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Many bacteria are able to survive in the presence of antibiotics in part because they possess pumps that can remove a broad range of small molecules; here, the structure of one such pump, AcrAB–TolC, is determined using X-ray crystallography and cryo-electron microscopy. AcrAB–TolC efflux pump structure Many bacteria are able to survive in the presence of antibiotics and other toxic compounds because they possess versatile energy-dependent transmembrane pumps. For example, the AcrAB–TolC efflux pump, which spans the inner and outer membranes of the bacterium, is able to transport a broad range of structurally unrelated small molecules/drugs out of some Gram-negative bacteria. The pump is comprised of an outer-membrane channel (TolC), a secondary transporter (AcrB; located in the inner membrane), and AcrA, a periplasmic protein that acts as a bridge for these two integral membrane proteins. In this paper, the authors solve an X-ray crystal structure of AcrB bound to AcrZ (a small protein that appears to alter the substrate preferences of AcrB) and a cryo-EM structure of the entire 771 kDa efflux pump. The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of these transporters form multicomponent ‘pumps’ that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component 1 , 2 , 3 , 4 , 5 , 6 , 7 . A model system for such a pump is the acridine resistance complex of Escherichia coli 1 . This pump assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins. The AcrAB–TolC efflux pump is able to transport vectorially a diverse array of compounds with little chemical similarity, thus conferring resistance to a broad spectrum of antibiotics. Homologous complexes are found in many Gram-negative species, including in animal and plant pathogens. Crystal structures are available for the individual components of the pump 2 , 3 , 4 , 5 , 6 , 7 and have provided insights into substrate recognition, energy coupling and the transduction of conformational changes associated with the transport process. However, how the subunits are organized in the pump, their stoichiometry and the details of their interactions are not known. Here we present the pseudo-atomic structure of a complete multidrug efflux pump in complex with a modulatory protein partner 8 from E. coli . The model defines the quaternary organization of the pump, identifies key domain interactions, and suggests a cooperative process for channel assembly and opening. These findings illuminate the basis for drug resistance in numerous pathogenic bacterial species.

There is increasing demand for safe and effective sanitizers for irrigation water disinfection to prevent transmission of foodborne pathogens to fresh produce. Here we compared the efficacy of pH-neutral electrolyzed oxidizing water (EOW), sodium hypochlorite (NaClO) and chlorine dioxide (ClO 2 ) against single and mixed populations of E. coli , Listeria and Salmonella under a range of pH and organic matter content. EOW treatment of the mixed bacterial suspension resulted in a dose-dependent (<1 mg/L free chlorine), rapid (<2 min) and effective (4–6 Log 10 ) reduction of the microbial load in water devoid of organic matter under the range of pH conditions tested (pH, 6.0, 7.0, 8.4 and 9.2). The efficacy of EOW containing 5 mg/L free chlorine was unaffected by increasing organic matter, and compared favourably with equivalent concentrations of NaClO and ClO 2 . EOW at 20 mg/L free chlorine was more effective than NaClO and ClO 2 in reducing bacterial populations in the presence of high (20–100 mg/L) dissolved organic carbon, and no regrowth or metabolic activity was observed for EOW-treated bacteria at this concentration upon reculturing in rich media. Thus, EOW is as effective or more effective than other common chlorine-based sanitizers for pathogen reduction in contaminated water. EOW’s other characteristics, such as neutral pH and ease of handling, indicate its suitability for fresh produce sanitation.

Drug efflux pumps confer resistance upon bacteria to a wide range of antibiotics from various classes. The expression of efflux pumps are also implicated in virulence and biofilm formation. Moreover, organisms can only acquire resistance in the presence of active drug efflux pumps. Therefore, efflux pump inhibitors (EPIs) are attractive compounds to reverse multidrug resistance and to prevent the development of resistance in clinically relevant bacterial pathogens. We investigated the potential of pure compounds isolated from plants to act as EPIs. In silico screening was used to predict the bioactivity of plant compounds and to compare that with the known EPI, phe‐arg‐β‐naphthylamide (PAβN). Subsequently, promising products have been tested for their ability to inhibit efflux. Plumbagin nordihydroguaretic acid (NDGA) and to a lesser degree shikonin, acted as sensitizers of drug‐resistant bacteria to currently used antibiotics and were able to inhibit the efflux pump‐mediated removal of substrate from cells. We demonstrated the feasibility of in silico screening to identify compounds that potentiate the action of antibiotics against drug‐resistant strains and which might be potentially useful lead compounds for an EPI discovery program. A powerful combination of in silico screening and bioassays were employed to identify phytochemicals which could inhibit the AcrB component of the AcrAB‐TolC drug efflux pump from Escherichia coli. Plumbagin and nordihydroguaretic acid sensitized drug‐resistant cells to antibiotics and were able to inhibit the efflux pump‐mediated removal of substrate from bacterial cells.

