Multidrug Resistance Phenotype Research Articles

Uropatógenos multirresistentes y con resistencia extendida a los antimicrobianos aislados en pacientes adultos de la comunidad de Barinas, Venezuela.

This study aimed to determine the frequency of multidrug-resis-tant (MDR) and extensively drug-resistant (XDR) uropathogens isolated from adult patients in Barinas City, Venezuela, in 2022. Of 1019 urine cultures from patients who attended the Barinas Clinical Microbiological Laboratory, 337 (33.07%) were selected according to inclusion criteria. The microbiological processing of urine was carried out through conventional methods. Antimicro-bial susceptibility tests and extended-spectrum beta-lactamase determination were performed using standardized phenotypic methods. The classification of MDR and XDR strains was conducted according to international criteria. Data were processed through descriptive statistics and frequency analysis. Of 337 urine cultures analyzed, 70.92% corresponded to female patients, and the age groups between 31 and 60 years were the most frequent. Of the uropathogens identified, 93.17% were represented by Enterobacterales, where Escherichia coli stood out with 87.54%, followed by Pseudomonasaeruginosa (2.67%) and Enterococcus faecalis (1.48%). Of the E. coli strains, 87.12% presented resis-tance to at least one antibiotic, with 46.78% MDR and 5.42% XDR. In general, more than half of the uropathogens identified were distributed either as MDR or XDR phenotypes. The results show the need to develop local research to im-prove empirical and targeted therapies in urinary tract infections, in addition to awareness actions for the rational use of antibiotics and epidemiological surveillance of multiresistant strains circulating in the region.

Moving towards a standardized definition of antimicrobial resistance: a comparison of the antimicrobial susceptibility profile of difficult-to-treat resistance (DTR) versus multidrug-resistant (MDR) Pseudomonas aeruginosa clinical isolates (CANWARD, 2016-2021)

Pseudomonas aeruginosa clinical isolates demonstrating difficult-to-treat resistance (DTR) and multidrug-resistant (MDR) phenotypes were evaluated by broth microdilution. Susceptibility was lower for all antimicrobials versus DTR relative to MDR isolates. Ceftazidime-avibactam, ceftolozane-tazobactam, and imipenem-relebactam susceptibility was 35.9%, 64.5%, and 47.0% for DTR isolates and 60.5%, 80.6%, and 71.5% for MDR isolates.

Determining the prevalence and genetic diversity of plasmid-mediated sulfonamide resistance in Escherichia coli from commercial broiler samples

Sulfonamides are commonly used antibacterials in commercial poultry, contributing toward the development of multidrug-resistant (MDR) phenotypes among Escherichia coli and that has emerged as global concern. The current study aimed to assess the sulfonamide resistance among isolated E. coli strains among commercial broilers. The bacterial strains were identified from fecal samples (n = 100) using selective media, followed by initial identification based on biochemical profiles. The susceptibility was determined by measuring the minimum inhibitory concentration (MIC) against sulfamethoxazole. The study also evaluated mobile genetic elements (MGEs), the mediators of antibiotic resistance, by amplification of plasmid DNA using specific primer PCR. Additionally, the isolates were subjected to multilocus sequence typing (MLST) analysis to investigate the genetic diversity among E. coli carrying sulfonamide resistance genes. The results revealed that 58% (58/100) E. coli strains were resistant to sulfonamides, with 36.20% (21/58) of the strains exhibiting an MIC breakpoint ≥512 µg/mL. PCR analysis showed that 42.85% (9/21) of the strains harbored the sul-1 gene, while 38.09% (8/21) carried the sul-2 gene, and 19.04% (4/21) had both genes. No isolate showed the presence of the sul-3 gene. Furthermore, class 1 and class 2 integrons were identified among 80.95% (17/21) and 19.04% (4/21) of the strains, respectively. MLST analysis confirmed that the strains belonged to sequence types (STs) including ST1638, ST155, ST48, ST350, ST23, ST156, and ST746. These findings underscore the diversity among E. coli strains in commercial poultry, which poses a significant risk.

PYED-1 Overcomes Colistin Resistance in Acinetobacter baumannii.

