Multi-resistant ST 258 Klebsiella pneumoniae presenting a hypermucoviscous phenotype

doi:10.21203/rs.3.rs-4854582/v1

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Multi-resistant ST 258 Klebsiella pneumoniae presenting a hypermucoviscous phenotype

https://doi.org/10.21203/rs.3.rs-4854582/v1

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To characterize phenotypically and genotypically a strain of Klebsiella pneumoniae ST258 isolated from an immunosuppressed patient treated at a University Hospital in Brazil, it was performed Antimicrobial Susceptibility Testing, Analysis of the Ability to form Biofilm, Genome Sequencing and String Test. The sample was characterized as a non-biofilm-forming, multi-resistant and hypermucoviscous strain. Genomic analysis revealed the presence of resistance genes, virulence genes and plasmids. This is the first report of hypervirulent hypermucoviscous KPC ST258 in Rio de Janeiro, Brazil. KPC ST258 represents a significant global health threat due to the limited available treatment to fight these infections. This observation poses a threat to the community and hospital environments, highlighting the need for increased surveillance to detect these strains.

Klebsiella pneumoniae ST258

Multi-resistant

hypermucoviscous

WGS

Klebsiella pneumoniae is a gram-negative, encapsulated, facultative anaerobic, rod-shaped bacterium from the Enterobacteriaceae family. It is an opportunistic pathogen that can cause urinary tract infections, bacteremia and pneumonia [1]. Is intrinsically resistant to ampicillin through the production of the enzyme β-lactamase, encoded by the blaSHV gene[2]. The WHO recognizes carbapenemase-producing K. pneumoniae (KPC) as a critical threat to public health [3].

MDR-hv Klebsiella spp. are both hypervirulent and resistant to multiple antimicrobials, in addition to undergoing an evolution that may lead to the emergence of phenotypically new strains [4]. They have diverse genetic origins, with cases reported, to date, in Asia (mainland China, Hong Kong, and Taiwan), Europe (France, Norway, United Kingdom, and Russia), Africa (Egypt) and America (Canada, United States and Brazil)[5].

The accumulation of mutations such as modifications in the quinolone resistance-determining region (QRDR mutations in gyrA and parC), changes in the porin gene (ompK36) and modifications leding to the overexpression of efflux pumps (OEP mutations: acrAB, oqxAB, ramA and rarA) has been reported in Klebsiella spp. MDR-hv with these mutations present resistance to several antimicrobials such as quinolones, carbapenems and tigecyclines [6].

Hypermucoviscous (HM) hypervirulent K. pneumoniae (hvKP) is prone to causing metastatic spread and life-threatening diseases. Initially described in the Asia-Pacific region, it is now identified in Western countries as well. The HM phenotype appears to enhance the virulence of hvKP strains and is often considered a substitute marker for improved virulence. This phenotype appears to depend on rmpA/rmpA2-associated enhanced production of polysaccharides and can frequently be identified by string-test [7].

Additionally, K. pneumoniae of sequence type 258 (ST258), known for producing carbapenemase (KPC), frequently infects hospitalized patients, leaving them with few therapeutic alternatives. As carbapenems represent the last line of defense against life-threatening bacterial infections, this poses a significant challenge for treatment [8].

On September 2, 2020, a 35-year-old immunosuppressed male was admitted to a University Hospital in Niteroi City, Rio de Janeiro, Brazil. According to his medical records, the patient presented with complaints of fever, cough, dyspnea, and seizure. These signs and symptoms began on August 30, 2020, four days prior to hospitalization. Upon admittion to the ICU (day 1), a preliminary examination showed an oxygen saturation (SO2) > 95%, hemoglobin level of 11,7 g/dL, hematocrit of 35,6%, platelet count of 223 x 10³/mm³.

The white blood cell counts tests displayed 6,0 x 10³/mm³ of lymphocytes and 8.0 x 103/mm³ of band (immature neutrophils). The patient tested negative for SARS-CoV-2 by RT-PCR five times.

