[beta]-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 [bla.sub.CMY] genes. Novel findings included [bla.sub.CMY]-positive Escherichia coli O157:H7 and a [bla.sub.SHV]-positive Salmonella isolate. CMY-positive isolates showed a ceftriaxone MIC [greater than or equal to] 2 [micro]g/mL.

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Extended-spectrum cephalosporins (ESC) are important for treating persons with severe Salmonella infections (1). This drug class is particularly important for pediatric therapy because fluoroquinolones are not approved for use in children. In 2000, 25% (8,153/32,022) of laboratory-confirmed Salmonella cases reported to the Centers for Disease Control and Prevention (CDC) occurred in children <5 years of age (2).

The National Antimicrobial Resistance Monitoring System (NARMS) for Enteric Bacteria began monitoring for resistance to cephalosporins and other drugs among human-derived Salmonella and Escherichia coli O157 isolates in 1996. Shigella was added to the surveillance in 1999. From 1996 to 1998, 15 (0.4%) of 4,093 non-typhi Salmonella isolates tested by NARMS were resistant to ESC, and none of the 675 E. coli O157 isolates tested were ESC resistant (3). Thirteen (87%) of 15 ESC-resistant Salmonella isolates exhibited a [bla.sub.CMY-2]-mediated mechanism of resistance (3,4), including 11 serotype Typhimurium, 1 Thompson, and 1 Newport. One S. Cubana isolate exhibited a [bla.sub.KPC-2] carbapenemase (5), and the remaining S. Typhimurium isolate exhibited a yet-uncharacterized extended-spectrum [beta]-lactamase (3). To determine the dynamics and mechanisms of cephalosporin resistance among species and serotypes, we examined the 2000 NARMS collection and determined mechanisms of ESC resistance in isolates exhibiting elevated cephalosporin MICs.

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 ([greater than or equal to] 16 [micro]g/mL), ceftiofur ([greater than or equal to] 4 [micro]g/mL), or ceftriaxone ([greater than or equal to] 16 [micro]g/mL).

Isoelectric Focusing (IEF) for [beta]-Lactamases

[beta]-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[degrees]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[degrees]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-[micro]L 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 [micro]g/mL solution of nitrocefin (Becton Dickinson, Franklin Lakes, N J, USA). Isoelectric points were estimated by comparison with the following standard [beta]-lactamases: TEM-12 (pI 5.25), SHV-3 (pI 7.0) and MIR-1 (pI 8.4).

Polymerase Chain Reaction (PCR) for [beta]-Lactamase Genes

For isolates that were IEF-positive for a [beta]-lactamase with a pI [greater than or equal to] 8.4, amplification of [bla.sub.CMY] genes was attempted. Internal primers were used to amplify a 369-bp portion of [bla.sub.CMY] genes from crude colony lysates. The forward primer anneals to nucleotide (nt) 271-289 of the 1,146-nt [bla.sub.CMY-2] sequence from Klebsiella pneumoniae (NCBI accession no. X91840) and has a sequence of 5'-GGCGTGTTGGGCGGCGATG-3'. The reverse primer anneals to nt 621-639 of [bla.sub.CMY-2] and has a sequence of 5'-CAGCGGAACCGTAATCCAG-3'. APBiotech Ready-to-Go

Beads (Piscataway, NJ, USA) were used to formulate 25-[micro]L reactions, which were run in an MJResearch thermocycler (Waltham, MA, USA) under the following conditions: 1 cycle of 94[degrees]C for 5 min followed by 25 cycles of: 94[degrees]C for 30 s, 59[degrees]C for 1 min, 72[degrees]C for 1 min. Amplicons were resolved by electrophoresis in 1% agarose gels. For isolates exhibiting a [beta]-lactamase with a pI = 8.0, [bla.sub.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).

Conclusions

In 2000, 2,152 non-Typhi Salmonella, Shigella, and E. coli O157 isolates were received and tested. Of these, 57 (2.6%) met the MIC criteria for additional testing to determine mechanisms of extended-spectrum cephalosporin resistance: 46 non-Typhi Salmonella isolates, 7 Shigella isolates (all S. sonnei), and 4 E. coli O157:H7 (Table). [bla.sub.CMY] genes were identified in 44 (96%) of the 46 Salmonella isolates. One S. Nienstedten isolate produced a [bla.sub.SHV] enzyme with a pI of 8.0. One S. Muenchen isolate with a cefoxitin MIC = 16 [micro]g/mL yielded no [beta]-1actamases by IEF. This isolate exhibited very low MIC of ceftriaxone and ceftiofur ([less than or equal to] 0.25 and [less than or equal to] 0.5 [micro]g/mL, respectively).

