Antimicrobial peptide 2K4L inhibits the inflammatory response in macrophages and Caenorhabditis elegans and protects against LPS-induced septic shock in mice.

2K4L is a rationally designed analog of the short α-helical peptide temporin-1CEc, a natural peptide isolated and purified from the skin secretions of the Chinese brown frog Rana chensinensis by substituting amino acid residues. 2K4L displayed improved and broad-spectrum antibacterial activity than temporin-1CEc in vitro. Here, the antibacterial and anti-inflammatory activities of 2K4L in macrophages, C. elegans and mice were investigated. The results demonstrated that 2K4L could enter THP-1 cells to kill a multidrug-resistant Acinetobacter baumannii strain (MRAB 0227) and a sensitive A. baumannii strain (AB 22933), as well as reduce proinflammatory responses induced by MRAB 0227 by inhibiting NF-κB signaling pathway. Similarly, 2K4L exhibited strong bactericidal activity against A. baumannii uptake into C. elegans, extending the lifespan and healthspan of the nematodes. Meanwhile, 2K4L alleviated the oxidative stress response by inhibiting the expression of core genes in the p38 MAPK/PMK-1 signaling pathway and downregulating the phosphorylation level of p38, thereby protecting the nematodes from damage by A. baumannii. Finally, in an LPS-induced septic model, 2K4L enhanced the survival of septic mice and decreased the production of proinflammatory cytokines by inhibiting the signaling protein expression of the MAPK and NF-κB signaling pathways and protecting LPS-induced septic mice from a lethal inflammatory response. In conclusion, 2K4L ameliorated LPS-induced inflammation both in vitro and in vivo.

You get what you test for: The killing effect of phage lysins is highly dependent on buffer tonicity and ionic strength.

The phage lysin field has done nothing but grow in the last decades. As a result, many different research groups around the world are contributing to the field, often with certain methodological differences that pose a challenge to the interpretation and comparison of results. In this work, we present the case study of three Acinetobacter baumannii-targeting phage lysins (wild-type endolysin LysMK34 plus engineered lysins eLysMK34 and 1D10) plus one lysin with broad activity against Gram-positive bacteria (PlySs2) to provide exemplary evidence on the risks of generalization when using one of the most common lysin evaluation assays: the killing assay with resting cells. To that end, we performed killing assays with the aforementioned lysins using hypo-, iso- and hypertonic buffers plus human serum either as the reaction or the dilution medium in a systematic manner. Our findings stress the perils of creating hypotonic conditions or a hypotonic shock during a killing assay, suggesting that hypotonic buffers should be avoided as a test environment or as diluents before plating to avoid overestimation of the killing effect in the assayed conditions. As a conclusion, we suggest that the nature of both the incubation and the dilution buffers should be always clearly identified when reporting killing activity data, and that for experimental consistency the same incubation buffer should be used as a diluent for posterior serial dilution and plating unless explicitly required by the experimental design. In addition, the most appropriate buffer mimicking the final application must be chosen to obtain relevant results.

Unveiling the Secrets of Acinetobacter baumannii: Resistance, Current Treatments, and Future Innovations.

Acinetobacter baumannii represents a significant concern in nosocomial settings, particularly in critically ill patients who are forced to remain in hospital for extended periods. The challenge of managing and preventing this organism is further compounded by its increasing ability to develop resistance due to its extraordinary genomic plasticity, particularly in response to adverse environmental conditions. Its recognition as a significant public health risk has provided a significant impetus for the identification of new therapeutic approaches and infection control strategies. Indeed, currently used antimicrobial agents are gradually losing their efficacy, neutralized by newer and newer mechanisms of bacterial resistance, especially to carbapenem antibiotics. A deep understanding of the underlying molecular mechanisms is urgently needed to shed light on the properties that allow A. baumannii enormous resilience against standard therapies. Among the most promising alternatives under investigation are the combination sulbactam/durlobactam, cefepime/zidebactam, imipenem/funobactam, xeruborbactam, and the newest molecules such as novel polymyxins or zosurabalpin. Furthermore, the potential of phage therapy, as well as deep learning and artificial intelligence, offer a complementary approach that could be particularly useful in cases where traditional strategies fail. The fight against A. baumannii is not confined to the microcosm of microbiological research or hospital wards; instead, it is a broader public health dilemma that demands a coordinated, global response.

