Introduction
Infections caused by antibiotic-resistant Enterobacteriaceae are difficult to treat because of their resistance to a broad-spectrum of antibiotics, such as 3rd and 4th generation cephalosporins [
1]. Resistance to cephalosporins is primarily due to the presence of extended spectrum β-lactamase (ESBL) enzymes. Carbapenems, including imipenem, meropenem, and ertapenem, are the drugs of choice for treating infections by ESBL producing Enterobacteriaceae; however, resistance to carbapenems has greatly increased worldwide during the last decade [
2]. Carbapenemase-producing Enterobacteriaceae (CPE) are an increasing threat to human health due to the limited treatment options for CPE infections such as febrile urinary tract infections (UTI), ventilator-associated pneumonia, abdominal sepsis, and bacteremia [
3]. Resistance to carbapenems is primarily attributed to acquired metallo-β-lactamase and serine carbapenemase enzymes (ESBL
carba); however, they may also be combined with other resistance mechanisms such as the loss of outer membrane porins that influence antibiotic uptake [
4]. An increase in the number of CPE cases has been reported in hospitals in Sweden and has therefore become notifiable according to the Swedish Communicable Diseases Act since 2012. CPE infections (both infections and carriers of CPE) have increased twofold in Sweden, from 47 cases (0.48/100,000 inhabitants) in 2014 to 115 cases (1.16/100,000 inhabitants) in 2015 [
5]. These numbers have further increased to 126 (1.26/100,000 inhabitants) in 2016. The most commonly identified CPE during 2014 was
Klebsiella pneumoniae (61%), followed by
Escherichia coli (33%) [
6].
Klebsiella spp. are ubiquitous in the environment, found in soil, surface water, and on plants [
7]. The most important clinical species,
K. pneumoniae and
Klebsiella oxytoca, are frequently the causative agents of nosocomial infections and have been associated with community-acquired infections such as bacteremia, pneumonia, meningitis, and UTI [
8,
9]. The carbapenemase genes,
blaOXA-48 (oxacillinase type-48),
blaKPC (
K. pneumoniae carbapenemase),
blaVIM (Verona integron encoded metallo-β-lactamase),
blaIMP (imipenemase), and
blaNDM (New Delhi metallo-β-lactamase), have been identified in
Klebsiella species [
10]. Moreover, the NDM-1 gene was first identified in a
K. pneumoniae isolated in Örebro, Sweden, from a patient previously hospitalized in New Delhi, India [
11].
Klebsiella spp. harboring carbapenemase genes often carry a variety of other antibiotic resistance genes conferring resistance to aminoglycosides and quinolones [
12]. Multidrug-resistant (MDR)
Klebsiella spp. that include resistance to carbapenems significantly limit the therapeutic options for CPE infections. Colistin and tigecycline are among the remaining drugs effective to treat infections caused by CPE. However, colistin resistance mediated by plasmid-borne
mcr-1 and
mcr-2 genes as well as tigecycline resistance due to efflux pump overexpression have been recently reported in Enterobacteriaceae [
13‐
16].
The majority of patients with CPE reported (both carriers or with infections) in Sweden have contracted the CPE abroad [
5]. In 71% of CPE cases in 2016, patients were reported to be colonized during international travels and only 27% have acquired CPE from domestic sources [
5]. With the increasing occurrence of infections with CPE, there is growing concern over the dissemination of CPE in the natural environment since that may have a direct or indirect effect on human health. Wastewater effluents from hospitals often release clinically relevant bacteria [
17], which increase the risk of dissemination of CPE into the environment. Moreover, carbapenemase-harboring bacteria have been reported from non-human sources including animal sewage and river water in both developing and developed countries [
17]. Currently, the situation of CPE in aquatic environments and wastewater effluent in Sweden is unknown; therefore, appropriate monitoring of wastewater effluents and environmental waters is ongoing.
The aim of this study was to evaluate the presence of CPE in wastewater and the recipient-river and lake water in Örebro, located in central Sweden, and to investigate the relationship of these bacteria to the clinical isolates obtained from patients in the same city by comparing the genotypic, phenotypic, and phyloproteomic profiles of the isolates.
