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Acinetobacter baumannii (A. baumannii) is considered a critical human pathogen due to multi-drug resistance and increased infections. As a result of the resistance of A. baumannii biofilms to antimicrobial agents, it is necessary to develop new biofilm control strategies. In the present study, we evaluated the efficacy of two previously isolated bacteriophage C2 phage, K3 phage and phage cocktail (C2 + K3 phage) as a therapeutic agent in combination with antibiotic (colistin) against biofilm of multidrug-resistant A. baumannii strains (n = 24). The effects of phage and antibiotics on mature biofilm were investigated simultaneously and sequentially in 24 and 48 h. The combination protocol was more effective than antibiotics alone in 54.16% of the strains in 24 h. The sequential application was more effective than the simultaneous protocol compared with the 24 h single applications. When the application of antibiotics and phages alone was compared with their combined administration in 48 h. The sequential and simultaneous applications were more effective than single applications in all strains except two. We observed that combination of phage and antibiotics could increase biofilm eradication and provides new insights into the use of bacteriophages and antibiotics in the treatment of biofilm-associated infections caused by antibiotic-resistant bacteria., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Banu KASKATEPE reports equipment, drugs, or supplies was provided by Ankara University., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Increasing drug resistance in Gram-negative bacteria presents significant health problems worldwide. Despite notable advances in the development of a new generation of β-lactams, aminoglycosides, and fluoroquinolones, it remains challenging to treat multi-drug resistant Gram-negative bacterial infections. Colistin (polymyxin E) is one of the most efficacious antibiotics for the treatment of multiple drug-resistant Gram-negative bacteria and has been used clinically as a last-resort option. However, the rapid spread of the transferable gene, mcr-1 which confers colistin resistance by encoding a phosphoethanolamine transferase that modifies lipid A of the bacterial membrane, threatens the efficacy of colistin for the treatment of drug-resistant bacterial infections. Colistin-resistant strains of Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae often reduce their susceptibility to other anti-Gram-negative bacterial agents. Thus, drugs effective against colistin-resistant strains or methods to prevent the acquisition of colistin-resistance during treatment are urgently needed. To perform cell-based screenings of the collected small molecules, we have generated colistin-resistant strains of E. coli, A. baumannii, K. pneumoniae, P. aeruginosa, and S. enterica Typhimurium. In-house MIC assay screenings, we have identified that rose bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) is the only molecule that displays unique bactericidal activity against these strains at low concentrations under illumination conditions. This article reports the antibacterial activity of a pharmaceutical-grade rose bengal against colistin-resistant Gram-negative bacteria., (© 2023. The Author(s), under exclusive licence to the Japan Antibiotics Research Association.)

Pseudomonas aeruginosa frequently becomes resistant to aminoglycosides by the acquisition of aminoglycoside modifying enzyme (AME) genes and the occurrence of mutations in the mexZ, fusA1, parRS, and armZ genes. We examined resistance to aminoglycosides in a collection of 227 P. aeruginosa bloodstream isolates collected over 2 decades from a single United States academic medical institution. Resistance rates of tobramycin and amikacin were relatively stable over this time, while the resistance rates of gentamicin were somewhat more variable. For comparison, we examined resistance rates to piperacillin-tazobactam, cefepime, meropenem, ciprofloxacin, and colistin. Resistance rates to the first four antibiotics were also stable, although uniformly higher for ciprofloxacin. Colistin resistance rates were initially quite low, rose substantially, and then began to decrease at the end of the study. Clinically relevant AME genes were identified in 14% of isolates, and mutations predicted to cause resistance were relatively common in the mexZ and armZ genes. In a regression analysis, resistance to gentamicin was associated with the presence of at least one gentamicin-active AME gene and significant mutations in mexZ, parS, and fusA1 . Resistance to tobramycin was associated with the presence of at least one tobramycin-active AME gene. An extensively drug-resistant strain, PS1871, was examined further and found to contain five AME genes, most of which were within clusters of antibiotic resistance genes embedded in transposable elements. These findings demonstrate the relative contributions of aminoglycoside resistance determinants to P. aeruginosa susceptibilities at a United States medical center. IMPORTANCE Pseudomonas aeruginosa is frequently resistant to multiple antibiotics, including aminoglycosides. The rates of resistance to aminoglycosides in bloodstream isolates collected over 2 decades at a United States hospital remained constant, suggesting that antibiotic stewardship programs may be effective in countering an increase in resistance. Mutations in the mexZ, fusA1, parR, pasS, and armZ genes were more common than acquisition of genes encoding aminoglycoside modifying enzymes. The whole-genome sequence of an extensively drug resistant isolate indicates that resistance mechanisms can accumulate in a single strain. Together, these results suggest that aminoglycoside resistance in P. aeruginosa remains problematic and confirm known resistance mechanisms that can be targeted for the development of novel therapeutics., Competing Interests: The authors declare no conflict of interest.

Colistin heteroresistance (HR) refers to a bacterial population comprised of several subpopulations with different levels of resistance to colistin. In this study, we discuss the classic form of HR, in which a resistant subpopulation exists within a predominantly susceptible population. We investigated the prevalence of colistin HR and its evolution into full resistance among 173 clinical carbapenem-resistant Acinetobacter baumannii isolates and examined the effect of HR on clinical outcomes. To determine HR, we performed population analysis profiling. Our results showed a high prevalence of HR (67.1%). To examine evolution of HR strains into full resistance, the HR strains were grown in colistin-containing broth, transferred onto colistin-containing plates, and colonies on these plates were transferred into colistin-free broth. Many of the HR strains (80.2%) evolved into full resistance, 17.2% reverted to HR, and 2.6% were borderline. We used logistic regression to compare 14-day clinical failure and 14-day mortality between patients infected by HR versus susceptible non-HR carbapenem-resistant A. baumannii. In the subgroup of patients with bacteremia, HR was significantly associated with 14-day mortality. IMPORTANCE To our knowledge, this is the first large-scale study to report on HR in Gram-negative bacteria. We described the prevalence of colistin HR in a large sample of carbapenem-resistant A. baumannii isolates, the evolution of many colistin HR isolates to a resistant phenotype following colistin exposure and withdrawal, and the clinical consequences of colistin HR. We found a high prevalence of HR among clinical carbapenem-resistant A. baumannii isolates; most evolved into a resistant phenotype following colistin exposure and withdrawal. In patients treated with colistin, evolution of HR A. baumannii into full resistance could lead to higher rates of treatment failure and contribute to the reservoir of colistin-resistant pathogens in health care settings., Competing Interests: The authors declare a conflict of interest. E.D.M. reports grants and/or personal fees from Pfizer, M.S.D., Rosh, Anglini, Nordic Pharma, and Sanofi-Aventis. G.L.D. reports grants and/or personal fees from Pfizer, Menarini, and M.S.D. K.S.K. reports grants and/or personal fees from Spero Therapeutics, Q.P.E.X., and MicuRx. M.P. reports grants and/or personal fees from Shionogi and Pfizer. Y.C. reports grants and/or personal fees from Allecra Therapeutics, Genentech, Nabriva Therapeutics, Pfizer, PPD, Q.P.E.X. Biopharma, Roche Pharmaceuticals, Spero Therapeutics, VenatoRX Pharmaceuticals. All other authors have no interests to declare.

Colistin is one of the last-resort antibiotics for treating infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However, mcr genes conferring resistance to colistin have been widely identified, which is considered a global threat to public health. Here, we investigated the prevalence and characteristics of mcr -harboring Escherichia coli strains isolated from humans, animals, and foods in China by PCR, antimicrobial susceptibility testing, conjugation experiments, molecular typing, genome sequencing, and bioinformatics analysis. In total, 135 mcr-1 -harboring E. coli isolates were acquired from 847 samples, and 6 isolates carried mcr-3 . Among them, 131 isolates were MDR bacteria. Sixty-five resistance genes conferring resistance to multiple antimicrobials were identified in 135 isolates. The diverse pulsed-field gel electrophoresis (PFGE) patterns and sequence types (STs) of mcr-1 -carrying isolates demonstrated that clonal dissemination was not the dominant mode of mcr-1 transmission. Seven types of plasmids were able to carry mcr-1 in this study, including IncI2, IncX4, IncHI2, p0111, IncY, and two hybrid plasmids. The genetic structures carrying mcr-1 of 60 isolates were successfully transferred into the recipient, including 25 IncI2 plasmids, 23 IncX4 plasmids, and an IncHI2 plasmid. mcr-1-pap2 was the dominant mcr-1 -bearing structure, followed by IS Apl1-mcr-1-pap2- IS Apl1 (Tn 6330 ) and IS Apl1-mcr-1-pap2 , among 7 mcr-1 -bearing structures of 135 isolates. In conclusion, IncI2, IncX4, and IncHI2 plasmids were the major vectors spreading mcr-1 from different geographical locations and sources. The prevalence of Tn 6330 may accelerate the transmission of mcr-1 . Continuous surveillance of mcr-1 and variants in bacteria is vital for evaluating the public health risk posed by mcr genes. IMPORTANCE The spread of polymyxin-resistant Enterobacteriaceae poses a significant threat to public health and challenges the therapeutic options for treating infections on a global level. In this study, mcr-1 -bearing ST10 E. coli was isolated from pigs, pork, and humans simultaneously, which demonstrated that ST10 E. coli was an important vehicle for the spread of mcr-1 among animals, foods, and humans. The high prevalence of mcr-1 -positive E. coli strains in pigs and pork and the horizontal transmission of mcr-1 -bearing plasmids in diverse E. coli strains suggest that pigs and pork are important sources of mcr-1 -positive strains in humans and pose a potential threat to public health. Additional research on the prevalence and characteristics of mcr-1 -positive E. coli is still required to facilitate early warning to improve polymyxin management in hospitals., Competing Interests: The authors declare no conflict of interest.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a devastating effect, globally. We describe, for the first time, the occurrence of carbapenem-resistant bacteria colonizing SARS-CoV-2 patients who developed hospital-associated infections with carbapenemase-producing, Gram-negative bacteria at some isolation centers of SARS-CoV-2 in the eastern part of Libya. In total, at first, 109 samples were collected from 43 patients, with the samples being recovered from oral ( n  = 35), nasal ( n  = 45), and rectal ( n  = 29) cavities. Strain identification was performed via matrix assisted laser desorption ionization-time of flight (MALDI-TOF). Antibiotic susceptibility testing was carried out on Mueller-Hinton agar, using the standard disk diffusion method. MIC determination was confirmed via E-TEST and microdilution standard methods. A molecular study was carried out to characterize the carbapenem and colistin resistance in Gram-negative bacterial strains. All of the positive results were confirmed via sequencing. Klebsiella pneumoniae ( n  = 32), Citrobacter freundii ( n  = 21), Escherichia coli ( n  = 7), and Acinetobacter baumannii ( n  = 21) were the predominant isolated bacteria. Gram-negative isolates were multidrug-resistant and carried different carbapenem resistance-associated genes, including NDM-1 (56/119; 47.05%), OXA-48 (15/119; 12.60%), OXA-23 (19/119; 15.96%), VIM (10/119; 8.40%), and the colistin resistance mobile gene mcr-1 (4/119; 3.36%). The overuse of antimicrobials, particularly carbapenem antibiotics, during the SARS-CoV-2 pandemic has led to the emergence of multidrug-resistant bacteria, mainly K. pneumoniae, A. baumannii, and colistin-resistant E. coli strains. Increased surveillance as well as the rational use of carbapenem antibiotics and, recently, colistin are required to reduce the propagation of multidrug-resistant strains and to optimally maintain the efficacy of these antibiotics. IMPORTANCE In this work, we describe, for the first time, the occurrence of carbapenem-resistant bacteria colonizing COVID-19 patients who developed hospital-associated infections with carbapenemase-producing, Gram-negative bacteria at some isolation centers of COVID-19 in the eastern part of Libya. Our results confirmed that the overuse of antimicrobials, such as carbapenem antibiotics, during the COVID-19 pandemic has led to the emergence of multidrug-resistant bacteria, mainly K. pneumoniae and A. baumannii, as well as colistin resistance., Competing Interests: The authors declare no conflict of interest.

