A Change In Which Variable Would Not Necessarily Change An Organismã¢â‚¬â„¢s Fitness?
Antibiotics (Basel). 2021 January; 10(1): 90.
Genome-Based Analyses of Fitness Effects and Compensatory Changes Associated with Acquisition of bla CMY-, bla CTX-Yard-, and bla OXA-48/VIM-1-Containing Plasmids in Escherichia coli
Michael Pietsch
aneRobert Koch Found, Department Infectious Diseases, Division Nosocomial Pathogens and Antimicrobial Resistances, Wernigerode Branch, 38855 Wernigerode, Deutschland; ed.ikr@mhcsteip (Thousand.P.); ed.ikr@yrefiefp (Y.P.)
Yvonne Pfeifer
1Robert Koch Institute, Department Infectious Diseases, Sectionalisation Nosocomial Pathogens and Antimicrobial Resistances, Wernigerode Branch, 38855 Wernigerode, Germany; ed.ikr@mhcsteip (M.P.); ed.ikr@yrefiefp (Y.P.)
Stephan Fuchs
2Robert Koch Institute, Department Methodology and Research Infrastructure, Partitioning Bioinformatics, 13353 Berlin, Deutschland; ed.ikr@sshcuf
Guido Werner
1Robert Koch Plant, Section Infectious Diseases, Partitioning Nosocomial Pathogens and Antimicrobial Resistances, Wernigerode Branch, 38855 Wernigerode, Germany; ed.ikr@mhcsteip (M.P.); ed.ikr@yrefiefp (Y.P.)
Received 2020 Nov 27; Accepted 2021 Jan 15.
Abstruse
(i) Background: Resistance plasmids are under selective conditions beneficial for the bacterial host, but in the absenteeism of selective pressure, this carriage may cause fitness costs. Compensation of this fitness burden is important to obtain competitive ability under antibiotic-free conditions. In this study, nosotros investigated fettle effects after a conjugative transfer of plasmids containing various beta-lactamase genes transferred into Escherichia coli. (two) Methods: Fourteen beta-lactamase-encoding plasmids were transferred from clinical donor strains to E. coli J53. Growth rates were compared for all transconjugants and the recipient. Selected transconjugants were challenged in long-term growth experiments. Growth rates were assessed at different time points during growth for 500 generations. Whole-genome sequencing (WGS) of initial and evolved transconjugants was determined. Results: Almost plasmid acquisitions resulted in growth differences, ranging from −iv.5% to 7.ii%. Transfer of a single bla CMY-16-conveying plasmid resulted in a growth burden and a growth benefit in contained mating. Long-term growth led to a compensation of fitness burdens and benefits. Analyzing WGS revealed genomic changes caused by Single Nucleotide Polymorphisms (SNPs) and insertion sequences over time. Conclusions: Fitness effects associated with plasmid acquisitions were variable. Potential compensatory mutations identified in transconjugants' genomes after 500 generations give interesting insights into aspects of plasmid–host adaptations.
Keywords: AmpC, ESBL, carbapenemase, fitness burden, compensatory mutation
1. Introduction
Microbial antibiotic resistance is a major claiming to public wellness. Resistance to nigh therapeutically important agents is increasing worldwide [1]. A generally discussed countermeasure to forestall and revert an increasing resistance development is to use antibiotics properly, thus reducing the pressure level to give resistant bacteria a selective reward over the susceptible population. In line with this argument, the question of a potential fitness brunt coming forth with resistance acquisition in bacteria is central. It is mostly gear up as a prerequisite that susceptible bacteria are fitter than their multidrug-resistant counterparts and thus, have an evolutionary advantage if selection pressure by antibiotic employ is released. Consequently, reduction of antibiotic use (reduced pressure) should immediately or with a certain delay lead to decreasing resistance rates and frequencies.
Mutation-based resistance such as ciprofloxacin resistance in enterobacteria or rifampicin resistance in mycobacteria and their effects on bacterial fettle and fitness compensation have been studied in greater detail, and corresponding results mainly questioned the textbook knowledge about resistance acquisition and fitness burdens [2,3,4]. If a fitness brunt was measurable at all, bacteria often and rapidly "adapted" to these challenges by selecting for populations demonstrating compensatory changes leading to fitness compensations [3,4,5,6,seven,8]. Whereas the effect has been described, its genetic backgrounds are much less understood. A lot of clinically important and trans-sectoral resistance bug come along with acquired resistance traits, like beta-lactamases, which are mainly located on mobile genetic elements, such as Integrative and Conjugative Elements (Ice) including plasmids, and which are horizontally acquired. This situation fifty-fifty complicates the above-described setting since fettle is influenced by resistance and plasmid acquisitions and since horizontal gene transfer itself may come along with an influence on fettle [9,10]. Many of these interacting factors and conditions were collated and reviewed recently [11].
