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Genomic Epidemiology of Global Carbapenemase-Producing Enterobacter spp., 2008-2014.

The emergence of carbapenem resistance is a major public health concern because these agents are regarded as one of the last effective therapies available for treating serious infections caused by Enterobacteriaceae (1). Carbapenemases are important causes of carbapenem resistance because they can be transferred between members of the Enterobacteriaceae. The most common carbapenemases among clinical Enterobacteriaceae are the Klebsiella pneumoniae carbapenemases (KPCs) (Amber class A), IMPs, VIMs, New Delhi metallo-[beta]-lactamase (NDMs) (class B or metallo-[beta]-lactamases), and oxacillin (OXA) 48-like (class D) enzymes (2).

Recent surveillance studies have shown that Enterobacter spp. are often the second or third most common Enterobacteriaceae species associated with carbapenemases (3,4). Typically, KPCs are common among Enterobacter spp. from the United States and South America (5). VIMs are often limited to Europe, NDMs to the Indian subcontinent, and OXA-48 to North Africa and the Middle East (5).

Comprehensive global data regarding the different Enterobacter species and molecular epidemiology are currently limited. We designed a study that used short-read whole-genome sequencing to describe the molecular characteristics and international distribution of Enterobacter spp. with different carbapenemases (n = 170) obtained from 2 global surveillance systems during 2008-2014.

Materials and Methods

Bacterial Isolates

We included 170 clinical, nonrepeat Enterobacter spp. collected from 2 global surveillance programs, namely the Merck Study for Monitoring Antimicrobial Resistance Trends (SMART) (2008-2014) and the AstraZeneca global surveillance program (2012-2014), presently known as the INFORM Global Surveillance Study of Antimicrobial Resistance (online Technical Appendix 1 Table 1, article/24/6/17-1648-Techapp1.xlsx; online Technical Appendix 2, The isolates initially underwent phenotypic identification and microdilution panel susceptibility testing, and all carbapenem-nonsusceptible isolates underwent molecular screening for [bla.sub.KPC] [bla.sub.VIM] [bla.sub.NDM] [bla.sub.OXA-4S]-like [bla.sub.IMF] and [bla.sub.GES] as described previously (6). We obtained a total of 142,226 Enterobacteriaceae from the period 2008-2014, and 6,457 (4.5%) were identified as Enterobacter spp.; 682 were nonsusceptible to 1 of the carbapenems, and 170/6,457 (2.6%) were positive for [bla.sub.KPC] [bla.sub.OXA-48] [bla.sub.NDMP] [bla.sub.VIM], and [bla.sub.IMP] and thus included in our study.

Whole-Genome Sequencing

We used the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) to prepare libraries for sequencing. We multiplexed and sequenced samples on an Illumina NextSeq500 for 300 cycles (151 bp paired-end).

Genomic Analysis

We obtained draft genomes by using SPAdes version 3.10.1 (7). We identified species based on the hsp60 gene sequences (8). We created whole-genome phylogenetic trees, including reference strains for identification of E. cloacae complex (9; online Technical Appendix 1 Table 2).

To define the presence of genes and their alleles, we accessed BLAST in combination with the following databases or typing schemes: BLAST (http://blast.ncbi.nlm.nih. gov/Blast), National Center for Biotechnology Information (NCBI) Beta-Lactamase Data Resources (http://www.ncbi., ResFinder (10), PlasmidFinder (11), and Enterobacter cloacae Multilocus Sequence Typing (MLST) Databases ( We classified integrons according to INTEGRALL (

Phylogenetic Analysis

We created a recombination-free, core single-nucleotide polymorphism (SNP)-based phylogenetic tree and identified SNPs by mapping the reads or aligning the genomes against E. xiangfangensis type strain LMG27195 (9) using the RedDog pipeline ( We removed recombination sites according to Gubbins (12) and removed prophages identified by PHAST (13). We included core SNPs and sites that were present in all genomes to create a maximum-likelihood tree using RAxML with the general time-reversible plus gamma substitution model (14). We visualized the tree by using iTOL version 3 (15).

To identify clades within certain sequence types (STs), we used a phylogeny-free population genetics approach of core SNPs, conducting hierarchal clustering analysis with the Bayesian Analysis of Population Structure program (16). We included all 1,048 available Enterobacter spp. genomes in the NCBI Reference Sequence Database ( as of June 20, 2017. An in silico MLST analysis identified 282 STs from 950 typeable genomes. We included a total of 201 genomes of STs 78, 90, 93, 105, 108, 114, and 171 for the clustering analysis (online Technical Appendix 1 Table 3). For each E. hormaechei subspecies or E. xiangfangensis, the hierarchal Bayesian Analysis of Population Structure clustering analysis (16) was conducted with 3 nested levels with a priori upper bound of the number of clusters between one fourth to one half of the total number of isolates. We defined clades by using the second level of clustering.

