Detection of Plasmid-Mediated Quinolone Resistance Genes in Clinical and Environmental Hospital Isolates of Klebsiella pneumoniae in Al-Najaf City pneumoniae Klebsiella

Aim of study : This study aimed to detected the presence of the plasmid mediated quinolone resistance genes in quinolone resistant Klebsiella pneumoniae isolates from clinical and environmental hospital samples. Methodology : A total of 195 clinical samples of different sources and 50 environmental hospital samples were collected from three main hospitals in Al-Najaf city .K. pneumoniae was identified depending on cultural and traditional biochemical tests, then confirmed by API 20E system. Phenotype detecting of quinolone resistance in K. pneumoniae isolates were carried out by growing on MacConkey agar supplemented with 10μg/ml Ciprofloxacin. Antibiotic susceptibility performed by disk diffusion. Quinolones resistant isolates were selected for molecular study for detecting aac (6 ')-Ib-cr, qepA, qnrS and qnrB as plasmid mediated resistance genes using Multiplex polymerase chain reaction. Results: Eighty-nine isolates were identified as .K.pneumoniae.Thirty–four(38%) resist ciprofloxacin(10μg/ml ) and were resistant to at least fourteen antibiotics to which they are tested. Hence, all isolates were considered to be multidrug resistant (MDR). The results of detection the plasmod mediated antibiotic resistance genes revealed the widely distribution aac (6 ')-Ib-cr gene alone or combined with qnrS gene in 14 (41.18%) KUFA JOURNAL FOR NURSING SCIENCES Vol.5 No. 2, May through August 2015 -2isolates ,or with qepA (2.94%) and 8.82% of bacterial isolates carried aac (6 ')-Ib-cr, qepA and qnrS whereas only one isolates (29.41%) that caused wound infection showed the presence of qnrB gene. Conclusions :High prevalence MDR K. pneumoniae harbouring PMQR mediated by aac(6`)-Ib-cr and qnrS . This study is the first trail to detect PMQR genes in clinical and environmental isolates of K.pneumoniae in Iraq. Recommendations:Further studies are necessary to understand the dissemination of plasmid mediated genes (qnr, aac(6`)-Ib-cr and qepA gene) and chromosomal resistance among K.pneumoniae and other common pathogenic bacteria. Keyword: Klebsiella pneumoniae, plasmid mediated quinolone resistance, qnrS, qnrB, aac(6`)-Ib-cr ,qepA,and Gram negative bacteria


INTRODUCTION
Fluoroquinolones have been frequently prescribed as empirical therapy against most hospital and community infections due to increased appearance of multiple drug resistant Gram negative bacteria including Klebsiella pneumoniae and to the disease severity (1) .With extensive clinical use of quinolones.Fluoroquinolone resistance has been a problem in clinical medicine for its limiting of available agents in the treatment of many types of infection (2) .
Quinolone resistance in the family Enterobacteriaceae is mostly attributed to the accumulation of mutations in the bacterial enzymes targeted by: DNA gyrase and DNA topoisomerase IV (3,4) .In addition Active efflux systems (acrAB-TolC) resulting in decreased intracellular accumulation of fluoroquinolones in K.pneumoniae (4) .Morever,Plasmidmediated quinolone resistance (PMQR) with the potential for horizontal transfer has been described along with three mechanisms: (i) a quinolone-protective mechanism encoded by the qnr genes (3) ; (ii) a modifying enzyme, aac(60)-Ib-cr and (iii) an efflux pump encoded by the qepA gene (5,6) .Plasmid-encoded quinolone resistance determinants confer low-level resistance, but their presence could potentially facilitate the evolution of the bacterial host toward higher levels of resistance by mutational alterations in type II topoisomerases (4) .K. pneumoniae strains represent an incredibly great epidemic potential and are one of the major sources of horizontally spreading antimicrobial resistance (2) .Fluoroquinolone resistant K.pneumoniae constitutes one of the most common Gram-negative bacteria showing multiple antibiotic resistance worldwide (2,3,5,6) .There is little information regarding in the occurrence of PMQR genes in Iraq .This study aimed to investigate occurrence of PMQR genes in ciprofloxacin resistant K.pneumoniae by multiplex PCR technique.

Sample collection:
A total of 245 samples were collected from different source that include urinary tract infection(89), Wound infection(54), Burn infection(32) and female genital tract infection (23),in addition to 50 swabs were taken from environmental hospital samples(operations and burn wards) during November , 2011 to February, 2012 from three hospitals :Al-Sadr Teaching, Al-Hakeem, and Al-Manathera in the Al-Najaf City.

Isolation and identification
Clinical and environmental hospital samples were cultured onto MacConkey agar and incubated for 18-24 h at 37 o C. All lactose-fermenting isolates were tested by morphologic characteristics and standard biochemical tests according to MacFaddin, (2000) (7) .Confirmation of K.pneumoniae was conducted using API20E system.

