A Novel Microbial Zearalenone Transformation through Phosphorylation

Zearalenone (ZEA) is a mycotoxin widely occurring in many agricultural commodities. In this study, a purified bacterial isolate, Bacillus sp. S62-W, obtained from one of 104 corn silage samples from various silos located in the United States, exhibited activity to transform the mycotoxin ZEA. A novel microbial transformation product, ZEA-14-phosphate, was detected, purified, and identified by HPLC, LC-MS, and NMR analyses. The isolate has been identified as belonging to the genus Bacillus according to phylogenetic analysis of the 16S rRNA gene and whole genome alignments. The isolate showed high efficacy in transforming ZEA to ZEA-14-phosphate (100% transformation within 24 h) and possessed advantages of acid tolerance (work at pH = 4.0), working under a broad range of temperatures (22–42 °C), and a capability of transforming ZEA at high concentrations (up to 200 µg/mL). In addition, 23 Bacillus strains of various species were tested for their ZEA phosphorylation activity. Thirteen of the Bacillus strains showed phosphorylation functionality at an efficacy of between 20.3% and 99.4% after 24 h incubation, suggesting the metabolism pathway is widely conserved in Bacillus spp. This study established a new transformation system for potential application of controlling ZEA although the metabolism and toxicity of ZEA-14-phosphate requires further investigation.     

Degradation of histamine by Bacillus polymyxa isolated from salted fish products

Histamine is the causative agent of scombroid poisoning, a foodborne chemical hazard. Histamine is degraded by the oxidative deamination activity of certain microorganisms. In this study, eight histamine-degrading bacteria isolated from salted fish products were identified as Rummeliibacillus stabekisii (1 isolate), Agrobacterium tumefaciens (1 isolate), Bacillus cereus (2 isolates), Bacillus polymyxa (1 isolate), Bacillus licheniformis (1 isolate), Bacillus amyloliquefaciens (1 isolate), and Bacillus subtilis (1 isolate). Among them, B. polymyxa exhibited the highest activity in degrading histamine than the other isolates. The ranges of temperature, pH, and salt concentration for growth and histamine degradation of B. polymyxa were 25–37°C, pH 5–9, and 0.5–5% NaCl, respectively. B. polymyxa exhibited optimal growth and histamine-degrading activity at 30°C, pH 7, and 0.5% NaCl in histamine broth for 24 hours of incubation. The histamine-degrading isolate, B. polymyxa, might be used as a starter culture in inhibiting histamine accumulation during salted fish product fermentation.     

Mycotoxin Removal by Lactobacillus spp. and Their Application in Animal Liquid Feed

The removal of mycotoxins from contaminated feed using lactic acid bacteria (LAB) has been proposed as an inexpensive, safe, and promising mycotoxin decontamination strategy. In this study, viable and heat-inactivated L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T cells were investigated for their ability to remove aflatoxin B₁ (AFB₁), ochratoxin A (OTA), zearalenone (ZEA), and deoxynivalenol (DON) from MRS medium and PBS buffer over a 24 h period at 37 °C. LAB decontamination activity was also assessed in a ZEA-contaminated liquid feed (LF). Residual mycotoxin concentrations were determined by UHPLC-FLD/DAD analysis. In PBS, viable L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T cells removed up to 57% and 30% of ZEA and DON, respectively, while AFB₁ and OTA reductions were lower than 15%. In MRS, 28% and 33% of ZEA and AFB₁ were removed, respectively; OTA and DON reductions were small (≤15%). Regardless of the medium, heat-inactivated cells produced significantly lower mycotoxin reductions than those obtained with viable cells. An adsorption mechanism was suggested to explain the reductions in AFB₁ and OTA, while biodegradation could be responsible for the removal of ZEA and DON. Both viable LAB strains reduced ZEA by 23% in contaminated LF after 48 h of incubation. These findings suggest that LAB strains of L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T may be applied in the feed industry to reduce mycotoxin contamination.     

