Introduction

Klebsiella pneumoniae belonging to Klebsiella spp. is one of the important species of Enterobacterale. As one of the most common opportunistic pathogens, it is also a major cause of hospital associated infections, ranking in the top three causative agents in most hospital settings [1]. As we know that extended β-lactams and carbapenem are high-efficiency broad-spectrum antibiotics with high sensitivity to Enterobacterale and are commonly used to treat K. pneumoniae infections. Therefore, with the antimicrobial agents being extensively used in clinical therapy, extended-spectrum β-lactam (ESBL)-producing and carbapenem-resistant K. pneumoniae (CRKp) have rapidly developed and disseminated, leading to subsequently increased morbidity and mortality. Thus, they were taken as a critical public health threat by The World Health Organization (WHO) [2].

It has been well known that blaCTX-M, blaTEM, blaSHV and blaOXA were the most frequent ESBLs, blaKPC, blaNDM and blaOXA-48 were the most common carbapenem-hydrolyzing β-lactamase (CHβLs) encoding genes widely prevalent in K. pneumoniae worldwide [3, 4]. In recent years, carbapenem-hydrolyzing oxacillinase, especially blaOXA-48-carrying K. pneumoniae has been increasingly reported worldwide, with Europe and Aisa being a hot spot for outbreaks of infection [5, 6], which endangers public health. However, information on the characterization of blaOXA-carrying K. pneumoniae is little. Unfortunately, the spread of blaOXA-48 gene in Iran, as an endemic country, has not been investigated and only one study has been investigated. Iran, Span, Greece and China are countries with high prevalence of blaOXA-48 gene [7]. So, in this study, we intended to know the changing epidemic characteristics and trends of blaOXA genes in K. pneumoniae which is helpful for the formulation of infection control measures.

Oxacillinases or OXA ß-lactamase was first recognized in the 1960s, displaying hydrolytic activity against penicillins and oxacillin. Afterwards, certain OXA ß-lactamases were found to inactivate the cephalosporins and carbapenems. To date, more than 940 types of OXA ß-lactamases have been found, showing heterogeneous substrate profiles. OXA ß-lactamases were divided into the 4 groups by Poirel and colleagues in 2010 [8]. Group I include OXA-1, OXA-2, and OXA-101 subgroups, belonging to acquired narrow-spectrum class D ß-lactamases without significant activity toward cephalosporins or the carbapenems. Group II is the acquired extended spectrum class D ß-lactamases hydrolyzing certain extended-spectrum cephalosporins (especially ceftriaxone and cefepime), which often originates from point mutations of narrow-spectrum class D ß-lactamases. However, structurally, certain extended-spectrum OXA enzymes are not related to the narrow-spectrum group I OXA-lactamases. Group III owns the ability to weakly hydrolyze carbapenems, but do not significantly hydrolyze the extend-spectrum cephalosporins. It is named carbapenem-hydrolyzing class D ß-lactamases and composed of 2 subgroups. One subgroup named the OXA-23-like enzymes, responsible for carbapenem resistance among Acinetobacter spp., the other subgroup is named the OXA-48-like enzymes, mainly found among the Enterobacterales, especially in K. pneumoniae. Group IV forms parts of the chromosomes of various non-fermenting Gram-negative bacteria, which includes the OXA-51-like subgroup in the Acinetobacter baumannii complex.

Molecular studies displayed a frequent distribution of blaOXA including blaOXA-1, blaOXA-2, blaOXA-48 etc. among K. pneumoniae [9]. Among the blaOXA-48-like CHβLs group, blaOXA-48, blaOXA-181, blaOXA-232, blaOXA-204, blaOXA-162, and blaOXA-244 are the most common enzymes identified, and some of them showed an epidemic state, for example, it has been reported that blaOXA-48 is endemic in Europe and Asia [5, 6]. The blaOXA-162 and blaOXA-244 (derivatives of blaOXA-48) were present in Europe [10]. The blaOXA-181 and blaOXA-232 were endemic in the Indian subcontinent and certain sub-Saharan African countries [11, 12]. Additionally, clonal dissemination of certain high-risk clones such as K. pneumoniae ST147, ST307, ST15, and ST14 play roles in the global dispersion of blaOXA-48-like CHβLs [12]. In recent years, detection of blaOXA-48 and clonal dissemination of blaOXA-48-carrying carbapenem-resistant Enterobacteriaceae (CRE) has been reported in China [13]. However, the distribution of blaOXA among global K. pneumoniae is unknown and the information available is quite limited.

