Detection of several carbapenems resistant and virulence genes in classical and hyper-virulent strains of Klebsiella pneumoniae isolated from hospitalized neonates and adults in Khartoum

Objective Klebsiella pneumoniae (K. pneumoniae) involves both community-acquired and nosocomial infections. It is responsible for a wide variety of infections, including infections of the urinary tract, pneumonia, bacteremia, meningitis, wound infection and purulent abscesses. We constructed this study to detect several carbapenems resistant and virulence genes in classical and hyper-virulent strains of K. pneumoniae isolated from hospitalized neonates and adults in Khartoum state. Results Seventy percent of the isolates were resistant to ceftazidime, 18(30%) to ciprofloxacin, 23(38.3%) to chloramphenicol, 24(40%) to gentamicin and 8% to imipenem, 35% were multidrug-resistant, and 7% extensively drug-resistant, all blood isolates (n = 14) were resistant to ceftazidime. entB was the most predominant virulence gene (93.3%), followed by mrkD (78.3%), kfu (60%), K2 (51.7%), magA (18.3%) and rmpA (5%). blaOXA-48 was the most predominant carbapenem-resistant gene (68.3%), followed by blaNDM (10%), blaKPC (8.3%), and blaIMP (3.3%). Eight hyper-virulent strains were positive for blaOXA-48 and two for blaNDM genes.


Introduction
Klebsiella pneumoniae (K. pneumoniae) is a non-motile, capsulated gram-negative rod about 1-2 µm long, and is a facultative anaerobe [1]. It is a common cause of urinary tract, soft-tissue, and central nervous system infections, in addition to endocarditis, and cases of severe bronchopneumonia, sometimes with chronic destructive lesions and multiple abscess formation in the lungs. In many cases, localized infections lead to bacteremia [1].
There are two types of K. pneumoniae strains "classic" (cKp), usually non-virulent, and drug-resistant gene producer and usually associated with hospital infections, while the other type is a hypervirulent (hvKp) drug-sensitive strain [2]. K. pneumoniae possesses different virulence and antimicrobial resistance genes associated with various clinical conditions [3].
Carbapenem-resistant hypervirulent strains of K. pneumonia are one of the most important organisms that cause fatal nosocomial infections [6]. Recently, increasing reports of resistance to carbapenem in healthcare-associated with K. pneumonia infections have been documented in Sudan [7][8][9]. The mortality rate of carbapenem-resistant K. pneumoniae bacteremia could reach 50% of cases [10].
However, to date, there are no published data in Sudan about the distribution and epidemiology of various types of Carbapenemases and virulence genes on hvKp and cKp strains circulating in Khartoum hospitals. This information is of great importance to understand their local epidemiology and to establish eradication and prevention procedures. Thus, this study was conducted to detect and to characterize the common virulence and carbapenem-resistant genes of hvKp and cKp strains isolated from hospitalized patients in different hospitals in Khartoum state.

Methods
A total of 60 isolates of Klebsiella pneumoniae were obtained from hospitalized patients (45 adults, and 15 neonates) in different hospitals of Khartoum State, during the period from January 2017 to March 2017. These isolates were collected and identified at hospitals as a part of their routine clinical procedure.

Bacterial identification
The isolates were re-identified by gram stain, standard biochemical methods (urease test, indole test, and carbohydrates fermentation test, motility test, and citrate utilization test) [11,12], and by K. pneumoniae speciesspecific primers (Table 1) targeting the 16S rRNA gene. Antibiotic susceptibility testing was done by the Kirby Bauer disc diffusion method on Mueller-Hinton agar, the following commonly used antibiotics for the treatment of K. pneumonia infection in Sudan were selected; ciprofloxacin (5 mcg), gentamicin (10 mcg), ceftazidime (30 mcg), imipenem (10 mcg), and chloramphenicol (30) (HiMedia Laboratories Pvt. Ltd. Mumbai, India), the results of sensitivity tests were interpreted according to Clinical And Laboratory Standards Institute (CLSI) guidelines [13]. E. coli ATCC 25922 and K. pneumonia (ATCC 700603) were used as quality control strains. Capsule stain was used to detect capsule [14]. String test was used to differentiate between hvKp and cKp strains: if the grown colonies of K. pneumoniae form a string > 5 mm in length using a sterile loop, this demonstrates the hypermucoviscosity phenotype [15].

DNA extraction and detection of virulent and resistant genes
DNA was extracted using the guanidine chloride method [16]. The DNA samples were stored at − 80 °C until used for PCR.
A primer sets targeting virulence, and carbapenemresistant genes of K. pneumoniae are shown in Table 1. The primers were dissolved according to manufacturer guidelines to prepare 10 pmol/μl in all PCR reactions.

PCR conditions
PCR was carried out in a 20 μl volume using the Maxime PCR PreMix kit (iNtRON Biotechnology, Seongnam, Korea), 1 μl of each forward and reverse primer (10 pmol/ μL), 2 μl of DNA, and then the volume was completed to 20 μl by distilled water. Four multiplex and single reaction PCR protocols were used for amplification of 16S rRNA, resistant and virulence genes, the initial melting temperature for all was 95 °C for 5 min, and a final extension was at 72 °C for 10 min. Details of annealing temperatures are listed in Table 1.

