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.

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.

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.

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.

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.

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.

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.

Post Trauma Acinetobacter Baumanii Meningitis Treatment Approach.

A. baumannii, which is said to be associated with nosocomial infections is considered a significant mortality risk if not adequately addressed. A. baumannii infections typically occur in the aftermath of surgery or trauma. Our patient developed complicated A. baumannii meningitis with lateral ventriculitis and a lumbar abscess post surgery after suffering from a fall. The patient was treated with a 21-day regimen of intrathecally administered colistin and polymyxin B. Following this therapeutic period, the patient's condition improved, ultimately leading to successful recovery and subsequent discharge. This case report serves to highlight the ability of intrathecal administration of antibiotics, that normally have limited potential of crossing the blood-brain-barrier, to lead to improved survival outcomes in multi-drug resistant nosocomial meningitis.

An Intra-Hospital Spread of Colistin-Resistant K. pneumoniae Isolates-Epidemiological, Clinical, and Genetic Analysis.

Background and Objective: Klebsiella pneumoniae appears to be a significant problem due to its ability to accumulate antibiotic-resistance genes. After 2013, alarming colistin resistance rates among carbapenem-resistant K. pneumoniae have been reported in the Balkans. The study aims to perform an epidemiological, clinical, and genetic analysis of a local outbreak of COLr CR-Kp. Material and Methods: All carbapenem-resistant and colistin-resistant K. pneumoniae isolates observed among patients in the ICU unit of Military Medical Academy, Sofia, from 1 January to 31 October 2023, were included. The results were analyzed according to the EUCAST criteria. All isolates were screened for blaVIM, blaIMP, blaKPC, blaNDM, and blaOXA-48. Genetic similarity was determined using the Dice coefficient as a similarity measure and the unweighted pair group method with arithmetic mean (UPGMA). mgrB genes and plasmid-mediated colistin resistance determinants (mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5) were investigated. Results: There was a total of 379 multidrug-resistant K. pneumoniae isolates, 88% of which were carbapenem-resistant. Of these, there were nine (2.7%) colistin-resistant isolates in six patients. A time and space cluster for five patients was found. Epidemiology typing showed that two isolates belonged to clone A (pts. 1, 5) and the rest to clone B (pts. 2-4) with 69% similarity. Clone A isolates were coproducers of blaNDM-like and blaOXA-48-like and had mgrB-mediated colistin resistance (40%). Clone B isolates had only blaOXA-48-like and intact mgrB genes. All isolates were negative for mcr-1, -2, -3, -4, and -5 genes. Conclusions: The study describes a within-hospital spread of two clones of COLr CR-Kp with a 60% mortality rate. Clone A isolates were coproducers of NDM-like and OXA-48-like enzymes and had mgrB-mediated colistin resistance. Clone B isolates had only OXA-48-like enzymes and intact mgrB genes. No plasmid-mediated resistance was found. The extremely high mortality rate and limited treatment options warrant strict measures to prevent outbreaks.

