Effect of honey bee forage plants in Tunisia on diversity and antibacterial potential of lactic acid bacteria and bifidobacteria from Apis mellifera intermissa and its products.

Lactic acid bacteria and bifidobacteria (LAB and Bifido), isolated from the gastrointestinal tract of Apis mellifera intermissa (BGIT), honey (H), propolis (P) and bee bread (BB) of hives set in different vegetations (wildflowers, caraway, orange blossom, Marrubium vulgare, Eucalyptus and Erica cinerea), were subjected to analysis of their antibacterial potential. Isolates able to inhibit Staphylococcus aureus were selected and identified with MALDI-TOF MS leading to 154 strains representing 12 LAB and Bifido species. Lactiplantibacillus plantarum, Pediococcus pentosaceus and Enterococcus faecalis were predominantly found in all matrices. BGIT showed the highest LAB and Bifido diversity with exclusive occurrences of five species (including Bifidobacterium asteroides and Limosilactobacillus fermentum). Honey was the second origin harboring an important variety of LAB species of which Apilactobacillus kunkeei and Enterococcus mundtii were characteristic of both H and BGIT. Principal components analysis revealed associations between antibacterial activities of LAB and Bifido, matrices and honey bee forage plants. Inhibition trends of S. aureus and Citrobacter freundii were highlighted with: L. plantarum from BGIT, P, H of bees feeding on E. cinerea; Pediococcus pentosaceus from BGIT, P, BB associated with E. cinerea; and Bifidobacterium asteroides from BGIT/orange blossom system. However, Enterococcus faecium associated with BGIT/Eucalyptus system antagonized Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. Our findings highlighted noteworthy effects of bee forage plants on the antibacterial activity of LAB and Bifido. Our approach could be useful to identify multiple conditions promoting antibacterial potency of LAB and Bifido under the combined effects of feeding plants and living matrices.

High Prevalence of blaNDM Among Carbapenem Non-Susceptible Klebsiella pneumoniae in a Tunisian Hospital First Report of blaNDM-9, blaKPC-20, and blaKPC-26 Genes.

Fifty-four carbapenem non-susceptible Klebsiella pneumoniae (CNSKP) isolates were collected from a Tunisian hospital over a period of 13 consecutive months. Carbapenemase production and the prevalence of carbapenemase-encoding genes were investigated using combined-disk test (CDT), modified Carba NP (mCarba NP) test, and UV-spectrophotometry method complemented by PCR experiments and sequencing. Carbapenemase production was detected by the mCarba NP test and CDT in 92.59% and 96.29% of the 54 CNSKP isolates, respectively; while imipenem hydrolysis was detected using UV-spectrophotometry in the crude extracts of 44 isolates. blaNDM, blaOXA-48-like, and blaKPC carbapenemase-encoding genes were found in 48, 31, and 22 isolates, respectively. Remarkably, blaNDM-9, blaKPC-20, and blaKPC-26 genes were reported. The co-occurrence of carbapenemase-encoding genes in a single isolate was detected in 62.96% of the isolates. The analysis of clonal relationships between the isolates by pulsed field gel electrophoresis revealed that the majority of them were genetically unrelated. Our investigation provides molecular data on enzymatic mechanism of carbapenem non-susceptibility among 54 CNSKP showing the dominance of blaNDM, and comprises the first identification of blaNDM-9, blaKPC-20, and blaKPC-26 genes in a Tunisia hospital.

Alpha-amylase and alphaglucosidase inhibitory properties, beta-galactosidase activity, and probiotic potential of lactic acid bacteria and bifidobacteria from Apis mellifera intermissa and its products.