Sequence Data Accession Numbers

We deposited the sequencing data in the DNA Data Bank of Japan and NCBI (NCBI BioProjects PRJNA259658 and PRJNA398291) databases (accession nos. DRA004879, SRP046977, and SRR2960053-SRR2960159 [SMART isolates] and SRR5939895-SRR5939952 [AstraZeneca isolates]). The sequences of new integrons or genetic environments described in this study were GenBank accession nos. LC224310-2, MF288916-351991, and MF327263-71.


Global Distribution of Carbapenemases among Enterobacter spp.

We included a total of 170 carbapenemase-producing Enterobacter strains in the study. The VIMs (VIM-1, 4, 5, 23, and 31; n = 51 [46 were only positive for VIM, and 5 co-produced OXA-48]) were the most common carbapenemase among this collection, followed by NDMs (NDM-1, 6, and 7; n = 43 [41 were positive only for NDM, 1 also co-produced OXA-48. and 1 co-produced KPC-2]); KPCs (KPC-2, 3, 4, and 5; n = 38 [37 were only positive for KPC, and 1 co-produced NDM]); OXA-48 (n = 31 [25 were only positive for OXA-48, 5 co-produced VIM, and 1 co-produced NDM]); and IMPs (IMP-1, 4, 8, 13, and 14; n = 14). Enterobacter spp. with [bla.sub.VIM] were mostly limited to Europe; isolates with [bla.sub.NDM] were present predominantly in the Balkans, India, and Vietnam; isolates with blaKPC were mainly found in the United States and South America; isolates with [bla.sub.OXA-48] were largely present in North Africa and the Middle East; and isolates with [bla.sub.IMP] occurred mostly in the Philippines, Taiwan, and Australia. The global distribution of isolates from this study was similar to what had previously been reported for other members of Enterobacteriaceae, especially Klebsiella spp. with carbapenemases (5,17).

E. aerogenes Distant from E. cloacae Complex

We identified 10 isolates as E. aerogenes. These results are described in online Technical Appendix 2.

E. xiangfangensis Identified as the Most Common Species

The E. cloacae complex (n = 160) from our study was obtained from intraabdominal (n = 69), urine (n = 56), skin and soft tissue (n = 19), blood (n = 2), and respiratory specimens (n = 14). We identified 8 species among E. cloacae complex (E. xiangfangensis [n = 65], E. hormaechei subsp. steigerwaltii [n = 47], E. cloacae cluster III [n = 14], E. cloacae subsp. cloacae [n = 13], E. cloacae cluster IV [n = 9], E. hormaechei subsp. oharae [n = 6], Enterobacter asburiae [n = 5], and Enterobacter kobei [n = 1]). These species were associated with different types of carbapenemases and showed global distribution (Figure 1; online Technical Appendix 2 Table 1). E. xiangfangensis was frequent in the Balkans (e.g., Croatia, Romania, and Serbia), whereas E. hormaechei subsp. steigerwaltii was mostly prevalent in Greece and Vietnam (online Technical Appendix 2 Table 1). This overrepresentation was attributable to the presence of particular STs among these species (online Technical Appendix 2 Table 2).

Dominant Sequence Types Identified among 4 Species in E. cloacae Complex

E. xiangfangensis from our study comprised 18 different STs, including 1 dominant ST, ST114 (19/65; 29%). E. hormaechei subsp. steigerwaltii comprised 15 different STs, including 2 dominant STs, ST90 (10/47; 21%) and ST93 (14/47; 30%). E. cloacae cluster III comprised 4 different STs, including 1 dominant ST, ST78 (10/14 [71%). All 6 of the E. hormaechei subsp. oharae isolates belonged to ST108 (Figure 1). The remaining species did not contain a dominant ST, and we found new STs among E. cloacae cluster IV (ST832 and ST834) and E. cloacae subsp. cloacae (ST835, ST836, and ST837).

Major and Minor Sequence Types among Enterobacter cloacae Complex

Among the E. cloacae complex, we identified 4 major STs ([greater than or equal to] 10 isolates/ST), ST114, ST93, ST90, and ST78. We also identified 2 minor STs (5-9 isolates/ST), ST105 and ST108.

ST114 (n = 19) from E. xiangfangensis was the most common ST and divided into 4 clades. Isolates representing 3 of the clades (ST114A, ST114B, and ST114C) were from this study, and isolates representing clade ST114D were from a different study (9; Figure 2; online Technical Appendix 2 Table 2). ST114 had a global distribution (Greece, Italy, Kuwait, Morocco, Romania, Serbia, Tunisia, and the United States) and was associated with different carbapenemases (VIM-1, VIM-4+OXA-48, NDM1, KPC-2, and OXA-48) (online Technical Appendix 2 Table 2). The largest clade (ST114A [n = 13]) was present in Serbia, Romania (with [bla.sub.NDM-1]), Tunisia, Morocco, and Kuwait (with [bla.sub.OXA-48]) (online Technical Appendix Table 2). Clade ST114B (n = 4) with [bla.sub.VIM-]1 was obtained from Greece and Italy, and clade ST114C (n = 2; 1 with [bla.sub.VIM-1] and 1 with [bla.sub.KPC-2]) was found in the United States. ST114 is a common global human Enterobacter clone (18) and is also present in companion animals (19). This international clone is associated with various antimicrobial resistance determinants (20) and was responsible for a prolonged nosocomial outbreak involving KPC-3 in the United States (21).