Comparative effectiveness and mortality of colistin monotherapy versus colistin-fosfomycin combination therapy for the treatment of carbapenem-resistant Enterobacteriaceae (CRE) infections: A propensity score analysis.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) infections pose a significant threat to global health due to limited treatment options and high mortality rates. Colistin-based regimens have emerged as a primary treatment approach, but the effectiveness and mortality outcomes of colistin monotherapy versus colistin-fosfomycin combination therapy remain uncertain. This study aims to compare the effectiveness and mortality of colistin monotherapy and colistin-fosfomycin combination therapy for CRE infections. Notably, our study is the first to undertake a comprehensive examination of the effectiveness and mortality outcomes between colistin monotherapy and colistin-fosfomycin combination therapy in the context of CRE infections. METHODS: A retrospective cohort study was conducted using data from patients diagnosed with carbapenem-resistant Enterobacteriaceae (CRE) infections at Nakornping Hospital during 2015 to 2022. Inverse probability weighting (IPW) was employed to create balanced cohorts of patients receiving either colistin monotherapy or colistin-fosfomycin combination therapy. The primary outcome measure was treatment effectiveness, assessed by 30-day mortality. Secondary outcome measures included clinical response, mortality at the end of treatment, and microbiologic response. Univariate and multivariate logistic regression analysis were employed after applying propensity score weighting using inverse probability of weighting (IPW). RESULTS: A total of 220 patients were included in the analysis, with 67 receiving colistin monotherapy and 153 receiving colistin-fosfomycin combination therapy. Propensity score weighting using IPW balanced the baseline characteristics between the two groups. The effectiveness of treatment, as measured by 30-day mortality, was not significantly different between the colistin monotherapy group and the colistin-fosfomycin combination therapy group (adjusted odds ratio [aOR] = 1.51, 95% confidence interval [CI]: 0.60-3.78, p = 0.383). Similarly, no significant difference was observed in the mortality at the end of treatment between the two groups (aOR = 1.26, 95% CI: 0.55-2.90, p = 0.576). The clinical response (aOR = 1.48, 95% CI: 0.61-3.59, p = 0.383) and microbiologic response (aOR = 0.66, 95% CI: 0.18-2.38, p = 0.527) were similar between the colistin monotherapy and colistin-fosfomycin combination therapy groups. CONCLUSION: The propensity score analysis among 220 matched patients showed comparable treatment effectiveness and mortality between colistin monotherapy and colistin-fosfomycin combination therapy for CRE infections. These results suggest that colistin monotherapy may be as effective as combination therapy. More prospective randomized controlled trials are needed to confirm these findings and establish optimal CRE treatment strategies.

Ineffectiveness of colistin monotherapy in treating carbapenem-resistant Acinetobacter baumannii Pneumonia: A retrospective single-center cohort study.

BACKGROUND: Acinetobacter baumannii, a common carbapenem-resistant gram-negative bacillus, usually causes nosocomial infections. Colistin has been used for carbapenem-resistant A. baumannii (CRAB) infections; however, only a few studies have evaluated colistin as a treatment option compared to appropriate controls. We investigated the effectiveness of colistin monotherapy in treating CRAB pneumonia compared to those treated without an active drug. METHODS: Adult patients (≥ 18 years) with CRAB isolated from respiratory specimens were screened from September 2017 to August 2022. Only patients with pneumonia treated with colistin monotherapy (colistin group) were included and compared to those without any active antibiotics (no active antibiotics [NAA] group). The primary and secondary outcomes were 30-day all-cause mortality and acute kidney injury within 30 days. The inverse probability of the treatment-weighted Cox proportional hazard model was used to compare mortality between groups. RESULTS: Among the 826 adult patients with CRAB in their respiratory specimens, 45 and 123 patients were included in the colistin and NAA groups, respectively. Most of the CRAB pneumonia (91.1%) cases were hospital-acquired pneumonia. The 30-day all-cause mortality rates in the colistin and NAA groups were 58.3% and 56.1%, respectively, and no difference was observed after adjustments (adjusted hazard ratio, 0.74; 95% CI, 0.47-1.17). The incidence of acute kidney injury was higher in the colistin group (65.3%) compared to the NAA group (39.0%) (P = 0.143). CONCLUSIONS: Colistin monotherapy did not significantly improve treatment outcomes for CRAB pneumonia. The development and evaluation of new antimicrobials for CRAB pneumonia should be advocated in clinical practice.

Clinical outcomes of colistin methanesulfonate sodium in correlation with pharmacokinetic parameters in critically ill patients with multi-drug resistant bacteria-mediated infection: A systematic review and meta-analysis.

