Genomic Diversity, Virulence, and Antimicrobial Resistance ofKlebsiella pneumoniae Strains from Cows and Humans

Yongqiang Yang,a,b,c Catherine H. Higgins,a Ibraheem Rehman,a Klibs N. Galvao,d Ilana L. Brito,e Marcela L. Bicalho,a

Jeongmin Song,f Hongning Wang,b Rodrigo C. Bicalhoa

aDepartment of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, USAbAnimal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu, ChinacSchool of Pharmaceutical Sciences (Shenzhen), Sun Yat‐sen University, Guangzhou, ChinadDepartment of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USAeNancy E. and Peter C. Meinig School of Biomedical Engineering, College of Engineering, Cornell University, Ithaca, New York, USAfDepartment of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA

ABSTRACT Klebsiella pneumoniae is a leading cause of severe infections in humansand dairy cows, and these infections are rapidly becoming untreatable due to theemergence of multidrug-resistant (MDR) strains. However, little is known about therelationship between bovine and human K. pneumoniae isolates at the genome pop-ulation level. Here, we investigated the genomic structures, pangenomic profiles, vir-ulence determinants, and resistomes of 308 K. pneumoniae isolates from humans anddairy cows, including 96 newly sequenced cow isolates. We identified 177 functionalprotein families that were significantly different across human and bovine isolates;genes expressing proteins related to metal ion (iron, zinc, and calcium) metabolismwere significantly more prevalent among the bovine isolates. Siderophore systemswere found to be prevalent in both the bovine and the human isolates. In addition,we found that the Klebsiella ferric uptake operon kfuABC was significantly moreprevalent in clinical mastitis cases than in healthy cows. Furthermore, on two dairyfarms, we identified a unique IncN-type plasmid, pC5, coharboring blaCTX-M-1 andmph(A) genes, which confer resistance to cephalosporins and macrolides, respec-tively. We provide here the complete annotated sequence of this plasmid.

IMPORTANCE We demonstrate here the genetic diversity of K. pneumoniae isolatesfrom dairy cows and the mixed phylogenetic lineages between bovine and humanisolates. The ferric uptake operon kfuABC genes were more prevalent in strains fromclinical mastitis cows. Furthermore, we report the emergence of an IncN-type plas-mid carrying the blaCTX-M-1 and mph(A) genes among dairy farms in the UnitedStates. Our study evaluated the genomic diversity of the bovine and human isolates,and the findings uncovered different profiles of virulence determinants among bo-vine and human K. pneumoniae isolates at the genome population level.

KEYWORDS antimicrobial resistance, genomic epidemiology, Klebsiella pneumoniae,virulence

Klebsiella pneumoniae is a Gram-negative, rod-shaped, encapsulated, facultativeanaerobe that is commonly found in the mouth, skin, and intestines of humans and

animals. As an opportunistic pathogen in humans, K. pneumoniae primarily attacksimmunocompromised individuals and has become a leading cause of community-acquired and hospital-acquired infections in humans (1). K. pneumoniae is a clinicallyimportant species and causes serious nosocomial infections, such as septicemia, pneu-monia, urinary tract infection, surgical site infection, and soft tissue infection (2).Nosocomial infections caused by K. pneumoniae are a leading cause of morbidity and

Citation Yang Y, Higgins CH, Rehman I, GalvaoKN, Brito IL, Bicalho ML, Song J, Wang H,Bicalho RC. 2019. Genomic diversity, virulence,and antimicrobial resistance of Klebsiellapneumoniae strains from cows and humans.Appl Environ Microbiol 85:e02654-18. https://doi.org/10.1128/AEM.02654-18.

Editor Christopher A. Elkins, Centers forDisease Control and Prevention

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Rodrigo C. Bicalho,rcb28@cornell.edu.

Received 2 November 2018Accepted 21 December 2018

Accepted manuscript posted online 4January 2019Published

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mortality in immunocompromised people, with fatality rates being upwards of 50%despite the use of appropriate antibiotic therapy (3, 4).