ST93 (n = 14), from E. hormaechei subsp. steigerwaltii, was the second most common ST in this collection and consisted of 1 clade (Figure 3; online Technical Appendix 2 Table 2). ST93 had a global distribution (Australia, Belgium, China, Romania, Spain, Thailand, United States, and Vietnam) and was associated with different carbapenemases (IMP-8, IMP-14, VIM-1, NDM-1, KPC-2, and OXA-48). ST93 was mostly present in Vietnam (n = 7), where it contained [bla.sub.NDM-1] and [bla.sub.OXA-48] (online Technical Appendix 2 Table 2).

ST90 (n = 10), from E. hormaechei subsp. steigerwaltii, and ST78 (n = 10), from E. cloacae cluster III, were the next most common STs in our collection. ST90 was divided into 3 clades; isolates from 2 of the clades (ST90B and ST90C) were from this collection, whereas isolates representing clade ST90A were from a different study (22; Figure 3; online Technical Appendix 2 Table 2). ST90C with [bla.sub.VIM-1] (n = 7) was from Greece, whereas clade ST90B showed an international distribution (ST90C with IMP-4 from Australia, KPC-2 from Canada, and NDM-1 from Romania).

ST78 from E. cloacae cluster III consisted of 1 clade. This ST was associated with VIM-1 (Greece, Italy, and Spain), IMP-4 (Philippines), IMP-8 (Taiwan), and OXA-48 (Turkey) (online Technical Appendix 2 Table 2).

The minor STs, including ST105 and ST108 (both with 6 isolates), were distinguished on the basis of their molecular epidemiology. ST105 from E. xiangfangensis belonged to a single clade and was only present in Croatia, where it contained [bla.sub.VIM-]1 All the E. hormaechei subsp. oharae isolates belonged to ST108, which was divided into 5 clades; isolates from 2 of the clades (ST108C and ST108D) were from this collection, whereas isolates representing the other clades were from different studies (23; Figure 4). Clade 108C (n = 4) was present in Spain with [bla.sub.VIM-1] (n = 2) and China with [bla.sub.IMP-1] (n = 2), and ST108D (n = 2) was found in Australia (with [bla.sub.IMP-4]) and Israel (with [bla.sub.OXA-48]).

[beta]-lactamases, Antimicrobial Resistance Determinants, and Plasmid Analysis

For each of the 170 isolates, we tabulated the study number, GenBank accession number, species, date, country of isolation, ST, and clade. The [beta]-lactamases, antimicrobial resistance determinants, plasmid replicon types, and plasmid STs are shown in online Technical Appendix 1 Table 1 and online Technical Appendix 2.

Genetic Environments Surrounding the Carbapenemase Genes

We were able to successfully characterize the immediate genetic environments surrounding the carbapenemase genes in 8/14 E. cloacae complex with IMP, 28/33 with KPC (including 4 novel structures named KPC-GE01, KPC-GE02, KPC-GE03, and KPC-GE04), 42/42 with NDM (including 4 novel structures named NDM-GE01, NDM-GE02, NDM-GE03, and NDM-GE04), 17/27 with OXA-48 (including 4 novel structures named OXA-GE01, OXA-GE02, OXA-GE03, and OXA-GE04), and 46/51 with VIM (including the novel integrons In1372, In1373, and In1374) (online Technical Appendix 2 Table 3). We have also described the novel structures found in E. aerogenes (online Technical Appendix 2).

The blaKPC were mainly associated with the Tn4401b isoform (including the 4 novel structures), whereas [bla.sub.OXA-48] was always associated with Tn1999 (including the 4 novel structures). Isolates with NDM contained ISA-ba125 upstream and [ble.sub.MBL] downstream of the [bla.sub.NDM], and the [bla.sub.VIM], and [bla.sub.IMP] were situated within diverse class I integrons from various countries (online Technical Appendix 2 Table 2).

Integrons Harboring [bla.sub.VIM-1] Circulating Locally within the Same or between Different STs in Spain, Greece, and Italy

In237 was present in ST78 (obtained in 2013) and ST90C (obtained in 2014) from the same institution in Greece. In916 was identified in ST78 (obtained in 2010) and ST114B (obtained in 2014) from the same institution in Italy. In624 was harbored in ST78, ST96, and ST108 from the same institution in Spain (all obtained in 2010). InS7 was detected in ST98, ST110, and ST141 from 2 different institutions in Greece (obtained in 2010 and 2014). In4873 was identified in ST114B from 2 different institutions in Greece (obtained in 2013) (online Technical Appendix 2 Tables 1, 2). In110 with [bla.sub.VIM-1] was present in ST105 from Croatia (obtained in 2013) and ST520 from Spain (obtained in 2012).

