Low-level Resistance Research Articles

In Vitro Characterization of Probiotic Potential of Limosilactobacillus fermentum against Salmonella Gallinarum Causing Fowl Typhoid.

Fowl typhoid, a septicaemic disease of poultry, is caused by Salmonella Gallinarum and leads to severe economic losses. The aim of the present study was to isolate, select and characterize indigenous probiotic lactobacilli with anti-Salmonella Gallinarum activity. A total 55 lactobacilli were isolated from the caeca and ileum parts of healthy chickens and identified to species level by 16S rDNA sequencing. All the isolates were initially screened for antimicrobial activity and selected isolates were further subjected to in vitro evaluation of probiotic properties. Lactobacilli isolates (n = 21) showed varying levels of activity (08-18 mm) against Salmonella Gallinarum. These selected isolates also showed tolerance to acidic conditions (pH 3 and 4). Out of these 21 isolates, 13 showed growth (>0.5 OD at 600 nm) 0.3% bile salts. Moreover, these isolates also had the ability of auto-aggregation (20.05 ± 0.62%-50.70 ± 1.40%), and co-aggregation with Salmonella Gallinarum (5.22 ± 0.21%-42.07 ± 0.70%). Results revealed that lactobacilli had a higher level of resistance to vancomycin (100%), streptomycin (100%), ciprofloxacin (95%), gentamicin (90%), doxycycline (90%), oxytetracycline (85%), and bacitracin (80%), and a lower level of resistance to penicillin (33%), erythromycin (28%), chloramphenicol (23%), fusidic acid (23%) and amoxicillin (4%). The Limosilactobacillus fermentum PC-10 and PC-76 were sensitive to most of the antibiotics. The overall results revealed that two Limosilactobacillus fermentum strains (PC-10 and PC-76) fulfill the in vitro selection criteria of probiotics, i.e, tolerance to low pH, resistance to bile salts, auto-aggregation, co-aggregation with Salmonella Gallinarum, and absence of acquired antibiotic resistance. The Limosilactobacillus fermentum PC-10 and PC-76 also inhibited the (>5 log10) growth of Salmonella Gallinarum in co-culture assay. It is concluded that Limosilactobacillus fermentum PC-10 and PC-76 may be further investigated and developed as anti-Salmonella Gallinarum probiotics for poultry.

Increased clonality among Neisseria gonorrhoeae isolates during the COVID-19 pandemic in Amsterdam, the Netherlands.

Distancing measures during the COVID-19 lockdown led to a temporary decrease of casual sex partners among clients of the Centre for Sexual Health (CSH) in Amsterdam, the Netherlands. We investigated the effect of this change on the genotypic and phenotypic distribution of Neisseria gonorrhoeae (Ng) isolates from CSH patients. From each Ng-positive patient we sequenced one isolate, resulting in 322 isolates which constituted two groups: 181 isolates cultured from 15 January to 29 February 2020 (before the first lockdown) and 141 cultured from 15 May to 30 June 2020 (during the first lockdown). Patient characteristics showed significantly more symptomatic patients and significantly fewer reported sex partners during the lockdown. Phenotypic data showed an increase in low-level azithromycin resistance and ceftriaxone susceptibility during the lockdown, and this remained after the study period. The diversity in sequence types (STs) decreased slightly during the lockdown. A shift occurred from ST 8156 being predominant before lockdown to ST 9362 during lockdown and a remarkably low median SNP distance of 17 SNPs was found between ST 9362 isolates obtained during lockdown. These findings reflect restricted travel and the change in sexual behaviour of CSH clients during the lockdown, with a potentially increased local transmission of the ST 9362 strain during this period, which led to genotypic and phenotypic changes in the Ng population. This shows that public health measures have far-reaching consequences and should be considered in the surveillance of other infectious diseases.

Abstract 6384: ATG-034, an LILRB4 antagonist antibody, reinvigorates dendritic cells and prevents tumor progression

