Systematic Comparison of Epidemic and Non-Epidemic Carbapenem Resistant Klebsiella pneumoniae Strains

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Systematic Comparison of Epidemic and Non-Epidemic Carbapenem Resistant Klebsiella pneumoniae Strains © 2021 the Authors Published version Koskinen, Katariina; Penttinen, Reetta; Örmälä-Odegrip, Anni-Maria; Giske, Christian G.; Ketola, Tarmo; Jalasvuori, Matti Koskinen, K., Penttinen, R., Örmälä-Odegrip, A.-M., Giske, C. G., Ketola, T., & Jalasvuori, M. (2021). Systematic Comparison of Epidemic and Non-Epidemic Carbapenem Resistant Klebsiella pneumoniae Strains. Frontiers in Cellular and Infection Microbiology, 11, Article 599924. https://doi.org/10.3389/fcimb.2021.599924 2021 Systematic Comparison of Epidemic and Non-Epidemic Carbapenem Resistant Klebsiella pneumoniae Strains Katariina Koskinen 1 , Reetta Penttinen 2 , Anni-Maria Örmälä-Odegrip 3 , Christian G. Giske 3,4 , Tarmo Ketola 1 and Matti Jalasvuori 1 * 1 Department of Biological and Environmental Science, Nanoscience Center, University of Jyva skyla , Jyva skyla , Finland, 2 Department of Biology, University of Turku, Turku, Finland, 3 Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden, 4 Department of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden Over the past few decades, extensively drug resistant (XDR) resistant Klebsiella pneumoniae has become a notable burden to healthcare all over the world. Especially carbapenemase-producing strains are problematic due to their capability to withstand even last resort antibiotics. Some sequence types (STs) of K. pneumoniae are significantly more prevalent in hospital settings in comparison to other equally resistant strains. This provokes the question whether or not there are phenotypic characteristics that may render certain K. pneumoniae more suitable for epidemic dispersal between patients, hospitals, and different environments. In this study, we selected seven epidemic and nonepidemic carbapenem resistant K. pneumoniae isolates for extensive systematic characterization for phenotypic and genotypic qualities in order to identify potential factors that precede or emerge from epidemic successfulness. Studied characteristics include growth rates and densities in different conditions (media, temperature, pH, resource levels), tolerance to alcohol and drought, inhibition between strains, ability to compensate pH, as well as various genomic features. Overall, there are clear differences between isolates, yet, only drought tolerance was found to notably associate with nonepidemic K. pneumoniae strains. We further report a preliminary study on the potential to control K. pneumoniae ST11 with an antimicrobial component produced by a nonepidemic K. pneumoniae. This component initially restricts bacterial growth, but stable resistance develops rapidly in vitro. Keywords: XDR Klebsiella pneumoniae, extended-spectrum beta-lactamase, epidemic, antibiotic resistance, virulence Frontiers in Cellular and Infection Microbiology | www.frontiersin.org February 2021 | Volume 11 | Article 5999241 Edited by: Rodnei Dennis Rossoni, Sao Paulo State University, Brazil Reviewed by: Tatiana Amabile De Campos, University of Brasilia, Brazil Jonatas Rafael De Oliveira, Anhembi Morumbi University, Brazil *Correspondence: Matti Jalasvuori matti.jalasvuori@jyu.fi Specialty section: This article was submitted to Bacteria and Host, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 28 August 2020 Accepted: 08 January 2021 Published: 23 February 2021 Citation: Koskinen K, Penttinen R, Örmälä-Odegrip A-M, Giske CG, Ketola T and Jalasvuori M (2021) Systematic Comparison of Epidemic and Non-Epidemic Carbapenem Resistant Klebsiella pneumoniae Strains. Front. Cell. Infect. Microbiol. 11:599924. doi: 10.3389/fcimb.2021.599924 ORIGINAL RESEARCH published: 23 February 2021 doi: 10.3389/fcimb.2021.599924 INTRODUCTION Klebsiella pneumoniae is a Gram-negative bacillus causing opportunistic infections outside of the gastrointestinal tract (Podschun and Ullman, 1998). Common conditions include pneumonia, urinary tract infections, wound infections, and less often liver abscess, meningitis, and septicemia. Some of the strains circulating in clinical settings are also showing increasingly virulent phenotypes (Pomakova et al., 2012;Shon et al., 2013). These strains are often characterized by hypermucoviscosity when cultivated on agar plates and they are more resilient against killing by serum or phagocytosis (Catalan-Najera et al., 2017). Moreover, extensively drug-resistant (XDR) among K. pneumoniae is increasing very rapidly compared to many other priority pathogens (World Health Organization, 