In veterinary medicine, K. pneumoniae is of interest because it commonly causesclinical mastitis in dairy herds. Mastitis is a highly prevalent disease in dairy cows andcreates an incontestable economic burden in the dairy industry worldwide, in additionto being a serious animal welfare issue because of the pain associated with theinfection (5). The economic losses associated with mastitis are predominantly due todecreased milk production, discarded milk, treatment costs, and premature culling (6, 7).Coliform organisms are among the highest-incidence organisms on farms with otherwiseexcellent udder health management. The implementation of programs for mastitis controlhas reduced the prevalence of important contagious pathogens, and currently, approxi-mately 40% of all clinical mastitis cases are associated with opportunistic Gram-negativebacteria, such as Klebsiella spp., Escherichia coli, Pseudomonas spp., and Pasteurella spp. (8,9, 10). Among the major mastitis pathogens causing mastitis, coliform organisms such asKlebsiella spp. and E. coli cause the most severe clinical cases, with greater milk loss and anaverage cost of more than $221.03 per case (11). Studies have shown differences in thepathogenicity of Klebsiella spp. as a mastitis pathogen and other Gram-negative organisms(12, 13). Klebsiella spp. cause longer intramammary infections than E. coli (14), more severeclinical episodes than Serratia spp. and E. coli (15, 16), and a greater milk production lossand risk of culling than intramammary infections caused by E. coli (17). Therefore, due to theseverity of bovine clinical mastitis, the low effectiveness of antibiotic treatments, and thelack of advancement in preventive measures against Klebsiella infection, K. pneumoniae is aparticularly concerning pathogen.

Whole-genome analysis is the ideal approach to building a robust phylogeny in recentlyemerged pathogens to explore their diverse backgrounds through the identification ofsingle nucleotide polymorphisms (SNPs) or other genetic variants. A recent pangenomicanalysis of K. pneumoniae isolates from humans and bovines found that genes namedregulators of mucoid phenotype A (rmpA) and A2 (rmpA2) as well as a cluster of sidero-phore genes (kfuABC and kvgAS) are associated with invasive infection in humans (18).However, the analysis of the genetic determinants of the bovine isolates was limited, andthe relatedness of the human and bovine K. pneumoniae isolates was not explored. A morecomprehensive study with a larger number of isolates from cows would allow for explo-ration of the relatedness of human isolates and bovine isolates.

Here, we assembled a collection of K. pneumoniae strains recovered from 143mastitic cows with various degrees of disease severity and sequenced the genomes of96 distinct isolates to compare their genomic structures, virulence factors, and antimi-crobial resistance (AMR) genes with those of the K. pneumoniae isolates studied by Holtet al. (18). We investigated the pangenomic gene functions of 308 isolates in total andidentified a group of virulence genes that had a different distribution between K.pneumoniae isolates collected from dairy cows and humans. Furthermore, we found aresistance plasmid not previously identified in the United States and clarified its geneticcontext according to the closed plasmid structure.

RESULTSDiversity and AMR profile of K. pneumoniae on dairy farms. We isolated 143

nonreplicate K. pneumoniae strains (see Fig. S1 and Data Set S1 in the supplementalmaterial) from mastitic dairy cows from four farms located in upstate New York.Molecular typing of the isolates using pulsed-field gel electrophoresis (PFGE) identified97 distinct PFGE groups, revealing a high genetic diversity within the isolate collection.Capsule locus typing of those 143 isolates showed that 127 (88.8%) were assigned to44 known capsule loci (Fig. S1). The capsule loci KN3 (30/143, 21%), K13 (15/143, 11%),and KN1 (10/143, 7%) were the most prevalent in our sample set, accounting forapproximately 39% of all infections. High genetic diversity was also reflected by thesequence types (STs) of those strains. Among the 96 newly sequenced strains, 46possessed new alleles of the housekeeping genes used for multilocus sequence typing

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(MLST), and no predominant STs were found. These new alleles were submitted to theMLST database and were assigned 43 new STs.