Antibiotic resistance has become more and more widespread over the recent decades, becoming a major global health problem and causing colistin to be increasingly used as an antibiotic of last resort. Acinetobacter baumannii, an opportunistic pathogen that has rapidly evolved into a superbug exhibiting multidrug-resistant phenotypes, is responsible for a large number of hospital infection outbreaks. With the intensive use of colistin, A. baumannii resistance to colistin has been found to increase significantly. In previous work, we identified a deflazacort derivative, PYED-1 (pregnadiene-11-hydroxy-16,17-epoxy-3,20-dione-1), which exhibits either direct-acting or synergistic activity against Gram-positive and Gram-negative species and Candida spp., including A. baumannii. The aim of this study was to evaluate the antibacterial activity of PYED-1 in combination with colistin against both A. baumannii planktonic and sessile cells. Furthermore, the cytotoxicity of PYED-1 with and without colistin was assessed. Our results show that PYED-1 and colistin can act synergistically to produce a strong antimicrobial effect against multidrug-resistant populations of A. baumannii. Interestingly, our data reveal that PYED-1 is able to restore the efficacy of colistin against all colistin-resistant A. baumannii isolates. This drug combination could achieve a much stronger antimicrobial effect than colistin while using a much smaller dosage of the drugs, additionally eliminating the toxicity and resistance issues associated with the use of colistin.

Evaluation of antibiotic resistance, toxin-antitoxin systems, virulence factors, biofilm-forming strength and genetic linkage of Escherichia coli strains isolated from bloodstream infections of leukemia patients

BackgroundOne of the most common complications in patients with febrile neutropenia, lymphoma, leukemia, and multiple myeloma is a bloodstream infection (BSI).ObjectiveThis study aimed to evaluate the antibiotic resistance patterns, virulence factors, biofilm-forming strength, and genetic linkage of Escherichia coli strains isolated from bloodstream infections (BSIs) of leukemia patients.MethodsThe study conducted in Iran from June 2021 to December 2022, isolated 67 E. coli strains from leukemia patients’ bloodstream infections in hospitals in two different areas. Several techniques including disk diffusion and broth microdilution were used to identify patterns of antibiotic resistance, microtiter plate assay to measure biofilm formation, and PCR to evaluate the prevalence of different genes such as virulence factors, toxin-antitoxin systems, resistance to β-lactams and fluoroquinolone antibiotics of E. coli strains. Additionally, the genetic linkage of the isolates was analyzed using the Enterobacterial Repeat Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) method.ResultsThe results showed that higher frequency of BSI caused by E. coli in man than female patients, and patients with acute leukemia had a higher frequency of BSI. Ampicillin and Amoxicillin-clavulanic acid showed the highest resistance, while Imipenem was identified as a suitable antibiotic for treating BSIs by E. coli. Multidrug-resistant (MDR) phenotypes were present in 22% of the isolates, while 53% of the isolates were ESBL-producing with the blaCTX-M gene as the most frequent β-lactamase gene. The fluoroquinolone resistance genes qnrB and qnrS were present in 50% and 28% of the isolates, respectively. More than 80% of the isolates showed the ability to form biofilms. The traT gene was more frequent than other virulence genes. The toxin-antitoxin system genes (mazF, ccdAB, and relB) showed a comparable frequency. The genetic diversity was detected in E. coli isolates.ConclusionOur results demonstrate that highly diverse, resistant and pathogenic E. coli clones are circulating among leukemia patients in Iranian hospitals. More attention should be paid to the treatment and management of E. coli bloodstream infections in patients with leukemia.

Antibiotic-Resistant Bacteria in Environmental Water Sources from Southern Chile: A Potential Threat to Human Health

Antimicrobial resistance (AMR) is a critical global issue affecting public and animal health. The overuse of antibiotics in human health, animal production, agriculture, and aquaculture has led to the selection of antibiotic-resistant strains, particularly in Gram-negative bacteria. Mutations and horizontal gene transfer play a significant role in the development of antimicrobial resistance, leading to the reduced efficacy of current antibiotics. Today, AMR in bacteria and antibiotic-resistance genes (ARGs) are increasingly recognized in multiple environmental sources, including recreational and irrigation waters. This study aims to identify Gram-negative bacteria from surface aquatic reservoirs in southern Chile and assess their susceptibility to clinically relevant antibiotics. Water samples were collected from four lakes, five rivers, one waterfall, and one watershed in southern Chile to isolate environmental Gram-negative bacilli (GNB). API-20E and MALDI–TOF were employed for bacterial identification. Kirby–Bauer disc diffusion tests and multiplex PCR were performed to determine their susceptibility profile. A total of 26 GNB strains were isolated from environmental water samples, predominantly belonging to the Pseudomonas (n = 9) and Acinetobacter (n = 7) genera. Among these strains, 96.2% were resistant to ampicillin and cefazoline, while 26.9% and 34.6% showed resistance to ceftazidime and cefepime, respectively. Additionally, 38.5% exhibited resistance to colistin. Two Enterobacter cloacae strains obtained from Cachapoal River (sixth region) and Villarrica Lake (ninth region), respectively, presented a multidrug-resistant (MDR) phenotype and carried at least two extended-spectrum β-lactamase (ESBL) genes. Thus, antibiotic-resistant GNB and ARGs were found in natural water reservoirs, raising concerns about the dissemination of resistance determinants among potentially pathogenic bacteria in environmental microbial communities.