Statistical analysis
Data of research was analyzed using SPSS. Frequencies and Chi square test was used for comparison of different correlations and associations between variables (p value ≤ 0.05).
There was no significant statistical association between the presence of virulence genes and carbapenems resistant genes except between entB and NDM (p-value = 0.005) ( Table 3). A total of 92% (43/47) of mrkD gene-positive isolates were positive for one or more carbapenem-resistant genes. There was a strongly significant association between the presence of mrkD and entb genes (p-value = 0.0005), they were co-existed in 46 isolates.

Discussion
In this study, eight hyper-virulent strains of K. pneumoniae were reported positive for carbapenems resistant genes (OXA-48 and NDM). The presence of these strains in the clinical setting will complicate clinical practice and will cause fatal nosocomial infections [6]. Although antimicrobial-resistant hvKP strains are rarely reported worldwide [17][18][19], here in Sudan, they appear to be more prevalent.
Eight neonatal blood isolates were multidrug-resistant, and three of them were extensively resistant to all antibiotics that were used. Consequently, the emergence of MDR pathogens would increase the mortality and morbidity and prolong hospitalization and cost of treatment [20].
All neonatal blood isolates (12) were resistant to ceftazidime. Ceftazidime-resistant Klebsiella pneumoniae (CRKP) in the pediatric oncology units of some Sudanese hospitals may be the cause of recent reports of high mortality rate associated with K. pneumoniae infections among this group in different Sudanese hospitals [21]. According to Schiappa [22], high resistance rates to ceftazidime could be due to the presence of a predominant enzyme (TEM-10) responsible for ceftazidime resistance in bloodstream isolates.
The isolates showed varying degrees of resistance to the other antibiotics; ciprofloxacin 30%, gentamicin 40%, and ceftazidime (70%). Resistance to these antibiotics may also be due to the presence of Extended-Spectrum Beta-lactamases (ESBLs) and other mechanisms like efflux pumps and porin mutations [23], which were not covered in this study. Although chloramphenicol is used as a treatment of choice for MDR gram-negative bacilli bacteria [24], 38% of our isolates were resistant to it, which may be caused by transferable enzymatic resistance to aminoglycosides, that is common in some hospitals [25].
In the current study, 94% (51/54) of the isolates harboring carbapenem-resistant genes were phenotypically susceptible to imipenem. This confirms what Walsh [26] said that this gene is not stable and relies upon other synergistic mechanisms to mediate resistance against carbapenems. In addition to imipenem, other antibiotics were analyzed in this study. Although five strains of K. pneumoniae in this study were resistant to imipenem, only three of them were positive for carbapenem-resistant genes (OXA-48), the rest two strains may possess other carbapenem-resistant genes not covered in this study or possessed another mechanism of resistance [27].
Of 46 K. pneumoniae isolates detected of having carbapenem-resistant genes, 10 had multiple genes cooccurring. This finding agrees with Ali & Omer [28] and Satir [29], which showed a multiplicity of genes in their isolates.
A total of 80% (4/5) of KPC and 100% (2/2) of IMP genes were positive among infant blood samples, and this may be due to organisms harboring these genes having a high ability to cause systemic infections, particularly in immunocompromised patients [30].
In this study, we found the essential gene for K. pneumoniae siderophores system entB gene is positive in 93.3% of all K. pneumoniae isolates, the rest (6.7%) of isolates that do not possess entB may contain other enterobactin (entA, C, D, E or F), or other siderophores systems like yersiniabactin or aerobactin as reported by Lawlor [20]. Furthermore, mrkD gene is presented in 78.3% of the isolate. This gene has been found to be important in adhesion, as reported by Chen et al. (2012) [30]. The rmpA gene was detected in 5% of isolates, in contrast with Aljanaby and Alhasani [20], who found the rmpA gene present in 62.5% of K. pneumoniae isolates. This difference may be attributed to its mode of inheritance as plasmid-mediated, as mentioned by [20], indicating the limited spread of this gene in our local strains in Sudan.
The capsular serotype gene K2 was present in 51.7% of isolates; the rest of isolates may contain other capsular serotypes, as mentioned by Ho [31]. This study showed that K2 is present in 80% of hypermucoviscous strains, indicating that there is a relationship between the presence of K2 gene and hypermucoviscous strains of K. pneumoniae, which is in agreement with the study by Guo [32] which found that K2 is the most common capsular serotype in hypermucoviscous strain. In contrast to other studies [20,[33][34][35], which found K1 was the most prevalent capsular serotype among hypermucoviscous K. pneumoniae.
The kfu gene (which codes for an iron uptake system) was present in 60% of isolates. The study showed no association between the presence of kfu gene and hypermucoviscosity. This finding disagrees with previous studies [20,36,37], which showed that kfu gene is associated with hypermucoviscosity phenotype, which may be attributed to diversity in geographical locations of studies.
The magA gene was found in 13.3% of isolates. The study showed no association between the presence of magA gene and hypermucoviscous strains. Although this gene is highly essential for K. pneumoniae, which confirms bacterial mucoviscosity, its prevalence among local isolates is not high, suggesting that other genes play a role in the formation of mucoviscosity [20].

Conclusion
The study reported for the first time in Sudan the following findings: 1. Presence of carbapenems resistant genes in hypervirulent strains of K. pneumoniae isolated from hospitalized patients. 2. Presence of MDR and XDR strains of K. pneumoniae in neonatal ward in some Sudanese hospitals.

Limitations
• Low sample size.
• DNA sequencing not done due to financial issues.