Blue light-mediated gene expression as a promising strategy to reduce antibiotic resistance in Escherichia coli.

The discovery of antibiotics has noticeably promoted the development of human civilization; however, antibiotic resistance in bacteria caused by abusing and overusing greatly challenges human health and food safety. Considering the worsening situation, it is an urgent demand to develop emerging nontraditional technologies or methods to address this issue. With the expanding of synthetic biology, optogenetics exhibits a tempting prospect for precisely regulating gene expression in many fields. Consequently, it is attractive to employ optogenetics to reduce the risk of antibiotic resistance. Here, a blue light-controllable gene expression system was established in Escherichia coli based on a photosensitive DNA-binding protein (EL222). Further, this strategy was successfully applied to repress the expression of ß-lactamase gene (bla) using blue light illumination, resulting a dramatic reduction of ampicillin resistance in engineered E. coli. Moreover, blue light was utilized to induce the expression of the mechanosensitive channel of large conductance (MscL), triumphantly leading to the increase of streptomycin susceptibility in engineered E. coli. Finally, the increased susceptibility of ampicillin and streptomycin was simultaneously induced by blue light in the same E. coli cell, revealing the excellent potential of this strategy in controlling multidrug-resistant (MDR) bacteria. As a proof of concept, our work demonstrates that light can be used as an alternative tool to prolong the use period of common antibiotics without developing new antibiotics. And this novel strategy based on optogenetics shows a promising foreground to combat antibiotic resistance in the future.

Combined antimicrobial action of streptomycin and terpenes against atypical mycobacteria isolated from fish.

The aim of this study was to investigate the antimycobacterial activity of 39 free terpenes and their activity in combination with streptomycin. Antimicrobial activity was first evaluated by screening 39 free terpenes at concentrations from 1.56 to 400 µg/mL. None of these exhibited positive effects against any of the nontuberculous mycobacteria (NTM) strains tested. However, six of the 39 terpenes (isoeugenol, nerol, (+)-α-terpineol, (1R)-(-)-myrtenol, (+)-terpinen-4-ol, and eugenol) were shown to enhance the activity of streptomycin against the NTM strains isolated from diseased ornamental fish.

The effect of diet composition on the diversity of active gut bacteria and on the growth of Spodoptera exigua (Lepidoptera: Noctuidae).

Gut microbiota plays a functional role in nutrition among several insects. However, the situation is unclear in Lepidoptera. Field studies suggest the microbiome may not be stable and is determined by diet, while in the laboratory, Lepidoptera are routinely reared on diet containing antibiotics with unknown effects on microbial communities. Furthermore, molecular approaches for the characterization of lepidopteran microbiomes rarely describe the metabolically active gut bacteria. The aim of this study was to evaluate how diet and antibiotics affect Spodoptera exigua (Hübner) growth and the diversity and activity of the gut bacteria community. We assessed how alfalfa and wheat germ-based diets affected larval growth, in the presence and absence of streptomycin. Alfalfa diet improved larval growth, pupal mass, and survival, but antibiotic was only beneficial in the wheat germ diet. We observed diet-driven changes in the gut bacterial communities. In the active community, the alfalfa colony was dominated by Enterococcus and Rhodococcus whereas in the wheat germ colony, only Enterococcus was present. In contrast, spore-forming Bacilli species were very common members of the DNA community. In both cases, streptomycin had a selective effect on the relative abundance of the taxa present. Our study highlights the importance of characterizing both the diversity and activity of the gut microbiota community. DNA-derived communities may include environmental DNA, spores, or non-viable bacteria, while RNA-derived communities are more likely to give an accurate representation of the diversity of active members that are potentially directly involved in the metabolic processes of the host.

Antibiotic resistance begets more resistance: chromosomal resistance mutations mitigate fitness costs conferred by multi-resistant clinical plasmids.

