Identification of extended-spectrum-beta-lactamases ( ESBLs ) , metallo-beta-lactamases ( MBLs ) , Amp-C and KPC ß-lactamases among Klebsiella pneumoniae isolated from adults and pediatric patients in Iran

Klebsiella pneumoniae is one of the major bacteria that cause acute infections. β-lactamases are main defensive mechanisms in bacteria against drugs. The aim of this study was to determine antibiotic resistance patterns and detection of extended-spectrum-beta-lactamase (ESBL), MBL, Amp-C and KPC β-lactamases among K. pneumoniae strains isolated from adults and infants. This descriptive study was done on 83 K. pneumoniae isolated from two hospitals. Antibiotic susceptibility tests were performed by Kirby-Bauer disc diffusion and broth microdilution methods according to Clinical Laboratory Standards Institute (CLSI) guidelines. ESBLs, MBL, Amp-C and KPC producing strains were detected by phenotypic confirmatory test, combination disk diffusion test (CDDT), Amp-C detection kit and Modified Hodge test, respectively. From 83 K. pneumoniae strains, 48 (57.5%), 3 (3.5%), 23 (28%) and 5 (6%) were ESBL, MBL, Amp-C and KPC positive, respectively. In this study, Fosfomycin and Tigecycline were more active than other antibiotics. The results demonstrated that incidence of ESBLs, Amp-C, MBLs and KPC was critical especially in infants. Therefore, detection of drug resistance patterns in K. pneumoniae and β-lactamases producing isolates is important for the prevention and control of these infections.


INTRODUCTION
Klebsiella pneumoniae is an opportunistic pathogen and a common cause of nosocomial infections such as pyogenic liver abscesses, meningitis, endophthalmitis, pneumonia, septicemias, urinary tract infections and soft tissue infections.Suitable options have been restricted for the treatment of infections with increase of multi-drug resistant (MDR) bacteria especially, K. pneumoniae nosocomial isolates (García-Sureda et al., 2011).It has been a very big problem treating infants infections due to MDR organisms (Roy et al., 2011).In some countries, many infant deaths occur as a result of MDR bacteria.Enzymes are major defensive mechanisms in some bac-teria against drugs (Drawz et al., 2010).The most clinically important enzymes are KPC-enzyme (Ambler class A), MBL enzymes (class B), Amp-C (class C) and ESBLs (class A), identified in K. pneumoniae as a source of nosocomial outbreaks in hospitals (Poirel et al., 2011;Thomson et al., 2010).ESBLs are currently reported as a major problem of drug resistance in K. pneumoniae (Ruiz de Alegría et al., 2011).ESBLs are enzymes that hydrolyze cephalosporins, penicillins and aztreonam and encoded by mobile elements such as plasmids.ESBL producing bacteria are usually resistant to drugs.Since ESBL genes are transmissible to other bacteria, it is necessary that ESBLs be tested for other bacteria in hospital and long-term care facility patient groups where ESBLs are encountered.Bacteria including both ESBL and AmpC enzymes are becoming more common (Thomson et al., 2010).
AmpC β-lactamases constitute a group of enzymes widely spread in Klebsiella pneumoniae.They inactivate cephalosporins such as ceftazidime, cefotaxime and cefpodoxime.Plasmid-mediated AmpC enzymes, which originated from chromosomal AmpC of variety organisms, have been reported since the 1980s (Mammeri et al., 2010).Carbapenem-hydrolyzing enzymes belonging to Ambler classes A, B, and D have been reported in the world among Klebsiella pneumoniae.Most of those bacteria are MDR.K. pneumoniae possessing the KPC β-lactamases is now spreading in some medical centers in USA and is being increasingly reported worldwide (Landman et al., 2009).KPC-producing isolates of K. pneumoniae and other bacteria have spread rapidly in the world especially in New York.Other reports have been described in other areas, including China, Greece, Demark, Argentina, Hungary, Brazil, Colombia, Finland, France, Italy, Germany, Sweden, Puerto Rico, Norway and the United Kingdom (Chen et al., 2011a).These strains typically possess an underlying ESBLs or Amp-C enzymes (Landman et al., 2009), which are a molecular class A serine enzyme belonging to functional group 2f, that can hydrolyze carbapenems, penicillins, cephalosporins and aztreonam.These enzymes can be inhibited by clavulanic acid and tazobactam (Fontana et al., 2010).The metallo-β-lactamases, which have been reported in the 1960s, were detected in some countries.In the 1990s, with the spread of enzymes encoding metallo-βlactamases located on mobile elements such as plasmids, these β-lactamases increased in K. pneumoniae such as New Delhi metallo-beta-lactamase (NDM), Imipenemase (IMP), Australia imipenemase (AIM), Seoul imipenemase (SIM), Verona integron-encoded-metallo-βlactamase (VIM), German imipenemase (GIM), Dutch imipenemase (DIM) and São Paolo metallo-β-lactamase (SPM) types (Cornaglia et al., 2011).
Recently, NDM-1 emerged as a global threat because the bacteria which possess this metallo-β-lactamase are resistant to almost all β-lactam antibiotics, aminoglycosides, fluoroquinolones and other classes of drug agents NDM-1 gene is located on plasmid or chromosome.The rapid emergence of NDM-1 has been related to transmissible plasmids which can transfer among dif-ferent isolates; subsequently, NDM-1 can spread throughout the world (Kus et al., 2011;Diene et al., 2011;Liang et al., 2011;Ong et al., 2011).So far, in Iranian studies on different bacteria, the emphasis has been on identification of Ambler class A and class D and also three reports on Ambler class B beta-lactamases (Fallah et al., 2011).The aim of this study was to determine antibiotic resistant patterns and detection of beta-lactamase types in K. pneumoniae isolated from hospitallized patients in Mofid Children and Taleghani hospitals, Tehran, Iran during 2012 year.