2014). In particular, infections caused by K. pneumoniae strains which have developed resistance against newer generations of b-lactams, such as carbapenems, can be hazardous and often lifethreatening. These carbapenemase genes are found to be abundant in bacteria originating from hospital environments although there are notable regional differences. Yet, sequence typing of the pathogens indicate that resistant K. pneumoniae strains can also disseminate globally between hospitals. WHO has classified carbapenemase-producing K. pneumoniae as an urgent threat (World Health Organization, 2017). Often, K. pneumoniae isolates are typed by utilizing partial sequences from seven housekeeping genes. These genes are part of the core genome and hence unlikely to be horizontally transferred between different K. pneumoniae strains. As such, sequence typing provides a rudimentary approach to identify genetic similarity among isolates of different sources of origin. It appears that certain sequence types (STs) have been more successful in dispersing between hospitals compared to other equally resistant strains. NDM-1 metallo-b-lactamase producing ST11 and 14 have been noted to be responsible of epidemics in various countries (Yong et al., 2009;Pitout et al., 2015;Samuelsen et al., 2017). Klebsiella pneumoniae carbapenemase (KPC) producing ST258 have even been referred as hyperepidemic clone (Bowers et al., 2015) and its epidemic potential has been further investigated in several meta-analyses (Dautzenberg et al., 2016). ST512, a single-locus variant of ST258, is also highly associated in epidemics globally (Conte et al., 2016). ST147 is also hazardous with numerous virulence and resistance genes (Turton et al., 2018). Despite of the notion that certain STs appear to be more prone for inter-hospital dispersal, it is still unclear what qualities alongside of pathogenicity, if any, may be responsible for this epidemic success. Majority of the surveillance attempts focus on resistance profiles and genotypic features (Giske et al., 2012). Genomic data also accumulates rapidly as whole genome sequences of many strains have become available (Holt et al., 2015). Yet, the phenotypic characteristics of differently successful K. pneumoniae STs are rarely studied in detail, or the phenotypic analysis focus on specifictraitssuchas hypermucoviscousity (Catalan-Najera et al., 2017). Comparison of the phenotypes of epidemic and non-epidemic strains could potentially reveal meaningful interactions between bacteria and their environment that contribute to the epidemic spread of XDR strains. In this study, we selected 14 carbapenem resistant K. pneumoniae strains isolated from patients hospitalized in USA, Sweden, UK, Greece, or India (Kitchel et al., 2009;Samuelsen et al., 2009;Kitchel et al., 2010;Samuelsen et al., 2011;Vading et al., 2011;Giske et al., 2012;Hasan et al., 2014). Five of these STs are continuously being detected in hospitals in multiple countries and can be considered as epidemiologically successful or epidemic (Table 1). The rest of the STs have made only seldom appearances and have rarely if at all dispersed to other hospitals and may hence be considered as non-epidemic STs. Here, we systematically determined and measured potentially relevant characteristics for these strains in order to reveal differences that may correlate and perhaps partly explain the epidemic success. MATERIALS AND METHODS Strains All 14 studied Klebsiella pneumoniae strains (see Table 1) were Illumina sequenced at Karolinska Institutet, Sweden. Genome sequences can be found from GenBank under BioProject id PRJNA680903. Sequences of EKP24 and NKP2 were also PacBio sequenced in University of Helsinki, Finland. Sequenced genomes were annotated by Rapid Annotation using Subsystem Technology (RAST, https://rast.nmpdr.org) and both secure and potential protein coding genes were mapped and the distribution of protein families were compared between the strains. Resistance genes were identified with ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/), prophages with Prophage Finder (https://omictools.com/ prophage-finder-tool), and CRISPR-regions with CRISPRfinder (https://crispr.i2bc.paris-saclay.fr). An algorithm was written to identify unannotated short open reading frames (ORFs) from the genome files (Supplementary File 1). The algorithm scans the genome for ORFs that have a potential ribosome binding site upstream of the start codon and is not overlapping with annotated genes. Growth Experiments Growth densities to each strain were measured in temperature of +37°C and room temperature with 230 rpm shaking or without shaking. Cells were grown overnight in 5 ml of LB