The 7 S. sonnei isolates included in the study met only the cefoxitin MIC criterion ([greater than or equal to] 16 [micro]g/mL). All 7 showed a ceftiofur MIC 1.0 [micro]g/mL or less, and a ceftriaxone MIC [less than or equal to] 0.25 [micro]g/mL. Six of these were also resistant to ampicillin, amoxicillin-clavulanate, and cephalothin. Each isolate was IEF-positive for a [beta]-lactamase enzyme with a pI [greater than or equal to] 8.4, but was polymerase chain reaction-negative for a [bla.sub.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 [greater than or equal to] 8.4. The pI in each case was 5.3 or 5.4. Plasmidborne [bla.sub.TEM-1] enzymes have been identified in several Salmonella serotypes (13).

Twenty-seven (61%) of 44 [bla.sub.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.sub.CMY] genes were found in 5 other Salmonella serotypes (Typhimurium, Heidelberg, Agona, Infantis, and Reading) in 2000. This contrasts with 1996-1998, when [bla.sub.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.sub.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.sub.CMY] in this E. coli serotype.

All 48 [bla.sub.CMY]-positive isolates (44 Salmonella and 4 E. coli O157:H7) exhibited a ceftiofur MIC [greater than or equal to] 8 [micro]g/mL; however, their ceftriaxone MIC ranged from 2 to 32 [micro]g/mL. Since the Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards) breakpoint for ceftriaxone resistance is 64 [micro]g/mL, none of these isolates were interpreted as ceftriaxone-resistant, and only 48% (23/48) were intermediate (16 or 32 [micro]g/mL). In contrast, all 48 CMY-producing isolates showed a cefoxitin MIC [greater than or equal to] 16 [micro]g/mL (intermediate or resistant according to CLSI guidelines).

NARMS sampling in 2000 showed that [bla.sub.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.sub.CMY]-positive Salmonella serotypes and the discovery of [bla.sub.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.sub.CMY] genes may be circulating among cattle. This remains to be proven and warrants more intensive study of [bla.sub.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.sub.CMY]-positive strains such as S. Newport MDRAmpC. Notably, isolates exhibiting this extended-spectrum cephalosporinase may show a ceftriaxone MIC as low as 2 [micro]g/mL, but MIC to ceftiofur and cefoxitin fall more reliably in the intermediate or resistant range. For this reason, we currently performed subsequent [beta]-lactamase analysis on any isolate exhibiting a ceftriaxone or ceftiofur MIC [greater than or equal to] 2 [micro]g/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 [beta]-lactamases, multidrug-resistant Salmonella isolates, bacteriophages, and other mobile genetic elements.

References

(1.) Hohmann EL. Nontyphoidal salmonellosis. Clin Infect Dis. 2001 ;32:263-9.

(2.) Centers for Disease Control and Prevention. PHLIS Salmonella 2000 annual summary. Division of Bacterial and Mycotic Diseases. 2001. [cited 2005 Jan 10]. Available at http://www.cdc.gov/ncidod/dbmd/ phlisdata/salmonella.htm#2000

(3.) Dunne EF, Fey PD, Kludt P, Reporter R, Mostashari F, Shillam P, et al. Emergence of domestically acquired ceftriaxone-resistant Salmonella infections associated with AmpC [beta]-1actamase. JAMA. 2000;284:3151-6.

(4.) Carattoli A, Tosini F, Giles WE Rupp ME, Hinrichs SH, Angulo FJ, et al. Characterization of plasmids carrying CMY-2 from expanded-spectrum cephalosporin-resistant Salmonella strains isolated in the United States between 1996 and 1998. Antimicrob Agents Chemother. 2002;46:1269-72.

(5.) Miriagou V, Tzouvelekis LS, Rossiter S., Tzelepi E, Angulo FJ, Whichard JM. Imipenem resistance in a Salmonella clinical strain due to plasmid-mediated class A carbapenemase KPC-2. Antimicrob Agents Chemother. 2003;47:1297-1300.