Abstract Background: Dendritic cells (DCs) are vital for initiating antigen-specific T cell-dependent antitumor immune responses. However, emerging evidence reveals that local DCs within the tumor microenvironment (TME) are tolerized to facilitate immune evasion. Reprogramming of DCs is recognized as a promising strategy for tumor immunotherapy. Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory receptor belonging to the LILR family which is mainly expressed on normal myeloid cells and myeloid-derived malignant cells. It is upregulated on tolerogenic DCs (tolDCs), which exhibit low expression levels of costimulatory molecules and resistance to DC maturation. Targeting LILRB4 to reprogram tolDCs has been reported to be a promising strategy for cancer treatment. Here we report the preclinical development of an LILRB4 antagonist antibody, ATG-034. Method: The protein-based and cell-based binding affinity of ATG-034 were measured using SPR, ELISA and FACS analysis. Fc receptor (FcR) stimulation assay was used to evaluate the ability of ATG-034 to block the interaction of LILRB4 with its ligand fibronectin. ATG-034-mediated enhancement of antigen presentation and co-stimulation ability of DCs was determined by FACS analysis for the change of surface expression of HLA-DR, HLA-ABC, CD86, and CD206. The mixed lymphocyte reaction (MLR) assay was used to assess the immunomodulatory potential of ATG-034. The in vivo antitumor efficacy of ATG-034 was evaluated in a radiation therapy-resistant murine Lewis lung carcinoma (LLC) syngeneic model. Results: ATG-034 binds to LILRB4 protein with a single-digit nM affinity. It potently reversed the fibronectin-mediated inhibition of FcR-driven TNF-α cytokine production. TolDCs were reprogrammed by ATG-034 to immunogenic DCs with significant upregulation of the expression of HLA-DR, HLA-ABC, CD86 and downregulation of the expression of CD206. Furthermore, ATG-034 remarkably reinvigorated tolDCs for priming T cell activation with noticeable upregulation of the expression of CD25 and the secretion of IFN-γ, enhancing antitumor immunity. In addition, 10 mg/kg ATG-034 significantly inhibited the LLC tumor growth in vivo with a TGI of 40.28%, while a benchmark antibody only demonstrated a 24.43% TGI. Conclusion: Our data show that ATG-034 reprograms tolDC, enhancing anti-tumor immunity, and demonstrates potent in vivo anti-tumor efficacy. Therefore, ATG-034 may be a promising strategy for the treatment of cancer. Citation Format: Ao Sun, Enlin Zheng, Huili Cao, Mengshi Cao, Suya Bai, Peng Chen, Linjie Tian, Jay Mei, Bo Shan, Bing Hou. ATG-034, an LILRB4 antagonist antibody, reinvigorates dendritic cells and prevents tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6384.

Molecular Characterization of katG and inhA Mutations by Genotype MTBDRplus Line Probe Assay To Guide Isoniazid and Ethionamide Use for Drug-Resistant Tuberculosis.

Introduction Drug-resistant tuberculosis (TB) continues to be a global health threat in all its forms. Significant resistance has been observed against isoniazid (INH), one of the most important therapeutic options for treating TB. Molecular testing methods such as line probe assay (LPA) provide rapid diagnosis and early management. Mutations in different genes can be detected, which indicate INH and ethionamide (ETH) drug resistance. We aimed to determine the frequency of these mutations in katG and inhA genes by LPA to guide INH and ETH use for drug-resistant TB. Materials and methods Two consecutive sputum samples were collected from each patient, followed by decontamination by N‑acetyl‑L‑cysteine and sodium hydroxide method. LPA was performed on the decontaminated samples by GenoType MTBDRplus, and the strips were analyzed. Results Out of the 3,398 smear-positive samples tested by LPA, valid results were found in 3,085 (90.79%) samples. Of the 3,085 samples, INH resistance was seen in 295 samples (9.56%), of which mono INH resistance was in 204 samples, and 91 were multidrug resistant. katG S315T was the most common mutation responsible for high-level INH resistance. At the same time, inhA c15t was the most common mutation associated with low-level INH resistance and ETH cross-resistance. The average turnaround time for the processing and reporting of samples was five days. Conclusions The high burden of INH resistance is alarming and can be a major obstacle to TB elimination. Although molecular methods have reduced the reporting time leading to early management of the patients still, a large knowledge gap persists.

Proteomic analysis of metronidazole resistance in the human facultative pathogen Bacteroides fragilis.

The anaerobic gut bacteria and opportunistic pathogen Bacteroides fragilis can cause life-threatening infections when leaving its niche and reaching body sites outside of the gut. The antimicrobial metronidazole is a mainstay in the treatment of anaerobic infections and also highly effective against Bacteroides spp. Although resistance rates have remained low in general, metronidazole resistance does occur in B. fragilis and can favor fatal disease outcomes. Most metronidazole-resistant Bacteroides isolates harbor nim genes, commonly believed to encode for nitroreductases which deactivate metronidazole. Recent research, however, suggests that the mode of resistance mediated by Nim proteins might be more complex than anticipated because they affect the cellular metabolism, e.g., by increasing the activity of pyruvate:ferredoxin oxidoreductase (PFOR). Moreover, although nim genes confer only low-level metronidazole resistance to Bacteroides, high-level resistance can be much easier induced in the laboratory in the presence of a nim gene than without. Due to these observations, we hypothesized that nim genes might induce changes in the B. fragilis proteome and performed comparative mass-spectrometric analyses with B. fragilis 638R, either with or without the nimA gene. Further, we compared protein expression profiles in both strains after induction of high-level metronidazole resistance. Interestingly, only few proteins were repeatedly found to be differentially expressed in strain 638R with the nimA gene, one of them being the flavodiiron protein FprA, an enzyme involved in oxygen scavenging. After induction of metronidazole resistance, a far higher number of proteins were found to be differentially expressed in 638R without nimA than in 638R with nimA. In the former, factors for the import of hemin were strongly downregulated, indicating impaired iron import, whereas in the latter, the observed changes were not only less numerous but also less specific. Both resistant strains, however, displayed a reduced capability of scavenging oxygen. Susceptibility to metronidazole could be widely restored in resistant 638R without nimA by supplementing growth media with ferrous iron sulfate, but not so in resistant 638R with the nimA gene. Finally, based on the results of this study, we present a novel hypothetic model of metronidazole resistance and NimA function.

