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The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing beta-Lactamasesof Potential Clinical Relevance

Gudeta, Dereje Dadi; Bortolaia, Valeria; Amos, Greg; Wellington, Elizabeth M. H.; Brandt, KristianKoefoed; Poirel, Laurent; Nielsen, Jesper Boye; Westh, Henrik; Guardabassi, LucaPublished in:Antimicrobial Agents and Chemotherapy

Link to article, DOI:10.1128/AAC.01424-15

Publication date:2016

Document VersionPublisher's PDF, also known as Version of record

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Citation (APA):Gudeta, D. D., Bortolaia, V., Amos, G., Wellington, E. M. H., Brandt, K. K., Poirel, L., ... Guardabassi, L. (2016).The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing beta-Lactamases of Potential ClinicalRelevance. Antimicrobial Agents and Chemotherapy, 60(1), 151-160. https://doi.org/10.1128/AAC.01424-15

The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing�-Lactamases of Potential Clinical Relevance

Dereje Dadi Gudeta,a Valeria Bortolaia,a Greg Amos,b Elizabeth M. H. Wellington,b Kristian K. Brandt,c Laurent Poirel,d

Jesper Boye Nielsen,e Henrik Westh,e,f Luca Guardabassia

Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmarka; School of Life Sciences, Universityof Warwick, Coventry, United Kingdomb; Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmarkc;Medical and Molecular Microbiology Unit, Department of Medicine, Faculty of Science, University of Fribourg, Fribourg, Switzerlandd; Department of ClinicalMicrobiology, University of Copenhagen, Hvidovre Hospital, Hvidovre, Denmarke; Institute of Clinical Medicine, Faculty of Health and Medical Sciences, University ofCopenhagen, Frederiksberg, Denmarkf

The origin of carbapenem-hydrolyzing metallo-�-lactamases (MBLs) acquired by clinical bacteria is largely unknown. We inves-tigated the frequency, host range, diversity, and functionality of MBLs in the soil microbiota. Twenty-five soil samples of differ-ent types and geographical origins were analyzed by antimicrobial selective culture, followed by phenotypic testing and expres-sion of MBL-encoding genes in Escherichia coli, and whole-genome sequencing of MBL-producing strains was performed.Carbapenemase activity was detected in 29 bacterial isolates from 13 soil samples, leading to identification of seven new MBLs inpresumptive Pedobacter roseus (PEDO-1), Pedobacter borealis (PEDO-2), Pedobacter kyungheensis (PEDO-3), Chryseobacte-rium piscium (CPS-1), Epilithonimonas tenax (ESP-1), Massilia oculi (MSI-1), and Sphingomonas sp. (SPG-1). Carbapenemaseproduction was likely an intrinsic feature in Chryseobacterium and Epilithonimonas, as it occurred in reference strains of differ-ent species within these genera. The amino acid identity to MBLs described in clinical bacteria ranged between 40 and 69%. Re-markable features of the new MBLs included prophage integration of the encoding gene (PEDO-1), an unusual amino acid resi-due at a key position for MBL structure and catalysis (CPS-1), and overlap with a putative OXA �-lactamase (MSI-1).Heterologous expression of PEDO-1, CPS-1, and ESP-1in E. coli significantly increased the MICs of ampicillin, ceftazidime, cef-podoxime, cefoxitin, and meropenem. Our study shows that MBL producers are widespread in soil and include four genera thatwere previously not known to produce MBLs. The MBLs produced by these bacteria are distantly related to MBLs identified inclinical samples but constitute resistance determinants of clinical relevance if acquired by pathogenic bacteria.

Soil is an important reservoir of antibiotic resistance determi-nants (1, 2). Several studies have shown that specific antibiotic

resistance genes of high clinical relevance may have originatedfrom environmental bacteria (3–6). However, the origins of resis-tance genes encoding carbapenem-hydrolyzing metallo-�-lacta-mases (MBLs) are largely unknown. MBLs are currently the mostcritical �-lactamases in clinical settings because they are insensi-tive to �-lactamase inhibitors and confer resistance to carbapen-ems, a �-lactam class of last-resort drugs used for treatment ofsevere bacterial infections (7).

Based on amino acid sequences, MBLs are classified as molec-ular class B �-lactamases and are further divided into B1, B2, andB3 subclasses (8). Subclass B1 and B3 MBLs bind two zinc ions(Zn1 and Zn2) in their active sites, employ different metal bindingamino acids (for the Zn2 ligand), and exhibit broad-spectrumactivity (9). Subclass B2 MBLs are mono-Zn enzymes whose ac-tivity is inhibited upon binding the second Zn (10) and havestrong preferences for carbapenem substrates (11). B1 and B3 en-zymes exist across different bacterial genera, whereas B2 enzymeshave been described only in members of the genera Serratia andAeromonas (10, 11).

The most common MBLs in clinical bacteria are NDM, VIM, andIMP (12). In addition, new types of MBLs are continuously emergingin pathogens from unknown reservoirs (13, 14). Identification of un-known MBL-encoding genes occurring in the environment is impor-tant for assessment of the human health risks associated with envi-ronmental development and transfer of carbapenem resistance (15,16). The aim of this study was to investigate the frequency, host range,

diversity, and functionality of MBLs in soil bacteria. Phenotypic andgenotypic characterization of the MBLs detected in the soil microbi-ota revealed the presence of an environmental reservoir of new car-bapenem-hydrolyzing MBLs of potential clinical relevance.

