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Regulators of colistin resistance mechanisms via chromosomal and plasmid-mediated pathways of lipopolysaccharide modifications in Enterobacteriaceae.
Source publication
Colistin regained global interest as a consequence of the rising prevalence of multidrug-resistant Gram-negative Enterobacteriaceae. In parallel, colistin-resistant bacteria emerged in response to the unregulated use of this antibiotic. However, some Gram-negative species are intrinsically resistant to colistin activity, such as Neisseria meningiti...
Citations
... Mg 2+ is also important for stabilizing the outer membrane structure of gram-negative bacteria by binding to negatively charged lipopolysaccharides (LPS) on the bacterial cell membrane [46]. These Mg 2+ -stabilized LPS moieties can be targeted by polymyxins [47], last-resort antibiotics for treating multidrugresistant bacterial infections [48,49] (Fig 3A). In Mg 2+ -limited conditions, bacteria activate dual two-component signaling pathways, PmrAB and PhoPQ, modifying lipid A of LPS with L-Ara4N (4-amino-4-deoxy-L-arabinose) and PEtN (phosphoethanolamine), leading to resistance to polymyxins [50][51][52][53] (Fig 3A). ...
Fungi and bacteria coexist in many polymicrobial communities, yet the molecular basis of their interactions remains poorly understood. Here, we show that the fungus Candida albicans sequesters essential magnesium ions from the bacterium Pseudomonas aeruginosa . To counteract fungal Mg 2 + sequestration, P . aeruginosa expresses the Mg 2 + transporter MgtA when Mg 2 + levels are low. Thus, loss of MgtA specifically impairs P . aeruginosa in co-culture with C . albicans , but fitness can be restored by supplementing Mg 2 + . Using a panel of fungi and bacteria, we show that Mg 2 + sequestration is a general mechanism of fungal antagonism against gram-negative bacteria. Mg 2 + limitation enhances bacterial resistance to polymyxin antibiotics like colistin, which target gram-negative bacterial membranes. Indeed, experimental evolution reveals that P . aeruginosa evolves C . albicans -dependent colistin resistance via non-canonical means; antifungal treatment renders resistant bacteria colistin-sensitive. Our work suggests that fungal–bacterial competition could profoundly impact polymicrobial infection treatment with antibiotics of last resort.
... ECA acts as a carbohydrate antigen found in various pathogenic strains of Enterobacteriaceae including K. pneumoniae [8]. The protein ArnA is involved in colistin (polymyxin E) resistance in various Gram-negative bacteria including K. pneumoniae by catalyzing the modification of lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) [9,10]. The downregulation of ArnA could reduce Ara4N addition to lipid A and retain the negative charge of the LPS. ...
The rising incidence of extensively drug-resistant (XDR) Klebsiella pneumoniae, including carbapenem- and colistin-resistant strains, leads to the limitation of available effective antibiotics. Miang, known as chewing tea, is produced from Camellia sinensis var. assamica or Assam tea leaves fermentation. Previous studies revealed that the extract of Miang contains various phenolic and flavonoid compounds with numerous biological activities including antibacterial activity. However, the antibacterial activity of Miang against XDR bacteria especially colistin-resistant strains had not been investigated. In this study, the compositions of phenolic and flavonoid compounds in fresh, steamed, and fermented Assam tea leaves were examined by HPLC, and their antibacterial activities were evaluated by the determination of the MIC and MBC. Pyrogallol was detected only in the extract from Miang and showed the highest activities with an MIC of 0.25 mg/mL and an MBC of 0.25–0.5 mg/mL against methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli ATCC 25922, colistin-resistant E. coli, and colistin-resistant K. pneumoniae. The effects on morphology and proteomic changes in K. pneumoniae NH54 treated with Miang extract were characterized by SEM and label-free quantitative shotgun proteomics analysis. The results revealed that Miang extract caused the decrease in bacterial cell wall integrity and cell lysis. The up- and downregulated expression with approximately a 2 to >5-fold change in proteins involved in peptidoglycan synthesis and outer membrane, carbohydrate, and amino acid metabolism were identified. These findings suggested that Miang containing pyrogallol and other secondary metabolites from fermentation has potential as an alternative candidate with an antibacterial agent or natural active pharmaceutical ingredient against XDR bacteria including colistin-resistant bacteria.
