Plasmid pUT-kmy, which consists of an R6K origin of replication, an mobRP4 origin of transfer, and a kanamycin resistance cassette [32] , was ligated with a sacB gene to generate plasmid pUT-KB for constructing revertants. Plasmid pUT-KB is a suicide vector containing a counter-selection marker, sacB , which originates from Bacillus subtilis [13]. When this gene is expressed on the integrated pUT-KB, it confers a sucrose-sensitivity phenotype, which enables positive selection with sucrose to detect the loss of the vector.
The allelic exchange method was used to restore the wild-type rpsL gene in the K. The 2. After double crossover occurred, the sucrose-resistant and kanamycin-sensitive colonies were selected, and the restorations of wild-type rpsL gene were confirmed via DNA sequencing. Competitive growth in vitro was performed as previously described [34] , [35] with minor modifications.
Each successive growth cycle was initiated by diluting the mixture 2 17 -fold into 4 ml of fresh LB broth. After the initial mixing and each growth cycle, appropriate dilutions of the mixture were plated on LB agar plates for colony counts. The number of parental streptomycin-susceptible bacteria was calculated as the total number of bacteria minus the number of streptomycin-resistant bacteria.
S t is called the selection coefficient at time t. S t is equal to 0 if there is no difference in fitness between the competing strains. If antibiotic resistance reduces bacterial fitness, S t is negative, while S t is positive if resistance increases bacterial fitness. The data are presented as relative bacterial fitness fit t , which was defined by Sander et al. Phagocytosis was measured using a standard assay [36]. The labeling of bacteria with fluorescein isothiocyanate was performed as described by Heinzelmann et al.
The percentage of neutrophils that had phagocytosed bacteria was counted at 15 and 30 min. An unincubated tube served as the 0 min time point.
The experimental procedures and FACS settings have been described previously [38]. The serum bactericidal activity was measured using the method described by Podschun et al. The cultures were washed and then diluted fold using phosphate buffered saline PBS.
To determine the number of viable bacteria, an aliquot of each bacterial suspension was removed immediately and after 3 hours of incubation. The number of viable bacteria was determined via dilution and plating on LB agar for colony counts. The culture was incubated until the mid-exponential growth phase, and the cells were then washed once, resuspended in PBS, and adjusted to the desired concentrations according to OD The actual concentrations were verified by plating the cells to determine viable counts.
Six mice for each group were injected intraperitoneally with 0. A two-tailed t -test or a log-rank test for survival analysis was used for statistical analysis. The K. Eighteen K. No streptomycin resistance was found to be caused by the rpsL mutations. Spontaneous mutants of K. The rpsL genes of all of these mutants were sequenced, and fourteen different mutations on the rpsL were obtained, which cause thirteen varied S12 protein Table 2.
The examination of streptomycin resistance for these different mutants using the E-test led to the identification of ten streptomycin-dependent mutants and four streptomycin-resistant mutants Table 2. The growth phenotype of the streptomycin-resistant mutants can be restored by replacing the wild-type rpsL to these mutants using an allelic exchange method Table 2 and Figure S1.
A highly sensitive in vitro growth competition experiment was used to further evaluate the fitness cost of the four streptomycin-resistant mutants Table 3. Competition assays were also performed for the four streptomycin-resistant mutants using their revertants as references, and the results demonstrated that the decreased fitness could be restored to original levels following complementation Table 3.
No significant difference was found between strain NVT and its four streptomycin-resistant mutants in the neutrophil phagocytosis assay Table 4. The results of serum bactericidal assay have been further validated by testing the revertant strains Table 4. The two serum-susceptible strains, the K42N and K42T mutants, were used to further investigate the contribution of each complement pathway to complement-mediated bacterial killing.
These results suggest that the alternative pathway has a crucial role in complement activation. To further assess the effects of the rpsL mutations on virulence, a mouse peritonitis model was used. The LD 50 of K. In the 18 K. The low-level resistance to streptomycin conferred by aadA can also be found in Salmonella and E.
No streptomycin resistance was found to be caused by rpsL mutations in any of the clinical strains of K. This result suggests that the mutations of S12 protein were uncommon in K. Compared with the parental strain in this study, the K42N and K42T mutants showed reduced virulence, while the K42R mutant showed similar virulence. These results are consistent with those reported in Salmonella typhimurium using the competition experiments in mice [27] and those reported in Erwinia carotovora using the potato tuber tests [26].