Overall, the AMR profiles were diverse among these strains, and 40% (57/143) wereresistant to one or more antimicrobial agents (Fig. S1; Data Set S1). The agent to whichthe highest prevalence of resistance was detected was streptomycin (29.4%, 42/143),followed by tetracycline (5.6%, 8/143) and gentamicin (4.2%, 6/143). Using the genomicdata for the 96 newly sequenced isolates from mastitic cows, we detected 17 AMR geneswith various prevalences (Fig. S2). The fosA (fosfomycin resistance), oqxAB (quinolonesresistance), blaAmpH (�-lactamase), and blaSHV (�-lactamase) genes were common resis-tance genes found in our isolate collection. The resistance to streptomycin in our isolatecollection was attributed to the strA and strB genes, located on an IncHI1B-type plasmid in27 of the 96 strains. Analysis of the genetic environment revealed that the strA and strBgenes were flanked by transposon Tn5393, the most common mobile element thatmediates the transmission of strAB genes across species (19).

Identification of an IncN-type plasmid carrying blaCTX-M-1 and mph(A). Wefound that four isolates were resistant to ceftiofur, with the MIC being 512 �g/ml.Among those, three strains that had genomic sequences were found to coharbor theblaCTX-M-1 (�-lactamase) and mph(A) (macrolide resistance) genes, which were locatedin the same contig in each strain. Comparison of the sequences by BLASTn analysisshowed that all three contigs shared a high degree of similarity with the sequence ofpL2-43 (GenBank accession no. KJ484641), an IncN-type plasmid found in an E. coliisolate recovered from a lamb in Switzerland in 2014 (20). Analysis of the plasmidsequences confirmed that they carried the same plasmid (named pC5) that coharboredthe blaCTX-M-1 and mph(A) genes. The complete sequence of pC5 (GenBank accessionno. MF953243) is 41,608 bp in size and carries 51 open reading frames (ORFs) (Fig. 1;Table S1). The blaCTX-M-1 gene is flanked by an ISEcp1 element in its upstream region,which is disrupted by an intact IS26 element (820 bp) in the opposite orientation. Thesequences between the blaCTX-M-1 and mph(A) genes (1,572 bp) contain two ORFs: oneORF carries a truncated mrx gene (1,272 bp), and the other ORF is similar to ORF477 butis truncated by an inverted right repeat (IRR) of the ISEcp1 element (23 bp). The mph(A)gene was mediated by another IS26 element with the same orientation. We then

FIG 1 Plasmid structures of pC5 and reference plasmids. (A) Circular genetic map of plasmid pC5 (GenBank accession no. MF953243) and reference plasmidspL2-43 (GenBank accession no. KJ484641), pKC394 (GenBank accession no. HM138652), pH1038-142 (GenBank accession no. KJ484634), pVI (GenBank accessionno. LT795508), pHHA45 (GenBank accession no. JX065630), pQNR2078 (GenBank accession no. HE613857), and pKC396 (GenBank accession no. HM138653). Themap was drawn using the BLAST Ring Image Generator (BRIG) program (http://sourceforge.net/projects/brig/). (B) Schematic presentation of major structuralfeatures of pC5 in comparison with the reference plasmids pL2-43, pKC394, pH1038-142, pVI, pHHA45, pQNR2078, and pKC396. Areas shaded in gray indicatehomologous regions of �99% nucleotide sequence identity in the plasmid scaffold regions. ORFs are portrayed by arrows to indicate the direction oftranscription and colored on the basis of their predicted gene functions. Antimicrobial resistance genes are indicated by red boxes. Blue boxes denote mobilegenetic elements. Green boxes indicate ORFs encoding hypothetical proteins. The figure is drawn to scale.