Resistance to beta-lactams in Enterobacteriaceae similar Escherichia coli, Klebsiella pneumoniae, Enterobacter cloaceae and Citrobacter freundii is mainly mediated by acquired resistance properties associated with the presence of beta-lactamases, including extended-spectrum-β-lactamases (ESBLs), plasmid-mediated AmpC beta-lactamases (pAmpCs) and carbapenemases [12,13,14]. The corresponding genes are mainly plasmid-located or reside on chromosomal ICEs. In some Enterobacterales, AmpC-type lactamase genes are genus- and/or species-specific, only their expression is depression controlled by a tightly regulated repressor. Promotor and repressor mutations could lead to a derepressed AmpC expression, for example in Enterobacter and Citrobacter, resulting in third generation cephalosporin resistance. Even so, this resistance characteristic is not transferable and thus has non received greater Public or Ane Wellness attention. The success to disseminate β-lactaman resistance widely amidst the different sectors is linked to distinct mobile vectors and/or bacterial lineages. Specific "beta-lactamase cistron/plasmid" and "beta-lactamase gene/plasmid/strain" combinations successfully disseminated in single sectors just are almost absent among others. Exemplarily, E. coli isolates of sequence type ST131 carrying a bla CTX-Grand-15 ESBL factor successfully spread amongst healthy humans and man patients worldwide, only are much less frequently encountered in wildlife and livestock [15]. In contrast, AmpC-type lactamases similar CMY-ii are prevalent among East. coli from livestock, specially chicken and poultry, just their prevalence varies amidst humans, with higher rates in Asia and comparably low rates in Europe [16,17,18]. Several High german studies assessed the prevalence of ESBL- and AmpC-mediated resistance among Eastward. coli in food animals, food and humans [19,20,21,22]. Every bit function of this interdisciplinary inquiry initiative, nosotros demonstrated a wider prevalence of bla CMY-ii as the most prevalent pAmpC-type beta-lactamase cistron amidst livestock and food, but less ofttimes amid humans in Frg [23]. The bla CMY-two gene was preferably located on plasmids of IncK and IncI types.
The preferred combination of certain "beta-lactamase gene/plasmid" or "beta-lactamase cistron/plasmid/strain" combinations potentially has a fitness groundwork. We hypothesized that such fitness effects may be measurable with a comparable uncomplicated experimental setting used in the present study. If plasmids would be adjusted to their bacterial host, then transferring them to a novel host should consequence in measurable fitness costs. We selected 47 donor strains of E. coli, K. pneumoniae, M. oxytoca and E. cloaceae possessing various pAmpC (bla CMY-2/16), ESBL (bla CTX-M-1/-14/-fifteen, bla SHV-12) and carbapenemase (bla OXA-48, bla VIM-one, bla NDM-1) genes. The resistance genes were transferred by broth mating into the E. coli J53 recipient. Transconjugants demonstrating highest growth differences compared to the recipient were selected for subsequent long-term growth experiments. Growth rates were assessed at dissimilar time points at the beginning, during and at the end of the long-term cultivation. Whole-genome sequences of initial and evolved transconjugants were compared to determine compensatory mutations to ameliorate initial fettle effects.
2. Results
two.one. Transfer Experiments
Birthday, 43 strains of Eastward. coli (north = 38), K. pneumoniae (n = 3), K. oxytoca (northward = 1) and E. cloacae (north = 1) were selected as donors for mating experiments, with E. coli J53 as a recipient (see Materials Department and Supplementary Table S1). Of these, fourteen donors transferred beta-lactamase-mediated resistance into E. coli J53 (Table 1). The transferred plasmids varied in size from 60 to 250 kb and belonged to different incompatibility groups. All transconjugants independent at least one ESBL, AmpC or carbapenemase gene, and 3 transconjugants additionally harbored the beta-lactamase gene bla TEM-1.
Table 1
Characteristics of the transconjugants (Kx) used for experiments on bacterial fitness.
| Isolate | Wildtype Host Species | Beta-Lactamase Factor | Plasmid Size (kb) | Replicon Type |
|---|---|---|---|---|
| RS039 K1 | East. coli | bla CTX-One thousand-1 | lxxx | IncI1 |
| RS292 K1 | E. coli | bla CTX-G-xv, bla TEM-1 | fourscore | IncFII |
| 17/xi K1 | E. coli | bla KPC-ii | 190 | IncA/C |
| 370/12 K1 | Grand. oxytoca | bla VIM-1 | 250 | IncFIB |
| 384/13 K2 | Thousand. pneumoniae | bla NDM-ane, bla CTX-M-fifteen, bla OXA-nine, bla TEM-1 | 110 | IncFII |
| 656/thirteen K2 | E. coli | bla SHV-12 | 100 | IncI1 |
| 531/12 K2 | E. coli | bla CMY-2 | 90 | IncI1 |
| 252/09 K3 * | E. coli | bla CMY-2 | 85, sixty | IncI1, IncFII |
| 346/12 K2 * | K. pneumoniae | bla OXA-48 | 60 | IncL/M-1 |
| RS165 K1 * | E. coli | bla CTX-M-14 | 70 | IncFII |
| 151/09 K2 * | E. cloacae | bla VIM-i | 100 | IncN/IncR |
| 104/15 K3 * | Eastward. coli | bla CTX-Yard-15 | 77 | IncF |
| 102/04 K1 * | East. coli | bla CMY-16, bla TEM-ane | 160 | IncA/C |
| 102/04 K2 * | E. coli | bla CMY-sixteen, bla TEM-1 | 160 | IncA/C |
The initial relative fitness load of the transconjugants after plasmid uptake compared to the plasmid-free recipient strain was adamant past growth experiments. The deviations of the relative fitness of the transconjugants to the plasmid-free recipient ranged from approximately −5% to +7%. For most transconjugants, the deviations ranged from 1–2% (Figure ane). Isolates with higher negative deviation in relative growth rate were 346/12 K2, 102/04 K2 and 151/09 K2. One isolate, 102/04 K1, showed a higher fitness compared to recipient E. coli J53.
Growth rates of the transconjugants (blue) relative to the growth rate of the plasmid-free recipient E. coli J53 Azir (yellowish). Isolates se-lected for further analysis are highlighted by a light blue background.
In addition to the growth rate, differences in growth beliefs were observed. Outside the exponential growth phase, which was used every bit a marker for the relative fitness adding, unlike curve progressions in the late log or stationary phase were observed (Supplementary Effigy S1). Strains with the highest growth rates did not always bear witness the highest endpoint optical density (OD) (see 102/04 K1). However, no meaning delay in the lag phase was observed. Simplified, information technology was causeless that with continuous and competitive growth, a college growth rate would outcompete other strains over time and lead to a fitness advantage and thus to higher abundance over fourth dimension. Therefore, the growth rate in the exponential growth stage was used every bit a marker for the fitness of the isolates.
