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Plasmids designed to alter the antibiotic resistance expressed by insertion mutations in Bacillus subtilis, through in vivo recombination.

Numerous insertion mutations conferring resistance to antibiotics are available in Bacillus subtilis. However, many of them have been constructed in vitro by inserting genes from the Staphylococcus aureus plasmids, pC194 or pE194, conferring resistance to chloramphenicol (Cm) or erythromycin (Er). Others are insertions of the Enterococcus faecalis Tn917 transposon conferring resistance to Er. This paucity of resistance markers has been limiting the possibilities of constructing and studying mutants carrying two or more of these mutations in the past. We constructed plasmids which can be used to change the antibiotic resistance expressed by preexisting chromosomal insertions, through transformation and homologous recombination. These vectors replace the pre-existing resistance to Cm or Er with new resistances to neomycin (Nm), phleomycin (Pm), spectinomycin (Sp) or tetracycline (Tc).

Bacillus subtilis↗

Antibiotic-resistance cassettes for Bacillus subtilis.

The genes encoding resistance to four different antibiotics (erythromycin, kanamycin, tetracycline and spectinomycin) were cloned in the polylinker of various Escherichia coli plasmid vectors. These cassettes can be inserted into cloned Bacillus subtilis (Bs) genes and used to create tagged chromosomal disruptions after recombination into Bs and selection in the presence of the appropriate antibiotic.

Bacillus subtilis↗

Novel series of plasmid vectors for gene inactivation and expression analysis in group A streptococci (GAS).

Ten novel streptococcal shuttle vectors for genomic integration and allelic replacements have been constructed based on plasmid pSF152. These vectors can replicate in E. coli, but not in streptococci because of the absence of a streptococcal origin of replication. The basic vector pFW5 (2.8 kb, aad9 spectinomycin-resistance marker) carries two multiple cloning sites MCS-I and MCS-II (10 and 15 restrictions sites, respectively) to either side of the aad9 resistance gene. Each MCS is flanked by transcription termination sites for stabilization of recombinant plasmids. In vector pFW6 the transcription terminator between aad9 and MCS-II was deleted. Plasmids pFW7 through pFW10 carry resistance genes for kanamycin, chloramphenicol, erythromycin, and tetracyclin instead of aad9. Vectors pFW11 and pFW12 are pFW5/6 derivatives harboring an improved synthetic aad9 promoter. In pFW-phoA and pFW-gfp, promoterless alkaline phosphatase and green fluorescent protein boxes were integrated into MCS-I. If streptococcal DNA fragments are cloned into MCS-I and MSC-II, these vectors can be used for specific allelic replacements in streptococci via double-crossover recombinations. Depending on the vector used, this event will not lead to polar effects, facilitating mutagenesis within operons. The vectors containing reporter boxes allow in vivo studies of gene expression and promoter activity in pathogenic streptococci and potentially, also in other Gram-positive bacteria.

Alkaline Phosphatase↗

Antimicrobial susceptibility of Edwardsiella tarda from the United States and Taiwan.

Edwardsiella tarda isolates from the United States and Taiwan were screened against 37 antimicrobial agents. These isolates were highly susceptible to the amino-glycosides, second and third generation cephalosporins, newer penicillins, nitrofurantoin, sulfamethoxazole/trimethoprim, and the quinolones. Source-related resistance was found for penicillin G, sulfadiazine, colistin, novobiocin, spectinomycin, ampicillin, tetracycline, and chloramphenicol. Generally, the Taiwan isolates were more resistant than the United States isolates.

Animals↗

Antibioresistance of Escherichia coli strains isolated in Morocco from chickens with colibacillosis.

