Significant Change In Susceptibility Research Articles

The role of aquaporins in osmotic cell lysis induced by Bacillus thuringiensis Cry1Ac toxin in Helicoverpa armigera

The insecticidal crystalline (Cry) and vegetative insecticidal (Vip) proteins derived from Bacillus thuringiensis (Bt) are used globally to manage insect pests, including the cotton bollworm, Helicoverpa armigera, one of the world's most damaging agricultural pests. Cry proteins bind to the ATP-binding cassette transporter C2 (ABCC2) receptor on the membrane surface of larval midgut cells, resulting in Cry toxin pores, and ultimately leading to cell swelling and/or lysis. Insect aquaporin (AQP) proteins within the membranes of larval midgut cells are proposed to allow the rapid influx of water into enterocytes following the osmotic imbalance triggered by the formation of Cry toxin pores. Here, we examined the involvement of H. armigera AQPs in Cry1Ac-induced osmotic cell swelling. We identified and characterized eight H. armigera AQPs and demonstrated that five are functional water channel proteins. Three of these (HaDrip1, HaPrip, and HaEglp1) were found to be expressed in the larval midgut. Xenopus laevis oocytes co-expressing the known Cry1Ac receptor HaABCC2 and each of the three HaAQPs displayed abnormal morphology and were lysed following exposure to Cry1Ac, suggesting a rapid influx of water was induced after Cry1Ac pore formation. In contrast, oocytes producing either HaABCC2 or HaAQP alone failed to swell or lyse after treatment with Cry1Ac, implying that both Cry1Ac pore formation and HaAQP function are needed for osmotic cell swelling. However, CRISPR/Cas9-mediated knockout of any one of the three HaAQP genes failed to cause significant changes in susceptibility to the Bt toxins Cry1Ac, Cry2Ab, or Vip3Aa. Our findings suggest that the multiple HaAQPs produced in larval midgut cells compensate for each other in allowing for the rapid influx of water in H. armigera midgut cells following Cry toxin pore formation, and that mutations affecting a single HaAQP are unlikely to confer resistance to Bt proteins.

Long exposure impact of antibiotics subinhibitory doses and silver nanoparticles on uropathogenic bacteria

Relevance. Although the primary purpose of using antibiotics is to treat infectious diseases, their misuse gradually leads to loss of their effectiveness. The aim of the current investigation was to explore the changes that occur in uropathogenic bacteria after long exposure to antimicrobials. Materials and Methods. We compared the effects of long-term exposure to ampicillin, cefazoline, kanamycin and silver nanoparticles (AgNPs) on susceptibility, biofilm formation and planktonic bacteria in 4 clinical uropathogenic strains namely Escherichia coli (UPEC), Staphylococcus aureus (S. aureus), Enterococcus faecalis (E. faecalis) and Streptococcus agalactiae (St. agalactiae). The minimum inhibitory concentrations (MIC) were determined using the microplate mircodilution method and bacteria were exposed to increasing concentrations of each antimicrobial (from MIC/2 to MIC) prepared in the brain heart infusion broth for 8 days. The susceptibility of bacteria to antibiotics was assessed using the Kirby Bauer disc diffusion method and the biofilm formation was assessed using crystal violet bacterial attachment assay. Results and Discussion. The data in this investigation highlight that long-term exposure to antimicrobials may induce changes in susceptibility to other antibiotics and biofilm formation in Uropathogenic strains. Indeed, exposure to ampicillin made E. faecalis resistant to ceftazidime and St agalactiae resistant to tetracycline, ceftazidime/clavulanate and ceftazidime. Following exposure to cefazolin, a significant decrease in susceptibility was observed in E. coli to ceftazidime/clavulanate and ceftazidime while S. aureus became resistant to ceftazidime/clavulanate, ceftazidime and to ceftriaxone. Similar variations were observed on St agalactiae and E. faecalis, which in addition to the 3 antibiotics above-mentioned, have become resistant to tetracycline. The most significant variations in susceptibility to antibiotics were observed following exposure to kanamycin: E. coli developed resistance to ceftazidime and a decrease in sensitivity was noted on ceftazidime/clavulanate while S. aureus, E. faecalis and St. agalactiae all 3 became resistant to ceftazidime/clavulanate and ceftazidime. In addition, except for E. coli all the bacteria in this investigation which had undergone successive passages in AgNPs developed resistance to ceftazidime/ clavulanate and ceftazidime. Bacteria exposed to ampicillin and cefazolin produced more biofilms than their respective controls. Conclusion. Long term exposure of uropathogens to antibiotics and AgNPs induces significant changes in susceptibility to other antibiotics and biofilm formation and antibiotics should therefore only be used when necessary.

