Characterization of bla CMY -Encoding Plasmids Among Salmonella Isolated in the United States in 2007

Abstract

Salmonella enterica is one of the most common bacterial causes of foodborne illness, and nontyphoidal Salmonella is estimated to cause ∼1.2 million illnesses in the United States each year. Plasmids are mobile genetic elements that play a critical role in the dissemination of antimicrobial resistance determinants. AmpC-type CMY β-lactamases (bla _CMY) confer resistance to extended-spectrum cephalosporins and β-lactam/β-lactamase inhibitor combinations and are commonly plasmid-encoded. A variety of plasmids have been shown to encode CMY β-lactamases and certain plasmids may be associated with particular Salmonella serotypes or environmental sources. In this study, we characterized bla _CMY β-lactamase-encoding plasmids among Salmonella isolates. Isolates of Salmonella from specimens collected from humans in 2007 were submitted to the Centers for Disease Control and Prevention National Antimicrobial Resistance Monitoring System laboratory for susceptibility testing. Three percent (65/2161) of Salmonella isolates displayed resistance to ceftriaxone (minimum inhibitory concentration [MIC] ≥4 mg/L) and amoxicillin/clavulanic acid (MIC ≥32 mg/L), a combination associated with the presence of a bla _CMY mechanism of resistance. Sixty-four (98.5%) isolates were polymerase chain reaction-positive for bla _CMY genes. Transformation and conjugation studies showed that 95% (61/64) of the bla _CMY genes were plasmid-encoded. Most of the bla _CMY-positive isolates were serotype Typhimurium, Newport, Heidelberg, and Agona. Forty-three plasmids were replicon type IncA/C, 15 IncI1, 2 contained multiple replicon loci, and 1 was untypeable. IncI1 plasmids conferred only the bla _CMY-associated resistance phenotype, whereas IncA/C plasmids conferred additional multi-drug resistance (MDR) phenotypes to drugs such as chloramphenicol, sulfisoxazole, and tetracycline. Most of the IncI1 plasmids (12/15) were sequence type 12 by plasmid multi-locus sequence typing. CMY β-lactamase-encoding plasmids among human isolates of Salmonella in the United States tended to be large MDR IncA/C plasmids or single resistance determinant IncI1 plasmids. In general, IncI1 plasmids were identified among serotypes commonly associated with poultry, whereas IncA/C plasmids were more likely to be identified among cattle/beef-associated serotypes.

Introduction

S almonella enterica is one of the most common bacterial causes of foodborne illness and nontyphoidal Salmonella (NTS) is estimated to cause ∼1.2 million illnesses in the United States each year (Scallan et al., 2011). A variety of serotypes cause human infections. Some serotypes such as Typhimurium are isolated from many different potential exposure sources, whereas others tend to be associated with certain food, environmental, or animal sources (Uzzau et al., 2000; Foley and Lynne, 2008; United States Department of Agriculture, 2008; Hoelzer et al., 2011). For example, serotype Dublin and Newport are often associated with cattle, whereas serotypes Heidelberg and Saintpaul are often associated with poultry (Foley and Lynne, 2008; United States Department of Agriculture, 2008). Fluoroquinolones and extended-spectrum cephalosporins (ESCs) are commonly used for the treatment of severe Salmonella infections. However, since fluoroquinolones may be associated with ligament and cartilage damage, ESCs such as ceftriaxone are commonly used for treatment of salmonellosis in children (Forsythe and Ernst, 2007).