Investigating the Effectiveness of Ceragenins against Acinetobacter baumannii to Develop New Antimicrobial and Anti-Adhesive Strategies.

A growing body of experimental data indicates that ceragenins (CSAs), which mimic the physicochemical properties of the host's cationic antimicrobial peptide, hold promise for the development of a new group of broad-spectrum antimicrobials. Here, using a set of in vivo experiments, we assessed the potential of ceragenins in the eradication of an important etiological agent of nosocomial infections, Acinetobacter baumannii. Assessment of the bactericidal effect of ceragenins CSA-13, CSA-44, and CSA-131 on clinical isolates of A. baumannii (n = 65) and their effectiveness against bacterial cells embedded in the biofilm matrix after biofilm growth on abiotic surfaces showed a strong bactericidal effect of the tested molecules regardless of bacterial growth pattern. AFM assessment of bacterial cell topography, bacterial cell stiffness, and adhesion showed significant membrane breakdown and rheological changes, indicating the ability of ceragenins to target surface structures of A. baumannii cells. In the cell culture of A549 lung epithelial cells, ceragenin CSA-13 had the ability to inhibit bacterial adhesion to host cells, suggesting that it interferes with the mechanism of bacterial cell invasion. These findings highlight the potential of ceragenins as therapeutic agents in the development of antimicrobial strategies against bacterial infections caused by A. baumannii.

Integration of individual preclinical and clinical anti-infective PKPD data to predict clinical study outcomes.

The AIDA randomized clinical trial found no significant difference in clinical failure or survival between colistin monotherapy and colistin-meropenem combination therapy in carbapenem-resistant Gram-negative infections. The aim of this reverse translational study was to integrate all individual preclinical and clinical pharmacokinetic-pharmacodynamic (PKPD) data from the AIDA trial in a pharmacometric framework to explore whether individualized predictions of bacterial burden were associated with the trial outcomes. The compiled dataset included for each of the 207 patients was (i) information on the infecting Acinetobacter baumannii isolate (minimum inhibitory concentration, checkerboard assay data, and fitness in a murine model), (ii) colistin plasma concentrations and colistin and meropenem dosing history, and (iii) disease scores and demographics. The individual information was integrated into PKPD models, and the predicted change in bacterial count at 24 h for each patient, as well as patient characteristics, was correlated with clinical outcomes using logistic regression. The in vivo fitness was the most important factor for change in bacterial count. A model-predicted growth at 24 h of ≥2-log10 (164/207) correlated positively with clinical failure (adjusted odds ratio, aOR = 2.01). The aOR for one unit increase of other significant predictors were 1.24 for SOFA score, 1.19 for Charlson comorbidity index, and 1.01 for age. This study exemplifies how preclinical and clinical anti-infective PKPD data can be integrated through pharmacodynamic modeling and identify patient- and pathogen-specific factors related to clinical outcomes - an approach that may improve understanding of study outcomes.

Nosocomial transmission of tet(x3), bla NDM-1 and bla OXA-97-carrying Acinetobacter baumannii conferring resistance to eravacycline and omadacycline, the Netherlands, March to August 2021.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an important pathogen causing serious nosocomial infections. We describe an outbreak of CRAb in an intensive care unit in the Netherlands in 2021. During an outbreak of non-resistant A. baumannii, while infection control measures were in place, CRAb isolates carrying highly similar bla NDM-1 - and tet(x3)-encoding plasmids were isolated from three patients over a period of several months. The chromosomal and plasmid sequences of the CRAb and non-carbapenemase-carrying A. baumannii isolates cultured from patient materials were analysed using hybrid assemblies of short-read and long-read sequences. The CRAb isolates revealed that the CRAb outbreak consisted of two different strains, carrying similar plasmids. The plasmids contained multiple antibiotic resistance genes including the tetracycline resistance gene tet(x3), and the bla NDM-1 and bla OXA-97 carbapenemase genes. We determined minimal inhibitory concentrations (MICs) for 13 antibiotics, including the newly registered tetracycline antibiotics eravacycline and omadacycline. The CRAb isolates showed high MICs for tetracycline antibiotics including eravacycline and omadacycline, except for minocycline which had a low MIC. In this study we show the value of sequencing multidrug-resistant A. baumannii for outbreak tracking and guiding outbreak mitigation measures.