For in-depth analyses, vii of the transconjugants (102/04 K1, 102/04 K2, RS165 K1, 252/09 K3, 346/12 K2, 151/09 K2 and 104/15 K3), expressing the virtually significant changes in growth, were selected for further experiments (highlighted in Figure 1).
2.2. Long-Term Growth Experiments
To identify potential fitness compensation furnishings, long-term growth experiments over 500 generations of the vii selected transconjugants were performed. The initially determined factor for the increment in generations per passage was viii.96. To reach generations G500, the cultures of the transconjugants were therefore inoculated consecutively for 56 days and sampled and analyzed at generations G200 and Thousand500 (Supplementary Table S2).
Since it was assumed that within the long-term cultures, a population diverseness acquired past different subpopulations could develop, iv randomly selected colonies of each plated long-term culture were selected and analyzed.
In general, all isolates showed a rather constant or strongly negative trend (102/04 K1 and 346/12 K2) of the relative fitness for generation Yard500 compared to G0. Interestingly, the relative fitness values determined for generation Thou200 were in some cases far above (RS165K1, +nine.2%) or far below (151/09 K2, −9.0%) the fitness mean of Yard0 to M500 (Effigy 2). The presence of beta-lactamase genes was tested positive by PCR in all selected long-term cultivated transconjugants in each generation. A change in the respective plasmid-mediated antimicrobial resistances was not observed, and respective resistance patterns remained stable over time (Supplementary Table S3).
Development of the relative fitness of transconjugants 102/04 K1, 102/04 K2 and RS165 K1 compared to the recipient during long-term cultivation for 500 generations. Aggregated values at the showtime bespeak (G0), subsequently 200 generations (G200) and 500 generations (Chiliad500) are shown. Fault confined draw standard deviations from three independent experiments (mean values from unmarried experiments based on technical triplicates).
two.3. Genome Reconstruction and Genome Comparisons
After 500 generations of long-term cultivation, three transconjugants with plasmids carrying bla CTX-1000-14 (RS165 K1) and bla CMY-16 (102/04 K1 and 102/04 K2) were selected for whole-genome comparing studies. 102/04 K1 and 102/04 K2 were called considering plasmid acquisition in G0 transconjugants demonstrated opposite fitness furnishings and RS165 K1 considering the G200 value showed a remarkable fitness proceeds (Effigy two). Generation G0 isolates of the transconjugants 102/04 K1 and 102/04 K2 were sequenced using PacBio technology to create a reference sequence. 102/04 K1/K2 isolates of generation M0 and one evolved transconjugant 102/04 K1 G500 were sequenced using Illumina MiSeq. RS165 K1 Thousand0 and iii evolved transconjugants of different generations of RS165 K1 (G200-1, K200-3, G500-iii) were sequenced using Illumina MiSeq. Whole-genome sequence analyses enabled the detection of base substitutions, deletions, insertions or modifications of other kinds in the chromosome or plasmids in the different isolates. Identified sequence modifications were examined for their possible influence on the fitness of the isolates.
SMRT sequencing and subsequent HGAP associates of 102/04 K1 and K2 resulted in two ring-closed sequences for each isolate. A blastn analysis confirmed and assigned these contigs to the expected chromosome and plasmid. The reconstruction of the genome sequences of the isolates of the latter generation based on Illumina reads was performed using the reconstructed sequences as a reference (Supplementary Tables S4 and S5).
The reconstructed genome and plasmid sequences of the isolates of the dissimilar generations of RS165 K1, and the iii 102/04 transconjugants, were examined for differences. Apart from a few SNPs and insertion sequences, the genome and plasmid sequences of the different transconjugants 102/04 K1, K2 and RS165 K1 were homologous to each other and did not show whatever genomic rearrangements (Effigy iii and Effigy 4).
Schematic presentation of the genetic changes of transconjugant isolates 102/04 K1 und K2 after long-term growth. Mutations in the chromosomes and amino acrid changes are marked at the respective positions. Base pair substitutions are marked by color. Positions of modified IS elements are highlighted by white bars. White arrows point towards changes in coding sequences.
Schematic presentation of the genetic changes of transconjugant isolates RS165 K1 after long-term growth. Mutations in the chromosomes and amino acid changes are marked at the respective positions. Base pair substitutions are marked past color. Positions of modified IS elements are highlighted past white bars. White arrows point towards changes in coding sequences.
Comparing of the three 102/04 transconjugant genomes showed a total of four chromosomal and i plasmidic SNP. In add-on, xi different insertion sites were found (10 on the chromosome and one on the plasmid sequence). Of these changes in the nucleotide sequence, viii were located in non-coding regions. Ane SNP caused a non-synonymous mutation of a poly peptide sequence and seven insertion sequences led to nonsense mutations of the encoded amino acid sequences due to premature stop codons. Detailed information on the private nucleotide sequence changes is shown in Figure iii. Most SNPs and insertion sites were observed between isolates 102/04 K1 and 102/04 K2, while isolates 102/04K1 G0 and K500, and 102/04 K2 G0 and One thousand500, were nearly identical in their SNPs and insertion sites (Effigy 3). However, seven SNPs or differences caused past insertions were also observed between these follow-up isolates. The inserted insertions' sequence (IS) elements belonged to the element IS10R in almost all cases.
