Prevalence and characterization of quinolone resistance and integrons in clinical Gram-negative isolates from Gaza strip, Palestine.

BACKGROUND: Gram-negative bacteria with quinolone resistance and extended-spectrum beta-lactamases (ESBLs) present significant treatment challenges. This study evaluated the prevalence and characteristics of quinolone resistance in Gram-negative strains, investigating the relationship between plasmid-mediated quinolone resistance (PMQR), ESBLs, and integrons. METHODS AND RESULTS: We collected 146 Gram-negative isolates from patients in three Palestinian hospitals. For quinolone resistance isolates, the presence and characterization of PMQR, ß-lactamase genes and integrons were studied by PCR and sequencing. Out of 146 clinical isolates, 64 (43.8%) were resistant to quinolones, with 62 (97%) being multidrug-resistant (MDR) and 33 (51.5%) ESBL-producers. PMQR-encoding genes were present in 45 (70.3%) isolates, including aac(6')-Ib-cr (26.6%), qnrA (18.8%), qnrS1 (20.8%), and qnrB (6.4%). BlaCTX-M genes were detected in 50% (32/64) of isolates, with blaCTX-M-15 being the most common. BlaTEM-1, blaSHV-1 and blaVIM genes were found in 13, 6, and 4 isolates, respectively. Class I integrons were found in 31/64 (48%) of isolates, with 14 containing gene cassettes conferring resistance to trimethoprim (dhfr17, dfrA12, dfrA1) and aminoglycosides resistance genes (aadA1, aadA2, aadA5, and aadA6). CONCLUSIONS: This study found a high rate of quinolone resistance, ESBL and integrons in clinical Gram-negative isolates from our hospitals. Urgent measures are crucial, including implementing an antimicrobial resistance surveillance system, to control and continuously monitor the development of antimicrobial resistance.

Antimicrobial activity and safety features assessment of Weissella spp. from environmental sources.

Weissella strains have been reported to be useful in biotechnological and probiotic determinations, and some of them are considered opportunistic pathogens. Given the widespread interest about antimicrobial susceptibilities, transmission of resistances, and virulence factors, there is little research available on such topics for Weissella. The aim of this study was to assess the safety aspects and antimicrobial potential of 54 Weissella spp. strains from different environmental sources. Antibiotic susceptibility, hemolytic activity, horizontal transfer, and antibacterial activity were studied, as well as the detection of biogenic amine BA production on decarboxylase medium and PCR was performed. All the strains were nonhemolytic and sensitive to chloramphenicol and ampicillin. Several strains were classified as resistant to fusidic acid, and very low resistance rates were detected to ciprofloxacin, tetracycline, streptomycin, lincomycin, erythromycin, and rifampicin, although all strains had intrinsic resistance to vancomycin, nalidixic acid, kanamycin, and teicoplanin. Two BA-producing strains (W. halotolerans FAS30 and FAS29) exhibited tyrosine decarboxylase activity, and just one W. confusa FS077 produced both tyramine and histamine, and their genetic determinants were identified. Ornithine decarboxylase/odc gene was found in 16 of the Weissella strains, although 3 of them synthesize putrescine. Interestingly, eight strains with good properties displayed antibacterial activity. Conjugation frequencies of erythromycin from Bacillus to Weissella spp. varied in the average of 3 × 10-9 transconjugants/recipient. However, no tetracycline-resistant transconjugant was obtained with Enterococcus faecalis JH2-2 as recipient. The obtained results support the safe status of Weissella strains, derived from environmental sources, when used as probiotics in animal feed.

Comparative Evaluation of the Capacity of Commercial and Autochthonous Saccharomyces cerevisiae Strains to Remove Ochratoxin A from Natural and Synthetic Grape Juices.

In this paper, we assessed the ability of two strains of Saccharomyces cerevisiae, in viable and dead forms, to remove ochratoxin A (OTA) from an artificially contaminated synthetic grape juice medium (SGM) (10 µg OTA/L) and a naturally contaminated grape juice (6.64 µg OTA/L). The first strain, named Levulin FB, is a commercial yeast used in making wine. The second, named SC5, is an autochthonous strain isolated from table grapes. OTA concentrations in juices before and after their contact with yeast cells were assessed. A significant decrease in OTA level (p < 0.05) in the SGM medium and in the natural grape juice was observed after 1 h of adding yeast cells (20 g/L) in viable and heat-treated forms. It was inferred that the dead forms of the two strains were more able to eliminate OTA than their viable forms in both media. This study demonstrates the potential application of an autochthonous yeast for the natural decontamination of grape juice from fungal toxins.

Biochemical, Biophysical and Functional Characterization of an Insoluble Iron Containing Hepcidin-Ferritin Chimeric Monomer Assembled Together with Human Ferritin H/L Chains at Different Molar Ratios.