Cell free preparations of probiotics exerted antibacterial and antibiofilm activities against multidrug resistant E. coli

The sharp increase in antibiotic resistance imposes a global threat to human health and the discovery of effective antimicrobial alternatives is needed. The use of probiotics to combat bacterial pathogens has gained a rising interest. Pathogenic Escherichia coli is causative of multiple clinical syndromes such as diarrheal diseases, meningitis and urinary tract infections. In this work, we evaluated the efficacy of probiotics to control multidrug-resistant E. coli and reduce their ability to form biofilms. Six E. coli resistant to at least five antibiotics (Ceftazidime, Ampicillin, Clarithromycin, Amoxicillin?+?Clavulanic Acid and Ceftriaxone) were isolated in this work. Preparations of cell-free spent media (CFSM) of six probiotics belonging to the genus Bifidobacterium and Lactobacillus which were grown in Man-Rogosa-Sharpe (MRS) broth exhibited strong antibacterial activity (inhibition zones of 11.77–23.10?mm) against all E. coli isolates. Two E. coli isolates, namely E. coli WW1 and IC2, which were most resistant to all antibiotics were subjected to antibiofilm experiments. Interestingly, the CFSM of MRS fermented by all probiotics resulted in inhibition of biofilm formation while B. longum caused highest inhibition (57.94%) in case of E. coli IC2 biofilms and L. plantarum was responsible for 64.57% reduction of E. coli WW1 biofilms. On the other hand, CFSM of skim milk fermented by L. helveticus and L. rhamnosus exhibited a slight inhibitory activity against IC2 isolate (inhibition percentage of 31.52 and 17. 68, respectively) while WW1 isolate biofilms was reduced by CFSM of milk fermented by B. longum and L. helveticus (70.81 and 69.49 reduction percentage, respectively). These results support the effective use of probiotics as antimicrobial alternatives and to eradicate biofilms formed by multidrug-resistant E. coli.     

Hygienic quality,adulteration of pork and histamine production by Raoultella ornithinolytica in milkfish dumpling

Ten milkfish dumpling products purchased from retail stores in southern Taiwan were collected to determine the occurrence of biogenic amines, histamine-forming bacteria, and adulteration of pork. This study showed the high contents of aerobic plate count (APC), total coliforms (TC) and Escherichia coli in tested milkfish dumpling samples, whereas the average content of various biogenic amines in all tested samples was < 1.6 mg/100 g (< 0.05 to 1.54 mg/100 g). Three histamine-producing bacterial strains (2 isolates of Raoultella ornithinolytica and 1 isolate of Enterobacter aerogenes) isolated from tested samples produced 276.6 ppm to 561.8 ppm of histamine in trypticase soy broth supplemented with 1.0% L-histidine (TSBH). Assay of multiplex polymerase chain reaction (PCR) revealed that the adulteration rates were 50% (5/10) for pork in milkfish dumplings. In addition, milkfish dumpling stuffing was inoculated with R. ornithinolytica at 5.0 log colony forming units (CFU)/g and stored at various temperatures from 4°C to 37°C to investigate bacterial growth and formation of histamine. The histamine contents quickly increased to higher than 50 mg/100 g in samples stored at 37°C and 25°C within 24 hours and 36 hours, respectively, as well as stored at 15°C within 48 hours. Therefore, bacterial growth and histamine formation were controlled by cold storage of the samples at 4°C.     

Elimination of Deoxynivalenol,Aflatoxin B1, and Zearalenone by Gram-Positive Microbes (Firmicutes)

