Molecular Epidemiology of Carbapenem-Resistant Klebsiella aerogenes in Japan.

Information regarding Klebsiella aerogenes haboring carbapenemase in Japan is limited. A comprehensive nationwide survey was conducted from September 2014 to December 2022, and 67 non-duplicate strains of carbapenem-resistant K. aerogenes were isolated from 57 healthcare facilities in Japan. Through genetic testing and whole-genome sequencing, six strains were found to possess carbapenemases, including imipenemase (IMP)-1, IMP-6, New Delhi metallo-ß-lactamase (NDM)-1, and NDM-5. The strain harboring blaNDM-5 was the novel strain ST709, which belongs to the clonal complex of the predominant ST4 in China. The novel integron containing blaIMP-1 featured the oxacillinase-101 gene, which is a previously unreported structure, with an IncN4 plasmid type. However, integrons found in the strains possessing blaIMP-6, which were the most commonly identified, matched those reported domestically in Klebsiella pneumoniae, suggesting the prevalence of identical integrons. Transposons containing blaNDM are similar or identical to the transposon structure of K. aerogenes harboring blaNDM-5 previously reported in Japan, suggesting that the same type of transposon could have been transmitted to K. aerogenes in Japan. This investigation analyzed mobile genetic elements, such as integrons and transposons, to understand the spread of carbapenemases, highlighting the growing challenge of carbapenem-resistant Enterobacterales in Japan and underscoring the critical need for ongoing surveillance to control these pathogens.

Impact of pH on citric acid antimicrobial activity against Gram-negative bacteria.

The antimicrobial activity of citric acid (CA) is often evaluated without pH adjustment or control and its impact on micro-organisms is better understood in acidic conditions. However, the biocidal action of the fully ionized CA molecule, predominantly available at higher pH, has not been previously investigated. The objective of this study was to evaluate the antimicrobial effect of high (10%) and low (1%) concentrations of CA, each adjusted over a wide range of pH values (4·5, 6·5 and 9·5) relative to the controls exposed to corresponding pH levels alone (no CA). The viability and morphology of Escherichia coli and Klebsiella aerogenes were evaluated using a culture-based enumeration assay in parallel with direct SEM imaging. Overall, the highest membrane damage and loss in viability were achieved with 10% CA at pH 9·5, which yielded at least 4·6 log10 CFU per ml (P < 0·001) reductions in both organisms. Insight into the superior efficacy of CA at high pH is proposed based on zeta potential measurements which reveal a more negatively charged bacterial surface at higher pH. This pH-dependent increase in surface charge may have rendered the cells potentially more sensitive towards chelants such as CA3- that interact with membrane-stabilizing divalent metals.

Colorimetric detection of residual quaternary ammonium compounds on dry surfaces and prediction of antimicrobial activity using bromophenol blue.

Controlling and monitoring the residual activity of quaternary ammonium compounds (QACs) are critical for maintaining safe yet effective levels of these agents in the environment. This study investigates the utility of bromophenol blue (BPB) as a safe, rapid and user-friendly indicator to detect in situ residual QACs dried on hard, non-porous surfaces, as well a means to assess their antimicrobial efficacy. At pH 7, BPB has a purple colour which turns blue upon its complexation with QACs such as didecyldimethylammonium chloride (DDAC). BPB itself has no antimicrobial properties up to 400 ppm. Within the range of 0-400 ppm, BPB colour change was tied to specific DDAC antimicrobial performances with a detection threshold of 100 ppm. BPB concentration and application volume could be adjusted such that a colour shift from purple to blue correlated with a set percent reduction (>99·9%) in test bacteria (Staphylococcus aureus and Klebsiella aerogenes). The BPB solutions developed in this study yielded similar colour shifts on polycarbonate and stainless steel surfaces and did not cross-react with chemical ingredients commonly found in sanitizers and disinfectant products. Overall, this study suggests that BPB provides a simple solution to safely monitor the post-application level and biocidal activity of residual dried QACs on surfaces.