Emergence of colistin-resistant Enterobacter cloacae and Raoultella ornithinolytica carrying the phosphoethanolamine transferase gene, mcr-9, derived from vegetables in Japan.

Colistin, a last-line antimicrobial agent, is recommended for the treatment of severe infections caused by multidrug-resistant (MDR) microorganisms. However, reports on plasmid-mediated mobilized colistin resistance (mcr) genes have prompted the importance of routine colistin resistance surveillance. Herein, we report the emergence of Enterobacter cloacae CST17-2 and Raoultella ornithinolytica CST129-1 carrying an mcr-9.1 gene in vegetables from Japan. A total of 308 colistin-resistant isolates were retrieved from 200 fresh vegetables in Hiroshima Prefecture, Japan. PCR detection of mcr-1 to mcr-9 was conducted. While none of the isolates detected positive for mcr-1 to mcr-8 genes, we found two (0.65%) positive strains, E. cloacae CST17-2 and R. ornithinolytica CST129-1, that harbored mcr-9.1 allele. These isolates were subjected to phenotypic susceptibility testing, whole-genome sequencing (WGS), PCR-based replicon typing, and conjugation experiment. We found that both isolates had high colistin resistance [minimum inhibitory concentration (MIC) 16 or >64 µg/mL] and showed MDR phenotypes. WGS of both isolates revealed mcr-9 on a plasmid of the IncHI2/HI2A backbone. The mcr-9-bearing plasmid, pCST17-2_1, was self-transferable, although the pCST129-1_1 plasmid was not. Despite being colistin-resistant, the so-called two-component regulatory operon, qseBC, which induces polymyxin resistance, was absent from the genetic arrangements downstream of mcr-9 in R. ornithinolytica CST129-1. Nonetheless, a conjugation experiment demonstrated that mcr-9 in a Raoultella-type background is capable of mediating colistin resistance. In silico genomic analysis and comparison revealed distinct genetic structures surrounding mcr-9, especially in the downstream vicinities. The E. cloacae CST17-2 strain is of sequence-type ST738, a sequence type that has emerged in mcr-9.1-containing E. cloacae. Remarkably, we report the first mcr-9-carrying colistin-resistant Enterobacteriaceae isolated from Japanese vegetables, which is a grave public health concern. Our findings highlight the importance of strict epidemiological monitoring to track and/or prevent further dissemination of mcr homologs across the vegetable industry.IMPORTANCEPlasmid-mediated mobile colistin-resistance genes have been recognized as a global threat because they jeopardize the efficacy of colistin in therapeutic practice. Here, we described the genetic features of two mcr-9.1-carrying Gram-negative bacteria with a colistin-resistant phenotype derived from vegetables in Japan. The colistin-resistant mcr-9.1, which has never been detected in vegetables, was located on a large plasmid in Enterobacter cloacae CST17-2 and Raoultella ornithinolytica CST129-1, suggesting a high chance of horizontal gene transfer. To the best of our knowledge, this is the first report of mcr-9 in R. ornithinolytica. This study indicates that fresh vegetables might be a potential source for the transmission of mcr-9 genes encoding resistance to frontline (colistin) and clinically relevant antimicrobials. The study also provides additional consideration for colistin use and the relevance of routine surveillance in epidemiological perspective to curb the continuous spread of mcr alleles.

