Prevalence and molecular characterization of colistin resistance in Pseudomonas aeruginosa isolates: insights from a study in Ardabil hospitals.

BACKGROUND: Pseudomonas aeruginosa is a common cause of nosocomial infections. However, the emergence of multidrug-resistant strains has complicated the treatment of P. aeruginosa infections. While polymyxins have been the mainstay for treatment, there is a global increase in resistance to these antibiotics. Therefore, our study aimed to determine the prevalence and molecular details of colistin resistance in P. aeruginosa clinical isolates collected between June 2019 and May 2023, as well as the genetic linkage of colistin-resistant P. aeruginosa isolates. RESULTS: The resistance rate to colistin was 9% (n = 18) among P. aeruginosa isolates. All 18 colistin-resistant isolates were biofilm producers and carried genes associated with biofilm formation. Furthermore, the presence of genes encoding efflux pumps, TCSs, and outer membrane porin was observed in all colistin-resistant P. aeruginosa strains, while the mcr-1 gene was not detected. Amino acid substitutions were identified only in the PmrB protein of multidrug- and colistin-resistant strains. The expression levels of mexA, mexC, mexE, mexY, phoP, and pmrA genes in the 18 colistin-resistant P. aeruginosa strains were as follows: 88.8%, 94.4%, 11.1%, 83.3%, 83.3%, and 38.8%, respectively. Additionally, down-regulation of the oprD gene was observed in 44.4% of colistin-resistant P. aeruginosa strains. CONCLUSION: This study reports the emergence of colistin resistance with various mechanisms among P. aeruginosa strains in Ardabil hospitals. We recommend avoiding unnecessary use of colistin to prevent potential future increases in colistin resistance.

Raw meat-based diet for pets: a neglected source of human exposure to Salmonella and pathogenic Escherichia coli clones carrying mcr, Portugal, September 2019 to January 2020.

BackgroundThe pet industry is expanding worldwide, particularly raw meat-based diets (RMBDs). There are concerns regarding the safety of RMBDs, especially their potential to spread clinically relevant antibiotic-resistant bacteria or zoonotic pathogens.AimWe aimed to investigate whether dog food, including RMBD, commercially available in Portugal can be a source of Salmonella and/or other Enterobacteriaceae strains resistant to last-line antibiotics such as colistin.MethodsFifty-five samples from 25 brands (21 international ones) of various dog food types from 12 suppliers were screened by standard cultural methods between September 2019 and January 2020. Isolates were characterised by phenotypic and genotypic methods, including whole genome sequencing and comparative genomics.ResultsOnly RMBD batches were contaminated, with 10 of 14 containing polyclonal multidrug-resistant (MDR) Escherichia coli and one MDR Salmonella. One turkey-based sample contained MDR Salmonella serotype 1,4,[5],12:i:- ST34/cgST142761 with similarity to human clinical isolates occurring worldwide. This Salmonella exhibited typical antibiotic resistance (bla TEM + strA-strB + sul2 + tet(B)) and metal tolerance profiles (pco + sil + ars) associated with the European epidemic clone. Two samples (turkey/veal) carried globally dispersed MDR E. coli (ST3997-complexST10/cgST95899 and ST297/cgST138377) with colistin resistance (minimum inhibitory concentration: 4 mg/L) and mcr-1 gene on IncX4 plasmids, which were identical to other IncX4 circulating worldwide.ConclusionSome RMBDs from European brands available in Portugal can be a vehicle for clinically relevant MDR Salmonella and pathogenic E. coli clones carrying genes encoding resistance to the last-line antibiotic colistin. Proactive actions within the One Health context, spanning regulatory, pet-food industry and consumer levels, are needed to mitigate these public health risks.

Assessing the synergistic potential of bacteriophage endolysins and antimicrobial peptides for eradicating bacterial biofilms.

Phage-encoded endolysins have emerged as a potential substitute to conventional antibiotics due to their exceptional benefits including host specificity, rapid host killing, least risk of resistance. In addition to their antibacterial potency and biofilm eradication properties, endolysins are reported to exhibit synergism with other antimicrobial agents. In this study, the synergistic potency of endolysins was dissected with antimicrobial peptides to enhance their therapeutic effectiveness. Recombinantly expressed and purified bacteriophage endolysin [T7 endolysin (T7L); and T4 endolysin (T4L)] proteins have been used to evaluate the broad-spectrum antibacterial efficacy using different bacterial strains. Antibacterial/biofilm eradication studies were performed in combination with different antimicrobial peptides (AMPs) such as colistin, nisin, and polymyxin B (PMB) to assess the endolysin's antimicrobial efficacy and their synergy with AMPs. In combination with T7L, polymyxin B and colistin effectively eradicated the biofilm of Pseudomonas aeruginosa and exhibited a synergistic effect. Further, a combination of T4L and nisin displayed a synergistic effect against Staphylococcus aureus biofilms. In summary, the obtained results endorse the theme of combinational therapy consisting of endolysins and AMPs as an effective remedy against the drug-resistant bacterial biofilms that are a serious concern in healthcare settings.

