp. with reduced fluoroquinolone susceptibility: a case series.

BACKGROUND: Salmonella spp. with reduced susceptibility to fluoroquinolones have higher than usual MICs to these agents but are still considered "susceptible" by NCCLS criteria. Delayed treatment response to fluoroquinolones has been noted, especially in cases of enteric fever due to such strains. We reviewed the ciprofloxacin susceptibility and clinical outcome of our recent enteric fever cases. METHODS: Salmonella enterica Serotype Typhi (S. Typhi) and Serotype Paratyphi (S. Paratyphi) blood culture isolates (1998-2002) were tested against nalidixic acid by disk diffusion (DD) and agar dilution (AD) and to ciprofloxacin by AD using NCCLS methods and interpretive criteria. Reduced fluoroquinolone susceptibility was defined as a ciprofloxacin MIC of 0.125-1.0 mg/L. The clinical records of patients treated with ciprofloxacin for isolates with reduced fluoroquinolone susceptibility were reviewed. RESULTS: Seven of 21 (33%) S. Typhi and S. Paratyphi isolates had reduced susceptibility to fluoroquinolones (MIC range 0.125-0.5 mg/L). All 7 were nalidixic acid resistant by DD (no zone) and by AD (MIC 128- >512 mg/L). The other 14 isolates were nalidixic acid susceptible and fully susceptible to ciprofloxacin (MIC range 0.015-0.03 mg/L). Five of the 7 cases were treated initially with oral ciprofloxacin. One patient remained febrile on IV ciprofloxacin until cefotaxime was added, with fever recurrence when cefotaxime was discontinued. Two continued on oral or IV ciprofloxacin alone but had prolonged fevers of 9-10 days duration, one was switched to IV beta-lactam therapy after remaining febrile for 3 days on oral/IV ciprofloxacin and one was treated successfully with oral ciprofloxacin. Four of the 5 required hospitalization. CONCLUSIONS: Our cases provide further evidence that reduced fluoroquinolone susceptibility of S. Typhi and S. Paratyphi is clinically significant. Laboratories should test extra-intestinal Salmonella spp. for reduced fluoroquinolone susceptibility.

Treatment with ciprofloxacin in children with typhoid fever.

We investigated the use, treatment results and safety of ciprofloxacin in the treatment of childhood typhoid and paratyphoid infections in an industrialized country. The study was retrospective, and the material consisted of children admitted to Hvidovre or Glostrup University Hospitals from 1991 to 1995, and treated with ciprofloxacin for a culture proven diagnosis of typhoid fever. 21 children were included, 18 had positive cultures for Salmonella typhi and 3 had positive cultures for S. paratyphi A. All isolates were fully susceptible to ciprofloxacin. The median duration of treatment was 10 d, median oral dose was 24 mg/kg/d and the median intravenous dose was 15 mg/kg/d. Within 4 d after start of treatment all subjects had a normal body temperature. No subjects had clinical or microbiological relapse and all stool cultures after end of treatment were negative. Adverse events were rare, but in 2 children a transient limb/ataxia or a period of confusion were recorded. Both children recovered within a few days without sequelae. We conclude that ciprofloxacin was effective and well tolerated for treatment of typhoid fever in children. The few adverse events that were recorded left no permanent sequelae, and were likely to be caused by the disease itself.

Efficacy of ciprofloxacin in enteric fever: comparison of treatment duration in sensitive and multidrug-resistant Salmonella.

The efficacy of two regimens of ciprofloxacin was compared in a randomized study conducted on 69 patients with enteric fever, 52.2% of whom had infection with multidrug-resistant (MDR) strains of Salmonella typhi or S. paratyphi. Patients were randomly assigned to two regimens (10 days versus 14 days) of ciprofloxacin (500 mg twice a day). The mean +/- SD time required for defervescence was similar for both regimens (4.2 +/- 1.9 days in the 10-day group and 4.9 +/- 2.6 days in the 14-day group). A 100% cure was observed in each treatment group and no serious side effects were observed. Relapse occurred in two patients (14-day regimen). Only one patient (14-day regimen) had growth of S. typhi in stool culture at the time of the first follow-up three days after completion of therapy. Follow-up studies on available patients on two, six, and 12 months after completion of therapy revealed that all patients had negative stool cultures for S. typhi and S. paratyphi. This study indicates that ciprofloxacin may be recommended as an initial therapy for enteric fever for adult men and nonpregnant and nonlactating women in areas where MDR strains of S. typhi and S. paratyphi are prevalent, and that 500 mg twice a day of the drug given for 10 days is as effective as 14 days at the same dosage.