MATERIALS AND METHODSSoil samples. A total of 25 soil samples recovered from six different geo-graphical locations (Algeria, United Kingdom, Germany, Denmark, Nor-way, and Spain) and soil uses (agricultural and nonagricultural) wereanalyzed, including 10 samples collected in previous studies (17–23) (Ta-ble 1). Fifteen samples were collected in this study from 0- to 15-cm depthusing sterile plastic bags and stored at �20°C. All soil samples werethawed at room temperature before processing.

Selective isolation of carbapenem-resistant Gram-negative bacte-ria. A combined approach, including direct plating and selective enrichmentin media with different nutrient contents, was used for isolation of carbap-

Received 19 June 2015 Returned for modification 30 August 2015Accepted 3 October 2015

Accepted manuscript posted online 19 October 2015

Citation Gudeta DD, Bortolaia V, Amos G, Wellington EMH, Brandt KK, Poirel L,Nielsen JB, Westh H, Guardabassi L. 2016. The soil microbiota harbors a diversity ofcarbapenem-hydrolyzing �-lactamases of potential clinical relevance. AntimicrobAgents Chemother 60:151–160. doi:10.1128/AAC.01424-15.

Address correspondence to Luca Guardabassi, lg@sund.ku.dk.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01424-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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enem-resistant bacteria. All media (here defined as “selective” media) weresupplemented with 8 �g/ml of vancomycin for inhibition of Gram-positivebacteria, 100 �g/ml of cycloheximide for inhibition of fungi, and 4 �g/ml ofmeropenem (MP) for selection of carbapenem-resistant bacteria (24). Aftersieving and dispersion by stirring and sonication (25), soil samples were seri-ally diluted up to 10�4 in sterile distilled water. These suspensions were usedfor direct plating of 200-�l aliquots per plate on selective brain heart infusionagar (BHI-A) (nutrient-rich medium) and on selective diluted nutrient brothagar (DNB-A) optimized for growth of taxonomically diverse oligotrophicsoil bacteria (25). In parallel, selective enrichment of meropenem-resistantbacteria was performed by adding aliquots of 1 ml undiluted soil bacterialsuspension separately to 99 ml of selective brain heart infusion broth (BHI-B)and 99 ml of selective diluted nutrient broth (DNB-B). After 48 h of incuba-tion at 25°C under shaking at 150 rpm, the enriched cultures were seriallydiluted up to 10�4, and each dilution was plated as described above. All theagar plates were incubated at 25°C and read daily for 2 weeks. Subsequently,all presumptive carbapenem-resistant isolates displaying unique colony mor-phologies (relative to size, surface texture, color, and margins) and differentmicroscopic appearances (relative to shape and size) were subcultured onselective BHI-A or DNB-A prior to storage in 15% glycerol at �80°C.

Media and antibiotics. Media and antibiotics were purchased fromDifco (Le Pont-de-Claix, France) and Sigma-Aldrich (Steinheim, Ger-many), respectively.

Phenotypic and genotypic characterization of carbapenem-resis-tant bacteria. The modified version of the CarbaNP test proposed byDortet et al. (26) was used for detection of carbapenemase activity inpresumptive carbapenem-resistant isolates from soil. Isolates not lysing in

Tris-HCl buffer (Thermo Scientific, Rockford, IL, USA) were sonicated(two cycles of 30 s at 40% amplitude) in 200 �l of Tris-HCl lysis bufferusing a Sonopuls Ultrasonic homogenizer (Bandelin Electronic GmbH &Co. KG). Imipenem-hydrolyzing isolates were identified by 16S rRNAgene sequencing using universal primers (see Table S1 in the supplemen-tal material). Isolates were assigned to bacterial species based on the 97%cutoff value of nucleotide identity proposed by Stackebrandt and Goebel(27). MBL phenotypic detection was done using MBL MP/MPI Eteststrips (bioMérieux, Marcy I’Etoile, France) containing MP at one extrem-ity and MP plus EDTA (MPI) at the other extremity and under conditionsreported below. Synergy between meropenem and EDTA was consideredindicative of MBL production.

Shotgun cloning. Identification of MBL-encoding genes was at-tempted for all the isolates displaying carbapenemase activity, exceptthose belonging to species or genera in which MBLs have been de-scribed previously (28). Genomic DNA (Easy-DNA kit; Invitrogen,Carlsbad, CA, USA) from the MBL producers was used for shotguncloning in Escherichia coli TOP10 (Invitrogen, Carlsbad, CA, USA) asdescribed previously (28). We used the high-copy-number vectorpZE21MCS (3) or the low-copy-number vector pCF430 (29) to maximizethe chance for cloning efficiency (pCF430 was used when cloning withpZE21MCS did not yield any MBL-producing clone). A total of 2 �l ofligation mixture was transformed into One Shot TOP10 Electrocomp E.coli (Invitrogen, Carlsbad, CA, USA) with a Gene Pulser II (Bio-Rad, CA,USA) according to the manufacturer’s guidelines. Libraries were screenedon Luria-Bertani agar (Difco, Le Pont-de-Claix, France) plates containing30 �g/ml of amoxicillin and 5 �g/ml of tetracycline (pCF430 resistance

TABLE 1 Description of soil samples and metallo-�-lactamase-producing isolates analyzed in this study

Soilidentifier Country Soil type Collection site Reference Metallo-�-lactamase-producing isolate(s)a

1 Denmark Nonagricultural Amagerfælled This study ND2 Denmark Nonagricultural Amagerfælled This study S. maltophilia (n � 3)