While diverse antibacterials are available in nature, each possesses their own strengths and limitations. One such antibacterial is colicins, proteinaceous toxins that are produced by strains of E. coli to subvert the growth or viability of other E. coli strains. Similarly, predatory bacteria, of which Bdellovibrio bacteriovorus is well-known, are microbes that actively predate on and consume other Gram-negative bacterial strains. While they are all quite active as antibacterials, they also present some limitations: rapid resistance development to colicins while predation does not completely kill their prey. Within this study, therefore, we evaluated the impact of two different colicins (colicin B [ColB] and colicin E5 [ColE5]) and B. bacteriovorus HD100 either individually or together against four clinical isolates of E. coli that are resistant to either colistin or carbapenem. While the ColB and ColE5 were quickly active when used alone, causing a significant loss in viability (>3-log) in susceptible populations after only 3 h, the pathogens always grew afterwards and had final cell densities that were similar with their respective controls. Predation with B. bacteriovorus HD100, in contrast, was most pronounced after 24 h (>3-log reduction in each pathogen viability but never complete). When combined, better killing efficiencies were observed with several of the pathogens, with complete eradication realized for two (<100 viable pathogens per mL). Given the diversity of colicins in nature and the broad-spectrum activities of B. bacteriovorus strains, the results presented here suggest there is a massive potential to control pathogens when they are used together. IMPORTANCE The coupled impact of drug resistance with reduced antibiotic development has placed humankind at a postantibiotic crossroads where antibiotic alternatives are desperately needed. Consequently, we discuss here the combined effectiveness of two vastly different classes of antibacterials, namely, colicins and a predatory bacterium (i.e.,Bdellovibrio bacteriovorus HD100), against two priority pathogenic groups, colistin- and carbapenem-resistant strains of E. coli. While each is effective in its own manner, these antibacterials also display limitations, i.e., the rapid appearance of mutations that confer resistance to the colicins while predatory bacteria do not completely kill their prey. Here, we show these limitations can be overcome using combined treatments of these antibacterials, with two pathogenic E. coli populations completely eradicated within 24 h. Given the diversity of colicins and the broad-spectrum activities of B. bacteriovorus strains, the results presented here suggests there is a massive potential to control pathogens when they are used together., Competing Interests: The authors declare no conflict of interest.

The envelope stress response (ESR) of Gram-negative enteric bacteria senses fluctuations in nutrient availability and environmental changes to avert damage and promote survival. It has a protective role toward antimicrobials, but direct interactions between ESR components and antibiotic resistance genes have not been demonstrated. Here, we report interactions between a central regulator of ESR viz. , the two-component signal transduction system CpxRA ( c onjugative p ilus e x pression), and the recently described mobile colistin resistance protein (MCR-1). Purified MCR-1 is specifically cleaved within its highly conserved periplasmic bridge element, which links its N-terminal transmembrane domain with the C-terminal active-site periplasmic domain, by the CpxRA-regulated serine endoprotease DegP. Recombinant strains harboring cleavage site mutations in MCR-1 are either protease resistant or degradation susceptible, with widely differing consequences for colistin resistance. Transfer of the gene encoding a degradation-susceptible mutant to strains that lack either DegP or its regulator CpxRA restores expression and colistin resistance. MCR-1 production in Escherichia coli imposes growth restriction in strains lacking either DegP or CpxRA, effects that are reversed by transactive expression of DegP. Excipient allosteric activation of the DegP protease specifically inhibits growth of isolates carrying mcr-1 plasmids. As CpxRA directly senses acidification, growth of strains at moderately low pH dramatically increases both MCR-1-dependent phosphoethanolamine (PEA) modification of lipid A and colistin resistance levels. Strains expressing MCR-1 are also more resistant to antimicrobial peptides and bile acids. Thus, a single residue external to its active site induces ESR activity to confer resilience in MCR-1-expressing strains to commonly encountered environmental stimuli, such as changes in acidity and antimicrobial peptides. Targeted activation of the nonessential protease DegP can lead to the elimination of transferable colistin resistance in Gram-negative bacteria. IMPORTANCE The global presence of transferable mcr genes in a wide range of Gram-negative bacteria from clinical, veterinary, food, and aquaculture environments is disconcerting. Its success as a transmissible resistance factor remains enigmatic, because its expression imposes fitness costs and imparts only moderate levels of colistin resistance. Here, we show that MCR-1 triggers regulatory components of the envelope stress response, a system that senses fluctuations in nutrient availability and environmental changes, to promote bacterial survival in low pH environments. We identify a single residue within a highly conserved structural element of mcr-1 distal to its catalytic site that modulates resistance activity and triggers the ESR. Using mutational analysis, quantitative lipid A profiling and biochemical assays, we determined that growth in low pH environments dramatically increases colistin resistance levels and promotes resistance to bile acids and antimicrobial peptides. We exploited these findings to develop a targeted approach that eliminates mcr-1 and its plasmid carriers., Competing Interests: The authors declare no conflict of interest.

Antimicrobial resistance is an increasing threat to human and animal health. Due to the rise of multi-, extensive, and pandrug resistance, last resort antibiotics, such as colistin, are extremely important in human medicine. While the distribution of colistin resistance genes can be tracked through sequencing methods, phenotypic characterization of putative antimicrobial resistance (AMR) genes is still important to confirm the phenotype conferred by different genes. While heterologous expression of AMR genes (e.g., in Escherichia coli) is a common approach, so far, no standard methods for heterologous expression and characterization of mcr genes exist. E. coli B-strains, designed for optimum protein expression, are frequently utilized. Here, we report that four E. coli B-strains are intrinsically resistant to colistin (MIC 8-16 μg/mL). The three tested B-strains that encode T7 RNA polymerase show growth defects when transformed with empty or mcr -expressing pET17b plasmids and grown in the presence of IPTG; K-12 or B-strains without T7 RNA polymerase do not show these growth defects. E. coli SHuffle T7 express carrying empty pET17b also skips wells in colistin MIC assays in the presence of IPTG. These phenotypes could explain why B-strains were erroneously reported as colistin susceptible. Analysis of existing genome data identified one nonsynonymous change in each pmrA and pmrB in all four E. coli B-strains; the E121K change in PmrB has previously been linked to intrinsic colistin resistance. We conclude that E. coli B-strains are not appropriate heterologous expression hosts for identification and characterization of mcr genes. IMPORTANCE Given the rise in multidrug, extensive drug, and pandrug resistance in bacteria and the increasing use of colistin to treat human infections, occurrence of mcr genes threatens human health, and characterization of these resistance genes becomes more important. We show that three commonly used heterologous expression strains are intrinsically resistant to colistin. This is important because these strains have previously been used to characterize and identify new mobile colistin resistance ( mcr ) genes. We also show that expression plasmids (i.e., pET17b) without inserts cause cell viability defects when carried by B-strains with T7 RNA polymerase and grown in the presence of IPTG. Our findings are important as they will facilitate improved selection of heterologous strains and plasmid combinations for characterizing AMR genes, which will be particularly important with a shift to Culture-independent diagnostic tests where bacterial isolates become increasingly less available for characterization., Competing Interests: The authors declare no conflict of interest.