Efficacy of adjunctive inhaled colistin and tobramycin for ventilator-associated pneumonia: systematic review and meta-analysis.

INTRODUCTION: Ventilator-associated pneumonia (VAP) presents a significant challenge in intensive care units (ICUs). Nebulized antibiotics, particularly colistin and tobramycin, are commonly prescribed for VAP patients. However, the appropriateness of using inhaled antibiotics for VAP remains a subject of debate among experts. This study aims to provide updated insights on the efficacy of adjunctive inhaled colistin and tobramycin through a comprehensive systematic review and meta-analysis. METHODS: A thorough search was conducted in MEDLINE, EMBASE, LILACS, COCHRANE Central, and clinical trials databases ( www. CLINICALTRIALS: gov ) from inception to June 2023. Randomized controlled trials (RCTs) meeting specific inclusion criteria were selected for analysis. These criteria included mechanically ventilated patients diagnosed with VAP, intervention with inhaled Colistin and Tobramycin compared to intravenous antibiotics, and reported outcomes such as clinical cure, microbiological eradication, mortality, or adverse events. RESULTS: The initial search yielded 106 records, from which only seven RCTs fulfilled the predefined inclusion criteria. The meta-analysis revealed a higher likelihood of achieving both clinical and microbiological cure in the groups receiving tobramycin or colistin compared to the control group. The relative risk (RR) for clinical cure was 1.23 (95% CI: 1.04, 1.45), and for microbiological cure, it was 1.64 (95% CI: 1.31, 2.06). However, there were no significant differences in mortality or the probability of adverse events between the groups. CONCLUSION: Adjunctive inhaled tobramycin or colistin may have a positive impact on the clinical and microbiological cure rates of VAP. However, the overall quality of evidence is low, indicating a high level of uncertainty. These findings underscore the need for further rigorous and well-designed studies to enhance the quality of evidence and provide more robust guidance for clinical decision-making in the management of VAP.

Practical synthesis of isoindolines yields potent colistin potentiators for multidrug-resistant Acinetobacter baumannii.

An efficient and practical one-pot synthesis of isoindolines from readily available starting materials was achieved under mild conditions by implementing an isoindole umpolung strategy. A variety of isoindolines were prepared with good to excellent yields. Biological screens of these identified compounds demonstrated that they are potent potentiators of colistin for multi-drug resistant Acinetobacter baumannii.

Genome analyses of colistin-resistant high-risk blaNDM-5 producing Klebsiella pneumoniae ST147 and Pseudomonas aeruginosa ST235 and ST357 in clinical settings.

