Antibiotic susceptibilities of Salmonella enterica serovar Typhi and S. enterica serovar Paratyphi A isolated from patients in Japan

The antibiotic susceptibilities of 62 strains of Salmonella enterica serovar Typhi and 37 strains of S. enterica serovar Paratyphi A were investigated with 18 antibiotics. Eighteen S. enterica serovar Typhi isolates and five S. enterica serovar Paratyphi A isolates were resistant to one or more antimicrobial agents, among which 10 S. enterica serovar Typhi isolates were nalidixic acid resistant and also showed decreased ciprofloxacin susceptibility.     

DNA sequence analysis of DNA gyrase and DNA topoisomerase IV quinolone resistance-determining regions of Salmonella enterica serovar Typhi and serovar Paratyphi A

The mutations that are responsible for fluoroquinolone resistance in the gyrA, gyrB, parC, and parE genes of Salmonella enterica serovar Typhi and serovar Paratyphi A were investigated. The sequences of the quinolone resistance-determining region of the gyrA gene in clinical isolates which showed decreased susceptibilities to fluoroquinolones had a single mutation at either the Ser-83 or the Asp-87 codon, and no mutations were found in the gyrB, parC, and parE genes.     

High-resolution melting assay for the detection of gyrA mutations causing quinolone resistance in Salmonella enterica serovars Typhi and Paratyphi

We developed a novel rapid assay to detect the gyrA mutations that cause quinolone resistance in typhoid and paratyphoid fever Salmonella spp. using high-resolution melting (Idaho Technology, Salt Lake City, UT) analysis of polymerase chain reaction amplicons. The presence of gyrA mutations led to small but consistent changes in amplicon melting temperatures that allowed quinolone-resistant isolates to be differentiated from susceptible ones.     

Clinically and Microbiologically Derived Azithromycin Susceptibility Breakpoints for Salmonella enterica Serovars Typhi and Paratyphi A

Azithromycin is an effective treatment for uncomplicated infections with Salmonella enterica serovar Typhi and serovar Paratyphi A (enteric fever), but there are no clinically validated MIC and disk zone size interpretative guidelines. We studied individual patient data from three randomized controlled trials (RCTs) of antimicrobial treatment in enteric fever in Vietnam, with azithromycin used in one treatment arm, to determine the relationship between azithromycin treatment response and the azithromycin MIC of the infecting isolate. We additionally compared the azithromycin MIC and the disk susceptibility zone sizes of 1,640 S. Typhi and S. Paratyphi A clinical isolates collected from seven Asian countries. In the RCTs, 214 patients who were treated with azithromycin at a dose of 10 to 20 mg/ml for 5 to 7 days were analyzed. Treatment was successful in 195 of 214 (91%) patients, with no significant difference in response (cure rate, fever clearance time) with MICs ranging from 4 to 16 μg/ml. The proportion of Asian enteric fever isolates with an MIC of ≤16 μg/ml was 1,452/1,460 (99.5%; 95% confidence interval [CI], 98.9 to 99.7) for S. Typhi and 207/240 (86.3%; 95% CI, 81.2 to 90.3) (P < 0.001) for S. Paratyphi A. A zone size of ≥13 mm to a 5-μg azithromycin disk identified S. Typhi isolates with an MIC of ≤16 μg/ml with a sensitivity of 99.7%. An azithromycin MIC of ≤16 μg/ml or disk inhibition zone size of ≥13 mm enabled the detection of susceptible S. Typhi isolates that respond to azithromycin treatment. Further work is needed to define the response to treatment in S. Typhi isolates with an azithromycin MIC of >16 μg/ml and to determine MIC and disk breakpoints for S. Paratyphi A.     

Decreased susceptibility to fluoroquinolones and gyrA gene mutation in the Salmonella enterica serovar Typhi and Paratyphi A isolated in Katmandu, Nepal, in 2003

Typhoid fever is the most common clinical diagnosis among febrile patients presenting to hospital in Katmandu. Salmonella enterica serovar Typhi (S. enterica serovar Typhi) and Salmonella enterica serovar Paratyphi A (S. enterica serovar Paratyphi A) with decreased susceptibility to fluoroquinolones and resistance to nalidixic acid are common in recent years. In the present study, we examined the in vitro susceptibility to fluoroquinolones and the presence of gyrA gene mutations in 30 clinical strains of S. Typhi and 39 of S. Paratyphi A, all of which were isolated in Katmandu, Nepal, in 2003. In those strains, we found that 73.3% and 94.9% of S. Typhi and S. Paratyphi A strains contained gyrA gene mutation, and showed the resistance to a quinolone, nalidixic acid, and decreased susceptibility to fluoroquinolones, ciprofloxacin, and levofloxacin. Although fluoroquinolones may still be useful as antibiotics for the treatment of typhoid fever, clinicians should be aware of the possibility of treatment failures of infections with S. Typhi and S. Paratyphi A strains with decreased susceptibility to fluoroquinolones.     