Minimum inhibitory concentration (MIC)
Antimicrobial drug susceptibilities were determined according to the guidelines of the CLSI by broth microdilution.Muller-Hinton broth (Merck, Germany) with distinct dose of imipenem, meropenem, Cefepime, Ampicillin, Piperacillin/Tazobactam, Cefotaxime, Ceftriaxone and Ceftazidime (GLAXO England Co and Himedia) was prepared.After shaking, 0.1 ml of diluted drug was added to each well of 96-well microtiter plates .Microbial suspensions were adjusted to 0.5 MacFarland and diluted in 1:10 with broth to yield 10 7 CFU/ml and to each well, 0.005 ml of the bacterial inoculums was seeded.Control line with no bacterial inoculation and Escherichia coli ATCC25922 was simultaneously maintained.Microplates were incubated at 37°C for 18 to 24 h.The lowest concentration of the drugs that produced no visible bacterial growth was reported as the MIC.

Phenotypic detection of MBL
Combination disk diffusion test (CDDT) was performed for identification of MBLs by imipenem, meropenem, doripenem and ertapenem (Mast Group, Merseyside, UK) alone and in combination with EDTA.
The inhibition zones of the Imipenem and Imipenem + EDTA, Meropenem and Meropenem + EDTA, Doripenem + EDTA, Ertapenem and Ertapenem + EDTA discs were compared after 18 h of incubation in air at 37°C.A zone diameter difference between the discs and discs + EDTA ≥7 mm was interpreted as a positive result for MBL production (Galani et al., 2008).

Phenotypic detection of ESBL
Detection of ESBLs was tested for all the isolates by Combination Disk Diffusion Test (CDDT) containing Ceftazidime (CAZ) and Cefotaxime (CTX) with CAZ 30 µg + CA 10 µg and CTX 30 µg + CA 10 µg per disc (Mast Group, Merseyside, UK).The zone of inhibitions was compared for the CTX, CAZ discs with that of the CAZ 30 µg + clavulanic (CA) 10 µg and CTX 30 µg + CA 10 disc.An increase in zone diameter of ≥5mm in the presence of clavulanic acid indicated the presence of ESBL in the test organism.E. coli ATCC 25922 and K. pneumoniae ATCC700603 were used as negative and positive controls for ESBL production, respectively.

Phenotypic detection of KPC
Identification of KPC enzyme was performed for all the K. pneumoniae by Modified Hodge Test.0.5 McFarland dilution containing E. coli ATCC 25922 was prepared in 5 ml of broth and then diluted in 1:10 by adding 0.5 ml of the 0.5 McFarland to 4.5 ml of Mueller Hinton broth (Merck, Germany).A lawn of the 1:10 dilution of E. coli ATCC 25922 was streaked to a Mueller Hinton agar (Merck, Germany) plate and allow to dry for 4 to 5 min.10 µg Ertapenem susceptibility disc (Mast Group, Merseyside, UK) was placed in the center of the test area.
In a line, test bacteria were streaked from the edge of the disc to the edge of the plate; and incubated for 18 to 24h at 37°C.After 18 to 24 h of incubation, they were examined for a clover leaf-type indentation at the intersection of the test bacteria and E. coli ATCC25922, within the zone of inhibition of the Ertapenem susceptibility disc.

