Last-resort Antibiotics Research Articles

A Review on Colistin Resistance: An Antibiotic of Last Resort.

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.

Molecular mechanisms of tigecycline-resistance among Enterobacterales.

The global emergence of antimicrobial resistance to multiple antibiotics has recently become a significant concern. Gram-negative bacteria, known for their ability to acquire mobile genetic elements such as plasmids, represent one of the most hazardous microorganisms. This phenomenon poses a serious threat to public health. Notably, the significance of tigecycline, a member of the antibiotic group glycylcyclines and derivative of tetracyclines has increased. Tigecycline is one of the last-resort antimicrobial drugs used to treat complicated infections caused by multidrug-resistant (MDR) bacteria, extensively drug-resistant (XDR) bacteria or even pan-drug-resistant (PDR) bacteria. The primary mechanisms of tigecycline resistance include efflux pumps' overexpression, tet genes and outer membrane porins. Efflux pumps are crucial in conferring multi-drug resistance by expelling antibiotics (such as tigecycline by direct expelling) and decreasing their concentration to sub-toxic levels. This review discusses the problem of tigecycline resistance, and provides important information for understanding the existing molecular mechanisms of tigecycline resistance in Enterobacterales. The emergence and spread of pathogens resistant to last-resort therapeutic options stands as a major global healthcare concern, especially when microorganisms are already resistant to carbapenems and/or colistin.

Electrochemical and Infrared Studies of a Model Bilayer of the Outer Membrane of Gram-Negative Bacteria and its Interaction with polymyxin─the Last-Resort Antibiotic.

A model bilayer of the outer membrane (OM) of Gram-negative bacteria, composed of lipid A and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), was assembled on the β-Tg modified gold (111) single crystal surface using a combination of Langmuir-Blodgett and Langmuir-Schaefer transfer. Electrochemical and spectroscopic methods were employed to study the properties of the model bilayer and its interaction with polymyxin. The model bilayer is stable on the gold surface in the transmembrane potential region between 0.0 and -0.7 V. The presence of Mg2+ coordinates with the phosphate and carboxylate groups in the leaflet of lipid A and stabilizes the structure of the model bilayer. Polymyxin causes the model bilayer leakage and damage in the transmembrane potential region between 0.2 and -0.4 V. At transmembrane potentials lower than -0.5 V, polymyxin does not affect the membrane integrity. Polymyxin binds to the phosphate and carboxylate groups in lipid A molecules and causes the increase of the tilt angle of acyl chains and the decrease of the tilt of the C═O bond. The results in this paper indicate that the antimicrobial activity of polymyxin depends on the transmembrane potential at the model bilayer and provides useful information for the development of new antibiotics.

Novel method for determination of colistin sulfate in human plasma by high-performance liquid chromatography-tandem mass spectrometry and its clinical applications in critically ill patients

Colistin is a last-resort antibiotic used for treating infections caused by carbapenem-resistant Gram-negative bacteria, particularly in critically patients, nevertheless its therapeutic window is narrow, and requires monitoring. A determination method suitable for clinical detection is conducive to ensure its efficacy and safety of patients with severe infection. We developed and validated a concise and accurate high-performance liquid chromatography-tandem mass spectrometry method for the determination of colistin A and B in human plasma. We used a Kinetex C18 column (50 mm × 2.1 mm, 2.6 μm) with acetonitrile (containing 0.1% formic acid) as the protein precipitant and water (containing 0.2% formic acid and 5 mmol/L ammonium formate) - acetonitrile (containing 0.2% formic acid) as the gradient elution. The calibration curves were linear over concentration ranges of 0.06–4.00 μg/mL (colistin A) and 0.1–7.0 μg/mL (colistin B). The precision, accuracy, matrix effect, extraction recovery, and stability were all validated. This method was applied to the therapeutic drug monitoring for 50 critically ill patients. The trough, peak, and average steady-state concentrations of these patients were 0.8 ± 0.4, 1.4 ± 0.5, and 1.0 ± 0.4 μg/mL, respectively. And the concentrations of colistin in human plasma were closely related to the patient's renal function.

