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Salmonella enterica persister cells form unstable small colony variants after in vitro exposure to ciprofloxacin.

ABSTRACT: Persistence phenotype and small colony variants (SCVs) can be part of a bacterial bet-hedging strategy for survival under environmental stresses, such as antimicrobial exposure. These phenotypes are of particular concern in persistent and relapsing infections, since cells resume to normal growth after cessation of the stressful condition. In this context, we found persisters and unstable SCVs as phenotypic variants of Salmonella enterica that were able to survive ciprofloxacin exposure. A high heterogeneity in persister levels was observed among S. enterica isolates grown under planktonic and biofilm conditions and exposed to ciprofloxacin or ceftazidime, which may indicate persistence as a non-multidrug-tolerant phenotype. Nevertheless, a comparable variability was not found in the formation of SCVs among the isolates. Indeed, similar proportions of SCV in relation to normal colony phenotype (NCP) were maintained even after three successive cycles of ciprofloxacin exposure testing colonies from both origins (SCV or NCP). Additionally, we found filamentous and dividing cells in the same scanning electron microscopy images from both SCV and NCP. These findings lead us to hypothesize that besides variability among isolates, a single isolate may generate distinct populations of persisters, where cells growing under distinct conditions may adopt different and perhaps complementary survival strategies.

SUBMITTER: Drescher SPM

PROVIDER: S-EPMC6510897 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Salmonella enterica persister cells form unstable small colony variants after in vitro exposure to ciprofloxacin.

Drescher Samara Paula Mattiello SPM Gallo Stephanie Wagner SW Ferreira Pedro Maria Abreu PMA Ferreira Carlos Alexandre Sanchez CAS Oliveira Sílvia Dias de SD

Scientific reports 20190510 1

Persistence phenotype and small colony variants (SCVs) can be part of a bacterial bet-hedging strategy for survival under environmental stresses, such as antimicrobial exposure. These phenotypes are of particular concern in persistent and relapsing infections, since cells resume to normal growth after cessation of the stressful condition. In this context, we found persisters and unstable SCVs as phenotypic variants of Salmonella enterica that were able to survive ciprofloxacin exposure. A high h ...[more]

PMID: 31076596

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Project description:Salmonella enterica strains are enteropathogenic bacteria that survive and proliferate within vacuolar compartments of epithelial and phagocytic cells. Recently, it has been reported that fibroblast cells are capable of restricting S. enterica serovar Typhimurium intracellular growth. Here, we show that prolonged residence of bacteria in the intracellular environment of fibroblasts results in the appearance of genetically stable small-colony variants (SCV). A total of 103 SCV isolates, obtained from four independent infections, were subjected to phenotypic analysis. The following phenotypes were observed: (i) delta-aminolevulinic acid auxotrophy; (ii) requirement for acetate or succinate for growth in glucose minimal medium; (iii) auxotrophy for aromatic amino acids; and (iv) reduced growth rate under aerobic conditions not linked to nutrient auxotrophy. The exact mutations responsible for the SCV phenotype in three representative isolates were mapped in the lpd, hemL, and aroD genes, which code for dihydrolipoamide dehydrogenase, glutamate-1-semyaldehyde aminotransferase, and 3-dehydroquinate dehydratase, respectively. The lpd, hemL, and aroD mutants had intracellular persistence rates in fibroblasts that were 3 to 4 logs higher than that of the parental strain and decreased susceptibility to aminoglycoside antibiotics. All three of these SCV isolates were attenuated in the BALB/c murine typhoid model. Complementation with lpd(+), hem(+), and aroD(+) genes restored the levels of intracellular persistence and antibiotic susceptibility to levels of the wild-type strain. However, virulence was not exhibited by any of the complemented strains. Altogether, our data demonstrate that similar to what it has been reported for SCV isolates of other pathogens, S. enterica SCV display enhanced intracellular persistence in eucaryotic cells and are impaired in the ability to cause overt disease. In addition, they also suggest that S. enterica SCV may be favored in vivo.