The emergence and dissemination of mobile colistin resistance (mcr) genes across the globe poses a significant threat to public health, as colistin remains one of the last line treatment options for multi-drug resistant infections. Environmental samples (157 water and 157 wastewater) were collected in Ireland between 2018 and 2020. Samples collected were assessed for the presence of antimicrobial resistant bacteria using Brilliance ESBL, Brilliance CRE, mSuperCARBA and McConkey agar containing a ciprofloxacin disc. All water and integrated constructed wetland influent and effluent samples were filtered and enriched in buffered peptone water prior to culture, while wastewater samples were cultured directly. Isolates collected were identified via MALDI-TOF, were tested for susceptibility to 16 antimicrobials, including colistin, and subsequently underwent whole genome sequencing. Overall, eight mcr positive Enterobacterales (one mcr-8 and seven mcr-9) were recovered from six samples (freshwater (n = 2), healthcare facility wastewater (n = 2), wastewater treatment plant influent (n = 1) and integrated constructed wetland influent (piggery farm waste) (n = 1)). While the mcr-8 positive K. pneumoniae displayed resistance to colistin, all seven mcr-9 harbouring Enterobacterales remained susceptible. All isolates demonstrated multi-drug resistance and through whole genome sequencing analysis, were found to harbour a wide variety of antimicrobial resistance genes i.e., 30 ± 4.1 (10-61), including the carbapenemases, bla OXA-48 (n = 2) and bla NDM-1 (n = 1), which were harboured by three of the isolates. The mcr genes were located on IncHI2, IncFIIK and IncI1-like plasmids. The findings of this study highlight potential sources and reservoirs of mcr genes in the environment and illustrate the need for further research to gain a better understanding of the role the environment plays in the persistence and dissemination of antimicrobial resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

In the face of the alarming rise in global antimicrobial resistance, only a handful of novel antibiotics have been developed in recent decades, necessitating innovations in therapeutic strategies to fill the void of antibiotic discovery. Here, we established a screening platform mimicking the host milieu to select antibiotic adjuvants and found three catechol-type flavonoids-7,8-dihydroxyflavone, myricetin, and luteolin-prominently potentiating the efficacy of colistin. Further mechanistic analysis demonstrated that these flavonoids are able to disrupt bacterial iron homeostasis through converting ferric iron to ferrous form. The excessive intracellular ferrous iron modulated the membrane charge of bacteria via interfering the two-component system pmrA / pmrB , thereby promoting the colistin binding and subsequent membrane damage. The potentiation of these flavonoids was further confirmed in an in vivo infection model. Collectively, the current study provided three flavonoids as colistin adjuvant to replenish our arsenals for combating bacterial infections and shed the light on the bacterial iron signaling as a promising target for antibacterial therapies.

Mobilized colistin resistance genes ( mcr ) may confer resistance to the last-resort antimicrobial colistin and can often be transmitted horizontally. mcr encode phosphoethanolamine transferases (PET), which are closely related to chromosomally encoded, intrinsic lipid modification PET (i-PET; e.g., EptA, EptB, CptA). To gain insight into the evolution of mcr within the context of i-PET, we identified 69,814 MCR-like proteins present across 256 bacterial genera (obtained by querying known MCR family representatives against the National Center for Biotechnology Information [NCBI] non-redundant protein database via protein BLAST). We subsequently identified 125 putative novel mcr -like genes, which were located on the same contig as (i) ≥1 plasmid replicon and (ii) ≥1 additional antimicrobial resistance gene (obtained by querying the PlasmidFinder database and NCBI's National Database of Antibiotic Resistant Organisms, respectively, via nucleotide BLAST). At 80% amino acid identity, these putative novel MCR-like proteins formed 13 clusters, five of which represented putative novel MCR families. Sequence similarity and a maximum likelihood phylogeny of mcr , putative novel mcr -like, and ipet genes indicated that sequence similarity was insufficient to discriminate mcr from ipet genes. A mixed-effect model of evolution (MEME) indicated that site- and branch-specific positive selection played a role in the evolution of alleles within the mcr-2 and mcr-9 families. MEME suggested that positive selection played a role in the diversification of several residues in structurally important regions, including (i) a bridging region that connects the membrane-bound and catalytic periplasmic domains, and (ii) a periplasmic loop juxtaposing the substrate entry tunnel. Moreover, eptA and mcr were localized within different genomic contexts. Canonical eptA genes were typically chromosomally encoded in an operon with a two-component regulatory system or adjacent to a TetR-type regulator. Conversely, mcr were represented by single-gene operons or adjacent to pap2 and dgkA , which encode a PAP2 family lipid A phosphatase and diacylglycerol kinase, respectively. Our data suggest that eptA can give rise to "colistin resistance genes" through various mechanisms, including mobilization, selection, and diversification of genomic context and regulatory pathways. These mechanisms likely altered gene expression levels and enzyme activity, allowing bona fide eptA to evolve to function in colistin resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gaballa, Wiedmann and Carroll.)

Background: We investigated the presence of heteroresistance against both tigecycline and colistin in Acinetobacter baumannii and then evaluated the effectiveness of combined antibiotic treatment given the existence of discrete tigecycline- and colistin-resistant subpopulations., Methods: We performed population analysis profiling (PAP) to evaluate the degree of composite heteroresistance in A. baumannii isolates, with the extent of this resistance quantified using subsequent antibiotic susceptibility testing. We then evaluated the amino acid sequence of PmrBAC and the relative mRNA expression levels of pmrB. Finally, we investigated the combined antibiotic efficacy of tigecycline and colistin in multiple-heteroresistant isolates using dual PAP and in vitro time-killing assays., Results: All tigecycline-heteroresistant A. baumannii isolates, with the exception of one colistin-resistant isolate, were also heteroresistant to colistin. Evaluations of the colistin-resistant subpopulations revealed amino acid alterations in PmrA and PmrB and increased expression of pmrB. All tigecycline-resistant subpopulations were susceptible to colistin, and all colistin-resistant subpopulations were susceptible to tigecycline. Dual PAP analysis using tigecycline and colistin showed no heteroresistance, and in vitro time-killing assays revealed that a combination of these two antibiotics effectively eliminated the bacterial cells., Conclusion: Our results suggest that multiple heteroresistance to tigecycline and colistin is highly prevalent among A. baumannii clinical isolates and that these resistant subpopulations exist independently in single multiple heteroresistant isolates. Therefore, our findings may explain the success of combined antibiotic therapies in these infections., (© 2023. The Author(s).)