BACKGROUND: Colistin is a viable option for multidrug resistant gram-negative bacteria emerged from inappropriate antibiotic use. Nonetheless, suboptimal colistin concentrations and nephrotoxicity risks hinder its clinical use. Thus, the aim of this study is to investigate clinical outcomes in correlation with pharmacokinetic differences and infection types in critically ill patients on intravenous colistin methanesulfornate sodium (CMS). METHODS: A systematic literature search of Embase, Google Scholars, and PubMed was performed to identify clinical trials evaluating pharmacokinetic parameters along with clinical outcomes of CMS treatment from inception to July 2023. The pooled analyses of clinical impact of CMS on nephrotoxicity, mortality, clinical cure, and colistin concentration at steady state (Css,avg) were performed. This study was registered in the PROSPERO (CRD 42023456120). RESULTS: Total of 695 critically ill patients from 17 studies were included. The mortality was substantially lower in clinically cured patients (OR 0.05; 95% CI 0.02 - 0.14), whereas the mortality rate was statistically insignificant between nephrotoxic and non-nephrotoxic patients. Inter-patient variability of pharmacokinetic parameters of CMS and colistin was observed in critically ill patients. The standard mean differences of Css,avg were statistically insignificant between clinically cure and clinically failure groups (standard mean difference (SMD) -0.25; 95% CI -0.69 - 0.19) and between nephrotoxic and non-nephrotoxic groups (SMD 0.67; 95% CI -0.27-1.61). The clinical cure rate is substantially lower in pneumonia patients (OR 0.09; 95% CI 0.01 - 0.56), and pharmacokinetic parameters pertaining to microbiological cure were different among strains. CONCLUSION: The mortality rate was substantially lower in clinically cured patients with CMS. However, no significant differences in Css,avg of colistin were examined to determine the impact of pharmacokinetic differences on clinical outcomes including mortality rate and nephrotoxicity risk. Nevertheless, the clinical cure rate is substantially lower in patients with respiratory infection than patients with urinary tract infection.

A molecular overview of the polymyxin-LPS interaction in the context of its mode of action and resistance development.

With the rising incidences of antimicrobial resistance (AMR) and the diminishing options of novel antimicrobial agents, it is paramount to decipher the molecular mechanisms of action and the emergence of resistance to the existing drugs. Polymyxin, a cationic antimicrobial lipopeptide, is used to treat infections by Gram-negative bacterial pathogens as a last option. Though polymyxins were identified almost seventy years back, their use has been restricted owing to toxicity issues in humans. However, their clinical use has been increasing in recent times resulting in the rise of polymyxin resistance. Moreover, the detection of "mobile colistin resistance (mcr)" genes in the environment and their spread across the globe have complicated the scenario. The mechanism of polymyxin action and the development of resistance is not thoroughly understood. Specifically, the polymyxin-bacterial lipopolysaccharide (LPS) interaction is a challenging area of investigation. The use of advanced biophysical techniques and improvement in molecular dynamics simulation approaches have furthered our understanding of this interaction, which will help develop polymyxin analogs with better bactericidal effects and lesser toxicity in the future. In this review, we have delved deeper into the mechanisms of polymyxin-LPS interactions, highlighting several models proposed, and the mechanisms of polymyxin resistance development in some of the most critical Gram-negative pathogens.

Antibiotic heteroresistance in Klebsiella pneumoniae: Definition, detection methods, mechanisms, and combination therapy.

Klebsiella pneumoniae is a common opportunistic pathogen that presents significant challenges in the treatment of infections due to its resistance to multiple antibiotics. In recent years, K. pneumoniae has been reported for the development of heteroresistance, a phenomenon where subpopulations of the susceptible bacteria exhibit resistance. This heteroresistance has been associated with increased morbidity and mortality rates. Complicating matters further, its definition and detection pose challenges, often leading to its oversight or misdiagnosis. Various mechanisms contribute to the development of heteroresistance in K. pneumoniae, and these mechanisms differ among different antibiotics. Even for the same antibiotic, multiple mechanisms may be involved. However, our current understanding of these mechanisms remains incomplete, and further research is needed to gain a more comprehensive understanding of heteroresistance. While the clinical recommendation is to use combination antibiotic therapy to mitigate heteroresistance, this approach also comes with several drawbacks and potential adverse effects. In this review, we discuss the definition, detection methods, molecular mechanisms, and treatment of heterogenic resistance, aiming to pave the way for more effective treatment and management in the future. However, addressing the problem of heteroresistance in K. pneumoniae represents a long and complex journey that necessitates comprehensive research efforts.