Two hundred and fifty eight isolates of Escherichia coli were made from autopsied chickens showing lesions of avian colibacillosis. Antibiograms showed high levels of resistance (greater than 40%) to sulphonamides (SSS), oxytetracycline (OT), trimethoprim + sulphamethoxazole (STX) and chloramphenicol (C). Medium frequencies of resistances (from 15 to 40%) were noted for streptomycin (S), spectinomycin (SPT), nalidixic acid (NA), oxolinic acid (OA), flumequine (UB) and enrofloxacine (ENR). For ampicillin (AM), gentamicin (GM), nitrofurans (FT), colistin (CS) and rifampin (RA) the frequencies of resistance were low (less than 15%). A linked resistance was observed for the 4 quinolones. A significant percentage of isolates (82.5%) were resistant to at least 2 antimicrobial agents. The most frequent antibiotypes were: C.OT.SSS.STX (4.65%), C.OT.SSS.STX.OA.NA.UB.ENR (4.65%), AM.S.C.OT.SSS.STX (4.26%) and OT.SSS.STX (3.87%).

Animals↗

Solid-phase clean-up and thin-layer chromatographic detection of veterinary aminoglycosides.

Chemical methods are needed to confirm the presence of antibiotics detected by microbial inhibition assays in fluids and tissues of farm animals. We have optimized the conditions for the isolation of hygromycin B with a copolymeric bonded solid-phase silica column followed by thin-layer chromatography (TLC) separation and detection of its fluorescence derivative after reaction with fluorescamine. The detection limit of the drug was 50 ng. Serum and plasma samples fortified with hygromycin B were acidified and passed through the copolymerized solid-phase columns previously conditioned with phosphate buffer. Hygromycin B was trapped in the columns and eluted with diethylamine-methanol and analyzed by TLC using acetone-ethanol-ammonium hydroxide as the developing solvent. Hygromycin B bands were derivatized at acidic pH with fluorescamine and visualized under ultraviolet light. Hygromycin B added to bovine plasma was detectable at 25, 50, 100, 250 and 500 ng/ml (ppb). Hygromycin B added to swine serum was detected at 50 ng/ml. However, the serum had to be deproteinized with trichloroacetic acid or acetonitrile prior to solid-phase extraction to gain accurate values. Neomycin and gentamicin (100 ng/ml aqueous solutions) could also be isolated with copolymeric solid-phase columns at a level of 50 ng. Gentamicin, neomycin, gentamicin, spectinomycin, hygromycin B and streptomycin could be separated by TLC, allowing multiresidue detection of these aminoglycosides. The respective RF values of 0.64, 0.56, 0.52, 0.33 and 0.20 indicate the separation of these five compounds. This procedure provides a rapid and sensitive method for the semi-quantitative estimation of aminoglycosides.

Animals↗

Studies on the antimicrobial activity of Nigella sativa seed (black cumin).

Filter paper discs impregnated with the diethyl ether extract of Nigella sativa seeds (25-400 micrograms extract/disc) caused concentration-dependent inhibition of Gram-positive bacteria represented by Staphylococcus aureus. Gram-negative bacteria represented by Pseudomonas aeruginosa and Escherichia coli (but not Salmonella typhimurium) and a pathogenic yeast Candida albicans. The extract showed antibacterial synergism with streptomycin and gentamicin and showed additive antibacterial action with spectinomycin, erythromycin, tobramycin, doxycycline, chloramphenicol, nalidixic acid, ampicillin, lincomycin and sulphamethoxyzole-trimethoprim combination. The extract successfully eradicated a non-fatal subcutaneous staphylococcal infection in mice when injected at the site of infection.

Animals↗

A ten-laboratory study of lomefloxacin (NY-198 or SC 47111) antimicrobial activity in Argentina.

The antimicrobial activity of a new fluoro-quinolone, lomefloxacin (NY-198, SC47111), was evaluated by standardized susceptibility testing methods at ten laboratories in Argentina. Lomefloxacin was found to be the most active drug against 1,316 recent clinical isolates compared directly to norfloxacin, co-trimoxazole, and gentamicin. Only 1.4% of Enterobacteriaceae were lomefloxacin-resistant (MIC greater than 4 micrograms/ml), and the lomefloxacin MIC90 for all staphylococci was 2 micrograms/ml, including methicillin-resistant isolates. Streptococci, enterococci, and some Pseudomonads (9% resistance) were less susceptible to lomefloxacin. Lomefloxacin was the most active against Neisseria gonorrhoeae strains compared to penicillin, cefuroxime, and spectinomycin. Among the tested Mycobacteria, only the nontuberculosis, slow growers had lomefloxacin MICs of greater than 4 micrograms/ml.