Mycotoxin contaminations in the feed and food chain are common. Either directly or indirectly, mycotoxins enter the human body through the consumption of food of plant and animal origin. Bacteria with a high mycotoxin elimination capability can reduce mycotoxin contamination in feed and food. Four Gram-positive endospore-forming bacteria (Bacillus thuringiensis AMK10/1, Lysinibacillus boronitolerans AMK9/1, Lysinibacillus fusiformis AMK10/2, and Rummeliibacillus suwonensis AMK9/2) were isolated from fermented forages and tested for their deoxynivalenol (DON), aflatoxin B1 (AFB1), and zearalenone (ZEA) elimination potentials. Notably, the contribution of bacterial cell wall fractions to the observed outstanding ZEA elimination rates was demonstrated; however, the ZEA elimination differed considerably within the tested group of Gram-positive bacteria. It is worth noting that the purified cell wall of L. boronitolerans AMK9/1, L. fusiformis AMK10/2 and B. thuringiensis AMK10/1 were highly efficient in eliminating ZEA and the teichoic acid fractions of B. thuringiensis AMK10/1, and L. fusiformis AMK10/2 could also be successfully used in ZEA binding. The ZEA elimination capacity of viable R. suwonensis AMK9/2 cells was outstanding (40%). Meanwhile, R. suwonensis AMK9/2 and L. boronitolerans AMK9/1 cells produced significant esterase activities, and ZEA elimination of the cell wall fractions of that species did not correlate with esterase activity. DON and AFB1 binding capabilities of the tested bacterial cells and their cell wall fractions were low, except for B. thuringiensis AMK10/1, where the observed high 64% AFB1 elimination could be linked to the surface layer (S-layer) fraction of the cell wall.     

Biodegradation of Ochratoxin A by Bacterial Strains Isolated from Vineyard Soils

Ochratoxin A (OTA) is a mycotoxin with a main nephrotoxic activity contaminating several foodstuffs. In the present report, five soil samples collected from OTA-contaminated vineyards were screened to isolate microorganisms able to biodegrade OTA. When cultivated in OTA-supplemented medium, OTA was converted in OTα by 225 bacterial isolates. To reveal clonal relationships between isolates, molecular typing by using an automated rep-PCR system was carried out, thus showing the presence of 27 different strains (rep-PCR profiles). The 16S-rRNA gene sequence analysis of an isolate representative of each rep-PCR profiles indicated that they belonged to five bacterial genera, namely Pseudomonas, Leclercia, Pantoea, Enterobacter, and Acinetobacter. However, further evaluation of OTA-degrading activity by the 27 strains revealed that only Acinetobacter calcoaceticus strain 396.1 and Acinetobacter sp. strain neg1, consistently conserved the above property; their further characterization showed that they were able to convert 82% and 91% OTA into OTα in six days at 24 °C, respectively. The presence of OTα, as the unique OTA-degradation product was confirmed by LC-HRMS. This is the first report on OTA biodegradation by bacterial strains isolated from agricultural soils and carried out under aerobic conditions and moderate temperatures. These microorganisms might be used to detoxify OTA-contaminated feed and could be a new source of gene(s) for the development of a novel enzymatic detoxification system.     

In Vitro Mechanism Assessment of Zearalenone Removal by Plant-Derived Lactobacillus plantarum BCC 47723

Zearalenone (ZEA) is a harmful secondary fungal metabolite, produced primarily by plant pathogenic fungi mostly belonging to the genus Fusarium. It is involved in reproductive disorders in animals since its structure is similar to the estrogen hormone. This induces precocious pubertal changes, fertility problems, and hyper estrogenic disorders. The main objectives of this study were to evaluate the ZEA removal capacity of plant-derived lactic acid bacteria (LAB) and to investigate the possible components and mechanisms involved in the removal of ZEA by physically and chemically treated plant-derived LAB. The bacterial cells were characterized using scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDS), Fourier transform infrared spectroscopy (FTIR), and the analysis of zeta potential, and hydrophobic index. Results revealed that 17 out of 33 plant-derived LAB exhibited ZEA removal from liquid medium. The percentage of removal ranged from 0.5–23% and Lactobacillus plantarum BCC 47723, isolated from wild spider flower pickle (Pag-sian-dorng), exhibited the highest removal. The alteration of proteins on L. plantarum BCC 47723 structure by Sodium dodecyl sulphate (SDS) treatment was positively affected on ZEA removal, whereas that of lipids on ZEA removal was negatively observed. Heat treatment influenced the higher ZEA adsorption. SEM images showed that the morphologies of modified bacterial cells were distinctly deformed and damaged when compared with untreated control. FTIR analysis indicated that the original functional groups, which included amide (C=O, C-N), carboxyl (C=O, C-O, O-H), methylene (C=C), and alcohol (O-H) groups, were not changed after ZEA adsorption. The zeta potential indicated that electrostatic interaction was not involved in the ZEA removal, while hydrophobicity was the main force to interact with ZEA. These findings can conclude that adsorption by hydrophobicity is the main mechanism for ZEA removal of plant-derived L. plantarum BCC 47723. The alteration of bacterial cell structure by heat treatment enhanced the efficiency of L. plantarum BCC 47723 for ZEA reduction. Its activity can be protected by the freeze-drying technique. Hence, plant-derived L. plantarum BCC 47723 can be considered as an organic adsorbent for ZEA reduction in food and feedstuff.     