Upregulation of outer membrane porin gene ompC contributed to enhancement of azithromycin susceptibility in multidrug-resistant Escherichia coli.

The outer membrane (OM) in gram-negative bacteria contains proteins that regulate the passive or active uptake of small molecules for growth and cell function, as well as mediate the emergence of antibiotic resistance. This study aims to explore the potential mechanisms for restoring bacteria to azithromycin susceptibility based on transcriptome analysis of bacterial membrane-related genes. Transcriptome sequencing was performed by treating multidrug-resistant Escherichia coli T28R with azithromycin or in combination with colistin and confirmed by reverse transcription-quantitative PCR (RT-qPCR). Azithromycin enzyme-linked immunosorbent assay (ELISA) test, ompC gene overexpression, and molecular docking were utilized to conduct the confirmatory research of the potential mechanisms. We found that colistin combined with azithromycin led to 48 differentially expressed genes, compared to azithromycin alone, such as downregulation of tolA, eptB, lpxP, and opgE and upregulation of ompC gene. Interestingly, the addition of colistin to azithromycin differentially downregulated the mph(A) gene mediating azithromycin resistance, facilitating the intracellular accumulation of azithromycin. Also, overexpression of the ompC elevated azithromycin susceptibility, and colistin contributed to further suppression of the Mph(A) activity in the presence of azithromycin. These findings suggested that colistin firstly enhanced the permeability of bacterial OM, causing intracellular drug accumulation, and then had a repressive effect on the Mph(A) activity along with azithromycin. Our study provides a novel perspective that the improvement of azithromycin susceptibility is related not only to the downregulation of the mph(A) gene and conformational remodeling of the Mph(A) protein but also the upregulation of the membrane porin gene ompC.IMPORTANCEUsually, active efflux via efflux pumps is an important mechanism of antimicrobial resistance, such as the AcrAB-TolC complex and MdtEF. Also, bacterial porins exhibited a substantial fraction of the total number of outer membrane proteins in Enterobacteriaceae, which are involved in mediating the development of the resistance. We found that the upregulation or overexpression of the ompC gene contributed to the enhancement of resistant bacteria to azithromycin susceptibility, probably due to the augment of drug uptakes caused and the opportunity of Mph(A) function suppressed by azithromycin with colistin. Under the combination of colistin and azithromycin treatment, OmpC exhibited an increased selectivity for cationic molecules and played a key role in the restoral of the antibiotic susceptibility. Investigations on the regulation of porin expression that mediated drug resistance would be important in clinical isolates treated with antibiotics.

In vitro activity of human defensins HNP-1 and hBD-3 against multidrug-resistant ESKAPE Gram-negatives of clinical origin and selected peptidoglycan recycling-defective mutants.