Coexistence of ß-lactamase genes and biofilm forming potential among carbapenem-resistant Acinetobacter baumannii in Lahore, Pakistan.

INTRODUCTION: Our goal was to investigate the antimicrobial resistance due to beta-lactamase genes and virulent determinants (biofilm-forming ability) expressed by Acinetobacter collected from health settings in Pakistan. A cross-sectional study was conducted for the molecular characterization of carbapenemases and biofilm-producing strains of Acinetobacter spp. METHODOLOGY: Two twenty-three imipenem-resistant Acinetobacter isolates were analyzed from 2020 to 2023.The combination disk test and modified hodge test were performed. Biofilm forming ability was determined by polystyrene tube assay. Multiplex polymerase chain reaction (PCR) for virulent and biofilm-forming genes, and 16S rRNA sequencing were performed. RESULTS: 118 (52.9%) carbapenem-resistant Acinetobacter (CR-AB) were isolated from wounds and pus, 121 (54.2%) from males, and 92 (41.2%) from 26-50-years-olds. More than 80% of strains produced ß-lactamases and carbapenemases. Based on the PCR amplification of the ITS gene, 174 (78.0%) CR-AB strains were identified from CR-Acinetobacter non-baumannii (ANB). Most CR-AB were strong and moderate biofilm producers. Genetic analysis revealed the blaOXA-23, blaTEM, blaCTX-M blaNDM-1 and blaVIM were prevalent in CR-AB with frequencies 91 (94.8%), 68 (70.8%), 19 (19.7%), 53 (55.2%), 2 (2.0%) respectively. Among virulence genes, OmpA was dominant in CR-AB isolates from wound (83, 86.4%), csuE 63 (80.7%) from non-wound specimens and significantly correlated with blaNDM and blaOXA genes. Phylogenetic analysis revealed three different clades for strains based on specimens. CONCLUSIONS: CR-AB was highly prevalent in Pakistan and associated with wound infections. The genes, blaOXA-23, blaTEM, blaCTX-M, and blaNDM-1 were detected in CR-AB. Most CR-AB were strong biofilm producers with virulent genes OmpA and csuE.

Molecular epidemiology of Acinetobacter baumannii during COVID-19 at a hospital in northern China.

BACKGROUND: The wide spread of carbapenem-resistance clones of Acinetobacter baumannii has made it a global public problem. Some studies have shown that the prevalence of Acinetobacter baumannii clones can change over time. However, few studies with respect to the change of epidemiological clones in Acinetobacter baumannii during Corona Virus Disease 2019 (COVID-19) were reported. This study aims to investigate the molecular epidemiology and resistance mechanisms of Acinetobacter baumannii during COVID-19. RESULTS: A total of 95 non-replicated Acinetobacter baumannii isolates were enrolled in this study, of which 60.0% (n = 57) were identified as carbapenem-resistant Acinetobacter baumannii (CRAB). The positive rate of the blaOXA-23 gene in CRAB isolates was 100%. A total of 28 Oxford sequence types (STs) were identified, of which the most prevalent STs were ST540 (n = 13, 13.7%), ST469 (n = 13, 13.7%), ST373 (n = 8, 8.4%), ST938 (n = 7, 7.4%) and ST208 (n = 6, 6.3%). Differently, the most widespread clone of Acinetobacter baumannii in China during COVID-19 was ST208 (22.1%). Further study of multidrug-resistant ST540 showed that all of them were carrying blaOXA-23, blaOXA-66, blaADC-25 and blaTEM-1D, simultaneously, and first detected Tn2009 in ST540. The blaOXA-23 gene was located on transposons Tn2006 or Tn2009. In addition, the ST540 strain also contains a drug-resistant plasmid with msr(E), armA, sul1 and mph(E) genes. CONCLUSION: The prevalent clones of Acinetobacter baumannii in our organization have changed during COVID-19, which was different from that of China. ST540 strains which carried multiple drug-resistant mobile elements was spreading, indicating that it is essential to strengthen the molecular epidemiology of Acinetobacter baumannii.