On September 14, 2020 (day 13), Acinetobacter baumannii was retrieved from tracheal aspirate and blood culture at the hospital’s clinical microbiology laboratory. On September 23, 2020 (day 21), blood tests showed a hemoglobin level of 6.1 g/dL and hematocrit of 19.0%, suggesting a case of anemia. Additionally, laboratory results showed 4 x 10³/mm³ of lymphocytes, 4 x 10³/mm³ of eosinophils, 4 x 10³/mm³ of monocytes, 15 x 10³/mm³ of band neutrophils. A Klebsiella pneumoniae was retrieved from the tracheal aspirate collected for the SARS-CoV-2 by RT-PCR surveillance test. K. pneumoniae colonies were retrieved from a MacConkey agar plate overnight incubation at 37°C. The colonies were glucose and lactose-positive, red, and dome-shaped with mucoid and stickiness aspects. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) (Bruker Daltonics, Bremen, Germany) further confirmed the Bacterial species identification. The Antimicrobial Sensitivity Test (AST) revealed resistance to Meropenem, Imipenem, Tazobactam, Cefazolin, Cefepime, Gentamicin, Amikacin, Tobramycin, Aztreonam, Ciprofloxacin, Levofloxacin, and Norfloxacin, which classified it as a multi-resistant strain. Analysis of Whole genome sequencing and assembly classified the sample as ST258 and revealed the presence of resistance genes against Aminoglycosides (aph(3)-Ib/ aph(6)-Id/ rmtB/aac(3)-IId/ aadA2/ aph(3)-Ia), ESBL ( fosA6/ bla_CTX−M−14/ bla_TEM−1B), Macrolides (mph(A)/ erm(42)), Chloramphenicol (catA1), Carbapenemase (bla_KPC−2), Sulfonamides (sul2), Tetracycline (tet(G)) and Trimetroprim (dfrA12). The Virulence Finder Database (VFDB) revealed a total of 54 virulence genes, including acrA, acrB (efflux pump); entA-F, fepA-D, and fepG (siderophore-iron uptake); finA-H (Adherence – Type I fimbriae); mrkA-D, mrkF, mrkH-I (Adherence – Type III fimbriae) and iutA (Aerobactin- iron uptake). Plasmids were observed (ColRNAI, IncFIB(K), IncFII(K), and IncN_1) and the following mutations were identified PmrB: T246A, PmrB: R256G (deleterious), and MgrB truncation: 23/47.

The isolate was not classified as biofilm-forming. However, it was String Test positive, exhibiting a dense filament greater than five millimeters upon touching the colony’s surface, indicating hypermucoviscosity (Fig. 1). The patient's medical records indicate that his death occurred on October 19, 2020.

Carbapenems play a crucial role in treating certain multidrug-resistant Gram-negative pathogens. The persistent spread of carbapenem-resistant bacteria, like ST258 K. pneumoniae, poses a significant worldwide health threat due to the scarcity of effective treatment options for these infections [7].

In a study carried out by Zafer and collaborators, the hypermucoviscous (HM) and hypervirulent phenotype was linked to the presence of IncHI1B plasmid associated with rmpA rmpA2, iutA, iroN, bla_CTX−M−15, bla_OXA−48, and bla_NDM−1, however, no sample was associated with the ST258 profile [11].

In this case report, a similar virulence and resistance profile (rmpA rmpA2, iutA, iroN, bla_CTX−M−14, bla_KPC−2) was found in an ST258 strain, corroborating the potential for transmission of resistance harboring the IncHI1B plasmid. Also, mutations in the chromosomal rcsA and rcsB genes, part of a signaling system regulating capsular biosynthesis, can also result in the HM phenotype.

To our knowledge, this is the first report of hypervirulent hypermucoviscous Klebsiella

pneumoniae ST258 in Rio de Janeiro, Brazil. The ST258 K. pneumoniae in this study exhibited a multidrug-resistance profile, including resistance to carbapenems and harboring one gene associated with this class of antimicrobial agents. This poses a threat, and surveillance to detect these strains must be considered. The precise mechanisms underlying the HM phenotype remain unknown in certain strains.

Sample

Klebsiella pneumoniae was retrieved from the tracheal aspirate of an immunosuppressed patient on the twenty-first day of hospitalization.

