NDM-1-Positive K. pneumoniae at a Teaching Hospital in Southwestern China: Clinical Characteristics, Antimicrobial Resistance, Molecular Characterization, Biofilm Assay, and Virulence

Background The emergence of the NDM-1-positive Klebsiella pneumoniae (K. pneumoniae) strains has led to limited therapeutic options for clinical treatment. Understanding the clinical characteristics, antimicrobial resistance, biofilm assay, and the virulence genes of these isolated strains is of great significance. Methods The polymerase chain reaction (PCR) was used to screen isolated NDM-1-positive K. pneumoniae. The clinical information of the patients was collected from medical records. The NDM-1-positive K. pneumoniae isolates were subjected to antimicrobial susceptibility testing and multilocus sequence typing. Sixty strains of NDM-1-negative K. pneumoniae isolated during the same period were collected as the control group for the virulence analysis. The virulence phenotype of the strains was preliminarily evaluated by the string test and crystal violet semiquantitative biofilm formation experiment. PCR combined with gene sequencing was used to detect common high toxicity capsule genes (K1, K2, K5, K20, K54, and K57) and common virulence-related genes (entB, ybtS, ureA, ycf, WabG, FimH, uge, iutA, KfuB, aerobactin, rmpA, magA, Alls, IrnN, and VatD). Results In the 30 nonduplicated NDM-1-positive K. pneumoniae isolates, 43.33% (13/30) of the patients had a history of a stay in the neonatal intensive care unit (NICU). All of the isolates exhibited multidrug resistance. Nine STs were identified, 77% (10/13) strains from the NICU were ST11. The NDM-1-positive K. pneumoniae string tests were all negative, and 35% (21/60) NDM-1-negative K. pneumoniae were positive. The ratios of NDM-1-positive K. pneumoniae isolates biofilm formation ability according to strong, medium, and weak classification were 67%, 23%, and 10%, respectively. NDM-1-negative K. pneumoniae isolates were 60%, 25%, and 15%, respectively. There was no statistical difference between the two groups (t = 0.61, P=0.2723). The virulence-associated genes with more than 80% of detection rates among the 30 NDM-1-positive K. pneumoniae isolates included entB (100%, 30/30), ybtS (93.33%, 28/30), ureA (90%, 27/30), ycf (83.33%, 25/30), and wabG (90%, 27/30). KfuB and iutA were detected at prevalence of 3.33% and 13.33%. vatD, allS, iroN, aerobactin, and rmpA were not detected. In the NDM-1-negative K. pneumoniae, all other 14 virulence genes except VatD were detected. After statistical analysis, FimH, WabG, ycf, iutA, kfuB, aerobactin, rmpA, and Alls virulence genes, P < 0.005, there was a statistical difference. Conclusion NDM-1-positive K. pneumoniae exhibited multidrug resistance, MLST typing is mainly ST11, there is small clonal dissemination in the NICU in the hospital, and the NDM-1-positive K. pneumoniae virulence genes carrier rate is lower than the NDM-1-negative K. pneumoniae virulence genes carrier rate.


