Incidence of antibiotic resistance in Salmonella typhimurium strains isolated in different epidemic situations

A total of 1800 S. typhimurium strains isolated in different regions of the USSR in 1968-1979 were studied. Of these strains, 68.6% were resistant to 6-8 antibiotics, 7.8% were resistant to 2-5 antibiotics, and 23.6% proved to be resistant to all antibiotics. The number of multiresistant strains sharply increased during the last 10 years. The strains isolated from different sources and in different epidemic situations considerably differed in their sensitivity to the action of antibiotics. The infective agents isolated from the hospital foci of salmonellosis were found to possess the maximum multiresistance. The study of the genetic nature of multiresistance showed that the multiresistance strains had a conjugative R-plasmid with resistance determinants CmTc, type Fin+, incompatibility group F1me. The phage typing of the strains with phages from the collection of the Tbilisi Research Institute for Vaccines and Sera revealed that these strains belonged to phagotype 2. The problems of the relationship between the biological properties of causative agents and the character of the epidemic process of the diseases caused by these agents are discussed.     

Comparative proteomic analysis of Staphylococcus aureus strains with differences in resistance to the cell wall-targeting antibiotic vancomycin

Three isogenic strains derived from a clinical vancomycin-intermediate Staphylococcus aureus isolate were examined by comparative protein abundance analysis. Subcellular fractionation was followed by protein separation in 2-DE gels and spot identification by MALDI-TOFTOF-MS and LC-MS/MS. Sixty-five significant protein abundance changes were determined. Numerous enzymes participating in the purine biosynthesis pathway were dramatically increased in abundance in strain VP32, which featured the highest minimal inhibitory concentration for vancomycin, compared to strains P100 and HIP5827. Peptidoglycan hydrolase LytM (LytM) and the SceD protein, a putative transglycosylase, were increased in abundance in the cell envelope fraction of strain VP32, whereas the enzyme D-Ala-D-Ala ligase was decreased in its cytosol fraction. Furthermore, penicillin-binding protein 2 (PBP2) had substantially higher activity in strain VP32 compared to that in strain HIP5827. LytM, PBP2 and D-Ala-D-Ala ligase catalyze reactions in the biosynthesis or the metabolism of cell wall peptidoglycan. It is plausible that expression and activity changes of these enzymes in strain VP32 are responsible for an altered cell wall turnover rate, which has been observed, and an altered peptidoglycan structure, which has yet to be elucidated for this highly vancomycin-resistant strain.     

Genomic comparison of Salmonella typhimurium DT104 with non-DT104 strains

DT104 emerged as a new branch of Salmonella typhimurium with resistance to multiple antimicrobials. To reveal some general genomic features of DT104 for clues of evolutionary events possibly associated with the emergence of this relatively new type of this pathogen, we mapped 11 independent DT104 strains and compared them with non-DT104 S. typhimurium strains. We found that all 11 DT104 strains contained three insertions absent in non-DT104 strains, i.e., the previously reported ST104, ST104B and ST64B. However, SGI-1, a genomic island known to be responsible for DT104 multidrug resistance, was not present in all DT104 strains examined in this study: one DT104 strain did not contain SGI-1 but carried a 144 kb plasmid, suggesting possible evolutionary relationships between the two DNA elements in the development of antimicrobial resistance.     

Reversible translocation of antibiotic resistance determinants in Salmonella ordonez.

Summary Salmonella ordonez (BM 2000) codes for kanamycin (Km, aphA), ampicillin (Ap), streptomycin (SmSp: aadA and Sm: aphC), chloramphenicol (Cm), tetracycline (Tc) and sulfornamide (Su) resistances and for production of colicin Ib (Cib). Genetical analysis by incompatibility testing, conjugation, transformation and physical studies using electron microscopy, agarose gel electrophoresis, led us to associate the Km and Cib characters to a 98.7 kilobase (kb) IncII plasmid (pIP565), and the Sm (aphC) and Su determinants to a 8.3 kb plasmid (pIP605). The ApCmSmSp(aadA)SuTc determinants were not associated in BM2000 S. ordonez with a plasmid structure. Following conjugation of S. ordonez to E. coli, the ApCmSmSpSuTc determinants were found stably associated with a single plasmid structure (pIP173, 127.5 kb) belonging to IncII group. Agarose gel electrophoresis of plasmid DNA restriction endonuclease digests and electron microscopy heteroduplex analysis showed that the acquisition of the ApCmSmSpSuTc determinants resulted from the insertion into pIP565 of a 28.8 kb DNA sequence. This sequence coding for ApCmSmSpSuTc resistances in S. ordonez could be translocated either to pIP565 plasmid or to several IncII plasmids but never to plasmids belonging to IncW, IncP or IncFII, suggesting the existence of specific sequences on the IncII receptor plasmids. Mereover, R-determinants were translocated back en bloc from pIP173 to the chromosome of a susceptible S. ordonez. The results were consistent with the presence in BM2000 S. ordonez chromosomal DNA of an integrated translocatable sequence encoding ApCmSmSpSuTc resistances. Such a structural association could account for the stability of these resistances in the Salmonella ordonez serotype.     

