Great Plasticity in a Great Pathogen: Capsular Types, Virulence Factors and Biofilm Formation in ESBL-Producing Klebsiella pneumoniae from Pediatric Infections in Uruguay

Klebsiella pneumoniae is widely recognized as an opportunistic hospital and community pathogen. It is one of the priority microorganisms included in the ESKAPE group, and its antibiotic resistance related to extended-spectrum β-lactamases (ESBL) is a global public health concern. The multi-drug resistance (MDR) phenotype, in combination with pathogenicity factors, could enhance the ability of this pathogen to cause clinical infections. The aim of this study was to characterize pathogenicity factors and biofilm formation in ESBL-producing K. pneumoniae from pediatric clinical infections. Capsular types, virulence factors, and sequence types were characterized by PCR. Biofilm formation was determined by a semiquantitative microtiter technique. MDR phenotype and statistical analysis were performed. The K24 capsular type (27%), virulence factors related to iron uptake fyuA (35%) and kfuBC (27%), and sequence types ST14 (18%) and ST45 (18%) were the most frequently detected. Most of the strains were biofilm producers: weak (22%), moderate (22%), or strong (12%). In 62% of the strains, an MDR phenotype was detected. Strains with K24 capsular type showed an association with ST45 and the presence of fyuA; strains with kfuBC showed an association with moderate or strong biofilm production and belonging to ST14. Weak or no biofilm producers were associated with the absence of kfuBC. The MDR phenotype was associated with the main ESBL gene, blaCTX-M-15. The high plasticity of K. pneumoniae to acquire an MDR phenotype, in combination with the factors exposed in this report, could make it even more difficult to achieve a good clinical outcome with the available therapeutics.


Introduction
Klebsiella pneumoniae is one of the priority pathogens included in the ESKAPE group [1], among the most challenging microorganisms to treat.It belongs to the critical group on the WHO priority pathogens list for research and development of new antibiotics [2].Recent studies have positioned this microorganism among the top five agents most associated with mortality worldwide, regardless of its susceptibility to antibiotics [3].Additionally, data from the Antimicrobial Resistance Collaborators group underscore the significance of pathogens responsible for more than 250,000 deaths associated with antimicrobial resistance (AMR) in 2019, where K. pneumoniae ranked third [4].
K. pneumoniae is the causative agent of a wide range of infections, including pneumonia, bacteremia and sepsis, meningitis, pyogenic liver abscesses, and urinary tract infections, among others [5].This microorganism is well-known as an opportunistic hospital and community pathogen, and the multi-drug resistance (MDR) phenotype it exhibits is a major global public health concern.The evolution of MDR in K. pneumoniae is largely driven by the acquisition of AMR genes, which are particularly prevalent among globally spread clones [6][7][8] and are often responsible for hospital outbreaks.Currently, AMR in this pathogen is mostly related to carbapenem and third-generation cephalosporin resistance [4].Different extended-spectrum β-lactamases (ESBL) conferring resistance to third-generation cephalosporins have been reported in our country, including those from the main CTX-M groups (CTX-M-15, CTX-M-2, CTX-M-9 and CTX-M-14, and CTX-M-8 [9][10][11]), SHVderived enzymes (SHV-2 and SHV-5 [9][10][11]), and carbapenemases conferring resistance to carbapenems (KPC-2 and NDM-1 [12]).Additionally, resistance to fluoroquinolones due to plasmid-mediated quinolone resistance (PMQR) mechanisms QnrA and QnrB, principally, and aminoglycosides Aac(6 ′ )Ib due to modifying enzymes have also been detected [9][10][11][12].Due to the high plasticity of this pathogen, in some cases, ESBL, carbapenemases, PMQR, and aminoglycosides modifying enzymes may coexist in the same clinical strain, leading to limited treatment options for patients involved [12].
In addition to AMR, the ability to cause serious infections in K. pneumoniae isolates is associated with other relevant factors, including capsular types (K-types), virulence factors, biofilm formation, sequence types, and clonal distribution, among the most significant [5,6,[13][14][15].
In relation to K-types, there are more than 130 predicted types, such as K2 (https: //bigsdb.pasteur.fr/klebsiella/,accessed on 13 December 2023), which are associated with strains causing invasive infections; K2 is particularly considered antiphagocytic and serum-resistant [6].The genes responsible for capsule production in K. pneumoniae are located in a chromosomal operon containing several genes involved in capsule production.Within this operon, the wzi gene encodes a surface protein involved in capsule attachment to the outer membrane.K-antigen typing is a combination of serological methods and sequencing of the wzi gene, where different wzi locus sequences are associated with specific K-antigens [5].
Other virulence factors related to the pathogenicity of this microorganism include fimbrial and non-fimbrial adhesins, iron-scavenging systems, and surface polysaccharides (capsule, as we mentioned).In relation to fimbrial adhesins, type 1 (fimH gene) and 3 fimbriae (mrkD gene) are the major adhesive structures characterized as pathogenicity factors [5].In terms of iron-scavenging systems, several siderophores are expressed in K. pneumoniae, including enterobactin, yersiniabactin, salmochelin, and aerobactin [5].
The capability of biofilm formation is another relevant pathogenic trait that some isolates may possess.In the report conducted by Zheng J.X. and colleagues, the association between type 1 and 3 fimbriae with the ability to enhance biofilm formation on urinary catheters is analyzed [14].
Among the different isolates, the identification of sequence types and clonal distribution is important.There are well-recognized successful clones harboring an MDR phenotype, such as ST11 and ST258, or ST14, ST15, ST17, and ST37, which have been associated with worldwide outbreaks in humans in recent years [15].
The aim of this exploratory study was to investigate pathogenicity factors and biofilm formation in ESBL-producing K. pneumoniae from pediatric clinical infections.

