Whole-Genome Sequencing Identifies In Vivo Acquisition of ablaCTX-M-27-Carrying IncFII Transmissible Plasmid as the Cause ofCeftriaxone Treatment Failure for an Invasive Salmonella entericaSerovar Typhimurium Infection

Bruce McCollister,a Cassandra V. Kotter,a Daniel N. Frank,a Taylor Washburn,a Michael G. Joblingb

University of Colorado Anschutz Medical Campus, Division of Infectious Disease, Aurora, Colorado, USAa; University of Colorado Anschutz Medical Campus,Department of Immunology and Microbiology, Aurora, Colorado, USAb

We report a case of ceftriaxone treatment failure for bacteremia caused by Salmonella enterica subsp. enterica serovar Typhimu-rium, due to the in vivo acquisition of a blaCTX-M-27-encoding IncFII group transmissible plasmid. The original �-lactamase-susceptible isolate ST882S was replaced by the resistant isolate ST931R during ceftriaxone treatment. After relapse, treatmentwas changed to ciprofloxacin, and the patient recovered. Isolate ST931R could transfer resistance to Escherichia coli at 37°C. Weused whole-genome sequencing of ST882S and ST931R, the E. coli transconjugant, and isolated plasmid DNA to unequivocallyshow that ST882S and ST931R had identical chromosomes, both having 206 identical single-nucleotide polymorphisms (SNPs)versus S. Typhimurium 14028s. We assembled a complete circular genome for ST931R, to which ST882S reads mapped with noSNPs. ST882S and ST931R were isogenic except for the presence of three additional plasmids in ST931R. ST931R and the E. colitransconjugant were ceftriaxone resistant due to the presence of a 60.5-kb IS26-flanked, blaCTX-M-27-encoding IncFII plasmid.Compared to 14082s, ST931R has almost identical Gifsy-1, Gifsy-2, and ST64B prophages, lacks Gifsy-3, and instead carries aunique Fels-2 prophage related to that found in LT2. ST882S and ST931R both had a 94-kb virulence plasmid showing >99%identity with pSLT14028s and a cryptic 3,904-bp replicon; ST931R also has cryptic 93-kb IncI1 and 62-kb IncI2 group plasmids.To the best of our knowledge, in vivo acquisition of extended-spectrum �-lactamase resistance by S. Typhimurium andblaCTX-M-27 genes in U.S. isolates of Salmonella have not previously been reported.

Greater than 1.4 million cases of nontyphoidal salmonellosisare estimated to occur each year in the United States, with

approximately 95% of cases resulting from foodborne transmis-sion (1). Although most infections result in mild to moderategastroenteritis that usually resolves with or without treatment,concurrent bacteremia is observed in approximately 6% of pa-tients and can be associated with serious disease, especially in in-fants aged �1 year, the elderly, and those with immunocompro-mised states such as HIV infection (2–5). Although routine use ofantimicrobial therapy is not generally recommended for the treat-ment of most Salmonella infections, such therapy may be lifesav-ing in persons with invasive disease.

Treatment of invasive salmonellosis has been compromised inrecent years due to the emergence of Salmonella isolates with sin-gle or multidrug resistance to a number of first-line agents (6, 7).Antimicrobial resistance in strains of nontyphoidal Salmonella(NTS) has been linked to the use of antimicrobial agents in live-stock (8–11). Consequently, agents such as fluoroquinolones andthird generation cephalosporins, such as ceftriaxone, have be-come treatment modalities of choice for therapy against severeSalmonella infections. In the United States before 1996, all re-ported cases of infection with ceftriaxone-resistant NTS isolateswere known or postulated to be acquired abroad (12). In 2000, thefirst reported case of a domestically acquired Salmonella infectionexpressing ceftriaxone resistance was reported in the United States(13). Since that time a number of investigations have revealed theongoing spread of ceftriaxone-resistant NTS isolates in both hu-mans and domesticated animals (14–16).

Resistance to ceftriaxone in NTS strains outside North Amer-

ica has generally been related to acquisition of plasmids contain-ing SHV-, TEM-, or CTX-M-encoding extended-spectrum �-lac-tamases (ESBLs) (17–19). In contrast, decreased susceptibility toceftriaxone observed in NTS isolates recovered from both humansand domesticated animals in the United States until recently hasbeen almost exclusively mediated through the production ofblaCMY-2, a Citrobacter freundii-derived ampC �-lactamase that isencoded on a variety of transmissible plasmids (20).

More recently, human NTS isolates harboring plasmids encod-ing ESBLs of the CTX-M family have been recovered sporadicallyin the United States, which were thought to be both domesticallyand nondomestically acquired (21). Consistent with these find-ings, recovery of blaCTX-M-expressing NTS in U.S. livestock pop-ulations has been subsequently reported (22). The blaCTX-M-typeenzymes are an emerging group of class A ESBLs initially reported

Received 29 July 2016 Returned for modification 27 August 2016Accepted 12 September 2016

Accepted manuscript posted online 26 September 2016

Citation McCollister B, Kotter CV, Frank DN, Washburn T, Jobling MG. 2016. Whole-genome sequencing identifies in vivo acquisition of a blaCTX-M-27-carrying IncFIItransmissible plasmid as the cause of ceftriaxone treatment failure for an invasiveSalmonella enterica serovar Typhimurium infection. Antimicrob AgentsChemother 60:7224 –7235. doi:10.1128/AAC.01649-16.

Address correspondence to Michael G. Jobling, michael.jobling@ucdenver.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01649-16.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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in the second half of the 1980s that have rapidly disseminated incertain Gram-negative bacilli, particularly in members of the En-terobacteriaceae family (23). These �-lactamases have become thepredominant family of ESBLs in many regions of the world (24).In clinical strains, blaCTX-M enzymes are encoded most commonlywithin plasmids that carry additional genes for resistance to otherantimicrobials, including aminoglycosides, chloramphenicol,trimethoprim-sulfamethoxazole, fluoroquinolones, tetracycline,and 16S RNA methylase genes, as well as housing other �-lacta-mase genes, with increasing reports in China and the developingworld (25–29), as well as the United States (30, 31).