Methods
Sampling locations
Environmental water samples were collected during October 2015 from Svartån River and Hjälmaren Lake near Örebro, Sweden, a city that reflects urban areas in Nordic countries. The Svartån River flows through Örebro city and is the recipient of the effluent water from Örebro wastewater treatment plant (WWTP) before it flows into Hjälmaren Lake. The WWTP serves a population of 140,000 people and processes an average of 45,000 m
3 of wastewater per day (
www.orebro.se). The untreated wastewater from Örebro hospital, veterinary clinics, household, and industries is transported via the sewage system to the WWTP. There are no pharmaceutical industries in Örebro city, and most of the agricultural activities are outside of the urban area. Surface water samples from the river were collected from upstream (59° 16′ 03.5″ N, 15° 08′ 49.9″ E) and downstream (59° 16′ 42.5″ N, 15° 15′ 41.5″ E) of Örebro city and WWTP. Surface water samples from Hjälmaren Lake (59° 16′ 40.2″ N, 15° 17′ 31.1″ E) were collected approximately 1 nautical mile downstream of WWTP effluent point. The influent and effluent water from Örebro WWTP was collected to analyze for the presence of CPE. Three separate water samples were collected in sterile 1-litre glass bottles from each location at different times of the day, transported and stored at 4 °C, and analyzed within 24 h.
CPE from a pre-existing collection at Örebro University Hospital were included in the study. The isolates were collected from patients during the years 2008 to 2015.
Bacterial isolation and identification
Water samples were filtered through 0.45 μm polyethylene sulfonate membrane filters (Sartorius Stedim Biotech, Sweden) and placed on selective chromogenic agar medium for carbapenem-resistant bacteria, chromID™ CARBA (bioMérieux, Marcy-l’Etoile, France) and chromID™ OXA-48 (bioMérieux, Marcy-l’Etoile, France). The agar plates were incubated for 18 h at 37 °C, and suspected CPE colonies were picked and streaked onto Chromocult® Coliform Agar (Merck, Darmstadt, Germany) to further identify the isolates as Enterobacteriaceae and to obtain pure cultures.
Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with Microflex LT system (Bruker Daltonik GmbH, Bremen, Germany) according to manufacturer’s instructions.
Analysis of carbapenemase production
Putative carbapenemase producers were screened for their susceptibility to meropenem and imipenem using the disk diffusion method according to the EUCAST specifications. The isolates with inhibition zone diameters of ˂ 25 mm and ˂ 23 mm for meropenem (10 μg) and imipenem (10 μg), respectively, were considered presumed carbapenemase producers [
18].
Presumed carbapenemase producers were further tested for their carbapenemase production using RAPIDEC® CARBA NP test (BioMérieux, Marcy-l’Etoile, France) according to manufacturer’s instructions [
19].
Antibiotic susceptibility testing
The antibiotic susceptibility of isolates was tested for six different classes of antibiotics: beta-lactams (imipenem, meropenem, cefotaxime, ceftazidime, piperacillin-tazobactam), aminoglycosides (gentamicin, amikacin, tobramycin), fluoroquinolone (ciprofloxacin), trimethoprim-sulfamethoxazole, tetracycline (tigecycline), and polymyxin B (colistin). The disk diffusion method was used according to the specifications of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [
20]. Minimum inhibitory concentrations were determined using ETEST® (bioMérieux, Marcy-l’Etoile, France) following manufacturer instructions. The plates were incubated at 37 °C for 18 h. EUCAST clinical breakpoint values for zone diameters (mm), and the MIC values were used to categorize the isolates as susceptible (S), intermediate (I), and resistant (R) [
21].
DNA isolation and whole genome sequencing
Genomic DNA of
Klebsiella spp. was isolated from an overnight culture in nutrient broth (Merck, Germany) using guanidinium thiocyanate-phenol-chloroform extraction method [
22]. DNA samples were sent to GATC Biotech (Konstanz, Germany) for whole genome sequencing (WGS). Short paired-end reads of 150 bp were generated using Illumina HiSeq. Quality trimming of the paired-end reads was performed with Trimmomatic version 0.32.3 [
23]. De novo genome assembly was performed using SPAdes Genome Assembler version 3.10.1 [
24]. The sequences have been submitted to GenBank ID: 451179 (
https://www.ncbi.nlm.nih.gov/bioproject/PRJNA451179).
Multilocus sequence typing and phylogenetic analysis
MLST was performed using MLST-1.8 Server provided by Center for Genomic Epidemiology [
25], and sequence types (STs) were confirmed using SRST2 [
26]. For genomic comparisons, we performed core genome SNP analysis and core genome gene-by-gene analysis. To increase the shared sequences for analysis, only
K. oxytoca were included in the analysis. Phylogenetic maximum likelihood tree based on core genome SNPs analysis was generated in Parsnp [
27] using the default parameters.
