β-Lactam Resistance and Enterobacteriaceae, United States

Extended-spectrum cephalosporins (ESC) are an important drug class for treating severe Salmonella infections. We screened the human collection from the National Antimicrobial Resistance Monitoring System 2000 for ESC resistance mechanisms. Of non-Typhi Salmonella tested, 3.2% (44/1,378) contained blaCMY genes. Novel findings included blaCMY-positive Escherichia coli O157:H7 and a blaSHV-positive Salmonella isolate. CMY-positive isolates showed a ceftriaxone MIC >2 µg/mL.


The Study
As NARMS participants, 17 state and local public health laboratories representing 40% of the US population submitted every tenth non-Typhi Salmonella isolate, every tenth Shigella isolate, and every fifth E. coli O157 isolate received in 2000 to CDC for antimicrobial susceptibility testing. Identification and serotyping were conducted at submitting laboratories. The MIC was determined for 17 antimicrobial agents at CDC by using partial range broth microdilution (Sensititre, Westlake, OH, USA). Isolates were chosen for further study based on the following MIC criteria: cefoxitin (>16 µg/mL), ceftiofur (>4 µg/mL), or ceftriaxone (>16 µg/mL). Isoelectric Focusing (IEF) for β β-Lactamases β-Lactamase content was determined for all isolates that met the MIC criteria for further study. The IEF methods of Rasheed et al. (6) were used with modification. Crude cellular protein extracts were prepared by pelleting 3-hour trypticase soy broth cultures (grown at 37°C with shaking at 300 rpm), resuspending in 0.2% sodium acetate at 5% of original culture volume, and freeze-thawing 4 times in a dry ice/ethanol bath and a 37°C water bath. Preparations were then diluted twofold with distilled water and placed on ice for 30 min with occasional swirling. The supernatant was collected after centrifuging for 30 min at maximum relative centrifugal force (14,000 rpm) in a Beckman 5417R microcentrifuge (Palo Alto, CA, USA). Three-to 5-µL aliquots of each preparation were resolved by focusing for 1.5 h on an Ampholine PAGplate polyacrylamide gel, pH range 3.5-9.5 (APBiotech, Piscataway, NJ, USA), according to manufacturer's instructions. Gels were stained by overlaying with a 500 µg/mL solution of nitrocefin (Becton Dickinson, Franklin Lakes, NJ, USA). Isoelectric points were estimated by comparison with the following standard β-lactamases: TEM-12 (pI 5.25), SHV-3 (pI 7.0) and MIR-1 (pI 8.4).

Polymerase Chain Reaction (PCR) for β β-Lactamase Genes
For isolates that were IEF-positive for a β-lactamase with a pI >8.4, amplification of bla CMY genes was attempted. Internal primers were used to amplify a 369-bp portion of bla CMY genes from crude colony lysates. The forward primer anneals to nucleotide (nt) 271-289 of the 1,146-nt bla CMY-2 sequence from Klebsiella pneumoniae (NCBI accession no. X91840) and has a sequence of 5′-GGCGT-GTTGGGCGGCGATG-3′. The reverse primer anneals to nt 621-639 of bla CMY-2 and has a sequence of 5′-CAGCG-GAACCGTAATCCAG-3′. APBiotech Ready-to-Go Beads (Piscataway, NJ, USA) were used to formulate 25-µL reactions, which were run in an MJResearch thermocycler (Waltham, MA, USA) under the following conditions: 1 cycle of 94°C for 5 min followed by 25 cycles of: 94°C for 30 s, 59°C for 1 min, 72°C for 1 min. Amplicons were resolved by electrophoresis in 1% agarose gels. For isolates exhibiting a β-lactamase with a pI = 8.0, bla SHV genes were amplified using primers 4 and 5 from Rasheed et al. (7) with Perkin-Elmer Amplitaq Gold 2X Master Mix (Boston, MA, USA).
The 7 S. sonnei isolates included in the study met only the cefoxitin MIC criterion (>16 µg/mL). All 7 showed a ceftiofur MIC 1.0 µg/mL or less, and a ceftriaxone MIC <0.25 µg/mL. Six of these were also resistant to ampicillin, amoxicillin-clavulanate, and cephalothin. Each isolate was IEF-positive for a β-lactamase enzyme with a pI>8.4, but was polymerase chain reaction-negative for a bla CMY gene. We suspect the resistance is related to overproduction of chromosomal ampC genes known to be present in Shigella species (8); however, porin deficiency (9) and penicillin-binding protein changes (10) are worth exploration as well. Efflux mechanisms (11,12) are possible, but multidrug-resistance pumps might be less likely since none of the 7 isolates were resistant to chloramphenicol, nalidixic acid, or ciprofloxacin, and only 4 were resistant to tetracycline.
Eight isolates (5 Salmonella and 3 S. sonnei) produced putative TEM enzymes in addition to an enzyme with a pI>8.4. The pI in each case was 5.3 or 5.4. Plasmidborne bla TEM-1 enzymes have been identified in several Salmonella serotypes (13).
Twenty-seven (61%) of 44 bla CMY -containing Salmonella in 2000 were serotype Newport. This finding coincides with emergence of a multidrug-resistant strain of S. Newport called MDRAmpC (14). MDRAmpC increased from 1% (1/77) of S. Newport isolates tested by NARMS in 1998 to 22% (27/124) in 2000 (15). In addition, bla CMY genes were found in 5 other Salmonella serotypes (Typhimurium, Heidelberg, Agona, Infantis, and Reading) in 2000. This contrasts with 1996-1998, when bla CMY was found in 3 serotypes (Newport, Typhimurium, and Thompson), which indicated that these genes or the mobile elements that house them have been disseminated. Furthermore, bla CMY genes were identified in each of the 4 E. coli O157:H7 isolates that met the MIC criteria in 2000. To our knowledge, this is the first report of bla CMY in this E. coli serotype.
NARMS sampling in 2000 showed that bla CMY genes continue to be the major mechanism of extended-spectrum cephalosporin resistance among non-Typhi Salmonella; other mechanisms of ESC are rare. The increasing diversity of bla CMY -positive Salmonella serotypes and the discovery of bla CMY genes in E. coli O157:H7 highlight the mobility of these mechanisms. This finding is not unexpected since these genes have been shown to be present on large plasmids, some of which are transferable by conjugation (4). Since S. Newport MDRAmpC and E. coli O157:H7 have been associated with bovine reservoirs, we hypothesize that bla CMY genes may be circulating among cattle. This remains to be proven and warrants more intensive study of bla CMY prevalence and movement in bovine production settings. Further research is also necessary to determine factors that contribute to dissemination of the mobile elements carrying these genes and selection of bla CMY -positive strains such as S. Newport MDRAmpC. Notably, isolates exhibiting this extended-spectrum cephalosporinase may show a ceftriaxone MIC as low as 2 µg/mL, but MIC to ceftiofur and cefoxitin fall more reliably in the intermediate or resistant range. For this reason, we currently performed subsequent β-lactamase analysis on any isolate exhibiting a ceftriaxone or ceftiofur MIC >2 µg/mL. This work was funded by an Interagency Agreement between the Food and Drug Administration and CDC.
Dr Whichard is a molecular biologist with the NARMS/FoodNet laboratory at CDC. Her research interests include β-lactamases, multidrug-resistant Salmonella isolates, bacteriophages, and other mobile genetic elements.