4-Quinolones↗

In vitro antibacterial activities of the fluoroquinolones PD 117596, PD 124816, and PD 127391.

Three new aminopyrrolidine-substituted fluorocyclopropyl quinolones--PD 117596, PD 124816, and PD 127391--were tested for in vitro antibacterial activity against 349 bacterial strains, which are primarily clinical isolates. The minimum inhibitory concentrations (MIC) in micrograms/ml required for greater than or equal to 90% of strains were 0.03-0.06 for staphylococci (26 strains); 0.06-0.25 for Streptococcus pyogenes, S. agalactiae, S. pneumoniae, and Enterococcus faecalis (80); less than or equal to 0.015 for Branhamella catarrhalis, Haemophilus influenzae, and Neisseria gonorrhoeae (42); 0.06 for Enterobacteriaceae (97); 0.125-0.25 for Acinetobacter spp. (14); 0.5 for Pseudomonas aeruginosa (20); 0.125-1.0 for Bacteroides fragilis (13); and 0.25-0.5 for anaerobic cocci (11). These activities were generally superior to that of ciprofloxacin, imipenem, ampicillin, penicillin G, oxacillin, cefazolin, ceftazidime, cefoxitin, cefsulodin, aztreonam, piperacillin, amikacin, spectinomycin, doxycycline, erythomycin, clindamycin, metronidazole, and vancomycin. The activities of the new quinolones were generally unchanged with light, 50% human serum, aerobic/anaerobic atmosphere, 5% sodium choate, cation supplementation, and 100-fold increased or decreased inoculum; as with other quinolones, potency was measurably diminished with decreasing pH (pH less than or equal to 6.0) and in 100% urine.

4-Quinolones↗

Cefetamet pivoxil in the treatment of uncomplicated gonorrhea.

We studied the efficacy and safety of cefetamet pivoxil (CAT), an oral aminothiazolyl cephalosporin, in a series of open, comparative multicenter studies in 207 women (four study centers) with uncomplicated gonorrhea, and summarized and pooled the results with those of earlier open dose-finding trials (360 men; six study centers). We compared single-dose treatment regimen of CAT--over the range of 400-1500 mg--with spectinomycin, thiamphenicol, ampicillin, or amoxicillin plus probenecid. The overall cure rates were 100% in 88 women treated with 1500 mg CAT and in 137 men treated with 1200 or 1500 mg CAT, 98% (114 of 116 men) in those treated with 800 or 1000 mg CAT, and 93% (42 of 45 men) in those treated with 400 or 500 mg CAT; the composite cure rate of the comparators was 97%. The tolerability of CAT (n = 428) compared favorably (1.8% adverse events) with that of the standard drugs (n = 139) (4.3% adverse events). Single-dose treatment with 1500 mg CAT is effective and safe in adults with uncomplicated gonorrhea.

Adolescent↗

Effect of aminoglycoside antibiotics on sodium transport across the Rana esculenta urinary bladder.

The aminoglycoside antibiotics inhibit the short circuit current (s.c.c.) in the urinary bladder of Rana esculenta. This effect is due to a direct inhibition on the sodium pump; ADH or db-AMPc are not able to reverse the inhibition of the sodium current, but are effective in stimulating the urea fluxes in the presence of antibiotics. In correlation with the number of the NH+2 groups in the antibiotic molecule there is a different pattern of inhibitory effect, i.e. neomycin greater than gentamycin greater than dihydrostreptomycin greater than kanamycin greater than spectinomycin.

Aminoglycosides↗

Transfer of transformed chloroplasts from Nicotiana tabacum to the Lycium barbarum plants.

Plastid transformation is an attractive technology for obtaining crop plants with new useful characteristics and for fundamental researches of plastid functioning and nuclear-plastid interaction. The aim of our experiments was to obtain plants with Lycium barbarum nucleus and transformed Nicotiana tabacum plastids. Plastome of previously engineered transplastomic tobacco plants contains reporter uidA gene and selective aadA gene that confers resistance to antibiotics spectinomycin and streptomycin. Asymmetric somatic hybridization was performed for transferring transformed tobacco plastids from transplastomic tobacco plants into recipient L. barbarum wild type plants. Hybrid L. barbarum plants containing transformed tobacco plastome with active aadA and uidA genes were obtained as a result of the experiments. The work shows the possibility of obtaining transplastomic plants by transferring the transformed plastids to remote species by using somatic hybridization technology. The developed technique is especially effective for obtaining transplastomic plants that have low regeneration and transformation ability.

