Characterization of multi-drug resistant Salmonella typhi from clinical specimens

Characterization of multidrug resistant Salmonella typhi from clinical specimens was investigated. Three hundred and sixty-four (364) clinical specimens of both stool and blood were collected from patients attending selected health facilities in Bauchi metropolis. The specimens were cultured using standard media, biochemical as well as serological tests for somatic H and O antigens were used to identify isolates. Antibiotics susceptibility test was performed using standard disk diffusion method, the isolates were screened for Multidrug resistance. The total of 9(2.4%) of the 364 specimens collected yielded positive for Salmonella typhi. of the Nine (9) positive Salmonella typhi 5(55.5%) were from females and 4(44.4%) from males. Typhoid fever was most frequent in children 5(55.5%) than in adults. There was no significant difference in isolates from the different health facilities with their p values >0.05. All isolates screened were resistant to more than three (3) antibiotics, of the total isolates, 88.8% were found to be resistant to ampicillin, 77.7% resistant to cotrimoxazole, and 88.8% resistant to chloramphenicol. However, resistance to ciprofloxacin by the isolates were not found. Therefore, ciprofloxacin remains the drug of choice for severe cases of typhoid fever, although caution should be exercised by clinicians in their prescription such that fluoroquinolones antibiotics therapy is used only in laboratory-proven cases of the disease and Salmonella-associated bacteremia to preserve its efficacy. To prevent occurrence of typhoid fever, good personal hygiene, provision of portable drinking water and environmental sanitation should be employed.


Introduction
In recent past, emergence of ever-increasing number of antibiotic resistant microbial strains has become a severe health threat to human-kind and one of the biggest challenges to global drug discovery programs [1].The inappropriate and over-use of antibiotics and other antimicrobials to treat infections and consequent antibiotic selection pressure are thought to be the major causative factors contributing to the appearance of strains with reduced susceptibility to antibiotics [2].
Typhoid fever is largely a disease of developing nations due to their poor standard of hygiene and unavailability of clean water.It is transmitted faeco-orally through contaminated food and water.Salmonella, a primary inhabitant of the gastrointestinal tract, is recognized as one of the most common causes of diarrhea infections worldwide, resulting in millions of infection and significant human death annually [3].People most at risk for serious complications due to Salmonella infections include older adults, pregnant women, infants, children, and people who have compromised immune systems [4].
Multidrug-resistant typhoid fever (MDRTF) is defined as typhoid fever caused by Salmonella typhi strains which are resistant to all the three first-line recommended drugs for treatment i.e. chloramphenicol; ampicillin and cotrimoxazole [5].Although the global burden of typhoid fever has reduced, emergence of multidrug-resistant Salmonella typhi (MDRST) is still a threat to public health.Despite the emergence of newer antibacterial drugs, enteric fever has continued to be a major health problem [5].Salmonella typhi gained resistance to antibiotics like ampicillin, ceftriaxone, and cotrimoxazole, besides developing resistance to efficacious drugs like ciprofloxacin.The aim of this study is to characterize multidrug resistance Samonella typhi from clinical specimen in selected health facilities.

Specimen collection
A total of 364 blood and stool samples was collected from the study areas to determine the presence of Salmonellae pathogens.Fluid Tetrathionate (8 ml) broth in Macartney bottles were used to collect 2ml of venous blood.About 1gram of stool sample was dispensed in nine (9 ml) of Selenite broth F (PART I and PART II) were used for pre enrichment and incubation was done at 370C for 48 hours and 24 hours respectively Turbidity was observed for both.

Isolation of Salmonella typhi from stool specimen
About 1 gram of faecal specimen was placed in Selenite F broth (Oxoid Limited, Hampshire, England) to allow for the multiplication of bacteria and incubated for 24 hrs at 37 °C, then sub cultured onto Salmonella-Shigella Agar, MacConkey agar and Brilliant Green agar, it was then incubated for 24 hrs at 37 °C.Typical colonies of Salmonella appear as pink colonies with or without black centers.Many cultures of Salmonella may produce colonies with large, glossy black centers or may appear as almost completely black colonies.The representative Salmonella colonies were characterized morphologically using Gram' staining method according standard protocols [6].

Isolation of Salmonella typhi from blood specimen
About 2 ml of venous blood will be drawn aseptically from each patient by cleaning the skin using tincture of iodine, and placed into 8 ml of tetrathionate broth (Oxoid Limited, Hampshire, England).Blood culture broths were incubated at 37 o C and checked for signs of bacterial growth daily for up to ten days.Bottles which showed signs of growth were sub-cultured onto Salmonella-Shigella Agar, Brilliant Green agar and MacConkey agar.Blood culture broths with no bacterial growth after seven days were also subcultured before being reported as Negative.

Biochemical screening
Biochemical test such as Sugar Fermentation, Urease, Hydrogen Sulphide production were carried out using standard procedures.