Objectives: Colistin-resistant Gram-negative pathogens have become a serious worldwide medical problem. This study was designed to reveal the effects of an intrinsic phosphoethanolamine transferase from Acinetobacter modestus on Enterobacterales., Methods: A strain of colistin-resistant A. modestus was isolated from a sample of nasal secretions taken in 2019 from a hospitalised pet cat in Japan. The whole genome was sequenced by next generation sequencing, and transformants of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae harbouring the phosphoethanolamine transferase-encoding gene from A. modestus were constructed. Lipid A modification in E. coli transformants was analysed using electrospray ionization mass spectrometry., Results: Sequencing of the entire genome revealed that the isolate harboured a phosphoethanolamine transferase-encoding gene, eptA&#95;AM, on its chromosome. Transformants of E. coli, K. pneumoniae, and E. cloacae harbouring both the promoter and eptA&#95;AM gene from A. modestus had 32-fold, 8-fold, and 4-fold higher minimum inhibitory concentrations (MICs) for colistin, respectively, than transformants harbouring a control vector. The genetic environment surrounding eptA&#95;AM in A. modestus was similar to that surrounding eptA&#95;AM in Acinetobacter junii and Acinetobacter venetianus. Electrospray ionization mass spectrometry analysis revealed that EptA&#95;AM modified lipid A in Enterobacterales., Conclusion: This is the first report to describe the isolation of an A. modestus strain in Japan and show that its intrinsic phosphoethanolamine transferase, EptA&#95;AM, contributes to colistin resistance in Enterobacterales and A. modestus., Competing Interests: Competing interests None declared., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: Antimicrobial-resistant bacteria of the order Enterobacterales are emerging threats to global public and animal health, leading to morbidity and mortality. The emergence of antimicrobial-resistant, livestock-associated pathogens is a great public health concern. The genera Enterobacter and Lelliottia are ubiquitous, facultatively anaerobic, motile, non-spore-forming, rod-shaped Gram-negative bacteria belonging to the Enterobacteriaceae family and include pathogens of public health importance. Here, we report the first draft genome sequences of a rare Lelliottia nimipressuralis strain MEZLN61 and two Enterobacter kobei strains MEZEK193 and MEZEK194 in Africa., Methods: The bacteria were isolated from environmental wastewater samples. Bacteria were cultured on nutrient agar, and the pure cultures were subjected to whole-genome sequencing. Genomic DNA was sequenced using an Illumina MiSeq platform. Generated reads were trimmed and subjected to de novo assembly. The assembled contigs were analysed for virulence genes, antimicrobial resistance genes, and extra-chromosomal plasmids, and multilocus sequence typing was performed. To compare the sequenced strains with other, previously sequenced E. kobei and L. nimipressuralis strains, available raw read sequences were downloaded, and all sequence files were treated identically to generate core genome bootstrapped maximum likelihood phylogenetic trees., Results: Whole-genome sequencing analyses identified strain MEZLN61 as L. nimipressuralis and strains MEZEK193 and MEZEK194 as E. kobei. MEZEK193 and MEZEK194 carried genes encoding resistance to fosfomycin (fosA), beta-lactam antibiotics (bla ACT-9 ), and colistin (mcr-9). Additionally, MEZEK193 harboured nine different virulence genes, while MEZEK194 harboured eleven different virulence genes. The phenotypic analysis showed that L. nimipressuralis strain MEZLN61 was susceptible to colistin (2 μg/mL), while E. kobei MEZEK193 (64 μg/mL) and MEZEK194 (32 μg/mL) were resistant to colistin., Conclusion: The genome sequences of strains L. nimipressuralis MEZLN6, E. kobei MEZEK193, and E. kobei MEZEK194 will serve as a reference point for molecular epidemiological studies of L. nimipressuralis and E. kobei in Africa. In addition, this study provides an in-depth analysis of the genomic structure and offers important information that helps clarify the pathogenesis and antimicrobial resistance of L. nimipressuralis and E. kobei. The detection of mcr-9, which is associated with very low-level colistin resistance in Enterobacter species, is alarming and may indicate the undetected dissemination of mcr genes in bacteria of the order Enterobacterales. Continuous monitoring and surveillance of the prevalence of mcr genes and their associated phenotypic changes in clinically important pathogens and environmentally associated bacteria is necessary to control and prevent the spread of colistin resistance., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The objective of this study was to develop a rapid prediction method for carbapenem-resistant Klebsiella pneumoniae (CRKP) and colistin-resistant K. pneumoniae (ColRKP) based on routine MALDI-TOF mass spectrometry (MS) results in order to formulate a suitable and rapid treatment strategy. A total of 830 CRKP and 1462 carbapenem-susceptible K. pneumoniae (CSKP) isolates were collected; 54 ColRKP isolates and 1592 colistin-intermediate K. pneumoniae (ColIKP) isolates were also included. Routine MALDI-TOF MS, antimicrobial susceptibility testing, NG-Test CARBA 5, and resistance gene detection were followed by machine learning (ML). Using the ML model, the accuracy and area under the curve for differentiating CRKP and CSKP were 0.8869 and 0.9551, respectively, and those for ColRKP and ColIKP were 0.8361 and 0.8447, respectively. The most important MS features of CRKP and ColRKP were m/z 4520-4529 and m/z 4170-4179, respectively. Of the CRKP isolates, MS m/z 4520-4529 was a potential biomarker for distinguishing KPC from OXA, NDM, IMP, and VIM. Of the 34 patients who received preliminary CRKP ML prediction results (by texting), 24 (70.6%) were confirmed to have CRKP infection. The mortality rate was lower in patients who received antibiotic regimen adjustment based on the preliminary ML prediction (4/14, 28.6%). In conclusion, the proposed model can provide rapid results for differentiating CRKP and CSKP, as well as ColRKP and ColIKP. The combination of ML-based CRKP with preliminary reporting of results can help physicians alter the regimen approximately 24 h earlier, resulting in improved survival of patients with timely antibiotic intervention., (Copyright © 2023 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Monophasic Salmonella Typhimurium (1,4,[5],12:i:-) is one of the leading Salmonella serovars causing human salmonellosis in Europe. It has been observed in Poland since 2008. This serovar is considered the one with the highest rate of mcr prevalence. This report presents a sequence characteristic of the multidrug-resistant (MDR) monophasic S. Typhimurium isolated from a pig faecal sample with the confirmed presence of the mcr-1.1 gene. The genome was assembled into the complete chromosome and 4 plasmids: IncHI2 (232 119 bp), IncFIB/IncFIC (133 901 bp), ColRNAI (6659 bp), and Col8282 (4066bp). The strain identified as ST34 carried multiple antimicrobial resistance genes located both on chromosome (tet(B)) and plasmids: mcr-1.1 and blaTEM-1B on ST4-IncHI2, and mef(B), blaTEM-1B, aadA1, qacL, dfrA12, aadA2, cmlA1, sul3, tet(M) on IncFIB/FIC. The mcr-1.1 gene was previously identified in E. coli deriving mainly from poultry, but this is the first case of the occurrence of mcr-positive Salmonella in Poland. The obtained results of analysis of the genome content draw attention to the problem of multidrug-resistant pathogens, especially in the context of resistance to colistin which is a last-resort antimicrobial., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: The resistance of Gram-negative bacteria to polymyxin B, caused by the plasmid-mediated colistin resistance gene mcr-1, which encodes a phosphoethanolamine transferase (MCR-1), is a serious threat to global public health. Therefore, it is urgent to find new drugs that can effectively alleviate polymyxin B resistance. Through the screening of 78 natural compounds, we found that cajanin stilbene acid (CSA) can significantly restore the susceptibility of polymyxin B to mcr-1 positive Escherichia coli (E. coli)., Purpose: In this study, we tried to evaluate the ability of CSA to restore the susceptibility of polymyxin B towards the E. coli, and explore the mechanism of sensitivity recovery., Study Design and Methods: Checkerboard MICs, time-killing curves, scanning electron microscope, lethal and semi-lethal models of infection in mice were used to assess the ability of CSA to restore the susceptibility of polymyxyn to E. coli. The interaction between CSA and MCR-1 was evaluated using surface plasmon resonance (SPR), and molecular docking experiments., Results: Here, we find that CSA, a potential direct inhibitor of MCR-1, effectively restores the sensitivity of E. coli to polymyxin B. CSA can restore the sensitivity of polymyxin B to drug-resistant E. coli, and the MIC value can be reduced to 1 μg/ml. The time killing curve and scanning electron microscopy results also showed that CSA can effectively restore polymyxin B sensitivity. In vivo experiments showed that the simultaneous use of CSA and polymyxin B can effectively reduce the infection of drug-resistant E. coli in mice. SPR and molecular docking experiments confirmed that CSA strongly bound to MCR-1. The 17-carbonyl oxygen and 12- and 18‑hydroxyl oxygens of CSA were the key sites binding to MCR-1., Conclusion: CSA is able to significantly restore the sensitivity of polymyxin B to E. coli in vivo and in vitro. CSA inhibits the enzymatic activity of the MCR-1 protein by binding to key amino acids at the active center of the MCR-1 protein., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Despite recent scientific advances, the global load of bacterial disease remains high and has been established against a backdrop of increasing antimicrobial resistance. Therefore, there is a pressing need for highly effective and natural antibacterial agents. In the present work, the antibiofilm effect provided by essential oils was evaluated. Of these, cinnamon oil extract showed potent antibacterial and antibiofilm activities against Staphylococcus aureus at an MBEC of 75.0 µg/mL. It was revealed that benzyl alcohol, 2-propenal-3-phenyl, hexadecenoic acid, and oleic acid were the major components of the tested cinnamon oil extract. In addition, the interaction between the cinnamon oil and colistin showed a synergistic effect against S. aureus . Cinnamon oil that had been combined with colistin was encapsulated by liposomes to enhance the essential oil's chemical stability, demonstrating a particle size of 91.67 nm, a PDI of 0.143, a zeta potential of -0.129 mV, and an MBEC of 50.0 µg/mL against Staphylococcus aureus . Scanning electron microscopy was employed to observe the morphological changes in the Staphylococcus aureus biofilm that was treated with the encapsulated cinnamon oil extract/colistin. As a natural and safe option, cinnamon oil exhibited satisfactory antibacterial and antibiofilm performance. The application of liposomes further improved the stability of the antibacterial agents and extended the essential oil release profile.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a difficult-to-treat nosocomial pathogen responsible for significant morbidity and mortality. Sulbactam-durlobactam (SUL-DUR), formerly ETX2514SUL, is a novel β-lactam-β-lactamase inhibitor designed specifically for the treatment of CRAB infections. The United States Food and Drug Administration (FDA) fast-track approval of SUL-DUR for the treatment of CRAB infections is currently pending after completion of the phase III ATTACK trial, which compared SUL-DUR to colistin, both in combination with imipenem-cilastatin (IMI) for patients with CRAB-associated hospital-acquired bacterial pneumonia, ventilator-associated pneumonia, and bacteremia. The results of this trial demonstrated that SUL-DUR was non-inferior to colistin for CRAB while also possessing a much more favorable safety profile. SUL-DUR was well-tolerated with the most common side effects being headache, nausea, and injection-site phlebitis. With the current landscape of limited effective treatment options for CRAB infections, SUL-DUR represents a promising therapeutic option for the treatment of these severe infections. This review will discuss the pharmacology, spectrum of activity, pharmacokinetics/pharmacodynamics, in vitro and clinical studies, safety, dosing, administration, as well as the potential role in therapy for SUL-DUR., (© 2023 Pharmacotherapy Publications, Inc.)

Objectives: To characterise 11 colistin- and carbapenem-resistant Acinetobacter baumannii isolates recently emerging in hospital settings., Methods: A. baumannii isolates were collected from hospitalised patients under colistin treatment in three countries of Southeast Europe: Turkey, Croatia, and Bosnia and Herzegovina. Isolates were identified using molecular methods., Results: Isolates from Turkey and Croatia belong to the sequence types ST195 or ST281 of the clone lineage 2, while the single isolate from Bosnia and Herzegovina belongs to the ST231 of clone lineage 1. All isolates turned out to be highly resistant to colistin (MIC ≥ 16 mg/L) and have point mutations in pmrCAB operon genes. The colistin-resistant isolate from Bosnia and Herzegovina had a unique P170L point mutation in the pmrB gene and the R125H point mutation in the pmrC gene. The L20S mutation in the pmrA gene was detected only in isolates from Croatia and has never been reported before in isolates from this country., Conclusion: Colistin resistance in A. baumannii in hospitalised patients receiving colistin treatment is a result of chromosomal mutations. The pattern of point mutations in pmrCAB genes suggests a spread of specific colistin-resistant isolates within the hospital., Competing Interests: Competing interests None to declare., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Colistin is a last resort drug for the treatment of multiple drug-resistant (MDR) Gram-negative bacterial infections. Rapid methods to detect resistance are highly desirable. Here, we evaluated the performance of a commercially available MALDI-TOF MS-based assay for colistin resistance testing in Escherichia coli at two different sites. Ninety clinical E. coli isolates were provided by France and tested in Germany and UK using a MALDI-TOF MS-based colistin resistance assay. Lipid A molecules of the bacterial cell membrane were extracted using the MBT Lipid Xtract Kit™ (RUO; Bruker Daltonics, Germany). Spectra acquisition and evaluation were performed by the MBT HT LipidART Module of MBT Compass HT (RUO; Bruker Daltonics) on a MALDI Biotyper® sirius system (Bruker Daltonics) in negative ion mode. Phenotypic colistin resistance was determined by broth microdilution (MICRONAUT MIC-Strip Colistin, Bruker Daltonics) and used as a reference. Comparing the results of the MALDI-TOF MS-based colistin resistance assay with the data of the phenotypic reference method for the UK, sensitivity and specificity for the detection of colistin resistance were 97.1% (33/34) and 96.4% (53/55), respectively. Germany showed 97.1% (33/34) sensitivity and 100% (55/55) specificity for the detection of colistin resistance by MALDI-TOF MS. Applying the MBT Lipid Xtract™ Kit in combination with MALDI-TOF MS and dedicated software showed excellent performances for E. coli. Analytical and clinical validation studies must be performed to demonstrate the performance of the method as a diagnostic tool., (© 2023. The Author(s).)