Chloroplasts↗

Class 2 integron-associated antibiotic resistance in Shigella sonnei isolates in Dakar, Senegal.

Forty-three Shigella sonnei isolates from adult patients with diarrhoea in Dakar were analysed for the presence of integrons. Isolates were resistant to sulphamethoxazole, trimethoprim, tetracycline, streptomycin and spectinomycin. A high prevalence of class 2 integrons (93%) was found. These integrons showed three distinct structures: a class 2 integron, part of the Tn7 family and its derivatives, carrying four cassettes in the order dfrA1-sat-aadA1-orfX; a truncated class 2 integron, without orfX; and a third type ca. 4 kb in size. These class 2 integrons probably play a role in the spread of multiresistance in S. sonnei isolates. To our knowledge, this is the first description of class 2 integrons in S. sonnei isolated in sub-Saharan Africa.

Adult↗

Characterisation of antimicrobial resistance patterns and class 1 integrons among Escherichia coli and Salmonella enterica serovar Choleraesuis strains isolated from humans and swine in Taiwan.

Escherichia coli isolates from humans (n=110) and swine (n=61) and Salmonella enterica serovar Choleraesuis isolates (n=95) from swine in southern Taiwan were characterised for antimicrobial resistance patterns and class 1 integrons. All E. coli isolates and S. Choleraesuis isolates were multidrug resistant and demonstrated high resistance to beta-lactams, aminoglycosides, tetracycline, sulfonamides, spectinomycin, chloramphenicol and nalidixic acid. By polymerase chain reaction and DNA sequencing, 104 (61%) E. coli isolates and 31 (33%) S. Choleraesuis isolates were found to carry class 1 integrons. The gene cassette array dfrA12-orfF-aadA2 was the most prevalent (24%) among the human and swine E. coli isolates, whilst the gene cassette array dfrA12-orfF-aadA2-sul1 was the most prevalent (24%) among S. Choleraesuis strains. For E. coil isolates, all class 1 integrons were located on conjugated plasmids. Meanwhile, human and swine E. coli isolates carrying identical gene cassettes were genetically unrelated. Our results revealed that multidrug resistance and class 1 integrons were widely present in E. coli and S. Choleraesuis isolates obtained in Taiwan and that class 1 integrons might play an important role in contributing to the horizontal transfer of antimicrobial resistance.

Animals↗

Antibiotic susceptibility profiles of new probiotic Lactobacillus and Bifidobacterium strains.

The antimicrobial susceptibilities and presence of plasmids in four new probiotic lactic acid bacteria (LAB) strains, Lactobacillus rhamnosus HN001 (DR20) HN067, Lactobacillus acidophilus HN017 and Bifidobacterium lactis HN019 (DR10), were determined. Resistance to 18 commonly used antibiotics was assessed by disk diffusion. The three Lactobacillus strains had similar antibiotic susceptibility profiles to those of Lactobacillus plantarum strain HN045 and two commercial probiotic Lactobacillus strains, GG and LA-1. The B. lactis strain HN019 had a similar profile to three commercial probiotic B. lactis strains (Bb12, HN049 and HN098). All 10 strains were sensitive to the Gram-positive spectrum antibiotics erythromycin and novobiocin, the broad-spectrum antibiotics rifampicin, spectinomycin, tetracycline and chloramphenicol and the beta-lactam antibiotics penicillin, ampicillin and cephalothin. By contrast, most strains were resistant to the Gram-negative spectrum antibiotics fusidic acid, nalidixic acid and polymyxin B and the aminoglycosides neomycin, gentamicin, kanamycin and streptomycin. All three L. rhamnosus strains (HN001, HN067 and GG) were resistant to vancomycin and several strains were also resistant to cloxacillin. Of the four new probiotic strains, only L. rhamnosus HN001 contained plasmids; however, a plasmid-free derivative of HN001 had the same antibiotic susceptibility profile as the parent strain.

