Susceptible Strains Research Articles

Mechanisms of Cefiderocol Resistance in Carbapenemase-Producing Enterobacterales: Insights from Comparative Genomics

Background/Objectives: Cefiderocol is a novel siderophore cephalosporin with potent in vitro activity against a broad spectrum of Gram-negative bacteria, including carbapenemase-producing Enterobacterales (CPE). However, the recent emergence of resistance in clinical settings raises important concerns regarding its long-term effectiveness. This study aims to investigate the genomic determinants associated with cefiderocol resistance in CPE isolates of human origin. Methods: Comparative genomic analyses were conducted between cefiderocol-susceptible and -resistant CPE isolates recovered from human clinical and epidemiological samples at a tertiary care hospital. Whole-genome sequencing, variant annotation, structural modelling, and pangenome analysis were performed to characterize resistance mechanisms. Results: A total of 59 isolates (29 resistant and 30 susceptible) were analyzed, predominantly comprising Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae. The most frequent carbapenemase gene among the resistant isolates was blaNDM, which was also present in a subset of susceptible strains. The resistant isolates exhibited a significantly higher burden of non-synonymous mutations in their siderophore receptor genes, notably within fecR, fecA, fiu, and cirA. Structural modelling predicted deleterious effects for mutations such as fecR:G104S and fecA:A190T. Additionally, porin loss and loop 3 insertions (e.g., GD/TD) in OmpK36, as well as OmpK35 truncations, were more frequent in the resistant isolates, particularly in high-risk clones such as ST395 and ST512. Genes associated with toxin–antitoxin systems (chpB2, pemI) and a hypothetical metalloprotease (group_2577) were uniquely found in the resistant group. Conclusions: Cefiderocol resistance in CPE appears to be multifactorial. NDM-type metallo-β-lactamases and missense mutations in siderophore uptake systems—especially in those encoded by fec, fhu, and cir operons—play a central role. These may be further potentiated by alterations in membrane permeability, such as porin disruption and efflux deregulation. The integration of genomic and structural approaches provides valuable insights into emerging resistance mechanisms and may support the development of diagnostic tools and therapeutic strategies.

Activation of the ROS/CncC Pathway Regulates Cytochrome P450 CYP4CJ2 to Confer Cycloxaprid Tolerance in Aphis gossypii.

The inducible P450 gene plays a key role in the response of insects to insecticide stress. However, the transcriptional regulatory mechanism of insecticide-inducible detoxification enzyme genes has not been well clarified. In the present study, a P450 gene, CYP4CJ2, could be induced by cycloxaprid in both susceptible and cycloxaprid-resistant Aphis gossypii strains. RNA interference (RNAi) and transgenic Drosophila results showed that this P450 gene contributes to cycloxaprid tolerance. Moreover, the results of RNAi, dual-luciferase reporter, and yeast one-hybrid assays verified that CYP4CJ2 is regulated by the transcription factor CncC. In addition, although exposure to cycloxaprid resulted in the accumulation of ROS in both susceptible and cycloxaprid-resistant strains, treatment of melon aphids with NAC reduced ROS levels and decreased CncC and CYP4CJ2 expression. These findings suggest that cycloxaprid-induced ROS accumulation activates the CncC pathway, leading to the upregulated expression of CYP4CJ2 and enhancement of cycloxaprid tolerance in melon aphids. These results deepened our understanding of the regulatory mechanism of insecticide-induced detoxification enzyme gene upregulation in insects.

Resistance to organophosphate and pyrethroid and their underlying mechanism in Aedes albopictus (Diptera: Culicidae) from dengue endemic sub-Himalayan West Bengal, India.

