Human Salmonella Infection Yielding CTX-M β-Lactamase, United States

To the Editor: In the United States most third-generation cephalosporin resistance among salmonellae is due to AmpC plasmid–mediated β-lactamases. Extended-spectrum β-lactamases (ESBLs) have rarely been reported (1). The CTX-M β-lactamases constitute a group of ESBL enzymes that are increasing in prevalence worldwide. Currently, the CTX-M enzymes are classified into 5 different subgroups on the basis of DNA sequence similarities (2). We report on a domestically acquired CTX-M­–producing Salmonella isolate in the United States. 
 
In 2003, public health laboratories in all US state health departments submitted every 20th non-Typhi Salmonella (NTS) isolate from humans to the Centers for Disease Control and Prevention (CDC) for susceptibility testing by the National Antimicrobial Resistance Monitoring System (NARMS). MICs were determined by broth microdilution and interpreted according to Clinical and Laboratory Standards Institute standards (www.clsi.org), when available. Resistance to cefquinome was defined as >32 mg/L. 
 
Among the 1,864 human NTS isolates submitted to NARMS in 2003, 105 (5.6%) displayed elevated MICs (>2 mg/L) to ceftriaxone or ceftiofur, third-generation cephalosporins used in human and veterinary medicine, respectively. Genomic DNA was prepared from the 105 isolates, and a PCR with degenerate primers capable of detecting all CTX-M enzymes identified a single positive S. enterica ser. Typhimurium (3). The isolate came from a stool sample collected in September 2003 from a white, non-Hispanic, US-born, 3-month-old boy who lived in the state of Georgia. The patient had diarrhea and fever for ≈1 week. Because neither the patient nor his family had traveled internationally in the 3 months before specimen collection, the infection appears to have been domestically acquired. The patient did not receive any antimicrobial agents before illness but was treated for 14 days with cefpodoxime. The infant recovered from the illness without complications. 
 
The isolate displayed resistance to β-lactams, aminoglycosides, phenicols, tetracyclines, and folate pathway inhibitors (Table). Two β-lactamases (isoelectric pH [pI] 7.5 and 8.8) were resolved by isoelectric focusing. 
 
 
 
Table 
 
MIC values of antimicrobial drugs for the Salmonella ser. Typhimurium isolate and its Escherichia coli DH10B transformant 
 
 
 
Group-specific PCR primers were used to characterize the presumed blaCTX-M gene (4). Primers TOHO1–2F and TOHO1–1R yielded a 351-bp product, confirming a group II blaCTX-M gene. To perform sequencing of the entire gene, a ClustalW alignment with representatives from group II was performed to identify primers (DNASTAR, Madison, WI, USA). The sequence of the gene was identical to the sequence of the blaCTX-M-5 gene detected in other isolates of S. enterica ser. Typhimurium (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"U95364","term_id":"2443464","term_text":"U95364"}}U95364 and {"type":"entrez-nucleotide","attrs":{"text":"AF286192","term_id":"10946250","term_text":"AF286192"}}AF286192) as well as to the kluA-2 gene of Kluyvera ascorbata (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AJ251722","term_id":"6580516","term_text":"AJ251722"}}AJ251722). 
 
The genetic environment of the blaCTX-M-5 gene was investigated by PCR specific for upstream insertion elements (ISEcp1, IS26, and ORF513) and the downstream sequence sul1 (5). Amplification with primer ISEcp1 and an internal blaCTX-M-5 primer yielded a PCR product of ≈350 bp. Sequencing confirmed presence of the 3′ end inverted repeat region of the ISEcp1. 
 
Presence of other β-lactamase–encoding genes (blaTEM, blaSHV, and blaOXA) was investigated by PCR (6–8). Amplification with primers OXA-1F and OXA-1R yielded a 595-bp product with a sequence consistent with that of blaOXA-1 (8). 
 
To determine whether the CTX-M enzyme was plasmid-borne, plasmids were extracted and transformed into electrocompetent Escherichia coli DH10B. The transformant exhibited resistance to cefotaxime but not to ceftazidime (Table). In addition, the transformant exhibited resistance to cefquinome and cefepime. The presence of a blaCTX-M gene was confirmed by PCR (3,4). The blaOXA gene could not be amplified from the E. coli transformant (8). 
 
A CTX-M–producing Salmonella isolate has been reported only once previously in the United States (9). This was in 1994, when an isolate of Salmonella ser. Typhimurium var. Copenhagen with a CTX-M-5 was recovered from a 4-month-old girl adopted from Russia; that infection was not domestically acquired (9). We compared the 1994 isolate and the isolate in this study by pulsed-field gel electrophoresis; the isolates showed distinct patterns. 
 
The ISEcp1 insertion sequence has been described as a flanking region of several blaCTX-M genes and has been implicated in the expression and mobilization of the genes (5). A recent study by Lartigue et al. showed that a CTX-M-2 progenitor in K. ascorbata could be mobilized and transferred to a conjugative E. coli plasmid by the ISEcp1B element; enhanced mobilization was observed in the presence of ceftazidime, cefotaxime, and piperacillin (10). 
 
