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Abstract Background: in Colombia, the law (Resolution 1382, 2013) prohibits the sale of milk that contains any antimicrobial drug residues, although no specific official screening tests and detection limits have been specified. At present, milk with positive results to both the Delvotest® and Snap® assays is simply rejected. To avoid contaminating bulk tanks with milk from individually treated cows, producers would benefit from having on-farm screening tests to conduct their own quality controls. In addition, on-site testing would allow farmers to check if the withdrawal times of commercially-available generic products are in accordance with labeled recommendations. Material and Methods: In this study, two commonly used rapid detection tests (Delvotest® and SNAP® specific for beta-lactams) were used on milk from 39 subclinical mastitic Holstein cows that were prescribed with daily intramuscular injections of a commercial suspension containing 8.000.000 IU of penicilin G (75% procaine penicilin G, 25% potasium penicillin) and 8 g of streptomycin sulfate, for a total of 4 days. Cows were individually milked and samples collected every 12 hours the day before, and for 3 days after the recommended withdrawal time of three days post-treatment. To inactivate the potential action of natural inhibitors of microbial growth that may be present in milk (ie., lysozyme and lactoferrin), the results of the Delvotest® were compared before and after milk samples were subjected to heat treatment (82°C for 5 minutes). Results: When the Delvotest® was used as per manufacturer's instructions (i.e., without heating), 7 of 39 cows were positive for one more day past the recommended withdrawal period. However, the results of the Snap® specific for beta-lactams and Delvotest® post-heating showed that only 2 of those 7 cows were positive, suggesting that 5 animals gave false positive results. For the 312 milk samples analyzed, a high degree of concordance was observed (Kappa coefficient=0.74±0.1) between the Snap® and Delvotest® post-heating. Conclusions: Considering that the streptomycin in this product is known to be eliminated faster than penicillin-G, the results suggest that the efficacy of the Delvotest® (after heat treatment) is similar to that of the Snap® beta-lactams for the detection of penicillin residues. However, when the Delvotest® was not preceded by heat treatment to inactivate potential natural inhibitors, it yielded a high number of false-positive results. The results also showed that in 95% (37/39) of the cows treated with this commercial product, the labeled instructions of a 3 day withdrawal period were adequate for compliance within the law.

We monitored the presence of Listeria monocytogenes in environmental sources and evaluated phenotypic and molecular characteristics of the isolates recovered. L. monocytogenes was isolated in 12 of the 107 samples from wild and farm environments, and from vegetation. Most isolates (83.3%) were of serotype 1/2a and the remainder (2) were of serotype 4b. All 12 isolates were susceptible to the whole range of antimicrobials tested. These 12 strains were carriers of the virulence genes prfA, hlyA, actA, plcA, plcB, inlA, inlB, inlC, and inlJ. The detection of the inlA gene in 4 strains using the PCR-RFLP suggests the potential of some of these strains to penetrate into epithelial cells of the intestinal barrier. Macrorestriction analysis also confirmed clonal identity of some environmental isolates with food and human isolates. These results indicate that the external environment is a source of potentially pathogenic strains of L. monocytogenes.

Diclofenac sodium, an antiinflammatory agent, exhibited remarkable inhibitory action against both drug sensitive and drug resistant clinical isolates of Mycobacterium tuberculosis, as well as other mycobacteria. This drug was tested in vitro against 45 different strains of mycobacteria, most of which were inhibited by the drug at 10-25 µg/ml concentration. When tested in vivo, diclofenac, injected at 10 µg/g body weight of a Swiss strain of white mice, could significantly protect them when challenged with 50 median lethal dose of M. tuberculosis H37 Rv 102. According to chi2 test, the in vivo data were highly significant (p<0.01). Diclofenac was further tested for synergism with the conventional antimycobacterial drug streptomycin against M. smegmatis 798. When compared with their individual effects, synergism was found to be statistically significant (p<0.05). By the checkerboard assessment procedure, the fractional inhibitory concentration index of this combination was found to be 0.37, confirming synergism. Diclofenac sódico, um agente antiinflamatório, mostrou ação inibitória marcante contra isolados clínicos de Mycobacterium tuberculosis sensíveis e resistentes à drogas, bem como contra outras micobactérias. A droga foi testada in vitro contra 45 cepas diferentes de micobactérias, sendo que a maioria foi inibida pela droga na concentração de 10-25 µg/ml. Quando testado in vitro, diclofenac injetado em ratos albinos da linhagem Swiss, na proporção de 10 µg/g de peso corporal, provocou proteção significativa dos animais desafiados com metade da dose letal de M. tuberculosis H37 Rv 102. De acordo com o teste chi2, os dados in vivo foram altamente significativos (p < 0,01). Diclofenac foi posteriormente testado quanto ao sinergismo com a droga antimicobacteriana convencional estreptomicina, frente a M. smegmatis 798. Quando comparado aos seus efeitos individuais, o sinergismo foi estatisticamente significativo (p < 0,05). Através da análise checkerboard, o índice fracional de concentração inibitória para essa combinação foi 0,37, confirmando o sinergismo.

