scholarly journals Application of CRISPR/Cas9 System for Plasmid Elimination and Bacterial Killing of Bacillus cereus Group Strains

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaojing Wang ◽  
Yufei Lyu ◽  
Siya Wang ◽  
Qingfang Zheng ◽  
Erling Feng ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
High Efficiency ◽  
Elimination Rate ◽  
Wild Strain ◽  
Prokaryotic Genome ◽  
Replication Initiation ◽  
Virulence Plasmid ◽  
Bacterial Killing ◽  
Significant Difference ◽  
Plasmid Elimination

The CRISPR-Cas system has been widely applied in prokaryotic genome editing with its high efficiency and easy operation. We constructed some “scissors plasmids” via using the temperature-sensitive pJOE8999 shuttle plasmid, which carry the different 20nt (N20) guiding the Cas9 nuclease as a scissors to break the target DNA. We successfully used scissors plasmids to eliminate native plasmids from Bacillus anthracis and Bacillus cereus, and specifically killed B. anthracis. When curing pXO1 and pXO2 virulence plasmids from B. anthracis A16PI2 and A16Q1, respectively, we found that the plasmid elimination percentage was slightly higher when the sgRNA targeted the replication initiation region (96–100%), rather than the non-replication initiation region (88–92%). We also tried using a mixture of two scissors plasmids to simultaneously eliminate pXO1 and pXO2 plasmids from B. anthracis, and the single and double plasmid-cured rates were 29 and 14%, respectively. To our surprise, when we used the scissor plasmid containing two tandem sgRNAs to cure the target plasmids pXO1 and pXO2 from wild strain B. anthracis A16 simultaneously, only the second sgRNA could guide Cas9 to cleave the target plasmid with high efficiency, while the first sgRNA didn't work in all the experiments we designed. When we used the CRISPR/cas9 system to eliminate the pCE1 mega-virulence plasmid from B. cereus BC307 by simply changing the sgRNA, we also obtained a plasmid-cured isogenic strain at a very high elimination rate (69%). The sterilization efficiency of B. anthracis was about 93%, which is similar to the efficiency of plasmid curing, and there was no significant difference in the efficiency of among the scissors plasmids containing single sgRNA, targeting multi-sites, or single-site targeting and the two tandem sgRNA. This simple and effective curing method, which is applicable to B. cereus group strains, provides a new way to study these bacteria and their virulence profiles.

scholarly journals POS0369 ELEVATED EXPRESSION OF TIM-3 ON NEUTROPHILS CORRELATES WITH DISEASE ACTIVITY AND SEVERITY OF ANKYLOSING SPONDYLITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 414.2-415
Author(s):  
X. Huang ◽  
T. W. Li ◽  
J. Chen ◽  
Z. Huang ◽  
S. Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ankylosing Spondylitis ◽  
Disease Activity ◽  
Immune Cells ◽  
Clinical Manifestations ◽  
Innate Immune ◽  
Innate Immune Cells ◽  
Bacterial Killing ◽  
Markers Of Inflammation ◽  
Significant Difference