Comparison of the four RS165 K1 isolates revealed 14 changes in the chromosome (Figure 4). The plasmid sequences did not show any differences. The chromosomal changes related to five SNPs, ii of which were located in noncoding regions and two of which led to non-synonymous mutations. The remaining SNP was located at position three (acceptor arm) of the tRNA Gln (glnV) in isolate RS165K1 G200-1. Furthermore, nine IS element insertions were observed, seven of which were localized in coding sequences and led to not-synonymous mutations. Generation G200 isolates showed different mutations amid each other. In the generation G500 isolate, identical mutations as in the G200 isolate were detected (Figure 4). Every bit in transconjugants 102/04 K1/K2, the majority of the differences in the nucleotide sequences were due to the insertion of IS10R at different positions in the chromosome.
3. Word
3.1. Comparative Growth Experiments
Measuring bacterial growth nether optimal conditions regarding temperature, aeration, rich nutrition conditions and without a competing flora is a quite artificial setting to make up one's mind bacterial fettle and for sure non the all-time model to mimic the natural habitat of a given bacterium. Consequently, competitive growth experiments to measure fettle furnishings should exist carried out nether the almost practical weather possible, meaning either in vitro or even ameliorate, performed nether in vivo conditions of an infection [24,25]. All the same, measuring growth dynamics and comparing plasmid-free and plasmid-containing isogenic strains allows a first and deep look into the circuitous interplay of plasmid (resistance cistron)–host interactions.
The horizontal spread of resistance genes has a high impact on the dissemination of a number of resistance properties of high public health impact. For Gram-negative leaner resistance to third generation cephalosporins mediated by ESBLs and pAmpC enzymes, resistance to carbapenems mediated by carbapenemases and resistance to colistin mediated by Mcr variants are of major importance. These resistance genes are horizontally caused and spread across bacterial genera and sectors [20,23,26,27]. The experimental setting presented here does not allow differentiating between fettle effects associated with plasmid and/or resistance cistron acquisition. We consider this every bit a rather theoretical problem since many of the analyzed resistance genes included in this study accept a strong association with a corresponding ICE or plasmid background and thus, resistance gene acquisition in nature is mainly and straight associated with conquering of a respective plasmid. In this regard, the experimental setup always measured the combined effect of a plasmid and pAmpC, ESBL or carbapenemase gene conquering.
Performing competitive growth experiments with progenitor and descendent strain variants in the same experimental setting, such as a combination of recipient and transconjugant cells, allow determining an firsthand and interconnected fitness outcome and measuring much lower fettle furnishings [28,29,30]. However, in a setting as described here, where conjugative plasmid transfer appears in liquid broth to a substantial amount, measuring growth rates in competitive experiments will always assess furnishings derived from de novo plasmid transfer and transconjugant propagation equally well. Thus, we decided to perform, determine and evaluate growth in single-growth experiments only.
As mentioned before, a simple comparative growth analysis under laboratory conditions in rich medium was chosen in the nowadays study to determine relative fitness. The fitness data of the generated transconjugants showed that the acquisition of a beta-lactamase gene-carrying plasmid did not necessarily take an effect on the fitness of the recipient E. coli J53. Only in about half of the transconjugants were negative growth effects, and in one case even a positive growth effect, observed (Figure 1). A correlation between plasmid size or replicon type on fettle could non be observed; due to the heterogeneous plasmid or transconjugants' collection, information technology was hard to conclude that a fitness effect derived from these parameters.
The plasmid size tin can, however, influence the growth behavior of E. coli, as it was described in a study by Smith et al. [31]. The authors observed in isogenic clones that the presence of larger plasmids resulted in a longer lag stage than that of smaller plasmids. A fitness effect of this kind was not demonstrated in the present study. Nevertheless, the observation of Smith et al. shows that, in addition to the growth rate, other factors must be considered for a comprehensive fettle characterization [32]. As described earlier, plasmid stability and fitness derived from deeper and detailed analyses of variable vectors constructed and transferred into an E. coli background showed an effect of the plasmid size on fettle [31]. These and similar studies hide the fact that the analyzed system with an artificial vector, that is not naturally hosted in E. coli, largely differs from a tight and evolutionary fine-tuned plasmid–host link, which may be associated with much lower or no fettle costs. For example, the transfer of a widely disseminated R1 plasmid (IncFII type) into an E. coli recipient did not reduce fettle of the transconjugants relative to the recipient. Long-term growth experiments were even capable of demonstrating a fettle advantage over fourth dimension [33]. Schaufler et al. [34] were capable of curing bla CTX-Thou-15-containing plasmids from epidemic E. coli strains, hence giving the opportunity to investigate fitness furnishings of the tightly regulated interconnection between a plasmid and its host subsequently loss of the respective plasmid. According to these and subsequent analyses [35], railroad vehicle of ESBL gene-containing plasmids did non come along with a fettle burden to the Due east. coli host, but rather with a fitness gain. Moreover, railroad vehicle of ESBL gene-carrying plasmids also increased virulence in the respective host. The association betwixt acquisition of resistance and increased virulence partly mediated via multicomponent genetic elements is a full general characteristic of larger ICEs, including plasmids [36,37].
Di Luca et al. [29] analyzed fettle costs associated with carbapenemase gene-conveying plasmids from K. pneumoniae (pG12-KPC-two; pG06-VIM-1) transformed into four Eastward. coli recipients of various clonal lineages (ST10, ST69, ST95, ST537) and phylogenetic groups, such every bit A, B2 and D. Relative fitness costs were mostly pocket-sized and in the range of 1.one% to 3.6% when compared between recipients and transformants. Fitness costs were dependent on the respective plasmid and the clonal background.