BACKGROUND: Colistin is a last-resort antibiotic used in extreme cases of multi-drug resistant (MDR) Gram-negative bacterial infections. Colistin resistance has increased in recent years and often goes undetected due to the inefficiency of predominantly used standard antibiotic susceptibility tests (AST). To address this challenge, we aimed to detect the prevalence of colistin resistance strains through both Vitek®2 and broth micro-dilution. We investigated 1748 blood, tracheal aspirate, and pleural fluid samples from the Intensive Care Unit (ICU), Neonatal Intensive Care Unit (NICU), and Tuberculosis and Respiratory Disease centre (TBRD) in an India hospital. Whole-genome sequencing (WGS) of extremely drug-resitant (XDR) and pan-drug resistant (PDR) strains revealed the resistance mechanisms through the Resistance Gene Identifier (RGI.v6.0.0) and Snippy.v4.6.0. Abricate.v1.0.1, PlasmidFinder.v2.1, MobileElementFinder.v1.0.3 etc. detected virulence factors, and mobile genetic elements associated to uncover the pathogenecity and the role of horizontal gene transfer (HGT). RESULTS: This study reveals compelling insights into colistin resistance among global high-risk clinical isolates: Klebsiella pneumoniae ST147 (16/20), Pseudomonas aeruginosa ST235 (3/20), and ST357 (1/20). Vitek®2 found 6 colistin-resistant strains (minimum inhibitory concentrations, MIC = 4 µg/mL), while broth microdilution identified 48 (MIC = 32-128 µg/mL), adhering to CLSI guidelines. Despite the absence of mobile colistin resistance (mcr) genes, mechanisms underlying colistin resistance included mgrB deletion, phosphoethanolamine transferases arnT, eptB, ompA, and mutations in pmrB (T246A, R256G) and eptA (V50L, A135P, I138V, C27F) in K. pneumoniae. P. aeruginosa harbored phosphoethanolamine transferases basS/pmrb, basR, arnA, cprR, cprS, alongside pmrB (G362S), and parS (H398R) mutations. Both strains carried diverse clinically relevant antimicrobial resistance genes (ARGs), including plasmid-mediated blaNDM-5 (K. pneumoniae ST147) and chromosomally mediated blaNDM-1 (P. aeruginosa ST357). CONCLUSION: The global surge in MDR, XDR and PDR bacteria necessitates last-resort antibiotics such as colistin. However, escalating resistance, particularly to colistin, presents a critical challenge. Inefficient colistin resistance detection methods, including Vitek2, alongside limited surveillance resources, accentuate the need for improved strategies. Whole-genome sequencing revealed alarming colistin resistance among K. pneumoniae and P. aeruginosa in an Indian hospital. The identification of XDR and PDR strains underscores urgency for enhanced surveillance and infection control. SNP analysis elucidated resistance mechanisms, highlighting the complexity of combatting resistance.

First detection and characterization of mcr-1 colistin resistant E. coli from wild rat in Bangladesh.

Colistin resistance is a global concern warning for a one health approach to combat the challenge. Colistin resistant E. coli and their resistance determinants are widely distributed in the environment, and rats could be a potential source of these isolates and resistant determinants to a diverse environmental setting. This study was aimed to determine the presence of colistin resistant E. coli (CREC) in wild rats, their antimicrobial resistance (AMR) phenotypes, and genotypic analysis of mcr-1 CREC through whole genome sequencing (WGS). A total of 39 rats were examined and CREC was isolated from their fecal pellets onto MacConkey agar containing colistin sulfate (1 µg/ mL). AMR of the CREC was determined by disc diffusion and broth microdilution was employed to determine MIC to colistin sulfate. CREC were screened for mcr genes (mcr-1 to mcr-8) and phylogenetic grouping by PCR. Finally, WGS of one mcr-1 CREC was performed to explore its genetic characteristics especially resistomes and virulence determinants. 43.59% of the rats carried CREC with one (2.56%) of them carrying CREC with mcr-1 gene among the mcr genes examined. Examination of seventeen (17) isolates from the CREC positive rats (n = 17) revealed that majority of them belonging to the pathogenic phylogroup D (52.94%) and B2 (11.76%). 58.82% of the CREC were MDR on disc diffusion test. Shockingly, the mcr-1 CREC showed phenotypic resistance to 16 antimicrobials of 8 different classes and carried the ARGs in its genome. The mcr-1 gene was located on a 60 kb IncI2 plasmid. On the other hand, ARGs related to aminoglycosides, phenicols, sulfonamides, tetracyclines and trimethoprims were located on a 288 kb mega-plasmid separately. The mcr-1 CREC carried 58 virulence genes including genes related to adhesion, colonization, biofilm formation, hemolysis and immune-evasion. The isolate belonged to ST224 and closely related to E. coli from different sources including UPEC clinical isolates from human based on cgMLST analysis. The current research indicates that rats might be a possible source of CREC, and the presence of mcr-1 and other ARGs on plasmid increases the risk of ARGs spreading and endangering human health and other environmental components through this infamous pest.

Epidemiology, resistance genomics and susceptibility of Acinetobacter species: results from the 2020 Spanish nationwide surveillance study.