Dengue cases are increasing every year in the sub-Himalayan part of West Bengal, where Aedes albopictus (Skuse 1894) has emerged as a predominant dengue vector. Dengue management heavily relies on the recurrent application of chemical insecticides such as temephos (larvicide) and deltamethrin (adulticide). However Ae. albopictus can quickly develop resistance which complicates their management. Thus, detailed population-specific studies on resistance status and mechanism of resistance are useful for designing an effective dengue management strategy. In this study, wild mosquitoes were collected and bioassays with temephos and deltamethrin, including synergist assays, were conducted to investigate the probable mechanism of resistance. Furthermore, carboxylesterase activity and level of monooxygenase were measured followed by real-time polymerase chain reaction analysis to assess expression patterns of CCE and CYP genes. Bioassays confirmed temephos and deltamethrin resistance in Ae. albopictus population is driven by metabolic detoxification. Biochemical assay recorded significantly elevated carboxylesterase activity and monooxygenase levels compared to a susceptible laboratory strain. This is further supported by the elevated relative expression of CCE and CYP genes with consistent expression of CYP6A8 and CCEae3a in these populations. A molecular docking study suggested that the conformation of the binding site for temephos and deltamethrin may enhance their potential metabolism. These findings align with the global trend of insecticide resistance development and highlight the urgent need for more sustainable and informed approaches to dengue vector management.

Restoration of Oxacillin susceptibility in MRSA strains by NPPB

Methicillin-resistant Staphylococcus aureus (MRSA) is a critical public health issue because of its resistance to multiple antibiotics, including β-lactams such as methicillin and oxacillin. To search for antibacterial sensitizers, we have investigated the synergistic antibacterial effects of the combination of the β-lactam antibiotic oxacillin and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) against the MRSA strains. Checkerboard assays showed the synergistic antibacterial efficacy of NPPB combined with oxacillin against five MRSA strains. In addition, the combined effect of NPPB and oxacillin was evaluated by disc diffusion assay and time-dependent growth assay in MRSA strain ATCC 33591, and real-time PCR results showed that the combined treatment decreased the expression levels of abcA and norA genes encoding efflux transporters. NPPB also decreased the oxacillin-induced biofilm formation of ATCC 33591. Field emission transmission electron microscopy showed that the combination treatment induced extensive damage to the bacterial cell wall and membrane, leading to cell lysis. NPPB and oxacillin treated to mice for one week did not cause toxicity on the liver and kidney functions. These findings highlighted the potential of combination therapy of oxacillin and NPPB to treat MRSA infection by enhancing antibiotic efficacy and overcoming resistance.

Evidence of microbiome contribution to the escalation of pyrethroid resistance in the major malaria vectors Anopheles gambiae s.s. and Anopheles funestus s.s

BackgroundExacerbation of pyrethroid resistance severely jeopardises the effectiveness of malaria vector control efforts. However, the mechanisms enabling the vectors to now survive exposure to very high doses of pyrethroids remain unclear. Here, using High-throughput sequencing of the 16 S ribosomal RNA gene coupled with antibiotic treatment, we provide evidence linking the mosquito microbiome to the escalation of pyrethroid resistance in major African malaria vectors, Anopheles gambiae (s.s.) and Anopheles funestus (s.s.).ResultsPhenotypic characterisation of An. gambiae (s.s.) and An. funestus (s.s.) populations revealed a high level of resistance to pyrethroid in both species, with mortality rates < 91% at 10x the diagnostic dose of each insecticide. A significant difference in bacterial composition was observed in An. gambiae s.s. between resistant mosquitoes exposed to 1X and 10X the diagnostic dose of permethrin, and the susceptible strains (PERMANOVA-F: 8.06; p = 0.02). The abundance of Pseudomonas_1 (Log2FC: 4.42, p = 0.0001) and Burkholderia_1 (Log2FC: 4.95, p = 0.001) bacteria were consistently associated with mosquitoes surviving 1X and 10X the diagnostic concentrations of permethrin, respectively, while Serratia_2 bacteria was mostly associated with insecticide susceptibility. In the An. funestus s.s. strain, there was no significant difference in bacterial alpha- and beta-diversity between the FUMOZ-R (exhibiting normal deltamethrin resistance) and FUMOZ-HR (selected for high deltamethrin resistance), suggesting a minimal impact of selection pressure on bacterial composition. However, in FUMOZ-HR, there was an increase in the abundance of Rahnella (Log2FC: 15.954, p = 9.73 E-12) and Leucobacter (Log2FC: 7.6, p = 0.008) bacteria, indicating their potential role in worsening deltamethrin resistance. Furthermore, treating resistant mosquitoes (both Anopheles species) with broad-spectrum bactericidal antibiotics (penicillin/streptomycin) via sugar solution increased their susceptibility to various diagnostic doses of permethrin and deltamethrin in WHO pyrethroid intensity bioassays.ConclusionOverall, our study emphasises the potential role of the microbiome in the escalation of insecticide resistance in Anopheles mosquitoes, identifying key bacterial strains associated with insecticide resistance and susceptibility. These candidate bacteria warrant further investigation to elucidate the mechanisms by which they contribute to the escalation of pyrethroid resistance.

