Salmonella Typhi (S. Typhi) causes typhoid fever, a major public health problem in developing countries [1Dyson Z A, Klemm E J, Palmer S, Dougan G. 2019; Antibiotic resistance and typhoid. Clin Infec Dis 68(Supplement_2): 165-70.
[http://dx.doi.org/10.1093/cid/ciy1111] , 2Engsbro AL, Riis Jespersen HS, Goldschmidt MI, et al. Ceftriaxone-resistant Salmonella enterica serotype Typhi in a pregnant traveller returning from Karachi, Pakistan to Denmark, 2019. Euro Surveill 2019; 24(21)
[http://dx.doi.org/10.2807/1560-7917.ES.2019.24.21.1900289] [PMID: 31138366] ]. Typhoid fever is common in areas where safe drinking water and sanitation are inadequate. More than 16 million new cases occur worldwide annually, resulting in approximately 600,000 deaths per year [1Dyson Z A, Klemm E J, Palmer S, Dougan G. 2019; Antibiotic resistance and typhoid. Clin Infec Dis 68(Supplement_2): 165-70.
[http://dx.doi.org/10.1093/cid/ciy1111] , 3Qamar FN, Azmatullah A, Kazi AM, Khan E, Zaidi AKM. A three-year review of antimicrobial resistance of Salmonella enterica serovars Typhi and Paratyphi A in Pakistan. J Infect Dev Ctries 2014; 8(8): 981-6.
[http://dx.doi.org/10.3855/jidc.3817] [PMID: 25116662] ]. The disease has been a major human pathogen for thousands of years, thriving in conditions of poor sanitation, crowding, and social chaos [3Qamar FN, Azmatullah A, Kazi AM, Khan E, Zaidi AKM. A three-year review of antimicrobial resistance of Salmonella enterica serovars Typhi and Paratyphi A in Pakistan. J Infect Dev Ctries 2014; 8(8): 981-6.
[http://dx.doi.org/10.3855/jidc.3817] [PMID: 25116662] -5Amicizia D, Arata L, Zangrillo F, Panatto D, Gasparini R. Overview of the impact of Typhoid and Paratyphoid fever. Utility of Ty21a vaccine (Vivotif®). J Prev Med Hyg 2017; 58(1): E1-8.
[PMID: 28515625] ]. Typhoid is transmitted mainly by the faecal-oral route [6Mahindroo J, Thanh DP, Nguyen TNT, et al. Endemic fluoroquinolone-resistant Salmonellaenterica serovar Kentucky ST198 in northern India. Microb Genom 2019; 5(7)
[http://dx.doi.org/10.1099/mgen.0.000275] [PMID: 31166889] , 7Singh Y, Saxena A, Bohra J, Kumar R, Kumar A, Saxena MK. Anti-microbial resistance in salmonella. J Basic Appl Sci 2019; 15: 27-31.
[http://dx.doi.org/10.29169/1927-5129.2019.15.03] ]. In most cases, an asymptomatic carrier of S. Typhi, or an individual who has recently recovered from the infection, continues to excrete large number of organisms in the stool for up to three months and contaminates food or water, either through direct food handling, through transfer of bacteria by flies and other insects, or by contamination of potable water [8Xu Z, Song Q, Li C, Zhan Y. Characterization of ciprofloxacin-resistant and ESBL-producing Salmonella enteric serotype Derby in Eastern China. BMC Microbiol 2019; 19(1): 61.
[http://dx.doi.org/10.1186/s12866-019-1434-6] [PMID: 30885127] -10Butler T. Treatment of typhoid fever in the 21st century: promises and shortcomings. Clin Microbiol Infect 2011; 17(7): 959-63.
[http://dx.doi.org/10.1111/j.1469-0691.2011.03552.x] [PMID: 21722249] ]. About 2-3% of patients are permanent carriers [11Zige DV, Ohimain EI, Sridhar MK. A community-based screening of asymptomatic typhoid carriers in Wilberforce Island, Bayelsa State, Nigeria. Int J Health Sci Res 2013; 3: 119-26.].