K. oxytoca JKo3 was selected as the reference genome. Neighbor-joining tree based on core genome MLST (allelic comparison) was performed in SeqSphere+ (Ridom Muenster, Germany) using default parameters, and
K. oxytoca JKo3 was selected as the reference genome. Phylogenetic trees were reconstructed in FigTree version 1.4.3 (Institute of Evolutionary Biology, University of Edinburgh). To provide a wider phylogenetic context, we included nine epidemiologically unrelated
K. oxytoca genomes in the analysis. The assembled genomes were randomly selected from Ensembl bacterial genome database (
bacteria.ensembl.org). The strain names and archive numbers are in Supplementary Table
S1.
Antibiotic resistance gene profiling
The antibiotic resistance gene profiling of isolates was performed with oligonucleotide microarray-based assay developed by Alere Technologies [
28,
29]. This was complemented with allelic identification of the antibiotic resistance genes (ARGs) from the assembled genomes using Resfinder, server 2.1 [
30]. For microarray-based identification of ARGs, fresh cultures from Mueller Hinton agar (BD, LePont de Claix, France) were inoculated to AMIES agar gel tubes (Copan, Brescia, Italy) and sent to Alere Technologies (Germany) for genotyping. The antibiotic resistance gene profiles of both clinical and environmental isolates were analyzed for clinically important carbapenemase genes along with narrow spectrum β-lactamase, ESBL, and other antibiotic resistance genes. The assay included the following carbapenemase genes,
blaKPC,
blaVIM,
blaNDM,
blaBIC,
blaDIM,
blaGES,
blaGOB,
blaPAM,
blaSFH,
blaSMB,
blaSME,
blaSPM,
blaTMB,
blaGIM,
blaIND,
blaKHM, as well as
blaOXA-23,
blaOXA-40,
blaOXA-48,
blaOXA-50,
blaOXA-51,
blaOXA-54, and
blaOXA-58, and the following ESBL genes,
blaCTX-M-1/M-15,
blaSHV and
blaTEM. In addition, genes conferring resistance to aminoglycosides (e.g.,
aac,
aad,
ant2,
aphA,
strA,
strB), quinolones (e.g.,
qepA,
qnrA,
qnrB,
qnrC,
qnrD,
qnrS), macrolides (
mph,
mrx), sulfonamides (
sul1,
sul2,
sul3), and trimethoprim (e.g.,
dfrA genes) were evaluated (Alere Technologies,
http://www.alere-technologies.com). Four genes related to two bacterial toxin-antitoxin systems higB-higA and splT-splA were also included in the assay.
Mass spectra analysis
A loop (approx. 1 μl) of freshly grown bacteria was suspended in ethanol (900 μl, 99.5%), centrifuged at 11,000×g for 2 min, and the pellet was air-dried at room temperature for 3 min. The pellet was incubated in 70% formic acid (20 μl) for 3 min at room temperature to lyse the cells and the released proteins were isolated with acetonitrile (20 μl). The mixture was vortexed and centrifuged at 11,000×g for 2 min. The supernatant was spotted onto a MALDI-TOF MS target plate with Bruker Matrix solution (1 μl). MALDI-TOF MS plates were air-dried at room temperature, and mass spectrometry was performed. Two replicate analyses were performed for each bacterial isolate.
The mass spectra of the bacterial proteins of 2000–20,000 mass-to-charge ratio (
m/
z) were analyzed using BioNumerics version 7.5 created by Applied Maths NV (
http://www.applied-maths.com). Default parameters for strict pre-processing of spectrum data using baseline subtraction, noise elimination, and curve smoothing were selected. Similarity comparison was performed with peak-based Pearson coefficient using default parameters, and phyloproteomic dendrogram was created.
