One-year surveillance for hypervirulent Klebsiella pneumoniae detected carbapenem-resistant superbugs

ABSTRACT Hypervirulent Klebsiella pneumoniae (hvKp) can cause infections in clinically healthy people, such as young and immunocompetent patients. Genes involved in the capsule synthesis or those encoding the siderophores have been adopted as predictors of hvKp. Certain sequence types, such as ST23 and ST86, have been associated with hvKp strains, too. The aim of this study was to investigate the presence of hvKp among 354 K. pneumoniae strains isolated from clinical samples of patients admitted to an Italian 900-bed hospital between 21 May 2021 and April 2022. All the isolates were screened by PCR for the amplification of virulence loci. Whole genome sequencing was performed in strains tested positive for at least one target gene. Thirteen out of 354 (3.7%) were hvKp. Five were wild type and belonged to the hypervirulent clones ST23, ST86, ST5, and ST375 and to the new clone ST6310. Six strains carried the blaKPC gene: three belonged to ST101, two to ST512, and one to ST395. Two isolates were ST147 and carried the blaNDM gene. Although hvKp isolation is not frequent, their presence should be systematically investigated to avoid the spreading of both virulent strains and strains with combined increase in virulence and resistance to antibiotics. PCR-based protocols are essential for surveillance of these strains, which do not always show a recognizable phenotype. Moreover, hvKp strains were isolated also from patients without history of recent foreign travels, indicating an increased spreading of these strains as well as an underestimated of their circulation so far. IMPORTANCE Klebsiella pneumoniae is a healthcare-associated pathogen frequently resistant to antibiotics. Hypervirulent strains of pneumoniae (hvKp) can spread from the primary site of infection to multiple sites causing life-threatening infections also in young otherwise healthy individuals. This study described the isolation of 13 isolates of K. pneumoniae with increased virulence in a large tertiary hospital over a 1-year period. Among them, eight strains were multidrug resistant and hypervirulent. Although these hypervirulent strains are still rare in Italy, their presence is particularly concerning since they can cause difficult-to-treat life-threatening infections. Moreover, not all the hypervirulent isolates were positive by the string test, so hvKp isolates were not always phenotypically detectable. Molecular biology techniques such as PCR amplification and next generation sequencing are therefore necessary for the detection of hvKp isolates, and surveillance programs exploiting molecular techniques are highly desirable.

Hypervirulent strains of K. pneumoniae (hvKp) can cause infections in young otherwise healthy individuals, differently from nosocomial multidrug resistant (MDR) K. pneumoniae.hvKp can disseminate from the primary site of infection to multiple sites causing life-threatening infections, such as hepatic abscesses, pneumonia, necrotizing fasciitis, endophthalmitis, meningitis, and sepsis (4).
Currently, the diagnosis of hvKp infections is based on clinical criteria, which includes unusual metastatic spread of K. pneumoniae infections, clusters of K. pneumoniae infections with increased severity and mortality.On the other hand, no single micro biological characteristic can define hvKp (6).Hypermucoviscosity is one of the main characteristics of hvKp strains, as most of these strains overexpress genes for capsular polysaccharides such as rmpA and magA, resulting in hypermucoid phenotype and higher resistance to phagocytosis and intercellular killing by neutrophils.Hypermuco viscosity is detected by string test, in which the colony is stretched with a loop.The formation of a viscous string longer than 5 mm is considered a positive string test (7).However, recent studies (8,9) have shown that hypermucosity is not an exclusive feature of hvKp, while conversely, not all hvKp isolates exhibit a hypermucoviscous phenotype.Most of hvKp isolates belong to sequence types ST23, ST25, ST65, ST86, and ST375 (6).
The presence of iron acquisition systems (e.g., aerobactin, salmochelin, yersiniabactin) involved in bacterial growth and survival is indicative of hvKp strains (4,6).Detection of genes such as iutA and iroN, which regulate iron acquisition systems, by PCR has been used in several studies (5,10).
Recently, the peg-344 gene has been included as a reliable, sensitive, and specific marker for the detection of hvKp such as aerobactin, salmochelin, rmpA, and rmpA2 (8,11).Indeed, PEG-344, whose function is still unclear, was proved to be required for maximal virulence in a pneumonia model (12).
The presence of both increased virulence and resistance in carbapenem-resistant hvKp limits the number of effective antimicrobial agents and therefore poses a serious challenge to treatment, infection control, and public health.
The aim of this study is to investigate the presence of kvKp isolates among K. pneumoniae strains isolated over a period of 1 year from patients at Fondazione IRCCS Policlinico San Matteo in Pavia (Italy).

