Minimum Inhibitory Concentrations Of Streptomycin Research Articles

Potential of bacteriophage PCT27 to reduce the use of agrochemicals to control Pectobacterium carotovorum subsp. Carotovorum in Chinese cabbage (Brassica pekinensis)

As antimicrobial resistance (AMR) continues to spread among bacteria and poses a serious threat to global health, the indiscriminate use of antibiotics in various fields, including agriculture, should be avoided. Here, we isolated a novel bacteriophage (phage), PCT27, specific for Pectobacterium carotovorum subsp. carotovorum (Pcc), a phytopathogen responsible for soft rot of crops, and investigated its potential as an adjunct to reduce the use of agrochemicals in Pcc control. Phage PCT27, which belongs to the family Autographiviridae, inhibited in vitro growth of Pcc within 1 h of infection by recognizing colanic acid as a host receptor. More than 90% of the virions rapidly adsorbed to the host cells within 3 min, supporting the efficient control of Pcc by PCT27. PCT27 supplementation reduced the minimum inhibitory concentration of streptomycin by 50% for Pcc control in vitro. Accordingly, PCT27 successfully complemented an antibiotic-based agrochemical in preventing Pcc infection in Chinese cabbage leaves and seedlings by reducing the dose of the agrochemical by a quarter. As a biocontrol agent for Pcc that complements agrochemicals, PCT27 can not only prevent economic losses due to crop decay but would also contribute to addressing the AMR crisis for sustainable agriculture and environmental safety.

Investigation of Efflux Pump Genes in Resistant Mycobacterium tuberculosis Complex Clinical Isolates Exposed to First Line Antituberculosis Drugs and Verapamil Combination

Tuberculosis (TB) is caused by Mycobacterium tuberculosis, still one of the most common life-threatening infectious diseases worldwide. Although drug resistance in M.tuberculosis is mainly due to spontaneous chromosomal mutations in genes encoding drug target or drug activating enzymes, the resistance cannot be explained only by these mutations. Low permeability of the cell wall, drug inactivating enzymes and especially efflux pumps (EPs) are other mechanisms of drug resistance in mycobacteria. Efflux pump inhibitors (EPIs) binding to M.tuberculosis EPs were shown to inhibit efflux of anti-TB drugs, to enhance M.tuberculosis killing, to reduce drug resistance and to produce synergistic effects with first line anti-TB drugs. In this study, we aimed to determine the minimum inhibitory concentration (MIC) of first-line anti-TB drugs in the presence of verapamil (VER) and the expression of 21 putative EP genes belonged to the ATP-binding cassette (ABC), major facilitator superfamily (MFS) and resistance-nodulation-division (RND) families which might have caused the resistance in nine M.tuberculosis complex clinical isolates resistant to all of the first line anti-TB drugs. MIC values of the isolates were determined in 96-well U-bottom plates by the resazurin microtiter test (REMA) method based on the color change principle. According to the determined MIC values of each isolate, freshly grown cultures in Middlebrook 7H9 broth were exposed to first-line anti-TB drugs and MIC of first-line anti-TB drugs in the presence of VER (½ MIC) at 37°C for 48 hours for RNA extraction. The non-drug exposed cultures were used as control. Total RNA was extracted using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and then treated with DNase I (Thermo Fischer Scientific Inc., Waltham, MA). Complementary DNA (cDNA) from the extracted RNAs was synthesized with the "First strand cDNA synthesis kit" (Thermo Fischer Scientific Inc., Waltham, MA) using oligo primers. The expression levels of efflux pump genes by quantitative realtime polymerase chain reaction (qRt-PCR) were performed using the QuantiTect SYBR Green Rt-PCR Kit (Qiagen, Germany). The housekeeping sigma factor gene sigA (Rv2703) was used as internal control in qRt‑PCR assays. Relative quantification of the clinical isolates was determined by the 2-∆∆Ct method by comparing the expression levels of efflux genes in cultures exposed to primary anti-TB drugs and VER with those of non-drug exposed cultures. MIC values of nine isolates by REMA method was determined between 32-512 μg/mL, 1-128 μg/mL, 2-32 μg/mL, 4-16 μg/mL and 15.62-250 μg/mL for streptomycin (SM), isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and VER, respectively. In the presence of ½ MIC VER, it was determined that the MIC of SM decreased 2-32 fold in eight isolates, the MIC of INH decreased by 2-8 fold in nine isolates, the MIC of RIF decreased by 2-16 fold in eight isolates, and the MIC of EMB decreased 2-4 fold in only five isolates. There was an increase in the expression of Rv1273c, Rv1456c, Rv1457 and Rv1819 efflux pump genes from the ABC family, Rv1634 and Rv0842 from the MFS family and Rv3823 efflux from the RND family in isolates exposed to ½ MIC of first-line anti-TB drugs stress. Rv1456c and Rv1819 were found to be associated with SM resistance, Rv1273c with EMB resistance, Rv1457, Rv0842 and Rv3823 with both RIF and EMB resistance, and Rv1634 with INH, RIF and EMB resistance. It was determined that there was a decrease in the expression levels of eight efflux pump genes from the ABC family (Rv1456c, Rv1457c, Rv1458c, Rv0194, Rv1272c, Rv1686c, Rv1687c, Rv1819c), six from MFS family (Rv0842, Rv0849, Rv1634, Rv2265, Rv2456c, Rv0876c) and two from RND family (Rv0507, Rv0676c) in isolates exposed to MIC of first-line anti-TB drugs in the presence of VER (½ MIC). Further studies with clinical isolates are needed to investigate the EPIs that can be used in alternative therapy and to determine the contribution of EPs to the development of resistance due to the increasing TB resistance.