The manifestation of a fitness brunt subsequently plasmid uptake in only a part of the transconjugants of the present written report contradicts the assumption that the conquering of a plasmid should always cause an initial negative fettle effect due to the additional burden of plasmid replication [32]. Based on a systematic review in which fettle costs due to acquired antimicrobial resistances were investigated, the authors Vogwill and MacLean were able to testify that although the uptake of a plasmid is often associated with fettle costs, in that location were also very little or no fitness costs in the studies investigated and a correlation between plasmid size and fettle load was non sufficiently proven [38]. The authors concluded that regular plasmid uptake and loss in combination with corresponding adaptation processes occurred in the evolutionary by of host organisms. Therefore, new plasmid uptake does not necessarily atomic number 82 to a loss of fitness, but merely if new plasmid–host combinations are created or the plasmids acquire new, non-adjusted genetic fragments, for instance, by horizontal gene transfer. This hypothesis is supported by the study of historical isolates from the Murray Collection, which showed that the "plasmid population" of isolates from the pre-antimicrobial period did not have a fundamentally different composition [39]. The absence of fitness costs in half of the investigated transconjugants in our written report could exist explained by a previously acquired plasmid–host accommodation. However, the observation of such unlike fitness values after uptake of plasmid p102/04 by Eastward. coli J53 (K1: 107.23%, K2: 95.58% fettle compared to plasmid-free E. coli recipient) implies that farther genetic effects accept or could take a far greater influence on fitness than plasmid–host adaptations.
In a previous study using the Gram-positive enterococci equally model organisms, it was shown that newly acquired ICEs including plasmids imposed an immediate biological cost in an Enterococcus faecium recipient [40]. Yet, the initial costs were mitigated through long term growth over 400 generations and beneficial plasmid–host associations quickly emerged. It was also demonstrated that the benign genetic changes were imprinted either on the plasmid or on the chromosome. 2d-round transconjugants either received the evolved resistance plasmid and thus, did non show a different growth beliefs, or demonstrated once again an initial fitness burden, suggesting that compensatory mutations were accumulated in the first-round transconjugants' chromosomes [twoscore].
3.ii. Genome Comparisons
AmpC-mediated fitness costs were mainly addressed in the context of mutation-based genetic changes leading to derepressed chromosomal AmpC genes which mediate extended beta-lactam resistances [41,42]. This is the first written report, we are aware of, which analyzed fitness effects of transferable bla CMY genes and plasmids, respectively. Remarkably, we achieved opposite furnishings when nosotros repeated the aforementioned experiment several times: transfer of the same CMY-ii-containing plasmid into E. coli K12 J53 resulted either in a fitness brunt or a fettle gain, suggesting that other as yet unknown circumstances may influence these biological consequences (see adjacent section).
The observed diametric fitness furnishings between the transconjugants 102/04 K1 and 102/04 K2 was with 11.6% departure in relative growth, surprisingly and unusually big. Since mutations in the transferred plasmids of the ii isolates could be excluded on the basis of the whole-genome information, it can be assumed that the different growth beliefs of the strains was caused by chromosomal mutations. Between transconjugants 102/04 K1 and 102/04 K2, eight sequence differences caused past three SNPs and 5 IS elements at different positions were observed. Of these, iii SNPs and three IS elements occurred in non-coding regions but could still have an affect on the gene expression. Interestingly, 102/04 K1 had a sspA factor truncated by one IS element. SspA (Stringent starvation protein A) is a stress protein expressed under glucose, nitrogen, phosphate or amino acid limiting conditions [43,44]. Several studies advise that sspA is important for the stress response during the stationary phase and under nutrient-limited conditions in Eastward. coli. The expression of sspA is positively regulated by the presence of relA and likewise increases with decreasing growth rate [43]. The exact role of SspA in transcription and cell physiology is still unclear. Williams et al. were able to evidence that a deletion of sspA during the exponential growth phase led to an altered expression of at least 11 proteins [43]. In addition, sspA mutants were found to be less fit than the wildtype under longer food-limiting conditions or long stationary phases [43]. Hansen et al. could also bear witness that sspA is necessary for the regulation of acrid tolerance in Eastward. coli [44]. Acid tolerance is an of import power of intestinal leaner to survive the low pH during gastric passage to the intestinal tract [45]. Therefore, the loss of sspA may give ascent to a presumed fitness reward under bogus growth weather, simply under natural conditions (e.g., in the human host), the loss of sspA can be expected to adversely touch on the fitness (or survivability) of Due east. coli.
In improver to sspA, oxyR was also plant truncated in 102/04 K1. OxyR is a Deoxyribonucleic acid-binding transcriptional regulator for oxidative stress and is indirectly involved in the regulation of more than forty different cistron products through its influence on mRNA stability [46].
three.3. Long-Term Growth Analysis
During long-term cultivation, a decreasing tendency in the relative fitness of the strains was evident from generations G200 to M500. This is peculiarly surprising considering the fact that, co-ordinate to observations in Richard Lenski'due south Long-term Experimental Evolution Project (LTEE), a permanent increase in the relative fettle of the isolates could be observed over sixty,000 generations [47]. Still, since a slight decrease in relative fettle was too observed in the passaged plasmid-free recipient strain Eastward. coli J53 Azir compared to generation K0, it can be assumed that other fitness-determining effects, which cannot be covered by the experimental approach used to mensurate fitness, affected the populations of the long-term cultures in the class of the experimental evolution. The discrepancy between expected fitness increase and observed fitness loss could probably accept been better represented past directly competitive growth experiments. In addition, the process of daily inoculating new media represents a major hurdle for less competitive clones. By inoculating low-book culture, clones with fitness factors other than a loftier growth rate, e.yard., a higher final OD, may be selected. Over this effect probably led to the displacement of the expectedly fitter clones. The observation that a high endpoint OD often does not occur in isolates with the highest growth rates supports this assumption.