The knockout of the nicotinic acetylcholine receptor subunit gene α1 (nAChR α1) through CRISPR/Cas9 technology exposes its involvement in the resistance of Spodoptera exigua to insecticides

Insect nicotinic acetylcholine receptors (nAChRs) are the directed targets of many insecticides. However, there have been no reports on the molecular characterization of the nAChR gene family or the causal association between nAChR α1 and resistance to insecticides in S. exigua, which is a significant agricultural pest. In this study, we identified a total of 9 candidate nAChR subunits in S. exigua, namely nAChR α1-α7 and nAChR β1-β2. For functional validation roles of Seα1 in insecticide resistance of S. exigua, we introduced a ∼ 1041-bp deletion of the Seα1 gene in a homozygous mutant strain (Seα1-KO) by CRISPR/Cas9 genome editing system, resulting in a premature truncation of the Seα1 protein and the subsequent loss of functional transmembrane (TM) 3 and TM4 elements. Compared with WH-S strain (wild-type strain), the Seα1-KO strain exhibited 2.62-folds resistant to trifluoropyrimidine, 8.3-folds resistant to dimehypo, and 5.28-folds resistant to dinotefuran, but no significant change in susceptibility to emamectin benzoate, spinetoram, lambda-cyhalothrin, permethrin and chlorpyrifos. Thus, this study has laid a solid foundation for investigating the role of nAChRs in S. exigua, and provides evidence for the crucial involvement of the α1 subunit in the mechanism of trifluoropyrimidine, dimehypo, and dinotefuran in S. exigua. Moreover, it provides a reference for the value of Seα1 subunit and its homologues in other species as insecticide targets.

Distribution, Trends, and Antimicrobial Susceptibility of Bacteroides, Clostridium, Fusobacterium, and Prevotella Species Causing Bacteremia in Japan During 2011-2020: A Retrospective Observational Study Based on National Surveillance Data.

The increasing prevalence of anaerobic bacteremia is a major concern worldwide and requires longitudinal monitoring. We present one of the largest and longest longitudinal studies on the prevalence and antimicrobial resistance of Bacteroides, Clostridium, Fusobacterium, and Prevotella spp. isolated from blood culture samples using national comprehensive surveillance data in Japan during 2011-2020 as part of the Japan Nosocomial Infections Surveillance. Data for 41 949 Bacteroides spp., 40 603 Clostridium spp., 7013 Fusobacterium spp., and 5428 Prevotella spp. isolates were obtained. The incidences of bacteremia caused by Bacteroides fragilis, Clostridium perfringens, and Fusobacterium nucleatum significantly increased during the period (P < .0001). Among the 20 species analyzed, 18 showed no significant changes in susceptibility over time, including B. fragilis, C perfringens, and F. nucleatum. However, resistance to clindamycin increased in B. thetaiotaomicron (P = .0312), and resistance to ampicillin increased in B. ovatus (P = .0008). Our comprehensive national surveillance data analysis demonstrated a continuous increase in the incidence of anaerobic bacteremia, particularly in B. fragilis, C. perfringens, and F. nucleatum. This may be linked to the increasing number of colorectal cancer cases or advancing methods for species identification and susceptibility testing, requiring cautious interpretation. The discovery of an upsurge in anaerobic bacteremia and potential alterations in susceptibility highlights the necessity for more extensive studies in this field.

Copper and nanostructured anatase rutile and carbon coatings induce adaptive antibiotic resistance

Contaminated surfaces are vehicles for the spread of infectious disease-causing microorganisms. A strategy to prevent their spread is applying antimicrobial coatings to surfaces. Both nanostructured anatase rutile and carbon (NsARC), a TiO2 formulation, and copper are examples of antimicrobial agents that are used in making or coating door handles and similar surfaces, to reduce microbial loads. Antimicrobial surfaces have been extensively tested for antimicrobial activity but not sublethal effects, such as exposure-associated multiple antibiotic resistance phenotypes usually caused by induction of efflux pump genes. The possibility of NsARC and copper inducing indicative efflux pump pathways was investigated by monitoring the expression of mScarlet fluorescent protein (FP) in two reporter strains of Escherichia coli. There was an increase in the expression of FP in the reporter strains exposed to NsARC and copper relative to the inert control composed of stainless steel. Furthermore we tested E. coli and Staphylococcus aureus following 8 h of exposure to NsARC for changes in resistance to selected antibiotics. E. coli that were exposed to NsARC became more susceptible to kanamycin but there was no significant change in susceptibility of S. aureus to any tested antibiotics. These findings suggests that even though NsARC and copper are antimicrobial, they also have some potential to cause unintended phenotypes.

