High diversity of beta-lactamases in the General Hospital Vienna verified by whole genome sequencing and statistical analysis

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High diversity of beta-lactamases in the General Hospital Vienna verified by whole genome sequencing and statistical analysis

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Q1

Ivan Barišic´ a,⇑, Dieter Mitteregger b, Alexander M. Hirschl b, Christa Noehammer a, Herbert Wiesinger-Mayr a a b

AIT Austrian Institute of Technology, Molecular Diagnostics, Muthgasse 11/2, 1190 Vienna, Austria Medical University of Vienna, Department of Laboratory Medicine, Division of Clinical Microbiology, Währinger Gürtel 18-20, 1090 Vienna, Austria

a r t i c l e

i n f o

Article history: Received 25 March 2014 Received in revised form 6 August 2014 Accepted 15 August 2014 Available online xxxx Keywords: Carbapenemase ESBL AmpC Rarefaction curve Whole genome sequencing Chao1

a b s t r a c t The detailed analysis of antibiotic resistance mechanisms is essential for understanding the underlying evolutionary processes, the implementation of appropriate intervention strategies and to guarantee efficient treatment options. In the present study, 110 b-lactam-resistant, clinical isolates of Enterobacteriaceae sampled in 2011 in one of Europe’s largest hospitals, the General Hospital Vienna, were screened for the presence of 31 b-lactamase genes. Twenty of those isolates were selected for whole genome sequencing (WGS). In addition, the number of b-lactamase genes was estimated using biostatistical models. The carbapenemase genes blaKPC-2, blaKPC-3, and blaVIM-4 were identified in carbapenem-resistant and intermediate susceptible isolates, blaOXA-72 in an extended-spectrum b-lactamase (ESBL)-positive one. Furthermore, the observed high prevalence of the acquired blaDHA-1 and blaCMY AmpC b-lactamase genes (70%) in phenotypically AmpC-positive isolates is alarming due to their capability to become carbapenem-resistant upon changes in membrane permeability. The statistical analyses revealed that approximately 55% of all b-lactamase genes present in the General Hospital Vienna were detected by this study. In summary, this work gives a very detailed picture on the disseminated b-lactamases and other resistance genes in one of Europe’s largest hospitals. Ó 2014 Published by Elsevier B.V.

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1. Introduction Already in 1940, Abraham et al. reported the inactivation of penicillin by an isolated bacterial enzyme that belongs to the very diverse group of b-lactamases comprising four different classes (Abraham and Chain, 1940). This classification system is based on the amino acid sequence similarity of the various enzymes (Ambler, 1980). Additionally, a functional classification system is used based on the different hydrolytic activity of the b-lactamases (Bush and Jacoby, 2010). Class A, C and D b-lactamases have a serine-based active site, and although they share very little DNA sequence similarity, 3D-structure analyses suggest a common protein precursor (Hall and Barlow, 2003, 2004). The catalytic mechanism involves the formation of an acyl-enzyme intermediate. Class B enzymes have a zinc-dependent reaction site and are also referred to as metallo-b-lactamases. The metal-ion is involved in

⇑ Corresponding author. Tel.: +43 66488390643; fax: +43 505504450. E-mail addresses: [email protected] (I. Barišic´), dieter.mitteregger@meduni wien.ac.at (D. Mitteregger), [email protected] (A.M. Hirschl), [email protected] (C. Noehammer), [email protected] (H. Wiesinger-Mayr).

the processing of the substrate and the intermediates (Page and Badarau, 2008). What all b-lactamases have in common is that they hydrolyse the core structure of the b-lactam antibiotics, the b-lactam ring. Today, more than 950 unique, naturally occurring b-lactamases have been described and their number is steadily increasing due to the careless use of antibiotics (Bush, 2010). Studies addressing this huge genetic diversity in clinical environments are rare and focus mainly on the detection of a handful of clinically highly abundant genes. One of the largest studies was conducted by Lascols et al., screening more than 1000 extended spectrum beta-lactamase (ESBL)-positive isolates worldwide for the presence of blaTEM, blaSHV, blaCTX-M, and blaKPC (Lascols et al., 2012). Although the high number of samples is impressive, such studies do not provide information on the statistical significance of the obtained data. Statistical models such as the species richness estimators ACE and Chao1 and the rarefaction curves are used in microbial ecology to estimate the total number of certain genes detectable within an experimental setup (Hartmann and Widmer, 2006; Pitta et al., 2010; Stevens and Ulloa, 2008). A main motivation behind these calculations is to avoid over- or under-sampling when addressing the genetic diversity of a habitat and to facilitate the comparison

http://dx.doi.org/10.1016/j.meegid.2014.08.014 1567-1348/Ó 2014 Published by Elsevier B.V.