Synthetic Human Lactoferrin Peptide hLF(1-11) Shows Antifungal Activity and Synergism with Fluconazole and Anidulafungin Towards Candida albicans and Various Non-Albicans Candida Species, Including Candidozyma auris

Introduction: Candidozyma auris (Cz. auris) has emerged globally, and diseases caused by it are associated with a mortality rate of 30–72%. This yeast is often multidrug-resistant and challenging to treat. A synthetic peptide, consisting of 11 amino acids of human lactoferrin (hLF1-11), offers a new therapy that is active against Candida albicans, non-albicans Candida yeasts, as well as Cz. auris. The current study examined the susceptibility of clinically relevant Candida species to hLF(1-11) in vitro and investigated the synergistic interaction of this peptide with fluconazole (FLU) and anidulafungin (ANI). Methods: Susceptibility of the yeasts to hLF(1-11) was tested with a microdilution method to determine minimum inhibitory concentrations (MICs). A total of 59 strains belonging to 16 species of Candida or Candidozyma were tested. The treatment cohort included 20 strains of Cz. auris originating from six different countries. Results: Mean MIC values of all susceptible strains ranged from 16.66 ± 6.46 μg/mL to 45.83 ± 10.21 μg/mL. There were no statistical differences in the susceptibility of hLF(1-11) for Cz. auris across geographic origins. In the combinatory tests, drugs acting together, the fractional inhibitory concentration indexes [FIC] &lt; 1.0, showed a synergistic or additive effect on the efficacy of FLU and ANI when used in combination with hLF(1-11). [FIC] indexes 1–2 were interpreted as intermediate. MIC values in combinatory use were 1–2 titer steps lower than when used alone. Conclusions: hLF(1-11) inhibits the growth of yeasts that belong to the genus Candida, including Cz. auris. The combinatory use may be further investigated to treat infections caused by resistant yeasts.

Nitric oxide and experimental autoimmune encephalomyelitis review.

Nitric oxide and experimental autoimmune encephalomyelitis review.

Thioquinazolinones as Antituberculosis Agents Targeting Phosphopantetheinyl Transferase.

The essential mycobacterial enzyme phosphopantetheinyl transferase (PptT) is a potential target for antituberculosis drugs. We here report the identification of (pyridin-3-ylmethyl)-substituted thioquinazolinones as hits in a screen against PptT and a structure-activity relationship (SAR) study of the chemotype. The resulting thioquinazolinones exert on-target whole-cell activity against Mtb in axenic culture and in macrophages with efficacy comparable to that of clinically used antituberculosis drugs. Increased susceptibility of a pptT hypomorphic strain of Mtb, resistance of PptT Trp170 mutant Mtb strains, and analysis of relevant mutations corroborated that the thioquinazolinones are on target, do not inhibit growth of other bacteria, are not cytotoxic to mammalian cells, did not bind to most mammalian receptors and ion channels tested, and did not react with glutathione. Mtb rapidly took up the thioquinazolinones and methylated them, affording products inactive against recombinant PptT.