Global Distribution of a Common NDM-1 Genetic Structure

The most common genetic structure immediately surrounding the [bla.sub.NDMs] (named NDM-GE-U.S.) in our collection was identical to that previously described on a 140.8 kb IncA/C plasmid (pNDM-U.S.; GenBank accession no. CP006661.1) found in K. pneumoniae ATCC BAA-2146 with [bla.sub.NDM-1] (24). This bacterium was isolated in 2010 from the urine of a US hospital patient who had previously received medical care in India (25). NDM-GE-U.S., a 3,063bp fragment consisting of [DELTA]ISAba125-[bla.sub.NDM-1]-[ble.sub.MBL]-trp-FdsbC, was present in 16/42 of NDM E. cloacae complex isolates among 14 different STs (88, 90B, 93, 114A, 279, 136, 182, 270, 435, 513, 524, 525, 609, and 832) obtained from Colombia, Romania, Philippines, Vietnam, South Africa, and Kenya (online Technical Appendix 2 Table 3).

We determined the sequence similarity of the isolates with NDM-GE-U.S. to previously sequenced plasmids in the GenBank database. The similarity to pNDM-U.S. ranged from 7% to 81%, suggesting that different plasmids contained NDM-GE-U.S. Twelve of the isolates showed high similarity (range 93%-100%) to pK518_NDM1, a 106.8-kb IncFII plasmid with [bla.sub.NDM-1] from China (GenBank accession no. CP023187). The remaining 4 isolates showed high similarity (range 98%-100%) with the 54-kb IncX3 plasmid pNDM-HN380 (n = 3) from China (26) and the 178.2-kb IncA/C plasmid p6234-178.193kb (n = 1) from the United States (GenBank accession no. CP010391).


The most common carbapenemase among Enterobacter spp. from our study was VIM, followed by NDM, KPC, OXA-48, and IMP. Carbapenemase-producing Enterobacter Spp. was dominated by 2 global species, namely E. xiangfangensis with 1 major clone (ST114) and E. hormaechei subsp. steigerwaltii with 2 major clones (ST90 and ST93). ST114 and ST90 were divided into different clades; some of the clades (e.g., 90C and 114B) were located in certain geographic regions affiliated with specific carbapenemases, whereas other clades (114A and 90B) were distributed globally in association with different types of carbapenemases.

The taxonomy of E. cloacae complex is confusing, and uncertainty still remains about what species make up this complex. In the early 2000s, Hoffmann and Roggenkamp (8) sequenced hsp60 and established 12 genetic clusters (I to XII) in E. cloacae complex. In 2005, the same authors further defined the taxonomy of E. cloacae complex and named cluster VII as E. hormaechei subsp. hormaechei, cluster VI as E. hormaechei subsp. oharae, and cluster VIII as E. hormaechei subsp. steigerwaltii (27). In 2014, Gu et al. (28) described a novel Enterobacter species obtained from sourdough in China named E. xiangfangensis, which clustered closest to E. hormaechei.

The first study that described the global distribution of E. cloacae clones was undertaken by Izdebski et al (18), who performed MLST on 173 cephalosporin-resistant E. cloacae isolates obtained from Israel and several countries in Europe. MLST identified 88 STs among this collection, with ST78, ST114, ST108, and ST66 being the most common and widespread clones. A ST78 isolate was positive for KPC-2, and a ST114 isolate was positive for VIM-1 (18). With the exception of this study from Izedebski et al (18), limited information is available regarding the global distribution of ST93, ST90, ST78, ST105, and ST108 and consists mainly of sporadic reports (29-32).

Chavda et al. (9) characterized 74 carbapenem-resistant Enterobacter spp. (more than half of the isolates were obtained from New Jersey, USA), and most possessed different [bla.sub.KPC]s, whereas only 2 isolates had [bla.sub.NDM-1]. E. xiangfangensis also dominated, and ST171 was the most common clone. ST171 was rare in our collection (n = 4) but did show genetic and geographic diversity. ST171 was divided into 3 clades: 171A, 171B, and 171C (online Technical Appendix 2 Figure). Clades 171B and 171C are associated with [bla.sub.KPC] from the United States and United Kingdom (online Technical Appendix 2 Figure). Clade 171B (n = 2) contained [bla.sub.KPC-2] from Colombia and [bla.sub.NDM-1] from Guatemala. Clade 171A (n = 1) with [bla.sub.NDM-1] was obtained from South Africa, and clade 171C with [bla.sub.KPC-3] was obtained from the United States.