media (+37°C, 230 rpm) and then transferred into 5 ml of fresh LB media in 1:5,000 ratio. Fresh cultures were grown in experimental settings of +37°C and 230 rpm or RT and 0 rpm for 20 h and growth densities were calculated as colony forming units (cfu)/ml. As a standard initial liquid culture for the growth curve experiments all the strains were cultured in 5 ml of 100% LB, +37°C, and 230 rpm overnight and then transferred into experimental settings. In order to test the effect of shaking, the initiating cultures were prepared then transferred into 5 ml of LB in 1:100 ratio and grown in experimental settings of +37°C and 230 rpm and +37°C Koskinen et al. Epidemic and Non-Epidemic Klebsiella pneumoniae Frontiers in Cellular and Infection Microbiology | www.frontiersin.org February 2021 | Volume 11 | Article 5999242 and 0 rpm. In growth curve experiments the effect of different media and varying concentrations and compositions of nutrients on growth was determined for each strain. One hundred percent LB was used throughout the experiments unless mentioned otherwise. Growth curves were measured in 10 and 1% LB, 100% BHI, and 100% of pure DMEM by diluting the initial culture in 1:100. Growth curves were measured at +37°C, 595 nm wavelength with Multiscan FC (Thermo Scientific) for 20 h in 5 min intervals and maximum growth and average growth rate were calculated. Survival in Acidic pH and Compensation Capacity Bacterial cells’ability to tolerate acidic surrounding pH and capability to compensate it by metabolism was measured for each strain in pH 3–7. Initial cultures were grown in 5 ml LB pH 7 at +37°C and 210 rpm overnight. Each strain was then transferred into 5 ml LB of either pH 3, pH 4, pH 5, pH 6, or pH 7 in 1:100 ratio and cultured in +37°C and 210 rpm. In pH 5–7 cultures were grown for 90 h and growth densities were calculated by plating in 16, 24, and 90 h. Then 1.5 ml of culture was filtered through 0.2 mm and supernatant pH was measured with Basic pH Meter (Denver Instruments) in 24 and 90 h. Cultures in pH 3–4 were shortened into 24 h experiment and growth densities were calculated in 16 and 24 h and supernatant pH was measured in 24 h. Growth curves were measured by diluting the initial pH 7 cultures in 1:100 ratio into LB pH of 3–7 and growth curves were measured at +37°C, 595 nm wavelength for 20 h in 5 min intervals. Cross-Strain Interactions In aim to study the dynamics all the strains were cultured separately, and their metabolic products secreted into surroundings were tested against other strains in cross-strain inhibition experiments. Cross-strain interactions were tested by collecting the media after overnight culturing at +37°C and 210 rpm. Overnight cultures were centrifuged first with 7,000 × g for 4 min, and the supernatant was centrifuged again with 10,000 × g for 1 min. Each strain was cross-plated with all the supernatants (overnight, +37°C), and the inhibition of the growth of each strain was observed. Supernatant Inhibition and Prophages A 4-week evolutionary experiment was designed to study appearance, persistence, and reversibility of putative colicin E3 resistance in sensitive EKP24 strain. For the first 2 weeks EKP24 was cultured with (n = 5) and without (n = 5) colicin E3 in 10% LB media supplemented with either NKP2 (containing colicin E3) or EKP24 (not containing colicin E3) supernatant filtrate (0.2 mm) in 1:4 ratio. Ten percent LB media was supplemented TABLE 1 | K. pneumoniae isolates used in the study. Isolate ST from database Isolation location No. of CRISPR loci No. of prophage regions Reference Virulence genes Capsule types Beta-lactamase NKP01 1534 37 USA 0 10 Kitchel et al., 2009 mrk K15K17K50K51K52 blaTEM-1B, blaKPC-2, blaSHV-11 NKP02 10924 334 USA 0 4 Kitchel et al., 2009 mrK N/A blaKPC-3, blaOKP-B-4, blaOXA-9, blaTEM-1A EKP03 70165 14 USA 3 10 Kitchel et al., 2009 irp, fyu, ybt, kfu, mrk K2 blaTEM-1A, blaSHV-28, blaKPC-3 EKP05 70708 258 USA 0 7 Kitchel et al., 2009 mrk N/A (K15K17K50K51K52) blaOXA-9, blaKPC-3, blaSHV-12 EKP08 2008025 11 USA 0 11 Kitchel et al., 2009 mrk K13 blaKPC-2, blaSHV-11 EKP10 AO-8053 512 Sweden (Israel*) 010Samuelsen et al., 2009 mrk N/A blaTEM-1A, blaOXA-9, blaSHV-11, blaKPC-3 EKP11 AO-15200 147 Sweden (Greece*) 26Samuelsen et al., 2011 mrk K64,K14 blaSHV-11, blaVIM-1 NKP18 VPKP389 36 Athens, Greece 110Hasan et al., 2014 irp, mrk, fyu, ybt k27 blaSHV-129, blaVIM-26 NKP20 VPKP229 17 Athens, Greece 08Hasan et al., 2014 irp, mrk, ybt k25 blaSHV-129, blaVIM-1 EKP22 N6 14 UK 2 6 Giske et al., 2012 mrk, ybt, kfu k2 blaCTX-M-15, blaSHV-11, blaTEM-1A, blaOXA-1, blaNDM-1, blaOXA-9 EKP24 ED502873 11 Sweden 0 6 Giske et al., 2012 irp, mrk, fyu, ybt N/A (K15K17K50K51K52) blaSHV-11, blaCTX-M-15, blaNDM-1 NKP25 N12 231 UK 1 4 Giske et al., 2012 kfu, mrk K51 blaSHV-1, blaTEM-1B, blaNDM-1, blaOXA-1 NKP28 B357 43 UK 1 8 Giske et al., 2012 mrk, kfu K30 blaCTX-M-15, blaDHA-1, blaCMY-6, blaSHV-11, blaOXA-9, blaNDM-1, blaTEM-1A NKP30 IR34 624 Chennai, India 13Giske et al., 2012 mrk K12, K29 blaTEM-1B, blaDHA-1, blaNDM-1, blaSHV-36, blaCTX-M-15, blaOXA-1 *Isolates associated with import from the country. Koskinen et al. Epidemic and Non-Epidemic Klebsiella pneumoniae Frontiers in Cellular and Infection Microbiology | www.frontiersin.org February 2021 | Volume 11 | Article 5999243 with 25 mg/ml of kanamycin and 150 mg/ml of ampicillin. After 2 weeks EKP24 cultured with the presence of colicin E3 were divided into two sets of samples (both n = 5). Other set was continued with colicin E3 exposure as described earlier. In the other set of samples, colicin E3 containing supernatant was replaced with EKP24 supernatant. Cultures were refreshed in 1:100 ratio three times and samples stored once a week with glycerol at −80°C. Development and persistence of colicin E3 resistance was determined by plating. Samples were taken at the beginning of the experiment, before division of colicin E3 exposed EKP24, and at the end of the experiment and were used for DNA extraction. DNA was isolated with DNeasy Blood & Tissue Kit (Qiagen) and sequenced with Illumina HiSeq. The observed reads were mapped to original PacBio-sequenced genome of NKP2 (described above) in order to detect the genetic variants. The variants developed under the exposure of colicin E3 were identified by filtering out those variants that were already present in the beginning of the experiment. The genetic analysis was performed with CLC Genomics Workbench v11 (Qiagen). Interactions between the putative colicin E3 producing NKP2 and susceptible EKP24 bacterial cells were also observed with confocal microscopy. Then 200 ml of 1% LB-agar was placed into a chamber of eight-chambered ibidi®ibiTreat m-Slide (Ibidi GmbH) covered with CID lid for µ-dishes (Ibidi GmbH), and 3ml of NKP2 and EKP24 were injected under the agar onto opposite sides. Encounter of the two strains was visualized with Nikon AR1 laser scanning confocal microscope with 60× water immersion objective and using Galvano scanner. Production of putative colicin E3 was further studied by growing NKP2 strain in different medias. NKP2 was grown in LB concentrations of 100, 10, and 1%, LB without tryptone, 100% Shieh (Song et al., 1988) and in 100% DMEM in +37°C and 200– 230 rpm overnight. NKP2 cultures were filtered through 0.2 mm filter and colicin E3 presence was determined by plating the supernatant with susceptible EKP24 strain. Alcohol Exposure All studied strains were exposed to multiple concentrations of ethanol (20, 50, 75, and 90%) and their ability to survive the exposure were measured with spectroscopy. In 30 s exposure experiment all the strains were first grown in 5 ml of LB (+37°C, 230 rpm, overnight) and then transferred in 1:10 ratio into fresh LB and left to grow overnight at +37°C on 96-well plate 100 ml per well to form biofilm. On the following day media was gently removed and replaced with 200 ml of ethanol in concentrations of 20, 50, 75, and 90%. After 30 s incubation in RT ethanol was replaced with 100 ml of LB and growth at +37°C was measured in 595 nm wavelength for 20 h in 5 min intervals. In ethanol evaporation experiment, cultures for 96-well plate were prepared as described earlier but volume was lowered into 35 ml per well. Ninety-six-plate cultures were incubated overnight at +37°C without the lid to let the excess media evaporate. Dried biofilms were exposed to 50 ml of ethanol (either 20, 50, 75, or 90%) and left to fully evaporate before addition of 200 mlofLB per well. Growth curve measurements was performed as earlier described. Drought Tolerance The capability to survive over long-lasting drought in a room air humidity was tested by culturing the strains on 96-well plate by transferring overnight grown culture (5 ml LB, +37°C, 210 rpm) in 1:10 ratio to LB. One hundred microliters per well was used with four replicates of each strain. Plates were incubated at +37°C for 3 days in order to grow biofilm. After 3 days, plates were relocated to RT and lids were removed to ensure total evaporation of media. After 12 days, 2 months and 6 months in drought, 200 ml of fresh LB was added into each well and the growth curves were measured at +37°C, 595 nm wavelength, 20 h in 5 min intervals. Morphological Characterization Morphological characteristics of the colonies were analyzed. In order to get single colonies, all strains were cultured in 5 ml LB in +37°C, 