Combined Forces Against Bacteria: Phages and Antibiotics

ABSTRACTBackground and AimIt is now known that bacteria are highly interactive and exhibit a range of complex cooperative behaviors, including conjugal plasmid transfer, toxins, swarming, drug resistance, toxin production, biofilm development, and other virulence traits. The development of phage–antibiotic synergy (PAS) could be a useful weapon against bacterial infections where antibiotics increase phage replication and antimicrobial activity. This study investigates the therapeutic potential of PAS in combating bacterial infections, focusing on the mechanisms and clinical implications.MethodsA comprehensive review of recent literature was conducted analyzing studies on PAS, including their effects on biofilm degradation, multidrug‐resistant (MDR) bacteria, and toxin‐producing pathogens. Key factors such as timing, dosing and compatibility of phage‐antibiotic combinations were examined.ResultsPAS showed promising results in various bacterial infections. The combination of phages and antibiotics restored the susceptibility of MDR strains, facilitated the degradation of biofilms and minimized the need for high doses of antibiotics, thereby reducing potential side effects. However, challenges such as the emergence of resistance and antagonistic interactions with certain combinations remain. Key factors influencing the efficacy of PAS include phage and antibiotic dosing, timing of administration, and the physiological state of the bacteria.ConclusionPAS has potential applications in the treatment of complex infections. Despite the promising results, further research is essential to standardize protocols, optimize therapeutic combinations and address safety concerns. Clinical trials focusing on phage selection, resistance management and patient‐specific treatments will be crucial for the translation of PAS into clinical practice.

PERTUSSIS- EPIDEMIOLOGY, DIAGNOSTICS, TREATMENT. LITERATURE REVIEW

Introduction and purpose. The bacterium Bordetella pertussis causes pertussis, an infectious respiratory disease. Despite the introduction of an expanded vaccination programme over the past decades, pertussis remains a public health concern. This literature review aims to compare clinical and laboratory diagnostics. Additionally, we will focus on the impact of the Covid-19 pandemic on the increase in cases of the disease. State of knowledge. The illness has three phases: an initial stage lasting one to two weeks; a second stage characterised by coughing fits lasting up to four weeks; and a final recovery phase. Although antibiotics used during the second stage reduce infectiousness, it is unclear whether they reduce the duration of illness. During the first two weeks, patients may experience flu-like symptoms. Culture testing is considered the gold standard, providing 100% specificity. This enables identification of the strain and antibiotic susceptibility testing. This method is most effective within the first two weeks of illness, while viable bacteria are still present.The number of cases fluctuates from year to year, but there has been a marked decrease among young children and an increase among adolescents and adults. Following the discontinuation of protective measures against SARS-CoV-2, significant pertussis outbreaks have occurred. Material and methods. This review analyzes the epidemiology, diagnosis, and treatment of whooping cough, with a particular focus on the years from 2019 to the present. The article was based on sources from scientific databases such as PubMed. Summary. Researchers have linked the increase in pertussis cases in recent years to the lifting of restrictions during the post-Covid period. It is therefore important to raise public awareness of the need for revaccination and to continue scientific research into the epidemiology and diagnosis of this disease.

Novel diamide insecticide, tetraniliprole, inhibits the population growth and development of Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) with different susceptibility levels, through its lethal and sublethal effects.

The tomato leaf miner, Tuta absoluta Meyrick (Lepidoptera: Gelechiidae), is a significant invasive species globally, inflicting considerable damage on tomato industry. Tetraniliprole is a new type of diamide insecticides, which can effectively control lepidoptera insects. In this study, we investigated both the acute toxicity and sublethal effects of tetraniliprole on T. absoluta populations exhibiting varying susceptibility levels. The results indicated that the LC50 of tetraniliprole for the susceptible strains (SX-S) and tetraniliprole-selection strains (Tet-R) were 0.31 and 21.22 mg·L-1, respectively. The Tet-R strain showed 68.45-fold higher resistance than the SX-S strain. Tetraniliprole exposure at LC10 and LC30 significantly prolonged larval duration in F0 generation SX-S and Tet-R strains. The pupal stage, pupal weight, and fecundity of the SX-S strain were significantly decreased, while only pupal weight was significantly reduced in the Tet-R strain. Moreover, LC30 of tetraniliprole significantly affected the r, λ, R0, and T of the Tet-R strain but had no significant impact on the SX-S strain. Additionally, sublethal tetraniliprole concentrations showed transgenerational effects on the F1 generation. The egg stage of the SX-S and Tet-R strains were significantly prolonged. Tetraniliprole significantly affected the R0, T and GRR of the SX-S strain, while only T was significantly affected in the Tet-R strain. In conclusion, tetraniliprole inhibited the growth and development of T. absoluta. Moreover, different susceptibility levels significantly influenced the sublethal effects and transgenerational effects of tetraniliprole on T. absoluta. These findings serve as a reference for the scientific use of tetraniliprole, and the IPM of T. absoluta.

