A longitudinal assessment of antimicrobial susceptibility among important pathogens collected as part of the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) in France between 2004 and 2012

Background Clinically important Gram-positive and -negative isolates were collected from patients in France between 2004 and 2012 as a part of the Tigecycline Evaluation and Surveillance Trial. Methods MICs were determined using methodology described by the Clinical and Laboratory Standards Institute. Results In total, 17,135 isolates were contributed by 29 medical centres; respiratory (25.1%) and cardiovascular (20.3%) sources predominated. High susceptibility was observed among Enterococcus spp. and Staphylococcus aureus (including methicillin-resistant S. aureus [MRSA]) to linezolid (100%), tigecycline (≥99.8%) and vancomycin (≥94.6%). The percentage of MRSA decreased from 34.3% in 2004 to 20.0% in 2009 before increasing to 34.7% in 2012. Vancomycin, linezolid, levofloxacin and carbapenems were highly active (≥99.6%) against Streptococcus pneumoniae; 3.2% were PRSP. Escherichia coli showed peak susceptibility to the carbapenems (≥99.9%), tigecycline (99.3%) and amikacin (97.9%); significant (p < 0.01) decreases in susceptibility were observed for ampicillin, cefepime and ceftriaxone between 2004 and 2012. ESBL production among E. coli increased from 3.0% (2004) to 14.9% (2012). High susceptibility was noted among Haemophilus influenzae to levofloxacin (100%), amoxicillin-clavulanate (99.2%), carbapenems (≥98.7%) and ceftriaxone (98.5%); β-lactamase production fluctuated with no notable trend between 18.1% (2007) and 27.7% (2011). Klebsiella spp. were highly susceptible to carbapenems (≥99.6%) and amikacin (≥96.4%); significant (p < 0.01) decreases in amoxicillin-clavulanate, cefepime, ceftriaxone, levofloxacin, piperacillin-tazobactam and tigecycline susceptibility were observed among K. pneumoniae between 2004 and 2012. Only imipenem was highly active (96.5% susceptible) against Acinetobacter baumannii. Imipenem and amikacin (87.7% and 87.1% susceptible) were the most active agents against P. aeruginosa; 10.2% of isolates were categorized as multidrug resistant. Conclusions Carbapenems, linezolid, tigecycline and vancomycin conserved good in vitro activity against most pathogens (according to their spectrum of activity) in France between 2004 and 2012. Electronic supplementary material The online version of this article (doi:10.1186/2047-2994-3-36) contains supplementary material, which is available to authorized users.


Background
France is home to one of the highest rates of antibiotic consumption and antimicrobial resistance in Europe [1], and has experienced rapidly changing trends of antimicrobial resistance in recent years. The European Antimicrobial Resistance Surveillance Network (EARS-Net) has reported significantly increasing levels of resistance in France [2], where 10.8% of Escherichia coli and 23.7% of Klebsiella pneumoniae isolates were reported to be intermediate or resistant to third-generation cephalosporins in 2012 (as compared with 1.9% in 2002 and 5.1% in 2005, respectively) [3]. Several programmes have been initiated to combat these increasing levels of resistance, including measures to control transmission of resistant pathogens, to promote the use of alcohol-based hand-rub solution in hospitals, to control/prevent the spread of emerging multidrug-resistant (MDR) organisms (i.e., vancomycinresistant enterococci [VRE], carbapenemase-producing Enterobacteriaceae) and to decrease antibiotic consumption [4]. These efforts have paid at least some dividends: declining levels of antimicrobial resistance have been reported in recent years among French isolates of Streptococcus pneumoniae to penicillin (from 36.2% in 2005 to 23.4% in 2012) and Staphylococcus aureus to methicillin (from 33.4% in 2001 to 19.1% in 2012) [3].
Tigecycline is a broad-spectrum antimicrobial agent which has been indicated for use in the treatment of complicated skin and skin structure infections (cSSTIs) and complicated intra-abdominal infections (cIAIS) (and in the USA, community-acquired bacterial pneumonia) [5]. The Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) is a global surveillance study which commenced in 2004, with the intention of monitoring the activity of the broad-spectrum glycylcycline tigecycline and a panel of comparator agents against an array of clinically important Gram-positive and Gram-negative organisms. In this study, we examine the activity of tigecycline and comparators against clinically important Gram-positive and Gram-negative pathogens collected from community and nosocomial patients in France between 2004 and 2012. This manuscript serves as an update to Rodloff et al. [6], who described a collection of isolates from France, Germany, Italy, Spain and the U.K. collected as a part of T.E.S.T. between 2004 and 2006, as well as Nørskov-Lauritsen et al. [7], who presented data on European isolates (including France) collected between 2004 and 2007.

