Multidrug-resistant Gram-negative Bacterial Infections Research Articles

Mechanisms of Gelofusine Protection against Polymyxin B-associated Renal Injury.

Polymyxins are a last-resort treatment option for multidrug-resistant Gram-negative bacterial infections, but they are associated with nephrotoxicity. Gelofusine was previously shown to reduce polymyxin-associated kidney injury in an animal model. However, the mechanism(s) of renal protection has not been fully elucidated. Here, we report the use of a cell culture model to provide insights into the mechanisms of renal protection. Murine epithelial proximal tubular cells were exposed to polymyxin B. Cell viability, polymyxin B uptake, mitochondrial superoxide production, nuclear morphology, and apoptosis activation were evaluated with or without concomitant gelofusine. A megalin-knockout cell line was used as an uptake inhibition control. Methionine was included in selected experiments as an antioxidant control. A polymyxin B concentration-dependent reduction in cell viability was observed. Increased viability was observed in megalin-knockout cells following comparable polymyxin B exposures. Compared to polymyxin B exposure alone, concomitant gelofusine and methionine significantly increased cell viability, reduced mitochondrial superoxide production, and improved nuclear morphology. Gelofusine, but not methionine, significantly reduced polymyxin B uptake and ratio of Bax/Bcl-2 protein (a biomarker of intrinsic apoptosis). Gelofusine and methionine were more effective at reducing renal cell injury in combination than either agent alone. The mechanisms of renal protection by gelofusine involve decreasing cellular drug uptake, reducing subsequent oxidative stress and apoptosis activation. These findings would be valuable for translational research into clinical strategies to attenuate drug-associated acute kidney injury.

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

BackgroundColistin 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).ResultsThis 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).ConclusionThe 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.

Membrane Permeability Dominates over Electrostatic Interactions in Dictating Drug Transport in Osmotically Shocked Escherichia coli.

To combat surging multidrug-resistant Gram-negative bacterial infections, better strategies to improve the efficacy of existing drugs are critical. Because the dual membrane cell envelope is the first line of defense for these bacteria, it is crucial to understand the permeation properties of the drugs through it. Our recent study shows that isosmotic conditions prevent drug permeation inside Gram-negative bacteria, Escherichia coli, while hypoosmotic stress enhances the process. Here, we unravel the reason behind such differential drug penetration. Specifically, we dissect the roles of electrostatic screening and low membrane permeability in the penetration failure of drugs under osmotically balanced conditions. We compare the transport of a quaternary ammonium compound malachite green in the presence of an electrolyte (NaCl) and a wide variety of commonly used organic osmolytes, e.g., sucrose, proline, glycerol, sorbitol, and urea. These osmolytes of different membrane permeability (i.e., nonpermeable sucrose and NaCl, freely permeable urea and glycerol, and partially permeable proline and sorbitol) clarify the role of osmotic stress in cell envelope permeability. The results showcase that under balanced osmotic conditions, drug molecules fail to penetrate inside E. coli cells because of low membrane permeabilities and not because of electrostatic screening imposed by the osmolytes. Contribution of the electrostatic interactions, however, cannot be completely overruled as at osmotically imbalanced conditions, drug transport across the bacterial subcellular compartments is found to be dependent on the osmolytes used.

A pharmacovigilance study of adverse events associated with polymyxins based on the U.S. Food and Drug Administration Adverse Event Reporting System (FAERS) database