We noted interesting associations and geographic distribution between genetic structures surrounding carbapenemase genes and clades, clones, and species. First, identical genetic structures were situated in various STs within the same or different institutions of the same country (e.g., NDM-GE01 with bla, in Vietnam; In87 and In237 with [bla.sub.VIM-1] in Greece; In916 with [bla.sub.VIM-1] in Italy; In624 with [bla.sub.VIM-1], in Spain; and NDM-GE03 with [bla.sub.NDM-1], in Guatemala). Second, identical genetic structure was present in different STs (ST105 and ST520), from different countries (e.g., In110 with [bla.sub.VIM-1] in Croatia and Spain). Third, different genetic structures were present in the same STs and clades obtained from different countries (e.g., ST78 with In237 from Greece, ST78 with In916 from Italy, ST78 with In624 from Spain, ST114A with NDM-GE02 from Serbia, and ST114A with pNDM-U.S. from Romania). Last, an identical genetic structure (NDM-GE-U.S.) was found in different global species, STs, and clades.

These associations demonstrate that certain mobile genetic elements with carbapenemase genes have the ability to move between clones and clades of Enterobacter spp. on a global scale. This ability is highlighted by ST78 with [bla.sub.VIM-1] within different integrons (In237, In916, and In624) that circulate between various countries (Greece, Italy, and Spain). As some STs are introduced into different countries, they apparently acquire the local genetic elements prevalent in that country. Of special concern is the description of a common NDM genetic structure, named NDM-GE-U.S., previously found on pNDM-U.S. and first described in a K. pneumoniae from the United States (24). NDM-GE-U.S. was present in different species, clones, and clades obtained from 6 countries spanning 4 continents. Sequence similarity analysis suggested that it was present on different types of plasmids (pK518_NDM1 and pNDM-HN380) among Enterobacter spp. with [bla.sub.NDM].

Our results support the current understanding that the carbapenem resistance pandemic is the consequence of circulating clones and the spread of mobile genetic elements. We found that certain clones and clades (ST78, ST90C, ST96, ST114A, ST114C, and ST141) containing particular genetic structures (In87, In624, In916, In237, NDM-GE01, NDM-GE02, and NDM-GE03) and carbapenemases were circulating locally within the same or between different institutions in certain countries (Greece, Guatemala, Italy, Spain, Serbia, and Vietnam). Other global clones and clades (ST90B, ST93, and ST108) contained various genetic structures and carbapenemases.

A limitation of this study was that plasmids harboring carbapenemases were not reconstructed because of the limitations of short-read sequencing (33). The characterization of plasmids is vital to fully comprehend the molecular epidemiology of Enterobacter spp. with carbapenemases, and a follow-up study using long-read sequencing is currently under way. In the meantime, our study highlights the importance of surveillance programs using whole-genome sequencing to provide insight into the characteristics and global distribution of clones and clades as well as their association with mobile genetic elements surrounding the different carbapenemase genes.

This work was supported by the John Mung Program from Kyoto University, Japan (Y.M.) and a research grant from the Calgary Laboratory Services (grant no. 10015169) to J.D.D.P. This work was also supported in part by National Institutes of Health grant nos. R01AI090155 (B.N.K.) and R21AI117338 (L.C.), and the Genome Center for Infectious Diseases grant no. U19AI110819 from the National Institutes of Health's National Institute of Allergy and Infectious Diseases (J.C.V.I.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funding organizations had no role in study design, data collection and interpretation, or the decision to submit the work for publication

Transparency declarations: J.D.D.P. had previously received research funds from Merck and AstraZeneca. P.B. is an employee of AstraZeneca, and M.M. is an employee of Merck. All other authors have nothing to declare.


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(29.) Bedenic B, Sardelic S, Luxner J, Bosnjak Z, Varda-Brkic D, Lukic-Grlic A, et al. Molecular characterization of class b carbapenemases in advanced stage of dissemination and emergence of class d carbapenemases in Enterobacteriaceae from Croatia. Infect Genet Evol. 2016;43:74-82. j.meegid.2016.05.011

(30.) Cao XL, Cheng L, Zhang ZF, Ning MZ, Zhou WQ, Zhang K, et al. Survey of clinical extended-spectrum beta-lactamase-producing Enterobacter cloacae isolates in a Chinese tertiary hospital, 2012-2014. Microb Drug Resist. 2017;23:83-9.

(31.) De Belder D, Faccone D, Tijet N, Melano RG, Rapoport M, Petroni A, et al. Novel class 1 integrons and sequence types in VIM-2 and VIM-11-producing clinical strains of Enterobacter cloacae. Infect Genet Evol. 2017;54:374-8. 10.1016/j.meegid.2017.07.019

(32.) Fernandez J, Montero I, Martinez O, Fleites A, Poirel L, Nordmann P, et al. Dissemination of multiresistant Enterobacter cloacae isolates producing OXA-48 and CTX-M-15 in a Spanish hospital. Int J Antimicrob Agents. 2015;46:469-74.

