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Comparison of susceptibility of Enterobacteriaceae causing community-onset urinary tract infection to isepamicin and amikacin by the disc diffusion method.

Byline: Yung-Chih. Wang, Ya-Sung. Yang, Ti-Yin, Shu-Chen. Kuo, Jung-Chung. Lin, Feng-Yee. Chang

Background: Enterobacteriaceae, common pathogens responsible for urinary tract infections are known to be susceptible to aminoglycosides. The emergence of resistant pathogens complicates antimicrobial regimen and becomes a challenge for clinicians. The aim of this study was to evaluate the susceptibility of clinically isolated Enterobacteriaceae to isepamicin and amikacin using disc diffusion method. Materials and Methods: A total of 234 Enterobacteriaceae isolates was collected and examined. Antimicrobial susceptibilities to gentamicin, amikacin, and isepamicin were assessed using disc diffusion method. The production of extended-spectrum [sz]-lactamase (ESBL) or AmpC [sz]-lactamase was also tested. The susceptibilities of the pathogens to isepamicin and amikacin were evaluated. Results: Two hundred and thirty-four Enterobacteriaceae isolates were found to be more susceptible to amikacin and isepamicin than to gentamicin. Of the isolates, 39 (16.7%) produced ESBL and 41 (17.5%) harbored AmpC [sz]-lactamase. The results revealed that amikacin and isepamicin exerted excellent antibacterial activity (94% vs. 93.6%) against all tested isolates. Isepamicin was effective against 89.7% ESBL-producing isolates and 92.7% of AmpC-producing isolates. The susceptibility to amikacin and isepamicin established by the disc diffusion method was mostly consistent with the overall agreement estimated 99.6%. Conclusions: Isepamicin showed excellent activities against infections caused by Enterobacteriaceae, including strains harboring ESBL or AmpC beta-lactamase. The susceptibility of tested isolated to isepamicin measured by disc diffusion method is comparable to that of amikacin.


Urinary tract infections (UTI) are among the most common bacterial infections in adults. [sup][1] Enterobacteriaceae have been reported to be the predominant pathogens causing UTI. [sup][1] Emergence of resistant enteric bacteria has significant clinical impact in mortality, length of hospital stay, and cost of therapy. [sup][2] The increasing incidence of UTI caused by extended-spectrum [sz]-lactamase (ESBL) or AmpC [sz]-lactamase producing Enterobacteriaceae has been of concerns to infection control practitioners and clinical microbiologist. [sup][2],[3],[4],[5],[6] The prevalence of ESBL and AmpC [sz]-lactamase among uropathogen has increased in last 10 years in the many geographical regions of the globe. [sup][3],[4],[5],[6]

Aminoglycosides are commonly prescribed for the infections caused by Gram-negative bacteria such as Enterobacteriaceae . Isepamicin, a broad-spectrum aminoglycoside, is mainly used in Asian and certain European countries. [sup][7],[8] It is effective against Gram-negative bacilli, including drug-resistance bacterial isolates, with similar susceptibility to amikacin. [sup][7],[8],[9] In Taiwan, a recent research has reported an increase in the resistance rates of Gram-negative pathogens to isepamicin that was introduced more than 10 years ago. [sup][10] It is suggested that antibiotic resistance pattern may change significantly in a certain period. The susceptibilities of clinical isolates to isepamicin require an in-depth re-evaluation for accuracy and effectiveness in a clinical setting.

Owing to the large number of patients hospitalized with UTI and to limited resources, disc diffusion with selected antimicrobial discs is used for routine susceptibility testing instead of automated microbroth dilution. However, the in vitro data of susceptibility testing of isepamicin using the antimicrobial discs are unavailable. It is not known whether the susceptibility to isepamicin, determined using the disc diffusion method, was comparable to susceptibility to amikacin among recent isolates. The aim of this study was to evaluate and compare the susceptibilities of clinical isolates from community-onset UTI to isepamicin and amikacin, using the disc diffusion method.

Materials and Methods

Study design and settings

This prospective study was conducted during January 2011 and December 2011 at the Taipei Veterans General Hospital, a 2900-bed tertiary-care teaching hospital located in Taipei, Taiwan. The criteria for UTIs were clinical signs and/or symptoms of UTIs (e.g., fever >38[degrees]C, frequency, urgency, dysuria, or suprapubic tenderness) without other well-identified cause; pyuria (i.e., urine specimen with ≥10 white blood cells/mm [sup]3 ) and a positive urine culture (≥1 x 10 [sup]5 CFU/mL of a uropathogen). Participants who were diagnosed with first-episode of UTI caused by Enterobacteriaceae identified by VITEK 2 system (bioMe?rieux, Marcy l'Etoile, France) were enrolled. Community-onset infection was defined as an infection diagnosed within 48 h after admission. Pregnant or lactating women and patients with complete obstruction of the urinary tract, perinephric or intrarenal abscess, and prostatitis were excluded. Of the Enterobacteriaceae species that were >50 isolates, 50 were randomly chosen and tested.