Whole-genome sequencing (WGS) has recently begun to beused as an epidemiological tool to analyze in vivo acquisition ofantibiotic resistance by pathogens, which has most frequently de-termined the cause to be development of chromosomal mutationsor rearrangements (32–36). We report here our use of whole-genome sequencing to analyze in vivo emergence of ceftriaxoneresistance in Salmonella enterica subsp. enterica serovar Typhimu-rium due to acquisition of a plasmid encoding blaCTX-M-27 duringtreatment for Salmonella bacteremia.

MATERIALS AND METHODSCase. A 68-year-old female of Ethiopian descent, who was receiving im-munosuppressant medication for rheumatoid arthritis, was admitted tothe hospital for stridor due to an enlarged goiter. She was brought to theemergency room by ambulance and intubated for airway protection. Shewas febrile on admission to 38.9°C (102°F), and two sets of blood cultureswere drawn (day 0 [d0]), which revealed no growth. On d2 of her hospi-talization, she spiked a fever to 39.1°C (102.4°F), and two sets of bloodcultures were obtained, which at 24 h were both positive for Gram-nega-tive rods, later identified as S. Typhimurium. She was started on cefepimeat 2 g every 8 h. Two sets of blood cultures obtained the following day (d3)remained negative. The isolate tested susceptible to ampicillin, ceftriax-one, ciprofloxacin, and trimethoprim-sulfamethoxazole by disk diffusionassay. The antibiotic regimen was narrowed to intravenous ceftriaxonegiven as 1 g daily once the results of antimicrobial sensitivity testing werereported (d5). Her fevers resolved with antibiotic therapy, but on d12 shedeveloped an increase in her peripheral white blood cell count to 15.5 �109 liter�1 without associated symptoms. Two sets of blood cultures wereobtained, and both sets were positive for S. Typhimurium at 24 h. Anti-microbial susceptibility testing on the isolate revealed the emergence ofantibiotic resistance to ampicillin and ceftriaxone. Her antibiotic regimenwas switched to intravenous ciprofloxacin (400 mg) every 12 h. No endo-vascular or occult source of infection was found by transthoracic echocar-diogram, computed tomography of the abdomen and pelvis, computedtomography with angiogram of the chest or whole-body positron emis-sion tomography-computed tomography. However, the computed to-mography of the abdomen and pelvis revealed evidence of wall thickeningin the ascending colon suggestive of an infectious colitis, although nostool specimens were obtained for culture throughout her hospitaliza-tion. Subsequent blood cultures (d13 to d16) were negative, and shewas discharged (d23) on intravenous ciprofloxacin to complete a6-week course of therapy.

Bacterial strains and susceptibility testing. The Salmonella isolateswere recovered from blood culture specimens incubated on the BD BactecFX (BD Diagnostic Systems, Sparks, MD) automated system. Positiveblood culture specimens were processed at the University of ColoradoHospital Microbiology Laboratory according to standard operating pro-cedures and bacterial isolates identified with the API 20E system (bio-Mérieux, Marcy l’Etoile, France). Confirmation of Salmonella spp. recov-ery and serotyping was performed by the Colorado Department of PublicHealth and Environment Laboratory.

Initial antimicrobial susceptibility testing (for ampicillin, ceftriaxone,ciprofloxacin, nalidixic acid, and trimethoprim-sulfamethoxazole) was

performed by disk diffusion on BBL Mueller-Hinton agar plates (BectonDickinson and Company, Sparks, MD) according to the methods of theClinical and Laboratory Standards Institute (CLSI) (37). After 16 to 18 hof incubation at 35°C in a non-CO2 incubator, inhibitory zone sizes wereread and interpreted in accordance with the CLSI M100-S23 supplement(37). Antimicrobial MICs were then determined for each bacterial isolatefollowing preparation of NUC51 panels and incubation in the MicroScanWalkAway automated system (Siemens, Munich, Germany) according tothe manufacturer’s instructions.

Matings. Conjugal transfer of resistance was determined using 1:10dilutions of fresh cultures from donor and recipient in prewarmed Luria-Bertani (LB) medium, followed by incubation without shaking for 90 minat 37°C. The numbers of donor, recipient, and transconjugant cells weredetermined by plating serial dilutions on selective (rifampin [Rif], strep-tomycin [Str], or ampicillin [Amp]) or differential (X-Gal [5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside]) LB agar and used to calculatethe numbers of transconjugants per donor. The recipients were Esche-richia coli ER1821R (a Rifr isolate [38] of ER1821 [restrictionless, lac�;New England BioLabs, Ipswich, MA]) or JM83 (Strr �lac) (39).

Genomic DNA prep and library construction. For genome sequenc-ing, DNA was extracted using the UltraClean Microbial DNA isolation kit(Mo Bio, Inc., Carlsbad, CA) and prepared for sequencing using the Nex-tera XT kit (Illumina, Inc., San Diego, CA). Four DNA libraries wereconstructed, three from genomic DNA prepared from the ST882S andST931R isolates, the E. coli Ampr transconjugant from a cross withST931R, and a plasmid DNA preparation from the latter strain. Multi-plexed sequencing of these libraries was done with a single run on anIllumina MiSeq using paired-end reads (2 � 300 bp; MiSeq reagent kit,v3). After demultiplexing, sequences were analyzed using Geneious v6.1.7software (BioMatters, Ltd., Auckland, New Zealand). De novo assembly ofeach data set was performed using a percentage of the total data appro-priate for the expected genome size by the Geneious assembler (40) rou-tine using the medium-low sensitivity parameters appropriate for whole-genome-sequencing (WGS) assembly. Using less data both minimizes theamount of memory required by the routine and may also increase theaverage contig size by reducing the contig fragmentation that can becaused by using too many reads. The routine loads data until the setpercentage cutoff has been reached. We and others have previously usedGeneious software to successfully assemble complete genomes for Esche-richia coli (41) and Bacillus subtilis (42). For analysis of the plasmid con-tent, paired-end reads were first mapped to the S. Typhimurium 14028sgenome NC_016856.1 (43), followed by de novo assembly of unused readsinto new contigs. Manual trimming and editing of terminally redundantcontig ends generated circular plasmid genomes (several additional smallcontigs corresponded to Salmonella prophage sequences not present inthe 14028s genome).

