The emergence of azithromycin-resistant Salmonella Typhi in Nepal

Abstract Background Typhoid fever remains a significant cause of morbidity and mortality in Asia and Africa. The emergence of azithromycin resistance in South Asia is concerning, as azithromycin is one of the last effective oral drugs for treating typhoid. Objectives To describe the molecular mechanism and phylogenetics of azithromycin-resistant (AzithR) Salmonella Typhi isolates from Patan Hospital, Kathmandu, Nepal. Methods Whole-genome sequences of three AzithR S. Typhi isolates (MIC >256 mg/L) were analysed and compared with a global collection to investigate the azithromycin resistance mechanism and phylogenetic structure. Clinical information is reported for one of the three patients infected with AzithR S. Typhi. Results The three AzithR isolates belonged to the H58 lineage and were genetically identical; they were distantly related to contemporaneous S. Typhi from Nepal and AzithR S. Typhi recently described in Bangladesh. Azithromycin resistance was mediated by a non-synonymous mutation in the acrB gene (R717L). The three AzithR isolates showed reduced susceptibility to ciprofloxacin (double mutation in the gyrA: S83F and D87G), and were susceptible to ampicillin, chloramphenicol and co-trimoxazole. Clinical information from one patient suggested non-response to azithromycin treatment. Conclusions This is the first molecular description of AzithR S. Typhi in Nepal. These organisms showed no phylogenetic link to AzithR S. Typhi in Bangladesh. Our data suggest that increasing use of azithromycin may pose a strong selective pressure driving the emergence of AzithR S. Typhi in South Asia. Further investigations are needed to evaluate treatment responses to azithromycin, predict evolutionary trajectories, and track the transmission of these organisms.


Abstract
Typhoid fever remains a significant cause of morbidity and mortality in Asia and Africa. The emergence of azithromycin resistance in South Asia is concerning, as azithromycin is one of the last effective oral drugs for treating typhoid. In mid-2019, three azithromycin-resistant (Azith R ) S. Typhi isolates were isolated from typhoid fever patients attending Patan Hospital, Kathmandu, Nepal. These organisms were whole genome sequenced and compared with a global collection. We found that the three Azith R isolates belonged to the H58 lineage and were genetically identical; they were distantly related to contemporaneous S. Typhi from Nepal and Azith R S. Typhi recently described in Bangladesh. Azithromycin resistance was mediated by nonsynonymous mutation in the acrB gene (R717L). Clinical information from one patient suggested non-response to azithromycin treatment.
Further investigations are needed to evaluate treatment responses to azithromycin, predict Azith R S.
Typhi's evolutionary trajectories, and track the transmission of these organisms.