Plasmids are the primary vectors of horizontal transfer of antibiotic resistance genes among bacteria. Previous studies have shown that the spread and maintenance of plasmids among bacterial populations depend on the genetic makeup of both the plasmid and the host bacterium. Antibiotic resistance can also be acquired through mutations in the bacterial chromosome, which not only confer resistance but also result in changes in bacterial physiology and typically a reduction in fitness. However, it is unclear whether chromosomal resistance mutations affect the interaction between plasmids and the host bacteria. To address this question, we introduced 13 clinical plasmids into a susceptible Escherichia coli strain and three different congenic mutants that were resistant to nitrofurantoin (ΔnfsAB), ciprofloxacin (gyrA, S83L), and streptomycin (rpsL, K42N) and determined how the plasmids affected the exponential growth rates of the host in glucose minimal media. We find that though plasmids confer costs on the susceptible strains, those costs are fully mitigated in the three resistant mutants. In several cases, this results in a competitive advantage of the resistant strains over the susceptible strain when both carry the same plasmid and are grown in the absence of antibiotics. Our results suggest that bacteria carrying chromosomal mutations for antibiotic resistance could be a better reservoir for resistance plasmids, thereby driving the evolution of multi-drug resistance.IMPORTANCEPlasmids have led to the rampant spread of antibiotic resistance genes globally. Plasmids often carry antibiotic resistance genes and other genes needed for its maintenance and spread, which typically confer a fitness cost on the host cell observed as a reduced growth rate. Resistance is also acquired via chromosomal mutations, and similar to plasmids they also reduce bacterial fitness. However, we do not know whether resistance mutations affect the bacterial ability to carry plasmids. Here, we introduced 13 multi-resistant clinical plasmids into a susceptible and three different resistant E. coli strains and found that most of these plasmids do confer fitness cost on susceptible cells, but these costs disappear in the resistant strains which often lead to fitness advantage for the resistant strains in the absence of antibiotic selection. Our results imply that already resistant bacteria are a more favorable reservoir for multi-resistant plasmids, promoting the ascendance of multi-resistant bacteria.

Genome-wide identification of fitness-genes in aminoglycoside-resistant Escherichia coli during antibiotic stress.

Resistance against aminoglycosides is widespread in bacteria. This study aimed to identify genes that are important for growth of E. coli during aminoglycoside exposure, since such genes may be targeted to re-sensitize resistant E. coli to treatment. We constructed three transposon mutant libraries each containing > 230.000 mutants in E. coli MG1655 strains harboring streptomycin (aph(3″)-Ib/aph(6)-Id), gentamicin (aac(3)-IV), or neomycin (aph(3″)-Ia) resistance gene(s). Transposon Directed Insertion-site Sequencing (TraDIS), a combination of transposon mutagenesis and high-throughput sequencing, identified 56 genes which were deemed important for growth during streptomycin, 39 during gentamicin and 32 during neomycin exposure. Most of these fitness-genes were membrane-located (n = 55) and involved in either cell division, ATP-synthesis or stress response in the streptomycin and gentamicin exposed libraries, and enterobacterial common antigen biosynthesis or magnesium sensing/transport in the neomycin exposed library. For validation, eight selected fitness-genes/gene-clusters were deleted (minCDE, hflCK, clsA and cpxR associated with streptomycin and gentamicin resistance, and phoPQ, wecA, lpp and pal associated with neomycin resistance), and all mutants were shown to be growth attenuated upon exposure to the corresponding antibiotics. In summary, we identified genes that are advantageous in aminoglycoside-resistant E. coli during antibiotic stress. In addition, we increased the understanding of how aminoglycoside-resistant E. coli respond to antibiotic exposure.

Combined transcriptomic and pangenomic analyses guide metabolic amelioration to enhance tiancimycins production.