Antimicrobial resistance genes harbored in invasive Acinetobacter calcoaceticus-baumannii complex isolated from Korean children during the pre-COVID-19 pandemic periods, 2015-2020.

Background: Acinetobacter baumannii (AB) has emerged as one of the most challenging pathogens worldwide, causing invasive infections in the critically ill patients due to their ability to rapidly acquire resistance to antibiotics. This study aimed to analyze antibiotic resistance genes harbored in AB and non-baumannii Acinetobacter calcoaceticus-baumannii (NB-ACB) complex causing invasive diseases in Korean children. Methods: ACB complexes isolated from sterile body fluid of children in three referral hospitals were prospectively collected. Colistin susceptibility was additionally tested via broth microdilution. Whole genome sequencing was performed and antibiotic resistance genes were analyzed. Results: During January 2015 to December 2020, a total of 67 ACB complexes were isolated from sterile body fluid of children in three referral hospitals. The median age of the patients was 0.6 (interquartile range, 0.1-7.2) years old. Among all the isolates, 73.1% (n=49) were confirmed as AB and others as NB-ACB complex by whole genome sequencing. Among the AB isolates, only 22.4% susceptible to carbapenem. In particular, all clonal complex (CC) 92 AB (n=33) showed multi-drug resistance, whereas 31.3% in non-CC92 AB (n=16) (P<0.001). NB-ACB showed 100% susceptibility to all classes of antibiotics except 3rd generation cephalosporin (72.2%). The main mechanism of carbapenem resistance in AB was the bla oxa23 gene with ISAba1 insertion sequence upstream. Presence of pmr gene and/or mutation of lpxA/C gene were not correlated with the phenotype of colistin resistance of ACB. All AB and NB-ACB isolates carried the abe and ade multidrug efflux pumps. Conclusions: In conclusion, monitoring and research for resistome in ACB complex is needed to identify and manage drug-resistant AB, particularly CC92 AB carrying the bla oxa23 gene.

A comprehensive investigation of the anion inhibition profile of a ß-carbonic anhydrase from Acinetobacter baumannii for crafting innovative antimicrobial treatments.

This study refers to the intricate world of Acinetobacter baumannii, a resilient pathogenic bacterium notorious for its propensity at antibiotic resistance in nosocomial infections. Expanding upon previous findings that emphasised the bifunctional enzyme PaaY, revealing unexpected γ-carbonic anhydrase (CA) activity, our research focuses on a different class of CA identified within the A. baumannii genome, the ß-CA, designated as 𝛽-AbauCA (also indicated as CanB), which plays a crucial role in the resistance mechanism mediated by AmpC beta-lactamase. Here, we cloned, expressed, and purified the recombinant 𝛽-AbauCA, unveiling its distinctive kinetic properties and inhibition profile with inorganic anions (classical CA inhibitors). The exploration of 𝛽-AbauCA not only enhances our understanding of the CA repertoire of A. baumannii but also establishes a foundation for targeted therapeutic interventions against this resilient pathogen, promising advancements in combating its adaptability and antibiotic resistance.

Lacticaseibacillus rhamnosus CRL 2244 secreted metabolites display killing and antibiotic synergistic activity against multi-drug resistant pathogens.