In this study, we firstly analyzed the distribution and phylogeny of blaOXA among global K. pneumoniae isolates. For the blaOXA-carrying strains, we further investigated the sequence type (ST) and the characteristics.

Materials and Methods

The acquisition and quality check of K. pneumoniae genomes

Aspera software was applied to download the K. pneumoniae genomes deposited in GenBank (https://www.ibm.com/aspera/connect/). A total of 12,356 complete genomes of K. pneumoniae strains were accessible. After the genome quality check by CheckM and Quest software, 11,429 qualified genomes were finally analyzed in this study.

Standardized genome annotations

All the 11,429 genomes were annotated by prokaryotic genome rapid annotation software Prokka to analyze the blaOXA genotype, thus to avoid differences in genomic predictions with different annotation methods and to get the strains carrying blaOXA.

Phylogenetic tree of blaOXA variants

Multiple sequence alignment for nucleotide sequences of the blaOXA gene was performed using Muscle [14], and then the GTRCAT replacement model was selected through the RaxML software [15]. In addition, 500 bootstrapping samples were set up to construct the preliminary phylogenetic tree, which was further imported into iTOL software to obtain the final one by setting bootstrap value being with less than 50 for the branches being deleted to obtain the final evolutionary tree [16].

Analysis of sequence types (STs)

Seven housekeeping gene sequences (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) and typing profile files of K. pneumoniae strains were obtained from MLST website (https://pubmlst.org). All the nucleotide sequences of 4386 blaOXA-carrying strains were analyzed using blastn tools in batches using housekeeping gene sequences as the database (parameters: value = 1e-5; identity = 100%; coverage = 100%).

Investigation of antimicrobial resistance genes among blaOXA-carrying K. pneumoniae

The distribution of other resistance genes including CHβLs encoding genes, aminoglycoside and fluoroquinolone resistance genes was investigated by blastn tools. All the nucleotide sequences of blaOXA-carrying strains were analyzed with the sequence of antimicrobial resistant genes as database using blastn, and then the results were filtered (parameters: identity >  = 90%; coverage >  = 90%; value = 1e-5).

Acquisition of strain meta information

Strain meta information such as isolation source, country, and date, etc. was extracted in batches from the downloaded gbk file by using perl script. Meanwhile, the corresponding cds, genes, contig quantity and genome length information of the genomes were also obtained from the prokka annotation file. All the information in addition to STs were integrated in an excel to facilitate the further analysis.

Results

OXA distribution among global K. pneumoniae strains

Among the 11,429 K. pneumoniae strains, 4386 ones carried a total of 5610 blaOXA variants which belonged to 27 genotypes of blaOXAs. The predominant was blaOXA-1 (n = 2891, 51.5%), followed by blaOXA-9 (n = 969, 17.3%), blaOXA-48 (n = 800, 14.3%), blaOXA-232 (n = 480, 8.6%), blaOXA-10 (n = 158, 2.8%), blaOXA-181 (n = 146, 2.6%), and blaOXA-2 (n = 108, 1.9%) (Fig. 1A). Variants including blaOXA-244 (n = 12, 0.2%), blaOXA-245 (n = 11, 0.2%), blaOXA-162 (n = 5, 0.1%), blaOXA-4 (n = 5, 0.1%), blaOXA-926 (n = 4, 0.1%), blaOXA-204 (n = 3, 0.1%), blaOXA-534 (n = 3, 0.1%), blaOXA-58 (n = 2, 0.03%), and blaOXA-320 (n = 2, 0.03%) were few. Whereas, the following variant was only detected in single strain which were blaOXA-21, blaOXA-392, blaOXA-517, blaOXA-519, blaOXA-663, blaOXA-796, blaOXA-827, blaOXA-427, blaOXA-544, blaOXA-436 and blaOXA-17.

