Virulence Genotype and Clinical Inuence of the Type VI Secretion System in Klebsiella Pneumoniae Isolates Causing Bloodstream Infections

Background Klebsiella pneumoniae (K. pneumoniae) causes bloodstream infection (BSI), which is responsible for a high rate of morbidity and mortality among different populations. In mainland China, data on the incidence and features of the T6SS gene cluster in K. pneumoniae is currently scarce. As a result, we conducted a prospective investigation to determine the involvement of the type VI secretion system (T6SS) in K. pneumoniae pathogenicity and antibiotic resistance. Method In this retrospective analysis, we enrolled 119 individuals who had been diagnosed with K. pneumoniae bloodstream infection and acquired demographic and clinical data from their medical records. The virulence genes rmpA, rmpA2, aerobactin, iroB, hcp, vgrG, and icmF were tested for K1 and K2, antimicrobial susceptibility. T6SS positive strains (N=20) were identied as having icmF, vgrG, and hcp, while T6SS negative strains (N=99) did not manifest the same. In this study, hvKP was dened as rmpA and aerobactin positivity. Five T6SS+ and ve T6SS- isolates were chosen for the competition, serum resistance, and biolm formation experiments to further gain insights regarding the microbiological properties of T6SS+ K. pneumoniae isolates.

1 Introduction cell-to-cell communication and signaling. The T6SS is used by Vibrio cholerae to cause variation in the cellular activity of the host that lowers the population of competing bacteria.
The goal of this study was to look at the involvement of the type VI secretion system (T6SS) in K. pneumoniae pathogenicity and antibiotic resistance. T6SS-producing and non-T6SS-producing K. pneumoniae clinical isolates were examined in terms of antimicrobial resistance and virulence characteristics. In vitro tests, for instance, competition, bio lm formation, and serum resistance assays were used to evaluate T6SS function.

Study designs and data collection
The First A liated Hospital of Anhui Medical University, a 3000-bed tertiary-level healthcare facility in Hefei, China, hosted this single-center prospective cohort study from July 2019 to January 2021. The current investigation was duly approved by the human ethics council of Anhui Medical University's First A liated Hospital. As no personal information was used in the study, patients were not required to provide a declaration of permission for submission.
The BacT/ALERT 3D system was used for culturing K. pneumoniae BSI, which was isolated from one or more sets of aseptically acquired blood culture bottles (Becton-Dickinson, Sparks, MD, USA). Blood was cultured in the microbiology lab employing a BacT/ALERT 3D system (Becton-Dickinson, Sparks, MD, USA). Matrix-associated laser desorption ionization-time of ight mass spectrometry (Vitek MS, Biomerieux, France) was utilized for the recognition of all isolates. Following the Clinical and Laboratory Standards Institute's recommendations, antibiotic susceptibility testing was done making use of the VITEK 2 (Card number: AST-GN13) system or the methods of Kirby-Bauer Disk Diffusion (Oxoid, UK) (CLSI). Escherichia coli ATCC25922 and K. pneumoniae ATCC700603 were employed as quality control isolates.

Clinical Data and De nitions
Medical data were used to acquire clinical information on 119 KP-infected patients. Immunosuppression was referred to as primary immunode ciency disorder and/or regular use of high-dose steroid therapy (prednisolone 10 mg/daily or comparable dose) for more than half a month, and/or immunosuppressive chemotherapy within the previous two months. Neutropenia was classi ed as a neutrophil count of fewer than 500 per liter. When symptoms and positive blood culture were gathered more than 48 hours following hospital admission, it was considered a nosocomial infection. MDR (multi-drug resistance) is referred to as acquired non-susceptibility to at least one antimicrobial entity from three or more groups.
The length of duration between the automated system's alert signal and the start of blood incubation and was assessed as time to positive (TTP). In case of the presence of more than one positive sample, we used the shortest TTP. Appropriate antibiotic therapy meant receiving a least one intravenous active antimicrobial treatment based on susceptibility results within 24 hours of collecting blood samples and before susceptibility ndings were available; In any other circumstance, unsuitable antimicrobial therapy was de ned [24].