... Over-the-counter dispensing of antibiotics is also common in Bangladesh and is a concern, especially when this involves 'Reserve' antibiotics [46][47][48][49] The use of colistin as an antibiotic of last resort is greatly threatened by its overuse and the associated rise of plasmid-borne mobile colistin resistance genes [50][51][52], spreading rapidly via horizontal gene transfer [53]. Resistance to colistin is generated by the chromosomally mediated modification of lipopolysaccharide (LPS) [54]. The acquisition of colistin resistance by a novel plasmid-mediated gene, mcr-1, was first described in Enterobacteriales from both farm-animal products and humans [55]. ...
Abstract
Colistin is a last-resort antimicrobial for treating multidrug-resistant Gram-negative bacteria. Phenotypic colistin resistance is highly associated with plasmid-mediated mobile colistin resistance (mcr) genes. mcr-bearing Enterobacteriaceae have been detected in many countries, with the emergence of colistin-resistant pathogens a global concern. This study assessed the distribution of mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes with phenotypic colistin resistance in isolates from diarrheal infants and children in Bangladesh. Bacteria were identified using the API-20E biochemical panel and 16s rDNA gene sequencing. Polymerase chain reactions detected mcr gene variants in the isolates. Their susceptibilities to colistin were determined by agar dilution and E-test by minimal inhibitory concentration (MIC) measurements. Over 31.6% (71/225) of isolates showed colistin resistance according to agar dilution assessment (MIC > 2 μg/mL). Overall, 15.5% of isolates carried mcr genes (7, mcr-1; 17, mcr-2; 13, and mcr-3, with co-occurrence occurring in two isolates). Clinical breakout MIC values (≥4 μg/mL) were associated with 91.3% of mcr-positive isolates. The mcr-positive pathogens included twenty Escherichia spp., five Shigella flexneri, five Citrobacter spp., two Klebsiella pneumoniae, and three Pseudomonas parafulva. The mcr-genes appeared to be significantly associated with phenotypic colistin resistance phenomena (p = 0.000), with 100% colistin-resistant isolates showing MDR phenomena. The age and sex of patients showed no significant association with detected mcr variants. Overall, mcr-associated colistin-resistant bacteria have emerged in Bangladesh, which warrants further research to determine their spread and instigate activities to reduce resistance.
Keywords: mobile colistin resistance; mcr gene; human-mcr; diarrheal infant patients; Bangladesh; MDR; antimicrobial stewardship programs
... In particular, the recent increase in Carbapenem-resistant organisms (CRO) led to an unprecedented increase in the use of Colistin, as most available antibiotics, including novel molecules, do not provide adequate coverage in all cases [2]. Otherwise, many Gram-negative bacilli (GNB) remain susceptible to Colistin, except few genera such as Proteus, Serratia, and Morganella, which possess intrinsic resistance through a variety of complex mechanisms [3]. Colistin is a bactericidal antibiotic that exerts its efect primarily through disruption of the bacterial cell membrane through its interaction with the lipopolysaccharide layer (LPS), which eventually leads to bacterial death [4,5]. ...
... Given its efectiveness, Colistin has also found its way into the veterinary and agricultural felds for both therapeutic and prophylactic uses [4]. Subsequently, Colistin resistance has progressed, induced by numerous mechanisms: chromosomally encoded resistance, mobile resistance, mutations, adaptation, and others that remain enigmatic to this day [3]. Tis presents complex challenges involving the use of Colistin in therapeutic settings, requiring rapid and accurate detection of pathogen susceptibility to the drug. ...
