Antibiotic Selection Pressure Research Articles

Antimicrobial resistance in nephrology.

The prevalence of antimicrobial resistance among many common bacterial pathogens is increasing. The emergence and global dissemination of these antibiotic-resistant bacteria (ARB) is fuelled by antibiotic selection pressure, inter-organism transmission of resistance determinants, suboptimal infection prevention practices and increasing ease and frequency of international travel, among other factors. Patients with chronic kidney disease, particularly those with end-stage renal disease who require dialysis and/or kidney transplantation, have some of the highest rates of colonization and infection with ARB worldwide. These ARB include methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus spp. and several multidrug-resistant Gram-negative organisms. Antimicrobial resistance limits treatment options and increases the risk of infection-related morbidity and mortality. Several new antibiotic agents with activity against some of the most common ARB have been developed, but resistance to these agents is already emerging and highlights the dire need for new treatment options as well as consistent implementation and improvement of basic infection prevention practices. Clinicians involved in the care of patients with renal disease must be familiar with the local epidemiology ofARB, remain vigilant for the emergence of novel resistance patterns and adhere strictly to practices proven to prevent transmission of ARB and other pathogens.

Antibiotic resistomes of healthy pig faecal metagenomes.

Antibiotic resistance reservoirs within food-producing animals are thought to be a risk to animal and human health. This study describes the minimum natural resistome of pig faeces as the bacteria are under no direct antibiotic selective pressure. The faecal resistome of 257 different genes comprised 56 core and 201 accessory resistance genes. The genes present at the highest relative abundances across all samples were tetW, tetQ, tet44, tet37, tet40, mefA, aadE, ant(9)−1, ermB and cfxA2. This study characterized the baseline resistome, the microbiome composition and the metabolic components described by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in healthy pig faeces, without antibiotic selective pressures. The microbiome hierarchical analysis resulted in a cluster tree with a highly similar pattern to that of the accessory resistome cluster tree. Functional capacity profiling identified genes associated with horizontal gene transfer. We identified a statistically significant positive correlation between the total antibiotic resistome and suggested indicator genes, which agree with using these genes as indicators of the total resistomes. The correlation between total resistome and total microbiome in this study was positive and statistically significant. Therefore, the microbiome composition influenced the resistome composition. This study identified a core and accessory resistome present in a cohort of healthy pigs, in the same conditions without antibiotics. It highlights the presence of antibiotic resistance in the absence of antibiotic selective pressure and the variability between animals even under the same housing, food and living conditions. Antibiotic resistance will remain in the healthy pig gut even when antibiotics are not used. Therefore, the risk of antibiotic resistance transfer from animal faeces to human pathogens or the environment will remain in the absence of antibiotics.

Dissemination of multiple carbapenem resistance genes in an in vitro gut model simulating the human colon.

Carbapenemase-producing Enterobacteriaceae (CPE) pose a major global health risk. Mobile genetic elements account for much of the increasing CPE burden. To investigate CPE colonization and the impact of antibiotic exposure on subsequent resistance gene dissemination within the gut microbiota using a model to simulate the human colon. Gut models seeded with CPE-negative human faeces [screened with BioMérieux chromID® CARBA-SMART (Carba-Smart), Cepheid Xpert® Carba-R assay (XCR)] were inoculated with distinct carbapenemase-producing Klebsiella pneumoniae strains (KPC, NDM) and challenged with imipenem or piperacillin/tazobactam then meropenem. Resistant populations were enumerated daily on selective agars (Carba-Smart); CPE genes were confirmed by PCR (XCR, Check-Direct CPE Screen for BD MAX™). CPE gene dissemination was tracked using PacBio long-read sequencing. CPE populations increased during inoculation, plateauing at ∼105 log10 cfu/mL in both models and persisting throughout the experiments (>65 days), with no evidence of CPE 'washout'. After antibiotic administration, there was evidence of interspecies plasmid transfer of blaKPC-2 (111742 bp IncFII/IncR plasmid, 99% identity to pKpQIL-D2) and blaNDM-1 (∼170 kb IncFIB/IncFII plasmid), and CPE populations rose from <0.01% to >45% of the total lactose-fermenting populations in the KPC model. Isolation of a blaNDM-1K. pneumoniae with one chromosomal single-nucleotide variant compared with the inoculated strain indicated clonal expansion within the model. Antibiotic administration exposed a previously undetected K. pneumoniae encoding blaOXA-232 (KPC model). CPE exposure can lead to colonization, clonal expansion and resistance gene transfer within intact human colonic microbiota. Furthermore, under antibiotic selective pressure, new resistant populations emerge, emphasizing the need to control exposure to antimicrobials.

METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS;

&#x0D; Introduction: The versatility of Staphylococcus aureus has been transformed as “Methicillin resistant Staphylococcus aureus”. The most challenging are the disastrous virulence patterns being expressed due to the selection pressure of antibiotics. For assessing the prevalence of methicillin resistant Staphylococcus aureus; screening by cefoxitin disc (30µg) diffusion method is still a realistic approach among conventional phenotypic methods, being applied in most of the laboratories. This reliable and feasible technique contributes significantly for MRSA detection. This study has been designed to evaluate the prevalence and identify the sensitivity pattern of methicillin resistant Staphylococcus aureus isolates from different clinical specimens in a tertiary care hospital. Study Design: Descriptive study. Setting: Pathology Department, Microbiology Laboratory, PGMI. Period: January 2015 to December 2015. Materials &amp; Methods: A total 713 clinical isolates of Staphylococcus aureus were processed. Identification and confirmation of Staphylococcus aureus was done by colony morphology on blood agar, gram stain, catalase, coagulase and DNA-ase tests. Screening for methicillin resistance was done using cefoxitin disc (30mg, OXOID); while different antibiotic discs were used to assess the sensitivity profile by Modified Kirby-Bauer Disc Diffusion method according to CLSI guidelines (2016). Results: Out of 713 Staphylococcus aureus isolates, 92 (12.90%) isolates were labelled as methicillin resistant by cefoxitin disc diffusion test. Out of 92 MRSA isolates, 57 (14.65%) were recovered from male patients and 35 (10.80%) from female patients. While, 60 (65.22%) MRSA isolates showed hemolysis on blood agar. Among 92 MRSA isolates, 41 (44.57%) were recovered from pus specimen. Resistance to trimethoprim/sulfamethoxazole was highest (65.22%) after penicillin (100%); while all the MRSA isolates were 100% sensitive to both vancomycin and linezolid. Conclusion: The prevalence of MRSA in hospital care settings is of great clinical concern. To combat this public health threat effectively, continuous surveillance of health-care associated infections, along with local antibiotic sensitivity pattern of MRSA; as well as formulation of a definite antibiotic policy is required.

A nonlinear time-series analysis approach to identify thresholds in associations between population antibiotic use and rates of resistance.

Balancing access to antibiotics with the control of antibiotic resistance is a global public health priority. At present, antibiotic stewardship is informed by a 'use it and lose it' principle, in which antibiotic use by the population is linearly related to resistance rates. However, theoretical and mathematical models suggest that use-resistance relationships are nonlinear. One explanation for this is that resistance genes are commonly associated with 'fitness costs' that impair the replication or transmissibility of the pathogen. Therefore, resistant genes and pathogens may only gain a survival advantage where antibiotic selection pressures exceed critical thresholds. These thresholds may provide quantitative targets for stewardship-optimizing the control of resistance while avoiding over-restriction of antibiotics. Here, we evaluated the generalizability of a nonlinear time-series analysis approach for identifying thresholds using historical prescribing and microbiological data from five populations in Europe. We identified minimum thresholds in temporal relationships between the use of selected antibiotics and incidence rates of carbapenem-resistant Acinetobacter baumannii (Hungary), extended-spectrum β-lactamase-producing Escherichia coli (Spain), cefepime-resistant E. coli (Spain), gentamicin-resistant Pseudomonas aeruginosa (France) and methicillin-resistant Staphylococcus aureus (Northern Ireland) in different epidemiological phases. Using routinely generated data, our approach can identify context-specific quantitative targets for rationalizing population antibiotic use and controlling resistance. Prospective intervention studies that restrict antibiotic consumption are needed to validate these thresholds.