The present study aimed to evaluate the probiotic potential, α-amylase and α-glucosidase inhibitory effects, and ß-galactosidase production of 19 non haemolytic lactic acid bacteria and bifidobacteria previously identified and isolated from honey bee gastrointestinal tract (BGIT) of Apis mellifera intermissa, honey, propolis and bee bread. The isolates were screened according to their high resistance to lysozyme and potent antibacterial activity. Our results indicated that among the 19 isolates, Limosilactobacillus fermentum BGITE12.2, Lactiplantibacillus plantarum BGITEC13, Limosilactobacillus fermentum BGITEC5.1 and Bifidobacterium asteroides BGITOB8, isolated from BGIT exhibited a good tolerance to 100 mg/mL lysozyme (> 82%), excellent tolerance to 0.5% bile salt [survival rate (SR) ≥ 83.19% ± 0.01], and a high SR (≥ 80.0%) under gastrointestinal tract conditions. The auto-aggregation ability was high (auto-aggregation index ranging from 67.14 ± 0.16 to 92.8% ± 0.03) for L. fermentum BGITE12.2, L. plantarum BGITEC13, and B. asteroides BGITOB8, and moderate for L. fermentum BGITEC5.1 (39.08% ± 0.11). Overall, the four isolates showed moderate co-aggregation capacity with pathogenic bacteria. They exhibited from moderate to high hydrophobicity towards toluene and xylene. The safety assessment revealed that the four isolates lacked gelatinase and mucinolytic activities. Also, they were susceptible to ampicillin, clindamycin, erythromycin, and chloramphenicol. Interestingly, the four isolates showed α-glucosidase and α-amylase inhibitory activities ranging from 37.08 ± 0.12 to 57.57% ± 0.1 and from 68.30 ± 0.09 to 79.42% ± 0.09, respectively. Moreover, L. fermentum BGITE12.2, L. plantarum BGITEC13, L. fermentum BGITEC5.1 isolates exhibited ß-galactosidase activity over a wide range of 52.49 ± 0.24-746.54 ± 0.25 Miller Units. In conclusion, our findings suggest that the four isolates could be potential candidates for probiotics with interesting functional properties.

Potential Probiotic Lactic Acid Bacteria with Anti-Penicillium expansum Activity from Different Species of Tunisian Edible Snails.

This study aimed to isolate lactic acid bacteria (LAB) from the digestive tract, meat and slime of edible snails (Helix lucorum, Helix aspersa and Eobania vermiculata) and investigate their antagonistic activity against Penicillium expansum. They were then characterized for their probiotic potential. Among 900 bacterial isolates, 47 LAB exhibiting anti-P. expansum activity were identified through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as Levilactobacillus brevis (25), Lactococcus lactis (3), Enterococcus faecium (12), Enterococcus faecalis (4), Enterococcus casseliflavus (1), and Enterococcus mundtii (2). Sixty-two percent of the strains were tolerant to 100 mg/L of lysozyme. Seventy two percent of the isolates were able to survive at pH 3 and most of them tolerate 2.5% bile salt concentration. Moreover, 23% of the strains displayed bile salt hydrolase activity. Interestingly, all strains were biofilm strong producers. However, their auto- and co-aggregation properties were time and pH dependent with high aggregative potentiality at pH 4.5 after 24 h. Remarkably, 48.94% of the strains showed high affinity to chloroform. The safety assessment revealed that the 47 LAB had no hemolytic activity and 64% of them lacked mucin degradation activity. All isolated strains were susceptible to gentamycin, streptomycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole. Overall, 43 LAB strains showed inhibitory activity against a broad spectrum of pathogenic Gram-positive and gram-negative bacteria, fungi, and yeast. Our findings suggest that L. brevis (EVM12 and EVM14) and Ent. faecium HAS34 strains could be potential candidates for probiotics with interesting antibacterial and anti-P. expansum activities.

Biochemical characterization of a novel extended-spectrum beta-lactamase from Pseudomonas aeruginosa 802.

Pseudomonas aeruginosa 802 was isolated at Rabta hospital in Tunis and was resistant to extended-spectrum cephalosporins and aztreonam. It produced a pI 7.6 extended-spectrum beta-lactamase (ESBL). The ESBL, named LBT 802, was purified to homogeneity by filtration on Sephadex G-75 followed by CM-Sepharose chromatography and high-performance liquid chromatography (HPLC) on a TSK-gel SP-5PW column. The LBT 802 enzyme had a molecular mass of 30 kDa. It showed a broad-substrate profile by hydrolyzing benzylpenicillin, ampicillin, cephalothin, cephaloridine, cefotaxime, ceftriaxone, and cefpirome but not ceftazidime, cefoxitin, imipenem, or aztreonam. The highest hydrolytic efficiency (Vmax/Km) was obtained for ampicillin, cephalothin, cephaloridine, and benzylpenicillin. Among extended-spectrum cephalosporins the best substrate was ceftriaxone followed by cefotaxime and cefpirome. LBT 802 activity was inhibited by clavulanic acid, sulbactam, imipenem, cefoxitin, and aztreonam. It showed its lowest Ki values for clavulanic acid, imipenem and sulbactam.