The dissimilar growth beliefs and genetic differences in isolates of one generation implied that different sub-lineages from the original clone of generation Grand0 developed. The mutations leading to these descendants established in the population could non be investigated due to the pocket-size sample size of the sequenced isolates. Withal, the detection of identical SNPs and insertion elements in RS165 K1 G200 and G500 indicated that the subclone RS165 K1 K200 had established itself in the population. Whole-genome sequencing of further isolates or the unabridged population could have provided further information on the composition of the population.
The adaption of cells to the caused plasmids and the growth weather condition could be deduced from the growth behavior of the individual transconjugants. In previous studies, information technology was shown that nether selective conditions, an adaptive development to the resistance gene-bearing plasmids occurred, which compensated for the fitness costs of acquiring plasmids [48,49,50]. Regulatory adaptive changes during co-evolution experiments occurred generally in the bacterial host chromosome and non in the plasmids [48,51,52,53]. The occurrence of almost all mutations in the bacterial host chromosome of the evolved transconjugants in the present study indicates towards chromosomal regulatory adaptations. Excluding the highly mobile IS elements, a maximum of 2–iv SNPs occurred in 500 generations of evolved isolates. In the comparable LTEE, 45 SNPs were observed during the first 20,000 generations, which did not occur linearly only were more abundant in the early generations [47]. Whether and which SNPs or inserted IS elements accept an effect on gene regulation and thus, on the fitness of the transconjugants of the present study, was non conclusively clarified. Studies by Harrison et al. and San Millan et al. have shown that fettle compensation can basically be caused past regulatory effects and re-create number reduction of plasmids [52,53]. In contrast, Porse et al. observed that positive adaptations were accomplished by deletions of large plasmid regions [54], which could be excluded in our study.
three.4. Limitations of the Study
The study has several limitations. First, various experimental settings be to measure bacterial fitness costs and we are well aware that this model practical here does not reverberate the situation multidrug-resistant pathogens see in their natural habitat in humans, patients, animals or the environment. This includes the different options of single growth and competitive growth experiments and modifications of growth weather, which has non been undertaken here (diet depletion, anaerobic conditions, etc.). 2d, we assessed the exponential growth rate as a mensurate to compare fitness, only we are well enlightened that besides other endpoints such as time to reach stationary phase or maximum cell density are other possible markers. Third, one limitation corresponds to a finding of this written report showing that identical experimental settings may lead to contrary results. It was demonstrated that bla CMY-16-mediated plasmid transfer may atomic number 82 to a fitness gain or loss in doing the same experiment twice. Thus, fitness effects may exist caused by side-effects associated with simply potentially contained from the measured and selected (resistance) plasmid transfer. Lastly, we were unable to experimentally confirm a causal link between the measured fitness gain and the described genetic changes in the transconjugants' genomes simply due to fact that the funding for this research project expired.
4. Materials and Methods
iv.1. Strains
Altogether, 43 strains of E. coli (n = 38), Grand. pneumoniae (n = 3), K. oxytoca (n = 1) and E. cloacae (n = 1) were selected equally donors from previous studies [nineteen,twenty,22,23] and from routine diagnostics sent for strain typing and beta-lactamase characterization to our laboratory (Supplementary Table S1). Majority of the selected donor strains (n = 26) were E. coli ST131 with bla CTX-M-15, while others were Eastward. coli strains possessing other bla CTX-K variants (bla CTX-Thousand-1, bla CTX-M-14), E. coli strains possessing bla CMY variants (bla CMY-2 and bla CMY-16) and E. coli strains possessing various carbapenemase genes (bla OXA-48, bla VIM-1, bla NDM-1). For mating experiments, a sodium azide-resistant (Azir) recipient strain E. coli J53 was used [55].
4.2. Transfer Experiments and Characterization of Transconjugants
Transfer of the beta-lactamase gene-carrying plasmids was conducted using broth mating and recipient strain Due east. coli J53 Azir. Transconjugants were cultivated on Luria-Bertani (LB) agar with cefotaxime (CTX, ane mg/L), cefoxtin (CXI, 10 mg/L), ampicillin (AMP, 50 mg/L) and 200 mg/Fifty sodium azide, morphologically checked and screened by PCR for presence of the respective beta-lactamase genes (Supplementary Tables S1 and S6). Furthermore, the plasmid DNA of the transconjugants was extracted and the replicon types of the transferred plasmids were characterized using the PBRT Kit ii.0 (Diatheva, Fano PU, Italy). The plasmid sizes were adamant by S1-nuclease brake and pulsed-field gel electrophoresis [56].
iv.3. Growth Experiments in Microtiter Plates
An overnight culture was adjusted to v × tenhalf dozen colony forming units (CFU)/mL in fresh LB-medium at 37 °C. Growth was measured in parallel in the Bioscreen C MBR photometer in a microtiter volume (Oy Growth Curves Ab Ltd., Helsinki, Finland). For this purpose, 200 μL of the jail cell break was pipetted in triplicate into the wells of a 96-well honeycomb plate (Oy Growth Curves Ab Ltd. (formerly: Bioscreen), Helsinki, Finland) and measured at 37 °C for twenty h. The optical density (OD600) was determined every 10 min. Before each OD measurement, the bacterial suspensions were mixed past short-vortexing. Reference strains were carried out on each plate to determine the relative fettle. The reference strains used were Due east. coli J53 Azir, and for the long-term growth experiments, additionally, the transconjugants of the respective Thou0 generation.