Aflatoxin B1 Degradation by a Pseudomonas Strain

Aflatoxin B₁ (AFB₁), one of the most potent naturally occurring mutagens and carcinogens, causes significant threats to the food industry and animal production. In this study, 25 bacteria isolates were collected from grain kernels and soils displaying AFB₁ reduction activity. Based on its degradation effectiveness, isolate N17-1 was selected for further characterization and identified as Pseudomonas aeruginosa. P. aeruginosa N17-1 could degrade AFB₁, AFB₂ and AFM₁ by 82.8%, 46.8% and 31.9% after incubation in Nutrient Broth (NB) medium at 37 °C for 72 h, respectively. The culture supernatant of isolate N17-1 degraded AFB₁ effectively, whereas the viable cells and intra cell extracts were far less effective. Factors influencing AFB₁ degradation by the culture supernatant were investigated. Maximum degradation was observed at 55 °C. Ions Mn²⁺ and Cu²⁺ were activators for AFB₁ degradation, however, ions Mg²⁺, Li⁺, Zn²⁺, Se²⁺, Fe³⁺ were strong inhibitors. Treatments with proteinase K and proteinase K plus SDS significantly reduced the degradation activity of the culture supernatant. No degradation products were observed based on preliminary LC-QTOF/MS analysis, indicating AFB₁ was metabolized to degradation products with chemical properties different from that of AFB₁. The results indicated that the degradation of AFB₁ by P. aeruginosa N17-1 was enzymatic and could have a great potential in industrial applications. This is the first report indicating that the isolate of P. aeruginosa possesses the ability to degrade aflatoxin.     

In vitro and intracellular activities of fosfomycin against clinical strains of Listeria monocytogenes

This study was designed to evaluate the potential role of fosfomycin as a therapeutic agent in human listeriosis. The in vitro activity of fosfomycin against 154 Listeria monocytogenes clinical isolates under conditions that mimic the induction of prfA expression was determined and was correlated with fosfomycin intracellular antimicrobial activity. In vitro, partial induction of prfA expression is achieved through bacterial growth in brain–heart infusion agar supplemented with activated charcoal (BHIC). A fosfomycin pharmacokinetic/pharmacodynamic breakpoint of ≤64 mg/L was estimated using a Monte Carlo simulation to assess the success of an intravenous fosfomycin dose of 300 mg/kg/day over 5000 individuals. Eighty strains (51.9%) were susceptible to fosfomycin in BHIC, with minimum inhibitory concentrations (MICs) of ≤64 mg/L; 13 strains (8.4%) had the epidemic clone (EC) marker. In addition, 27 strains (17.5%) had a three doubling dilutions reduction in the MIC from ≥1024 mg/L to 128 mg/L (96–128 mg/L by Etest). The fosfomycin modal MIC is lower under prfA expression. However, this effect is smaller in terms of clinical categorisation of isolates and can be influenced by the serotype and clonal type. In A549 cells, the reductions in bacterial inocula of the two susceptible isolates studied after 1 h and 24 h of incubation with fosfomycin at 0.5× the human maximum serum concentration (C_max) were 45.8% and 46.6%, and 93.8% and 99.1%, respectively. Slightly higher reductions were found with fosfomycin at 1× C_max. The resistant strain tested showed significantly lower reductions in all assays.     

