Detection of blaNDM -Metallo-?-Lactamase Genes in Klebsiella pneumonia Strains Isolated From Burn Patients in Baghdad Hospitals

Abbas Atyia Hammoudi,Azhar Noori Hussein,Mohammed Shamkhi Jebur
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Keywords : Burn Patients, blaNDM -Metallo-?-Lactamase Genes, Klebsiella pneumonia.
Medical Journal of Babylon  13:4 , 2017 doi:1812-156X-13-4
Published :16 July 2017

Abstract

From the period from March to August 2016, 210 swabs were collected from the burn patients hospitalized in different hospitals in Baghdad City: Al-Karama Teaching Hospital, Special Burn Hospital, Central Teaching Laboratories, Child protection Teaching Hospital, Imam Ali Hospital. Out of 210 clinical isolates, 42 (37.5 %) had been shown a single isolated of pathogenic bacteria K. pneumoniae and the others were belonged to other bacteria and mixed growth isolates. Identification of all isolates were carried out depending on macroscopic, microscopic characterizations, conventional biochemical tests and Api 20E system.Metallo-? lactamase (MBL) enzymes were screen by two phenotypic methods(Meropenem-EDTA double disks method and Modified Hodg test). Susceptibility testing were used with The following antibiotic disks:Imipenem,Meropenem,Ceftazidime,Cefotaxime,Pipracillin,Gentamicin,Amikacin andCiprofloxacin.The percentage of resistance isolates were as followed:Imipenem (21.42%),Meropenem (19.04%),Ceftazidime(69.04%), Cefotaxime (85.71%), Pipracillin(85.71%), Gentamicin (26.19%), Amikacin (19.04 %) andCiprofloxacin(59.52%).The percentage of the prevalence of blaNDM-1 and blaNDM-2 genes in K. pneumoniae isolates from burn patients in Baghdad hospitals were as followed:20(100 %) forblaNDM-1 genes and 6 (30 %) for blaNDM-2 genes.

Introduction

Klebsiella pneumoniaewas an opportunistic gram-neg¬ative pathogenic bacterium associated with a range of nosocomial infections (e.g. septicemia, pneumonia, bacteremia, meningitis, urinary tract, burn and wound infec¬tions) [1]. Furthermore it was the most medically important species of the genus Klebsiella. In recent years, Klebsiella have become important pathogens in nosocomial infections [2]. It was also a potential community – acquired pathogen [3]. Antibiotic therapies are widely used for treating infectious diseases. Nowadays, antibiotic-resistant bacteria are a great concern of worldwide public health [4]. The problem of antimicrobial resistance is highlighted by a recent increase of carbapenem-resistant K. pneumoniae, which has largely been driven by the emergence and spread of mobile genetic elements carrying carbapenemase resistance genes including the metallo-beta-lactamase [5, 6].Meropenem and imipenem are carbapenems that remain active against organisms carrying most Ambler classes of ?-lactamases which include many Gram-negative bacilli, including Klebsiella spp. One of the major mechanisms of carbapenem resistance in this pathogen is the production of carbapenem hydrolyzing ?-lactamases. These specific groups of ?-lactamases are categorized into class B metallo ?-lactamases (MBLs) includingImipenemase (IMP)and Verona integrin encoded metallob-lactamase (VIM), New Delhi metallo-beta-lactamase (NDMs) and class D (Oxacillinases) includingOXA-23-like, OXA-24/40-like and OXA-58 [7, 8]. The new MBL, New Delhi metallo-?-lactamase (NDM-1), initially reported in K. pneumoniae and E. coli recovered from a Swedish patient who was previously hospitalized in India in 2008 [9]. The rapid emergence spread of NDMpositive bacteria has a complex epidemiology involving a variety of harboring species (principally Klebsiella pneumoniaeand E. coli), inter-strain, inter-species, and inter-genus transmission,which has been related to a diverse moveable plasmid that can be transferred from one bacteria to another, from man to man and even from country to country in more than 40 countries worldwide [10,11]. The bacteria with NDM-1 gene are known as superbugs and public health must pay more attention to them [12]. Many phenotypic, genotypic, phylogenic and molecular methods used to detect the production of enzymes by bacteria that responsible about drug resistant which causes increased morbidity and mortality among patients with infections caused by these bacteria and increased healthcare costs due to the extended hospital stay [13]. In recent years, many Iraqi patients were travelled to India and to other countries for medical care purpose which may helped in acquiring NDM gene. In Iraq there were no information about the occurrence of NDMK. pneumoniae producing clinical isolates. So the proposed aim of this study was to detect MBL genes blaNDM-1,2among resistant isolates of K. pneumoniae ob¬tained from burn patients in Baghdad Hospitalsbypolymerase chain reaction(PCR).

