A 7.8-kbp fragment of chromosomal DNA from a region controlling multiple antibiotic resistance (Mar) in Escherichia coli has been sequenced. Within the fragment is a potential divergent promoter region including marO, which contains two pairs of direct repeats, suggesting possible operator-regulatory sites. To the left of marO (region I) are one or two transcriptional units with three putative open reading frames (ORFs) encoding 64, 157, and 70 amino acids. To the right (region II) is a transcriptional unit containing three putative ORFs (0RF125/144, 0RF129, and 0RF72). Of six independent Mar mutants, four had mutations within the ORF encoding the first putative protein (0RF125/144) downstream of marO, including three different single-point mutations and an IS2 insertion. One of the other mutations occurred in marO (20-bp duplication), and the other occurred in a site in marO or 0RF144 (a 1-bp change). All six mutations led to increased transcription of the region II transcript. High-copy-number plasmids containing marO and the adjacent 0RF125/144 region from a wild-type source but not from a Mar mutant reduced the antibiotic resistance of a Mar mutant to levels comparable to those of wild-type cells. High-copy-number plasmids containing wild-type marO alone caused an increase in resistance to tetracycline, chloramphenicol, and norfloxacin in a wild-type strain. The nature of the Mar mutations and the results of the complementation studies suggest that 0RF125/144 encodes a repressor (designated MarR) which acts at marO. The second ORF (0RF129), designated marA, would encode a protein, MarA, whose sequence shows strong similarity to those of a family of positive transcriptional regulators. A TnS insertion in marA inactivated the multiresistance phenotype of Mar mutants. The function of 0RF72, designated marB, encoding the third putative protein in the operon, and that of other ORFs detected within the 7.8-kb fragment have not yet been determined.

BackgroundThe impact of food animals as a possible reservoir for extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae, and the dissemination of such strains into the food production chain need to be assessed. In this study 334 fecal samples from pigs, cattle, chicken and sheep were investigated at slaughter. Additionally, 100 raw milk samples, representing bulk tank milk of 100 different dairy farms, 104 minced meat (pork and beef) samples and 67 E. coli isolates from cattle E. coli mastitis were analyzed.ResultsAs many as 15.3% of the porcine, 13.7% of the bovine, 8.6% of the sheep and 63.4% of the chicken fecal samples yielded ESBL producers after an enrichment step. In contrast, none of the minced meat, none of the bulk tank milk samples and only one of the mastitis milk samples contained ESBL producing strains. Of the total of 91 isolates, 89 were E. coli, one was Citrobacter youngae and one was Enterobacter cloacae. PCR analysis revealed that 78 isolates (85.7%) produced CTX-M group 1 ESBLs while six isolates (6.6%) produced CTX-M group 9 enzymes. Five detected ESBLs (5.5%) belonged to the SHV group and 2 isolates (2.2%) contained a TEM-type enzyme. A total of 27 CTX-M producers were additionally PCR-positive for TEM-beta-lactamase. The ESBL-encoding genes of 53 isolates were sequenced of which 34 produced CTX-M-1, 6 produced CTX-M-14, 5 produced CTX-M-15 and also 5 produced SHV-12. Two isolates produced TEM-52 and one isolate expressed a novel CTX-M group 1 ESBL, CTX-M-117. One isolate--aside from a CTX-M ESBL-- contained an additional novel TEM-type broad-spectrum beta-lactamase, TEM-186.ConclusionsThe relatively high rates of ESBL producers in food animals and the high genetic diversity among these isolates are worrisome and indicate an established reservoir in farm animals.

Stable chromosomal multiple-antibiotic-resistant (Mar) mutants of Escherichia coli, derived by exposing susceptible cells to low concentrations of tetracycline or chloramphenicol, express cross-resistance to structurally unrelated antibiotics. The entire resistance phenotype is reversed to susceptibility by insertion of transposon Tn5 into a locus, designated marA, near 34 min on the chromosome (A. M. George and S. B. Levy, J. Bacteriol. 155:541-548, 1983). Strains in which 39 kbp of chromosomal DNA, including marA, had been deleted were unable to produce Mar mutants. The deletion strain could be complemented in trans by introduction of intact marA+ on plasmid F'506. Junction fragments from a strain containing marA::Tn5 were cloned, exploiting kanamycin resistance on Tn5 for selection. They were used as probes to search a phasmid library of E. coli K-12 for recombinants containing the marA+ region. Two phasmids which contained regions hybridizing to this probe were identified and shown to complement delta marA in a deletion strain. From one phasmid, several marA-containing fragments were cloned: those of greater than or equal to 7.8 kbp restored the ability to form Mar mutants in a deletion strain. These Mar mutants were shown to be dependent on the cloned marA fragment. Chromosomal as well as recombinant Mar mutants showed increased expression of a marA-specific mRNA species of about 1.4 kb, which was barely or not detectable in wild-type strains. Exposure of mutants and, to a lesser extent, parental strains to tetracycline or chloramphenicol resulted in elevated levels of mRNA which hybridized to the marA probe. These results indicate that the marA locus is needed for production of Mar mutants and is regulated, responding to at least two antibiotics to which it controls resistance.

One of the currently most relevant resistance mechanisms in Enterobacteriaceae is the production of enzymes that lead to modern expanded-spectrum cephalosporin and even carbapenem resistance, mainly extended-spectrum ␤-lactamases (ESBLs) and carbapenemases. A worrisome aspect is the spread of ESBL and carbapenemase producers into the environment. The aim of the present study was to assess the occurrence of ESBL-and carbapenemase-producing Enterobacteriaceae and to further characterize ESBL-and carbapenemase-producing Enterobacteriaceae in rivers and lakes in Switzerland. ESBL-producing Enterobacteriaceae were detected in 21 (36.2%) of the 58 bodies of water sampled. One river sample tested positive for a carbapenemase-producing Klebsiella pneumoniae subsp. pneumoniae strain. Seventy-four individual strains expressing an ESBL phenotype were isolated. Species identification revealed 60 Escherichia coli strains, seven Klebsiella pneumoniae subsp. pneumoniae strains, five Raoultella planticola strains, one Enterobacter cloacae strain, and one Enterobacter amnigenus strain. Three strains were identified as SHV-12 ESBL producers, and 71 strains carried genes encoding CTX-M ESBLs. Of the 71 strains with CTX-M ESBL genes, 8 isolates expressed CTX-M-1, three produced CTX-M-3, 46 produced CTX-M-15, three produced CTX-M-55, one produced CTX-M-79, six produced CTX-M-14, and four produced CTX-M-27. Three of the four CTX-M-27 producers belonged to the multiresistant pandemic sequence type E. coli B2:ST131 that is strongly associated with potentially severe infections in humans and animals.

A highly sensitive and specific method, termed PCR/NheI, for the detection of genes coding for SHV extended-spectrum beta-lactamases (ESBL) in clinical isolates is presented. It is based on polymerase chain reaction (PCR) amplification of the blaSHV genes, followed by restriction with NheI. Due to the glycine (positive 238) (SHV-non-ESBL)-->serine (position 238) (SHV-ESBL) mutation, only PCR fragments from the genes coding for SHV-ESBLs were cleaved. A commercially available test for ESBLs, the E test ESBL, identified 52% of our 29 clinical isolates carrying blaSHV-ESBL genes as ESBL producers.