Efflux pump effects on Mycobacterium tuberculosis drug resistance

Resistance and tolerance to antituberculosis drugs have become serious problems in disease treatment. This multi-phase study investigated the contributions of efflux pumps to Mycobacterium tuberculosis drug resistance. In the first phase, the minimum inhibitory concentration (MIC) levels of antibiotics were determined. In the second phase, MIC levels were determined in the presence of the efflux pump inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP), verapamil, reserpine and thioridazine. In the third phase, MIC levels were reduced in 6 M. tuberculosis isolates in the presence of efflux pump inhibitors to determine the expression of putative efflux pump genes by reverse transcriptase-polymerase chain reaction (RT-PCR). MIC levels of fluoroquinolones decreased in 6 (6.52%) isolates, MIC of rifampicin in 4 (4.34%), and MIC of streptomycin in 3 (3.26%) in the presence of efflux pump inhibitors reserpine, CCCP and verapamil. The efflux pump inhibitors CCCP, verapamil, and reserpine changed MICs 2- to 16-fold. Overexpression of all 15 efflux pump genes was observed in 6 isolates with a reduction in MIC values in the presence of efflux pump inhibitors. The overexpression of efflux-related genes in resistant isolates suggests that efflux pumps are associated with resistance development.

Changpingibacter yushuensis gen. nov., sp. nov., isolated from fluvial sediment in Qinghai Tibet Plateau of China.

Two facultatively anaerobic, short rod-shaped, non-motile, Gram-stain-positive, unknown bacterial strains (JY-X040T and JY-X174) were isolated from fluvial sediments of Tongtian River in Yushu Tibetan Autonomous Prefecture, Qinghai province, China. Cells formed translucent, gray, round and convex colonies, with a diameter of less than 0.5 mm after 5 days of incubation at 30°C on brain heart infusion-5% sheep blood agar. The 16S rRNA gene sequence similarity between strain JY-X040T and Fudania jinshanensis 313T is 93.87%. In the four phylogenetic trees constructed based on the 16S rRNA gene and 423 core genes, the two isolates form an independent branch, phylogenetically closest to F. jinshanensis 313T, but could not be classified as a member of the genus Fudania or any other genus of the family Arcanobacteriaceae. The DNA G + C content of strain JY-X040T was 57.8%. Calculation results of average nucleotide identity, digital DNA-DNA hybridization value and amino acid identity between strain JY-X040T and F. jinshanensis 313T are 69.9%, 22.9%, and 64.1%. The major cellular fatty acids were C16:0 (23%) and C18:1ω9c (22%). The cell-wall peptidoglycan type was A5α (L-Lys-L-Ala-L-Lys-D-Glu). The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and four unidentified components. The whole-cell sugars contained rhamnose and ribose. MK-10(H4) was the sole respiratory quinone. The minimum inhibitory concentration of streptomycin was 32 μg/ml. All physiological, biochemical, chemotaxonomic and genomic characteristics support that strains JY-X040T and JY-X174 represent members of a novel species in a new genus, Changpingibacter yushuensis gen. nov., sp. nov. The type strain is JY-X040T (GDMCC 1.1996T = KCTC 49514T).