A growing increase in the number of serious infections caused by multidrug resistant bacteria (MDR) is challenging our society. Despite efforts to discover novel therapeutic options, few antibiotics targeting MDR have been approved by the Food and Drug Administration (FDA). Lactic acid bacteria have emerged as a promising therapeutic alternative due to their demonstrated ability to combat MDR pathogens in vitro. Our previous co-culture studies showed Lacticaseibacillus rhamnosus CRL 2244 as having a potent killing effect against carbapenem-resistant Acinetobacter baumannii (CRAB) strains. Here we report that cell-free conditioned media (CFCM) samples obtained from Lcb. rhamnosus CRL 2244 cultures incubated at different times display antimicrobial activity against 43 different pathogens, including CRAB, methicillin-resistant Staphylococcus aureus (MRSA) and carbapenemase Klebsiella pneumoniae (KPC)-positive strains. Furthermore, transwell and ultrafiltration analyses together with physical and chemical/biochemical tests showed that Lcb. rhamnosus CRL 2244 secretes a <3 kDa metabolite(s) whose antimicrobial activity is not significantly impaired by mild changes in pH, temperature and various enzymatic treatments. Furthermore, sensitivity and time-kill assays showed that the bactericidal activity of the Lcb. rhamnosus CRL 2244 metabolite(s) enhances the activity of some current FDA approved antibiotics. We hypothesize that this observation could be due to the effects of Lcb. rhamnosus CRL 2244 metabolite(s) on cell morphology and the enhanced transcriptional expression of genes coding for the phenylacetate (PAA) and histidine catabolic Hut pathways, metal acquisition and biofilm formation, all of which are associated with bacterial virulence. Interestingly, the extracellular presence of Lcb. rhamnosus CRL 2244 induced the transcription of the gene coding for the CidA/LgrA protein, which is involved in programmed cell death in some bacteria. Overall, the findings presented in this report underscore the promising potential of the compound(s) released by Lcb. rhamnosus CRL2244 as an alternative and/or complementary option to treat infections caused by A. baumannii as well as other MDR bacterial pathogens.

Genetic alterations in the pmrAB two-component system and lipid A biosynthesis genes of polymyxin-resistant Acinetobacter baumannii isolates.

The rate of pandrug-resistant Acinetobacter baumannii strains is on the rise in all continents. This bacterium can acquire resistance to all antibiotics, even to colistin. Alterations in the lipid A or/and the two-component pmrAB were earlier detected in colistin resistance. We investigated and analyzed two strains of A. baumannii (ABRC1 and ABRC2) isolated from two patients admitted to intensive care unit with a septic shock. Both strains were resistant to all tested antibiotics including colistin with a MIC >256 mg L-1. Colistin resistance genes (pmrA, pmrB, lpxA, lpxC, lpxD, and lpsB) of two strains (ABRC1 and ABRC2) were investigated by PCR and sequencing. Obtained nucleic acid sequences were aligned with reference sequences of ATCC 19606 and 17987. In this study two amino acid mutations, N287D in the lpxC gene and E117K in the lpxD gene, were detected in both ABRC1 and ABRC2 strains. ABRC1 had an additional H200L mutation in the pmrA gene. Both colistin resistant strains harbored the same A138T mutation in the pmrB gene. The ABRC2 strain also had an alteration in the kinase domain, specifically an R263S substitution of the histidine kinase domain. Three identical mutations were found in the lpsB gene of both A. baumannii strains: Q216K + H218G + S219E. As a result, a newly deduced protein sequence in both ABRC1 and ABRC2 strains differed from those described in ATCC 17978 and 19606 strains was determined. Colistin resistance is multifactorial in A. baumannii. In our study we detected novel mutations in colistin resistant A. baumannii clinical isolates.

Evaluating virulence features of Acinetobacter baumannii resistant to polymyxin B.

The increasing resistance to polymyxins in Acinetobacter baumannii has made it even more urgent to develop new treatments. Anti-virulence compounds have been researched as a new solution. Here, we evaluated the modification of virulence features of A. baumannii after acquiring resistance to polymyxin B. The results showed lineages attaining unstable resistance to polymyxin B, except for Ab7 (A. baumannii polymyxin B resistant lineage), which showed stable resistance without an associated fitness cost. Analysis of virulence by a murine sepsis model indicated diminished virulence in Ab7 (A. baumannii polymyxin B resistant lineage) compared with Ab0 (A. baumannii polymyxin B susceptible lineage). Similarly, downregulation of virulence genes was observed by qPCR at 1 and 3 h of growth. However, an increase in bauE, abaI, and pgAB expression was observed after 6 h of growth. Comparison analysis of Ab0, Ab7, and Pseudomonas aeruginosa suggested no biofilm formation by Ab7. In general, although a decrease in virulence was observed in Ab7 when compared with Ab0, some virulence feature that enables infection could be maintained. In light of this, virulence genes bauE, abaI, and pgAB showed a potential relevance in the maintenance of virulence in polymyxin B-resistant strains, making them promising anti-virulence targets.

LysJEP8: A promising novel endolysin for combating multidrug-resistant Gram-negative bacteria.