210 rpm overnight. Cultures were diluted into 10 −6 in water and plated onto LB-agar plates. Plates were incubated at +37°C for overnight and the colonies were photographed. Strains EKP5, EKP3 and EKP22 were found to produce translucent colonies along with the traditional colonies. These translucent colonies were further cultivated by transferring one colony to a fresh LB-agar plate and incubated at +37°C overnight or transferred into liquid culture of 5 ml LB and cultured at +37°C and 210 rpm overnight before plating in 10 −6 dilution onto the new LB-plates. Growth densities were calculated from these plates. From the same liquid culture used for the growth density definition, 1:100 dilutions were made into LB and growth curves were measured in +37°C, 595 nm wavelength, 20 h in 5 min intervals. Statistical Analyses To explore if epidemic or non-epidemic strains can be characterized by their capabilities, we performed discriminant analysis using MASS package (Venables & Ripley, 2002)inR (version. 3.3.2). Effects of individual variables on discriminant function were tested by regressing predicted values against original variables. Overall performance of discriminant function was addressed by Bayesian logistic regression of epidemic status against predicted values of discriminant function using Stan with R (McElreath, 2016). RESULTS Selection of Strains and Genomic Analysis We selected 14 K. pneumoniae strains for detailed phenotypic and genomic analysis in an attempt to identify characteristics that may potentially associate with epidemic STs. The strains were abbreviated either as EKP or NKP for Epidemic and Nonepidemic K. pneumoniae, respectively, and the strain number was derived from an internal naming system. The strains and their key genomic traits are listed in Table 1. Note that two epidemic STs are represented twice (ST14 and ST11), but their genetic features differ from one another and were thus selected for phenotypic studies in order to evaluate whether the phenotypes of different strains of a single ST are similar. Koskinen et al. Epidemic and Non-Epidemic Klebsiella pneumoniae Frontiers in Cellular and Infection Microbiology | www.frontiersin.org February 2021 | Volume 11 | Article 5999244 Epidemic and non-epidemic STs are not grouping together when the genomic regions used for sequence typing are used to infer genetic relationship (Figure 1). As such, the epidemic strains do not appear to share a common ancestor that diverged from nonsuccessful strains. Therefore, epidemic success is not likely to be linked to a single vertically inherited (genetic) trait, which evolved once. This however does not exclude the possibility that traits preceding epidemic spread are transferred horizontally between strains of different STs. Intriguingly, strains EKP3 and EKP22 (both ST14) and EKP8 and EKP24 (both ST11) are phenotypically different regardless of the same ST (Supplementary Figure 1), sometimes being even the opposite phenotypic extremes out of all strains. Colony morphologies of all strains are highly similar (Supplementary Figure 2). The genomes of all the strains were annotated and their overall gene contents compared. Based on the annotation, epidemic and non-epidemic strains appear to be generally uniform metabolically and functionally, hence providing no apparent genome-level design differences to explain epidemic qualities (Table 2). Neither the presence or absence of CRISPR system or the number of CRISPR loci appear to be linked with epidemic successfulness. Also, the number of mobile elements such as prophages or plasmids do not associate specifically with either group. The most obvious potentially explanatory features, i.e. virulence genes and antibiotic resistance genes, are similar between epidemic and non-epidemic K. pneumoniae despite of differences among individual strains (Tables 1 and 2). We further speculated that some generally overlooked features such as short open reading frames (ORFs) of length 30 to 150 nucleotides could possibly be linked with epidemic qualities. These genes are rarely identified as coding regions with automated annotation algorithms despite of the fact that they are sometimes transcribed and translated and may reflect recent adaptations to new life strategies or specific conditions (that may be related to epidemic spread). We prepared an algorithm to extract all short ORFs which are preceded by a (near-)perfect ribosome biding site and which do not overlap with existing annotated ORFs (Python code is available in Supplementary File 1). On average, approximately 200 unannotated ORFs were extracted from the sequences. Yet, while putative short genes are common, their count is