Please cite this article in press as: Barišic´, I., et al. High diversity of beta-lactamases in the General Hospital Vienna verified by whole genome sequencing and statistical analysis. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.08.014

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of the genetic diversity of different habitats despite varying sample numbers. Although the dissemination of b-lactamase genes in Europe follows global trends, regional differences can be observed with a higher incidence of b-lactamase-positive strains in southern parts of the continent. The question remains if this gradient exists due to varying healthcare systems, climate conditions, travel behaviour or other factors. Central Europe is located in between the borders of this north-south antibiotic resistance gradient and thus may be an indicator for future trends. The presence of the b-lactamase genes blaTEM, blaSHV, blaCTX-M, blaOXA-1, blaKPC, blaVIM, and blaNDM was already confirmed in Austria, but the investigations focused only on the detection of these genes and information on the presence of other b-lactamases such as plasmid-mediated AmpCs, OXA-48, etc. is missing (Duljasz et al., 2009; Eisner et al., 2006; Heller et al., 2012; Hoenigl et al., 2012; Huemer et al., 2011; Prelog et al., 2008; Zarfel et al., 2011a,b). In the present study, we selected 110 b-lactam-resistant clinical isolates of Enterobacteriaceae from the General Hospital Vienna and screened them for a wide range of b-lactamase genes. These phenotypically ESBL-, AmpC- or carbapenemase-positive isolates were analysed by PCR and the detected genes were Sanger-sequenced, in parallel, the isolates were also characterised with a new padlock probe-based detection assay identifying 31 b-lactamase genes (Barisic et al., 2013). After these characterisations, 20 isolates were selected for whole genome sequencing (WGS) using the IonTorrent platform, and in the subsequent sequence data analyses, the isolates were not only screened for b-lactamases but also other antibiotic resistance genes. WGS of an isolate was performed if Sanger-sequencing identified a new gene not listed in Genbank or if the observed antibiotic resistance phenotype could not be explained using PCR and the padlock probe assays. Subsequently, the obtained DNA sequence data was used for statistical calculations estimating the total number of the b-lactamase genes in the General Hospital Vienna.

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2. Materials and methods

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2.1. Susceptibility testing and phenotypical detection of resistance mechanisms

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The clinical isolates were tested for antibiotic susceptibility on Mueller-Hinton agar plates, using the disc diffusion test according to the EUCAST (European Committee on Antimicrobial Susceptibility Testing) recommendations (versions 1.2 and 1.3, The European Committee on Antimicrobial Susceptibility Testing – EUCAST 2011). In the case of resistance to third generation cephalosporins and/or cefoxitin in disc diffusion testing, Enterobacteriaceae known to possibly harbour plasmid-encoded AmpC enzymes based on phenotypical testing were screened for the presence of AmpC b-lactamases by a combined-disc test (AmpC ID Confirm Kit; Rosco Diagnostica A/S, Taastrup, DK) according to the manufacturer’s instructions. In organisms that produce an inducible chromosomal AmpC b-lactamase, identification alone was indicative of AmpC production (Thomson, 2010). As in primary disc diffusion testing, extended-spectrum cephalosporins (cefuroxime 30 lg, cefpodoxime 10 lg, cefotaxime 5 lg, cefepime 30 lg,) were put around a central amoxicillin-clavulanate (20 lg-10 lg) disc in a way to enable inhibitor-based synergisms. ESBL-producing Enterobacteriaceae could be recognized based upon observation of this defining criterion (Moland and Thomson, 1994). In the case of cefoxitin resistance and lacking synergism phenomena, a 20 mm-double-disc synergy test between an amoxicillin-clavulanate and a cefepime disc was performed to avoid missing ESBL-positive phenotypes masked by concomitant AmpC production (Garrec et al., 2011). According to EUCAST antimicrobial wild-type distributions of

microorganisms (http://mic.eucast.org/Eucast2/SearchController/ search.jsp?action=performSearch&BeginIndex=0&Micdif=dif&NumberIndex=50&Antib=177&Specium=-1&Discstrength=10), meropenem inhibition zone diameter 6 0.25 mm in primary disc diffusion testing was chosen to screen isolates with elevated carbapenem minimal inhibitory concentrations (MIC), which had to undergo confirmatory phenotypic testing in cases where meropenem Etest (bioMérieux, Marcy-l’Étoile, France) revealed MICs P 0.5 mg/l (http://mic.eucast.org/Eucast2/SearchController/search.jsp?action =performSearch&BeginIndex=0&Micdif=mic&NumberIndex=50& Antib=177&Specium=-1). Ambler class A b-lactamases, metallo-blactamases, or AmpC b-lactamases in combination with efflux pumps or reduced permeability were recognized phenotypically by KPC + MBL Confirm ID Kit (Rosco Diagnostica A/S, Taastrup, DK), while Ambler class D b-lactamases where detected by the modified Hodge test (Lee et al., 2010). The KPC + MBL Confirm ID Kit identifies a reduces permeability with AmpC b-lactamases only and not with class A and B b-lactamases. The minimum inhibitory concentration (MIC) values were only recorded for meropenem in carbapenemase-positive isolates.

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2.2. Bacterial isolates and DNA extraction

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For this study, 110 phenotypically ESBL-, AmpC- and carbapenemase-positive clinical isolates collected at the Division of Clinical Microbiology of the Medical University and General Hospital Vienna between January and September 2011 were genetically characterised (Supplementary Table 1). These consisted of 56 consecutively selected ESBL-positive bacterial strains from an equal number of different patients between January and April 2011 and 54 isolates comprising b-lactamases other than ESBLs consecutively selected between June and September 2011. The ethical clearance to carry out this study was obtained from the ethics committee of the Medical University of Vienna (approval number: 2137_001). The isolates were grown on BD Columbia blood agar plates with 5% sheep blood (BD Diagnostic Systems, Sparks, USA) at 37 °C overnight. Cells were transferred with an inoculation loop into 1.5 ml microcentrifuge tubes with 500 ll ddH2O, incubated at 100 °C for ten minutes and plunged into liquid nitrogen for one minute. The thermal cell lysis was repeated two more times. Upon centrifugation at 13,000 rpm for 1 min, the supernatant was used for the subsequent PCR and the padlock probe assay as DNA template. For the WGS experiments, genomic DNA was extracted using the NucleoSpin Tissue Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions.

