Genotyping and Virulence Analysis of Drug Resistant Clinical Klebsiella pneumoniae isolates in egypt

Klebsiella pneumoniae is a highly drug-resistant human pathogen responsible for a variety of serious infections. Integrons, mobile genetic elements capable of integrating antibiotic resistance genes, and the capsule are important virulence factors that increase bacteria resistance to phagocytosis and antimicrobial agents. Molecular typing is an effective tool for identifying the likely etiology of infection. This study aimed to investigate the presence of the rmpA, wcaG, intI1, intI2, and intI3 virulence genes in clinical Klebsiella pneumoniae isolates, and explore their molecular genotypes by using eRiC-PCR. Fifty Klebsiella pneumoniae strains were isolated from various specimens. Antimicrobial resistance was evaluated by using the disc diffusion method. Five genes were amplified by conventional PCR. Genotyping was performed molecularly by using ERIC-PCR. Forty-seven isolates were multi-drug resistant. in all, 18%, 36%, and 98% of the 50 K. pneumoniae isolates were positive for rmpA, wcaG, and intI1 genes, respectively; however, all isolates were negative for intI2 and intI3 genes. Dendogram analysis of the ERIC-PCR results showed 49 distinct patterns, arranged in five clusters. Our study demonstrates high levels of antibiotic resistance and virulence among clinical isolates of K. pneumoniae. Such resistance reflects a growing problem for public health. Further, the presence of integrons increases the horizontal spread of antibiotic resistance and virulence genes among bacterial isolates. The ERIC-PCR technique is an effective method for molecular typing and epidemiological studies of hospital-acquired infections.


Journal of Pure and Applied Microbiology iNtRODuCtiON
Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative rod-shaped highly virulent bacterium, capable of causing serious diseases in humans including, pneumonia, liver abscesses, urinary tract infections, and life-threatening septicemia [1][2][3] . The bacterium is a major cause of nosocomial infections in Egypt 4 . K. pneumoniae currently manifests growing resistance worldwide to several important antimicrobial agents including, beta-lactam antibiotics, fluoroquinolones, and aminoglycosides. This increase in antimicrobial resistance constitutes an arising problem globally 5 . A primary mechanism for acquisition of resistance of K. pneumoniae is through integrons. These mobile genetic elements integrate and express antibiotic resistance genes. Integrons are carried and transferred by plasmids and transposons; facilitate spread by horizontal gene transfer within bacterial populations 6 . Five integron classes were defined based on difference in base sequences of integrase enzyme genes (int). Class 1 is the predominant among the five integron classes and is widely detected in Gram-negative bacteria, including K. pneumoniae. Structurally, class 1 integrons consist of two conserved regions: 3' conserved segment and 5' conserved segment, in addition to internal gene cassettes coding for antimicrobial resistance genes. Class 2 integrons can be detected occasionally in K. pneumoniae; in contrast, class 3 integrons are rarely documented. More than one hundred and thirty different cassettes that confer resistance against important antibiotic classes e.g.: ß-lactams, fluoroquinolones, aminoglycosides, and macrolides have been discovered 7 . The polysaccharide capsule is an important factor for bacterial virulence. The capsule describes bacterial mucoid phenotype and defines resistance to phagocytosis and host defense factors. For example, hypermucoid K. pneumoniae isolates are related to the incidence of invasive syndromes 8 . The plasmid gene rmpA activates transcription of the wzyKpK1 cps locus that in turn enhances capsule synthesis in K. pneumoniae 2 . Moreover, the wcaG virulence gene is gene located in transposable chromosomal region. This gene is responsible for synthesis of K. pneumoniae capsules, and is also helps in the conversion of mannose to fucose that may enhance the bacterial resistance to phagocytosis 9 .
Virulence analysis and molecular typing techniques are robust tools distinguish for identifying the probable source of infection, determining the genetic relationships in nosocomial infection outbreaks, and assisting in management and treatment of MDR K. pneumoniae infections 10 . Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) is a molecular technique that is used to estimate genetic diversity among enterobacteriacae family members. ERIC sequences are 126 bp long, noncoding, and conserved. ERIC sequences occur in variable positions and numbers in bacteria, and the ERIC technique can be used to evaluate genetic differences among bacterial isolates. The use of the ERIC-PCR for investigation of the diversity of bacterial isolates is rapid, sensitive, and consistent 11,12 . This study was aimed to investigate the presence of the rmpA, wcaG, and class 1, 2, and 3 integron virulence genes in clinical K. pneumoniae isolates, and to assess correlations among antibiotic resistance and virulence genes. Moreover, our isolates were molecularly genotyped by using the ERIC-PCR technique.

