Abstract
This study focused on the characterization of 19 hypermucoviscous Klebsiella pneumoniae strains, that were identified from 26 hypermucosal strains. In order to identify hypermucoviscous strains of K. pneumoniae, the string test was applied. This phenotype is known in the literature as one of the virulence factors of this species together with the production of biofilm and other hypervirulence factor genes such as: rmpA, rmpA2, iucA, iroB, peg-344. We also investigated presence of magA gene that correlates with the hyper-production of capsule of K1 serotype. Of the strains under study, 13 out of 19 harboured at least one virulence factor.
Sequence type (ST) was determined in order to identify known high-risk clones or new emerging high-risk clones and their variability in a single clinical setting. Important STs found among these strains were ST65 and ST29. Carbapenem resistance was also investigated and 4 out of 19 strains harboured at least a carbapenemase: one strain harboured a KPC enzyme alone, one strain carried a KPC and an OXA-48 like, one strain produced OXA-48-like alone, and the last strain harboured two metallo-β-lactamases (VIM-1 and NDM-5) plus OXA-48-like. In particular, this latter strain belongs to ST383, which was recently reported in Northern Italy as a hypervirulent and XDR strain.
The global spread of hypervirulent K. pneumoniae is an important epidemiological issue that should be considered in diagnostic and therapeutic managements of patients with K. pneumoniae infections.
Introduction
In recent years, Klebsiella pneumoniae has gained notoriety as an infectious agent due to its increase in number of serious infections and its increasing resistance to antibiotic therapy. Furthermore, additional genetic traits associated with hypervirulence, and antibiotic resistance have recently been identified that make K. pneumoniae infection an increasingly emergency problem [1].
It is important to differentiate between classical K. pneumoniae (cKp) strains that typically cause nosocomial infections, now found worldwide and hypervirulent (hvKp) strains that cause severe, community-acquired systemic infections in otherwise healthy individuals, an emergency that initially worried only Asian countries but it is now rapidly expanding [2].
In 1986, Liu et al. [3] reported the first seven clinical cases of invasive K. pneumoniae infection in community individuals who presented with a liver abscess in the absence of biliary tract disease and these K. pneumoniae strains were defined as hypervirulent. Notably, hvKp strains cause primary liver abscesses in patient populations that do not appear to have any underlying liver disease. In turn, liver abscesses can give rise to a number of other secondary infections due to hematogenous spread from the liver [4].
To date, there are four major classes of virulence factors that have been characterized well in K. pneumoniae: capsule, including the production of hypercapsule in hvKp strains; lipopolysaccharide (LPS); siderophores; and type 1 and 3 fimbriae. Siderophores play an important role in virulence, and hv K. pneumoniae encodes for salmochelin, aerobactin, other than enterobactin, and yersiniabactin, the last of which is also produced by cKp strains. Salmochelin is a form of c-glucosylated enterobactin encoded by the iroA gene. It is present in only about 2–4% of nosocomial K. pneumoniae strains but is much more prevalent in hv K. pneumoniae strains. Aerobactin is a citrate-hydroxamate siderophore encoded by the iucA gene; it is expressed rarely by classical nosocomial clinical isolates of K. pneumoniae but it is present in 93–100% of hvKp isolates [2].
Currently, magA is defined as an essential gene for the formation of the biosynthesis of K1 capsular polysaccharides but it is not an independent specific virulence gene in strains of K. pneumoniae that cause liver abscesses [5].
Clinical laboratories are currently still in the midst of studying the differentiation between hvKp and cKp. Increased capsule production and aerobactin production have been identified as specific virulence factors for the hypermucoviscous phenotype; in addition to these elements, it has been shown that several biomarkers and quantitative production of siderophores accurately predict hvKp strains [6].
Klebsiella pneumoniae is known for its propensity to collect resistance plasmids. Extensive use of carbapenems has led to the emergence and rapid spread of carbapenemase-producing strains. The types of carbapenemases prevalent in K. pneumoniae are: K. pneumoniae carbapenemase (KPC)-type enzymes; New Delhi metallo-β-lactamase (NDM)-type enzymes; and oxacillinase (OXA)-type enzymes, mainly OXA-48 [7].
The aim of this study is the identification of hypermucoviscous and hypervirulent strains through the molecular and phenotypic characterization of 19 isolates of multidrug-resistant (MDR) and non-MDR K. pneumoniae. This work stems from an ongoing health emergency, since the failure to recognize the increasingly dangerous factors of hypervirulence and resistance could lead to underestimate the risky circulation of these strains.
Materials and methods
Bacterial strains
The study included a collection of 26 strains of K. pneumoniae that have been isolated at Microbiology and Virology Hospital Service in Verona, Italy in the year 2021 and collected from blood cultures and abscesses during routine clinical analysis. All strains were identified by Maldi-tof Vitek MS (BioMérieux, France).
Antimicrobial susceptibility determination
Antimicrobial susceptibility tests for carbapenems were performed by disc diffusion method following the EUCAST (European Committee on Antimicrobial Susceptibility Testing) guideline. The results were interpreted using the breakpoints table of Enterobacterales on EUCAST's website [8].