Optimization of preparative HPLC method for the purification of thymol from the essential oil of Trachyspermum ammi (L.).

Optimization of preparative HPLC method for the purification of thymol from the essential oil of Trachyspermum ammi (L.).

Selection for florfenicol resistance at sub-MIC concentrations in Pasteurella multocida.

Antimicrobial resistance is a global threat to both human and animal health, and transfer of resistance between these spheres is recognised as a key concern for all species. Selection for resistance at sub-inhibitory antimicrobial concentrations has been characterised for some bacteria-antimicrobial combinations but there is little data from non-laboratory strains, and veterinary antimicrobials and bacterial species. Here, we demonstrate a minimum selective concentration of 0.06 mg/L (1/6 xMIC) for florfenicol in wild-type Pasteurella multocida, through competition experiments between a susceptible strain and a floR-resistant mutant. We also show that sub-inhibitory concentrations of florfenicol do not appear to significantly select for de novo resistance in P. multocida and present the challenges with adapting experimental protocols between bacterial species. These results have important implications for antimicrobial resistance selection at sub-inhibitory concentrations, method development for within-species differentiation in novel bacterial species, and application to policy regarding antimicrobial contamination in animal-feed.

New Quipazine Derivatives Active Against Drug-Resistant Oncogenic Helicobacter pylori Strains with Biofilm

Helicobacter pylori (H. pylori) is regarded as a significant risk factor for gastritis, peptic ulcer disease, and gastric cancer. However, the increasing resistance of H. pylori strains has resulted in low eradication rates and ineffective treatments. Herein, we report on identification of a new quipazine derivative—compound 9c (N-(3-chlorobenzyl)-2-(piperazin-1-yl)quinolin-4-amine), which displayed antibacterial properties (MIC range 2–4 µg/mL) against H. pylori CagA-positive reference strains associated with an increased risk of gastric cancer, including metronidazole-resistant ATCC 43504, clarithromycin-resistant ATCC 700684 and susceptible J99 strain, as well as clinical, multidrug-resistant isolate (3CML, resistant to clarithromycin, metronidazole and levofloxacin). Compound 9c showed bacteriostatic activity (MBC/MIC ratio > 4), demonstrated antibiofilm-forming properties and prevented auto-aggregation of microbial cells. It also displayed an additive effect in ½ MIC (2 µg/mL) when administered with clarithromycin and/or metronidazole. Compound 9c had no impact on gut microbiota reference strains of S. aureus, E. coli, E. faecalis and L. paracasei as well as no hemolytic activity against sheep erythrocytes. Finally, by reducing the viability of the SNU-1 human gastric cancer cell line (IC50 = 3.28 μg/mL), compound 9c might offer important implications regarding the oncogenic characteristics of cagA+ H. pylori strains.

Increase in insecticide susceptibility after sublethal exposure to deltamethrin in the German cockroach (Blattodea: Ectobiidae).

The German cockroach, Blattella germanica (L.), is a major urban insect pest primarily controlled by insecticides, including pyrethroid sprays, fipronil baits, and indoxacarb baits. However, widespread pyrethroid resistance increases the probability that cockroaches will survive treatments in the field. Many insecticides are applied concurrently or repetitively on a periodic basis, meaning survivors have the chance to be re-treated, but any lasting physiological effects from initial survival may impact the performance of subsequent treatments. We investigated the effects of sublethal deltamethrin exposure on the susceptibility of susceptible (UCR) and resistant (Ryan) strains of German cockroaches. Ineffective pyrethroid treatment was simulated by treating cockroaches with the LD20 of deltamethrin. Survivors were treated again with the same dose of deltamethrin, a food source containing the LC50 of fipronil, or a food source containing the LC50 of indoxacarb. Both strains of cockroaches experienced greater mortality when treated with deltamethrin 3 d (49.8% to 67.6%), after the sublethal exposure. No impact on fipronil or indoxacarb susceptibility was observed after pre-treatment with deltamethrin. Because surviving a deltamethrin treatment did not induce tolerance to deltamethrin or other insecticides, this suggests no within-generation drawback of current conventional management practices of applying multiple treatments in the same site. Furthermore, sequential exposure to deltamethrin caused greater mortality than expected, indicating that this insecticide may remain useful even if initial efficacy is low. Comprehensive examinations of additional resistant strains treated with formulated products are warranted to understand whether these effects may impact field control.