Draft genome sequences of rare Lelliottia nimipressuralis strain MEZLN61 and two Enterobacter kobei strains MEZEK193 and MEZEK194 carrying mobile colistin resistance gene mcr-9 isolated from wastewater in South Africa

Antimicrobial-resistant bacteria of the order Enterobacterales are emerging threats to global public and animal health, leading to morbidity and mortality. The emergence of antimicrobial-resistant, livestock-associated pathogensis a great public health concern. The genera Enterobacter and Lelliottia are ubiquitous, facultatively anaerobic, motile, non-spore-forming, rod-shaped Gram-negative bacteria belonging to the Enterobacteriaceae family and include pathogens of public health importance. Here, we report the first draft genome sequences of a rare Lelliottia nimipressuralis strain MEZLN61 and two Enterobacter kobeistrains MEZEK193 and MEZEK194 in Africa. The bacteria were isolated from environmental wastewater samples. Bacteria were cultured on nutrient agar, and the pure cultures were subjected to whole-genome sequencing. Genomic DNA was sequenced using an Illumina MiSeq platform. Generated reads were trimmed and subjected to de novo assembly. The assembled contigs were analysed for virulence genes, antimicrobial resistance genes, and extra-chromosomal plasmids, and multilocus sequence typing was performed. To compare the sequenced strains with other, previously sequenced E. kobei and L. nimipressuralis strains, available raw read sequences were downloaded, and all sequence files were treated identically to generate core genome bootstrapped maximum likelihood phylogenetic trees. Whole-genome sequencing analyses identified strain MEZLN61 as L. nimipressuralis and strains MEZEK193 and MEZEK194 as E. kobei. MEZEK193 and MEZEK194 carried genes encoding resistance to fosfomycin (fosA), beta-lactam antibiotics (blaACT-9), and colistin (mcr-9). Additionally, MEZEK193 harboured nine different virulence genes, while MEZEK194 harboured eleven different virulence genes. The phenotypic analysis showed that L. nimipressuralis strain MEZLN61 was susceptible to colistin (2 μg/mL), while E. kobei MEZEK193 (64 μg/mL) and MEZEK194 (32 μg/mL) were resistant to colistin. The genome sequences of strains L. nimipressuralis MEZLN6, E. kobei MEZEK193, and E. kobei MEZEK194 will serve as a reference point for molecular epidemiological studies of L. nimipressuralis and E. kobeiin Africa. In addition, this study provides an in-depth analysis of the genomic structure and offers important information that helps clarify the pathogenesis and antimicrobial resistance of L. nimipressuralis and E. kobei. The detection of mcr-9, which is associated with very low-level colistin resistance in Enterobacter species, is alarming and may indicate the undetected dissemination of mcr genes in bacteria of the order Enterobacterales. Continuous monitoring and surveillance of the prevalence of mcr genes and their associated phenotypic changes in clinically important pathogens and environmentally associated bacteria is necessary to control and prevent the spread of colistin resistance.

RdxA Diversity and Mutations Associated with Metronidazole Resistance of Helicobacter pylori.

Metronidazole (MNZ) is administered as first-line antibiotic for Helicobacter pylori eradication therapy; however, increasing resistance to MNZ impaired the efficacy. Increasing the dose of MNZ was recommended to overcome low-level resistance, but it was difficult to determine MNZ resistance level simply based on the rdxA gene mutation. In this study, the rdxA sequences of 511 clinical H. pylori strains were analyzed to assess the genotypes associated with MNZ resistance. We observed that the prevalences of rdxA sequences with missense, nonsense, and frameshift mutations were 70.25, 11.35, and 17.03%, respectively. Regarding the amino acid substitutions, T31E, H53R, D59N, L62V, S88P, G98S/N, R131K, and V172I were present in most strains regardless of the resistance phenotype. The correlation analysis showed R16H/C, Y47C, A67V/T, and V204I substitutions were associated with MNZ resistance. The mutation resulting in RdxA truncation was observed in 36.29% of the resistant strains, and 83.45% of these strains displayed high-level MNZ resistance (MIC > 256 μg/mL). Moreover, all strains with truncated mutation positions before amino acid 70 expressed high-level MNZ resistance. Our results indicated that most amino acid mutations probably contributed to the sequence diversity of RdxA, while R16H/C, Y47C, A67V/T, and V204I were potentially helpful to identify resistant strains. Although it was difficult to determine the mutations associated with MNZ resistance, the prediction of high-level resistance based on truncated characteristics of RdxA might be an important approach, which can effectively avoid H. pylori eradication therapy with unreasonable of MNZ dose increases for patients with high-level drug resistance. IMPORTANCE The increasing resistance to metronidazole impaired the efficacy of Helicobacter pylori eradication, and increasing the dose of metronidazole was recommended to overcome low-level resistance. For patients infected with highly resistant strains, the current empirical treatments, which generally used metronidazole in double doses or more, appeared impossibly to overcome the resistance and would only increase the incidence of adverse effects. Our results indicated that high-level metronidazole resistance was predominant, and almost half of the patients with high-level drug resistance could avoid usage of metronidazole based on the truncated mutations of RdxA sequences, which can effectively avoid H. pylori eradication therapy with unreasonable increases in the metronidazole dose.

