Title : Phenotypic changes in salmonella typhi
Abstract:
Salmonella enterica serotype Typhi (Hereafter referred to as S.Typhi) is the causative agent of Typhoid. Evolution of drug resistance in S.Typhi, initially as MDR strains (Strains resistant to Chloramphenicol, Ampicillin, and co-cotrimoxazole) to resistance to Fluoroquinolones after a decade. Currently, ceftriaxone (3rd cephalosporin) has been the drug of choice for treatment. Recently there have been reports from different parts of the world and from Gujarat of Ceftriaxone resistant S.Typhi because of the acquisition of blaCTX-M 15.1 The bacilli exhibited Bipolar staining (Safety-pin appearance). With this phenotypic change, we hypothesize its possible association with the resistance pattern.
Introduction: Salmonella is a genus of Gram-negative bacilli of the family Enterobacteriaceae. It has two species Salmonella enterica and Salmonella bongarii. S.enterica is further divided into six subspecies2 that contain over 2600 serotypes3. Salmonella species are non-spore-forming, mostly motile with peritrichous flagella, measuring 0.7-1.5 µm in diameter and 2 - 5 µm in length.4 The Centers for Disease Control and Prevention (CDC) is currently using the Salmonella nomenclature system suggested by the World Health Organization (WHO) Collaborating Centre as a nomenclature system.: Species: Salmonella enterica serotype Typhi.5
The evolution of drug resistance in Salmonella Typhi is a great public health concern. Multi-Drug Resistance (MDR) that emerged in 1990 is defined, in the case of S.Typhi, as resistance to Chloramphenicol, Amoxicillin, and Co-cotrimoxazole. This was followed by a decreased susceptibility to Fluoroquinolones and high-level fluoroquinolone resistance emerged and spread throughout the world.6 Clinicians relied, then, more on third-generation cephalosporins and Azithromycin. Recently there have been reports from different parts of the world and from Gujarat of Ceftriaxone resistant S.Typhi because of the acquisition of blaCTX-M 15.1
Clinicians in Vadodara, during recent upsurge of S.Typhi cases, experienced that even in cases of Ceftriaxone susceptible Salmonella Typhi infections the patients required prolonged treatment (up to two weeks) to render the patient afebrile.
We observed a conspicuous phenotypic change in S. Typhi bacilli in the gram-stained smear made directly from positive blood cultures.
Material and methods: The study was carried out at the Central Laboratory of Parul Sevashram Hospital of Parul Institute of Medical Sciences and Research. Our Central Laboratory is NABL accredited and the blood cultures are performed on an automated system. Identification and Drug Susceptibility of the bacterial Isolates is done on VITEK 2.
Result: The study comprised of 25 Blood culture positive typhoid cases in last three months. Recently our city (Vadodara) experienced a sudden upsurge in the cases of Typhoid and the cases were more severe requiring prolonged antibiotic treatment.
We observed that Gram's stained smears made from positive blood culture bottles, revealed conspicuous changes in the bacilli. The bacilli showed Bipolar staining (Safety pin appearance) under the Oil immersion
Discussion: We hypothesize an association between emerging phenotypic change in the bacilli and the changes in their tolerance to antibiotics and requiring prolonged antibiotic therapy.
Since the 1990s, it has been well-accepted that a key component of the pathogenesis of B. pseudomallei is its ability to survive intracellularly in both phagocytic and non-phagocytic cells.7 Our findings open a new window for further genotypic studies to explore the association between phenotypic change and acquisition of virulence factors.
Recent trends in S typhi bacteremia: The temporal change in trends of antimicrobial resistance in Salmonella typhi bacteremia has been observed in the state of Gujarat in the last few months. Cephalosporins have been the mainstay of management of MDR S Typhi infections for the past several years. Perhaps cephalosporins failed as empirical therapy in a substantial number of cases as we observed a rise in ESBL XDR S.Typhi infections in recent outbreaks. Also, the children especially required longer hospital stays due to delayed fever defervescence. Average days for fever defervescence were observed to be 6-8 days of Intravenous therapy. Prolonged fever in most children was observed to be due to immune dysregulation or exaggerated immune response as we ruled out other causes, such as persistent bacteremia, deep-seated abscesses, or secondary HLH. Culture-proven S typhi bacteremia was also observed in children who were vaccinated with 2 doses of Typhoid conjugate vaccine. Relapse and complications were observed in those who were treated with inappropriate antibiotics and for inadequate duration. Distant complications such as osteomyelitis and spondylodiscitis were also observed in children treated for enteric fever.
Traditionally, a Bipolar staining pattern was used for probable identification of Burkholderia species especially pseudomallei in blood culture in patients with community-acquired pneumonia or B.cepacia in case of VAP. The other bacilli having similar staining patterns include Yersinia pestis and Fransisella tolerances.
Since the 1990s, it has been well-accepted that a key component of the pathogenesis of B. pseudomallei is its ability to survive intracellularly in both phagocytic and non-phagocytic cells7Our findings foster a probable association of the phenotypic characteristic with the acquisition of some virulence factors by S.Typhi and open a new window for further genotypic studies to find factors responsible for the intracellular survival of these strains.
Conclusion: This is the first observation study regarding the phenotypic change in the staining property of Salmonella Typhi. We hypothesize an association between the phenotypic change and change in the behavior of typhoid infections. We suggest further studies in S.Typhi virulence mechanisms and to develop new guidelines for the treatment of typhoid to tackle the menace.
Ethical statement: The study was approved by the institutional ethics committee, with reference no. IEC-INT/2022/Study-134, dated April 12, 2022. The patient gave informed consent for the publication.