Sample collection, isolation, and identification
Seventy-five different samples were collected from patients at Damanhour General Hospital, El-Behira, Egypt from February to April 2018. The samples were collected from wound swabs, sputum, blood, endotracheal tube (ETT) aspirates, urine, and bedsore swabs. Samples were cultured on MacConkey agar plates for the detection of K. pneumoniae. Lactose fermenting mucoid colonies were Gram stained and subjected to several biochemical tests, including triple sugar iron agar, indole, methyl red, Voges Proskauer, citrate, oxidase, and catalase 13 . The identification of K. pneumoniae isolates was confirmed to the species level by using the automated vitek 2 system (Bio-Merieux, l'Etoile, France).

DNA extraction
DNA was extracted directly by using the boiling method with some modifications 16 . Briefly, 4_5 pure bacterial colonies of each isolate cultivated on nutrient agar plates were suspended in 500 μl of sterile water in a sterile Eppendorf tube, and heated at 95°C for 10 min. Bacterial suspensions were cooled on ice for 5 min, then centrifuged at 14,000 rpm for 5 min. The supernatants were transferred to sterile Eppendorf tubes and stored at_20°C.
Molecular detection of wcaG, rmpA, and class 1, 2, and 3 integrons genes by conventional PCR in our clinical K. pneumoniae isolates All the DNA extracts were tested for five virulence genes (rmpA, wcaG, intI1, intI2, and intI3) by using a thermal cycler (Veriti, Applied Biosystems, Lincoln Foster City, California, USA) and five pairs of primers (Table 1). PCR amplicons were then resolved on 1.5 % agarose gel stained with ethidium bromide, and visualized via ultraviolet illumination. Cycling conditions were: initial denaturation at 94°C for 5 min; followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 54°C for 1 min, and extension at 72°C for 1 min, and a final extension at 72°C for 7 min 9 . Molecular genotyping of K. pneumoniae isolates by using eRiC-PCR ERIC-PCR typing was performed by using the primer ERIC2 (5-AAGT AAGTGACTGGGGTGAGCG-3) and with cycling conditions of: initial incubation at 94°C for 15 min., followed by 40 cycles at 94°C for 1 min, 37°C for 1 min, and final elongation at 72°C for 8 min. PCR products were resolved on 1.5% agarose gel stained with ethidium bromide, and visualized via ultraviolet illumination 12 . The gel electrophoresis results were analyzed by using the TotalLab Quant Analysis software (Version 1.0., TotalLab Ltd. United Kingdom) following the manufacturer's instructions.

Statistical analysis
Correlations between the antibiotic resistance of the K. pneumoniae isolates, virulence genes, and ERIC genotypes were statistically determined by using Chi square test and Monte Carlo method by using the IPM SPSS software package version 20.0 (Armonk, NY:IBM Corp).

Results
Fifty (66.7%) of 75 collected clinical samples were initially identified as Klebsiella species by their growth appearance on MacConkey agar plates and their morphological and biochemical characteristics. Mucoid lactose fermenting colonies appeared as Gram negative rods upon Gram staining. Biochemically, all isolates were oxidase negative, catalase positive, indole negative, methyl red negative, Voges Proskauer positive, and citrate positive. On triple sugar iron agar slants, all isolates produced acid butt and slant with gas and no H 2 S. These samples were confirmed as K. pneumoniae by using the automated vitek 2 system.
Further, nine (18%) of the 50 K. pneumoniae isolates were positive for the rmpA gene and 18 (36%) for the wcaG. Forty-nine (98%) isolates were positive for the intI1 gene ( Fig. 2 and  3); however, all (100%) K. pneumoniae isolates were negative for intI2 and intI3 genes. Only one isolate (no.47) was negative for all genes. The remaining forty-nine isolates showed four virulence patterns (Table 3). Significant correlations were found between the intI1 gene and resistance to aztreonam and ceftriaxone (p-value ≤ 0.05), and between the rmpA gene and resistance to gentamicin and amikacin (p-value ≤ 0.05). No significant correlations were observed between the wcaG gene and resistance to any antibiotic (p-value < 0.05).
The 49 genotypically positive isolates were genotyped by using ERIC-PCR, followed by analysis with TotalLab Quant Analysis software (Version 1.0., TotalLab Ltd. United Kingdom). This analysis used the Dice method for comparison and the UPGMA method for clustering. Dendogram analysis of K. pneumoniae isolates showed forty-nine distinct patterns, arranged in five clusters. These clusters contained 11, 11, 10, 6, and 11 of the 49 K. pneumoniae isolates, respectively (Fig. 4).
A significant correlation was found between the presence of the wcaG gene and ERIC genotypes (p-value ≤ 0.05), however, no such correlation was found for rmpA or intI1 (p-value < 0.05).