Assessment of insecticide resistance of Aedes aegypti (Diptera: Culicidae) populations to insect growth regulator pyriproxyfen, in the northeast region of Brazil.

Vector control has been an essential strategy in Brazil to manage vector-borne diseases, and the use of insecticides plays an important role in this effort. Pyriproxyfen (PPF) has become a common insect growth regulator used to control juvenile stages of mosquitoes by disturbing their growth and development. This study assesses the susceptibility and resistance status of Brazilian Ae. aegypti populations that previously showed low resistance levels to PPF. Eggs of Ae. aegypti were collected from six cities located in the northeast states of Ceará (Quixadá, Icó, and Juazeiro do Norte), and Bahia (Itabuna, Brumado, and Serrinha). We used the Ae. aegypti Rockefeller strain as an experimental control and a strain known to be susceptible to insecticides. Inhibition of emergence rates by 50% of Ae. aegypti populations varied from 0.0098-0.046 µg/L. Mosquitoes from Icó, Serrinha, and Brumado showed low resistance levels [resistance ratio (RR50) = 2.33, 4.52, and 4.83, respectively], whereas moderate levels of resistance were detected in populations from Juazeiro do Norte (RR50=5.83) and Itabuna (RR50=7.88). Aedes aegypti collected from the Quixadá population showed a high resistance level to pyriproxyfen (RR50=11). The evolution of resistance in Brazilian Ae. aegypti populations to PPF can compromise vector control efforts. Continuous monitoring of insecticide resistance in Ae. aegypti is essential for making timely management decisions for effective vector control and management.

Comparison of Antimicrobial Resistance among Commensal Escherichia coli Isolated from Retail Table Eggs Produced by Laying Hens from the Cage and Non-Cage Housing Systems in Western Australia.

Antimicrobial resistance (AMR) has become a global public health concern in recent decades. Although several investigations evaluated AMR in commensal and pathogenic bacteria from different foods of animal origin in Australia, there is a lack of studies that compared AMR in commensal E. coli isolated from retail table eggs obtained from different laying hen housing systems. This study aimed to determine AMR and differences in AMR patterns among E. coli isolates recovered from retail table eggs sourced from caged and non-caged housing systems in Western Australia. Commensal E. coli isolates were tested for susceptibility to 14 antimicrobials using a broth microdilution method. Clustering analyses and logistic regression models were applied to identify patterns and differences in AMR. Overall, there were moderate to high frequencies of resistance to the antimicrobials of lower importance used in Australian human medicine (tetracycline, ampicillin, trimethoprim, and sulfamethoxazole) in the isolates sourced from the eggs of two production systems. All E. coli isolates were susceptible to all critically important antimicrobials except the very low level of resistance to ciprofloxacin. E. coli isolates from eggs of non-caged systems had higher odds of resistance to tetracycline (OR = 5.76, p < 0.001) and ampicillin (OR = 3.42, p ≤ 0.01) compared to the isolates from eggs of caged systems. Moreover, the number of antimicrobials to which an E. coli isolate was resistant was significantly higher in table eggs from non-caged systems than isolates from caged systems' eggs. Considering the conservative approach in using antimicrobials in the Australian layer flocks, our findings highlight the potential role of the environment or human-related factors in the dissemination and emergence of AMR in commensal E. coli, particularly in retail table eggs of non-cage system origin. Further comprehensive epidemiological studies are required to better understand the role of different egg production systems in the emergence and dissemination of AMR in commensal E. coli.