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compared the genetic background of pC5 with that of seven publicly available plasmidsequences with high similarity (coverage, �90%; identity, �99%), all of which wereidentified in E. coli strains in European countries (20, 21, 22, 23) (Table 1). They allmaintained a typical IncN plasmid backbone scaffold, including a replicon and genesinvolved in plasmid stability, mutagenesis enhancement, antirestriction functions, andconjugative transfer (24). The difference among these plasmids was in the mosaicregion that carried resistance genes and in the mobile genetic elements represented(Fig. 1). Plasmid pC5 shared an identical genetic neighborhood of the blaCTX-M-1 andmph(A) genes with pHHA45, pKC394, and pVI. However, in plasmid pL2-43, the reverseposition of mph(A)-IS26 was the main structural difference from our plasmid.

Phylogeny of K. pneumoniae from cows and humans. We compared 308 ge-nomes from K. pneumoniae KpI isolates, including 123 isolates from dairy cows and 185isolates from humans. We identified a total of 92,409 core genome SNPs among these308 genomes and built a maximum likelihood (ML) phylogenetic tree based on theseSNPs (Fig. 2). The results revealed a deep branching and scattered population structurethat was broadly classified into 169 distinct phylogenetic lineages. The 123 bovineisolates had diverse population structures and did not form specific clusters. Further-more, the population structure of the 123 bovine isolates was highly mixed with the185 human isolates, and there were 27 lineages containing both human and bovineisolates. We assessed the clonality and clades of 294 isolates with assigned STs from the308 strains as minimum-spanning trees (Fig. 3). Of these, we identified 190 distinct STs.The most prevalent STs were ST23 (n � 10 isolates), ST43 (n � 8), ST11 (n � 7), ST107(n � 7), ST15 (n � 6), ST199 (n � 6), ST14 (n � 5), ST20 (n � 5), and ST48 (n � 5).Furthermore, ST34, ST35, ST37, ST43, ST65, ST107, ST133, ST290, ST294, ST309, andST791 were found in both human and bovine isolates.

Pangenomic analysis identified distinct proteins between cow and humanisolates. We then investigated the functions of all genes across the 308 human andbovine isolates. In total, there were 1,705,306 open reading frames (ORFs) in the 308genomes, with an average of 5,536 ORFs in each genome. All the ORFs could beclassified into 68,420 clusters by sequence identity, which included 24,753 clustersharboring multiple sequences and 43,667 clusters with singleton genes. Functionannotation against the Kyoto Encyclopedia of Genes and Genomes (KEGG) databaseshowed that 72% of the representative genes were assigned to 9,407 distinct KEGG hitswith known functions among different functional categories (Fig. S3; Table S2). Thesefunctions included environmental information processing, genetic information process-ing, cellular processes, carbohydrate metabolism, amino acid metabolism, metabolismof cofactors and vitamins, energy metabolism, human disease-related functions, en-zyme families, and nucleotide metabolism.

We then calculated the distribution of these KEGG assignments. There were 1,800and 2,392 functional units found in all bovine isolates and human isolates, respectively.Among them, 2,684 functional units were found at a prevalence of �95% in bothbovine and human isolates. Nonetheless, there were 177 functional units with signifi-cant differences between the human and bovine isolates (P � 0.0001, �2 test) (Fig. S4;Table S3). Proteins related to metal ion (iron, zinc, and calcium) metabolism weresignificantly more prevalent in the bovine isolates; examples included the Fe3� dicitratetransport protein FecA (81.3% versus 43.2%), the zinc protease PqqL (65.9% versus 7%),and the calcium permeable stress-gated cation channel protein TMEM63 (60.2% versus44.3%). However, the reverse situation was true for proteins related to heavy metalion-related transport; for example, 29.7% of the human isolates but only 2.4% of thebovine isolates carried the mercuric ion transport protein MerT.