Samples
Fondazione IRCCS Policlinico San Matteo is a 900-bed hospital, located in Lombardy region of Italy, where Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae has been endemic since 2013 (16).In the study period, 1,822 K. pneumo niae isolates were collected during the routines of the bacteriology laboratory from a total of 627 patients.In this study, we searched for hvKp strains in 354 non-repetitive clinical K. pneumoniae isolates (one per patient) collected from patients at Fondazione IRCCS Policlinico San Matteo between May 2021 and April 2022.The selected 354 strains included strains with characteristics associated with virulence and strains from invasive infections such as bloodstream infections.In addition, we wanted to evaluate the presence of strains with both virulence and resistance characteristics that had never been reported in our hospital.Therefore, extended spectrum beta-lactamase (ESBL)producing and carbapenem-resistant strains, hereafter referred to as MDR, were also considered.In detail, the 354 strains included: • 21 K. pneumoniae strains positive by string test and isolated from any clinical specimen; • 225 K. pneumoniae MDR strains negative by string test and isolated from any clinical specimen except for blood cultures; and • 108 K. pneumoniae wild-type isolates obtained from blood cultures.
The type of specimens and the type of patient from which the 354 K. pneumoniae isolates have been isolated, as well as the resistance profile of the isolates, are shown in Table 1.
MALDI-TOF (Bruker Daltonics GmbH, Bremen, Germany) equipped with Bruker Biotyper 3.1 database was used for species identification.The antibiotic resistance profile was determined by the BD-Phoenix instrument (Becton Dickinson, USA), using the NMIC-505 panel.

PCR amplification, sequencing, and genome analysis
Genomic DNA was extracted with a Blood and Tissue kit (QIAGEN, Düsseldorf, Germany) following the manufacturer's instructions.PCR reactions were performed to investigate the presence of iutA, iroN loci (10), rmpA (17), and peg-344 (18).
The genomes of the isolates positive for at least one virulence gene were sequenced.Short reads were obtained by Illumina MiSeq with a 2 × 150 paired-end run, after Nextera XT library preparation (Illumina Inc., San Diego, USA).Genomes were assembled using Shovill 1.1.Kleborate (19) was used to assess the sequence type and to search for virulence and resistance genes.The presence of plasmids was evaluated with Plasmid Finder (20) and manually curated.
A coreSNP-based phylogeny was performed for isolates belonging to ST147, since an outbreak caused by K. pneumoniae ST147 started in Tuscany region of Italy in 2018 and continued until 2021 (21).The data set consisted of 181 strains of K. pneumoniae ST147 from the outbreak (21,22) and the two isolates from this study.A genomic background was constructed by retrieving the 20 most similar high-quality genomes (N = 94) to those in the data set from the BV-BRC database (https://www.bv-brc.org/)according to k-mer content similarity (MASH).The P-DOR pipeline (https://github.com/SteMIDIfactory/P-DOR) was used to align all genomes in the data set to a reference (573.4026 in BV-BRC) and extract coreSNPs.Maximum likelihood phylogeny was inferred with RAxML (using 100 bootstrap resamples) on the resulting coreSNP alignment using the general time reversible model, as suggested by ModelTest-NG, with ascertainment bias correction (23).  of the 20 patients from whom the 20 isolates were obtained are shown in Table 2.The median age of the 20 patients was 64.35 years (range: 37-81), and 11 were female.Nine strains (2.54%) were isolated from rectal swabs and were resistant to carbape nems, with eight strains bearing blaKPC and one bearing blaNDM encoding for the New Delhi metallo-beta-lactamase (NDM).Only one isolate from rectal swabs was string test positive.The remaining 11 strains were isolated mainly from blood cultures (n = 3; 0.8%), bronchoalveolar lavage (BAL) (n = 2; 0.56%), and urine (n = 2; 0.56%).Four of 11 strains had KPC carbapenemase and one strain had NDM.The characteristics of all the 20 genomes are shown in Table 3.