A reduced virulence of the streptomycin-resistant K87R mutant was further found in this study. Compared with the parental strain, the K87R mutant showed a fold increase in the LD 50 in a mouse peritonitis model, while the K42N and K42T mutants showed a 1,fold increase. The slower growth rate may cause them to be more easily eliminated by the immune system in vivo. In the serum bactericidal assay, the K42N and K42T mutants were found to become susceptible to serum, while the alternative pathway should be the crucial role in complement activation.
Whether this result is due to different levels of protein expression caused by the rpsL mutations requires further study, while this difference has been found between the K42T mutant and its parental strain in E. Becoming serum-susceptible should explain the lowest virulence found for the K42N and K42T mutants compared with the parental strain, as well as the K42R and K87R mutants, which all showed serum resistance. Based on the in vitro and animal experiments, the K42R mutant only showed a small fitness cost in a highly sensitive in vitro growth competition experiment.
This mutant is also found at the highest frequency in streptomycin-resistant clinical isolates of Mycobacterium tuberculosis , while the K87R mutant was also prevalent [9] , [45]. Because the properties of Arginine are very similar to that of Lysine, such as they both are positively-charged amino acids, this substitution could cause the subtle effect on protein structure and function [46]. Compared with the parental strain, all four streptomycin-resistant mutants exhibited reduced fitness, and three of them showed decreased virulence, while the ten streptomycin-dependent mutants needed streptomycin to maintain their growth.
Only the K42R mutant showed similar pathogenicity to its parental strain as previously described in other Enterobacteriaceae [26] , [27] , [47]. This similar pathogenicity can also been found in a highly virulent K. In conclusion, the streptomycin resistance caused by rpsL mutations usually made the bacteria less competitive than its wild-type strain in the absence of streptomycin. As Asn and Thr are uncharged polar side chain amino acids the mutation gid p. Furthermore, we found genetic variants in Rv and Cytochrome p, both previously associated with antimicrobial resistance phenotypes Raman and Chandra, ; Gupta et al.
It will be interesting to in the future further address all these novel candidates at the functional level. Within the largest transmission cluster of STR resistant isolates TC1 , we found differences in growth in the presence of STR suggesting a continuing process of adaptation.
Interestingly, isolates 6C1, 5D1 and 1C8 were genetically indistinguishable in the sense that they were not separated by any high confidence genetic variant. Yet, whether isolates 6C1 and 5D1 showed delayed growth in the presence of a high These findings suggest that the phenotype observed in 6C1 and 5D1, and not in 1C8, might be drug tolerance associated with recent or transient genetic variants within the isolates Wallis et al. Drug tolerance mechanisms are elusive, challenging to study and prone to technical- and culture-related artifacts Lee et al.
Our analysis of low frequency variants in NGS data and Sanger re-sequencing support that 1C8 differs from 6C1 and 5D1 by having one additional cytosine in the homopolymeric cytosine tract of the glpK gene. This polymorphism was found in four isolates from the studied cohort including two with low STR resistance. Nonetheless, it was also found in two STR susceptible isolates.
Of note, the possible role of glpK in M. It is thus tempting to speculate that changes homopolymeric cytosine tract of glpK may be related to the observed phenotype of low-to-intermediate STR resistance, a hypothesis that will need to be further addressed in future studies Bellerose et al.
The highest level of STR resistance was found in the isolate 2H3 that diverged from the other TC1 isolates by harboring one mutation in the gene bioF2 p. Although a SNP in an adjacent position of bioF2 p.
The gene bioF2 is likely involved in M. Mycobacteria rely primarily on de novo synthesis of biotin, an important micronutrient serving as an essential cofactor for several enzymes Park et al. Consequently, affecting biotin biosynthesis might influence bacilli growth. Thus, bioF2 p. It will be relevant to test this hypothesis in future experiments using genetically engineered mutants targeting these bioF2 residues.
Importantly, the phenotype of high STR resistance of the isolate 2H3 found in in vitro cultures was maintained when using the infection model of mouse BMDM in which the bacilli grow within the controlled intracellular microenvironment of the macrophage phagosome. STR resistance has not been perceived as a major clinical threat since this antibiotic was replaced in first line TB treatment by more efficient combinations of other antimicrobials.
The mechanisms underlying this process remain elusive, but likely involve secondary mutations that compensate the fitness cost of resistance to more than one drug. If this hypothesis proves correct, it is of great relevance to survey and increase the efforts to control the transmission of STR resistant strains. In this context, our findings raise concerns on the potential inaccuracy of molecular diagnostic methods to predict STR resistance and the necessity to combine phenotypic and genotypic analysis to generate more comprehensive DR mutation databases.
Also, our results propose the modulation of biotin biosynthesis as a novel putative compensatory mechanism for STR resistance. The datasets presented in this study can be found in online repositories. DR prepared the figures, tables, and drafted the manuscript.