Extensive diversity of virulence genes among bovine and human isolates. Intotal, we detected 173 virulence genes in the 308 isolates (Table S4). These included135 and 170 virulence genes found in the bovine and human isolates, respectively, ofwhich 132 genes were common to both types of isolates (Fig. 4). The enterobactin loci,adhesion-related gene clusters, secretion system-related gene clusters, and fimbria

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gene clusters were found in all isolates. Of note, the average number of virulence genesper isolate was significantly higher for the human isolates than for the bovine isolates(70 versus 62; P � 0.0001, Kruskal-Wallis test) (Fig. 4). The virulence genes associatedwith colibactin, aerobactin, allantoinase, yersiniabactin, microcin, salmochelin, and thermpA and rmpA2 genes were rare (0.5% to 5%) among the bovine isolates, whereas theywere found at increased frequency in the human isolates (9% to 35%) (Fig. 4).Furthermore, the combination of colibactin, aerobactin, allantoinase, yersiniabactin,microcin, salmochelin, and the rmpA and rmpA2 genes was frequent in K. pneumoniaeisolates from humans. Among all 123 bovine isolates, we also investigated the distri-bution of virulence genes in healthy cows and mastitic cows. All the virulence genesfound in the healthy group were present in either the subclinical mastitis or clinicalmastitis cases. However, 26 virulence genes were exclusive to the subclinical mastitiscases and 6 were exclusive to the clinical mastitis cases (Fig. 4). Notably, we found thatthe Klebsiella ferric uptake operon kfuABC was significantly more prevalent (P � 0.05, �2

test) in clinical mastitis cases than in subclinical mastitis cases and healthy cows (39%,20%, and 11%, respectively; P � 0.0336, �2 test).

DISCUSSION

The emergence and transmission of multidrug-resistant and virulent K. pneumoniaestrains can cause severe, untreatable infections in humans and animals. However, dueto insufficient data at the population level, the diversity of AMR genes of K. pneumoniaein cows is still unknown. In this study, we identified an IncN-type plasmid, pC5,coharboring the extended-spectrum �-lactamase (ESBL) gene blaCTX-M-1 and the mac-rolide phosphotransferase gene mph(A) in four distinct isolates on two farms. Ceftiofuris one of the most commonly used antibiotics in both adult and young dairy cattle in the

FIG 2 Phylogeny of core genome SNPs in 308 genomes of K. pneumoniae KpI isolates from humans and dairy cows. Thecore genome SNPs were identified by mapping sequencing reads against a reference genome (K. pneumoniae strainNTUH-K2044, GenBank accession no. NC_012731.1). The RAxML program was used to calculate the phylogenetic tree toconstruct a maximum likelihood phylogeny.

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United States, whereas macrolides are mostly used in young dairy cattle (�20 months ofage) for the treatment of respiratory disease (https://www.aphis.usda.gov/animal_health/nahms/dairy/downloads/dairy07/Dairy07_is_AntibioticUse.pdf). The use of ceftiofurand/or macrolides may promote the dissemination of pC5-like plasmids. Seven plas-

FIG 3 Minimum-spanning trees of 294 K. pneumoniae isolates from humans and dairy cows by MLST type. The 294 isolates that had assigned STs were derivedfrom 308 K. pneumoniae isolates from humans and dairy cows. Each node within the tree represents a single ST. The size of the nodes is proportional to thenumber of K. pneumoniae isolates belonging to that ST. The length of the branch between each node is proportional to the number of distinct alleles of sevenhousekeeping genes that differ between the two linked nodes. The 30 STs with the highest numbers of isolates are labeled with different colors.

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mids that had been previously identified in E. coli either in humans or in animals inEuropean countries shared a high similarity with pC5. In the United States, CTX-M-like-producing Enterobacteriaceae strains were first reported in 2003 (25). CTX-M-15 is themost common CTX-M enzyme found in Enterobacteriaceae in humans in the UnitedStates (26), whereas CTX-M-1 is rare. A previous study found 1 CTX-M-1-producingisolate among 208 ESBL-producing K. pneumoniae isolates from humans and 1 CTX-M-1-producing isolate among 163 ESBL-producing E. coli isolates from humans (27). Thewide geographical and source distribution of pC5-like plasmids in European countriesindicated that such IncN plasmids are responsible for the dissemination of blaCTX-M-1

and other resistance genes. Our study reports the emergence of this plasmid in theUnited States.