Background:Ankylosing spondylitis (AS) is a type of common, chronic inflammatory disease that compromises the axial skeleton and sacroiliac joints, causing inflammatory low back pain and progressive spinal stiffness, over time some patients develop spinal immobility and ankylosis which can lead to a decrease in quality of life. The last few decades, evidence has clearly indicated that neutrophil also plays key roles in the progression of AS. However, the immunomodulatory roles and mechanisms of neutrophils in AS are poorly understood. T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) has been reported as an important regulatory molecule, expressed and regulated on different innate immune cells, plays a pivotal role in several autoimmunity diseases. Recent study indicates that Tim3 is also expressed on neutrophils. However, the frequency and roles of Tim3-expressing neutrophils in AS was not clear.Objectives:In this study, we investigated the expression of Tim3 on neutrophils in AS patients and explored the correlation between the level of Tim3-expressing neutrophils and the disease activity and severity of AS.Methods:Patients with AS were recruited from Guangdong Second Provincial General Hospital (n=62). Age/sex-matched volunteers as Healthy controls (HC) (n=39). The medical history, clinical manifestations, physical examination, laboratory measurements were recorded. The expression of costimulatory molecules including programmed death 1 (PD-1), Tim-3 on neutrophils were determined by flow cytometry. The mRNA expression of PD-1 and Tim-3 was determined by real-time PCR. The levels of Tim3-expressing neutrophils in AS patients were further analyzed for their correlation with the markers of inflammation such as ESR,CRP,WBC and neutrophil count(NE), as well as disease activity and severity of AS. The expression of Tim3 on neutrophils was monitored during the course of treatment (4 weeks).Results:The expression of Tim3 on neutrophils in patients with AS was increased compared to the HC (Figure 1A). However, significant difference was observed in the frequency of PD-1-expressing neutrophils between AS patients and HC (Figure 1B). The expression analysis of Tim-3 mRNA, but not PD-1, confirmed the results obtained from flow cytometry (Figure 1C). The level of Tim3-expressing neutrophils in patients with AS showed an positive correlation with ESR, CRP and ASAS-endorsed disease activity score (ASDAS) (Figure 1D). Moreover, the frequency of Tim3-expressing neutrophils in active patients(ASDAS≥1.3) was increased as compare with the inactive patients (ASDAS<1.3) (Figure 1E). As shown in Figure 1F, the frequency of Tim3-expressing neutrophils decreased after the treatment.Conclusion:Increased Tim-3 expression on neutrophils may be a novel indicator to assess disease activity and severity in AS, which may serves as a negative feedback mechanism preventing potential tissue damage caused by excessive inflammatory responses in AS patients.References:[1]Han, G., Chen, G., Shen, B. & Li, Y., Tim-3: an activation marker and activation limiter of innate immune cells. FRONT IMMUNOL 4 449 (2013).[2]Vega-Carrascal, I. et al., Galectin-9 signaling through TIM-3 is involved in neutrophil-mediated Gram-negative bacterial killing: an effect abrogated within the cystic fibrosis lung. J IMMUNOL 192 2418 (2014).Figure 1.(A,B)The expression of Tim3 and PD-1 on neutrophils in AS and HC were determined by flow cytometry.(C) The expression of Tim3 and PD-1 on neutrophils in AS and HC were determined by RT-PCR.(D)The correction between Tim3-expressing neutrophils and ESR,CRP,ASDAS.(E) The expression of Tim3 on neutrophils in active and inactive patients.(F) Influence of treatment on the frequency of Tim3-expressing neutrophils.Disclosure of Interests:None declared

Susceptibility of Aedes Aegypti Against Various Insecticides in Lahore, Pakistan

2021 ◽  
Author(s):  
Farkhanda Manzoor ◽  
Rooma Adalat ◽  
Tallat Anwar Faridi ◽  
Wafa Fatima ◽  
Muhammad Moazzam ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Wild Strain ◽  
Effective Vaccine ◽  
P Value ◽  
Significant Difference ◽  
Incomplete Coverage ◽  
Wild Strains ◽  
New Strategies ◽  
Lahore City ◽  
Indoor And Outdoor