String test
The string test is a simple, rapid, and readily available screening method that confirms a known virulence factor of K. pneumoniae, the hypermucoviscous phenotype. Furthermore, not all mucoid strains of K. pneumoniae show a positive string test, therefore a colonial mucoid appearance does not equate to a hypermucoviscous phenotype [9]. This phenomenon highlights a clear difference between the classic capsular mucoid strains and the hypermucoviscous variants [10]. The test is positive when there is formation of a viscous string of in > 5 mm in length when a loop is used to stretch the colony on an agar plate [11].
Hypervirulence factor detection
Hypervirulence factors of hypermucoviscous strains were detected by Polymerase Chain Reaction (PCR) using primers and thermal profiles reported earlier [12]. The marker genes used to distinguish hvKp from cKp are: rmpA/rmpA2 encoding regulator of mucoid phenotype (hypermucoid); iucA gene in the locus of aerobactin siderophore; iroB gene in the locus of salmochelin siderophore; and peg-344, which is a putative transporter. To determine the presence of magA gene, a PCR was performed using primers and conditions already reported [13].
Carbapenemase detection
The CARBA NP phenotypic test [14] was used for the rapid detection of carbapenemase production in Enterobacterales. Molecular characterization of carbapenemase genes (blaVIM, blaIMP, blaNDM, blaKPC and blaOXA-48) was performed by PCR [15].
Multilocus Sequence Typing (MLST)
MLST analysis was conducted through simplex PCRs for seven different housekeeping genes as described on the Institute Pasteur website [16, 17]. The MLST PCR products were purified through the QIAquick PCR Purification Kit and then sequenced at Eurofins Genomics (Germany, Ebersberg) in order to obtain sequence-typing (ST).
In vitro biofilm production
The production of biofilm was also analysed through the crystal violet test performed on a microtiter plate, and the optical density was measured in the plate at 550 nm through a fluorescence microplate reader. The results obtained were then subjected to the analysis described by Stepanovic et al. [18].
Results and discussion
To date K. pneumoniae is recognized as an urgent threat to human health due to the emergence of multidrug-resistant strains associated with hospital outbreaks as well as due to hypervirulent strains associated with severe community-acquired infections. Therefore, the convergence of virulence and resistance genes could potentially soon lead to the emergence of invasive, untreatable infections caused by K. pneumoniae [1–4].
All 26 K. pneumoniae isolates were selected in this study on the basis of their hypermucosal aspect and were analysed through the string test screening method. Strains with a string >5 mm were classified as a hypermucoviscous (hmv) phenotype; this was shown in 19 out of the 26 strains. The incorporation of the string test into the daily practice of microbiological surveillance could lead to more appropriate diagnostic tools. At the same time, we must remember that non-hmvKp positive strains can also be highly virulent when organisms possess hypervirulence genes [19].
These 19 hypermucoviscous strains were further investigated for virulence factors, and MLST was performed in order to check for high-risk clones; all data are summarized in Table 1.
Virulence factors, antimicrobial susceptibility, carbapenemases and sequence types of the 19 hypermucoviscous strains under study
| Strains | String test | Carba NP | Carbapenemases | ST | Hypervirulence factors | magA | Biofilm | Kirby-Bauer Disk Diffusion Susceptibility | ||
| Imipenem | Meropenem | Ertapenem | ||||||||
| AMP 3291 | + | – | ND | 65 | iucA, iroB, peg-344, rmpA, rmpA2 | Strong | S | S | S | |
| AMP 3678 | + | – | ND | 86 | iucA, iroB, peg-344, rmpA, rmpA2 | Strong | S | S | S | |
| AMP 3679 | + | – | ND | 65 | iucA, iroB, peg-344, rmpA, rmpA2 | Strong | S | S | S | |
| AMP 3680 | + | – | ND | 1,224 | None | Strong | S | S | S | |
| AMP 3681 | + | – | ND | 45 | None | + | Strong | S | S | S |
| AMP 3682 | + | – | ND | ND | None | + | Strong | S | S | S |
| AMP 3683 | + | – | ND | 380 | iucA, iroB, peg-344, rmpA, rmpA2 | + | Strong | S | S | S |
| AMP 3866 | + | – | ND | 29 | iucA, iroB, rmpA, rmpA2 | Strong | S | S | S | |
| AMP 3872 | + | – | ND | 17 | None | Strong | S | S | S | |
| AMP 3875 | + | + | KPC | 35 | None | Strong | R | I | R | |
| AMP 3876 | + | – | ND | 86 | None | Strong | S | S | S | |
| AMP 3877 | + | – | ND | 29 | iucA, iroB, peg-344, rmpA, rmpA2 | Strong | S | S | S | |
| AMP 3878 | + | – | ND | 10 | None | + | Strong | S | S | S |
| AMP 3880 | + | – | ND | 65 | iucA, iroB, peg-344, rmpA, rmpA2 | Strong | S | S | S | |
| AMP 3881 | + | – | ND | 111 | None | Strong | S | S | S | |
| MDR 444 | + | + | OXA-48-like | 395 | None | + | Strong | S | I | R |
| MDR 1682 | + | + | VIM-1 NDM-5 OXA-505 | 383 | rmpA2 | Strong | R | R | R | |
| MDR 2145 | + | + | KPC OXA-48-like | 101 | None | Strong | R | R | R | |
| MDR 3605 | + | – | ND | 307 | rmpA2 | + | Strong | S | S | S |
ND = Not Determined; S = Susceptible; R = Resistant; I = Susceptible at increase dosage
ST = sequence type
All 19 strains, following the Stepanovic criteria for classification [18], resulted in strong biofilm producers. This factor will contribute to these strains' tolerance of antibiotics [2].