(33.) Arredondo-Alonso S, Willems RJ, van Schaik W, Schurch AC. On the (im)possibility of reconstructing plasmids from whole-genome short-read sequencing data. Microb Genom. 2017;3:e000128.

December 2016: Zoonotic Infections

* Investigation of and Response to 2 Plague Cases, Yosemite National Park, California, USA, 201 5

* Anomalous High Rainfall and Soil Saturation as Combined Risk Indicator of Rift Valley Fever Outbreaks, South Africa, 2008-2011

* Cutaneous Granulomas in Dolphins Caused by Novel Uncultivated Paracoccidioides brasiliensis

* Vertebrate Host Susceptibility to Heartland Virus

* Whole-Genome Characterization and Strain Comparison of VT2f-Producing Escherichia coli Causing Hemolytic Uremic Syndrome

* African Horse Sickness Caused by Genome Reassortment and Reversion to Virulence of Live, Attenuated Vaccine Viruses, South Africa, 2004-2014

* Streptococcus agalactiae Serotype IV in Humans and Cattle, Northern Europe

* Effect of Live-Poultry Market Interventions on Influenza A(H7N9) Virus, Guangdong, China

* Infectious Dose of Listeria monocytogenes in Outbreak Linked to Ice Cream, United States, 2015

* Baylisascaris procyonis Roundworm Seroprevalence among Wildlife Rehabilitators, United States and Canada, 2012-2015

* Electrolyte and Metabolic Disturbances in Ebola Patients during a Clinical Trial, Guinea, 2015

* Genetically Different Highly Pathogenic Avian Influenza A(H5N1) Viruses in West Africa, 2015

* Highly Pathogenic Reassortant Avian Influenza A(H5N1) Virus Clade in Poultry, Bhutan

* Horizontal Transmission of Chronic Wasting Disease in Reindeer

* Highly Divergent Dengue Virus Type 2 in Traveler Returning from Borneo to Australia

* Unusual Ebola Virus Chain of Transmission, Conakry, Guinea, 2014-2015

* Human Infection with Novel Spotted Fever Group Rickettsia Genotype, China, 2015

Gisele Peirano, [1] Yasufumi Matsumura, [1] Mark D. Adams, [2] Patricia Bradford, Mary Motyl, Liang Chen, Barry N. Kreiswirth, Johann D.D. Pitout

[1] These authors contributed equally to this article.

[2] Current affiliation: Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA.

Dr. Peirano is a research associate at Calgary Laboratory Services and the University of Calgary. Her main research interests revolve around the detection and molecular epidemiology of antimicrobial drug resistance mechanisms among gram-negative bacteria.

Author affiliations: University of Calgary, Calgary, Alberta, Canada (G. Peirano, J.D.D. Pitout); Kyoto University Graduate School of Medicine, Kyoto, Japan (Y. Matsumura); J. Craig Venter Institute, La Jolla, California, USA (M.D. Adams); AstraZeneca Pharmaceuticals LP, Waltham, Massachusetts, USA (P. Bradford); Merck & Co., Inc., Rahway, New Jersey, USA (M. Motyl); Rutgers University, Newark, New Jersey, USA (L. Chen, B.N. Kreiswirth); University of Pretoria, Pretoria, South Africa (J.D.D. Pitout)


Address for correspondence: Johann D.D. Pitout, Calgary Laboratory Services, #9, 3535 Research Rd NW, Calgary, AB T2L 2K8, Canada; email:

Caption: Figure 1. Phylogenetic tree of the different species and sequence types among 160 Enterobacter cloacae complex isolates identified from Enterobacter spp. isolates collected in the Merck Study for Monitoring Antimicrobial Resistance Trends, 2008-2014, and the AstraZeneca global surveillance program, 2012-2014. The tree is rooted with E. cloacae complex Hoffmann cluster IX (Chavda group R) strain 35,699. A total of 369,123 core single-nucleotide polymorphisms were found; 4,010 were used to draw the tree (after phages and recombination sites were excluded). KPC, Klebsiella pneumoniae carbapenemase; NDM, New Delhi metallo-[beta]-lactamase; OXA, oxacillin; ST, sequence type; -, information missing; *, isolate identified in another study. Scale bar indicates nucleotide substitutions per site.
Figure 2. Phylogenetic tree of the different clades among 40
Enterobacter xiangfangensis ST14 isolates identified from
Enterobacter spp. isolates collected in the Merck Study for
Monitoring Antimicrobial Resistance Trends, 2008-2014, and the
AstraZeneca global surveillance program, 2012-2014. The tree is
rooted with E. hormaechei subsp. hormaechei isolate ATCC49162. A
total of 317,867 core single-nucleotide polymorphisms were found;
27,705 were used to draw the tree (after phages and recombination
sites were excluded). The isolates from other studies were negative
for carbapenemases. KPC, Klebsiella pneumoniae carbapenemase; NDM,
New Delhi metallo-[beta]-lactamase; OXA, oxacillin; ST, sequence
type; -, information missing. Scale bar indicates nucleotide
substitutions per site.