Gut colonization with vancomicyn-resistant enterococci among patients with hematologic malignancies

BackgroundVancomycin-resistant enterococci (VRE) are well known agents that colonize the gastrointestinal tract of immunocompromised patients, especially those with hematologic malignancies. The aim of the current study was to determine the incidence of and risk factors for colonization with VRE among patients with hematologic malignancies.MaterialsFor a nine-month period, all patients admitted to the Hematology ward at University Hospital in Pleven, Bulgaria who had hematologic malignancy and duration of hospitalization of more than 48 h were screened for colonization with VRE. The data collected from patients and their medical records during the entire hospital stay included: demographic characteristics, clinical information and information about all antimicrobials used. A longitudinal study was used to assesses the risk factors and statistical analysis was performed using SPSS version 27.0.ResultsA total of 119 patients were enrolled in the study. Colonization with VRE was established in 18 of them. One patient carried two species, resulting in a total of 19 VRE: 12 Enterococcus gallinarum, 4 Enterococcus casseliflavus, 2 Enterococcus faecium and 1 Enterococcus faecalis. VanA phenotype, with high-level resistance of vancomycin (MIC ≥ 256 μg/ml) and teicoplanin (MIC = 96 μg/ml), was demonstrated by one E. faecium, which carried vanA. The other E. faecium and E. faecalis expressed low-level resistance to vancomycin (MICs: 8 μg/ml and 12 μg/ml), susceptibility to teicoplanin (MICs = 0.5 μg/ml) and vanB was detected. All E. gallinarum and E. casseliflavus showed low-level resistance to vancomycin and susceptibility to teicoplanin. E. gallinarum strains were positive for vanC1 and E. casseliflavus for vanC2. Only two patients were colonized with vanA or vanB enterococci and the rest 16 were positive for vanC. The univariate analysis revealed that patient’s age (70–79 years; p = 0.025) and multiple myeloma (p = 0.001) are risk factors for VRE acquisition among the investigated patients. In addition, the multivariate analysis confirmed that patient’s age (70–79 years) is an independent risk factor for VRE colonization.ConclusionsOur results showed that 15.1% of patients with hematologic malignancies were colonized with VRE. There was a distinct prevalence of vanC enterococci. Among the analyzed risk factors, advanced age and multiple myeloma contributed to VRE acquisition.

Cytotoxicity and Antimicrobial Resistance of Aeromonas Strains Isolated from Fresh Produce and Irrigation Water

The genus Aeromonas has received constant attention in different areas, from aquaculture and veterinary medicine to food safety, where more and more frequent isolates are occurring with increased resistance to antibiotics. The present paper studied the interaction of Aeromonas strains isolated from fresh produce and water with different eukaryotic cell types with the aim of better understanding the cytotoxic capacity of these strains. To study host-cell pathogen interactions in Aeromonas, we used HT-29, Vero, J774A.1, and primary mouse embryonic fibroblasts. These interactions were analyzed by confocal microscopy to determine the cytotoxicity of the strains. We also used Galleria mellonella larvae to test their pathogenicity in this experimental model. Our results demonstrated that two strains showed high cytotoxicity in epithelial cells, fibroblasts, and macrophages. Furthermore, these strains showed high virulence using the G. mellonella model. All strains used in this paper generally showed low levels of resistance to the different families of the antibiotics being tested. These results indicated that some strains of Aeromonas present in vegetables and water pose a potential health hazard, displaying very high in vitro and in vivo virulence. This pathogenic potential, and some recent concerning findings on antimicrobial resistance in Aeromonas, encourage further efforts in examining the precise significance of Aeromonas strains isolated from foods for human consumption.

Character of wound microflora and resistance to antibiotics: monitoring results 2012–2020

The aim of the study was to improve the microbiological diagnosis of surgical infections, collect and analyze the data on the spectrum of surgical microflora and monitoring of antibiotic resistance in the period from 2012 to 2020.The research material was the separation of surgical wounds of patients, cultures of gram-negative and gram-positive bacteria isolated from the biological material of patients by the bacteriological method, the results of testing the sensitivity of bacterial cultures to antibiotics by diffusion into agar and using semi-automatic analyzers VITEK-2.A 9-year multicenter monitoring of the species composition of bacteria associated with wound infections revealed a high frequency of isolation of a number of conditionally pathogenic microorganisms – Escherichia spp., Klebsiella spp., Pseudomonas spp., Acinetobacter spp., Enterococcus spp., Streptococcus spp., Staphylococcus spp., Citrobacter spp., Proteus spp. In the period from 2012 to 2020, the amount of information entered into the computer database about these bacterial strains increased 1.4–2.0 times. Monitoring of bacterial resistance to antibiotics during this period showed a high level and dynamics to 3rd generation cephalosporins, carbapenems, and other classes. The lowest levels of resistance were found among the strains of Escherichia and the highest – Klebsiella, Pseudomonas and Acinetobacter. Staphylococci are characterized by high percentage of methicillin-resistant strains.Gram-negative (Escherichia, Klebsiella, Pseudomonas, Acinetobacter) and gram-positive (Enterococci, Streptococci, Staphylococci) are detected in the surgical patients with UTI with a high frequency. Surgical pathogens are characterized by a high level of resistance to 3rd generation cephalosporin, carbapenems, and other antibiotics. Long-term monitoring of bacterial resistance to antibiotics makes it possible to increase the effectiveness of laboratory diagnostics of surgical infections, to assess a resistance level of pathogens, an antibacterial therapy strategy and a medical aid quality.