Objectives: The emergence of carbapenem-resistant Enterobacter cloacae complex (CR-ECC) has posed significant global challenges to the clinical treatment of healthcare-associated infections. This study reports the clonal outbreak of NDM-1-producing Enterobacter hormaechei (E. hormaechei) with the coexistence of tmexCD2-toprJ2 and mcr-9 in China., Methods: During the outbreak (January 2018 - December 2021), 15 non-repetitive multidrug-resistant E. hormaechei strains were obtained from 13 patients in a tertiary hospital. Antimicrobial susceptibility testing, plasmid stability, plasmid conjugation, plasmid fitness evaluation, colistin induction, whole-genome sequencing, and bioinformatics analysis were performed. A phylogenetic tree was constructed based on single nucleotide polymorphisms of core genomes to illustrate the evolutionary dynamics of mcr-9-carrying E. hormaechei strains worldwide., Results: The 15 E. hormaechei strains belonged to the high-risk international clone ST78 and co-harboured tmexCD2-toprJ2 and bla NDM-1 , of which 12 E. hormaechei strains carried the mcr-9 gene. Whole-genome sequencing analysis revealed that tmexCD2-toprJ2 and bla NDM-1 coexisted on the IncFIB/IncFII-type plasmid, which could be transferred to Escherichia coli J53 by conjugation and had a significant effect on host fitness. The mcr-9 gene was located between two insertion sequences, IS903B and IS1R, but lacked the two-component system regulatory gene qseBC, which might be the reason for all mcr-9-positive E. hormaechei strains remaining susceptible to colistin. The expression of mcr-9 was not inducible in strains confirmed by colistin induction assays. Phylogenetic analysis illustrated the silent spread and rapid evolution of mcr-9-carrying E. hormaechei worldwide., Conclusion: This study enriched the epidemiological and genomic characterisation of the coexistence of tmexCD2-toprJ2 and mcr-9 in ST78 CR-ECC isolates and demonstrated that they could prolong clonal dissemination in a tertiary hospital in China. Continuous epidemiological surveillance and molecular characterisation of CR-ECC should be conducted to monitor the evolution of CR-ECC around the world., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Aim: We investigated the therapeutic capacity of the isolated Klebsiella bacteriophage NK20 against pandrug-resistant strains. Moreover, we assessed the impact of resistance development on the overall therapeutic outcome both in vitro and in vivo., Main Methods: The pandrug-resistant K. pneumoniae Kp20 is used as a host strain for the isolation of bacteriophages using sewage samples. Spot assay was then used to compare the spectra of the isolated phages, while kinetic and genomic analysis of the phage with the broadest spectrum was assessed. Antibacterial potential of the phage was assessed using turbidimetric assay and MIC with and without colistin. Finally, the therapeutic efficacy was evaluated in vivo using a rat respiratory infection model., Key Findings: The isolated lytic bacteriophage (NK20) showed a relatively broad spectrum and an acceptable genomic profile. In vitro antibacterial assay revealed bacterial resistance development after 12 h. Colistin inhibited bacterial regrowth and reduced pandrug-resistant strains' colistin MICs. Despite the isolation of resistant clones, intranasal administration of NK20 significantly (p < 0.05) reduced the bacterial load in both the pulmonary and blood compartments and rescued 100 % of challenged rats. Histological and immunological analysis of treated animals' lung tissue revealed less inflammation and lower TNF-α and caspase-3 expression., Significance: NK20 is a promising candidate that rescued rats from untreatable, pan-drug-resistant K. pneumoniae Kp20. Moreover, it steers the evolution of resistant mutants with higher sensitivity to colistin and less virulence, opening the door for using phages as sensitizing and anti-virulence entities rather than direct killer., Competing Interests: Declaration of competing interest The authors declare that the research was conducted with no financial or commercial relationships that could be explained as a potential conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Treating multidrug-resistant infections has increasingly relied on last-resort antibiotics, including polymyxins, for example colistin. As polymyxins are given routinely, the prevalence of their resistance is on the rise and increases mortality rates of sepsis patients. The global dissemination of plasmid-borne colistin resistance, driven by the emergence of mcr-1, threatens to diminish the therapeutic utility of polymyxins from an already shrinking antibiotic arsenal. Restoring sensitivity to polymyxins using combination therapy with sensitizing drugs is a promising approach to reviving its clinical utility. Here we describe the ability of the biotin biosynthesis inhibitor, MAC13772, to synergize with colistin exclusively against colistin-resistant bacteria. MAC13772 indirectly disrupts fatty acid synthesis (FAS) and restores sensitivity to the last-resort antibiotic, colistin. Accordingly, we found that combinations of colistin and other FAS inhibitors, cerulenin, triclosan and Debio1452-NH 3 , had broad potential against both chromosomal and plasmid-mediated colistin resistance in chequerboard and lysis assays. Furthermore, combination therapy with colistin and the clinically relevant FabI inhibitor, Debio1452-NH 3 , showed efficacy against mcr-1 positive Klebsiella pneumoniae and colistin-resistant Escherichia coli systemic infections in mice. Using chemical genomics, lipidomics and transcriptomics, we explored the mechanism of the interaction. We propose that inhibiting FAS restores colistin sensitivity by depleting lipid synthesis, leading to changes in phospholipid composition. In all, this work reveals a surprising link between FAS and colistin resistance., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The management of severe neurologic infections due to multidrug-resistant (MDR) Klebsiella pneumoniae infection remains a challenge. Limited antibiotic treatment regimens make treatment of severe MDR K. pneumoniae infection more difficult. We describe a patient who developed severe meningitis and ventriculitis after craniotomy caused by MDR K. pneumoniae and was effectively treated with the administration of multichannel applications (intravenous, intrathecal and aerosol inhalation) of colistin sulfate. This case provides clinical evidence that the intrathecal, intravenous and aerosol inhalation of colistin sulfate by multichannel application can be a last resort in refractory intracranial infection by MDR K. pneumoniae .

Background: Pseudomonas aeruginosa is a significant cause of infection in burn wounds. Antibiotics are widely used to treat infectious diseases, and alongside their therapeutic benefits, they can damage host cells. Significant side effects, such as nephrotoxicity and neurotoxicity, are observed in 60% of patients treated with colistin. Therefore, using a suitable alternative instead of antibiotics is paramount. This study aimed to investigate the effects of phage therapy and antibiotic therapy on memory function in rats with P. aeruginosa infected burn wounds., Methods: Adult male rats were divided into three groups: (1) infected without treatment (control), (2) infected and treated with colistin antibiotic (3,000,000 international units/kg/day), and (3) infected and treated with 100 μL of phage suspension (approximately 10 9 PFU/mL). In all animals, after anesthesia, a third-degree burn was created in the back area. One hour later, treatment was performed for seven consecutive days. Passive avoidance test, novel object recognition test, locomotion activity, hippocampal neuron count, and oxidative stress measurement in blood serum were performed., Results: In antibiotic-treated group memory recall, recognition index, number of healthy neurons in CA1, CA2, and CA3 hippocampus areas and the amounts of MDA, and FRAP significantly decreased compared with the control group. The phage-treated group was not shown any harmful effect on the memory process, number of healthy hippocampal neurons, and showed more positive effects in blood serum examinations compared with the antibiotic group., Conclusions: Phage therapy could be a safe and effective alternative to antibiotics in the treatment of burn-related infections., (© 2023 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Objectives: In veterinary medicine, colistin has been widely used as therapeutic and prophylactic agent, and for growth promotion. However, colistin has been re-introduced into treatment of human MDR bacterial infections. We assessed the characteristics and spread of plasmid-borne colistin resistance among healthy pigs, workers with animal-contact and their household members in Thailand., Methods: WGS and MIC data of 146 mcr-positive isolates from a cross-sectional One Health study were analysed. Long-read sequencing and conjugation were performed for selected isolates., Results: mcr-carrying isolates were detected in 38% of pooled-pig samples and 16% of human faecal samples. Of 143 Escherichia coli and three Escherichia fergusonii, mcr-1, mcr-3, and mcr-9 variants were identified in 96 (65.8%), 61 (41.8%) and one (0.7%) isolate, respectively. Twelve E. coli co-harboured two mcr variants (mcr-1 and mcr-3). Clonal transmission was detected in five out of 164 farms. mcr-1 was mostly harboured by epidemic IncX4 and IncHI1 plasmids (89.9%). Conversely, mcr-3 was harboured by a range of different plasmids. Comparative plasmid studies suggested IncP and IncFII plasmids as possible endemic mcr-3 plasmids in Asian countries. Moreover, mcr-3 was associated with different mobile genetic elements including TnAs2, ISKpn40 and IS26/15DI. Detected genetic signatures (DRs) indicated recent mcr-3 transpositions, underlining the mobilizable nature of the mcr-3 cassette., Conclusions: The epidemiology of mcr and the possible evolution of successful plasmids and transposition modules should be carefully monitored. Of special concern is the growing number of different horizontal gene transferring pathways encompassing various transposable modules the mcr genes can be shared between bacteria., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Background. Klebsiella variicola was once recognised as a benign plant-endosymbiont but recent case reports suggest that it is a newly emerging Gram-negative pathogen related to opportunistic infection of multiple sites in humans. Methods. Antimicrobial susceptibility testing was performed using broth microdilution method. To identify colistin resistance mechanisms, phoPQ , pmrAB , and mgrB were sequenced and their mRNA expression was analysed using quantitative real-time PCR. In addition, we tried to detect crrAB and mcr . The lipid A moieties of colistin-susceptible and -resistant isolates were analysed using MALDI-TOF. Results. Among the two K. variicola isolates, one is colistin-resistant, and another is colistin-susceptible. The colistin-resistant K. variicola isolate showed no mutations in phoPQ, pmrAB , and mgrB , and crrAB and mcr were not identified. However, its phoQ and pbgP expression was significantly higher and amino-arabinosylated lipid A with hexa-acylated species in lipopolysaccharide was identified. Conclusions. We found that colistin resistance in K. variicola was mediated by the modification of lipid A. Although the isolate was obtained from faecal samples of healthy adults, colistin-resistant K. variicola challenges public health as an opportunistic pathogen.