Antimicrobial resistance (AMR) poses a global threat to public health. While antibiotic overuse is a primary driver, emerging evidence suggests that non-antibiotic medications (NAMs) may also contribute. This concern is particularly relevant in residential aged care facilities (RACFs), where both NAMs and antibiotics are frequently used. We investigated whether nine commonly used NAMs in RACFs, including ibuprofen, diclofenac, acetaminophen, furosemide, metformin, atorvastatin, tramadol, temazepam, and pseudoephedrine at gut-relevant concentrations, enhance ciprofloxacin-induced mutagenesis in Escherichia coli . Our findings showed that ibuprofen and acetaminophen significantly increased mutation frequency and conferred high-level ciprofloxacin resistance. Whole-genome sequencing identified mutations in GyrA, MarR, and AcrR, with the latter two correlated with overexpression of AcrAB-TolC drug efflux pump. Co-exposure to two NAMs further elevated mutation rates and ciprofloxacin resistance levels. This study underscored the overlooked role of NAMs in driving AMR and highlighted the need to reassess polypharmacy risks in aged care settings.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a significant opportunistic human pathogen, posing a considerable threat to public health due to its antimicrobial resistance and limited treatment options. The incidence of CRPA is high in the Philippines; however, genomic analysis of CRPA in this setting is limited. Here, we provide the phenotypic and molecular characterization of 35 non-duplicate CRPA obtained from three tertiary hospitals in Metro Manila, Philippines, from August 2022 to January 2023. Six sequence types (STs), including international high-risk clones ST111 and ST357, were identified. This article highlights the first report in the Philippines on the identification of P. aeruginosa harboring Klebsiella pneumoniae Carbapenemase-2 (KPC-2), coproduced with Verona Integron-encoded Metallo-beta-lactamase-2 (VIM-2) and Oxacillinase-74 (OXA-74). Notably, this is also the first report of KPC in the Philippines identified in P. aeruginosa. New Delhi Metallo-beta-lactamase-7 (NDM-7), coproduced with Cefotaxime-Munich-15 (CTX-M-15) and Temoneira-2 (TEM-2), was also identified from a novel ST4b1c. The relentless identification of NDM in the Philippines’ healthcare setting poses a significant global public health risk. The initial detection of the P. aeruginosa strain harboring KPC exacerbated the situation, indicating the inception of potential dissemination of these resistance determinants within P. aeruginosa in the Philippines.

There are very few articles in the literature describing continuous models of bacterial infections that mimic disease pathogenesis in humans and animals without using separate cohorts of animals at each stage of disease. In this work, we developed bioluminescent mouse models of partial-thickness scald wound infection and sepsis that mimic disease pathogenesis in humans and animals using a recombinant luciferase-expressing Staphylococcus aureus strain (Xen29). Two days post-scald wound infection, mice were treated twice daily with a 2% topical mupirocin ointment for 7 days. For sepsis experiments, mice were treated intraperitoneally with 6 mg/kg daptomycin 2 h and 6 h post-infection and time to moribund monitored for 72 h. Consistent bacterial burden data were obtained from individual mice by regular photon intensity quantification on a Xenogen IVIS Lumina XRMS Series III biophotonic imaging system, with concomitant significant reduction in photon intensities in drug-treated mice. Post-mortem histopathological examination of wounds and bacterial counts in blood correlated closely with disease severity and total flux obtained from Xen29. The bioluminescent murine models provide a refinement to existing techniques of multiple bacterial enumeration during disease pathogenesis and promote animal usage reduction. The models also provide an efficient and information-rich platform for preclinical efficacy evaluation of new drug classes for treating acute and chronic human and animal bacterial infections.

