Resistance prevalence profile of Klebsiella pneumoniae in the Intensive Care Units of Al-Shatby

Klebsiella pneumoniae has been identified as an opportunistic pathogen associated with both community-acquired and nosocomial infections mainly among patients admitted to the Intensive care units (ICUs). Some resistant genes were transferred vertically or horizontally within many microbial communities, directly between bacteria through plasmids or integrin. Different techniques including; phenotypic and genetic ones were used to evaluate the presence of β-lactamases among the isolated strains of K. pneumoniae . This bacterial species is the most commonly isolated pathogen (95 isolates) from all the examined samples (51.35%). Current results revealed that 30 and 14 strains of K. pneumoniae are positive to Extended spectrum β-lactamase (ESBL) production, and AmpC β-lactamase producers, respectively. On the other hand, modified carbapenem inactivation and modified Hodge test (MHT) were used to assess the carbapenem resistant strains. It is observed that all the β-lactamase producers’ strains are also carbapenemase producers, whereas only nine strains (30%) are MHT positive. The Polymerase Chain Reaction (PCR) technique revealed that TEM, BETA, NDM and IPM genes are found on the bacterial plasmid (100%). However the presence of the β-lactamase genes on the bacterial DNA varied among the different strains. The presence of the resistance genes on the bacterial plasmid may signify the resistance acquired upon the previous exposure of this bacterium to the different antibiotics. The aims of the current work were to isolate K. pneumonia from Al-Shatby hospital ICUs, to determine the incidence of its β-lactamases, and to decide the frequency of acquisition of 12 different genes among ESBL K. pneumoniae isolates.


Introduction
During the past decades, the emerging problem of ESBL producing bacteria has received a great attention. Doi et al., (2013) reported that the most commonly important ways by which the Gramnegative bacteria can resist β-lactam antibiotics are through the production of enzymes (β-lactamase enzymes) capable of hydrolyzing the β-lactam ring of the antibiotics.
According to Falagas et al., (2014), K. pneumoniae carbapenemase (KPC) enzyme is the most commonly encountered enzyme among these isolates. Lutgring and Limbago, (2016) study revealed that the mechanisms of carbapenem resistance in the family Enterobacteriaceae are multifarious. They consist of βlactamases carbapenem hydrolyzing enzymes construction, and resistance in line for the combination of other factors including ESBLs or AmpC βlactamases hyper-production. The Carbapenemase Producing Carbapenem-Resistant Enterobacteriaceae (CP-CRE) can spread rapidly, and their detection may warrant implementation of more-intensive infection control interventions than would be employed for none-CP-CRE (CDC. 2013). In addition, Tamma et al., (2016) reported that CP-CRE has more hazardous influence than none-CP-CRE. The Gram-negative βlactamases are regulated by several genes such as; blaCTX-M, blaTEM and blaSHV (Monstein et al., 2007).
The Ambler's molecular classification is based on the nucleotide and amino acid sequences of the βlactamases enzymes. Four classes (A, B, C and D) are documented as conferring to Ambler's classification. The classes (A, C and D) have serine deposit, while the class B has cysteine or histidine residue singly or together (Ambler, 1980;Papp-Wallace et al., 2018). This study aimed to evaluate the incidences of the resistant MDR, ESBL, and CRKP genes of the K. pneumoniae isolates recovered from the ICUs of Al-Shatby Pediatric Hospital, Alexandria, Egypt.

Sampling and collection of the bacterial isolates
This prospective cohort study was conducted over 7 months from the 1 st of January to the 31 th of July, 2017, at the Surgical sites ICU, Neonates ICU "NICU", and Pediatric ICU "PICU", of Al-Shatby Pediatric Hospital. The different clinical samples were collected from the mid-stream urine, blood, and from the broncho-alveolar lavage (BAL).
All the bacterial isolates used throughout this work were kindly provided and phenotypically identified by the Surveillance Microbiology Department Strain Bank, at Al-Shatby Pediatric Hospital, Alexandria, Egypt. The strains of Klebsiella ATCC 13883, Klebsiella 1705 (ESBL +ve), Klebsiella 1706 (ESBL -ve), and Escherichia coli ATCC 25922, were kindly provided by the Naval Medical Research Unit (NAMRU) no.3, Cairo, Egypt.