Low level of antifungal resistance in Candida species recovered from Iranian HIV-associated oral infection.

Oral candidiasis (OC) is the most frequent opportunistic fungal infection, which is a predictive indicator of immunosuppression and disease progression among people living with HIV/AIDS (PLWHA). In the present study, 109 Candida isolates were collected from 94 PLWHA afflicted with oral Candida infection (OCI) following highly active antiretroviral therapy (HAART). The susceptibility profiles of Candidaspp. to six antifungal agents were evaluated using CLSI broth microdilution. The prevalence of OCI was 34.06%. The susceptibility profile of Candidaspp. revealed 100% sensitivity to caspofungin, while 6.4%, 5.4%, 24.5%, and 2.8% of Candida isolates showed resistance or nonwild-type MICs to fluconazole, itraconazole, posaconazole, and amphotericin B, respectively. Notably, 15.9% of patients and 3.7% of isolates showed mixed Candida infections and multidrug resistance, respectively. The low-level resistance to antifungal agents observed in the present study may be explained by the fact that none of the participants had prior and prolonged exposure to these antifungals. However, more focus should be placed on the mechanisms of reduced susceptibility and low-level resistance in Candida species since they can serve as stepping stones to developing clinical resistance. Alongside this, it seems a must to understand the local epidemiology of Candida spp. and their susceptibility pattern.

Characterization difference of typical KL1, KL2 and ST11-KL64 hypervirulent and carbapenem-resistant Klebsiella pneumoniae.

Almost all the formation of hypervirulent and carbapenem-resistant Klebsiella pneumoniae follow two major patterns: KL1/KL2 hvKP strains acquire carbapenem-resistance plasmids (CR-hvKP), and carbapenem-resistant Klebsiella pneumoniae (CRKP) strains obtain virulence plasmids (hv-CRKP). These two patterns may pose different phenotypes. In this study, three typical resistance and hypervirulent K. pneumoniae (KL1, KL2, and ST11-KL64), isolating from poor prognosis patients, were selected. Compared with ST11-KL64 hv-CRKP, KL1/KL2 hypervirulent lineages harbor significantly fewer resistance determinants and exhibited lower-level resistance to antibiotics. Notably, though the blaKPC gene could be detected in all these isolates, KL1/KL2 hvKP strain did not exhibit corresponding high-level carbapenem resistance. Unlike the resistance features, we did not observe significant virulence differences between the three strains. The ST11-KL64 hv-CRKP (1403) in this study, showed similar mucoviscosity, siderophores production, and biofilm production compared with KL1 and KL2 hvKP. Moreover, the hypervirulent of ST11-KL64 hvKP also verified with the human lung epithelial cells infection and G. mellonella infection models. Moreover, we found the pLVPK-like virulence plasmid and IncF blaKPC-2 plasmid was crucial for the formation of hypervirulent and carbapenem-resistant K. pneumoniae. The conservation of origin of transfer site (oriT) in these virulence and blaKPC-2 plasmids, indicated the virulence plasmids could transfer to CRKP with the help of blaKPC-2 plasmids. The co-existence of virulence plasmid and blaKPC-2 plasmid facilitate the formation of ST11-KL64 hv-CPKP, which then become nosocomial epidemic under the antibiotic stress. The ST11-KL64 hv-CPKP may poses a substantial threat to healthcare networks, urgent measures were needed to prevent further dissemination in nosocomial settings.

Animal Model Studies, Antibiotic Resistance and Toxin Gene Profile of NE Reproducing Clostridium perfringens Type A and Type G Strains Isolated from Commercial Poultry Farms in China