AZ_661       ST114     OXA-48,VIM-4    Kuwait     2013
AZ 662       ST114     OXA-48,VIM-4    Kuwait     2013
SMART710     ST114     OXA-48          Tunisia    2012
ems          ST114     --              UK         2014
e2473        ST114     --              UK         2014
e716         ST114     --              UK         2014
AZ 801       ST114     NDM-1           Romania    2014
SMART 1160   ST114     OXA-48          Tunisia    2014
SMART 1109   ST114     OXA-48          Morocco    2013
SMART 1020   ST114     NDM-1           Serbia     2013
SMART661     ST114     NDM-1           Serbia     2012
SMART1269    ST114     NDM-1           Serbia     2014
SMART1261    ST114     NDM-1           Serbia     2014
SMART 1263   ST114     NDM-1           Serbia     2014
SMART1262    ST114     NDM-1           Serbia     2014
SMART1270    ST114     NDM-1           Serbia     2014
eSS2         ST114     --              UK         2014
GN02645      ST114     --              USA        2008
GN02667      ST114     --              USA        2008
SMART27      ST114     VIM-1           USA        2009
BIDMC100     ST114     KPC-2           --         2014
SMART416     ST114     KPC-2           USA        2011
MGH_3        ST114     KPC-3           --         --
MGHJ14       ST114     KPC-3           --         --
MGH177       ST114     --              USA        2015
MGH1S9       ST114     --              USA        2015
ell30        ST114     --              UK         2014
6627         ST114     --              UK         2014
el873        ST114     --              UK         2014
el879        ST114     --              UK         2014
e629         ST114     --              UK         2014
SMART1420    ST114     VIM-1           Italy      2014
MRSM 17626   ST114     VIM-1           USA        2013
AZ_623       ST114     VtM-1           Greece     2013
SMART1147    ST114     VIM-1           Greece     2013
AZ_622       ST114     VIM-1           Greece     2013
34399        ST114     KPC-3           USA        2011
ARLG-2824    ST114     KPC-3           USA        2015
AR LG-2974   ST114     KPC-3           USA        2015
ARLG-2967    ST114     KPC-3           USA        2013

Figure 3. Phylogenetic tree of the different clades among 51
Enterobacter hormaechei subsp. steigerwaltii ST90 and ST93 isolates
identified from Enterobacter spp. isolates collected in the Merck
Study for Monitoring Antimicrobial Resistance Trends, 2008-2014,
and the AstraZeneca global surveillance program, 2012-2014. The
tree is rooted with E. hormaechei subsp. hormaechei isolate
ATCC49162. A total of 317,867 core single-nucleotide polymorphisms
were found; 27,705 were used to draw the tree (after phages and
recombination sites were excluded). The isolates from other studies
were negative for carbapenemases. Clades are grouped by color. KPC,
Klebsiella pneumoniae carbapenemase; NDM, New Delhi
metallo-[beta]-lactamase; OXA, oxacillin; ST, sequence type; -,
information missing. Scale bar indicates nucleotide substitutions
per site.

