Epidemiology and molecular characteristics of the type VI secretion system in Klebsiella pneumoniae isolated from bloodstream infections

Abstract Background The type VI secretion system (T6SS) has been identified as a novel virulence factor. This study aimed to investigate the prevalence of the T6SS genes in Klebsiella pneumoniae‐induced bloodstream infections (BSIs). We also evaluated clinical and molecular characteristics of T6SS‐positive K pneumoniae. Methods A total of 344 non‐repetitive K. pneumoniae bloodstream isolates and relevant clinical data were collected from January 2016 to January 2019. For all isolates, T6SS genes, capsular serotypes, and virulence genes were detected by polymerase chain reaction, and antimicrobial susceptibility was tested by VITEK® 2 Compact. MLST was being conducted for hypervirulent K. pneumoniae (HVKP). Results 69 (20.1%) were identified as T6SS‐positive K. pneumoniae among 344 isolates recovered from patients with BSIs. The rate of K1 capsular serotypes and ten virulence genes in T6SS‐positive strains was higher than T6SS‐negative strains (P = .000). The T6SS‐positive rate was significantly higher than T6SS‐negative rate among HVKP isolates. (P = .000). The T6SS‐positive K. pneumoniae isolates were significantly more susceptible to cefoperazone‐sulbactam, ampicillin‐sulbactam, cefazolin, ceftriaxone, cefotan, aztreonam, ertapenem, amikacin, gentamicin, levofloxacin, and ciprofloxacin (P < 0.05). More strains isolated from the community and liver abscess were T6SS‐positive K. pneumoniae (P < .05). Multivariate regression analysis indicated that community‐acquired BSIs (OR 2.986), the carriage of wcaG (OR 10.579), iucA (OR 2.441), and p‐rmpA (OR 7.438) virulence genes, and biliary diseases (OR 5.361) were independent risk factors for T6SS‐positive K. pneumoniae‐induced BSIs. Conclusion The T6SS‐positive K. pneumoniae was prevalent in individuals with BSIs. T6SS‐positive K. pneumoniae strains seemed to be hypervirulent which revealed the potential pathogenicity of this emerging gene cluster.


| INTRODUC TI ON
Klebsiella pneumoniae is an important pathogen causing bloodstream infections (BSIs). According to the China Antimicrobial Surveillance Network (CHINET), the isolation rate of K. pneumoniae in blood was 15.3%, second only to Escherichia coli. In recent years, scholars have discovered a new type of K. pneumoniae called hypervirulent K. pneumoniae (HVKP). 1,2 Compared with classic K. pneumoniae (CKP), HVKP was characterized by causing severe invasive community-acquired infections with metastatic spread in immunocompetent individuals. 3,4 Usually, hypervirulent strains were resistant to most antimicrobials. 5 However, multidrug-resistant HVKP strains were increasingly reported recently. The emergence of this superbug could cause severe fatal infections in both the hospital and the community. [6][7][8][9] Bacterial secretion systems are ubiquitous; until now, eight types of secretion systems have been described (T1SS, T2SS, T3SS, T4SS, T5SS, T6SS, T7SS, and T9SS). By secreting proteins as virulence factor, bacteria can attack other microorganisms, evade the host immune system, cause tissue damage, and invade host cells. 10 The type VI secretion system (T6SS) is a transmembrane complex which is used to deliver effectors to hosts or target bacteria. The action process is similar to the puncture mechanism used for phage tail contraction. An effector-loaded needle is injected into the target cell. 11,12 As an important virulence factor, T6SS plays a key role in colonization competition and infection of bacteria. Several intestinal pathogens use T6SS to antagonize symbiotic intestinal E.coli promoting colonization and disease progression. 13 T6SS in Campylobacter jejuni has been shown to be important for adhesion and invasion of host cells in vitro. 14 K. pneumoniae T6SS contributes to bacterial competition, cell invasion, type-1 fimbriae expression, and in vivo colonization. 15 T6SS has been identified as a novel virulence factor. There were less reports about the characteristics of BSIs caused by K. pneumoniae expressing T6SS genes. So, the purpose of this study was to investigate the distribution of the T6SS genes and clinical and molecular characteristics in K. pneumoniae-induced BSIs.