(6.) Rasheed JK, Anderson GJ, Yigit H, Queenan AM, Domenech-Sanchez A, Swenson JM, et al. Characterization of the extended-spectrum beta-lactamase reference strain, Klebsiella pneumoniae K6 (ATCC 700603), which produces the novel enzyme SHV-18. Antimicrob Agents Chemother. 2000;44:2382-8.

(7.) Rasheed JK, Jay C, Metchock B, Berkowitz F, Weigel L, Crellin J, et al. Evolution of extended-spectrum [beta]-lactam resistance (SHV-8) in a strain of Escherichia coli during multiple episodes of bacteremia. Antimicrob Agents Chemother. 1997;41:647-53.

(8.) Bergstrom S, Olsson O, Normark S. Common evolutionary origin of chromosomal beta-lactamase genes in enterobacteria. J Bacteriol. 1982;150:528-34.

(9.) Kar AK, Ghosh AS, Chauhan K, Ahamed J, Basu J, Chakrabarti P, et al. involvement of a 43-kilodalton outer membrane protein in [beta]-1actam resistance of Shigella dysenteriae. Antimicrob Agents Chemother. 1997; 41:2302-4.

(10.) Ghosh AS, Kar AK, Kundu M. Alterations in high molecular mass penicillin-binding protein 1 associated with beta-lactam resistance in Shigella dysenteriae. Biochem Biophys Res Commun. 1998; 248:669-72.

(11.) George AM, Levy SB. Amplifiable resistance to tetracycline, chloramphenicol, and other antibiotics in Escherichia coli: involvement of a non-plasmid-determined efflux of tetracycline. J Bacteriol. 1983; 155:531-40.

(12.) Alekshun MN, Levy SB. Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. Antimicrob Agents Chemother 1997;41:2067-75.

(13.) Llanes C, Kirchgesner V, Plesiat E Propagation of TEM- and PSE-type [beta]-1actamases among amoxicillin-resistant Salmonella spp. isolated in France. Antimicrob Agents Chemother. 1999;43:2430-6.

(14.) Centers for Disease Control and Prevention. Outbreak of multidrug-resistant Salmonella Newport--United States, January-April 2002. MMWR Morb Mortal Wkly Rep. 2002;51:545-8.

(15.) Gupta A, Fontana J, Crowe C, Bolstorff B, Stout A, Van Duyne S, et al. Emergence of multidrug-resistant Salmonella enterica serotype Newport infections resistant to expanded-spectrum cephalosporins in the United States. J Infect Dis. 2003;188:1707-16.

Jean M. Whichard,* Kevin Joyce,* Paul D. Fey, ([dagger]) Jennifer M. Nelson,* Frederick J. Angulo,* and Timothy J. Barrett*

* Centers for Disease Control and Prevention, Atlanta, Georgia, USA; and ([dagger]) University of Nebraska Medical Center, Omaha, Nebraska, USA

Address for correspondence: Jean M. Whichard, MS G29, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30333, USA; fax: [phone]; email: [e-mail]

Table. Characterization of isolates exhibiting increased MIC to
extended-spectrum [beta]-lactams among 2000 NARMS *

                          Total no. met          Total
                          MIC criteria/      PCR-positive
                            total no.        for [bla.sub.
Isolate/serotype           tested (%)          CMY] (%)

Salmonella
  Newport                  27/124 (22)         27 (100)
  Typhimurium             11/303 (3.6)         11 (100)
  Heidelberg               3/79 (3.8)           3 (100)
Other nontyphoidal
Salmonella (Agona,
Infantis, Muenchen,
Nienstedten,
Reading)                  5/872 (0.57)          3 (60)
Shigella sonnei            7/367 (1.9)             0
Escherichia coli
O157:H7                   4/407 (0.98)          4 (100)

Total                    57/2,152 (2.6)         48 (84)

                              Total
                          IEF-positive
                             for pl            No. that
                          [greater than      produce other
                          or equal to]          [beta]-
Isolate/serotype             8.4 (%)          lactamases

Salmonella
  Newport                   27 (100)               1
  Typhimurium               11 (100)               2
  Heidelberg                 3 (100)               1
Other nontyphoidal
Salmonella (Agona,
Infantis, Muenchen,
Nienstedten,
Reading)                     3 (60)                1
Shigella sonnei              7 (100)               3
Escherichia coli
O157:H7                      4 (100)               0

Total                        55 (96)               8

* NARMS, National Antimicrobial Resistance Monitorinq System;
PCR, polvmerase chain reaction; IEF, isoelectric focusing.