Introduction
As far as humans are concerned, K. pneumoniae is a frequently-isolated bacterial pathogen that colonizes the oropharynx, skin, or gastrointestinal tract. It causes various infections, including bacteraemia, pneumonia, urinary tract infections, suppurative infections, and cholangitis, especially to patients suffering from underlying disease conditions such as diabetes mellitus [1,2].
e New Delhi metallo-β-lactamase (NDM) is a metalloβ-lactamase able to hydrolyze almost all β-lactams [3]. NDM-1 was first identified in a K. pneumoniae strain isolated from a Swedish patient who had been hospitalized in New Delhi, India, in 2008 [4]. NDM-positive strains cause a variety of infections that have been reported to be associated with high mortality rates [5]. NDM-positive strains have been found worldwide, resulting in a significant challenge for clinical management and public health [6]. Twenty-four NDM variants have been identified in more than 60 species of 11 bacterial families, and several variants have shown enhanced carbapenemase activity [7]. Among the 24 NDM variants, NDM-1 has the widest host spectrum identified so far and has been found in a number of species belonging to 11 bacterial families. K. pneumoniae and Escherichia coli are the predominant carriers of blaNDM-1 [8].
Research has shown that K. pneumoniae with the NDM-1 genotype was the primary cause of neonatal carbapenem resistant sepsis in China [9]. Fuursted, et al. have reported that K. pneumoniae carrying NDM-1 have an intrinsic virulence potential [10]. Researchers also confirmed that bacteria can increase drug resistance through a variety of drug resistance mechanisms, while improving its pathogenicity [11].
Virulence factors are important in colonization, invasion, and development of infection, and the virulence factors such as lipopolysaccharides (LPS), capsule, siderophores, and fimbriae in K. pneumoniae have been well characterized to date [12].
Previous studies have focused on the risk factors of blaNDM-1 and the surrounding environment of genes. Our previous studies have shown that NDM-1 can be expressed on chromosomes and plasmids, and NDM-1-positive K. pneumoniae is multidrug resistant and can spread easily [13]. However, little is known about the virulence factor characteristics of NDM-1-positive K. pneumoniae. In this study, the clinical infection characteristics, drug resistance characteristics, and MLST homology typing of NDM-1positive K. pneumoniae were analyzed, and NDM-1-negative K. pneumoniae was used as a control group. e phenotype and virulent genes were compared, and new ideas for the research on infection control, clinical treatment, and the pathogenicity of NDM-1-positive K. pneumoniae were proposed. e isolates were identified as K. pneumoniae strains by using the VITEK-2 System (bioMe'rieux). Also, PCR was performed to detect blaNDM-1 (Forward 5′-GGGCAGTCGCTTC-CAACGGT-3′, Reverse 5′-GTAGTGCTCAGTGTCGG-CAT-3′) [14]. Clinical information was collected, including demographics, underlying medical conditions, clinical presentations, and antimicrobial therapy. is study was approved, and informed consent was acquired from the patients involved in this study.

Antimicrobial Susceptibility Testing.
e antimicrobial susceptibility testing was performed with a VITEK-2 automated microbiology analyzer platform (bioMérieux, Marcy l'Etoile, France) to examine the sensitivity of NDM-1positive K. pneumoniae against common antibiotics. e minimal inhibitory concentration (MIC) of imipenem was further verified by the E-test method according to the guideline recommended by the Clinical and Laboratory Standards Institute (CLSI, 2018), and the MIC of colistin B was further verified by the microdilution broth method according to the guideline recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2018). E. coli ATCC25922 was used as a control strain for the antimicrobial susceptibility testing.

String
Test. K. pneumoniae strains were incubated overnight on blood agar. A single colony was touched with a loop and stretched outward. e length of the viscous string was pulled upward and measured. A positive string test result was defined as a string longer than 5 mm. e string test was repeated three times for each strain, and the final result was determined [15].

Biofilm Formation Assay.
In brief, 10 μl of the 0.5 McFarland bacterial standard and 190 μl of the Luria-Bertani (LB) broth were inoculated into the wells of a 96well microplate, with three wells per strain, and the microplate was incubated at 37°C for 24 h. ereafter, the LB broth was removed, and the bacterial cells were stained with 200 μl of 0.1% crystal violet at room temperature for 15 min and, then, the due was removed. e plate was washed with distilled water and, then, dried. e absorbance was measured with a microplate reader set at 590 nm after adding 200 μl of ethanol for 10 min into the wells. e yield of biofilm formation of the strains was 2 Canadian Journal of Infectious Diseases and Medical Microbiology interpreted as follows: OD > 0.5 as strong-producing, 0.2 ≤ OD ≤ 0.5 as moderate-producing, and OD < 0.2 as weak-producing [16].