7X multiplexed, optofluidic detection of nucleic acids for antibiotic-resistance bacterial screening.

Rapid and accurate diagnosis of bacterial infections resistant to multiple antibiotics requires development of new bio-sensors for differentiated detection of multiple targets. This work demonstrates 7x multiplexed detection for antibiotic-resistance bacterial screening on an optofluidic platform. We utilize spectrally multiplexed multi-spot excitation for simultaneous detection of nucleic acid strands corresponding to bacterial targets and resistance genes. This is enabled by multi-mode interference (MMI) waveguides integrated in an optofluidic device. We employ a combinatorial three-color labeling scheme for the nucleic acid assays to scale up their multiplexing capability to seven different nucleic acids, representing three species and four resistance genes.

Dual Valorization of Olive Mill Wastewater by Bio-Nanosynthesis of Magnesium Oxide and Yarrowia lipolytica Biomass Production.

This research investigates an efficient dual valorization of olive mill wastewater in the biosynthesis of magnesium oxide nanoparticles and in the depollution of the effluent by Yarrowia lipolytica growth evaluation. After removal of polyphenols, the recovered biophenols were reacted with the magnesium precursor to provide magnesium oxide nanoparticles. In order to confirm the biosynthesized magnesium oxide nanoparticles, several analyses were undertaken. The Fourier transform infrared spectrum gives a broad absorption at 658 cm-1 confirming the presence of the magnesium oxide nanoparticles, while the UV/VIS absorption spectroscopy reveals an intense transition with a maximum absorption at 300 nm. The X-ray diffraction and transmission electron microscopy analyses show that nanoparticles are in pure cubic crystalline with spherical and hexagonal shapes (average size is 19.4 nm). The zeta potential analysis illustrates a negative potential proving a good stability of the biosynthesized nanoparticles. Nanoparticles were assigned for their in vitro antibacterial activity against Escherichia coli, Enterobacter aerogenes, Salmonella typhimurium, Staphylococcus cohnii, and Bacillus niacini. The evaluation of the growth of Yarrowia lipolytica on the recovered olive mill wastewater after removal of polyphenols yielded 3.2 g/L of the Yarrowia biomass in 72 h without nutriment additions, providing an important decrease of chemical oxygen demand (73 %).

Next-Generation-Sequencing-Based Hospital Outbreak Investigation Yields Insight into Klebsiella aerogenes Population Structure and Determinants of Carbapenem Resistance and Pathogenicity.

Klebsiella aerogenes is a nosocomial pathogen associated with drug resistance and outbreaks in intensive care units. In a 5-month period in 2017, we experienced an increased incidence of cultures for carbapenem-resistant K. aerogenes (CR-KA) from an adult cardiothoracic intensive care unit (CICU) involving 15 patients. Phylogenomic analysis following whole-genome sequencing (WGS) identified the outbreak CR-KA isolates to group together as a tight monoclonal cluster (with no more than six single nucleotide polymorphisms [SNPs]), suggestive of a protracted intraward transmission event. No clonal relationships were identified between the CICU CR-KA strains and additional hospital CR-KA patient isolates from different wards and/or previous years. Carbapenemase-encoding genes and drug-resistant plasmids were absent in the outbreak strains, and carbapenem resistance was attributed to mutations impacting AmpD activity and membrane permeability. The CICU outbreak strains harbored an integrative conjugative element (ICE) which has been associated with pathogenic Klebsiella pneumoniae lineages (ICEKp10). Comparative genomics with global K. aerogenes genomes showed our outbreak strains to group closely with global sequence type 4 (ST4) strains, which, along with ST93, likely represent dominant K. aerogenes lineages associated with human infections. For poorly characterized pathogens, scaling analyses to include sequenced genomes from public databases offer the opportunity to identify emerging trends and dominant clones associated with specific attributes, syndromes, and geographical locations.

Spheroplast-Mediated Carbapenem Tolerance in Gram-Negative Pathogens.

Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied and yet potentially widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen Vibrio cholerae can tolerate exposure to the typically bactericidal ß-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread ß-lactam tolerance is. Here, we tested a panel of clinically significant Gram-negative pathogens for their response to the potent, broad-spectrum carbapenem antibiotic meropenem. We show that clinical isolates of Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae, but not Escherichia coli, exhibited moderate to high levels of tolerance of meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered wild-type morphology and growth upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in clinically significant bacterial species, and we suggest that this could contribute to treatment failure associated with these organisms.

Anti-pathogenic, antibiofilm, and technological properties of fermented food associated Staphylococcus succinus strain AAS2.

The present investigation was aimed to determine the anti-pathogenic, antibiofilm, and technological properties of fermented food associated Staphylococcus succinus strain AAS2. The anti-pathogenic attribute of cell-free neutralized supernatant (CFNS) of strain AAS2 was assessed against food-borne and enteric pathogens that revealed promising activity against Staphylococcus aureus and Enterobacter aerogenes with high arbitrary unit of 220.25 ± 3.3 and 170.2 ± 4.6 AU/mL, respectively. Furthermore, the antibiofilm and time-kill assay of CFNS of strain AAS2 depicted remarkable reduction in biofilm formation of indicator pathogens in a dose-dependent manner. Moreover, time-kill assay data revealed the drastic reduction in the viability (log cfu/mL) of S. aureus and E. aerogenes in the presence of varied minimum inhibitory concentration ranges of CFNS. The distinct technological properties of strain AAS2 were demonstrated using standard methodologies. Reported results estimated moderate level of exopolysaccharide (41.3 ± 0.6 mg/L) and lipase production (8.3 ± 0.3 mm), followed by remarkable autolytic (30.1 ± 1.2-43.1 ± 1.3%), catalase (13.82 ± 0.3 AU), and nitrate reductase (10.25 ± 0.3 mM nitrite/mg dry weight) activities under standard conditions. Most importantly, the strain cleared the specific in vitro safety assessment tests. The described anti-pathogenic and technological traits of strain AAS2 paved the way to utilize it in pharmaceutical as well as food processing industries as starter/adjunct culture.

Structural investigation of some novel synthesized Schiff base Transition metal complexes derived from drug together with Antimicrobial study.

A new series of copper (II), cobalt (II), zinc (II), nickel (II), manganese (II), iron (II) complexes with a novel Schiff base were synthesized by the condensation of sulphadizine and thiophene-2-carbaldehyde.The ligand and its complexes were characterized by using diverse instrumental procedures like microanalysis, thermo gravimetric examination and spectroscopy. The integrated ligand and its metal complexes were subjected to antibacterial studies. These studies demonstrated the enhanced activity of metal complexes against reported microbes with respect to the Schiff base.

Hydromethanolic extract of Rehum emodi exhibits significant antimicrobial activity against acute gastroenteriti bacterial strains.

Rehum emodi is an important medicinal herbal and has been reported to exhibit tremendous pharmacological potential. The present study was designed to evaluate the antibacterial activity of hydromethanolic extract of rhizome of Rehum emodi against the acute gastroenteriti bacterial strains. The antimicrobial activity was determined by micro-dilution method. Antioxidant activity was determined by DPPH assay and cytotoxicity by MTT assay. Phytochemical analysis was carried out by LC/MS analysis. The results of the present study showed that hydromethanolic extract of rhizome of Rehum emodi (REE) exhibited significant antimicrobial activity against the gastroenteriti bacterial strains. The MIC values ranged from 25 µg/ml to 125 µg/ml. Moreover, the cytotoxicity of the REE was evaluated against the human breast cell line FR-2 and it was observed that REE exerted minimal cytotoxic effects on these cells with an IC50 of 250 µg/ml indicating that this extract is non-toxic to human cells. The phytochemical analysis revealed the presence of several secondery metabolites such as anthroquinones (anthrone, emodin, aloe emodin and rhein) flavonoids (quercetin, and naringenin) and phenolics (sinapinic acid and gallic acid) which could potentially be responsible for the activity of the extract. In conclusion REE could potentially prove to be useful in the treatment of acute gastroenteritis.

