Host microbiome associated low intestinal acetate correlates with progressive NLRP3-dependent hepatic-immunotoxicity in early life microcystin-LR exposure.

BACKGROUND: Microcystins (MCs), potent hepatotoxins pose a significant health risk to humans, particularly children, who are more vulnerable due to higher water intake and increased exposure during recreational activities. METHODS: Here, we investigated the role of host microbiome-linked acetate in modulating inflammation caused by early-life exposure to the cyanotoxin Microcystin-LR (MC-LR) in a juvenile mice model. RESULTS: Our study revealed that early-life MC-LR exposure disrupted the gut microbiome, leading to a depletion of key acetate-producing bacteria and decreased luminal acetate concentration. Consequently, the dysbiosis hindered the establishment of a gut homeostatic microenvironment and disrupted gut barrier function. The NOD-like receptor family pyrin domain - containing 3 (NLRP3) inflammasome, a key player in MC-induced hepatoxicity emerged as a central player in this process, with acetate supplementation effectively preventing NLRP3 inflammasome activation, attenuating hepatic inflammation, and decreasing pro-inflammatory cytokine production. To elucidate the mechanism underlying the association between early-life MC-LR exposure and the progression of metabolic dysfunction associated steatotic liver disease (MASLD), we investigated the role of acetate binding to its receptor -G-protein coupled receptor 43 (GPR43) on NLRP3 inflammasome activation. Our results demonstrated that acetate-GPR43 signaling was crucial for decreasing NLRP3 protein levels and inhibiting NLRP3 inflammasome assembly. Further, acetate-induced decrease in NLRP3 protein levels was likely mediated through proteasomal degradation rather than autophagy. Overall, our findings underscore the significance of a healthy gut microbiome and its metabolites, particularly acetate, in the progression of hepatotoxicity induced by early life toxin exposure, crucial for MASLD progression. CONCLUSIONS: This study highlights potential therapeutic targets in gut dysbiosis and NLRP3 inflammasome activation for mitigating toxin-associated inflammatory liver diseases.

Host gut resistome in Gulf War chronic multisymptom illness correlates with persistent inflammation.

Chronic multisymptom illness (CMI) affects a subsection of elderly and war Veterans and is associated with systemic inflammation. Here, using a mouse model of CMI and a group of Gulf War (GW) Veterans' with CMI we show the presence of an altered host resistome. Results show that antibiotic resistance genes (ARGs) are significantly altered in the CMI group in both mice and GW Veterans when compared to control. Fecal samples from GW Veterans with persistent CMI show a significant increase of resistance to a wide class of antibiotics and exhibited an array of mobile genetic elements (MGEs) distinct from normal healthy controls. The altered resistome and gene signature is correlated with mouse serum IL-6 levels. Altered resistome in mice also is correlated strongly with intestinal inflammation, decreased synaptic plasticity, reversible with fecal microbiota transplant (FMT). The results reported might help in understanding the risks to treating hospital acquired infections in this population.

Environmental Microcystin exposure in underlying NAFLD-induced exacerbation of neuroinflammation, blood-brain barrier dysfunction, and neurodegeneration are NLRP3 and S100B dependent.

Nonalcoholic fatty liver disease (NAFLD) has been shown to be associated with extrahepatic comorbidities including neuronal inflammation and Alzheimer's-like pathology. Environmental and genetic factors also act as a second hit to modulate severity and are expected to enhance the NAFLD-linked neuropathology. We hypothezied that environmental microcystin-LR (MC-LR), a toxin produced by harmful algal blooms of cyanobacteria, exacerbates the neuroinflammation and degeneration of neurons associated with NAFLD. Using a mouse model of NAFLD, exposed to MC-LR subsequent to the onset of fatty liver, we show that the cyanotoxin could significantly increase proinflammatory cytokine expression in the frontal cortex and cause increased expression of Lcn2 and HMGB1. The above effects were NLRP3 inflammasome activation-dependent since the use of NLRP3 knockout mice abrogated the increase in inflammation. NLRP3 was also responsible for decreased expression of the blood-brain barrier (BBB) tight junction proteins Occludin and Claudin 5 suggesting BBB dysfunction was parallel to neuroinflammation following microcystin exposure. An increased circulatory S100B release, a hallmark of astrocyte activation in MC-LR exposed NAFLD mice also confirmed BBB integrity loss, but the astrocyte activation observed in vivo was NLRP3 independent suggesting an important role of a secondary S100B mediated crosstalk. Mechanistically, conditioned medium from reactive astrocytes and parallel S100B incubation in neuronal cells caused increased inducible NOS, COX-2, and higher BAX/ Bcl2 protein expression suggesting oxidative stress-mediated neuronal cell apoptosis crucial for neurodegeneration. Taken together, MC-LR exacerbated neuronal NAFLD-linked comorbidities leading to cortical inflammation, BBB dysfunction, and neuronal apoptosis.