Our study sequenced 96 strains which belonged to different PFGE groups, and 46 ofthese were new STs. This indicates that there is no dominant strain responsible forinfections with K. pneumoniae in cows. We built a representative pool of genomes frombovine and human strains comprising more than 300 genomes. Both the human andbovine isolates shared a large number of functional modules to form a core genomethat may contribute to the maintenance of bacterial life. However, environmentalchanges and selection pressures have led to differences in some specific functionsamong the human and bovine isolates. For example, we found that proteins involvedin metal ion (iron, zinc, and calcium) metabolism were significantly more prevalent in

FIG 4 Virulence genes in 308 K. pneumoniae KpI isolates. (A) Number of virulence genes per isolate. Box plots indicate the average number of virulence genesin 123 bovine strains and 185 human strains. The means are shown as horizontal bars, with values of 62 and 70 for the bovine and human isolates, respectively.The P value was calculated using the Kruskal-Wallis test. (B) Distribution of virulence genes in 123 bovine isolates and 185 human isolates. Each number indicatesthe number of different virulence genes found within each group. (C) Frequency of gene clusters among 123 bovine isolates and 185 human isolates. The Pvalues were calculated using the chi-square test. (D) Distribution of virulence genes in newly sequenced clinical K. pneumoniae KpI isolates. Each numberindicates the number of different virulence genes found within each group. (E) Distribution of virulence genes in 123 bovine isolates. Each number indicatesthe number of different virulence genes found within each group.

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bovine isolates. Iron, such as Fe3�, is sequestered by transferrin and lactoferrin (28). Theconcentration of iron in vivo is critical for the control of cellular metabolism in bacteria,and insufficient iron abolishes bacterial growth (29). To grow successfully in hosttissues, bacteria must be able to obtain metal ions from host transport proteins.

The pathogenicity of clinical K. pneumoniae strains is caused by various virulencedeterminants. We found that human isolates carried more virulence genes than bovineisolates. Virulence genes related to siderophore biosynthesis, fimbria proteins, adhe-sion, and secretion systems were found to be prevalent in both bovine and humanisolates. The most common siderophore in cows is enterobactin. However, somesiderophore systems (e.g., aerobactin, colibactin, allantoinase, yersiniabactin, microcin,and salmochelin systems) were significantly more prevalent in human isolates. Further-more, previous studies showed that the ferric uptake operon kfuABC genes weresignificantly associated with tissue invasion in humans (18, 30). Our study confirmedthat kfuABC is also associated with clinical mastitis cases in dairy cows. The secretedsiderophores could grab iron from transferrin, lactoferrin, or ferritin and support thegrowth and infection of K. pneumoniae in hosts (31). Our results provide informationrelated to the genomic structure, the virulence profile, and the resistome of K. pneu-moniae isolates from cows and humans.

MATERIALS AND METHODSSampling design and bacterial strains. This work was conducted between January and August

2017 at four commercial dairy farms, which were eligible for inclusion in the study if they met thefollowing criteria: they had a herd size of �500 lactating cows, they participated in monthly Dairy HerdImprovement Association (DHIA) testing, they had an accurate recording of clinical mastitis cases, andthey were willing to participate. All the farms were located in Cayuga County of New York State, and thefarms were located inside a 15-mi-diameter circular region. Each dairy farm produced its own supply offorages, corn, grass, and alfalfa silage and purchased concentrated feed (corn, soybean meal, etc.) froma local vendor. Additionally, veterinary care was provided by a different veterinary clinic for each one ofthe four farms. Ceftiofur is routinely used on all four dairy farms. A known presence of Klebsiella clinicalmastitis was also required to ensure that an appropriate number of Klebsiella isolates could be collectedfrom each farm. Medians for this sample of herds were 1,057 lactating cows (range, 524 to 1,466 lactatingcows), 12,150 kg of milk/cow per year (range, 11,773 to 13,091 kg of milk/cow per year), and a bulk tanksomatic cell count of 140,500 cells/ml (range, 100,000 to 267,000 cells/ml).