Antibacterial, and antioxidant potentials of non-cytotoxic extract of Trichoderma atroviride.

Trichoderma species are a rich source of metabolites, but less known for biomedical potential. This work deals with antibacterial and antioxidant potentials of intracellular non-cytotoxic metabolites, extracted from Trichoderma atroviride (KNUP001). A total of 53 fractions was collected by column chromatography and tested for cytotoxicity by MTT assay. Only one fraction (F41) was found to be non-toxic to Vero cells with 95.4 ±â€¯0.61% of survival. The F41 was then subjected to chemical analysis, antibacterial and antioxidant assays. The F41 at 500 µg ml-1 showed the total antioxidant of 48.70 ±â€¯2.90%, DPPH radical scavenging activity of 37.25 ±â€¯2.25, nitric oxide (NO) radical scavenging activity of 54.55 ±â€¯1.95 and H2O2 radical scavenging activity of 43.75 ±â€¯3.21. The F41 at 25 µg ml-1 displayed antibacterial activity against E. coli (14.25 ±â€¯0.25 mm), Proteus mirabilis (10.40 ±â€¯0.60 mm), and Enterobacter aerogenes (5.60 ±â€¯0.40 mm). GC-MS analysis revealed the dominant presence of oleic acid C 18.1 (63.18%), n-hexadecanoic acid (6.17%), and ethyl oleate (4.93%) in the F41, and hence these fatty acids are likely responsible for the antioxidant and antibacterial activities of F41. Hence, further investigation deserves on purification and characterization of the active metabolites from T. atroviride strain KNUP001 towards developing molecular leads to effective antibacterial drugs, and non-toxic to host cells.

Antimicrobial 1,3,4-trisubstituted-1,2,3-triazolium salts.

A series of 1,3,4-trisubstituted-1,2,3-triazolium bromide salts were prepared by efficient two-step sequences of azide-alkyne cycloaddition and benzylic substitution. The antimicrobial activity of each triazolium salt and correlating triazole precursor was evaluated using a minimum inhibitory concentration (MIC) assay. MIC activities as low as 1 µM against Gram-positive bacteria, 8 µM against Gram-negative bacteria and 4 µM against fungi were observed for salt analogs, while neutral triazoles were inactive. Analogs representing selective and broad-spectrum antimicrobial activity were each identified. MIC structure-activity relationships observed within this motif indicate that the presence of cationic charge and balance of overall hydrophobicity are strongly impactful, while benzyl vs. aryl substituent identity and variation of substituent regiochemistry are not.

Antimicrobial activity and toxicity of gold nanoparticles: research progress, challenges and prospects.

Gold nanoparticles are emerging materials that exhibit characteristics distinct from those of traditional materials and that have promising potential for application in the fields of chemistry, physics, biology and medicine. During the past decades, numerous studies on the antimicrobial activity and toxicity of gold nanoparticles have been published. With respect to antimicrobial activity, gold nanoparticles conjugated with small molecules, such as antibiotics, drugs, vaccines and antibodies, are more efficient than individual nanoparticles and molecules. Regarding the toxicity effects, results are often unclear and conflicting because of the lack of a standard experimental method; various studies have used different approaches, administration routes and doses, and similar experiments may lead to different conclusions. To provide a systematic overview of and insight in the current knowledge for researchers committed to this filed, we discuss the recent research advances related to the antimicrobial activity and toxicity of gold nanoparticles, both in vitro and in vivo, and identify major issues that require further study. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper discusses the recent research progress on antimicrobial activity and toxicity of gold nanoparticles and provides general insights into the field for researchers committed to this field.

Evaluation of the Rapid Polymyxin NP Test for Polymyxin B Resistance Detection Using Enterobacter cloacae and Enterobacter aerogenes Isolates.