Trend of plasmid-mediated quinolone resistance genes at the Children's Hospital in Tunisia.

The prevalence of plasmid-mediated quinolone resistance genes [qnr, aac(6')-Ib-cr and qepA] was sought among Enterobacteriaceae strains obtained from the Children's Hospital of Tunis (Tunisia). Non-duplicate isolates (n = 278) with resistance to extended-spectrum cephalosporins and collected in 2003, 2007, 2008 and 2009 were screened for qnr genes. Forty (14.4 %) isolates were qnr positive and were screened for the presence of the aac(6')-Ib-cr and qepA genes. qnrB was detected in 21 Klebsiella pneumoniae, 11 Escherichia coli and 6 Enterobacter cloacae isolates. Sequence analysis of the qnrB amplicons revealed variants including 24 qnrB1, 11 qnrB2 and 3 qnrB6. qnrS (qnrS1 allele) was detected only in K. pneumoniae isolates, either alone (two isolates) or with the qnrB gene (one isolate). The qnrA, qnrC and qnrD genes were not found in any of the 278 isolates. No qnr-positive isolates carried the qepA gene. Pyrosequencing results showed that aac(6')-Ib-cr, a variant of the aac(6')-Ib gene, was present in 31 qnr-positive isolates (21 K. pneumoniae isolates, seven Escherichia coli isolates and three Enterobacter cloacae isolates). aac(6')-Ib was also found either alone (two isolates) or in association with aac(6')-Ib-cr (one isolate). Of the 40 qnr-positive isolates, 92.5, 82.5, 57.5, 85 and 82.5 % were non-susceptible to nalidixic acid, ciprofloxacin, levofloxacin, ofloxacin and norfloxacin, respectively, and all were extended-spectrum ß-lactamase producers. Random amplified polymorphic DNA-PCR typing of these isolates showed 16, 8 and 5 different genotypes in K. pneumoniae, Escherichia coli and Enterobacter cloacae isolates, respectively. Our study highlights the high prevalence of qnr in association with aac(6')-Ib-cr among Enterobacteriaceae isolates, even from children, who are patients not overtreated with quinolones.

Emergence and dominance of CTX-M-15 extended spectrum beta-lactamase among Escherichia coli isolates from children.

Of forty-seven extended-spectrum cephalosporin-resistant Escherichia coli isolates, collected from children at the Children's Hospital in 2006 (Tunis, Tunisia), we analyzed 32 isolates that were genotypically different by enterobacterial repetitive intergenic consensus -polymerase chain reaction. For all isolates, the double-disk diffusion test revealed synergy between clavulanate and cefotaxime and/or ceftazidime, suggesting the production of extended-spectrum beta-lactamases. Polymerase chain reaction experiments, performed on plasmid DNA, and sequencing revealed the presence of bla(TEM-1B) (26 isolates, 81%), bla(TEM-34(IRT-6)) (3 isolates, 9%), bla(SHV-12) (2 isolates, 6%), and bla(CTX-M-15) (31 isolates, 97%). Further, the insertion sequence ISEcp1 was found upstream from the bla(CTX-M-15) gene in 11 isolates. The bla genes were found alone or in various combinations in a single isolate. bla(TEM-1B) and bla(CTX-M-15) genes were detected in 26 out of the 32 isolates. Three isolates harbored both bla(TEM-34(IRT-6)) and bla(CTX-M-15). bla(SHV-12) was identified either alone or with bla(CTX-M-15) in a single isolate. Our investigation showed the dominance of CTX-M-type extended-spectrum beta-lactamases, with CTX-M-15 particularly common, and to our best knowledge, this is the first report of the coexistence of CTX-M-15 and IRT-6 in E. coli isolates from children in Tunisia.