Low nutrient levels as drinking water conditions can reduce the fitness cost of efflux pump-mediated ciprofloxacin resistance in Pseudomonas aeruginosa

The long-term persistence of antibiotic resistance in the environment, especially in drinking water, is a public health concern. Expression of an efflux pump, an important mechanism of resistance to antibiotics, usually confers a fitness cost in bacteria. In this study, we aimed to determine why antibiotic resistance conferred by overexpression of an efflux pump persisted in low-nutrient environments (TOC < 10 mg/L) such as drinking and source water in which antibiotic selective pressure might be very low or even absent. Competition experiments between wild-type Pseudomonas aeruginosa and ciprofloxacin-resistant mutants revealed that the fitness cost of ciprofloxacin resistance significantly decreased (p < 0.05) under low-nutrient (0.5 mg/L total organic carbon (TOC)) relative to high-nutrient (500 mg/L TOC) conditions. Mechanisms underlying this fitness cost were analyzed. The mexD gene expression in resistant bacteria (cip_3 strain) was significantly lower (p < 0.05) in low-nutrient conditions, with 10 mg/L TOC ((8.01 ± 0.82)-fold), than in high-nutrient conditions, with 500 mg/L TOC ((48.89 ± 4.16)-fold). Moreover, rpoS gene expression in resistant bacteria ((1.36 ± 0.13)-fold) was significantly lower (p < 0.05) than that in the wild-type strain ((2.78 ± 0.29)-fold) under low-nutrient conditions (10 mg/L TOC), suggesting a growth advantage. Furthermore, the difference in metabolic activity between the two competing strains was significantly smaller (p < 0.05) in low-nutrient conditions (5 and 0.5 mg/L TOC). These results suggest that nutrient levels are a key factor in determining the persistence of antibiotic resistance conferred by efflux pumps in the natural environment with trace amounts or no antibiotics.

Blood stream infections by carbapenem resistant bacteria: critical perspectives from Indian tertiary care hospita

Purpose: Emergence of Multi-Drug Resistance (MDR) Gram Negative Bacilli (GNB) is increasing at a faster pace, and carbapenems are the drug of choice for treatment. But rampant use of these agents has given rise to Carbapenem-Resistant Enterobacteriaceae (CRE). CRE causes wide range of infections, with Blood Stream Infection (BSI) associated with highest mortality. Thereby leaving colistin as the only available option for treatment. Our study was aimed, to measure the occurrence of CRE BSI in our institute. Methods & Materials: A prospective observational study was performed over six months (July-December 2017). Blood culture was performed by BacT/ALERT automated system followed by identification of isolates and susceptibility testing by MALDI-TOF (Vitek-MS) and VITEK 2 system (bioMerieux, Inc., Durham, NC). The clinical characters, demography and resistance profiles of patients with culture proven CRE BSI were studied. Results: Among the 53 patients, the mean (SD) age was 48.94 ± 20.66 years, with a male preponderance (M: F = 2.5:1). On admission, 62.3% patients received antibiotics. Secondary bacteremia and ICU admission seen in 88.7% and 71.7% respectively. Median (IQR) time to bacteremia (since admission), stay in ICU and hospital was 10 (11.5), 7 (11.5) and 19(18.5) days respectively. Diabetes mellitus (23) was the most common comorbid condition. Among the isolates, Klebsiella pneumoniae was the most common (26) followed by Acinetobacter baumannii complex (14). Lower respiratory tract (24.5%) was found to be the most common source followed by hepatobiliary tract (17%). Median (IQR) for Pitt's bacteremia (<24 hr of bacteremia) and SOFA score (on admission) was 4(4) and 7(8) respectively. About 96.2% of the isolates were sensitive to colistin and 47.2% to tigecycline. As per sensitivity, combination of drugs were started but Colistin was given in 43.4% cases. Adverse event was seen in 73.9% patients received colistin. Favorable outcome was not seen in 41.5% patients. Conclusion: CRE causing BSI is a significant threat to public health, warranting increased efforts towards strategies focusing on prevention, detection and infection control practices. Severe underlying conditions, use of invasive procedures and antibiotic selection pressure may predispose the patient to acquire CRE infection. Efficient management of underlying condition with optimal antibiotic may better the outcome.

Electrokinetic remediation of antibiotic-polluted soil with different concentrations of tetracyclines.