ABSTRACT Background Polymyxins have been regarded as last-line treatment for multidrug-resistant gram-negative bacterial infections. Nonetheless, concerns regarding toxicity persist. This study aimed to explore and compare potential adverse events (AEs) between colistin and polymyxin B (PMB). Methods Outpatient antibiotic use associated with acute upper respiratory infections in China: a nationwide cross-sectional study Polymyxins-related AEs were retrieved from the U.S. Food and Drug Administration Adverse Event Reporting System between 2004 and 2022. Potential signals were estimated by the reporting odds ratio (ROR), and subgroup analyses were preformed to adjust for potential factors in AEs with significant disproportionality. Results Analysis of 3,915 records involving 718 patients revealed a higher disproportionality of renal and urinary disorders (ROR 1.62, 95% CI 1.01–2.59) and acute kidney injury (ROR 1.75, 95% CI 1.07–2.87) with colistin treatment. Conversely, colistin exhibited a lower risk for neurotoxicity (ROR 0.47, 95% CI 0.30–0.73). Seven cases of skin hyperpigmentation were reported with PMB, whereas none were reported with colistin. Over 80% of cases involving polymyxin-related AEs occurred during the first two weeks of therapies, with a median onset time of 4.5 days. Conclusions Outpatient antibiotic use associated with acute upper respiratory infections in China: a nationwide cross-sectional study Patients received colistin displayed a higher potential risk of nephrotoxicity but a lower risk of neurotoxicity. Clinicians should be vigilant in monitoring the AEs of hyperpigmentation disorders induced by PMB.

Decoding the Structure-Function Relationship of the Muramidase Domain in E. coli O157.H7 Bacteriophage Endolysin: A Potential Building Block for Chimeric Enzybiotics.

Bacteriophage endolysins are potential alternatives to conventional antibiotics for treating multidrug-resistant gram-negative bacterial infections. However, their structure-function relationships are poorly understood, hindering their optimization and application. In this study, we focused on the individual functionality of the C-terminal muramidase domain of Gp127, a modular endolysin from E. coli O157:H7 bacteriophage PhaxI. This domain is responsible for the enzymatic activity, whereas the N-terminal domain binds to the bacterial cell wall. Through protein modeling, docking experiments, and molecular dynamics simulations, we investigated the activity, stability, and interactions of the isolated C-terminal domain with its ligand. We also assessed its expression, solubility, toxicity, and lytic activity using the experimental data. Our results revealed that the C-terminal domain exhibits high activity and toxicity when tested individually, and its expression is regulated in different hosts to prevent self-destruction. Furthermore, we validated the muralytic activity of the purified refolded protein by zymography and standardized assays. These findings challenge the need for the N-terminal binding domain to arrange the active site and adjust the gap between crucial residues for peptidoglycan cleavage. Our study shed light on the three-dimensional structure and functionality of muramidase endolysins, thereby enriching the existing knowledge pool and laying a foundation for accurate in silico modeling and the informed design of next-generation enzybiotic treatments.

A systematic evaluation of population pharmacokinetic models for polymyxin B in patients with liver and/or kidney dysfunction.

Polymyxin B (PMB) is considered a last-line treatment for multidrug-resistant (MDR) gram-negative bacterial infections. Model-informed precision dosing with population pharmacokinetics (PopPK) models could help to individualize PMB dosing regimens and improve therapy. However, the external prediction ability of the established PopPK models has not been fully elaborated. This study aimed to systemically evaluate eleven PMB PopPK models from ten published literature based on a new independent population, which was divided into four different populations, patients with liver dysfunction, kidney dysfunction, liver and kidney dysfunction, and normal liver and kidney function. The whole data set consisted of 146 patients with 391 PMB concentrations. The prediction- and simulation-based diagnostics and Bayesian forecasting were conducted to evaluate model predictability. In the overall evaluation process, none of the models exhibited satisfactory predictive ability in both prediction- and simulation-based diagnostic simultaneously. However, the evaluation of the models in the subgroup of patients with normal liver and kidney function revealed improved predictive performance compared to those with liver and/or kidney dysfunction. Bayesian forecasting demonstrated enhanced predictability with the incorporation of two to three prior observations. The external evaluation highlighted a lack of consistency between the prediction results of published models and the external validation dataset. Nonetheless, Bayesian forecasting holds promise in improving the predictive performance of the models, and feedback from therapeutic drug monitoring is crucial in optimizing individual dosing regimens.

Efficacy of Random Forest Models in Predicting Multidrug-Resistant Gram-Negative Bacterial Nosocomial Infections Compared to Traditional Logistic Regression Models.