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2.3. DNA amplification, Sanger-sequencing and padlock probes assay

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Amplification reactions were conducted using both new primers listed in Supplementary Table 2 and published primers (Supplementary Table 3) to double-check the PCR results. The new primers were designed using the software package ARB (Ludwig et al., 2004) and quality checked using the NetPrimer software (Premier Biosoft, 2012). The final primer concentration was 1 lM in the PCR mix. The reagents of the Mastermix 16S Basic PCR kit (Molzym, Bremen, Germany) were used at a total volume of 50 ll. One microlitre of the cell lysate was added per reaction. The thermal cycling was carried out by an initial denaturation step at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing for 30 s (annealing temperatures listed in Supplementary Tables 2 and 3), elongation at 72 °C for 80 s, and a final elongation cycle at 72 °C for 10 min. Negative controls without DNA template were used in all experiments. The PCR products were subsequently purified according to the manufacturer’s protocols using the MinElute PCR Purification kit (Qiagen, Hilden,

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Germany) and the MinElute Gel Extraction kit (Qiagen), respectively and finally bi-directionally Sanger-sequenced (Microsynth, Balgach, Switzerland). Additionally, the clinical isolates were analysed using the multiplex padlock probe assay designed to detect the 33 clinically most prevalent b-lactamase genes. The experimental details are described in a previous publication (Barisic et al., 2013). In brief, the protocol started with the enzymatic digestion of 10 ll of the genomic DNA by the restriction enzymes AluI and HpyF3I followed by the digested DNA being incubated with padlock probes which were ligated in the presence of a DNA target. To enrich the captured DNA target, two rolling circle amplifications (RCA) were performed interrupted by an enzymatic digestion. This amplification reaction is also referred to as circle-to-circle amplification (C2CA). Then, the RCA-products were fluorescently labelled using a linear PCR and the labelled DNA was transferred to a microarray slide. Upon microarray hybridisation, the slide was washed, scanned and processed using the GenePix Pro 6.0 software. The microarray data was analysed using Microsoft Excel 2007.

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2.4. Whole genome analysis

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WGS of an isolates was performed if a b-lactamase gene not listed in Genbank was identified or if any b-lactamase gene responsible for the observed phenotype was not detected. The bacterial genomes were sequenced using the IonTorrent PGM platform (Life Technologies, Carlsbad, USA) according to the manufacturer’s instructions. The Ion Xpress Plus Fragment Library Kit was used to enzymatically shear 100 ng of the genomic DNA. The target fragment size was 400 bp. Subsequently, the fragmented DNA was processed using the Ion DNA Barcoding kit (Life Technologies) and its size selected using the E-Gel SizeSelect 2% Agarose kit (Life Technologies). The size distribution of the DNA fragments was analysed using the High Sensitivity Kit (Agilent, Santa Clara, USA). Further sample processing was performed using the Ion OneTouch Kit (Life Technologies). Finally, the amplified DNA was sequenced using the 318 chip (Life Technologies). The single reads obtained were de novo assembled using MIRA 3.9.9, which is part of the Assembler plugin on the Ion Torrent server. Subsequently, the contigs were aligned to a reference genome of the corresponding species using Mauve and submitted to the RAST analysis platform (Aziz et al., 2008; Darling et al., 2004). RAST is an automated annotation platform for bacterial genomes. The annotated genes were screened in RAST for antibiotic resistance genes and their genetical context. Additionally, the contigs were analysed using the ResFinder web-service (Zankari et al., 2012).

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2.5. Diversity analyses

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BLAST analyses and sequence comparisons in ARB were performed to classify the identified sequences and calculate a phylogenetic tree of detected b-lactamases using ARB. The biostatistical software EstimateS 9.1 was used to calculate the rarefaction curve, Chao1 and ACE values (Colwell, 2013). The rarefaction curves were calculated using 100 randomisations. Species richness estimators ACE and Chao1 estimate the point when further sampling efforts would not result in the discovery of new species. These asymptotic estimators use the information on the distribution of rare species for their calculations. In this study, we used these species richness estimators to calculate the number of different b-lactamase genotypes and b-lactamase genes in the General Hospital Vienna. The genetic diversity of phenotypically ESBL-, AmpC-, or generally b-lactamase-positive (including also carbapenemases) isolates was compared. The input parameters for the estimators were the following (Fig. S1A): a b-lactamase gene-group was defined as the set of genes that was detected by a single primer pair. A b-lactamase gene coded for a unique amino

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acid sequence. A b-lactamase genotype harboured a unique combination of b-lactamase genes. The workflow of the statistical analyses is illustrated in Fig. S1B. Furthermore, data matrices comprising the results from the study of Lascols et al. were created and also analysed using EstimateS 9.1.

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2.6. Nucleotide sequence accession numbers

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The nucleotide sequences determined in the present study using Sanger-sequencing were submitted to GenBank under the accession numbers JX268601 to JX268788. The draft genomes are accessible under the accession numbers CBWA010000001-CBW A010000440 (#1, Escherichia coli), CBWB010000001-CBWB01000 0408 (#5, E. coli), CBWC010000001-CBWC010000367 (#9, E. coli), CBWD010000001-CBWD010001888 (#10, K. pneumoniae), CBWE0 Q2 10000001-CBWE010000084 (#15, M. morganii), CBWG01000000 1-CBWG010000204 (#25, E. coli), CBWH010000001-CBWH010000 132 (#29, E. coli), CBWI010000001-CBWI010000427 (#33, K. pneumoniae), CBWJ010000001-CBWJ010000196 (#35, E. coli), CBWU01 0000001-CBWU010000990 (#39, K. pneumoniae), CBWK0100000 01-CBWK010000894 (#43, K. pneumoniae), CBWL010000001-CB WL010000957 (#46, K. pneumoniae), CBWM010000001-CBWM010 000898 (#53, K. pneumoniae), CBWN010000001-CBWN010000196 (#C7, E. coli), CBWO010000001-CBWO010000348 (#C8, E. cloacae), CBWP010000001-CBWP010000097 (#C11, E. coli), CBWQ0100000 01-CBWQ010000979 (#C21, K. pneumoniae), CBWR010000001-CB WR010000121 (#C41, E. coli), CBWT010000001-CBWT010001186 (#C56, E. coli).

