Background
Third-generation cephalosporin-resistant and carbapenem-resistant
Klebsiella pneumoniae are important health-care pathogens associated with increased morbidity and mortality among high-risk patients. Recent data from the European Antimicrobial Resistance Surveillance network (EARS-Net) for health-care associated pathogens (from invasive infections) constitute a rising number of third-generation cephalosporin-resistant and carbapenem-resistant
K. pneumoniae all over Europe [
1]. In the period 2012 to 2015 some countries, such as Spain, Portugal and Croatia, experienced rising trends of carbapenem resistance still at a low level (< 4%) but triggering the overall European trend. In other countries such as Romania, Italy, and Greece, the rates were already much higher and, in addition, partly showed an increasing trend (Romania). In contrast to this, the majority of European countries, including Germany demonstrated rates below 1% without showing any significant development in recent years. Thus, Germany could be considered as a low-prevalence country according to EARS-Net data regarding the occurrence and spread of carbapenem-resistant
K. pneumoniae. However, the National Reference Centre (NRC) for Gram-negative nosocomial pathogens in Germany is challenged by analysing an increasing number of several thousand carbapenem-resistant
Enterobacteriaceae,
Pseudomonas aeruginosa and
Acinetobacter each year, starting with only a few hundred isolates back in 2008/2009. In 2015 and 2016 diagnostic laboratories throughout Germany sent 1148 and 1317 carbapenem-resistant
K. pneumoniae isolates to the NRC, respectively, and exactly 51% of them possessed a carbapenemase [
2,
3]. The most frequent carbapenemase type in these isolates was OXA-48, followed by KPC-2/− 3 and NDM-1.
Since May 2016, Acinetobacter spp. and Enterobacteriaceae with carbapenem non-susceptibility are mandatorily notifiable to the national health authorities in Germany. From September 2016 to August 2017, 4028 notifications were reported. The most prevalent notified pathogen was K. pneumoniae (1147, 29%). Notifications of carbapenem-resistant K. pneumoniae came from all over Germany with a range of 3 to 267 notifications per federal state. For 293 (26%) of those, a carbapenemase was reported. The most common specified carbapenemases were OXA-48-like (53%), KPC-2/− 3 (17%) and NDM-1 (12%)(A.R., A.v.L., M.A.S., unpublished data).
With this study we aimed at describing a population snapshot of carbapenemase-producing K. pneumoniae isolated from hospitalized patients in Germany. We compiled a representative subset of 107 carbapenemase-producing K. pneumoniae collected between 2008 and 2014 from all over Germany and harbouring carbapenemases OXA-48, KPC-2, KPC-3 and NDM-1. We performed whole genome sequencing to (i) infer the phylogenetic relatedness of corresponding isolates including identification of the multi-locus sequence type (MLST); (ii) extract β-lactamase- and virulence gene content; (iii) deduce the capsule type (wzi typing); and (iv) reconstruct the plasmid content. Finally, we compared our results with K. pneumoniae isolates from clinical and ambulatory settings in Europe and worldwide (carbapenemase-producers were in the minority).
Discussion
More than half of the study sample (58/105; 2×
K. variicola) belonged to the prominent and worldwide distributed clonal group CG258, including ST258, ST512, ST11, ST395, and ST340. In collections from other countries and continents ST258 and ST11 were the most frequent sequence types within CG258 [
26]. Isolates of ST258 are worldwide frequently associated with KPC-2, a finding which was also confirmed for the situation in Germany. SNP-based analyses of ST258/KPC-2 and ST512/KPC-3 showed a clear genetic separation of isolates from Europe and the United States (Additional file
1: Figure S4). This indicates a circulation of KPC clones in Europe. The first outbreak due to
K. pneumoniae-ST258 with KPC-2 was reported in 2008 in Germany, and the index patient had been previously hospitalized in Greece [
27]. Routine screening for carbapenemase-producing
K. pneumoniae is mandatory at hospital admission in Germany only as part of a risk-based adapted approach (considering certain risk factors such as having received hospital treatment in a high-risk country) and thus patients with undetected colonization may contribute to their further spread within Germany [
8].