Detection of virulence genes, capsular serotypes, and T6SS genes
A commercial DNA extraction kit was used to extract DNA from K. pneumoniae isolates (Sangon Biotech, Shanghai, China). From an overnight culture, 1mL of bacterial suspension matched comparable to 0.5 McFarland was made and centrifuged. DNA was extracted from the pellet according to the kit's instructions and then resolved in 100µL TE buffer.
PCR-based detection of biomarker genes employed 12.5 µl of 2× Spark Taq PCR Master Mix (Solely Bio,Shandong China), 1 µl of forward primer, 1 µl of reverse primer (10 pmol/µl primer stock), 1 µl of genomic DNA, and 9.5 µl of water. PCR was performed using a Biometra thermocycler with the cycling conditions mentioned as follows: First step: 94.0°C for 3 min; Second step: 94.0°C for 30 s; Third step: primer-speci c annealing temperature for 30 s; Forth step: 72°C for 1 min; Fifth step: repeating steps 2-4 for 35 cycles; Sixth step: 72.0°C for 10 min; Seventh step: holding at 4°C. 1% agarose gel was used to resolve the ampli ed products. Additional le 1: Table S1 lists all the primers employed.

Bio lm assay
Bio lm formation tests were carried out as described earlier, with a number of tweaks [25]. The OD600 was used to standardize overnight cultures. The suspension was inoculated into the wells of a polystyrene 96-well at-bottom plate using a 200-µL aliquot and left to incubate for 18 hours at 37°C. The medium was then taken from the plates, and each well was thoroughly washed three times with water. After 30 minutes of air-drying, crystal violet solution (1%, 150 µL) was applied to individual wells for 15 minutes. The colorant was discarded after staining the adherent cells, and 200 µL of distilled water was used to rinse the wells three times in order to eliminate any surplus stain. For 0.5 hours, the plates were air-dried. In 200 µL of 100 % ethanol, the dye absorbed by the adhering cells was solubilized. As a negative control, sterile LB was employed. The stained adherent bacteria and control wells had their OD600 values tested. Three separate studies, each with three replicate wells, were carried out. Bio lm production positive was de ned as a strain with an absorbance value greater than 2 times that of the blank absorbance.

Serum resistance assay
The serum resistance analyses were carried out as elaborated earlier, with minimal modi cations [26]. Dilution 1:100 of overnight bacterial cultures was carried out into fresh LB media (10 mL) and the diluted specimens were allowed to incubate until an OD600 of 0.5 was achieved. The culture was then rinsed in phosphate-buffered saline (PBS) for 1 mL followed by resuspension in 1 mL of PBS. Following that, 100 µL of the bacterial suspension were combined with 300 µL of normal human serum (NHS). The suspensions of serum-bacteria were mixed and then set to incubate at 37°C for 3 hours. A 100-µL aliquot of each culture was obtained prior to and following the 3-h period of incubation, diluted serially, and plated for calculating the serum bactericidal effect. The serum bactericidal effect was calculated using the ratio of serum-bacteria suspension CFUs to bacterial suspension CFUs without NHS. All studies were carried out in triplicate, and the ndings are expressed as percent survival.

In vitro competition assay
Strains of the attacker (K. pneumoniae) and prey (E. coli MG1655) were cultured in LB overnight at 37°C. Each culture was then diluted 1:100 in 10 mL of LB broth after being standardized to an OD600 of 0.5. The suspensions of bacteria were mixed in a 2:1 attacker: prey ratio and cultured for 20 hours at 180 rpm and 37°C. The determination of cells count for each strain was carried out via pouring their serial 10-fold dilutions onto LB agar plates in the presence or absence of ampicillin (50 mg/L) [27][28]. An in vitro competitive index was calculated to measure the relative tness of K. pneumoniae isolates against E. coli strain (CI). The CI was calculated as the ratio of ampicillin-resistant K. pneumoniae to ampicillinsusceptible CFUs E. coli. A total of ten competition experiments were carried out.