Effects of Three Commonly Used Herbicides on Bacterial Antibiotic Resistance

The spread of antibiotic resistance has become a serious threat to global public health. Recently, several studies have found that non-antibiotic chemicals can promote the generation and spread of bacterial antibiotic resistance. However, the effects of herbicides on the antibiotic resistance of bacteria remain unclear. In this study, Escherichia coli DH5α was used as the model strain to explore the effects of three commonly used herbicides (glyphosate, glufosinate, and dicamba) on the antibiotic resistance under soil environmental concentrations. The results showed that herbicide exposure affected the sensitivity of E. coli DH5α to antibiotics and significantly improved the resistance of E. coli DH5α to gentamicin (glyphosate > dicamba > glufosinate). After 30 d of herbicide exposure, the E. coli mutant strains enhanced the resistance to tetracycline, chloramphenicol, and aminoglycoside antibiotics, and the minimum inhibitory concentration of streptomycin was increased by 19.8 times. The whole-genome sequencing results illustrated that herbicides induced several previously well-characterized mutations associated with membrane proteins (ompF and papC), fimbriae proteins (yraH), and ribosomes (rpsL) related to antibiotic resistance. Together, the results showed that herbicides can enhance the antibiotic resistance of bacteria via inducing genetic mutations, thereby promoting the potential risk of the spread of antibiotic resistance genes in the environment.

Isovalerylshikonin, a new resistance-modifying agent from Arnebia euchroma, supresses antimicrobial resistance of drug-resistant Staphylococcus aureus

Antimicrobial resistance is the greatest threat to the treatment of bacterial infectious diseases. The development of resistance-modifying agents (RMAs) represents a promising strategy to mitigate the spread of bacterial antimicrobial resistance. In this study, a natural product, isovalerylshikonin (IVS), was isolated from Arnebia euchroma, a traditional Chinese medicine herb, that exhibited marginal antibacterial activity against drug-resistant Staphylococcus aureus RN4220, with a minimum inhibitory concentration (MIC) of 16 mg/L. In addition, a synergistic effect between IVS and streptomycin (STM) was detected by the microdilution antimicrobial chequerboard assay, with a reduction in the MIC of STM by up to 16-fold against strain RN4220. A bacterial ethidium bromide efflux assay and reverse transcription PCR were performed to investigate the synergistic mechanism. IVS significantly inhibited bacterial efflux and expression of msrA mRNA in vitro. A murine peritonitis/sepsis model was employed to test the in vivo synergistic activity of IVS and STM. IVS synergistically decreased bacterial counts with STM in peritoneal, spleen and liver tissue and increased mouse survival with STM in 7 days. The acute toxicity of IVS was tested and the 50% lethal dose (LD50) of IVS with a single exposure was 2.584 g/kg in mice. Overall, IVS, a low-toxicity RMA, exhibited synergistic antibacterial activities in vitro and in vivo against drug-resistant S. aureus. The effects were mediated by suppression of msrA mRNA expression and reduced bacterial efflux. In addition, these data support that IVS is a potential RMA against microbial resistance caused by the MsrA efflux pump.

Rapid Detection of Streptomycin-Resistant Mycobacterium tuberculosis by rpsL-Restriction Fragment Length Polymorphism.

Background:Molecular methods for the detection of drug-resistant tuberculosis (DR-TB) are potentially more rapid than conventional culture-based drug susceptibility testing, facilitating the commencement of appropriate treatment for patients with DR-TB. The aim of this study was to evaluate and develop polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays for the detection of mutations within rpsL, and for the determination of streptomycin (STR) resistance in Mycobacterium tuberculosis.Materials and Methods:Clinical specimens were collected from individuals with suspected TB referred to the TB Center of Isfahan, from which 205 M. tuberclosis were isolated and identified by conventional phenotypic methods. The minimum inhibitory concentration of STR for all isolates was determined using the proportion method and 10 isolates were recognized as STR resistant M. tuberculosis. The effect of genetic alterations in the rpsL gene for these resistant isolates were investigated by PCR-RFLP method.Results:Three (30%) isolates showed point mutation at codon 43 by RLFP analysis.Conclusion:Our results suggest that RFLP analysis of the rpsL gene is useful for the rapid prediction of STR resistant strains of M. tuberculosis.