| Isolates and Clinical data collection
In this study, a total of 344 non-repetitive K. pneumoniae bloodstream isolates and relevant clinical data were collected from January 2016 to January 2019. Isolates were recovered from samples with positive for blood culture, after separation and cultivation, and then identified by MALDI-TOF MS (Bruker). The distinction between community-acquired and hospital-acquired BSIs was determined by the time of detection of K. pneumoniae in blood cultures.
Within 48 hours after admission was defined as community-acquired BSIs. But over 48 hours into inpatient admission and infections correlated with the presence of medical devices was defined as hospital-acquired BSIs. 5,16 Meanwhile, the following clinical information of the patients was collected from medical records, like age, gender, origin of bacteremia, personal history, underlying disease, and clinical outcomes.

| Detection of T6SS genes, capsular serotypes, and virulence genes
The presence of capsular serotypes and virulence genes was detected by polymerase chain reaction (PCR) as previously described. 17 Intracellular proliferative F family proteins (IcmF), valine-glycine repeat protein (VgrG), and hemolysin-coregulated protein (Hcp) were indicated to be core proteins of the T6SS. 15 To identify the T6SS genes in K. pneumoniae, PCR was performed using primer pairs designed specifically for icmF, vgrG, and hcp in this study. Genomic DNA of K. pneumonia was extracted by boiling method. PCR products were electrophoresed in 1.0% agarose gel, and they were visualized using a Gel Doc ™ XR image analysis station (Bio-Red) to judge whether the gene was positive. Strains positive for p-rmpA and iroB and iucA were designated as HVKP. 18 icmF, vgrG, and hcp are all positive were designated as T6SS-positive in this study. All primers used were listed in Table 1.

| Multilocus sequence typing (MLST) and eBURST
MLST was performed for all HVKP through amplification, sequencing, and analyzing seven housekeeping genes for K. pneumoniae, in-

| Statistical analysis
Categorical variable analysis was used by Chi-square test or Fisher's exact test. Student's t test or the Mann-Whitney U test was used to analyze the measurement data. A P value < .05 was considered statistically significant. The virulence and clinical characteristics were summarized, and the risk factors of T6SS-positive K. pneumoniae-induced BSIs were determined by logistic regression analysis. All variables with P values < .1 were incorporated into a multivariate model using a backward approach. All data analysis was performed by SPSS software (version 25.0). According to data analysis, the prevalence of K1 capsular serotype in T6SS-positive strains was higher than T6SS-negative strains (P = .000).

| Distribution of T6SS genes, capsular serotypes, and virulence genes
But K20 and K54 were not detected in the T6SS-positive strains.

| Antimicrobial resistance of T6SS-positive and T6SS-negative K. pneumoniae bloodstream isolates
Generally, all tested antimicrobial resistance of T6SS-positive K.
pneumoniae was lower than that of T6SS-negative strains. Except for natural resistance to ampicillin, the highest resistance rate was K. pneumoniae to ampicillin-sulbactam. Fortunately, it can be seen from Table 4 that the current resistance rate to tigecycline was low to 1.5%. The T6SS-positive K. pneumoniae isolates were significantly more susceptible to cefoperazone-sulbactam, ampicillin-sulbactam, cefazolin, ceftriaxone, cefotan, aztreonam, ertapenem, amikacin, gentamicin, levofloxacin, and ciprofloxacin (P < .05). Especially, the detection rate of carbapenem-resistant K. pneumoniae (CR-KP) in the T6SS-positive strain was also lower (P < .05). A summary of these results was shown in Table 4.  Currently, T6SS has been identified as a virulence factor, which can inject enzymes, toxins, or other proteins into competing bacteria or host cells, and secrete proteins as virulence factors. 26 As an important core protein of the T6SS, VgrG forms a cell-puncturing tip and Hcp forms a tail-tube structure for transport effector proteins. 27 VgrG was not only a directly interact device, but also a secreted protein of T6SS, which exerted virulent infections. 28,29 When VgrG was separated from the Hcp tube, the secreted proteins of T6SS were also released into the host cell through the Hcp tube. 30 IcmF were the conservatively integrated inner membrane proteins of T6SS and responsible for delivering effector proteins to target cells. 31  Abbreviations: CR-KP, carbapenem-resistant Klebsiella pneumoniae;ESBL+, producing extendedspectrum beta-lactamase.

