Use of antibiotic disks to evolve drug-resistant bacteria.

The methods used to generate antibiotic-resistant bacterial strains can be labour-intensive, costly, lengthy and/or prone to plate-to-plate variation. We propose a simple, inexpensive and easily replicated method to expose bacteria to a continuous gradient of antibiotic concentration, providing an environment of positive selective pressure for evolution of antibiotic-resistant strains.

The Relationship Between Agar Thickness and Antimicrobial Susceptibility Testing.

Antimicrobial susceptibility testing can be done using solid or liquid-based medium. Solid-based assays are easy and inexpensive; they are limited by not being as quantitative as liquid-based assays. Agar depth can influence the accuracy of plate-based assays and it is assumed the basis of this effect is antimicrobial agent diffusion. We tested this assumption by using ETEST® to quantitate the relationship between agar depth and minimum inhibitory concentration and zone of inhibition.

Examining changes in bacterial abundance in complex communities using next-generation sequencing is enhanced with quantitative PCR.

Changes in the composition of microbial communities are often examined using high-throughput sequencing (HTS). Here we analyzed bar-coded Illumina sequencing data from a previous work describing the microbial community found in the sputum of a cystic fibrosis patient by itself or in combination with qPCR to measure the absolute abundance of Pseudomonas aeruginosa and Burkholderia multivorans. Through this comparison we were able to determine the computational analysis accuracy of sequencing data to measure the relative abundance of specific taxa. While no correlation was found between relative abundance and absolute abundance of P. aeruginosa or B. multivorans, we did find conclusions derived from HTS data alone differed from those derived from a combination of HTS and qPCR. Our results highlight the importance of using qPCR with HTS when characterizing organisms in microbial communities having a dominant taxon.

Analysis of changes in diversity and abundance of the microbial community in a cystic fibrosis patient over a multiyear period.

The evolution of pulmonary disease in cystic fibrosis (CF) usually begins when bacteria get trapped in mucus in the lungs and become established as a chronic infection. While most CF patients experience periods of stability, pulmonary exacerbations (PEs) can occur multiple times per year and result in permanent damage to the lungs. Little is known of the shift from a period of stability to a PE, but this shift is likely to be attributed to changes in the bacterial community. Here, we identified changes in the lung microbiota to determine if they reflect patient health, indicate the onset of exacerbations, or are related to antibiotic treatment. In contrast to most bacterial studies on CF, we collected weekly samples from an adult CF patient over a period of 3 years and performed quantitative PCR (qPCR) and Illumina sequencing on those samples. While many DNA-based studies have shown the CF microbiota to be relatively stable, we observed an increase in the total bacterial abundance over time (P < 0.001), while the number of different taxa (bacterial richness) and the number of different taxa and their abundances (diversity) significantly decreased over time (P < 0.03), which was likely due to repeated antibiotic exposure. Using genus-specific primers with qPCR, we observed an increase in the abundance of Burkholderia multivorans, a CF-associated pathogen, prior to the occurrence of a PE (P = 0.006). Combining these DNA-based techniques with frequent sampling identified a potential initiator for exacerbations and described a response of the CF microbiota to time and antibiotic treatment not observed in previous CF microbiota studies.

Mechanical homogenization increases bacterial homogeneity in sputum.

Sputum obtained from patients with cystic fibrosis (CF) is highly viscous and often heterogeneous in bacterial distribution. Adding dithiothreitol (DTT) is the standard method for liquefaction prior to processing sputum for molecular detection assays. To determine if DTT treatment homogenizes the bacterial distribution within sputum, we measured the difference in mean total bacterial abundance and abundance of Burkholderia multivorans between aliquots of DTT-treated sputum samples with and without a mechanical homogenization (MH) step using a high-speed dispersing element. Additionally, we measured the effect of MH on bacterial abundance. We found a significant difference between the mean bacterial abundances in aliquots that were subjected to only DTT treatment and those of the aliquots which included an MH step (all bacteria, P = 0.04; B. multivorans, P = 0.05). There was no significant effect of MH on bacterial abundance in sputum. Although our results are from a single CF patient, they indicate that mechanical homogenization increases the homogeneity of bacteria in sputum.