Dengue fever is an arbo-viral infection, widespread all over the world. In 21th Century, there is no safe affordable and effective vaccine accessible yet; vector control is that most effective method for the control of the disease Objective: To determine the susceptibility status of wild and laboratory strains larvae and adults of Aedes aegypti against different group of insecticides in Lahore city. Method: From Lahore sites, larvae were collected where insecticides used for wild strain at high frequency and quantity. The Insectary of National Institute of Malaria Research and Training (NIMRT), Lahore, Pakistan, adults and larvae were collected for laboratory strain.The laboratory strains for larvae bioassays were used. The mosquitoes populations indoor and outdoor collected in 2009, hatched from larvae into adults insectary in Lahore, Pakistan. During this study, four major insecticides groups are used which include Pyrethroids (Deltamethrine 2.5% SC), Neonicotenoids (Imidacloprid 5% SC), Phenyl-pyrazoles (Fipronil 2.5% EC) and Organophosphates dichlorvos (DDVP 50% EC). For data analysis, Minitab statistical software (Version 13.20) used for data expressed as mean ± S.E.M from bioassays. By using EPA Probit, LC was estimated with 95% confidence. The 50 statistically significant p value was <= 0.05. For comparing the concentrations of insecticides, Duncan's multiple range tests was used with significant difference (5% level) using at New Costat. Results: Different location of Lahore samples, Imidacloprid the most toxic to Aedes aegypti's wild strains on the other hand while Fipronil was also active for wild larval samples. Deltamethrine showed least activity against both adults and larval strains. The susceptibility of the eld strains was lower than laboratory strains; resistant ratio varies from insecticide to insecticide. In reporting results, mosquitos' population was resistance because of infrequent and incomplete coverage. Conclusions: This study concluded that Pyrethroids and agriculture pest control play role in indirect growth of insecticides' classes. Based on this study it is suggested that by using new strategies to prevent and delay in growth of insecticides will helpful in Lahore, city, Pakistan.

scholarly journals Pharmacokinetics of intramuscularly administered aminosidine in healthy subjects.

1997 ◽  
Vol 41 (5) ◽  
pp. 982-986 ◽  
Author(s):  
T P Kanyok ◽  
A D Killian ◽  
K A Rodvold ◽  
L H Danziger
Keyword(s):  
Healthy Subjects ◽  
Randomized Clinical Trials ◽  
Elimination Rate ◽  
Pharmacokinetic Parameters ◽  
Multiple Drug ◽  
Time Data ◽  
Time Curve ◽  
First Order ◽  
Significant Difference

Aminosidine is an older, broad-spectrum aminoglycoside antibiotic that has been shown to be effective in in vitro and animal models against multiple-drug-resistant tuberculosis and the Mycobacterium avium complex. The objective of this randomized, parallel trial was to characterize the single-dose pharmacokinetics of aminosidine sulfate in healthy subjects (eight males, eight females). Sixteen adults (mean [+/- standard deviation] age, 27.6 +/- 5.6 years) were randomly allocated to receive a single, intramuscular aminosidine sulfate injection at a dose of 12 or 15 mg/kg of body weight. Serial plasma and urine samples were collected over a 24-h period and used to determine aminosidine concentrations by high-performance liquid chromatographic assay. A one-compartment model with first-order input, first-order output, and a lag time (Tlag) and with a weighting factor of 1/y2 best described the data. Compartmental and noncompartmental pharmacokinetic parameters were estimated with the microcomputer program WinNonlin. One subject was not included (15-mg/kg group) because of the lack of sampling time data. On average, subjects attained peak concentrations of 22.4 +/- 3.2 microg/ml at 1.34 +/- 0.45 h. All subjects had plasma aminosidine concentrations below 2 microg/ml at 12 h, and all but two subjects (one in each dosing group) had undetectable plasma aminosidine concentrations at 24 h. The dose-adjusted area under the concentration-time curve from 0 h to infinity of aminosidine was identical for the 12- and 15-mg/kg groups (9.29 +/- 1.5 versus 9.29 +/- 2.2 microg x h/ml per mg/kg; P = 0.998). Similarly, no significant differences (P > 0.05) were observed between dosing groups for peak aminosidine concentration in plasma, time to peak aminosidine concentration in plasma, Tlag, apparent clearance, renal clearance, elimination rate constant, and elimination half-life. A significant difference was observed for the volume of distribution (0.35 versus 0.41 liters/kg; P = 0.037) between the 12 and 15 mg/kg dosing groups. Now that comparable pharmacokinetic profiles between dosing groups have been demonstrated, therapeutic equivalency testing via in vitro pharmacokinetic and pharmacodynamic modelling and randomized clinical trials in humans should be conducted.

