Antimicrobial Resistance to Cefotaxime and Ertapenem in Enterobacteriaceae: the Effects of Altering Clinical Breakpoints

Introduction: The Clinical and Laboratory Standards Institute (CLSI) updated its antimicrobial susceptibility testing interpretation criteria for Enterobacteriaceae. This study assessed the effects of clinical breakpoint changes in the CLSI 2009 to 2012 guidelines on antibiotic susceptibility testing reports. Methodology: In total, 2,076 non-duplicate clinical isolates of Enterobacteriaceae were analyzed. The disk diffusion method was used for susceptibility testing. The CLSI 2009-12 clinical breakpoints were applied to determine susceptibility of cefotaxime and ertapenem. Combined-disk testing was used for phenotypic confirmation of extended-spectrum beta–lactamase (ESBL) production. Conclusions: Breakpoints changes in the updated CLSI guidelines resulted in higher resistance rates to cefotaxime and ertapenem. In addition, the effects were different in individual Enterobacteriaceae species. As a result, clinicians may opt to use alternative antimicrobial agents. Upon implementation of the newer CLSI guidelines, laboratories should be aware of the possible consequences and closely monitor the effects.


Introduction
Antimicrobial susceptibility testing is one of the most important tasks in clinical microbiology laboratories.It is not only a valuable guide for antibiotic therapy but also an important epidemiological tool to monitor resistant organisms.
Hence, antimicrobial susceptibility testing should be performed according to generally accepted and clinically relevant guidelines.
Several laboratories, particularly in countries without a national antibiotic susceptibility testing system, have been using the Clinical and Laboratory Standards Institute (CLSI) guidelines to interpret antibiotic-sensitivity testing.Recently, the CLSI updated and included its recommendations into the CLSI 2010, 2011 and 2012 guidelines, which include major changes in the antibiotic susceptibility testing for Enterobacteriaceae.Inhibition zone diameter breakpoints for Enterobacteriaceae defining susceptibility to third-generation cephalosporins and carbapenems are significantly higher in the CLSI 2010/11 version compared to the CLSI 2009 breakpoints [1][2][3].Recently, the CLSI recommended slightly lower zone diameter susceptibility breakpoints for ertapenem in its 2012 updates, while breakpoints for third-generation cephalosporins remained unchanged [4].In addition, with the increase of cephalosporin breakpoints in the CLSI 2010-12 guidelines, it is no longer necessary to detect extended-spectrum beta-lactamase (ESBL) producers and to adjust all cephalosporin sensitivity results to resistance, as previously recommended by CLSI 2009 guideline.
Nevertheless, a controversy arose with regard to the appropriateness of current recommendations.Some investigators continue to support the importance of knowing the ESBL status in an isolate to ensure proper therapy [5,6].Especially in ESBL-endemic areas, where therapeutic options are very limited due to associated co-resistance in other classes of antimicrobial agents [7,8].Moreover, in a survey on 144 infectious disease specialists, most respondents reported that they would not treat Enterobacteriaceae infections based on the current breakpoints alone without testing for ESBL [9].Some experts even suggested that clinical laboratories should not use the most recent CLSI breakpoints [9,10].
Adoption of new guidelines and breakpoints can have significant effects on antibiotic susceptibility testing reports, with concomitant changes in antibiotic prescriptions by clinicians.Prior to the implementation of new guidelines in diagnostic microbiology laboratories, the impact of changes in antibiotic susceptibility testing reports needs to be evaluated.
This study compared the new standards with respect to changes in cefotaxime and ertapenem among Enterobacteriaceae isolates.

Clinical isolates
Between March 2011 and June 2011, the study collected consecutive non-duplicate clinical isolates of Enterobacteriaceae in our clinical microbiological laboratory.Our clinical laboratory primarily serves a 1500-bed veterans hospital in central Taiwan.Identification to the species level was performed by routine laboratory methods including the Vitek 2 system (bioMérieux, Marcy l'Etoile, France).

Susceptibility testing
The Kirby-Bauer disk diffusion test was used for susceptibility testing.Cefotaxime and ertapenem were tested during the study period.Antibiotics discs were obtained from Becton Dickinson (BD Diagnostic Systems, Cockeysville, USA).Susceptibility testing was done on Mueller-Hinton agar (BD Diagnostic Systems, Cockeysville, USA) using McFarland 0.5 from overnight cultures followed by incubation at 35℃ for 16-18 hours.Inhibition zone diameters were determined and recorded by two experienced microbiologists.