Analysis of Salmonella Agona and Salmonella Weltevreden in Malaysia by PCR fingerprinting and antibiotic resistance profiling

Forty-eight strains of Salmonella enterica subsp. enterica serovar Agona and 33 strains of Salmonella enterica subsp. enterica serovar Weltevreden were characterized by random amplified polymorphic DNA (RAPD) fingerprinting using 3 different arbitrary primer, Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) and antimicrobial susceptibility testing. By using RAPD, 81 strains (44 strains of S. Agona and 33 strains of S. Weltevreden) can be clustered into 14 groups and 6 single isolates whereas ERIC-PCR produced 7 clusters and 3 single isolates. Thirteen antimicrobial agents were used and all the isolates were resistant to erythromycin and showed Multiple Antimicrobial Resistance indexes, ranging from 0.08 to 0.62. Poultry still remain as the common reservoir for multi-drug-resistant Salmonella. On the other hand, vegetables contaminated with S. Weltevreden showed a gain in antimicrobial resistance. Besides that, consistent antibiograms were observed from S. Weltevreden isolated at Kajang wet market on 2000/08/02.     

Characterization of antibiotic resistance genes linked to class 1 and 2 integrons in strains of Salmonella spp. isolated from swine

The aim of this study was to characterize the antibiotic resistance profiles, the integron-associated resistance determinants, and the potential ability of transferring these determinants by conjugation in Salmonella enterica isolated from swine. Fifty-four strains of Salmonella spp. were isolated from healthy swine. The percentages of resistance, determined by the plate dilution method were as follows: oxytetracycline (41%), streptomycin (39%), sulphamethoxazol+trimethoprim (19%), enrofloxacin-ciprofloxacin (13%), and amoxicillin (0%). The most important resistance serovars were Salmonella Branderburg, Salmonella Derby, Salmonella Typhimurium, and Salmonella Heidelberg. The oxytetracycline-resistant strains amplified the genes tetA (36%), tetB (64%); and the strains resistant to streptomycin and trimethoprim amplified the genes aadA1 (100%) and dfrA1 (100%), respectively. None of the fluoroquinolone-resistant strains amplified the gene qnr. Ten strains amplified the class 1 integron harboring the cassette aadA1. Six strains amplified the class 2 integron harboring the cassettes dfrA1, sat1, and aadA1. The conjugation assays showed that 2 strains transferred the tetA and aadA1 genes and the class 1 integron to a recipient strain. Taken together, the results obtained in this study show a high percentage of resistance in and the presence of integrons in strains of S. enterica isolated from swine. This information should support the implementation of regulations for the prudent use of antimicrobial agents in food-producing animals.     

Genomic diversification among archival strains of Salmonella enterica serovar typhimurium LT7

To document genomic changes during long periods of storage, we analyzed Salmonella enterica serovar Typhimurium LT7, a mutator strain that was previously reported to have higher rates of mutations compared to other serovar Typhimurium strains such as LT2. Upon plating directly from sealed agar stabs that had been stocked at room temperature for up to four decades, many auxotrophic mutants derived from LT7 gave rise to colonies of different sizes. Restreaking from single colonies consistently yielded colonies of diverse sizes even when we repeated single-colony isolation nine times. Colonies from the first plating had diverse genomic changes among and even within individual vials, including translocations, inversions, duplications, and point mutations, which were detected by rare-cutting endonuclease analysis with pulsed-field gel electrophoresis. Interestingly, even though the colony size kept diversifying, all descendents of the same single colonies from the first plating had the same sets of detected genomic changes. We did not detect any colony size or genome structure diversification in serovar Typhimurium LT7 stocked at -70 degrees C or in serovar Typhimurium LT2 stocked either at -70 degrees C or at room temperature. These results suggest that, although colony size diversification occurred during rapid growth, all detected genomic changes took place during the storage at room temperature and were carried over to their descendents without further changes during rapid growth in rich medium. We constructed a genomic cleavage map on the LT7 strain that had been stocked at -70 degrees C and located all of the detected genomic changes on the map. We speculated on the significance of mutators for survival and evolution under environmentally stressed conditions.     

