Klebsiella pneumoniae : characteristics of carbapenem resistance and virulence factors *

Klebsiella pneumoniae, known as a major threat to public health, is the most common factor of nosocomial and community acquired infections. In this study, 50 K. pneumoniae clinical specimens isolated from bronchial, urea, blood, catheter, rectal, bile, tracheal and wound cultures were collected. These isolates were identified and carbapenem resistance was determined via an automated system, CHROMagar Orientation and CHROMagar KPC. The carbapenemase gene regions (blaIMP, blaVIM, blaOXA, blaNDM and blaKPC) and presence of virulence factors (magA, k2A, rmpA, wabG, uge, allS, entB, ycfM, kpn, wcaG, fimH, mrkD, iutA, iroN, hly ve cnf-1) of these isolates were determined by using Multiplex-PCR. The OXA-48 carbapenemase gene regions were determined in 33 of 50 K. pneumoniae strains. In addition, NDM-1 resistance in one, OXA-48 and NDM-1 resistance in four unusual K. pneumoniae isolates were detected. Virulence gene regions that were encountered among K. pneumoniae isolates were 88% wabG, 86% uge, 80% ycfM and 72% entB, related with capsule, capsule lipoprotein and external membrane protein, responsible for enterobactin production, respectively. Even though there was no significant difference between resistant and sensitive strains due to the virulence gene regions (P≥0.05), virulence factors in carbapenem resistant isolates were found to be more diverse. This study is important for both, to prevent the spread of carbapenem resistant infections and to plan for developing effective treatments. Moreover, this study is the first detailed study of the carbapenem resistance and virulence factors in K. pneumoniae strains.

The aim of this study was to identify the genotypes of capsules, mucoviscosity, adhesins and other virulence factors of K. pneumoniae strains isolated from clinical specimens and to evaluate the association among potential virulence factors, carbapenem resistance and infection types.

METHODS
Bacterial strains and identification.50 Klebsiella pneumoniae strains, obtained from clinical specimens including bronchial, urea, blood, catheter, rectal, bile, tracheal and wound infections, were collected from six different hospitals, in Ankara, Antalya, Istanbul, Kayseri, between 2010 and 2014, and were included in this study.Isolates were defined with the use of an automatized system (Vitek-32 System, bioMerrieux-France), CHROMagar Orientation (CHROMagar Company, Paris, France) and conventional phenotypic methods (classical biochemical properties, such as Gram staining, hemolysis of blood agar, string test, IMViC tests, lactose fermentation, ornithine decarboxylase and motility tests).
PCR products were analyzed by electrophoresis in a 1.8% agarose gel at 150 V for 2 h in 1 × TBE (89 mM Tris, 89 mM Boric Acid and 2 mM EDTA) containing 0.05 mg/L ethidium bromide and using Gel Logic 200 Molecular Imaging System (Kodak; Rochester).
Data analysis.Clinical data were analyzed using "Minitab v17.1.0"software package for Windows.Fisher's Exact Test were performed.A difference was considered highly significant when p ≤ 0.05.

RESULTS AND DISCUSSION
In this study, we have demonstrated that expressions of carbapenem resistance and presence of virulence genes in K. pneumoniae are weakly correlated in clinical specimens.Despite that, virulence factors in carbapenem resistant isolates were found to be more diverse.

