Abundance and phenotypic diversity of Escherichia coli isolates with diminished susceptibility to expanded-spectrum cephalosporins in faeces from healthy food animals after slaughter.

Antimicrobial resistance (AR) is an increasing phenomenon but its quantitative estimation remains controversial. The classical resistance percentage approach is not well suited to detect either emergence or low levels resistance. One option is to shift the focus from strains to hosts. This approach is applied to test for phenotypic diversity associated with diminished susceptibility to expanded-spectrum cephalosporins (DSESC) in faecal Escherichia coli from healthy food animals in Spain. We performed E. coli enumeration in faecal samples of broilers (82 pooled samples) and pigs (80 pooled samples) at the slaughterhouse level, using Coli-ID plates alone and supplemented with cefotaxime at two levels (1 and 8 microg/ml). Antimicrobial susceptibility of isolates was tested by the agar diffusion method. Clustering was carried out using these numerical values and Ward and UPGMA methods. When using plates supplemented with 1 microg/ml of cefotaxime for DSESC E. coli detection, 93% (76/82) of broiler pooled samples and 36% (29/80) pig pooled samples tested positive. When using 8 microg/ml of cefotaxime, 67% (55/82) of broilers and 13% (10/80) of pigs were positive. Nevertheless, the relative abundance of this phenotype was low in both animal species (range 0-4.3%). Irrespective of the clustering method (Ward or UPGMA), a noticeable phenotypic diversity was detected, especially from the plates containing 1 microg/ml of cefotaxime. We concluded that: (a) E. coli with phenotype DSESC are common in broilers and pigs but are less frequent in pigs, and (b) the host approach is the most appropriate method for antimicrobial resistance assessment when null or very low levels of antimicrobial resistant bacteria are expected.

Beta-lactamase characterization in Escherichia coli isolates with diminished susceptibility or resistance to extended-spectrum cephalosporins recovered from sick animals in Spain.

A total of 1439 Escherichia coli isolates from sick animals were received from the Spanish Network of Veterinary Antimicrobial Resistance Surveillance (VAV) from 1997 to 2001. Antimicrobial susceptibility tests were performed and diminished susceptibility to cefotaxime and ceftazidime was identified in 2.5% and 2.8% of the isolates, respectively. Beta-lactamase characterization was carried out in the group of 20 E. coli isolates with both characteristics. The MIC ranges of different beta-lactams showed by these 20 isolates were as follows (in microg/ml): ampicillin (64-->256), amoxicillin-clavulanic acid (4-64), ticarcillin (8-->128), cefazolin (32-->256), cefoxitin (4-->128), cefotaxime (1-64), ceftazidime (2-->64), ceftriaxone (0.5-64), imipenem (< or = 0.06-0.25), and aztreonam (2-->32). TEM, SHV, CMY, and FOX beta-lactamase genes were analyzed by PCR and sequencing. The beta-lactamase genes detected were the following ones (number of isolates): bla(TEM-1b) (3), bla(TEM-1a) (1), bla(TEM-30f) (2), bla(TEM-1b) + bla(CMY-2) (2), and bla(SHV-12) (1). Sequences of the promoter and/or attenuator region of the chromosomal ampC gene were studied in all the 20 isolates. Mutations at position -42 or -32 were detected in 16 isolates and these mutations were associated with the presence of a TEM type beta-lactamase in 6 isolates. Besides, a high variety of plasmidic beta-lactamases was detected including TEM-30 and CMY-2. To our knowledge, this is the first time that TEM-30 beta-lactamase has been detected in E. coli isolates of animal origin.

Detection and characterization of extended-spectrum beta-lactamases in Salmonella enterica strains of healthy food animals in Spain.

OBJECTIVES: To carry out the characterization of the genes encoding extended-spectrum beta-lactamases (ESBLs) and their genetic environments in four expanded-spectrum cephalosporin-resistant Salmonella enterica isolates (serovars: two Virchow, one Enteritidis, one Rissen) recovered during the monitoring programmes performed in Spain by the VAV Network from faecal samples of pigs, poultry and laying hens at the slaughterhouse level. METHODS: The presence and characterization of ESBL genes as well as their genetic environments in the four S. enterica isolates were investigated by PCR and sequencing. The presence of other resistance genes was also analysed by PCR and sequencing. RESULTS: Three avian S. enterica isolates (two Virchow and one Enteritidis) harboured the bla(CTX-M-9) gene combined with bla(TEM-1b). The bla(CTX-M-9) gene was included in these three isolates in a class 1 integron with the following 5'-->3' structure: integron 1 variable region (dfrA16-aadA2 gene cassettes)-qacEDelta1-sul1-orf513-bla(CTX-M-9)-orf3-like-orf1005. The sul2 gene was also detected in these three bla(CTX-M-9)-containing isolates and tet(A) in one of them. The two serovar Virchow isolates showed an indistinguishable PFGE pattern, although they were recovered from different animal species (broiler and laying hen). A porcine ESBL-positive isolate (serovar Rissen) harboured the bla(SHV-12) gene combined with bla(TEM-1b). This bla(SHV-12)-containing isolate also harboured the tet(A), aadA and sul1 genes. CONCLUSIONS: The emergence of ESBL-producing S. enterica isolates among food animals is described for the first time in Spain, with those of the CTX-M group being the predominant ESBLs detected.

Genetic basis for dissemination of armA.

OBJECTIVES AND METHODS: armA is a novel plasmid-borne 16S rRNA methyltransferase that confers high-level resistance to 4,6-disubstituted deoxystreptamines. Recently, we have isolated from a high-level broad-spectrum aminoglycoside-resistant Escherichia coli animal isolate a plasmid, pMUR050, that bore the armA gene. In order to elucidate the genetic basis for the spread of armA, we have determined the complete nucleotide sequence of pMUR050. RESULTS: armA was borne by a complex transposon composite flanked by two direct repeats of IS26. The transposon composite included a class one integron with sul1 for resistance to sulphonamides and ant3''9 conferring resistance to spectinomycin-streptomycin, and a macrolide efflux pump and mefE/mel conferring high-level resistance to erythromycin. We identified in GenBank that another plasmid, pCTX-M3, from a Polish Citrobacter freundii human isolate, bore the same genetic structure, including armA. CONCLUSIONS: armA is present in human and animal isolates within a novel transposon composite. Further spread of armA between bacteria of diverse origin is to be expected.

armA and aminoglycoside resistance in Escherichia coli.

We report armA in an Escherichia coli pig isolate from Spain. The resistance gene was borne by self-transferable IncN plasmid pMUR050. Molecular analysis of the plasmid and of the armA locus confirmed the spread of this resistance determinant.

Monitoring and characterization of extended-spectrum beta-lactamases in Escherichia coli strains from healthy and sick animals in Spain in 2003.

Genes encoding CTX-M-14, CTX-M-9, CTX-M-1, CTX-M-32, SHV-12, TEM-52, or CMY-2 beta-lactamases were detected in 21 Escherichia coli strains recovered during 2003 from sick animals (11 of 459 [2.4%] strains) and healthy animals (10 of 158 [6.3%] strains) in Spain. Twelve of these strains harbored bla(CTX-M) genes and showed unrelated pulsed-field gel electrophoresis patterns.