Relationship between clinical efficacy for pulmonary MAC and drug-sensitivity test for isolated MAC in a recent 6-year period

There are a few recent reports about the relationship between the clinical effect and drug-sensitivity test. We investigated the relationship between the clinical efficacy of treatment for pulmonary Mycobacterium avium complex (MAC) and drug-sensitivity test for isolated MAC by comparison between data from 2005 to 2007 and from 2008 to 2010. We studied 60 patients who satisfied diagnostic criteria of nontuberculous mycobacterial infection established by the American Thoracic Society in 2007 and who received combination therapy using rifampicin (RFP), ethambutol (EB), streptomycin (SM), and clarithromycin (CAM). Average CAM dosage was increased from the early (517 mg/day) to the later (800 mg/day) period. Sputum conversion rate increased from 63% in the early period to 83% in the later period. Clinical improvement also increased from 38% in the early period to 53% in the later period. The causative microorganisms isolated were M. avium in 35 patients and M. intracellulare in 25. In both periods, isolated MAC strains showed excellent minimum inhibitory concentration (MIC) for CAM. Regarding the relationship between clinical efficacy and MICs of RFP, EB, CAM, and SM, most patients with good clinical effects showed low MIC for CAM in both periods. Good clinical efficacy, including the sputum conversion rate, was obtained with an increased dose of CAM in the later period. We speculate that the increased dose of CAM influenced the good clinical effect in both periods.

Isolation and Characterization of Burkholderia gladioli from Orchids in Hawaii.

Bacterial diseases of orchids continue to be serious problems. Bacterial strains were isolated from orchid plants exhibiting disease symptoms in Hawaii. Small to large leaf spots with or without water-soaking or soft rots were observed on various orchid genera, including Dendrobium, Oncidium, and Miltonia spp. and hybrids. Bacteria isolated and cultured from the lesions were tentatively identified using analytical profile index (API) strips and standard physiological and biochemical tests, and confirmed by species-specific polymerase chain reaction and sequencing of the 16S rRNA gene. The variation in pathogenic, morphological, cultural, and molecular characteristics of the orchid isolates also was evaluated. In our studies, a gramnegative, aerobic, rod-shaped bacterium that produced pale yellow, opaque, round colonies with entire margins on nutrient broth yeast extract agar (NBY) was isolated consistently from diseased orchid plants. On yeast dextrose calcium carbonate agar, the isolates produced brownishyellow, nonmucoid colonies, with the majority of the strains secreting a diffusible yellow or tan pigment into the media. The bacterium was identified as Burkholderia gladioli. Molecular analysis indicated very little diversity in the 16S rDNA gene. Testing B. gladioli isolates using media containing copper or streptomycin indicated varying levels of resistance (copper resistant = Cur; streptomycin resistant, Smr), with approximately 75% of the strains resistant to copper and 94% of the strains resistant to streptomycin. The minimum inhibitory concentration (MIC) of cupric sulfate among Cur strains ranged from 50 to 1,000 μg/ml and the MIC of streptomycin was 50 to 100 μg/ml for all Smr B. gladioli strains tested. Field and laboratory data suggest the frequent use of these chemicals in nurseries may have inadvertently resulted in the development of copper and streptomycin resistance in B. gladioli from orchids.

Aminoglycoside efflux in Pseudomonas aeruginosa: involvement of novel outer membrane proteins.

The expression of tripartite multidrug efflux pumps such as MexA-MexB-OprM in Pseudomonas aeruginosa contributes to intrinsic resistance to a wide variety of antimicrobials, including beta-lactams, chloramphenicol, macrolides, quinolones, and tetracycline. The MexX-MexY linker-pump combination has been shown to be involved in intrinsic resistance to aminoglycosides, but the identity of the cognate outer membrane channel component remains under debate. Fourteen uncharacterized OprM homologs identified in the genome of P. aeruginosa were examined as candidates for this role by assessing the minimum inhibitory concentrations (MICs) of aminoglycosides in P. aeruginosa strain PAK knockout mutants lacking the corresponding genes. Insertional inactivation of OpmG, OpmI, and OpmH resulted in decreases of various degrees in the MICs of streptomycin, kanamycin, and gentamicin. When reintroduced into P. aeruginosa on multicopy plasmids, OpmG was able to complement the susceptibility of an opmG::miniTn5 mutant; however, cloned opmH, the proposed ortholog of Escherichia coli tolC according to our phylogenetic analysis, was able to only partially complement the opmH::miniTn5 mutant. Mini-microarray hybridization analysis demonstrated that opmG disruption does not affect expression of OpmI or OpmH (ruling out such indirect effects on aminoglycoside resistance); however, opmH disruption did have possible effects on expression of OpmG and OpmI. Based on the data, we propose that OpmG is a major outer membrane efflux channel involved in aminoglycoside efflux in P. aeruginosa PAK and that OpmI, its most related paralog, may share an overlapping function.