Virulence-Associated
Genes. e primer sequences for capsular serotyping and the virulence genes are listed in Supplementary Materials. Capsular serotypes, including K1, K2, K5, K20, K54, and K57, were determined using the methods described previously [17,18]. e fifteen virulenceassociated genes, including aerobactin, iroN, kfuB, rmpA, alls, ybtS, ureA, uge, wabG, ycf, entB, iutA, aerobactin, vatD, magA, and fimH, were determined by PCR using the primers described previously [19]. e categorical variables were listed as percentages and evaluated using the Chi-square test or Fisher's exact test. e continuous data were expressed as mean ± standard deviation (mean ± SD) or median (25th-75th percentile) appropriately and analyzed using Student's t-test or the Mann-Whitney U test. A P value <0.05 was considered statistically significant. All tests were two-tailed.  Table 1. In the 30 nonduplicated NDM-1-positive K. pneumoniae isolates, the patients' median length of hospitalization was 49 days. 43.3% (13/30) of the patients had a history of a stay in the neonatal intensive care unit (NICU). Of these patients, 63.3% (19/30) were males. All, but five patients, received invasive treatment prior to infection with NDM-1-positive K. pneumoniae, including central venous catheters and invasive mechanical ventilators. 70% (21/30) had been treated by carbapenems (imipenem and meropenem).

Antimicrobial Susceptibility Testing.
e antimicrobial resistance rates of the 30 NDM-1-positive K. pneumoniae isolates are shown in Table 2.
ere was no statistical difference between the two groups (t � 0.61, P � 0.2723).