This study evaluates whether random forest (RF) models are as effective as traditional Logistic Regression (LR) models in predicting multidrug-resistant Gram-negative bacterial nosocomial infections. Data were collected from 541 patients with hospital-acquired Gram-negative bacterial infections at two tertiary-level hospitals in Urumqi, Xinjiang, China, from August 2022 to November 2023. Relevant literature informed the selection of significant predictors based on patients' pre-infection clinical information and medication history. The data were split into a training set of 379 cases and a validation set of 162 cases, adhering to a 7:3 ratio. Both RF and LR models were developed using the training set and subsequently evaluated on the validation set. The LR model achieved an accuracy of 84.57%, sensitivity of 82.89%, specificity of 80.10%, positive predictive value of 84%, negative predictive value of 85.06%, and a Yoden index of 0.69. In contrast, the RF model demonstrated superior performance with an accuracy of 89.51%, sensitivity of 90.79%, specificity of 88.37%, positive predictive value of 87.34%, negative predictive value of 91.57%, and a Yoden index of 0.79. Receiver operating characteristic curve analysis revealed an area under the curve of 0.91 for the LR model and 0.94 for the RF model. These findings indicate that the RF model surpasses the LR model in specificity, sensitivity, and accuracy in predicting hospital-acquired multidrug-resistant Gram-negative infections, showcasing its greater potential for clinical application.

Signaling through the Salmonella PbgA-LapB regulatory complex activates LpxC proteolysis and limits lipopolysaccharide biogenesis during stationary-phase growth.

Salmonella enterica serovar Typhimurium (S. Typhimurium) controls lipopolysaccharide (LPS) biosynthesis by regulating proteolysis of LpxC, the rate-limiting enzyme and target of preclinical antibiotics. PbgA/YejM/LapC regulates LpxC levels and controls outer membrane (OM) LPS composition at the log-to-stationary phase transition. Suppressor substitutions in LPS assembly protein B (LapB/YciM) rescue the LPS and OM integrity defects of pbgA-mutant S. Typhimurium. We hypothesized that PbgA regulates LpxC proteolysis by controlling LapB's ability to bind LpxC as a function of the growth phase. According to existing models, when nutrients are abundant, PbgA binds and restricts LapB from interacting with LpxC and FtsH, which limits LpxC proteolysis. However, when nutrients are limited, there is debate whether LapB dissociates from PbgA to bind LpxC and FtsH to enhance degradation. We sought to examine these models and investigate how the structure of LapB enables salmonellae to control LpxC proteolysis and LPS biosynthesis. Salmonellae increase LapB levels during the stationary phase to promote LpxC degradation, which limits lipid A-core production and increases their survival. The deletion of lapB, resulting in unregulated lipid A-core production and LpxC overabundance, leads to bacterial growth retardation. Tetratricopeptide repeats near the cytosol-inner membrane interface are sufficient for LapB to bind LpxC, and remarkably, LapB and PbgA interact in both growth phases, yet LpxC only associates with LapB in the stationary phase. Our findings support that PbgA-LapB exists as a constitutive complex in S. Typhimurium, which differentially binds LpxC to control LpxC proteolysis and limit lipid A-core biosynthesis in response to changes in the environment.IMPORTANCEAntimicrobial resistance has been a costly setback for human health and agriculture. Continued pursuit of new antibiotics and targets is imperative, and an improved understanding of existing ones is necessary. LpxC is an essential target of preclinical trial antibiotics that can eliminate multidrug-resistant Gram-negative bacterial infections. LapB is a natural LpxC inhibitor that targets LpxC for degradation and limits lipopolysaccharide production in Enterobacteriaceae. Contrary to some studies, findings herein support that LapB remains in complex instead of dissociating from its presumed negative regulator, PbgA/YejM/LapC, under conditions where LpxC proteolysis is enhanced. Advanced comprehension of this critical protein-lipid signaling network will lead to future development and refinement of small molecules that can specifically interfere.