K-Types, Virulence Factors, and Hypermucoviscosity
We characterized 17 different K-types in the studied collection.The most frequent K-type was K24 in 11/40 strains.K2 was found in only 4/40 isolates (Table 1).
The virulence factors mrkD, wabG, and ureA were identified in all strains.Only one strain was negative for fimH or uge, strain 26 was fimH negative, and strain 30 was uge negative (data not shown).The fyuA gene was present in 14/40 strains, and the kfuBC gene was present in 11/40 strains (Table 1).The genes magA, allS, rmpA, cf29a, iutA, and chuA were not identified in the collection.
The string test was negative, and none of the isolates was considered to be a hypermucoviscous isolate.

Biofilm
Biofilm formation was detected in 23/40 of the strains, which were categorized as strong (n = 5), moderate (n = 9), or weak (n = 9) biofilm producers (Table 1, Figure 1).A wide variety of K-types were detected among biofilm producers, and none of them prevailed.Most of the strong biofilm producers were positive for the ABC iron transport system kfuBC, and only one was also positive for the yersiniabactin receptor fyuA.Three of the involved isolates came from invasive samples (blood).In moderate biofilm producers, only three isolates were positive for kfuBC, and one of them was also positive for fyuA; most of the strains in this group were obtained from urine samples.In relation to isolates defined as weak biofilm producers, fyuA was more prevalent than in the other groups.The strains involved here were obtained from either invasive or non-invasive samples.One strain was positive for fyuA/kfuBC and was recovered from a cerebrospinal fluid sample.No biofilm producers accumulated most of the fyuA-positive strains.In terms of ESBL production, we identified bla CTX-M-15 among the isolates that were not biofilm producers in almost half of the cases.However, in the other groups, none ESBL prevailed (Table 1).
strong (n = 5), moderate (n = 9), or weak (n = 9) biofilm producers (Table 1, Figure 1).A wide variety of K-types were detected among biofilm producers, and none of them prevailed.Most of the strong biofilm producers were positive for the ABC iron transport system kfuBC, and only one was also positive for the yersiniabactin receptor fyuA.Three of the involved isolates came from invasive samples (blood).In moderate biofilm producers, only three isolates were positive for kfuBC, and one of them was also positive for fyuA; most of the strains in this group were obtained from urine samples.In relation to isolates defined as weak biofilm producers, fyuA was more prevalent than in the other groups.The strains involved here were obtained from either invasive or non-invasive samples.One strain was positive for fyuA/kfuBC and was recovered from a cerebrospinal fluid sample.No biofilm producers accumulated most of the fyuA-positive strains.In terms of ESBL production, we identified blaCTX-M-15 among the isolates that were not biofilm producers in almost half of the cases.However, in the other groups, none ESBL prevailed (Table 1).

