Plasmid Pulsed-field Gel Electrophoresis Research Articles

Genetic variation in the conjugative plasmidome of a hospital effluent multidrug resistant Escherichia coli strain

Bacteria harboring conjugative plasmids have the potential for spreading antibiotic resistance through horizontal gene transfer. It is described that the selection and dissemination of antibiotic resistance is enhanced by stressors, like metals or antibiotics, which can occur as environmental contaminants. This study aimed at unveiling the composition of the conjugative plasmidome of a hospital effluent multidrug resistant Escherichia coli strain (H1FC54) under different mating conditions. To meet this objective, plasmid pulsed field gel electrophoresis, optical mapping analyses and DNA sequencing were used in combination with phenotype analysis. Strain H1FC54 was observed to harbor five plasmids, three of which were conjugative and two of these, pH1FC54_330 and pH1FC54_140, contained metal and antibiotic resistance genes. Transconjugants obtained in the absence or presence of tellurite (0.5 μM or 5 μM), arsenite (0.5 μM, 5 μM or 15 μM) or ceftazidime (10 mg/L) and selected in the presence of sodium azide (100 mg/L) and tetracycline (16 mg/L) presented distinct phenotypes, associated with the acquisition of different plasmid combinations, including two co-integrate plasmids, of 310 kbp and 517 kbp. The variable composition of the conjugative plasmidome, the formation of co-integrates during conjugation, as well as the transfer of non-transferable plasmids via co-integration, and the possible association between antibiotic, arsenite and tellurite tolerance was demonstrated. These evidences bring interesting insights into the comprehension of the molecular and physiological mechanisms that underlie antibiotic resistance propagation in the environment.

Identification and Characterization of CTX-M-Producing Shigella Isolates in the United States

Shigellosis is a major source of gastroenteritis throughout the world (14). Extended-spectrum β-lactamases (ESBLs), including cefotaximases (CTX-M), confer resistance to extended-spectrum cephalosporins and significantly compromise the treatment options for shigellosis. Numerous ESBLs have been described among Enterobacteriaceae (2, 8, 13); however, only a single CTX-M-producing Shigella isolate has been reported in the United States (10). From 1999 to 2007, 3,880 Shigella isolates were screened for antimicrobial susceptibility to 14 to 17 antimicrobials by broth microdilution (Sensititre; Trek Diagnostics, Westlake, OH). Six isolates displayed decreased susceptibility (MIC ≥ 2 mg/liter) to ceftriaxone (Table ​(Table1).1). The six case-patients included three males and two females (gender information was unavailable for one patient), and the median age was 3 years (range, 1 to 8 years). Additional details were available for five patients. Three of the five (60%) were hospitalized, and one was admitted twice. One patient had an adopted sibling from Russia but had not traveled herself. The second patient traveled to a neighboring state prior to illness onset, and the third reported no travel. Of the nonhospitalized patients, one was an asymptomatic adoptee from China and the second reported no travel. Two patients received antimicrobial therapy: ceftriaxone, cefotaxime, and trimethoprim-sulfamethoxazole for one patient, azithromycin for the other patient. TABLE 1. Characterization of CTX-M-positive Shigella isolates, transformants, and CTX-M-encoding plasmidsa PCR analysis was used to screen the six isolates for 13 different classes or groups of bla genes, and PCR results were confirmed by DNA sequencing (1, 5, 11, 12, 16, 18-21). Four isolates were positive for the blaCTX-M-15 gene, while two were positive for the blaCTX-M-14 gene (Table ​(Table1).1). All four blaCTX-M-15 isolates were PCR positive for non-ESBL blaTEM-1 genes. Both blaCTX-M-14 isolates were PCR positive for non-ESBL blaOXA-1 genes, and a single isolate was positive for both blaTEM-1 and blaOXA-1. By pulsed-field gel electrophoresis (PFGE) analysis, all three S. sonnei and all three S. flexneri isolates demonstrated distinct patterns (data not shown) (15). All six blaCTX-M genes were determined to be plasmid encoded (6). The non-ESBL β-lactamases (OXA-1, TEM-1) did not transfer and were not encoded on the same CTX-M plasmids. All three S. sonnei plasmids and two of the S. flexneri plasmids harbored only the CTX-M-associated resistance. The remaining S. flexneri plasmid contained additional determinants conferring resistance to trimethoprim-sulfamethoxazole and gentamicin. All three S. sonnei plasmids were incompatibility type IncI1 and approximately 90 kb in size (plasmid pulsed-field gel electrophoresis) (Table ​(Table1)1) (4). Plasmid multilocus sequence typing (pMLST) identified them as novel sequence types designated as ST31 complex. The plasmid from AM22451 contained several point mutations in one allele, necessitating the ST32 designation within the ST31 clonal complex (http://pubmlst.org/plasmid) (7). Of the three S. flexneri plasmids, the blaCTX-M-15-positive plasmid was a 165-kb IncA/C plasmid, while the two blaCTX-M-14-positive plasmids were identical 75-kb IncFII plasmids. CTX-M-14 and CTX-M-15 are the most common types of cefotaximases identified among Shigella isolates (9, 17, 22), and IncI1 plasmids carrying CTX-M-15 have been already described in Escherichia coli and Salmonella isolates from Australia, France, and the United Kingdom (3). The emergence of CTX-M-producing Shigella isolates in the United States is concerning and necessitates continued resistance surveillance.