This study investigated the efficacy of electrokinetic remediation of soils polluted with different concentrations of tetracyclines (TCs). Three widely used TCs (oxytetracycline, chlortetracycline, and tetracycline) were selected, and concentrations of 0, 5, 10, 20, and 50mg/kg (C0, C5, C10, C20, C50) were selected for comparison. Antibiotic-polluted soils with no electric field served as controls. The average removal rates of TCs in different treatments ranged from 25 to 48% after 7-day remediation. The contributing ratios of electrokinetics to TCs removal varied from 22 to 84%. The concentrations of NH4+ increased in soils and electrolytes, which indicated the decomposition of TCs in the electric field. The highest removal amount of TCs was obtained in the C50 treatment, due to efficient reactions of TCs with oxidative radicals generated during the electrolysis. The fluctuant range of pH in the electrolytes was decreased with increasing concentration of TCs, while the soil pH was increased. The removal rate of antibiotic-resistant bacteria (ARB) in the C5 treatment was significantly higher than that in other treatments. The abundance of antibiotic resistance genes (ARGs) increased with the concentrations of TCs in soils. It might result from the induction of increasing selective pressure of antibiotics. Significant removal of ARGs occurred in the C50 treatment (38-60%). In terms of controlling ARB and ARGs, which were more resistant, the electrokinetic technology showed advantageous effects. Above all, electrokinetic technology provides an effective remediation method, especially for TC-polluted soil with a concentration of 20-50mg/kg.

Antimicrobial susceptibility and genotyping of Mycoplasma pneumoniae isolates in Beijing, China, from 2014 to 2016

BackgroundThe presence of macrolide-resistant Myocplasma pneumoniae has been frequently reported in recent years, especially in China. In this study, we investigated the antimicrobial susceptibility and genotype against M. pneumoniae isolates from 2014 to 2016, Beijing.MethodsWe investigated the activities of four antibiotics against 81 M. pneumoniae isolates in vitro. All isolates were amplification of domains II and V of the 23S rRNA gene and the L4 and L22 ribosomal protein fragments. All isolates were genotyped with duplex real-time PCR, MLVA and VNTR detection in p1 gene.ResultsThe macrolide resistance rate was 65.4% (53/81). Each of the macrolide-resistant M. pneumoniae isolates was resistant to erythromycin (Minimum Inhibitory Concentration, MIC, ≥256 μg/ml) and azithromycin (MIC, 2–64 μg/ml), but susceptible to tetracycline and levofloxacin in vitro. Fifty two macrolide-resistant isolates harbored the A2063G mutation, and only 1 macrolide-resistant isolates harbored the A2064G mutation in domain V of the 23S ribosomal RNA gene. The C162A, A430G, and T279C mutations in the L4 and L22 ribosomal protein genes were not responsible for macrolide resistance, but they were related to the particular genotype of M. pneumoniae. 95.7% of type 1 isolates (45/47) were macrolide-resistance, and 23.5% of the type 2 isolates (8/34) were macrolide-resistance. Type 2 M. pneumoniae macrolide-resistance rate was 50.6% higher than that of the previous reports in China. The eight macrolide-resistant type 2 M. pneumoniae isolates were belong to 3/5/6/2 and 3/5/7/2 MLVA genotypes.ConclusionTo our knowledge, this phenomenon likely resulted from a combination of genotype shifting from type1 to type 2 and antibiotic selection pressure in M. pneumoniae in China in recent years. The increase of resistance in type 2 is not due to the spread of same clone. However, the relationship between genotype shifts and macrolide resistance in M. pneumoniae needs to be further verified with more extensive surveillance data.

Mycoplasmas under experimental antimicrobial selection: The unpredicted contribution of horizontal chromosomal transfer.

Horizontal Gene Transfer was long thought to be marginal in Mycoplasma a large group of wall-less bacteria often portrayed as minimal cells because of their reduced genomes (ca. 0.5 to 2.0 Mb) and their limited metabolic pathways. This view was recently challenged by the discovery of conjugative exchanges of large chromosomal fragments that equally affected all parts of the chromosome via an unconventional mechanism, so that the whole mycoplasma genome is potentially mobile. By combining next generation sequencing to classical mating and evolutionary experiments, the current study further explored the contribution and impact of this phenomenon on mycoplasma evolution and adaptation using the fluoroquinolone enrofloxacin (Enro), for selective pressure and the ruminant pathogen Mycoplasma agalactiae, as a model organism. For this purpose, we generated isogenic lineages that displayed different combination of spontaneous mutations in Enro target genes (gyrA, gyrB, parC and parE) in association to gradual level of resistance to Enro. We then tested whether these mutations can be acquired by a susceptible population via conjugative chromosomal transfer knowing that, in our model organism, the 4 target genes are scattered in three distinct and distant loci. Our data show that under antibiotic selective pressure, the time scale of the mutational pathway leading to high-level of Enro resistance can be readily compressed into a single conjugative step, in which several EnroR alleles were transferred from resistant to susceptible mycoplasma cells. In addition to acting as an accelerator for antimicrobial dissemination, mycoplasma chromosomal transfer reshuffled genomes beyond expectations and created a mosaic of resistant sub-populations with unpredicted and unrelated features. Our findings provide insights into the process that may drive evolution and adaptability of several pathogenic Mycoplasma spp. via an unconventional conjugative mechanism.