Plasmids are mobile genetic elements that play a critical role in the dissemination of antimicrobial resistance determinants. AmpC-type CMY β-lactamases (bla _CMY) confer resistance to ESCs and β-lactam/β-lactamase inhibitor combinations and are commonly plasmid-encoded (Dunne et al., 2000). A variety of plasmids can carry these resistance genes and these plasmids can be differentiated based on their replicon features (Carattoli et al., 2002, 2005). Plasmids may be associated with certain serotypes of Salmonella (Carattoli et al., 2005; Hopkins et al., 2006; Folster et al., 2010). In the United States, the most common AmpC-type β-lactamase among Enterobacteriaceae is the CMY-2 β-lactamase (Philippon et al., 2002). Several reports have described bla _CMY-2 plasmids belonging to incompatibility/replicon (inc/rep) group A/C, F, I1, L/M, and P (Blanc et al., 2008; Mataseje et al., 2009; Call et al., 2010; Folster et al., 2010). However, the most commonly reported bla _CMY-2 encoding plasmids have been IncA/C and IncI1 (Hopkins et al., 2006; Baudry et al., 2009; Folster et al., 2010). IncA/C plasmids are widespread among Enterobacteriaceae, commonly confer multi-drug resistance (MDR), and have been found among diverse animal, environmental, and human clinical settings (Lindsey et al., 2009; Mulvey et al., 2009). Recently, IncA/C plasmids have been shown to mobilize the Salmonella Genomic Island 1 (Douard et al., 2010). IncI1 plasmids are commonly associated with Escherichia coli or Salmonella from avian or poultry sources and healthcare settings (Baudry et al., 2009). IncI1 plasmids are characterized by the presence of a type IV pilus operon, which some pathogenic bacteria use to adhere and invade eukaryotic cells (Kim and Komano, 1997). Recent studies have shown that IncI1 plasmids are more frequently associated with pathogenic than commensal isolates of avian E. coli (Johnson et al., 2007). Several studies have characterized bla _CMY plasmids from non-human sources, including IncI1 plasmids in Salmonella serotype Kentucky from poultry and IncA/C and IncI1 plasmids in Typhimurium from cattle, poultry, and swine (Fricke et al., 2009; Glenn et al., 2011; Sugawara et al., 2011). Additional studies have shown homology between bla _CMY-2 IncI1 plasmids carried by E. coli and Salmonella isolated from animals and humans (Winokur et al., 2001; Daniels et al., 2007). Taken together, this suggests that transmission of bla _CMY-2-positive isolates to humans may be through contaminated food sources or animal contact.

To better understand the source and transmission of bla _CMY β-lactamase plasmids, we identified and characterized these plasmids among Salmonella isolates from humans in the United States in 2007. We compared various characteristics of these plasmids, including replicon type, conjugative ability, size, and additional antimicrobial resistance conferred, to the serotypes of the original isolates to identify possible patterns of distribution, transmission, and source.

Materials and Methods

Isolate collection and testing

Isolates of Salmonella from specimens collected from ill persons were obtained by clinical laboratories in the United States and forwarded to state public health laboratories. Participating state public health laboratories serotyped and submitted every twentieth NTS to the Centers for Disease Control and Prevention (CDC) National Antimicrobial Resistance Monitoring System (NARMS) laboratory for susceptibility testing. Broth microdilution (Sensititre^®; Trek Diagnostics, Westlake, OH) was used to determine the minimum inhibitory concentrations (MIC) for 15 antimicrobial agents: amikacin, ampicillin, amoxicillin-clavulanic acid, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim-sulfamethoxazole. Resistance was defined by Clinical and Laboratory Standards Institute (CLSI) interpretive standards, when available (CLSI, 2011). For ceftiofur and streptomycin, where no CLSI interpretive criteria for human isolates exist, the resistance breakpoint used is 8 and 64 mg/L, respectively (Centers for Disease Control and Prevention, 2009). Testing was performed according to the manufacturer's instructions and with the following quality control strains: E. coli ATCC 25922, Staphylococcus aureus ATCC 29213, E. coli ATCC 35218, and Pseudomonas aeruginosa ATCC 27853. Salmonella isolates that displayed resistance to ceftriaxone and amoxicillin/clavulanic acid, indicating the presence of an AmpC class β-lactamase, were chosen for further study.