Genome analysis. Single-nucleotide polymorphism (SNP) analysis wasdone using the CSIPhyologeny web server with default settings (https://cge.cbs.dtu.dk/services/CSIPhylogeny/) or output from the mapped consensusfrom within the Geneious program. Another server on the same site, Res-Finder (https://cge.cbs.dtu.dk/services/ResFinder/) (44), was used to de-tect antibiotic resistance determinants in assembled plasmid genomesprior to annotation. Genome-level comparisons were done using progres-siveMauve (45), which reorders and maps WGS contig data sets to com-plete genomes, producing a scalable graphic output from whole-genomedown to base-pair resolution. Replicon typing made use of the PubMLSTwebsite (http://pubmlst.org/plasmid/) developed by Keith Jolley (46) andsited at the University of Oxford. The development of that website wasfunded by the Wellcome Trust.

Accession number(s). Chromosome and plasmid genomes have beenassigned GenBank accession numbers CP016385 to CP016390.

RESULTSAntimicrobial susceptibility testing. Initial testing using Kirby-Bauer disc diffusion assay indicated that ST882S was susceptible to

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all antimicrobials tested but that ST931R was resistant to ampicil-lin and ceftriaxone. MICs for 16 antibiotics tested against the sus-ceptible and resistant S. Typhimurium isolates and the E. coli par-ent and transconjugant strain are listed in Table 1. The emergenceof ampicillin and ceftriaxone resistance observed by Kirby-Bauerdisk diffusion assay in the ST931R Salmonella isolate was againobserved by MIC testing, as MICs to ampicillin and ceftriaxoneincreased at least 4-fold and 8-fold, respectively, compared to theinitial, susceptible ST882S Salmonella isolate. Resistance to othercephalosporins, including cefazolin, cefotaxime, ceftazidime, andcefepime, also developed in this isolate. The addition of clavulanicacid (CLA) to cefotaxime and ceftazidime reversed the resis-tance observed for these two antibiotics, suggesting the pres-ence of an ESBL-type enzyme. Resistance to members of otherclasses of antibiotics was not noted, since no discernible MICchanges to ertapenem, gentamicin, levofloxacin, tetracycline,or trimethoprim-sulfamethoxazole were observed between thetwo clinical Salmonella isolates. Taken together, these findings ledus to hypothesize that the ST931R isolate emerged directly fromthe ST882S isolate by acquisition of ceftriaxone resistance withinthe patient during antibiotic therapy.

Isolate ST931R encodes a transmissible ESBL resistance de-terminant. Log-phase cultures of ST931R (Ampr) and E. coli

ER1821R (Rifr) transferred the ESBL phenotype (selected asAmpr) from ST931R to ER1821R at a frequency of 7.0 � 10�3

donor/recipient. Semiquantitative analyses (data not shown)showed that the transconjugant could retransmit Ampr at similarfrequencies to E. coli JM83 (Strr), demonstrating that the trans-missible factor itself is conjugation proficient. Detection of plas-mid DNA using a commercial miniprep kit was inconclusive, buta modified alkaline lysis protocol using polyethylene glycol pre-cipitation showed that the transconjugant possessed a single largeplasmid that we named pESBL931. MIC testing of the E. colistrains showed that the transconjugant’s ampicillin and cephalo-sporin resistance pattern matched that shown by the resistant Sal-monella isolate. Together, these data show that the acquisition of asingle ESBL-encoding plasmid was necessary and sufficient to ex-plain the emergence of ampicillin and ceftriaxone resistance in theclinical Salmonella isolate.

Genome analysis. To further analyze the plasmid and confirmthe genetic relatedness of the ST882S and ST931R Salmonella iso-lates, we used next-generation DNA sequencing to assemble thesequences of the isolated plasmid DNA, the ceftriaxone-resistantE. coli transconjugant, and the full genomes (chromosome andplasmidome) of the ST882S and ST931R isolates. The results ofsequencing and assembly are summarized in Table 2.

TABLE 1 Microscan MIC reports for susceptible and resistant Salmonella and E. coli strains

Agenta

Microscan MIC (�g/ml)b

Salmonella ST882S(susceptible)

Salmonella ST931R(resistant)

E. coli ER1821R(susceptible)

Transconjugant E. coli ER1821R(resistant)

AMP �8 <16 �8 <16AMP-SUL �8/4 16/8 �8/4 16/8AMX-CLA �8/4 �8/4 �8/4 �8/4Cefazolin �8 <16 �8 <16Ceftriaxone �8 <32 �8 <32Cefotaxime �2 <32 �2 <32Cefotaxime-CLA �0.5 �0.5 �0.5 �0.5Ceftazidime �1 <16 �1 <16Ceftazidime-CLA �0.5 �0.5 �0.5 �0.5Cefepime �4 <16 �4 <16PIP-TZB �16 �16 �16 �16Ertapenem �2 �2 �2 �2Gentamicin �4 �4 �4 �4Levofloxacin �2 �2 �2 �2Tetracycline �4 �4 �4 �4SXT �2/38 �2/38 �2/38 �2/38a AMP, ampicillin; CLA, clavulanic acid; AMX, amoxicillin; SUL, sulbactam; PIP-TZB, piperacillin-tazobactam; SXT, trimethoprim-sulfamethoxazole.b Boldface indicates MIC levels that were reported as resistant, except for AMP-SUL, which was reported as intermediate.

TABLE 2 Whole-genome sequence assembly data for all DNA libraries

Sample

No. of paired MiSeqreads (% used for denovo analyses)

No. of contigs of 500 bp(no. of plasmid contigs)

Avg foldcoveragea

Largestcontig (bp) N50 (bp)

No. of contigs with asize �N50

pESBL933 DNA 480,734 (20) 2b (1) 350 69,103 69,103 1E. coli ER1821R(pESBL931) 1,705,087 (22) 84 (4p)c 20.0 1.1* 269,473 103,211 16S. Typhimurium ST882S 1,950,898 (20) 74 (2) 18.8 0.6* 365,590 136,132 12S. Typhimurium ST931R 1,621,490 (24) 84 (5) 15.5 0.2* 271,829 97,525 15a That is, for fractions of the full-read data set used for de novo assembly; coverages for mapping to assembled genomes used the whole data set. *, means for the ten largestnonplasmid contigs the standard deviations.b The second contig coverage only matched IS5 sequences from E. coli chromosome five times.c The “p” denotes partial incomplete or misassembled contigs that nevertheless fully cover the pESBL931 plasmid.