S. Typhi is a gram-negative enteric bacillus, a member of Enterobacteriaceae: ferments glucose, reduces nitrate, and is oxidative negative [12Ong SY, Pratap CB, Wan X, et al. The Genomic Blueprint of Salmonella enterica subspecies enterica serovar Typhi P-stx-12. Stand Genomic Sci 2013; 7(3): 483-96.
[http://dx.doi.org/10.4056/sigs.3286690] [PMID: 24019994] ]. This facultatively anaerobic bacillus possesses three major antigens: H or flagellar antigen; O or somatic antigen; and Vi or virulence antigen. Clinical diagnosis of typhoid is confirmed by culture of the organism from blood, duodenal secretions, or stool [4Ugboko H, De N. Mechanisms of Antibiotic resistance in Salmonella typhi. Int J Curr Microbiol Appl Sci 2014; 3(12): 461-76., 13Ismail A. New advances in the diagnosis of typhoid and detection of typhoid carriers. Malays J Med Sci 2000; 7(2): 3-8.
[PMID: 22977383] ]. Excretion of S. Typhi in the stool is more likely with higher blood bacterial counts, and children tend to have higher bacteraemia than adults [14Ramyil MCS, Ogundeko TO, Idyu II, Ameh JM. Use of stool culture as a determinant parameter of enteric fever in adults attending Bingham University Teaching Hospital Jos, Nigeria. Clin Med Res 2014; 3: 31.]. Fluoroquinolones such as ciprofloxacin are recommended by the World Health Organisation (WHO) as they are the most effective orally active, inexpensive, and well-tolerated drugs for the treatment of typhoid fever [15Nüesch-Inderbinen M, Abgottspon H, Sägesser G, Cernela N, Stephan R. Antimicrobial susceptibility of travel-related Salmonella enterica serovar Typhi isolates detected in Switzerland (2002-2013) and molecular characterization of quinolone resistant isolates. BMC Infect Dis 2015; 15: 212.
[http://dx.doi.org/10.1186/s12879-015-0948-2] [PMID: 25963025] ]. Ciprofloxacin is the current first drug of choice for diarrheal cases in Zimbabwe according to the Ministry of Health and Child Care guidelines [16Ministry of Health and Child Care. 2011.https://srhr.org/abortion-policies/documents/countries/03-Zimbabwe-Essential-Medicines-List-2011.pdf].

S. Typhi has gained resistance to antibiotics like ampicillin, ceftriaxone, and co-trimoxazole, besides developing resistance to efficacious drugs like ciprofloxacin. The emergence of multidrug resistance to the commonly used antibiotics has complicated the treatment [17Sehra D, Sehra S, Ralia P, Sehra ST. An altered drug resistance pattern in Salmonella typhi. American J Infec Dis and Microbio 2013; 1: 84-5.
[http://dx.doi.org/10.12691/ajidm-1-5-1] , 18Zaki SA, Karande S. Multidrug-resistant typhoid fever: a review. J Infect Dev Ctries 2011; 5(5): 324-37.
[http://dx.doi.org/10.3855/jidc.1405] [PMID: 21628808] ]. The pathogenesis of S. Typhi depends upon a large number of factors controlled by arrays of genes that synergise into actual virulence of the bacteria [19Foysal MJ, Momtaz F, Haque MH, Hossain MN, Rahman MM, Prodhan SH. Detection of lipase and elastase in typhoid and UTI patients caused by salmonella typhimurium in the eastern part of Bangladesh. Asian Jr of Microbiol Biotech Env Sc 2016; 18(4): 815-20., 20Everest P, Wain J, Roberts M, Rook G, Dougan G. The molecular mechanisms of severe typhoid fever. Trends Microbiol 2001; 9(7): 316-20.
[http://dx.doi.org/10.1016/S0966-842X(01)02067-4] [PMID: 11435104] ].

In the case of Harare, Zimbabwe, the city experienced a rise in cases of typhoid fever in Mabvuku and Tafara, both high-density suburbs, in January 2010. From October 2011 to June 2012, there was another typhoid outbreak, whose epicentre was the Harare high-density township of Dzivarasekwa [21Muti M, Gombe N, Tshimanga M, et al. Typhoid outbreak investigation in Dzivaresekwa, suburb of Harare City, Zimbabwe, 2011. Pan Afr Med J 2014; 18: 309.
[http://dx.doi.org/10.11604/pamj.2014.18.309.4288] [PMID: 25469202] , 22Chaminuka L, Nyatsanza TD. An assessment of water shortages and coping mechanisms of Harare residents: A case of Msasa Park and Dzivaresekwa Extension. IOSR J Agric Vet Sci 2013; 4(3): 21-35.
[http://dx.doi.org/10.9790/2380-0432135] ]. Such high-density areas of Zimbabwe typically comprise crowded households with high numbers of people (Fig. 1a) while low-density areas are more affluent and less crowded (Fig. 1b). There were unsubstantiated reports of another typhoid outbreak in parts of Harare in the first quarter of 2016, which is the subject of our current study. Hence this study was carried out to determine ciprofloxacin resistance of S. Typhi isolated at the National Microbiology Reference Laboratory (NMRL) in Harare, Zimbabwe between January and April 2016.