4.4. Long-Term Growth Experiments
Based on the initial fitness characterization, seven transconjugants with characteristic fitness effects (102/04 K1, 102/04 K2, 252/09 K2, 104/15 K3, RS165 K1, 151/09 K2, 346/12 K2) were identified, which were subsequently cultivated for 500 generations and investigated for possible fitness compensation effects. The initial inoculum of long-term cultivation experiments resulted from a unmarried colony of a respective transconjugant, which was suspended into five mL LB medium and incubated at 37 °C in a shaking incubator at 140 rpm. Afterward about 24 h, 10 μL of the bacterial break was transferred into v mL fresh LB medium and incubated again for 24 h. An inoculation book of 10 μL resulted in an increment of viii.96 generations per passage (24 h). The cultivation was carried out over 500 generations. Every 100 generations, bacterial civilization was sampled and cryopreserved at −80 °C. Cultures of isolates after 200 (K200) and 500 generations (M500) were examined for possible changes in their fitness and antimicrobial susceptibilities. For this purpose, the jail cell suspension was plated in a dilution of 10−6 each onto LB agar and LB agar containing selective antibiotics (CXI: 10 mg/Fifty, AMP: 50 mg/50). From the antibody-containing agar plates, iv colonies per each generation were selected and examined for their fettle backdrop past growth measurements, antibody susceptibility testing by goop microdilution for xiii antibiotics (AMP, CTA, CTZ, CXI, GEN, KAN, AMI, STR, NAL, CMP, CIP, MER, TRS; see EUCAST abbreviations) according to EUCAST (European Commission on Antimicrobial Susceptibility Testing) recommendations and the presence of beta-lactamase genes past PCR.
iv.5. Determining Growth Characteristics and Relative Fitness
To make up one's mind the relative fitness, the growth rate in the exponential phase was used equally a fettle parameter. For this purpose, the growth rates μtx (time 10) were first determined for all sequent measurement points using the following formula with ODi at tone and OD2 at t2:
The exponential growth rate was determined past an initial series of experiments with the plasmid-free recipient East. coli J53 Azir. For the present experimental conditions, a threshold of μtx ≥ 0.5 (h−1) was defined as exponential growth. To exclude measurement errors or outliers outside the exponential growth phase, the additional status was defined that μtx at t10 is only assigned to the exponential phase if μtx-1 at tx-one and μtx+1 at tx+1 are also to a higher place the threshold. The growth rate μ for the entire exponential stage was then determined by the mean value of all growth rates, μtx, that run across the weather and are above the threshold. The respective triplicates of an isolate (= technical triplicate) were combined to a mean (average) value and the standard divergence was determined. Using the growth rate, μ, the relative fettle of each isolate compared to the recipient, transconjugants of the unlike generations or wildtype strains were calculated. The relative growth rate was adamant by the growth charge per unit of a transconjugant μ divided past the growth charge per unit of the recipient E. coli J53 Azir (multiplied past 100%). The growth rates of E. coli J53 Azir, the wildtype or transconjugant G0 isolates were used to make up one's mind reference fitness values. Deviations from these growth rates resulted in the relative fettle loss/gain in percent (%). All measurements were performed at least in biological triplicates, resulting in a mean (average) value (of the mean values of the technical triplicates).
iv.half-dozen. Isolation of DNA, Plasmid DNA and Genomic Deoxyribonucleic acid for Sequencing
Genomic Deoxyribonucleic acid for PCR screening of resistance genes was isolated by uncomplicated heating and subsequent centrifugation. Genomic Dna for side by side-generation sequencing (NGS) was isolated with the DNeasy Blood and Tissue Kit (Qiagen GmbH, Hilden, Germany) using a standard protocol. Genomic Deoxyribonucleic acid for unmarried-molecule real-time (SMRT) sequencing was isolated with a Genomic-tip 100/Grand Kit (Qiagen GmbH). The quality, quantity and purity of the Deoxyribonucleic acid were determined by agar gel electrophoresis, photometrically using a BioPhotometer (Eppendorf, Hamburg, Germany) and in a QuBit four fluorometer (Thermo Fisher Scientific, Inc., Waltham, MA, United states of america) with a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Inc.). Plasmid Dna was extracted by the Plasmid Mini Kit (Qiagen GmbH, Hilden, Germany). Sizes of plasmids were assessed subsequently agarose gel electrophoresis compared to plasmids of known sizes using reference strains E. coli V517 and E. coli R222.
four.7. Dna Sequencing by NGS and SMRT Sequencing
For subsequent bioinformatic analyses, vii dissimilar 102/04 K1 (10000, G500-1) and K2 (M0) isolates as well as RS165 K1 (G0, G200-1, G200-3, G500-3) isolates of different generations were sequenced using Illumina whole-genome sequencing. For reference purposes, the M0 transconjugants of 102/04 K1 and 102/04 K2 were long-read sequenced using SMRT sequencing. Sequencing libraries were generated using the Nextera XT DNA Library Preparation Kits (Illumina, Inc., San Diego, CA, USA), every bit specified by the manufacturer. NGS sequencing was performed at the Core Facility of the Robert Koch Institute MF2, on a MiSeq arrangement (Illumina, Inc.) using the MiSeq v3 Reagent Kit (Illumina, Inc.) with two × 300 bp paired-end mode. SMRT Sequencing was executed on a Pacific Biosciences apparatus RS II at GATC Biotech AG (at present: MWG Eurofins, Konstanz, Deutschland). The sample grooming (DNA extraction) was performed co-ordinate to the specifications of the service provider and as described above.
four.8. Whole-Genome Information Analysis
The quality of the generated Illumina raw data was checked using the programme FastQC (https://github.com/s-andrews/FastQC). The quality of the raw data was improved by filtering out or shortening reads with poor-quality parameters. Trimmomatic was applied to filter and exclude raw reads of poor quality, if necessary [57]. For subseqeunt de novo assembly of raw data, the parameter "maxinfo 15:0.v" was called. All farther parameters were used every bit default settings. The raw data had an average Phred value of more than 30 and the average read quality could exist increased to a PHRED score of more than 35 later on trimming. Illumina raw data were assembled using SPAdes (five.3.ten.1) [58]. The PacBio whole-genome data (102-04 K1 G0, 102-04 K2 Yard0) were assembled by the service provider using the HGAP software (5.three), resulting in two contigs per sequencing: one contig covering the complete chromosome and 1 covering the plasmid. Polishing and band closing of long-read data were conducted past aligning of trimmed Illumina read data (parameter "slidingwindow 4:15") on the HGAP3-generated contigs using Unicycler [59]. The assignment of the contigs as chromosomal or plasmid sequence was done by a BLAST assay. The poly peptide coding sequence segments of the consensus sequences of chromosome and plasmid were annotated using RAST annotation servers [threescore].