Fig. 1
figure 1

Distribution of K. pneumoniae blaOXA variants and sequence types

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Phylogenetic relationship of blaOXA variants among global K. pneumoniae

The phylogenetic tree showed that blaOXA variants could be divided into 8 clades (Fig. 2). Among them, clade A was composed of 10 variants and all of them belonged to carbapenem-hydrolyzing class D ß-lactamase (Group III, blaOXA-48-like subgroup). Clade B belonged to Group IV (blaOXA-51-like subgroup). Clade C belonged to Group II with the activity of acquired extended spectrum class D ß-lactamases, amongst them, blaOXA-10-type oxacillinase, with weak carbapenemases being previously found [17]. Natural variant of blaOXA-10 was reported to own increased carbapenemase activity and high-level expression of blaOXA-10 and blaOXA-10 derivative blaOXA-663 can confer carbapenem resistance when expressed at sufficiently high levels in the OmpK36 deficiency background [18], indicating that clade C may own the potential to develop to be group III. In clade D, blaOXA-544 derived from blaOXA-2 family, as well as blaOXA-21 all belonged to Group 1, acquired narrow-spectrum oxacillinase without significant activity toward cephalosporins or the carbapenems [19]. The blaOXA variants within Clade H may belong to Group I considering the activity of blaOXA-1 and blaOXA-4, although the hydrolyzing activity of blaOXA-534, blaOXA-329, blaOXA-320 and blaOXA-796 remained unknown. Clade E, F and G were composed of single blaOXA variant, where, blaOXA-9 and blaOXA-926 were narrow-spectrum oxacillinase [20], blaOXA-427 was a new plasmid-borne carbapenem-hydrolysing class D β-lactamase in Enterobacteriaceae [21]. Overall, a relatively independent evolutionary relationship between carbapenem-hydrolyzing oxacillinase blaOXA and non-carbapenem-hydrolyzing oxacillinase blaOXA were observed. However, several blaOXA within Group I and II may develop carbapenem-hydrolyzing activity under the selective pressure of antimicrobial agents.

Fig. 2
figure 2

Phylogenetic tree based on SNP sequence of global blaOXA variants

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The sequence types of blaOXA-carrying K. pneumoniae

A total of 300 different STs were identified among the 4386 blaOXA-carrying K. pneumoniae strains. The predominant type was ST11 (n = 477, 10.9%), followed by ST258 (n = 410, 9.4%), ST15 (n = 367, 8.4%), ST307 (n = 300, 6.8%), ST147 (n = 292, 6.7%), ST16 (n = 266, 6.1%) and other STs (Figure S1). In addition, 94 strains were novel or unknown STs (Table S1).

For strains producing different blaOXA variants, 2845 blaOXA-1-carrying K. pneumoniae strains were assigned into 209 STs. Amongst them, ST11 (n = 372, 13.1%) was the predominant one, followed by ST307 (n = 287,10.2%) (Fig. 1B). Meanwhile, 957 blaOXA-9-carrying K. pneumoniae strains belonged to 74 STs, with ST258 being accounting for the majority (367/957 38.4%), followed by ST16 (126/957 13.2%) (Fig. 1C). The most extensively studied blaOXA-48-carrying K. pneumoniae were assigned into 91 STs, with ST11 (136/794, 17.1%) and ST101 (103/794, 13.0%) being the leading STs.

Additionally, the predominant blaOXA variant of 477 ST11 strains was blaOXA-1 (n = 378, 79.2%) and the main blaOXA variant of 410 ST258 strains was blaOXA-9 (n = 370, 90.2%). The distribution of blaOXA-1 in ST11 (378/477, 79.2%) was significantly higher than that in ST258 (24/410, 5.9%), the difference was statistically significant (p = 0.000). Meanwhile, the prevalence of blaOXA-9 in ST258 (370/410, 90.2%) was obviously higher than that in ST11 (48/477, 10.1%), the difference was also statistically significant (p = 0.000).