Establishment and application of recombinase polymerase amplification combined with a lateral flow dipstick for the detection of mcr-1 in uncultured clinical samples

The rapid dissemination of the mcr-1 gene via plasmid-mediated transfer has raised concerns regarding the efficacy of colistin as a last-resort treatment for multidrug-resistant Gram-negative bacterial infections. Current mcr-1 gene detection methods mainly focus on cultured bacteria, which is a complex and time-consuming process requiring skilled personnel, making it unsuitable for field analysis. A rapid detection technique combining recombinase polymerase amplification with a lateral flow dipstick targeting uncultured clinical samples was developed. This new method targeting the mcr-1 gene region (23 232-23 642 bp, no. KP347127.1) achieved a low detection limit of 10 copies/μL. The whole process was carried out with high specificity and was completed within 20 min. The evaluation assay was conducted using 45 human faecal samples; 16 strains yielded a 98% accuracy, closely matching antimicrobial susceptibility outcomes. The novel method integrates nucleic acid extraction, isothermal amplification, and a test assay, suggesting the potential for timely colistin resistance surveillance in frontline disease control and healthcare settings, supporting future prevention and clinical standardization efforts.

One Health surveillance of colistin-resistant Enterobacterales in Belgium and the Netherlands between 2017 and 2019.

Colistin serves as the last line of defense against multidrug resistant Gram-negative bacterial infections in both human and veterinary medicine. This study aimed to investigate the occurrence and spread of colistin-resistant Enterobacterales (ColR-E) using a One Health approach in Belgium and in the Netherlands. In a transnational research project, a total of 998 hospitalized patients, 1430 long-term care facility (LTCF) residents, 947 children attending day care centres, 1597 pigs and 1691 broilers were sampled for the presence of ColR-E in 2017 and 2018, followed by a second round twelve months later for hospitalized patients and animals. Colistin treatment incidence in livestock farms was used to determine the association between colistin use and resistance. Selective cultures and colistin minimum inhibitory concentrations (MIC) were employed to identify ColR-E. A combination of short-read and long-read sequencing was utilized to investigate the molecular characteristics of 562 colistin-resistant isolates. Core genome multi-locus sequence typing (cgMLST) was applied to examine potential transmission events. The presence of ColR-E was observed in all One Health sectors. In Dutch hospitalized patients, ColR-E proportions (11.3 and 11.8% in both measurements) were higher than in Belgian patients (4.4 and 7.9% in both measurements), while the occurrence of ColR-E in Belgian LTCF residents (10.2%) and children in day care centres (17.6%) was higher than in their Dutch counterparts (5.6% and 12.8%, respectively). Colistin use in pig farms was associated with the occurrence of colistin resistance. The percentage of pigs carrying ColR-E was 21.8 and 23.3% in Belgium and 14.6% and 8.9% in the Netherlands during both measurements. The proportion of broilers carrying ColR-E in the Netherlands (5.3 and 1.5%) was higher compared to Belgium (1.5 and 0.7%) in both measurements. mcr-harboring E. coli were detected in 17.4% (31/178) of the screened pigs from 7 Belgian pig farms. Concurrently, four human-related Enterobacter spp. isolates harbored mcr-9.1 and mcr-10 genes. The majority of colistin-resistant isolates (419/473, 88.6% E. coli; 126/166, 75.9% Klebsiella spp.; 50/75, 66.7% Enterobacter spp.) were susceptible to the critically important antibiotics (extended-spectrum cephalosporins, fluoroquinolones, carbapenems and aminoglycosides). Chromosomal colistin resistance mutations have been identified in globally prevalent high-risk clonal lineages, including E. coli ST131 (n = 17) and ST1193 (n = 4). Clonally related isolates were detected in different patients, healthy individuals and livestock animals of the same site suggesting local transmission. Clonal clustering of E. coli ST10 and K. pneumoniae ST45 was identified in different sites from both countries suggesting that these clones have the potential to spread colistin resistance through the human population or were acquired by exposure to a common (food) source. In pig farms, the continuous circulation of related isolates was observed over time. Inter-host transmission between humans and livestock animals was not detected. The findings of this study contribute to a broader understanding of ColR-E prevalence and the possible pathways of transmission, offering insights valuable to both academic research and public health policy development.

Resensitizing tigecycline- and colistin-resistant Escherichia coli using an engineered conjugative CRISPR/Cas9 system.