Quantitative susceptibility mapping reveals alterations of dentate nuclei in common types of degenerative cerebellar ataxias.

The cerebellar nuclei are a brain region with high iron content. Surprisingly, little is known about iron content in the cerebellar nuclei and its possible contribution to pathology in cerebellar ataxias, with the only exception of Friedreich’s ataxia. In the present exploratory cross-sectional study, quantitative susceptibility mapping was used to investigate volume, iron concentration and total iron content of the dentate nuclei in common types of hereditary and non-hereditary degenerative ataxias. Seventy-nine patients with spinocerebellar ataxias of types 1, 2, 3 and 6; 15 patients with Friedreich’s ataxia; 18 patients with multiple system atrophy, cerebellar type and 111 healthy controls were also included. All underwent 3 T MRI and clinical assessments. For each specific ataxia subtype, voxel-based and volumes-of-interest-based group analyses were performed in comparison with a corresponding age- and sex-matched control group, both for volume, magnetic susceptiblity (indicating iron concentration) and susceptibility mass (indicating total iron content) of the dentate nuclei. Spinocerebellar ataxia of type 1 and multiple system atrophy, cerebellar type patients showed higher susceptibilities in large parts of the dentate nucleus but unaltered susceptibility masses compared with controls. Friedreich’s ataxia patients and, only on a trend level, spinocerebellar ataxia of type 2 patients showed higher susceptibilities in more circumscribed parts of the dentate. In contrast, spinocerebellar ataxia of type 6 patients revealed lower susceptibilities and susceptibility masses compared with controls throughout the dentate nucleus. Spinocerebellar ataxia of type 3 patients showed no significant changes in susceptibility and susceptibility mass. Lower volume of the dentate nuclei was found to varying degrees in all ataxia types. It was most pronounced in spinocerebellar ataxia of type 6 patients and least prominent in spinocerebellar ataxia of type 3 patients. The findings show that alterations in susceptibility revealed by quantitative susceptibility mapping are common in the dentate nuclei in different types of cerebellar ataxias. The most striking changes in susceptibility were found in spinocerebellar ataxia of type 1, multiple system atrophy, cerebellar type and spinocerebellar ataxia of type 6. Because iron content is known to be high in glial cells but not in neurons of the cerebellar nuclei, the higher susceptibility in spinocerebellar ataxia of type 1 and multiple system atrophy, cerebellar type may be explained by a reduction of neurons (increase in iron concentration) and/or an increase in iron-rich glial cells, e.g. microgliosis. Hypomyelination also leads to higher susceptibility and could also contribute. The lower susceptibility in SCA6 suggests a loss of iron-rich glial cells. Quantitative susceptibility maps warrant future studies of iron content and iron-rich cells in ataxias to gain a more comprehensive understanding of the pathogenesis of these diseases.

Cadherin Protein Is Involved in the Action of Bacillus thuringiensis Cry1Ac Toxin in Ostrinia furnacalis.

Transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal proteins have been extensively planted for insect pest control, but the evolution of Bt resistance in target pests threatens the sustainability of this approach. Mutations of cadherin in the midgut brush border membrane was associated with Cry1Ac resistance in several lepidoptera species, including the Asian corn borer, Ostrinia furnacalis, a major pest of maize in Asian–Western Pacific countries. However, the causality of O. furnacalis cadherin (OfCad) with Cry1Ac resistance remains to be clarified. In this study, in vitro and in vivo approaches were employed to examine the involvement of OfCad in mediating Cry1Ac toxicity. Sf9 cells transfected with OfCad showed significant immunofluorescent binding with Cry1Ac toxin and exhibited a concentration-dependent mortality effect when exposed to Cry1Ac. The OfCad knockout strain OfCad-KO, bearing homozygous 15.4 kb deletion of the OfCad gene generated by CRISPR/Cas9 mutagenesis, exhibited moderate-level resistance to Cry1Ac (14-fold) and low-level resistance to Cry1Aa (4.6-fold), but no significant changes in susceptibility to Cry1Ab and Cry1Fa, compared with the original NJ-S strain. The Cry1Ac resistance phenotype was inherited as autosomal, recessive mode, and significantly linked with the OfCad knockout in the OfCad-KO strain. These results demonstrate that the OfCad protein is a functional receptor for Cry1Ac, and disruption of OfCad confers a moderate Cry1Ac resistance in O. furnacalis. This study provides new insights into the mode of action of the Cry1Ac toxin and useful information for designing resistance monitoring and management strategies for O. furnacalis.