Antimicrobial resistance (AMR) is an escalating global health crisis, driven by the overuse and misuse of antibiotics. Multidrug-resistant Gram-negative bacteria, such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, are particularly concerning due to their high morbidity and mortality rates. In this context, endolysins, derived from bacteriophages, offer a promising alternative to traditional antibiotics. This study introduces LysJEP8, a novel endolysin derived from Escherichia phage JEP8, which exhibits remarkable antimicrobial activity against key Gram-negative members of the ESKAPE group. Comparative assessments highlight LysJEP8's superior performance in reducing bacterial survival rates compared to previously described endolysins, with the most significant impact observed against P. aeruginosa, and notable effects on A. baumannii and K. pneumoniae. The study found that LysJEP8, as predicted by in silico analysis, worked best at lower pH values but lost its effectiveness at salt concentrations close to physiological levels. Importantly, LysJEP8 exhibited remarkable efficacy in the disruption of P. aeruginosa biofilms. This research underscores the potential of LysJEP8 as a valuable candidate for the development of innovative antibacterial agents, particularly against Gram-negative pathogens, and highlights opportunities for further engineering and optimization to address AMR effectively.

Evaluating the antibacterial effect of meropenem-loaded chitosan/sodium tripolyphosphate (TPP) nanoparticles on Acinetobacter baumannii isolated from hospitalized patients.

BACKGROUND: Acinetobacter baumannii is a health threat due to its antibiotic resistance. Herein, antibiotic susceptibility and its association with the Toxin-antitoxin (TA) system genes in A. baumannii clinical isolates from Iran were investigated. Next, we prepared meropenem-loaded chitosan nanoparticles (MP-CS) and investigated their antibacterial effects against meropenem-susceptible bacterial isolates. METHODS: Out of 240 clinical specimens, 60 A. baumannii isolates were assessed. Antibiotic resistance of the isolates against conventional antibiotics was determined alongside investigating the presence of three TA system genes (mazEF, relBE, and higBA). Chitosan nanoparticles were characterized in terms of size, zeta potential, encapsulation efficiency, and meropenem release activity. Their antibacterial effects were assessed using the well diffusion method, minimum inhibitory concentration (MIC), and colony-forming unit (CFU) counting. Their cytotoxic effects and biocompatibility index were determined via the MTT, LDH, and ROS formation assays. RESULTS: Ampicillin, ceftazidime, and colistin were the least effective, and amikacin and tobramycin were the most effective antibiotics. Out of the 60 isolates, 10 (16.7%), 5 (8.3%), and 45 (75%) were multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR), respectively. TA system genes had no significant effect on antibiotic resistance. MP-CS nanoparticles demonstrated an average size of 191.5 and zeta potential of 27.3 mV alongside a maximum encapsulation efficiency of 88.32% and release rate of 69.57%. MP-CS nanoparticles mediated similar antibacterial effects, as compared with free meropenem, against the A. baumannii isolates with significantly lower levels of meropenem. MP-CS nanoparticles remarkably prevented A549 and NCI-H292 cell infection by the A. baumannii isolates alongside demonstrating a favorable biocompatibility index. CONCLUSION: Antibiotic-loaded nanoparticles should be further designed and investigated to increase their antibacterial effect against A. baumannii and assess their safety and applicability in vivo settings.

Distribution and molecular characterization of carbapenemase-producing gram-negative bacteria in Henan, China.

This study aimed to investigate the epidemiological characteristics and trends over time of carbapenemase-producing (e.g., KPC, NDM, VIM, IMP, and OXA-48) Gram-negative bacteria (CPGNB). Non-duplicated multi-drug resistant Gram-negative bacteria (MDRGNB) were collected from the First Affiliated Hospital of Zhengzhou University from April 2019 to February 2023. Species identification of each isolate was performed using the Vitek2 system and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry according to the manufacturer's instructions. PCR detected carbapenem resistance genes in the strains, strains carrying carbapenem resistance genes were categorized as CPGNB strains after validation by carbapenem inactivation assay. A total of 5705 non-repetitive MDRGNB isolates belonging to 78 different species were collected during the study period, of which 1918 CPGNB were validated, with the respiratory tract being the primary source of specimens. Epidemiologic statistics showed a significant predominance of ICU-sourced strains compared to other departments. Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa were the significant CPGNB in Henan, and KPC and NDM were the predominant carbapenemases. Carbapenem-resistant infections in Henan Province showed an overall increasing trend, and the carriage of carbapenemase genes by CPGNB has become increasingly prevalent and complicated. The growing prevalence of CPGNB in the post-pandemic era poses a significant challenge to public safety.