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Chromosomal relatedness of isolates ST882S and ST931R. Toexamine the relatedness of the Salmonella isolates, we used CSI-Phylogeny, a single-nucleotide polymorphism (SNP) web server,to map one 300-bp read set each from the ST882S and ST931Rdata sets to 14028s, the first fully virulent S. Typhimurium ge-nome sequenced to completion (NC_016856.1 [43]). Thisyielded 206 identical SNPs for both data sets at a reference ge-nome coverage of 97.5%. Mapping both the ST882S and ST931Rpaired reads to the S. Typhimurium 14028s genome using thevariant/SNP call routine in Geneious reported 260 identical SNPsfor both ST882S and ST931R read sets (198 SNPs in common withan additional 62 SNPs not called by CSIPhylogeny, using the de-fault parameters). Together, these two complementary methodsshow that the ST882S and ST931R chromosomal genomes cannotbe distinguished from one another and support our hypothesisthat the ST931R isolate arose from the ST882S isolate by acquisi-tion of the pESBL931 plasmid encoding resistance to ceftriaxone.

Further proof for this conclusion came from a complete ge-nome sequence for the ST931R chromosome that we assembledfrom the consensus sequence called by mapping the paired-readdata set to the 14028s genome and replacing discordant regions(mostly insertion sequences [IS] or prophage insertions or dele-tions) with corresponding regions from contigs with perfectflanking homology from a de novo assembly. Illumina library in-serts are generally too short to unequivocally assemble across re-gions of identity larger than 1 kb, such as some insertion sequencesand rRNA operons. These made up the majority of the termini ofthe contigs in the de novo assembly. Mapping to a closely relatedcomplete genome can provide a framework from which to assem-ble a complete genome. We expand on this in Text S1 in the sup-plemental material. Subsequent minor manual editing of singlepolymorphisms (mostly in prophage or duplicated genes or rRNAloci) in the consensus versus 14082s produced a single circularchromosome of 4,852,920 bp with 61-fold average coverage (us-ing the complete data set). The S data set remapped to this Rgenome with 95-fold average coverage, no gaps, and zero SNPs. Ade novo assembly of unused ST882S reads from this mapping(83,870 paired and 9,273 mostly low quality unpaired) producedonly two significant-sized contigs that corresponded to the viru-lence plasmid and p3904, and many other small low-coverage(�27 reads) and low-quality contigs, mostly of 4 reads or less.Only 1,222 unused paired reads of low quality remained, and low-quality unused unpaired reads increased to 9,585. In addition, atthe genome level, progressiveMauve completely aligned the 74contigs (500 bp) from a de novo assembly of ST882S paired readsto the ST931R consensus genome, with four ambiguities and fourunaligned contigs. Four contigs had incorrectly joined noncontig-uous segments at IS200 homologies. Two unaligned contigs cor-responded to the virulence plasmid and p3904, and two contigs(1.0 and 2.5 kb) corresponded to fragments of chimeric rRNAloci. This indicates that no additional genetic determinants werepresent in ST882S and confirmed that the S and R chromosomes,virulence plasmids and p3904 genomes can reasonably be said tobe identical. Notably, both ST882S and ST931R have the identicalSNP, 290950C, in the rrnH locus that is not found by a BLASTsearch in any other Salmonella genome (nonredundant/nt refseqor WGS). Full coverage for ST882S reads over the whole of theST931R genome and lack of any substantial high-quality novelcontigs from the ST882S unused reads after mapping additionally

indicates that we did not miss any gross genome rearrangementsby using 14028s as a guide genome.

While ST882S and ST931R are highly similar to 14082s, theirprophage profiles differ, having a unique Fels-2 prophage notpresent in 14028s, while ST882S/ST931R lack the Gifsy-3 pro-phage found in 14028s. These prophages are named after the in-stitute (Fels [47]) or its location (Gif sur Yvette [48]) of the dis-covering laboratories. We could find no complete closed genomefor an S. Typhimurium isolate with an identical prophage profile,but, we did find a contig data set (based on the almost completeidentity of their virulence plasmids, noted in the next section) foran isolate, CVM N42450, that appears to be the closest availablegenomic match to ST882S and ST931R. We analyzed SNPs be-tween the genomes of ST931R and the contig set for isolate CVMN42450 (JYZN01) using CSIPhylogeny. This reported only 46SNPs between these two isolates, compared to the 206 SNPsseen versus 14082s; 19 of these SNPs are shared by both com-parators. Thus, the genome of ST931R is more closely related tothat of CVM N42450, an S. Typhimurium strain isolated fromchicken breast in California in 2012, than it is to 14028s. AprogressiveMauve alignment of the 126 CVM N42450 contigsto the ST931R genome showed complete congruity except forwhat appears to be a misassembly of contig 27 (at the repeatregion flanking the clustered regularly interspaced short palin-dromic repeat (CRISPR)-cas locus; marked with an asterisk inFig. 1). Figure 1 aligns the four best-characterized completegenomes with the highest symmetric identity scores in the NCBI ge-nome neighbor report for CVM N42450 (http://www.ncbi.nlm.nih.gov/genome/neighbors/152?genome_assembly_id�260322#) withthe ST931R and CVM N42450 genomes. Prophage loci are markedwith colored boxes. All pathogenic isolates have an ST64B-relatedprophage and all except CVM N42450 and ST882S/ST931R(which have completely syntenic prophage loci) differ at one ormore other prophage loci. Although a more detailed analysis ofthe ST882S/ST931R genome is beyond the scope of this study, abrief overview of the ST882S/ST931R genome is given in Text S1the supplemental material. The presence of a complete virulenceplasmid (see next section) and the ST64B prophage is consistentwith the invasive phenotype (49, 50) and, having been isolatedfrom patient blood cultures, the ST882S/ST931R isolates clearlyhave a full complement of virulence genes necessary to produce aninvasive infection.

Plasmidomes of ST882SS and ST931R. Plasmid-related con-tigs were identified in all data sets based on the presence of termi-nally repeated sequences and increased data coverage (indicativeof increased copy number) relative to the genomic contigs. Boththe ST882S and the ST931R genomic data independently assem-bled contigs that produced identical 93,855-bp and 3,904-bp cir-cular plasmids. The larger plasmid was 99.9% identical to the93,832-bp 14028s virulence plasmid CP001362 (51), with only fiveSNPs (two intergenic, two silent, and an F200L substitution inSpvD), a 24-bp expansion of a repeat sequence (11 versus 7repeats of CCTGTT) and a 1-bp deletion in a putative trans-glycosylase gene. A BLAST search of the WGS database with theDNA sequence, including the 1-bp deletion, found a singlecomplete match to a contig from the genome assembly of a2012 S. Typhimurium isolate, CVM N42450, from California(WGS NZ_JYZN01). The ST882S/ST931R virulence plasmid is100% identical to eight contigs (99.8% coverage) for CVMN42450, covering all of the above SNPs and the deletion; unfor-

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tunately, coverage for the repeat sequence is lacking. The smallerplasmid was identical to pEC147-3 (JX238454), a cryptic plasmiddiscovered in a plasmidome analysis of a set of ESBL-producingEscherichia coli isolates from urinary tract infections (52).