Polymerase chain reaction amplification and sequencing of bla _CMY

For each isolate, DNA was isolated by lysing the bacteria at 95°C and collecting the supernatant following centrifugation for 10 min at 20,000 g (Sorvall RC5B Plus, SS-34 rotor; Thermo Fischer Scientific Inc., Waltham, MA). Polymerase chain reaction (PCR) reactions contained 1× Hot Start PCR Master Mix (Qiagen Inc., Valencia, CA), 0.4 μM of each primer, 5 μL template DNA, and sterile PCR water to a final volume of 50 μL. Thermal cycling was performed using the following conditions: 15 min at 95°C, followed by 30 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 90 s. To determine the presence or absence of bla _CMY genes, primers ampC1 and ampC2, previously shown to amplify the bla _CMY gene, were used (Winokur et al., 2001). These primers and internal primers CMYSEQ1 (5′-GGTTGCAGGACGCGTCTG-3′) and CMYSEQ2 (5′-CCGCAATGGACTCCGGGC-3′) were used to sequence the entire bla _CMY gene. Sequencing was performed using Big Dye version 3.1 (Applied Biosystems, Foster City, CA) and sequence reactions are cleaned with Centri-sep plates (Princeton Separations, Adelphia, NJ). The reactions are electrophoresed through POP-7 polymer (Applied Biosystems) on a 3730 DNA Analyzer (Applied Biosystems) equipped with a 48-capillary, 50 cm array. Sequence analysis was performed using Lasergene 8 software (DNASTAR Inc, Madison, WI).

Plasmid purification and characterization

Purified plasmid DNA was used to transform laboratory E. coli strains, to separate the bla _CMY plasmids from other plasmid types before plasmid inc/rep testing, plasmid pulsed-field gel electrophoresis (pPFGE), and plasmid multi-locus sequence typing (pMLST). Plasmids were purified using the QiaFilter Midi kit (Qiagen Inc.), following a modified manufacturer's protocol as previously described (Folster et al., 2010). Electroporation of each plasmid into E. coli DH10B Electromax competent cells (Invitrogen, Carlsbad, CA) was performed as previously described (Folster et al., 2010). Cells were plated on LB agar plates containing 100 mg/L of ampicillin or 4 mg/L ceftriaxone (Sigma-Aldrich, St. Louis, MO). Plasmids were re-purified from a single bla _CMY PCR-positive transformant to isolate a single plasmid from each isolate. Purification was performed as described above with the additional modification of growing the cells overnight in 25 mL of LB broth with 100 mg/L of ampicillin or 4 mg/L ceftriaxone. Conjugation experiments were performed as previously described using a sodium azide-resistant J53 E. coli strain as recipient (gift from G. Jacoby) (Martinez-Martinez et al., 1998). Transconjugants were selected on LB containing 200 mg/L sodium azide and 4 mg/L of ceftriaxone. A single colony from each transformation experiment was PCR-screened for the bla _CMY gene to confirm successful conjugation.

Plasmid size was determined using a pPFGE protocol (Folster et al., 2010). Plasmids from E. coli strains PDK9 and V517, which contain several plasmids that range in size from 2.2 to 220 kb, were also extracted and used as size standards on the gels (Macrina et al., 1978).

Plasmid PCR-based replicon typing (PBRT) was performed as previously described (Carattoli et al., 2005). pMLST was performed on IncI1 plasmids as previously described (Garcia-Fernandez et al., 2008). No pMLST scheme is available for IncA/C plasmids. Sequences were submitted to the plasmid multi locus sequence type (pMLST) web page (http://pubmlst.org/plasmid/) and the sequence type (ST) type was determined.

Results

Identification of bla _CMY β-lactamase positive Salmonella isolates

In 2007, 2163 Salmonella isolates were screened for antimicrobial susceptibility by NARMS. Sixty-five (3%) displayed resistance to ceftriaxone (MIC ≥4 mg/L) and amoxicillin/clavulanic acid (MIC ≥32 mg/L). Sixty-four (98.5%) were PCR-positive for bla _CMY genes.

Characterization of bla _CMY β-lactamase plasmids

To determine if these bla _CMY genes were plasmid or chromosomally located, plasmids were purified and electroporated into competent E. coli DH10B strains, selected on 100 mg/L ampicillin, and PCR-screened for the bla _CMY gene. Fifty-nine bla _CMY-containing plasmids were successfully transferred by this method. Two purified plasmids produced ampicillin-resistant colonies but were bla _CMY negative, suggesting the presence of additional β-lactamase-producing plasmids. For these, the electroporation experiment was modified by selection with 4 mg/L ceftriaxone. Both purified plasmids produced bla _CMY PCR-positive colonies when ceftriaxone selection was used. In total, 61/64 (95%) of the bla _CMY genes were located on plasmids. The serotypes of the 61 isolates included 19 Typhimurium, 18 Newport, 7 Heidelberg, 7 Agona, 3 Dublin, 2 Typhimurium Var O:5-, and a single Bredeney, Enteritidis, [I 4, 12:i-], Ohio, and Saintpaul (Table 1). One bla _CMY gene from one isolate from each serotype was PCR-amplified, purified, and sequenced to confirm 100% homology to the bla _CMY-2 gene. The purified plasmids were further characterized (Table 1). Incompatibility replicon typing demonstrated that most had either IncA/C or IncI1 plasmids. One plasmid contained two replicon loci (IncFIB+P) and one plasmid contained three loci (IncFIB+A/C+P). One plasmid could not be identified by the current PBRT scheme.