Multidrug‐resistant (MDR) bacterial infections constitute one of the top global public health threats. This study investigated the potential of gallium liquid metal nanoparticles (GaLM NPs) as a new agent against MDR pathogens. GaLM NPs was bactericidal against methicillin‐resistant Staphylococcus aureus (MRSA) isolates and a vancomycin‐intermediate S. aureus reference strain (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values between 39 and 156 μg/mL). The bactericidal activity of GaLM NPs was supported by transmission electron microscopy showing marked ultrastructural changes in the cell envelope of MRSA USA300. GaLM NPs were bacteriostatic against selected Gram‐negative (Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae) reference strains and isolates (MICs between 39 and 625 μg/mL). Furthermore, GaLM NPs demonstrated additive and bacteriostatic activity when combined with sub‐inhibitory concentrations of colistin against P. aeruginosa isolates. Additionally, GaLM NPs showed anti‐biofilm activity against MRSA USA300 (minimum biofilm eradication concentration of 625 μg/mL); morphology changes of GaLM NPs‐treated cells was demonstrated by scanning electron microscopy. Finally, GaLM NPs was safe to human epidermal keratinocyte cell line at 1024 µg/mL (6.5 × MIC). We conclude that GaLM NPs warrant further exploration for the effective treatment of Gram‐positive or Gram‐negative infections both alone and in combination with antimicrobial drugs. Gallium liquid metal nanoparticles (GaLM NPs) are promising agents for treating multidrug‐resistant Gram‐positive and Gram‐negative bacterial infections.(A), Preparation of GaLM NPs suspension; (B, C), Morphology of GaLM NPs without and with F127, scale bar: 500 nm; (D), Diameter distribution of GaLM NPs; (E), TEM showing effects of GaLM NPs treatment on morphology of MRSA USA300; (F), SEM showing eradication of MRSA USA 300 biofilms by GaLM NPs.

The increasing rate of carbapenem-resistant bacteria within healthcare environments is an issue of great concern that needs urgent attention. This resistance is driven by metallo-β-lactamases (MBLs), which can catalyse the hydrolysis of almost all clinically available β-lactams and are resistant to all the clinically utilized β-lactamase inhibitors. In this study, an uncharacterized MBL is identified in a multidrug resistant isolate of the opportunistic pathogen, Chryseobacterium indologenes . Sequence analysis predicts this MBL (CIM-1) to be a lipoprotein with an atypical lipobox. Characterization of CIM-1 reveals it to be a high-affinity carbapenemase with a broad spectrum of activity that includes all cephalosporins and carbapenems. Results also shown that CIM-1 is potentially a membrane-associated MBL with an uncharacterized lipobox. Using prediction tools, we also identify more potentially lipidated MBLs with non-canonical lipoboxes highlighting the necessity of further investigation of lipidated MBLs. An uncharacterized metallo-β-lactamase identified in a multidrug-resistant Chryseobacterium indologenes has an atypical lipobox and is potentially lipidated. It has a high-affinity for β-lactams including the last-resort antibiotics, carbapenems.

Stenotrophomonas maltophilia is a multidrug‐resistant (MDR), Gram‐negative bacterium intrinsically resistant to beta‐lactams, including last‐resort carbapenems. As an opportunistic pathogen, it can cause serious healthcare‐related infections. This study assesses the prevalence, resistance profiles, and genetic diversity of S. maltophilia isolated from residential aged care facilities (RACFs). RACFs are known for their overuse and often inappropriate use of antibiotics, creating a strong selective environment that favors the development of bacterial resistance. The study was conducted on 73 S. maltophilia isolates recovered from wastewater and facility swab samples obtained from three RACFs and a retirement village. Phenotypic and genotypic assessments of the isolates revealed high carbapenem resistance, exemplifying their intrinsic beta‐lactam resistance. Alarmingly, 49.3% (36/73) of the isolates were non‐wild type for colistin, with minimum inhibitory concentration values of > 4 mg/L, and 11.0% (8/73) were resistant to trimethoprim‐sulfamethoxazole. No resistance mechanisms were detected for either antimicrobial. Genotypic assessment of known lineages revealed isolates clustering with Sm17 and Sm18, lineages not previously reported in Australia, suggesting the potential ongoing spread of MDR S. maltophilia. Lastly, although only a few isolates were biocide tolerant (2.7%, 2/73), their ability to grow in high concentrations (64 mg/L) of triclosan is concerning, as it may be selecting for their survival and continued dissemination. This study marks the first assessment of the prevalence, resistome, and genomic characteristics of multidrug‐resistant (MDR) Stenotrophomonas maltophilia in residential aged care facilities. MDR S. maltophilia were found in all sampled facilities, with colistin non‐wild type and trimethoprim‐sulfamethoxazole resistant isolates recovered. Many isolates also exhibited high tolerance to triclosan and benzalkonium chloride. Notably, our findings indicate the continued emergence of S. maltophilia, with lineages not previously identified in Australia observed in this study. This underscores the urgent need for comprehensive surveillance and effective control measures.

A generally applicable approach to analyze intact membrane protein complexes by mass spectrometry is reported. This method allows subunit stoichiometry, lipid binding and the effects of post-translational modifications on complex formation to be explored. We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we released the intact complex largely devoid of detergent. This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.