Distribution characteristics of blaOXA among global K. pneumoniae

The blaOXA-9 was the earliest blaOXA variants in the genome submitted in USA in 2001 albeit blaOXA-1 was the first reported one. Since the first identification of blaOXA-48-carrying K. pneumoniae isolates in 2001, the number of blaOXA-carrying K. pneumoniae isolates collected from all the specimen types had basically shown an increasing trend till 2014 and had been stable since then (Fig. 3). The predominant blaOXA-1, blaOXA-9 and blaOXA-48 variants showed the same trend, which should be of concern.

Fig. 3
figure 3

The number of global K. pneumoniae blaOXA variants detected from 2001 to 2021

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The main host of blaOXA-carrying K. pneumoniae isolates was homo sapiens (2696/4386, 61.5%), followed by water-related host (60/4386, 1.4%), animal-related host (45/4386, 1.0%) and plant-related host (20/4386, 0.5%). The isolates of homo sapiens were mainly derived from blood, respiratory, alimentary tract, and urinary tract infections (Table 1).

Table 1 Source distribution and ratio of global blaOXA-carrying K. pneumoniae strains in homo sapiens
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The most predominant blaOXA-1-carrying K. pneumoniae strains were distributed widely around the world except Northern America, which was relatively rare. The blaOXA-9-carrying K. pneumoniae strains were mostly found in Northern America rather than other regions, especially, ST258 was mainly distributed in this area. The most studied blaOXA-48-carrying K. pneumoniae strains were mainly distributed in Europe and Asia. The blaOXA-232-carrying K. pneumoniae strains and blaOXA-181-carrying K. pneumoniae strains were almost found in Asian countries (Fig. 4).

Fig. 4
figure 4

Global distribution of blaOXA variants and sequence types of blaOXA-carrying K. pneumoniae strains

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The distribution of antimicrobial resistance determinants among global blaOXA-carrying K. pneumoniae

Among the 4386 blaOXA-carrying K. pneumoniae strains, blaKPC (n = 985, 22.5%) and blaNDM (n = 732, 16.7%) were the main blaCHßLs with blaVIM (n = 135, 3.1%) and blaIMP (n = 28, 0.6%) also being identified. Although aph (n = 5645, 128.7%), aad (n = 2927, 66.7%) and aac (n = 2742, 62.5%) were the predominant aminoglycoside resistance genes, rmt (n = 347, 7.9%), armA (n = 332, 7.6%), and ant (2’)-Ia (n = 99, 2.3%) were also found. qnr (n = 2340, 53.4%), oqxAB (n = 4131, 94.2%), aac(6’)-Ib-cr (n = 49, 1.1%) and qepA (n = 12, 0.3%) were the fluoroquinolone resistance genes.

In detail, the blaNDM-1 (n = 618, 84.1%) and blaKPC-3 (n = 586, 59.6%) were the most common subtypes of blaKPC and blaNDM respectively. Moreover, rmtF1 and rmtB1 were the main variants of rmt gene. qnrB1 and qnrS1 were the major subtypes of qnr gene (Fig. 5).

Fig. 5
figure 5

Subtypes of antimicrobial resistance genes among global blaOXA-carrying K. pneumoniae strains

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Discussion

Current evidence suggests that compared to other Gram-negative opportunists, K. pneumoniae has a wider ecological distribution, significantly greater antimicrobial resistance gene diversity and a higher plasmid burden [22]. In recent years, with the carbapenem being frequently used in clinical treatment. Carbapenem-hydrolyzing oxacillinase has been increasingly found, with the outbreaks of infection being reported in Mediterranean and China [2, 5, 6]. As we know that carbapenem-hydrolyzing oxacillinase was commonly prevalent in Enterobacterale, especially in K. pneumoniae [22]. So the analysis of the distribution of blaOXA among the global K. pneumoniae based on the genomes could help us to fully illustrate the characteristics of blaOXA-carrying K. pneumoniae, which is quite useful for formulation and implementation of infection control measures.