The emergence of plasmid-encoded tet(X4) and mcr-1 isolated from human and animal sources has affected the treatment of tigecycline and colistin, and has posed a significant threat to public health. Tigecycline and colistin are considered as the "last line of defense" for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections, so there is an urgent need to find a method that can resensitize tet(X4)-mediated tigecycline-resistant and mcr-1-mediated colistin-resistant bacteria. In this study, we developed a glutamate-based, chromosomal, plasmid-balanced lethal conjugative CRISPR/Cas9 system, which can simultaneously resensitize tet(X4)-mediated tigecycline-resistant and mcr-1-mediated colistin-resistant Escherichia coli. The counts of tigecycline- and colistin-resistant bacteria decreased to 1% in vivo after the resensitization system was administered. This study opens up new pathways for the development of CRISPR-based tools for selective bacterial pathogen elimination and precise microbiome composition change.

A Real-World Pharmacovigilance Study of Ceftazidime/Avibactam: Data Mining of the Food and Drug Administration Adverse Event Reporting System Database.

Ceftazidime/avibactam (CAZ/AVI) is a combination of a well-known third-generation, broad-spectrum cephalosporin with a new beta-lactamase inhibitor that has been approved for the treatment of various infectious diseases (especially multidrug-resistant Gram-negative bacterial infections) by the Food and Drug Administration (FDA). The current study extensively assessed CAZ/AVI-related adverse events (AEs) in the real world through data mining of the FDA Adverse Event Reporting System (FAERS) database to better understand toxicities. The signals of CAZ/AVI-related AEs were quantified using disproportionality analyses, including the reporting odds ratio, the proportional reporting ratio, the Bayesian confidence propagation neural network, and the multi-item gamma Poisson shrinker algorithms. Out of 10,114,815 records retrieved from the FAERS database, 628 cases were identified, where CAZ/AVI was implicated as the primary suspect drug. A total of 61 preferred terms with significant disproportionality that simultaneously met the criteria of all four algorithms were retained. Several unexpected safety signals may also occur, including melena, hypernatremia, depressed level of consciousness, brain edema, petechiae, delirium, and shock hemorrhagic. The median onset time for AEs associated with CAZ/AVI was 4 days, with most cases occurring within 3 days after CAZ/AVI initiation.

Liposomal Formulation Reduces Transport and Cell Uptake of Colistin in Human Lung Epithelial Calu-3 Cell and 3D Human Lung Primary Tissue Models.

Respiratory tract infections caused by multi-drug-resistant (MDR) bacteria have been a severe risk to human health. Colistin is often used to treat the MDR Gram-negative bacterial infections as a last-line therapy. Inhaled colistin can achieve a high concentration in the lung but none of aerosolized colistin products has been approved in the USA. Liposome has been reported as an advantageous formulation strategy for antibiotics due to its controlled release profile and biocompatibility. We have developed colistin liposomal formulations in our previous study. In the present study, the cellular uptake and transport of colistin in colistin liposomes were examined in two human lung epithelium in vitro models, Calu-3 monolayer and EpiAirway 3D tissue models. In both models, cellular uptake (p < 0.05) and cellular transport (p < 0.01) of colistin were significantly reduced by the colistin liposome compared to the colistin solution. Our findings indicate that inhaled colistin liposomes could be a promising treatment for extracellular bacterial lung infections caused by MDR Pseudomonas aeruginosa (P. aeruginosa).

Synergistic effect of tryptamine-urea derivatives to overcome the chromosomally-mediated colistin resistance in Klebsiella pneumoniae

Colistin is considered as the last-resort antibiotics to treat multi-drug resistant Gram-negative bacterial infections in humans. However, the clinical use of colistin was limited because of the apparition of chromosomal mutations and mobile colistin resistance genes in bacterial isolates. One promising strategy is to combine existing antibiotics with promising non-antibiotics to overcome the widespread emergence of antibiotic-resistant pathogens. Moreover, colistin resistance would be regulated by two component systems PhoP/PhoQ which leads to permanent synthesis of cationic groups compensating for Mg2+ deficiency. In this study, the synthesis of a small library of tryptamine urea derivatives was carried out. In addition, antibiotic susceptibility, antibiotic adjuvant screening and checkerboard assays were used to investigate the antibacterial activity of these synthesized compounds and the potential synergistic activity of their combination with colistin. Conformational analysis of the docked binding modes of the active compound in the predicted binding pocket of bacterial response regulator PhoP were carried out, to see if the active compound inhibits PhoP which is involved in colistin resistance. Finally, hemolytic activity studies have been conducted on the most active compound.