Drug efflux and lipid A modification by 4-L-aminoarabinose are key mechanisms of polymyxin B resistance in the sepsis pathogen Enterobacter bugandensis

A concern with the ESKAPE pathogen, Enterobacter bugandensis, and other species of the Enterobacter cloacae complex, is the frequent appearance of multidrug resistance against last-resort antibiotics, such as polymyxins. Here, we investigated the responses to polymyxin B (PMB) in two PMB-resistant E. bugandensis clinical isolates by global transcriptomics and deletion mutagenesis. In both isolates, the genes of the CrrAB-regulated operon, including crrC and kexD, displayed the highest levels of upregulation in response to PMB. ∆crrC and ∆kexD mutants became highly susceptible to PMB and lost the heteroresistant phenotype. Conversely, heterologous expression of CrrC and KexD proteins increased PMB resistance in a sensitive Enterobacter ludwigii clinical isolate and in the Escherichia coli K12 strain, W3110. The efflux pump, AcrABTolC, and the two component regulators, PhoPQ and CrrAB, also contributed to PMB resistance and heteroresistance. Additionally, the lipid A modification with 4-L-aminoarabinose (L-Ara4N), mediated by the arnBCADTEF operon, was critical to determine PMB resistance. Biochemical experiments, supported by mass spectrometry and structural modelling, indicated that CrrC is an inner membrane protein that interacts with the membrane domain of the KexD pump. Similar interactions were modeled for AcrB and AcrD efflux pumps. Our results support a model where drug efflux potentiated by CrrC interaction with membrane domains of major efflux pumps combined with resistance to PMB entry by the L-Ara4N lipid A modification, under the control of PhoPQ and CrrAB, confers the bacterium high-level resistance and heteroresistance to PMB.

Identification of kinase modulators as host-directed therapeutics against intracellular methicillin-resistant Staphylococcus aureus.

The increasing prevalence of antimicrobial-resistant Staphylococcus aureus strains, especially methicillin-resistant S. aureus (MRSA), poses a threat to successful antibiotic treatment. Unsuccessful attempts to develop a vaccine and rising resistance to last-resort antibiotics urge the need for alternative treatments. Host-directed therapy (HDT) targeting critical intracellular stages of S. aureus emerges as a promising alternative, potentially acting synergistically with antibiotics and reducing the risk of de novo drug resistance. We assessed 201 ATP-competitive kinase inhibitors from Published Kinase Inhibitor Sets (PKIS1 and PKIS2) against intracellular MRSA. Seventeen hit compounds were identified, of which the two most effective and well-tolerated hit compounds (i.e., GW633459A and GW296115X) were selected for further analysis. The compounds did not affect planktonic bacterial cultures, while they were active in a range of human cell lines of cervical, skin, lung, breast and monocyte origin, confirming their host-directed mechanisms. GW633459A, structurally related to lapatinib, exhibited an HDT effect on intracellular MRSA independently of its known human epidermal growth factor receptor (EGFR)/(HER) kinase family targets. GW296115X activated adenosine monophosphate-activated protein kinase (AMPK), thereby enhancing bacterial degradation via autophagy. Finally, GW296115X not only reduced MRSA growth in human cells but also improved the survival rates of MRSA-infected zebrafish embryos, highlighting its potential as HDT.

The Combination of Membrane Disruption and FtsZ Targeting by a Chemotherapeutic Hydrogel Synergistically Combats Pathogens Infections.

The emergence of multidrug-resistant (MDR) bacteria poses a significant challenge to global health. Due to a shortage of antibiotics, alternative therapeutic strategies are urgently needed. Unfortunately, colistin, the last-resort antibiotic, has unavoidable nephrotoxicity and hepatotoxicity, and its single killing mechanism is prone to drug resistance. To address this challenge, a promising combinatorial approach that includes colistin, a membrane-disrupting antimicrobial agent, and chelerythrine (CHE), a FtsZ protein inhibitor is proposed. This approach significantly reduces antibiotic dose and development of resistance, leading to almost complete inactivation of MDR pathogens in vitro. To address solubility issues and ensure transport, the antimicrobial hydrogel system LNP-CHE-CST@hydrogel, which induced reactive oxygen species (ROS) and apoptosis-like cell death by targeting the FtsZ protein, is used. In an in vivo mouse skin infection model, the combination therapy effectively eliminated MDR bacteria within 24 h, as monitored by fluorescence tracking. The findings demonstrate a promising approach for developing multifunctional hydrogels to combat MDR bacterial infections.