The use of immune compounds as antimicrobial adjuvants is a classic idea recovering timeliness in the current antibiotic resistance scenario. However, the activity of certain antimicrobial peptides against ESKAPE Gram-negatives has not been sufficiently investigated. The objective of this study was to determine the activities of human defensins HNP-1 and hBD-3 alone or combined with permeabilizing/peptidoglycan-targeting agents against clinical ESKAPE Gram-negatives [Acinetobacter baumannii (AB), Enterobacter cloacae (EC), Klebsiella pneumoniae (KP), and acute/chronic Pseudomonas aeruginosa (PA)]. Lethal concentrations (LCs) of HNP-1 and hBD-3 were determined in four collections of multidrug resistant EC, AB, KP, and PA clinical strains (10-36 isolates depending on the collection). These defensins act through membrane permeabilization plus peptidoglycan building blockade, enabling that alterations in peptidoglycan recycling may increase their activity, which is why different recycling-defective mutants were also included. Combinations with physiological lysozyme and subinhibitory colistin for bactericidal activities determination, and with meropenem for minimum inhibitory concentrations (MICs), were also assessed. HNP-1 showed undetectable activity (LC > 32 mg/L for all strains). hBD-3 showed appreciable activities: LC ranges 2-16, 8-8, 8->32, and 8->32 mg/L for AB, EC, KP, and PA, being PA strains from cystic fibrosis significantly more resistant than acute origin ones. None of the peptidoglycan recycling-defective mutants showed greater susceptibility to HNP-1/hBD-3. Combination with colistin or lysozyme did not change their bactericidal power, and virtually neither did meropenem + hBD-3 compared to meropenem MICs. This is the first study comparatively analyzing the HNP-1/hBD-3 activities against the ESKAPE Gram-negatives, and demonstrates interesting bactericidal capacities of hBD-3 mostly against AB and EC. IMPORTANCE: In the current scenario of critical need for new antimicrobials against multidrug-resistant bacteria, all options must be considered, including classic ideas such as the use of purified immune compounds. However, information regarding the activity of certain human defensins against ESKAPE Gram-negatives was incomplete. This is the first study comparatively assessing the in vitro activity of two membrane-permeabilizing/peptidoglycan construction-blocking defensins (HNP-1 and hBD-3) against relevant clinical collections of ESKAPE Gram-negatives, alone or in combination with permeabilizers, additional peptidoglycan-targeting attacks, or the blockade of its recycling. Our data suggest that hBD-3 has a notable bactericidal activity against multidrug-resistant Acinetobacter baumannii and Enterobacter cloacae strains that should be considered as potential adjuvant option. Our results suggest for the first time an increased resistance of Pseudomonas aeruginosa strains from chronic infection compared to acute origin ones, and provide new clues about the predominant mode of action of hBD-3 against Gram-negatives (permeabilization rather than peptidoglycan-targeting).

Characterisation of colistin resistance in Gram-negative microbiota of pregnant women and neonates in Nigeria.

A mobile colistin resistance gene mcr was first reported in 2016 in China and has since been found with increasing prevalence across South-East Asia. Here we survey the presence of mcr genes in 4907 rectal swabs from mothers and neonates from three hospital sites across Nigeria; a country with limited availability or history of colistin use clinically. Forty mother and seven neonatal swabs carried mcr genes in a range of bacterial species: 46 Enterobacter spp. and single isolates of; Shigella, E. coli and Klebsiella quasipneumoniae. Ninety percent of the genes were mcr-10 (n = 45) we also found mcr-1 (n = 3) and mcr-9 (n = 1). While the prevalence during this collection (2015-2016) was low, the widespread diversity of mcr-gene type and range of bacterial species in this sentinel population sampling is concerning. It suggests that agricultural colistin use was likely encouraging sustainment of mcr-positive isolates in the community and implementation of medical colistin use will rapidly select and expand resistant isolates.

Molecular epidemiology and mechanisms of carbapenem and colistin resistance in Klebsiella and other Enterobacterales from treated wastewater in Croatia.