similar in epidemic and nonepidemic strains (data not shown). Phenotypic Qualities of the Strains It is possible that epidemic spread selects for or is preceded by specific phenotypic traits. These traits may not necessarily be linked with any particular genetic feature as there may be several mutational pathways to acquire the quality and hence, they may be difficult to identify with genetic or genomic comparisons. As such, we listed a number of measurable phenotypes that may be linked with epidemic success. The traits, their speculated association with epidemic dispersal, and the variables used in this study are listed in Table 3. The original data from measurements is available in Supplementary File 2. FIGURE 1 | Phylogenetic distance based on Sequence Type sequences. Koskinen et al. Epidemic and Non-Epidemic Klebsiella pneumoniae Frontiers in Cellular and Infection Microbiology | www.frontiersin.org February 2021 | Volume 11 | Article 5999245 TABLE 2 | Genomic analysis of individual strains based on RAST-annotation. EKP03 EKP05 EKP08 EKP10 EKP11 EKP22 EKP24 NKP01 NKP02 NKP18 NKP20 NKP25 NKP28 NKP30 Cell Wall and Capsule 217 230 218 230 234 214 220 216 196 214 217 217 193 227 Capsular and extracellular polysacchrides 38 54 42 54 57 38 41 38 42 36 37 37 39 49 Gram-negative cell wall components 91 90 91 90 90 38 90 89 66 90 88 92 69 92 Cell wall and capsule—no subcategory 88 86 85 86 87 85 89 89 88 87 88 88 85 86 Virulence, Disease, and Defense 148 150 141 155 156 154 136 146 143 154 144 168 134 129 Adhesion 77777777777777 Toxins and superantigens 00000000000000 Bacteriocins, ribosomally synthesized antibacterial peptides 12 12 12 12 12 12 12 12 12 12 12 12 12 12 Resistance to antibiotics and toxic compounds 121 123 114 128 129 127 109 119 116 127 117 141 107 102 Virulence, disease, and defense 00000000000000 Detection 00000000000000 Invasion and intracellular resistance 88888888888888 Phages, Prophages, Transposable elements, Plasmids 47 51 62 57 81 49 44 29 78 78 66 19 77 8 Phage family-specific subsystems 00000000000000 Transposable elements 00400007000050 Phages, prophages 47 50 57 57 80 48 43 20 77 77 66 19 70 8 Phages, prophages, transposable elements, plasmids—no subcategory 01101112110010 Pathogenicity islands 00000000000000 Gene transfer agent 00000000000000 Plasmid related functions 00000000000000 Membrane Transport 231 267 214 246 289 346 238 293 247 310 270 325 279 234 Protein secretion system, Type II 19 19 19 19 19 19 19 19 19 19 19 19 19 19 ABC transporters 78 72 65 72 78 79 60 71 68 69 78 71 71 75 Protein secretion system, Type VII (Chaperone/Usher pathway, CU) 20 25 20 25 19 20 25 25 19 20 20 28 26 22 Protein translocation across cytoplasmic membrane 77777777777777 Protein secretion system, Type V 00000000002002 Protein secretion system, Type I 50000500050000 Cation transporters 24 24 22 24 24 24 24 24 23 24 24 25 22 22 Protein secretion system, Type III 00000000000000 Protein secretion system, Type VI 19 17 16 17 14 19 15 14 0 14 15 14 13 22 Protein secretion system, Type VIII (Extracellular nucleation/precipitation pathway, ENP) 00000000000000 Protein and nucleoprotein secretion system, Type IV 21 68 28 46 92 137 49 97 73 114 67 125 87 29 Iron Acquisition and Metabolism 80 77 67 76 74 80 68 76 74 67 74 70 69 67 Siderophores 18 21 20 20 18 18 20 20 22 18 18 18 17 19 Iron acquisition and metabolism— no subcategory 62 56 47 56 56 62 48 56 52 49 56 52 52 48 Iron transpot 00000000000000 Motility and Chemotaxis 12 10 9 10 11 13 8 10 11 10 11 10 10 10 Magnetotaxis 00000000000000 Motility and chemotaxis—no subcategory 12 10 9 10 11 13 8 10 11 10 11 10 10 10 Flagellar motility in Prokaryota 00000000000000 Social motility and nonflagellar swimming in bacteria 00000000000000 Regulation and Cell Signaling 173 167 165 167 168 173 175 178 174 180 170 172 171 174 Quorum sensing and biofilm formation 13 13 13 13 13 13 13 13 13 13 13 13 13 13 Regulation of virulence 98888888888888 Programmed cell death and toxinantitoxin systems 16 18 15 17 17 18 23 23 17 19 20 18 19 24 DNA Metabolism 155 131 126 140 173 157 145 151 138 144 143 133 142 142 CRISPs 70007700000007 Koskinen et al. Epidemic and Non-Epidemic Klebsiella pneumoniae Frontiers in Cellular and Infection Microbiology | www.frontiersin.org February 2021 | Volume 11 | Article 5999246 Supplementary Figure 1 summarizes the results for epidemic and non-epidemic strains. We carried out a discriminant analysis for the measured phenotypic data. The analysis associated several phenotypic measurements with discriminant function. The discriminant function is a combination of linear effects of variables that give best separation of the data to distinct classes, in this study