Polymyxin resistance is an increasing problem worldwide. Currently, determining susceptibility to polymyxins is problematic and lengthy. Polymyxins diffuse poorly into agar, potentially giving inaccurate disk diffusion and Etest results. A rapid screening test (2 h) for the detection of polymyxin resistance in Enterobacteriaceae, developed by P. Nordmann and L. Poirel (rapid polymyxin NP test) in 2016, detects glucose metabolization in the presence of polymyxin E (PE) and PB via pH-induced color change. The sensitivity and specificity were 99.3 and 95.4%, respectively, with results obtained in ≤2 h. Our goal was to evaluate this test using PB against larger numbers of Enterobacter A total of 143 nonduplicate Enterobacter isolates (102 E. cloacae complex, 41 E. aerogenes) were tested, including 136 collected from Ochsner Health System patients from March to May 2016 and 7 previously determined PB-resistant E. cloacae isolates from JMI Laboratories. MICs were determined via broth microdilution. For the rapid polymyxin NP test, a color change from orange to yellow is positive; a weak/no color change is deemed negative after 4 h. Of 143 Enterobacter isolates, 25 were determined to be PB resistant by broth microdilution (MIC > 2 µg/ml), including all 7 JMI isolates. Of these 25, 7 were positive by the rapid polymyxin NP test (included 3/7 JMI isolates). All 118 isolates determined to be PB susceptible by broth microdilution were NP test negative. The sensitivity and specificity for the rapid polymyxin NP test were 25 and 100%, respectively, compared to broth microdilution. Although the rapid polymyxin NP test is a much faster method (2 to 4 h) for polymyxin resistance determination compared to broth microdilution (16 to 20 h), our study indicates that it may be subject to limitations when testing Enterobacter.

Motuporamine Derivatives as Antimicrobial Agents and Antibiotic Enhancers against Resistant Gram-Negative Bacteria.

Dihydromotuporamine C and its derivatives were evaluated for their in vitro antimicrobial activities and antibiotic enhancement properties against Gram-negative bacteria and clinical isolates. The mechanism of action of one of these derivatives, MOTU-N44, was investigated against Enterobacter aerogenes by using fluorescent dyes to evaluate outer-membrane depolarization and permeabilization. Its efficiency correlated with inhibition of dye transport, thus suggesting that these molecules inhibit drug transporters by de-energization of the efflux pump rather than by direct interaction of the molecule with the pump. This suggests that depowering the efflux pump provides another strategy to address antibiotic resistance.

Detection of New Delhi Metallo-Beta-Lactamase (Encoded by blaNDM-1 ) in Enterobacter aerogenes in China.

BACKGROUND: The increase in blaNDM-1 in Enterobacteriaceae has become a major concern worldwide. In previous study, we investigated clonal dissemination and mechanisms of resistance to carbapenem in China. METHODS: We carried out retrospective surveillance for blaNDM-1 among carbapenem-resistant enterobacter strains, which were isolated from patients at our hospital by bacterial strains selection, antimicrobial susceptibility testing, species identification, and molecular detection of resistance gene. RESULTS: We found three blaNDM-1 -positive isolates which were identified as Enterobacter aerogenes in clinical patients in China. The blaNDM-1 -positive Enterobacter aerogenes isolates were first found. CONCLUSION: It is important to mandate prudent usage of antibiotics and implement infection control measures to control the spread of these resistant blaNDM-1 -positive strains.

Isolation and characterization of antibiotic producing bacterial strains from red soil of Himalayan region of Pakistan.