Hepcidin and ferritin are key proteins of iron homeostasis in mammals. In this study, we characterize a chimera by fusing camel hepcidin to a human ferritin H-chain to verify if it retained the properties of the two proteins. The construct (HepcH) is expressed in E. coli in an insoluble and iron-containing form. To characterize it, the product was incubated with ascorbic acid and TCEP to reduce and solubilize the iron, which was quantified with ferrozine. HepcH bound approximately five times more iron than the wild type human ferritin, due to the presence of the hepcidin moiety. To obtain a soluble and stable product, the chimera was denatured and renatured together with different amounts of L-ferritin of the H-chain in order to produce 24-shell heteropolymers with different subunit proportions. They were analyzed by denaturing and non-denaturing PAGE and by mass spectroscopy. At the 1:5 ratio of HepcH to H- or L-ferritin, a stable and soluble molecule was obtained. Its biological activity was verified by its ability to both bind specifically cell lines that express ferroportin and to promote ferroportin degradation. This chimeric molecule showed the ability to bind both mouse J774 macrophage cells, as well as human HepG2 cells, via the hepcidin-ferroportin axis. We conclude that the chimera retains the properties of both hepcidin and ferritin and might be exploited for drug delivery.

The role of dextran production in the metabolic context of Leuconostoc and Weissella Tunisian strains.

High molecular weight dextrans improve the rheological properties of fermented products and have immunomodulatory and antiviral activity. We report on 5.84 × 107-2.61 × 108 Da dextrans produced by Leuconostoc lactis AV1n, Weissella cibaria AV2ou and Weissella confusa V30 and FS54 strains. Dextransucrases catalyze dextran synthesis by sucrose hydrolysis concomitant with fructose generation. The four bacteria have dextransucrases with molecular weight of about 160 kDa detected by zymograms. Each bacterium showed different interplay of dextran production and metabolic fluxes. All bacteria produced lactate, and AV2ou apart, synthesized mannitol from fructose. FS54 hydrolyzed dextran blue and the concentration of dextran produced by this bacterium decreased during the stationary phase. The AV1n binding to Caco-2 cells and polystyrene plates was higher under conditions for dextran synthesis. Thus, this is the first instance of a Weissella dextranase, associated with a dextransucrase ability, and of a positive influence of dextran on adhesion and aggregation properties of a bacterium.

Isolation, Characterization and Chemical Synthesis of Large Spectrum Antimicrobial Cyclic Dipeptide (l-leu-l-pro) from Streptomyces misionensisV16R3Y1 Bacteria Extracts. A Novel 1H NMR Metabolomic Approach.

Streptomyces is the most frequently described genus of Actinomycetes, a producer of biologically active secondary metabolites. Indeed, the Streptomyces species produces about 70% of antibiotics and 60% of antifungal molecules used in agriculture. Our study was carried out with the goal of isolating and identifying antimicrobial secondary metabolites from Streptomyces misionensisV16R3Y1 isolated from the date palm rhizosphere (southern Tunisia). This strain presented a broad range of antifungal activity against Fusarium oxysporum, Aspergillus flavus, Penicillium expansum, Aspergillus niger, Candida albicans, Candida metapsilosis, and Candida parapsilosis and antibacterial activity against human pathogenic bacteria, including Escherichia fergusonii, Staphylococcus aureus, Salmonella enterica, Enterococcus faecalis, Bacillus cereus and Pseudomonas aeruginosa. The purification procedure entailed ethyl acetate extract, silica gel column, and thin layer chromatography. Based on 1H NMR metabolomic procedure application, also supported by the GC-MS analysis, cyclic dipeptide (l-Leucyl-l-Proline) was identified as the major compound in the bioactive fraction. In order to confirm the identity of the active compound and to have a large quantity thereof, a chemical synthesis of the cyclic dipeptide was performed. The synthetic compound was obtained with a very good yield (50%) and presented almost the same effect compared to the extracted fraction. This study indicates for the first time that Streptomyces misionensis V16R3Y1 exhibits a broad spectrum of antimicrobial activities, produced cyclic dipeptide (l-Leucyl-l-Proline) and might have potential use as a natural agent for pharmaceutical and agri-food applications.

Different Modes of Regulation of the Expression of Dextransucrase in Leuconostoc lactis AV1n and Lactobacillus sakei MN1.