Statistical analysis
In the case of continuous variables, medians and interquartile ranges (IQR) were used, whereas, for categorical variables, relative and absolute frequencies were employed. The Student's t-test or the Mann-Whitney U test, as well as the 2 test or Fisher's exact test, were used for the comparison of categorical and continuous parameters. The log-rank test was carried out to examine differences between groups, and Kaplan-Meier survival curves were employed for survival analysis. Patients who were not evaluable for crude mortality analysis were those who were discarded for aggressive antimicrobial therapy due to endstage illness. All statistical tests were two-tailed, and statistical signi cance was referred to as a P-value equivalent to 0.05. The SPSS statistical package v.24.0 (SPSS Inc., Chicago, IL, USA) was employed to conduct the analyses, and Prism software v.9.0 (GraphPad Inc., La Jolla, CA, USA) was used to create the visualizations.

Study population and patient characteristics
In the duration of the study, one hundred and thirty inpatients with a positive K. pneumoniae blood culture were included. Eleven of them were disquali ed ( ve cases were newborns, four cases were infected with other bacteria, two cases had incomplete information). As a result, 119 instances were nally examined in this work.

Microbiological characteristics and antimicrobial susceptibility
Apart from natural resistance to ampicillin, K. pneumoniae had the highest rate of resistance towards piperacillin, whereas tigecycline resistance was as low as 0.8%. T6SS-positive K. pneumoniae strains also had lower rates of antimicrobial resistance than T6SS-negative bacteria. With the exception of minocycline, tigecycline, and ceftazidime averbactam, practically all antibiotic drugs had signi cantly greater resistance rates for T6SS negatives than for T6SS positives. 24 (20.2 %) of the 119 K. pneumoniae clinical isolates were found to be ESBL-producing. Seventy-eight strains (65.5%) were found to be multidrug-resistant bacteria (MDR), which were shown to be more prevalent in the T6SS-negative group (71.7% vs 35.0%, P = 0.002). Carbapenem-resistant K. pneumoniae (CRKP) was also found in lower numbers in the T6SS-positive strain (20.0% vs 45.4%, P = 0.03). Table 2 has more information on these ndings.    Table 1 shows that the overall 30-day mortality rate was 32.8 % (39 patients), with 66.7 % (26 patients) dying within the rst seven days of being admitted to the hospital. There were no signi cant variations in early (7-day) mortality according to the T6SS genotype. Late mortality among patients infected with T6SS + isolates, on the other hand, was 10% compared to 37.4% among patients infected with T6SS strains (P = 0.01).

Crude Impact of TTSS Genotype in Mortality
The survival curve (Fig. 1A) revealed no signi cant differences in the 7-day cumulative likelihood of mortality between T6SS-positive and T6SS-negative KP-BSI isolates (log-rank P = .46). Meanwhile, there was no signi cant difference in cumulative survival until day 30 (Fig. 1B) between the KP episodes that were positive or negative for T6SS (log-rank P = .38).

Bio lm formation, competitive index, and serum resistance
T6SS + and T6SS-strains had their relative bio lm-forming activity calculated, and individual isolates were investigated in triplicate. T6SS-positive KP isolates' mean OD600 value was not substantially different from T6SS-negative isolates (mean ± standard deviation [SD], 0.059 ± 0.005 vs. 0.08 ± 0.04; P = 0.303) (Fig. 2A). After 3 h of incubation in serum, T6SS-positive isolates revealed no signi cant changes in survival rate compared to T6SS-negative isolates (40.95% vs. 21.73%; P = 0.35) (Fig. 2B). In vitro competition experiments were also carried out to calculate the CI to see if T6SS + isolates could outcompete other bacteria. The result shown that T6SS-positive isolates were more competitive during joint incubation than T6SS-negative isolates (Fig. 2C).