Impact of CLSI Break Point Changes Over the Past Decade on Antimicrobial Susceptibility in Gram-Negative Bacteria

Background: Over the past decade, the CLSI has updated susceptibility break points for several antimicrobial agents. The purpose of this study was to evaluate the impact of these changes against gram-negative bacteria at our academic medical center. Methods: In this retrospective, IRB-approved study, we collected consecutive, nonduplicate clinical isolates of Enterobacter cloacae, Escherichia coli, Klebsiella aerogenes, K. oxytoca, K. pneumoniae, and Pseudomonas aeruginosa for the past decade (2010–2019) at our academic medical center and 3 adult ICUs. Susceptibility testing was performed using the BD Phoenix automated system. For these isolates, susceptibilities for 7 β-lactams (aztreonam, ceftriaxone, ceftazidime, cefepime, piperacillin/tazobactam, ertapenem, and meropenem) and 2 fluoroquinolones (levofloxacin, ciprofloxacin) were calculated based upon CLSI break points in 2010 and current CLSI break points in 2020. Any change &gt;5% in susceptibility was deemed significant for this analysis. Results: In 17.5% of Enterobacteriales isolates tested, at least 1 antimicrobial demonstrated significant decline. Ertapenem was the most commonly affected antimicrobial (45% of the isolates) followed by ceftriaxone (35%) and cefepime (25%). Susceptibilities of aztreonam, ceftazidime, and meropenem were not affected for any of the Enterobacteriales. The most common organism demonstrating a significant impact on change in susceptibility among the Enterobacteriales was E. cloacae (41.7% of the time) followed by E. aerogenes (20.8%), K. oxytoca (12.5%), K. pneumoniae (8.3%) and E. coli (4.2%). Most of the impact was observed hospital-wide (33.3%), followed closely by the MICU (28.6%), the NSICU (23.8%) and the CVICU (14.3%). For P. aeruginosa, the impact of the antimicrobial break-point changes on susceptibility was more pronounced than the Enterobacteriales. Overall, 93.8% of the time there was a significant decline in antimicrobial susceptibility. Each antimicrobial (ciprofloxacin, levofloxacin, meropenem, and piperacillin/tazobactam) demonstrated a significant decline in susceptibility hospital-wide and in each ICU except for the susceptibility of meropenem in the NSICU. Conclusions: Changes in break points had a significant impact on the susceptibility of all antimicrobials for P. aeruginosa at our institution, both hospital-wide and in the adult ICUs. Although the impact was less for the Enterobacteriales, ertapenem, ceftriaxone, and cefepime demonstrated significant susceptibility changes, especially with E. cloacae. Understanding and evaluating the impact of the break-point changes may lead to changes in empiric therapy in other institutions.Funding: NoDisclosures: None

Knockout of Two Cry-Binding Aminopeptidase N Isoforms Does Not Change Susceptibility of Aedes aegypti Larvae to Bacillus thuringiensis subsp. israelensis Cry4Ba and Cry11Aa Toxins.

Simple SummaryThe midgut aminopeptidase N (APN) isoforms have been identified as the binding receptor of insecticidal Cry toxins in numerous insects, including the major arbovirus vector Aedes aegypti (Ae. aegypti). However, whether the Cry-binding APN acts as an essential functional receptor to mediate Bacillus thuringiensis subsp. israelensis (Bti) toxicity in Ae. aegypti larvae remains to be determined. In this study, our results provide the direct molecular evidence demonstrating that two Cry-binding APN isoforms (AeAPN1 and AeAPN2) did not play a key role in mediating Bti Cry4Ba and Cry11Aa toxicity in Ae. aegypti larvae.The insecticidal Cry4Ba and Cry11Aa crystal proteins from Bacillus thuringiensis subsp. israelensis (Bti) are highly toxic to Ae. aegypti larvae. The glycosylphosphatidylinositol (GPI)-anchored APN was identified as an important membrane-bound receptor for multiple Cry toxins in numerous Lepidoptera, Coleoptera, and Diptera insects. However, there is no direct molecular evidence to link APN of Ae. aegypti to Bti toxicity in vivo. In this study, two Cry4Ba/Cry11Aa-binding Ae. aegypti GPI-APN isoforms (AeAPN1 and AeAPN2) were individually knocked-out using CRISPR/Cas9 mutagenesis, and the AeAPN1/AeAPN2 double-mutant homozygous strain was generated using the reverse genetics approach. ELISA assays showed that the high binding affinity of Cry4Ba and Cry11Aa protoxins to the midgut brush border membrane vesicles (BBMVs) from these APN knockouts was similar to the background from the wild-type (WT) strain. Likewise, the bioassay results showed that neither the single knockout of AeAPN1 or AeAPN2, nor the simultaneous disruption of AeAPN1 and AeAPN2 resulted in significant changes in susceptibility of Ae. aegypti larvae to Cry4Ba and Cry11Aa toxins. Accordingly, our results suggest that AeAPN1 and AeAPN2 may not mediate Bti Cry4Ba and Cry11Aa toxicity in Ae. aegypti larvae as their binding proteins.