The incidence of infections caused by resistant Gram-negative pathogens has become a critical factor in public health due to the limitation of therapeutic options for the control of infections caused, especially, by Enterobacteriaceae (Escherichia coli and Klebsiella pneumoniae), Pseudomonas spp., and Acinetobacter spp. Thus, given the increase in resistant pathogens and the reduction of therapeutic options, polymyxins were reintroduced into the clinic. As the last treatment option, polymyxins were regarded as the therapeutic key, since they were one of the few classes of antimicrobials that had activity against multidrug-resistant Gram-negative bacilli. Nonetheless, over the years, the frequent use of this antimicrobial has led to reports of resistance cases. In 2015, mcr (mobile colistin resistance), a colistin resistance gene, was described in China. Due to its location on carrier plasmids, this gene is characterized by rapid spread through conjugation. It has thus been classified as a rising threat to public health worldwide. In conclusion, based on several reports that show the emergence of mcr in different regional and climatic contexts and species of isolates, this work aims to review the literature on the incidence of the mcr gene in Brazil in different regions, types of samples identified, species of isolates, and type of carrier plasmid., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

In this study, we report the carbapenemase-encoding genes and colistin resistance in Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii , and Pseudomonas aeruginosa in the second year of the COVID-19 pandemic. Clinical isolates included carbapenem-resistant K. pneumoniae , carbapenem-resistant E. coli , carbapenem-resistant A. baumannii , and carbapenem-resistant P. aeruginosa . Carbapenemase-encoding genes were detected by PCR. Carbapenem-resistant K. pneumoniae and carbapenem-resistant E. coli isolates were analyzed using the Rapid Polymyxin NP assay. mcr genes were screened by PCR. Pulsed-field gel electrophoresis and whole-genome sequencing were performed on representative isolates. A total of 80 carbapenem-resistant E. coli , 103 carbapenem-resistant K. pneumoniae , 284 carbapenem-resistant A. baumannii , and 129 carbapenem-resistant P. aeruginosa isolates were recovered. All carbapenem-resistant E. coli and carbapenem-resistant K. pneumoniae isolates were included for further analysis. A selection of carbapenem-resistant A. baumannii and carbapenem-resistant P. aeruginosa strains was further analyzed (86 carbapenem-resistant A. baumannii and 82 carbapenem-resistant P. aeruginosa ). Among carbapenem-resistant K. pneumoniae and carbapenem-resistant E. coli isolates, the most frequent gene was bla NDM (86/103 [83.5%] and 72/80 [90%], respectively). For carbapenem-resistant A. baumannii , the most frequently detected gene was bla OXA-40 (52/86, 60.5%), and for carbapenem-resistant P. aeruginosa , was bla VIM (19/82, 23.2%). For carbapenem-resistant A. baumannii , five indistinguishable pulsotypes were detected. Circulation of K. pneumoniae New Delhi metallo-β-lactamase (NDM) and E. coli NDM was detected in Mexico. High virulence sequence types (STs), such as K. pneumoniae ST307, E. coli ST167, P. aeruginosa ST111, and A. baumannii ST2, were detected. Among K. pneumoniae isolates, 18/101 (17.8%) were positive for the Polymyxin NP test (two, 11.0% positive for the mcr-1 gene, and one, 5.6% with disruption of the mgrB gene). All E. coli isolates were negative for the Polymyxin NP test. In conclusion, K. pneumoniae NDM and E. coli NDM were detected in Mexico, with the circulation of highly virulent STs. These results are relevant in clinical practice to guide antibiotic therapies considering the molecular mechanisms of resistance to carbapenems.

Introduction: Pseudomonas aeruginosa is an opportunistic pathogen, which causes healthcare-associated infections in immunosuppressed patients. They exhibit resistance to multiple classes of antibiotics via various mechanisms such as the over-expression of efflux pumps, decreased production of the outer membrane protein (D2 porin), over-expression of the chromosomally encoded AmpC cephalosporinase, modification of drugs, and mutation(s) at the target site of the drug. The bacteria also develop antibiotic resistance through the acquisition of resistance genes carried on mobile genetic elements. Limited data on phenotypic as well as genotypic characterization of MDR P. aeruginosa in Nepal infers the needs for this study. This study was carried out to determine the prevalence rate of metallo-β-lactamase (MBL-producer) as well as colistin resistant multidrug resistant (MDR) P. aeruginosa in Nepal and also to detect MBL, colistin resistance, and efflux pump encoding genes i.e. bla NDM-1 , mcr-1 and MexB respectively in MDR P. aeruginosa isolated from clinical samples., Methods/methodology: A total of 36 clinical isolates of P. aeruginosa were collected. All bacterial isolates were phenotypically screened for antibiotic susceptibility using Kirby Bauer Disc Diffusion method. All the multidrug resistant P. aeruginosa were phenotypically screened for MBL producer by Imipenem-EDTA combined disc diffusion test (CDDT). Similarly, MIC value for colistin was also determined by broth microdilution method. Genes encoding carbapenemase (bla NDM-1 ), colistin resistant (mcr-1) and efflux pump activity (MexB) were assayed by PCR., Results: Among 36 P. aeruginosa, 50% were found to be MDR among which 66.7% were found to be MBL producer and 11.2% were found to be colistin resistant. Among MDR P. aeruginosa, 16.7%, 11.2% and 94.4% were found to be harbouring bla NDM-1 , mcr-1 and MexB genes respectively., Conclusion: In our study, carbapenemase production (encoded by bla NDM-1 ), colistin resistant enzyme production (encoded by mcr-1), and expression of efflux pump (encoded by MexB) are found to be one of the major causes of antibiotic resistance in P. aeruginosa. Therefore, periodic phenotypic as well as genotypic study in Nepal on P. aeruginosa would provide the scenario of resistance pattern or mechanisms in P. aeruginosa. Furthermore, new policies or rules can be implemented in order to control the P. aeruginosa infections., (© 2023. The Author(s).)

A bacterial species is considered to be intrinsically resistant to an antimicrobial when nearly all of the wild-type isolates (i.e., those without acquired resistance) exhibit minimum inhibitory concentration (MIC) values that are sufficiently high such that susceptibility testing is unnecessary, and that the antimicrobial should not be considered for therapy. Accordingly, knowledge of intrinsic resistance influences both the selection of treatment regimens and the approach to susceptibility testing in the clinical laboratory, where unexpected results also facilitate the recognition of microbial identification or susceptibility testing errors. Previously, limited data have suggested that Hafnia spp. may be intrinsically resistant to colistin. We evaluated the in vitro activity of colistin against 119 Hafniaceae that were isolated from human samples: 75 (63%) from routine clinical cultures and 44 (37%) from stool samples of travelers undergoing screening for antimicrobial resistant organisms. Broth microdilution colistin MICs were ≥4 μg/mL for 117 of 119 (98%) isolates. Whole-genome sequencing of 96 of the isolates demonstrated that the colistin-resistant phenotype was not lineage-specific. 2 of the 96 (2%) isolates harbored mobile colistin resistance genes. Compared to whole-genome sequencing, VITEK MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and VITEK 2 GN ID failed to consistently distinguish between Hafnia alvei, Hafnia paralvei, and Obesumbacterium proteus. In conclusion, using a reference antimicrobial susceptibility testing method and a genetically diverse collection of isolates, we found Hafnia spp. to be intrinsically resistant to colistin. The recognition of this phenotype will help inform rational approaches by which to perform antimicrobial susceptibility testing and therapy for patients with infections that are caused by Hafnia spp., Competing Interests: The authors declare a conflict of interest. S.E.T., R.C.L., and V.M.P. receive royalties from UpToDate. R.C.L. also received payments for editorial services from the CDC Foundation. V.M.P. is a member of the Clinical and Laboratory Standards Institute Subcommittee on Antimicrobial Susceptibility Testing.

Background and Objectives : Developing effective treatment outcomes for multidrug-resistant Acinetobacter baumannii (MRAB) infections, with colistin and tigecycline the current frontline therapies, is challenging, because of the risk of renal toxicity and low blood concentrations of active ingredients when administered intravenously. This study aimed to identify the effect of combination therapy using conventional antimicrobial agents that are used for treating drug-resistant bacteria and the additional synergistic effect of four probiotic culture extracts isolated from the human body and Lactobacillus preparations. Materials and Methods : The antimicrobial combination and synergistic effect of adding Lactobacillus extract against 33 strains of A. baumannii isolated from pus, urine, and other specimens submitted to the Department of Laboratory Medicine of a university hospital, located in Gyeonggi-do, Korea, was investigated over a 3-year period between January 2017 and December 2019. Results : Antimicrobial susceptibility tests on bacteria isolated in clinical practice demonstrated that 26 strains (79%) were MRAB, while multi-locus sequence typing indicated that ST191 was the predominant type (45%; n = 15). Checkerboard test results demonstrated that combination therapy using meropenem and colistin had the highest synergistic effect (fractional inhibitory concentration index = 0.5), while the time-kill assay test using Lactobacillus spp. culture extract exhibited an inhibitory effect within 1 h and complete inhibition of MRAB within 3 h. Lactobacillus paracasei exhibited the fastest antimicrobial reactivity and longest sustained antimicrobial activity. Conclusion : These findings provide useful foundational data for an appropriate combination of colistin with other antimicrobial agents for treating MRAB infection in clinical settings, and the use of various probiotic culture extracts to reduce the required dosage, and therefore toxicity of colistin.