Clinical scores (mild, severe, and culled/dead) for mastitis were assessed after diagnosis according tothe severity scoring system previously described (32). Milk samples were collected from one affectedquarter of mastitis cases aseptically on the farm and cultured using an on-farm culture system (AccuMast;FERA Animal Health, Ithaca, NY) in the diagnostic laboratory of the farm. Plates with Klebsiella-positivecolonies were sent to the laboratory for further analysis.

Antimicrobial susceptibility testing. All Klebsiella isolates were examined for susceptibility toantimicrobial agents using the Bauer-Kirby disk diffusion method following the Clinical and LaboratoryStandards Institute (CLSI) recommendations (33). A double-disk synergy test for screening ESBL-producing isolates was carried out as described previously (34). An additional determination of theceftiofur MIC was carried out for those ESBL-producing isolates. E. coli ATCC 25922 was used as thecontrol strain.

PFGE. Pulsed-field gel electrophoresis (PFGE) was conducted in accordance with the Pulse Netprotocol (www.cdc.gov/pulsenet) provided by the Centers for Disease Control and Prevention (CDC). ThePFGE patterns were analyzed and clustered using BioNumerics (version 7.6) software (Applied Maths,Kortrijk, Belgium). Different PFGE clusters were represented in alphabetical order. Every band differencewithin a PFGE cluster resulted in adding a numerical order to the pulsed-field cluster.

Capsule locus typing. Isolation of genomic DNA from every strain was performed using a PowerSoilDNA isolation kit (MO BIO Laboratories, Inc, Carlsbad, CA) according to the manufacturer’s instructions.A PCR targeting the capsule polysaccharide synthesis (cps) region in K. pneumoniae was used to identifythe capsular types in this study (35). For the strains that had whole-genome sequencing data, we alsoused the Kaptive tool to identify the whole-capsule synthesis locus (K-locus), based on assembly scaffolds(36).

Whole-genome sequencing. In total, 96 K. pneumoniae isolates with distinguishable PFGE profileswere subjected to whole-genome sequencing. DNA libraries were sequenced on a MiSeq 2000 platform(Illumina, Inc., San Diego, CA). The quality of the original reads was evaluated using the FASTQC tool. TheFASTX-trimmer and Trimmomatic tools were used to do the trimming of sequencing reads (37). Thesequencing reads were assembled into contigs using the SPAdes (version 3.10) algorithm (38), andplasmid sequences were assembled using the plasmidSPAdes algorithm (39). Moreover, the genomes of185 K. pneumoniae KpI isolates from humans hospitalized with septicemia, pneumonia, and woundinfection from several countries and 49 K. pneumoniae KpI isolates from bovines were accessed from aprevious study (18). The bovine isolates from the study of Holt et al. (18) included 20 isolates from cowswith clinical mastitis, 10 isolates from cows with subclinical mastitis, and 19 fecal or rumen isolates.