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3. Results

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3.1. Distribution of b-lactamases in Enterobacteriaceae

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A total of 657 isolates of Enterobacteriaceae were identified by phenotypic tests as ESBL-, AmpC- or carbapenemase-positive in the General Hospital Vienna in 2011. In the present study, 110 isolates from 110 different patients were analysed, where in 93 of these clinical strains, 208 b-lactamase DNA sequences comprising 12 b-lactamase gene-groups and 29 b-lactamase genes were detected using PCR, the padlock probe assay and WGS (Table 1). b-Lactamase genes of all four classes were detected which is indicating the high genetic diversity in the hospital and is also illustrated in a phylogenetic tree (Fig. S2). Two carbapenem-resistant (Meropenem MIC > 8 mg/l) and three isolates with decreased carbapenem susceptibility (Meropenem MIC > 2 mg/l) were among the consecutively sampled isolates comprising AmpC- and carbapenemase-positives. The carbapenem resistance was mediated in two K. pneumoniae isolates by blaKPC-2, in one E. coli strain by blaKPC-3 and in one E. cloacae isolate by blaVIM-4. No carbapenemase genes could be identified in the E. cloacae #C8 isolate with decreased carbapenem susceptibility. Thus, the whole genome of this strain was sequenced. The strain #C8 was most closely related to E. cloacae subsp. cloacae NCTC 9394 according to the BLAST analysis (86% query coverage, 98% sequence similarity). The yet uncharacterized blaACT-16 variant and a chromosomal AmpC gene (97% sequence similarity to the AmpC gene of E. cloacae subsp. cloacae NCTC 9394) were identified as the only b-lactamase genes in the isolate #C8. The lack of functional porins or the presence of efflux pumps can significantly reduce the amount of b-lactams in the periplasm, and thus, increase MIC values in the presence of AmpC b-lactamases resulting in a carbapenemase-positive phenotype (Jacoby, 2009). The translated ompC nucleotide sequence lacked the first 14 amino acids having its start codon at amino acid position 15, and thus, the isolate #C8 had a mutated ompC gene explaining the decreased carbapenem susceptibility.

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Table 1 Detected b-lactamase genes and their observed frequencies. The b-lactamase-positive isolates include AmpC-, ESBL-, carbapenemase-, and unclassified b-lactamase-positive strains. Frequency of observed b-lactamase genes in Gene-groups Genes

93 b-lactamase-positive isolates

ACT

1

54 ESBL-positive isolates

5 carbapenemase-positive isolates 1

blaACT-16

1

CMY-2 group blaCMY-2 blaCMY-4 blaCMY-42 blaCMY-109

14

CTX-M-1 group blaCTX-M-1 blaCTX-M-15 blaCTX-M-55

32

CTX-M-9 group blaCTX-M-14 blaCTX-M-27 blaCTX-M-38

18

DHA

21

1 12

2

10 2 1 1

8 2 1 1

2

30 5 26 1

1 3 26 1

1

17 9 3 6

8 3 6 19

blaDHA-1 KPC

32 AmpC-positive isolates

2

21

19

2

3

3 2 1

blaKPC-2 blaKPC-3 OXA-1 group blaOXA-1

37

OXA-9 group blaOXA-9

1

OXA-24 group blaOXA-72

1

SHV

2 1 18 18

1

1 1

1

1 18

blaSHV-1 blaSHV-2 blaSHV-5 blaSHV-11 blaSHV-12 blaSHV-26 blaSHV-33

17

3 4 1 27 2 2 1

2 1 1

1 9

blaTEM-1 blaTEM-30 blaTEM-32 blaTEM-135

4 3 4 1 7 1

18

39

VIM

19 1

40

TEM

19

37

28

36 1 1 1

6 1 1 1

2 28

2

1

1

blaVIM-4

1

1

Table 2 Observed versus calculated number of b-lactamase gene-groups, genes and genotypes in Enterobacteriaceae in the General Hospital Vienna obtained by the species richness estimators. ESBL-positives

b-Lactamase-positives

Gene-groups Genes b-Lactamase genotypes

332 333 334 335 336 337 338 339 340

AmpC-positives

Observed

ACE

Chao1 ± SD

Observed

ACE

Chao1 ± SD

Observed

ACE

Chao1 ± SD

12 29 43

25 53 94

18 ± 7 50 ± 16 109 ± 41

9 19 25

11 26 44

10 ± 2 31 ± 13 46 ± 16

5 11 13

5 21 48

5±0 23 ± 17 61 ± 58

A high prevalence (70%) of the plasmid-mediated AmpC b-lactamases CMY (blaCMY-2, blaCMY-4, blaCMY-42, blaCMY-109) and DHA-1 was observed in the phenotypically AmpC-positive isolates. blaCMY-109 is a novel gene variant identified in the phenotypically AmpC-positive E. coli isolate #C11 from a stool sample. The gene was initially identified using PCR and Sanger sequencing, the sequence of this novel gene was confirmed using WGS, and the gene number blaCMY-109 was assigned by George Jacoby (Lahey Clinic Medical Center). It is most closely related to blaCMY-84 (99%