Comparison of CLSI 2009-2012 breakpoints
Inhibition zone diameters were interpreted according to the CLSI 2009-12 recommended zone diameter breakpoints for Enterobacteriaceae (Table 1).The CLSI 2009 susceptibility breakpoint for cefotaxime was ≥ 23 mm which increased to ≥ 26 mm based on the CLSI 2010-12 guidelines.The CLSI 2009 susceptibility breakpoint for ertapenem was ≥ 19 mm using CLSI 2009, which increased to ≥ 23 mm using CLSI 2011 and reduced to ≥ 22 mm with CLSI 2012.Rates of susceptibility to cefotaxime and ertapenem measured using 2009 CLSI breakpoints were compared with those obtained using 2010-12 CLSI breakpoints [1,3,4].

E. coli
As observed in the trends of Enterobacteriaceae, resistance rate to cefotaxime increased from 13.8% with CLSI 2009 to 27.0% with CLSI 2010-12.Resistance rates to ertapenem remained at 0%.

E. cloacae, E. aerogenes
The resistance rate to cefotaxime in E. cloacae and E. aerogenes increased from 16.5% and 17.1% to 22.8% and 29.3% with CLSI 2009 and CLSI 2010-12, respectively.The resistance rates to ertapenem in E. cloacae and E. aerogenes changed from 0% with CLSI 2009 to 2.4% and 4.9% with CLSI 2011 and to 1.6% and 2.4% with CLSI 2012, respectively.
Depending on the bacterial species, there were different effects on susceptibility.Similar susceptibility patterns were noted for both ESBLpositive and ESBL-negative E. coli (Table 3

Discussion
In this study, we found that breakpoint changes in the CLSI guidelines resulted in higher resistance rates to cefotaxime and ertapenem.In addition, the effects vary among individual Enterobacteriaceae species.
Our findings, in agreement with other studies, suggest that the application of new CLSI standards results in significantly more Enterobacteriaceae being reported resistant to extended-spectrum cephalosporins and carbapenems [12].As a consequence, clinicians will most likely prescribe other antimicrobial classes.As the amount of antibiotic used and the development of resistance are generally linked, increased reports of Enterobacteriaceae resistant to third-generation cephalosporins and carbapenems may cause greater usage of other antibiotics and changes in antibiograms [13,14].Further clinical studies are required to determine the long-term effects on the antimicrobial stewardship policy and the trends in antibiotic resistance.
In addition, application of higher diameter breakpoints will increase the number of multipledrugresistant Enterobacteriaceae, which is defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories, including carbapenems and extended spectrum cephalosporins [15].With the implementation of CLSI 2010-12 guidelines, Enterobacteriaceae will increasingly be reported as multidrug-resistant and carbapenemresistant, resulting in higher rates of patient isolation as well as higher costs arising from increased hospital hygiene measures and the requirement for confirmatory tests in the laboratory for multidrugresistant Enterobacteriaceae.
The effects of the breakpoints changes from CLSI 2009 to CLSI 2010-2012 differ among Enterobacteriaceae species: for instance, while the resistance rate for cefotaxime in K. oxytoca remains almost unchanged, isolates of E. coli are more frequently reported as resistant to cefotaxime using the CLSI 2010/2011 criteria (Table 2).This finding is probably related to the different distribution in natural diameter among individual Enterobacteriaceae species as compared with the breakpoints, which are defined for the whole Enterobacteriaceae family (Figures 1a  and 1b).For instance, 13.2% of E. coli had intermediate susceptibility to cefotaxime using the CLSI 2009 guideline, and were classified as resistant in the 2010-12 guidelines.In contrast, none of the K. oxytoca isolates had a zone diameter of 15-22 mm.Regarding the uniform resistant breakpoint definitions for both drugs and species, the lower average diameters of E.coli readings explain the greater increase in cefotaxime resistance rates compared to those of K. oxytoca.Other investigators also found a more limited change in the susceptibility to cefotaxime among K. oxytoca between the CLSI 2009 and CLSI 2010-12 recommendations [6].We suggest that species-adapted breakpoints for Enterobacteriaceae species would eliminate artifacts and improve interpretation of antibiotic susceptibility testing.
The CLSI 2010 guidelines have led to a fundamental change of antibiotic susceptibility testing results for ESBL-producing isolates.With the updated interpretive criteria, it is no longer necessary to perform routine ESBL testing and edit results in confirmed ESBL-producers.The adoption of the new CLSI guidelines raises the question on how sensitive and specific CLSI 2010-12 breakpoints are for thirdgeneration cephalosporins in the detection of ESBL.Overall, the study shows that CLSI 2010-12 breakpoints successfully designated a larger number of ESBL-positive isolates as cefotaxime-resistant.Other studies also demonstrated the high sensitivity of the CLSI ESBL screening breakpoint for cefotaxime [16].
Implementation of the CLSI 2011/12 interpretive criteria may have increased the carbapenem resistance rate in some Enterobacteriaceae species.Generally, ertapenem were active against more than 90% of Enterobacteriaceae isolates; however, there were wide variations in the ertapenem susceptibility between Enterobacteriaceae species in this study.Previous studies showed that the rate of ertapenem resistance in Enterobacteriaceae isolates worldwide was low based on the CLSI 2011 criteria [17].More recently, Huang et al. reported that the resistance rate may be higher in some Enterobacteriaceae species and certain countries [18].In our study, only 77.4% of ESBL-positive K. pneumoniae isolates were susceptible to ertapenem according to the CLSI 2011 criteria, while ertapenem was active in more than 99% of E. coli isolates.Some Enterobacteriaceae species demonstrated a substantial change with different criteria.The rates of susceptibility to ertapenem among Enterobacteriaceae as measured by the CLSI 2009 and 2011 criteria differed by more than 5% among ESBL-positive K. pneumoniae (15.1%),E. cloacae (9.4%), C. freundii (6.3%) and E. cloacae (7.4%); however, when the results were interpreted by the CLSI 2012 criteria, only ESBL-positive K. pneumoniae differed by more than 5%.There are wide variations in the speciesdependent reduction in susceptibility among countries.In Hong Kong, Proteus spp., Enterobacter spp., Providencia spp.and Morganella spp.had the greatest reduction in the imipenem susceptibility rate with the new CLSI breakpoints [19].
This study was limited to the moderate to high antimicrobial resistance found in Taiwan [20].The increase in resistance rates due to changes in the guidelines here reported, may be more obscure compared to changes seen in populations with lower levels of resistance, since diameter distributions are shifted to lower mean values.Another limitation of this study was the inclusion of only cefotaxime and ertapenem.In order to better understand the importance of other antibiotics in clinical practice, more studies are needed to investigate the impact of the breakpoint change in a complete antibiogram.Furthermore, in this study the underlying resistance mechanisms were not identified.Some studies have reported data suggesting that different resistance mechanisms can have varying effects on categorization based on the old and new interpretive criteria [21,22].In addition, the identification of ESBL production was performed by using only the combination disk diffusion method.Some ESBLproducing organisms may be missed with the phenotypic confirmatory test.The application of genotypic confirmatory tests or Etest ESBL strips is useful for in vitro confirmation of ESBL [23].