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Phylogenetic analysis. The core genome SNPs were identified by mapping sequence reads againsta reference genome (K. pneumoniae strain NTUH-K2044, GenBank accession no. NC_012731.1) using theBurrows-Wheeler aligner (40). The plasmid sequences in K. pneumoniae strain NTUH-K2044 were ex-cluded. The Genome Analysis Toolkit (GATK) was used for base quality score recalibration, indelrealignment, and duplicate removal (41) and also for SNP and indel discovery, along with genotyping,using standard hard filtering parameters or variant quality score recalibration according to GATK bestpractices recommendations (42, 43). The alleles at all filtered SNP sites were concatenated to form amultiple-sequence alignment of all 308 K. pneumoniae KpI genomes. To construct a maximum likelihoodphylogeny of the K. pneumoniae isolates, the RAxML program was used to calculate the phylogenetic treefrom the combined alignment file with 1,000 bootstraps (44). The lineages of the phylogenetic tree weredefined using the BAPS (version 6.0) program (45). We used Interactive Tree of Life (iTOL) (46) and FigTree(http://tree.bio.ed.ac.uk/software/figtree/) software to visualize and edit the phylogenetic tree.

Genome annotation and taxonomy. For each de novo assembled genome, coding sequences werepredicted using the Prodigal (version 2.6) program (47) and annotated using the rapid prokaryoticgenome annotation tool Prokka (48). We concatenated all ORFs in all genomes into a single file. We thenclustered all the ORFs into clusters that represented a group of highly similar proteins by using theUCLUST algorithm in the USEARCH program with a 90% identity cutoff (49). We extracted the seedsequence for every cluster group in order to do functional annotation, using the DIAMOND program (50),against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The descriptive statistics offunctional genes in bovine and humans were calculated in JMP Pro (version 12) software (SAS InstituteInc., Cary, NC) using the chi-square test.

Identification of virulence and AMR genes. We detected known virulence genes and sequenceconservation using a direct read mapping approach with the SRST2 program with a coverage cutoff of90% (51). The reference sequence of virulence genes and alleles was retrieved from the K. pneumoniaeBIGSdb database (https://bigsdb.pasteur.fr/), the virulence factor database (VFDB) (52), and the Patho-genFinder database (53). Taxonomic investigation of virulence among genomic sequences was per-formed with the MG-RAST (version 4.0.2) metagenomics analysis server (http://metagenomics.anl.gov) byuploading the raw reads of sequencing to the server. The associations of virulence genes with clinicalcases were calculated in SAS (version 9.3) software (SAS Institute Inc., Cary, NC).

The AMR genes were identified by use of the SRST2 program (51) according to the ResFinderdatabase (54). The MLST of newly sequenced K. pneumoniae isolates was identified using the MLST(version 1.8) program (55). The new alleles were submitted to the MLST database (https://bigsdb.pasteur.fr/klebsiella/klebsiella.html) to assign new STs. Plasmid replicon types were detected using the Plasmid-Finder (version 1.3) program (56).

Accession number(s). The genome assemblies of newly sequenced strains and the plasmid se-quence of pC5 (GenBank accession no. MF953243) have been deposited in the NCBI database underBioProject no. PRJNA414542 and PRJNA400493, respectively.

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/AEM

.02654-18.SUPPLEMENTAL FILE 1, PDF file, 1 MB.SUPPLEMENTAL FILE 2, XLSX file, 0.3 MB.SUPPLEMENTAL FILE 3, XLSX file, 0.02 MB.SUPPLEMENTAL FILE 4, XLSX file, 0.02 MB.SUPPLEMENTAL FILE 5, XLSX file, 0.05 MB.

ACKNOWLEDGMENTSThis work was supported by Agriculture and Food Research Initiative competitive

grant no. 2013-67015-21233 from the USDA National Institute of Food and Agriculture.We thank the team of curators at the Institut Pasteur MLST system (Paris, France) for

importing novel alleles, profiles, and/or isolates at https://bigsdb.pasteur.fr/, SvetlanaFerreira Lima for help in whole-genome sequencing, Helen Korzec and Martin Zinicolafor collecting the samples, Erika Korzune Ganda for help in data analysis, Qi Sun andMinghui Wang for assistance with genomic analysis, Julie Siler for help in antimicrobialsusceptibility testing, Martin Wiedmann for providing the Salmonella sp. strain H9812used in PFGE typing, Eva Heinz for help in capsule locus identification, and DarylHenderson for help in preparing the manuscript.

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