Sequence Types and Pulse Types
A total of 20 different STs were detected, with the most frequently being ST14 (n = 7) and ST45 (n = 7); 13/20 of the identified STs were represented by only one isolate (Table 1).Global optimal eBURST analysis revealed a great phylogenetic diversity and a scattered distribution among the characterized STs in this report.However, some of them are related to each other.For example, ST258 is a single locus variant (SLV) of ST11, ST14 and ST15 are SLVs of each other and triple locus variants of ST11, ST16 and ST20 are SLVs of ST17, and ST443 is a double locus variant of ST17 (Figure 2).
Six out of seven of the ST14 isolates were categorized as moderate or strong biofilm producers, whereas the same proportion, but in the absence of biofilm production, was detected in ST45 (n = 6).In ST14, K2 isolates were the most frequent, and the presence of the ABC iron transport system kfuBC was detected in three cases related to invasive infections (blood or cerebrospinal fluid).All ST45 strains belonged to K24 K-type, with fyuA being positive in many of these strains, and urine was the main source (Table 1).
The UPGMA analysis of PFGE resulted in 28 different pulse types (PT), data not shown.Most PTs (n = 21) were represented by a single isolate, and no dominant PT was detected (Table 1).and ST45 (n = 7); 13/20 of the identified STs were represented by only one isolate (Table 1).Global optimal eBURST analysis revealed a great phylogenetic diversity and a scattered distribution among the characterized STs in this report.However, some of them are related to each other.For example, ST258 is a single locus variant (SLV) of ST11, ST14 and ST15 are SLVs of each other and triple locus variants of ST11, ST16 and ST20 are SLVs of ST17, and ST443 is a double locus variant of ST17 (Figure 2).

Antibiotic Susceptibility
The MDR phenotype was found in 25/40 strains when data were analyzed using either EUCAST-2012 or EUCAST-2023 guidelines.The MIC 50 , MIC 90, and susceptibility values were interpreted for each antibiotic (Table 2).Amikacin resistance increased from 10% (n = 4) to 52% (n = 21) when the data were analyzed using the latter guidelines.(Table 1).

Statistical Results
We statistically investigated significant associations (p < 0.05) for the main variables in each group (Table 3).Isolates from the K24 capsular type were associated with the presence of the yersiniabactin receptor fyuA and were related to ST45.The presence of the virulence factor fyuA was associated with isolates from ST45 and with isolates harboring bla CTX-M-15 .Strains positive for the ABC iron transport system kfuBC were more likely to be strong or moderate biofilm producers and being from ST14; weak or no biofilm producers were associated with the absence of kfuBC.Being a strong or moderate biofilm producer was associated with ST14.Finally, an MDR phenotype was associated with the main ESBL gene bla CTX-M-15 , while bla CTX-M-2 was associated with strains not exhibiting an MDR phenotype (Table 3).The statistical results remained consistent when data were interpreted according to EUCAST-2023.
The wide variety of capsular types characterized is in accordance with the polyclonal distribution of the studied isolates, not being able to identify any predominant pulse type.
an appropriate treatment option in the cases of urine isolates with MIC values of 40 mg/L.This antibiotic is recommended for a wide range of infections, including urinary tract infections, respiratory infections, and enteric infections (https://www.eucast.org/accessed on 13 January 2024).
Although we were expecting to find some particular K-type, virulence factor, biofilm production (strong or moderate), sequence type, or ESBL type when the sample came from an invasive infection, we did not find any relation in this sense.This could be related to the small number of samples and also to some clinical reasons like comorbidities (for example, immunosuppression) or the nature of some infections (for example, nosocomial ones).
4.2.K-Types, Virulence Factors, and Hypermucoviscosity Characterization K-types were defined according to the wzi alleles by comparing the partial 447 bp sequences of this gene with the K. pneumoniae wzi database (https://bigsdb.pasteur.fr/klebsiella/ accessed on 13 December 2023).If the designated wzi allelic type correlated with a single capsular serotype in the database, the serotype was used for the description of the capsular types (e.g., K2, K17).If it correlated with multiple or non-capsular serotypes, the wzi allelic type was used (e.g., wzi50, wzi86) [33].
The hypermucoviscous feature of strains was examined by the string test, as previously described [34].

Biofilm Quantification
Bacterial ability to produce biofilm was assessed using a semiquantitative microtiter technique.Briefly, the strains were grown overnight in Luria Bertani (LB) broth at 37 • C under static conditions.Twenty microliters from this culture were inoculated into 180 µL of LB in 96-well flat-bottomed polystyrene microtiter plates and then incubated for 48 h at 37 • C without shaking.Planktonic bacteria were removed, and the attached bacteria were washed three times with phosphate-buffered saline (PBS).The plates were stained with 1% crystal violet (CV) for 15 min at room temperature.Then, the excess dye was removed with three subsequent PBS washes, and CV was solubilized with 200 µL of 95% ethanol.The biofilm biomass was measured by optical density at 590 nm (OD590) using a Microplate Reader (Varioskan, Thermo Scientific).Strains were classified as follows: OD ≤ ODc = no biofilm producer; ODc < OD ≤ (2 × ODc) = weak biofilm producer; (2 × ODc) < OD ≤ (4 × ODc) = moderate biofilm producer; and (4 × ODc) < OD = strong biofilm producer; where ODc were control wells with media without bacteria [35].

