om as , F e r na n d o G on zález Candelas, SeqCOVID-SPAIN consortium, Tanja Stadler & Richard A. NeherThis is a PDF file of a peer-reviewed paper that has been accepted for publication. Although unedited, the content has been subjected to preliminary formatting. Nature is providing this early version of the typeset paper as a service to our authors and readers. The text and figures will undergo copyediting and a proof review before the paper is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

This widespread occurrence of ESBL-producing Enterobacteriaceae suggests that the community could act as a reservoir and that food could contribute to the spread of these strains.

No abstract

In this study, we investigated the population structure of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in Spain and determined possible associations between specific multilocus sequence typing (MLST) types and ESBL types. Ninety-two ESBL-producing E. coli isolates from 11 Spanish hospitals were studied. The predominant ESBLs in this collection were CTX-M-14 (45.7%), SHV-12 (21.7%) and CTX-M-9 (20.6%). Phylogenetic groups and MLST types were studied. Thirty-seven isolates (40.2%) belonged to phylogroup A, 26 (28.3%) to group B1, 13 (14.1%) to group B2 and 16 (17.4%) to group D. Fifty-six sequence types (STs) were identified, but ST131 (eight isolates) and ST167 (five isolates) were the most prevalent. The most common ST complexes were ST10 (13 isolates; 14.3%) and ST23 (10 isolates; 11%). Escherichia coli ST131 carried six different ESBLs (CTX-M-1, CTX-M-9, CTX-M-10, CTX-M-14, CTX-M-15 and SHV-12), E. coli ST10 complex carried five ESBLs and E. coli ST23 complex carried four ESBLs. A great diversity of MLST types was observed among Spanish ESBL-producing E. coli isolates.

A new CTX-M-type ␤-lactamase (CTX-M-9) has been cloned from a clinical cefotaxime-resistant Escherichia coli strain. Despite the close identity that exists between the CTX-M-9 and Toho-2 ␤-lactamases (88%), the 35 amino acids located between residues Ala-185 and Ala-219 are totally different in both enzymes. Outside of this region there are only six amino acids substitutions between both proteins.Not long after the beginning of the use of the extendedspectrum ␤-lactam antibiotics, extended-spectrum ␤-lactamases (ESBLs) were detected in Europe and the United States and have now become a serious problem around the world. ESBLs are most often derivatives of TEM or SHV enzymes. However, there is a small growing family of plasmid-mediated ESBLs of Ambler class A that are not closely related to TEM or SHV ␤-lactamases but that show homology to chromosomal ␤-lactamases of Klebsiella oxytoca, including CTX-M-1 (MEN-1) (3, 5-7), CTX-M-2 (4, 6), CTX-M-3 (14), CTX-M-4 (12, 13), 20), Toho-1 (15), Toho-2 (17), and two different enzymes, both designated CTX-M-5, which will be referred to here as CTX-M-5 (8) and 20). In this report we present a new ␤-lactamase closely related to the ␤-lactamases in this family.In 1996, a cefotaxime-resistant Escherichia coli strain (785-D) against which synergy of cefotaxime with clavulanic acid was found was detected by a conventional disk diffusion susceptibility test. The strain was isolated from the urine of a 65-year-old woman who had a urinary tract infection and diabetes mellitus type 2. Two months before the isolation, the patient underwent a nephroureterectomy because of a renal carcinoma.The MICs of the ␤-lactam antibiotics were determined by the Etest (Biodisk, Solna, Sweden). The ␤-lactamase crude cell extracts were prepared from 250-ml cultures in Luria-Bertani (LB) broth (Oxoid, Basingstoke, United Kingdom). Washed, centrifuged cell pellets were subjected to three cycles of 15-s sonication treatments at 4°C, and the supernatants of the sonic extracts were frozen at Ϫ20°C until they were tested. ␤-Lactamases were characterized initially by isoelectric focusing as described previously (2) in polyacrylamide gels with a pH gradient from 4 to 11 (SERVALYT 4-9 T, 9-11 T; Serva, Heidelberg, Germany). Enzyme activities in the gel were detected by the iodometric method (2).Substrate hydrolysis in sonic extracts was monitored spectrophotometrically with a Biochrom 4060 spectrophotometer (Pharmacia, Uppsala, Sweden) as described previously (19).Conjugation studies were performed in a solid medium as described previously (19) by using E. coli 785-D (susceptible to kanamycin) and E. coli HB101 (Nal r Kan r ) as the donor and the recipient, respectively. Kanamycin (50 g/ml) and cefotaxime (4 g/ml) were used for transconjugant selection. One of the transconjugants (E. coli MSP492) was used for further experiments.Extraction of plasmid DNA was by the alkaline lysis procedure reported previously (18). Cloning of the cefotaxime resistance gene was as follows: plasmid DNA from E. coli MSP492 was pa...