Table 1.

Serotype of Salmonella Isolates and Characterization of Their Corresponding bla _CMY Plasmids

Serotype	No. of isolates	Conjugative	Inc/rep type	Approximate size (kb)	Add. Resistance ^a
Typhimurium	1	8/12	A/C	220	FIS, TET
	9			100–165	CHL, FIS, TET
	1			230	CHL, FIS, KAN, TET
	1			240	CHL, FIS, GEN, SXT, TET
	1	1/1	FIB+A/C+P	190	CHL, FIS, KAN, SXT, TET
	6	5/6	I1	100–190	None
Newport	18	6/18	A/C	90–160	CHL, FIS, TET
Heidelberg	7	7/7	I1	90–105	None
Agona	2	3/7	A/C	120, 150	CHL, FIS, TET
	2			120, 145	CHL, FIS, KAN, TET
	3			155, 155, 160	CHL, FIS, KAN, SXT, TET
Dublin	3	0/3	A/C	100, 170, 190	CHL, FIS, KAN, TET
Typhimurium var. O:5-	2	2/2	I1	110	None
			FIB+P	220	CHL, FIS, KAN, SXT, TET
Bredeney	1	0/1	A/C	165	CHL, FIS, TET
Enteritidis	1	1/1	A/C	100	CHL, FIS, KAN, TET
I 4, 12:i:-	1	1/1	I1	105	None
Ohio	1	1/1	unknown	150	CHL, FIS, TET
Saintpaul	1	1/1	I1	110	None

All transformants were resistant to ampicillin, amoxicillin/clavulanic acid, cefoxitin, and ceftiofur.

CHL, chloramphenicol; FIS, sulfisoxazole; GEN, gentamicin; KAN, kanamycin; SXT, trimethoprim-sulfamethoxazole; TET, tetracycline.

Additional drugs tested: AMI, amikacin; CIP, ciprofloxacin; NAL, nalidixic acid.

To determine the transferability of these plasmids under more natural conditions, conjugation experiments were performed. More than half (32/61) of the plasmids were transferred successfully by conjugation (Table 1). Fifteen of 16 IncI1 plasmids were conjugative, whereas 19 of the 45 IncA/C plasmids were successfully conjugated (Table 1). At least one study suggests that IncA/C plasmids possessing bla _CMY may have lost their self-conjugative ability; however, these plasmids may be mobilized by the presence of additional conjugative plasmids (Poole et al., 2009). Based on pPFGE results, the IncA/C plasmid sizes varied from 90 to 240 kb, whereas the IncI1 plasmid sizes ranged from 100 to 190 kb. To determine if these plasmids conferred additional resistance phenotypes, the electroporated E. coli DH10B transformants were tested for antimicrobial susceptibility. All transformants demonstrated bla _CMY-associated resistance (ampicillin, amoxicillin/clavulanic acid, ceftiofur, and ceftriaxone). All IncA/C plasmids conferred resistance to two to five additional antimicrobial agents. The most common additional resistance phenotypes among the transformants were chloramphenicol, sulfisoxazole, and tetracycline. None of the IncI1 transformants demonstrated resistance beyond the bla _CMY-associated resistance phenotype. Both plasmids with multiple replicon loci conferred resistance to five additional antimicrobials, whereas the unknown type plasmid conferred resistance to three additional antimicrobials (Table 1).

pMLST of the bla _CMY-IncI1 plasmids

To further characterize the bla _CMY-IncI1 plasmids, pMLST was performed. Most (12/15) were ST 12 and were identified in serotypes Heidelberg, Typhimurium, and Typhimurium Var O:5- (Table 2). One serotype Heidelberg isolate contained an ST23 plasmid, one Typhimurium isolate contained an ST2 plasmid, and the I 4,12:i:- isolate contained an ST26 plasmid.