The ST931R genome data set identified three other plasmidgenomes not present in the ST882S genome data set. First weidentified pESBL931, a 68,117-bp IncFII (2:A�:B�) group plas-mid (alleles determined from pubmlst.org/plasmid). This se-quence was analyzed with ResFinder and a single �-lactamaseresistance gene, identical to an ESBL-type blaCTX-M-27 determi-nant (53), was identified. In addition, two IncI plasmid repli-cons were assembled, a 93,202-bp IncI1 (3:4:17:3:3) plasmidand a 60,496-bp IncI2 plasmid. The pESBL931 plasmid DNAdata set assembled a single large contig producing a 68,117-bpcircular genome identical to that from the ST931R genome assem-bly. The total genomic data set for E. coli carrying pESBL931 wasfirst mapped to an E. coli K-12 genome (MG1655) and then, byde novo assembly of the unused reads, we assembled a68,117-bp circular plasmid identical to the independent assem-blies of pESBL931. From this data set, we have also assembledand published a complete genome for the E. coli recipient strainER1821R (38). All data sets from Ampr samples assembledidentical 68,117-bp pESBL931 plasmids. A graphical depictionof pESBL931 is shown in Fig. 2.

Relative plasmid copy number can be estimated by mappingthe full read data set back to the circular contigs and comparingthe ratio of read coverages for each plasmid contig versus thecomplete chromosome. This assumes plasmids and chromosomesbehave similarly at each step in the process (e.g., DNA isolation,library preparation or amplification steps). Average coverage isremarkably consistent; for example, the top 10 non-plasmid con-

tigs for the ST931R de novo assembly (over 2 million bases) gave anaverage coverage of 15.5 0.2 (Table 2), with an average standarddeviation of 5.6 0.2 (36% 1%). For the ST931R genomes, thecalculated plasmid copy numbers (rounded to the nearest wholenumber to emend false precision) versus 61-fold coverage for thegenome were 3� (virulence plasmid and p931IncI1), 5� (p3904),7� (pESBL931), and 9� (p931IncI2). Relative copy numbers forthe virulence plasmid and p3904 versus the ST882S genome weresimilar at 2� and 4�, respectively, at 95-fold genome coverage.Interestingly, the copy number for pESBL931 in E. coli was 6-foldlower than in ST931R, at only 1� to 2� (135-fold versus 90-foldgenome coverage), although this difference was not reflected inthe MIC levels, which were the same for both strains (Table 1).

Together, these genome assemblies suggest that ST931R arosefrom ST882S by the concurrent acquisition of all three plasmids invivo, concomitant with acquisition of ceftriaxone resistance. Noother antibiotic resistance determinants were found to be encodedby any of the other plasmids, a finding consistent with the MICdata for the S and R isolates, although the IncI1 plasmid encodes anovel microcin highly similar to mccPDI (54). mccPDI is respon-sible for the proximity-dependent inhibition phenotype (55) ex-pressed by a group of cattle-adapted E. coli and hypothesized, byvirtue of being able to inhibit a wide variety of E. coli strains, to givethese strains a selective advantage in vivo. A brief characterizationof these plasmids is presented in Text S1 in the supplemental ma-terial.

Analysis of pESBL931 and context of the blaCTX-M-27 gene.The blaCTX-M-27 locus encoded by pESBL931 is flanked by directrepeats of IS26 in reverse orientation (i.e., tnpA encoded on thereverse strand relative to blaCTX-M-27). Internal to the IS26 ele-ments is an ISEcp1 fragment 5= of the blaCTX-M-27 allele that likely

FIG 1 progressiveMauve alignment of the ST931R genome with representative S. Typhimurium genomes. Genomes are aligned on the empty ST64B locus ofLT2 (bottom panel). Locally colinear blocks (conserved segments that appear to be internally free from genome rearrangements) are shown in different colors;their boundaries generally coincide with prophage loci. Almost all variability between genomes is due to these prophage loci and occurs in the central 2 Mb. Eachpanel corresponds to a single genome, with the strain name at bottom left and base pair numbering across the top. Red ticks on the CVM N42450 genome showcontig boundaries (all others are complete circular genomes). Colored boxes identify homologous prophages (linked between genomes by solid lines), with redboxes showing unique prophages. Smaller black boxes show the CRISPR-cas-associated repeat that appears misassembled in CVM N42450 (marked with anasterisk). To the right is shown the percent identities (symmetrical and gapped) of each comparator genome to CVM N42450 taken from the genome neighborprofile. Citations for published genomes are SL1344 (FQ312003.1) (106), UK-1 (NC_016863.1) (107), 14082s (NC_016856.1) (43), and LT2 (NC_003197.1)(51).

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provides a high-strength promoter (56), a complete IS903D ele-ment 3= of blaCTX-M-27, followed by an amino-terminally trun-cated iroN reading frame, Tn2-related sequences, and a secondcopy of IS26. This organization is also found in many blaCTX-M-24

and blaCTX-M-65 loci (29) (Fig. 2). This arrangement and insertionsite are identical to those found in a group of ESBL-encodingplasmids from China, typified by plasmids pHN7A8 (JN232517)(29) and pXZ (JF927996) (57). However, the blaCTX-M loci in these

plasmids encode different CTX-M alleles and also have additionalgenes encoding resistance to fosfomycin and aminoglycosides,also flanked by direct repeats of IS26 in the same reverse configu-ration (Fig. 2).