... Tis highlights the importance of rapid determination of susceptibility to Colistin in performing the appropriate selection of antibiotics early on. Tis is particularly challenging due to the fact that the broth microdilution method has multiple pitfalls related to the design of the test [5] and is not suitable for routine use in daily microbiology laboratory workfows [3]. Most traditional AST methods, including BMD, require long turnaround times (TAT) that reach 72 hours from sampling [20]. ...
Background
Colistin (Polymyxin E) has reemerged in the treatment of MDR Gram-negative infections. Traditional Colistin AST methods have long turnaround times and are cumbersome for routine use. We present a SEM-AST technique enabling rapid detection of Colistin resistance through direct observation of morphological and quantitative changes in bacteria exposed to Colistin.
Methods
Forty-four Gram-negative reference organisms were chosen based on their Colistin susceptibility profiles. Bacterial suspensions of ∼10⁷ CFU/mL were exposed to Colistin at EUCAST-ECOFF, with controls not exposed, incubated at 37°C, and then sampled at 0, 15, 30, 60, and 120 minutes. Phosphotungstic Acid (PTA) staining was applied, followed by SEM imaging using Hitachi TM4000PlusII-Tabletop-SEM at ×2000, ×5000 and ×7000 magnifications. Bacterial viability analysis was performed for all conditions by quantifying viable and dead organisms based on PTA-staining and morphologic changes.
Results
We identified a significant drop in the percentage of viable organisms starting 30 minutes after exposure in susceptible strains, as compared to nonsignificant changes in resistant strains across all tested organisms. The killing effect of Colistin was best observed after 120 minutes of incubation with the antibiotic, with significant changes in morphologic features, including bacterial inflation, fusion, and lysis, observed as early as 30 minutes. Our observation matched the results of the gold standard-based broth microdilution method.
Conclusions
We provide an extended application of the proof of concept for the utilization of the SEM-AST assay for Colistin for a number of clinically relevant bacterial species, providing a rapid and reliable susceptibility profile for a critical antibiotic.
... Additionally, Strain 3007's resistance to gentamicin is associated with the aac(3)-IVa gene. Both strains exhibit significant colistin resistance (MIC > 128 mg/L), consistent with previous findings of inherent resistance in this genus [17]. ...
... Furthermore, these strains displayed resistance to critical antibiotics such as tigecycline, colistin, and carbapenem. Colistin and tigecycline resistance appears common in this genus, but the underlying mechanisms are yet to be elucidated [17]. Interestingly, Strain 3007 demonstrated resistance to imipenem, attributed to the presence of DHA-1 beta-lactamase. ...
Providencia genus is known to harbor certain opportunistic pathogens capable of causing human infections. Here, we report two strains of multidrug-resistant bacteria initially identified as Providencia rettgeri by mass spectrometry, but genome analysis revealed their ANI (79.84–84.20%) and dDDH (21.1–25.6%) values to fall below the accepted species threshold for known Providencia species. We therefore propose that these isolates be recognized as a novel species, Providencia xianensis sp. nov. Alarmingly, both strains, isolated from locations far apart, exhibited resistance to last-resort antibiotics, indicating their possible wide distribution, underscoring the urgency for immediate attention and enhanced surveillance for this emerging multidrug-resistant pathogen.
... Moreover, bacteria are increasingly becoming resistant to multiple antimicrobial agents, making it challenging to treat infectious diseases with the limited availability of antibiotics [2]. Colistin, an older antibiotic often referred to as the "last-resort drug," has been traditionally employed in the management of Gramnegative bacterial infections that are resistant to several antibiotics [3,4]. However, one major problem is that many Gram-negative bacteria now have genes that are resistant to colistin, which reduces the number of effective antibiotics available. ...