BackgroundAs increasing antibiotic resistance in Acinetobacter baumannii poses a global healthcare challenge, understanding its evolution is crucial for effective control strategies.AimWe aimed to evaluate the epidemiology, antimicrobial susceptibility and main resistance mechanisms of Acinetobacter spp. in Spain in 2020, and to explore temporal trends of A. baumannii.MethodsWe collected 199 single-patient Acinetobacter spp. clinical isolates in 2020 from 18 Spanish tertiary hospitals. Minimum inhibitory concentrations (MICs) for nine antimicrobials were determined. Short-read sequencing was performed for all isolates, and targeted long-read sequencing for A. baumannii. Resistance mechanisms, phylogenetics and clonality were assessed. Findings on resistance rates and infection types were compared with data from 2000 and 2010.ResultsCefiderocol and colistin exhibited the highest activity against A. baumannii, although colistin susceptibility has significantly declined over 2 decades. A. non-baumannii strains were highly susceptible to most tested antibiotics. Of the A. baumannii isolates, 47.5% (56/118) were multidrug-resistant (MDR). Phylogeny and clonal relationship analysis of A. baumannii revealed five prevalent international clones, notably IC2 (ST2, n = 52; ST745, n = 4) and IC1 (ST1, n = 14), and some episodes of clonal dissemination. Genes bla OXA-23, bla OXA-58 and bla OXA-24/40 were identified in 49 (41.5%), eight (6.8%) and one (0.8%) A. baumannii isolates, respectively. ISAba1 was found upstream of the gene (a bla OXA-51-like) in 10 isolates.ConclusionsThe emergence of OXA-23-producing ST1 and ST2, the predominant MDR lineages, shows a pivotal shift in carbapenem-resistant A. baumannii (CRAB) epidemiology in Spain. Coupled with increased colistin resistance, these changes underscore notable alterations in regional antimicrobial resistance dynamics.

Influence of PhoPQ and PmrAB two component system alternations on colistin resistance from non-mcr colistin resistant clinical E. Coli strains.

BACKGROUND: The current understanding of acquired chromosomal colistin resistance mechanisms in Enterobacterales primarily involves the disruption of the upstream PmrAB and PhoPQ two-component system (TCS) control caused by mutations in the regulatory genes. Interestingly, previous studies have yielded conflicting results regarding the interaction of regulatory genes related to colistin resistance in Escherichia coli, specifically those surrounding PhoPQ and PmrAB TCS. RESULTS: In our study, we focused on two clinical non-mcr colistin-resistant strains of E. coli, TSAREC02 and TSAREC03, to gain a better understanding of their resistance mechanisms. Upon analysis, we discovered that TSAREC02 had a deletion (Δ27-45) in MgrB, as well as substitutions (G206R, Y222H) in PmrB. On the other hand, TSAREC03 exhibited a long deletion (Δ84-224) in PhoP, along with substitutions (M1I, L14P, P178S, T235N) in PmrB. We employed recombinant DNA techniques to explore the interaction between the PhoPQ and PmrAB two-component systems (TCSs) and examine the impact of the mutated phoPQ and pmrB genes on the minimum inhibitory concentrations (MICs) of colistin. We observed significant changes in the expression of the pmrD gene, which encodes a connector protein regulated by the PhoPQ TCS, in the TSAREC02 wild-type (WT)-mgrB replacement mutant and the TSAREC03 WT-phoP replacement mutant, compared to their respective parental strains. However, the expressions of pmrB/pmrA, which reflect PmrAB TCS activity, and the colistin MICs remained unchanged. In contrast, the colistin MICs and pmrB/pmrA expression levels were significantly reduced in the pmrB deletion mutants from both TSAREC02 and TSAREC03, compared to their parental strains. Moreover, we were able to restore colistin resistance and the expressions of pmrB/pmrA by transforming a plasmid containing the parental mutated pmrB back into the TSAREC02 and TSAREC03 mutants, respectively. CONCLUSION: While additional data from clinical E. coli isolates are necessary to validate whether our findings could be broadly applied to the E. coli population, our study illuminates distinct regulatory pathway interactions involving colistin resistance in E. coli compared to other species of Enterobacterales. The added information provided by our study contribute to a deeper understanding of the complex pathway interactions within Enterobacterales.

In vitro activity of antibiotics potentially effective against difficult-to-treat strains of Gram-negative rods: retrospective study.

