In Vitro Synergism of Ciprofloxacin and Cefotaxime against Nalidixic Acid-Resistant Salmonella enterica Serotypes Paratyphi A and Paratyphi B

Paratyphoid fever is considered an emerging systemic intracellular infection caused by Salmonella enterica serotypes Paratyphi A, B, and C. We performed in vitro time-kill studies on three clinical isolates of nalidixic acid-resistant Salmonella serotype Paratyphi (NARSP) with different concentrations of ciprofloxacin and cefotaxime to identify combinations of antibiotics with synergistic activity against paratyphoid fever. Furthermore, we identify the frequency of mutations to ciprofloxacin, cefotaxime, and rifampin resistance and also sequenced the gyrA, gyrB, parC, and parE genes to identify the cause of resistance in NARSP. When the activity of ciprofloxacin at 0.75× MIC (0.012 to 0.38 μg/ml) with cefotaxime at the MIC (0.125 to 0.25 μg/ml) against all three NARSP isolates was investigated, synergy was observed at 24 h, and the bacterial counts were reduced by >3 log₁₀ CFU/ml. This synergy was elongated for up to 72 h in two out of three isolates. When ciprofloxacin at 0.75× MIC (0.012 to 0.38 μg/ml) was combined with cefotaxime at 2× MIC (0.25 to 0.50 μg/ml), synergy was prolonged for up to 72 h in all three isolates. Both Salmonella serotype Paratyphi A isolates carried single mutations in codon 83 of the gyrA gene and codon 84 of the parC gene that were responsible for their reduced susceptibility to ciprofloxacin, while no mutations were found in the gyrB or parE gene. The ciprofloxacin-plus-cefotaxime regimen was very effective in reducing the bacterial counts at 24 h for all three isolates, and this combination therapy may be helpful in reducing the chance of the emergence of fluoroquinolone-resistant mutants in patients with severe paratyphoid fever.Enteric fever (typhoid fever and paratyphoid fever) is a systemic intracellular infection caused by Salmonella enterica serotype Typhi or Salmonella enterica serotype Paratyphi A, B, and C. Paratyphoid fever caused by S. Paratyphi is considered an emerging disease, as its incidence has increased alarmingly in recent years (29), causing more asymptomatic infections than S. Typhi. Now enteric fever is a major public health problem in many countries. It is endemic in the Indian subcontinent, Southeast Asia, and Africa, causing at least 22 million cases of illness each year and 200,000 deaths (21), and more than 5 million of these cases were due to S. Paratyphi A (19). According to the findings of a 12-month survey by Ochiai et al. (20), 14% of the cases of enteric fever in Indonesia were caused by S. Paratyphi; these rates were 15% in Pakistan, 24% in India, and 64% in China. Shirakawa et al. (28) reported that in Kathmandu, Nepal, enteric fever caused by S. Paratyphi A is more prevalent than that caused by S. Typhi. The outbreak of enteric fever due to S. Paratyphi in Guangxi Province in southeast China and the increasing rate of isolation of serotype Paratyphi in other areas may signal that S. Paratyphi A is an emerging pathogen in Asia (20).Since 1997, cases of infection with nalidixic acid-resistant Salmonella serotype Typhi (NARST) with decreased susceptibility to ciprofloxacin have been reported in many parts of the world and are now common in the Indian subcontinent and Southeast Asia (23). In Europe and Wales, high-level fluoroquinolone resistance in salmonellae is associated with foreign travel, especially to Southeast and South Asia (11). Some patients with typhoid fever caused by NARST that is susceptible to fluoroquinolones in vitro, according to current Clinical and Laboratory Standards Institute (CLSI) interpretive criteria, can show a delayed response to ciprofloxacin or treatment failure or can even be refractory to treatment both clinically and bacteriologically (26). The failure of treatment with fluoroquinolones in the cases of S. Typhi and S. Paratyphi A in the Indian subcontinent and Southeast and Central Asia due to decreased susceptibility to ciprofloxacin is now a serious concern (30). Fluoroquinolone-resistant S. Paratyphi A has already been reported in Japan (1), as has the relapse of an S. Paratyphi B infection in a child after treatment with ciprofloxacin (7). Fluoroquinolone resistance in most bacterial isolates is due to mutations in the genes encoding DNA gyrase (gyrA, gyrB) or DNA topoisomerase IV (parC, parE) (10). Additionally, increased efflux activity is also a major mechanism of resistance in many Gram-negative bacteria, including Salmonella (9). Currently, however, there is no treatment of choice for patients infected with multidrug-resistant (MDR) and nalidixic acid-resistant Salmonella enterica serotype Paratyphi (NARSP) (24). Indeed, clinicians recommend either the longer-duration use of high-dose fluoroquinolone or the expanded-spectrum drugs cephalosporin or azithromycin, or the combination of these regimens, for the treatment of NARST and NARSP infections. To date, however, no time-kill studies have demonstrated the efficacy of such combination therapy for NARSP infections.We conducted time-kill studies on three clinical isolates, including an isolate from a patient at Chosun University Hospital infected with S. Paratyphi A and one isolate each of S. Paratyphi A and B obtained from the South Korea Center for Disease Control and Prevention (KCDC). In addition, we investigated the association of quinolone resistance with mutations in the genes coding for gyrase A and topoisomerase IV in these isolates.