Antimicrobial sensitivity pattern and multi-drug resistance screening
The susceptibility testing was carried out by disc diffusion method using Mueller Hinton agar and it was tested in vitro for susceptibility to the following antibiotics (OXOID Ltd., UK) suggested by WHO,2010, ampicillin (A,10 µg), amoxicillin (AMC,30 µg), augmentin (AUG,15 µg), amikacin (AK, 10 µg), ceftriaxone (CFX,30 µg), ceftazidine (CTX, 30 µg),ciprofloxacin (CFX, 10 µg), ,neomycin (N, 30 µg), erythromycin (E, 25 µg),chloramphenicol (C, 30 µg), naliixic acid (NA, 30µg), and imipenem (I,10 µg) this is based on [6] This was conducted using the method of [7].Using a sterile wire loop, 3-5 well isolated colonies were picked and emulsified in nutrient broth.The prepared turbidity was matched with a turbidity standard (0.5 McFarland) to have an equivalent suspension.Sterile swab was used to inoculate the suspension by streaking on the prepared and dried Mueller Hinton agar plate evenly.It was then allowed to stay for 3-5 minutes.Sterile forceps were used to place the antimicrobial discs on the inoculated plates.Within 30 minutes after applying the disc, the plates were incubated at 35 O C for 16-18 hours.Zone diameter for ATCC 25922 was compared with NCCLS Published Limits; Interpretative chart was then used to interpret the zone sizes of Inhibition.Result was recorded as susceptible, intermediate susceptible, or resistant based on the Zones sizes of each antimicrobial disc used [6], [2].This present study indicates that Females had the highest percentage (55.5%)while males had the least (44.4%)Table 2.This is in agreement with report of [8] which could be attributed to the fact that females are closer to edible items than their male counterparts.It also indicated that outpatients (77.7%) had the most percentage of Salmonella typhi isolated than the in patients (22.2%).The frequency of occurrence of S. typhi across age group in selected health facilities is represented in table 3. Age group 0-9 had the highest frequency of occurrence and age group 60-69 had the least.There was no significant difference between the age group and the number of isolates as p>0.05.[9] showed that prevalence of typhoid fever in endemic areas is considered high in n school aged children and young adults.Older adults are presumably relatively resistant due to frequent boosting of immunity.

Figure 1 Frequency of occurrence of different isolates
Figure 1 shows frequency of different enteric bacteria isolates with Escherichia coli having the highest occurrence at 63.4% and the least was Enterobacter at 2.2%.S. typhi was found to be 5.5% out of the total number of samples collected.Resistance to ampicillin, chloramphenicol, cotrimoxazole and ciprofloxacin was observed in this study, the resistance could be as a result of indiscriminate use of antibiotics.The inclusion of preventive doses of antimicrobial agents in poultry feed as growth promoters is often associated with the development of resistance in enteric bacteria.During this study, of the total isolates, 88.8% were found to be resistant to Ampicillin, 77.7% resistant to Cotrimoxazole, and 88.8% resistant to Chloramphenicol.However, resistance to Ciprofloxacin by the isolates were not found.The use of antimicrobials for growth-promotion, prophylaxis and treatment of animal of animal's food increases the prevalence of resistance in human pathogens.High susceptibility of Salmonella typhi was observed against fluoroquinolones (ciprofloxacin).This finding is similar to a report by [10].However, in Lagos, Nigeria, [11] reported 18% reduced susceptibility of Salmonella spp.To ofloxacin and ciprofloxacin.The high susceptibility of enteric bacteria to the fluoroquilones recorded in this study may be connected to relatively high cost of ciprofloxacin [11].Therefore, fluoroquinolones are not used indiscriminately because not many could afford them.

Conclusion
Multidrug resistant Salmonella typhi (MDR) is progressively increasing compared to earlier reports in the country.
Children account for the highest percentage of Salmonella typhi isolated from the study group.The high incidence rate suggest that challenges of portable water availability exist in the community.Apart from contaminated water, faecaloral route of transmission may also be a reason for the preponderance in children of Salmonella infection in children.Resistance to first line antibiotics (Ampicillin, Chloramphenicol and Cotrimozaxole) suggest possible abuse of drugs and consequent high resistance in the community.Resistance to Ciprofloxacin was not recorded which may be due to less frequent use in infection treatment because of their costs.Ciprofloxacin still remain the drug of choice but caution should be taken by clinicians to avoid resistance of Salmonella typhi to the antibiotic.

Table 1
Distribution of specimens collected and percentage of Salmonella typhi isolated from blood and stool according to different health facilities in the study area

Table 2
Gender and patient group distribution pattern of specimens collected and S. typhi isolated in the study area [2]alue (x 2 ) > 0.85 Table1indicates the distribution of specimens and percentages of Salmonella typhi from blood and stool in different health facilities.The table shows that there was no significant difference (p>0.05) between blood and stool samples.Stool had the highest number of positive samples 5(55.5%) and blood had the least 4(44.4%).The isolation of S. typhi from stools and bloods is suggestive of typhoid fever.Widal test which is a serological test is the most widely requested by clinicians.The test has only moderate sensitivity and specificity.It can be negative in up to 30% of culture-proven cases of typhoid fever.This may be because of prior antibiotic therapy that has blunted the antibody response.On the other hand, S. typhi shares O and H antigens with other Salmonella serotypes and has cross-reacting epitopes with other Enterobacteriacae, and this can lead to false-positive results.Hence, the gold standard for Salmonella typhi identification is in blood and stool cultures[2].

Table 3
Frequency of occurrence of Salmonella typhi across age group and selected health facilities in the study area

Table 4
Antibiotics susceptibility pattern of S typhi isolated from the study area