Impact of Breathing Pattern and Nebulization on Expelled Viral Content During Mechanical Ventilation Using an Ex Vivo Porcine Lung System.

BACKGROUND: Little is known about the fate of expelled viral particulates during the aerosolization of inhaled medications during mechanical ventilation. We hypothesized that breathing patterns that generate a greater degree of shear stress and turbulent air flow will produce a greater concentration of exhaled viral RNA with the presence of a nebulizer during mechanical ventilation. METHODS: Eight ex vivo pig lungs were utilized as the physiological model. Each lung was dedicated to a specific breathing pattern that consisted of tidal breathing, respiratory distress, cough, and sneeze. Breath simulations were carried out through a commercial mechanical ventilator. Ninety mL of a bacteriophage stock at a concentration of 108 PFU/mL were introduced into the lungs during a 10-min sample collection session. The number of viral particles collected in exhalate was measured using quantitative polymerase chain reaction. The impact of breathing pattern on measured viruses was analyzed through two-way analysis of variance. RESULTS: The interaction effect between nebulization and breath pattern on exhaled viral quantity was not statistically significant P = .80, partial η2 = 0.167. The analysis of the main effects indicated that the effects of the breathing pattern and nebulization phase were not statistically significant P = .26, partial η2 = 0.519; P = .98, partial η2 = 0, respectively. There were no statistically significant differences among the breathing patterns related to measurable viral RNA. Coughing produced the most measurable increase in measured viral quantity during the nebulization phase and non-nebulization phase with a mean exhaled viral quantity (3.5 × 105 ng/µL [95% CI 1.6 × 105-5.5 × 105] and 2.7 × 105 ng/µL [95% CI 7.1 × 103-5.5 × 105], respectively). Tidal breathing with the presence of a nebulizer and respiratory distress without a nebulizer produced the lowest measured viral quantities (M = 1.1 × 105 ng/µL [95% CI -1.7 × 105 to 3.9 × 105]; M = 1.2 × 105 ng/µL [95% CI -1.6 × 105 to 4.0 × 105]). CONCLUSIONS: In this ex vivo porcine model, the introduction of a nebulizer did not increase the mean viral RNA captured throughout all of the breathing patterns.

Burkholderia multivorans Exhibits Antibiotic Collateral Sensitivity.

Burkholderia multivorans is a member of the Burkholderia cepacia complex whose members are inherently resistant to many antibiotics and can cause chronic lung infections in patients with cystic fibrosis. A possible treatment for chronic infections arises from the existence of collateral sensitivity (CS)-acquired resistance to a treatment antibiotic results in a decreased resistance to a nontreatment antibiotic. Determining CS patterns for bacteria involved in chronic infections may lead to sustainable treatment regimens that reduce development of multidrug-resistant bacterial strains. CS has been found to occur in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Here, we report that B. multivorans exhibits antibiotic CS, as well as cross-resistance (CR), describe CS and CR networks for six antibiotics (ceftazidime, chloramphenicol, levofloxacin, meropenem, minocycline, and trimethoprim-sulfamethoxazole), and identify candidate genes involved in CS. Characterization of CS and CR patterns allows antibiotics to be separated into two clusters based on the treatment drug to which the evolved strain developed primary resistance, suggesting an antibiotic therapy strategy of switching between members of these two clusters.

Reciprocal antibiotic collateral sensitivity in Burkholderia multivorans.

Antibiotic collateral sensitivity (CS) occurs when a bacterium that acquires resistance to a treatment drug exhibits decreased resistance to a different drug. Here we identify reciprocal CS networks and candidate genes in Burkholderia multivorans. Burkholderia multivorans was evolved to become resistant to each of six antibiotics. The antibiogram of the evolved strain was compared with the immediate parental strain to determine CS and cross-resistance. The evolution process was continued for each resistant strain. CS interactions were observed in 170 of 279 evolved strains. CS patterns grouped into two clusters based on the treatment drug being a ß-lactam antibiotic or not. Reciprocal pairs of CS antibiotics arose in ≥25% of all evolved strains. A total of 68 evolved strains were subjected to whole-genome sequencing and the resulting mutation patterns were correlated with antibiograms. Analysis revealed there was no single gene responsible for CS and that CS seen in B. multivorans is likely due to a combination of specific and non-specific mutations. The frequency of reciprocal CS, and the degree to which resistance changed, suggests a long-term treatment strategy; when resistance to one drug occurs, switch to use of the other member of the reciprocal pair. This switching could theoretically be continued indefinitely, allowing life-long treatment of chronic infections with just two antibiotics.

