Prevalence of Salmonella typhi in among febrile patients in a tertiary care hospital of South West Rajasthan.

Background: Salmonella enterica, serotype typhi, remains the predominant Salmonella species causing enteric fever in India. The mode of Salmonella typhi transmission is considered to be predominantly vehicle-borne through contaminated water or food. In India, the incidence of Salmonella typhi occurs between the months of April and June (dry season) followed by July and September (monsoon season). Typhoid fever may be difficult to distinguish clinically from other febrile illnesses and if left untreated, intestinal, neuropsychiatric, and other complications develop in some patients. Objective: The aim of this study was to determine the prevalence of S. typhi in bloodstream infections and its antimicrobial susceptibility pattern among patients with febrile illness. Methodology: Febrile patients admitted in the hospital who were prescribed blood culture tests and whose samples were sent to microbiology laboratory were included in the study. All blood samples (average 5 mL for adults and 2-3 mL for pediatric age group) were immediately inoculated into Bac-T ALERT aerobic blood culture bottles containing sodium polyethanol sulfonate as an anticoagulant (0.025%). If growth was isolated, isolated colony characteristics of growth and Gram stain were assessed. On Gram staining, typical nonlactose fermenting Gram negative bacilli were further subjected to species identification and detection of antimicrobial susceptibility pattern on the VITEK2.
Results: In this study period, a total of 511 blood culture (paired) samples were processed, out of which 47 isolates of Salmonella were obtained. Among these isolates, 33 (70.23%) were from males, and 14 (29.77%) were from females. Amongst these, 35 (74.4%) patients were from rural, 8 (17%) were from subrural, and 4 (8.5%) were from urban areas. Out of the total 47 isolates of Salmonella, 42 (89.36%) were Salmonella typhi, 2 (4.25%) were Salmonella paratyphi A and B each, and 1 (2.12%) was Salmonella enterica. Antimicrobial susceptibility pattern of Salmonella isolates revealed that all the isolates of Salmonella species were highly susceptible (95%-100%) to third generation cephalosporins (ceftazidime, ceftriaxone, cefepime, cefoperazone-sulbactam) and other higher antibiotics such as betalactamase inhibitors - piperacillin tazobactam (95%-100%) and Ticarcillin-clavulanic acid (100%). They were also highly susceptible (100%) to carbapenams (imipenem, merpenem, doripenem, and ertapenem) but showed a fairly decreased susceptibility was towards nalidixic acid with 15% for Salmonella typhi and 50% for other Salmonella isolates.
Conclusion: Surging drug-resistant Salmonella enterica cases, the level of resistance was not as high as predicted in our study population. Multidrug-resistant (MDR) trends may vary; therefore, drug susceptibility testing side-by-side to empirical therapy is mandatory, especially in developing countries where there is a practice of self-medication. Copyright:

Introduction

Enteric fever is prevalent world over and continues to be a major public health problem in developing countries. Annual mortality from typhoid has increased by 39% between 1990 and 2020.[1] Infections with S. typhi are responsible for approximately 20 million new cases of typhoid each year, globally.[2] In India, Salmonella enterica, serotype Typhi, remains the predominant Salmonella species causing enteric fever.[3]

The mode of Salmonella typhi transmission is considered to be largely indirect and predominantly vehicle-borne through contaminated water or food.[4] The role of water as a vehicle for typhoid fever has been appreciated since the late 1800s.[5] The risk for infection is high in low- and middle-income countries where typhoidal Salmonella is endemic and that have poor sanitation and lack of access to safe food and water.[6] Its epidemiology is also affected by seasonal variations.[78] In India, the peak incidence of Salmonella typhi occurs between the months of April and June (dry season) followed by July and September (monsoon season).[9] Typhoid fever may be difficult to distinguish clinically from other febrile illnesses and if left untreated, intestinal, neuropsychiatric, and other complications develop in some patients.

Culturing the bacteria from body fluids is the definitive test for the diagnosis of typhoid fever although inconclusive serological methods such as the Widal test are commonly employed in many health care settings.[1011] A specific diagnosis of typhoid requires access to a competent laboratory that can process blood cultures and such laboratories are uncommon in resource-poor regions. Microbiologic culture of blood or bone marrow remains the mainstay of laboratory diagnosis.[13]

Moreover, Salmonella species are increasingly evolving antimicrobial resistance to several commonly used antimicrobial agents. Although fluoroquinolones (FQ) are the drugs of choice to treat invasive Salmonella infections, decreased susceptibility to ciprofloxacin is increasing quickly worldwide.[14] The problem of MDR Salmonella has increased the challenge in the management of the disease in endemic regions by increasing morbidity and mortality as well as the cost of treatment. However, to control the spread of typhoid fever, surveillance for S. typhi and the assessment of antimicrobial susceptibility is essential. Therefore, the aim of this study was to determine the prevalence of S. typhi in bloodstream infections and its antimicrobial susceptibility pattern among patients with febrile illness in the South-Western part of Rajasthan wherein there are challenging lifestyle habits, and lack of awareness about personal hygiene is also prevalent

Materials and Methods

Study area, duration and design

A cross-sectional retrospective hospital-based study was performed in a multispecialty tertiary care center in South-Western Rajasthan for a period of 17 months from 1 March, 2019 to 31 August, 2020.

