Incidence of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in patients with urinary tract infection

ABSTRACT CONTEXT AND OBJECTIVES: Resistant bacteria are emerging worldwide as a threat to favorable outcomes from treating common infections in community and hospital settings. The present investigation was carried out to study the incidence of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in patients with urinary tract infection in different seasons of the year, in order to determine the prevalence of the genes blaTEM, blaSHV and blaCTX-M, which are responsible for ESBL production among ESBL-producing K. pneumoniae, in three cities in Iran, and to investigate the antimicrobial susceptibility pattern of K. pneumoniae in different seasons. DESIGN AND SETTING: Retrospective study carried out among patients with urinary tract infections in five hospitals in Iran. METHOD: Two hundred and eighty-eight clinical isolates of K. pneumoniae were collected between March 2007 and April 2008 from five hospitals in three cities in Iran. ESBLs were identified by phenotypic and genotypic methods. ESBL-producing Klebsiella pneumoniae were evaluated against non-beta-lactam antibiotics. Genes coding for ESBLs (blaSHV, TEM and CTX-M) were screened. RESULTS: Among the 288 clinical isolates of K. pneumoniae, 37.7%, 46.7% and 15.6% were obtained from hospitals in Ilam, Tehran and Tabriz, respectively, of which 39.4%, 50.7% and 45.8% were ESBL-producing K. pneumoniae in Ilam, Milad and Emam Reza hospitals, respectively. CONCLUSION: According to the results from this study, resistance to third-generation cephalosporins is higher during the cold months than during the warm months.


INTRODUCTION
Resistant bacteria are emerging worldwide as a threat to beneficial outcomes from treating common infections in community and hospital settings. 1The introduction of third-generation cephalosporins into clinical practice in the early 1980s was heralded as a major breakthrough in the fight against beta-lactamase-mediated bacterial resistance to antibiotics.These cephalosporins had been developed in response to the increased prevalence of beta-lactamases in certain organisms (e.g.ampicillin-hydrolyzing TEM-1 and SHV-1 beta-lactamases in Escherichia coli and Klebsiella pneumoniae), as well as in response to the spread of such beta-lactamases into new hosts (e.g.Haemophilus influenzae and Neisseria gonorrhoeae).The third-generation cephalosporins were not only effective against most beta-lactamase-producing organisms, but also had the advantage of lower nephrotoxic effects than shown by aminoglycosides and polymyxins.However, plasmid-encoded beta-lactamases capable of hydrolyzing the extended-spectrum cephalosporins were soon reported, in 1983. 2 Extended-spectrum beta-lactamases (ESBLs) are known as extended-spectrum because they are able to hydrolyze a broader spectrum of beta-lactam antibiotics than the simple parent betalactamases from which they are derived.Such ESBLs also have the ability to inactivate beta-lactam antibiotics containing an oxyimino group, such as oxyimino-cephalosporins (e.g.ceftazidime, ceftriaxone or cefotaxime) or oxyimino-monobactam. 3 Furthermore, they are not active against cephamycins and carbapenems.Generally, they are inhibited by beta-lactamase inhibitors such as clavulanate and tazobactam.ESBLs have been found in a wide range of Gram-negative rods.However, the vast majority of strains expressing these enzymes belong to the Enterobacteriaceae family. 4K.
pneumoniae and E. coli stand out as the major ESBL producers. 5rong selective pressure due to the use of beta-lactam drugs has resulted in the emergence of ESBL-producing strains.7][8] The plasmids that harbor genes encoding ESBLs frequently contain other genes encoding mechanisms of resistance to aminoglycoside and cotrimoxazole. 9ESBLs are clinically important because they destroy cephalosporins, the workhorse hospital antibiotics that are given as first-line agents to many severely ill patients.Delayed recognition and inappropriate treatment of severe infections caused by ESBL producers acting on cephalosporins have been associated with increased mortality. 10It is important to study the prevalence of K. pneumoniae in Iran, since it is a country with four seasons.

OBJECTIVE
This study was carried out to evaluate the incidence of extendedspectrum beta-lactamase-producing K. pneumoniae in patients with urinary tract infection in different seasons, in order to determine the prevalence of the genes blaTEM, blaSHV and blaCTX-M, which are responsible for ESBL production among ESBL-producing K. pneumoniae, in three cities in Iran, and to investigate the antimicrobial susceptibility pattern of K. pneumoniae in different seasons.

Bacterial isolates
Two hundred and eighty-eight clinical isolates of K. pneumoniae associated with urinary tract infections, which were isolated in five hospitals in three cities in Iran between March 2007 and April 2008, were studied.The cities and hospitals included in this study were: Ilam (Emam Khomaini, Mostafa Khomaini and Ghaem Hospitals), Tabriz (Emam Reza Hospital) and Tehran (Milad Hospital).

