Analysis of Trends in Resistance to Fluoroquinolones and Extended Spectrum Beta-Lactams among Salmonella Typhi Isolates Obtained from Patients at Four Outpatient Clinics in Nairobi County, Kenya

Typhoid fever caused by the bacterium Salmonella enterica serovar Typhi (S. Typhi) causes an estimated 25 million illnesses and approximately 200,000 deaths annually mostly in developing countries. Although the management of typhoid fever has been effectively through antibiotic treatment, S. Typhi is increasingly becoming resistant to the currently recommended drugs. This study utilized a quasi-experimental design focusing on archived samples to describe antimicrobial susceptibility patterns of S. Typhi and determine the genetic basis of resistance to the two most commonly used classes of antimicrobials. A total sample size of 287 isolates of S. Typhi isolates stored in −80 ̊C freezer at the Centre for Microbiology Research was utilized. Isolates were subjected to antimicrobial susceptibility testing to commonly available antimicrobials using disk diffusion method, then analyzed for trends in resistance to fluoroquinolones and extended spectrum beta lactams. Among the 287 isolates 158 (55.5%) were found to be Multi Drug Resistant (MDR). This implied that these isolates were resistant to all first line classes of treatment such as ampicillin, chloramphenicol and sulfamethoxazole-trimethroprim. In addition to this, these isolates were also resistant to at least one of the currently recommended drugs of choice, either a β-lactam or a fluoroquinolone. This study observed resistances at 18.2% and 15.4% to fluoroquinolones and cephalosporins respectively. PCR results revealed presence of blaTEM, blaINT and blaCTX-M genes coding for resistance to β-lactams in 80% of the isolates that had combined resistance to β-lactams and fluoroquinolones. It is likely that How to cite this paper: Kavai, S.M., Kangogo, M., Muigai, A.W.T. and Kariuki, S. (2018) Analysis of Trends in Resistance to Fluoroquinolones and Extended Spectrum Beta-Lactams among Salmonella Typhi Isolates Obtained from Patients at Four Outpatient Clinics in Nairobi County, Kenya. Advances in Microbiology, 8, 578-588. https://doi.org/10.4236/aim.2018.87038 Received: June 4, 2018 Accepted: July 27, 2018 Published: July 30, 2018 Copyright © 2018 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access

freezer at the Centre for Microbiology Research was utilized. Isolates were subjected to antimicrobial susceptibility testing to commonly available antimicrobials using disk diffusion method, then analyzed for trends in resistance to fluoroquinolones and extended spectrum beta lactams. Among the 287 isolates 158 (55.5%) were found to be Multi Drug Resistant (MDR). This implied that these isolates were resistant to all first line classes of treatment such as ampicillin, chloramphenicol and sulfamethoxazole-trimethroprim. In addition to this, these isolates were also resistant to at least one of the currently recommended drugs of choice, either a β-lactam or a fluoroquinolone. This study observed resistances at 18.2% and 15.4% to fluoroquinolones and cephalosporins respectively. PCR results revealed presence of bla TEM , bla INT and bla CTX-M genes coding for resistance to β-lactams in 80% of the isolates that had combined resistance to β-lactams and fluoroquinolones. It is likely that How to cite this paper: Kavai