Exploration of high-yield mechanism is important for further titer improvement of valuable antibiotics, but how to achieve this goal is challenging. Tiancimycins (TNMs) are anthraquinone-fused enediynes with promising drug development potentials, but their prospective applications are limited by low titers. This work aimed to explore the intrinsic high-yield mechanism in previously obtained TNMs high-producing strain Streptomyces sp. CB03234-S for the further titer amelioration of TNMs. First, the typical ribosomal RpsL(K43N) mutation in CB03234-S was validated to be merely responsible for the streptomycin resistance but not the titer improvement of TNMs. Subsequently, the combined transcriptomic, pan-genomic and KEGG analyses revealed that the significant changes in the carbon and amino acid metabolisms could reinforce the metabolic fluxes of key CoA precursors, and thus prompted the overproduction of TNMs in CB03234-S. Moreover, fatty acid metabolism was considered to exert adverse effects on the biosynthesis of TNMs by shunting and reducing the accumulation of CoA precursors. Therefore, different combinations of relevant genes were respectively overexpressed in CB03234-S to strengthen fatty acid degradation. The resulting mutants all showed the enhanced production of TNMs. Among them, the overexpression of fadD, a key gene responsible for the first step of fatty acid degradation, achieved the highest 21.7 ± 1.1 mg/L TNMs with a 63.2% titer improvement. Our studies suggested that comprehensive bioinformatic analyses are effective to explore metabolic changes and guide rational metabolic reconstitution for further titer improvement of target products. KEY POINTS: • Comprehensive bioinformatic analyses effectively reveal primary metabolic changes. • Primary metabolic changes cause precursor enrichment to enhance TNMs production. • Strengthening of fatty acid degradation further improves the titer of TNMs.

The profile of genome-wide DNA methylation, transcriptome, and proteome in streptomycin-resistant Mycobacterium tuberculosis.

Streptomycin-resistant (SM-resistant) Mycobacterium tuberculosis (M. tuberculosis) is a major concern in tuberculosis (TB) treatment. However, the mechanisms underlying streptomycin resistance remain unclear. This study primarily aimed to perform preliminary screening of genes associated with streptomycin resistance through conjoint analysis of multiple genomics. Genome-wide methylation, transcriptome, and proteome analyses were used to elucidate the associations between specific genes and streptomycin resistance in M. tuberculosis H37Rv. Methylation analysis revealed that 188 genes were differentially methylated between the SM-resistant and normal groups, with 89 and 99 genes being hypermethylated and hypomethylated, respectively. Furthermore, functional analysis revealed that these 188 differentially methylated genes were enriched in 74 pathways, with most of them being enriched in metabolic pathways. Transcriptome analysis revealed that 516 genes were differentially expressed between the drug-resistant and normal groups, with 263 and 253 genes being significantly upregulated and downregulated, respectively. KEGG analysis indicated that these 516 genes were enriched in 79 pathways, with most of them being enriched in histidine metabolism. The methylation level was negatively related to mRNA abundance. Proteome analysis revealed 56 differentially expressed proteins, including 14 upregulated and 42 downregulated proteins. Moreover, three hub genes (coaE, fadE5, and mprA) were obtained using synthetic analysis. The findings of this study suggest that an integrated DNA methylation, transcriptome, and proteome analysis can provide important resources for epigenetic studies in SM-resistant M. tuberculosis H37Rv.

Influence of Bacterial Contamination and Antibiotic Sensitivity on Cryopreserved Sperm Quality of Indian Red Jungle Fowl.

Aims: Bacterial contamination may occur in feces during collection and processing of semen. Bacteria not only compete for nutrients with spermatozoa but also produce toxic metabolites and endotoxins and affect sperm quality. The aim of the present study was to investigate the effect of antibiotic supplementation on the sperm quality of Indian red jungle fowl, estimation and isolation of bacterial species and their antibiotic sensitivity. Materials and Methods: Semen was collected and initially evaluated, diluted, and divided into six experimental extenders containing gentamicin (2.5 µg/mL), kanamycin (31.2 µg/mL), neomycin (62.5 mg/mL), penicillin (200 U/mL), and streptomycin (250 µg/mL), and a control having no antibiotics were cryopreserved and semen quality was evaluated at post-dilution, post-cooling, post-equilibration, and post-thawing stages (Experiment 1). A total aerobic bacterial count was carried out after culturing bacteria (Experiment 2) and subcultured for antibiotic sensitivity (Experiment 3). Results: It was shown that penicillin-containing extender improved semen quality (sperm motility, plasma membrane integrity, viability, and acrosomal integrity) compared with the control and other extenders having antibiotics. The bacteria isolated from semen were Escherichia coli, Staphylococcus spp., and Bacillus spp. Antibiotic sensitivity results revealed that E. coli shows high sensitivity toward neomycin, kanamycin, and penicillin. Staphylococcus spp. shows high sensitivity toward streptomycin, neomycin, and penicillin. Bacillus spp. shows high sensitivity toward kanamycin and penicillin. Conclusions: It was concluded that antibiotics added to semen extender did not cause any toxicity and maintained semen quality as that of untreated control samples, and penicillin was identified as most effective antibiotic. It is recommended that penicillin can be added to the semen extender for control of bacterial contamination without affecting the semen quality of Indian red jungle fowl.