This cross-sectional study was conducted after permission by the Harare Hospital’s Clinical Director and ethical approval from Africa University Research Ethics Committee (AUREC). The study was carried out at the National Microbiology Reference Laboratory (NMRL), located at Harare Central Hospital in Zimbabwe. NMRL receives stool or blood specimens from medical facilities located all over Zimbabwe and provides confirmatory and biochemical typing of pathogenic isolates. Upon receipt, stool and blood samples from Harare laboratories were cultured on Blood Agar (BA) or deoxycholate Citrate Agar (DCA), as appropriate. Kligler Iron Agar (KIA) and Lysine Iron Agar (LIA) were then used for presumptive identification and isolates were further screened using Vi and D slide serology (Fig. 2a).

Demographics and clinical data recovered from Harare patients suspected to be suffering from typhoid were noted. Isolates from stool and blood samples, for the confirmed cases, received during the period from January 2016 to April 2016, were retrieved. The Mueller–Hinton agar disk diffusion method was subsequently used to determine antimicrobial susceptibility profiles of the 35 confirmed S. Typhi isolates against ciprofloxacin (Fig.2b). Results were interpreted according to Clinical and Laboratory Standards guidelines (Table 1).

Fig. (1) shows a) high-density layout versus b) low-density suburb house in Zimbabwe.

Fig. (2) Flow chart showing isolation, identification, and sensitivity testing for Salmonella Typhi isolates at NMRL. Steps before this current study are shown by the line marked a) Pre-study. Sensitivity testing was carried out on 35 confirmed S. Typhi isolates in the current study, marked b.

All isolates came from blood or stool cultures of 402 diarrheal cases from Harare Health facilities. In this study, 41 isolates reacted positively with Kligler iron agar, lysine iron agar, and were citrate negative, which is suggestive of S. Typhi. The Salmonella antisera Poly H, Poly O, Monofactor 9, 12, Vi, and dH reacted positively with 35 of the 41 isolates, confirming their identity as S. Typhi. The location of origin for the different samples and the numbers of confirmed S. Typhi isolates per site is shown in Table 2.

Most of the areas with positive cases of S. Typhi are high-density areas. Only two (6%) of the cases were from low-density suburbs (Eastview and Greendale). Thirty-five isolates out of 402 suspected and tested specimens in this study reacted positively with Salmonella Antisera (Poly H, poly O, Monofactor 9,12, Vi, and dH) which confirms their identity as S. Typhi. For the confirmed S. Typhi, resistance to cipro-floxacin was observed in a quarter of isolates (Table 1).

Most high-density areas, where S. Typhi positive specimens originated are overpopulated and have regular water cuts resulting in a decrease in hygiene [21Muti M, Gombe N, Tshimanga M, et al. Typhoid outbreak investigation in Dzivaresekwa, suburb of Harare City, Zimbabwe, 2011. Pan Afr Med J 2014; 18: 309.
[http://dx.doi.org/10.11604/pamj.2014.18.309.4288] [PMID: 25469202] , 22Chaminuka L, Nyatsanza TD. An assessment of water shortages and coping mechanisms of Harare residents: A case of Msasa Park and Dzivaresekwa Extension. IOSR J Agric Vet Sci 2013; 4(3): 21-35.
[http://dx.doi.org/10.9790/2380-0432135] ]. Over 25% of S. Typhi isolates characterised are resistant to ciprofloxacin. Our results agree with overall trends reported prior to this account, with an increase in resistance to ciprofloxacin from 0% in 2012, 25% in 2015 to 22% in 2017 [23Mashe T, Gudza-Mugabe M, Tarupiwa A, et al. Laboratory characterisation of Salmonella enterica serotype Typhi isolates from Zimbabwe, 2009-2017. BMC Infect Dis 2019; 19(1): 487.
[http://dx.doi.org/10.1186/s12879-019-4114-0] [PMID: 31151421] ]. The reason for the increase in ciprofloxacin resistance in Zimbabwe may be its over-use as a broad-spectrum antibiotic to treat many infectious diseases [23Mashe T, Gudza-Mugabe M, Tarupiwa A, et al. Laboratory characterisation of Salmonella enterica serotype Typhi isolates from Zimbabwe, 2009-2017. BMC Infect Dis 2019; 19(1): 487.
[http://dx.doi.org/10.1186/s12879-019-4114-0] [PMID: 31151421] , 24Yang HY, Nam YS, Lee HJ. Prevalence of plasmid-mediated quinolone resistance genes among ciprofloxacin-nonsusceptible Escherichia coli and Klebsiella pneumoniae isolated from blood cultures in Korea. Can J Infect Dis Med Microbiol 2014; 25(3): 163-9.
[http://dx.doi.org/10.1155/2014/329541] [PMID: 25285114] ]. Though fluoroquinolone resistance is chromosomally mediated, selective pressures exerted by the overuse of these drugs may see such isolates becoming more common in the future [24Yang HY, Nam YS, Lee HJ. Prevalence of plasmid-mediated quinolone resistance genes among ciprofloxacin-nonsusceptible Escherichia coli and Klebsiella pneumoniae isolated from blood cultures in Korea. Can J Infect Dis Med Microbiol 2014; 25(3): 163-9.
[http://dx.doi.org/10.1155/2014/329541] [PMID: 25285114] ].