Identification of the bacterial isolates
The morphological features of each bacterial isolate was studied (Noor et al., 2014), and then identified biochemically (Barrow and Feltham, 1993). Additional identification for the selected Klebsiella isolates was carried out using Thermo Fisher Scientific RapIDTM ONE System (REF: R8311006); which was an identification system based on enzyme technology, and Vitek 2 automated system (bioMerieux, Marcy l'Etoile, France), at the Medical Research Center (MRC), Faculty of Medicine, Alexandria University, Alexandria, Egypt.

Evaluation of the prevalence of resistance among the K. pneumoniae strains
The disc-diffusion assay (Bauer et al., 1959) was evaluated using different groups of common antibiotics including; aminopenicillins, 1st generation cephalosporins, aminoglycosides (tobramycin), ureidopenicillins, trimethoprim-sulfamethoxazole, and monobactams, and then incubated at 35± 2˚C for 24 h. Novel Research in Microbiology Journal, 2019

Detection of the Extended-spectrum βlactamase in K. pneumoniae
The Double-disc synergy test (DDST) (Jaspal et al., 2013) and the CLSI confirmatory test [Recommends a phenotypic confirmatory combineddisk test for ESBL production in Enterobacteriaceae. It consists of measuring the growth-inhibitory zones around both cefotaxime (CTX) and ceftazidime (CAZ) disks with or without clavulanate (CA)] (Aggeliki et al., 2014), were used for detection of the ESBL. The DDST was carried out using Cephalosporins discs which were placed in the petri plates next to a centrally placed disc of clavulanic acid (amoxicillin/ clavulanic acid 20/ 10 μg), according to CLSI. (2015). AmpC βlactamases were clinically important cephalosporinases and Cefoxitin (Jacoby, 2009).

Determination of the K. pneumoniae resistance genes using molecular techniques
The DNA of the selected K. pneumoniae isolates was extracted using Thermo Scientific GeneJET Genomic DNA Purification Kit, while the plasmids of these isolates were extracted using GEBRI kit. To detect the presence of β-lactamases genes, normalization of the DNA and the plasmid concentrations were performed to avoid the dissimilarities between both concentrations before use. About twelve primers were designed (Table 1) and the genes used to mark the β-lactamase class A were; TEM, CTX, KPC, Beta and SHV. The β-lactamase Class B (carbapenemase encoding) genes were; VIM, IMP and NDM, whereas the β-lactamase class C encoding gene was FOX. Finally, the β-lactamase class D encoding genes were OXA-10, OXA-24 and OXA-58 (Pérez-Pérez and Hanson, 2002;Mariana et al., 2004).

Sample collection and identification of the bacterial isolates
A total of 1470 samples were collected from 949 ICU hospitalized patients that were distributed as follows: Surgical, PICU and NICU (11.59, 15.91 and 72.49%, respectively) of the patients admitted. A total of 1217 out of 1470 samples are considered sterile, whereas 68 samples are considered as contaminated samples. The most commonly isolated pathogen from all the examined samples is Klebsiella sp. representing 51.35% (95 out of the total 185 isolated pathogens), and is distributed as 5.27, 25.26 and 69.47% in urine, BAL and blood samples, respectively. The Vitek 2 and RapID™ one systems were used for identification of Klebsiella sp., and they confirmed their identification as K. pneumoniae with > 99.9% confidence level. Results in Table (2) revealed that the incidence of Gram-negative bacterial isolates detected in the ICUs' is 69.18%, while it is 18.91% for Gram-positive bacteria. The incidence of the isolated bacterial spp. revealed that Klebsiella sp. is the most commonly isolated pathogen followed by Candida albicans, Coagulase negative Staphylococcus (CONS), and Pseudomonas aeruginosa with; 51.35, 11.89, 7.57 and 6.49%, respectively.

Evaluation of prevalence of resistance among the K. pneumoniae strains
The prevalence of antibiotics resistance among the isolated strains of K. pneumoniaea revealed that they are resistant to aminopenicillins, 1 st generation cephalosporins, aminoglycosides (tobramycin), ureidopenicillins, trimethoprim-sulfamethoxazole, and monobactams. The K. pneumonia strains exhibited high resistance pattern to colistin and meropenem antibiotics (81 and 55.7%, respectively), as clear in Table (3), Fig. (1). These results may represent a high risk and provide very limited options for treatment of bacterial infections. However, it may raise an alert for a surveillance program to monitor the bacterial trends and antibiotics resistance patterns in Egypt.