Effect of streptomycin sulphate on the nitrification system in activated sludge: insight into nitrification characteristic, antibiotic resistance gene and microbial community.

Wastewater with residual streptomycin sulphate usually contains high content of ammonia-nitrogen. However, the biological removal process of ammonia-nitrogen under streptomycin sulphate circumstance was unclear. In this study, short-term and long-term effects of streptomycin sulphate on biological nitrification systems, including AOB, NOB, SAOR, SNOR and SNPR, were evaluated comprehensively. The results indicated IC50 for AOB and NOB were 7.5 and 6.6 mg/L. SAOR and SNPR could be decreased to 3.43 ± 0.52 mg N/(g MLSS·h) and 0.24 ± 0.03 mg N/(g MLSS·h) while the addition of streptomycin sulphate was 10 mg/L. When streptomycin sulphate addition was stopped, nitrification ability recovered slightly, SAOR and SNPR increased to 9.37 ± 0.36 mg N/(g MLSS·h) and 1.66 ± 0.49 mg N/(g MLSS·h), respectively. The protein of EPS increased gradually during the acclimatization process, and the maximal protein value was 68.24 mg/g MLSS on the 100th day, however, no significant change of polysaccharose was observed during the acclimatization process. High abundance of ARGs and intI1 was detected in effluent and sludge of the biological treatment system. The maximal relative abundance of aadA1 in the sludge appeared on the 140th day, and increased by 0.99 orders of magnitude. Biological diversity decreased significantly during the acclimatization process, relative abundance of nitrosomonas was changed from 9.07% to 38.68% on the 61st day, while relative abundance of nitrobacter was changed from 1.30% to 0.64%. It should be noted that relative abundances of nitrosomonas and nitrobacter were reduced to 16.17% and 0.25% on the 140th day. This study would be helpful for nitrogen removal in wastewater with antibiotic.

Whole-genome sequencing and transcriptome-characterized mechanism of streptomycin resistance in Vibrio parahaemolyticus O10: K4.

Streptomycin resistance in V. parahaemolyticus has been widespread in both clinical and environmental isolates. Therefore, it is of great significance to characterize the mechanism of streptomycin resistance in V. parahaemolyticus. O10:K4 has emerged and becoming the new dominant serotype since 2020. In this study, we isolated a total of 36 strains of V. parahaemolyticus O10:K4 from 2020 to 2022 and found that more than half of them were resistant to streptomycin. We obtained streptomycin resistant and sensitive strains by detecting the resistance profiles. Whole-genome sequencing showed that VP_RS10735 and VP_RS05605 were the predominant mutations in streptomycin resistant O10:K4 clinical isolates. In addition, this study provided global insight into the characteristics of the transcriptome signature of streptomycin resistance, revealing that efflux transporters play a key role in streptomycin resistance. Finally, we found that streptomycin resistant strain was more virulent than sensitive strain. The results of this study should advance our understanding of the mechanisms of aminoglycoside resistance.

Evaluation of two colorimetric microplate microtiter tests for susceptibility testing of clinical isolates of Mycobacterium tuberculosis to first-line antituberculosis drugs.