Susceptibility to antimicrobial preparations of Klebsiella pneumoniae strains isolated from patients in a multidisciplinary hospital

Introduction. The extraordinary genetic potential of microorganisms has benefited from human overuse antibiotics to develop multiple resistance mechanisms. The large size and heterogeneity of the Russian population, the presence of territories with significant differences in demographic, cultural, and socio-economic indicators, the features of the management and availability of medical care are significant factors influencing the spread of resistance genes to antibacterial therapy of some strains of microorganisms.
 Materials and methods. Susceptibility to twenty one antimicrobial agents of one hundred twenty eight Klebsiella pneumoniae isolates, isolated from various loci of patients in a multidisciplinary hospital, including 16% of strains from the lower respiratory tract, 44% from wounds and wound discharge, and 40% from urine, was assessed.
 Results. Among isolates from urine, 20% of isolates were found to have the multidrug resistance (MDR) phenotype, 42% had the extreme resistance phenotype (XDR). Among the isolates of Kl. pneumoniae from purulent wounds, 18% had the MDR phenotype, and 43% had the XDR phenotype. Among the isolates of Kl. Рneumoniae from the lower respiratory tract, 37% had the MDR phenotype, 40% had the XDR phenotype. There are no pan-resistant strains in all groups. 
 The presence of genes for metallo-beta-lactamase (VIM, IMP, NDM groups) and serine carbapenemase (bovine and OXA-48) in molecular genetic study by real-time PCR of isolated Kl. pneumoniae was found in 73.4% of cases. 
 The results of determining the sensitivity of the isolates revealed a low activity of amoxicillin / clavulanic acid, III and IV generation cephalosporins (ceftazidime, cefotaxime, cefepime). 86.7% of isolates are resistant to drugs of the aminoglycoside group (amikacin), and 100% to gentamicin. Of the group of carbapenems, meropenem showed the highest activity – 26.6%, about 7% were sensitive to ertapenem. From the group of fluoroquinolones, sensitivity ranged from 20 to 30%. The highest activity of all drugs was shown by ticarcillin/clavulanate – 33.3%.
 Limitations. The criteria for inclusion in the group of examination and selection of biomaterial were the presence of previous massive antibacterial therapy in the anamnesis, the presence of catheters, drains, etc.
 Conclusions. There was established a high proportion of strains Kl. pneumoniae with the phenotype of multiple antibiotic resistance.

Characterization of Virulence Genes Associated with Type III Secretion System and Biofilm Formation in Pseudomonas aeruginosa Clinical Isolates.

Pseudomonas aeruginosa is a common pathogen with an increasing multidrug resistance (MDR) phenotype. Its virulence determinants include many factors such as antimicrobial resistance, biofilm formation, and type III secretion system (T3SS) which correlate with disease severity. There are no reports regarding the virulence features of P. aeruginosa in Cyprus. The aim of this study was to investigate the frequency and distribution of selected virulence-encoding genes and evaluate the biofilm formation potential as well as antibiotic resistance rates of isolates in the region. One hundred clinical P. aeruginosa isolates were obtained from clinical specimens and were identified using standard microbiological techniques. Antimicrobial susceptibility was assessed using the VITEK-2 system and biofilm quantification was performed by the microtiter plate assay with crystal violet staining. The presence of algD, exoU, exoT, and exoS was evaluated using polymerase chain reaction(PCR). Among all isolates, 35% were strong biofilm former, 28% were moderate biofilm former, 19% were weak biofilm former, and 18% were non-biofilm former. The rates of MDR and extensive drug resistance (XDR) were 26% and 1%. PCR analysis indicated that 93% of the isolates were algD positive. T3SS genes exoT, exoS, and exoU were detected in 91%, 63%, and 32% of the isolates, respectively. There was a high frequency of exoT + /exoS + genotype (61%), whereas exoT + /exoU + (32%) and exoS + /exoU + (2%) genotypes were relatively uncommon. This study reports the first dataset on the molecular profile of P. aeruginosa in Cyprus. Our results demonstrated that most strains have the biofilm-forming capacity with an algD-positive genotype and the majority carry exoT and exoS with a high frequency of exoT + /exoS + genotype.

Glycolipids from Sargassum filipendula, a Natural Alternative for Overcoming ABC Transporter-Mediated MDR in Cancer.