Translational genomics to develop a Salmonella enterica serovar Paratyphi A multiplex polymerase chain reaction assay

The use of pathogen genome sequence data for the control and management of infections remains an ongoing challenge. We describe a broadly applicable, web-enabled approach that can be used to develop bacteria-specific polymerase chain reaction (PCR) assays. Salmonella enterica Paratyphi A has emerged as a major cause of enteric fever in Asia. Culture-based diagnosis is slow and frequently negative in patients with suspected typhoid and paratyphoid fever, potentially compromising patient management and public health. We used the MobilomeFINDER web-server to perform in silico subtractive hybridization, thus identifying 43 protein-coding sequences (CDSs) that were present in two Paratyphi A strains but not in other sequenced Salmonella genomes. After exclusion of 29 CDSs found to be variably present in Paratyphi A strains by microarray hybridization and grouping of remaining CDSs by genomic location, four dispersed targets (stkF, spa2473, spa2539, hsdM) were used to develop a highly discriminatory multiplex PCR assay. All 52 Paratyphi A strains within the diverse panel investigated produced one of two pathognomonic four-band signatures. Given rapid and ongoing expansion of DNA and comparative genomics databases, our universally accessible web-server-supported do-it-yourself approach offers the potential to contribute significantly to the rapid development of species-, serovar-, or pathotype-specific PCR assays targeting pre-existing and emerging bacterial pathogens.     

伤寒沙门菌sRNA S2959增强细菌动力和生物膜形成能力

目的研究伤寒沙门菌小RNA(sRNA) S2959对细菌动力以及生物膜形成能力的影响。方法利用Red重组系统制备伤寒沙门菌sRNA S2959缺陷株(△S2959),同时制备sRNA S2959缺陷株的空载体株(△S2959-pBAD)和回补株(△S2959-p S2959)等相关菌株。通过sRNA S2959缺陷株和野生株的生长曲线实验观察sRNA S2959缺失后对细菌生长的影响;通过细菌sRNA S2959缺陷株、空载体株、回补株的泳动能力实验和生物膜实验观察sRNA S2959对细菌动力以及生物膜形成能力的影响;再用定量PCR(q PCR)方法检测细菌鞭毛相关基因在sRNA S2959相关菌株中的表达水平。结果成功制备sRNA S2959缺陷株和回补株; sRNA S2959缺陷株和野生株在LB和TSB液体培养基中的生长曲线无明显差异;与野生株相比,sRNA S2959缺陷株的泳动能力和生物膜形成能力下调(P0.05),而回补株的泳动能力和生物膜形成能力较缺陷株显著增加(P0.05); q PCR结果显示,高表达sRNA S2959后,细菌鞭毛相关基因(flh D、fli A和flj B)的mRNA水平分别上调大约4、3和6倍。结论伤寒沙门菌sRNA S2959促进鞭毛的表达,进一步上调伤寒沙门菌动力以及生物膜的形成能力。     

Molecular analysis of incHI1 antimicrobial resistance plasmids from Salmonella serovar Typhi strains associated with typhoid fever

The first outbreak of multidrug-resistant (MDR) typhoid fever in Vietnam was in 1993, and by 1995 nearly 90% of cases were MDR. Plasmid HCM1, sequenced in full, is an incHI1 plasmid from Salmonella enterica serovar Typhi strain CT18, isolated in Vietnam in 1993. Restriction analysis shows that pHCM1 shares a restriction fragment length polymorphism (RFLP) pattern with plasmids isolated from the first outbreak and 10 of 17 MDR plasmids isolated from sporadic cases occurring at the same time in Vietnam. A core region of pHCM1 has significant DNA sequence similarity to plasmid R27, isolated in 1961 from S. enterica in the United Kingdom. There are five regions of DNA in pHCM1 which are not present in R27. Two of these are putative acquisition regions; the largest is 34.955 kbp in length and includes sequences of several antibiotic resistance genes and several insertion sequences. The borders of this region are defined by two identical IS10 left elements, associated with an inversion of DNA or with a truncated Tn10 element. The second, smaller region is 14.751 kbp and carries a trimethoprim resistance gene dfr14A cassette associated with a class 1 integrase. In 1993 to 1994, restriction analysis revealed some variations in the structures of Salmonella serovar Typhi MDR plasmids which were mapped to the two putative acquisition regions and three smaller variable regions. In 1996 a single RFLP type, RFLP7, was found to carry the dfrA7 and sul-1 genes, which were not present on R27 or pHCM1. This plasmid type appears to have a selective advantage over other plasmids with the same resistance phenotype.     