Pulsed-Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST) Characterization
For strains collected in the first period, XbaI-PFGE to detect pulse types (PT) and MLST to detect sequence types (ST) were performed [10].Sequence typing was conducted using the guidelines described in the K. pneumoniae MLST database (https://bigsdb.pasteur.fr/klebsiella/ accessed on 13 December 2023).Both PFGE and MLST analyses for the strains collected in the second period were previously performed and published [10].PFGE results from both periods (n = 40) were interpreted using the unweighted pair-group method with an arithmetic mean (UPGMA), and the ST distribution for all strains was visualized with the global optimal eBURST [36] using the dataset available at the MLST database (https://bigsdb.pasteur.fr/klebsiella/accessed on 13 December 2023).

Statistical Analysis
We analyzed associations between the different variables studied: capsular types, virulence factors, biofilm production, sequence types, ESBL, MDR phenotype, and type of infection.Nominal variables were compared using the Chi-square test for variables divided into categories [38].A two-tailed p-value less than 0.05 was considered statistically significant.Statistical analyses were performed using SPSS 23.0 software (IBM SPSS Inc., Chicago, IL, USA).The associations were investigated with the aim of generating future hypotheses for studies with a more clinical approach.

Conclusions
Among the great diversity we were able to detect between the factors analyzed in ESBL-producing K. pneumoniae in this report, we were able to highlight interesting findings.We characterized the most frequent K-type, K24, in association with isolates belonging to ST45 and the presence of the fyuA yersiniabactin receptor.Isolates with the virulence factor kfuBC (ABC iron transport system) showed an association with the capability of being a strong or moderate biofilm producer and belonging to ST14.Remarkably, strains that were weak or had no biofilm producers were associated with the absence of kfuBC.It might be possible that kfuBC plays a role in moderate or strong biofilm formation.The MDR phenotype was high, and it was associated with the main ESBL gene, bla CTX-M-15 .The high prevalence of the MDR phenotype could be partially explained by the additional resistance mechanisms [aac(6 ′ )Ib-cr/qnrB] in isolates harboring bla CTX-M-15 .
We believe that future clinical research is important to evaluate the association between the different clinical presentations and factors studied in this report.
Despite our collection consisting of only 40 isolates from many years ago, it is worth highlighting that the results presented here represent the first findings on this topic in our country.These findings could contribute to our understanding of the characteristics of ESBL-producing K. pneumoniae clinical infections in the pediatric population.
The high plasticity of K. pneumoniae emphasizes the importance of continuous surveillance, which could result not only in better use of available therapeutic resources but also in the control of this microorganism in hospital settings.

Figure 1 .
Figure 1.Biofilm formation of the 40 K. pneumoniae strains studied.The graphic shows the values (mean and standard deviation) of optical density at 590 nm (OD590 nm) of crystal violet obtained for each strain.Dashed lines at 0.2, 0.4, and 0.8 express the threshold value for each biofilm capability formation category: no biofilm producer (NO), weak (W), and moderate (M) biofilm, respectively.Values above 0.8 were considered as strong (S) biofilm formation.

Figure 1 .
Figure 1.Biofilm formation of the 40 K. pneumoniae strains studied.The graphic shows the values (mean and standard deviation) of optical density at 590 nm (OD590 nm) of crystal violet obtained for each strain.Dashed lines at 0.2, 0.4, and 0.8 express the threshold value for each biofilm capability formation category: no biofilm producer (NO), weak (W), and moderate (M) biofilm, respectively.Values above 0.8 were considered as strong (S) biofilm formation.

Figure 2 .
Figure 2. Population snapshot of K. pneumoniae with groups defined at single locus variant (SLV) level and indicating the distribution of the 20 sequence types found in this work (represented in red dots).Snapshot generated by goeBURST v.1.2.1 software using a dataset downloaded from https://bigsdb.pasteur.fr/klebsiella/(accessed on 13 December 2023).

Figure 2 .
Figure 2. Population snapshot of K. pneumoniae with groups defined at single locus variant (SLV) level and indicating the distribution of the 20 sequence types found in this work (represented in red dots).Snapshot generated by goeBURST v.1.2.1 software using a dataset downloaded from https://bigsdb.pasteur.fr/klebsiella/(accessed on 13 December 2023).

Table 1 .
Main features of K. pneumoniae isolates.Data are sorted in increasing order by K-type/wzi.

Table 2 .
Minimum inhibitory concentration data and antibiotic susceptibility percentages for K. pneumoniae isolates.

Table 3 .
Statistical associations between main variables detected in K. pneumoniae isolates.Statistical associations were analyzed for the main variables in each group.