In this review an overview is given on antibiotic resistance (AR) mechanisms with special attentions to the AR genes described so far preceded by a short introduction on the discovery and mode of action of the different classes of antibiotics. As this review is only dealing with acquired resistance, attention is also paid to mobile genetic elements such as plasmids, transposons, and integrons, which are associated with AR genes, and involved in the dispersal of antimicrobial determinants between different bacteria.

In the past decade, various methods have been developed for the identification and typing of prokaryotic and eukaryotic organisms at the DNA level. These methods differ in their taxonomic range, discriminatory power, reproducibility, and ease of interpretation and standardization (62,67,86,87,101,106,110,116). The ideal genotyping method produces results that are invariable from laboratory to laboratory and allows unambiguous comparative analyses and the establishment of reliable databases.One of the newest and most promising methods is amplifiedfragment length polymorphism (AFLP) analysis (11,118,122), developed by Keygene BV, Wageningen, The Netherlands. This method combines universal applicability with high powers of discrimination and reproducibility (45). An increasing number of reports describe the use of AFLP analysis for plant and animal genetic mapping, medical diagnostics, phylogenetic studies, and microbial typing. This minireview describes the principles, advantages, and disadvantages of AFLP analysis and summarizes its applications in different fields. PRINCIPLE OF AFLPIn the nomenclature of Vaneechoutte (110), AFLP analysis belongs to the category of selective restriction fragment amplification techniques, which are based on the ligation of adapters (i.e., linkers and indexers) to genomic restriction fragments followed by a PCR-based amplification with adapterspecific primers. For AFLP analysis ( Fig. 1), only a small amount of purified genomic DNA is needed; this is digested with two restriction enzymes, one with an average cutting frequency (like EcoRI) and a second one with a higher cutting frequency (like MseI or TaqI). Double-stranded oligonucleotide adapters are designed in such a way that the initial restriction site is not restored after ligation, which allows simultaneous restriction and ligation, while religated fragments are cleaved again. An aliquot is then subjected to two subsequent PCR amplifications under highly stringent conditions with adapter-specific primers that have at their 3Ј ends an extension of one to three nucleotides running into the unknown chromosomal restriction fragment. An extension of one selective nucleotide amplifies 1 of 4 of the ligated fragments, whereas three selective nucleotides in both primers amplify 1 of 4,096 of the fragments. The PCR primer which spans the average-frequency restriction site is labeled. After polyacrylamide gel electrophoresis a highly informative pattern of 40 to 200 bands is obtained. The patterns obtained from different strains are polymorphic due to (i) mutations in the restriction sites, (ii) mutations in the sequences adjacent to the restriction sites and complementary to the selective primer extensions, and (iii) insertions or deletions within the amplified fragments.Since the original publication by Vos et al. in 1995 (118) several enzyme combinations have been used, such as EcoRI, PstI, HindIII, or ApaI combined with MseI or TaqI. For animal genomes EcoRI and TaqI appear to be the most suitable (2). Alternative AFLP typing proce...

The minimum inhibitory concentrations (MICs) of 6 different antibiotics (chloramphenicol, clindamycin, erythromycin, streptomycin, tetracycline and vancomycin) were determined for 143 strains of lactic acid bacteria and bifidobacteria using the Etest. Different MICs were found for different species and strains. Based on the distribution of these MIC values, most of the strains were either susceptible or intrinsically resistant to these antibiotics. However, the MIC range of some of these antibiotics showed a bimodal distribution, which suggested that some of the tested strains possess acquired antibiotic resistance. Screening for resistance genes was performed by PCR using specific primers, or using a DNA microarray with around 300 nucleotide probes representing 7 classes of antibiotic resistance genes. The genes identified encoded resistance to tetracycline [tet(M), tet(W), tet(O) and tet(O/W)], erythromycin and clindamycin [erm(B)] and streptomycin [aph(E) and sat(3)]. Internal portions of some of these determinants were sequenced and found to be identical to genes described in other bacteria. All resistance determinants were located on the bacterial chromosome, except for tet(M), which was identified on plasmids in Lactococcus lactis. The contribution of intrinsic multidrug transporters to the antibiotic resistance was investigated by cloning and measuring the expression of Bifidobacterium breve genes in L. lactis.