sequence similarity) and blaCMY-66 (99%), and clusters into the blaCMY-2 group with 14 amino acid substitutions distributed over the whole gene in comparison to blaCMY-2. Interestingly, in AmpCpositive isolates blaCMY was only present in combination with blaTEM (n = 8) or as only b-lactamase gene (n = 4), and all blaTEM subtypes (blaTEM-30, blaTEM-32, blaTEM-135) other than blaTEM-1 were only observed in strains carrying a blaCMY gene (Supplementary Table 4). The blaDHA-1 gene was identified in 43% of the phenotypically AmpC-positive isolates and beside one ESBL-positive

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M. morganii isolate (#15), all genes were detected in K. pneumoniae isolates. As expected, the CTX-M genes were the dominant resistance mechanism in the ESBL-positive isolates (84%). Initially, no CTX-M genes could be detected in five ESBL-positive isolates using PCR but were identified by subsequent WGS. Furthermore, the ESBL-positive K. pneumoniae isolate (#22) comprised the carbapenemase gene blaOXA-72 which is the first occurrence of this gene in a member of the family of Enterobacteriaceae. The gene was found in combination with the other b-lactamase genes blaCTX-M-15, blaTEM-1 and blaSHV-33 using PCR and Sanger-sequencing. Despite the presence of a carbapenemase gene, the strain was characterised phenotypically as ESBL-positive. In one ESBL-positive K. pneumoniae (#39) isolate the phenotype could not be resolved using WGS either. In summary, we are reporting the emergence of several b-lactamase genes in Austria: blaACT-16, blaCMY-2, -4, -42, -109, blaCTX-M-27, -38, blaDHA-1, blaOXA-9, blaOXA-72, blaSHV-26, -33, blaTEM-32, -50, -135, and blaVIM-4.

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3.2. Occurrence of other resistance mechanisms

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The WGS data of the 20 sequenced isolates was also screened for antibiotic resistance genes other than b-lactamases using the Web tool ResFinder. The technical sequencing results such as total read numbers, coverage, etc., are shown in Supplementary Table 5. Resistance mechanisms against eight additional classes of antibiotics were detected (Supplementary Table 6) where one of the most important among the detected genes is aac(60 )–lb–cr having been detected in 45% of the sequenced isolates. The gene is coding a bifunctional enzyme which is active against aminoglycosides and fluoroquinolones. Arr genes coding for the ADP-ribosyltransferases responsible for rifampicin inactivation are rather rarely in E. coli identified resistance mechanisms. The arr-3 gene was identified in the E. coli isolate #C41, and thus, this is the first report of this gene in E. coli. Furthermore, the fosA gene conferring fosfomycin resistance was identified in 40% of the sequenced isolates and was highly abundant in the sequenced K. pneumoniae isolates (75%) but also detected once in E. coli (#C56) and in E. cloacae (#C8). The high incidence of this resistance gene was surprising due to the infrequent clinical use of fosfomycin. The erythromycin resistance methyltransferase gene erm(42) confers high resistance to lincosamides and low to moderate resistance to macrolide and streptogramin B antibiotics. It was only detected in members of the family Pasteurellaceae and the species Mycobacterium massiliense so far (Michael et al., 2012). The only known gene that is related to erm(42) was erm(41) with a nucleotide sequence similarity of 95%. The other erm genes did not have any significant sequence similarities. In this study, the erm gene was found on a plasmid in the ESBL-positive M. morganii isolate #15, and thus, we are reporting erm(42) in a member of the order Enterobacteriales for the first time. Interestingly, the sequenced isolates had an average of 11 different resistance genes against five different classes of antibiotics. The minimum number of different resistance genes observed in isolates analysed using WGS was two, the highest number was 18 (Supplementary Tables 4 and 6).

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3.3. b-Lactamase diversity in the General Hospital Vienna

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In total, 43 different b-lactamase genotypes were detected. The b-lactamase genotype most frequently detected was observed 17 times in AmpC-positive K. pneumoniae isolates comprising blaDHA1, blaOXA-1, and blaSHV-11 (Supplementary Table 4). The second most common genotype contained blaCTX-M-15 and blaOXA-1 in ESBLpositive E. coli (n = 8). Other genotypes were less dominant and identified only four times or less. The MLST analysis based on the WGS data and the Web tool MLST 1.7 (Larsen et al., 2012) revealed