epidemic and non-epidemic strains. This approach revealed highly significant (posterior values did not overlap with zero) likelihood for a given trait to belong to either epidemic or nonepidemic group (resolved with Bayesian logistic regression between predicted values of discriminant function and epidemic status, Bayesian R 2 = 45%, Figure 2). Regressing predicted values of discriminant function against original variables indicated especially strong role of various measurements of drought tolerance in discriminant function (Figure 3) and non-epidemic strains with high discriminant function score were clearly more drought tolerant. Cross-Inhibition and Antibacterial Potential of a Putative Colicin We further studied the cross-strain inhibition given that the epidemic successfulness could emerge from the ability of epidemic strains to suppress non-epidemic strains during spread between hospitals or hosts (Figure 4). The inhibition, when detected, was determined with a dilution series to be either due to molecular activity or prophage induction (diluted phages produce distinct plaques unlike inhibiting molecules). Pairwise inhibition was infrequent and no general pattern between epidemic and non-epidemic strains was identifiable. As a curiosity, we selected one cross-inhibiting strain pair for more detailed analysis. NKP2 produces a component into its medium that inhibits EKP24 (ST11). Given the wide dispersal of ST11 K. pneumoniae and its association with NDM-1 encoding plasmids and hypervirulence (Gu et al., 2017), the inhibiting factor could provide a possible way to control these strains. However, the strain used in this study is not hypervirulent and therefore assessing direct applicability against hypervirulent strains was not conducted. Genomic comparison of NKP2 and EKP24 revealed the presence of genes for Colicin E3 in NKP2 that were absent from EKP24. Hence, Colicin E3 was hypothesized to be the inhibiting molecule. Co-culturing of these strains in the same medium demonstrates that EKP24 is unable to multiply. We further studied the adaptation of EKP24 to the continuous presence of the hypothesized Colicin E3 by serially culturing EKP24 in the presence of NKP2 medium extract for 4 weeks (n = 5). These cultures were refreshed three times a week. After 2 weeks, we removed the selection from five TABLE 3 | Studied characteristics and their hypothesized association with epidemic capability. Trait/quality Variables/factors used in this study Relevance Virulence genes Number/type Number of virulence genes may directly affect the strain’s potential to cause infections Antibiotic resistances Number/type Antibiotic resistance can compromise treatment, thus causing prolonged infections and increase the time during which bacteria disperse Growth rate/density Max growth rate (r), Max growth density (K) Faster and more dense growth may increase the bacterial load in the surrounding environment and thus its epidemic potential Growth temperature 22°C (room temperature), 37°C Growth differences in room temperature vs 37°C may reflect trade-offs in withinand outside-host environments and thus its adaptation to the environment vs the host Growth in different media LB, DMEM, BHI Potential to grow in different nutrient environments may provide bacteria more opportunities to proliferate in alternative habitats and thus provide possibilities to survive outside the host Growth in different nutrient levels 1, 10, 100% L Potential to grow in varying nutrient levels may provide advantage in different environments and hence affect its dispersal to new hosts Growth in varying pH pH 3, pH 4, pH 5, pH 6, pH 7 Bacteria may be exposed to different pH in the environment and the host (phagocytosis, skin, gastrointestinal tract) and the sensitivity to pH may decrease the changes for dispersal Potential to compensate surrounding pH pH 3, pH 4, pH 5 Potential to modify the surrounding microenvironment may play a crucial role in the bacterial chances to adapt to fluctuating environmental pH and hence affect its dispersal Resilience in EtOH 20, 50, 75, 90% EtOH Survival in the presence of alcohol containing sanitizers may directly affect the persistence of the strain in the environment and hence influence its potential to get transmitted Survival in the absence of water 14 days, 2 months, 6 months The potential to withstand drought can increase the timespan during which pathogen remains viable in hospital environment and hence affect its changes to get transmitted to new hosts Recovery after drought 14 days, 2 months, 6 months Faster recovery after drought can provide bacteria increased potential to colonize or infect new hosts Growth in mixed/ spatially