Chemotherapy is a widely used strategy to treat cancer, a disease that causes millions of deaths each year. However, its efficacy is reduced by the overexpression of ABC transporters, which are proteins that expel the drugs used in chemotherapy and involved in the multidrug resistance (MDR). Glycolipids have been identified as potential inhibitors of ABC transporters. Algae of the genus Sargassum contain high levels of glycolipids, making them a promising therapeutic alternative against the MDR phenotype. Sargassum filipendula glycolipids were obtained by exhaustive maceration with chloroform/methanol, purified by column and thin layer chromatography, and then characterized by FTIR, NMR, and LC-MS. Cell viability by PI labeling and inhibition of ABC transporters were analyzed by flow cytometry. Assessment of resistance reversal was determined by MTT assay. Ten sulfoquinovosylglycerol-type compounds were found, and six of them are reported for the first time. In particular, moiety 4 (GL-4) showed strong and moderate inhibitory activity against ABCC1 and ABCB1 transporters respectively. Treatment of GL-4 in combination with the antineoplastic drug vincristine sensitized Lucena-1 cell model to drug and reversed the MDR phenotype. This is the first report of glycolipids isolated from S. filipendula capable of inhibiting ABC transporters and thus overcoming acquired drug resistance.

Repurposing carvacrol, cinnamaldehyde, and eugenol as potential anti-quorum sensing agents against uropathogenic Escherichia coli isolates in Alexandria, Egypt

BackgroundUrinary tract infections represent one of the most frequent hospital and community-acquired infections with uropathogenic Escherichia coli (UPEC) being the main causative agent. The global increase in the emergence of multidrug-resistant (MDR) UPEC necessitates exploring novel approaches. Repurposing natural products as anti-quorum sensing (QS) agents to impede bacterial virulence is gaining momentum nowadays. Hence, this study investigates the anti-QS potentials of carvacrol, cinnamaldehyde, and eugenol against E. coli isolated from urine cultures of Egyptian patients.ResultsAntibiotic susceptibility testing was performed for 67 E. coli isolates and 94% of the isolates showed MDR phenotype. The usp gene was detected using PCR and accordingly, 45% of the isolates were categorized as UPEC. Phytochemicals, at their sub-inhibitory concentrations, inhibited the swimming and twitching motilities of UPEC isolates, with eugenol showing the highest inhibitory effect. The agents hindered the biofilm-forming ability of the tested isolates, at two temperature sets, 37 and 30 °C, where eugenol succeeded in significantly inhibiting the biofilm formation by > 50% at both investigated temperatures, as compared with untreated controls. The phytochemicals were shown to downregulate the expression of the QS gene (luxS) and critical genes related to motility, asserting their anti-QS potential. Further, the combinatory activity of the phytoproducts with five antibiotics was assessed by checkerboard assay. The addition of the phytoproducts significantly reduced the minimum inhibitory concentrations of the antibiotics and generated several synergistic or partially synergistic combinations, some of which have not been previously explored.ConclusionsOverall, carvacrol, cinnamaldehyde, and eugenol could be repurposed as potential anti-QS agents, which preferentially reduce the QS-based communication and attenuate the cascades of gene expression, thus decreasing the production of virulence factors in UPEC, and eventually, subsiding their pathogenicity. Furthermore, the synergistic combinations of these agents with antibiotics might provide a new perspective to circumvent the side effects brought about by high antibiotic doses, thereby paving the way for overcoming antibiotic resistance.

Effects of the chemosensitizer verapamil on P-glycoprotein substrate efflux in rainbow trout hepatocytes

The ATP-dependent membrane transporter P-glycoprotein (P-gp) is associated with resistance to a wide variety of chemical substrates, as well as the multi-drug resistance (MDR) phenotype in mammals. Less is known regarding P-gp's function and relevance in teleosts; this study expanded the range of known substrates and the inhibitory effects of a model chemosensitizer verapamil. The P-gp-mediated uptake and efflux dynamics of 5 known mammalian substrates (berberine, cortisol, doxorubicin, rhodamine 123 [R123], and vinorelbine) were examined in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes with and without co-exposure to varying doses of verapamil. Initial substrate uptake rates (pmol/106 cells/min) varied widely and were in order: berberine (482 ± 94) > R123 (364 ± 67) > doxorubicin (158 ± 41) > cortisol (20.3 ± 5.9) > vinorelbine (15.3 ± 3.5). Initial efflux rates (pmol/106 cells/min) were highest in berberine (464 ± 110) > doxorubicin (341 ± 57) > R123 (106 ± 33) > cortisol (26.6 ± 6.1) > vinorelbine (9.0 ± 2.4). Transport of vinorelbine and R123 is verapamil sensitive, but verapamil had no effect on transport of berberine, cortisol, or doxorubicin. Cortisol and doxorubicin showed evidence of high P-gp affinity, thus displacing verapamil from their shared P-gp binding site. Cortisol, doxorubicin, R123, and vinorelbine transport by rainbow trout P-gp was confirmed, while berberine could not be confirmed or excluded as a substrate. Binding sites and affinities were similar between mammalian and trout P-gp for doxorubicin, R123, and vinorelbine, while fish P-gp had a higher affinity for cortisol than mammalian P-gp. This study demonstrated that the range of substrates, as well as binding sites and affinities, of fish P-gp are well-aligned with those in mammals.