Antibiotic resistance of Salmonella strains isolated from fish and crustaceans

A total of 240 Salmonella strains, 158 from 730 fish samples and 82 from 276 crustacean samples, obtained during a 2 year period, were examined for resistance to 10 antibiotics. More than 90% of the strains were resistant to bacitracin, penicillin and novobiocin. The least resistance was observed to chloramphenicol (6·7%) and nalidixic acid (12%). Multiple antibiotic resistance indexing of the strains showed that more than 95% originated from high risk sources of contamination such as poultry, swine, cattle and human environments where antibiotics are often used.     

Profiling antibiotic resistance in Escherichia coli strains displaying differential antibiotic susceptibilities using Raman spectroscopy

The rapid identification of antibiotic resistant bacteria is important for public health. In the environment, bacteria are exposed to sub-inhibitory antibiotic concentrations which has implications in the generation of multi-drug resistant strains. To better understand these issues, Raman spectroscopy was employed coupled with partial least squares-discriminant analysis to profile Escherichia coli strains treated with sub-inhibitory concentrations of antibiotics. Clear differences were observed between cells treated with bacteriostatic (tetracycline and rifampicin) and bactericidal (ampicillin, ciprofloxacin, and ceftriaxone) antibiotics for 6 hr: First, atomic force microscopy revealed that bactericidal antibiotics cause extensive cell elongation whereas short filaments are observed with bacteriostatic antibiotics. Second, Raman spectral analysis revealed that bactericidal antibiotics lower nucleic acid to protein (I₈₁₂/I₈₃₀) and nucleic acid to lipid ratios (I₁₄₈₃/I₁₄₅₂) whereas the opposite is seen with bacteriostatic antibiotics. Third, the protein to lipid ratio (I₂₉₃₆/I₂₈₈₅ and I₂₉₃₆/I₂₈₅₀) is a Raman stress signature common to both the classes. These signatures were validated using two mutants, Δlon and ΔacrB, that exhibit relatively high and low resistance towards antibiotics, respectively. In addition, these spectral markers correlated with the emergence of phenotypic antibiotic resistance. Overall, this study demonstrates the efficacy of Raman spectroscopy to identify resistance in bacteria to sub-lethal concentrations of antibiotics.     

Molecular characterization of antibiotic resistance in clinical Salmonella typhi isolated in Ghana

Fifty-eight clinical Salmonella typhi strains isolated from patients suspected of suffering from typhoid fever were obtained at the Korle-Bu Teaching Hospital and the Noguchi Memorial Institute for Medical Research, both located in Ghana, Africa. Each isolate was examined for susceptibility to ampicillin, chloramphenicol, streptomycin, tetracycline, and trimethoprim/sulfamethoxazole by the disk diffusion assay. Five of the isolates were resistant to all five antibiotics while 10 isolates were resistant to ampicillin, chloramphenicol, and trimethoprim/sulfamethoxazole, which are considered 'first line' antibiotics in the treatment of typhoid fever. Thirty-four isolates were resistant to at least one of the antibiotics tested and 62% of these isolates possessed conjugable plasmids belonging to incompatibility group IncHI. Ninety percent of the conjugable plasmids conferred a multiple drug-resistant phenotype on the strains harboring them. Additionally, 14 strains contained plasmids that were transformable and six of them encoded multiple drug resistance. Our findings indicate that multiple drug resistance to the 'first line' antibiotics in S. typhi may be more prevalent in Africa than previously thought.     