Sample collection
A volume of urine measured using the calibrated loop method was inoculated into nutrient agar medium for colony counting.
Densities of single-potential K. pneumoniae greater than or equal to 10 4 CFU/ml (colony forming units per milliliter) were interpreted as positive for urinary tract infection, while the test was repeated if the result was 10 2 -10 4 CFU/ml.Results less than 10 2 CFU/ml were interpreted as negative for urinary tract infection. 11ine specimens were cultured for isolation of K. pneumoniae in blood agar and MacConkey agar media.

ESBL detection
ESBL production was detected by using the disk diffusion method.In keeping with the Clinical and Laboratory Standards Institute (CLSI) recommended guidelines, ESBL screening was performed by means of disk diffusion using ceftazidime (30 µg), cefotaxime (30 µg), ceftriaxone (30 µg), aztreonam (30 µg) and cefpodoxime (30 µg) disks.The ESBL phenotype was confirmed by means of the double disk diffusion method, using antibiotic disks containing a combination of cephalosporin plus clavulanic acid, in conjunction with the corresponding cephalosporin disk alone.

Polymerase chain reaction amplification of blaTEM, blaSHV and blaCTX-M genes
The polymerase chain reaction was carried out using the following primers: blaTEM (forward 5'-GAGTATCAACATTTC-CGTGTC-3' , reverse 5'-TAATCAGTGAGGCACCTTCTC-3'); blaSHV (forward 5'-AAGATCCACTATCGCCCAGCAG-3' , reverse 5'-AAGATCCACTATCGC CCAGCAG-3'); 15 and blaCTX-M (forward 5'-ACGCTGTTGTTAGGAAGTG-3' , reverse 5'-TTGAGGCTGGGTGAAGT-3'). 16e blaSHV gene was amplified under the following conditions: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 56 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 10 min.The blaTEM gene was amplified under the following conditions: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 45 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 10 min.The blaCTX-M gene was amplified under the following conditions: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 seconds annealing at 48 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 10 min.The amplicons were run on 1% agarose gel.The gels were stained with ethidium bromide, and bands observed at the desired position were photographed using an ultraviolet light transilluminator.
The statistical analyses to determine the frequency of antimicrobial resistance were descriptive and performed using the Statistical Package for the Social Sciences (SPSS), version 13.

Polymerase chain reaction results
The frequencies of ESBL genes varied among the different hospitals in different seasons.In total, 104, 22 and 17 isolates were positive for blaSHV, blaCTX-M and blaTEM genes, respectively.In the hospitals in Ilam, out of the eight K. pneumoniae isolates with blaTEM, 12.5% (n = 1), 12.5% (n = 1), 25% (n = 2) and 50% (n = 4) were obtained in the spring, summer, fall and winter, respectively.