Background: Drug-resistance tuberculosis (TB) is one of the most important global public health problems. Accurate and rapid drug-susceptibility testing is critical for the effective treatment of TB patients. Various colorimetric methods are used for anti-TB drug-susceptibility testing (DST) and minimum inhibitory concentration (MIC) determination. This study was conducted to evaluate the resazurin microtiter assay (REMA) and malachite green decolorization assay (MGDA). Methods: A total of 65 Mycobacterium tuberculosis strains isolated from patients with suspected TB using REMA and malachite green microtiter assay methods were tested against streptomycin (SM), isoniazid (INH), rifampicin (RIF), and ethambutol (ETB). The Mycobacterial Growth Indicator Tube 960 DST method was accepted as the gold standard in the evaluation of test results. Results: The sensitivity of REMA and MGDA tests was found to be 87.5% and 62.5% for INH, respectively. RIF and SM sensitivity for both tests was 100%. While ETB sensitivity was 81.8 for the REMA test, this rate was 60% for the MGDA test. Specificity of both tests varied between 92.5% and 98.2% according to the drug types. Conclusion: REMA and MGDA are a simple, rapid, and low cost. They can be used as an alternative test for drug-susceptibility testing and MIC determination. Extensive studies and standardization are needed for the routine use of both tests.

Probing the effects of streptomycin on Brassica napus germination and assessing its molecular interactions using extensive molecular dynamics (MD) simulations.

Antibiotics are chemical compounds that are used to treat and prevent disease in humans and animals. They have been used in animal feed for over 60 years and are widely used in industrial farming. Antibiotics can have negative environmental impacts, including the potential to contribute to the development of antibiotic-resistant organisms. They can enter the environment through various pathways, including the manufacturing process, the direct application of antibiotic-laden manure to fields, and through grazing animals. Antibiotics that are given to animals can be excreted from where they can enter soil and groundwater which enable their entry in plants. Streptomycin is an antibiotic that is used against a range of gram-positive and gram-negative bacteria, but its use has led to the development of antibiotic resistance in some pathogens. It has also been shown to have negative impacts on a range of plant species, including tobacco, tomato, and wheat. Although, the major effect of streptomycin on plant physiology have been studied, the molecular mechanisms at play are barely understood in plant body. In current study, we examined the impact of streptomycin on germination of Brassica napus and then using docking, MM-GBBSA and MD simulations identified key proteins that interact with streptomycin by performing rigorous computational screening of 106 different proteins. Our finding suggest that streptomycin might be interacting with acyl-CoA oxidases, protochlorophyllide reductase B and leucoanthocyanidin dioxygenase based on simulation and docking analysis.

In vitro culture and morphology of Neoechinorhynchus buttnerae (Eoacanthocephala: Neoechinorhynchidae) collected from the intestine of tambaqui (Colossoma macropomum) farmed in the Brazilian Amazon.

The tambaqui (Colossoma macropomum) is a species of great economic importance for fish farming in the Brazilian Amazon, and acanthocephaliasis caused by Neoechinorhynchus buttnerae (Golvan 1956) represents an obstacle to its production due to it causing severe morphological damage to the intestinal mucosa, thus impairing the absorption of nutrients and causing weight loss in the fish. Therefore, the establishment of in vitro protocols for evaluation of anthelmintic drugs is the first step to development of effective measures for in vivo control of this endoparasite. The present study evaluated the in vitro survival of N. buttnerae maintained in Eagle's minimum essential medium under different culture conditions. Three assays were carried out to evaluate whether temperature, supplementation with the antibiotics penicillin and streptomycin, and culture medium replacement or no replacement would influence the motility and morphology of the acanthocephalans. The results of the Kaplan-Meier analysis indicated that the use of culture in minimum essential medium together with penicillin and streptomycin prolonged the parasite's survival when kept at temperatures of 24 °C or 28 °C. We describe herein for first time an alternative protocol that is ideal for the in vitro culture of N. buttnerae. As such, this protocol ensures greater reliability in further in vitro studies with N. buttnerae.

Three genes controlling streptomycin susceptibility in Agrobacterium fabrum.