Mechanisms of cytotoxic activity of pyrrole-carboxamides against multidrug-resistant tumor cell sublines

Introduction. Mitotic poisoning agents (MPAs) affecting the dynamic state of the microtubules, are the well-known and effective chemotherapeutic agents. Mitotic poisoning agents are binding to the microtubules, and thereby interfere with tubulin polymerization or depolymerization dynamic state, resulting in the cell cycle arrest in M-phase (mitotic catastrophe) and subsequent apoptotic cell death. We reported previously about potent cytotoxic activities against the pyrrole-carboxamides (PCs) (PC-61 and PC-84) against broad spectrum of cancer cell lines, including triple negative breast cancer, lung and prostate cancer.Aim. To examine the cytotoxic activities of PC-61 and PC-84 against multidrug-resistant cancer cell lines indicated above.Materials and methods. Studу was performed on the triple-negative paclitaxel-resistant breast cancer cell line HCC1806 Tx-R and doxorubicin-resistant osteosarcoma SaOS-2 Dox-R cell line.Results. The cytotoxic activity of PCs was due to the inhibition of tubulin polymerization. Immunofluorescence staining data revealed PC’s ability to interfere with tubulin’s assembly in multidrug-resistant cancer cell lines. As an outcome of inhibition of tubulin polymerization, PCs induced cell cycle arrest in M-phase, and further led to apoptotic cell death of cancer cells.Conclusion. Collectively, we demonstrated potent cytotoxic activity of PCs against cancer cell lines with multidrug-resistant phenotype, which arising the possibilities to develop novel and effective anti-tumor agents that belongs to mitotic poisoning agents

Multiple and multidrug resistance in Botrytis cinerea: molecular mechanisms of MLR/MDR strains in Greece and effects of co-existence of different resistance mechanisms on fungicide sensitivity.

Botrytis cinerea is a high-risk pathogen for fungicide resistance development. Within the fungal populations, strains have developed multiple mutations in different target genes leading to multiple resistance (MLR) or mutations associated with overexpression of efflux transporters leading to multidrug resistance (MDR). These types of resistance are a major threat, and their successful management is a major challenge. The current study was initiated to a) determine frequencies of MLR/MDR strains in populations originating from several crops, b) identify the types of MDR that occur in Greece, and c) determine interactions between MLR and MDR at the level of sensitivity to botryticides. The frequencies of MLR/MDR phenotypes were determined in 515 isolates subjected to bioassays using discriminatory concentrations of thiophanate-methyl, iprodione, cyprodinil, fenhexamid, boscalid, fluopyram, fludioxonil, pyraclostrobin, and tolnaftate. Interestingly, 7.8% and 31.3% of isolates from strawberry and rootstock seedlings were resistant to every single fungicide class, while MDR phenotypes from strawberries, rootstocks, and tomatoes accounted for 26%, 87%, and 13.4%, respectively. The MLR and MDR isolates were further molecularly analyzed regarding genes erg27, sdhB, Bcpos5, and Mrr1, responsible for resistance to fenhexamid, boscalid and fluopyram, cyprodinil, and MDR, respectively. The different mutations' presence was determined along with a new mutation in Mrr1 leading to MDR. MDR isolates were characterized as MDR1 or MDR1h based on the presence of a 3-bp deletion in Mrr1. MDR1h was predominant in isolates from rootstocks and MDR1 from tomatoes and strawberries, whereas the most frequent target-site mutations were F412S (erg27), H272R (sdhB), and L412F (Bcpos5). To determine whether the accumulation of target-site mutations along with MDR mutations exhibits an additive effect concerning fungicide resistance, the sensitivity of isolates possessing the predominant target-site mutations was calculated in both the presence and the absence of MDR-associated mutations. EC50 in cyprodinil and boscalid increased to about twofold in the presence of MDR mutations, while there was no difference for fenhexamid. In conclusion, MLR/MDR frequencies are notably high in heavily treated crops in Greece, and the combination of MLR and MDR mutations leads to even higher fungicide resistance levels, highlighting the importance of resistance management.