Discussion
In the present study, carbapenemase-producing
K. oxytoca were isolated from sewage and river waters running through Örebro city in central Sweden. To the best of our knowledge, this was the first CPE isolated from the environment in Sweden, thus prior to 2015, no isolates were reported. This is in contrast to other European countries that have reported health-care associated CPE since 2011 [
31]. Only two carbapenemase-producing
Klebsiella isolates were recovered from the environment, indicating that the levels of CPE in environmental waters remain very low. However, the presence of these resistant bacteria in the river, even at low levels, remains a concern. Although carbapenemase-producing
K. oxytoca were found in Svartån River, they were not detected in the downstream lake water, which suggests that CPE may be present at even lower levels or completely absent in the lake due to the increased dilution or die-off under environmental conditions. Environmental
K. oxytoca isolates, E1 and E2, harbored
blaVIM-1 and
blaIMP-29, respectively, and also showed phenotypic resistance to either one or two carbapenem antibiotics (Table
2). These carbapenemase genes are the most commonly identified worldwide and
blaVIM-1 is predominant in Europe, while
blaIMP alleles are prevalent in Asia [
32]. As none of the isolates from patients in Örebro University Hospital were identified with
blaIMP-29, this implies that the source of CPE in the wastewater most likely originate from the community.
Only six cases of CPE infections were reported in Örebro County between 2008 and 2015, and the majority of these patients had likely contracted their resistant bacteria from abroad rather than domestic acquisition in the Swedish hospital environment. The clinical
K. pneumoniae isolates H1 and H2 harbored
blaNDM-1 and
blaKPC-3, respectively, and the bla
NDM type metallo-β-lactamases are highly prevalent in the Indian sub-continent [
33,
34], while bla
KPC carbapenemases have worldwide prevalence [
35‐
38]. These isolates harbored antibiotic resistance genes for a broad range of antibiotics as well as showed functional resistance to most of the antibiotics tested in this study. The
K. pneumoniae H1 co-harbored other ESBL genes such as
blaCTX-M-15, which is frequently detected in
blaNDM-1-positive Enterobacteriaceae [
39] and also the predominant ESBL type in Örebro County [
40]. Strains H4 and H6 only carried
blaVIM-1, and these had high genome similarity to the strain from river (E1) indicating that this isolate might be widespread in the community.
Resistance to carbapenems was due to the presence of functional carbapenemase genes; however, other mechanisms that were not analyzed in this study could also contribute the final phenotype. These mechanisms include the overexpression of efflux-pumps and downregulation or modification of outer-membrane proteins such as ompK and phoE [
41]. Most carbapenemase genes are found on mobile plasmids that can be exchanged between the members of Enterobacteriaceae through horizontal gene transfer [
42]. Some plasmids carry carbapenemase genes along with toxin-antitoxin systems that increase plasmid prevalence by post-segregational killing of daughter cells that lack the plasmid [
43]. Thus, antibiotic selection pressure is not always necessary to maintain antibiotic resistance plasmids in a bacterial population. The recipient WWTP of the hospital and municipal wastewater is a major source of antibiotic-resistant Enterobacteriaceae, and the treatment process often reduces but does not eliminate these organisms [
44]. Complex and nutrient-rich microbial environment in the WWTP may provide ideal conditions for the expansion of antibiotic resistance into natural environments [
45] and may help to disseminate plasmids with toxin-antitoxin systems in a bacterial population without any selection pressure. It is interesting to note that our strains did not have the higB-higA and splT-splA toxin-antitoxin genes. However, there is a possibility of the presence of other toxin-antitoxin systems that were not analyzed in this study.
In countries with low to middle-income economies, untreated wastewaters from households and pharmaceutical industries are discharged into natural environments without prior treatment, and this has been shown to increase antibiotic-resistant bacteria in the environment [
46]. In Sweden, however, the regulations for wastewater disposal have been strict since the approval of the Environmental Protection Act in 1969. Our study revealed the presence of CPE in the Swedish aquatic environment, albeit at low levels, regardless of the stringent regulations for antibiotic use. Although, the study is based on the comparison of a small set of strains, the presence of related CPE in both hospital and associated river and lake environments is an important observation. This shows the possibility of transmission of CPE from hospital to aquatic environment since the effluent wastewater from hospital is transported through the drainage system into the WWTP without prior treatment. In addition to the hospital, the community and veterinary clinics are other potential sources of CPE that may reach the aquatic environment, with the WWTP being the distributor. The clinical isolates carrying carbapenemase genes were most probably acquired from abroad and not generated at the hospital, suggesting that alternate strategies must be employed to avoid the further environmental contamination and transmission of MDR in the community. Although WWTP plays an important role to minimize the organic pollution from the wastewater, they can be ultimate points of dissemination for MDR bacteria into natural environments [
47]. Therefore, WWTP must apply processes for eradicating bacteria, including MDR bacteria from wastewater before discharging into the environment. Thus, attempts to reduce the spread of CPE to the aquatic environment in Sweden and surrounding countries are of vital importance since the presence of CPE in these niches is still rare.