Karen E. Preston

Nucleotide Sequence of the Chromosomal ampC Gene of Enterobacter aerogenes

ABSTRACT The AmpC β-lactamase gene and a small portion of the regulatoryampR sequence of Enterobacter aerogenes 97B were cloned and sequenced. The β-lactamase had an isoelectric point of 8 and conferred cephalosporin and cephamycin resistance on the host. The sequence of the cloned gene is most closely related to those of theampC genes of E. cloacae and C. freundii.

Relatedness of coagulase-negative staphylococci causing bacteremia in low-birthweight infants.

OBJECTIVE To investigate coagulase-negative staphylococcus (CONS) causing bacteremia in a neonatal intensive care unit (NICU). DESIGN A 14-month retrospective review of 47 infants in the NICU with CONS bacteremia was undertaken to determine CONS glycocalyx production, plasmid pattern, total DNA restriction fragment polymorphism, and clinical risk factors. RESULTS The isolates included 32 Staphylococcus epidermidis, six Staphylococcus haemolyticus, four Staphylococcus warneri, four Staphylococcus saprophyticus, and one Staphylococcus hominis. Sixty-five percent of S epidermidis produced glycocalyx; other species did not. Oxacillin resistance (52%) and the antibiograms of the CONS were consistent with other units in the hospital. Five similar CONS plasmid patterns were found among 16 isolates; 31 isolates had unique patterns. Extractions of total DNA from these isolates were digested using HindIII, HaeIII, and BstEII. Those with similar restriction fragment length patterns could not linked as nosocomially transmitted among infants with bacteremia. CONCLUSION Our observations suggest that multiple strains of CONS infect infants in the NICU who have similar risk factors. Although current infection control practices limit transmission of a pathogen, they do not prevent CONS bacteremias.

Survey of Plasmid-Associated Genetic Markers in Enterobacteriaceae with Reduced Susceptibilities to Cephalosporins

ABSTRACT Clinical isolates of Enterobacteriaceae with reduced susceptibilities to cephalosporins were collected from 1993 to 2000. The organisms were screened for the extended-spectrum β-lactamase (ESBL) phenotype, and plasmid extracts were screened for genetic markers by hybridization. A blaTEM 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 blaSHV, int, aac(3)-Ia, dfrA1, IS6100, tetA, IncM markers, and Anon 13, a marker for the Klebsiella pneumoniae chromosomal sequences that flank blaSHV-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 β-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.

Increased oxacillin activity associated with glycopeptides in coagulase-negative staphylococci

Vancomycin resistance in methicillin-resistant staphylococci presents a potential therapeutic problem. In order to understand the impact of low-level vancomycin resistance in coagulase-negative Staphylococci, stepwise selection of vancomycin resistance was accomplished by growingstaphylococcus haemolyticus in culture media with increasing concentrations of vancomycin. A >40-fold increase in susceptibility to β-lactam antibiotics was observed. No obvious alterations in the growth curve, the presence of themecA gene, total DNA restriction fragment length polymorphism (RFLP), β-lactamase production, or the crude protein fraction were detected in thestaphylococcus haemolyticus-derived clones when compared to the original isolate. The proportion of the oxacillin-heteroresistant population also remained similar. A comparable phenomenon occurred with the selection ofstaphylococcus epidermidis exhibiting low-level resistance to vancomycin. Additionally, it was observed that clinical isolates of coagulase-negative Staphylococci grown in the presence of sub-minimum inhibitory concentrations of either vancomycin or teicoplanin lost their high-level resistance to oxacillin. Checkerboard tests showed that the combination of vancomycin and oxacillin was synergistic for two isolates ofStaphylococcus haemolyticus, two of four isolates ofStaphylococcus epidermidis, and one isolate ofStaphylococcus hominis.

Recurrent bacterial peritonitis caused by Neisseria cinerea in a chronic ambulatory peritoneal dialysis (CAPD) patient

We present an unusual case of recurrent (chronic ambulatory peritoneal dialysis) CAPD-associated peritonitis caused by Neisseria cinerea. Using DNA restriction fragment length polymorphism (RFLP) analysis, we determined that the recurrent infection was caused by reinfection with a different N. cinerea strain rather than relapse with the index strain and that the probable origin of the reinfecting organism was the patients upper respiratory tract.

Chromosomal sequences from Klebsiella pneumoniae flank the SHV-5 extended-spectrum β-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.