P. roseus strain SI-33 (n � 1; PEDO-1)3 Denmark Agricultural Copenhagen (unfertilized) 17 S. maltophilia (n � 3)4 Denmark Nonagricultural Hygum (control site) 18 J. lividum (n � 1)5 Denmark Agricultural Holeby 19 ND6 Denmark Agricultural Lundgaard 20 ND7 Denmark Agricultural Højgaard (Askov) 20 ND8 UK Nonagricultural Warwickshire (Stockton) 21 C. piscium strain Stok-1(n � 1; CPS-1); E. tenax

strain Stok-2 (n � 1; ESP-1)P. kyungheensis strain Stok-3 (n � 1; PEDO-3)

9 Germany Agricultural Dossenheim (control site) 23 S. maltophilia (n � 1)10 Germany Agricultural Dossenheim (control site) 23 ND11 Germany Agricultural Dossenheim (plantomycin treated) 23 ND12 Algeria Nonagricultural Djelfa 22 ND13 Spain Agricultural Abanilla This study ND14 Spain Agricultural Abanilla This study M. oculi strain SB1-3 (n � 1, MSI-1)15 Spain Agricultural Abanilla This study S. maltophilia (n � 1)16 Spain Agricultural Abanilla This study S. maltophilia (n � 1)17 Spain Agricultural Abanilla This study ND18 Spain Agricultural Abanilla This study ND19 Spain Agricultural Abanilla This study S. maltophilia (n � 1)20 Spain Agricultural Abanilla This study ND21 Spain Agricultural Abanilla This study ND22 Spain Agricultural Abanilla This study S. maltophilia (n � 1)23 Norway Nonagricultural Ossian Sars Nature Reserve This study Sphingomonas sp. strain ALS-13. (n � 1; SPG-1)

S. maltophilia (n � 1)J. lividum (n � 1)P. borealis strain ALS-14 (n � 1; PEDO-2)

24 Norway Nonagricultural Ny-Ålesund This study J. lividum (n � 1)25 Norway Nonagricultural Ny-Ålesund This study J. lividum (n � 4)

S. maltophilia (n � 3)a n, number of isolates; ND, not detected.

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marker) or 50 �g/ml of kanamycin (pZE21MCS resistance marker).Amoxicillin was used for screening libraries to avoid false-negative results(30). Amoxicillin-resistant transformants were screened for carbapen-emase production by the modified CarbaNP test as described above. DNAinserts from recombinant plasmids harbored by carbapenemase-produc-ing transformants (recombinant clones) were amplified by PCR (see Ta-ble S1 in the supplemental material) and Sanger sequenced (Macrogen,Seoul, Republic of Korea).

WGS and bioinformatics analysis. Whole-genome sequencing(WGS) was performed on soil MBL-producing strains belonging tospecies for which MBLs have not been described. DNA was extractedusing a DNeasy blood and tissue kit (Qiagen, Düsseldorf, Germany),and normalization of the DNA concentration was performed usingQubit (Invitrogen, Paisley, United Kingdom). Libraries were gener-ated using a Nextera XT DNA Sample Preparation kit (Illumina, CA,USA), and sequencing was performed on an Illumina MiSeq benchtopsequencer (Illumina, CA, USA) using a MiSeq Reagent kit v2 (300cycles) and two 150-bp paired-end reads. Reads from sequencing wereassembled using Velvet v1.0.11 (http://www.ebi.ac.uk/~zerbino/velvet/) and VelvetOptimiser (http://bioinformatics.net.au/software.velvetoptimiser.shtml). Putative MBL-encoding genes in the WGSdata were detected by ARG-ANNOT (31). The MBL three-dimen-sional (3D) structure was predicted with the protein-modeling serverPhyre2 v2.0 (32). In the contigs containing the putative MBL-encodinggenes, open reading frames (ORFs) were predicted and annotated us-ing CLC main workbench v6.8.2 (Qiagen, Aarhus, Denmark), andeach predicted protein was used as a query sequence (BLASTP) tosearch similar conserved domains in the NCBI sequence database.MBLs displaying maximum identity to the query sequences were usedfor amino acid alignments (ClustalW2) and phylogenetic-tree con-struction (FigTree v1.4.2 [http://tree.bio.ed.ac.uk/software/figtree/]).

Screening of MBL production in reference strains. MBL productionwas screened in 17 validly described reference strains displaying close 16SrRNA gene identity (�90%) to selected MBL producers isolated from soilin order to determine whether it was an intrinsic property within mem-bers of the same genus. Pedobacter, Sphingomonas, Chryseobacterium, andMassilia strains were purchased from the CCUG strain collection(Gothenburg, Sweden) and Epilithonimonas strains from the DSMZstrain collection (Braunschweig, Germany). Reference strains werescreened by PCR using primers (see Table S1 in the supplemental mate-rial) targeting the MBL-encoding genes identified through cloning andwhole-genome sequencing. The MBL activity and MIC of meropenemwere determined as described above. MBL production in reference strainsdisplaying meropenem MIC values of �0.5 �g/ml was confirmed by usingCarbaNP test II (33).

MIC determination. The MIC of meropenem was determined byEtest (bioMérieux, Marcy I’Etoile, France) in MBL-producing soil iso-lates, reference strains, and recombinant clones. The Etest was performedaccording to the manufacturer’s instructions, with the exception of theincubation temperature, set at 25°C for MBL-producing soil isolates andreference strains. The MICs of a broader range of �-lactams were deter-mined in the recombinant clones by broth microdilution using the Sen-sititre ESBL plate format (Trek Diagnostic Systems, OH, USA) accordingto standard procedures (http://www.eucast.org/).