Introduction
Typhoid fever which is a prolonged bacteremic illness with or without diarrhea [1] is caused by the bacteria Salmonella enterica serovar Typhi (S. Typhi). Salmonella Typhi is primarily adapted to humans as the primary host [2]. Typhoid fever is mainly a disease of adults and older children [3] over ten years of age and is commonly spread through the fecal-oral route via food, drinks, and contaminated water. Human carriers can also contaminate water or food and spread to other people through ingestion of the contaminated materials [4], [5].
There is great variation in the clinical course of typhoid fever and this ranges from fever to marked multi-organ system toxaemia [6]. Generally, Typhoid fever is characterized by abdominal pains, sore throat, malaise, nausea, vomiting, weakness, dizziness, and fever. Chronic infection or complicated typhoid infection also sometimes causes confusion [7]. Patients who develop severe disease thereafter range from 10% -15% [8].
In endemic regions, lack of nonspecific features such as diarrhoea, vomiting, or predominantly respiratory symptoms may lead to misdiagnosis of typhoid fever. This is because typically it has an incubation period of 10 -14 days.
It is also mostly associated with prolonged low-grade fever, headache, malaise, myalgia, dry cough, anorexia, and nausea. In sub-Saharan Africa, the incidence of typhoid fever was estimated at 725 cases/100,000 persons in 2010. However, West and Central Africa lacked studies of incidence data [2] [9]. In a single study in Kibera slums in Kenya, among children 2 to 8 years of age, incidence was estimated at 520 per 100,000 person-years of observation [5].
Multidrug resistant clones of S. Typhi are a huge challenge in management of the infection in Kenya. Previous studies recorded a prevalence of 77.2% multidrug resistant S. Typhi isolates collected from hospitals within Nairobi County over the period 1997-2007 [10]. Currently over a third of S. Typhi isolates in many endemic areas are multidrug resistant. They equally have diminished susceptibility to fluoroquinolones, which have been the drugs of choice for MDR cases over the last ten years [3]. Other alternative drugs including extended spectrum β-lactams are also becoming less effective with the growing problem of resistance [11]. The MDR S. Typhi is a major problem especially in poor informal settlements in Kenya. In these areas, effective alternative antimicrobials are too expensive to be afforded by majority of the population or else unavailable to the general public [5].

Bacterial Isolates
The study utilized a quasi-experimental design analyzing archived isolates from blood and rectal swabs that were obtained from an ongoing surveillance study.
The isolates were stored in −80˚C freezers located at Centre for Microbiology Research, KEMRI.
A total of 287 isolates were used of which 90 were from Mukurukwa Njenga Briefly, the archived vials containing S. Typhi isolates were removed from the −80˚C freezer and allowed to thaw at room temperature. A loop-full of the isolate was inoculated using the streak plate method on Mac Conkey agar (Oxoid).
Incubation was done at 37˚C for 24 hours. S. Typhi was confirmed by serotyping tests using the slide agglutination technique whereby polyvalent antisera O, followed by antisera 9, 12, Vi and d (Murex Diagnostics, Dartford, UK).

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing was performed on the isolates using the Kirby-Bauer disc diffusion technique [12]. The inoculum for susceptibility test-

Data Analysis
Data analysis was done using the SAS software, version 9.3 (SAS Institute).
Chi-square test was applied for P-value derivation for analysis of patient Metadata and risk factors associated with Typhoid Fever. (P < 0.05) was considered statistically significant. WHO-net program was used to analyze trend for resistance with data presented in bar graphs.

Prevalence of MDR in S. Typhi
In the 287 S. Typhi utilized in this study, the overall prevalence of MDR was 55.5%. Resistance to fluoroquinolones and β-lactams was observed at 18.2% and 15.4% respectively. 34 isolates had combined resistance to fluoroquinolones and β-lactams.

Selected Meta Data on the Patients with MDR S. Typhi
A total of 11 (32.4%) of these isolates came from female patients while 23 (67.6%) came from male patients. 21 (61.7%) of the isolates were obtained from stool samples ( Figure 1) while 13 (39.3%) were obtained from blood samples ( Figure 2). Among this 25 (73.5%) were obtained from patients who had fever at the time they visited the clinic while 9 (26.5%) tested negative for fever. A totalof 15 (67.6%) S. Typhi obtained from stool samples were MDR, in addition to being MDR, they also had combined resistance to β-lactams and fluroquinolones. Most resistance was observed against nalidixic acid (14.4%) and amoxicillin clavulanate (13.8%). Ciprofloxacin and cefpodoxime had the lowest level of resistance at 0.6% each (Figure 1). Amoxillinclavulanate had the highest resistance level (15.6%) in patients who ≤5 years of age (Table 3). This was closely followed by nalidixic Acid (11.3%).
Patients in the bracket of 6 -10 years had S. Typhi that showed highest resistance to nalidixic acid (17.3). This was similar to the patients in the 11 -16 age brackets (33.3%).