Renoprotective effects of crocin against colistin-induced nephrotoxicity in a rat model.

Colistin is used to treat multidrug-resistant gram-negative bacterial infections. It increases the membrane permeability of kidney cells, leading to kidney toxicity. Crocin, a carotenoid found in saffron, has anti-oxidant and nephroprotective properties. The present study aimed to explore the potential renoprotective effects of crocin against colistin-induced nephrotoxicity. Six groups of male Wistar rats were utilized: 1- Control (0.5 ml of normal saline, 10 days, IP); 2- Crocin (40 mg/kg, 10 days, IP); 3-Colistin (23 mg/kg, 7 days, IP); 4-6 Colistin (23 mg/kg, 7 days, IP)+ crocin (10, 20, 40 mg/kg, 10 days, IP). On day 11, rats were sacrificed and their blood and kidney samples were collected to measure creatinine, blood urea nitrogen (BUN), glutathione (GSH) levels, malondialdehyde (MDA), and histopathological alterations. Colistin caused a significant increase in BUN, creatinine, and MDA, and a decrease in GSH compared to the control group. It also led to congested blood vessels, glomerular shrinkage, and medullary tubular degeneration. Co-administration of crocin with colistin resulted in a significant decrease in BUN and creatinine, increased GSH levels, and ameliorated the histopathological alterations compared to the colistin group. No significant difference was found between the control group and the crocin (40 mg/kg) group. It might be suggested that colistin can induce kidney damage by inducing oxidative stress. However, crocin shows protective effects against colistin-induced renal injury by acting as an anti-oxidant. Hence, crocin can be used as a supplement to reduce tissue and biochemical damage caused by colistin injection.

Phenotypic and genotypic characterization of colistin-resistant Escherichia Coli with mcr-4, mcr-5, mcr-6, and mcr-9 genes from broiler chicken and farm environment

BackgroundColistin is an antibiotic used as a last-resort to treat multidrug-resistant Gram-negative bacterial infections. Colistin had been used for a long time in veterinary medicine for disease control and as a growth promoter in food-producing animals. This excessive use of colistin in food animals causes an increase in colistin resistance. This study aimed to determine molecular characteristics of colistin-resistant Escherichia coli in broiler chicken and chicken farm environments.ResultsFour hundred fifty-three cloacal and farm environment samples were collected from six different commercial chicken farms in Kelantan, Malaysia. E. coli was isolated using standard bacteriological methods, and the isolates were tested for antimicrobial susceptibility using disc diffusion and colistin minimum inhibitory concentration (MIC) by broth microdilution. Multiplex PCR was used to detect mcr genes, and DNA sequencing was used to confirm the resistance genes. Virulence gene detection, phylogroup, and multilocus sequence typing (MLST) were done to further characterize the E. coli isolates. Out of the 425 (94%; 425/453) E. coli isolated from the chicken and farm environment samples, 10.8% (48/425) isolates were carrying one or more colistin-resistance encoding genes. Of the 48 colistin-resistant isolates, 54.2% (26/48) of the mcr positive isolates were genotypically and phenotypically resistant to colistin with MIC of colistin ≥ 4 μg/ml. The most prominent mcr gene detected was mcr-1 (47.9%; 23/48), followed by mcr-8 (18.8%; 9/48), mcr-7 (14.5%; 7/48), mcr-6 (12.5%; 6/48), mcr-4 (2.1%; 1/48), mcr-5 (2.1%; 1/48), and mcr-9 (2.1%; 1/48) genes. One E. coli isolate originating from the fecal sample was found to harbor both mcr-4 and mcr-6 genes and another isolate from the drinking water sample was carrying mcr-1 and mcr-8 genes. The majority of the mcr positive isolates were categorized under phylogroup A followed by phylogroup B1. The most prevalent sequence typing (ST) was ST1771 (n = 4) followed by ST206 (n = 3). 100% of the mcr positive E. coli isolates were multidrug resistant. The most frequently detected virulence genes among mcr positive E. coli isolates were ast (38%; 18/48) followed by iss (23%; 11/48). This is the first research to report the prevalence of mcr-4, mcr-5, mcr-6, mcr-7, and mcr-8 genes in E. coli from broiler chickens and farm environments in Malaysia.ConclusionOur findings suggest that broiler chickens and broiler farm environments could be reservoirs of colistin-resistant E. coli, posing a risk to public health and food safety.