Insecticide resistance monitoring of Aphis spiraecola (Hemiptera: Aphididae) to neonicotinoids and abamectin in a major apple-growing region.

Aphis spiraecola is a significant pest of fruit trees worldwide, causing substantial yield and economic losses in apple production. The control of A. spiraecola primarily depends on chemical pesticides; however, long-term field monitoring of insecticide resistance is lacking. This study assessed the susceptibility of a laboratory-susceptible strain (SS) of A. spiraecola to abamectin, imidacloprid, thiamethoxam, and acetamiprid. The LC50 values of A. spiraecola to abamectin, imidacloprid, thiamethoxam, and acetamiprid were 0.13, 1.44, 1.76, and 1.19mg L-1, respectively. The resistance levels of 8 A. spiraecola field populations in a major apple-growing region were investigated in 2021 to 2023. The resistance ratios (RR) varied in different years and regions. The resistance level of A. spiraecola in the XP (Xingping) region to abamectin was the highest (RR = 42.08) in 2021. Most field populations displayed low or moderate levels of resistance to 3 neonicotinoids. However, the field population of A. spiraecola in LQ (Liquan) region had a high resistance level to thiamethoxam in 2021 with an LC50 of 109.63mg L-1 and a resistance ratio of 62.29. The resistance levels of A. spiraecola to 4 pesticides were found to be decreasing in 3 yr. Compared to the SS strain, the field populations of A. spiraecola showed no mutations in nAChR, GABA receptor, or GluCl, suggesting that target site insensitivity may not contribute to the resistance. This study can contribute to the rational use of chemical insecticides for controlling A. spiraecola, thereby reducing economic losses in apple production and minimizing environmental pollution.

Rapid emergence of resistance to broad-spectrum direct antimicrobial activity of avibactam.

Avibactam (AVI) is a diazabicyclooctane (DBO) β-lactamase inhibitor used clinically in combination with ceftazidime. At concentrations higher than those typically achieved in vivo, it also has broad-spectrum direct antibacterial activity against Enterobacterales strains, including metallo-β-lactamase-producing isolates, mediated by inhibition of penicillin-binding protein 2 (PBP2). This activity has some mechanistic similarities to that of more potent novel DBOs (zidebactam and nacubactam) in late clinical development. We found that resistance to AVI emerged readily, with a mutation frequency of 2 × 10-6 to 8 × 10-5. Whole-genome sequencing of resistant isolates revealed a heterogeneous mutational target that permitted bacterial survival and replication despite PBP2 inhibition, in line with prior studies of PBP2-targeting drugs. While such mutations are believed to act by upregulating the bacterial stringent response, we found a similarly high mutation frequency in bacteria deficient in components of the stringent response, although we observed a different set of mutations in these strains. Although avibactam-resistant strains had increased lag time, suggesting a fitness cost that might render them less problematic in clinical infections, there was no statistically significant difference in growth rates between susceptible and resistant strains. The finding of rapid emergence of resistance to avibactam as the result of a large and complex mutational target adds to our understanding of resistance to PBP2-targeting drugs and has potential implications for novel DBOs with potent direct antibacterial activity, which are being developed with the goal of expanding cell wall-active treatment options for multidrug-resistant gram-negative infections.IMPORTANCEAvibactam (AVI) is the first in a class of novel β-lactamase inhibitor antibiotics called diazabicyclooctanes (DBOs). In addition to its ability to inhibit bacterial β-lactamase enzymes that can destroy β-lactam antibiotics, we found that AVI had direct antibacterial activity, at concentrations higher than those used clinically, against even highly multidrug-resistant bacteria. This activity is the result of inhibition of the bacterial enzyme penicillin-binding protein 2 (PBP2). Resistance to other drugs that inhibit PBP2 occurs through mutations that involve upregulation of the bacterial "stringent response" to stress. We found that bacteria developed resistance to AVI at a high rate, as a result of mutations in stringent response genes. We also found that bacteria with impairments in the stringent response could still develop resistance to AVI through different mutations. Our findings indicate the importance of studying how resistance will emerge to newer, more potent DBOs in development and early clinical use.