Poultry necrotic enteritis (NE) is a complex and multifactorial disease caused by Clostridium perfringens types. Earlier, the disease was prevented and/or controlled through the addition of in-feed antibiotics and antimicrobial growth promoters (AGPs). The ban on the use of these agents as feed additives has been a major reason for re-emergence of this disease leading to huge economic losses to the world poultry industry. Understanding the pathogenesis of NE by developing an effective experimental model remains challenging and lacks consistency owing to the involvement of several critical factors involved in causing lesions of disease in the field. In this study, locally characterized C. perfringens types, i.e., ACP (toxinotype A), and GCP (toxinotype G), obtained from NE outbreaks on commercial farms in China (2020-2022), were used to experimentally induce NE in Specific-Pathogen-Free (SPF) chicks. The lesion scores observed on day 20 were 1.9 ± 1.10 (GCP strain) and 1.5 ± 1.08 (ACP strain), and both had significant difference as compared to the control group. The inclusion of fishmeal in addition to oral clostridial dose, i.e., fishmeal (day 7 onward) + Clostridia (7.5 × 108 cfu/mL consecutively for 04 days) induced a lesion score of 2.0 ± 1.15 in respective groups. Use of coccidia (Eimeria necatrix) on day 9 followed by clostridia challenge enhanced the lesion scores to 2.5 ± 1.08 and 2.2 ± 1.23 for type G and type A strains, respectively. When both predisposing factors (coccidia + fish meal) were given together, i.e., fishmeal (day 7 onward) and coccidia (day 9) along with clostridia, the lesion scores were 3.2 ± 1.22 (GCP + coccidia + fish meal) and 3.0 ± 1.15 (ACP + coccidia + fish meal). These results were significantly different from group 1 (ACP) and 2 (GCP), in which only C. perfringens was used to induce NE. The clinical signs as well as histopathological lesions in experimentally induced groups were found similar as reported in the literature. The two type G strains identified in this study were also used for susceptibility testing against various drugs. Both strains were found to be resistant to amikacin, doxycycline, metronidazole, neomycin, nystatin, polymyxin B, streptomycin, and tetracycline. Variable susceptibility was seen against ceftriaxone, florfenicol, gentamicin, and kanamycin drugs. Amoxicillin, ampicillin, cefotaxime, ciprofloxacin, enrofloxacin, ofloxacin, and penicillin were effective drugs based upon their low level of resistance and therefore they might be preferred over other antimicrobial agents for proper treatment/prophylaxis of NE infections. Further studies are needed to study the pathogenesis of NE in detail in experimentally induced models along with continuous monitoring of the resistance pattern of C. perfringens strains in the field.

Prevalence and Characterization of Beta-Lactam and Carbapenem-Resistant Bacteria Isolated from Organic Fresh Produce Retailed in Eastern Spain

Fresh fruits and vegetables are potential reservoirs for antimicrobial resistance determinants, but few studies have focused specifically on organic vegetables. The present study aimed to determine the presence of third-generation cephalosporin (3GC)- and carbapenem-resistant Gram-negative bacteria on fresh organic vegetables produced in the city of Valencia (Spain). Main expanded spectrum beta-lactamase (ESBL)- and carbapenemase-encoding genes were also detected in the isolates. One hundred and fifteen samples were analyzed using selective media supplemented with cefotaxime and meropenem. Resistance assays for twelve relevant antibiotics in medical use were performed using a disc diffusion test. A total of 161 isolates were tested. Overall, 33.5% presented multidrug resistance and 16.8% were resistant to all β-lactam antibiotics tested. Imipenem resistance was observed in 18% of isolates, and low resistance levels were found to ceftazidime and meropenem. Opportunistic pathogens such as Acinetobacter baumannii, Enterobacter spp., Raoultella sp., and Stenotrophomonas maltophilia were detected, all presenting high rates of resistance. PCR assays revealed blaVIM to be the most frequently isolated ESBL-encoding gene, followed by blaTEM and blaOXA-48. These results confirm the potential of fresh vegetables to act as reservoirs for 3GC- and carbapenem-producing ARB. Further studies must be carried out to determine the impact of raw organic food on the spread of AMRs into the community.

Antimicrobial resistance of Salmonella strains isolated from food products of animal origin in Ukraine between 2018-2021

Salmonellosis is a zoonotic infection of humans and animals. In recent years, an increase in the number of resistant strains of Salmonella from animals and animal products have been recorded. We conducted microbiological testing and antibiotic resistance profile of 34 Salmonella strains isolated from foods of animal origin between 2018 to 2021 in Ukraine. Twenty isolates were successfully assigned to six different serovars [S. Enteritidis (n=13, S. Infantis (n=2), S. Schwarzengrund (n=1), S. Livingstone (n=2), S. Vuadens (n=1) and S. Hadar (n=1)], and 14 isolates remained untyped. Most of the isolated strains were recovered from poultry products. The strains were tested for six antibacterial groups: quinolones, aminoglycosides, β-lactams, cephalosporins, phenols, and dihydrofolate reductase inhibitors by disc diffusion method. The highest level of resistance was recorded to 4th generation cephalosporins, e.g., cefepime (88.2%). The lowest level of resistance was to cefotaxime and ceftriaxone (5.9%). No resistance was found to imipenem and aminoglycoside antibiotics, particularly gentamicin and amikacin. The study results are of concern because antibiotic-resistant bacteria and their genes can cause human infections by penetration and transmission at any stage of the food production cycle. Further research is required to monitor the contamination of food products of animal origin with salmonellosis pathogens, including antibiotic-resistant strains.

Resistance of Black Aspergilli Species from Grape Vineyards to SDHI, QoI, DMI, and Phenylpyrrole Fungicides.