| D ISCUSS I ON
*A P value < .05 was considered to be statistically significant TA B L E 4 Antimicrobial resistance of T6SS-positive and T6SS-negative K pneumoniae bloodstream isolates T6SS-positive. Based on this standard, our study indicated that the frequency of T6SS genes among K. pneumoniae bloodstream isolates was 20.1%, which was lower than K. pneumoniae isolated from pyogenic liver abscess (PLA) (88.1%) and the intestinal (41.5%). 15 As a key virulence factor, K1 and K2 were most associated with hypervirulent of all capsular serotypes among K. pneumoniae. 32 We used PCR to test six common high-virulence-associated capsular serotypes. K1 was most frequently in K. pneumoniae bloodstream isolates, followed by K2. Analysis revealed that detection rate of K1 in T6SS-positive strains was significantly higher than T6SS-negative strains. In addition, there was a study demonstrated that T6SS genes contributes to the development of meningitis caused by K1 E. coli. 33 Taken together, T6SS-positive K. pneumoniae strain seems to have a strong virulence potential.
The virulence of the T6SS-positive K. pneumoniae strains was further supported by this study that the positive rates of the virulence The antimicrobial resistance rates of T6SS-positive K. pneumoniae strains in this study were lower than T6SS-negetive. But carbapenem-resistant, tigecycline-resistant, and ESBL-producing K.
pneumoniae still existed. As we know, hypervirulent strains were usually sensitive to antimicrobials. Also, HVKP were difficult to obtain or lose resistance-associated plasmids, and capsules can also affect the horizontal spread of resistant genes. 44 However, the studies about ESBLs-producing, carbapenemase-resistant, even NDM-1 HVKP strains have been reported increasingly in recent years. [6][7][8][9]45 Once hypervirulence and high resistance characteristics are combined, it will undoubtedly become a great threat to public health.
HVKP was characterized by causing severe and spreadable community-acquired infections like liver abscess in young healthy people. 46,47 Analysis of clinical characteristics showed that T6SSpositive K. pneumoniae was more easily acquired from the community, which was also a manifestation of hypervirulent. More T6SS-positive K. pneumoniae were isolated from patients with liver abscess. There was a study about K. pneumoniae isolated from PLA claimed that T6SS genes aid interspecies and intraspecies antibacterial competitiveness, mediate in the transcriptional expression of type-1 fimbriae, and promote the occurrence of liver abscesses. 15 Besides, it was worth noting that biliary disease seemed to be related to T6SS-positive K. pneumoniae. However, biliary tract infections were mostly caused by CKP in previous studies. 48 The coordinated regulation of T6SS and the bile efflux transporter ensuring C. jejuni survival during exposure to the upper range of physiological concentrations of deoxycholicacid. 14 The adaptive mechanism may also exist in T6SS-positive K. pneumoniae, and relevant researches are required to corroborate the association between T6SS-positive K.
pneumoniae and biliary disease.
In conclusion, the prevalence of T6SS genes is high among K.
pneumoniae BSIs. T6SS-positive strains exhibit hypervirulent properties and potential pathogenicity. Community-acquired infections, the carriage of wcaG, iucA, and p-rmpA virulence genes, and biliary diseases were independent risk factors, of T6SS-positive K. pneumoniae-induced BSIs. This study introduced the molecular and clinical characteristics of T6SS-positive K. pneumoniae isolated from BSIs which make clinicians aware of the importance in epidemiologic surveillance of this gene cluster. Furthermore, it can also contribute to the in-depth study about virulence mechanism of K. pneumoniae.

ACK N OWLED G M ENTS
We thank all the staff in the Microbiology Department of Xiangya Hospital for their kind help. This work was supported by the National Natural Science Foundation of China (81672066).

CO N FLI C T S O F I NTE R E S T
The author reports no conflicts of interest in this work.

E TH I C A L A PPROVA L
The study was approved by the Ethics Committee of Xiangya Hospital, Central South University. No informed consent was taken because this study was retrospective, and it did not cause additional medical procedure.