In 2008, a case of L3/4 vertebral osteomyelitis due to Salmonella Paratyphi A was first reported with bacteriological confirmation in Dubai, United Arab Emirates [12]; however, it was not described if the isolate exhibited resistance to nalidixic acid and macrolides.
In this case, we carried out NGS to elucidate the mechanism underlying antibiotic resistance in Salmonella Paratyphi A due to the emerging development of osteomyelitis during intravenous ceftriaxone treatment. As of late, there have been no studies involving the use of NGS in identifying the mechanism underlying treatment failure for Salmonella Paratyphi A.
A recent study reported that 40% of Salmonella isolates in Chennai, India have an MIC of > 0.5 uL/mL against ceftriaxone [13].
There is a wide variety of serotypes and susceptibility results among Salmonella spp. isolated from clinical specimens in Korea [14]. The three most common Salmonella serotypes are Enteritidis, Typhimurium, and Infantis. These Salmonella strains had resistance rates of 38.7% to ampicillin, 23.0% to chloramphenicol, 8.2% to cefotaxime, 8.6% to ceftriaxone, and 6.3% to trimethoprim-sulfamethoxazole [14]. Another study showed the same result, where the major serotypes isolated in Jeollanam-do, Korea, were Salmonella Enteritidis and Salmonella Typhimurium, where a total of 22 different serotypes were identified, and the major serotypes were Salmonella Enteritidis (116 strains, 42.0%) and Salmonella Typhimurium (60 strains, 21.7%). The highest resistance was observed in response to nalidixic acid (43.4%), followed by ampicillin (40.5%) and tetracycline (31.6%) [15]. Resistance to nalidixic acid was detected in 81.0% of Salmonella Enteritidis isolates. Multidrug resistance was detected in 43.3% of Salmonella spp. Salmonella Enteritidis and Salmonella Typhimurium presented the highest resistance (98.3%) and multidrug resistance (73.3%) rates, respectively [15]. A recent report similar to our case showed that a patient infected with Salmonella Paratyphi A was treated with ceftriaxone, but his symptoms remained; therefore, his treatment was changed to azithromycin [7]. Even though the MIC of azithromycin was not elevated (8 mg/L), azithromycin treatment failed. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) states that the wild-type isolates of Salmonella Typhi have an MIC ≤16 mg/L. In our case, the MIC of the isolate showed that it was azithromycin-resistant (MIC > 16 mg/L) (Table 2).
Based on our NGS results, azithromycin resistance genes such as mph and mef were not found; however, it was suggested that the mechanism of resistance to azithromycin was due to a RND antibiotic efflux pump.
In our previous study, a combination of ciprofloxacin and cefotaxime showed synergistic effects against nalidixic acid-resistant Salmonella Paratyphi A and B. This combination appears to be more effective than monotherapy and may help reduce the chances that fluoroquinolone-resistant mutants will emerge in patients with severe typhoid fever [16, 17]. In this case, we treated with ciprofloxacin and cefotaxime, and the patient’s clinical features including back pain and ESR level decreased.
Olaquindox-resistant isolates were found to contain the gene combination oqxAB, which encodes an RND family efflux pump, confers resistance to olaquindox quinolones and chloramphenicol, and reduces susceptibility to other antibiotics [18]. OqxAB, a plasmid-mediated RND efflux pump conferring resistance to multiple antibiotics, was found in Salmonella isolates recovered from food samples. The overall OqxAB-positive rate of Salmonella typhimurium strains was 29% (159 out of 546 isolates), and the yearly rates were 0, 13, 26, 32, 36, 39, and 42% during the years 2005 to 2011, respectively. OqxAB was also found to be associated with multidrug resistance in S. typhimurium isolates from Hong Kong and from the Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention (ICDC), Chinese Center for Disease Control and Prevention, Beijing, China. Among the S.typhimurium isolates of the OqxAB-positive group, 94% (Hong Kong) and 98% (ICDC) were resistant to ciprofloxacin (MIC = 2 mg/L); the corresponding resistance rate in the OqxAB-negative S. typhimurium isolates from Hong Kong and ICDC was only 11% [19].
Our NGS study showed that expression of multiple RND family efflux pumps such as MdsC, CRP, and SdiA, which may be related to quinolone resistance, may have been responsible for the failure of ceftriaxone treatment (Table 2). The upregulation of endogenous SdeXY-HasF-mediated efflux has been reported to be associated with tigecycline resistance in Serratia marcescens, along with increases in MIC for tetracycline, ciprofloxacin, and cefpirome [20]. The overexpression of the BmeB efflux pump has also been reported to cause low-to-intermediate-level clinically relevant fluoroquinolone resistance and can be coupled with GyrA substitutions to cause high-level fluoroquinolone resistance. Finally, it also contributes to high-level clinically relevant resistance to beta-lactams [21]. Our case also showed that both the amino acid substitution GyrA S83F and the expression of multiple RND family efflux pumps led to high-level resistance to quinolone. No resistance genes in Salmonella Paratyphi A related to ceftriaxone, such as CTX-M, CMY-2, or other extended-spectrum beta-lactamases were identified using NGS. However, there was no response or even progression to vertebral osteomyelitis to treatment with third-generation cephalosporins after 18 days of the initial treatment. The GyrA S83F substitution and the expression of multiple RND family efflux pumps may have contributed to the failure of treatment with ceftriaxone, even though the MIC of the isolate to ceftriaxone was less than 1. In our case, there was a possibility that the early onset of metastatic spondylitis accounted for the failure of treatment with a third-generation cephalosporin because the treatment duration was not long enough. However, further studies on RND antibiotic efflux pumps are necessary to truly identify it as the cause of third-generation cephalosporin treatment failure.
In 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.