Bacteriology and antimicrobial susceptibility testing

Antimicrobial susceptibilities to gentamicin, amikacin, and isepamicin were assessed using disc diffusion method and interpreted according to the manufacturer's instructions and the guidelines of the Clinical and Laboratory Standards Institute (CLSI), [sup][11] respectively. ESBL production screening was performed using the CLSI ESBL confirmatory test with discs containing cefotaxime (CTX; 30 [micro]g) and ceftazidime (CAZ; 30 [micro]g), with and without clavulanic acid (CLA; 10 [micro]g) on Mueller-Hinton agar plates. [sup][12] A ≥5 mm increase in the diameter of inhibition zone in the presence of CLA confirmed phenotypically ESBL production. Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were used as respectively negative and positive controls, as well as routine quality controls. As AmpC production could interfere ESBL detection, a modified disc potentiation test using 3-aminophenylboronic acid (APB; 400 [micro]g) was conducted to validate ESBL production in isolates that harbor plasmid-mediated or chromosomal AmpC. [sup][13],[14] A ≥5 mm increase in the inhibition zone diameter of CTX/CLA and/or CAZ/CLA discs tested in combination with APB (CTX/CLA/APB and/or CAZ/CLA/APB) versus CTX and/or CAZ discs containing APB (CTX/APB and/or CAZ/APB) was considered positive for ESBL production. Similarly, a ≥5 mm increase in the inhibition zone diameter of CTX/CLA and/or CAZ/CLA discs tested in combination with APB (CTX/CLA/APB and/or CAZ/CLA/APB) versus CTX/CLA and/or CAZ/CLA discs was considered positive for AmpC production.

Category agreement definition

All isolates were tested in parallel for susceptibility to isepamicin and amikacin. For categorical agreement, using amikacin as the standard, very major errors were defined as those in which a susceptible result was obtained for amikacin and a resistant result was obtained for isepamicin, major errors were defined as those in which a resistant result was obtained for amikacin and a susceptible result was obtained for isepamicin, minor errors were defined as those in which result for one of the agents was intermediate and the other agent was susceptible or resistant.


Antimicrobial susceptibility of the studied isolates

A total of 234 Enterobacteriaceae isolates was obtained and examined in this study. E. coli , K. pneumoniae , and Proteus mirabilis were the major pathogens tested (50 of 234 isolates, 21.4%), followed by Serratia marcescens (19 of 234 isolates, 8.1%). Isepamicin and amikacin exhibited similar antimicrobial activity (94% vs. 93.6%) with excellence against tested isolates except one ESBL-producing E. coli isolate [Table 1]. The susceptible rate of K. pneumoniae to the both antibiotics was 84%. Our data showed that Providencia stuartii exhibited a resistance rate of 33.3% to amikacin and isepamicin. 89% of ESBL-producing isolates were susceptible to amikacin and isepamicin. Noticeably, the susceptibility of ESBL-producing isolates to gentamicin was only 69.2% (27 of 39 isolates) [Table 2]. The results revealed that the susceptibility rates of AmpC-producing isolates to amikacin and isepamicin were 92.7% whereas 73.2% of isolates were susceptible to gentamicin [Table 3].{Table 1}{Table 2}{Table 3}

Category agreement

The overall agreement between amikacin and isepamicin was 99.6% [Table 4]. The Pearson correlation coefficient between amikacin and isepamicin was 0.94. The very major error rate was 0.42%, whereas the major and minor error rates were zero. There was one ESBL-producing strain interpreted as susceptible to amikacin, but resistant to isepamicin. When isepamicin was used as a standard, the major error rate was 0.42% and very major and minor errors resulted in nil.{Table 4}


Community-onset UTIs account for more than 8 million physician visits and over 1 million hospital admissions in the United States annually. [sup][15] The microbial spectrum of uncomplicated UTIs consists mainly of E . coli (75-95%), with occasional other species of Enterobacteriaceae , such as P. mirabilis and K. pneumoniae , and Staphylococcus saprophyticus. [sup][2],[3],[15] Other Gram-negative and Gram-positive species are rarely isolated in uncomplicated UTIs. Current guidelines recommend empirical treatment with a drug before the availability of its in vitro activity against the uropathogen. [sup][3],[16] These therapeutic strategies are based on the narrow and predictable spectrum of etiologic pathogens and their susceptibility patterns. Since the resistance patterns of uropathogens causing uncomplicated UTIs vary considerably between regions and countries, a specific treatment recommendation may not be universally suitable for all regions or countries. Therefore, local antimicrobial susceptibility patterns of uropathogens in particular should be considered in empirical antimicrobial selection for uncomplicated UTIs. [sup][3],[16]