Network meta-analysis of antibiotic resistance patterns in gram-negative bacterial infections: a comparative study of carbapenems, fluoroquinolones, and aminoglycosides

IntroductionAntimicrobial resistance poses a grave global threat, particularly with the emergence of multidrug-resistant gram-negative bacterial infections, which severely limit treatment options. The increasing global threat of antimicrobial resistance demands rigorous investigation, particularly concerning multidrug-resistant gram-negative bacterial infections that present limited therapeutic options. This study employed a network meta-analysis, a powerful tool for comparative effectiveness assessment of diverse antibiotics. The primary aim of this study was to comprehensively evaluate and compare resistance patterns among widely used antibiotic classes, namely carbapenems, fluoroquinolones, and aminoglycosides, for combating gram-negative pathogens.MethodsWe searched PubMed, Web of Sciences, Scopus, Scholarly, Medline, Embase, and Cochrane databases up to August 27, 2023. Studies showing antibiotic resistance in clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii exposed to carbapenems, fluoroquinolones, and aminoglycosides were included. This study determined treatment-specific resistance percentages and ranked these treatments based on resistance using a random-effects network meta-analysis technique. To investigate the impact of the study and pathogen features, subgroup and meta-regression analyses were performed. Risk ratios and 95% confidence intervals (CIs) were calculated using a network meta-analysis (NMA) incorporating both direct and indirect evidence. Clinical improvement, cure, microbiological eradication, and death from any cause were the primary outcomes. Nephrotoxicity was a secondary result.ResultsThe analysis included 202 publications and 365,782 gram-negative isolates. The NMA included data from 20 studies and 4,835 patients. Carbapenems had the lowest resistance rates throughout the pathogen spectrum, with resistance percentages of 17.1, 22.4, and 33.5% for Enterobacteriaceae, P. aeruginosa, and A. baumannii, respectively. For the same infections, aminoglycosides showed resistance rates of 28.2, 39.1, and 50.2%, respectively. Fluoroquinolones had the highest resistance rates at 43.1, 57.3, and 65.7%, respectively. Unexpectedly, resistance to all three antibiotic classes has increased over time, with multidrug resistance being the most prevalent.ConclusionThis extensive network meta-analysis provides an overview of the patterns of resistance throughout the world and how they are changing. The most effective choice is still carbapenems, but the increasing resistance highlights the critical need for multimodal therapies to protect antibiotic effectiveness against these powerful gram-negative infections.

Evaluation of Antibiotic Resistance Mechanisms in Gram-Negative Bacteria.