Purpose: As an under-explored biomaterial, bacterial biofilms have a wide range of applications in the green synthesis of nanomaterials. The biofilm supernatant of Pseudomonas aeruginosa PA75 was used to synthesize novel silver nanoparticles (AgNPs). BF75-AgNPs were found to possess several biological properties., Methods: In this study, we biosynthesized BF75-AgNPs using biofilm supernatant as the reducing agent, stabilizer, and dispersant and investigated their biopotential in terms of antibacterial, antibiofilm, and antitumor activities., Results: The synthesized BF75-AgNPs demonstrated a typical face-centered cubic crystal structure; they were well dispersed; and they were spherical with a size of 13.899 ± 4.036 nm. The average zeta potential of the BF75-AgNPs was -31.0 ± 8.1 mV. The BF75-AgNPs exhibited strong antibacterial activities against the methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase Escherichia coli (ESBL-EC), extensively drug-resistant Klebsiella pneumoniae (XDR-KP), and carbapenem-resistant Pseudomonas aeruginosa (CR-PA). Moreover, the BF75-AgNPs had a strong bactericidal effect on XDR-KP at 1/2 × MIC, and the expression level of reactive oxygen species (ROS) in bacteria was significantly increased. A synergistic effect was observed when the BF75-AgNPs and colistin were used for the co-treatment of two colistin-resistant XDR-KP strains, with fractional inhibitory concentration index (FICI) values of 0.281 and 0.187, respectively. Furthermore, the BF75-AgNPs demonstrated a strong biofilm inhibition activity and mature biofilm bactericidal activity against XDR-KP. The BF75-AgNPs also exhibited a strong antitumor activity against melanoma cells and low cytotoxicity against normal epidermal cells. In addition, the BF75-AgNPs increased the proportion of apoptotic cells in two melanoma cell lines, and the proportion of late apoptotic cells increased with BF75-AgNP concentration., Conclusion: This study suggests that BF75-AgNPs synthesized from biofilm supernatant have broad prospects for antibacterial, antibiofilm, and antitumor applications., Competing Interests: The authors report no conflicts of interest in this work., (© 2023 Xia et al.)

Phage lytic enzymes are promising antimicrobial agents. In this study, an endolysin derived from vB&#95;AbaM&#95;PhT2 (vPhT2), was identified. This endolysin represented the conserved lysozyme domain. Recombinant endolysin (lysAB- vT2) and hydrophobic fusion endolysin (lysAB-vT2-fusion) were expressed and purified. Both endolysins showed lytic activity against bacterial crude cell wall of Gram-negative bacteria. The MIC of lysAB-vT2-fusion was 2 mg/ml corresponding to 100 µM, while the MIC of lysAB-vT2 was more than 10 mg/ml (400 µM). Combination of lysAB-vT2-fusion with colistin, polymyxin B or copper was synergistic against A. baumannii (FICI value as 0.25). Antibacterial activity of lysAB-vT2-fusion plus colistin at the fractional inhibitory concentrations (FICs) revealed that it can inhibit Escherichia coli, Klebsiella pneumoniae and various strains of extremely drug-resistant A. baumannii (XDRAB) and phage resistant A. baumannii. The lysAB- vT2-fusion still retained its antibacterial activity after incubating the enzyme at 4, 20, 40 and 60 °C for 30 min. The lysAB-vT2-fusion could inhibit the mature biofilm, and incubation of lysAB-vT2-fusion with T24 human cells infected with A. baumannii led to a partial reduction of LDH release from T24 cells. In summary, our study highlights the antimicrobial ability of engineered lysAB-vT2-fusion endolysin, which can be applied for the control of A. baumannii infection., (© 2023. Crown.)

Background: While the treatment of ESBL-producing Enterobacterales osteomyelitis relies on carbapenems, the optimal regimen for OXA48 types remains unclear. We evaluated the efficacy of ceftazidime/avibactam in different combinations in an experimental model of OXA-48-/ESBL-producing Escherichia coli osteomyelitis., Methods: E. coli pACYC184 is a clinical strain harbouring blaOXA-48 and blaCTX-M-15 inserts, with 'increased exposure susceptibility' to imipenem (MIC, 2 mg/L), gentamicin (MIC, 0.5 mg/L), colistin (MIC, 0.25 mg/L), ceftazidime/avibactam (MIC, 0.094 mg/L) and fosfomycin (MIC, 1 mg/L), and resistance to ceftazidime (MIC, 16 mg/L). Osteomyelitis was induced in rabbits by tibial injection of 2 × 108 cfu of OXA-48/ESBL E. coli. Treatment started 14 days later for 7 days in six groups: (1) control, (2) colistin 150.000 IU/kg subcutaneously (SC) q8h, (3) ceftazidime/avibactam 100/25 mg/kg SC q8h, (4) ceftazidime/avibactam + colistin, (5) ceftazidime/avibactam + fosfomycin 150 mg/kg SC q12h, (6) ceftazidime/avibactam + gentamicin 15 mg/kg intramuscularly (IM) q24h. Treatment was evaluated at Day 24 according to bone cultures., Results: In vitro, time-kill curves of ceftazidime/avibactam in combination showed a synergistic effect. In vivo, compared with controls, rabbits treated with colistin alone had similar bone bacterial density (P = 0.50), whereas ceftazidime/avibactam alone or in combinations significantly decreased bone bacterial densities (P = 0.004 and P < 0.0002, respectively). Bone sterilization was achieved using ceftazidime/avibactam in combination with colistin (91%) or fosfomycin (100%) or gentamicin (100%) (P < 0.0001), whereas single therapies were not different from controls. No ceftazidime/avibactam-resistant strains emerged in rabbits treated, regardless of the combination., Conclusions: In our model of E. coli OXA-48/ESBL osteomyelitis, ceftazidime/avibactam in combination was more effective than any single therapy, whatever the companion drug used (gentamicin or colistin or fosfomycin)., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We monitored swine-derived Escherichia coli on a Japanese farm where colistin had been used for the treatment of diseases caused by bacteria and investigated colistin resistance and the presence of mcr-1 in 36 E. coli strains isolated before and after the withdrawal of colistin use. Through the withdrawal of colistin use on the farm, the prevalence of colistin-resistant and mcr-1-positive E. coli was markedly reduced but not eradicated because mcr-1 had been maintained in multiple plasmids and various sequence types of nonpathogenic E. coli carried in healthy swine. The monitoring of sequence types of mcr-1-positive E. coli is expected to be important for controlling colistin resistance in swine or other animals.

Aim: Frozen, breaded chicken products have been implicated in Salmonella outbreaks, and may be incorrectly perceived as ready-to-eat, leading to mishandling or undercooking by consumers. This study aimed to assess the prevalence of Salmonella and antimicrobial resistant (AMR) Escherichia coli in these products., Methods and Results: Samples of frozen, raw, or partly cooked, coated chicken products were collected between April and July 2021 from retailers in the UK and tested for Salmonella spp., generic E. coli, extended spectrum beta-lactamase-producing, colistin-resistant, and carbapenem-resistant E. coli. One isolate of each bacterial type from each sample was selected for minimum inhibitory concentration determination for a range of antimicrobials. Salmonella was detected in 5 of 310 (1.6%) samples, identified as Salmonella Infantis in three samples and Salm. Java in two. One Salm. Infantis isolate was multidrug resistant, while the other Salmonella isolates were each resistant to at least one class of antimicrobials. Generic E. coli were detected in 113 samples (36.4%), with multidrug resistance being demonstrated in 20.0% of these. Escherichia coli with the ESBL phenotype were detected in 15 (4.8%) of samples and the AmpC phenotype in 2 (0.6%). A colistin-resistant E. coli was isolated from one sample; this possessed the mcr-1 gene. No carbapenem-resistant E. coli were detected. The five Salmonella-positive samples from this study, together with 20 Salmonella-positive products from an earlier study in 2020/2021, were cooked according to the manufacturers' instructions. Following cooking, Salmonella was not detected in any samples., Conclusions: This survey demonstrates continued contamination of frozen, coated chicken products with Salmonella, and provides data on the prevalence of AMR in these products., (© Crown copyright 2023.)

Carbapenem-resistant Acinetobacter baumannii-calcoaceticus complex (CRAB) is one of the top-priority pathogens for new antibiotic development. Unlike other antibiotic-resistant threats, none of the available therapies have been shown to consistently reduce mortality or improve patient outcomes in clinical trials. Antibiotic combination therapy is routinely used in clinical practice; however, the preferred combination has not been defined. This narrative review focuses on evidence-based solutions for the treatment of invasive CRAB infections. We dissect the promise and perils of traditional agents used in combination, such as colistin, sulbactam, and the tetracyclines, and offer clinical pearls based on our interpretation of the available data. Next, we investigate the merits of newly developed β-lactam agents like cefiderocol and sulbactam-durlobactam, which have demonstrated contrasting results in recent randomized clinical trials. The review concludes with the authors' perspective on the evolving treatment landscape for CRAB infections, which is complicated by limited clinical data, imperfect treatment options, and a need for future clinical trials. We propose that effective treatment for CRAB infections requires a personalized approach that incorporates host factors, the site of infection, pharmacokinetic-pharmacodynamic principles, local molecular epidemiology of CRAB isolates, and careful interpretation of antibiotic susceptibility testing results. In most clinical scenarios, a dose-optimized, sulbactam-based regimen is recommended with the addition of at least one other in vitro active agent. Should sulbactam-durlobactam receive regulatory approval, recommendations will need to be re-evaluated with the most recent evidence., Competing Interests: Potential conflicts of interest . R. K. S. has served as a consultant for Allergan, Cidara, Entasis, GlaxoSmithKline, Melinta, Menarini, Merck, Pifzer, Shionogi, Utility, and Venatorx, and has received investigator-initiated funding from Merck, Melinta, Roche, Shionogi, and Venatorx. D. L. P. has served as a consultant for Accelerate, Biomerieux, Entasis, Lysovant, Merck, Pfizer, Shionogi, and Venatorx, and has received research grants from Merck, Pfizer, and Shionogi. P. D. T. reports no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: The dissemination of polymyxin resistance represents a significant threat to public health. Materials & methods: Sequence-based typing was performed by 53 mcr-1 Escherichia coli isolates using fumC / fimH  (CH) genes to characterize clones spreading from pig farming. Furthermore, 12 isolates had their whole genome sequenced for phylogenetic study. Results: The isolates were classified into 22 distinct CH types, and two novel CH types (CH41-1578 and CH4-1579) and one sequence type (ST12652) was also described. According to phylogenetic study, both multilocus sequence typing and CH methods grouped the isolates similarly. Conclusion: Our findings suggest that the dissemination of the mcr-1 gene in pig farming has occurred mainly by horizontal gene transfer, and CH typing proved to be a good tool to characterize E. coli clones.