DISCUSSION
The development of extended-spectrum cephalosporins in the early 1980s was regarded as a major addition to the therapeutic armamentarium in the fight against beta-lactamase-mediated bacterial resistance. 17Regrettably, the emergence of K.
pneumoniae presenting resistance to ceftazidime and other cephalosporins has seriously compromised the efficacy of these life-saving antibiotics.
The data obtained from clinical samples of K. pneumoniae now show high antibiotic resistance.The resistance to antibiotics shown by Klebsiella has increased worldwide. 18Carbapenems are the drugs of choice for many infections caused by Gram-positive and Gram-negative bacteria. 19Imipenem has been found to be the most effective antibiotic.Our results indicated that all isolates were susceptible to imipenem.This is consistent with the findings of Alzahrani and Akhtar from Saudi Arabia. 20Gram-negative  pathogens harboring ESBLs have caused numerous outbreaks of infections and are becoming an increasing therapeutic problem in many countries.The incidence of ESBL-producing strains among clinical isolates has been steadily increasing over the past years, thus resulting in limitations to the therapeutic options. 21BLs are now a significant problem in hospitalized patients throughout the world.The prevalence of ESBLs among clinical isolates varies worldwide, and the patterns are rapidly changing over time. 22Patients suffering from infections caused by ESBLproducing organisms are at higher risk of treatment failure when broad-spectrum beta-lactam antibiotics are used.Therefore, it is recommended that any organisms experimentally confirmed as ESBL producers should be reported as resistant to the entire broad-spectrum beta-lactam antibiotic, regardless of any susceptibility test results. 14The phenotypic test only presumptively identifies the presence of ESBL.The task of identifying the specific ESBLs present in clinical isolates is more complicated.The easiest and most common molecular method used to detect the presence of a beta-lactamase belonging to a family of enzymes is the polymerase chain reaction, using oligonucleotide primers that are specific for the beta-lactamase gene.These primers are usually chosen to anneal to regions where various point mutations are not known to occur. 23e current study was conducted during different seasons and in hospitals in different parts of Iran.To the best of our knowledge, this study was the first to cover different seasons and hospitals all at once.In the hospitals in Ilam, comparison between different seasons revealed that the highest resistance to third-generation cephalosporins among patients with urinary tract infections was for ceftriaxone in the winter (77.5%).The results showed that ESBL production by K. pneumoniae was higher in winter than in the other seasons.Interestingly, similar results recurred for ciprofloxacin, cotrimoxazole and amikacin.The dominant gene responsible for ESBL production was blaSHV (n = 32) and the lowest frequency observed was for blaTEM (n = 8).As expected, the highest frequency of these three genes responsible for ESBL production was observed in the winter.Ceftazidime and ceftriaxone resistance was highest in patients with urinary tract infections at the screening stage.In these patients, the highest levels of ESBL-producing K. pneumoniae (42.65%) and amikacin resistance were found in winter (41.4%) while resistance to ciprofloxacin (27.8%) and cotrimoxazole (50%) was greater in the fall than in the other seasons.The results reflected the observation that blaSHV was the dominant gene responsible for ESBL production, followed by blaSHV and blaCTX-M.The frequencies of all three genes in the winter were more than in the other seasons, while the lowest frequency was in the summer.Our findings demonstrated that K. pneumoniae presented greater resistance to antibiotics and higher ESBL production during the cold seasons than during the warm seasons.
In patients with urinary tract infections in Emam Reza Hospital, the highest resistance to third-generation cephalosporins was found for ceftriaxone (88.8%) in the winter.The ESBL production by K. pneumoniae was 52.4%, while resistance to amikacin was 36.4% and to ciprofloxacin, 9%, in the winter.Resistance to cotrimoxazole was 22.2% in the fall, which was higher than in the other seasons.
Climatic factors may also be important in the pathogenesis of urinary tract infections.There have been several reports suggesting that there is higher incidence of such infections in cold weather.Most studies suggest that these infections are particularly likely to occur when patients dress in a manner inappropriate to the weather conditions. 24Our results showed variations in the incidence rate throughout the year, with a decrease in warm seasons.One possible explanation, suggested by Stansfeld, is that upper respiratory tract infections may precede urinary tract infections.Since coughs and colds are more common in the winter months, the same would apply to urinary tract infections. 25 In Saudi Arabia in 2007, out of 400 K. pneumoniae isolates investigated, 55% were positive for ESBLs, 97.3% for blaSHV and 84.1% for blaTEM genes.The resistance rates to cefotaxime and ceftazidime were 97% and 95%, respectively.All the ESBL-producing isolates were susceptible to imipenem. 29The results from Milad Hospital, Emam Reza Hospital and the hospitals in Ilam also showed high frequency of blaSHV, while the frequency of blaTEM was lower than in the study done in Saudi Arabia.Our study showed that imipenem was an effective antibiotic for K.
pneumoniae, which is in agreement with Al-Agamy et al. in 2007 in Saudi Arabia. 29n Dokuz Eylul University Hospital in Turkey, 38% of Enterobacteria isolates were ESBL positive. 30A similar result was found in hospitals in Ilam (39.4% positive for ESBLs) while in Milad and Emam Reza Hospitals, the frequency of ESBL-producing K.
pneumoniae was higher than in Dokuz Eylul University Hospital.
In the latter, 52.7% showed blaTEM and 74.3% blaSHV, while the high frequency of blaSHV and low frequency of blaTEM were again found in Milad and Emam Reza Hospitals and the hospitals in Ilam.

CONCLUSION
According to the results from this study, resistance to third-generation cephalosporins was generally higher during the cold months than during the warm months.Furthermore, resistance to cefpodoxime was almost equal to that of aztreonam, while resistance to other cephalosporins used in this study varied considerably.
Our study showed that K. pneumoniae recovered from urine specimens produced a higher number of ESBLs.It also showed resistance towards fluoroquinolones, aminoglycosides and cotrimoxazole.
The gene predominantly responsible for ESBL production was blaSHV.The levels of ESBLs produced in Milad Hospital, in Tehran, were much higher than those in other hospitals covered in this study.In all the hospitals, imipenem was used as an effective antibiotic against ESBL-producing K. pneumoniae.Resistance to ciprofloxacin, which was commonly seen in the cold seasons, was low, but resistance to cotrimoxazole and amikacin was found to be almost the same but higher than the resistance to ciprofloxacin.Strict antibiotic policies should be adopted in hospitals, in order to estimate the impact of higher resistance among bacteria and to take steps towards reducing this resistance.

Table 1 ,
while the antibiotic susceptibility pattern towards other antibiotic panels is shown in in hospitals in Ilam, Tehran and Tabriz, respectively.Out of the total number of K. pneumoniae isolates available for this study, 18.75% (n = 54), 12.84% (n = 37), 33.3% (n = 96) and 35.1% (n = 101) were isolated in the spring, summer, fall and winter, respectively.The number of K. pneumoniae isolates in different seasons (spring, summer, fall and winter) from different hospitals and the corresponding ESBL patterns are presented in

Table 2 .
The numbers of K. pneumoniae isolates were found to be high in the fall and winter seasons.

Table 1 .
Incidence of Klebsiella pneumoniae and antimicrobial susceptibility patterns for ESBL detection in different seasons

Table 2 .
Antimicrobial susceptibility testing on ESBL-producing Klebsiella pneumoniae in different seasons