Phenotypic detection of Amp-C
The detection kit for Amp-C was developed by mast group containing 3 discs-labeled A, B and C. 1: Disc A contains Cefpodoxime and an Amp-C enzyme inducer.2: Disc B contains Cefpodoxime, an Amp-C enzyme inducer and an ESBL enzyme inhibitor.3: Disc C contains Cefpodoxime, an Amp-C enzyme inducer, an ESBL enzyme inhibitor, and an Amp-C enzyme inhibitors.ESBL enzyme inhibitor is included the disc set (discs B and C) for identification of the existence of Amp-C enzyme when the bacteria are also co-producing ESBL enzyme.Bacteria were tested employing on plates of Mueller Hinton agar (Merck, Germany).
The discs were located on the inoculated agar surface and incubated at 37°C for 18 to 24 h, and after incubation the zones of inhibition were measured; zones with a ≥5mm increase in diameter around disc C compared with both discs A and B were considered positive for Amp-C existence (Halstead et al., 2012).

Statistical analysis
Our study is descriptive and experimental; therefore to analyze the results, we used MINITAB16 software.P.value and confidence intervals are <0.05 and 95%, respectively.

DISCUSSION
Effective use of microbiology laboratories is essential for controlling the spread of multi-drug resistant (MDR) pathogens and thereby decreasing the need to use drugs.Infection control is very beneficial to patients and also decreases mortality and morbidity rate in the world.The experiment in diagnostic microbiology is vital for detecting many resistant bacteria.Many bacteria may not be recognized because they have false resistance or susceptibility in conventional tests.This can lead to patients receiving unsuitable drugs and contribute to the spread of the bacteria to other patients.Therefore, the identification of such "Cryptic"resistance is so vital.Due to the fact that susceptibility assays may be unreliable, beneficial assays are required to detect the resistant mechanisms involved.These mechanisms are AmpC βlactamases, extended-spectrum-beta-lactamases (ESBLs) and carbapenemases of molecular classes A and B especially KPC and NDM-1 (Thomson et al., 2010).This study exhibits that Fosfomycin and Tigecycline were more active than the other antibiotics.Leone et al. (2012) study showed that of 136 K. pneumoniae, 37.1% were MDR and ESBL producers and K. pneumoniae strains were resistant to fluoroquinolones,  gentamycin and 71% were resistant to imipenem.This study shows that 48 (57.5%), 3 (3.5%),23 (28%) and 5 (6%) were ESBL, MBL, Amp-C and KPC positive, respectively.ESBL-producing K. pneumoniae were obtained from 24 adults and 33 pediatric patients in two hospitals.
This study confirms the relevant role of ESBL-KP as a pathogen in neonatal units.In the Taleghani Hospital, of 28 positive isolates, eight of ESBL-producing strains were   In a research in Spain, the results revealed that 133 adults and 29 pediatric patients were infected with ESBLproducing K. pneumoniae (Ruiz de Alegría et al., 2011).The wide use of drugs might facilitate the spread of antibiotic resistance, and thus control of beta-lactamases; especially, ESBL-producing K. pneumoniae within infant units should be considered as a precedence.Shigemoto et al showed that of 2,113 isolates of K. pneumoniae, 52 were ESBL-positive.Among the ESBL-positive K. pneumoniae, five isolates were also MBL positive (Shigemoto et al., 2012).In this study, 2 (4.4%) of adults and 1 (2.6%) of infants were MBL positive and 14 (31.3%) of adults and 9 (23.7%) of infants were Amp-C positive.The prevalence of Amp-C in Iran and the entire world is unknown, because laboratories have problem detecting this resistant mechanism accurately.Rodriguez-Bano et al showed that 100 patients were infected with plasmid-mediated Amp-C beta-lactamases including 17 Amp-C-producing K. pneumoniae (Rodríguez-Baño et al., 2012).
Our study is the first on infants that harbour types KPC and MBL as well as on ESBL + AmpC + KPC and ESBL + MBL + AmpC in adults and children of Iran.Microbiology laboratories must be able to identify resistant bacteria in a timely manner, especially those that have false susceptibility in vitro to antibiotics that may be considered for therapy of infected adults and infants.
Bacteriological excellence is needed more than ever, and it is vital that ESBLs enzymes, AmpC and carbapenemases be promptly and accurately detected.

Table 2 .
Antimicrobial susceptibility testing results of 83 isolates of Klebsiella pneumoniae collected from Mofid Children and Taleghani Hospitals, Tehran, Iran.

Table 3 .
Microbiological activities of various antimicrobial agents against 83 K. penumoniae isolates.

Table 4 .
Number and percent β-lactamases present in Klebsiella pneumoniae collected from Mofid Children and Taleghani Hospitals, Tehran, Iran.