Fungicide applications constitute a management practice that reduces the size of fungal populations and by acting as a genetic drift factor, may affect pathogen evolution. In a previous study, we showed that the farming system influenced the population structure of the Aspergillus section Nigri species in Greek vineyards. The current study aimed to test the hypothesis that the differences in the population structure may be associated with the selection of fungicide-resistant strains within the black aspergilli populations. To achieve this, we determined the sensitivity of 102, 151, 19, and 22 for the A. uvarum, A. tubingensis, A. niger, and A. carbonarious isolates, respectively, originating either from conventionally-treated or organic vineyards to the fungicides fluxapyroxad-SDHIs, pyraclostrobin-QoIs, tebuconazole-DMIs, and fludioxonil-phenylpyrroles. The results showed widespread resistance to all four fungicides tested in the A. uvarum isolates originating mostly from conventional vineyards. In contrast, all the A. tubingensis isolates tested were sensitive to pyraclostrobin, while moderate frequencies of only lowly resistant isolates were identified for tebuconazole, fludioxonil, and fluxapyroxad. Sequencing analysis of the corresponding fungicide target encoding genes revealed the presence of H270Y, H65Q/S66P, and G143A mutations in the sdhB, sdhD, and cytb genes of A. uvarum resistant isolates, respectively. No mutations in the Cyp51A and Cyp51B genes were detected in either the A. uvarum or A. tubingensis isolates exhibiting high or low resistance levels to DMIs, suggesting that other resistance mechanisms are responsible for the observed phenotype. Our results support the initial hypothesis for the contribution of fungicide resistance in the black aspergilli population structure in conventional and organic vineyards, while this is the first report of A. uvarum resistance to SDHIs and the first documentation of H270Y or H65Q/S66P mutations in sdhB, sdhD, and of the G143A mutation in the cytb gene of this fungal species.

Proteus mirabilis isolated from untreated hospital wastewater, Ibadan, Southwestern Nigeria showed low-level resistance to fluoroquinolone and carried qnrD3 on Col3M plasmids.

Untreated wastewater emanating from healthcare facilities are risk factors for the spread of antimicrobial resistance (AMR) at the human-environment interface. In this study, we investigated the determinants of resistance in three multidrug resistant strains of Proteus mirabilis isolated from untreated wastewater collected from three government owned hospitals in Ibadan, Nigeria. Despite showing low-level resistance to ciprofloxacin, whole genome sequencing revealed the transferable mechanism of quinoloneresistance (TMQR)gene qnrD3 carried on Col3M plasmids in all the isolates. Core genome phylogenetic analysis showed the isolates are closely related differing from each other by ≤ 23 single nucleotide polymorphisms (SNP). Further, they shared the closest evolutionary relationship with isolates from China. Similarly, the Col3M plasmids is most closely related to p3M-2A found in P. vulgaris 3M isolated from the intestine of shrimps in China. This to the best of our knowledge is the first report of Col3M plasmids carrying qnrD3 in environmental bacterial isolates. Our results indicate a possible silent spread of this important plasmid associated with the dissemination of qnrD3 in Nigeria, and further highlights the important role played by untreated wastewater from healthcare facilities in the spread of AMR in low- and middle-income countries.

P450-mediated resistance in Myzus persicae (Sulzer) (Hemiptera: Aphididae) reduces the efficacy of neonicotinoid seed treatments in Brassica napus.

The prophylactic use of seeds treated with neonicotinoid insecticides remains an important means of controlling aphid pests in canola (Brassica napus) crops in many countries. Yet, one of the most economically important aphid species worldwide, the peach potato aphid (Myzus persicae), has evolved mechanisms which confer resistance to neonicotinoids, including amplification of the cytochrome P450 gene, CYP6CY3. While CYP6CY3 amplification has been associated with low-level resistance to several neonicotinoids in laboratory acute toxicity bioassays, its impact on insecticide efficacy in the field remains unresolved. In this study, we investigated the impact of CYP6CY3 amplification on the ability of M. persicae to survive neonicotinoid exposure under laboratory and semi-field conditions. Three M. persicae clones, possessing different copy numbers of CYP6CY3, were shown to respond differently when exposed to the neonicotinoids, imidacloprid and thiamethoxam, in laboratory bioassays. Two clones, EastNaernup209 and Osborne171, displayed low levels of resistance (3-20-fold), which is consistent with previous studies. However, in a large-scale semi-field trial, both clones showed a surprising ability to survive and reproduce on B. napus seedlings grown from commercial rates of neonicotinoid-treated seed. In contrast, an insecticide-susceptible clone, of wild-type CYP6CY3 copy number, was unable to survive on seedlings treated in the same manner. Our findings suggest that amplification of CYP6CY3 in M. persicae clones substantially impairs the efficacy of neonicotinoid seed treatments when applied to B.napus. These findings highlight the potentially important real-world implications of resistances typically considered to be 'low level' as defined through laboratory bioassays. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.