Antimicrobial Profiles for the S. Typhi against β-Lactams and Fluoroquinolones
The most common resistant phenotype was ampicillin (64.9%), sulfamethoxazole-trimethroprim (59.6%), and chloramphenicol, (51.7%) ( Figure   3). Resistance was also observed against nalidixic Acid (16.2%) and amoxicillin clavaulate (11.3%). cephalosporin's' had a low resistance compared to other antimicrobials (Table 4).      There was no significant association between the Samples the S. Typhi were obtained from and the gender of the patients the samples came from in relation to contracting Typhoid fever (Table 1). There also was no significant relation-

Discussion
The overall prevalence of MDR S. Typhi in this study was 55.5%. In 1997-1999 a study done in Kenya from hospital isolates found the MDR in S. Typhi to be 65% [14]. While another study carried out in the same study sites in 2010 had the prevalence of MDR S. Typhi at 70% [3]. The prevalence in this study clearly shows a decrease in the MDR S. Typhi. Variations in prevalence rates over time can be attributed to environmental, economic and social settings of a community especially the ones involved these studies, including different patterns of antimicrobial use for treatment of typhoid and other endemic bacterial diseases over time.
Characterization of outbreaks caused by MDR S. Typhi in Asia and some parts of Africa has been well documented [5] [10]. Typhoid fever outbreaks in Sub-Saharan Africa are rarely documented hence data on prevalence and antimicrobial susceptibility trends are scarce [10].
Therefore the observed MDR S. Typhi in Kenya though showing a decrease over time is still something to worry about as S. Typhi are multidrug resistant to most of the commonly available antimicrobials.
Data from this study confirms there is rise in resistance in the recommended drugs for Typhoid Fever such as nalidixic acid (16.2%) and amoxicillin clavulanate (11.3%). The increased prevalence of MDR S. Typhi strains resistant to nalidixic acid should be of public health concern as it is a marker for possible complete fluoroquinolone resistance arising [15].
Resistance was encoded on PARC and PARE genes. In our settings cases of MDR infections are managed using fluoroquinolones, such as ciprofloxacin and norfloxacin. These have been widely used in the last twenty years. Currently, most patients who turned positive for Typhoid fever emerging from slow fever and other symptoms had the clinicians recommend ciprofloxacin as the drug of choice [16].
However with the current MDR prevalence of S. Typhi to drugs of choice, it is expected that the resistance to ciprofloxacin would eventually rise if proper measures are not taken to counter resistance [17]. In this study, cephalosporins had a low resistance compared to other antimicrobials. We observed bla CTX-M , bla TEM , bla INT which encode resistance to extended spectrum beta lactams.
However for the recommended drugs of treatment for typhoid fever, absolutely no resistance should have been observed. This implies that these drugs could still be effective. In cases where there is uncertainty about the susceptibility of a strain or indeed the diagnosis, it is advisable to select an extended spectrum cephalosporin such as ceftriaxone as the safest choice especially if the patient is admitted to hospital as resistance is currently unlikely [18]. It is important to note that resistance to azithromycin which is another effective oral option for uncomplicated typhoid diseasewas less than 10%. In order to secure its continued effectiveness its use should be prudent for management of typhoid disease [19].
Even in settings in the United States, where Typhoid fever is not endemic, pa-

Conclusion
This study observed very high resistance to ampicillin, sulfamethoxazole-trimethroprim, choramphenicol and tetracycline which were the most commonly available antimicrobials in our settings. Resistance was also observed in the β-lactams especially amoxicillin clavulanate and nalidixic acid.
Emerging resistance to nalidixic acid may be a precursor to low level resistance to flouroquinolones, a potentially worrisome situation as this is the current drug of choice for treatment of typhoid fever in Kenya.