Influence of analysis conditions for antimicrobial susceptibility test data on susceptibility rates.

To support effective antibiotic selection in empirical treatments, infection control interventions, and antimicrobial resistance containment strategies, many medical institutions collect antimicrobial susceptibility test data conducted at their facilities to prepare cumulative antibiograms. To evaluate how the setpoints of duplicate isolate removal period and data collection period affect the calculated susceptibility rates in antibiograms. The Sakai City Medical Center is a regional core hospital for tertiary emergency medical care with 480 beds for general clinical care. In this study, all the Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae isolates collected at the Sakai City Medical Center Clinical Laboratory between July 2013 and December 2018 were subjected to antimicrobial susceptibility tests and the resulting data was analyzed. The longer the duplicate isolate removal period, the fewer the isolates are available for every bacterial species. Differences in the length of the duplicate isolate removal period affected P. aeruginosa susceptibility rates to β-lactam antibiotics by up to 10.8%. The setpoint of the data collection period affected the antimicrobial susceptibility rates by up to 7.3%. We found that a significant change in susceptibility could be missed depending on the setting of the data collection period, in preparing antibiogram of β-lactam antibiotics for P. aeruginosa. When referring to antibiograms, medical professionals involved in infectious disease treatment should be aware that the parameter values, such as the duplicate isolate removal period and the data collection period, affect P. aeruginosa susceptibility rates especially to β-lactam antibiotics. And antibiogram should be updated within the shortest time period that is practically possible, taking into account restrictions such as numbers of specimen.

CRISPR-mediated gene knockout reveals nicotinic acetylcholine receptor (nAChR) subunit α6 as a target of spinosyns in Helicoverpa armigera.

The spinosyn insecticides (spinosad and spinetoram) have been intensively used to control a wide range of agricultural pests. However, resistance to spinosyns has evolved in several agricultural pests. Disruption of the nicotinic acetylcholine receptor subunit α6 (nAChRα6) has been associated with high levels of resistance to spinosyns in both field and laboratory-selected strains of several insect pests. Among the 12 nAChR subunits of Helicoverpa armigera, Haα6 has the closest sequence similarity (66.02%) to Haα7. Here we used CRISPR-mediated knockouts to evaluate the role of two nAChR subunits (Haα6 and Haα7) of H. armigera in toxicity of spinosyns. Individual knockouts of Haα6 and Haα7 were created utilizing CRISPR/Cas9 system in H. armigera. The Haα6 knockout (Haα6-KO) strain exhibited high levels of resistance to spinosad (531-fold) and spinetoram (1105-fold) compared with the wild-type parent SCD strain, whereas the Haα7 knockout (Haα7-KO) strain showed no significant susceptibility changes to both spinosyns. Genetic analyses demonstrated that resistance to spinosad conferred by knockout of Haα6 was autosomal, incompletely recessive and tightly linked to the disruption mutation of Haα6. Both Haα6-KO and Haα7-KO strains had no significant effects on susceptibility to other four insecticides including emamectin benzoate, beta-cypermethrin, chlorantraniliprole and indoxacarb. Our results provide in vivo functional evidence for Haα6 as a target of spinosyns in H. armigera, and little or no role of Haα7 in mediating toxicity of spinosyns. The results are valuable to the development of resistance monitoring and management methods for spinosyn resistance in H. armigera. © 2020 Society of Chemical Industry.

Sulfoxaflor efficacy in the laboratory against imidacloprid-resistant and susceptible populations of the green peach aphid, Myzus persicae: Impact of the R81T mutation in the nicotinic acetylcholine receptor