Peritoneal Fluid and Solute Transport with Different Polyglucose Formulations

1998 ◽  
Vol 18 (2) ◽  
pp. 193-203 ◽  
Author(s):  
Tao Wang ◽  
Olof Heimbürger ◽  
Hui-Hong Cheng ◽  
Jonas Bergström ◽  
Bengt Lindholm
Keyword(s):  
Solute Transport ◽  
Peritoneal Cavity ◽  
Peritoneal Fluid ◽  
Absorption Rate ◽  
Glucose Solution ◽  
Ex Vivo ◽  
Elimination Rate ◽  
Fluid Absorption ◽  
Significant Difference ◽  
Glucose Polymer

Objective To study peritoneal fluid and solute transport characteristics using different polyglucose solutions with and without the addition of glucose. Design Thirty-one rats were divided into three groups. A 4-hour dwell study with frequent dialysate and blood samples was performed in each rat using 25 mL of 7.5% polyglucose solution (PG, n = 11),7.5% polyglucose + 0.35% glucose solution (PG1, n = 12), or 3.75% polyglucose + 1.93% glucose solution (PG2, n = 8). Radiolabeled human albumin (RISA) was added to the solutions as an intraperitoneal volume (IPV) marker. In addition, polyglucose degradation was evaluated ex vivo over 24 hours. Experimental Animals Thirty-one male Sprague Dawley rats (300 g) were used. Main Outcome Measures Fluid and solute (glucose, urea, sodium, potassium, and total protein) transport characteristics as well as changes in dialysate osmolality were evaluated. Results The IPV was higher in the PG1 and PG2 groups than in the PG group during the first 2 hours of the dwell. The IPV, in fact, decreased during the first hour of the dwell in the PG group. However, the net ultrafiltration at 4 hours tended to be lower in the PG2 (3.2 ± 1.5 mL) group compared to the PG (5.1 ± 2.3 mL) and the PG1 groups (5.2 ± 2.1 mL) (p = 0.07), and no significant difference was found between the PG and PG1 groups. Adding glucose to the PG solution increased the RISA elimination rate (KE, representing the fluid absorption rate from the peritoneal cavity): 25.5 ± 8.2, 37.5 ± 12.2, and 42.5 ± 8.9 μL/ min for the PG, PG1, and the PG2 group, respectively, p < 0.01. Dialysate osmolality (Dos) increased with the dwell time in the PG and PG1 groups but decreased in the PG2 group. The increase in Dos was partially due to the degradation of glucose polymer, which was supported by the marked increase in osmolality over 24 hours of incubation of PG solution with peritoneal fluid, ex vivo. The diffusive mass transport coefficient for the investigated solutes did not differ among the three groups (except for glucose, which was significantly lower in the PG group). The sieving coefficient for sodium was significantly higher in the PG group compared to the PG1 group (p < 0.05). Conclusion Our results suggest that, although there was an initial decrease in the intraperitoneal dialysate volume, significant amounts of fluid can be removed by polyglucose solution during a single 4-hour dwell in rats, despite the low osmolality of the solution. The positive fluid removal induced by the PG solution is partially due to the lower fluid absorption rate associated with this solution and may, to some extent, also be due to the degradation of glucose polymer within the peritoneal cavity, resulting in increased dialysate osmolality. The addition of glucose to the polyglucose solution does not seem to improve ultrafiltration in a 4-hour dwell in the rat model. However, the peritoneal fluid absorption rate may be increased, and peritoneal transport of glucose and sodium may be altered, by adding glucose to the polyglucose solution.