Main Text
Typhoid fever is a life-threatening systemic infection predominantly caused by Salmonella enterica serovar Typhi (S. Typhi). Though the disease has been controlled in developed countries, it continues to cause significant morbidity and mortality in resource-poor settings in Asia and Africa.
Effective antimicrobial therapy is essential to avoid deaths and serious complications. However, S.
Typhi has continually evolved resistance to antimicrobials used for its treatment, posing a constant clinical challenge and likely exacerbating disease burden. 1 Multi-drug resistance (MDR; resistance to chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole) first evolved in S. Typhi in the late 1980s, followed by fluoroquinolone resistance in the 1990s. 2 Third-generation cephalosporins have since been used for typhoid treatment, but the emergence of extensively-drug resistant (XDR; MDR plus resistance to fluoroquinolones and third-generation cephalosporins) S. Typhi in Pakistan 3 has reduced the clinical efficacy of these drugs and raises concerns regarding the imminent spread of untreatable S. Typhi. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 12, 2020. . https://doi.org/10.1101/2020.08.07.20166389 doi: medRxiv preprint Azithromycin is effectively the last remaining oral antimicrobial to treat typhoid fever. 4 Although azithromycin resistance in S. Typhi has rarely been reported, an increasing reliance on this drug over the last decade has led to the emergence of azithromycin-resistant (Azith R ) S. Typhi in South Asia. A recent study in Bangladesh indicated that azithromycin resistance (MIC >32 µg/ml) in S. Typhi was associated with a non-synonymous mutation (R717Q) in the acrB gene, which encodes an efflux pump. 5 There are limited data on clinical responses to azithromycin in Azith R S. Typhi-infected patients. Here, we report the genomics, antimicrobial resistance profiles, and phylogenetic relatedness of three Azith R S. Typhi isolates isolated from typhoid fever outpatients visiting a hospital in Nepal.
We report clinical manifestations and azithromycin response data for one of the patients.
Typhoid fever is frequently managed in the outpatient department (OPD) of the hospital and blood culture is routinely performed following routine microbiological procedures when enteric fever is suspected. 6 Antimicrobial susceptibility testing is performed by a modified Bauer-Kirby disc diffusion, with Etests Ò to determine MICs (bioMérieux, France); results are interpreted using CLSI guidelines. 7 In August and September 2019, routine microbiological diagnostic procedures identified three patients with Azith R S. Typhi attending the OPD. Clinical information was only available for one of three patients infected with Azith R S. Typhi.
On 25 August 2019, a 28-year-old male from Nakkhu (Lalitpur District) presented to the OPD following four days of anorexia and persistent fever despite three days of paracetamol use. General and systemic examinations were normal except for a fever of 38.9°C on presentation. Investigations revealed a haemoglobin count of 13.7 g/dl, total white blood cell count of 8.6 thousand/µl with increased neutrophils (DLC: N-83, L-17), platelet count of 195 thousand/µl, and C-reactive protein of 18.7 mg/dl. Urine microscopy and analysis were normal and blood culture was performed. A clinical diagnosis of enteric fever was made and oral azithromycin (1g once/day) was administered. The patient was asked to return for the blood culture reports after 72 hours. On day 2 of culture, his blood culture was positive for S. Typhi, which was found to be Azith R (6mm zone of inhibition on disc diffusion, MIC >256 µg/ml). The patient did not return for the scheduled 72-hour follow-up, but was traced on day seven of treatment. On day seven, the patient reported a fever of 38.3°C lasting for two days. Physical examination showed no abnormalities, but laboratory examinations were repeated due to the previous blood culture results. Repeat laboratory analysis showed a haemoglobin count of 13.5 g/dl, total white blood cell count of 6.9 thousand/µl, continued increased neutrophils (DLC: N-77, L-23), platelet count of 219 thousand/µl, and C-reactive protein of 23 mg/dl. A repeat blood culture was performed and an Azith R S. Typhi was again isolated. The patient was admitted and administered intravenous ceftriaxone. The patient became afebrile after 48 hours of ceftriaxone treatment and was discharged after 7-days of intravenous ceftriaxone. Two additional Azith R S. Typhi isolates displaying identical resistance phenotypes were identified from a 53-year-old female from Nakkhu, Lalitpur (25 th August 2019) and a 26-year-old male from Ramshatol, Ramechhap (9 th September 2019); no clinical information was available for these patients. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 12, 2020. . https://doi.org/10.1101/2020.08.07.20166389 doi: medRxiv preprint Genomic and phylogenetic analyses showed that these Azith R S. Typhi isolates and the contemporaneous Azith S isolate from this study belonged to the H58 lineage. The three Azith R isolates were genetically identical and differed from the Azith S isolate by 42 SNPs; the three Nepali Azith R isolates were also distantly related to all other H58 isolates from the global collection ( Figure   1), and were not linked to the Azith R H58 isolates recently reported in Bangladesh. 5 Given that most previously published Nepali H58 isolates grouped within sub-lineage II (including the Azith S isolate from this study), the Nepali Azith R isolates likely evolved from an older H58 variant, with a phylogenetic distance between the Azith R isolates and H58 sub-lineage II of at least 27 SNPs.