Impact of the Epithelial Lining Fluid Milieu on Amikacin Pharmacodynamics Against Pseudomonas aeruginosa

BackgroundEven though nebulised administration of amikacin can achieve high epithelial lining fluid concentrations, this has not translated into improved patient outcomes in clinical trials. One possible reason is that the cellular and chemical composition of the epithelial lining fluid may inhibit amikacin-mediated bacterial killing.ObjectiveThe objective of this study was to identify whether the epithelial lining fluid components inhibit amikacin-mediated bacterial killing.MethodsTwo amikacin-susceptible (minimum inhibitory concentrations of 2 and 8 mg/L) Pseudomonas aeruginosa isolates were exposed in vitro to amikacin concentrations up to 976 mg/L in the presence of an acidic pH, mucin and/or surfactant as a means of simulating the epithelial lining fluid, the site of bacterial infection in pneumonia. Pharmacodynamic modelling was used to describe associations between amikacin concentrations, bacterial killing and emergence of resistance.ResultsIn the presence of broth alone, there was rapid and extensive (> 6 − log₁₀) bacterial killing, with emergence of resistance identified in amikacin concentrations < 976 mg/L. In contrast, the rate and extent of bacterial killing was reduced (≤ 5 − log₁₀) when exposed to an acidic pH and mucin. Surfactant did not appreciably impact the bacterial killing or resistance emergence when compared with broth alone for either isolate. The combination of mucin and an acidic pH further reduced the rate of bacterial killing, with the maximal bacterial killing occurring 24 h following initial exposure compared with approximately 4–8 h for either mucin or an acidic pH alone.ConclusionsOur findings indicate that simulating the epithelial lining fluid antagonises amikacin-mediated killing of P. aeruginosa, even at the high concentrations achieved following nebulised administration.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40268-021-00344-5.     

Inhibition of colonic motility and defecation by RS-127445 suggests an involvement of the 5-HT2B receptor in rodent large bowel physiology

Background:

5-HT_2B receptors are localized within the myenteric nervous system, but their functions on motor/sensory neurons are unclear. To explore the role of these receptors, we further characterized the 5-HT_2B receptor antagonist RS-127445 and studied its effects on peristalsis and defecation.

Experimental approach:

Although reported as a selective 5-HT_2B receptor antagonist, any interactions of RS-127445 with 5-HT₄ receptors are unknown; this was examined using the recombinant receptor and Biomolecular Interaction Detection technology. Mouse isolated colon was mounted in tissue baths for isometric recording of neuronal contractions evoked by electrical field stimulation (EFS), or under an intraluminal pressure gradient to induce peristalsis; the effects of RS-127445 on EFS-induced and on peristaltic contractions were measured. Faecal output of rats in grid-bottom cages was measured over 3 h following i.p. RS-127445 and separately, validation of the effective doses was achieved by determining the free, unbound fraction of RS-127445 in blood and brain.

Key results:

RS-127445 (up to 1 µmol·L⁻¹) did not interact with the 5-HT₄ receptor. RS-127445 (0.001–1 µmol·L⁻¹) did not affect EFS-induced contractions of the colon, although at 10 µmol·L⁻¹ the contractions were reduced (to 36 ± 8% of control, n= 4). RS-127445 (0.1–10 µmol·L⁻¹) concentration-dependently reduced peristaltic frequency (n= 4). RS-127445 (1–30 mg·kg⁻¹), dose-dependently reduced faecal output, reaching significance at 10 and 30 mg·kg⁻¹ (n= 6–11). In blood and brain, >98% of RS-127445 was protein-bound.