Variation of the Spore Population of a Natural Source Strain of Bacillus cereus in the Presence of Inosine

2004 ◽  
Vol 67 (5) ◽  
pp. 934-938 ◽  
Author(s):  
J. COLLADO ◽  
A. FERNÁNDEZ ◽  
M. RODRIGO ◽  
A. MARTÍNEZ
Keyword(s):  
Heat Resistance ◽  
Bacillus Cereus ◽  
Wild Strain ◽  
Natural Source ◽  
Spore Concentration ◽  
Factors Affecting ◽  
Germination Process ◽  
Germination Temperature ◽  
Spore Population ◽  
Spore Culture

The heat resistance of a wild strain of Bacillus cereus spores isolated from liquid egg was characterized, and the effect of the nutritional germinant inosine on the spore population was then studied, considering different factors such as germination temperature, inosine concentration, and age of spore culture. The heat resistance clearly indicates that these spores can survive mild heat treatments such as those used for cooked refrigerated food of extended durability or liquid egg, posing safety problems for these foods with temperature abuse. The germination study indicates that temperature, spore age, and the interaction between the two were the factors affecting the level of spores remaining after the germination process. No significant differences were found for the three inosine concentrations used in the study (1, 5, and 10 mM). The highest reduction in the spore concentration was reached at 30° C after 120 min, although the reduction in the spore counts at germination temperatures of 4 and 8° C was also considerable.

High Efficiency Recovery of Hematopoietic Progenitors from Peripheral Blood Harvests after Short- and Long-Term Cryopreservation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5275-5275
Author(s):  
Ulrich Denz ◽  
Dagmar Wider ◽  
Antonia Mueller ◽  
Monika Engelhardt
Keyword(s):  
Suspension Culture ◽  
Peripheral Blood ◽  
High Efficiency ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Viable Cells ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Group A

Abstract Introduction: Transplantation of functional hematopoietic stem cells (HSC) using peripheral blood (PB), bone marrow (BM) or cord blood (CB) cells is widely used to treat malignant and nonmalignant disorders. Because long-term cryopreservation is performed for PB, BM and CB cells, and these are often used years after cell harvests, the implementation of a quality-assurance is a major requirement to ensure graft safety for clinical use. Methods: We assessed the efficiency of recovery of viable HSC from 37 patients (pts; n=20 NHL, n=6 Hodgkin, n=9 MM, n=2 AML) and 6 allogeneic-donors (AD) with stored PBSC samples. All pts had received an auto-PBSCT between 1992–2004. Stored PBSC samples used in this analysis had been cryopreserved for a median of 5.6 years (y; range: 1.3–12). We determined post-thawing recovery, cell viability, ex vivo expansion potential, CD34+ numbers, CFU growth in methylcellulose culture and LTC-ICs. Viable cells were determined by trypan blue and propidium iodide via FACS analysis, CFUs in 0.9% methylcellulose (supplemented with IMDM, 30% FCS and EPO, IL-3+GM-CSF) and LTC-IC as previously described. Pts and AD were analyzed as a total group and within 3 subgroups of: A) ‘long-term’ cryopreservation: n=21 PBSC harvests had a median cryopreservation of 9.5y (8–12), B) ‘short-term’ cryopreservation: n=16 harvests had a 2.9y (1.3–5.6) cryopreservation period, and C) n=6 pts showing delayed engraftment (EG) or early death after auto-PBSCT: the cryopreservation in these 6 pts was 2.7y (2.2–3.5). Cryopreservation results were correlated with clinical results and EG. Results: Hematopoietic EG in group A and B was prompt with WBC&gt;1000/μl and platelets&gt;20,000/μl on d10–11 post PBSC reinfusion. EG in group C was delayed albeit 4.3x106 CD34+ cells/kg bw (2.1–8.6) had been retransfused (WBC&gt;1000/μl + platelets&gt;20,000/μl: d+13 post PBSC infusion, non-platelet-EG &gt;20,000/μl before death: n=5). Primary cause of death in group C was progressive disease in 3 and serious infections in 5 pts. Group A showed 74.3% viable cells post-thawing in PBSC grafts. Median number of CD34+ cells were 2.9%. Median numbers of CFU-C, BFU-E and GEMM were 36, 60 and 7, respectively. This was comparable with results in group B, showing 70% viable cells post-thawing, CD34+ cells of 4.2% and CFUs of 43, 75 and 6, respectively (p&gt;0.05). Proliferative capacity was intact in both groups after 7 days of suspension culture, generating CFU-C, BFU-E and GEMM of 67, 29 and 1, respectively. In group C, viable cells were present in only 58% and median CFU-C, BFU-E and GEMM were 21, 5 and 0, respectively (p&lt;0.05). After 7 days of suspension culture, total CFUs were 5 (&lt;5% as compared to group A+B). Mean CFU-Cs before and after LTC-IC were 9 and 8 after LTC-IC culture in group C, whereas these were 18 and 16 in group A (p&lt;0.05). Thus, the percentage of viable cells, CFUs and LTC-ICs was preserved after long-term cryopreservation (group A), showed no significant difference between group A+B, but were decreased in group C. Conclusions: We show that human PBSC can be stored for more than a decade without apparent loss of HSC activity and can be efficiently retrieved. These results reinforce that expiration dates cannot be set for safely stored cryopreserved HSC. Assessment of CD34+ cell numbers, clonogenic potential via methylcellulose and LTC-IC assays are clinically relevant, since they may correlate with clinical outcome. Thus, these hematopoietic assays are valuable to assess the quality of cryopreservation and possibly also outcome of PBSCT.