This Salmonella isolate’s resistance to cefepime and cefquinome, fourth-generation cephalosporins, is troubling. Cefquinome is not approved for use in the United States but has been used in Europe for treating food animals since 1994. ESBLs, including CTX-M enzymes, are more common in Europe than in the United States (1). Further studies are warranted to clarify the extent to which the use of cefquinome has contributed to high CTX-M prevalence in Europe. 
 
In conclusion, we report a domestically acquired CTX-M–producing Salmonella isolate in the United States. Because third-generation cephalosporins are important for treating invasive Salmonella infections, continued monitoring of ESBL-producing bacteria is important.

and human-to-human virus transmission leaves areas for further work.
These results support previous fi ndings that knowledge about avian infl uenza, especially about prevention and human-to-human transmission, has scope for improvement (4,5). Persons in Europe reported that they have little ability to prevent themselves from getting avian infl uenza (6). Previous research in the Lao People's Democratic Republic examined how consumers' knowledge of avian infl uenza risk reduced the likelihood that consumers will substitute poultry for other foods during an avian infl uenza crisis. This research indicates the importance of informing persons about consumption and transmission-related risks to reduce the likelihood of unnecessary behavioral changes that can cause larger macrolevel market effects (7).
The state of knowledge about avian infl uenza in Europe during the outbreak in the spring of 2006 leaves room for further public health information campaign efforts, especially those that increase consumers' understanding of consumption-related avian infl uenza risks. Persons in Europe appear to be aware of culling procedures and the risks of touching infected birds but have a more limited understanding of how avian infl uenza in their region should infl uence their consumption patterns. To the Editor: In the United States most third-generation cephalosporin resistance among salmonellae is due to AmpC plasmid-mediated β-lactamases.

Elias Mossialos and Caroline Rudisill
Extended-spectrum β-lactamases (ESBLs) have rarely been reported (1). The CTX-M β-lactamases constitute a group of ESBL enzymes that are increasing in prevalence worldwide. Currently, the CTX-M enzymes are classifi ed into 5 different subgroups on the basis of DNA sequence similarities (2). We report on a domestically acquired CTX-M -producing Salmonella isolate in the United States.
In 2003, public health laboratories in all US state health departments submitted every 20th non-Typhi Salmonella (NTS) isolate from humans week. Because neither the patient nor his family had traveled internationally in the 3 months before specimen collection, the infection appears to have been domestically acquired. The patient did not receive any antimicrobial agents before illness but was treated for 14 days with cefpodoxime. The infant recovered from the illness without complications.
Group-specifi c PCR primers were used to characterize the presumed bla CTX-M gene (4). Primers TOHO1-2F and TOHO1-1R yielded a 351bp product, confi rming a group II bla CTX-M gene. To perform sequencing of the entire gene, a ClustalW alignment with representatives from group II was performed to identify primers (DNASTAR, Madison, WI, USA). The sequence of the gene was identical to the sequence of the bla CTX-M-5 gene detected in other isolates of S. enterica ser. Typhimurium (GenBank accession nos. U95364 and AF286192) as well as to the kluA-2 gene of Kluyvera ascorbata (GenBank accession no. AJ251722).
The genetic environment of the bla CTX-M-5 gene was investigated by PCR specifi c for upstream insertion elements (ISEcp1, IS26, and ORF513) and the downstream sequence sul1 (5). Amplifi cation with primer ISEcp1 and an internal bla CTX-M-5 primer yielded a PCR product of ≈350 bp. Sequencing confi rmed presence of the 3′ end inverted repeat region of the ISEcp1.
To determine whether the CTX-M enzyme was plasmid-borne, plasmids were extracted and transformed into electrocompetent Escherichia coli DH10B. The transformant exhibited resistance to cefotaxime but not to ceftazidime (Table). In addition, the transformant exhibited resistance to cefquinome and cefepime. The presence of a bla CTX-M gene was confi rmed by PCR (3,4). The bla OXA gene could not be amplifi ed from the E. coli transformant (8).
A CTX-M-producing Salmonella isolate has been reported only once previously in the United States (9). This was in 1994, when an isolate of Salmonella ser. Typhimurium var. Copenhagen with a CTX-M-5 was recovered from a 4-month-old girl adopted from Russia; that infection was not domestically acquired (9). We compared the 1994 isolate and the isolate in this study by pulsed-fi eld gel electrophoresis; the isolates showed distinct patterns.

1958
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008 The ISEcp1 insertion sequence has been described as a fl anking region of several bla CTX-M genes and has been implicated in the expression and mobilization of the genes (5). A recent study by Lartigue et al. showed that a CTX-M-2 progenitor in K. ascorbata could be mobilized and transferred to a conjugative E. coli plasmid by the ISEcp1B element; enhanced mobilization was observed in the presence of ceftazidime, cefotaxime, and piperacillin (10).
This Salmonella isolate's resistance to cefepime and cefquinome, fourth-generation cephalosporins, is troubling. Cefquinome is not approved for use in the United States but has been used in Europe for treating food animals since 1994. ESBLs, including CTX-M enzymes, are more common in Europe than in the United States (1). Further studies are warranted to clarify the extent to which the use of cefquinome has contributed to high CTX-M prevalence in Europe.
In conclusion, we report a domestically acquired CTX-M-producing Salmonella isolate in the United States. Because third-generation cephalosporins are important for treating invasive Salmonella infections, continued monitoring of ESBL-producing bacteria is important.