structured population 0 rpm, 230 rpm Growth as a differently structured population may play a role in various stages of infection and persistence in the environment and may therefore affect the strain’s potential to disperse Number of plasmids Number/Inc-type Plasmids often carry genes that benefit the bacterium in specific conditions and hence their number may be related to survival in various conditions inside and outside the host Genomic prophages Number/Inc-type Activation of prophages may cause infections in competing K. pneumoniae strains and may therefore provide the prophage carrying strain an advantage in situations where several strains occupy the same environment Strain-specific inhibition Pairwise inhibition Production of bacteriocins or other antimicrobials may inhibit the growth of competing strains and thus may hinder the potential for sensitive strains to disperse into environments or hosts with other K. pneumoniae strains Short ORFs in the genome Number of ORFs Number of short open reading frames in the genome may reflect bacterial adaptive history and hence may be linked to epidemic potential Koskinen et al. Epidemic and Non-Epidemic Klebsiella pneumoniae Frontiers in Cellular and Infection Microbiology | www.frontiersin.org February 2021 | Volume 11 | Article 5999247 additional replicates. Resistance to NKP2 medium (with hypothesized Colicin E3) emerged already during the first culture transfer, and it remained stable even after the removal of selection. Re-sequencing of three putative Colicin E3 resistant samples revealed a prevalent mutation in Aerobactin siderophore receptor IutA. This mutation was absent in a culture that was not exposed to NKP2 extract. IutA has been shown to serve as a receptor for cloacin DF13 (Van Tiel-Menkveld et al., 1982), which is homologous to Colicin E6 and E3 (Akutsu et al., 1989). As such, we hypothesize that NKP2 extract rapidly selected for IutA mutants. Also, it is worth noting that NKP2 medium was not observed to inhibit the growth of the other ST11 strain EKP8, hence showing narrow activity. Altogether, the hypothesized Colicin E3 does not appear to provide efficient antimicrobial activity against drug-resistant K. pneumoniae strains even when the targeted strain is initially sensitive to the colicin. DISCUSSION K. pneumoniae has become one of the priority drug-resistant pathogens in hospital settings worldwide. Some K. pneumoniae STs are more prevalent compared to others, which provokes the question whether there are qualities in these genetically related groups that have made them more potent for dispersal. Here, we studied multiple phenotypic and genotypic characteristics of seven epidemic and non-epidemic carbapenem resistant K. pneumoniae isolates that emerged from different parts of the world. None of the specific genetic qualities associated uniformly with epidemic or non-epidemic strains. Overall, this again suggests that sequence typing is not an optimal approach for inferring qualities of individual pathogens. In other words, there are K. pneumoniae strains that are relatively different from one another for their specific characteristics while still grouping together in ST-analyses. Phenotypic characterization revealed indication that, while genetic differences were minute based on ST-analyses, phenotypic differences that separate epidemic and non-epidemic K. pneumoniae strains do exist. Such apparently controversial result could emerge if phenotypes are strongly dictated by genetic differences other than indicated by core genome-based ST-analyses or even by epigenetic modifications (Casadesus and Low, 2006). While most of the studied factors could not explicitly help explain epidemic qualities, the strong association of drought tolerance with epidemically non-successful strains could give some insights on the dissemination. All the isolates in this study were able to withstand 6 months of dryness. Interestingly, nonepidemic strains generally recovered faster and into higher density after the drought, which presents the possibility that non-epidemic strains may have qualities that provide them opportunities to cause infections in specific cases, for example, after long-term residence on surfaces (Kramer et al., 2006). Bacteria have multiple ways to protect itself during the drought. For example, effective biofilm formation is a major protection mechanism in bacterial FIGURE 2 | Predicted Linear discriminant values in epidemic (red) and non-epidemic (green) K. pneumoniae strains. Koskinen et al. Epidemic and Non-Epidemic Klebsiella pneumoniae Frontiers in Cellular and Infection Microbiology | www.frontiersin.org February 2021 | Volume 11 | Article 5999248