Antibiotic resistance has emerged as a significant global public health issue, driven by the rapid adaptation of microorganisms to commonly prescribed antibiotics. Colistin, previously regarded as a last-resort antibiotic for treating infections caused by Gram-negative bacteria, is increasingly becoming resistant due to chromosomal mutations and the acquisition of resistance genes carried by plasmids, particularly the mcr genes. The mobile colistin resistance gene (mcr-1) was first discovered in E. coli from China in 2016. Since that time, studies have reported different variants of mcr genes ranging from mcr-1 to mcr-10, mainly in Enterobacteriaceae from various parts of the world, which is a major concern for public health. The co-presence of colistin-resistant genes with other antibiotic resistance determinants further complicates treatment strategies and underscores the urgent need for enhanced surveillance and antimicrobial stewardship efforts. Therefore, understanding the mechanisms driving colistin resistance and monitoring its global prevalence are essential steps in addressing the growing threat of antimicrobial resistance and preserving the efficacy of existing antibiotics. This review underscores the critical role of colistin as a last-choice antibiotic, elucidates the mechanisms of colistin resistance and the dissemination of resistant genes, explores the global prevalence of mcr genes, and evaluates the current detection methods for colistin-resistant bacteria. The objective is to shed light on these key aspects with strategies for combating the growing threat of resistance to antibiotics.
... However, the use of colistin as a last resort antibiotic is greatly threatened by the rise of plasmid-borne mobile colistin resistance gene [58][59][60], spreading rapidly via horizontal gene transfer [61]. Resistance to colistin is generated by the chromosomally mediated modification of lipopolysaccharide (LPS) [62]. Acquisition of colistin resistance by a novel plasmid-mediated gene, mcr-1, was first described in Enterobacteriales from both farm-animal products and humans [63]. ...
Colistin is a last-resort antimicrobial for treating multidrug-resistant Gram-negative bacteria. Phe-notypic colistin resistance is highly associated with plasmid-mediated mobile colistin resistance (mcr) genes. mcr-bearing Enterobacteriaceae have been detected in many countries, with the emergence of colistin-resistant pathogens a global concern. This study assessed the distribution of mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes with the phenotypic colistin resistance in isolates from diarrheal infants and children in Bangladesh. Bacteria were identified using the API-20E biochemical panel and 16s rDNA gene sequencing. Polymerase chain reac-tions detected mcr gene variants in the isolates. Their susceptibilities to colistin were determined by agar dilu-tion and E-test by minimal inhibitory concentration (MIC) measurements. Over 30.0% (69/225) of isolates showed colistin resistance by agar dilution assessment (MIC> 2.0 μg/mL). Overall, 15.5% of isolates carried mcr genes (7, mcr-1; 17, mcr-2; 13, mcr-3; and co-occurrence occurred in 2 isolates). Clinical breakout MIC val-ues (≥ 4 μg/mL) were associated with 91.3% of mcr-positive isolates. The mcr-positive pathogens include twenty Escherichia spp., five Shigella flexneri, five Citrobacter spp., two Klebsiella pneumoniae, and three Pseudo-monas parafulva. mcr-genes appeared to be significantly associated with phenotypic colistin resistance phe-nomena (p=0.000), with 100% colistin-resistant isolates showing MDR phenomena. Age and sex of patients showed no significant association with detected mcr variants. Overall, mcr-associated colistin-resistant bacte-ria have emerged in Bangladesh, which warrants further research to determine their spread and instigate ac-tivities to reduce resistance.
... For K. pneumoniae, mutations in pmrC, pmrD, mgrB and its promotor region, crrAB, yciM, lpxM and arnA were additionally explored. For Enterobacter spp., mutations in mgrB and its promotor were also considered [11,35]. Virulence genes were functionally classified according to the VFDB [31]. ...
Background
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.
Methods
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.
Results
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.
Conclusions
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.
... Polymyxins, which are cationic and branched cyclic decapeptides, were discovered in the 1940s, and they are still used to treat multi-drug resistant Gram-negative bacteria including Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli. However, their use in the clinic is limited due to severe adverse effects such as neurotoxicity (6), and their potency against other clinically important Gram-negative bacteria is not outstanding (7). The latter is due to the rapid acquisition of resistance, formation of persister cells, or dissemination of resistance genes such as mcr-1 by horizontal gene transfer (8). ...