Leuconostoc lactis AV1 strain isolated from a Tunisian avocado was characterized as a dextran producer. The promoter P dsrLL and the dsrLL gene encoding the DsrLL dextransucrase responsible for the dextran synthesis were transcriptionally fused to the mCherry coding gene generating the pRCR20 plasmid. Upon plasmid transfer, both AV1n and the dextran non-producing Leuconostoc mesenteroides CM70 became red due to expression of the mCherry from the P dsrLL-dsr-mrfp transcriptional fusion. Characterization of the polymers present in cultures supernatants revealed that the DsrLL encoded from pRCR20 in the recombinant bacteria was able to synthesize dextran. The production of dextran by the DsrLL in AV1n increased in response to low temperature, reaching 10-fold higher levels at 20°C than at 37°C (4.15 g/L versus 0.41 g/L). To analyze if this stress response includes activation at the transcriptional level and if it was only restricted to Leuconostoc, AV1n was transformed with plasmids carrying either the P dsrLL -mrfp fusion or the P dsrLS of Lactobacillus sakei MN1 fused to the mrfp gene, and the influence of temperature and carbon source on expression from the Dsr promoters was monitored by measurement of the mCherry levels. The overall expression analysis confirmed an induction of expression from P dsrLL upon growth at low temperature (20°C versus 30°C and 37°C) in the presence of sugars tested (sucrose, glucose, maltose, and fructose). In addition, the presence of sucrose, the substrate of Dsr, also resulted in activation of expression from P dsrLL . A different behavior was detected, when expression from P dsrLS was evaluated. Similar levels of fluorescence were observed irrespectively of the carbon source or temperature, besides a sequential decrease at 30°C and 20°C, when sucrose was present in the growth medium. In conclusion, the two types of regulation of expression of Dsr presented here revealed two different mechanisms for environmental adaptation of Leuconostoc and Lactobacillus that could be exploited for industrial applications.

Inhibitory Effect of Lactobacillus plantarum FL75 and Leuconostoc mesenteroides FL14 against Foodborne Pathogens in Artificially Contaminated Fermented Tomato Juices.

Tomatoes and tomato based-foods contain beneficial microorganisms and various organic acids that have important nutritional values for human. The objective of this study was to access the physiochemical properties of fermented tomatoes juices and to evaluate the competitiveness of lactic acid bacteria (LAB) against Listeria monocytogenes, Listeria innocua, and Salmonella spp., in artificially contaminated tomato juice. Microbial counting (LAB, fungi Salmonella spp., and Listeria spp.) was performed after fermentation and weekly during storage. Different organic acids (Lactic, succinic, and acetic) and ethanol were also monitored using HPLC method. Color parameters were also determined. The results showed an increase of lactic and acetic acid content, during fermentation and storage of juices inoculated with Lactobacillus plantarum and Leuconostoc mesenteroides at 25°C. Besides, citric acid and ethanol revealed higher content at the end of storage compared to that registered at 4°C. The pH from tomatoes juices decreased from an initial value of 4.5 to below 3.2. Alongside, foodborne pathogen population was significantly suppressed in tomatoes juices when the samples were coinoculated with LAB strains. Moreover, the inhibition of Salmonella species was faster compared to that of Listeria. After four weeks of storage at 4°C, Lb. plantarum and Lc. mesenteroides showed high survival rate, while pathogenic bacteria, yeasts, and molds cell numbers decreased drastically in all the contaminated vials. This work highlights the efficiency of Lb. plantarum and Lc. mesenteroides as potential starters for developing nutritious and safe fermented tomato juice products.

Functional Probiotic Assessment and In Vivo Cholesterol-Lowering Efficacy of Weissella sp. Associated with Arid Lands Living-Hosts.

The research and the selection of novel probiotic strains from novel niches are receiving increased attention on their proclaimed health benefits to both humans and animals. This study aimed to evaluate the functional properties of Weissella strains from arid land living-hosts and to select strains with cholesterol-lowering property in vitro and in vivo, for use as probiotics. They were assessed for acid and bile tolerance, antibiotic susceptibility, membrane properties, antibacterial activity, antiadhesive effect against pathogens to host cell lines, and cholesterol assimilation in vitro. Our results showed that the majority of strains revealed resistance to gastrointestinal conditions. All the strains were nonhemolytic and sensitive to most of the tested antibiotics. They also exhibited high rates of autoaggregation and some of them showed high coaggregation with selected pathogens and high adhesion ability to two different cell lines (Caco-2 and MIM/PPk). Particularly, W. halotolerans F99, from camel feces, presented a broad antibacterial spectrum against pathogens, reduced Enterococcus faecalis and Escherichia coli adhesion to Caco-2 cells, and was found to reduce, in vitro, the cholesterol level by 49 %. Moreover, W. halotolerans F99 was evaluated for the carbohydrate utilization as well as the serum lipid metabolism effect in Wistar rats fed a high-cholesterol diet. W. halotolerans F99 showed an interesting growth on different plant-derivative oligosaccharides as sole carbon sources. Compared with rats fed a high-fat (HF) diet without Weissella administration, total serum cholesterol, low-density lipoprotein cholesterol, and triglycerides levels were significantly (p<0.001) reduced in W. halotolerans F99-treated HF rats, with no significant change in high-density lipoprotein cholesterol HDL-C levels. On the basis of these results, this is the first study to report that W. halotolerans F99, from camel feces, can be developed as cholesterol-reducing probiotic strain. Further studies may reveal their potential and possible biotechnological and probiotic applications.