A process that we refer to as control by epistasy of synthesis (CES process) occurs during chloroplast protein biogenesis in Chlamydomonas reinhardtii: the synthesis of some chloroplast-encoded subunits, the CES subunits, is strongly attenuated when some other subunits from the same complex, the dominant subunits, are missing. Herein we investigate the molecular basis of the CES process for the biogenesis of the cytochrome b6f complex and show that negative autoregulation of cytochrome f translation occurs in the absence of other complex subunits. This autoregulation is mediated by an interaction, either direct or indirect, between the 5' untranslated region of petA mRNA, which encodes cytochrome f, and the C-terminal domain of the unassembled protein. This model for the regulation of cytochrome f translation explains both the decreased rate of cytochrome f synthesis in vivo in the absence of its assembly partners and its increase in synthesis when significant accumulation of the C-terminal domain of the protein is prevented. When expressed from a chimeric mRNA containing the atpA 5' untranslated region, cytochrome f no longer showed an assembly-dependent regulation of translation. Conversely, the level of antibiotic resistance conferred by a chimeric petA-aadA-rbcL gene was shown to depend on the state of assembly of cytochrome b6f complexes and on the accumulation of the C-terminal domain of cytochrome f. We discuss the possible ubiquity of the CES process in organellar protein biogenesis

The assessment of detection sensitivity of five microbial inhibition tests (MITs), STAR (screening test for antibiotic residues) with the test strain Bacillus subtilis BGA, Delvotest SP-NT, Total Antibiotics, Kalidos TB, and Kalidos MP with the test strain Bacillus stearothermophilus var. calidolactis to five aminoglycosides (AMGs), gentamicin, neomycin, streptomycin, kanamycin, and spectinomycin in fortified milk samples were studied. The sensitivity of MITs to AMGs was evaluated on the basis of experimental determination of detection limits (LODs) of MITs for AMGs. The LODs of these tests were compared with the maximum residue limits (MRLs) established for milk by the Commission Regulation (EU) No. 37/2010. LODs of STAR for AMGs in fortified milk samples were at the levels of MRL for neomycin (1.50 microg/g), gentamicin (0.10 microg/g), streptomycin (0.20 microg/g) and kanamycin (0.15 microg/g). Spectinomycin (0.20 microg/g) was not detected at the level of MRL. The LODs determined by Delvotest SP-NT, Total Antibiotics and Kalidos MP were comparable, but only gentamicin and neomycin were reliably detected at the levels of MRL. Kalidos TB was more sensitive to AMGs than Delvotest SP-NT, Total Antibiotics and Kalidos MP. Gentamicin, neomycin and streptomycin were detected at the levels of MRL.