Discussion
Klebsiella. pneumoniae is a common nosocomial pathogen that is causing widespread concern due to multidrug resistance and the recent emergence of hypervirulent strains in clinical settings. Capsule, Pili, lipopolysaccharide (LPS), and iron carriers are now known to be pathogenic factors [29]. RmpA is a plasmid-based virulence element in K. pneumoniae that regulates capsular polysaccharide production [30]. Enterobactin, yersiniabactin, salmochelin, and aerobactin are the four iron-absorbing molecules (iron carriers) found in K. pneumoniae [31]. Aerobactin is thought to be the primary virulence factor of hvKP since it is involved in iron transport, proliferation, and virulence in K. pneumoniae [32]. String test in combination with aerobactin and the rmpA index improved the detection rate of hvKP, according to Li et al.
In this investigation, strains positive for rmpA and aerobactin were classi ed as hvKP, and the results revealed that 26 (21.8%) of the strains were hvKP. T6SSpositive strains were de ned as those that were positive for icmF, vgrG, and hcp in a prior investigation. Based on these criteria, our research found that T6SS genes were present in 16.8% of K. pneumoniae bloodstream isolates. However, only patients with bloodstream infections were included in this investigation, not individuals with chronic infections who might have been colonized by mature K. pneumoniae colonies. T6SS positive strains had a greater detection rate of K1 capsular serotypes and four virulence genes than T6SS negative bacteria, according to the ndings. The incidence of hvkp was substantially greater in T6SS-positive strains in comparison to the T6SS-negative strains. This research backed up the pathogenicity of the T6SS-positive KP strains.
Antimicrobial sensitivity was found in hypervirulent bacteria in previous research. T6SS-positive K. pneumoniae strains had lower antimicrobial resistance rates than T6SS-negative bacteria in this investigation. With the exception of minocycline, tigecycline, and ceftazidime averbactam, practically all antibiotic drugs had signi cantly greater resistance rates for T6SS negatives than for T6SS positives.
The percentage of multidrug-resistant, carbapenem-resistant, and ESBL-producing K. pneumoniae, on the other hand, was extremely high. Multidrug resistance levels linked with this species have skyrocketed, posing a global threat, in particular for carbapenemase-producing K. pneumoniae. The antibacterial activity of T6SS in K. pneumoniae, on the other hand, is little understood. A study found that -lactam antibiotics, at a sub-inhibitory concentration increased T6SS-dependent killing by stimulating the production and secretion of the CRKP HS11286 T6SS. As a result of the antibiotic stress, CRKP HS1186 outperforms the T6SS/multidrug-resistant strain in terms of growth superiority.
We investigated the risk variables impacting the prognosis of K. pneumoniae BSI in this study, which included a complete clinical and microbiological evaluation. The signi cant death rate associated with this infection is con rmed by our ndings, with 21.8% and 32.8% of patients dying within the rst 7 and more fatal than bacteremia produced by other germs.
T6SS+ isolates had stronger bio lm-forming activity and survived longer when present in normal human serum than T6SS-isolates, according to previous research. However, the ndings of our study on K. pneumoniae isolates contradict this concept. The differences between K. pneumoniae and other bacteria could be due to species-speci c characteristics. It's unclear whether T6SS+ isolates' bio lm-forming ability and serum resistance are related to the T6SS's own function. A number of investigations have shown that the T6SS is involved in bio lm production in a variety of bacteria; however, certain bacteria do not need the T6SS to create bio lms. Type 3 pili and capsular polysaccharides (CPs) are the most critical surface features of K. pneumoniae that take part in the production process [34][35]. Pili mediate long-term adherence, whereas CPs in uence intercellular communication and bio lm structure in the end. The function of bio lm can also be affected by mutations in certain K. pneumoniae genes [36].
In this investigation, we discovered that T6SS+ isolates outperformed T6SS-isolates in terms of outcompeting E. coli. In a vast majority of T6SS-carrying bacteria, the signi cance of the T6SS in intraspecies and interspecies bacterial competition has been recognized as a key characteristic of the system. The T6SS has been shown to direct effector proteins to other bacteria of the likes of P. aeruginosa, V. cholerae, and S. marcescens, allowing the organism to get involved in competition more successfully against other bacteria in its growing environment [37][38][39][40]. Nevertheless, the antibacterial properties of the T6SS in K. pneumoniae have yet to be discovered. The T6SS renders a tness advantage for T6SS+ clinical isolates of K. pneumoniae, according to our ndings.

Conclusion
T6SS-positive K. pneumoniae was common in people who had BSIs. T6SS-positive K. pneumoniae strains apparently looked hypervirulent. In comparison to T6SS-negative isolates, K. pneumoniae isolates with T6SS-positive might outcompete Escherichia coli. T6SS+ isolates, on the other hand, did not show stronger bio lm-forming activity or a higher survival rate in the presence of normal human serum in