Overexpression of Efflux Pumps, Mutations in the Pumps' Regulators, Chromosomal Mutations, and AAC(6')-Ib-cr Are Associated With Fluoroquinolone Resistance in Diverse Sequence Types of Neonatal Septicaemic Acinetobacter baumannii: A 7-Year Single Center Study.

This study investigates susceptibility toward three fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin), multiple fluoroquinolone-resistance mechanisms, and epidemiological relationship of neonatal septicaemic Acinetobacter baumannii. Previous studies on fluoroquinolone resistance in A. baumannii focused primarily on ciprofloxacin susceptibility and assessed a particular mechanism of resistance; a more holistic approach was taken here. Epidemiological relationship was evaluated by Multi Locus Sequence Typing. Minimum Inhibitory Concentrations of fluoroquinolones was determined with and without efflux pump inhibitors. Overexpression of efflux pumps, resistance-nodulation-cell-division (RND)-type, and multidrug and toxic compound extrusion (MATE)-type efflux pumps were evaluated by reverse transcriptase-qPCR. Mutations within regulatory proteins (AdeRS, AdeN, and AdeL) of RND-pumps were examined. Chromosomal mutations, presence of qnr and aac(6')-Ib-cr were investigated. A. baumannii were highly diverse as 24 sequence-types with seven novel STs (ST-1440/ST-1441/ST-1481/ST-1482/ST-1483/ST-1484/ST-1486) were identified among 47 A. baumannii. High resistance to ciprofloxacin (96%), levofloxacin (92%), and particularly moxifloxacin (90%) was observed, with multiple mechanisms being active. Resistance to 4th generation fluoroquinolone (moxifloxacin) in neonatal isolates is worrisome. Mutations within GyrA (S83L) and ParC (S80L) were detected in more than 90% of fluoroquinolone-resistant A. baumannii (FQRAB) spread across 10 different clonal complexes (CC1/CC2/CC10/CC25/CC32/CC126/CC149/CC216/CC218/CC513). Efflux-based FQ resistance was found in 65% of FQRAB with ≥2 different active pumps in 38% of strains. Overexpression of adeB was highest (2.2-34-folds) followed by adeJ, adeG, and abeM. Amino acid changes in the regulators (AdeRS/AdeN/AdeL) either as single or multiple substitutions substantiated the overexpression of the pumps. Diverse mutations within AdeRS were detected among different CCs whereas mutations within AdeN linked to CC10 and CC32. Chromosomal mutations and active efflux pumps were detected simultaneously among 64% of FQRAB. Presence of aac(6')-Ib-cr was also high (74% of FQRAB) but qnrS were absent. As most FQRABs had chromosomal mutations, this was considered predominant, however, isolates where pumps were also active had higher MIC values, establishing the critical role of the efflux pumps. The high variability of FQ susceptibility among FQRAB, possessing the same set of mutations in gyrA, parC, and efflux pump regulators, was also noted. This reveals the complexity of interpreting the interplay of multiple resistance mechanisms in A. baumannii.

Acinetobacter baumannii transfers the blaNDM-1 gene via outer membrane vesicles.

Objectives: To investigate the transmission of the gene encoding New Delhi metallo-ß-lactamase-1 ( bla NDM-1 ) through outer membrane vesicles (OMVs) released from an Acinetobacter baumannii strain (A_115). Methods: Isolation and purification of OMVs by density gradient from a carbapenem-resistant clinical strain of A. baumannii harbouring plasmid-mediated bla NDM-1 and aac(6')-Ib-cr genes was performed. DNA was purified from the OMVs and used for PCR and dot-blot analysis. Vesicles treated with DNase I and proteinase K were used to transform A. baumannii ATCC 19606 and Escherichia coli JM109 strains. MIC values for the transformants were determined, followed by PCR and restriction digestion of plasmids. PFGE was done for A_115 and transformants of ATCC 19606 and JM109. Results: The A. baumannii strain (ST 1462) released vesicles (25-100 nm) during in vitro growth at late log phase. PCR and dot-blot analysis confirmed the presence of bla NDM-1 and aac(6')-Ib-cr genes in intravesicular DNA. bla NDM-1 and aac(6')-Ib-cr genes were transferred to both the A. baumannii ATCC 19606 and E. coli JM109 recipient cells. The transformation frequency of the purified OMVs was in the range of 10 -5 -10 -6 and gradually reduced with storage of OMVs. The sizes of the plasmids in the transformants and their restriction digestion patterns were identical to the plasmid in A_115. The transformants showed elevated MIC values of the ß-lactam group of antibiotics, which confirmed the presence of a bla NDM-1 -harbouring plasmid. Conclusions: This is the first experimental evidence of intra- and inter-species transfer of a plasmid harbouring a bla NDM-1 gene in A. baumannii via OMVs with high transformation frequency.

Carbapenem Resistance in Acinetobacter baumannii and Other Acinetobacter spp. Causing Neonatal Sepsis: Focus on NDM-1 and Its Linkage to ISAba125.