Analysis of the carbapenemase gen regions
OXA-48 was first identified from K.pneumoniae in Turkey (Poirel et al., 2004) and spread of OXA-48 producing K.pneumoniae in the European countries and Mediterranean area has been observed (Nordmann et al., 2011).NDM-1 (New Delhi metallo-β-lactamase), one of the most clinically significant carbapenemase producer, was first reported in New Delhi, India (Yong et al., 2009), followed by several case reports in United Kingdom, Pakistan and now worldwide (Dortet et al., 2012).At the present time, co-producing NDM-1 and OXA group carbapenemases have been reported in Morocco (Abouddihaj et al., 2012), Oman (Dortet et al., 2012), Singapore (Balm et al., 2013) and the United States (Doi et al., 2014).
In this study, two different multiplex PCR reaction mixtures were defined for five resistance genes (blaIMP, blaVIM, blaOXA, blaNDM and blaKPC) and were used to study 50 K.pneumoniae strains.Among these isolates, only oxacillinase (OXA-48) gene was
Co-producing NDM-1 and OXA-48 carbapenemases (one K. pneumoniae strain) in Turkey was reported by Alp et al., (2013).In this study, four K. pneumoniae strains were found to produce both, NDM-1 and OXA-48.It is obvious that this resistance occurrence had increased in the last three years in Turkey.
There was no isolate detected containing the magA, k 2 A, cnf-1, hly and allS genes.The magA (mucoviscosity-associated gene A and specific to K1 capsule serotype), k 2 A (specific to K2 capsule serotype) and allS (associated with allantoin metabolism) genes play a decisive role in the pathogenesis of liver abscess (Fang et al., 2004;Ku et al., 2008).The absence of these genes indicated that there are no liver or abscesses specimens in this study.Study by Chou et al., 2004 andCompain et al. (2014) also supports this situation.Besides this, the presence of cnf-1, hly and allS in Klebsiella are reported to be absent in other studies (Mamlouk et al., 2006;Yu et al., 2008).
Capsule associated genes (wabG, uge and ycfM) promote infection by resistance to phagocytosis (Cortés et al., 2002).These genes were commonly found in K. pneumoniae isolates, they seem to be at the basis of pathogenicity of K. pneumoniae.In this study, virulence gene regions that we encountered among K. pneumoniae isolates were wabG (in 88% of isolates), uge (86%), ycfM (80%) and entB (72%), encoding the capsule, capsule lipoprotein, external membrane protein and enterobactin production, respectively (Fig. 8).These rates are consistent with previous studies reporting that K. pneumoniae clinical strains were producers of virulence factors (El Fertas-Aissani et al., 2013).
According to distribution of virulence genes of K. pneumoniae strains, the most diversity in virulence was seen in urine and tracheal specimens, as shown in Fig. 9.This situation is closely related to the urinary tract infections and pneumonia caused frequently in humans.In addition, it was found that nine different virulence factors were present in rectal swab specimens which were recently isolated from pediatric colonization of patients.
The overall virulence factor productions among carbapenem resistant (n = 34) and carbapenem susceptible (n = 16) K. pneumoniae strains are shown in Table 3.These results indicate that there was no significant difference between resistant and sensitive strains due to the virulence gene regions (P ≥ 0.05).
Forty virulence profiles were defined and when virulence and carbapenemase gene profiles were analyzed, virulence factors in carbapenem-resistant isolates were found to be more diverse, as shown in Table 4. Clinical K. pneumoniae strains express two types of fimbrial adhesins; type 1 and type 3 fimbriae (Podschun and Ullmann, 1998).While type 1 fimbriae, encoding fimH, play an important role in urinary tract infections caused by these strains, type 3 fimbriae, encoding mrkD, promote biofilm devel-  (Struve et al., 2009).Besides it, siderophores encoding entB, iutA and iroN, are iron binding proteins and they also promote biofilm formation (May and Okabe, 2011;El Fertas-Aissani et al., 2013).In this study, total fimbrial adhesins (fimH, mrkD and kpn) were observed in 42 strains (84%) and siderophores (entB, iutA and iroN) were observed in 40 strains (80%) (Table 4).This situation shows that these virulence factors are important for Klebsiella pathogenicity.
It is interesting to note that the carbapenem resistance strain no.91 has only one virulence factor (uge), but carbapenem susceptible strains no.53, 55, 56, 57 and 95 have at least seven virulence factors.In addition, however, 55--57 and 56-78 groups have the same virulence factors, although the carbapenem resistance or clinical source of the strains in the same group are different.This situation shows that there is no correlation among carbapenem resistance, virulence factors and infection types.
It is known that virulence factors and antibiotic resistance are generally considered to play a significant role in bacterial pathogenesis (Beceiro et al., 2013).Many studies have reported that virulence factors are associated with antibiotic resistance in pathogenic bacteria (Arısoy et al., 2008;El Fertas-Aissani et al., 2013), however, the present study indicates that there is no significant correlation among virulence factors, carbapenem resistance and infections types.Recently, a few studies have  indicated that quorum sensing affects these mechanisms (Yang et al., 2012;Wang et al., 2013).Consequently, the study presented here demonstrated that virulence factors, antibiotic resistance and quorum sensing molecules should be considered in a collective manner in further studies on bacterial pathogenesis for developing effective treatments.

Figure 3 .
Figure 3. Determined virulence gene regions in K. pneumoniae strains

Figure 8 .
Figure 8. Distribution of virulence genes of K. pneumoniae strains."n" is the number of isolates that were found to possess a given gene; "%" represents n as the percenatge of the 50 strains studied.