Acquisition of a second multi-drug resistance-encoding element by IncM1 plasmid pACM130 abolished conjugative transfer.

Within the IncM plasmid family there is a lineage that has a transposon Tn1721-based multiple-resistance island inserted in the backbone gene mucB. So far, this group includes R1215, p202c, pIGT15, pARM26, and pACM1, from Europe and the USA. A new member of this group, pACM130, was isolated at the same American hospital as pACM1 and has a similar resistance island, but also carries a copy of Tn1331 that interrupts the traY gene in the conjugation operon. The conjugative phenotype of this plasmid has been abolished, though pACM130 could be mobilized by an intact traY cloned into a laboratory vector and transformed into the same donor bacterium.

The complete nucleotide sequence of the multi-drug resistance-encoding IncL/M plasmid pACM1.

The 89,977 bp nucleotide sequence of pACM1, isolated from a 1993 outbreak strain of cephalosporin-resistant Klebsiella oxytoca, has been completed and assigned GenBank accession number KJ541681. The plasmid has a single 31,842 bp mosaic multi-drug resistance-encoding (MDR) region comprising the mer resistance module of Tn1696, two integrons with a total of seven cassettes, one complete copy each of IS1R and IS26, and the bla(SHV-5)-carrying Tn2003 (with defective IS26 termini), all within a Tn1721-like element inserted into the mucB gene of the IncL/M plasmid backbone. The Tn1721-Tn1696 combination resembles sequence found in the chromosomal MDR islands of some Acinetobacter baumannii isolates. Among the completely sequenced IncL/M resistance plasmids, the Tn1721-based MDR region is unique, but data from older studies suggest that this type of plasmid was widespread in the 1990s. Since resistance gene dosage is affected by plasmid copy number (PCN), we used a relatively simple new "efficiency-corrected" qPCR assay to measure the PCN of pACM1. There are approximately three copies per chromosome in an Escherichia coli DH5α host, and two in the original Klebsiella oxytoca isolate. We could not find similar PCN data for other medically important plasmids for comparison. The study of this plasmid property and its effect on resistance levels should be facilitated in the future by the availability of qPCR instruments and complete genome sequences.

Risk factors for surgical site infection after cardiac surgery: the role of endogenous flora.

OBJECTIVE: The study's objective was to assess predictors of surgical site infection (SSI) after cardiac surgery and the relationship of perioperative nasal carriage of Staphylococcus species with the development of SSI. METHODS: Surveillance for infections was performed, and anterior nares cultures of patients who underwent cardiac surgery were obtained. Preoperative risk factors were analyzed, and staphylococcal isolates from nares and SSI were compared using pulsed-field gel electrophoresis. RESULTS: Twelve patients had 14 SSIs (5.7 infections/100 surgeries). Two risk factors were significantly associated with SSI: smoking (P = .002, confidence interval(95) 1.1-1.4, relative risk = 1.3) and increased body mass index (P = .003, confidence interval(95) 2.8-99.8, relative risk = 16.8). A total of 5 of 8 infected patients (62.5%) for whom nares cultures were available had identical strains in their nares and SSI. CONCLUSION: Smoking and body mass index were predictors of SSI. Approximately 2 of 3 infected patients for whom nares cultures were obtained had an SSI that was likely from an endogenous source.

Risk factors associated with extended-spectrum beta-lactamase-producing organisms at a tertiary care hospital.