A similar increase in resistance has been observed in many countries around the world. A study on S. Typhi isolates in the United Kingdom discovered a decrease in susceptibility to ciprofloxacin to 23%, in 1999 [25Threlfall EJ, Ward LR. Decreased susceptibility to ciprofloxacin in Salmonella enterica serotype typhi, United Kingdom. Emerg Infect Dis 2001; 7(3): 448-50.
[http://dx.doi.org/10.3201/eid0703.017315] [PMID: 11384525] ]. Other studies recommended that possible alternative drugs to treat ciprofloxacin-resistant S. Typhi such as ceftriaxone or cefotaxime could be considered in the treatment and management of typhoid fever [26Khanal B, Sharma SK, Bhattacharya SK, Bhattarai NR, Deb M, Kanungo R. Antimicrobial susceptibility patterns of Salmonella enterica serotype typhi in eastern Nepal. J Health Popul Nutr 2007; 25(1): 82-7.
[PMID: 17615907] ]. The decrease in ciprofloxacin susceptibility was also observed in Cambodian adults [27Vlieghe ER, Phe T, De Smet B, et al. Azithromycin and ciprofloxacin resistance in Salmonella bloodstream infections in Cambodian adults. PLoS Negl Trop Dis 2012; 6(12)e1933.
[http://dx.doi.org/10.1371/journal.pntd.0001933] [PMID: 23272255] ]. In another research done in South Africa, 18% of the isolates were resistant to ciprofloxacin [28Keddy KH, Smith AM, Sooka A, Ismail H, Oliver S. Fluoroquinolone-resistant typhoid, South Africa. Emerg Infect Dis 2010; 16(5): 879-80.
[http://dx.doi.org/10.3201/eid1605.091917] [PMID: 20409393] ]. A similar trend was observed on 70 isolates in Bangladesh [29Mannan A, Shohel M, Rajia S, Mahmud NU, Kabir S, Hasan I. A cross sectional study on antibiotic resistance pattern of Salmonella typhi clinical isolates from Bangladesh. Asian Pac J Trop Biomed 2014; 4(4): 306-11.
[http://dx.doi.org/10.12980/APJTB.4.2014C770] [PMID: 25182556] ]. The resistance of S. Typhi is mainly due to the accumulation of mutation in the quinolone resistance- determining regions of DNA gyrase and topoisomerase IV genes which are target site alterations [30Rushdy AA, Mabrouk MI, Abu-Sef FA, Kheiralla ZH, Mohamed Abdel-All S, Saleh NM. Contribution of different mechanisms to the resistance to fluoroquinolones in clinical isolates of Salmonella enterica. Braz J Infect Dis 2013; 17(4): 431-7.
[http://dx.doi.org/10.1016/j.bjid.2012.11.012] [PMID: 23742803] ].