Three hundred and ten enterococcal isolates (178 Enterococcus faecium, 68 E. durans, 49 E. faecalis, 8 E. italicus, 3 E. gallinarum, 3 E. casseliflavus, and 1 E. hirae) from Slovak Bryndza cheese were evaluated for susceptibility to nine antimicrobial agents (vancomycin, teicoplanin, ampicillin, streptomycin, gentamicin, erythromycin, rifampicin, nitrofurantoin, and ciprofloxacin). All enterococcal isolates from Bryndza cheese were susceptible to ampicillin, streptomycin, gentamicin, vancomycin, and teicoplanin as determined by the disk diffusion method. Vancomycin resistance genes vanA and vanB were not detected. Resistance rates of enterococcal isolates to rifampicin, erythromycin, ciprofloxacin, and nitrofurantoin were 24, 26, 2, and 1%, respectively. Thirty-six % of E. faecium isolates and 22% of the E. faecalis isolates were resistant to erythromycin. Resistance to rifampicin was similar in E. faecium (31%) and E. faecalis (29%). Both E. faecium and E. faecalis strains showed the same resistance to ciprofloxacin (2%). E. durans isolates showed low levels of resistance to rifampicin, erythromycin, ciprofloxacin, and nitrofurantoin (1-4%). Forty-eight (30 %) of the E. faecium isolates, two (3%) of the E. durans isolates, and six (12%) of the E. faecalis isolates exhibited multidrug resistance. The highest frequency of resistant enterococci was observed in Bryndza produced in winter season.

A total of 126 Salmonella spp. isolates from pigs belonging to 13 serotypes (Typhimurium, Derby, Infantis, Enteritidis, Agona, Kaapstad, London, Montevideo, Bredeney, Give, Oritamerin, Schwarzengrund and Tennessee) were tested for sensitivity to 14 antibiotics. Resistance to 1-8 antibiotics was demonstrated in 64 isolates, classified into 7 serotypes with the most frequent being Salmonella typhimurium (n=54). S. typhimurium strains were found to be the most resistant to streptomycin (91.5%), sulphonamides (88.1%), ampicillin (86.4%), tetracycline (84.7%) and chloramphenicol (83.0%), displaying the ACSSuT phenotype. In all strains of this phenotype (n=27), the gene for integrase (int1) and resistance genes blaPSE-1, floR, aadA2, sul1 and tetG were detected by PCRs. In some of the strains, additional resistance to amoxycillin/clavulanic acid, sulphamethoxazole/trimethoprim, nalidixic acid and enrofloxacin was found.

The antimicrobial susceptibility of 30 clinical and 30 food Bacillus cereus isolates was determined. All isolates were susceptible to streptomycin, ciprofloxacin and gentamicin, 90 % of them to clindamycin and vancomycin, and 67 % to erythromycin. All isolates were resistant to amoxicillin with clavulanic acid, ampicillin, cefotaxime, ciprofloxacin, cloxacillin, cefotaxime with clavulanic acid and penicillin. The MIC values (determined by E-tests) were 48-256 mg/L for ampicillin, 0.19-1.5 mg/L for gentamicin, 0.125-1.0 mg/L for clindamycin, 0.047-4.0 mg/L for erythromycin and 1.5-16 mg/L for vancomycin. The MICs 4.6-18.75 g/L were observed for penicillin using the microdilution method. The presence of metallo-beta-lactamases was detected by E-test for 100 % of strains. Nonhemolytic diarrheal enterotoxin (NHE) was produced by 98.3 % of strains, while 31.7 % of them produced hemolytic diarrheal enterotoxin (HBL). Clinical isolates produced 10 % more HBL than food isolates. The psychrotrophic strains isolated from food samples produced NHE at 6.5 deg C in 73 % of cases.

The study was aimed at the assessment whether foodstuffs contaminated with Bacillus cereus may concurrently be vectors of spreading resistance. The contamination of foodstuffs with B. cereus strains was found in 31 % of dairy and in 28 % of meat products tested. Only one product from skimmed milk was contaminated. High-fat milk products that were heat-treated during the technological process (87 samples), as well as heat-treated meat products (65 samples), were contaminated significantly frequently (63 % and 48 % of the samples, respectively, P0.01). Almost all B. cereus isolates displayed low susceptibility to ampicillin, cephalothin, and to oxacillin. Except for streptomycin (STR) resistance, resistance to other 8 antimicrobial agents occurred sporadically. The STR resistant isolates came particularly from spreading butter (8 samples, P0.05). It was established that the same samples were contaminated with two subpopulations of B. cereus with different STR resistances. The frequent occurrence of B. cereus in foodstuffs with either fat content and/or subject to heat treatment in processing makes these products risky. However, our study did not confirm that foodstuffs contaminated with B. cereus were concurrently vectors of transmissible resistance genes.