BACKGROUND: In 1995, beta-lactam inhibitor combinations replaced third-generation cephalosporins as empirical therapy in an effort to manage extended-spectrum beta-lactamase (ESBL) resistance. This study investigated the relationship between antibiotic usage and ESBL organisms from 1994 through 2002 using epidemiological and molecular analysis. METHODS: A case-control study of 119 patients with ESBL organisms and 132 patients with non-ESBL organisms was conducted. Demographics, co-morbidities, device utilization and antibiotic use were analysed for all patients and infected patients only (cases = 75, controls = 83). Both exposure and degree of exposure (in grams) to antibiotics were included. A dot blot hybridization technique was used to identify genes in plasmid extracts from the ESBL organisms. RESULTS: Ventilator days OR 1.1 (1.06, 1.15) P < 0.001, adult respiratory distress syndrome (ARDS) OR 3.1 (1.0, 9.7) P = 0.05, prior aminoglycoside use OR 2.7 (1.2, 6.1) P = 0.02, prior third-generation cephalosporin use OR 7.2 (2.6, 20) P < 0.001, and prior trimethoprim/sulfamethoxazole use OR 8.8 (3.1, 26) P < 0.001 were significantly associated with ESBL organisms by multivariate analysis. All models were concordant with a significant association of ventilator days, third-generation cephalosporins and trimethoprim/sulfamethoxazole with ESBL organisms. beta-Lactamase inhibitor combinations were not associated with ESBL organisms. Hybridization of plasmid extracts demonstrated that 95% of the ESBL organisms carried intI1, a mobile DNA element with a sulphonamide-resistance (R) gene and a frequent carrier of other R factors. Genes for specific types of trimethoprim-R and aminoglycoside-R were present in 26% and 40% of the extracts, respectively. CONCLUSIONS: These data indicate that, besides patient risk factors and third-generation cephalosporins, other antibiotics may provide selective pressures in maintaining ESBL organisms due to multiple resistance genes on plasmids. beta-Lactamase inhibitor combinations appear to be an acceptable substitute to third-generation cephalosporins in strategies to control ESBL organisms.

Chromosomal sequences from Klebsiella pneumoniae flank the SHV-5 extended-spectrum beta-lactamase gene in pACM1.

The nucleotide sequence was determined for Anon 13, a 1250-bp SmaI fragment located approximately 2.8 kb downstream from bla(SHV-5) in pACM1. Anon 13 is 99% identical to a segment of the unpublished sequence of the Klebsiella pneumoniae chromosome. Genes of the K. pneumoniae sequence are undefined, but conceptual amino acid translations of two ORFs in Anon 13 are homologous to L-fuculose-1-phosphate aldolase (FucA) and a conserved hypothetical protein present in the chromosomes of several species of bacteria. In addition, restriction mapping indicates that the region of homology between the K. pneumoniae chromosome and pACM1 is as least 7.9 kb and includes both Anon 13 and bla(SHV). These observations demonstrate the chromosomal origin of the bla(SHV-5) on pACM1.

The SHV-5 extended-spectrum beta-lactamase gene of pACM1 is located on the remnant of a compound transposon.

The SHV-5 extended-spectrum beta-lactamase gene of pACM1 was previously shown to reside on a segment of DNA ( approximately 7.9 kb) homologous to part of the Klebsiella pneumoniae chromosome. Regions of pACM1 overlapping the ends of the homology were sequenced. A defective copy of IS26 was found on each side of, and immediately adjacent to, the homology. The copies were oriented as direct repeats reminiscent of the compound transposon Tn2680. Other mobile elements and a putative mutagenesis gene, several of which were also defective, were also located in the vicinity of the homology. An intact precursor to the transposon remnant might have contributed to the dissemination of the SHV-5 gene.

Survey of plasmid-associated genetic markers in enterobacteriaceae with reduced susceptibilities to cephalosporins.

Clinical isolates of Enterobacteriaceae with reduced susceptibilities to cephalosporins were collected from 1993 to 2000. The organisms were screened for the extended-spectrum beta-lactamase (ESBL) phenotype, and plasmid extracts were screened for genetic markers by hybridization. A bla(TEM) probe was derived from pUC19; other probes were derived from pACM1, the plasmid responsible for the first known appearance of an ESBL in our institution. These probes included bla(SHV), int, aac(3)-Ia, dfrA1, IS6100, tetA, IncM markers, and Anon 13, a marker for the Klebsiella pneumoniae chromosomal sequences that flank bla(SHV-5). There were 42 hybridization patterns among 237 isolates. Patterns designated pACM1-like occurred in 44% of the isolates (eight species) and were always associated with the clavulanic acid (CA)-susceptible ESBL phenotype. The TEM marker was not predictive of the ESBL phenotype. Mapping indicated the presence of an SHV marker and up to 7.5 kb of its flanking chromosomal sequences in three non-IncM plasmids obtained in transformation experiments. We theorize that this DNA segment spread to other plasmids from pACM1-like sources. CA insensitivity became more frequent with time and was usually associated with either the TEM marker or the absence of both bla markers. One plasmid-encoded enzyme with characteristics of an AmpC beta-lactamase was observed in a transformant lacking both TEM and SHV markers. Although SHV type ESBLs were a continuing source of reduced susceptibility to cephalosporins in our institution, organisms with different resistance mechanisms were added to the hospital microflora in later years. These changes might be related, in part, to ESBL control strategies implemented in 1995.