Transfer of quinolone resistance gene qnrA1 to Escherichia coli through a 50 kb conjugative plasmid resulting from the splitting of a 300 kb plasmid.

OBJECTIVES: To analyse the in vitro transfer of the qnrA1 gene by a 50 kb (pSZ50) self-transferable plasmid that derives from a 300 kb plasmid (pSZ300) and to determine the complete nucleotide sequence of plasmid pSZ50. METHODS: Extended-spectrum ß-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes of an Escherichia coli clinical isolate were analysed. Plasmid analysis included conjugation and selection on seven antibiotics examined by antimicrobial susceptibility testing, RFLP comparison, Southern hybridization, incompatibility group identification and shotgun sequencing. RESULTS: The E. coli 5509 isolate carries the genes encoding the ESBL CTX-M-15 and the quinolone resistance determinants qnrA1, qnrB2 and aac(6')-Ib-cr on a 300 kb plasmid. Seven transfer resistances were analysed by conjugation under two conditions (30 and 37°C), leading to two distinct transconjugant phenotypes with different resistances. Transconjugants of phenotype A harboured a 300 kb plasmid named pSZ300 that conferred resistance to eight antibiotics and harboured the qnrA1, aac(6')-Ib-cr and bla(CTX-M-15) genes. Transconjugants of phenotype B were resistant to three antibiotics and they harboured the qnrA1 gene on an ≈ 50 kb plasmid named pSZ50. Both plasmids were self-transferable at a frequency of 1 × 10(-3). Plasmid pSZ300 was typed to be both an IncF and IncN plasmid, whereas pSZ50 corresponded only to type IncN. Fingerprinting and Southern hybridization showed that plasmid pSZ50 derived from pSZ300. The complete nucleotide sequence of plasmid pSZ50 was determined (51556 bp) and 55 open reading frames were predicted. The qnrA1 gene was identified in a tandem duplicate inside a sul1-type integron structure. CONCLUSIONS: The plasmid pSZ300 represented a fusion of two replicons (IncF and IncN), and our observations suggest that the plasmid pSZ50 (IncN) may split and transfer antibiotic resistance determinants. This mechanism could be advantageous in the dissemination of antibiotic resistance genes.

[Molecular study of methicillin-resistant Staphylococcus haemolyticus in a Mexican hospital]. / Estudio molecular de Staphylococcus haemolyticus resistente a meticilina en un hospital de México.

OBJECTIVE: To perform the molecular characterization of methicillin-resistant Staphylococcus haemolyticus (MRSH) clinical isolates from patients in a Mexican hospital. METHODS: Sixty three Staphylococcus ssp. isolates collected from September 2000 to October 2002 were analyzed. Antimicrobial susceptibility was determined by disk diffusion method and the presence of the mecA gene was detected by PCR technique. Isolates characterization was carried out by pulsed field gel electrophoresis (PFGE). RESULTS: The frequency of S. haemolyticus was 25.5% (18 of 63 clinical isolates), all S. haemolyticus isolates were methicillin-resistant and they were positive for the mecA gene. A major pattern (A) with 8 subtypes was identified. This clone was distributed during the 20 months period. Most of them were isolated from the surgery (55%) and pediatric services (27.5%). CONCLUSION: The methicillin-resistant S. haemolyticus permanence as pathogen in this hospital, suggest the implementation of control programs in order to decrease the prevalence of this multiresistant pathogen.

Clonal and horizontal dissemination of Klebsiella pneumoniae expressing SHV-5 extended-spectrum beta-lactamase in a Mexican pediatric hospital.

One hundred eighty-four clinical isolates of Klebsiella pneumoniae were recovered from August 1996 to October 1997 at the Pediatric Hospital of the Instituto Mexicano del Seguro Social in Mexico City, Mexico. Most of the isolates were collected from the neonatal intensive care unit and infant wards, which are located on the same floor of the hospital. Isolates were genotypically compared by pulsed-field gel electrophoresis with XbaI restriction of chromosomal DNA. Of 184 clinical isolates, 91 belonged to cluster A and comprised three subtypes (A1, A2, and A3), while 93 isolates, comprising two minor clones, B (10 isolates) and C (7 isolates), and 76 unique patterns, were considered unrelated isolates (URI). Susceptibility patterns were indistinguishable in both groups. Fifty extended-spectrum beta-lactamase-producing isolates, including 34 from clone A and 16 from URI, were examined for further studies. Molecular and genetic analysis showed that 47 of 50 clinical isolates expressed the SHV-5 beta-lactamase. This enzyme, in combination with TEM-1, was encoded in a >or=170-kb conjugative plasmid. Results indicate that dissemination of this resistance was due to clonal and horizontal spread.