Bacterial resistance surveillance is one of the main outputs of microbiological laboratories and its results are important part of antimicrobial stewardship (AMS). In this study, the susceptibility of specific bacteria to selected antimicrobial agents was tested. The susceptibility of 90 unique isolates of pathogens of critical priority obtained from clinically valid samples of ICU patients in 2017-2021 was tested. 50% of these fulfilled difficult-to-treat resistance (DTR) criteria and 50% were susceptible to all antibiotics included in the definition. 10 Enterobacterales strains met DTR criteria, and 2 (20%) were resistant to colistin (COL), 2 (20%) to cefiderocol (FCR), 7 (70%) to imipenem/cilastatin/relebactam (I/R), 3 (30%) to ceftazidime/avibactam (CAT) and 5 (50%) to fosfomycin (FOS). For Enterobacterales we also tested aztreonam/avibactam (AZA) for which there are no breakpoints yet. The highest MIC of AZA observed was 1 mg/l, MIC range in the susceptible cohort was 0.032-0.064 mg/l and in the DTR cohort (incl. class B beta-lactamase producers) it was 0.064-1 mg/l. Two (13.3%) isolates of Pseudomonas aeruginosa (15 DTR strains) were resistant to COL, 1 (6.7%) to FCR, 13 (86.7%) to I/R, 5 (33.3%) to CAT, and 5 (33.3%) to ceftolozane/tazobactam. All isolates of Acinetobacter baumannii with DTR were susceptible to COL and FCR, and at the same time resistant to I/R and ampicillin/sulbactam. New antimicrobial agents are not 100% effective against DTR. Therefore, it is necessary to perform susceptibility testing of these antibiotics, use the data for surveillance (including local surveillance) and conform to AMS standards.

An update on antibacterial AlkylGuanidino Ureas: Design of new derivatives, synergism with colistin and data analysis of the whole library.

Antimicrobial resistance (AMR) represents one of the most challenging global Public Health issues, with an alarmingly increasing rate of attributable mortality. This scenario highlights the urgent need for innovative medicinal strategies showing activity on resistant isolates (especially, carbapenem-resistant Gram-negative bacteria, methicillin-resistant S. aureus, and vancomycin-resistant enterococci) yielding new approaches for the treatment of bacterial infections. We previously reported AlkylGuanidino Ureas (AGUs) with broad-spectrum antibacterial activity and a putative membrane-based mechanism of action. Herein, new tetra- and mono-guanidino derivatives were designed and synthesized to expand the structure-activity relationships (SARs) and, thereby, tested on the same panel of Gram-positive and Gram-negative bacteria. The membrane-active mechanism of selected compounds was then investigated through molecular dynamics (MD) on simulated bacterial membranes. In the end, the newly synthesized series, along with the whole library of compounds (more than 70) developed in the last decade, was tested in combination with subinhibitory concentrations of the last resort antibiotic colistin to assess putative synergistic or additive effects. Moreover, all the AGUs were subjected to cheminformatic and machine learning analyses to gain a deeper knowledge of the key features required for bioactivity.

Phenotypic and genotypic characterization of extended spectrum beta-lactamase producing E. coli harboring carbapenem and colistin-resistant genes from poultry farms in Egypt.

Background: eEscherichia coli (E. coli) bacteria that produce extended spectrum beta-lactamase (ESBL) is associated with a high prevalence of human illnesses worldwide. The emergence of resistance to carbapenem and colistin compounds poses further challenges to the treatment options for these illnesses. This study aimed to evaluate the phenotypic and genotypic pattern of resistance to carbapenem and colistin in ESBL-producing E. coli. Escherichia coli isolates collected from the respiratory tract of chickens in El-Sharkia government, Egypt. Methods: A total of 250 lung samples were collected from 50 poultry farms. These samples were then subjected to isolation, identification, and serotyping of E. coli. The presence of antimicrobial resistance was identified by disc diffusion testing. The occurrence of ESBL phenotypes was also assessed using the double disc synergy method. PCR/sequencing techniques were employed to examine the presence of ESBL (ß-lactamase (bla)-TEM, blaSHV, and blaCTX-M), colistin (mcr-1), and carbapenem (blaNDM, blaVIM, and blaKPC) resistance genes. Results: The findings revealed that 140 out of 250 (56%) were identified as E. coli. All E. coli isolates had a high level of multi-antimicrobial resistance (MAR) with an index value greater than 0.2, and 65.7% of them were confirmed to produce ESBL. Out of the 92 ESBL phenotypes, 55 (59.7%), 32 (34.7%), 18 (19.6%), and 37 (40.2%) isolates harbor b laTEM-3, b laSHV-4, b laCTX-M-1, a nd blaCTX-M-14 genes, respectively. The blaNDM-1 gene was identified in all 40 phenotypes that exhibited resistance to carbapenem, accounting for 28.5% of all strains of E. coli and 43.4% of ESBL isolates. The VIM and KPC genes were not detected in any of the samples. Furthermore, there was a significant prevalence of the mobilized colistin resistance (mcr)-1 gene, with 64 (69.5%) of the ESBL isolates exhibiting this gene. Conclusion: The prevalence of ESBL-producing E. coli, particularly those resistant to carbapenem and colistin, poses a significant public health risk in society.

Multidrug-resistant Stenotrophomonas maltophilia in residential aged care facilities: An emerging threat.