Nucleotide sequence accession numbers. The nucleotide sequencesdescribed in this study were deposited in the GenBank nucleotide se-quence database with accession numbers KP109675 to KP109681 and inthe Comprehensive Antibiotic Research Database (CARD [http://aac.asm.org/content/57/7/3348.full]) to enhance early detection in clinical iso-lates by WGS, which is becoming a routine tool for strain typing in clinicalmicrobiology.

RESULTSFrequency and classification of carbapenemase-producing bac-teria isolated from soil. Meropenem-resistant Gram-negative

bacteria were isolated from all 25 soil samples analyzed in thisstudy, leading to a total of 130 isolates. Carbapenemase activitywas detected by CarbaNP test in 29 of these isolates, originatingfrom 13 soil samples, including both agricultural and nonagricul-tural soils (Table 1). These carbapenemase-producing isolates be-longed to the genera Pedobacter, Epilithonimonas, Sphingomonas,Massilia, Chryseobacterium, Janthinobacterium, and Stenotroph-omonas (Table 1). All carbapenemases were classified as MBLsbased on synergy between meropenem and EDTA. Based on16S rRNA gene sequence identity, seven strains were assignedto species or genera that were not known to produce MBLsprior to this study: Pedobacter roseus strain SI-33 (n � 1), Pedo-bacter borealis strain ALS-14 (n � 1), Pedobacter kyungheensisstrain Stok-3 (n � 1), Chryseobacterium piscium strain Stok-1 (n �1), Epilithonimonas tenax strain Stok-2 (n � 1), Massilia oculistrain SB1-3 (n � 1), and Sphingomonas sp. strain ALS-13 (n � 1)(Table 2). The Sphingomonas strain was not assigned to any spe-cies, as the closest species (Sphingomonas hakookensis) displayed16S rRNA gene sequence identity below the cutoff value of 97%(95.71%). The remaining 22 strains were related to Stenotroph-omonas maltophilia and Janthinobacterium lividum, which areknown MBL producers (28).

Genetic context of MBL-encoding genes in soil bacteria .New MBL-encoding genes were identified in presumptive C. pis-cium strain Stok-1 (annotated as blaCPS-1), E. tenax strain Stok-2(blaESP-1), and P. roseus strain SI-33 (blaPEDO-1) by using shotguncloning in E. coli TOP10 cells, and their genetic context was fur-ther examined by WGS (see Table S2 in the supplemental mate-rial). The blaCPS-1 gene was located between two genes encodingputative pirin-C protein (Orfa2) and NADPH-dependent FMNreductase (Orfa3) (Fig. 1A). The blaESP-1 gene was identified on anoperon encoding a putative diphosphomevalonate decarboxylase(Orfb1), two hypothetical proteins (Orfb2 and Orfb3), and a pu-tative bleomycin resistance protein (Orfb4) (Fig. 1B; see Table S3in the supplemental material). The blaPEDO-1 gene was locatedbetween 275-bp (Nr1) and 440-bp (Nr2) noncoding DNA se-quences (Fig. 1C). An operon encoding putative phage tail sheathproteins (Ptp1 and Ptp2), phage tail tube protein (Pttp), andphage base plate (Pbp) was found upstream of Nr1. Nr2 contained16-bp inverted repeats located 57 bp downstream of the blaPEDO-1

stop codon, followed by two genes encoding putative YVTN�-propellin repeat-containing proteins (YVTN1 and YVNT2). Anadditional operon encoding acriflavine resistance protein (AcrB),resistance nodulation cell division protein (RND), and RND fam-ily efflux transporter MFP subunit (MFP) was identified down-stream of the �-propellin repeats (Fig. 1C; see Table S3 in thesupplemental material).

MBL-encoding genes from presumptive P. borealis strainALS-14 (blaPEDO-2), P. kyungheensis strain Stok-3 (blaPEDO-3),Sphingomonas sp. strain ALS-13 (blaSPG-1), and M. oculi strainSB1-3(blaMSI-1) were identified by WGS, since the shotgun clon-ing approach was unsuccessful with these strains. The blaPEDO-2

gene was flanked by genes encoding a putative nuclease (Orfc3)and a putative transcriptional regulator (Orfc4) (Fig. 1D; see Ta-ble S3 in the supplemental material), whereas blaPEDO-3 was lo-cated between two genes encoding a putative GNAT family acetyl-transferase (Orfd3 and Orfd2) (Fig. 1E). The blaSPG-1 gene wasflanked by genes encoding a putative phosphopantothenate syn-thase (Orfe1) and a hypothetical protein (Orfe2) (Fig. 1F; see Ta-ble S3 in the supplemental material). The 3= end of blaMSI-1 over-

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lapped the 5= end of a gene encoding a putative OXA �-lactamase,which was annotated as blaMSI-OXA (Fig. 1G). Genes encoding aputative hypothetical protein and a putative argininosuccinatesynthase were detected upstream of blaMSI-1 and downstream ofblaMSI-OXA, respectively (data not shown).

