Antibiotic resistance trends in high-risk lineages of Salmonella enterica serovar Typhi: A study spanning pre to post COVID-19 pandemic.

OBJECTIVES: The aim of this study was to understand the status of extensively drug-resistance (XDR) genotype in Salmonella enterica serotype Typhi (S. Typhi) recovered during the pre to post COVID-19 pandemic period using Multiplex PCR. METHODS: A longitudinal descriptive study was carried out during five years. Antibiotic susceptibility testing was performed according to the Clinical Laboratory Standards Institute antimicrobial susceptibility testing guidelines. The identification of S. Typhi, the detection of their high-risk lineages and XDR genotype was done using single nucleotide polymorphism-based multiplex PCR. RESULTS: A total of four hundred nine (n = 409) S. Typhi isolates were recovered during pre to post COVID-19 pandemic period. Among them, 30.81% belonged to the pre COVID-19 period while 69.19% to the post COVID-19 period. Different trends in antibiotic resistance in S. Typhi isolates with high prevalence of XDR-S. Typhi were observed. However, there was comparatively different frequency of their occurrence among the S. Typhi isolates recovered during pre to post COVID-19 pandemic period. Multiplex PCR showed that the majority of S. Typhi isolates were the H58 haplotype or genotype 4.3.1 which contained XDR genotype. CONCLUSIONS: The increasing episodes of XDR-S. Typhi causing typhoid fever in endemic areas is alarming. The antibiotic resistance in food and water borne pathogens greatly contribute to the dissemination of the antimicrobial resistance in pathogenic bacteria, which has now been considered as a global concern.

The dynamics of colicin E9 release from Escherichia coli in native conditions.

Colicin (Col) plasmid contains colicin encoding genes arranged in an operon controlled by an SOS inducible promoter. Therefore, any external stresses to the host cell can induce the expression of the downstream genes in the Col operon, including a lysis gene. The lysis protein is involved in the extracellular release of colicin through lysis of the producer cells, which causes a decline in culture turbidity. However, it is not yet known that E. coli cells with the native pColE9-J plasmid hold the same level of cell death at the population level following a set of induced conditions. In this study, using a mitomycin C sensitivity assay along with a live dead staining method of detection, we showed that the native pColE9-J plasmid, which unusually carries an extended Col operon (ColE9) containing two lysis genes, did not confer a rapid decline in the culture turbidity following induction with mitomycin C. Interestingly a subset of the cells suffered perturbation of their outer membrane, which was not observed from single lysis mutant (∆celE or ∆celI) cells. This observed heterogeneity in the colicin E9 release leading to differential outer membrane perforation may bring a competitive advantage to these cells in a mixed population.

Heterogeneity in the spontaneous induction of the promoter of the ColE9 operon in Escherichia coli.

Spontaneous production of E colicins is known to occur in only a small fraction of colicinogenic population. The current study aimed to determine if the same holds true for the production of colicin E9 in real time, by investigating the induction dynamics of the promoter of the ColE9 operon which results in the expression of the ColE9 activity and functional genes. A novel fluorescent reporter was constructed which carries the fusion of the ColE9 promoter and the gfpmut2 gene in a low copy number plasmid that was compatible with the native ColE9-J plasmid. Using the fluorescent reporter construct in the non colicinogenic E. coli cells, the induction of the ColE9 promoter was investigated. The current study demonstrates that the spontaneous induction of the ColE9 promoter occurs in a heterogenous manner and this heterogeneity is maintained in a bacterial population for several generations suggesting that it is unlikely due to any irreversible mutation in the bacterial culture. Furthermore, the same investigations were repeated using the colicin E9 producing E. coli cells. Flow cytometry analysis revealed that 7.1 ± 0.68% of the colicin E9 producing E. coli cells expressed GFP albeit only 2.45 ± 0.30% was observed from non colicinogenic E. coli cells. The considerable increase in the number of the fluorescent cells was likely due to the DNase activity of colicin E9 produced by their clonemates, resulting the auto-induction, which can be abolished with the inactivation of the DNase activity of the colicin E9.

In vitro growth inhibition and cytotoxicity of Euphorbia caducifolia against four human cancer cell lines and its phytochemical characterisation.

Several Euphorbia species have been used in folklore as cancer remedies, however, scientific studies on the cytotoxicity (in vitro studies) of Euphorbia caducifolia are lacking. In present study, anticancer potential of E. caducifolia aerial parts ethanol extract and its fractions were evaluated against human lung (NCI-H460), breast (MCF-7), prostate (PC-3) and cervical (HeLa) cancer cell lines, using sulphorhodamine-B in vitro cytotoxicity (in vitro studies) assay. The ethanol extract demonstrated growth inhibitory effect against all aforementioned cancer cell lines with IC50, 19-135 µg/mL and LC50, ~220 µg/mL, and its petroleum ether fraction obtained on bioactivity guided fraction showed highest activity with IC50, 28-70 µg/mL and LC50, 71 µg/mL against NCI-H460 and MCF-7 cell lines. Its phytochemicals were analysed by gas chromatography-mass spectrometry (GC-MS). The present study provides scientific justification for its traditional use against cancer.