K. pneumoniae
Kleborate analyses showed that 13 (3.64%)strains had virulence score above 3 and seven of them were string test positive (Table 4).One strain isolated from a blood culture belonged to ST23 and had the highest virulence score.This strain is the only one in this study displaying aerobactin, salmochelin, yersiniabactin, and colibactin.
Eight strains meet the definition of convergent antimicrobial resistant and virulent strains as proposed by Lam et al. (19), having virulence score ≥3 and resistance score ≥1 (Table 3).Three strains belonging to ST101 isolated from rectal swabs and one strain ST147 isolated from a rectal swab had a virulence score of 4 and had aerobactin, salmochelin, and yersiniabactin.The three strains ST101 were KPC producing and the strain ST147 was NDM producing.The other four convergent antimicrobial resistant and virulent strains had a virulence score of 3 and belonged to ST147, ST395, and ST512.
A phylogeny that included the two strains ST147 isolated in this study and the 181 ST147 strains from the outbreak that occurred in Tuscany (21,22) is shown in Fig. 1.One of the two strains of this study (SanMatteo_6445) clustered in a monophyletic clade with three other strains from the study by Martin et al. (21) and the common branch is highly supported (100/100 bootstraps).Evaluation of clonal relatedness by core-genome SNPs reveals that clones were overall highly related to the cluster (SNP range: [19][20].As for other strains in the same cluster, the FIB(pQil)-type plasmid (pQil-NDM-147Tu; 56,064 bp; GenBank accession number CP071030) was identified.This plasmid carried the bla NDM-1 gene, along with other resistant determinants such as aacA4-cr, bla OXA-1 , and bla CTX-M-15 .In addition, the HIB-FIB(Mar) plasmid (pVIR-147Tu; 341,914 pb; GenBank accession number CP071028) was found.This one carried several resistant determinants such as armA, sul1 and sul2, bla CTX-M-15 , and virulence ones such as rmpADC, iucABCD-iutA, and peg-344.On the contrary, the other strain (SanMatteo_6337) clustered with high branch support (94/100 bootstraps) with four strains from the BV-BRC DB isolated in Russia between 2018 and 2019 (SNP range: 41-104).The SanMatteo_6337 strain did not carry the FIB(pQil)-type plasmid and carried the HIB-FIB(Mar)-type plasmid, instead.