Conclusions and implications:

High-protein binding of RS-127445 indicates that relatively high doses are required for efficacy. The results suggest that 5-HT_2B receptors tonically regulate colonic motility.     

No Evidence for a Culturable Bacterial Tetrodotoxin Producer in Pleurobranchaea maculata (Gastropoda: Pleurobranchidae) and Stylochoplana sp. (Platyhelminthes: Polycladida)

Tetrodotoxin (TTX) is a potent neurotoxin found in the tissues of many taxonomically diverse organisms. Its origin has been the topic of much debate, with suggestions including endogenous production, acquisition through diet, and symbiotic bacterial synthesis. Bacterial production of TTX has been reported in isolates from marine biota, but at lower than expected concentrations. In this study, 102 strains were isolated from Pleurobranchaea maculata (Opisthobranchia) and Stylochoplana sp. (Platyhelminthes). Tetrodotoxin production was tested utilizing a recently developed sensitive method to detect the C9 base of TTX via liquid chromatography—mass spectrometry. Bacterial strains were characterized by sequencing a region of the 16S ribosomal RNA gene. To account for the possibility that TTX is produced by a consortium of bacteria, a series of experiments using marine broth spiked with various P. maculata tissues were undertaken. Sixteen unique strains from P. maculata and one from Stylochoplana sp. were isolated, representing eight different genera; Pseudomonadales, Actinomycetales, Oceanospirillales, Thiotrichales, Rhodobacterales, Sphingomonadales, Bacillales, and Vibrionales. Molecular fingerprinting of bacterial communities from broth experiments showed little change over the first four days. No C9 base or TTX was detected in isolates or broth experiments (past day 0), suggesting a culturable microbial source of TTX in P. maculata and Stylochoplana sp. is unlikely.     

Aspergillus flavus Growth Inhibition and Aflatoxin B1 Decontamination by Streptomyces Isolates and Their Metabolites

Aflatoxin B₁ is a potent carcinogen produced by Aspergillus flavus, mainly during grain storage. As pre-harvest methods are insufficient to avoid mycotoxin presence during storage, diverse curative techniques are being investigated for the inhibition of fungal growth and aflatoxin detoxification. Streptomyces spp. represent an alternative as they are a promising source of detoxifying enzymes. Fifty-nine Streptomyces isolates and a Streptomyces griseoviridis strain from the commercial product Mycostop^®, evaluated against Penicillium verrucosum and ochratoxin A during previous work, were screened for their ability to inhibit Aspergillus flavus growth and decrease the aflatoxin amount. The activities of bacterial cells and cell-free extracts (CFEs) from liquid cultures were also evaluated. Fifty-eight isolates were able to inhibit fungal growth during dual culture assays, with a maximal reduction going down to 13% of the control. Aflatoxin-specific production was decreased by all isolates to at least 54% of the control. CFEs were less effective in decreasing fungal growth (down to 40% and 55% for unheated and heated CFEs, respectively) and aflatoxin-specific production, with a few CFEs causing an overproduction of mycotoxins. Nearly all Streptomyces isolates were able to degrade AFB₁ when growing in solid and liquid media. A total degradation of AFB₁ was achieved by Mycostop^® on solid medium, as well as an almost complete degradation by IX20 in liquid medium (6% of the control). CFE maximal degradation went down to 37% of the control for isolate IX09. The search for degradation by-products indicated the presence of a few unknown molecules. The evaluation of residual toxicity of the tested isolates by the SOS chromotest indicated a detoxification of at least 68% of AFB₁’s genotoxicity.     

Isoprenyl phenolic ethers from the termite nest-derived medicinal fungus Xylaria fimbriata

Seven new isoprenyl phenolic ethers, namely fimbriethers A–G (1–7), were isolated from the fermented broth of the termite nest-derived medicinal fungus Xylaria fimbriata YMJ491. Their structures were determined by spectroscopic data analysis and compared with those reported. The effects of all the isolates at a concentration of 100 μM on the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells were evaluated, and all of them exhibited NO production inhibitory activity with E_max values ranging from 4.6 ± 2.0% to 49.7 ± 0.5% without significant cytotoxicity. In addition, these seven compounds did not alter phenylephrine-induced vasocontraction in isolated intact thoracic aortic rings from C57BL/6J mouse, indicating 1–7 were not involved in the regulation of endothelial NOS-mediated NO production.     