Stenotrophomonas maltophilia is a multidrug-resistant (MDR), Gram-negative bacterium intrinsically resistant to beta-lactams, including last-resort carbapenems. As an opportunistic pathogen, it can cause serious healthcare-related infections. This study assesses the prevalence, resistance profiles, and genetic diversity of S. maltophilia isolated from residential aged care facilities (RACFs). RACFs are known for their overuse and often inappropriate use of antibiotics, creating a strong selective environment that favors the development of bacterial resistance. The study was conducted on 73 S. maltophilia isolates recovered from wastewater and facility swab samples obtained from three RACFs and a retirement village. Phenotypic and genotypic assessments of the isolates revealed high carbapenem resistance, exemplifying their intrinsic beta-lactam resistance. Alarmingly, 49.3% (36/73) of the isolates were non-wild type for colistin, with minimum inhibitory concentration values of > 4 mg/L, and 11.0% (8/73) were resistant to trimethoprim-sulfamethoxazole. No resistance mechanisms were detected for either antimicrobial. Genotypic assessment of known lineages revealed isolates clustering with Sm17 and Sm18, lineages not previously reported in Australia, suggesting the potential ongoing spread of MDR S. maltophilia. Lastly, although only a few isolates were biocide tolerant (2.7%, 2/73), their ability to grow in high concentrations (64 mg/L) of triclosan is concerning, as it may be selecting for their survival and continued dissemination.

Combining deep learning and droplet microfluidics for rapid and label-free antimicrobial susceptibility testing of colistin.

Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a last resort therapy with extremely drug resistant Gram-negative bacteria. Here, we developed a rapid, easy and miniaturized colistin susceptibility assay based on microfluidics, which allows for culture and high-throughput analysis of bacterial samples. Specifically, a simple microfluidic platform that can easily be operated was designed to encapsulate bacteria in nanoliter droplets and perform a fast and automated bacterial growth detection in 2 h, using standardized samples. Direct bright-field imaging of compartmentalized samples proved to be a faster and more accurate detection method as compared to fluorescence-based analysis. A deep learning powered approach was implemented for the sensitive detection of the growth of several strains in droplets. The DropDeepL AST method (Droplet and Deep learning-based method for AST) developed here allowed the determination of the colistin susceptibility profiles of 21 fast-growing Enterobacterales (E. coli and K. pneumoniae), including clinical isolates with different resistance mechanisms, showing 100 % categorical agreement with the reference broth microdilution (BMD) method performed simultaneously. Direct AST of bacteria in urine samples on chip also provided accurate results in 2 h, without the need of complex sample preparation procedures. This method can easily be implemented in clinical microbiology laboratories, and has the potential to be adapted to a variety of antibiotics, especially for last-line antibiotics to optimize treatment of patients infected with multi-drug resistant strains.

A challenging case of carbapenem resistant Klebsiella pneumoniae-related pyogenic liver abscess with capsular polysaccharide hyperproduction: a case report.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a major public health problem, necessitating the administration of polymyxin E (colistin) as a last-line antibiotic. Meanwhile, the mortality rate associated with colistin-resistant K. pneumoniae infections is seriously increasing. On the other hand, importance of administration of carbapenems in promoting colistin resistance in K. pneumoniae is unknown. CASE PRESENTATION: We report a case of K. pneumoniae-related pyogenic liver abscess in which susceptible K. pneumoniae transformed into carbapenem- and colistin-resistant K. pneumoniae during treatment with imipenem. The case of pyogenic liver abscess was a 50-year-old man with diabetes and liver transplant who was admitted to Abu Ali Sina Hospital in Shiraz. The K. pneumoniae isolate responsible for community-acquired pyogenic liver abscess was isolated and identified. The K. pneumoniae isolate was sensitive to all tested antibiotics except ampicillin in the antimicrobial susceptibility test and was identified as a non-K1/K2 classical K. pneumoniae (cKp) strain. Multilocus sequence typing (MLST) identified the isolate as sequence type 54 (ST54). Based on the patient's request, he was discharged to continue treatment at another center. After two months, he was readmitted due to fever and progressive constitutional symptoms. During treatment with imipenem, the strain acquired blaOXA-48 and showed resistance to carbapenems and was identified as a multidrug resistant (MDR) strain. The minimum inhibitory concentration (MIC) test for colistin was performed by broth microdilution method and the strain was sensitive to colistin (MIC < 2 µg/mL). Meanwhile, on blood agar, the colonies had a sticky consistency and adhered to the culture medium (sticky mucoviscous colonies). Quantitative real-time PCR and biofilm formation assay revealed that the CRKP strain increased capsule wzi gene expression and produced slime in response to imipenem. Finally, K. pneumoniae-related pyogenic liver abscess with resistance to a wide range of antibiotics, including the last-line antibiotics colistin and tigecycline, led to sepsis and death. CONCLUSIONS: Based on this information, can we have a theoretical hypothesis that imipenem is a promoter of resistance to carbapenems and colistin in K. pneumoniae? This needs more attention.