Detection of the Extended-Spectrum βlactamase in K. pneumonia using (DDST) and CLSI confirmatory assays
In a trail to recognize the ESBL producing K. pneumonia strains, the double disc synergy (DDST) and CLSI examinations were used. Results in Fig. (2 a,  b) demonstrated that 27 and 30 out of the 95 of isolated K. pneumoniae strains are ESBL producers using the DDST, while the CLSI confirmatory assays presenting; 28.4 and 31.5%, respectively.

Screening for the AmpC β-lactamase production
In the current study, 14 out of 30 (46.6%) of ESBL positive K. pneumoniae strains are considered as AmpC β-lactamase producing bacteria as shown in Fig. (2c).

Phenotypic detection of carbapenemases production by K. pneumoniae
In a trial to detect carpabenmase production by the bacteria under investigation, a confirmatory test known as Modified Carbapenem Inactivation Method (mCIM) (Virginia et al., 2017) was evaluated. Results presented in Fig. (3) revealed that all the 30 strains of the tested bacteria are Extended spectrum β-lactamase (ESBL) positive, and are carpabenemase producers. About 9 out of the 30 ESBL K. pneumoniae strains are proved to be carbapenem-resistant Klebsiella pneumoniae (CRKP) positive. The MHT is considered positive, because the E. coli 25922 strain showed a clover leaf-like indentation along with K. pneumoniae growth within the meropenem diffusion inhibition zone.

Screening for the β-lactamase genes in the K. pneumoniae strains using PCR
The PCR technique was used to amplify the genes of the 9 selected K. pneumoniae strains, where strain 1-9 proved to be Multi drug resistant (MDR), ESBL and CRKP, and were compared to the reference strains (ESBL +ve 1705, ESBL -ve 1706, and bacteria with ATCC 13883) (Fig. 4-7). It is interesting to note that the TEM and BETA genes are detected on the plasmid in all the 9 tested strains of K. pneumoniae. However the percentage of their presence on the DNA varied among the K. pneumoniae strains (TEM and BETA,44.44 and 33.33%,respectively). Results revealed that NDM and IPM genes are present (100%) on the plasmid in all the tested K. pneumoniae strains. However their percentages are variable i.e. VIM and NDM are present in 11.11 and 66.66% on the DNA, respectively. The presence of the FOX gene on the DNA and the plasmid of all tested strains are variable (11.11 and 55.55%, respectively). The OXA-10 and OXA-24 genes are variably detected on the plasmid (22.22 and 11.11%, respectively). The current results demonstrated that the ICU's patients harbored most of the carbapenem-resistant strains, with the highest percentage of carbapenemases resistance genes are detected in K. pneumoniae.

Discussion
Recently, Labib et al. (2018) reported that 291 isolates of Gram-negative bacteria out of 1420 patients admitted to the PICU with a mortality rate of 37.1% were detected in 2 PICUs at the Cairo University Hospitals, Egypt. However, Klebsiella sp. (36.0%) was the most frequently isolated microorganism. Similarly, Amer et al. (2017) detected the distribution of carbapenem resistance in a survey study within the different patient's samples (BAL, blood, CVP and skin and soft tissues). The recorded percentage of carbapenem resistance distribution in the K. pneumoniae strains was 39%. Previously, Ejaz, (2013) illustrated that in a total number of 710 K. pneumoniae; only 214 (30.1%) were ESBL positive, while Saied et al., (2011) revealed that ESBL was detected in 79% of the K. pneumonia, and 39% of E. coli using the DDST test.
The TEM-type gene has an average Mw of 1100 bp, while SHV-gene has 868 bp as reported by Polse et al., (2016). However, Trung et al., (2015) reported that KPC gene has an average Mw of 412 bp, while the BETA gene (686) detection was done by using a specific primer designed according to Klebsiella sp. information documented in the GenBank. On the other hand, the VIM-, NDM-and IMP-type genes have an average Mw of 500, 475 and 432 bp; respectively, which were similar to the findings of Shanthi et al., (2014). The FOX-type gene has an average Mw of 190 bp, which was similar to the molecular weight recorded by Sorour et al., (2008). Similar to the current findings, Najar et al., (2013) reported that OXA-24 and OXA-58 genes have average Mw of 564 and 246 bp, respectively.

Conclusion
The current results of incidences of the ESBL and CRKP of K. pneumoniae in the ICU's at Al-Shatby Pediatric Hospital, Alexandria, Egypt, may pave the way for a surveillance program to monitor the resistance patterns of this pathogenic bacterium, thus Novel Research in Microbiology Journal, 2019 provide a clear vision towards the correct treatment options.