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The variation in manure-amended soil survival capability among 18 Escherichia coli O157 strains (8 animal, 1 food, and 9 human isolates) was studied using a single sandy soil sample and a single sample of cattle manure as the inoculum carrier. The virulence profiles of E. coli O157 strains were characterized by detection of virulence determinants (73 genes, 122 probes in duplicate) by using the Identibac E. coli genotyping DNA miniaturized microarray. Metabolic profiling was done by subjecting all strains to the Biolog phenotypic carbon microarray. Survival times (calculated as days needed to reach the detection limit using the Weibull model) ranged from 47 to 266 days (median, 120 days). Survival time was significantly higher for the group of human isolates (median, 211 days; minimum [min.], 71; maximum [max.], 266) compared to the group of animal isolates (median, 70 days; min., 47; max., 249) (P ‫؍‬ 0.025). Although clustering of human versus animal strains was observed based on pulsed-field gel electrophoresis (PFGE) patterns, no relation between survival time and the presence of virulence genes was observed. Principal component analysis on the metabolic profiling data revealed distinct clustering of short-and long-surviving strains. The oxidization rate of propionic acid, ␣-ketobutyric acid, and ␣-hydroxybutyric acid was significantly higher for the long-surviving strains than for the short-surviving strains. The oxidative capacity of E. coli O157 strains may be regarded as a phenotypic marker for enhanced survival in manure-amended soil. The large variation observed in survival is of importance for risk assessment models.

The analysis of the developmental pattern of the aA-, aB-, PSI-, PB2-, ,GB3-, PA3/A1-, and ps-crystallin genes during fetal and postnatal development of the rat shows that the differential regulation of crystallin synthesis relies on differential gene shutdown rather than differential gene activation; that is, all crystallin genes are active during early development but turn off at different stages. The only two exceptions to this rule are the aB-and pscrystallin genes. The aB-crystallin gene transcript becomes first detectable at 18 days of fetal development, while the ps-crystallin gene appears to be active only in the postnatal period.We also determined the absolute numbers of the aA-, aB-, BBl-, pB2-, PB3-, PA3/A1-, ps-, and y-crystallin gene transcripts present in the lens at various times after birth. Comparison of these RNA data with the published protein data shows that the aB-and flB2-crystallin RNAs are relatively overrepresented, suggesting the possibility that these two RNA species are not used as efficiently as other crystallin mRNAs. Examination of the known (hamster) aB-crystallin sequence and elucidation of the (rat) PB2-crystallin sequence yielded no evidence for aberrant codon usage. These two RNAs have one sequence motif in common: they are the only crystallin mRNAs in which the translation initiation codon is preceded by CCACC.The abundant water-soluble proteins of the mammalian lens are encoded by three gene families, the a-, p-and ycrystallin gene families (for review see [l, 21). The a-crystallin gene family has only two members, the aA-and aB-crystallin genes. The protein products of these two genes form the large a-crystallin complex. The p-crystallin gene family is known to have at least six members, of which only four, namely PA3/1-, the PBl-, the BB2-, and the pB3-crystallin sequences, have been cloned. The ,!I-crystallins are found as hetero-oligomers in the lens. The third crystallin gene family, the y-crystallin genes, is evolutionarily related to the fi-crystallin gene family. In the rat the y-crystallin gene family has seven members. Six of these are closely related and encode the monomeric ycrystallins. The seventh codes for the more distantly related monomeric ps-crystallin (for review, see [3]). It has been previously shown that the six y-crystallin genes are all turned on during the early embryonic development of the rat or mouse lens but are differentially shut off during postnatal development [4, 51. As the y-crystallins and the P-crystallins derive from the same ancestral sequence, it is of interest to determine whether the developmental expression of the p-crystallin genes follows the same pattern as that of the y-crystallin genes. If so, these two gene families might have inherited the same mechanism of gene activation. We show that the all members of these two gene Families, except for the ps-crystallin gene, are indeed activated during early embryonic development of the rat lens. We further show that the activation of the aBcrystallin gene lags behind that of the aA-cr...