Surveillance of tigecycline activity tested against clinical isolates from a global (North America,Europe, Latin America and Asia-Pacific) collection (2016)

Tigecycline and comparators were tested by the reference broth microdilution method against 33 348 non-duplicate bacterial isolates collected prospectively in 2016 from medical centres in the Asia-Pacific (3443 isolates), Europe (13 530 isolates), Latin America (3327 isolates) and the USA (13 048 isolates). Among 7098 Staphylococcus aureus isolates tested, >99.9% were inhibited by ≤0.5?mg/L tigecycline (MIC_50/90, 0.06/0.12?mg/L), including >99.9% of methicillin-resistant S. aureus and 100.0% of methicillin-susceptible S. aureus. Tigecycline was slightly more active against Enterococcus faecium (MIC_50/90, 0.03/0.06?mg/L) compared with Enterococcus faecalis (MIC_50/90, 0.06/0.12?mg/L) and its activity was not adversely affected by vancomycin resistance when tested against these organisms. Tigecycline potency was comparable for Streptococcus pneumoniae (MIC_50/90, 0.03/0.06?mg/L), viridans group streptococci (MIC_50/90, 0.03/0.06?mg/L) and β-haemolytic streptococci (MIC_50/90, 0.06/0.06?mg/L) regardless of species and penicillin susceptibility. Tigecycline was active against Enterobacteriaceae (MIC_50/90, 0.25/1?mg/L; 97.8% inhibited at ≤2?mg/L) but was slightly less active against Enterobacteriaceae isolates expressing resistant phenotypes: carbapenem-resistant Enterobacteriaceae (MIC_50/90, 0.5/2?mg/L; 98.0% susceptible); multidrug-resistant (MIC_50/90, 0.5/2?mg/L; 93.1% susceptible); and extensively drug-resistant (MIC_50/90, 0.5/4?mg/L; 87.8% susceptible). Tigecycline inhibited 74.4% of 888 Acinetobacter baumannii isolates at ≤2?mg/L (MIC_50/90, 2/4?mg/L) and demonstrated good in vitro activity against Stenotrophomonas maltophilia (MIC_50/90, 1/2?mg/L; 90.6% inhibited at ≤2?mg/L) Tigecycline was active against Haemophilus influenzae (MIC_50/90, 0.12/0.25?mg/L) regardless of β-lactamase status. Tigecycline represents an important treatment option for resistant Gram-negative and Gram-positive bacterial infections.     

Co-Cultivation of Two Bacillus Strains for Improved Cell Growth and Enzyme Production to Enhance the Degradation of Aflatoxin B1

Bacillus sp. H16v8 and Bacillus sp. HGD9229 were identified as Aflatoxin B₁ (AFB₁) degrader in nutrient broth after a 12 h incubation at 37 °C. The degradation efficiency of the two-strain supernatant on 100 μg/L AFB₁ was higher than the bacterial cells and cell lysate. Moreover, degradations of AFB₁ were strongly affected by the metal ions in which Cu²⁺ stimulated the degradation and Zn²⁺ inhibited the degradation. The extracellular detoxifying enzymes produced by co-cultivation of two strains were isolated and purified by ultrafiltration. The molecular weight range of the detoxifying enzymes was 20–25 kDa by SDS-PAGE. The co-culture of two strains improved the total cell growth with the enhancement of the total protein content and detoxifying enzyme production. The degradation efficiency of the supernatant from mixed cultures increased by 87.7% and 55.3% compared to Bacillus sp. H16v8 and HGD9229, individually. Moreover, after the degradation of AFB₁, the four products of the lower toxicity were identified by LC-Triple TOF-MS with the two proposed hypothetical degradation pathways.     