In this study, we found that there was a rapid increase in global blaOXA-carrying K. pneumoniae strains since the first blaOXA was identified [23]. Overall, blaOXA-1 variants were widely distributed around the world. The blaOXA-9 variants were mostly prevalent in Northern America. Whereas, blaOXA-48 variants were mainly reported in Europe and Asia which was in accordance with previous studies [5, 6]. Notably, as the blaCHßLs, albeit blaOXA-48, blaOXA-232 and blaOXA-181 were not the most prevalent ones in our study, the emergence of them in different parts of the world has been reported and such genes were found to be most likely underreported due to problems with the laboratory detection of these enzymes [10]. Moreover, we found an increasing trend and wider distribution of the blaOXA-48 CHßLs, which is consistent with the previous reports [13], indicating an escalating threat of CRKp.

The phylogenetic tree showed a diversity of blaOXA variants identified within K. pneumonae. Functionally, all the blaOXA-variants could be divided into carbapenem-hydrolyzing oxacillinase and non-carbapenem-hydrolyzing one. Within the 29 blaOXA variants, 12 out of them have been identified to be blaCHßLs, albeit the detailed hydrolyzing activities of 5 variants remained unknown, the increasing diversity and activity towards carbapenem of these blaOXA-variants remind us of the continual change trend under the selective pressure of antimicrobial agents.

Diverse STs identified in our study indicated genetic diversity for blaOXA-carrying K. pneumonaie and a broad host for blaOXA variants. Of great concern, high-risk epidemic clones ST11 and ST258 were the predominant STs in our study which have also been the most common host for blaCHßLs [24]. As we know that the prevalence of ST11 is high in the continent of Europe, in Greece and Spain, as well as in China [2, 24], and the ST11 clone was reported not only to be the dominant host for blaKPC and blaNDM [25, 26], but also associated with blaOXA-48-like genes (e.g., blaOXA-163, blaOXA-181) [27, 28]. Although blaOXA-1 was the main variant among ST11 clones, the frequent occurrence of blaOXA-48 within global high-risk clone ST11 in our study indicates us that there may be a consistent distribution of blaOXA-blaCHßLs within K. pneumonaie. Additionally, albeit multiple blaOXA variants were detected in the ST11 strains, a simultaneous occurrence of blaOXA-48 and blaOXA-1 was frequently found, further suggesting the compatibility between these 2 variants within ST11 clone. It has been well known that ST258 clones was the predominant host for blaKPC in European and American countries [24], Fortunately, blaOXA-9 was the main variant within ST258 clone in our study, without other blaOXA variants concurrence, which was consistent with the previous study [29, 30], probably indicating a better fitness between blaOXA-9 and ST258 clones.

Of great concern, more than half of the blaOXA-carrying K. pneumonaie carried blaCHßLs. The frequent occurrence of blaNDM-1 was mainly among blaOXA-1 producing K. pneumonaie, and high prevalence of blaKPC-3 was predominantly within blaOXA-1 and blaOXA-9 carrying one. which may indicate a better fitness between blaOXA-1 and blaNDM-1, and an association between blaOXA-1/ blaOXA-9 with blaKPC-3. Moreover, almost all the blaOXA-carrying K. pneumonaie carried at least a fluoroquinolone resistance gene, especially oqxAB, suggesting that these genes may play an important role in the fluoroquinolone resistance of K. pneumonaie. Additionally, albeit rmt and armA were not the mainly prevalent aminoglycoside resistance gene, such genes frequently should attract our attention based on the conferred resistance to clinically important amikacin.

There are several limitations. First, part of strain information uploaded to GenBank was incomplete, this may result in statistical bias. Second, some blaOXA-variants within K. pneumoniae have never been reported and the detailed activity remained unknown, since we could just get the genome without the exact strain, so it is hard for us to further analyze the hydrolyzing activity.

In summary, blaOXA-1 and blaOXA-9 were the main blaOXA among global K. pneumoniae with ST11 and ST258 being the predominant STs. blaOXA evolved rapidly towards blaCHßLs under the selective pressure of antimicrobial agents with blaOXA-48 and blaOXA-232 group being increasingly found.