A key to effective insect pest management and insecticide resistance management is to provide growers with a range of new tools as potential alternatives to existing compounds or approaches. Sulfoxaflor (Isoclast™ active) is a new sulfoximine insecticide which is active on a broad range of sap-feeding insects, including species that have reduced susceptibility to currently used insecticides, such as imidacloprid from the neonicotinoid class. Sulfoxaflor (SFX) and imidacloprid (IMI) were tested in laboratory bioassays to compare the susceptibility of field populations of green peach aphid, Myzus persicae (Sulzer), exhibiting varying degrees of resistance involving an alteration (R81T) to the insect nicotinic acetylcholine receptor. The LC50 values for M. persicae exposed to SFX ranged from 0.09 to 1.31 (mg litre−1), whereas when the same populations were exposed to IMI the LC50 values ranged from 0.6 to 76.2 (mg litre−1). M. persicae were significantly more sensitive to SFX as compared to IMI for nine of the 13 populations tested. For M. persicae populations confirmed to be homozygous susceptible (ss) or heterozygous rs) for the R81T point mutation, there was no significant differences in the observed LC50 values for either SFX or IMI relative to the susceptible reference population (15LP1). However, in all M persicae populations that were homozygous (rr) for the R81T point mutation, susceptibility was significantly less to IMI as compared to the reference population with resistance ratios ranging from 22.1 to 63.5-fold. In contrast, only one homozygous resistant population (15MP9) exhibited a statistically significant change in susceptibility (RR = 10-fold) to SFX as compared to the reference population, which was far less than the 56-fold observed for imidacloprid in that same population. Thus, this study indicates there is no specific correlation between the laboratory efficacy of SFX and IMI in field collected populations in Spain displaying varying degrees of resistance to IMI. Furthermore, the presence of target site resistance in M. persicae to IMI, in the form of the R81T mutation, does not a priori translate to a reduction in sensitivity to sulfoxaflor. Consequently, SFX can be an effective tool for use in insect pest management programs for green peach aphid. These data also serve as a baseline reference for green peach aphid sensitivity to SFX prior to commercial uses in Spain.

Functional validation of nicotinic acetylcholine receptor (nAChR) α6 as a target of spinosyns in Spodoptera exigua utilizing the CRISPR/Cas9 system.

The beet armyworm, Spodoptera exigua, is a serious agricultural pest that is primarily controlled using chemical insecticides. Recently, resistance to the insecticide spinosad has been described in S. exigua field populations. To date, there has been no functional evidence proving the involvement of the nicotinic acetylcholine receptor (nAChR) α6 mutation in spinosad resistance in S. exigua. In this study, using the CRISPR/Cas9 genome-editing system, a homozygous strain (Seα6-KO) with approximately 1760-bp deletion within Seα6 in S. exigua causing a premature truncation of Seα6 was successfully constructed. Insecticide bioassays showed that Seα6-KO exhibited 373-fold higher resistance to spinosad and 850-fold higher resistance to spinetoram compared to WH-S strain with the same genetic background but showed no significant change in susceptibility to emamectin benzoate and chlorantraniliprole. Genetic analysis revealed that Seα6-KO is inherited as an incompletely recessive trait. The results clearly demonstrated the functional role of Seα6 in resistance to spinosyn insecticides and provide an example of using genome editing to verify a target premature truncation associated with resistance.

Understanding selection of antimicrobial resistance in biofilms through experimental evolution

Background. Biofilms possess several important properties which differentiate them from free-living cells, including their response to selective pressures. This work demonstrates a link between exposure to non-therapeutic antimicrobials and selection for antibiotic resistance in experimentally evolved biofilms of Pseudomonas aeruginosa. Methods. Biofilms of P. aeruginosa PA14 were experimentally evolved through successive subculture under periodically increasing antimicrobial stress. Beginning at 0.25-times MIC, biofilms were exposed to benzalkonium chloride, ciprofloxacin, colistin, zinc or copper sulfate. When the antimicrobial stress resulted in growth failure, the last successful passage was phenotyped. Biofilm formation was quantified via crystal violet uptake and MICs determined by broth microdilution according to EUCAST guidelines. Results. Experimental evolution selected for mutants which produced approximately four-fold more biomass than the parenteral strain. Both ciprofloxacin and chloramphenicol readily selected for mutants able to survive &gt;1024-times MIC. Conversely, no change in susceptibility was observed for either metal. However, despite zinc sulfate not selecting for decreased susceptibility to itself, cross-resistance to both colistin and meropenem was observed in the zinc-passaged lineages. Benzalkonium chloride did not select for a significant change in susceptibility when grown planktonically. However, when assayed as a biofilm, a 5-fold decrease in benzalkonium chloride susceptibility was observed, indicating a biofilm-specific mechanism of antimicrobial tolerance. Conclusion. Mutants with an increased capacity to form biofilms and decreased antimicrobial susceptibility were successfully selected. Furthermore, zinc sulfate, a common feed additive, was able to select for decreased susceptibility to clinically-relevant antibiotics, lending to concerns that ubiquitous non-therapeutic antimicrobials may act as non-canonical drivers of antibiotic resistance.

A Study on Acinetobacter baumannii and Staphylococcus aureus Strains Recovered from the Same Infection Site of a Diabetic Patient.