Streptomycin (Sm) is a commonly used antibiotic for its efficacy against diverse bacteria. The plant pathogen Agrobacterium fabrum is a model for studying pathogenesis and interkingdom gene transfer. Streptomycin-resistant variants of A. fabrum are commonly employed in genetic analyses, yet mechanisms of resistance and susceptibility to streptomycin in this organism have not previously been investigated. We observe that resistance to a high concentration of streptomycin arises at high frequency in A. fabrum, and we attribute this trait to the presence of a chromosomal gene (strB) encoding a putative aminoglycoside phosphotransferase. We show how strB, along with rpsL (encoding ribosomal protein S12) and rsmG (encoding a 16S rRNA methyltransferase), modulates streptomycin sensitivity in A. fabrum. IMPORTANCE The plant pathogen Agrobacterium fabrum is a widely used model bacterium for studying biofilms, bacterial motility, pathogenesis, and gene transfer from bacteria to plants. Streptomycin (Sm) is an aminoglycoside antibiotic known for its broad efficacy against gram-negative bacteria. A. fabrum exhibits endogenous resistance to somewhat high levels of streptomycin, but the mechanism underlying this resistance has not been elucidated. Here, we demonstrate that this resistance is caused by a chromosomally encoded streptomycin-inactivating enzyme, StrB, that has not been previously characterized in A. fabrum. Furthermore, we show how the genes rsmG, rpsL, and strB jointly modulate streptomycin susceptibility in A. fabrum.

A Novel IncX Plasmid Mediates High-Level Oxytetracycline and Streptomycin Resistance in Erwinia amylovora from Commercial Pear Orchards in California.

Isolates of the fire blight pathogen Erwinia amylovora with high-level resistance to oxytetracycline (minimal inhibitory concentration [MIC] > 100 µg/ml) and to streptomycin (MIC > 100 µg/ml) were recovered from four commercial pear orchards in California between 2018 and 2020. The two representative oxytetracycline- and streptomycin-resistant (OxyTcR-SmR) strains 32-10 and 33-1 were as virulent as the antibiotic susceptible strain 13-1 in causing blossom blight of pear and were recovered more than 50% of the time 7 days after co-inoculation to pear flowers with strain 13-1. In the field, inoculation of strain 32-10 to pear flowers that were pretreated with oxytetracycline at 200 µg/ml did not reduce disease compared with an untreated control. Four OxyTcR-SmR strains were subjected to draft genome sequencing to identify the genetic determinants of antibiotic resistance and their location. A 43.6-kb IncX plasmid, designated pX11-7, was detected in each of the four strains, and this plasmid encoded the tetracycline-resistance gene tetB and the streptomycin-resistance gene pair strAB within a large putatively mobile genetic element consisting of the transposon Tn10 that had inserted within the streptomycin-resistance transposon Tn6082. We also determined that pX11-7 was conjugative and was transferred at a rate that was 104 to 105 higher into an E. amylovora strain isolated in California compared with an E. amylovora strain that was isolated in Michigan. The occurrence of high levels of resistance to both oxytetracycline and streptomycin in E. amylovora strains from commercial pear orchards in California significantly limits the options for blossom blight management in these locations.

Role of multidrug resistance and co-resistance on a high percentage of streptomycin resistance in Escherichia coli isolated from chicken meats in Japan.

We focused on streptomycin resistance because of the high percentage of streptomycin-resistant Escherichia coli concerning the amount used of streptomycin. Antimicrobial resistance and horizontal transfer were identified in 117 isolates of coliform bacteria from chicken meat to identify the factors that increase streptomycin resistance. Escherichia (45 isolates) was the predominant genus. Most streptomycin-resistant Escherichia isolates were resistant to other antimicrobials (17/18), suggesting that using various antimicrobials could select streptomycin-resistant Escherichia isolates. Resistance was transferred from 7 out of the 18 streptomycin-resistant isolates. The transconjugants acquired strA/strB (7/7), blaTEM (5/7), aphA1 (5/7), tetB (3/7), dfrA14 (1/7) and/or dfrA17 (1/7). The co-resistance of streptomycin resistance with other resistances would also increase streptomycin resistance.