Aminoglycosides are high potent, broad-spectrum antibiotics widely used for the treatment of life-threating infections, including the UTIs. [sup][9],[17] The emergence of resistant strains has reduced the significance of aminoglycosides in empiric therapies. [sup][9],[17] Amikacin and isepamicin, lately developed aminoglycosides are active against strains that have developed resistance towards earlier aminoglycosides. Among the various resistance mechanisms, the aminoglycoside-modifying enzymes play a critical role in aminoglycoside resistance. [sup][9],[17] There are three major types of aminoglycoside-modifying enzymes, the N -acetyltransferases (AACs), O -nucleotidyltransferases, and O -phosphotransferases. [sup][8],[9],[17] The different aminoglycoside-modifying enzymes produced by the pathogens contribute to the resistance of the pathogens to each kind of aminoglycoside. The type 1 6'-AAC has been reported to confer around 30% of the total resistance to aminoglycosides in the USA and the Europe, particularly among the Enterobacteriaceae . [sup][8],[9],[17] Isepamicin, one of the latest aminoglycosides, is moderately inactivated by type 1 6'-AAC and stable to the type 2 6'-AAC. [sup][8],[9],[17] This may explain the relatively better activity of isepamicin in comparison to other aminoglycoside antibiotics. [sup][9]

Isepamicin is a widely used newly developed aminoglycoside in numerous Asian countries ad some European countries; however, the relevant clinical data are limited. It has been reported to be active in vitro against Gram-negative bacteria that are resistant to amikacin and other aminoglycosides. [sup][7],[9] In the present study, isepamicin was shown to exert excellent activity against most of the tested isolates. Among enteric bacteria, K. pneumoniae and P. stuartii were found to be more resistant to isepamicin in comparison with amikacin and gentamycin. However, further researches are needed to investigate the antibiogram of clinically available bacteria to isepamicin. There were several reports describing the antimicrobial activity of isepamicin against the resistant strains in Taiwan [sup][18],[19],[20],[21] [Table 5]. A recent study conducted in 2003 reports that the isepamicin has significant antimicrobial activity against Enterobacteriaceae (susceptibility 78-97%). [sup][18] Moreover, isepamicin has been reported to be effective against 81.3% of ESBL-producing E. coli and 73.3% of ESBL-producing K. pneumoniae. [sup][19] It is concluded that isepamicin is highly active against blood isolates of Enterobacteriaceae . In Taiwan, the ESBL and AmpC beta-lactamase producing Enterobacteriaceae have emerged as important pathogens in past few years. [sup][10] Whether isepamicin has activity against these resistant strains is unknown. Our results show that isepamicin exerts excellent antimicrobial activities against resistant strains harboring ESBL or AmpC beta-lactamase. It is suggested that isepamicin represents a therapeutic option against the drug-resistance Enterobacteriaceae infections.{Table 5}

Disc diffusion testing is known for its feasibility and cost-effectiveness. [sup][11] In comparison with minimal inhibitory concentration testing, it is especially suitable for common pathogens, such as those associated with UTIs. Currently, limited clinical data are available for assessing the susceptibilities of isepamicin using disc diffusion, leading to restricted clinical use of isepamicin. The results of disc diffusion test revealed a high correlation between amikacin and isepamicin. It is suggested that the susceptibility of bacteria to amikacin may be considered as guidance on the use of isepamicin against infections.

The present study, to our best knowledge, is the first study describing the efficiency of isepamicin and amikain against Enterobacteriaceae species using the disc diffusion method. We also demonstrated the excellent activity of isepamicin against the drug-resistant Enterobacteriaceae isolated from UTIs. There are few limitations of this study. First of all, this single hospital study with relative small number of isolates could not indicate the real prevalence of resistance pattern of Enterobacteriaceae in the region. Second, all the isolates in our study were obtained from the UTI. The results of susceptibility of the antibiotics against the isolates may differ from those obtained from different infectious origins.


The present study shows that isepamicin could be a good choice against infections caused by Enterobacteriaceae including resistant strains. The susceptibility results to amikacin and isepamicin examined using disc diffusion method are comparable, suggesting that isepamicin is interchangeable with amikacin due to high agreement among the isolates from community-onset UTIs.


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Title Annotation:Original Article
Author:Wang, Yung-Chih; Yang, Ya-Sung; Ti-Yin; Kuo, Shu-Chen; Lin, Jung-Chung; Chang, Feng-Yee
Publication:Journal of Medical Sciences
Article Type:Report
Geographic Code:9TAIW
Date:Nov 1, 2014
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