Among the most problematic bacteria with clinical relevance are the carbapenem-resistant Enterobacterales (CRE), as there are very limited options for their treatment. Treated wastewater can be a route for the release of these bacteria into the environment and the population. The aim of this study was to isolate CRE from treated wastewater from the Zagreb wastewater treatment plant and to determine their phenotypic and genomic characteristics. A total of 200 suspected CRE were isolated, 148 of which were confirmed as Enterobacterales by MALDI-TOF MS. The predominant species was Klebsiella spp. (n = 47), followed by Citrobacter spp. (n = 40) and Enterobacter cloacae complex (cplx.) (n = 35). All 148 isolates were carbapenemase producers with a multidrug-resistant phenotype. Using multi-locus sequence typing and whole-genome sequencing (WGS), 18 different sequence types were identified among these isolates, 14 of which were associated with human-associated clones. The virulence gene analysis of the sequenced Klebsiella isolates (n = 7) revealed their potential pathogenicity. PCR and WGS showed that the most frequent carbapenemase genes in K. pneumoniae were blaOXA-48 and blaNDM-1, which frequently occurred together, while blaKPC-2 together with blaNDM-1 was mainly detected in K. oxytoca, E. cloacae cplx. and Citrobacter spp. Colistin resistance was observed in 40% of Klebsiella and 57% of Enterobacter isolates. Underlying mechanisms identified by WGS include known and potentially novel intrinsic mechanisms (point mutations in the pmrA/B, phoP/Q, mgrB and crrB genes) and acquired mechanisms (mcr-4.3 gene). The mcr-4.3 gene was identified for the first time in K. pneumoniae and is probably located on the conjugative IncHI1B plasmid. In addition, WGS analysis of 13 isolates revealed various virulence genes and resistance genes to other clinically relevant antibiotics as well as different plasmids possibly associated with carbapenemase genes. Our study demonstrates the important role that treated municipal wastewater plays in harboring and spreading enterobacterial pathogens that are resistant to last-resort antibiotics.

Antimicrobial resistance and public and animal health risks associated with pathogenic Escherichia coli isolated from calves.

We aimed to determine the antimicrobial susceptibility profile of pathogenic Escherichia coli strains isolated from fecal samples of calves and buffalo calves (2008-2013), in Minas Gerais, Brazil, as well as the frequency of O157 gene and strains carrying extended-spectrum beta-lactamases (ESBL) and mobile colistin resistance (mcr) genes. E. coli strains (n=518) were tested for susceptibility against ten antimicrobials. Tetracycline was the antimicrobial with the highest resistance rate (382/518), followed by ampicillin (321/518), sulfamethoxazole/trimethoprim (312/518), chloramphenicol (192/518), gentamicin (126/518), ciprofloxacin (148/518), cefazolin (89/518), colistin (54/518) and cefoxitin (34/518). Multidrug resistance (MDR) was observed in 381/518 isolates. No strain harbored mcr or O157 genes, whereas 19/99 were ESBL positive. The most prevalent pathotype and phylogroup were STEC and B1, respectively. Age, EHEC pathotype and resistance to aminoglycoside and cephem were significantly associated with MDR in the multivariate model. Overall, E. coli strains showed high rates of resistance to penicillin, tetracyclines and folate inhibitors, in addition to an alarming rate of MDR and ESBL-producing strains.

Inhibition of inosine metabolism of the gut microbiota decreases testosterone secretion in the testis.

Growing evidence indicates that gut microbiota is involved in the regulation of the host's sex hormone levels, such as through interfering with the sex hormone metabolism in the intestine. However, if gut microbiota or its metabolites directly influence the sex hormone biosynthesis in the gonad remains largely unknown. Our previous study showed that colistin, as a narrow-spectrum antibiotic, can significantly downregulate the serum testosterone levels and thus enhance the antitumor efficiency of anti-PD-L1 in male mice; however, the underlying mechanism for the regulation of the host's testosterone levels remains uninvestigated. In the present study, we analyzed the impact of colistin on the immune microenvironment of the testis as well as the composition and metabolism of gut microbiota in male mice. Our results showed that colistin has an impact on the immune microenvironment of the testis and can downregulate serum testosterone levels in male mice through inhibition of Akkermansia, leading to destroyed inosine metabolism. Supplement with inosine can restore testosterone secretion probably by prompting the recovery of the intestinal mucus barrier and the serum lipopolysaccharides levels. All these findings reveal a new pathway for the regulation of the host's sex hormone levels by gut microbiota.IMPORTANCEThis study demonstrates that exposure to even narrow-spectrum antibiotics may affect the host's testosterone levels by altering the gut microbiota and its metabolites. Our findings provide evidence that some specific gut bacteria have an impact on the sex hormone biosynthesis in the testis.