Using SimulATe to model the effects of antibiotic selective pressure on the dynamics of pathogenic bacterial populations.

Antibiotics are notable weapons in fighting bacteria. Nowadays, however, the effectiveness of antibiotics is severely hindered by the increasing levels of antibiotic resistances in pathogenic bacterial populations, which can persist due to the selective pressure caused by antibiotic exposure. Arguably, the main cause of antibiotic resistances endurance in nature is antibiotic misuse, such as via overusing, inappropriate prescribing as well as the uncontrolled use in agriculture and livestock. There is also a lack of knowledge on appropriate antibiotic usage by the general public. Public scientific literacy and more research on therapeutic practices are fundamental to tackle this problem. Here, we present SimulATe a software which allows the simulation of the effects of antibiotic therapies on bacterial populations during human infections. This software can be used to develop students’ scientific literacy, using infections and antibiotic treatments as context to engage students in scientific practices, and discussions on antibiotic treatment onset and duration or on its use in immunosuppressed or critically ill individuals. SimulATe’s features also allow it to be used for research purposes allowing the simulation of real scenarios and exploration of their outcomes across the parameters’ landscape.

Deciphering of microbial community and antibiotic resistance genes in activated sludge reactors under high selective pressure of different antibiotics

Currently, the effects of high antibiotic concentrations on the performance of microbiota and antibiotic resistance genes (ARGs) in activated sludge (AS) process are not well characterized. Lab-scale batch reactors were performed to evaluate the dynamics of microbial community and ARGs in response to six antibiotics at different concentrations using high-throughput sequencing-based 16S rRNA gene and metagenomic analyses. The presence of antibiotics remarkably decreased the microbial diversity, caused a great change of the microbiota structure, and exerted a selective pressure on the enrichment of potential antibiotic resistant bacteria (ARB), such as Arthrobacter, Thauera, Geothrix, Rudaea, Aridibacter, Conexibacter, Terrimonas, etc. High antibiotic selective pressures increased ARG abundance but simultaneously reduced ARG number. In total, 491 ARG subtypes belonging to 20 ARG types were detected and kanamycin treatment showed the highest ARG abundances. A core set of 54 ARG subtypes that accounted for 66.7%–99.6% of the total ARG abundances were shared by all samples. The increase of the abundances of both corresponding and non-corresponding ARGs under a specific antibiotic treatment revealed the collateral effects of antibiotic selective pressure. Microbial community may play an important role in the composition of ARGs. Network analysis indicated that both internal-type and external-type of ARGs exhibited higher non-random co-occurrence incidences and 18 genera were speculated as the possible hosts for multiple ARGs. This study deciphered the profiles and relationships between microbial community and ARGs in AS process treating wastewater with high antibiotic concentrations and could provide helpful guidance for controlling the development and dissemination of ARB and ARGs.