Materials and methods

Isolation and Identification
During the period from March to August 2016, 42 K. pnuemoniae strains were isolated from 210 swabs of burn patients hospitalizedin different hospitals in Baghdad City: Al-Karama Teaching Hospital, Special Burn Hospital, Central Teaching Laboratories, Child protection Teaching  Hospital, Imam Ali Hospital. Specimens were collected by sterile swabs after the removal of dressing and cleaning the wound surface by 70% alcohol. The isolation and identification of K. pnuemoniaefrom wound specimens were streaked on blood agar, MacConkey agar and Eosin methylene blue(EMB) agar (Biomark Lab. Pune. India) and incubated at 37? for 24hrs. The isolates were identified as K. pnuemoniaeby manual biochemical tests that were used in accordance with the manufacturer’s instructions;  based on Gram staining, catalase test, oxidase test, triple sugar iron (TSI) fermentation, Indole test,Voges- Proskauer (VP) test, Methyl red (MR) test, Simmons Citrate test,Urease test, motility test, and string test [14].For final confirmation, biochemical tests embedded in the API-20E biochemical kit system (Bio-Merieux, France).

Antimicrobial Susceptibility Testing
The susceptibility pattern of isolates to different antibiotics were examined using disk diffusion method (Kirby-Bauer) on Muller-Hinton agar plates(Biomark Lab.,Pune. India) according to guidelines of CLSI [15]. The antimicrobial disks were included: Imipenem (10?g), Meropenem (10?g), Ceftazidime(30?g), Cefotaxime (30?g),  Pipracillin (100?g), Gentamicin (10?g), Amikacin (30?g)  and Ciprofloxacin (5µg) ( MAST Co. UK). Pseudomonas aeruginosa ATCC27853 were used as a control strain[16].




Results

Bacterial strains, antibiotic susceptibility and MBL phenotypic test. In this study a total of 210 sample swabs of clinical isolates of burn wound infections were cultured , examined and identified. Out of 210 clinical isolates, 42 (37.5 %) had been shown a single isolated of pathogenic bacteria K. pneumonia and the others were belonged to other bacteria: 36 (32.14%) Pseudomonas spp., 20 (17.86%) E. coli, 10(8.93%) S.aureus and 4 (3.57 %) Proteus spp.,while mixed growth isolates frequency as the following:K. pneumoniae and Pseudomonas spp.64 (65.31),Pseudomonas spp. and E. coli 18 (18.37%), K. pneumoniae and E. coli 7(7.14%), Pseudomonas spp. and Proteus spp. 4 (4.08 %), K. pneumoniae and S.aureus 3 (3.06 %) and Proteus spp. and E. coli 2 (2.04 %). In a local study done by Mohammed(2007),who isolated K. pneumoniae from burn wound infection (36.7%) 20[21]; whileAssal, (2010) isolated K. pneumoniae from wound (31.25%).These results were agreement with this study. Kehinedet.al(2004) also found that Klebsiella spp. (34.4%) was the most common isolate from infected burn wounds [22].K. pneumoniae associated with hospital-acquired infection accounting for 34–36% of cases of K. pneumoniae bacteremia [23]. Antibiotic Susceptibility Testing Antimicrobial resistance to the carbapenems(e.g. imipenem and meropenem) mediated by metallo-?lactamase(MBL) enzymes hasremarkable clinical implications since the carbapenems are usually the last options of treatment for bacterial infections caused by multidrug resistant organisms (e.g. producers of extended spectrum B-lactamases) [24].Eight antibiotic disks were used in this study included two types of Carbapenems antibiotics; Imipenem (IPM), Meropenem (MEM) and two types of third generation Cephalosporins included; Ceftazidime (CAZ), Cefotaxime (CTX). Table(1) summarizes the results of antibiotic susceptibility test and reflects forty-two isolates were resistance to the following antibiotics; Imipenem (21.42%), Meropenem (19.04%), Ceftazidime (69.04%),Cefotaxime(85.71%),Pipracillin(85.71%)),gentamicin(26.19%),Amikacin(19.04%)andCiprofloxacin(59.52). Furthermore, some isolates exhibited intermediate susceptibility to Imipenem(9.52%), Meropenem(4.76%), Ceftazidime(11.90%), Pipracillin (4.76%), Amikacin(4.76%) and Ciprofloxacin(2.38%).While some isolates showed susceptibility to the antibiotics as the following: Gentamycin and Amikacin(78.57%), Meropenem(76.19%), Imipenem(69.04%), Ciprofloxacin(38.09%), Ceftazidime(19.04%)and both Cefotaxime, Pipracillin (9.52%). A high degree of resistance to the tested antibiotics was noted among the bacteria isolates especially to the third-generation cephalosporins; Cefotaxime(85.71%), Ceftazidime (69.04%),this results of the study agreement with Ejikeugwuet al., 24[25]who reportedthat K. pneumoniae show a resistance rates for CTX were (61.5 %), CAZ(38.5 %)and Fluoroquinolones (Ciprofloxacin; CIP show a resistance rate(53.8 %). While the carbapenems used, IPM and MEM, the resistance rates of the K. pneumonia was (12.8 %),(7.7 %) respectively.

