Background
The
Klebsiella oxytoca species complex (KoSC) is a group of Gram-negative bacilli within the order
Enterobacterales consisting of nine species [
1‐
3] including
Klebsiella michiganensis [
4],
Klebsiella grimontii [
5] and
Klebsiella pasteurii [
6]. KoSC are the second most common
Klebsiella group identified as the cause of clinical infections in humans after the
Klebsiella pneumoniae species complex (KpSC) [
7]. Like its sister complex KpSC, KoSC has the ability to persist in a variety of niches, including wet environments [
8,
9], hostile environments such as handwashing soaps [
10,
11], prosthetic material like central venous catheters [
12], and within gastrointestinal flora [
13], all of which contribute to its ability to cause opportunistic infections in healthcare settings.
Acquired antimicrobial resistance (AMR) is an emerging concern for KoSC. There are now several reports of multi-drug resistant (MDR)
K. oxytoca outbreaks carrying either carbapenemases or extended-spectrum beta-lactamases (ESBLs), which confer third-generation cephalosporin resistance (3GCR) [
1,
9,
14]. In part, this resistance is due to a large shared gene pool with KpSC [
1] that can act as a potent source and reservoir of new AMR genes [
15]. Additionally, 3GCR can emerge through point mutations in the promoter region of the chromosomal
blaOXY gene (which normally confers only ampicillin resistance), leading to over-production and an ESBL phenotype [
16,
17]. The most well-characterised virulence factors in KoSC is the kleboxymycin-biosynthetic gene cluster that encodes for the cytotoxin kleboxymycin or tilivallin, both of which are implicated in the pathological changes caused by antibiotic-associated haemorrhagic colitis [
3]. Most of the remaining virulence information about KoSC has been extrapolated from established
K. pneumoniae data [
1], often without phenotypic evidence.
Despite its clinical relevance and the increasing concerns about AMR, little is known about the epidemiology and broader population structure of KoSC within healthcare institutions. Like KpSC, members of the KoSC remain difficult to distinguish both phenotypically and by matrix-assisted laser desorption/ionization mass spectrometry (MALDI–TOF) [
11,
18], and as such are usually reported as
K. oxytoca by clinical laboratories.
Here, we describe the clinical characteristics of a collection of 239 KoSC infection isolates from hospitalised patients within a single tertiary hospital network across a 15-month period. We sequenced a subset of 92 isolates, including all invasive isolates, to characterise their genetic diversity, assess for evidence of nosocomial transmission, and determine the genetic context of acquired AMR determinants.
Discussion
To our knowledge, this is the first description of the population structure of a non-outbreak collection of isolates identified as
K. oxytoca from a single hospital network. Isolates from urine were most common, followed by isolates from respiratory and wound specimens, similar to what has been observed for a collection of KpSC infections from the same hospital across a similar time period [
39].
We preferentially sequenced hospital onset isolates, invasive isolates and isolates with acquired drug resistance, and found that isolates identified as
K. oxytoca by the clinical laboratory instead represented four distinct species, with significant diversity within each. Amongst invasive isolates, we found that
K. oxytoca and
K. michiganensis were most common, however these species were also the most common across the whole collection. As we did not sequence a representative sample of isolates from all specimen types, we are unable to draw conclusions regarding differences in the rates of urinary tract infections and gastrointestinal carriage for the different species within the KoSC. Improvement in laboratory species identification in the KoSC will be key for bettering our understanding of the clinical syndromes and the likelihood of acquired resistance associated with each species. Models for correctly identifying KoSC species using MALDI-TOF now exist [
40,
41], however these models aren’t yet implemented in the MALDI-TOF instrument databases. Whilst spectra can be exported from the instruments and analysed independently, this requires significant additional expertise.
Consistent with earlier studies, we found the virulence factor kleboxymycin gene cluster in all four species in our data, though not conserved in any one species [
42]. We did not identify any
K. pneumoniae virulence factors apart from yersiniabactin in our isolate collection, in contrast to some previous studies [
1,
43]. Given our decision to focus on nosocomial isolates we may have been less likely to identify hypervirulent isolates associated with community-acquired infections, although we did include all invasive isolates with community onset. These findings suggest that these key
K. pneumoniae virulence factors, apart from yersiniabactin, are uncommon in KoSC. Significantly more work is required to characterise novel virulence factors in KoSC and this will require both phenotypic and genotypic studies.
We observed a marked amount of genomic diversity in our sampled population, collected over a single year from one hospital. The amount of diversity found (where most STs were represented by a single genome) was similar to that found in an earlier study that examined a 10-year collection of
K. oxytoca genomes from multiple clinical labs in the UK and Ireland [
1]. Our collection did not include any of the globally distributed STs (ST2 or ST9) [
44], or ST315 which was responsible for a
K. michiganensis outbreak in a neonatal care unit in Australia [
11]. There was limited evidence of strain transmission in our collection, with only one likely transmission event, of an isolate with an acquired MDR plasmid including
blaIMP−4. However, this event involved patients that were admitted two months apart, and to separate wards, with no overlap in admission times. This may suggest an environmental reservoir or an unrecognised intermediate patient or healthcare worker with carriage or unsampled infection. This low frequency of identified transmission is consistent with previous work in
K. pneumoniae demonstrating most infections are from a patient’s own flora [
23]. Previous studies of KoSC have shown close relatedness between environmental and clinical specimens suggesting a possible environmental source for infection [
1,
11]. We found a number of isolate pairs in our collection with > 30 but < 105 SNVs, consistent with transmission from an environmental source. Further work sequencing environmental and screening isolates from within the community and hospital will provide context to describe the relative contribution of environmental isolates to carriage and clinical infection and additionally allow for some assessment of candidate virulence factors.
Within our collection AMR was quite rare—only six isolates had acquired multi-drug resistance that included ESBLs. Notably four of these isolates had also acquired the carbapenemase
blaIMP-4. Of these four carbapenemase isolates, two carried an identical IncL/M plasmid that has previously been described in
Enterobacter cloacae from Sydney [
38]. The remaining two isolates carried an IncHI2A plasmid that has been found to be circulating in multiple species within our hospital [
36], and is highly similar to a plasmid previously described in Brisbane [
45], consistent with the previously described widespread dissemination of
blaIMP-4 within the Enterobacteriaceae along Australia’s east coast [
46‐
49]. Internationally, IncHI2A plasmids carrying
mcr-9 with
blaIMP-4 or
blaNDM-1 have also been described in
Klebsiella species and other Enterobacteriaceae, suggesting that these plasmids are widespread [
50‐
52]. Given that these plasmids are distributed across multiple species in the KoSC and STs, this suggests regular horizontal transfer of plasmids amongst species within this family.
This study provides insight into the clinical epidemiology and population structure of KoSC within a single hospital network, and has identified significant population diversity within what was identified by the clinical laboratory as simply K. oxytoca. K. oxytoca was not an uncommon pathogen in our hospital, and whilst AMR was rare, the MDR plasmids identified in our isolates indicate that KoSC has access to the same plasmid reservoir as KpSC, providing the potential for any isolate to become highly drug-resistant with the acquisition of a single plasmid. Future genomic studies will increase our understanding of both community and environmental population structures, which will allow us to further investigate virulence factors and other attributes of successful nosocomial isolates.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.