Can flow cytometric measurements of reactive oxygen species levels determine minimal inhibitory concentrations and antibiotic susceptibility testing for Acinetobacter baumannii?

Current antimicrobial susceptibility testing (AST) requires 16-24 hours, delaying initiation of appropriate antibiotics. Hence, there is a need for rapid AST. This study aims to develop and evaluate the feasibility of a rapid flow cytometric AST assay to determine minimum inhibitory concentration (MIC) for carbapenem-resistant Acinetobacter baumannii (CRAB). Antibiotic exposure causes increased intracellular reactive oxygen species (ROS) in bacteria. We hypothesized that ROS can be used as a marker to determine MIC. We assessed three CRAB clinical isolates across fifteen antibiotics at various concentrations in a customized 96-well microtiter plate. The antibiotics assessed include amikacin, beta-lactams (ampicillin/sulbactam, aztreonam, cefepime, ceftolozane/tazobactam, doripenem, imipenem, meropenem, and piperacillin/tazobactam), levofloxacin, polymyxin B, rifampicin, trimethoprim/sulfamethoxazole, and tetracyclines (tigecycline and minocycline). These clinical CRAB isolates were assessed for ROS after antibiotic treatment. Increased ROS levels indicated by increased RedoxSensorTM Green (RSG) fluorescence intensity was assessed using flow cytometry (FCM). MIC was set as the lowest antibiotic concentration that gives a ≥1.5-fold increase in mode RSG fluorescence intensity (MICRSG). Accuracy of MICRSG was determined by comparing against microtiter broth dilution method performed under CLSI guidelines. ROS was deemed accurate in determining the MICs for ß-lactams (83.3% accuracy) and trimethoprim/sulfamethoxazole (100% accuracy). In contrast, ROS is less accurate in determining MICs for levofloxacin (33.3% accuracy), rifampicin (0% accuracy), amikacin (33.3% accuracy), and tetracyclines (33.3% accuracy). Collectively, this study described an FCM-AST assay to determine antibiotic susceptibility of CRAB isolates within 5 hours, reducing turnaround time up to 19 hours.

In-vitro evaluation of different antimicrobial combinations with and without colistin against carbapenem-resistant Acinetobacter baumannii clinical isolates.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) infections are one of the most common causes of nosocomial infections and have high mortality rates due to difficulties in treatment. In this study, the in vitro synergistic interactions of the colistin (CT)-meropenem (MEM) combination and patient clinical outcomes were compared in CRAB-infected patients that receive CT-MEM antimicrobial combination therapy. In addition, in vitro synergistic interactions of MEM-ertapenem (ETP), MEM-fosfomycin (FF) and CT-FF antimicrobial combinations were investigated. Finally, the epsilometer (E) test and checkerboard test results were compared and the compatibility of these two tests was evaluated. METHODS: Twenty-one patients were included in the study. Bacterial identification was performed with MALDI-TOF, and antimicrobial susceptibility was assessed with an automated system. Synergy studies were performed using the E test and checkerboard method. RESULTS: For the checkerboard method, the synergy rates for CT-MEM, MEM-FF, MEM-ETP and CT-FF were 100%, 52.3%, 23.8% and 28.5%, respectively. In the E test synergy tests, synergistic effects were detected for two isolates each in the CT-MEM and CT-FF combinations. Microbial eradication was achieved in nine (52.9%) of the 17 patients that received CT-MEM combination therapy. The agreement between the E test and the checkerboard test was 6.5%. CONCLUSIONS: A synergistic effect was found with the checkerboard method for the CT-MEM combination in all isolates in our study, and approximately 70% of the patients benefited from treatment with this combination. In addition, more than half of the isolates showed a synergistic effect for the MEM-FF combination. Combinations of CT-MEM and MEM-FF may be options for the treatment of CRAB infections. However, a comprehensive understanding of the potential of the microorganism to develop resistant mutants under applied exposures, as well as factors that directly affect antimicrobial activity, such as pharmacokinetics/pharmacodynamics, is essential for providing treatment advice. We found a low rate of agreement between the E test method and the checkerboard test method in our study, in contrast to the literature. Comprehensive studies that compare clinical results with methods are needed to determine the ideal synergy test and interpretation method.