All of these plasmids are related to R100 from Shigella flexneri,the type plasmid for the IncFII group (NC_002134.1). pESBL931is most similar to pXZ (57) (JF927996) and pHN3A11 (JX997935)(58), plasmids found in ESBL-producing E. coli strains in pets and

FIG 2 Physical map of pESBL931 with comparison of its blaCTX-M-27 resistance module to other similar blaCTX-M modules and plasmids. The lower portion ofthe figure shows a circular map of the 68,117-bp plasmid pESBL931, with distinct regions of the plasmid as boxes on the circle (red, blaCTX-M-27 module; light blue,toxin/antitoxin [pemIK and hok/mok] and stability [stbBA] loci; white, tra, conjugal transfer region; light green, rep, plasmid replication genes). Open readingframes are shown on two tracks inside the circle as solid red arrows/triangles pointing in the direction of transcription (sense strand, followed by antisensestrand), except that blaCTX-M-27 is shown in yellow, and putative transposases are shown in black with �iroN in gray. The inner track shows the deviation fromthe average %GC. Tracks were generated by CGView server (108). The upper portion expands (blue lines) and compares the pemIK-stbBA region (these genesflank the resistance module) of pESBL931, with putative ancestral plasmid 2009C-3133 (CP013026) encoding a complete yjcA gene. The plasmid name, size ofthe region expanded, and the full plasmid size are given on the left. R100 has a simple insertion (not shown to scale) of an IS1-flanked (red boxes) resistancemodule into yjcA; all other plasmids have deleted the 5= portion of and 1,375 bp distal to yjcA, shown as a black box. Other IS elements are shown as differentcolored boxes (blue, IS26; dark green, IS903B; orange, ISEcp1; white, fragments of Tn2 or Tn1722). Open yellow arrows indicate resistance genes; black-filledarrows indicate transposase genes of IS elements. Unlabeled genes or elements correspond to similarly labeled elements in pESBL931. Open red arrows denoteplasmid backbone genes. Text to the right shows the status of two plasmid backbone loci that may contain an intron in ycjA (note, this gene is distinct from yjcA) or acomplete Tn10 or IS10 scar into parB; wt, wild type (no insertion); wtvar, no insert but variant gene sequence. Blue arrows point to these loci on the circular map.

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food animals from China. This IncFII plasmid backbone is a com-mon vector for multiple drug resistance cassettes worldwide, oftenwith identical insertions at this locus. The R100 plasmid, alsoknown as NR1, was isolated in Japan in the 1950s and is the arche-typical drug resistance R plasmid (59). It possesses an IS1-flankedmultidrug resistance (MDR) determinant (known as Tn2670) in-serted into the yjcA gene, and a full copy of Tn10 at another loca-tion (ydeB-yefA) on the plasmid. yjcA is a nonessential gene lo-cated between the plasmid addiction system pemIK and plasmidstability/partition stbBA loci (Fig. 2).

Figure 2 also shows this region from a plasmid from a Shiga-toxinogenic E. coli isolate 2009C-3133 (CP013026) that appears tohave complete uninterrupted locus. An additional plasmidpEC743_3 (NZ_CP015072.1) has a minimal IS1-IS26-based dele-tion/insertion at this locus but lacks any antibiotic resistancegenes. The pESBL931 blaCTX-M-27 locus is a recombinational in-sertion of an IS26-flanked cassette at this site, and the related MDRplasmid loci in pHN7A8/pXZ can be obtained from this by re-combinational insertion of another IS26-based cassette encodingthe fos, blaTEM1b, and rmtB genes. The backbones of all these plas-mids are highly related but differ from R100 at two other sites asnoted by He et al. (29). Some may have a 2.3-kb group II intronnear ycjA (unfortunately easily confused with yjcA, the site of theblaCXT-M-27 locus). The second site has either a wild-type gene oran IS10 inverted repeat-related scar (Fig. 2), where R100 has anintact Tn10 element. A more detailed analysis of these rearrange-ments is given in Text S1 in the supplemental material.

DISCUSSIONIn vivo acquisition of pESBL931. We report a case of ceftriaxonetreatment failure for a Salmonella enterica serovar Typhimuriumisolate due to the in vivo acquisition of a blaCTX-M-27-encodingIncFII:2 transmissible plasmid, during antibiotic therapy, from anunidentified donor organism. In vivo acquisition of antibiotic re-sistance is rarely documented; in the last 20 years, we found onlythree reports that identified putative in vivo acquisition of ESBLresistance due to conjugative plasmid transfer during therapy withcefotaxime or ceftriaxone, in three other Salmonella serovars: En-teritidis in 1995 (60), Anatum in 2003 (61), and Oranienburg in2016 (62). However, as is the case in this study, none of thesereports could identify the source of resistance, although the firsttwo reports speculated that the occurrence of similar resistancepatterns or resistance alleles in other isolates in the clinical envi-ronment indicated a likely source for the plasmid donor. A fewother reports document varied antibiotic resistance acquisitionduring Salmonella infections from nosocomial (63) or inpatientisolates of other Gram-negative pathogens (64–66). Most drug-resistant Salmonella isolates are suggested to arise from foods ofanimal origin, potentially from the use of antimicrobial agents inlivestock (13, 67, 68). However, the development of antibioticresistance within Salmonella strains as part of human fecal carriagehas also been observed. In a community outbreak of a susceptibleS. Typhimurium strain, ca. 15% of persons receiving ampicillintherapy had ampicillin-resistant isolates identified subsequentlyin their stool specimens (69). As in animals, fecal carriage in hu-mans of commensal microbiota that harbor resistance determi-nants, including blaCTX-M genetic elements, has been observed.Thus, normal human microbiota likely is an additional pool ofgenetic resistance determinants from which resistant Salmonellaisolates can emerge. In support of this model, several studies have

documented the possible in vivo transfer of plasmid-mediated an-tibiotic resistance between Salmonella isolates and resident hu-man commensal microbes (64, 70). Although providing a prece-dent for in vivo acquisition of antibiotic resistance, we cannotdefinitively identify a source of the pESBL931 plasmid.