SMART935     ST204    KPC-5            Puerto Rico    2013
SMART39S     ST204    IMP-8            Taiwan         2011
SMART1234    ST133    IMP-4            Australia      2014
SMART1141    ST62     VIM-4            Hungary        2013
SMART1385    ST62     KPC-2            Argentina      2014
SMART685     ST62     KPC-2            Argentina      2014
AZ BB4       ST521    OXA-48           Jordan         2012
SMART1414    ST190    OXA-48           Turkey         2014
AZ.626       ST461    KPC-3            USA            2014
el605        ST93     OXA-48           Belgium        2013
SMART 1411   ST93     --               UK             2014
e432         ST93     NDM-1            Romania        2014
e783         ST93     --               UK             2014
e153         ST93     --               UK             2014
SMART201     ST93     --               UK             2014
MGH_15       ST93     KPC-2            USA            2010
ECN1H2       ST93     KPC-3            --             --
MGH_6        ST93     KPC-2            USA            2012
CAV1169      ST93     KPC-2            --             --
CAV1668      ST93     KPC-2            --             --
CAV1311      ST93     KPC-2            USA            2012
CAV1411      ST93     KPC-2            USA            2012
MGH_10       ST93     KPC-2            USA            2011
AZ. 598      ST93     KPC-2            USA            2011
SMAKT1276    ST93     NDM-1            --             --
5MART557     ST93     VIM-1            Austria        2012
AZ_605       ST93     IMP-14           Thailand       2014
SMART657     ST93     VIM-1            Spain          2011
SMART312     ST93     IMP-8            China          2012
SMART1488    ST93     NDM-1            Vielram        2012
SMART851     ST93     NDM-1            Vietnam        2010
SMART599     ST93     NDM-1            Vietnam        2014
SMART601     ST93     NDM-1            Vietnam        2014
AZ-756       ST93     OXA-48           Vietnam        2012
SMART1458    ST93     NDM-1, OXA-48    Vietnam        2011
SMART1256    ST93     NDM-1            Vietnam        2011
SWART1351    ST141    VIM-1            Greece         2014
SMART1139    ST141    VIM-1            Greece         2014
SMART134B    ST88     VIM-1            Italy          2014
AZ-578       STUB     KPC-2, NDM-1     Colombia       2014
SMART1009    ST88     KPC-2            Colombia       2013
SMART1267    ST88     KPC-2            Colombia       2014
AZ-564       ST535    KPC-2            USA            2012
SMART1009    ST110    KPC-2            Argentina      2013
SMART1286    ST110    VIM-1            Greece         2014
SMART1267    ST110    VIM-1            Greece         2014
AZ-564       ST175    VIM-23           Mexico         2012
SMART1078    ST510    KPC-2            Colombia       2013
SMART539     ST510    KPC-2            Colombia       2011
SMART446     ST514    VIM-1            Italy          2011
DSM16691     ST906    --               Belgium        -
E248         ST90     --               UK             2014
E2743        ST90     --               UK             2014
InSali10     ST90     --               Portugal       2013
e2260        ST90     --               UK             2014
e2144        ST90     --               UK             2014
SMART1291    ST90     IMP-4            Australia      2014
EcLI         ST90     IMP-4            Australia      2013
SMART1291    ST90     NDM-1            Romania        2013
EclI         ST90     KPC-2            Ecuador        2011
SMART1118    ST90     --               UK             2014
UAH2         ST90     --               UK             2014
e298         ST90     --               UK             2014
e795         ST90     --               UK             2014
e884         ST90     --               UK             2014
e2284        ST90     VIM-4            UK             2014
e1356        ST90     --               UK             2014
e2352        ST90     --               UK             2014
e2129        ST90     VIM-1            Greece         2014

Figure 4. Phylogenetic tree of the different clades among 39
Enterobacter hormaechei subsp. oharae ST108 isolates identified
from Enterobacter spp. isolates collected in the Merck Study for
Monitoring Antimicrobial Resistance Trends, 2008-2014, and the
AstraZeneca global surveillance program, 2012-2014. The tree was
rooted with E. hormaechei subsp. hormaechei isolate ATCC49162. A
total of 317,867 core single-nucleotide polymorphisms were found;
27,705 were used to draw the tree (after phages and recombination
sites were excluded). The isolates from other studies were negative
for carbapenemases. Clades are grouped by color. KPC, Klebsiella
pneumoniae carbapenemase; NDM, New Delhi metallo-[beta]-lactamase; OXA,
oxacillin; ST, sequence type; -, information missing. Scale bar
indicates nucleotide substitutions per site.

ST108:941726609    ST108 --         South Africa    --
1202_ECLO          ST108 --         USA             --
el 251             ST108 --         UK              2014
SMART267           ST108 VIM-1      Spain           2010
SMART268           ST108 VIM-1      Spain           2010
AZ_601             ST108 IMP-1      China           2012
AZ 602             ST108 IMP-1      China           2012
e813               ST108 --         UK              2014
e289               ST10B --         UK              2014
el774              ST108 --         UK              2014
e774               ST108 --         UK              2014
e2276              ST106 --         UK              2014
e633               ST108 --         UK              2014
el337              ST108 --         UK              2014
DSM 16687 *        ST108 --         Germany         --
e247               ST108 --         UK              2014
el28S              ST108 --         UK              2014
SMART 1260         ST108 IMP-4      Australia       2014
el518              ST108 --         UK              2014
el764              ST108 --         UK              2014
AZ_656             ST108 OXA-48     Israel          2013
e965               ST108 --         UK              2014
el0B3              ST108 --         UK              2014
e985               ST108 --         UK              2014
GN04225            ST108 --         USA             2011
GN04369            ST108 --         USA             2011
61222              ST108 --         UK              2014
el227              ST108 --         UK              2014
e1198              ST108 --         UK              2014
e559               ST108 --         UK              2014
e894               ST108 --         UK              2014
e452               ST108 --         UK              2014
e1028              ST108 --         UK              2014
e1386              ST108 --         UK              2014
61772              ST108 --         UK              2014
e2024              ST108 --         UK              2014
e341               ST108 --         UK              2014
61331              ST108 --         UK              2014
e978               ST108 --         UK              2014
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Article Details
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Title Annotation:RESEARCH
Author:Peirano, Gisele; Matsumura, Yasufumi; Adams, Mark D.; Bradford, Patricia; Motyl, Mary; Chen, Liang;
Publication:Emerging Infectious Diseases
Article Type:Report
Date:Jun 1, 2018
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