Study population and inclusion criteria

All the febrile patients enrolled in the hospital who had been prescribed blood culture tests by the treating physicians with their samples received in the microbiology laboratory were included in the study.

Exclusion criteria

All the patients not consenting for blood culture, who had received antibiotics within 1 week before presentation, or those presenting on more than one time during the study period were excluded from the study.

Sample size and ethical consideration

A total of 511 patients blood samples (paired) were collected and received in the Central Lab Microbiology Department for blood culture during the study period. Written informed consent was taken, and ethical clearance was taken from the ethical committee of the hospital.

Procedure

Under all aseptic precautions by using 70% alcohol followed by povidone iodine and then subsequently with alcohol (Triple Swab technique), all blood samples were collected (venous site). Blood culture bottles containing brain heart infusion broth were immediately cleaned with 70% alcohol, and the collected blood samples (average 5 mL for adults and 2–3 mL for pediatric age group) were immediately sent to the microbiology lab for further processing. These blood culture bottles were further inoculated into an automated 3D blood culture system Bac-T ALERT which contained 0.025% of sodium polyethanol sulfonate as an anticoagulant for detection of growth, which gives color-coded alarms when detected positive. These bottles were incubated for a period of a maximum of 5 days after which the sample was labeled sterile.

On bottles flagging positive, Gram-stain was done, and the microorganism was identified microscopically. A positive flagged bottle was sub-cultured on Blood agar and Mac Conkey agar subsequently and incubated overnight at 37°C following standard procedures. If any bacterial growth was isolated, along with colony characteristics Gram stain was assessed. All those showing typical nonlactose fermenting Gram negative bacilli were further subjected to species identification and detection of antimicrobial susceptibility pattern on the VITEK 2 which is a fully automated Advanced Expert Phenotypic System.

A 0.5 McFarland suspension of the colonies was prepared and processed following standard operating procedures. Antimicrobial susceptibility pattern and the extended-spectrum beta-lactamase (ESBL) status were determined by the latest Clinical and Laboratory Standard Institution guidelines.

Results

During the study period, a total of 511 blood culture (paired) samples were processed, out of which 47 isolates of Salmonella were obtained. Among these isolates, 33 (70.23%) were from males and 14 (29.77%) were from females. Amongst these, 35 (74.4%) patients were from rural, 8 (17%) were from subrural, and 4 (8.5%) were from urban areas. Out of the total 47 isolates of Salmonella, 42 (89.36%) were Salmonella typhi, 2 (4.25%) were Salmonella paratyphi A and B each, and 1 (2.12%) was Salmonella enterica [Table 1].

Table 1

Various Salmonella Spp. solated from blood samples

Organism	No. of isolates
Salmonella typhi	42
Salmonella paratyphi A	2
Salmonella parathyphi B	2
Salmonella enterica	1

The antimicrobial susceptibility pattern of the Salmonella isolates revealed that all the isolates of Salmonella species were highly susceptible (95%–100%) to thirdgeneration cephalosporins (ceftazidime, ceftriaxone, cefepime, cefoperazone-sulbactam) and other higher antibiotics such as betalactamase inhibitors – piperacillin tazobactam (95%–100%) and ticarcillin–clavulanic acid (100%). They were also highly susceptible (100%) to carbapenams (imipenem, merpenem, doripenem, and ertapenem) but showed a fairly decreased susceptibility was towards nalidixic acid with 15% for Salmonella typhi and 50% for other Salmonella isolates. The sensitivity pattern of Salmonella isolates for FQ was also found to be greatly reduced for ciprofloxacin, which was only 34% for Salmonella typhi, and all other isolates of S. paratyphi A, S. paratyphi B, and Salmonella enterica were fully resistant (100%) to it. Whereas for levofloxacin, there was intermediate susceptibility (50%–60%) amongst all.

For cotrimoxazole, fairly good sensitivity (70%) was observed amongst Salmonella typhi isolates and Salmonella paratyphi A and B, and Salmonella enterica were found to be fully susceptible (100%) to it as depicted in [Table 2].