Aminoglycoside-Resistant Streptococcus and Enterococcus Species Isolated from Bovine Mammary Secretions

A total of 117 isolates representing four Streptococcus species and 20 isolates representing two Enterococcus species from bovine mammary secretions were examined for resistance to streptomycin, kanamycin, and gentamicin. Resistance to streptomycin (85.4%) was most prevalent, followed by kanamycin (19%) and gentamicin (2.2%). Minimum inhibitory concentration of streptomycin for most organisms examined ranged from 16 to 250µg/ml. For kanamycin, the minimum inhibitory concentration for most organisms was .5 to 62.5µg/ml. Minimum inhibitory concentration for gentamicin was lower than for kanamycin. Two strains each of Streptococcus uberis and Enterococcus faecalis had minimum inhibitory concentration for streptomycin of >2000µg/ml, and two strains of E. faecalis had similar minimum inhibitory concentration for kanamycin. The minimum bactericidal concentration for all organisms was one to four times higher than minimum inhibitory concentration. None of the organisms evaluated was found to carry plasmids. Transfer of antibiotic resistance in plasmid-free strains was not achieved by mobilization with the aid of plasmid pAD1, indicating the absence of conjugative transposons. These data suggest that expression of aminoglycoside resistance of Streptococcus and Enterococcus species of bovine origin is likely due to factors other than plasmids or conjugative transposons.

Effects of low concentrations of antibiotics on Escherichia coli adhesion.

We have previously shown that subinhibitory concentrations of antibiotics may influence the adhesion of Escherichia coli SS142 to human epithelioid tissue culture cells. This report shows that these effects are not limited to E. coli SS142 or to our tissue culture system. Most of the 10 E. coli strains studied showed decreased adhesion to Intestine 407 tissue culture cells after growth in 25% of the minimum inhibitory concentration of streptomycin, tetracycline, trimethoprimsulfametrole, chloramphenicol, and clindamycin. Nalidixic acid at 25% of the minimum inhibitory concentration caused an increase of adhesion. The hemagglutinating activity of the five hemagglutinating strains and the adhesiveness of E. coli SS142 to human buccal cells were similarly affected by low concentrations of the above-mentioned antibiotics. We conclude that E. coli adhesion to human epithelioid tissue culture cells is a valid model of bacterial adhesion because of its high accuracy and reproducibility.

Susceptibility of Mycoplasma pneumoniae to antibiotics in vitro.

The antibiotic susceptibility of laboratory strains, Mac and FH, and isolates of Mycoplasma pneumoniae was determined in broth media. With the Mac strain, the minimum inhibitory concentration (MIC) increased with the increase in inoculum size for most antibiotics and the final minimum inhibitory concentration as determined during a month's incubation did for all antibiotics. The antibiotics studied were classified by the MIC into three groups; the first group with the order of 1/100 μg/ml (erythromycin, josamycin, and leucomycin), the second with the order of 1/10 μg/ml (oleandomycin, tetracycline, and spiramycin), and the third with the order of 1 or 10 μg/ml (lincomycin, chloramphenicol, and streptomycin). With the FH strain, the MIC of streptomycin was much lower, showing that the Mac strain was resistant to streptomycin. The susceptibility of 152 isolates from 99 patients to erythromycin, josamycin, tetracycline, and chloramphenicol was essentially similar to that of the Mac strain except for two isolates, which had been isolated from a patient during erythromycin therapy and exhibited resistance to erythromycin and josamycin. The MIC of streptomycin was distributed at 1 μg/ml in 93% of the isolates and at 100 μg/ml or over in 6%. It was considered that streptomycin-resistant strains occur in M. pneumoniae. Of nine patients who had been given erythromycin, tetracycline, chloramphenicol, or Clotaon for more than a week, the isolates after therapy showed decreased susceptibility to erythromycin in one patient and did not to the antibiotics given in the other eight patients.