Carbapenem-resistant determinants and their surrounding genetic structure were studied in Acinetobacter spp. from neonatal sepsis cases collected over 7 years at a tertiary care hospital. Acinetobacter spp. (n = 68) were identified by ARDRA followed by susceptibility tests. Oxacillinases, metallo-ß-lactamases (MBLs), extended-spectrum ß-lactamases and AmpCs, were detected phenotypically and/or by PCR followed by DNA sequencing. Transconjugants possessing the bla NDM-1(New Delhi metallo-ß-lactamase) underwent further analysis for plasmids, integrons and associated genes. Genetic environment of the carbapenemases were studied by PCR mapping and DNA sequencing. Multivariate logistic regression was used to identify risk factors for sepsis caused by NDM-1-harboring organisms. A. baumannii (72%) was the predominant species followed by A. calcoaceticus (10%), A. lwoffii (6%), A. nosocomialis (3%), A. junni (3%), A. variabilis (3%), A. haemolyticus (2%), and 14TU (2%). Fifty six percent of the isolates were meropenem-resistant. Oxacillinases present were OXA-23-like, OXA-58-like and OXA-51-like, predominately in A. baumannii. NDM-1 was the dominant MBL (22%) across different Acinetobacter spp. Isolates harboring NDM-1 also possessed bla (VIM-2, PER-1, VEB-2, CTX-M-15), armA, aac(6')Ib, aac(6')Ib-cr genes. bla NDM-1was organized in a composite transposon between two copies of ISAba125 in the isolates irrespective of the species. Further, OXA-23-like gene and OXA-58-like genes were linked with ISAba1 and ISAba3 respectively. Isolates were clonally diverse. Integrons were variable in sequence but not associated with carbapenem resistance. Most commonly found genes in the 5' and 3'conserved segment were aminoglycoside resistance genes (aadB, aadA2, aac4'), non-enzymatic chloramphenicol resistance gene (cmlA1g) and ADP-ribosylation genes (arr2, arr3). Outborn neonates had a significantly higher incidence of sepsis due to NDM-1 harboring isolates than their inborn counterparts. This study demonstrates the significance of both A. baumannii and other species of Acinetobacter in cases of neonatal sepsis over an extended period. Oxacillinases and bla NDM-1 are the major contributors to carbapenem resistance. The dissemination of the bla NDM-1 is likely linked to Tn125 in diverse clones of the isolates.

A reliable phenotypic assay for detection of ESBLs and AmpCs in MBL-producing gram-negative bacteria with the use of aminophenylboronic acid, dipicolinic acid and cloxacillin.

ESBLs and AmpCs may escape detection when they coexist with metallo-ß-lactamases such as New Delhi Metallo-ß-lactamases-1. In this study a combination disk assay was established using cefotaxime, cefotaxime/clavulanic acid, cefotaxime/clavulanic acid/cloxacillin, cefoxitin and cefoxitin/phenylboronic acid/cloxacillin on Mueller Hinton agar supplemented with dipicolinic acid for determination of ß-lactamases in the presence of NDM-1.

A five-year experience of carbapenem resistance in Enterobacteriaceae causing neonatal septicaemia: predominance of NDM-1.

Treatment of neonatal sepsis has become a challenge with the emergence of carbapenemase-producing bacteria. This study documents the trend of carbapenem susceptibility in Enterobacteriaceae that caused septicaemia in neonates over a five year period (2007-2011) and the molecular characterisation of Enterobacteriaceae resistant to carbapenems and cephalosporins. Hundred and five Enterobacteriaceae including Escherichia coli (n = 27), Klebsiella pneumoniae (n = 68) and Enterobacter spp. (n = 10) were isolated from blood of septicaemic neonates followed by antibiotic susceptibility tests, determination of MIC values, phenotypic and genotypic detection of ß-lactamases. Carbapenem was the most active antimicrobial tested after tigecycline. CTX-M type was the most prevalent ESBL throughout the period (82%). New Delhi Metallo-ß-lactamase-1 (NDM-1), which is a recent addition to the carbapenemase list, was the only carbapenemase identified in our setting. Fourteen percent of the isolates possessed blaNDM-1. Carbapenem non-susceptibility was first observed in 2007 and it was due to loss of Omp F/Ompk36 in combination with the presence of ESBLs/AmpCs. NDM-1 first emerged in E. coli during 2008; later in 2010, the resistance was detected in K. pneumoniae and E. cloacae isolates. NDM-1-producing isolates were resistant to other broad-spectrum antibiotics and possessed ESBLs, AmpCs, 16S-rRNA methylases, AAC(6')-Ib-cr, bleomycin resistant gene and class 1 integron. Pulsed field gel electrophoresis of the NDM-1-producing isolates indicated that the isolates were clonally diverse. The study also showed that there was a significantly higher incidence of sepsis caused by NDM-1-harbouring isolates in the male sex, in neonates with low birth weight and neonates born at an extramural centre. However, sepsis with NDM-1-harbouring isolates did not result in a higher mortality rate. The study is the first to review the carbapenem resistance patterns in neonatal sepsis over an extended period of time. The study highlights the persistence of ESBLs (CTX-Ms) and the emergence of NDM-1 in Enterobacteriaceae in the unit.