Sequences of the new MBLs identified in soil bacteria. Thepredicted amino acid sequences of the seven new MBLs from soilwere analyzed, and their evolutionary relationship with previouslydescribed MBLs was shown by phylogenetic-tree analysis (Fig. 2).All zinc-coordinating amino acid residues of subclass B3 MBLswere conserved in PEDO-1, PEDO-2, SPG-1, CPS-1, MSI-1, andESP-1, with the exception of a His-to-Gln substitution at position

116 (standard numbering system) in �-lactamases PEDO-1,PEDO-2, CPS-1, and ESP-1, which was previously reported inGOB �-lactamases (34) (Fig. 3). Similarly to all GOB �-lactamases(GOB-1 to GOB-18), a Met residue was present at position 221 inPEDO-1, PEDO-2, and ESP-1 �-lactamases instead of the Ser res-idue found at this position in other subclass B3 �-lactamases (8).Remarkably, CPS-1 �-lactamase displayed a previously unob-served Leu residue at position 221 (Fig. 3). Based on multipleamino acid sequence alignment, PEDO-1, PEDO-2, CPS-1, andESP-1 �-lactamases showed maximum identity (56 to 75%) toGOB-1 �-lactamase from Elizabethkingia meningoseptica (for-merly Chryseobacterium meningosepticum) (34) (see Table S4 in

TABLE 2 Phenotypic and genotypic traits of metallo-�-lactamase-producing soil strains and closely related reference strains

Soil strainMeropenemMIC (�g/ml)

Reference strain with closest 16S rRNA gene identity to soil straina

Name (accession no.)

16S rRNAidentity(%)

CarbaNP testresult

Meropenem MIC(�g/ml)

P. roseus strain SI-33 8 P. roseus CL-GP80T (DQ112353) 99.78 ND NDP. kyungheensis THGT17 (JN196132) 98.40 ND NDP. borealis DSM19626 (EU030687) 98.13 ND NDP. agri PB92T (EF660751) 98.00 Negative 8P. heparinus CCUG 12810 (AJ438172) 96.29 Negative 0.125P. africanus CCUG 39353 (AJ438171) 95.40 Negative 0.5P. boryungensis CCUG 60024 (HM640986) 95.32 Negative 0.125P. saltans CCUG 39354 (AJ438173) 91.31 Negative 0.064

P. kyungheensis strain Stok-3 �32 P. kyungheensis THGT17 (JN196132) 98.16 ND NDP. agri PB92T (EF660751) 97.92 Negative 8P. roseus CL-GP80T (DQ112353) 97.46 ND NDP. borealis DSM19626 (EU030687) 97.31 ND NDP. heparinus CCUG 12810 (AJ438172) 95.69 Negative 0.125P. africanus CCUG 39353 (AJ438171) 95.62 Negative 0.5P. boryungensis CCUG 60024 (HM640986) 95.17 Negative 0.125P. saltans CCUG 39354 (AJ438173) 90.71 Negative 0.064

P. borealis strain ALS-14 �32 P. borealis DSM19626T (EU030687) 98.94P. agri PB92T (EF660751) 98.52 Negative 8P. kyungheensis THGT17T (JN196132) 97.85P. roseus CL-GP80T (DQ112353) 97.46P. heparinus CCUG 12810 (AJ438172) 95.77 Negative 0.125P. africanus CCUG 39353 (AJ438171) 95.47 Negative 0.5P. boryungensis CCUG 60024 (HM640986) 94.88 Negative 0.125P. saltans CCUG 39354 (AJ438173) 90.64 Negative 0.064

E. tenax strain Stok-2 4 E. tenax DSM 16811 (AF493696) 98.24 Positive 3E. lactis DSM19921 (EF204460) 98.00 Positive 3

C. piscium strain Stok-1 4 C. piscium CCUG 51923 (AM040439) 98.58 Positive 3C. balustinum CCUG 13228 (M58771) 98.58 Positive 1.5C. scophthalmum CCUG 33454 (AJ271009) 98.07 Positive 3

Sphingomonas sp. strain ALS-13 �32 S. hankookensis CCUG 57509 (FJ194436) 95.71 Positive �32S. aquatilis CCUG 48825 (AF131295) 94.93 Positive 8S. adhaesiva CCUG 27819 (D16146) 94.57 Positive NDS. paucimobilis CCUG 6518 (D16144) 94.21 Negative 0.047S. jaspsi CCUG 53607 (AB264131) 94.20 Positive NDS. echinoides CCUG 2870 (AB021370) 94.13 Positive ND

M. oculi strain SB1-3 0.5 M. oculi CCUG 43427AT (FR773700) 98.94M. timonae CCUG 45783 (U54470) 97.14 Negative 0.25

a ND, not determined, as the strain was not available or did not grow on Mueller-Hinton medium.

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the supplemental material). SPG-1 showed the highest amino acididentity (47%) to CAU-1 �-lactamase from Caulobacter crescentus(35), and MSI-1 �-lactamase displayed approximately 40% iden-tity to SMB-1 and AIM-1 �-lactamases from Serratia marcescens(13) and Pseudomonas aeruginosa (36), respectively (see Table S4in the supplemental material). MSI-1 (class B �-lactamase homo-logue) and MSI-OXA (class D �-lactamase homologue) displayedthe highest amino acid identity (64 to 66%) to putative domains inMassilia timonae strain CCUG 45783, where the two domainswere fused to form a putative bifunctional MBL-OXA enzyme.The two domains in the reference strain were annotated as MT-1and MT-OXA (Fig. 1H). MT-1 displayed a predicted 3D structuresimilar to that of SMB-1 from S. marcescens, whereas MT-OXAand MSI-OXA resembled FUS-1 (OXA-85), a narrow-spectrumclass D �-lactamase identified in Fusobacterium nucleatum subsp.polymorphum (37). All metal-coordinating amino acid residues ofsubclass B1 MBLs were conserved in PEDO-3 (data not shown).PEDO-3 showed the highest amino acid identity (55%) toJOHN-1 from Flavobacterium johnsoniae (38) (see Table S5 in thesupplemental material).