Discovery of Novel Small Molecule Growth Inhibitors to Manage Pseudomonas Leaf Spot Disease on Peppers (Capsicum sp.).

Pseudomonas leaf spot (PLS) disease in peppers caused by Pseudomonas syringae pv. syringae (Pss) is an emerging seedborne phytopathogen. Pss infection can severely reduce the marketable yield of peppers in favorable environmental conditions and cause significant economic losses. The intensive use of copper-sulfate and streptomycin-sulfate to control PLS and other bacterial diseases is associated with antimicrobial-resistant Pss strains, making these control methods less effective. So, there is an urgent need to develop novel antimicrobials effective against Pss in peppers. Several studies, including those done in our laboratory, have shown that small molecule (SM) antimicrobials are ideal candidates as they can be effective against multidrug resistant bacteria. Therefore, our study aims to identify novel SM growth inhibitors of Pss, assess their safety, and evaluate their efficacy on Pss-infected pepper seeds and seedlings. Using high-throughput screening, we identified 10 SMs (PC1 to PC10) that inhibited the growth of Pss strains at 200 µM or lower concentrations. These SMs were effective against both copper- and streptomycin-resistant as well as biofilm-embedded Pss. These SMs were effective against other plant pathogens (n = 22) at low concentrations (<200 µM) and had no impact on beneficial phytobacteria (n = 12). Furthermore, these SMs showed better or equivalent antimicrobial activity against Pss in infested pepper seeds and inoculated seedlings compared with copper-sulfate (200 µM) and streptomycin (200 µg/ml). Additionally, none of the SMs were toxic to pepper tissues (seeds, seedlings, or fruits), human Caco-2 cells, and pollinator honeybees at 200 µM. Overall, the SMs identified in this study are promising alternative antimicrobials for managing PLS in pepper production.

Heterogeneity among Clinical Intestinal Escherichia coli Isolates upon Acquired Streptomycin Resistance.

Escherichia coli isolates from inflammatory bowel disease (IBD) patients are often multidrug resistant, including to streptomycin. Streptomycin resistance (StrR) mutations can alter bacterial behavior, which may influence intestinal disease. We generated a spontaneous StrR strain of the intestinal adherent-invasive E. coli (AIEC) strain NC101. Whole-genome sequencing revealed a single missense mutation in rpsL that commonly confers StrR, rpsL-K43N. StrR NC101 exhibited a striking loss of aggregation and significantly increased motility, behaviors that can impact host-microbe interactions. Behavioral changes were associated with reduced transcription of csgA, encoding the biofilm component curli, and increased transcription of fliC, encoding flagellin. Scanning electron microscopy (SEM) detailed morphologic changes consistent with the observed alterations in multicellular behavior. Because intestinal E. coli isolates exhibit remarkable strain-specific differences, we generated spontaneous StrR mutants of 10 clinical E. coli phylotype B2 strains from patients with IBD, colorectal cancer, and urinary tract infection. Out of these 10 StrR clinical strains, two had altered colony morphology on Congo red agar (suggesting changes in extracellular products), and three had significant changes in motility. These changes were not associated with a particular rpsL mutation nor with the presence of virulence genes encoding the inflammation-associated E. coli metabolites yersiniabactin or colibactin. We conclude that common mutations in rpsL, which confer StrR, can differentially alter disease-associated phenotypes across intestinal E. coli strains. These findings highlight the heterogeneity among seemingly similar intestinal E. coli strains and reveal the need to carefully study the strain-specific effects of antibiotic resistance mutations, particularly when using these mutations during strain selection studies. IMPORTANCE We demonstrate that StrR, commonly acquired through a single point mutation in rpsL (a gene encoding part of the 30S bacterial ribosome), strikingly alters the morphology and behavior of a key intestinal AIEC strain, NC101. These changes include remarkably diminished aggregation and significantly increased motility, traits that are linked to AIEC-defining features and disease development. Phenotypic changes were heterogeneous among other StrR clinical E. coli strains, underscoring the need to evaluate the strain-specific effects of commonly acquired antibiotic resistance mutations. This is important, as the results of studies using mutant StrR Enterobacteriaceae strains (e.g., for cloning or in vivo selection) may be confounded beyond our demonstrated effects. Long term, these findings can help researchers better distinguish the contribution of specific E. coli traits to functional changes in the microbiota. Evaluating these strain-level differences could provide insight into the diversity of IBD symptoms and lead to improved therapies for microbiota-driven intestinal disorders.