First report of multidrug-resistant carbapenemase-producing Aeromonas caviae co-harboring mcr-3.43 and mcr-7.2.

Hospital sewage serves as a crucial reservoir for antibiotic resistance genes. As colistin and carbapenems are the last-resort antibiotics, the emergence of their resistance genes has become a significant concern in clinical settings. In this study, we found that two novel mcr alleles (mcr-3.43 and mcr-7.2) with two carbapenemase genes (blaNDM-1 and blaKPC-2) were encoded in a single Aeromonas caviae strain isolated from hospital sewage. Our phylogenetic analysis revealed that the mcr-3.43 gene clustered with mcr-3.17 (with 95.55% amino acid identity), while the mcr-7.2 gene clustered with mcr-7.1 (with 68.68% amino acid identity). BLAST search against GenBank showed that mcr-7.2 was exclusively detected in Aeromonas spp. Mobile genetic elements were not found in the genetic context of mcr-7.2, suggesting that the dissemination of mcr-7.2 in Aeromonas spp. may be dependent on vertical transfer or recombination. The blaNDM-1 was adjacent to a recombinase gene and flanked by two IS91 elements, indicating a potential mobilization mechanism mediated by recombination and/or ISs. The blaKPC-2 gene was located on an IncU plasmid and adjacent to an ISKpn6. In summary, our study provides evidence for Aeromonas spp. as one of the potential reservoirs of colistin and carbapenem resistance genes.IMPORTANCEThe study discovered two novel mcr genes (mcr-3.43 and mcr-7.2) and two carbapenemase genes (blaNDM-1 and blaKPC-2) in a single Aeromonas caviae strain retrieved from hospital sewage. Using phylogenetic analysis and comparative data evaluation, the study revealed the genetic relatedness and dissemination potential of the detected resistance genes. With the exclusive discovery that mcr-7.2 is only present in Aeromonas spp. and the lack of mobile genetic elements in its genetic context, there is a strong indication of limited dissemination. The identification of these four resistance genes in a single strain of Aeromonas provided valuable insights into their potential presence in this genus. This study revealed that hospital sewage functions as a significant reservoir for antibiotic resistance genes, including colistin and carbapenem resistance genes.

Private and well drinking water are reservoirs for antimicrobial resistant bacteria

Water quality testing does not recognise antimicrobial resistance (AMR) and is often limited to indicators of faecal contamination Escherichia coli and Enterococcus species. In Europe, data on AMR in drinking water is scarce. In Ireland, as in many countries, household drinking water is supplied via mains or via private wells or water schemes. Using citizen science, we identified Irish private drinking water supplies as reservoirs of antimicrobial resistant bacteria (ARB). Gram-negative (n = 464) and Gram-positive (n = 72) bacteria were isolated. We identified instances of potentially opportunistic ARB such as Enterobacter cloacae, Acinetobacter baumannii and Enterococcus species. We report reservoirs of multidrug resistance in Enterococcus casseliflavus, E. cloacae, E. coli, Stenotrophomonas maltophilia, and Serratia rubidaea. We also identified linezolid-resistant Enterococcus in Irish drinking water. Linezolid is a last-resort antibiotic used to treat vancomycin-resistant Enterococcus sp. Additionally, we identified mobile AMR in three water samples, two of which were carried on IncF group, one on IncQ and five on Col-like plasmids. Our work suggests that private drinking water is a potential sink and source of AMR pathogens. This highlights a value of drinking water surveillance in a One Health framework as the surveillance would provide information regarding the movement and persistence of ARB and ARGs that are able to survive in drinking water and subsequently have the opportunity to be mobilised through humans; linking the environment to the human and potentially threatening human health.

Emergence of pandrug-resistant carbapenemase-producing Enterobacterales in dogs and cats: a cross-sectional study in Egypt.