Enhancing effect of natural adjuvant, panduratin A, on antibacterial activity of colistin against multidrug-resistant Acinetobacter baumannii.

Colistin- and carbapenem-resistant Acinetobacter baumannii is a serious multidrug resistant (MDR) bacterium in clinical settings. Discovery of new antibacterial drugs against MDR is facing multiple challenges in drug development. Combination of known antibiotics with a robust adjuvant might be an alternative effective strategy for MDR treatment. In the study herein, we report an antibiotic adjuvant activity of a natural compound panduratin A from fingerroot (Boesenbergia rotunda) as a potent adjuvant to colistin. The present study investigated the antibiotic adjuvant effect of panduratin A against 10 colistin- and carbapenem-resistant A. baumannii. Antibacterial activities were tested by broth microdilution method. Biofilm assay was used to determine the efficacy of panduratin A in biofilm formation inhibition on two representative strains Aci46 and Aci44. Genomic and transcriptomic analyses of colistin- and carbapenem-resistant A. baumannii strains were used to identify potential resistance and tolerance mechanism in the bacteria. Panduratin A-colistin combination showed an increased effect on antibacterial in the A. baumannii. However, panduratin A did not improve the antibacterial activity of imipenem. In addition, panduratin A improves anti-biofilm activity of colistin against Aci44 and Aci46, the colistin- and carbapenem-resistant A. baumannii. Panduratin A markedly enhances bactericidal and anti-biofilm activity of colistin against colistin- resistant A. baumannii. Based on genome comparisons, single nucleotide polymorphism (SNP) patterns in six genes encoding biofilm and lipid A biosynthesis were shared in Aci44 and Aci46. In Aci44, we identified a partial sequence of pmrB encoding a polymyxin resistant component PmrB, whereas a full length of pmrB was observed in Aci46. RNA-seq analyses of Aci44 revealed that panduratin A-colistin combination induced expression of ribosomal proteins and oxidative stress response proteins, whereas iron transporter and MFS-type transporter systems were suppressed. Panduratin A-colistin combination could promote intracellular reactive oxygen species (ROS) accumulation could lead to the cidal effect on colistin-resistant A. baumannii. Combination of panduratin A and colistin showed a significant increase in colistin efficacy against colistin- resistant A. baumannii in comparison of colistin alone. Genomic comparison between Aci44 and Aci46 showed mutations and SNPs that might affect different phenotypes. Additionally, based on RNA-Seq, panduratin A-colistin combination could lead to ROS production and accumulation. These findings confirmed the potency of panduratin as colistin adjuvant against multidrug resistant A. baumannii.

Characterization of Acinetobacter baumannii-calcoaceticus complex isolates and microbiological outcome for patients treated with sulbactam-durlobactam in a phase 3 trial (ATTACK).

Acinetobacter baumannii-calcoaceticus complex (ABC) causes severe, difficult-to-treat infections that are frequently antibiotic resistant. Sulbactam-durlobactam (SUL-DUR) is a targeted ß-lactam/ß-lactamase inhibitor combination antibiotic designed to treat ABC infections, including those caused by multidrug-resistant strains. In a global, pathogen-specific, randomized, controlled phase 3 trial (ATTACK), the efficacy and safety of SUL-DUR were compared to colistin, both dosed with imipenem-cilastatin as background therapy, in patients with serious infections caused by carbapenem-resistant ABC. Results from ATTACK showed that SUL-DUR met the criteria for non-inferiority to colistin for the primary efficacy endpoint of 28-day all-cause mortality with improved clinical and microbiological outcomes compared to colistin. This report describes the characterization of the baseline ABC isolates from patients enrolled in ATTACK, including an analysis of the correlation of microbiological outcomes with SUL-DUR MIC values and the molecular drivers of SUL-DUR resistance.

A sophisticated virulence repertoire and colistin resistance of Citrobacter freundii ST150 from a patient with sepsis admitted to ICU in a tertiary care hospital in Uganda, East Africa: Insight from genomic and molecular docking analyses.