Modelling environmental antibiotic-resistance gene abundance: A meta-analysis

The successful treatment of infectious diseases heavily relies on the therapeutic usage of antibiotics. However, the high use of antibiotics in humans and animals leads to increasing pressure on bacterial populations in favour of resistant phenotypes. Antibiotics reach the environment from a variety of emission sources and are being detected at relatively low concentrations. Given the possibility of selective pressure to occur at sub-inhibitory concentrations, the ecological impact of environmental antibiotic levels on microbial communities and resistance levels is vastly unknown. Quantification of antibiotic-resistance genes (ARG) and of antibiotic concentrations is becoming commonplace. Yet, these two parameters are often assessed separately and in a specific spatiotemporal context, thus missing the opportunity to investigate how antibiotics and ARGs relate. Furthermore, antibiotic (multi)resistance has been receiving ever growing attention from researchers, policy-makers, businesses and civil society. Our aim was to collect the limited data on antibiotic concentrations and ARG abundance currently available to explore if a relationship could be defined in surface waters, sediments and wastewaters. A metric of antibiotic selective pressure, i.e. the sum of concentrations corrected for microbial inhibition potency, was used to correlate the presence of antibiotics in the environment to total relative abundance of ARG while controlling for basic sources of non-independent variability, such as country, year, study, sample and antibiotic class. The results of this meta-analysis show a significant statistical effect of antibiotic pressure and type of environmental compartment on the increase of ARG abundance even at very low levels. If global environmental antibiotic pollution continues, ARG abundance is expected to continue as well. Moreover, our analysis emphasizes the importance of integrating existing information particularly when attempting to describe complex relationships with limited mechanistic understanding.

Immunization Against Staphylococcus aureus Infections.

Background: Infections caused by Staphylococcus aureus continue to plague surgical patients, whether as surgical site infections or other nosocomial infections that complicate surgical care. The only meaningful methods available to decrease the risk of developing such infections are topical skin antisepsis (pre-operative skin preparation) and peri-operative antibiotic prophylaxis, neither of which offer a panacea. Alternatives to the latter are sought so as to minimize antibiotic selection pressure as a factor in the increasing problem of antimicrobial drug resistance. This review considers the possibility that immunization against S. aureus may offer a viable alternative for prophylaxis. Methods: Review and synthesis of pertinent English-language medical literature. Results: Vaccination against viral pathogens has been in successful clinical use for more than two centuries and was instrumental in the eradication of smallpox and the near-elimination of diseases such as poliomyelitis. Vaccinations against a limited number of bacterial pathogens (e.g., Bordetella pertussis, Clostridium tetanii, Corynebacterium diphtheriae, Haemophilus influenzae type b, Neisseria meningiditis, Streptococcus pneumoniae) have also been introduced with success, whereas others against bacteria are in development (C. difficile, Pseudomonas aeruginosa, S. aureus). Vaccination against S. aureus infection is in current veterinary use (e.g., to prevent mastitis among dairy cattle) but has not been successful to date in human beings despite multiple attempts, although development continues. Conclusions: Because of its complex microbiology, including multiple virulence factors and the ability to evade host immune surveillance, S. aureus presents numerous antigenic targets for vaccine development. Failure of two prior single-antigen vaccines in clinical trials has led to the consensus that future vaccine candidates must be directed against multiple antigens. Two distinct four-antigen vaccines are in clinical trials, but efficacy is yet to be determined.

2074. Performance and Impact Evaluation of Direct Rapid Antibiotic Susceptibility Testing on Antibiotic Treatment Accuracy in Clinical Setting

BackgroundTimely and effective antibiotics treatment is crucial in early period of bacteremia. Antibiotic susceptibility testing (AST) is essential for choosing an optimal antibiotics treatment, but conventional AST requires 2 days from confirmation of blood culture positivity. Direct rapid antibiotic susceptibility testing (dRAST) based on microfluidic agarose cannel chip technology determines antibiotic susceptibility by time lapse imaging in 6 hours. We evaluated the performance of dRAST to improve selection of adequate antibiotic in clinical practice settings.MethodsTwo hundred eighty-three patients with positive blood culture (BC) bottles were included for analysis. BC bottles from these patients were processed by current microbiology analyzer: Microscan for Gram positive strains and VITEK2 for Gram-negative strains. At the same time, AST was performed using dRAST. The susceptibility results were reported to infectious diseases specialists who determine optimal antibiotics based on AST results. We compared the time differences and accuracy of dRAST with those of conventional method.ResultsOf 283 patients, 117 (41.5%) patients were infected with Gram positive bacteria, 163 (57.4%) patients were infected with Gram negative bacteria and 3 (1.1%) patients were infected with Gram-positive and -negative bacteria. The total turnaround time for conventional method and dRAST from blood culture collection was 78.3 ± 27.0 and 55.9 ± 18.9 hours, respectively. Seventy-seven of 95 (81.1%) patients who received ineffective or suboptimal antibiotic treatment after confirming the results of Gram stain and 81 of 86 (94.2%) patients who received unnecessary broad-spectrum antibiotic treatment could have received adjusted optimal treatment based on dRAST.ConclusionThe use of dRAST system would accelerate earlier effective antibiotic administration and reduce the antibiotic selective pressure in patients with bacteremia.Disclosures J. Choi, QuantaMatrix Inc.: Employee, equity interest. S. Han, QuantaMatrix Inc.: Employee, equity interest. D. Y. Kim, QuantaMatrix Inc.: Board Member, equity interest. S. Kwon, QuantaMatrix Inc.: Board Member, equity interest.