Antimicrobial susceptibility of 128 Salmonella enterica serovar typhi and paratyphi A isolates from northern India

Most systemic Salmonella enterica serovar typhi and paratyphi A infections diagnosed in the United States (up to 70%) are acquired during travel to regions of high endemicity. Increasing resistance to agents commonly used for the treatment of such infections (including multidrug resistant isolates) is being reported from several areas of the world (Southeast Asia, Africa, Latin America). Since regional differences in susceptibility patterns may exist, we sought to determine the frequency of antimicrobial resistance among blood and stool isolates (n = 128) from patients in Northwestern India. Salmonella enterica serotype typhi (n = 101) isolates from 14 patients were susceptible to all agents tested. Among 55 isolates with single drug resistance, 44 (81%) were resistant to chloramphenicol. Multidrug resistant (>/=3 drugs) Salmonella enterica was more common in pediatric patients (10 of 30) compared to adults (10 of 71 patients; p = 0.05). All isolates (S. enterica serovar typhi and serovar paratyphi A) were susceptible to ciprofloxacin and ceftriaxone. Travelers to Northwestern India may still receive trimethoprim-sulfamethoxazole, or ciprofloxacin for effective chemoprophylaxis if indicated. Ceftriaxone and ciprofloxacin remain favorable choices for treatment of patients with enteric fever in this region.     

Chromosome-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhi

A salmonella genomic island, designated SGI11, was found in 18 of 26 multidrug-resistant Salmonella enterica serovar Typhi isolates from Bangladesh. SGI11 was an IS1 composite transposon and carried 7 resistance genes that conferred resistance to 5 first-line antimicrobials. Eleven of the 18 SGI11-carrying S. Typhi isolates had developed resistance to high levels of ciprofloxacin.     

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.     

Highly resistant Salmonella enterica serovar Typhi with a novel gyrA mutation raises questions about the long-term efficacy of older fluoroquinolones for treating typhoid fever

As a consequence of multidrug resistance, clinicians are highly dependent on fluoroquinolones for treating the serious systemic infection typhoid fever. While reduced susceptibility to fluoroquinolones, which lessens clinical efficacy, is becoming ubiquitous, comprehensive resistance is exceptional. Here we report ofloxacin treatment failure in typhoidal patient infected with a novel, highly fluoroquinolone-resistant isolate of Salmonella enterica serovar Typhi. The isolation of this organism has serious implications for the long-term efficacy of ciprofloxacin and ofloxacin for typhoid treatment.     

Plasmid-Mediated Resistance to Cephalosporins in Salmonella enterica Serovar Typhi