scholarly journals Biocontrol ofPhytophthora infestans, Fungal Pathogen of Seedling Damping Off Disease in Economic Plant Nursery

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
B. Loliam ◽  
T. Morinaga ◽  
S. Chaiyanan
Keyword(s):  
Fungal Pathogen ◽  
Fungal Pathogens ◽  
High Efficiency ◽  
Peat Moss ◽  
Economic Plant ◽  
Damping Off ◽  
Termite Mounds ◽  
Plant Nursery ◽  
Significant Difference ◽  
Infected Tomato

This research aims to control Seedling damping off disease in plants by using antagonistic actinomycetes against the causative fungi.Phytophthora infestanswas isolated from the infected tomato plant seedling obtained from an economic plant nursery in Amphoe Pak Chong, Nakhon Ratchasima Province, Thailand. The chitinolyticStreptomyces rubrolavendulaeS4, isolated from termite mounds at the grove of Amphoe Si-Sawat, Kanchanaburi Province, Thailand, was proven to be the most effective growth inhibition of fungal pathogens tested on potato dextrose agar. Tomato and chili seedlings that colonized with antagonisticS. rubrolavendulaeS4 were grown inP. infestansartificial inoculated peat moss. Percents of noninfested seedling in fungal contaminated peat moss were compared to the controls with uninoculated peat moss. InP. infestanscontaminated peat moss, the percents of survival of tomato and chili seedling were significantly increased () from 51.42 to 88.57 and 34.10 to 76.71 for theS. rubrolavendulaeS4 treatment, respectively. TheS. rubrolavendulaeS4 also showed high efficiency equivalent to fungicide, metalaxyl with no significant difference (). It was clearly demonstrated thatS. rubrolavendulaeS4 can prevent the tomato and chili seedling damping off disease in economic plant nurseries.

Comparison of Tobramycin Pharmacokinetics After Administration by Cris and a Traditional Intravenous Piggyback Infusion

1995 ◽  
Vol 29 (5) ◽  
pp. 465-469
Author(s):  
Vincent F Mauro ◽  
Lori R Jacobs ◽  
Laurie S Mauro ◽  
Rodger D MacArthur ◽  
Donald B White
Keyword(s):  
Maximum Concentration ◽  
Elimination Rate ◽  
Random Order ◽  
Open Label ◽  
Medical College ◽  
Primary Endpoints ◽  
Significant Difference ◽  
Secondary Endpoints ◽  
Open Label Trial ◽  
Infusion System