The Effect of Inoculum Size on Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis.

Phenotypic drug susceptibility testing (DST) requires a standardized amount of inoculum to produce reproducible susceptibility results. The most critical step in the application of DST in Mycobacterium tuberculosis isolates is the preparation of the bacterial inoculum. In this study, the effect of bacterial inoculum prepared in various McFarland turbidities on primary antituberculosis drug susceptibility of M. tuberculosis strains was investigated. Five standard ATCC strains (ATCC 27294 [H37Rv], ATCC 35822 [izoniazid-resistant], ATCC 35838 [rifampicin-resistant], ATCC 35820 [streptomycin-resistant], ATCC 35837 [ethambutol-resistant]) were tested. Inoculums of McFarland standard of 0.5, 1, 2, 3, and 1:100 dilutions of 1 McFarland standard of each strain were used. The effect of inoculum size on DST results was determined by the proportion method in Lowenstein-Jensen (LJ) medium and nitrate reductase assay (NRA) in the LJ medium. In both test methods, the increase in inoculum size did not affect the DST results of the strains. On the contrary, DST results were obtained more rapidly as a result of the use of dense inoculum. DST results obtained in all McFarland turbidities were found to be 100% compatible with the recommended amount of inoculum, 1:100 dilution of 1 McFarland standard (inoculum size of gold standard method). In conclusion, the use of a high amount of inoculum did not change the drug susceptibility profile of tuberculosis bacilli. Minimizing manipulations during the inoculum preparation phase of susceptibility testing, this outcome will decrease the need for equipment and make the test application easier, particularly in developing countries. IMPORTANCE During DST application, it can be challenging to evenly homogenize TB cell clumps with lipid-rich cell walls. These experiments must be carried out under Biosafety Level-3 (BSL-3) laboratory conditions with personal protective equipment and taking safety precautions because the procedures applied at this stage cause the formation of bacillus-laden aerosols and carry a serious risk of transmission. Considering this situation, this stage is important given that it is not possible to establish a BSL-3 laboratory in poor and developing countries. Reducing the manipulations to be applied during the preparation of bacterial turbidity will minimize the risk of aerosol formation. Perhaps there will be no need to do these steps for susceptibility tests in these countries or even in developed countries.

Uptake of the Plant Agriculture-Used Antibiotics Oxytetracycline and Streptomycin by Cherry Radish─Effect on Plant Microbiome and the Potential Health Risk.

Antibiotics are used to control certain bacterial diseases in plant agriculture. Understanding antibiotic uptake by edible vegetables after application and associated risks on plant microbiome and human health is critical. In this study, oxytetracycline and streptomycin, the two most commonly used antibiotics in plant agriculture, were applied to cherry radish via continuous soil drenching to study their translocations into plant tissues, influence on radish microbiome, and the potential health risk to mice. The results demonstrated that oxytetracycline induced hormesis in radish plants and both antibiotics were translocated into the leaves, fruits, and roots of radishes from the soil, with significantly higher plant uptake of streptomycin than oxytetracycline. Interestingly, the proportion of culturable oxytetracycline or streptomycin-resistant bacteria in the antibiotic-accumulated radish tissues was significantly higher than that in the antibiotic-free radish tissues, although both bacterial and fungal communities in different radish tissues were not affected by the accumulated antibiotics, demonstrating that antibiotic application could enrich antibiotic resistance in the plant microbiome. Feeding mice with antibiotics-accumulated radish tissues did not show significant effects on the weight and blood glucose levels of mice. Overall, this study provides important insights into the risk of using antibiotics in plant agriculture.