DisCussiON
Klebsiella pneumoniae is a dangerous pathogen that causes serious wound, urinary tract, respiratory tract infections, meningitis, and bacteremia. K. pneumoniae is a common cause of nosocomial infections, and serious outbreaks are reported in hospitals along with increased  morbidity and mortality rates. The uncontrolled use of antibiotics for treating K. pneumoniae infections has resulted in increased bacterial resistance and the emergence of MDR strains 12,17 .
Forty-seven (94%) of our 50 K. pneumoniae isolates were MDR. In contrast, lower percentages (54%, 38%, 81.5%, and 90%) of MDR were reported in other studies [17][18][19] . The high percentage of MDR in our isolates may be due to overuse of antibiotics in Egypt and the incidence of class 1 integrons in 98% of our isolates. These integrons spread antibioticresistant genes by horizontal gene transfer. High resistance percentages ranging from 92% to 100% were exhibited for penicillins and cephalosporins, including ampicillin, ceforuxime, amoxicillin/ clavulanic acid, ceftazidime, ceftriaxone, and cefepime, and lower percentages were reported in other studies 17,19,20 . High level of resistance was also displayed in this study to imipenem, co-trimoxazole, aztreonam, and ciprofloxacin; however, lower percentages were reported by other studies 17,19,20 . Moderate incidence of resistance (54%, 46%, and 40%) was exhibited for amikacin, tetracycline, and chloramphenicol, respectively, in this study. Such incidence is similar to previous reports 19,20 . Alternatively, 28% of our isolates were resistant to gentamycin; however, a higher percentage was reported by Farivar et al. 19 . Finally, all isolates in this study were sensitive to colistin; in contrast, Farivar et al. 19 reported a lower percentage (83%). Overall, the incidence of antibiotic resistance among our isolates was high compared to previous studies.
We found that 18 (36%) out of isolates were positive for the wcaG gene by conventional PCR. Lower percentages (6.5% and 23%) were reported by Zhang et al. in 7,9,22 . Significant correlations were found between the intI1 gene and resistance to aztreonam and ceftriaxone (p-value ≤ 0.05). In contrast, Derakhshan et al. reported a positive association between the intI1 gene and resistance to cefotaxime, ceftriaxone, ceftazidime, amoxicillinclavulanic acid, aztreonam, ciprofloxacin, tobramycin, tetracycline, co-trimoxazole, gentamicin, and cefepime. In our study, significant correlations were also found between presence of the rmpA gene and resistance to gentamicin and amikacin (p-value ≤ 0.05); however, Derakhshan et al. reported a positive association between this gene and resistance to amoxicillin-clavulanic acid, tobramycin, and gentamicin. In contrast, we observed no significant correlations between the wcaG gene and resistance to any antibiotic (p value < 0.05); nevertheless, Derakhshan et al. reported a positive association between the gene and resistance to all antibiotics assessed, except imipenem 9 . Significant correlations confirm that virulence genes rmpA and intI1 in the bacterial genome often co-occur antimicrobial resistance.
The diversity in the ERIC patterns observed in the study may reflect the non-clonal distribution of virulent K. pneumoniae strains. Our data support the results of Wasfi et al. as their Dendrogram analysis of ERIC genotyping revealed 21 distinct patterns among 28 isolates divided into three clusters (A-C) containing 12/28, 9/28, and 7/28 of the isolates, respectively 12 21,26 . Such genetic variation among K. pneumoniae strains was also reported by other studies in Iran, Taiwan, Russia, and Algeria [27][28][29][30] .
In conclusion, our study demonstrates a high incidence of antibiotic resistance and virulence among Klebsiella pneumoniae clinical isolates, reflecting the continuing threat to public health that needs to be followed up continuously. The presence of class 1 integrons increases the risk of spreading antibiotic resistance genes and virulence factors through horizontal gene transfer. Moreover, the ERIC-PCR technique is a