Carbapenem resistance in reference strains. Among the 17purchased reference strains, carbapenemase activity was detectedin five out of six Sphingomonas species and in all Chryseobacterium(n � 3) and Epilithonimonas (n � 2) species tested. All these ref-erence strains produced class B �-lactamase, as demonstrated by

inhibition of carbapenemase activity by EDTA using CarbaNP testII. In contrast, no carbapenemase activity was found in the fivePedobacter species and the M. timonae reference strain (Table 2).The MICs of meropenem were determined for only 14 strains,since three Sphingomonas reference strains did not grow on Mu-eller-Hinton agar. The MICs ranged between 1.5 and �32 instrains displaying carbapenemase activity, whereas lower MICs(0.047 to 0.5 �g/ml) were observed for strains without carbapen-emase activity, with the exception of Pedobacter agri PB92, forwhich the MIC was 8 �g/ml.

PCR screening of the reference strains using primers targetingthe seven new MBL-encoding genes revealed the presence of (i)blaCPS-1 in Chryseobacterium balustinum and Chryseobacteriumscophthalmun but not in the C. piscium reference strain CCUG51923; (ii) blaESP-1 in both E. tenax and Epilithonimonas lactis ref-erence strains; and (iii) blaMSI-1 in the M. timonae reference strainCCUG 45783. The remaining four genes (blaPEDO-1, blaPEDO-2,blaPEDO-3, and blaSPG-1) were not detected in any reference strain.

Heterologous expression of MBL-encoding genes in E. coli.The effects of heterologous expression of ESP-1, CPS-1, andPEDO-1 on �-lactam susceptibility were assessed by comparingthe MICs of a broad range of �-lactams between the three carbap-enemase-producing recombinant clones and the wild-type E. coliTOP10. Expression of these MBL-encoding genes conferred resis-tance to ampicillin, cefoxitin, cefpodoxime, and ceftazidime alone

FIG 1 Genetic organization of metallo-�-lactamase-encoding genes from presumptive C. piscium strain Stok-1 (A), E. tenax strain Stok-2 (B), P. roseus strainSI-33 (downstream inverted repeats are underlined) (C), P. borealis strain ALS-14 (D), P. kyungheensis strain Stok-3 (E), Sphingomonas sp. strain ALS-13 (F), M.oculi strain SB1-3 (G), and M. timonae CCUG 45783 with putative bifunctional (PBF) �-lactamase (H).

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or combined with clavulanic acid and determined an increase inthe MICs of cefazolin, cefotaxime (with or without clavulanicacid), and meropenem below the clinical breakpoints (Table 3).The MICs of ceftriaxone and piperacillin-tazobactam were in-creased by expression of ESP-1, but not by expression of CPS-1and PEDO-1. None of the MBLs isolated from soil bacteria af-fected the levels of susceptibility to cefepime.

DISCUSSION

This study shows that carbapenem-hydrolyzing MBLs are wide-spread in soil, as indicated by the recovery of carbapenemase-producing bacteria in 52% of the samples tested. Seven new car-bapenemases were detected in a wide range of bacterial hostsranging from Bacteroidetes (Pedobacter, Chryseobacterium, andEpilithonimonas) to Proteobacteria (Sphingomonas and Massilia).Our study is the first to report MBLs in members of the generaPedobacter, Sphingomonas, Epilithonimonas, and Massilia. Al-though the occurrence of presumptive MBLs has been sporadi-cally reported in environmental bacteria (3, 28, 39), previousstudies were based on either metagenomics, which does not allowidentification of the host bacteria, or culture on nutrient-rich me-

dia, which are known to inhibit growth of a large proportion ofsoil bacteria (25). Moreover, most of the studies did not show thehydrolytic activity of presumptive MBLs toward carbapenems.Knowledge of the bacterial hosts, the genetic organization ofMBL-encoding genes, and the activities of the enzymes is useful topredict the likelihood of horizontal gene transfer to clinically rel-evant species, as well as to facilitate detection of the possible emer-gence of new resistance genes in clinical settings (15, 16).

Phylogenetic-tree and amino acid sequence analyses showedthat the seven MBLs detected in soil belonged to either subclass B1(PEDO-1) or B3 (PEDO-1, PEDO-2, CPS-1, ESP-1, MSI-1, andSPG-1). These new MBLs were distantly related to the most fre-quently detected MBLs in clinical isolates, with predicted aminoacid identities ranging from 40 to 69%. Closely related Pedobacterspecies produced different MBL subclasses, highlighting the diver-sity of MBLs within the genus. MSI-1 in the M. oculi strain wasphylogenetically related to THIN-B, a resident carbapenemaseproduced by J. lividum (28). These two MBLs shared commonancestors with acquired SMB-1 and AIM-1 carbapenemases pro-duced by S. marcescens (13) and P. aeruginosa (35), respectively(Fig. 2). This may suggest that blaSMB-1 and blaAIM-1 could have

FIG 2 Unrooted phylogenetic tree of subclass B1 (red lines) and subclass B3 (blue lines) MBLs. MBLs isolated from clinical and environmental samples areindicated by stars and triangles, respectively. GenBank/EMBL sequence database accession numbers for the previously known MBLs are as follows: IND-1,AF099139; CGB-1, AF339734; EBR-1, AF416700; JOHN-1, AY028464; BlaB1, AF189298; IMP-1, ADI87504; SIM-1, AAX76774; KHM-1, BAH16555; GIM-1,CAF05908; MUS-1, AF441286; TUS-1, AF441287; NDM-1, CAZ39946; VIM-1, CAC35170; SPM-1, CAD37801; FIM-1, JX570731; AIM-1, AM998375; THIN-B,CAC33832; SMB-1, AB636283; CAU-1, AJ308331; BJP-1, NP_772870; L1, ABO60992; POM-1, EU315252; GOB-1, AAF04458; and FEZ-1, CAB96921.