The escalating crisis of multidrug resistance is raising the fear of untreatable Gram-negative infections and killing a substantial number of patients. The underpopulated antibiotic drug development pipelines drive polymyxins (polymyxin B and colistin) as crucial therapeutic options. However, the cumbersome synthesis process and inefficient cyclization method limit the efficient preparation of polymyxin core scaffolds in the development of polymyxin derivatives. Here, we innovatively applied a substitution reaction between bromobenzene and sulfhydryl to cyclize colistin core scaffolds. The reaction was mild and efficient, improving the total yield of the compound from less than 10% to 55.90%. Nearly 30 novel derivatives with thioether bond-mediated cyclic scaffolds were designed and synthesized. Evaluation of antibacterial activities and biological properties revealed that many new compounds that are stable in mouse plasma possess high antimicrobial potency against Gram-negative bacteria and display no hemolytic toxicity. Our optimal peptide PE-2C-C8-DH eradicated Acinetobacter baumannii within 24 h in vitro, and had lower acute toxicity and significant therapeutic effects on mice infected with Pseudomonas aeruginosa, which deserves further development., (© 2023. The Author(s), under exclusive licence to the Japan Antibiotics Research Association.)

Emergence and dissemination of resistance to last-resort antibiotics such as carbapenem and colistin is a growing, global health concern. Wastewater treatment plants (WWTPs) link human activities and the environment, can act as reservoirs and sources for emerging antibiotic resistance, and likely play a large role in antibiotic resistance transmission. The aim of this study was to investigate occurrence and characteristics of colistin- and carbapenem-resistant Escherichia coli (CCREC) in wastewater and sludge samples collected over a one-year period from different functional areas of an urban WWTP in Jinan city, Shandong, China. A total of 8 CCREC were isolated from 168 samples with selective agar and PCR, corresponding to high prevalence of 4.8%, co-harboring carbapenem resistance genes (bla NDM ) and colistin resistance gene (mcr-1) and subsequently whole-genome sequenced. Additionally, all isolates were multidrug-resistant by antimicrobial susceptibility testing and carried a variety of antibiotic resistance genes. Two isolates carrying virulence genes associated with avian pathogenic E. coli were identified, one belonging to the high-risk clone O101:H9-ST167. Southern blotting was used to characterize CCREC isolates and plasmids carrying bla NDM -genes or mcr-1 could be transferred to a recipient strain E. coli J53 by in vitro conjugation assays. Resistance to other antibiotic classes were sporadically co-transferred to the transconjugant. Transposition of and mcr-1-carrying element from a transferable IncHI2-plasmid was observed among two CCREC clones isolated within 4 days of each other. The occurrence of multidrug-resistant CCREC capable of transferring their antibiotic resistance genotypes via conjugative plasmids is alarming. WWTPs bring bacteria from different sources together, providing opportunities for horizontal exchange of DNA among compatible hosts. Further dissemination of the colistin-, carbapenem-, or both colistin- and carbapenem resistant E. coli could lead to a serious threat to public health., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Acinetobacter baumannii is an important opportunistic pathogen known for its high levels of resistance to many antibiotics, particularly those considered last resorts such as colistin and carbapenems. Plasmids of this organism are increasingly associated with the spread of clinically important antibiotic resistance genes. Although A. baumannii is a ubiquitous organism, to date, most of the focus has been on studying strains recovered from clinical samples ignoring those isolated in the environment (soil, water, food, etc.). Here, we analysed the genetic structures of eight novel plasmids carried by an environmental colistin-resistant A. baumannii (strain E-072658) recovered in a recycled fibre pulp in a paper mill in Finland. It was shown that E-072658 carries a new variant of the mcr-4 colistin resistance gene ( mcr-4.7 ) in a novel Tn 3- family transposon (called Tn 6926 ) carried by a novel plasmid p8E072658. E-072658 is also resistant to sulphonamide compounds; consistent with this, the sul2 sulphonamide resistance gene was found in a p dif module. E-072658 also carries six additional plasmids with no antibiotic resistance genes, but they contained several p dif modules shared with plasmids carried by clinical strains. Detailed analysis of the genetic structure of all eight plasmids carried by E-072658 showed a complex evolutionary history revealing genetic exchange events within the genus Acinetobacter beyond the clinical or environmental origin of the strains. This work provides evidence that environmental strains might act as a source for some of the clinically significant antibiotic resistance genes.

Escherichia coli is a common pathogen in human and veterinary clinical infection. With antibiotic resistance including colistin resistance increasing globally, few antibiotic treatments are available for use against multidrug-resistant strains of E. coli. Given such circumstances, bacteriophage (phage) therapy is once again being considered as a potential alternative or adjunct to antibiotic therapy. Here, we isolated 52 phages from 816 samples from pig, chicken and duck farms in 4 provinces in China and identified a novel Escherichia phage, vB&#95;EcoStr-FJ63A, from pig feces. Morphological observation showed that phage vB&#95;EcoStr-FJ63A had an icosahedral capsid and an inflexible tail. Whole-genome sequencing revealed a double-stranded DNA genome of 168,157 bp (including 271 coding sequences) with a GC content of 40.29%. Bioinformatic analysis classified phage vB&#95;EcoStr-FJ63A as a Krischvirus, belonging to Straboviridae. The phage was relatively stable at pH 4-10 and below 60℃. It was lytic against a wide variety of colistin-resistant strains of E. coli from various animals, with one-step growth curves showing a latent period of 30 min and burst size of ∼11 PFU per infected cell. Maximum bactericidal activity was achieved within 2 h. No antibiotic resistance or virulence genes were detected in the phage genome. Further studies are warranted to develop phage vB&#95;EcoStr-FJ63A as a potential biocontrol agent against colistin-resistant E. coli., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Colistin (polymyxin E) is a group of cationic antimicrobial cyclic peptides and is recognized as a last-resort defense against lethal infections with carbapenem-resistant pathogens. In addition to the plasmid-borne mobilized phosphoethanolamine (PEA) transferases, the functional expression of lipid A-modifying enzymes encoded on chromosomes has been attributed to intrinsic bacterial colistin resistance. However, the mechanisms of colistin resistance in Riemerella anatipestifer remain unknown. Herein, the GE296&#95;RS09715 gene-encoded Lipid A PEA transferases (RaEptA) was identified in R. anatipestifer. Genetic and structural analyses revealed that the amino acid sequence of RaEptA shared 26.6%-33.1% similarities with the family of Lipid A PEA transferases (EptA) and MCR-like proteins and have defined 12 residues that contribute to the formation of phosphatidylethanolamine (PE)-recognizable cavities. Comparative analyses of colistin resistance in RA-LZ01 and RA-LZ01ΔRaEptA showed the level of colistin has fallen from 96 μg mL -1 down to 24 ~ 32 μg mL -1 . Site-directed mutagenesis assay of the PE-binding cavity and expression of the mutants reveals that K309-rRaEptA can remodel the surface of Escherichia coli and rendering it resistant to colistin, suggesting this point-mutation of P309K is necessary for EptA-mediated lipid A modification. Moreover, the virulence of RA-LZ01ΔRaEptA was attenuated compared with RA-LZ01 both in vivo and vitro. Taken together, the results represent the RaEptA involved in the colistin resistance and pathogenicity, and the P309K mutation might alter bacterial adaptation and increase the spread of colistin resistance from R. anatipestifer to other gram-negative bacteria. The findings of this study suggest another scenario for the spread of colistin resistance genes and should be considered by a wide audience., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Persistent antibiotic use results in the rise of antimicrobial resistance with limited or no choice for multidrug-resistant (MDR) and extensively drug resistant (XDR) bacteria. This necessitates a need for alternative therapy to effectively combat clinical pathogens that are resistant to last resort antibiotics. The study investigates hospital sewage as a potential source of bacteriophages to control resistant bacterial pathogens. Eighty-one samples were screened for phages against selected clinical pathogens. Totally, 10 phages were isolated against A. baumannii, 5 phages against K. pneumoniae, and 16 phages were obtained against P. aeruginosa. The novel phages were observed to be strain-specific with complete bacterial growth inhibition of up to 6 h as monotherapy without antibiotics. Phage plus colistin combinations reduced the minimum-biofilm eradication concentration of colistin up to 16 folds. Notably, a cocktail of phages exhibited maximum efficacy with complete killing at 0.5-1 μg/ml colistin concentrations. Thus, phages specific to clinical strains have a higher edge in treating nosocomial pathogens with their proven anti-biofilm efficacy. In addition, analysis of phage genomes revealed close phylogenetic relations with phages reported from Europe, China, and other neighbouring countries. This study serves as a reference and can be extended to other antibiotics and phage types to assess optimum synergistic combinations to combat various drug resistant pathogens in the ongoing AMR crisis., Competing Interests: Declaration of Competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The extracellular capsule is a virulence factor present in many facultative pathogens, but its role in antimicrobial resistance remains controversial. To shed light on this debate, we tested six antibiotics on four Klebsiella pneumoniae species complex strains. Noncapsulated strains exhibited increased tolerance to polymyxins, but not to other antibiotics, as measured using the MIC. Our results urge caution on the use of therapeutic agents that target the capsule and may result in selection for its inactivation.