Dissemination of Multidrug-Resistant, Extensively Drug Resistant and Pandrug-Resistant Pseudomonas aeruginosa Isolates among In-Patients and Out-Patients in a Multi-Profile Health Care Settings

Pseudomonas aeruginosa is one of the most life-threatening pathogens, especially in healthcare settings, and a main contributor to multi-drug resistance (MDR), extensive-drug resistance (XDR), and pan-drug resistant (PDR) phenotypes. However, there is limited data on the degree of resistance of these isolates in this region. This study seeks to determine the distribution of MDR, XDR, and PDR Pseudomonas aeruginosa strains from different patient groups. A total of five hundred (500) non-duplicated strains of Pseudomonas aeruginosa of human clinical samples were collected from the Microbiology Laboratory Unit of Alex Ekwueme Teaching Hospital Abakaliki. The isolates were identified and re-characterized by standard microbiology techniques. MDR, XDR, and PDR were determined using the Kirby–Bauer disk diffusion method, and the results were analyzed using the Clinical Laboratory Standard Institute (CLSI) zone diameter breakpoints. The result shows that the overall resistant phenotype was MDR 50. 7%, XDR 20.5%, and PDR 9.6% while in samples from in-patients and out-patients, resistant phenotype proportions were MDR 43.2%, XDR 32.4%, PDR 10.1% and MDR 61.2%, XDR 29.7%, and PDR 18.5% respectively. Worrisomely, only a few tested antimicrobial agents (Amikacin, cefepime) were active against the test organism, presenting a limited therapeutic option. It is therefore imperative to establish strong regulative measures and guidelines that would help in curtailing the increasing dissemination of these superbugs in healthcare institutions in Nigeria.

Microbial Primer: Multidrug efflux pumps.

Multidrug efflux pumps are molecular machines that sit in the bacterial cell membrane and pump molecules out from either the periplasm or cytoplasm to outside the cell. While involved in a variety of biological roles, they are primarily known for their contribution to antibiotic resistance by limiting the intracellular accumulation of antimicrobial compounds within bacteria. These transporters are often overexpressed in clinical isolates, leading to multidrug-resistant phenotypes. Efflux pumps are classified into several families based on their structure and understanding the characteristics of each family is important for the development of novel therapies to restore antibiotic potency.

Within-Host Diversity of Coagulase-Negative Staphylococci Resistome from Healthy Pigs and Pig Farmers, with the Detection of cfr-Carrying Strains and MDR-S. borealis.

The ecology and diversity of resistome in coagulase-negative staphylococci (CoNS) from healthy pigs and pig farmers are rarely available as most studies focused on the livestock-associated methicillin-resistant S. aureus. This study aims to characterize the antimicrobial resistance (AMR) mechanisms, intra-host species diversity (more than one species in a host), and intra-species AMR diversity (same species with more than one AMR profile) in CoNS recovered from the nasal cavities of healthy pigs and pig farmers. One-hundred-and-one CoNS strains previously recovered from 40 pigs and 10 pig farmers from four Spanish pig farms were tested to determine their AMR profiles. Non-repetitive strains were selected (n = 75) and their AMR genes, SCCmec types, and genetic lineages were analyzed by PCR/sequencing. Of the non-repetitive strains, 92% showed a multidrug resistance (MDR) phenotype, and 52% were mecA-positive, which were associated with SCCmec types V (46.2%), IVb (20.5%), and IVc (5.1%). A total of 28% of the pigs and pig farmers had intra-host species diversity, while 26% had intra-species AMR diversity. High repertoires of AMR genes were detected, including unusual ones such as tetO, ermT, erm43, and cfr. Most important was the detection of cfr (in S. saprophyticus and S. epidermidis-ST16) in pigs and pig farmers; whereas MDR-S. borealis strains were identified in pig farmers. Pig-to-pig transmission of CoNS with similar AMR genes and SCCmec types was detected in 42.5% of pigs. The high level of multidrug, within-host, and intra-species resistome diversity in the nasal CoNS highlights their ability to be AMR gene reservoirs in healthy pigs and pig farmers. The detection of MDR-S. borealis and linezolid-resistant strains underscore the need for comprehensive and continuous surveillance of MDR-CoNS at the pig farm level.

Genome-wide transcriptional analyses of Clarireedia jacksonii isolates associated with multi-drug resistance.