Identification, Antimicrobial Susceptibility and Clinical Significance of Klebsiella variicola Strains

Klebsiella variicola strains are Gram-negative rod-shaped bacteria that usually cause bloodstream, urinary and respiratory tract infections. The aim of the study was to identify K. variicola strains, evaluate the susceptibility of strains to selected antimicrobials, and detect their resistance mechanisms to β-lactams. Strain identification was performed using the mass spectrometry method. DNA sequencing was performed for selected strains. Susceptibility to selected antimicrobials was assessed using an automated method. The presence of an antimicrobial resistance mechanism and genes encoding ESβL was determined using the double-disc synergy test and genotypic methods. Most of the 108 analyzed strains were susceptible to imipenem (99.1%), meropenem (96.3%) and amikacin (96.3%). Over 12% of strains produced ESβL and were multidrug-resistant. Although K. variicola strains remain susceptible to antibiotics, there is a constant need to monitor their susceptibility to selected antimicrobials. The isolation of multidrug-resistant K. variicola strains underscores the critical importance of accurate species identification. This species may be clinically significant, as certain strains can also produce enzymes that pose significant threats today.

A multi-assay assessment of insecticide resistance in Culex pipiens (Diptera: Culicidae) informs a decision-making framework

Insecticide resistance (IR) is an increasing problem globally, making control of vector-borne diseases more difficult. Reduced susceptibility to permethrin in Culex pipiens, an important vector for West Nile virus, has been reported across the US based on a standardized laboratory method: the CDC bottle bioassay. This bioassay uses a rapid phenotypic outcome to reveal evidence for IR, but how this translates to the effectiveness of formulated products used in an operational setting is unclear. Therefore, other methods for IR monitoring are recommended to quantify IR or evaluate formulated products against field populations in real-world conditions. To compare some of the available methods, we collected populations of Cx. pipiens from six sites in the Northwest Mosquito Abatement District (Cook Co., Illinois), and used a susceptible laboratory strain of Cx. pipiens as a control, to test for IR to pyrethroids using CDC bottle bioassays, caged field trials, and topical applications. CDC bottle bioassays suggested that Cx. pipiens from this area exhibit IR to both etofenprox and Sumithrin®. Caged field trials with ultra-low volume Anvil® 10 + 10 (Sumithrin®) demonstrated resistance to the product and underscored the need for inclusion of a susceptible control to differentiate IR from inadequate product distribution. Topical applications revealed low to high levels of resistance to synergized and unsynergized pyrethroids (etofenprox, Sumithrin®, and deltamethrin) in all field populations. Based on these data, we provide a new decision-making tree for mosquito control professionals which will guide selection of the most optimal assay for IR surveillance based on their goals, needs, and resources.

Matrix-based DNA Extraction for Targeted Next-Generation Sequencing on Decontaminated Sputum Samples.

Next-generation sequencing (NGS) is now recognized as a powerful tool for timely and accurate drug-resistant tuberculosis (DR-TB) diagnosis. Targeted NGS (tNGS) offers a streamlined approach by focusing on specific genes associated with drug resistance, bypassing the need for traditional culture-based methods with turnaround times ranging from weeks to months. The World Health Organization (WHO) has recommended tNGS as a valuable strategy for improving tuberculosis (TB) diagnosis to guide treatment and improve treatment outcomes, particularly in resource-limited settings. Among the WHO-recommended tNGS assays, we have selected a method that provides rapid and comprehensive drug susceptibility testing, lineage determination, and strain typing. While standardized DNA extraction methods are available, they can be time-consuming and labor-intensive. To address this challenge, we optimized a simplified, matrix-based DNA extraction protocol in combination with magnetic bead purification. This method offers a rapid and efficient approach for extracting DNA directly from decontaminated sputum sediments, enabling rapid downstream tNGS analysis. By streamlining the DNA extraction process from sputum sediment, this protocol could facilitate wider adoption of tNGS in routine clinical settings, ultimately contributing to improved patient outcomes and lending to global TB control efforts.