Table 2

Sensitivity pattern of Salmonella species

Antibiotics	S. typhi (n=42)	S. paratyphi A (n=2)	S. paratyphi B (n=2)	S. enterica (n=1)
Amikacin	0 (0%)	0 (0%)	0 (0%)	0 (0%)
Gentamicin	0 (0%)	0 (0%)	0 (0%)	0 (0%)
Ciprofloxacin	14 (34%)	0 (0%)	1 (50%)	0 (0%)
Levofloxacin	25 (60%)	1 (50%)	1 (50%)	0 (0%)
Piperacillin/Tazobactum	39 (95%)	2 (100%)	2 (100%)	1 (100%)
Ceftazidime	39 (95%)	2 (100%)	2 (100%)	1 (100%)
Cefepime	42 (100%)	2 (100%)	2 (100%)	1 (100%)
Cefoperazone/Salbactum	39 (95%)	2 (100%)	2 (100%)	1 (100%)
Cefriaxone	39 (95%)	2 (100%)	2 (100%)	1 (100%)
Imipenam	42 (100%)	2 (100%)	2 (100%)	1 (100%)
Meropenam	42 (100%)	2 (100%)	2 (100%)	1 (100%)
Doripenem	42 (100%)	2 (100%)	2 (100%)	1 (100%)
Ertapenem	42 (100%)	2 (100%)	2 (100%)	1 (100%)
Ticarcillin/Clavulanic acid	42 (100%)	2 (100%)	2 (100%)	1 (100%)
Aztreonam	42 (100%)	2 (100%)	2 (100%)	1 (100%)
Trimethoprim/Sulfamethoxazole	29.4 (70%)	2 (100%)	2 (100%)	1 (100%)
Minocycline	42 (100%)	2 (100%)	2 (100%)	1 (100%)
Colistin	42 (100%)	2 (100%)	2 (100%)	1 (100%)
Nalidixic acid	6 (15%)	1 (50%)	1 (50%)	1 (100%)
Ofloxacin	42 (100%)	2 (100%)	2 (100%)	1 (100%)

Discussion

Salmonella serovar typhi and Salmonella serovar paratyphi A are the major agents of enteric fever. Changing trends of antimicrobial susceptibility pattern has been observed throughout different geographic regions of India which mandates constant surveillance and evaluation.

In the current study, out of the total Salmonella spp. isolated, the majority (89.36%) were Salmonella typhi followed by Salmonella paratyphi A and B (4.25% each). These findings were similar to the findings of the study done by Patil et al.[9] in 2019 which reported S. typhi isolates three times higher than S. paratyphi A isolates (76.5% vs. 23.5%). Altogether a study done by Misra et al.[11] in 2016 stated that S. typhi was the predominant isolate at 67%, followed by 18% S. paratyphi A and 10% were of S. typhimurium. Another study done by Khadaka et al.[10] reported S. typhi (68.8%) as a major isolate followed by S. paratyphi A (31.1%). The majority of the Salmonella isolates were from the rural patients which probably can be attributed to the poor faeco-oral hygiene and water sanitation conditions in these areas.

Salmonella isolates in the current study showed a decreased susceptibility towards FQ, especially Ciprofloxacin. As amongst Salmonella typhi isolates, only 34% of them were sensitive, among Salmonella paratyphi A isolates, 50% were sensitive, whereas all the isolates of Salmonella enterica and Salmonella paratyphi B were resistant to Ciprofloxacin. In studies done by Patil et al.,[9] only 32.9% of Salmonella typhi were susceptible to Ciprofloxacin, and Misra et al.[11] stated that 15% of Salmonella typhi and 16.7% of Salmonella paratyphi A isolates were reported susceptible to Ciprofloxacin.

However, a fairly good sensitivity pattern was observed for Cephalosporins (95%–100%) which is similar to findings reported in the studies done by Patil et al.[9] In a study by Khadaka et al.,[10] all the isolates showed good sensitivity patterns to third-generation cephalosporins. Furthermore, in the current study, Cotrimoxazole showed 70% sensitivity to Salmonella typhi isolates, whereas Salmonella paratyphi A and B and Salmonella enterica were fully susceptible (100%) which is very much similar to the findings stated by Khadaka et al.[10] (100%).

The susceptibility towards Nalidixic acid in the current study was greatly reduced to 15% for Salmonella typhi and 50% for Salmonella paratyphi A and B which is similar to the findings reported by Misra et al.[11] with 12.6% susceptibility and by Khadaka et al.[10] with 6.7% suceptibility Rahman MA et al.[12] also with 15.4%.

Conclusion

This study concludes the re-emergence of susceptibility pattern of Salmonella strains to various broad-spectrum antibiotics. A major concern about FQdecreased susceptibility and cephalosporins such as cefepime, ceftriaxone on the other hand being, more susceptible, emphasizes the need for continuous evaluation and judicious use of antimicrobials, considering the ever-changing landscape. A surge in drug-resistant Salmonella enterica cases and even the level of resistance was not as high as predicted in our study population. Looking at the trends of multidrug resistance scenarios, thus drug susceptibility testing side-by-side to empirical therapy is mandatory, especially in developing countries where there is a practice of self-medication. Referring to our findings, higher susceptibility of Salmonella enterica to the conventional anti-typhoidal drugs was attributed compared to macrolides and fluoroquinolones. Therefore, reconsideration of these antibiotics as implicated therapies could be useful in clinical management. Henceforth, more prospective studies are warranted or should be encouraged to correlate the clinical outcome of treatment based on in vitro antimicrobial susceptibility patterns of Salmonella isolates in typhoid cases.

Limitation of study

Among self-medicated cases, FQ-sensitive isolates might have failed to grow contributing to the lower incidence; therefore, the exact incidence of the disease could be even greater than observed. Besides, lacking the molecular laboratory set-up (presumed as a necessity for high-quality data in clinical studies) in our settings was another drawback as the blood culture has limited sensitivity.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.