DISCUSSION
This study reports the isolation of 13 isolates of hypervirulent K. pneumoniae, based on sequence type and the presence of virulence genes such as aerobactin and salmochelin.Six out of 13 strains were isolated from rectal swab (colonization), while seven strains caused infections.The 13 strains had different characteristics.Two strains belonged to ST23 and hvKp ST86, respectively, and caused bloodstream infections in two patients who had not recently traveled abroad.hvKp ST23 was reported for the first time in Italy in 2014 (24), and in our hospital, it was isolated from urine and BAL samples between 2017 and 2018 (25).However, this sequence type is very uncommon in Italy and only five genomes of K. pneumoniae ST23 were found in the BV-BRC database as isolated in Italy, including the 6453 isolate of this study and the isolate from a BAL sample of the same patient (last accessed on 30 March 2023).ST23 strain isolated in this study was wild type but hvKp  ST23 bearing carbapenemases were described in several European countries also from blood and respiratory samples (3,24).Equally rare is the ST86, which presence is documented in Italy so far only by the presence in BV-BRC databases of three genomes of K. pneumoniae ST86, including strain hvKp_4 of this study (reported in the BV_BRC database as 10028588).In Europe, ST86 hvKp strains have already been reported in Spain where three isolates out of 878 were found to be hypervirulent ST86 in a 7-year study (2) and in France where three out  of 59 K. pneumoniae isolates were hypervirulent ST86 in a 5-year study (3).In France, carbapenem-resistant hvKp bearing OXA-48 have been isolated as well (26).
Other sequence types frequently associated with hypervirulence, such as ST5 and ST375, were found in this study.ST5 was previously isolated in Italy in a multicentric study (27).This strain, as well as hvKp_19 of this study, bears virulence factors such as ICEKp1, aerobactin, salmochelin, and rmpADC hypermucoidy locus.Only four genomes belonging to ST375 were found in the BV-BRC database as isolated in Europe, none in Italy.Hypervirulent ST375 was previously isolated in Japan (28) from liver abscesses of two patients.The ST375 isolate of this study was isolated from a liver biopsy, too, and as the isolates from Japan, it carried aerobactin and rmpA genes.
The new sequence type ST6310, a single locus variant of ST25 for mdh gene, was described for the first time in this study.Strain hvKp_2, which belonged to ST6310, had aerobactin, salmochelin, and rmpA loci and it was isolated from a patient who had lived in China.
hvKp isolates belonging to well-known MDR high-risk clones such as ST101, ST147, ST395, and ST512 were also found in this study.hvKp are usually susceptible to most antibiotics, but the ability of Klebsiella to acquire genetic elements leads to the emergence of MDR-hvKp clones (4,13).Most MDR strains of this study did not have multiple virulence genes, except for three strains belonging to ST101 and one from ST147 that bore a wider range of virulence genes.The latter ST147 strain resulted closely related to the strains that caused a large hospital outbreak in the Tuscany region of Italy.Indeed, this isolate along with the strains described in the study of Di Pilato et al. are part of subclade of ST147, called ST147-vir, characterized by the presence of the ybt-encoding element ICEKpn3 and the pQil-NDM-147Tu-like and the pVIR-147Tu-like plasmids (22).The sequence type 147 is a high-risk clone associated with hospital-acquired infections worldwide and mediating the global spread of NDM-like metallo-beta-lactamase genes (29).Therefore, it is crucial to track the dissemination of these potential successful clones in hospital settings and understand their evolution to avoid the spread of resistant and virulent determinants, especially those determinants carried on plasmids, such as bla NDM-1 .
In conclusion, this study described the isolation of 13 isolates of K. pneumoniae with increased virulence in a large tertiary hospital over a 1-year period.Although the isolation of these strains is not frequent, their presence is particularly concerning, as some of these isolates are not only hypervirulent but also MDR, effectively limiting the therapeutic options in case of infections.
Not all the hypervirulent isolates were positive by the string test, so hvKp are not always phenotypically detectable.Molecular biology techniques such as PCR amplification and next generation sequencing are therefore necessary for the detection of hvKp isolates.Surveillance programs exploiting PCR for the detection of hvKp isolates are desirable also because virulence genes are often carried on mobile genetic elements, which can be transferred within and between different bacterial species.In addition, the presence of hvKp should be systematically investigated in both wild-type and MDR strains, to avoid the spreading of strains with both increased virulence and resistance to antibiotics.Finally, hvKp strains were also isolated from patients with no history of recent travels abroad, suggesting an increased prevalence or an undetected circulation of hvKp isolates.

FIG 1
FIG 1 Maximum likelihood phylogeny of Klebsiella pneumoniae ST147 based on SNP cores obtained using the P-DOR pipeline (https://github.com/SteMIDIfactory/P-DOR).The two strains of this study are indicated in red, while 181 genomes ST147 from the outbreak that occurred in Tuscany reported by Martin et al.

TABLE 1
Type of specimens and the type of patient from which the 354 K. pneumoniae isolates have been isolated a

Criterion of selection Number of samples for the criterion Inpatients Outpatients Type of specimen Number of samples Wild type ESBL KPC NDM VIM Other
a The resistance profile of the isolates is also shown.b NA was used to indicate that zero strains have that characteristic.

TABLE 2
Characteristics of the 20 patients from whom Klebsiella pneumoniae with increased virulence was isolated a

Strain number Isolation date Sample Sex Age Hospital ward Outcome String test Resistance mechanisms PCR rmpA PCR iutA PCR iroN PCR peg-344
a Outcome for the patients colonized by K. pneumoniae with increased virulence is indicated with NA (Not Applicable).

TABLE 4
Characteristics of the 20 strains that were sequenced a