Objective: To compare the administration pharmacokinetics of a 30-minute intravenous piggyback (ivpb) infusion of tobramycin with those of controlled-release infusion system (CRIS) using a 20-mL vial at rates of 60 and 120 mL/h. Design: Randomized, controlled, crossover, prospective, open-label trial. Setting: Medical college-affiliated hospital. Participants: Eight healthy volunteer men between the ages of 22 and 24 years weighing between 60 and 90 kg. Interventions: Volunteers received, in random order, tobramycin sulfate 2 mg/kg iv on 3 occasions separated by 1 week. The drug was administered using a 50-mL ivpb infusion at 100 mL/h for 30 minutes, and with the CRIS using a 20-mL vial with flow rates of 60 mL/h for 1 hour (slow) and 120 mL/h for 1 hour (fast). Main Outcome Measures: Primary endpoints were area under the time–concentration curve (AUC), time to reach maximum concentration (tmax), and maximum concentration (Cmax). Secondary endpoints were elimination rate constant (ke), clearance (Cl), and half-life (t1/2). Results: Six volunteers successfully completed the trial. The tmax values observed following fast CRIS and ivpb were 28 ± 8 and 32 ± 4 minutes, respectively, and not significantly different from each other. Both occurred significantly earlier than the tmax associated with slow CRIS (44 ± 7 min). The Cmax values observed following ivpb (11.2 ± 1.5 mg/L) and slow CRIS (10.9 ± 0.9 mg/L) administration were not significantly different from each other, but both were significantly lower than that of fast CRIS (13.4 ± 1.5 mg/L). The AUCs of slow and fast CRIS were 29.8 ± 4.8 and 31.2 ± 3.8 mg/L•h, respectively, and were not significantly different from each other. The AUC of fast CRIS was significantly greater than that observed with ivpb (27.4 ± 4.3 mg/L•h). No significant difference in ke (fast CRIS 0.32 ± 0.03 h-1; slow CRIS 0.33 ± 0.04 h-1; ivpb 0.34 ± 0.0 h-1) was observed among any of the methods. Conclusions: CRIS administration of tobramycin resulted in higher AUCs than did ivpb administration. Compared with ivpb, fast CRIS resulted in a higher Cmax, but the tmax values of fast CRIS and ivpb administration were not statistically different. Compared with ivpb, slow CRIS resulted in a more delayed tmax, but the Cmax values of slow CRIS and ivpb were not statistically different.

scholarly journals The Stability Region of the Large Virulence Plasmid ofShigella flexneri Encodes an Efficient Postsegregational Killing System

2000 ◽  
Vol 182 (9) ◽  
pp. 2416-2421 ◽  
Author(s):  
Sameera Sayeed ◽  
Lucretia Reaves ◽  
Lyndsay Radnedge ◽  
Stuart Austin
Keyword(s):  
Plasmid Stability ◽  
Shigella Flexneri ◽  
Plasmid Vector ◽  
Inhibitory Effect ◽  
Replication Initiation ◽  
Virulence Plasmid ◽  
Additional Element ◽  
Host Chromosome ◽  
The Stability ◽  
Postsegregational Killing

ABSTRACT The large virulence plasmid pMYSH6000 of Shigella flexneri contains a determinant that is highly effective in stabilizing otherwise unstable plasmids in Escherichia coli. Expression of two small contiguous genes, mvpAand mvpT (formerly termed STBORF1 and STBORF2), was shown to be sufficient for stability. Mutations in mvpT abolished plasmid stability, and plasmids expressing only mvpT killed the cells unless mvpA was supplied from a separate plasmid or from the host chromosome. When replication of a plasmid carrying the minimal mvp region was blocked, growth of the culture stopped after a short lag and virtually all of the surviving cells retained the plasmid. Thus, the mvp system stabilizes by a highly efficient postsegregational killing (PSK) mechanism, withmvpT encoding a cell toxin and mvpA encoding an antidote. The regions that surround the mvp genes in their original context have an inhibitory effect that attenuates plasmid stabilization and PSK. The region encompassing the mvpgenes also appears to contain an additional element that can aid propagation of a pSC101-based plasmid under conditions where replication initiation is marginal. However, this appears to be a relatively nonspecific effect of DNA insertion into the plasmid vector.

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