Overview of Klebsiella Pneumoniae as a Nosocomial Pathogen and ESBL Producing Strains in Iran

Corresponding Author: Fariba Akrami Department of Microbiology, Faculty of Medicine, Babol University of Medical Sciences, Babol, IR Iran, Email: Fariba4820@gmail.com Abstract: Klebsiella pneumoniae is one of the most important human bacterial pathogens with an extensive range of community and hospital acquired infections that may lead to morbidity and mortality. Evaluating the prevalence and epidemic sources of infections and the pathogenicity mechanism of bacteria can be investigated by various typing methods. The emergence of multi-drug resistant strains and extended-spectrum ß-lactamase (ESBL) producing isolates has already become a great challenge in nosocomial infection incidence. There are several reports on ESBL isolates of K. pneumoniae in Iran. However, our aim is a comprehensive analysis on ESBL isolates K. pneumoniae from different parts of Iran which has not yet been performed.


Introduction
Klebsiella pneumoniae is one of the most abundant species of the Klebsiella genus that causes complications such as urinary tract infections, Ventilator-Associated Pneumonia (VAP), sepsis and endophthalmitis in Asia and America. Emergence of multi-drug resistant strains has already become a great challenge in nosocomial infection incidence (Pokra et al., 2016;Kashani and Eliott, 2013). In 1883, Friedlander, the German microbiologist and pathologist, isolated the encapsulated bacilli from a patient with pneumonia. The bacterium was initially called Friedlander's bacillus but was renamed Klebsiella due to Edwin Klebs. Currently, the Klebsiella genus is classified among the five predominant common gram negative pathogens that could lead to nosocomial infections (Horan et al., 1988). Klebsiella oxytoca, Klebsiella rhinoscleromatis and Klebsiella ozaenae are the main subspecies of K. pneumoniae based on nucleic acid hybridization (Sakazaki et al., 1989). In addition, Klebsiella terrigena, Klebsiella ornithinolytica, Klebsiella planticola and Klebsiella aerogenes are known other species (Izard et al., 1981;Gavini et al., 1986;Iyer et al., 2017). Nowadays, more than 50% of these strains are isolated from wound, respiratory and urinary tract infections (Podschun and Ullmann, 1994). There are several reports on ESBL isolates of K. pneumoniae in Iran. However, a comprehensive analysis of ESBL isolates of K. pneumoniae from different parts of Iran has not yet been performed. The searches were done according to several English and Persian databases including PubMed, Scopus, Isi, Iranmedex and SID to identify studies addressing ESBL isolates of K. pneumoniae in Iran during the past decade.

Genomic Structure
In Holt and colleagues' study, more than 300 isolates of Klebsiella pneumoniae strains were investigated based on whole-genome sequencing method that lead to KpI (K. pneumoniae), KpII (K. quasipneumoniae) and KpIII (K. variicola) as the three main distinct species, of which K. pneumoniae is the most significant one in human infections (Fig. 1). The most important gene clusters are associated with various virulence factors, regulators of mucoid phenotype (rmpA, rmpA2), siderophore systems, the ferric uptake operon kfuABC, the two-component regulator kvgAS and an allantoinase gene cluster. The three chromosomal core genes are classified as LEN βlactamases, SHV and OKP. On the other hand both FosA and oqxAB that associate in resistance to fosfomycin and quinolones have been transferred horizontally from Escherichia coli (Holt et al., 2015;Chen et al., 2014).

Cell Structure, Metabolism and Natural Habitat
The most vital metabolic pathways of K. pneumoniae are recapitulated as glycolysis, tricarboxylic acid, oxidation of fatty acids and creatine phosphate (Dong et al., 2012). Moreover K. pneumoniae is able to produce 2-butanone from glucose in 2-3 butanediol synthesis process (Chen et al., 2015).  (Zheng et al., 2014) K. pneumoniae isolates from different sources such as environmental specimens, wastewater, soil, plants and mammalian mucosa, can also be found in the intestinal tract, nasopharynx and membrane surfaces as a saprophytic pathogen. It should be noted that indiscriminate antibiotic treatment is leading to the emergence of multidrug resistant strains (Tullus et al., 1988).