Diabetic foot ulcer infections are frequently polymicrobial in nature and exhibit increased morbidity and mortality, as well as, treatment failures. Interactions between Acinetobacter baumannii and Staphylococcus aureus were studied, which showed strain-dependent changes in growth and antibiotic susceptibility. This study examined the interactions between two clinical strains of A. baumannii (1929) and S. aureus (1928) that were recovered from skin and soft tissues of a diabetic patient. When S. aureus 1928 and A. baumannii 1929 were co-cultured together, there was no significant decrease in growth in either clinical strains, indicating that both strains can co-exist in the same site of infection. Additionally, neither strains experienced statistically significant changes in susceptibility. These findings highlight that these two pathogens can be found in the same niche of infection, which may lead to more aggressive outcome of the infection.

Dependence of resting-state fMRI fluctuation amplitudes on cerebral cortical orientation relative to the direction of B0 and anatomical axes

Functional magnetic resonance imaging (fMRI) is now capable of sub-millimetre scale measurements over the entire human brain, however with such high resolutions each voxel is influenced by the local fine-scale details of the cerebral cortical vascular anatomy. The cortical vasculature is structured with the pial vessels lying tangentially along the grey matter surface, intracortical diving arterioles and ascending venules running perpendicularly to the surface, and a randomly oriented capillary network within the parenchyma. It is well-known that the amplitude of the blood-oxygenation level dependent (BOLD) signal emanating from a vessel depends on its orientation relative to the B0-field. Thus the vascular geometric hierarchy will impart an orientation dependence to the BOLD signal amplitudes and amplitude differences due to orientation differences constitute a bias for interpreting neuronal activity. Here, we demonstrate a clear effect of cortical orientation to B0 in the resting-state BOLD-fMRI amplitude (quantified as the coefficient of temporal signal variation) for 1.1 mm isotropic data at 7T and 2 mm isotropic at 3T. The maximum bias, i.e. the fluctuation amplitude difference between regions where cortex is perpendicular to vs. parallel to B0, is about +70% at the pial surface at 7T and +11% at 3T. The B0 orientation bias declines with cortical depth, becomes progressively smaller closer to the white matter surface, but then increases again to a local maximum within the white matter just beneath the cortical grey matter, suggesting a distinct tangential network of white matter vessels that also generate a BOLD orientation effect. We further found significant (negative) biases with the cortex orientation to the anterior-posterior anatomical axis of the head: a maximum negative bias of about −30% at the pial surface at 7T and about −13% at 3T. The amount of signal variance explained by the low frequency drift, motion and the respiratory cycle also showed a cortical orientation dependence; only the cardiac cycle induced signal variance was independent of cortical orientation, suggesting that the cardiac induced component of the image time-series fluctuations is not related to a significant change in susceptibility. Although these orientation effects represent a signal bias, and are likely to be a nuisance in high-resolution analyses, they may help characterize the vascular influences on candidate fMRI acquisitions and, thereby, may be exploited to improve the neuronal specificity of fMRI.

Knockout of three aminopeptidase N genes does not affect susceptibility of Helicoverpa armigera larvae to Bacillus thuringiensis Cry1A and Cry2A toxins.

Bacillus thuringiensis (Bt) insecticidal toxins have been globally utilized for control of agricultural insects through spraying or transgenic crops. Binding of Bt toxins to special receptors on midgut epithelial cells of target insects is a key step in the mode of action. Previous studies suggested aminopeptidase N1 (APN1) as a receptor or putative receptor in several lepidopteran insects including Helicoverpa armigera through evidence from RNA interefence‐based gene silencing approaches. In the current study we tested the role of APNs in the mode of action of Bt toxins using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR‐associated protein 9‐mediated gene knockout. Three APN genes (HaAPN1, HaAPN2 and HaAPN5) were individually knocked out in a susceptible strain (SCD) of H. armigera to establish three homozygous knockout strains. Qualitative in vitro binding studies indicated binding of Cry1Ac or Cry2Ab to midgut brush border membrane vesicles was not obviously affected by APN knockout. Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain. This suggests that the three HaAPN genes we tested may not be critical in the mode of action of Cry1A or Cry2A toxins in H. armigera.

Improvement of gram-negative susceptibility to fluoroquinolones after implementation of a pre-authorization policy for fluoroquinolone use: A decade-long experience.