One of the most important emerging health problems is the increasing role of animals in the rapid global rise in resistance to last-resort antibiotics, such as carbapenems. However, there is limited information on the role of pet animals in harboring and spreading pandrug-resistant (PDR) carbapenemase-producing Enterobacterales (CPE), especially in Egypt. This cross-sectional study was conducted to screen for CPE in healthy and diseased pets using phenotypic and molecular methods and the NG-Test CARBA 5 immunochromatographic assay. Rectal swabs were collected from 62 dogs and 48 cats, incubated overnight in tryptic soy broth containing 10 μg of meropenem disc and subsequently cultured on MacConkey agar supplemented with meropenem (1 mg/L). Sixty-six isolates (60.6%), including 56 Klebsiella pneumoniae, seven Escherichia coli, and three K. oxytoca isolates, were confirmed to be carbapenem-resistant Enterobacterales (CRE) by the disc diffusion method, broth microdilution test, CNPt-direct, and PCR assay targeting carbapenemase genes. Forty-three (65.2%) dogs and 23 (34.8%) cats carried CPE. Of these, 35 (70.0%) were healthy (including 27 dogs and 8 cats) and 31 (52.5%) were diseased (including 16 dogs and 15 cats). bla OXA-181 was the most common gene detected (42/66, 63.6%), followed by bla IMP (40/66, 60.6%), bla OXA-48-like (29/66, 43.9%), bla KPC and bla VIM (20/66, 30.3% each), and bla NDM (17/66, 25.8%). The identified genotypes were bla KPC-2, bla IMP-1, bla VIM-1, bla NDM-1, and bla NDM-5. The CARBA 5 assay showed higher sensitivity and specificity for the detection of NDM, OXA and KPC than that for VIM and IMP genes. Antimicrobial resistance profiles of CRE isolates revealed 20 PDR, 30 extensively drug-resistant (XDR), and 16 multidrug-resistant (MDR) phenotypes. This study provides evidence of colonization with PDR CPE in dogs and cats. To manage the infection or colonization of pets in veterinary clinical settings, extended surveillance systems should be considered, and the use of critical antibiotics should be strictly controlled.

Redirecting pantoprazole as a metallo-beta-lactamase inhibitor in carbapenem-resistant Klebsiella pneumoniae.

The development of resistance to carbapenems in Klebsiella pneumoniae due to the production of metallo-β-lactamases (MBLs) is a critical public health problem because carbapenems are the last-resort drugs used for treating severe infections of extended-spectrum β-lactamases (ESBLs) producing K. pneumoniae. Restoring the activity of carbapenems by the inhibition of metallo-β-lactamases is a valuable approach to combat carbapenem resistance. In this study, two well-characterized clinical multidrug and carbapenem-resistant K. pneumoniae isolates were used. The sub-inhibitory concentrations of pantoprazole and the well-reported metallo-β-lactamase inhibitor captopril inhibited the hydrolytic activities of metallo-β-lactamases, with pantoprazole having more inhibiting activities. Both drugs, when used in combination with meropenem, exhibited synergistic activities. Pantoprazole could also downregulate the expression of the metallo-β-lactamase genes bla NDM and bla VIM. A docking study revealed that pantoprazole could bind to and chelate zinc ions of New Delhi and Verona integron-encoded MBL (VIM) enzymes with higher affinity than the control drug captopril and with comparable affinity to the natural ligand meropenem, indicating the significant inhibitory activity of pantoprazole against metallo-β-lactamases. In conclusion, pantoprazole can be used in combination with meropenem as a new strategy for treating serious infections caused by metallo-β-lactamases producing K. pneumoniae.

Comparison of tet(X4)-containing contigs assembled from metagenomic sequencing data with plasmid sequences of isolates from a cohort of healthy subjects.