Microbiology and Epidemiology
K. pneumoniae belonging to the family Enterobacteriaceae is a Gram-negative, non-motile, encapsulated, lactose fermenting, facultative anaerobic, rod-shaped bacterium that can grow in potassium cyanide citrate with no growth at 10°C (Goetz et al., 1995). The main source of clinical infections is gastrointestinal tract infection and also hospital staff's hands, though the most outbreaks are found on neonatal wards (Montgomerie, 1979).

Pathogenic Factors
The most important virulence factors are as follows: • Capsule: K. pneumoniae capsule structure which consists of repeating sugar units (4-6) which completely including uronic acid residues. This polysaccharide capsule has ability for attachment and producing biofilm formation, however, has A 366 B Klebsiella pneumoniae strains 5422, 5,424, 761bp Translation, ribosomal structure and biogenesis (215) RNA processing and modification (1) Transcription (498) Replication, recombination and repair (210) Chromatin structure and dynamics (2) Cell cycle control, cell division (70) Defense mechanisms (172) Signal transduction mechanisms (216) Cell wall/membrane/envelope biogenesis (258) Cell motility (81) Intracellular traffickingrt (110) Posttranslational modification (182) Energy production and conversion (366) Carbohydrate transport and metabolism (679) Amino acid transport and metabolism (747) Nucleotide transport and metabolism (116) Coenzyme transport and metabolism (246) Lipid transport and metabolism (145) Inorganic ion transport and metabolism (525) Secondary metabolites biosynthesis (180) General function prediction only (874) Function unknown ( enough capacity to provide resistance to desiccation, preserves from phagocytosis against polymorphonuclears and granulocytes and also from serum bactericidal effect and activation of the C3b complement (Magill et al., 2014;March et al., 2013). On the other hand, strains with repetitive sequences of mannose-a-2/3-mannose or lrhamnose-a-2/3-l-rhamnose have less pathogenicity than others, until now 78 various capsular serotypes were defined (Hsu et al., 2013) • Fimbriae: K. pneumonia has type 1 and 3 of pili, in which type 1 pili mediates hemagglutination of guinea pig erythrocytes, has the ability to interact with D-mannose residues of glycoprotein receptors on host cells and salivary and genital membrane surfaces (Gupta et al., 2003;Firon et al., 1984), whereas type 3 pili has capacity to mannoseresistant agglutination of human erythrocytes which were treated with tannic acid. This type of pilus consists of MrkA (original) and MrkD (adhesion) subunits that depends on mrkABCDF operon and responsible for biofilm formation, binding to tracheal epithelium, renal and lung tissue cells (Babu et al., 1986;Ares et al., 2017). Ares et al. conducted a study that confirms the essential role of H-NS protein in the regulation of type 3 polysaccharide capsule of K. pneumoniae (Ares et al., 2017) • Outer Membrane Proteins (OMP): These proteins have a critical function in materials transport and pathogenicity. The role of OmpA as a eukaryotic cell adhesion, serum resistance and protects the bacteria against galectin-3 is noticeable (Ares et al., 2016). Moreover, OmpK35 and OmpK36 have been reported as a two main outer membrane porins, which are homologous as OmpF and OmpC. It should be mentioned that both OmpK35 and OmpK36 are related with Extended-spectrum ßlactamase and associated with carbapenem resistance strains in K. pneumoniae respectively (Llobet et al., 2009;Tsai et al., 2011) • Phospholipase activity: Lery et al. study indicates the role of phospholipase (Dpld1) as a new virulence factor in K. pneumoniae (Lery et al., 2014) • Siderophore: these high-affinity iron-chelating compounds which were secreted by many microorganisms are required for bacterial growth, reproduction and spread of infection especially during pneumonia inflammation and bacterial dissemination. This event depends on the activation of the master transcription factor hypoxia inducible factor-1(HIF-1) protein and also inducing cytokine secretion (Holden et al., 2016) Typing Methods Evaluating the prevalence and epidemic sources of infections and the pathogenicity mechanism of bacteria can be investigated by various typing methods, like PFGE, MLST, RAPD, Rep-PCR and etc: • Pulsed-field gel electrophoresis (PFGE): is one of the most common techniques for identifying the epidemiological and nosocomial source infections (Holden et al., 2016;de Souza Lopes et al., 2005)  bacteriocin typing are the most common methods and are used as the best typing for this bacteria (Berrazeg et al., 2013;Slopek et al., 1967;Rennie and Duncan, 1974) Antibiotic Resistance K. pneumoniae is naturally resistant against several antibiotic agents such as penicillin, ampicillin, amoxicillin, oxacillin, carbenicillin due to frequency of ß-lactamase genes (Ørskov and Ørskov, 1984;da Silva et al., 2012;Chambers, 2000). Resistance to ß-lactamase and carbapenem antibiotics is associated through a range of ß-lactamase, such as strains SHV, TEM, CTX-M and carbapenemase respectively (Chaves et al., 2001). Strains which are harboring SHV-1 and TEM-1 may be resistant to piperacillin or first-generation cephalosporin (Grundmann et al., 2010;Girlich et al., 2000;Lemozy et al., 1995). Moreover, ESBL producing strains were reported for the first time in Germany that are responsible for resistance to cephalosporins such as cefotaxime, ceftriaxone and ceftazidime and monobactams (aztreonam) (Nicolas-Chanoine, 1997; Knothe et al., 1983). Due to this issue the prevalence of antibiotic-resistant A. baumannii strains have increased in Iran and this may cause significant clinical problems. In addition, the AmpC gene was also identified in K. pneumoniae strains, albeit in another form called MIR-1, which is 90% similar to Enterobacter cloacae. This gene contains FOX-1, FOX-2, FOX-3, CMY-2, CMY-4, CMY-8, MOX-1, MOX-2, DHA-1, DHA-2, LAT-1, LAT-2 and ACC-1 (Jacoby and Sutton, 1991;Philippon et al., 2002). These strains are resistant to aminopenicillins, carboxypenicillins and ureidopenicillins, while these classes of genes are not well able to hydrolysis with cefepime or a carbapenem. Relevant studies that were performed in different region of Iran are described in Table 1.