Discussions

Bacterial strains, antibiotic susceptibility and MBL phenotypic test. In this study a total of 210 sample swabs of clinical isolates of burn wound infections were cultured , examined and identified. Out of 210 clinical isolates, 42 (37.5 %) had been shown a single isolated of pathogenic bacteria K. pneumonia and the others were belonged to other bacteria: 36 (32.14%) Pseudomonas spp., 20 (17.86%) E. coli, 10(8.93%) S.aureus and 4 (3.57 %) Proteus spp.,while mixed growth isolates frequency as the following:K. pneumoniae and Pseudomonas spp.64 (65.31),Pseudomonas spp. and E. coli 18 (18.37%), K. pneumoniae and E. coli 7(7.14%), Pseudomonas spp. and Proteus spp. 4 (4.08 %), K. pneumoniae and S.aureus 3 (3.06 %) and Proteus spp. and E. coli 2 (2.04 %). In a local study done by Mohammed(2007),who isolated K. pneumoniae from burn wound infection (36.7%) 20[21]; whileAssal, (2010) isolated K. pneumoniae from wound (31.25%).These results were agreement with this study. Kehinedet.al(2004) also found that Klebsiella spp. (34.4%) was the most common isolate from infected burn wounds [22].K. pneumoniae associated with hospital-acquired infection accounting for 34–36% of cases of K. pneumoniae bacteremia [23]. Antibiotic Susceptibility Testing Antimicrobial resistance to the carbapenems(e.g. imipenem and meropenem) mediated by metallo-?lactamase(MBL) enzymes hasremarkable clinical implications since the carbapenems are usually the last options of treatment for bacterial infections caused by multidrug resistant organisms (e.g. producers of extended spectrum B-lactamases) [24].Eight antibiotic disks were used in this study included two types of Carbapenems antibiotics; Imipenem (IPM), Meropenem (MEM) and two types of third generation Cephalosporins included; Ceftazidime (CAZ), Cefotaxime (CTX). Table(1) summarizes the results of antibiotic susceptibility test and reflects forty-two isolates were resistance to the following antibiotics; Imipenem (21.42%), Meropenem (19.04%), Ceftazidime (69.04%),Cefotaxime(85.71%),Pipracillin(85.71%)),gentamicin(26.19%),Amikacin(19.04%)andCiprofloxacin(59.52). Furthermore, some isolates exhibited intermediate susceptibility to Imipenem(9.52%), Meropenem(4.76%), Ceftazidime(11.90%), Pipracillin (4.76%), Amikacin(4.76%) and Ciprofloxacin(2.38%).While some isolates showed susceptibility to the antibiotics as the following: Gentamycin and Amikacin(78.57%), Meropenem(76.19%), Imipenem(69.04%), Ciprofloxacin(38.09%), Ceftazidime(19.04%)and both Cefotaxime, Pipracillin (9.52%). A high degree of resistance to the tested antibiotics was noted among the bacteria isolates especially to the third-generation cephalosporins; Cefotaxime(85.71%), Ceftazidime (69.04%),this results of the study agreement with Ejikeugwuet al., 24[25]who reportedthat K. pneumoniae show a resistance rates for CTX were (61.5 %), CAZ(38.5 %)and Fluoroquinolones (Ciprofloxacin; CIP show a resistance rate(53.8 %). While the carbapenems used, IPM and MEM, the resistance rates of the K. pneumonia was (12.8 %),(7.7 %) respectively.

Conclusions

The study had shown the spreading of blaNDMK. pneumoniae isolates among patients with burn infections. The rapid spread of blaNDM genes of K. pneumoniae isolates among patients with burn infections in this study poses an increased threat to hospitalized patients in Iraq and more importantly, avoiding misuse , overuse of antibiotics may converse the undesired effects of multidrug resistant and NDMproducing bacteria. Furthermore we would expect more NDM variants to be discovered in the next years in Iraq.

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