Antimicrobial Susceptibility Testing (AST)

Colonies were suspended in sterile saline solution until they reached 0.5 McFarland. Then, confluent sowing was carried out on Müeller-Hinton agar medium (Kasvi, Paraná, Brazil). Antimicrobial discs (amoxicillin/clavulanic acid 20ug/10ug, ceftazidime 30ug, cefepime 30ug, cefotaxime 30ug, ceftriaxone 30ug, meropenem 10ug, imipenem 10ug, aztreonam 30ug, ciprofloxacin 5ug, levofloxacin 5ug, amikacin 30ug, gentamicin 10ug, trimethoprim-sulfamethoxazole 1.25/23.75ug and tetracycline 30ug) were placed on the plate and incubated at 37 ºC for 18 hours. The reading was carried out by measuring the complete diameter of the inhibition halo (CLSI, 2023) [9].

Biofilm Growth Using Crystal Violet Assay: This test was performed according to[10] with some adaptations. 200 µL of the inoculum diluted in Tryptic Soy Broth (TSB, Sigma-Aldrich, Saint Louis, Massachusetts, USA), was added to each of the three wells of a 96-well flat-bottom polystyrene plate (NUNC - ThemoFisher Scientifc, Waltham, Massachusetts, USA), and incubated for 24h at 37 ºC. After that the contents of each well were removed and the wells were washed with phosphate-buffered saline (PBS). The plate was dried and stained with 200µL of crystal violet, followed by incubation at room Temperature (RT) for 15 minutes. The plate was washed, dried and 200µL of absolute alcohol was added to each well. After 30 minutes, the plates were read in an ELISA (Enzyme Linked Immuno Sorbent Assay) microplate reader at 560 nm. The cutoff point was defined by the equation: cutoff point = OD (Mean White) + (3x Standard Deviation (White)). The results will be interpreted as follows: Non-biofilm forming (0) - OD(a) ≤ cutoff point; Weak biofilm former (+) - cutoff point < OD(a) ≤ 2 x cutoff point; Moderate biofilm former (++) − 2 x cutoff point < OD(a) ≤ 4 x cutoff point; Strong biofilm former (+++) − 4 x cutoff point < OD(a). Any negative OD value must be presented as zero.

String test

The bacterial sample was cultured on McConkey agar plate (Kasvi, Paraná, Brazil) and incubated for 24 hours at 37°C. After bacterial growth, the formation of a viscous filament was verified by touching the surface of a colony with a sterile loop. The formation of a filament larger than 5 millimeters when touching the colony and moving the loop upwards caracterizes the strain as hypermucoviscous [7].

DNA Extraction and Next Generation Sequencing

Bacterial DNA was extracted using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany) and quantified using the QuantiFluor ONE ds DNA system (Promega, Wisconsin, USA). The sequencing library was created using the Nextera DNA Flex (Illumina, California, USA), and was quantified by fluorometry using the QuantiFluor® ONE ds DNA system. After dilution, the library was sequenced using the NextSeq 1000/2000 P2 (200 Cycles) or NextSeq 2000 (Illumina, California, USA). Sequence analysis was carried out using the CABGen v1.09 platform [8] and The Virulence Finder Database (VFDB).

ACKNOWLEDGEMENTS

This work was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro – FAPERJ (Projeto REDES: E26/211.554/2019; E-26/201.328/2021), and by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brazil (CAPES) Finance Code 001.

AVAILABILITY OF DATA AND MATERIALS

Sequence data that support the findings of this study have been deposited in the BioSample Database in the National Center for Biotechnology Information’s with the primary accession code SAMN43145338

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The study was approved by the ethical committee in Research from the Medicin Faculty to the Universidade Federal Fluminense (CEP – FM/UFF), being registered under CAAE number 26823614.2.0000.5243. The studies were conducted following the local legislation and institutional requirements. The participants provided their informed consent to participate in this study.

ADITIONAL INFORMATIONS:

CONFLICT OF INTEREST

The authors declare no conflict of interest.

COMPETING INTEREST

The authors declare no competing interests.

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No competing interests reported.

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Multi-resistant ST 258 Klebsiella pneumoniae presenting a hypermucoviscous phenotype

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