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408 409 410 411

5

the presence of 3 isolates (#29, #35, #C41) belonging to the same sequence type ST-131 whereas other sequence types were only present once (Supplementary Table 6). In total, 72% of the b-lactamase genes that were detected using WGS (excluding chromosomal AmpC genes) were located on plasmids (Supplementary Table 4). Among the 22 plasmid groups detected using the WGS data and the PlasmidFinder Web tool (Carattoli et al., 2014), the most frequently encountered ones were FIA (n = 8/20; AP001918), FIB(K) (n = 9/20; JN233704) and FII(K) (n = 8/20; CP000648) which in general do not comprise b-lactamases (Supplementary Table 7). Also the genetic context of the detected b-lactamase genes was analysed identifying dominant IS26 transposons comprising blaOXA-1 and aac(60 )–lb–cr, blaCTX-M associated ISEcp1 transposons and Tn2 and Tn3 transposons comprising blaTEM-1. The transposon of the isolates #43, #46 and #53 shared the same resistance region (Supplementary Table 7). Additionally, the total number of b-lactamases present in the General Hospital Vienna was estimated using the statistical algorithms Chao1, ACE and rarefaction curves (Table 2). In general, AmpC-positive isolates showed a lower genetic diversity of b-lactamases than ESBL-positive ones (Fig. 1). The rarefaction curve in Fig. 1A showing the number of genegroups in AmpC-positive isolates is the only one approaching the horizontal asymptote prior to the extrapolation point, which shows that the majority of the b-lactamase genes in these isolates were detected and that further analyses in the General Hospital Vienna would not reveal any new gene-groups which is also supported by the Chao1 and ACE models (Table 2). However, further analyses of all b-lactamase-positive isolates including especially carbapenemase-positive will lead to the discovery of several undetected gene-groups in the hospital. Depending on the estimation model, 48% (ACE) and 67% (Chao1) of the total b-lactamase gene-groups present in the hospital were detected in this study. In addition, the estimations reveal that 55% (ACE) and 58% (Chao1) of the total b-lactamase genes have been identified. The genes present in ESBL-positive isolates were largely identified (73% ACE; 61% Chao1), whereas in AmpC-positives 52% (ACE) and 48% (Chao1) of the b-lactamase genes were detected, respectively. In contrast, the observed b-lactamase genotype diversity was too high to calculate reliable estimations, which is reflected by the high standard deviations (Table 2) and the linear slope structures prior to the extrapolation point (Fig. 1C). As a consequence, the extrapolation of the rarefaction curves is not reliable apparent from the crossing-over of the ESBL and AmpC lines. Yet, the high number of observed b-lactamase genotypes (n = 43) among the 110 isolates and the evidence of many additional b-lactamase genotypes in the hospital represent an interesting observation raising questions regarding the responsible genetical dynamics.

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3.4. Global diversity analyses of ESBLs

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The presented biostatistical calculations are very useful to determine appropriate sample numbers and thus, avoid underand over-sampling, where in particular, the latter leads to unnecessary high costs and loss of time. The data from a large-scale study of Lascols et al. was used to illustrate the advantages of the used biostatistical models in more detail (Lascols et al., 2012). In their study, they analysed 1093 phenotypically ESBL-positive isolates from 35 countries obtained from 2008 to 2009 and screened the isolates for the presence of the genes of CTX-M group-1, -2, -8/ 25, -9, SHV, TEM, and KPC. Our rarefaction curve, Chao1 and ACE values showed that the lowest genetic diversity of ESBL genes was observed in Asia, and that all ESBL gene-groups and genotypes (gene-group based resolution) present in the investigated environments in Asia were identified (Fig. 2A and C and Table 3). Furthermore, the rarefaction curves illustrate that the analysed sample

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A

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Number of gene-groups

18 16 β-Lactamase-positive ESBL-positive AmpC-positive

14 12 10 8 6 4 2

In contrast, more isolates should have been screened to address the complete genetic diversity of ESBL genes in Africa, North America, the Middle East, and the South Pacific. The highest b-lactamase diversity was observed in Latin America. Unfortunately, no meaningful estimations can be calculated from the African data due to the low sample number. The varying distribution of ESBL genes in E. coli and K. pneumoniae isolates was surprising. The b-lactamase diversity in K. pneumoniae isolates was significantly higher compared to the average diversity in all species and in E. coli, respectively (Fig. 2B and D). Interestingly, although more than 1000 isolates were analysed a higher sample number is necessary to address the complete b-lactamase diversity in clinical ESBL-positive isolates, which is especially the case for K. pneumoniae isolates (Table 3).

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4. Discussion

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4.1. b-Lactamases in Austria

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Today, clinical microbial research is focusing especially on carbapenemases, which create therapeutical problems due to the lack of alternative potent b-lactam antibiotics. The carbapenemase genes blaVIM-4, blaKPC-2 and blaKPC-3 detected in this study are commonly found in carbapenem-resistant isolates across Europe (Nordmann et al., 2012). However, the emergence of blaVIM-4 was observed in Austria for the first time, although local studies have demonstrated the presence of blaVIM-1 in E. cloacae and blaVIM-2 in Pseudomonas aeruginosa (Duljasz et al., 2009; Heller et al., 2012). The presence of KPC in K. pneumoniae and K. oxytoca was confirmed in South-Eastern Austria, but no sequence analyses were performed, thus, it remains unclear if blaKPC-2 and blaKPC-3 are new in Austria (Hoenigl et al., 2012; Zarfel et al., 2011b). In addition, blaKPC genes were not identified in E. coli in Austria so far. blaOXA-48 and blaNDM-1 were not detected in this study although the presence of blaNDM-1 was already confirmed in South-Eastern Austria (Zarfel et al., 2011a). Furthermore, we report for the first time the occurrence of the carbapenemase blaOXA-72 in Enterobacteriaceae, which was only observed in Acinetobacter baumannii so far. The gene was identified in a K. pneumoniae isolate together with blaCTX-M-15, blaTEM-1 and blaSHV-33 and despite the presence of blaOXA-72, an ESBL-positive phenotype was determined. This isolate will be analysed in detail in a subsequent study. In the E. cloacae isolate #C8 with the blaACT-16 gene, a discrepancy was observed between the phenotype (metallo-b-lactamase, due to synergism of meropenem with the chelating agent dipicolinic acid in a disc diffusion test) and the genotype. The metallo-b-lactamase-positive phenotype remained unexplained upon phenotypical re-testing. The minimal inhibitory concentration (MIC) of meropenem was 2 mg/l i.e., sensitive according to EUCAST and intermediate sensitive according to current CLSI clinical breakpoints, respectively. This strain was, prior to WGS, additionally tested with the commercial kit Check-MDR CT103 (Check-Points, Wageningen, Netherlands) detecting also the AmpC b-lactamase ACT/MIR (data not shown). However, the Check-MDR CT103 kit did not identify the blaACT-16 or any additional gene and the detectable blaACT genes are not described in detail by the manufacturer. Thus, it remains unclear if the kit is targeting blaACT-16. Although no known carbapenemase genes were identified by WGS, the presence of a gene encoding a mutated OmpC porin was confirmed, which in combination with the AmpC gene blaACT-16 resulted in the observed phenotype. The deletion of 14 amino acids at the start of the OmpC protein might influence the 3D structure of the porin significantly but structure analyses have to be conducted to determine the impact of the deletion in detail. The high incidence of plasmid mediated AmpC genes among AmpC-positive pathogens (70%) in this study was unexpected,