Anti-Bacterial Agents↗

Prevalence and antibiotic-resistance of Salmonella isolated from beef sampled from the slaughterhouse and from retailers in Dakar (Senegal).

A study was made of Salmonella contamination in beef sampled from a slaughterhouse and from retailers in Dakar, Senegal. The serotypes as well as antibiotic-resistance patterns of the Salmonella isolates were determined. A total of 435 meat samples (236 from the slaughterhouse, 199 from retailers) were tested. Among them, 275 (63%) were positive for Salmonella, 43% (101/236) from the slaughterhouse and 87% (174/199) from the retailers. Furthermore, 97% of the investigated retailers had at least one beef sample contaminated by Salmonella. The 286 Salmonella isolates were divided into 51 serotypes. The most prevalent serotypes were Salmonella bredeney (25%), S. muenster (8%), S. waycross (7%), S. corvallis (4%) and S. kentucky (4%). About 62% of the isolates were resistant to nitrofurans. Resistance rates were lower to streptomycin (22%), sulfamethoxazole (15%), spectinomycin (1%), chloramphenicol (1%), and tetracycline (0,4%) while low-level resistance to quinolones was detected. About 16% of the Salmonella strains were multiresistant to two or more antibiotic families. Finally, ten resistance profiles have been identified. This study shows the huge spread of Salmonella in the beef production chain in Dakar, Senegal. Finally, this study provides the very first data about Salmonella prevalence in sub-saharian Africa.

Abattoirs↗

Antibiotic resistance of lactic acid bacteria and Bifidobacterium spp. isolated from dairy and pharmaceutical products.

The outlines of antibiotic resistance of some probiotic microorganisms were studied. This study was conducted with the double purpose of verifying their ability to survive if they are taken simultaneously with an antibiotic therapy and to increase the selective properties of suitable media for the isolation of samples containing mixed bacterial populations. We isolated from commercial dairy and pharmaceutical products, 34 strains declared as probiotics, belonging to the genera Bifidobacterium and Lactobacillus, and 21 strains of starter culture bacteria. All the microorganisms have been compared by electrophoresis of the soluble proteins for the purpose of identifying them. A Multiplex-PCR with genus- and species-specific primers was used to detect for Bifidobacterium animalis subsp. lactis presence. All bifidobacteria were B. animalis subsp. lactis except one Bifidobacterium longum. Sometimes the identification showed that the used strain was not the one indicated on the label. The lactobacilli were Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus delbrueckii subsp. bulgaricus. The streptococci were all Streptococcus thermophilus. The minimal inhibitory concentration (MIC) of 24 common antibiotic substances has been valued by the broth microdilution method. All tested strains were susceptible to ampicillin, bacitracin, clindamycin, dicloxacillin, erytromycin, novobiocin, penicillin G, rifampicin (MIC(90) ranging from 0.01 to 4 microg/ml); resistant to aztreonam, cycloserin, kanamycin, nalidixic acid, polymyxin B and spectinomycin (MIC(90) ranging from 64 to >1000 microg/ml). The susceptibility to cephalothin, chloramphenicol, gentamicin, lincomycin, metronidazole, neomycin, paromomycin, streptomycin, tetracycline and vancomycin was variable and depending on the species.

Animals↗

A nosocomial outbreak of Serratia marcescens producing inducible Amp C-type beta-lactamase enzyme and carrying antimicrobial resistance genes within a class 1 integron.

We investigated an outbreak of Serratia marcescens in the adult intensive care unit of the University Hospital of Napoli. The outbreak involved 13 cases of infection by S. marcescens over a nine-month period and was caused by a single pulsed-field gel electrophoresis clone. The epidemic strain was multiply antibiotic resistant, producing an inducible Amp C-type beta-lactamase enzyme and carrying the trimethoprim-resistance gene and the adenyltransferase gene, which confers resistance to streptomycin and spectinomycin, within a class 1 integron. Antimicrobial therapy with beta-lactams was associated with S. marcescens acquisition in the intensive care unit.

Adult↗