Table 2.

Plasmid Multi-Locus Sequence Type of the IncI1 bla _CMY Plasmids

		Allele ^a
Serotype	No. of isolates	ardA	pilL	repI	sogS	trbA/pndC	ST
Heidelberg	6	4	1	1	4	3	12
Typhimurium	5	4	1	1	4	3	12
Typhimurium var. O:5-	1	4	1	1	4	3	12
I 4, 12:i:-	1	4	1	1	2	13	26
Typhimurium	1	2	1	1	2	3	2
Heidelberg	1	2	1	1	1	3	23

Five plasmid alleles are used to define the sequence type.

ST, sequence type.

Discussion

The rise in ESC resistance in the United States is well documented and is due to the spread of the plasmid-encoded bla _CMY-2 gene (Carattoli et al., 2002; Philippon et al., 2002; Blanc et al., 2008; Baudry et al., 2009). In this study, we identified and characterized 61 bla _CMY β-lactamase plasmids found among Salmonella isolated from humans in the United States in 2007. The majority of the plasmids were either incompatibility type IncA/C (n=42) or IncI1 (n=16). Two plasmids contained multiple replicon loci and one plasmid was untypeable by the current PBRT scheme. Both IncA/C and IncI1 plasmids are common carriers of bla _CMY β-lactamases, and both incompatibility types were observed in our previous study of bla _CMY β-lactamases among Salmonella Heidelberg isolates (Baudry et al., 2009; Folster et al., 2010). As previously observed, all of the bla _CMY IncA/C plasmids conferred additional antimicrobial resistance phenotypes and were multi-drug resistant (MDR), whereas none of the bla _CMY IncI1 plasmids conferred additional resistance beyond the bla _CMY-associated phenotype. The most common bla _CMY IncA/C plasmid conferred additional resistance to chloramphenicol, sulfisoxazole, and tetracycline. Resistance to gentamicin, kanamycin, and trimethoprim-sulfamethoxazole was rarely observed. Plasmid size varied within each replicon type; IncA/C plasmids were 100–240 kb, whereas IncI1 plasmids were 90–190 kb. Plasmid size did not correlate with the number of additional resistance phenotypes conferred, suggesting that the size differences observed were not due to the addition/deletion of antimicrobial resistance cassettes.

The most common serovars containing the bla _CMY β-lactamase plasmids were Typhimurium (n=19), Newport (n=18), Heidelberg (n=7), and Agona (n=7). Additional serovars more rarely observed were Dublin, Typhimurium Var O:5-, Bredeney, Enteritidis, I 4,12:i:-, Ohio, and Saintpaul. Specific serotypes appeared to carry bla _CMY β-lactamase exclusively on either IncI1 or IncA/C type plasmids. Serovars commonly associated with cattle and beef products, such as Newport and Dublin, carried bla _CMY exclusively on IncA/C plasmids, whereas serotypes commonly associated with poultry and poultry products, such as Heidelberg, I 4, 12:i:-, and Saintpaul, carried bla _CMY exclusively on IncI1 plasmids (Table 1) (Uzzau et al., 2000; United States Department of Agriculture, 2008). One exception was serotype Agona isolates, commonly associated with turkey and turkey products, which carried bla _CMY β-lactamases exclusively on MDR-IncA/C plasmids. However, Agona isolates have historically been associated with many different animals, including cattle, swine, and chicken. Several cephalosporin-resistant isolates of serotype Agona were documented from cattle sources in the United States in 2002–2004 (Frye et al., 2008). For serotype Typhimurium, which is ubiquitous among food animal sources, including cattle and poultry, we identified both IncA/C and IncI1 bla _CMY plasmids among our isolates, but never both replicon types in a single isolate. Further work is necessary to determine if the replicon type of bla _CMY-plasmids may indicate the possible source of the isolate.

Overall, these data suggest that different environmental factors may select for specific plasmid replicon types. It would be important to understand whether adhesion and virulence factors, such as those associated with the IncI1 plasmids, favor maintenance in poultry niches and/or transmission through the food chain to humans. The importance of antimicrobial selective pressures in selection, maintenance, and dissemination of bla _CMY plasmids, including those IncA/C plasmids that confer resistance to multiple classes of drugs beyond the β-lactams, would be important to understand as well. Because this study was limited to Salmonella isolates from humans, we can only hypothesize about selection pressures on bla _CMY β-lactamase plasmid type in other hosts. Further studies on Salmonella isolates from human, retail meat, and animal sources can more fully explore whether certain plasmid replicon types can be linked to specific sources.