Project description:Background & objectivesInfection with Salmonella enterica serovar Typhi (hereafter S. Typhi) is an important public health problem in India. There has been an increase in the number of reported clinical failures to ciprofloxacin treatment but the data on possible mechanism of failure are limited. One mechanism that has been widely reported and found associated with ciprofloxacin resistance, is the mutations in target genes in QRDR (quinolone resistance determining region). It is hypothesized that mutations in DNA gyrase or topoisomerase IV result in therapeutic failure under selective pressure of antibiotic while the patient is on treatment. We undertook in vitro sequential selection studies to expose the clinical isolates of S. Typhi to different concentration of ciprofloxacin to study the role of antibiotic selective pressure in the development of mutations in QRDR.MethodsTotal 26 clinical isolates were divided in to two parts: part I included six isolates obtained from three patients with relapse of enteric fever and part II included 20 isolates with different ciprofloxacin MIC levels. For in vitro induction of mutation experiment, five S. Typhi isolates were selected which included three NAS (nalidixic acid sensitive) and 2 NAR (nalidixic acid resistant) S. Typhi. These isolates were grown under increasing concentrations of ciprofloxacin and mutations acquired in QRDR of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) were investigated by sequencing.ResultsFor the isolates included in the part I of the study, it was found that the MIC to ciprofloxacin increased in the isolates obtained during the relapse of enteric fever as compare to the first isolate. All isolates had single mutation in gyrA gene at S83 without additional mutation in the second isolate. In the second part of the study, the nine isolates with varying MICs to ciprofloxacin also had single mutation in gyrA gene at S83 and another six had triple mutations, two mutations in gyrA gene (at S83 and D87) and one mutation in parC gene (at S80). In in vitro induction of mutation experiment, all mutated isolates showed triple mutation (two mutation in gyrA and one in parC gene) while no mutations were found in wild isolates.Interpretation & conclusionsUpon exposure to the step-wise increased concentration of ciprofloxacin, isolates become more tolerant to the ciprofloxacin and showed 2-4 fold higher MICs without new mutation after 8 μg/ml. So the accumulation of mutations under continuous ciprofloxacin pressure and tolerance of the mutant isolates led to the clinical failure. These results also suggested that there could be another mechanism responsible for resistance.

Project description:BackgroundSmall colony variants (SCVs), constituting a slow-growing subpopulation of bacteria that facilitates persistence in lethal environmental conditions, are able to revert to the phenotype of rapid growth for further proliferation and transmission. Salmonella enterica serotype Typhimurium is one of the most important foodborne pathogens. This study investigated the genetic mechanisms how SCVs induced by streptomycin reverted to the fast-growing phenotype and the phenotypic changes of SCVs among their complete life cycle in S. Typhimurium.MethodsSalmonella Typhimurium SCVs were obtained by streptomycin treatment and their revertants were collected in the absence of antibiotics. The fitness, antimicrobial susceptibility, biofilm formation, and the biofilm-related genes expression were analyzed in comparison to their wild type strain, and the whole genome sequencing was performed to identify the genetic changes in the life cycle of S. Typhimurium SCVs.ResultsSmall colony variants were characterized by an increased antimicrobial resistance to streptomycin (64-fold), imipenem (twofold), and gentamicin (fourfold). A significant increase in biofilm production with higher expression of csgB was observed in SCVs (P < 0.01). The genetic alterations of all SCVs occurred in ubiE gene (coenzyme Q8 and menaquinone synthesis) with frameshift mutations. However, all fast-growing revertants again lost the trait of increased biofilm production (P > 0.05), in which two modes of the genetic changes for reversing to the rapidly growing form were observed: four revertants harbored a secondary mutation in ubiE, which reinstated most of the amino acid sequence of the ubiE, and other four revertants harbored a mutation in prfB.ConclusionsSalmonella Typhimurium could switch to the phenotype of SCVs under the treatment of streptomycin by a mutation in ubiE, partially combined with increased production of biofilm, and these SCVs could escape from growth restriction by a compensatory mutation in prfB or a new mutation in ubiE. These findings may contribute to establishing phenotype-directed treatments against SCVs of S. Typhimurium.