Multidrug-resistant Gram-negative bacterial infections are exponentially increasing, posing one of the most urgent global healthcare and economic threats. Due to the lack of new therapies, the World Health Organization classified these bacterial species as priority pathogens in 2017, known as ESKAPE pathogens. This classification emphasizes the need for urgent research and development of novel targeted therapies. The majority of these priority pathogens are Gram-negative species, which possess a structurally dynamic cell envelope enabling them to resist multiple antibiotics, thereby leading to increased mortality rates. Despite 6 years having passed since the WHO classification, the progress in generating new treatment ideas has not been sufficient, and antimicrobial resistance continues to escalate, acting as a global ticking time bomb. Numerous efforts and strategies have been employed to combat the rising levels of antibiotic resistance by targeting specific resistance mechanisms. These mechanisms include antibiotic inactivating/modifying enzymes, outer membrane porin remodelling, enhanced efflux pump action, and alteration of antibiotic target sites. Some strategies have demonstrated clinical promise, such as the utilization of beta-lactamase inhibitors as antibiotic adjuvants, as well as recent advancements in machine-based learning employing artificial intelligence to facilitate the production of novel narrow-spectrum antibiotics. However, further research into an enhanced understanding of the precise mechanisms by which antibiotic resistance occurs, specifically tailored to each bacterial species, could pave the way for exploring narrow-spectrum targeted therapies. This review aims to introduce the key features of Gram-negative bacteria and their current treatment approaches, summarizing the major antibiotic resistance mechanisms with a focus on Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Additionally, potential directions for alternative therapies will be discussed, along with their relative modes of action, providing a future perspective and insight into the discipline of antimicrobial resistance.

RNA-based antibiotic susceptibility testing of tmexCD-toprJ-mediated tigecycline resistance in Klebsiella pneumoniae

The emergence and prevalence of plasmid-encoded RND-type efflux pump TMexCD-TOprJ severely compromise tigecycline treatment, which is recognized as the last resort for multidrug-resistant (MDR) Gram-negative bacterial infections. There is an urgent need for rapid antibiotic susceptibility testing (AST) that can simultaneously identify the genotype and phenotype of tmexCD-toprJ-positive bacteria. Through characterizing transcriptional profiling responses of tmexCD-toprJ-positive and -negative strains after exposure to 2 μg/mL tigecycline, here we identified 12 differentially RNA biomarkers and developed an RNA-based AST (RBAST) to distinguish tmexCD-toprJ-positive and -negative K. pneumoniae. These mRNA biomarkers were successfully validated in tigecycline exposure time variations, concentration shifts, and other tmexCD-toprJ variants. In addition, a group of clinical isolated strains was effectively distinguished using RBAST, with an accuracy of over 94% during 3 h test period. Our work highlights the potential of RNA transcripts as biomarkers for rapid AST, which will contribute to deploying effective antibiotic regimens in clinical practice.

Multidrug-resistant Gram-negative bacterial infections and associated factors in a Kenyan intensive care unit: a cross-sectional study

BackgroundPatients admitted to intensive care units (ICU) are at risk of Gram-negative bacteria (GNB) infections, especially those caused by multidrug-resistant (MDR) isolates, increasing morbidity, mortality, and healthcare costs. However, epidemiological surveillance data on MDR bacteria to inform infection prevention and control (IPCs) interventions is limited in our study setting. Here we assessed the prevalence and factors associated with GNB infections in ICU- patients admitted in our study setting.MethodsThis was a hospital-based cross-sectional study among patients admitted to ICU at the Nairobi West Hospital, Kenya, between January and October 2022. Altogether, we recruited 162 patients, excluding those hospitalized for less than 48 h and declining consent, and collected demographics and clinical data by case report form. Blood, wound and throat swab, ascetic tap, stool, urine, tracheal aspirate, and sputum samples were collected cultured. Isolates identity and antimicrobial susceptibility were elucidated using the BD Phoenix system.ResultsThe prevalence of GNB infections was 55.6%, predominated by urinary tract infections (UTIs). We recovered 13 GNB types, with Escherichia coli (33.3%) and Klebsiella pneumoniae (31.1%) as the most common isolates. Factors associated with GNB infections were a history of antibiotic use (aOR = 4.23, p = 0.001), nasogastric tube use (NGT, aOR = 3.04, p = 0.013), respiratory tract (RT, aOR = 5.3, p = 0.005) and cardiovascular (CV, aOR = 5.7, p = 0.024) conditions. 92% of the isolates were MDR,predominantly Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.ConclusionWe report a high prevalence of MDR-GNB infections, predominated by UTI, in ICU, whereby patients with a history of antibiotic use, using the NGT, and having RT and CV conditions were at increased risk. To improve the management of ICU-admitted patients, continuous education, training, monitoring, evaluation and feedback on infection prevention and control are warranted in our study setting.