Plasmid-mediated quinolone resistance confers a form of reduced susceptibility, which is thought to promote the accumulation of the target site mutation [31Strahilevitz J, Jacoby GA, Hooper DC, Robicsek A. Plasmid-mediated quinolone resistance: a multifaceted threat. Clin Microbiol Rev 2009; 22(4): 664-89.
[http://dx.doi.org/10.1128/CMR.00016-09] [PMID: 19822894] ]. The increased resistance of S. Typhi could reflect the overuse of ciprofloxacin in the treatment of typhoid as well as in other unrelated infections. Incomplete treatment may also be a contributing factor to the development of resistance [23Mashe T, Gudza-Mugabe M, Tarupiwa A, et al. Laboratory characterisation of Salmonella enterica serotype Typhi isolates from Zimbabwe, 2009-2017. BMC Infect Dis 2019; 19(1): 487.
[http://dx.doi.org/10.1186/s12879-019-4114-0] [PMID: 31151421] , 26Khanal B, Sharma SK, Bhattacharya SK, Bhattarai NR, Deb M, Kanungo R. Antimicrobial susceptibility patterns of Salmonella enterica serotype typhi in eastern Nepal. J Health Popul Nutr 2007; 25(1): 82-7.
[PMID: 17615907] , 32Yang YA, Chong A, Song J. Why is eradicating typhoid fever so challenging: Implications for vaccine and therapeutic design. Vaccines (Basel) 2018; 6(3): 45.
[http://dx.doi.org/10.3390/vaccines6030045] [PMID: 30042307] ]. This might be the reason why there is a high proportion of ciprofloxacin-resistant isolates in the areas studied. In addition, an increase in antimicrobial resistance has been attributed to the selection pressure created by the uncontrolled use of antimicrobials in animal husbandry. The lack of strict control on antimicrobial usage increases the risk of dissemination of foodborne microbes harbouring arrays of resistance genes [33Weam B, Abraham M, Doiphode S, et al. Foodborne bacterial pathogens associated with the risk of gastroenteritis in the state of qatar. Int J Health Sci (Qassim) 2016; 10(2): 197-207.
[PMID: 27103902] ].

Questions still remain regarding the origin of antimicro-bial-resistant mutations and transmission routes of antimicro-bial-resistant pathogens across populations. Antimicrobial resistance monitoring by bacterial whole-genome and 16S ribosomal RNA sequencing is a rapidly growing field and it is reported that such methods are likely to impact public health [34Laudadio I, Fulci V, Stronati L, Carissimi C. Next-generation metagenomics: Methodological challenges and opportunities. OMICS 2019; 23(7): 327-33.
[http://dx.doi.org/10.1089/omi.2019.0073] [PMID: 31188063] ]. This is because molecular research and environmental metagenomics are more holistic approaches for studying the development of antimicrobial resistance mutations. Hence, metagenomic profiles could aid the prediction of antimicrobial resistance from clinical specimens. By leveraging information collected in this field, it is possible to characterize the “resistome” in human samples, from diverse geographical regions.

Table 1
Inhibition zone diameter standards for interpreting results after S. Typhi susceptibility testing for ciprofloxacin, according to Clinical and Laboratory Standards guidelines (2013)

Table 2
Geographic location of origin and sensitivity patterns of S. Typhi isolates

The rise in antimicrobial resistance to the first-line drug used for typhoid treatment in Zimbabwe implies that the drug should be used with due care. According to the ciprofloxacin susceptibility patterns of S. Typhi isolated, antimicrobial resistance must be monitored regularly in order to check effective drug use. When a strain of microorganism acquires resistance to a drug, another drug must be found to treat the resistant infection effectively. Drugs like ceftriaxone and nalidixic acid should be considered for the treatment of typhoid as they are less commonly used in the population and no resistance has been documented. New antibiotics also need to be developed to deal with the global problem of antimicrobial drug resistance. The WHO and the Drugs for Neglected Diseases Initiative (DNDi) are working in collaboration on a partnership to develop novel antibiotics, as well as promoting their responsible use and ensuring equitable access [35Renwick MJ, Simpkin V, Mossialos E. 2016. Available from: https://www.ncbi.nlm.nih .gov/books/NBK447334/].

Larger research involving national data over longer periods of time should be carried out. Whole-genome sequencing to determine the presence of the virulent genes capable of cell invasion and infection, sequencing to check the DNA gyrase mutation, antimicrobial sensitivity assays to monitor first-, second- and third-line drugs and evidence-based vaccination programs for typhoid prophylaxis should be introduced. The global problem of antimicrobial resistance needs to be understood through the use of novel methods, for example designing new drugs informed by genotypic relatedness and geographic and phylogenetic relationships of isolates.

There was a limited number of isolates encountered during the period of study and limited resources to investigate the virulent genes. It is recommended that more focus be given to genotyping methods when studying the development of antimicrobial resistance mutations.