Sepsis and multidrug resistance comprise a complex of factors attributable to mortality among intensive care unit (ICU) patients globally. Pathogens implicated in sepsis are diverse, and their virulence and drug resistance remain elusive. From a tertiary care hospital ICU in Uganda, we isolated a Citrobacter freundii strain RSM030 from a patient with sepsis and phenotypically tested it against a panel of 16 antibiotics including imipenem levofloxacin, cotrimoxazole and colistin, among others. We sequenced the organism's genome and integrated multilocus sequencing (MLST), PathogenFinder with Virulence Factor analyzer (VFanalyzer) to establish its pathogenic relevance. Thereafter, we combined antiSMASH and PRISM genome mining with molecular docking to predict biosynthetic gene clusters (BGCs), pathways, toxin structures and their potential targets in-silico. Finally, we coupled ResFinder with comprehensive antibiotic resistance database (CARD) to scrutinize the genomic antimicrobial resistance profile of the isolate. From PathogenFinder and MLST, this organism was confirmed to be a human pathogen (p = 0.843), sequence type (ST)150, whose virulence is determined by chromosomal type III secretion system (T3SS) (the injectosome) and plasmid-encoded type IV secretion system (T4SS), the enterobactin biosynthetic gene cluster and biofilm formation through the pgaABCD operon. Pathway and molecular docking analyses revealed that the shikimate pathway can generate a toxin targeting multiple host proteins including spectrin, detector of cytokinesis protein 2 (Dock2) and plasmalemma vesicle-associated protein (PLVAP), potentially distorting the host cell integrity. From phenotypic antibiotic testing, we found indeterminate results for amoxicillin/clavulanate and levofloxacin, with resistance to cotrimoxazole and colistin. Detailed genome analysis revealed chromosomal beta lactam resistance genes, i.e. blaCMY-79, blaCMY-116 and blaTEM-1B, along with multiple mutations of the lipopolysaccharide modifying operon genes PmrA/PmrB, pmrD, mgrA/mgrB and PhoP/PhoQ, conferring colistin resistance. From these findings, we infer that Citrobacter freundii strain RSM030 is implicated in sepsis and resistance to standard antibiotics, including colistin, the last resort.

NhaA: A promising adjuvant target for colistin against resistant Escherichia coli.

The emergence and widespread of multidrug-resistant Gram-negative bacteria have posed a severe threat to human health and environmental safety, escalating into a global medical crisis. Utilization of antibiotic adjuvants is a rapid approach to combat bacterial resistance effectively since the development of new antimicrobial agents is a formidable challenge. NhaA, driven by proton motive force, is a crucial secondary transporter on the cytoplasmic membrane of Escherichia coli. We found that 2-Aminoperimidine (2-AP), which is a specific inhibitor of NhaA, could enhance the activity of colistin against sensitive E. coli and reverse the resistance in mcr-1 positive E. coli. Mechanistic studies indicated that 2-AP induced dysfunction in cytoplasmic membrane through the suppression of NhaA, leading to metabolic inhibition and ultimately enhancing the sensitivity of E. coli to colistin. Moreover, 2-AP restored the efficacy of colistin against resistant E. coli in two animal infection models. Our findings reveal the potential of NhaA as a novel target for colistin adjuvants, providing new possibilities for the clinical application of colistin.

The NLRP3 Inflammasome Is a Major Cause of Acute Renal Failure Induced by Polypeptide Antibiotics.

Drug-induced acute renal failure (ARF) is a public health concern that hinders optimal drug therapy. However, pathological mechanisms of drug-induced ARF remain to be elucidated. Here, we show that a pathological process of drug-induced ARF is mediated by proinflammatory cross-talk between kidney tubular cells and macrophages. Both polymyxin B and colistin, polypeptide antibiotics, frequently cause ARF, stimulated the ERK and NF-κB pathways in kidney tubular cells, and thereby upregulated M-CSF and MCP-1, leading to infiltration of macrophages into the kidneys. Thereafter, the kidney-infiltrated macrophages were exposed to polypeptide antibiotics, which initiated activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome. Interestingly, blockade of the NLRP3 activation clearly ameliorated the pathology of ARF induced by polypeptide antibiotics, suggesting that a combination of the distinct cellular responses to polypeptide antibiotics in kidney tubular cells and macrophages plays a key role in the pathogenesis of colistin-induced ARF. Thus, our results provide a concrete example of how drugs initiate ARF, which may give insight into the underlying pathological process of drug-induced ARF.