This study characterized a cephalosporin-resistant Salmonella enterica serovar Typhi isolate. The organism possessed a plasmid encoding the CTX-M-15 extended-spectrum-β-lactamase. This plasmid is the determinant for the phenotype of cephalosporin resistance and is transferrable among Enterobacteriaceae.Typhoid fever is a systemic infection caused by Salmonella enterica serovar Typhi and has become a predominantly travel-associated disease in developed countries (3, 6). Fluoroquinolones have been the most effective drugs for treatment since the spread of multidrug-resistant (resistant to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole) S. Typhi strains. Furthermore, in the era of emergence of fluoroquinolone-resistant S. Typhi, it appears that alternative antimicrobial agents are necessary to ensure therapy for typhoid fever (7, 8). Actually, expanded-spectrum cephalosporins and macrolide antibiotics are considered to be viable alternatives (12). However, a few cephalosporin-resistant S. Typhi strains, which produce extended-spectrum β-lactamases (ESBLs), have been reported (1, 9, 11).Forty-nine clinical isolates of S. Typhi were collected from 48 patients with typhoid fever in 2008. All isolates were identified by regional public health centers and sent to the Department of Bacteriology I, National Institute of Infectious Diseases. Isolates were routinely phage typed by the standard technique with the phage set kindly provided by the Health Protection Agency, London, United Kingdom, and MICs of antimicrobials were determined by the broth microdilution method. The results were interpreted according to the criteria of the Clinical and Laboratory Standards Institute (CLSI) (2, 5). One isolate with the phage type E1 (080049Ty) displayed a high level of resistance to ceftazidime, cefotaxime, and ceftriaxone. This isolate was also resistant to nalidixic acid, showed reduced susceptibility to fluoroquinolones, and was susceptible to imipenem, aztreonam, kanamycin, gentamicin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole. The MICs of ceftazidime and cefotaxime decreased significantly in the presence of clavulanic acid at a fixed concentration of 4 mg liter⁻¹ (Table ​(Table1).1). A plasmid profile of the resistant S. Typhi isolate showed one specific band, which was absent in the susceptible strain (080048Ty) with the same phage type, E1 (Fig. ​(Fig.11 A). The plasmid DNA prepared by alkaline lysis with SDS was dissolved in Milli-Q water at a suitable concentration and was used for electroporation of Escherichia coli W3110. Transformants were selected on LB agar containing ampicillin. The resultant transformant produced a β-lactam resistance pattern similar to that produced by S. Typhi isolate 080049Ty (Table ​(Table1).1). These results suggest that a certain gene(s) located on the plasmid conferred the phenotype of resistance against expanded-spectrum cephalosporins. The presence of the gene conferring the ESBL phenotype was verified by PCR from the plasmid DNA with the primer pair for detection of the CTX-M-type β-lactamase gene (5′-CAAGCGCAGGTGGGCGACAGCAC-3′ and 5′-CTTTTGCCGTCTAAGGCGATAAAC-3′; data not shown) and subsequent Southern hybridization of undigested plasmid DNA with a digoxigenin-labeled probe generated with DIG-High Prime (Roche Diagnostics) according to the manufacturer''s instructions (Fig. ​(Fig.1B).1B). In addition, sequence analysis revealed that this bla_CTX-M was 100% identical to bla_CTX-M-15 (data not shown). Most of the bla_CTX-M-15 genes have been identified in Enterobacteriaceae, mainly in E. coli and Klebsiella spp., and CTX-M-15-producing E. coli strains were isolated from healthy people as well (4, 10). Therefore, the cephalosporin-resistant S. Typhi isolate in this study may have acquired the transferable plasmid containing bla_CTX-M-15 from another enteric bacterium in the patient''s intestine. E. coli W3110 harboring the above transformed plasmid with bla_CTX-M-15 was used as the donor of the plasmid in the transconjugation analysis to confirm the transferability. The expanded-spectrum cephalosporin-susceptible S. Typhi isolate 080048Ty was used as the recipient strain; this strain was also resistant to nalidixic acid. A mixture of each bacterial culture was incubated overnight at 37°C. Transconjugants were isolated on LB agar plates supplemented with ampicillin and nalidixic acid, and the presence of a plasmid with the same size as that of the donor was verified (Fig. ​(Fig.1).1). The doubly resistant colonies appeared with frequencies of 10⁻⁴ to 10⁻³ (defined as the number of ampicillin- and nalidixic acid-resistant S. Typhi colonies divided by the number of nalidixic acid-resistant S. Typhi colonies). These results indicate the transfer of a plasmid containing bla_CTX-M-15 from E. coli to S. Typhi at a significant frequency.Open in a separate windowFIG. 1.Plasmid profiles of cephalosporin-resistant S. Typhi (080049Ty; lane 1), cephalosporin-susceptible S. Typhi (080048Ty; lane 2), an E. coli transformant (lane 3), and an S. Typhi transconjugant (lane 4). (A) Staining by ethidium bromide. (B) Southern blot analysis with a digoxigenin-labeled probe specific for CTX-M-15.

TABLE 1.

MICs for the strains used in this study

Emergence of multidrug-resistant Salmonella enterica serovar typhi in Korea

A chloramphenicol-resistant strain of Salmonella enterica serovar Typhi was first noted in Korea in 1992, when a resistant isolate was detected in a returned traveler. Continued isolation of multidrug-resistant (MDR) strains thereafter in other settings prompted a retrospective analysis of laboratory records and phenotypic and genotypic analyses of 12 chloramphenicol-resistant isolates. Among these, one isolate was resistant only to chloramphenicol, and the other isolates were also resistant to ampicillin and co-trimoxazole. MDR was transferred by conjugation from 9 of the 11 isolates. PCR showed that all isolates had an incompatible group HI1 plasmid, and oriT was detected in 10 isolates, which included strains with an unsuccessful transfer of resistance. All of the ampicillin-resistant isolates had a beta-lactamase band of pI 5.4 and bla(TEM) alleles. A PCR amplicon from an isolate showed that the sequences were identical to those of bla(TEM-1), suggesting that all isolates had a TEM-1 beta-lactamase. All isolates had class 1 integrons: 10 isolates had integrons of ca. 1.2 kb with dhfr7 gene cassettes, and 1 isolate had an integron of ca. 2.3 kb with aacA4 and bla(OXA-1)-like gene cassettes. The pulsed-field gel electrophoresis patterns of 7 of 11 MDR isolates were identical and indistinguishable from those reported for isolates in India and Indonesia. In conclusion, some of the MDR strains in Korea are related to those in other Asian countries. Susceptibility testing became necessary for selection of antimicrobial agents for the optimal treatment of patients with the emergence of MDR Salmonella serovar Typhi in Korea.