498

483 484 485 486 487 488 489 490 491 492 493 494 495

0 0

B

200 400 600 800 Number of β-lactamase sequences

1000

50 45

Number of genes

40 35 30 25 20 15 10 5 0 0

200 400 600 800 Number of β-lactamase sequences

1000

Number of β-lactamase genotypes

C 120 100

80

60

40

20

0 0

Q4

476 477 478 479 480 481

200

400 600 800 Number of clinical isolates

1000

Fig. 1. Rarefaction curve analyses of the detected b-lactamases in the General Hospital Vienna. The observed number of gene-groups (A) and genes (B) was plotted versus the number of detected b-lactamase sequences. The number of different b-lactamase genotypes was plotted versus the number of the characterised clinical isolates harbouring b-lactamase genes. The solid lines illustrate the observed b-lactamase diversity, the dashed lines the extrapolated diversity.

number from Asia was higher than necessary because the curves is approximating the horizontal asymptote after 200 characterised isolates but more than 400 isolates were screened. In Europe and Latin America, no further gene-groups are expected to be identified within the chosen experimental setup either, indicating that Europe was also over-sampled regarding the detectable gene-groups.

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500 1000 1500 Number of β-lactamase sequences

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Fig. 2. Rarefaction curve analyses of the detected b-lactamases from a global analysis of ESBL-positive isolates conducted by Lascols et al. The graphs illustrate the blactamase diversity in different geographical regions (A and C) and according to species (B and D), respectively. The number of b-lactamase genotypes ((A) gene-group resolution; (B) gene resolution) was plotted versus the number of the characterised clinical isolates harbouring b-lactamase genes. The observed number of gene-groups (C) and genes (D) was plotted versus the number of detected b-lactamase sequences. The solid lines illustrate the observed b-lactamase diversity, the dashed lines the extrapolated diversity. Table 3 Observed versus calculated number of ESBL gene-groups, genes and genotypes using species richness estimators applied to the data collected by Lascols et al. Observed

545 546 547

ACE

Chao1 ± SD

Gene-groups

Global Asia Europe Latin America North America South Pacific

7 4 6 7 5 4

7 4 6 7 6 5

Genotypes (gene-group resolution)

Global Asia Europe Latin America North America South Pacific

18 7 11 15 7 6

21 7 13 18 8 8

22 ± 5 7±0 13 ± 2 17 ± 2 8±2 8±4

Genotypes (gene resolution)

All species E. coli K. pneumoniae

60 36 45

91 50 82

98 ± 22 57 ± 16 111 ± 44

Genes

All Species E. coli K. pneumoniae

37 25 31

48 29 50

54 ± 15 43 ± 24 73 ± 39

because these were thought to be less disseminated (Jacoby, 2009). Furthermore, this is the first report of blaDHA-1 and blaCMY genes in Austria. Although other blaCMY genes such as blaCMY-2 and blaCMY-4

7±0 4±0 6±0 7±0 5±0 4±0

are globally prevalent in AmpC-positive pathogens, the blaCMY-42 gene was only identified once in Germany so far (Hentschke et al., 2011). The specific danger regarding plasmid-mediated

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AmpC genes concerns the possibility of pathogens to develop a carbapenem resistance (most likely affecting ertapenem) upon porin loss (Quale et al., 2006; Kohler et al., 1999; Gutierrez et al., 2007). An interesting observation was made in connection with the molecular detection methods. Initially, the characterisation with PCR and bi-directional Sanger-sequencing resulted in five new blaCTX-M genes previously not observed but the putative new blaCTX-M genes were identified upon WGS as the result of PCR or sequencing errors. Thus, a fraction of the high number of the single point mutations among b-lactamases identified using PCR and bidirectional Sanger-sequencing might be the result of technical errors.