Our patient reported consuming raw beef 5 days prior to theonset of Salmonella bacteremia, the only identifiable epidemio-logic risk factor for Salmonella acquisition in this patient. Theclosest genome match to ST882S/ST931R, CVM N14540, is a S.Typhimurium that has recently been found elsewhere in the foodchain. Raw beef, if contaminated at some point in the food chainor preparation, could also be a potential source of an ESBL donor.ESBL-positive E. coli has increasingly been found in the food chain(22, 71), and the biosample information for the pESBL931-relatedblaCTX-M-27 encoding plasmid pTC_N37410PS (CP007652) re-ports the source as a 2012 isolate from U.S. cattle. An alternativeand equally plausible source for the ESBL donor is the patient’sown gut flora. The patient’s case history details evidence of wallthickening in the ascending colon, suggestive of an infectious coli-tis, a likely primary source of infection for S. Typhimurium. Wecan envisage enteric ST882S encountering an ESBL donor in thegut milieu, followed by a secondary invasive infection of ST931Rduring antibiotic treatment. Carriage by healthy (72) and gastro-intestinal-symptomatic (73) adults of ESBL-positive E. coli andblaCTX-M-27 in particular (74) has been documented, althoughspecific plasmids were not identified. Unfortunately, the diagnos-tic regime for our patient did not require stool cultures, whichmay in retrospect have been used to identify an ESBL donor or-ganism.

ESBL plasmids CTX-M and Salmonella. Currently, pESBL931is the only annotated and complete blaCTX-M-27 plasmid inGenBank (accessed 8/31/2016) from an organism other than E.coli. A recent entry (KX008967) appears to be a complete butunannotated multidrug resistant plasmid from Shigella sonneiwith a blaCTX-M-27 identical DNA sequence. There is only oneother complete and annotated blaCTX-M-27 plasmid sequence,from an E. coli isolated from a patient in Thailand in 2012(pEC732-2, NZ_CP015140 [unpublished data]). pEC732-2 has adifferent plasmid backbone and a divergent blaCTX-M-27 locus,with an identical IS1R insertion site 3= of pemK, but a truncatedISEcp1 preceding the blaCTX-M-27 gene. This is followed by a partialIS903 sequence linked to an unrelated 18-kb IS26-flanked MDRregion in a different and hybrid IncF I/II replicon. A plasmid verysimilar to pESBL931 (pTC_N37410PS, CP007652 [unpublisheddata]; a 2012 E. coli isolate from Texas cattle) has an incompletebut syntenic blaCTX-M-27 locus (from pemK to stbA) with 34N=sreplacing approximately 1.8 kb of pESBL931 sequence (coveringalmost all of IS1R-Tn2-IS26) 3= of the blaCTX-M-27 gene, a 60-bpdeletion 5= of ISEcp1, and four small insertions (36, 1, 1 and 22 bp)in the distal IS26 element that frameshift the tnpA gene.

Information in general on flanking sequences for mostblaCTX-M-27 loci is similarly sparse. Of the 32 sequences forblaCTX-M-27 in GenBank as of June 2016, only 5 have significantflanking sequence data; all available flanking sequences have anISEcp1 fragment (with variable spacing) 5= of the blaCTX-M-27 alleleand part or all of an IS903 element 3= of blaCTX-M-27. Most are alsofollowed by an amino-terminally truncated iroN reading frame(an organization also found in many CTX-M-24 and 65 loci),followed by a second IS26 (Fig. 2). The bla-IS903D-�iroN associ-ation was first found in a 1999 Klebsiella isolate blaCTX-M-19 locus

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associated with a complete ISEcp1 element and which appeared tobe inserted within a Tn1721 element (75). Although the pemIK-IS1R-IS26-IsEcp1-blaCTX-M-IS903-�iroN gene order is commonto several CTX-M loci (e.g., M14, M24, and M65), the order of thegenes 3= of blaCTX-M-27-IS903D (i.e., �iroN-=Tn2=-IS26) appearsrestricted to and can be considered a marker of blaCTX-M-27 loci. Ina PCR-based study of local Japanese E. coli ESBL isolates, Mat-sumura (76) noted six distinct arrangements to the blaCTX-M-27

flanking sequences, almost all (174/176) flanked by inverted re-peats of IS26 in contrast to the locus in pESBL931 that isflanked by direct repeats. Details of the type of plasmid orinsertion location were not presented. This arrangement may bepeculiar to the local environment; the only other instance of ablaCTX-M27 locus flanked by inverted repeats of IS26 is a contigbeginning and ending with partial IS26 elements (and thus nogenomic context; NZ_JVGK01000036.1) from a urine isolate of E.coli, collected and sequenced as part of a longitudinal study of allisolates from an intensive care unit in Washington, USA (77).

ESBL, CTX-M, and blaCTX-M-27 in Salmonella. In the UnitedStates, most third-generation cephalosporin resistance among hu-man Salmonella isolates has historically been due to a plasmid-mediated blaCMY-2 (AmpC-type) �-lactamases (14, 78, 79), andwhile NTS with ESBL determinants (including blaCTX-M) are in-creasingly common in other parts of the world (19, 80–82), theystill remain rare in the United States (83). blaCTX-M determinantsin NTS infections are now being reported in the United States (84)and have increased significantly in veterinary and food isolates inthe rest of the world in recent years (85–87). blaCTX-M-encodingNTS were first isolated in the late 1980s in Tunisia (88) and in 1990in Argentina and concurrently in Europe (89). The first U.S. oc-currence of blaCTX-M of any type in NTS in humans was found inS. Typhimurium in 2003 (90), and three more cases were reportedin 2007 (21). In 2005, the first publication specifically reportingblaCTX-M-27 in NTS (serovar Livingston) appeared, occurring in anosocomial outbreak among neonates in Tunisia in 2002; theseisolates all showed high-level resistance to ceftriaxone (91). Tworecent reports from China found blaCTX-M-27 plasmids (all withvaried but non-FII Inc group replicons) in serovar Indiana isolatesin ducks from 2009 to 2010, reported in 2014 (28) and in Indianaand Typhimurium serovars from pigs and chickens in 2014, re-ported in 2016 (92), which now identifies blaCTX-M-27 as the mostcommon ESBL determinant in food-producing animals in China.A single case of an Indiana serovar with blaCTX-M-27 from a 2011neonate infection (from 21 ESBL isolates of human and 133 iso-lates of animal origin) was also reported in 2016 from China (93).The blaCTX-M-27 allele was first identified in 2003 from a 2000French clinical isolate of E. coli (pathotype not stated) (53). Itdiffers from blaCTX-M-14 at a single residue (D240G), a substitutionthat is also found in blaCTX-M-15 and blaCTX-M-16 loci. Compared toblaCTX-M-14, blaCTX-M-27 has a decreased Km for ceftazidime, pro-ducing a corresponding increase in MIC (53). F-group plasmidswith blaCTX-M-27 alleles are much more common in E. coli isolates(94), and blaCTX-M-27 is now found frequently in the ST131 groupof E. coli (74, 76, 95, 96), although these studies also note that thecontext of the loci are limited or infrequently reported.