Diversity of Vaginal Lactic Acid Bacterial Microbiota in 15 Algerian Pregnant Women with and without Bacterial Vaginosis by using Culture Independent Method.

INTRODUCTION: Bacterial Vaginosis (BV) is the most common lower genital tract disorder among women of reproductive age (pregnant and non-pregnant) and a better knowledge of Lactobacillus species richness in healthy and infected vaginal microbiota is needed to efficiently design better probiotic products to promote the maintenance of normal flora which will help prevent bacterial vaginosis. AIM: To evaluate and compare the diversity of lactic acid bacterial species in pregnant women with and without BV. MATERIALS AND METHODS: A pilot study was carried out during November-2014 to March-2015 in University Badji Mokhtar, Annaba, Algeria. Vaginal swabs were collected from 15 pregnant women aged between 19 and 35 years (mean 27.6 years; n=15) living in the East of Algeria visiting Gynecology service, hospital Abdallah Nouaouria- El bouni, Annaba. Vaginal samples were gram-stained, and scored by the Nugent method. The cohort included cases of women with healthy "normal" vaginal flora, infected flora with bacterial vaginosis and women with "intermediate" flora. The vaginal LAB community from pregnant women was identified by culture independent method based on Denaturing Gradient Gel Electrophoresis (DGGE), with the 16S rRNA gene sequencing. RESULTS: A majority of LAB affiliated to the genus Lactobacillus was found in "normal" and "intermediate" flora (87.5% and 43.75% respectively), while a majority of LAB affiliated to the genus Enterococcus was identified in women with bacterial vaginosis and intermediate flora (60% and 46.75% respectively). Our results showed that the presence of Lactobacillus iners and Lactobacillus delbruekii promotes stability of the vaginal microbiota. CONCLUSION: This result confirms the findings of previous studies suggesting that the occurrence of predominant Lactobacillus negatively correlates with bacterial vaginosis incidence and their current use as probiotics. Lactobacillus iners and Lactobacillus delbruekii can be defined as critical for defense of the vagina. In addition, Enterococcus feacalis can be considered as an indicator of imbalance of the vaginal ecosystem.

Pentachlorophenol degradation by Janibacter sp., a new actinobacterium isolated from saline sediment of arid land.

Many pentachlorophenol- (PCP-) contaminated environments are characterized by low or elevated temperatures, acidic or alkaline pH, and high salt concentrations. PCP-degrading microorganisms, adapted to grow and prosper in these environments, play an important role in the biological treatment of polluted extreme habitats. A PCP-degrading bacterium was isolated and characterized from arid and saline soil in southern Tunisia and was enriched in mineral salts medium supplemented with PCP as source of carbon and energy. Based on 16S rRNA coding gene sequence analysis, the strain FAS23 was identified as Janibacter sp. As revealed by high performance liquid chromatography (HPLC) analysis, FAS23 strain was found to be efficient for PCP removal in the presence of 1% of glucose. The conditions of growth and PCP removal by FAS23 strain were found to be optimal in neutral pH and at a temperature of 30 °C. Moreover, this strain was found to be halotolerant at a range of 1-10% of NaCl and able to degrade PCP at a concentration up to 300 mg/L, while the addition of nonionic surfactant (Tween 80) enhanced the PCP removal capacity.

Diversity and antimicrobial properties of lactic acid bacteria isolated from rhizosphere of olive trees and desert truffles of Tunisia.

A total of 119 lactic acid bacteria (LAB) were isolated, by culture-dependant method, from rhizosphere samples of olive trees and desert truffles and evaluated for different biotechnological properties. Using the variability of the intergenic spacer 16S-23S and 16S rRNA gene sequences, the isolates were identified as the genera Lactococcus, Pediococcus, Lactobacillus, Weissella, and Enterococcus. All the strains showed proteolytic activity with variable rates 42% were EPS producers, while only 10% showed the ability to grow in 9% NaCl. In addition, a low rate of antibiotic resistance was detected among rhizospheric enterococci. Furthermore, a strong antibacterial activity against plant and/or pathogenic bacteria of Stenotrophomonas maltophilia, Pantoea agglomerans, Pseudomonas savastanoi, the food-borne Staphylococcus aureus, and Listeria monocytogenes was recorded. Antifungal activity evaluation showed that Botrytis cinerea was the most inhibited fungus followed by Penicillium expansum, Verticillium dahliae, and Aspergillus niger. Most of the active strains belonged to the genera Enterococcus and Weissella. This study led to suggest that environmental-derived LAB strains could be selected for technological application to control pathogenic bacteria and to protect food safety from postharvest deleterious microbiota.