Strategy for extracting DNA from clay soil and detecting a specific target sequence via selective enrichment and real-time (quantitative) PCR amplification.

We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, microg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil.

Rapid emergence of a ceftazidime-resistant Burkholderia multivorans strain in a cystic fibrosis patient.

BACKGROUND: Burkholderia multivorans poses a serious health threat to cystic fibrosis patients due to innate resistance to multiple antibiotics and acquisition of resistance to a range of antibiotics due to the frequent use of antibiotics to treat chronic infections. Monitoring antibiotic susceptibility is crucial to managing patient care. We identified the rapid emergence of a ceftazidime-resistant strain in a single patient within four days during a hospitalization for treatment of an exacerbation. METHODS: B. multivorans was isolated from expectorated sputum samples using Burkholderia cepacia selective agar. A macrodilution assay was performed on all isolates to determine the minimum inhibitory concentration of ceftazidime. Approximately 4000 colonies were scored to identify the percent of ceftazidime-resistant colonies. Extracted DNA was used to determine the total bacterial counts and abundance of B. multivorans using quantitative PCR. RESULTS: An increase from no detectable B. multivorans ceftazidime-resistant colonies to over 75% of all colonies tested occurred within a four-day period. The resistant population remained dominant in 6 of the 8 samples in the following 17 months of the study. qPCR revealed an association between change in the percent of resistant colonies and abundance of B. multivorans, but not of total bacteria. No association was found between the acquisition of resistance to ceftazidime and other antibiotics commonly used to treat B. multivorans infections. CONCLUSIONS: The rapid emergence of a ceftazidime-resistant by B. multivorans strain occurred during a hospitalization while under selective pressure of antibiotics. The resistant strain maintained dominance in the B. multivorans population which resulted in an overall decline in a patient health and treatment efficacy.

The use of open-ended problem-based learning scenarios in an interdisciplinary biotechnology class: evaluation of a problem-based learning course across three years.

Use of open-ended Problem-Based Learning (PBL) in biology classrooms has been limited by the difficulty in designing problem scenarios such that the content learned in a course can be predicted and controlled, the lack of familiarity of this method of instruction by faculty, and the difficulty in assessment. Here we present the results of a study in which we developed a team-based interdisciplinary course that combined the fields of biology and civil engineering across three years. We used PBL scenarios as the only learning tool, wrote the problem scenarios, and developed the means to assess these courses and the results of that assessment. Our data indicates that PBL changed students' perception of their learning in content knowledge and promoted a change in students' learning styles. Although no statistically significant improvement in problem-solving skills and critical thinking skills was observed, students reported substantial changes in their problem-based learning strategies and critical thinking skills.

Viable but nonculturable bacteria are present in mouse and human urine specimens.

The presence of viable but nonculturable bacteria in human clean-catch and mouse bladder-isolated urine specimens was investigated. Viable but nonculturable bacteria are alive but do not give rise to visible growth under nonselective growth conditions. Urine specimens obtained from human female volunteers with or without an active urinary tract infection were found to contain, on average, significantly more viable than culturable forms of bacteria. Additional support for the presence of viable but nonculturable cells in urine specimens considered sterile was obtained from examination of urine specimens obtained directly from the bladder of healthy mice. Because the viability assay used to study the viable but nonculturable condition is by necessity growth independent, and hence indirect, the accuracy of this assay that scores cells with intact cell membranes as being viable was studied. Greater than 95% of Escherichia coli cells exposed to lethal doses of UV irradiation were found to lose their membrane integrity within a day, a time frame similar to that used to examine urine specimens. These data suggest that viable but nonculturable cells can occur within regions of the urinary tract previously considered sterile.