Phenotypic Detection of Fluoroquinolones Resistance
Preliminary screening of K. pneumoniae isolates resistance to ciprofloxacin was carried out using pick and patch method on MacConkey agar supplemented with 10 µg/ml ciprofloxacin , incubated at 37 ̊C for 18-24h .

b) Agarose Gel Electrophoresis
Agarose gel electrophoresis for pcr products parallel to a molecular marker (promega/ USA effective size range: 100 to 1500 bp) was performed for detecting and evaluating size products.Finally, the gel was photographed using Biometra gel documentation system .

Figure(1):Antibiotics susceptibility profile of 34 isolates of quinolone resistant Klebsiella pneumoniae recovered from three hospitals in Al-Najaf City
Our study showed that all ciprofloxacin resistant K.pneumoniae isolates were resistant to all quinolones antibiotic tested (Levofloxacin, Gatifloxacin, Norfloxacin, Nalidixic acid) and Multiple antibiotic resistance which showed highly resistance to at least more than 14 antibiotic while 88% isolates were susceptible to Carbapenem (Imipenem and Meropenem)as shown in Figure (1).Table (2) shows significant high percentage (97%) of fluroquinolone resistant Klebsiella pneumoniae isolates were found to carry PMQ genes.Ninty -four percent (32/34) of quinolones resistant K. pneumoniae were found to carry aac(6`)-Ib-cr gene either alone(14/34 isolates) or in combination(18/34 isolates).Plasmid mediated quinolone resistance gene , qepA was detected in four isolates , collected from urinary tract infections ,listed in table (3).qnrS, has been amplified in17 isolates collected from different clinical and environmental sources in combination either with aac(6`)-Ib-cr gene or with both aac(6`)-Ib-cr and qnrS genes
This study is relatively in agreement with a study carried out in General Al-Nasseriyia Hospital by Nakkash and Al-Husseiny (2008) that isolated 99 isolates of K. pneumoniae represented by 61 isolates from surgical wound infections and 38 from hospital environment (13) .and a study by al-Al-Sehlawi (2012 ) showed that 39.16% Klebsiella.spp isolates obtained from clinical samples and environmental hospital samples in the Holly Najaf city (14) .While, Fayroz-Ali (2012) found that Klebsiella.spp was the second most common (16.8%) organism isolated from urine samples from patients after E.coli, (15) and Al Rediany (2012) detected Klebsiella spp. in 20 samples(16%) of burn infection and 105 (84%) samples from wound infection in Al-Najaf hospitals (16) .
The wide spread for Klebsiella spp.may be due to the ability of this bacteria to survive under unsuitable environmental conditions since it has a thick polysaccharide capsule, and different mechanisms of antibiotic resistance (17) .
The percentage of K. pneumoniae is variable in the different studies, this may be attributed to drug overuse, difference of diagnostic methods and the hospital policy in management of such cases.Moreover, geographic climatic and hygienic factors may also be correlated with the relative variability of results among different area (18) .
Antibiotic susceptibility test showed that all ciprofloxacin resistant isolates considered to be multidrug resistant (MDR) they resist to at least fourteen antibiotic to which they are tested but Carbapenems (imipenem and meropenem) were the most efficient antibiotics against K. pneumoniae isolates due to high susceptibility rate.This is an expected result in K. pneumoniae which recorded in local studies ( 14,16 ) while was lower than that reported by other local studies which reported that the susceptibility of K.pneumoniae isolates collected from clinical and environmental samples to imipenem was (100%) (16,19,20) .
The increase in the rate of quinolone resistance is high in Najaf hospitals (19,20) and the resistance may be multi-factorial, this resistance may be result from the tendency for bacteria to develop resistance and subtherapeutic concentrations of the drug (15) .
A variant of aminoglycoside amnoacetyl transferase enzyme(aac(6´)-Ib-cr) is capable to modify ciproflocxacin and reducing its activity has been reported to be widely prevalent around the world and to be circulated with qnr genes or and qepA gene (5,21) .There is substantial increase in quinolone resistance associated with aac(6´)-Ib-cr gene are found in14 (41.14%),K.pneumoniae isolates, and this gene shows a combination with qnrS and qepA 13 (38.32%),1(29.41%),respectively.In addition to combination aac(6´)-Ib-cr with qepA and qnrS 3( 8.82 ).
The percentage of qnrs positive isolates agreed with Chines study by Cai et al. (2011) that identified qnrS in 18.9% of 37K.pneumoniae isolates but qnrB gene was not detected in their study. (22)ut of 64 Enterobacteriaceae isolates collected from Kuwait hospitals, 3 (4.7%)were positive for a qnrB gene (10) .In Japan, six qnrB genes were detected in K. pneumoniae, K.oxytoca, Escherichia.coli, Citrobacter freundii, Proteus mirabilis and P.fluorescence from zoo reptiles and falcons (23) .In Chennai , qnrB gene was detected in 48% of 23 multi drug resistant isolates of K. pneumoniae ( 5) .But in our study amplification product of qnrB gene was observed in one isolates .thismay be associated to the geographical distribution of qnr genes.