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Updated: Sep 22, 2025
High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages
Published on: August 11, 2014
Macrophage Cell Lines and Murine Infection by Salmonella enterica Serovar Typhi L-Form Bacteria
Debayan Ganguli1, Swarnali Chakraborty1, Suparna Chakraborty1
1ICMR-National Institute of Cholera and Enteric diseases, Beleghata, Kolkata, West Bengal, India.
Abstract:
Antibiotic resistance of pathogenic bacteria has emerged as a major threat to public health worldwide. While stable resistance due to the acquisition of genomic mutations or plasmids carrying antibiotic resistance genes is well established, much less is known about the temporary and reversible resistance induced by antibiotic treatment, such as that due to treatment with bacterial cell wall-inhibiting antibiotics such as ampicillin. Typically, ampicillin concentration in the blood and other tissues gradually increases over time after initiation of the treatment. As a result, the bacterial population is exposed to a concentration gradient of ampicillin during the treatment of infectious diseases. This is different from in vitro drug testing, where the organism is exposed to fixed drug concentrations from the beginning until the end. To mimic the mode of antibiotic exposure of microorganisms within host tissues, we cultured the wild-type, ampicillin-sensitive Salmonella enterica serovar Typhi Ty2 strain (S. Typhi Ty2) in the presence of increasing concentrations of ampicillin over a period of 14 days. This resulted in the development of a strain that displayed several features of the so-called L-form of bacteria, including the absence of the cell wall, altered shape, and lower growth rate compared with the parental form. Studies of the pathogenesis of S. Typhi L-form showed efficient infection of the murine and human macrophage cell lines. More importantly, S. Typhi L-form was also able to establish infection in a mouse model to the extent comparable to its parental form. These results suggested that L-form generation following the initiation of treatment with antibiotics could lead to drug escape of S. Typhi and cell to cell (macrophages) spread of the bacteria, which sustain the infection. Oral infection by the L-form bacteria underscores the potential of rapid disease transmission through the fecal-oral route, highlighting the need for new approaches to decrease the reservoir of infection.
Insights
Antibiotic treatment can induce temporary bacterial resistance. Salmonella Typhi developed cell-wall deficient L-forms, which infected macrophages and mice, suggesting a new mechanism for persistent infection and transmission.
Area of Science:
- Microbiology
- Infectious Diseases
- Bacterial Pathogenesis
Background:
- Antibiotic resistance is a global public health threat.
- Stable resistance mechanisms are well-studied, but temporary, reversible resistance induced by antibiotics is less understood.
- Bacterial cell wall-inhibiting antibiotics like ampicillin create concentration gradients in vivo, unlike fixed concentrations in vitro.
Purpose of the Study:
- To investigate temporary resistance in Salmonella Typhi (S. Typhi) induced by ampicillin exposure.
- To mimic in vivo antibiotic concentration gradients during bacterial culture.
- To characterize the resulting bacterial forms and their pathogenic potential.
Main Methods:
- Cultured ampicillin-sensitive S. Typhi Ty2 with increasing ampicillin concentrations over 14 days.
- Characterized the resulting bacterial strain for cell wall presence, morphology, and growth rate.
- Assessed the pathogenicity of the modified S. Typhi in murine and human macrophage cell lines and a mouse infection model.
Main Results:
- Developed an S. Typhi strain with L-form characteristics: absent cell wall, altered shape, and reduced growth rate.
- The S. Typhi L-form efficiently infected macrophage cell lines.
- The S. Typhi L-form established infections in mice comparable to the parental strain.
Conclusions:
- Antibiotic-induced L-form generation may allow S. Typhi to evade treatment.
- L-forms can spread via cell-to-cell transmission (macrophages), sustaining infection.
- Oral infectivity of L-forms suggests fecal-oral transmission, necessitating new control strategies.