Recently discovered tet(X) gene variants have provided new insights into microbial antibiotic resistance mechanisms and their potential consequences for public health. This study focused on detection, analysis, and characterization of Tet(X4)-positive Enterobacterales from the gut microbiota of a healthy cohort of individuals in Singapore using cultivation-dependent and cultivation-independent approaches. Twelve Tet(X4)-positive Enterobacterales strains that were previously obtained from the cohort were fully genome-sequenced and comparatively analyzed. A metagenomic sequencing (MS) data set of the same samples was mined for contigs that harbored the tet(X4) resistance gene. The sequences of tet(X4)-containing contigs and plasmids sequences were compared. The presence of the resistance genes floR and estT (previously annotated as catD) was detected in the same cassette in 10 and 12 out of the 12 tet(X4)-carrying plasmids, respectively. MS detected tet(X4)-containing contigs in 2 out of the 109 subjects, while cultivation-dependent analysis previously reported a prevalence of 10.1%. The tet(X4)-containing sequences assembled from MS data are relatively short (~14 to 33 kb) but show high similarity to the respective plasmid sequences of the isolates. Our findings show that MS can complement efforts in the surveillance of antibiotic resistance genes for clinical samples, while it has a lower sensitivity than a cultivation-based method when the target organism has a low abundance. Further optimization is required if MS is to be utilized in antibiotic resistance surveillance.IMPORTANCEThe global rise in antibiotic resistance makes it necessary to develop and apply new approaches to detect and monitor the prevalence of antibiotic resistance genes in human populations. In this regard, of particular interest are resistances against last-resort antibiotics, such as tigecycline. In this study, we show that metagenomic sequencing can help to detect high abundance of the tigecycline resistance gene tet(X4) in fecal samples from a cohort of healthy human subjects. However, cultivation-based approaches currently remain the most reliable and cost-effective method for detection of antibiotic-resistant bacteria.

Multidrug-resistant Enterococcus faecium strains enter the Norwegian marine environment through treated sewage.

This study aimed to understand the antibiotic resistance prevalence among Enterococcus spp. from raw and treated sewage in Bergen city, Norway. In total, 517 Enterococcus spp. isolates were obtained from raw and treated sewage from five sewage treatment plants (STPs) over three sampling occasions, with Enterococcus faecium as the most prevalent (n = 492) species. E. faecium strains (n = 307) obtained from the influent samples, showed the highest resistance against quinupristin/dalfopristin (67.8%). We observed reduced susceptibility to erythromycin (30.6%) and tetracycline (6.2%) in these strains. E. faecium strains (n = 185) obtained from the effluent samples showed highest resistance against quinupristin/dalfopristin (68.1%) and reduced susceptibility to erythromycin (24.9%) and tetracycline (8.6%). We did not detect resistance against last-resort antibiotics, such as linezolid, vancomycin, and tigecycline in any of the strains. Multidrug-resistant (MDR) E. faecium strains were detected in both influent (2.3%) and effluent (2.2%) samples. Whole genome sequencing of the Enterococcus spp. strains (n = 25) showed the presence of several antibiotic resistance genes, conferring resistance against aminoglycosides, tetracyclines, and macrolides, as well as several virulence genes and plasmid replicons. Two sequenced MDR strains from the effluents belonged to the hospital-associated clonal complex 17 and carried multiple virulence genes. Our study demonstrates that clinically relevant MDR Enterococcus spp. strains are entering the marine environment through treated sewage.

Phylogenomic and phenotypic analyses highlight the diversity of antibiotic resistance and virulence in both human and non-human Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative, opportunistic pathogen that causes infections in the immunocompromised. With a high incidence of muti-drug resistance, carbapenem-resistant A. baumannii is designated as a priority 1 pathogen by the WHO. The current literature has expertly characterized clinical isolates of A. baumannii. As the challenge of these infections has recently been classified as a One Health issue, we set out to explore the diversity of isolates from human and non-clinical sources, such as agricultural surface water, urban streams, various effluents from wastewater treatment plants, and food (tank milk); and, importantly, these isolates came from a wide geographic distribution. Phylogenomic analysis considering almost 200 isolates showed that our diverse set is well-differentiated from the main international clones of A. baumannii. We discovered novel sequence types in both hospital and non-clinical settings and five strains that overexpress the resistance-nodulation-division efflux pump adeIJK without changes in susceptibility reflected by this overexpression. Furthermore, we detected a bla ADC-79 in a non-human isolate despite its sensitivity to all antibiotics. There was no significant differentiation between the virulence profiles of clinical and non-clinical isolates in the Galleria mellonella insect model of virulence, suggesting that virulence is neither dependent on geographic origin nor isolation source. The detection of antibiotic resistance and virulence genes in non-human strains suggests that these isolates may act as a genetic reservoir for clinical strains. This endorses the notion that in order to combat multi-drug-resistant infection caused by A. baumannii, a One Health approach is required, and a deeper understanding of non-clinical strains must be achieved.IMPORTANCEThe global crisis of antibiotic resistance is a silent one. More and more bacteria are becoming resistant to all antibiotics available for treatment, leaving no options remaining. This includes Acinetobacter baumannii. This Gram-negative, opportunistic pathogen shows a high frequency of multi-drug resistance, and many strains are resistant to the last-resort drugs carbapenem and colistin. Research has focused on strains of clinical origin, but there is a knowledge gap regarding virulence traits, particularly how A. baumannii became the notorious pathogen of today. Antibiotic resistance and virulence genes have been detected in strains from animals and environmental locations such as grass and soil. As such, A. baumannii is a One Health concern, which includes the health of humans, animals, and the environment. Thus, in order to truly combat the antibiotic resistance crisis, we need to understand the antibiotic resistance and virulence gene reservoirs of this pathogen under the One Health continuum.