In contrast to ST258, carbapenemase-producing ST11 (CG258) isolates are quite frequent in Asia. In our collection we found only one ST11 isolate indicating that this ST has so far occurred only rarely in Germany. ST395 isolates, representing another CG258 type, appeared three times in our collection and were always associated with OXA-48. OXA-48-producing
K. pneumoniae isolates of ST395 were also found recently in other European countries, such as France, the Netherlands and Hungary [
28‐
32].
The present collection also contained a cluster of ST101 isolates (
n = 16), 15 of which were OXA-48 producers. Although the two external collections included in the phylogenetic analysis did not contain ST101
K. pneumoniae, the combination ST101-OXA-48 is not unique to Germany. Clinical isolates with these characteristics were described in neighbouring European countries in recent years, such as Czech Republic, France and Belgium [
28,
33,
34], and also all over Europe and North Africa [
29,
35]. ST101 isolates seem to be especially distributed in Europe, and also mainly associated with OXA-48 [
36]. Two further clusters were formed by ST147 and ST15 isolates producing mainly OXA-48 but also NDM-1 and/or KPC-2 (Fig.
3). Both clonal lineages of
K. pneumoniae are well known as colonizers or the cause of infection in human patients; outbreaks due to ST147 with OXA-48 have been reported in Germany and ST15 with OXA-48 was also found in pets in a German veterinary clinic [
37,
38].
Due to the presence of the
blaLEN gene and the
gyrA sequence two of the 107 isolates were classified as
K. variicola (phylogroup Kpn III). The same MLST scheme is used for all three phylogroups and all species
K. variicola, K. pneumoniae and
Klebsiella quasipneumoniae, respectively [
36]. The OXA-48 producing
K. variicola isolate of ST906 represents a very rare ST and ST/carbapenemase combination. ST906 was first defined in a set of strains from an Israeli clinic [
39]. However, the authors failed to proof the species
K. variicola.
K. variicola isolates of ST347 were described in previous studies from China, Japan, Greece and Sweden [
40‐
43]. However, only the Greek isolate was a carbapenemase producer (KPC-2) [
42]. In general, reports about carbapenemase-producing
K. variicola are rare; we found a report about OXA-181-producing
K. variicola from vegetables imported from Asia to Europe [
44] and NDM-producing isolates originating from a river in China [
45]. In a very recent study performed in the US an ST906-isolate producing NDM-1 was also identified as
K. variicola [
46]
.
One isolate was clearly separated from the rest of our collection regarding the presence of virulence genes: the OXA-48-producing ST23 isolate no. 316/15, carrying capsule type K1 and containing all but one tested virulence markers including different siderophore genes (Additional file
3: Table S3). ST23 isolates are known to be hypervirulent and can cause severe infections in apparently healthy individuals [
47,
48]. In particular, ST23 isolates expressing the K1 capsule were frequently associated with cases of community-acquired pyogenic liver abscesses and occurred predominantly in Asian countries [
24,
48]. Our single ST23 isolate originated from a tracheal secretion suggesting a respiratory tract infection. Subsequent molecular analyses by
XbaI macrorestriction and pulsed field gel electrophoresis of isolates from the same diagnostic laboratory revealed that this isolate belonged to a cluster of seven isolates that occurred in the period February 2012 to July 2012. The sample isolates originated from three blood cultures, one urine sample and several swabs. No further clinical or patient information was available for these isolates. Hypervirulent strains of
K. pneumoniae often express a typical hypermucoviscous phenotype and demonstrate a small spectrum of resistances, mainly only to penicillins, e.g. ampicillin. Our ST23 isolate contained various β-lactamase genes, including classical β-lactamases (
blaSHV-11,
blaTEM-1,
blaOXA-1), ESBLs (
blaCTX-M-15) and carbapenemases (
blaOXA-48). A quick, so-called ‘string test’ for identifying a hypermucoviscous phenotype was negative, possibly associated with the premature STOP codon in the
rmpA2 gene (regulator of the mucoid phenotype A gene). However, the string test gives only a hint and is not reliable enough to predict a corresponding clinical behaviour. In addition, we do not know whether this mutation was also prevalent in the other clonally related isolates that were not available for a genome-based analysis. The combination of hypervirulence and resistance to last-line therapeutics is highly worrisome and has just recently raised serious public health concerns [
49‐
51].