Meropenem–nacubactam activity against AmpC-overproducing and KPC-expressing Pseudomonas aeruginosa in a neutropenic murine lung infection model

Nacubactam is a novel non-β-lactam diazabicyclooctane β-lactamase inhibitor under development for the treatment of serious Gram-negative infections. This study assessed the efficacy of human-simulated epithelial lining fluid (ELF) exposure of nacubactam in combination with meropenem against AmpC-overproducing (n=4) and Klebsiella pneumoniae carbapenemase (KPC)-expressing (n=3) Pseudomonas aeruginosa isolates in the neutropenic murine lung infection model. Meropenem, nacubactam and meropenem–nacubactam (1:1 concentration ratio) minimum inhibitory concentrations (MICs) were determined in triplicate using broth microdilution. Regimens that provided ELF profiles mimicking those observed in humans given nacubactam 2 g q8h (1.5-h infusion) alone and in combination with a subtherapeutic ELF exposure of meropenem were administered 2 h after inoculation. Efficacy was assessed as the change in log₁₀ colony-forming units (CFU)/lung at 24 h compared with 24-h meropenem monotherapy. Meropenem, nacubactam and meropenem–nacubactam MICs were 8–>64, 128–>256 and 2–16 mg/L, respectively. Meropenem and nacubactam monotherapy groups demonstrated bacterial growth over 24 h for each isolate. Against AmpC-overproducing and KPC-expressing P. aeruginosa isolates, meropenem–nacubactam resulted in −2.73±0.93 and −4.35±1.90 log₁₀CFU/lung reduction, respectively, relative to meropenem monotherapy. Meropenem–nacubactam showed promising in-vivo activity against meropenem-resistant P. aeruginosa, indicative of a potential role for the treatment of infections caused by these challenging pathogens.     

Synergistic activity of colistin with azidothymidine against colistin-resistant Klebsiella pneumoniae clinical isolates collected from inpatients in Greek hospitals

BackgroundNew antibiotics are urgently needed to treat multi-drug resistant infections; however, production of novel antibiotics is diminishing. Synergistic combination drug therapy to enhance the activity of available antibiotics may improve management of patients with resistant infections.MethodsColistin-resistant Klebsiella pneumoniae isolates were collected from inpatients in 10 Greek hospitals and used to study combination activity of colistin plus azidothymidine. Combination activity was evaluated with the sum of fractional inhibitory concentrations (ΣFIC) using the mini checkerboard broth microdilution method.ResultsA hundred individual strains were tested. Synergistic activity was noted in 79% (79/100) of isolates and additive activity in the remaining 21% (21/100). ΣFIC₅₀ and ΣFIC₉₀ were 0.28 and 0.56, respectively.ConclusionColistin with azidothymidine exhibited promising synergistic activity against colistin-resistant Klebsiella pneumoniae isolates warranting further investigation of the combination.     

The histamine content of dried flying fish products in Taiwan and the isolation of halotolerant histamine-forming bacteria

Thirty dried flying fish products were purchased from fishing village stores in Taiwan and tested to detect the presence of histamine and histamine-forming bacteria. Except for histamine and cadaverine, the average content of various biogenic amines in the tested samples was less than 3.5 mg/100 g. Eight (26.6%) dried flying fish samples had histamine levels greater than the United States Food and Drug Administration guideline of 5 mg/100 g for scombroid fish and/or scombroid products, whereas four (13.3%) samples contained more than the hazard action level of 50 mg/100 g. One histamine-producing bacterial isolate was identified as Staphylococcus xylosus by 16S rDNA sequencing with polymerase chain reaction amplification. This isolate was capable of producing 507.8 ppm of histamine in trypticase soy broth supplemented with 1.0% l-histidine (TSBH). The S. xylosus isolate was a halotolerant bacterium that had a consistent ability to produce more than 300 ppm of histamine at 3% sodium chloride concentration in TSBH medium after 72 hours.