Occurrence and mechanisms of tigecycline resistance in carbapenem- and colistin-resistant Klebsiella pneumoniae in Thailand

Tigecycline has been regarded as one of the most important last-resort antibiotics for the treatment of infections caused by extensively drug-resistant (XDR) bacteria, particularly carbapenem- and colistin-resistant Klebsiella pneumoniae (C-C-RKP). However, reports on tigecycline resistance have been growing. Overall, ~ 4000 K. pneumoniae clinical isolates were collected over a five-year period (2017–2021), in which 240 isolates of C-C-RKP were investigated. Most of these isolates (91.7%) were resistant to tigecycline. Notably, a high-risk clone of ST16 was predominantly identified, which was associated with the co-harboring of blaNDM-1 and blaOXA-232 genes. Their major mechanism of tigecycline resistance was the overexpression of efflux pump acrB gene and its regulator RamA, which was caused by mutations in RamR (M184V, Y59C, I141T, A28T, C99/C100 insertion), in RamR binding site (PI) of ramA gene (C139T), in MarR (S82G), and/or in AcrR (L154R, R13Q). Interestingly, four isolates of ST147 carried the mutated tet(A) efflux pump gene. To our knowledge, this is the first report on the prevalence and mechanisms of tigecycline resistance in C-C-RKP isolated from Thailand. The high incidence of tigecycline resistance observed among C-C-RKP in this study reflects an ongoing evolution of XDR bacteria against the last-resort antibiotics, which demands urgent action.

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.

Bavachin Rejuvenates Sensitivity of Colistin against Colistin-Resistant Gram-Negative Bacteria.

The emergence of plasmid-mediated colistin resistance threatens the efficacy of colistin as a last-resort antibiotic used to treat infection caused by Gram-negative bacteria (GNB). Given the shortage of new antibiotics, the discovery of adjuvants to existing antibiotics is a promising strategy to combat infections caused by multidrug-resistant (MDR) GNB. This study was designed to investigate the potential synergistic antibacterial activity of bavachin, a bioactive compound extracted from the Psoralea Fructus, combined with colistin against MDR GNB. Herein, the synergistic efficacy in vitro and the therapeutic efficacy of colistin combined with bavachin in vivo were evaluated. The synergistic mechanism was detected by fluorescent probe and the transcript levels of mcr-1. Bavachin combined with colistin showed an excellent synergistic activity against GNB, as the FICI ≤ 0.5. In contrast to colistin alone, combination therapy dramatically increased the survival rate of Galleria mellonella and mice in vivo. Moreover, the combination of bavachin and colistin significantly reduced the amount of bacterial biofilm formation, improved the membrane disruption of colistin and inhibited mcr-1 transcription. These findings show that bavachin is a potential adjuvant of colistin, which may provide a new strategy to combat colistin-resistant bacteria infection with lower doses of colistin.

Isolation, Identification, and Antibiogram of Colistin-resistant Acinetobacter baumannii from Rivers in and around Kathmandu Valley