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FIG 3 Alignment of amino acid sequences of subclass B3 metallo-�-lactamases newly described in this study with previously described MBLs. Metal-bindingamino acids (12) are indicated with arrowheads. Position 221 is indicated with a star. Leu 221 in CPS-1 is boxed. GenBank/EMBL sequence database accessionnumbers are as follows: GOB-1, AAF04458; L1, ABO60992; FEZ-1, CAB96921; CAU-1, AJ308331; BJP-1, NP772870; and AIM-1, M998375.

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been acquired from members of Oxalobacteraceae, to which bothMassilia and Janthinobacterium belong.

Remarkable features were observed in some of the new MBLs.CPS-1 contains an unusual, not previously reported Leu residue atposition 221. Position 221 is critical for MBL structure and catal-ysis, as previously shown by the decrease in resistance to ampicil-lin, cefotaxime, and imipenem obtained by in vivo replacement ofMet221 by Cys, Ser, His, Gln, Asp, Glu, and Ile in the GOB enzyme(40). As CPS-1 is related (68%) to GOB enzymes, we hypothesizethat the Leu221 residue has an impact on the catalytic efficiency ofCPS-1 for �-lactam antibiotics. The blaPEDO-1 gene was associatedwith genes encoding putative phage proteins showing structuraland functional homology to phage tail assembly proteins of bac-teriophage T4 (41, 42). Moreover, the gene encoding this MBLwas located between strands of noncoding DNA regions contain-ing inverted repeats, which have been suggested to mediate pro-tein-primed replication of bacteriophages and rolling-circletransposition (43). Altogether, these observations strongly suggestthat blaPEDO-1 is located on a prophage integrated into the chro-mosome of P. roseus strain SI-33. This may have implications forpotential acquisition of blaPEDO-1 by other bacterial species, asphages are known to mobilize antibiotic resistance genes amongenvironmental bacteria (44, 45). Finally, MSI-1 overlapped a pu-tative OXA-type �-lactamase, and both predicted ORFs appearedto be driven by a single promoter. A similar genetic organizationwas detected by examining the published sequence of the M. ti-monae CCUG 45783 reference strain. In this case, the MBL andOXA domains were fused into a putative bifunctional MBL-OXAenzyme (protein accession number WP_005669365.1), althoughhydrolytic activity toward carbapenems associated with MBL pro-duction was not observed. To our knowledge, fusion of class B andclass D �-lactamases has never been reported prior to this study,although the existence of bifunctional enzymes for other classes of�-lactamases has been described previously (38). Possible acqui-sition of bifunctional �-lactamases by clinically relevant species isa reason for concern due to the broad substrate preferences thatsuch enzymes may retain (46).

Analysis of reference strains closely related to the MBL-pro-ducing species identified in this study revealed that carbapen-emase production might be intrinsic to Chryseobacterium and Epi-

lithonimonas, as it was detected in all species tested within thegenera. In contrast, all Pedobacter and single Sphingomonas andMassilia reference strains did not show carbapenemase activity.Most (five out of six) of the Sphingomonas reference strains and C.piscium CCUG 51923 showed carbapenemase activity but did notharbor the MBL-encoding genes found in soil Sphingomonas sp.strain ALS-13 (blaSPG-1) and Chryseobacterium strain Stok-1(blaCPS-1), respectively. This suggests heterogeneity of the MBLswithin these genera, likely including as-yet-undescribed MBLtypes. Although none of the Pedobacter reference strains producedcarbapenemase and harbored blaPEDO1-3, P. agri PB92T showed ahigh (8 �g/ml) meropenem MIC, and analysis of available WGSdata (accession number AJLG00000000) indicated that it har-bored a putative MBL (GenBank protein accession numberWP_010603234.1, displaying 57% and 73% amino acid identity toPEDO-1 and PEDO-2, respectively. Based on 3D structure predic-tion (data not shown), this putative MBL showed significant sim-ilarity to the X-ray structure of FEZ-1 carbapenemase producedby Fluoribacter (Legionella) gormanii (47). However, no carbapen-emase activity was detected in P. agri PB92T, suggesting that theputative MBL is not optimally expressed under the conditions ofthe CarbaNP test. Alternatively, other mechanisms of resistancemay be responsible for the high meropenem MIC value observedfor the strain.