Eradication of a multiple drug resistant Salmonella paratyphi A causing meningitis with ciprofloxacin.

A previously healthy breast-fed baby was admitted at 10 days of age to a hospital in the north of Pakistan with diarrhoea and fever. He was treated for suspected sepsis with intravenous cefotaxime and tobramycin. Cultures of blood and faeces at that time proved negative. At 12 days of age, seizures began and examination of CSF revealed evidence of pyogenic meningitis but bacteria were neither seen microscopically nor isolated in culture. Ceftazidime was substituted for cefotaxime and carbenicillin was given also. Since the baby's condition continued to deteriorate with persistent fever, vomiting and recurrent seizures, he was transferred to the Aga Khan University Hospital, Karachi. Examination of CSF there confirmed the diagnosis of pyogenic meningitis and revealed Gram-negative bacteria. Cultures of CSF and faeces yielded Salmonella paratyphi A but the blood culture was negative. The isolate was found to be multiple antimicrobially resistant but sensitive to ciprofloxacin. Treatment with this drug was therefore started 3 days after the baby's admission to the Aga Khan Hospital. Within 36 h, improvement was observed. From then onwards, the baby made a progressive recovery and was healthy when seen at 7 months of age.

Failure of ciprofloxacin to eradicate convalescent fecal excretion after acute salmonellosis: experience during an outbreak in health care workers.

OBJECTIVE: To determine the efficacy of ciprofloxacin therapy in eradicating convalescent fecal excretion of salmonellae after acute salmonellosis. DESIGN: Randomized, placebo-controlled, double-blind trial of ciprofloxacin, with prospective follow-up of nonparticipants. SETTING: An acute care community hospital experiencing an outbreak of salmonellosis. PATIENTS: Twenty-eight health care workers developed acute infection with Salmonella java; 15 participated in a placebo-controlled trial of ciprofloxacin, beginning on day 9 after infection. INTERVENTIONS: Eight patients were randomly assigned to receive ciprofloxacin, 750 mg, and 7 patients to receive placebo; both were administered orally twice daily for 14 days. Nonparticipants who received therapy were placed on the same ciprofloxacin regimen. MEASUREMENTS AND MAIN RESULTS: Study participants had follow-up stool cultures every 3 days initially and then weekly for 3 weeks; nonparticipants were followed until three consecutive cultures were negative. All eight ciprofloxacin recipients showed eradication of S. java from stool cultures within 7 days of beginning therapy (compared with 1 of 7 placebo recipients), and their stool cultures remained negative up to 14 days after discontinuing therapy (P less than 0.01). However, 4 of 8 relapsed; their stool cultures became positive between 14 and 21 days after therapy. In addition, 3 of 3 hospitalized patients treated with ciprofloxacin who did not participate in the controlled trial also relapsed. Thus, the total relapse rate was 7 of 11 (64%; 95% CI, 31% to 89%). In 4 of these 7 patients, relapse was associated with a longer duration of fecal excretion of salmonellae than that of the placebo group. Relapse could not be explained on the basis of noncompliance, development of resistance, or presence of biliary disease. CONCLUSIONS: Despite its excellent antimicrobial activity against salmonellae and its favorable pharmacokinetic profile, ciprofloxacin at a dosage of 750 mg orally twice daily had an unacceptably high failure rate in patients with acute salmonellosis and may have prolonged fecal excretion of salmonellae. The late occurrence of relapses indicates the need to obtain stool cultures up to 21 days after therapy to document fecal eradication in acute salmonellosis.

Open study of ciprofloxacin in enteric fever.

Thirty adult patients with typhoid or paratyphoid fever were treated with ciprofloxacin. All patients were cured with eradication of the causative organism. No major adverse reactions were seen. Ciprofloxacin is an effective agent for the treatment of enteric fever.