Role of the Multidrug Resistance Efflux Pump MexCD-OprJ in the Pseudomonas aeruginosa Quorum Sensing Response.

Multidrug efflux pumps constitute a category of antibiotic resistance determinants that are a part of the core bacterial genomes. Given their conservation, it is conceivable that they present functions beyond the extrusion of antibiotics currently used for therapy. Pseudomonas aeruginosa stands as a relevant respiratory pathogen, with a high prevalence at hospitals and in cystic fibrosis patients. Part of its success relies on its low susceptibility to antibiotics and on the production of virulence factors, whose expression is regulated in several cases by quorum sensing (QS). We found that overexpression of the MexCD-OprJ multidrug efflux pump shuts down the P. aeruginosa QS response. Our results support that MexCD-OprJ extrudes kynurenine, a precursor of the alkyl-quinolone signal (AQS) molecules. Anthranilate and octanoate, also AQS precursors, do not seem to be extruded by MexCD-OprJ. Kynurenine extrusion is not sufficient to reduce the QS response in a mutant overexpressing this efflux pump. Impaired QS response is mainly due to the extrusion of 4-hydroxy-2-heptylquinoline (HHQ), the precursor of the Pseudomonas Quinolone Signal (PQS), leading to low PQS intracellular levels and reduced production of QS signal molecules. As the consequence, the expression of QS-regulated genes is impaired and the production of QS-regulated virulence factors strongly decreases in a MexCD-OprN P. aeruginosa overexpressing mutant. Previous work showed that MexEF-OprJ, another P. aeruginosa efflux pump, is also able of extruding kynurenine and HHQ. However, opposite to our findings, the QS defect in a MexEF-OprN overproducer is due to kynurenine extrusion. These results indicate that, although efflux pumps can share some substrates, the affinity for each of them can be different. Although the QS response is triggered by population density, information on additional elements able of modulating such response is still scarce. This is particularly important in the case of P. aeruginosa lung chronic infections, a situation in which QS-defective mutants are accumulated. If MexCD-OprJ overexpression alleviates the cost associated to triggering the QS response when un-needed, it could be possible that MexCD-OprJ antibiotic resistant overproducer strains might be selected even in the absence of antibiotic selective pressure, acting as antibiotic resistant cheaters in heterogeneous P. aeruginosa populations.

Evaluation of kanamycin and neomycin resistance in Lactobacillus plantarum using experimental evolution and whole-genome sequencing

High antibiotic resistance in the probiotic bacterium Lactobacillus plantarum was achieved using experimental evolution by subculture in the presence of kanamycin and neomycin. After 35 d in the presence of antibiotic, the minimum inhibitory concentration (MIC) of L. plantarum to kanamycin and neomycin were 1024 μg/mL and 256 μg/mL, respectively, which were 64-fold higher than the original isolates. These highly resistant L. plantarum isolates were then subcultured in the absence of antibiotics for 30 d and MIC values decreased to 128 μg/mL and 32 μg/mL at 10 d and 15 d, respectively, but remained constant thereafter. MIC values following subculture in the absence of antibiotic were still above the cut-off value for resistance, indicating that the acquisition of resistance was an irreversible process. Non-synonymous mutations of the resistant isolates were detected by whole-genome sequencing. Of these mutated genes regulating three structural variants (SV) including major facilitator family protein, the ABC transporter substrate binding protein and histidine kinase, were recovered to original state in the absence of antibiotic selection pressure indicating that these mutations played an important role in increasing kanamycin and neomycin resistance in L. plantarum. An evaluation of mobility of resistance genes showed that none of the resistance genes were located on mobile elements in the genome indicating that horizontal gene transfer to other bacteria was impossible. This study provides a way to evaluate antibiotic resistance in L. plantarum regarding its potential safety concern before application in food industry.