No mobile genetic elements were detected in the regionsadjacent to the MBL-encoding genes, except in blaPEDO-1. Sim-ilarly, progenitors of genes encoding CTM-X and OXA-48�-lactamases, now widespread in clinical Gram-negative iso-lates worldwide, were detected on the chromosome of environ-mental isolates of Kluyvera and Shewanella in the absence of mo-bile genetic elements (5, 6). Interestingly, downstream of theblaESP-1 gene from E. tenax Stok-2 there was a gene encoding aputative bleomycin resistance protein that has been previouslyreported downstream of blaNDM-1 (48, 49), suggesting a possi-ble evolutionary relationship between ESP-1 and NDM-1. Ithas been suggested that environmental opportunistic patho-gens may facilitate transfer of resistance genes between the en-vironment and clinical settings (3). Based on the recent resis-tome risk ranking proposal (16), blaPEDO-1 could qualify forresistance readiness condition 2 (RESCon 2), i.e., potentially

TABLE 3 Effects of heterologous expression of metallo-�-lactamase-encoding genes from E. tenax strain Stok-2 (blaESP-1), C. piscium strain Stok-1(blaCPS-1), and P. roseus strain SI-33 (blaPEDO-1) on MICs of �-lactams in E. coli TOP10

Antimicrobial agent

EUCAST clinicalbreakpoint(�g/ml)b

MIC (�g/ml)

E. coli E. coli pESP-1 E. coli pCPS-1 E. coli pPEDO-1

Meropenema �8 0.032 0.5 0.094 0.094Ceftazidime �4 �0.25 32 4 16Cefazolin � �8 �16 �8 16Cefepime �4 �1 �1 �1 �1Cefoxitin � 8 �64 64 64Cefpodoxime �1 0.5 32 4 2Cefotaxime �2 �0.25 2 0.5 0.5Ceftriaxone �2 �1 2 �1 �1Ampicillin �8 �4 256 64 64Piperacillin-tazobactam �16 �4 16 �4 �4Ceftazidime-clavulanic acid � �0.25 16 4 8Cefotaxime-clavulanic acid � 0.12 2 0.25 0.5a MIC was determined by Etest.b �, data could not be obtained.

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transferable resistance determinants with known mechanismsharbored by a nonpathogen. The remaining MBL-encodinggenes could be grouped as RESCon5, a group conferring resis-tance to currently used antibiotics but not located on mobilegenetic elements.

The new metallo-�-lactamase-encoding genes isolated fromsoil bacteria displayed poor activity against meropenem aftercloning into E. coli (Table 3). Previous studies have shown thatmost of the clinically important MBLs, such as VIM (50, 51) andIMP (52), confer reduced susceptibility, but not resistance, tomeropenem on E. coli laboratory strains despite their high cata-lytic efficiency toward carbapenems (7, 9). This could be due toadditional mechanisms (e.g., porin loss and efflux pumps) or�-lactamases that may correspond to the carbapenem resistancephenotype in the original host (53) or to the high permeabilitycoefficient of carbapenems in E. coli (54).

In conclusion, this study expands the current knowledge ofthe occurrence, host range, diversity, and functionality ofMBL-encoding genes in the soil microbiota. MBL productionwas observed for the first time in members of the genera Pedo-bacter, Epilithonimonas, Sphingomonas, and Massilia, revealingthe existence of seven new MBLs belonging to subclass B1 or B3enzymes. Some of the MBLs produced by soil bacteria dis-played remarkable features, such as integration of the MBL-encoding gene (blaPEDO-1) into prophage, an unusual aminoacid residue at a key position for MBL structure and catalysis(Leu221 in CPS-1), or overlap of blaMSI-1 with a putative OXA-like �-lactamase-encoding gene. These enzymes have not yetemerged in clinical settings but constitute potential carbap-enem resistance determinants in pathogenic bacterial species,as demonstrated by their ability to confer resistance to ampi-cillin and various cephalosporins, as well as reduced suscepti-bility to carbapenems, once expressed in E. coli.

ACKNOWLEDGMENTS

K.K.B. acknowledges the Center for Environmental and Agricultural Mi-crobiology (CREAM) funded by the Villum Foundation. We thank GeirWing Gabrielsen for providing us the soil samples from Svalbard, Nor-way; Rob Willems for providing imipenem/cilastatin; and Roh SeongWoon for the kind gift of Pedobacter agri PB92.

FUNDING INFORMATIONThis study was supported by grant HEALTH-F3-2011-282004 (EvoTAR)from the European Union.

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Erratum for Gudeta et al., The Soil Microbiota Harbors a Diversity ofCarbapenem-Hydrolyzing �-Lactamases of Potential Clinical Relevance

Dereje Dadi Gudeta,a Valeria Bortolaia,a Greg Amos,b Elizabeth M. H. Wellington,b Kristian K. Brandt,c Laurent Poirel,d

Jesper Boye Nielsen,e Henrik Westh,e,f Luca Guardabassia

Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmarka; School of Life Sciences, Universityof Warwick, Coventry, United Kingdomb; Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmarkc;Medical and Molecular Microbiology Unit, Department of Medicine, Faculty of Science, University of Fribourg, Fribourg, Switzerlandd; Department of ClinicalMicrobiology, University of Copenhagen, Hvidovre Hospital, Hvidovre, Denmarke; Institute of Clinical Medicine, Faculty of Health and Medical Sciences, University ofCopenhagen, Frederiksberg, Denmarkf

Volume 60, no. 1, p. 151–160, 2016. Page 156, Figure 2 legend, line 1: “subclass B1 (red lines) and subclass B3 (blue lines) MBLs” shouldread “subclass B1 (blue lines) and subclass B3 (red lines) MBLs.”

Page 156, right column, lines 10 and 11: “B1 (PEDO-1)” should read “B1 (PEDO-3).”

Citation Gudeta DD, Bortolaia V, Amos G, Wellington EMH, Brandt KK, Poirel L,Nielsen JB, Westh H, Guardabassi L. 2016. Erratum for Gudeta et al., The soilmicrobiota harbors a diversity of carbapenem-hydrolyzing �-lactamases ofpotential clinical relevance. Antimicrob Agents Chemother 60:2599.doi:10.1128/AAC.00375-16.

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