The identification of sequence modifications of "later on-generation/long-time cultivated" strains compared to the generation G0 strains, was conducted by using the long-read-based reconstructed G0 strains 102/04 K1 and 102/04 K2 equally a reference. In item, de novo assembled Illumina read-based contigs were aligned and orientated with the proprietary Geneious Mapper (Parameter: Medium–Depression-Sensitivity, no iterations) on the reconstructed reference generation 10000 genomes of 102/04 K1 and 102/04 K2 (Geneious v10.0.five (Biomatters, Ltd., Auckland, New Zealand)) [61]. This procedure enabled a better identification of transpositions and insertions in the genome. Occurring insertions or variations from the reference were compared past blastn against the NCBI Genbank Nucleotide Collection (nr/nt). Mobile genetic elements, such as insertion sequences (IS), were identified and annotated in detail using the ISfinder database (https://isfinder.biotoul.fr) [62]. The final consensus sequence, created by using the reference-oriented contigs, including identified insertions and deletions, was then re-checked for sequence consistency by realigning the reads on the consensus. Finally, the sequences of the generation G0 and the long-time cultivated strains were compared using Mauve (http://darlinglab.org/mauve/mauve.html) [63]. The same procedure was used to reconstruct and compare the plasmid sequences. Annotations of the references were transferred on the newly reconstructed sequences. Resistance genes, plasmid replicon genes and selected insertion sequences were annotated using the PlasmidFinder one.three, ResFinder 3.0 and ISfinder databases (http://www.genomicepidemiology.org/) [64,65].
five. Conclusions
Horizontal gene transfer appears frequently in nature and new "resistance factor–plasmid–host" combinations resulting from a unmarried genetic event may arise regularly, and some of these new combinations may be fitter than their resistance plasmid-complimentary progenitors. Every bit the reservoir of transferable pAmpC-type resistance is a big problem in animal farming and in humans in some parts of the world, information technology cannot be excluded that a new variant appears one time that successfully spreads across all sectors. This study showed the complexity of the resistance problem in terms of acquisition, fitness effects and biological consequences of horizontal resistance factor and mobile element transfer studied later on an in vitro broth mating of beta-lactamase-, and particularly bla CMY-conveying, plasmids transferred between Enterobacterales isolates. We showed that performing the same experiment twice could lead to contrary effects in terms of fitness brunt or gain associated with an acquisition of a bla CMY-16-harboring plasmid in an E. coli host (Figure 1). Nosotros observed fettle compensation over hundreds of generations from both sides, starting from either a fettle loss or a fitness proceeds associated with resistance plasmid conquering (Effigy ii). Using a combination of long-read and short-read sequencing information, we were capable of identifying genomic changes appearing during growth of selected transconjugants for 500 generations in liquid culture (Effigy 3 and Figure 4). Still, when assessing and evaluating these genomic changes over time, nosotros were unable to accost direct and putatively causative links betwixt measured fitness effects and insertions of mobile DNA or unmarried nucleotide changes in the evolved transconjugants. We assume that the effects of these genomic changes might be indirect and regulatory, a hypothesis that requires farther experimental proof in time to come experiments.
Acknowledgments
Nosotros thank colleagues at the Sequencing Core Facility MF2 at the Robert Koch Establish for performing Illumina sequencing and MWG Eurofinns for generating PacBio sequences. A special thanks to Sibylle Mueller-Bertling for performing the goop mate conjugation assays.
Supplementary Materials
The post-obit are available online at https://www.mdpi.com/2079-6382/10/1/90/s1, Tabular array S1: Characteristics of clinical donor strains and the corresponding transconjugants used for fitness experiments, Tabular array S2: Fitness values of the evolved transconjugants from long-term growth experiments, Table S3: Antibiotic susceptibilities (MICs in mg/Fifty) of transconjugants of the different generations, detected by broth microdilution, Table S4: Results of de novo associates of the reads of isolates 102/04 K1 G0 und K2 Yard0, Tabular array S5: Results of read mapping of illumina data for evolved transconjugants of RS165 K1 onto the reference sequence of 102/04 K1 Thousand0, Tabular array S6: PCR primer pairs used in this study, Effigy S1: Growth curves of 7 selected transconjugants with beta-lactamase gene-conveying plasmids vs. the plasmid-free recipient E. coli J53 Azir.
Writer Contributions
M.P., Y.P. and One thousand.West. designed the report and selected the donor and recipient strains. Thou.P. performed virtually of the experiments, determined all the data and calculated the statistics. S.F. supported the WGS-based analyses. G.W. and Yard.P. wrote the manuscript, and all authors reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding
The study was supported by a grant from the Federal Ministry of Teaching and Research for the project RESET (grant no. 01KI1013).
Institutional Review Lath Statement
Not applicative.
Informed Consent Statement
Non applicable.
Conflicts of Involvement
The authors accept no conflicts of interest to declare.
Footnotes
Publisher's Annotation: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832316/
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