Parallel Integration and Chromosomal Expansion of Metabolic Pathways.

Robust fermentation of biomass-derived sugars into bioproducts demands the reliable microbial expression of metabolic pathways. Plasmid-based expression systems may suffer from instability and result in highly variable titers, rates, and yields. An established mitigation approach, chemical induced chromosomal expansion (CIChE), expands a singly integrated pathway to plasmid-like copy numbers while maintaining stability in the absence of antibiotic selection pressure. Here, we report parallel integration and chromosomal expansion (PIACE), extensions to CIChE that enable independent expansions of pathway components across multiple loci, use suicide vectors to achieve high-efficiency site-specific integration of sequence-validated multigene components, and introduce a heat-curable plasmid to obviate recA deletion post pathway expansion. We applied PIACE to stabilize an isopentenol pathway across three loci in E. coli DH1 and then generate libraries of pathway component copy number variants to screen for improved titers. Polynomial regressor statistical modeling of the production screening data suggests that increasing copy numbers of all isopentenol pathway components would further improve titers.

Mathematical modelling of vancomycin-resistant enterococci transmission during passive surveillance and active surveillance with contact isolation highlights the need to identify and address the source of acquisition

BackgroundClinical studies and mathematical simulation suggest that active surveillance with contact isolation is associated with reduced vancomycin-resistant enterococci (VRE) prevalence compared to passive surveillance. Models using pre- and post-intervention data that account for the imperfect observation and serial dependence of VRE transmission events can better estimate the effectiveness of active surveillance and subsequent contact isolation; however, such analyses have not been performed.MethodsA mathematical model was fitted to surveillance data collected pre- and post-implementation of active surveillance with contact isolation in the haematology-oncology ward. We developed a Hidden Markov Model to describe undetected and observed VRE colonisation/infection status based on the detection activities in the ward. Bayesian inference was used to estimate transmission rates. The effectiveness of active surveillance was assumed to be via increased detection and subsequent contact isolation of VRE positive patients.ResultsWe estimated that 31% (95% credible interval: 0.33–85%) of the VRE transmissions were due to cross-transmission between patients. The ratio of transmission rates from patients with contact isolation versus those without contact isolation was 0.33 (95% credible interval: 0.050–1.22).ConclusionsThe majority of the VRE acquisitions in the haematology-oncology ward was estimated to be due to background rates of VRE, rather than within ward patient to patient acquisition. The credible interval for cross-transmission was wide which results in a large degree of uncertainty in the estimates. Factors that could account for background VRE acquisition include endogenous acquisition from antibiotic selection pressure and VRE in the environment. Contact isolation was not significantly associated with reduced VRE transmission in settings where the majority of VRE acquisition was due to background acquisition, emphasising the need to identify and address the source of acquisition. As the credible interval for the ratio of VRE transmission in contact isolated versus non-contact isolated patients crossed 1, there is a probability that the transmission rate in contact isolation was not lower. Our finding highlights the need to optimise infection control measures other than active surveillance for VRE and subsequent contact isolation to reduce VRE transmission. Such measures could include antimicrobial stewardship, environmental cleaning, and hand hygiene.

Pansusceptible Escherichia coli isolates obtained from faeces of free-ranging Baird’s tapirs (Tapirus bairdii) suggests a low selective pressure for resistance determinants in the northwestern region of the Talamanca Mountain Range, Costa Rica

The main objective of this study was to analyse the antimicrobial susceptibility profile of Escherichia coli isolates obtained from faecal samples of free-ranging Baird's tapirs (Tapirus bairdii) in the northwestern region of the Talamanca Mountain Range, Costa Rica. Faecal samples were collected by opportunistic search of the study area from February-September 2017 during seven field expeditions. Escherichia coli isolates were recovered using selective and differential MacConkey agar medium and were subjected to biochemical identification and antimicrobial susceptibility testing using a VITEK®2 Compact automated system and the AST-N279 card. A total of 60 E. coli isolates were obtained from 63 faecal samples. Following evaluation of nine different antimicrobial classes, 98% (59/60) of the isolates were characterised as pansusceptible; only 1 isolate presented resistance to nalidixic acid. We propose that the commensal intestinal microbiota of free-ranging Baird's tapirs in this area remains isolated from antibiotic selective pressure, probably because seven different protected areas converge, thus giving a possible low anthropogenic activity to the region.