Emerging multidrug resistance (MDR) in Clarireedia spp. is a huge challenge to the management of dollar spot (DS) disease on turfgrass. Insight into the molecular basis of resistance mechanisms may help identify key molecular targets for developing novel effective chemicals. Previously, a MDR isolate (LT586) of C. jacksonii with significantly reduced sensitivities to propiconazole, boscalid, and iprodione, and a fungicide-sensitive isolate (LT15) of the same species were isolated from creeping bentgrass (Agrostis stolonifera L.). The present study aimed to further explore the molecular mechanisms of resistance by using genome-wide transcriptional analyses of the two isolates. A total of 619 and 475 differentially expressed genes (DEGs) were significantly down and upregulated in the MDR isolate LT586, compared with the sensitive isolate LT15 without fungicide treatment. Three hundreds and six and 153 DEGs showed significantly lower and higher expression in the MDR isolate LT586 than those in the sensitive isolate LT15, which were commonly induced by the three fungicides. Most of the 153 upregulated DEGs were xenobiotic detoxification-related genes and genes with transcriptional functions. Fifty and 17 upregulated DEGs were also commonly observed in HRI11 (a MDR isolate of the C. jacksonii) compared with the HRS10 (a fungicide-sensitive isolate of same species) from a previous study without and with the treatment of propiconazole, respectively. The reliability of RNA-seq data was further verified by qRT-PCR method using a few select potentially MDR-related genes. Results of this study indicated that there were multiple uncharacterized genes, possibly responsible for MDR phenotypes in Clarireedia spp., which may have important implications in understanding the molecular mechanisms underlying MDR resistance.

Clinical Characteristics, Antimicrobial Resistance, Virulence Genes and Multi-Locus Sequence Typing of Non-Typhoidal Salmonella Serovar Typhimurium and Enteritidis Strains Isolated from Patients in Chiang Mai, Thailand.

Non-typhoidal salmonellosis (NTS) caused by ingesting Salmonella enterica contaminated food or drink remains a major bacterial foodborne disease. Clinical outcomes of NTS range from self-limited gastroenteritis to life-threatening invasive NTS (iNTS). In this study, we isolated Salmonella spp. from the stool and blood of patients hospitalized at Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand, between 2016-2021 (a total of 395 cases). Then, serovar Typhimurium and Enteritidis were identified and further characterized by multiplex PCR, and multi-locus sequence typing. Our data show that multidrug resistance (MDR) sequence type 34 (ST34) and ST11 are the predominant sequence types for serovars Typhimurium and Enteritidis, respectively. Most S. Typhimurium ST34 lacks spvB, and most S. Enteritidis ST11 harbor sseI, sodCI, rpoS and spvB genes. NTS can be found in a wide range of ages, and anemia could be a significant factor for S. Typhimurium infection (86.3%). Both S. Typhimurium (6.7%) and S. Enteritidis (25.0%) can cause iNTS in immunocompromised patients. S. Typhimurium conferred MDR phenotype higher than S. Enteritidis with multiple antibiotic resistance indexes of 0.22 and 0.04, respectively. Here, we characterized the important S. Typhimurium, S. Enteritidis, and human clinical factors of NTS within the region.

Multidrug resistance phenotype and its relation to stem cell characteristics in chronic myeloid leukemia

The present work aimed to evaluate the expression profile of genes related to stem cells (SC) characteristics during the acquisition of the multidrug resistance (MDR) phenotype in the chronic myeloid leukemia (CML). For this, the K562 (non MDR) and FEPS (MDR) cell lines were used. K562 cells had resistance induced by exposure to daunorubicin (DNR), and induction was confirmed by flow cytometry with an increase in ABCB1 expression in K562 cells treated at the highest concentration. Real-time PCR gene expression analysis showed a direct relationship in the expression of OCT4 and ABCB1 genes, with an increase in ABCB1 expression after exposure to DNR, followed by an increase in OCT4 gene expression. This direct relationship was confirmed in the MDR FEPS cells that had the ABCB1 gene silenced. For the ALOX5 gene, we observed an inverse relationship with ABCB1, with a decrease in the expression of ALOX5 in the DNR-transformed K562 cells, and an increase in the expression of this gene when ABCB1 was silenced in the FEPS cells. Thus, during the acquisition of the MDR phenotype by the K562 cells, it was possible to observe that there is an increase in the expression of ABCB1, accompanied by the expression of OCT4, while the expression of ALOX5 is decreased.