Context of the other plasmids unique to ST931R and sharedwith ST882S. Plasmids related to the other two IncI group plas-mids found in ST931R have received little study since the shuffloncharacteristic of this group of plasmids were first reported in 1987for IncI1 (97) and 1990 for IncI2 (98). Twenty-five years later,

with the widespread use of whole-genome sequencing, coverage ofthis group of plasmids has exploded through recognition of theirrole in spread of antibiotic resistance, particularly ESBL �-lacta-mases—with over 200 PubMed articles relating to IncI1 plasmidsin the last decade; IncI2 plasmids have only recently been discov-ered as vectors for ESBL and other resistance determinants (99),with more than half of relevant Pubmed articles published since2013. Both sets of plasmids were historically associated with E. colibut are increasingly found in NTS isolates (80, 100). Alarmingly,ESBL-encoding IncI2 plasmids are now spreading the colistin re-sistance determinant mcr-1 in China (101) and Europe (102). Thefirst U.S. case of colistin resistance due to mcr-1 occurred with anE. coli carrying a novel MDR blaCTX-M-55 IncF plasmid earlier thisyear (103). Although both Salmonella IncI plasmids were cryptic,the ST931R IncI1 plasmid is most similar to pTF2 (KJ563250), ablaCTX-M-1-encoding plasmid in a Danish E. coli isolate from anunpublished study (99% identity with 89% coverage). TheST931R IncI2 plasmid is most similar (99% identity with 90%coverage) to an unpublished cryptic plasmid (CP011294) from aEuropean S. enterica subsp. diarizonae 60:r:z causing diarrhea andsepsis with fatal outcome. A plasmid identical to the 3,904-bpplasmid found in both ST882S and ST931R was originally discov-ered in an ESBL-positive (IncF blaTEM-1 and IncI1 blaCTX-M-9)urinary tract E. coli isolate (52); almost identical plasmids havebeen found in an E. coli isolate causing septicemia (104) and in anMDR NTS serovar Heidelberg (105) isolate from turkey meat inCanada (as the largest of four small plasmids, along with an MDR-IncHI2 plasmid). These matches support the idea that the originalST882S Salmonella isolate had already encountered plasmid-bear-ing E. coli prior to the acquisition of pESBL931 and the IncI plas-mids.

Conclusion. This study is the first to use whole-genome se-quencing to unequivocally determine the in vivo acquisition ofplasmid-determined antibiotic resistance by an originally suscep-tible Salmonella as the cause of relapse. This is an infrequentlydocumented and unusual but clinically significant observation. Inthis case, multiple plasmids were transferred to the pathogen,probably simultaneously. Since the particular ESBL determinantinvolved, blaCTX-M-27, is uncommon in Salmonella in the UnitedStates and is most often found in E. coli isolates, the most likelysource was an E. coli donor, possibly from a food source or thepatient’s own intestinal flora. This study highlights the impor-tance of plasmid biology in the epidemiology of enteric infectionsand the spread of antibiotic resistance in human pathogens.

FUNDING INFORMATIONA portion of this work, including some of the efforts of Michael G. Jobling,was funded by HHS | NIH | National Institute of Allergy and InfectiousDiseases (NIAID) (AI-31940). Conjugation experiments were performedby M.G.J. while supported by NIH grant AI-31940 to Randall Holmes.The remainder of his contribution and those of all other authors receivedno specific contribution from any funding agency in the public, commer-cial, or not-for-profit sectors.

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Erratum for McCollister et al., Whole-Genome Sequencing Identifies In VivoAcquisition of a blaCTX-M-27-CarryingIncFII Transmissible Plasmid as theCause of Ceftriaxone Treatment Failurefor an Invasive Salmonella entericaSerovar Typhimurium Infection

Bruce McCollister,a Cassandra V. Kotter,a Daniel N. Frank,a Taylor Washburn,a

Michael G. Joblingb

University of Colorado Anschutz Medical Campus, Division of Infectious Disease, Aurora, Colorado, USAa;University of Colorado Anschutz Medical Campus, Department of Immunology and Microbiology, Aurora,Colorado, USAb

Volume 60, no. 12, p. 7224 –7235, 2016. Due to a typesetting error, several symbolsin Table 1 are incorrect. The correct table appears below. Citation McCollister B, Kotter CV, Frank DN,

Washburn T, Jobling MG. 2017. Erratum forMcCollister et al., Whole-genome sequencingidentifies in vivo acquisition of a blaCTX-M-27-carrying IncFII transmissible plasmid as thecause of ceftriaxone treatment failure for aninvasive Salmonella enterica serovarTyphimurium infection. Antimicrob AgentsChemother 61:e00009-17. https://doi.org/10.1128/AAC.00009-17.

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

TABLE 1 Microscan MIC reports for susceptible and resistant Salmonella and E. coli strains

Agenta

Microscan MIC (�g/ml)b

SalmonellaST882S(susceptible)

SalmonellaST931R(resistant)

E. coliER1821R(susceptible)

TransconjugantE. coli ER1821R(resistant)

AMP �8 >16 �8 >16AMP-SUL �8/4 16/8 �8/4 16/8AMX-CLA �8/4 �8/4 �8/4 �8/4Cefazolin �8 >16 �8 >16Ceftriaxone �8 >32 �8 >32Cefotaxime �2 >32 �2 >32Cefotaxime-CLA �0.5 �0.5 �0.5 �0.5Ceftazidime �1 >16 �1 >16Ceftazidime-CLA �0.5 �0.5 �0.5 �0.5Cefepime �4 >16 �4 >16PIP-TZB �16 �16 �16 �16Ertapenem �2 �2 �2 �2Gentamicin �4 �4 �4 �4Levofloxacin �2 �2 �2 �2Tetracycline �4 �4 �4 �4SXT �2/38 �2/38 �2/38 �2/38aAMP, ampicillin; CLA, clavulanic acid; AMX, amoxicillin; SUL, sulbactam; PIP-TZB, piperacillin-tazobactam; SXT,trimethoprim-sulfamethoxazole.

bBoldface indicates MIC levels that were reported as resistant, except for AMP-SUL, which was reported asintermediate.

ERRATUM

crossm

March 2017 Volume 61 Issue 3 e00009-17 aac.asm.org 1Antimicrobial Agents and Chemotherapy