MS and NSO supervised the study, planned the experiments, and wrote the final version of the manuscript. All authors read and approved the final manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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The ability of exogenous streptomycin application to control disease caused by C. Blister symptoms were observed on tomato leaves inoculated with all three C. No blistering was observed in the water control at 7 and 14 dpi Fig. Taken together, these data demonstrate that the streptomycin-resistant strains identified in the laboratory are unlikely to be controlled by streptomycin application on tomato.
Streptomycin is a common antibiotic used in disease management, including management of bacterial canker on tomato Hausbeck et al. Prolonged application has led to streptomycin resistance in both M.
In this study, we identified three C. Streptomycin targets the S12 protein of the ribosomal 30S subunit and interferes with protein synthesis Chiou and Jones Mutations in rpsL , which encodes the S12 protein, can cause resistance to streptomycin McManus et al. In the genera Erwinia and Xanthomonas , the mutations at th or th nucleotide in rpsL resulted in streptomycin resistance Chiou and Jones ; Zhang et al. Similar streptomycin-resistant C. We also generated strain BTR-2, which harbors a nonsynonymous SNP at the th nucleotide in rpsL gene, but the growth of this strain was impaired compared with the wild type.
This is inconsistent with the results in Xanthomonas oryzae pv. Moderate resistance to streptomycin is also related to streptomycin-inactivating enzymes and efflux pumps McManus et al. For example, in Streptomyces griseus and Streptomyces glaucescens , the phosphotransferase APH 6 can inactivate streptomycin Shaw et al. The ykkCD efflux pump, which belongs to a small multidrug resistance family, has been reported to confer streptomycin and chloramphenicol resistance in Bacillus subtilis and E.
Identical alleles are present in both resistant and susceptible strains Jia et al. In strain TX, transposon mutagenesis and targeted knockouts of gene or led to a loss of streptomycin resistance. In streptomycin-resistant and -sensitive C. We were not able restore the resistance by complementation of or in individual knockouts.
This may be owing to the use of plasmids for complementation as opposed to chromosomal insertion, resulting in overexpression of each gene.
Alternatively, other genes may be involved in streptomycin resistance, potentially because of the accumulation of SNPs induced by the stress of electroporation in strain TX Although C. We identified three strains isolated in from Hebei Province in China exhibiting streptomycin resistance.
It is possible that strong selective pressure because of streptomycin application in this province led to the emergence of streptomycin resistance.
Additional studies investigating the origin of streptomycin resistance should be performed to identify genes associated with resistance, and these studies may help predict the efficacy of streptomycin application and can monitor potential resistance spread.
Search for more papers by this author. Shree P. Add to favorites Download Citations Track Citations. View article. Abstract Clavibacter michiganensis is the causal agent of bacterial canker of tomato, which causes significant economic losses because of the lack of resistant tomato varieties. View as image HTML. TABLE 2. Candidate resistance-related genes in strain TX Source Function Gene identification Genome annotation Putative streptomycin phosphotransferase , Genome annotation Predicted resistant gene , , , Comparative analyses ABC transporter gene , , , Comparative analyses RND a multidrug efflux pump , , Transposon insertion site Hypothetical protein , Candidate resistance-related genes in strain TX SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing.
Differential contribution of Clavibacter michiganensis ssp. Plant Pathol. Effects of temperature, plant age, inoculum concentration, and cultivar on the incubation period and severity of bacterial canker of tomato. Plant Dis. Molecular analysis of high-level streptomycin resistance in Erwinia amylovora. Phytopathology Proteomic analysis of resistance mediated by Rcm 2.
Plant-Microbe Interact. Streptomycin resistance in pulmonary tuberculosis. BMJ Clavibacter : A new genus containing some phytopathogenic coryneform bacteria, including Clavibacter xyli subsp. Comparative efficiency of chemical compounds for in vitro and in vivo activity against Clavibacter michiganensis subsp. Crop Prot.
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Evaluation of tomato breeding lines resistant to bacterial canker. Streptomycin, a substance exhibiting antibiotic activity against gram-positive and gram-negative bacteria. Fiji: An open-source platform for biological-image analysis. Methods Streptomycin resistance in Erwinia amylovora. Prokka: Rapid prokaryotic genome annotation. Bioinformatics Screening for new sources of resistance to Clavibacter michiganensis subsp. Euphytica Bacterial canker of tomato: Current knowledge of detection, management, resistance, and interactions.
Molecular genetics of aminoglycoside resistance genes and familial relationship of the aminoglycoside-modifying enzyme.
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