Detection of induced synthesis of colicin E9 using ColE9p::gfpmut2 based reporter system.

The majority of colicin operons are regulated by an SOS response inducible promoter (SOS promoter), located at upstream of the colicin operons. Therefore, colicin synthesis is induced by DNA damaging agents like mitomycin C (MMC) because the resulting DNA damage switches on the SOS response in bacteria. In this study, we have described the strategy for fusion of the SOS promoter of the colicin E9 operon (ColE9p) with a promoterless green fluorescent reporter gene (gfpmut2). We observed that the ColE9p-gfpmut2 is inducible by MMC which confirmed that the ColE9p-gfpmut2 is sensitive to SOS response inducing agents. The data implies that the ColE9p-gfpmut2 based reporter system is suitable for monitoring the ColE9 synthesis induced by SOS response inducing agents including antibiotics. Using green fluorescent protein expression from the ColE9p-gfpmut2 as an indicator of ColE9 synthesis; we have investigated, first time, the inducing effects of cephalexin antibiotic on ColE9 synthesis. Our data demonstrated that the cephalexin has potential to induce ColE9 synthesis from E. coli JM83 host cells albeit the level of this induction is very low hence its detection required a highly sensitive method.

Pattern of induction of colicin E9 synthesis by sub MIC of Norfloxacin antibiotic.

The presence of dual SOS boxes in the regulatory region of the most of colicin operons confines synthesis of colicin to times of stress, presumably to reduce the cost of its production. However, in presence of certain inducing agents, such as antibiotics, this tight control of colicin operon is usually lost. Although synthesis of most of colicins is known to be regulated by SOS response of host cell, different patterns of induction from distinct colicins against various inducing agents have been shown in recent years. In this study, we investigated the induction pattern of enzymatic colicin E9 (ColE9) synthesis following treatment with various concentrations (sub MICs) of the Norfloxacin (NOR) using pSBM23 construct which carries transcriptional fusion of SOS inducible promoter of pColE9 (ColE9p) and a fluorescent reporter gene (gfpmut2) into kanamycin resistant pColE9-J plasmid. Flow cytomtry analysis of the Escherichia coli cells containing pSBM23, following treatment with various concentrations showed that the SOS response mediated induction of the synthesis of ColE9 happens in a dose-dependent manner. In summary, our results suggest that the presence, even in a minute amount, of SOS response inducing agents such as fluoroquinolone antibiotic in natural habitat of colicinogenic population can promote such a costly antagonistic behaviour of microbes.

Resonance Rayleigh scattering, second-order scattering and frequency doubling scattering spectra of copper(II)-Flutamide system with anionic surfactants and its analytical application.

A simple, sensitive, and rapid method based on ion association, for the determination of FLD has been developed. Flutamide (FLD) can react with Cu(II) to form 1:1 cationic chelate at pH 2.2-7.0 Mclivaine buffer medium, which can further react with anionic surfactants (AS) such as sodium dodecyl sulfate (SDS), sodium lauryl sulfonate (SLS) and sodium dodecylbenzene sulfonate (SDBS) to form 1:1 ion-association complexes. As a result, the resonance Rayleigh scattering (RRS), second-order scattering (SOS) and frequency doubling scattering (FDS) were enhanced to the highest degree. The maximum RRS, SOS and FDS wavelengths of three ion-association complexes were located at 345/345 nm, 610/305 nm and 430/860 nm, respectively. The increments of scattering intensity (ΔI) were directly proportional to the concentration of FLD in certain ranges. The detection limits (3σ) of FLD for SDS, SLS and SDBS systems were 1.9 ng ml(-1), 2.1 ng ml(-1) and 2.2 ng ml(-1)(RRS method), 2.4 ng ml(-1), 2.7 ng ml(-1) and 2.6 ng ml(-1) (SOS method) and 2.3 ng ml(-1), 2.4 ng ml(-1) and 2.5 ng ml(-1) (FDS method), separately. The sensitivity of RRS method was higher than those of FDS and SOS methods. The optimum conditions of RRS method and the influence factors, the composition and the reaction mechanism have been discussed. Since the method is highly selective, it does not interference concomitant substances. These methods were applied successfully for the determination of FLD in pharmaceutical formulation and urine samples.