The five main loci, namely iroB, iucA, peg-344, rmpA, and rmpA2, which according to the literature can differentiate and therefore define a hypervirulent strain with respect to the classical forms of K. pneumoniae, have been selected. Through molecular characterization, it was found that seven strains of the study displayed at least 4 hypervirulence genes. According to what is reported in the literature, a K. pneumoniae strain is defined as hypervirulent if it has at least four of the aforementioned genes [12]. Figure 1 reports the distribution of virulence genes in the strains under study.
Virulence factor distribution among the strains in this study
Citation: Acta Microbiologica et Immunologica Hungarica 69, 4; 10.1556/030.2022.01908
However, it should also be taken into account that the strains MDR 1682 and MDR 3605 carry the rmpA2 gene alone, which according to the ECDC risk assessment [1] is one of the major factors of hypervirulence.
According to the PCR results for the magA gene, which is responsible for the capsular serotype K1, 6 out of 19 strains (31.6%) resulted positive. This result is consistent with literature, which reports that K1 capsular serotype together with K2 are the two types of capsule mostly related to hypervirulence [10], since capsule serotyping is a very long procedure that includes different type K and type O antigens, these strains cannot be defined with certainty as K1 serotype. However, this test can still be a good start in the K. pneumoniae capsule serotyping procedure.
Carbapenemase-producing strains were screened through the CarbaNP test, and only 4 out of 19 strains resulted positive (21.05%). All CarbaNP-positive strains were subjected to molecular analysis through various PCRs. Carbapenemases were detected in different combinations: KPC alone; OXA-48 alone; KPC and OXA-48 together; and one strain harboured VIM-1, NDM-5, and OXA-48-like. The last is considered also a potential pathotype since it harbours the rmpA2 gene, while the other three strains harbouring carbapenemases did not carry rmpA2. The three carbapenemases harboured by the MDR 1682 strain were sequenced and found to be VIM-1, NDM-5, and OXA-505 enzymes. NDM-5 is a variant recently found in Italy, in Escherichia coli strains isolated in Tuscany [20].
MLST was performed in order to identify ST linked to the hmv Kp pattern, and to check if ST43 was present as indicated by the ECDC [1]. We have not found ST43; however, we were able to detect other STs present in our hospital setting that can act as emerging clones. Noteworthy also are the multiple STs that we found among hmv strains and harbouring hypervirulence factors. Three strains were characterized as ST65, which, according to the literature, is precisely linked to the hypervirulence of the K2 capsular serotype strains and has caused severe and fatal infections in clinical setting in China [21]. Two strains, on the other hand, belong to ST29, which is commonly associated with extended-spectrum β-lactamase-encoding genes (in particular, blaCTX−M−15), but rarely has carbapenemase genes [22]. The AMP 3678 strain was identified as ST86, which corresponds to the ST found in the first case of community-acquired pneumonia due to an hmv strain of K. pneumoniae [23]. The strain AMP 3683 showed ST380, which is considered an emerging ST in severe community-acquired infections [24].
Among seven strains with five or four hypervirulence genes, we found four STs, most of them already correlated with hypervirulence and considered as emerging clones. This was a surprising and worrying result, since these strains are more widespread than is generally believed and they can easily combine with multidrug resistance.
Another important finding is that the strain harbouring the three carbapenemases belong to ST383. At the beginning of 2022, NDM-1/5- and OXA-48-co-producing, extensively drug-resistant hypervirulent K. pneumoniae strains were detected in Northern Italy, more specifically at the San Raffaele hospital in Milan, and these strains were ST383 [25].
Among the strains that do not harbour the virulence factors taken into consideration, we find ST101 and ST307. These STs have been identified in Southern Italy in serious bloodstream infections and are emerging high-risk carbapenems-resistant clones of K. pneumoniae. Furthermore, these clones appear to be potentially extremely virulent [26].
In conclusion, the global spread of hvKp is an important epidemiological change that should be considered in the diagnostic and therapeutic management of patients with K. pneumoniae infections. Unfortunately, there is high concern about the possibility of a further combination of virulence and resistance, thus leading to severe and untreatable infections in healthy individuals, which would be extremely difficult to manage.
The emergence of these high-risk clones and the global spread of these strains has left physicians with very few treatment options. Therefore, it is essential to have new protocols for strengthened screening control in order to limit the spread of multidrug-resistant and hypervirulent K. pneumoniae strains. New strategies need to be further explored as therapeutic options in the treatment of infections caused by both classical and hypervirulent strains of K. pneumoniae.
Conflict of interests
None.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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