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4.2. Resistance genes detected with WGS

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The WGS data revealed a high number of different antibiotic resistance genes among the 20 sequenced isolates. In average, every isolate harboured 11 different resistance genes against >5 different antibiotic classes which is representing a serious limitation regarding treatment options. The co-existence of resistance genes targeting aminoglycoside and quinolone antibiotics in b-lactamase-positive strains was already observed in other studies but large differences regarding the frequencies of some genes were reported (Habeeb et al., 2013; Luo et al., 2011). In b-lactamasepositive isolates from Pakistan, aac(60 )–lb–cr was identified in 96% of the cases and qnr in 88%, respectively. In contrast, isolates from China (aac(60 )–lb–cr 13% and qnr 44%) and Korea (aac(60 )– lb–cr 34% and qnr 62%) harboured these genes less frequently (Luo et al. 2011; Shin et al., 2009). The incidence of aac(60 )–lb–cr observed in the present study (45%) was in-between these former results, while the detection rate of qnr genes (20%) was significantly lower. Furthermore, we could not confirm the frequent co-existence of these two genes in our study (22%), which was previously stated in other reports (Luo et al., 2011; Jiang et al., 2008). Two genes were observed in this study in a species previously unknown in this respect. The arr-3 gene conferring resistance to rifampicin was not reported in E. coli so far, but in K. pneumoniae and an environmental Aeromonas species (Marti and Balcazar, 2012; Ahmed and Shimamoto, 2011). The emergence of a rifampicin resistance gene reservoir is alarming due to the importance of this antibiotic in anti-mycobacterial therapy and in the treatment of orthopaedic device-related infections (Vergidis et al., 2011; Sendi and Zimmerli, 2012). Furthermore, the erm(42) gene conferring high resistance to lincosamides and low to moderate resistance to macrolide and streptogramin B was observed in a member of the order Enterobacteriales (M. morganii isolate #15) for the first time (Desmolaize et al., 2011). This finding is remarkable as members of Enterobacteriaceae already display intrinsic resistance against these classes of antibiotics. The gene was previously only detected in members of the family Pasteurellaceae and the species M. massiliense. Additionally, a surprisingly high prevalence of the fosA gene (40%) conferring fosfomycin resistance was detected. In two Asian studies analysing the presence of plasmid-mediated fosfomycin resistance genes among b-lactamase-positive isolates, a significantly lower number of fosA genes (Korea 2%, Japan 3.6%) was observed (Lee et al., 2012; Sato et al., 2013). The dominant fosfomycin resistance gene in these studies was fosA3. To our knowledge, the only report from Europe analysing plasmid-mediated fosfomycin resistance from clinical isolates was published in 1997, in which the incidence of the fosA and fosB genes in the screened fosfomycin-resistant bacteria was 8% (Arca et al., 1997). Further research is necessary to clarify if the high incidence of fosA observed in the present study is a local phenomenon or a European trend.

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4.3. Analyses of resistance gene diversity

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To our best knowledge, this is the first study conducting genetic diversity estimations of antibiotic resistance genes in a clinical environment. The rarefaction curve, Chao1 and ACE values are used to estimate the total number of species in an investigated habitat since it is highly improbable to exactly determine the total number of the species experimentally. Furthermore, the species richness estimators are likewise used to calculate the incidence of genes in a given habitat. They have already been used to estimate the number of a wide range of different genes and gene cassettes in various environments (Koenig et al., 2008; Matsui et al., 2011; Zaprasis et al., 2010). The calculations are based on an obvious correlation including the number of samples and the observed species. At the start of an analysis of a community very rich in species, it is likely that with every sampled individual a new species is identified. After intensive sampling, it becomes less likely to discover species not already encountered, because most individuals have been recorded. Theoretically, all present species are documented in a perfectly sampled habitat, and the sampling of new individuals will not lead to the discovery of new species. This perfectly documented habitat plotted as a graph with the number of observed species on the y-axis and the sampled individuals on the x-axis would result in a jagged curve, also referred to as accumulation curve, with a horizontal asymptote. Rarefaction curves are the result of repeated randomisations of the sample order, and thus, are smooth curves corresponding to an accumulation curve. They are used to compare the species diversity of different habitats and communities despite varying sample numbers. Our data show that the majority of b-lactamase genes disseminated in the General Hospital Vienna in 2011 were detected but that the total number of different b-lactamase genotypes remained unclear. This indicates that the pathogenic environment in the hospital is in general not dominated by a certain clonal strain or single, highly mobile and large genetic elements conferring antibiotic resistance which is also supported by the facts that only one sequence type (ST-131; n = 3) was detected more than once among the sequenced isolates, that none of the isolates shared the same plasmid composition (Supplementary Table 6) and that only two transposons with more than one b-lactamase gene were detected (Supplementary Table 7). Furthermore, these results implicate that single resistance genes are frequently exchanged among pathogenic bacteria, leading to the high number of genotypes. This suggestion is also supported by the observation that the large majority of the b-lactamase genes identified by WGS were flanked by mobile elements among which the ISEcp1, IS26, Tn2 and Tn3 transposons were dominant. The high rate of genetic resistance exchange is also reflected by the high diversity of other resistance genes among the investigated strains (Supplementary Table 6). Additional conclusions that can be drawn from the rarefaction curve analyses concern the higher b-lactamase diversity observed in ESBL-positive isolates compared to AmpC-positives (Fig. 1). This difference can be explained by a higher number of enzymes leading to an ESBL-positive phenotype or a higher flexibility of genetic elements comprising ESBL genes. The benefit of the biostatistical estimations was also illustrated using the data from Lascols et al. The over-sampling in Asia and the high b-lactamase diversity among K. pneumoniae isolates are the most remarkable aspects of these statistical analyses (Fig. 2). Asia was probably over-sampled due to the researchers’ disbelief that only so few different b-lactamase genes were present in the investigated hospitals. However, if the statistical tests would have been conducted during the study, the experimental resources could have been redirected in order to cover the under-sampled regions Africa, Middle East and the South Pacific.

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Acknowledgements

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The authors want to thank the technical staff at the AIT and the General Hospital Vienna for their contributions to this work and Viktoria Vasalik for her help on the manuscript.

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Appendix A. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2014.08 .014.

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