Novel Coumarins Derivatives for A. baumannii Lung Infection Developed by High-Throughput Screening and Reinforcement Learning.

With the increasing resistance of Acinetobacter baumannii (A. baumannii) to antibiotics, researchers have turned their attention to the development of new antimicrobial agents. Among them, coumarin-based heterocycles have attracted much attention due to their unique biological activities, especially in the field of antibacterial infection. In this study, a series of coumarin derivatives were synthesized and screened for their bactericidal activities (Ren et al. 2018; Salehian et al. 2021). The inhibitory activities of these compounds on bacterial strains were evaluated, and the related mechanism of the new compounds was explored. Firstly, the MIC values and bacterial growth curves were measured after compound treatment to evaluate the antibacterial activity in vitro. Then, the in vivo antibacterial activities of the new compounds were assessed on A. baumannii-infected mice by determining the mice survival rates, counting bacterial CFU numbers, measuring inflammatory cytokine levels, and histopathology analysis. In addition, the ROS levels in the bacterial cells were measured with DCFH-DA detection kit. Furthermore, the potential target and detailed mechanism of the new compounds during infection disease therapy were predicted and evidenced with molecular docking. After that, ADMET characteristic prediction was completed, and novel, synthesizable, drug-effective molecules were optimized with reinforcement learning study based on the probed compound as a training template. The interaction between the selected structures and target proteins was further evidenced with molecular docking. This series of innovative studies provides important theoretical and experimental data for the development of new anti-A. baumannii infection drugs.

Study of MIC of silver and zinc oxide nanoparticles, strong and cost-effective antibacterial against biofilm-producing Acinetobacter baumannii in Shiraz, Southwest of Iran.

BACKGROUND: Acinetobacter baumannii resistant strains lead to increased mortality, treatment costs, and an increase in the length of hospitalization. Nowadays, nanoparticles are considered a substitute for antibiotics. This study aimed to determine the MIC of Silver (Ag) and Zinc Oxide (ZnO) Nanoparticles (NPs) on Biofilm-Producing Acinetobacter baumannii and determine the relationship between MIC and frequency of efflux pump genes in cutaneous specimens in Shiraz, Southwest Iran in 2021-2022. METHODS: In this study, specimens were collected from April 2021 to June 2022 at Namazi and Faqihi Hospitals in Shiraz. Investigation of biofilm production in multidrug resistance (MDR) isolates was done by the microtiter plate method. Synthesized nanoparticles were characterized by UV-vis spectrum, X-ray diffraction (XRD), and electron microscopy. The MIC of AgNPs and ZnONPs for isolates was done using the method described in the CLSI guideline (2018). The antibacterial effect of MIC of NPs on inanimate objects was done by colony counts. The prevalence of efflux pump genes (adeR, adeC, adeA, abeM, adeK, adeI) was also investigated by PCR technique. RESULTS: The highest ceftriaxone resistance (68%) and lowest colistin resistance (7%) were identified. 57% of isolates were MDR. In addition, 71.9% could produce biofilm and 28.1% of isolates could not produce biofilm. The average size of AgNPs and ZnONPs in the present study is 48 and < 70 nm, respectively. The nanoparticles were spherical. The MIC and the MBC of the ZnONPs were in the range of 125 to 250 µg/mL respectively. Also, for AgNPs, the MIC and the MBC were in the range of 62.5 to 250 µg/ml, respectively. AbeM gene had the highest frequency and the AdeK gene had the lowest frequency. Statistical analysis showed that there is a relationship between the frequency of adeA, adeC, and adeM genes with the MIC of AgNPs and ZnONPs. CONCLUSION: According to the results of the present study, inanimate objects such as scalpels in contact with AgNPs (6000 µg/ml for 240 min) or ZnONPs (5000 µg/ml for 120 min) can be free of biofilm producing Acinetobacter baumannii  with efflux pump genes.