Use of impedance measurements to estimate numbers of antibiotic resistant Salmonella strains

Impedance detection times were compared with traditional plating methods for enumerating antibiotic-resistant strains of Salmonella stanley, Salm. thompson and Salm. infantis grown in laboratory medium and pork slurry. The correleation of log₁₀ counts of salmonellas with detection times was highly significant ( r = -0·96 for broth and r = -0·94 for slurries. The confidence limits (± og₁₀ 1·0 for broth and ± log₁₀ 1·65 for slurry) indicated that detection times could reliably be used as a rapid means of enumerating salmonellas when large numbers of counts of known strains are required for growth studies. Use of antibiotic-resistant strains also permitted their selective detection by impedance from the natural spoilage flora of pork slurry when the same antibiotics were incorporated in the detection medium.     

Bacteriocinogeny and antibiotic resistance of naturally occurring strains of Streptococcus mutans

We studied bacteriocinogenies and antibiotic resistances of naturally occurring strains of Streptococcus mutans isolated from different sources. It was found that naturally occurring strains from humans and rats were distinguished by their bacteriocin susceptibilities and production. We had a few incidences of tetracycline resistant strains only among those isolated from rats. It is quite interesting that none of the resistant strains we collected produced bacteriocins.     

Annual trends in antibiotic resistance of nosocomial Acinetobacter baumannii strains and the effect of synergistic antibiotic combinations

Acinetobacter baumannii is becoming increasingly resistant to antibiotics often requiring combination therapy. Annual changes of resistance to selected antimicrobials of 150 A. baumannii strains, isolated as nosocomial pathogens between 1994 and 2000 were investigated. The synergistic effects of antimicrobials were studied using a microdilution checkerboard technique in eight selected isolates resistant to third-generation cephalosporins and beta-lactam/beta-lactamase inhibitor combinations and to at least one aminoglycoside. Rates of resistance of cefepime, ceftazidime, ampicillin/sulbactam, amikacin and ciprofloxacin (before 1996 and between 1996-2000) were 29.7% - 72.6, 37.8% - 81.4%, 35.1% - 72.6%, 8.1% - 56.6%, 5.4% - 46.0% respectively (p < 0.001 for each one). Synergy was observed in at least one of the combinations of antibiotics from seven of eight isolates (87%), no antagonism was detected with any combination. Ceftazidime-amikacin (50%) and ampicillin/sulbactam-tobramycin (50%) were the most effective combinations. Due to the effectiveness of sulbactams to Acinetobacter, ampicillin/sulbactam-tobramycin combination is recommended as the first line of choice.     

Compensatory adaptation to the deleterious effect of antibiotic resistance in Salmonella typhimurium

Most chromosomal mutations that cause antibiotic resistance impose fitness costs on the bacteria. This biological cost can often be reduced by compensatory mutations. In Salmonella typhimurium, the nucleotide substitution AAA42 --> AAC in the rpsL gene confers resistance to streptomycin. The resulting amino acid substitution (K42N) in ribosomal protein S12 causes an increased rate of ribosomal proofreading and, as a result, the rate of protein synthesis, bacterial growth and virulence are decreased. Eighty-one independent lineages of the low-fitness, K42N mutant were evolved in the absence of antibiotic to ameliorate the costs. From the rate of fixation of compensated mutants and their fitness, the rate of compensatory mutations was estimated to be > or = 10-7 per cell per generation. The size of the population bottleneck during evolution affected fitness of the adapted mutants: a larger bottleneck resulted in higher average fitness. Only four of the evolved lineages contained streptomycin-sensitive revertants. The remaining 77 lineages contained mutants that were still fully streptomycin resistant, had retained the original resistance mutation and also acquired compensatory mutations. Most of the compensatory mutations, resulting in at least 35 different amino acid substitutions, were novel single-nucleotide substitutions in the rpsD, rpsE, rpsL or rplS genes encoding the ribosomal proteins S4, S5, S12 and L19 respectively. Our results show that the deleterious effects of a resistance mutation can be compensated by an unexpected variety of mutations.     

转基因植物中抗生素抗性基因的安全性评价

20世纪80年代以来,植物转基因技术取得突飞猛进的进展。转基因植物大量出现,带来了巨大的经济效益和社会效益。与此同时,转基因植物的释放可能带来的风险也越来越受到人们的重视,抗生素抗性基因是筛选转基因植物的常用基因,其安全性引起了人们的普遍关注,作者就转基因植物中抗生素抗性基因的安全性作一综述。