Regarding AMR, none of the Azith R H58 isolates carried an acquired AMR gene, but harboured a nonsynonymous mutation in the acrB gene (STY0519), changing arginine (R) to leucine (L) at codon 717. R717L and R717Q mutations in acrB have been found to confer Azith R in Bangladeshi S.
Paratyphi A and S. Typhi, respectively. 5 Furthermore, the Nepali Azith R H58 isolates exhibited double mutations in the gyrA gene (S83F, D87G), leading to reduced fluoroquinolone susceptibility; no parC mutations were observed. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 12, 2020. . https://doi.org/10.1101/2020.08.07.20166389 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 12, 2020. . https://doi.org/10.1101/2020.08.07.20166389 doi: medRxiv preprint The emergence of Azith R S. Typhi warrants further detailed clinical investigation to better understand the correlation between in vitro azithromycin susceptibility and clinical responses to azithromycin treatment. In vitro resistance to azithromycin does not often agree with in vivo effectiveness because the drug is mostly concentrated intracellularly. 17 A previous study found no difference in response to azithromycin treatment among patients infected with S. Typhi exhibiting azithromycin MICs from 4-16 µg/ml. 18 However, S. Typhi isolates with azithromycin MICs > 16 µg/ml are rare and clinical data on treatment responses in such organisms are lacking. Here, we report three patients infected with Azith R S. Typhi with MIC >256 µg/ml, one of whom was given oral azithromycin 1g once/day. Though patient adherence to initial treatment cannot be ensured, clinical investigation suggests that the patient might not have adequately responded to azithromycin treatment and experienced microbiological failure. Further clinical and epidemiological investigations are needed to examine the increase of azithromycin resistance in S. Typhi in South Asia, limit its transmission, and improve empiric antimicrobial regimes.
Our data show that azithromycin resistance mutations at codon 717 (acrB gene) have independently emerged across distantly related lineages, suggesting that increasing use of azithromycin for treating typhoid fever may impose a strong selective pressure driving the emergence and spread of Azith R S. Typhi. Convergent evolution towards azithromycin resistance in S. Typhi is of particular concern, as the development of azithromycin resistance mutations in XDR S. Typhi will eventually result in potentially untreatable infections. Further studies are needed to understand the driving forces and fitness effects of such resistance mutations. Typhoid conjugate vaccine (TCV) should be introduced in Nepal and other endemic countries to reduce the selective pressures induced by antimicrobial usage.
All three Azith R S. Typhi isolates in this study were genetically identical, phylogenetically unrelated to all contemporary Nepali S. Typhi, and exhibited double mutations in the gyrA gene. We is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 12, 2020. . https://doi.org/10.1101/2020.08.07.20166389 doi: medRxiv preprint hypothesize these patients may have been infected from the same source. Additionally, the infecting organisms originate from an H58 variant that has been replaced by the dominant H58 sub-lineage II. 9 This observation suggests this variant may be circulating at a low prevalence in the human population or have been maintained in a reservoir separate from the environmental transmission cycle, such as through asymptomatic carriage in the gallbladder. These data suggest these patients could have been infected from a chronic carrier within whom the infecting S. Typhi has evolved resistance to azithromycin.
In conclusion, we report three outpatients infected with highly azithromycin-resistant (MIC >256 µg/ml) S. Typhi in Nepal. One patient was given oral azithromycin 1g once/day and experienced prolonged fever until rescue treatment with ceftriaxone. All three Azith R isolates were genetically identical H58 variants that were phylogenetically distinct from other contemporaneous Nepali S.
Typhi. Azithromycin resistance was mediated by a chromosomal mutation R717L in the arcB gene.
Further clinical and epidemiological investigations are required to track their transmission and evaluate clinical responses in patients infected with these important pathogens.

Acknowledgments
We would like to thank all members of the Molecular Epidemiology group at Oxford University Clinical Research Unit (OUCRU) in Vietnam; Microbiology Laboratory at Patan Hospital and doctors, nurses and health assistants of Patan Hospital Emergency and Outpatient Clinics.

Financial Support
No funding was involved in the study.

Conflicts of interest
The authors declare no competing interests. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 12, 2020. . https://doi.org/10.1101/2020.08.07.20166389 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 12, 2020. . https://doi.org/10.1101/2020.08.07.20166389 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 12, 2020. . https://doi.org/10.1101/2020.08.07.20166389 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 12, 2020. . https://doi.org/10.1101/2020.08.07.20166389 doi: medRxiv preprint