To further characterize the IncI1 bla _CMY β-lactamase plasmids, pMLST was performed (http://pubmlst.org/plasmid/) (Garcia-Fernandez et al., 2008). Currently, there are 131 IncI1 plasmids in the database grouped into 48 different STs. The majority of our plasmids (12/15) were ST12 and this included plasmids from three different serotypes (Heidelberg, Typhimurium, and Typhimurium var. O:5-). All 10 of the previously described IncI1 bla _CMY-2 plasmids from serotype Heidelberg isolated from humans in the United States were ST12 (Folster et al., 2010). The pMLST database has 12 additional ST12 plasmids documented, including a bla _CMY-2 plasmid found in a Salmonella Kentucky isolate from poultry and bla _CMY-2 plasmids isolated from human, animal, and environmental E. coli and Salmonella spp. from Canada (Fricke et al., 2009; Mataseje et al., 2010). We identified a single ST2 plasmid from a Typhimurium isolate, an ST23 plasmid from a Heidelberg isolate, and an ST26 plasmid from an I 4,12:i:- isolate. There are 13 ST2 plasmids in the pMLST database and all are bla _CMY-2 plasmids carried by E. coli or Salmonella ser. Heidelberg isolates from human, dog, and environmental sources (Garcia-Fernandez et al. 2008; Mataseje et al., 2010). There is a single ST23 plasmid in the database, a bla _CMY-2 plasmid identified from a human clinical E. coli isolate and six ST26 plasmids, including two bla _CMY-2 plasmids from human clinical E. coli isolated in the UK and one bla _CTX-M plasmid from E. coli isolated from poultry in Italy (Mataseje et al., 2010). These data support the conclusion that similar IncI1 type bla _CMY plasmids are found within Salmonella and E. coli, and these isolates are commonly derived from poultry sources (Fricke et al., 2009; Mataseje et al., 2010). However, more representative pMLST data are necessary to draw firm conclusions.

Overall, the bla _CMY IncA/C plasmids were more similar in size and combination of additional antimicrobial profiles conferred within a given serotype than across different serotypes. This suggests that transmission of these plasmids may occur more frequently within a population composed of a single serotype than across populations composed of different serotypes. Another possibility is that clonal expansion could be amplifying specific resistance plasmid-containing members of a given serotype. In contrast, the majority of bla _CMY IncI1 plasmids were very similar, including ST, across different serotypes. This suggests the possible transmission of bla _CMY IncI1 plasmids among different serotypes. If it is ultimately confirmed that certain bla _CMY β-lactamase plasmids are more common in certain animal sources (IncA/C in cattle vs. IncI1 in poultry), then it will be important to systematically compare farming, transport, and food processing practices among different food animal production systems to better understand factors that lead to dissemination of these plasmids within and among various serotypes of Salmonella.

Conclusions

ESCs are used in both humans and animals and the rise in ESC resistance in the United States is concerning. This report confirms previous findings that resistance in Salmonella is primarily due to plasmid-mediated production of CMY β-lactamases. More importantly, this report offers a comprehensive study of the characteristics of bla _CMY plasmids among Salmonella isolated from humans in the United States during a single year. This report also describes the observed pattern of plasmid characteristics in relation to the Salmonella serotypes carrying these plasmids. While we offer a possible hypothesis for the observed plasmid replicon type and serotype relationships observed, additional studies are necessary to further elucidate the mechanisms involved in plasmid transmission among human, animal, food, and environmental sources.

Footnotes

Acknowledgments

We thank the NARMS participating public health laboratories for submitting the isolates, Anne Whitney for DNA sequencing, Alessandra Carattoli for the plasmid incompatibility typing control strains, George Jacoby for the sodium azide-resistant J53 strain used in conjugation experiments, and Maria Karlsson for her critical review. This work was supported by an interagency agreement between CDC and the FDA Center for Veterinary Medicine.

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Disclosure Statement

No competing financial interests exist.

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