The emergence of multi drug resistant microbial pathogens has become a global health challenge and set a dire requirement of searching new effective antimicrobials. Soil is an ultimate reservoir of biologically active micro flora, which harbors trillions of microbial strains producing compounds of commercial interest. Hence aim of the present study was an attempt to isolate and identify the antibiotic producing microbial strains from the red soil of Himalayan an unexplored region of Pakistan. In this study from 10 different soil samples only one bacterial strain was isolated capable of antimicrobial activity. Strain was identified by biochemical characteristics and final identification was done by API 20 NE kit which showed 99% homology with P. aeruginosa. Hence the strain was identified as P. aeruginosa S2. Antibacterial and antifungal activity of the P. aeruginosa S2 showed that Staphylococcus aureus was extremely sensitive to it with a zone of inhibition of 42mm. Staphylococcus epidermidis, Enterobacter aerogenes, Aspergillus fumigatus and Candida albicans were also inhibited by the isolated strain. Effect of Glycerol, Copper sulphate (CuSo4), Sodium sulphate (Na2SO4) and Glycerol on antibiotic production was also evaluated by supplementing growth media with these chemicals. Pseudomonas aeruginosa was grown in bulk quantity using solid state fermentation and crude extract was prepared using organic solvents and subjected to silica gel column chromatography for purification of active compound. Purified compound showed antibacterial against human pathogens. The unexplored Kashmir Himalayas are of great significance because of its richness in biodiversity and need to be explored for isolation and characterization of native microbes for biologically active secondary metabolites. This untouched region may be considered as hub of new antimicrobials and may have applications in natural product-based drug discovery.

Evaluation of different pretreatment protocols to detect accurately clinical carbapenemase-producing Enterobacteriaceae by MALDI-TOF.

OBJECTIVES: Carbapenemase-resistant bacteria are increasingly spreading worldwide causing public concern due to their ability to elude antimicrobial treatment. Early identification of these bacteria is therefore of high importance. Here, we describe the development of a simple and robust protocol for the detection of carbapenemase activity in clinical isolates of Enterobacteriaceae, suitable for routine and clinical applications. METHODS: The final protocol involves cellular lysis and enzyme extraction from a defined amount of bacterial cells followed by the addition of a benchmark drug (e.g. the carbapenem antibiotic imipenem or ertapenem). Carbapenem inactivation is mediated by enzymatic hydrolysis (cleavage) of the ß-lactam common structural motif, which can be detected using MALDI-TOF MS. RESULTS: A total of 260 strains were studied (208 carbapenemase producers and 52 non-carbapenemase producers) resulting in 100% sensitivity and 100% specificity for the KPC, NDM and OXA-48-like PCR-confirmed positive isolates using imipenem as benchmark. Differences between the benchmark (indicator) antibiotics imipenem and ertapenem, buffer constituents and sample preparation methods have been investigated. Carbapenemase activity was further characterized by performing specific inhibitor experiments. Intraday and interday reproducibility (coefficient of variation) of the observed hydrolysis results were 15% and 30%, respectively. A comparative study of our extraction method and a recently published method using whole bacterial cells is presented and differences are discussed. CONCLUSIONS: Using this method, an existing carbapenemase activity can be directly read from the mass spectrum as a ratio of hydrolysed product and substrate, setting an important step towards routine application in clinical laboratories.

Synthesis of novel sulfonamide analogs containing sulfamerazine/sulfaguanidine and their biological activities.

Sulfamerazine and sulfaguanidine are clenched with p-nitrobenzoyl chloride and the products obtained are reduced to NaxS in ethanol-water. Novel sulfonamides (6a-g and 9a-g) were synthesized by the reaction of these reduced products (4 and 8) with various sulfonyl chlorides (5a-g). The structures of these compounds were characterized using spectroscopic analysis (IR, (1)H-NMR, (13)C-NMR and HRMS) technique. Antimicrobial activity of sulfonamides (3, 4, 7, 8, 6a-g and 9a-g) was evaluated by the agar diffusion method. These compounds showed antimicrobial activity against tested microorganism strains (Gram-positive bacteria, clinic isolate and yeast and mold). Compounds 9d, 9e, 9a, 6d and 6e showed particularly antimicrobial activity against tested Gram-positive (Bacillus cereus and B. subtilis) and Gram-negative (Enterobacter aerogenes) bacteria.