Project description:UNLABELLED:We investigated the application capabilities of a laser optical sensor, BARDOT (bacterial rapid detection using optical scatter technology) to generate differentiating scatter patterns for the 20 most frequently reported serovars of Salmonella enterica. Initially, the study tested the classification ability of BARDOT by using six Salmonella serovars grown on brain heart infusion, brilliant green, xylose lysine deoxycholate, and xylose lysine tergitol 4 (XLT4) agar plates. Highly accurate discrimination (95.9%) was obtained by using scatter signatures collected from colonies grown on XLT4. Further verification used a total of 36 serovars (the top 20 plus 16) comprising 123 strains with classification precision levels of 88 to 100%. The similarities between the optical phenotypes of strains analyzed by BARDOT were in general agreement with the genotypes analyzed by pulsed-field gel electrophoresis (PFGE). BARDOT was evaluated for the real-time detection and identification of Salmonella colonies grown from inoculated (1.2 × 10(2) CFU/30 g) peanut butter, chicken breast, and spinach or from naturally contaminated meat. After a sequential enrichment in buffered peptone water and modified Rappaport Vassiliadis broth for 4 h each, followed by growth on XLT4 (~16 h), BARDOT detected S. Typhimurium with 84% accuracy in 24 h, returning results comparable to those of the USDA Food Safety and Inspection Service method, which requires ~72 h. BARDOT also detected Salmonella (90 to 100% accuracy) in the presence of background microbiota from naturally contaminated meat, verified by 16S rRNA sequencing and PFGE. Prolonged residence (28 days) of Salmonella in peanut butter did not affect the bacterial ability to form colonies with consistent optical phenotypes. This study shows BARDOT's potential for nondestructive and high-throughput detection of Salmonella in food samples. IMPORTANCE:High-throughput screening of food products for pathogens would have a significant impact on the reduction of food-borne hazards. A laser optical sensor was developed to screen pathogen colonies on an agar plate instantly without damaging the colonies; this method aids in early pathogen detection by the classical microbiological culture-based method. Here we demonstrate that this sensor was able to detect the 36 Salmonella serovars tested, including the top 20 serovars, and to identify isolates of the top 8 Salmonella serovars. Furthermore, it can detect Salmonella in food samples in the presence of background microbiota in 24 h, whereas the standard USDA Food Safety and Inspection Service method requires about 72 h.

Project description:Active efflux pump is a primary fluoroquinolone resistant mechanism of clinical isolates of Salmonella enterica serovar Typhimurium. RamA is an essential element in producing multidrug resistant (MDR) S. enterica serovar Typhimurium. The aim of the present study was to elucidate the roles of RamA on the development of ciprofloxacin, the first choice for the treatment of salmonellosis, resistance in S. enterica serovar Typhimurium. Spontaneous mutants were selected via several passages of S. enterica serovar Typhimurium CVCC541 susceptible strain (ST) on M-H agar with increasing concentrations of ciprofloxacin (CIP). Accumulation of ciprofloxacin was tested by the modified fluorometric method. The expression levels of MDR efflux pumps were determined by real time RT-PCR. In ST and its spontaneous mutants, the ramA gene was inactivated by insertion of the kan gene and compensated on a recombinant plasmid pGEXΦ(gst-ramA). The mutant prevention concentration (MPC) and mutant frequencies of ciprofloxacin against ST and a spontaneous mutant in the presence, absence and overexpression of RamA were tested. Four spontaneous mutants (SI1-SI4) were obtained. The SI1 (CIP MICs, 0.1 mg/L) without any target site mutation in its quinolone resistant determining regions (QRDRs) and SI3 (CIP MICs, 16 mg/L) harboring the Ser83→Phe mutation in its QRDR of GyrA strains exhibited reduced susceptibility and resistance to multidrugs, respectively. In SI1, RamA was the main factor that controlled the susceptibility to ciprofloxacin by activating MdtK as well as increasing the expression level of acrAB. In SI3, RamA played predominant role in ciprofloxacin resistance via increasing the expression level of acrAB. Likewise, the deficiency of RamA decreased the MPCs and mutant frequencies of ST and SI2 to ciprofloxacin. In conclusion, the expression of RamA promoted the development of ciprofloxacin resistant mutants of S. enterica serovar Typhimurium. The inhibition of RamA could decrease the appearance of the ciprofloxacin resistant mutants.

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Salmonella enterica persister cells form unstable small colony variants after in vitro exposure to ciprofloxacin.

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Salmonella enterica persister cells form unstable small colony variants after in vitro exposure to ciprofloxacin.

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