Background: Acinetobacter baumannii, an opportunistic Gram-negative pathogen, poses an escalating threat in clinical settings due to the rise of multidrug-resistant infections. Despite its clinical significance, there exists a considerable gap in understanding its environmental dissemination.
 Aims and Objectives: The primary objective is to examine the distribution of A. baumannii and its antibiotic resistance in river ecosystems. Specifically, we aim to identify strains resistant to Colistin, a last-resort antibiotic, and elucidate the susceptibility patterns to other antibiotics.
 Materials and Methods: Water samples from 10 rivers were collected and subjected to analysis using Leeds Acinetobacter Agar Base and a series of biochemical tests. Antibiotic susceptibility testing, focusing on Colistin resistance, was performed using standard procedures.
 Results: Out of the 284 isolated strains, 14 (4.9%) exhibited resistance to Colistin, while demonstrating varying susceptibility patterns to other antibiotics. Notably, Gentamycin showed effectiveness against resistant strains (14.28%), while Ceftazidime resistance was complete. Colistin-sensitive strains displayed high susceptibility to Ciprofloxacin (84.44%) and lower susceptibility to Chloramphenicol (53.33%). Carbapenem susceptibility was observed across all isolates.
 Conclusion: The study underscores a concerning environmental presence of multidrug-resistant A. baumannii in rivers around Kathmandu Valley, with Sundarijal being the exception. The findings emphasize the necessity of scrutinizing environmental reservoirs for pathogen spread, advocating for heightened awareness of potential health implications beyond clinical settings. Urgent attention is needed to comprehend and counteract the emergence and dissemination of antibiotic resistance, necessitating comprehensive strategies and continued surveillance

Azomycin Orchestrate Colistin-Resistant Enterobacter cloacae Complex's Colistin Resistance Reversal In Vitro and In Vivo.

The Enterobacter cloacae complex (ECC) is a group of nosocomial pathogens that pose a challenge in clinical treatment due to its intrinsic resistance and the ability to rapidly acquire resistance. Colistin was reconsidered as a last-resort antibiotic for combating multidrug-resistant ECC. However, the persistent emergence of colistin-resistant (COL-R) pathogens impedes its clinical efficacy, and novel treatment options are urgently needed. We propose that azomycin, in combination with colistin, restores the susceptibility of COL-R ECC to colistin in vivo and in vitro. Results from the checkerboard susceptibility, time-killing, and live/dead bacterial cell viability tests showed strong synergistic antibacterial activity in vitro. Animal infection models suggested that azomycin-colistin enhanced the survival rate of infected Galleria mellonella and reduced the bacterial load in the thighs of infected mice, highlighting its superior in vivo synergistic antibacterial activity. Crystal violet staining and scanning electron microscopy unveiled the in vitro synergistic antibiofilm effects of azomycin-colistin. The safety of azomycin and azomycin-colistin at experimental concentrations was confirmed through cytotoxicity tests and an erythrocyte hemolysis test. Azomycin-colistin stimulated the production of reactive oxygen species in COL-R ECC and inhibited the PhoPQ two-component system to combat bacterial growth. Thus, azomycin is feasible as a colistin adjuvant against COL-R ECC infection.

Emergence of carbapenem resistant gram-negative pathogens with high rate of colistin resistance in Egypt: A cross sectional study to assess resistance trends during the COVID-19 pandemic

The current study investigated the temporal phenotypic and genotypic antimicrobial resistance (AMR) trends among multi-drug resistant and carbapenem-resistant Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa recovered from Egyptian clinical settings between 2020 and 2021. Bacterial identification and antimicrobial sensitivity of 111 clinical isolates against a panel of antibiotics were performed. Molecular screening for antibiotic resistance determinants along with integrons and associated gene cassettes was implemented. An alarming rate (98.2%) of these isolates were found to be phenotypically resistant to carbapenem. Although 23.9 % K. pneumoniae isolates were phenotypically resistant to colistin, no mobile colistin resistance (mcr) genes were detected. Among carbapenem-resistant isolates, blaNDM and blaOXA-48-like were the most prevalent genetic determinants and were significantly overrepresented among K. pneumoniae. Furthermore, 84.78% of K. pneumoniae isolates co-produced these two carbapenemase genes. The plasmid-mediated quinolone resistance genes (qnrS and qnrB) were detected among the bacterial species and were significantly more prevalent among K. pneumoniae. Moreover, Class 1 integron was detected in 82% of the bacterial isolates. This study alarmingly reveals elevated resistance to last-resort antibiotics such as carbapenems as well as colistin which impose a considerable burden in the health care settings in Egypt. Our future work will implement high throughput sequencing-based antimicrobial resistance surveillance analysis for characterization of novel AMR determinants. This information could be applied as a step forward to establish a robust antibiotic stewardship program in Egyptian clinical settings, thereby addressing the rising challenges of AMR.