A number of recent reports have suggested that the
wzi locus is frequently exchanged between
K. pneumoniae isolates [
52,
53] and, as such, could be putatively genetically linked to other, horizontally exchanged markers, such as plasmid-located carbapenemase genes. In particular, the highly successful ST258 clone seemed to be a hybrid strain with the
cps locus (containing the
wzi gene) located next to a recombination hotspot [
54]. Although KPC-2/3, OXA-48 and NDM-1 genes are commonly located on plasmids, a chromosomal integration is also reasonable, at least for some carbapenemase genes. It has been described recently, that
blaKPC-2 could also be chromosomally determined via IS-mediated Tn
4401 transposition into specific chromosomal loci, especially observed in CG258 isolates (ST11, ST258, ST340) [
55]. Although we could not properly elucidate plasmid structures by assembling Miseq reads, we suggest a plasmid localization of the carbapenemase genes in most cases based on the sum of assembled and interpreted NGS data. The finding of IncFII sequences in KPC-producing CG258 isolates and IncL/M sequences in all OXA-48 producers supports our suggestion because these associations of carbapenease genes and plasmid replicon types are well known [
53,
56].
ST258 isolates (our collection and genomes of international isolates included in the study) contained four
wzi alleles;
wzi_29 (
n = 26);
wzi_154 (
n = 4);
wzi_83 (
n = 1) and
wzi_150 (
n = 1). Similar distributions were described in two recent studies performed in the US [
52,
57] The association within ST258 isolates of
cps1/
wzi_29 with
blaKPC-2 and
cps2/
wzi_154 with
blaKPC-3, respectively, was identified for our study sample of German clinical isolates but was also described for isolates from other studies and countries [
54,
58,
59]. Bowers et al., supposed that the association of
cps1 in KPC-2-producing
K. pneumoniae isolates and of
cps2 in KPC-3-positive isolates is derived from the fact that the point mutations in
blaKPC leading to a differentiation into
blaKPC-2 and
blaKPC-3 appeared at the same time when the two clades with different
cps loci diverged [
58]. All of the ST512 isolates in our study contained
blaKPC-3 and
wzi-154; which is identical to all
blaKPC-3-positive
K. pneumoniae isolates of ST258 in a recent study performed in Italy. Thus, our results presented for German isolates confirm previous findings performed on
blaKPC-positive CG258 isolates (ST258, ST512) from Italy [
59]. In all ST512 isolates from Italy only
wzi_154 was present. This exclusive association among a comprehensive set of 461
K. pneumoniae isolates collected all over the country led the authors of the previous study to assume that ST512 isolates may have originated from ST258-
cps2-clade strains [
59].
Limitations
Our study has some limitations. First, strain sending to reference laboratories is voluntary and taking this into consideration we cannot exclude any bias in our strain collection, which served as the basis to select representative isolates for further genome-based analysis. Second, clinical and patient data available with the strains submitted were limited. We could not perform any analysis in this regard, nor could we provide additional clinical and patient information to the data that are already provided. Third, we are well aware that even a collection of about 100 strains representing 12 federal states in Germany and a timeframe of 7 years does not allow statistically significant assumptions (which we have not calculated and specified as such). Fourth, it has already been addressed in previous papers that plasmid content is difficult to reconstruct with Miseq data, thus the location of the carbapenemase gene on a specific plasmid could and, in consequence, a potential spread of distinct carbapenemase gene-carrying plasmids not be identified. Correspondingly, the putative link between plasmid types and carbapenemase genes remains to a certain extent hypothetical. Fifth, in the last 2 years, data from the NRC showed a rapid increase of
Enterobacteriaceae (including
K. pneumoniae) isolates producing VIM-1 in Germany [
2], but this trend could not be considered when this study was initiated.