Conclusions
Widespread implementation of the new CLSI standards for antibiotic susceptibility testing make results more comparable worldwide.However, further evidence-based clinical studies should validate the proposed guideline changes.In the implementation stage of the new CLSI standards, laboratories and clinicians need to be aware of the implications of modified antibiotic susceptibility testing reports in clinical practice, such as the effects on antibiotic prescription.In addition, to ensure early detection of emerging resistance, species-related zone breakpoints should be considered.

Figure 1 .
Figure 1.Distribution of zone diameters for cefotaxime and CLSI clinical breakpoints in E. coli (Fig 1a) and Klebsiella oxytoca (Fig 1b).Vertical lines represent the zone diameter breakpoints defined in the CLSI 2009 and CLSI 2010 guidelines.

Table 1 .
CLSI 2009-12 susceptibility testing breakpoints of Enterobacteriaceae Categories without value: interpretation guidelines were not changed compared to the previous version.S, susceptible; I, intermediate; R, resistant.

Table 2 .
(continued) Susceptibility of Enterobacteriaceae clinical isolates according to CLSI 2009-12 S, susceptible; I, intermediate; R, resistant; Categories without value: interpretation guidelines were not changed compared to the previous version.
).Only minor reductions in susceptibility of E. coli to cefotaxime and ertapenem were observed when comparing CLSI 2009 and CLSI 2010-12 breakpoints.However, the rates of susceptibility to cefotaxime and ertapenem among K. pneumoniae changed to a greater extent, particularly in ESBL-positive K. pneumoniae.Susceptibilities of cefotaxime against ESBL-positive K. pneumoniae were 3.7% and 0% for CLSI 2009 and CLSI 2010 breakpoints, respectively.Susceptibilities of ertapenem against ESBL-positive K. pneumoniae were 92.5%, 77.4%, and 81.1% for CLSI 2009, CLSI 2010 and CLSI 2012 breakpoints, respectively.In addition, three of the 40 K. oxytoca isolates and two of the 117 P. mirabilis isolates were ESBL-positive.One of the three ESBL-positive K. oxytoca isolates were susceptible to cefotaxime with CLSI 2009 breakpoints and became intermediate susceptible to cefotaxime with CLSI 2010-12 breakpoints.Two ESBL-positive P. mirabilis isolates were intermediate susceptible to cefotaxime with CLSI 2009 breakpoints and were classed as resistant with CLSI 2010-12 breakpoints.All ESBL-positive K. oxytoca and P. mirabilis isolates were susceptible to ertapenem.