Antibiotic-resistant Gram-negative bacteria remain a globally leading cause of bacterial infection-associated mortality, and it is imperative to identify novel therapeutic strategies. Recently, the advantage of using antibacterials selective against Gram-negative bacteria has been demonstrated with polymyxins that specifically target the lipopolysaccharides of Gram-negative bacteria. However, the severe cytotoxicity of polymyxins limits their clinical use. Here, we demonstrate that polymyxin B nonapeptide (PMBN), a polymyxin B derivative without the terminal amino acyl residue, can significantly enhance the effectiveness of commonly used antibiotics against only Gram-negative bacteria and their persister cells. We show that although PMBN itself does not exhibit antibacterial activity or cytotoxicity well above the 100-fold minimum inhibitory concentration of polymyxin B, PMBN can increase the potency of co-treated antibiotics. We also demonstrate that using PMBN in combination with other antibiotics significantly reduces the frequency of resistant mutant formation. Together, this work provides evidence of the utilities of PMBN as a novel potentiator for antibiotics against Gram-negative bacteria and insights for the eradication of bacterial persister cells during antibiotic treatment.
IMPORTANCE
The significance of our study lies in addressing the problem of antibiotic-resistant Gram-negative bacteria, which continue to be a global cause of mortality associated with bacterial infections. Therefore, identifying innovative therapeutic approaches is an urgent need. Recent research has highlighted the potential of selective antibacterials like polymyxins, which specifically target the lipopolysaccharides of Gram-negative bacteria. However, the clinical use of polymyxins is limited by their severe cytotoxicity. This study unveils the effectiveness of polymyxin B nonapeptide (PMBN) in significantly enhancing the eradication of persister cells in Gram-negative bacteria. Although PMBN itself does not exhibit antibacterial activity or cytotoxicity, it remarkably reduces persister cells during the treatment of antibiotics. Moreover, combining PMBN with other antibiotics reduces the emergence of resistant mutants. Our research emphasizes the utility of PMBN as a novel potentiator to decrease persister cells during antibiotic treatments for Gram-negative bacteria.
... The arnBCADTEF operon encodes for a set of enzymes that modify lipopolysaccharides (LPS), which are a major component of the outer membrane of gram-negative bacteria. The 4-amino-4-deoxy-l-arabinose (l-Ara4N) chemical groups are specifically added to the lipid A portion of LPS by the arnBCADTEF operon 24 . This can inhibit the binding of colistin to the bacterial membrane and hence reduce the drug's effectiveness. ...
The increasing antimicrobial resistance in Providencia stuartii (P. stuartii) worldwide, particularly concerning for immunocompromised and burn patients, has raised concern in Bangladesh, where the significance of this infectious opportunistic pathogen had been previously overlooked, prompting a need for investigation. The two strains of P. stuartii (P. stuartii SHNIBPS63 and P. stuartii SHNIBPS71) isolated from wound swab of two critically injured burn patients were found to be multidrug-resistant and P. stuartii SHNIBPS63 showed resistance to all the 22 antibiotics tested as well as revealed the co-existence of blaVEB-6 (Class A), blaNDM-1 (Class B), blaOXA-10 (Class D) beta lactamase genes. Complete resistance to carbapenems through the production of NDM-1, is indicative of an alarming situation as carbapenems are considered to be the last line antibiotic to combat this pathogen. Both isolates displayed strong biofilm-forming abilities and exhibited resistance to copper, zinc, and iron, in addition to carrying multiple genes associated with metal resistance and the formation of biofilms. The study also encompassed a pangenome analysis utilizing a dataset of eighty-six publicly available P. stuartii genomes (n = 86), revealing evidence of an open or expanding pangenome for P. stuartii. Also, an extensive genome-wide analysis of all the P. stuartii genomes revealed a concerning global prevalence of diverse antimicrobial resistance genes, with a particular alarm raised over the abundance of carbapenem resistance gene blaNDM-1. Additionally, this study highlighted the notable genetic diversity within P. stuartii, significant informations about phylogenomic relationships and ancestry, as well as potential for cross-species transmission, raising important implications for public health and microbial adaptation across different environments.