Due to concerns over increasing fluoroquinolone (FQ) resistance among gram-negative organisms, our stewardship program implemented a preauthorization use policy. The goal of this study was to assess the relationship between hospital FQ use and antibiotic resistance. Retrospective cohort. Large academic medical center. We performed a retrospective analysis of FQ susceptibility of hospital isolates for 5 common gram-negative bacteria: Acinetobacter spp., Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Primary endpoint was the change of FQ susceptibility. A Poisson regression model was used to calculate the rate of change between the preintervention period (1998-2005) and the postimplementation period (2006-2016). Large rates of decline of FQ susceptibility began in 1998, particularly among P. aeruginosa, Acinetobacter spp., and E. cloacae. Our FQ restriction policy improved FQ use from 173 days of therapy (DOT) per 1,000 patient days to <60 DOT per 1,000 patient days. Fluoroquinolone susceptibility increased for Acinetobacter spp. (rate ratio [RR], 1.038; 95% confidence interval [CI], 1.005-1.072), E. cloacae (RR, 1.028; 95% CI, 1.013-1.044), and P. aeruginosa (RR, 1.013; 95% CI, 1.006-1.020). No significant change in susceptibility was detected for K. pneumoniae (RR, 1.002; 95% CI, 0.996-1.008), and the susceptibility for E. coli continued to decline, although the decline was not as steep (RR, 0.981; 95% CI, 0.975-0.987). A stewardship-driven FQ restriction program stopped overall declining FQ susceptibility rates for all species except E. coli. For 3 species (ie, Acinetobacter spp, E. cloacae, and P. aeruginosa), susceptibility rates improved after implementation, and this improvement has been sustained over a 10-year period.

Ten years of MON 810 resistance monitoring of field populations of Ostrinia nubilalis in Europe

AbstractFrom 2005 to 2015, Ostrinia nubilalis were collected in the most important maize‐growing areas in Europe where MON 810 was cultivated. The susceptibility of these O. nubilalis collections to the Cry1Ab protein was determined using overlay bioassays and compared to that of reference (control) strains. Larvae that died or did not moult after 7 days were used to calculate a moulting inhibition concentration (MIC). Two different batches of Cry1Ab protein were used over the course of this study. Between 2005 and 2015, 145 collections of O. nubilalis from 14 areas were analysed. The Cry1Ab susceptibility of populations from different geographic regions differed only slightly across years. The greatest variability in the MIC50 for field samples collected from 2005 to 2011 and tested with batch 1 was 6.6‐fold in 2006. For field‐collected O. nubilalis, the difference between MIC50 values of the most susceptible and most tolerant samples was 13.1‐fold for this period. For samples collected in 2012–2015 and tested with batch 2, the greatest variability was 4.1‐fold in 2014. A diagnostic concentration (MIC99) was calculated for batch 1 (48 ng/cm2) using the results from all the collections in 2005–2012. Bridging experiments indicated that the diagnostic concentration for batch 2 was 28 ng/cm2. From 2006 onwards, no O. nubilalis reached the 2nd larval stage when the diagnostic concentration of either batch of Cry1Ab was used. Only one insect collected from Romania in 2012 and two insects collected as reference strain from Spain in 2015 survived exposure to a dosage of 20 ng/cm2, and none of these larvae survived on MON 810 maize. Our results indicate that there has been no significant change in susceptibility to Cry1Ab in European populations of O. nubilalis over the period 2005–2015.

Impact of the pneumococcal conjugate vaccine on serotype distribution of adult non-invasive Streptococcus pneumoniae isolates in Tokai region, Japan, 2008-2016.

The introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in February 2010 markedly reduced the burden of invasive pneumococcal disease and changed serotype distribution in Japan. In November 2013, PCV7 was replaced by the 13-valent pneumococcal conjugate vaccine (PCV13). We investigated the serotype distribution and susceptibility trends of non-invasive Streptococcus pneumoniae isolates collected from adult patients. A total of 504 pneumococcal isolates were collected during 4 periods between 2008 and 2016 (Period 1; between June 2008 and April 2009, Period 2; between September 2010 and March 2011, Period 3; between October 2011 and March 2012, Period 4; between August 2015 and January 2016). The coverage of PCV7 and PCV13 significantly decreased from 38.6% and 60.5% in Period 1 to 6.6% and 31.1% in Period 4. This change was mainly due to a large decrease in the frequency of serotype 19F, 6B, and 14. Serotype 3 was the most frequently isolated, and gradually increased. Additionally, non-PCV13 serotypes 11A, 33F, and 35B significantly increased. Most of the PCV7 serotypes 19F, 23F, 6B, and 14 had mutations of penicillin-binding protein genes and macrolide resistance genes, and these serotypes showed low susceptibilities to cefdinir and clarithromycin. On the other hand, a significant change in susceptibility to various antimicrobial agents was not observed throughout the study period, except for decreased susceptibility to carbapenems. Continuous surveillance studies of pneumococcal serotype changes and drug susceptibility are necessary in future.