Discussion
K. pneumoniae causes a wide range of infections both in the community and health-care setting leading to increased morbidity and mortality [20]. e patients infected with Canadian Journal of Infectious Diseases and Medical Microbiology NDM-1-positive K. pneumoniae in this study have a large age span, mainly from newborns and elderly patients, suggesting that people with poor immunity and underlying diseases are susceptible to it. Ten of the 13 neonatal patients are premature and low birth weight infants, which may be related to their immune insufficiency, low phagocytic ability of white blood cell, and underdeveloped skin barrier. In addition, the 25 of 30 patients underwent invasive examinations, indicating that device intervention may weaken the patient's immunity. e specimens in this study were mainly derived from the sputum, suggesting that NDM-1-positive K. pneumoniae is more likely to spread through the respiratory tract. e sputum specimens are likely to cause environmental pollution and the spread of contact, so they should to be treated with strict disinfection measures to prevent its spread in the hospital. e 30 patients had injected antibiotics before we separated the NDM-1-positive K. pneumoniae strains from them. Antibiotics are mainly cephalosporins antibiotics and penicillium carbon alkene. An in vitro susceptibility test showed that it was highly resistant to β-lactam drugs and β-lactamase inhibitor, but sensitive to aminoglycoside and fluoroquinolone, which is consisted with the literature that aminoglycoside is still recognized as a first-line therapy for treatment of K. pneumoniae infection [21]. In theory, strains carrying blaNDM-1 should be sensitive to aztreonam, but drug susceptibility results showed that treatment with aztreonam alone had a poor curative effect. Except for polymyxin B and tigecycline, it was highly resistant to a majority of clinical antibiotics. It is because that blaNDM-1 is mainly disseminated by plasmid IncA/C, which always carries a variety of resistance genes resulting in the emergence of antibiotics resistance [5]. Although aztreonam is stable against MBLs, NDM-1-positive K. pneumoniae strains usually have ESBLs or AmpC enzymes that are able to hydrolyze aztreonam. Aztreonam alone, therefore, has limited clinical utility against NDM-producing strains. In this study, all strains are sensitive to tigecycline and polymyxin B, which is consistent with Darey's research showing that polymyxin alone can treat infections caused by NDM-1positive bacteria [22]. e use of colistin has also been hampered by the neurological adverse effects and occurrence of renal toxicity [23,24]. ere is evidence that polymyxinbased combinations may be more effective than polymyxin  alone, so it is usually recommended to combine polymyxin with other antibiotics [25]. e 30 NDM-1-positive K. pneumoniae strains in this study were typed by MLST, and there were 9 types in total, which mainly is ST11. In China, most of the NDM-1-positive strains have been found belonging to different ST types and being scattered. ST11, ST14, ST15, and ST147 strains are relatively common NDM-positive K. pneumoniae lineages and have been found in multiple countries across several continents, almost all of which were isolated from humans [7]. In medical institutions and wards, if more than 3 cases of homogenous nosocomial infections occur in a short time, it is called nosocomial infection outbreak. In terms of time, the NO.10 strain was the first screened strain in the NICU ward. e patient was admitted to the hospital in January 24, 2018, and stayed for 20 days, which is overlapped with the 11 th , 12 th , and 13 th strains at the same time. Afterwards, the 14 th and 16 th strains were overlapped with the 12 th strain again. ese 6 strains were genotyped by MLST, which shows 5 strains of them are ST11. Considering that the NDM-1 is located in the plasmid and easy to spread, it suggests that there is a clonal spread of ST11 NDM-1-positive K. pneumoniae strains in the NICU.
At present, most studies define the positive strain in the string test as hvKP. In this experiment, NDM-1-positive K. pneumoniae string tests were all negative, while the positive rate of the string test in the NDM-1-negative group was as high as 53.3%. It preliminarily indicates that NDM-1-  negative K. pneumoniae may be more virulent. However, some scholars believe that the string test cannot be used as a criterion for judging whether the K. pneumoniae has high virulence. Biofilm can enhance the defense ability of bacteria and resist the killing effect of antibiotics. e results of this study showed that all strains formed biofilms at varying degrees. Although NDM-1-positive K. pneumoniae does not show enhanced biofilm forming ability, the formation of biofilm will prolong the disease, so we still need to be vigilant.
Capsular polysaccharide (CPS), as one of the most important virulence factors of K. pneumoniae, can resist the phagocytosis of macrophages and neutrophils. Among them, K1, K2, K5, K20, K54, and K57 are recognized as highly virulent serotype [12]. In this test, NDM-1-positive K. pneumoniae did not detect the abovementioned 6 capsular serotypes.
is indicates that when the bacteria acquires drug resistance, the gene expression ability of common high-virulence capsule serotypes will be weakened, and it is also possible that resistant bacteria express other capsule serotypes that have not been detected. e 4 of 6 types of NDM-1-negative K. pneumoniae high-virulence capsular serotypes were detected, mainly K1 and K2. In this experiment, magA and rmpA were not detected in the NDM-1-positive K. pneumoniae group. 15% of NDM-1-negative K. pneumoniae carry rmpA, and 12% carry magA, which is basically consistent with the drawing experiment and the detection rate of high-virulence capsule serotypes. e positive rate in the NDM-1-negative group is higher than that in the control group. ere are many virulence factors related to pathogenicity in K. pneumoniae. e basis of them is mainly the capsular polysaccharide and iron uptake system. In addition, it also includes high mucus, lipopolysaccharide, and fimbriae-related (type I fimbriae and type III Pili), and biofilm formation. e 30 strains of NDM-1positive K. pneumoniae are most widely distributed in entB, ureA, ybtS, FimH, uge, and ycf. ese virulence genes are related to bacterial adhesion, iron uptake, and anti-phagocytosis. Although the positive rate of NDM-1-positive K. pneumoniae in the drawing experiment and common capsular serotypes is zero, it still expresses many important virulence genes.
Once K. pneumoniae inserts the virulence gene into the drug-resistant plasmid, it will make it a highly resistant, highly virulent, and easily spread strain. Some literatures have reported that highly virulent and resistant strains have begun to appear all over the world. In 2016, a case of blaNDM-1-positive ST231 CR-hvKP was reported in India. Also, a case of blaNDM-1-positive ST23 type CR-hvKP was reported in Europe in 2017 [26]. ere were also several cases of CR-hvKP carrying blaNDM-1 reported in China [27,28].
In summary, compared with the control group, the positive rate of NDM-1-positive K. pneumoniae in the string test and common high-virulence capsular serotypes, as well as the distribution of these virulence genes, are significantly reduced, which is consistent with Montanari's claim that bacteria lose some virulence genes in order to obtain resistance genes for optimal adaptability [29]. It indicates that although NDM-1-positive K. pneumoniae has increased drug resistance, its pathogenicity may be weaker. However, the NDM-1-positive K. pneumoniae still expresses many important virulence factors.

Data Availability
All data that were used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.