Treatment
As shown in Table 1, reported Klebsiella resistance rates in Iran ranged as high as 96%. and as seen in Fig. 2, mean multidrug resistance rates generally increased over time and the last set of isolates collected in Iran were more resistant to all antibiotics (30%). The highest rates of resistance were observed towards β-lactam antibiotics (ceftriaxone, cefotaxime, piperacillin, ceftazidime, cefepime, aztreonam and ampicillin). Also, most of the isolates from all over the country were still sensitive to imipenem, meropenem, tazocin, piperacillintazobactam and amikacin and the imipenem is still a effective drug in Iran.

Prevention and Control
According to conducted studies, identifying the risk factors and mechanisms of drug resistance is related to various enzymes that are produced, including ESBLs, MBLs, KPC and Amp-C belonging to Ambler A, B and C groups. Identification of these resistance factors will lead to the pivotal proper treatment. Direct contact limitation between patients and healthy people, following patients under treatment and compliance with individual health are the critical strategies for controlling the outbreak infections. and Cefpodoxime throat and other samples Maleki et al. (2018) 25.5% blaCTX-M (92%) Cefotaxime 2013 urine Isfahan (Maleki et al., 2018) and blaTEM (76%) and Ceftazidime

Fig. 2: Percentage of ESBL strains over time in Iran
Loss of this porin may be one of the factors contributing to antimicrobial resistance among ESBL-producing K. pneumoniae and may favor the selection of additional mechanisms of resistance. Microbiology laboratories must be able to identify resistant bacteria in a timely suitable manner, especially those that are falsely susceptible in vitro to antibiotics. Bacteriological excellence is needed more than ever (57).

Conclusion
There is a relatively high prevalence of drug resistant K. pneumoniae isolates in Iran. This review showed that the prevalence of ESBL-producing K. pneumoniae varies in different regions of Iran and the capital city of Iran (Tehran,) has a higher incidence of ESBL compared to northern regions and the western cities. Thus, a high degree of awareness among physicians and microbiologists, active infection control committees, appropriate antimicrobial therapy, improvement of hygiene conditions and monitoring of drug resistant isolates are urgently needed in order to better control the emergence and spread of ESBL K. pneumonia isolates in hospital settings.

Ethical Consideration
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely addressed by the authors.