Methods
Between 2004 and 2012 there were 29 centres in France. The majority of these centres were university hospitals. No centres contributed in all 9 study years. Three centres contributed in 8 years, two in 7 years, four in 6 years, six in 5 years, three in 4 years, three in 3 years, five in 2 years, and three in a single year.

Bacterial isolates
Each centre was required to submit a minimum of 65 Gram-positive isolates and 135 Gram-negative isolates, including at least 25 S. aureus, 15 Enterococcus spp., 15 S. pneumoniae, 10 Streptococcus agalactiae, 25 Klebsiella spp., 25 E. coli, 25 Enterobacter spp., 20 Pseudomonas aeruginosa, 15 Acinetobacter spp., 15 H. influenzae and 10 Serratia spp. isolates. Each submitted isolate had to be considered by the contributing centre to be of clinical significance as the probable causative agent of a hospitalor community-acquired infection. All body sites were considered acceptable isolate sources for this study, including body fluid, central nervous system, cardiovascular system, gastro-intestinal, genito-urinary (no more than 25% of isolates from any centre), head, ears, eyes, nose and throat, integument, lymph, muscular, reproductive, respiratory, skeletal or medical instruments (i.e. catheters, drains, forceps, probes). No banked or stored isolates or duplicate isolates from a single patient were accepted into the T.E.S.T. study. Isolate inclusion was independent of patient age, sex, antimicrobial use and/or medical history.
All isolates were sent to a single reference laboratory, International Health Management Associates (IHMA, Schaumburg, IL), which was responsible for organism collection and transport and organism identification confirmation and development. IHMA also undertook creation and management of a centralized isolate database. Quality control (QC) checks were carried out by IHMA on approximately 10% of isolates annually.
Extended-spectrum β-lactamase (ESBL) production among E. coli and Klebsiella spp. was identified by IHMA using cefotaxime (30 μg), cefotaxime-clavulanic acid (30/10 μg), ceftazidime (30 μg), and ceftazidimeclavulanic acid (30/10 μg) discs [10]. A positive ESBL result was designated by an increase of ≥5 mm in the inhibition zone on the combination disc compared with the corresponding cephalosporin disc. Discs were manufactured by Oxoid, Inc. (Ogdensburg, NY, USA); Mueller-Hinton agar was produced by Remel, Inc. (Lenexa, KS, USA). H. influenzae isolates were tested for β-lactamase production using locally preferred methodologies. Multidrug resistance was defined as resistance to three or more classes of antimicrobial agent, and only included antimicrobials with available breakpoints. Daily QC testing was performed using QC strains Enterococcus faecalis ATCC 29212, S. aureus ATCC 29213, S. pneumoniae ATCC 49619, E. coli ATCC 25922, P. aeruginosa ATCC 27853 and H. influenzae ATCC 49247 and ATCC 49766, as appropriate. QC strains used for ESBL testing were K. pneumoniae ATCC 700603 (ESBLpositive) and E. coli ATCC 25922 (ESBL-negative), while P. aeruginosa (ATCC 27853) was used for the QC of ceftazidime and cefotaxime discs. Information on T.E.S.T. study protocols can be found online [5].
Longitudinal data were examined for statistically significant changes in susceptibility between 2004 and 2012 using the Cochran Armitage Trend Test. A positive change reflected a statistically significant decrease in susceptibility, while a negative change indicated that susceptibility had increased significantly. A p < 0.01 was used in this analysis as a cut-off value for statistical significance (a significance value of p < 0.05 was not used here as computing a high volume of statistical tests can lead to significant results purely by chance; setting a lower significance value greatly reduces the chance of this happening).

S. aureus
All (N = 2229) S. aureus isolates were susceptible to linezolid, tigecycline and vancomycin, including MRSA isolates, while 93.7% were susceptible to minocycline ( Table 1). The percentage of S. aureus identified as MRSA in France decreased from 34.3% in 2004 to 20.0% in 2009, but increased to 34.7% in 2012; the average MRSA rate over the total course of the study was 28.3% (Table 3). There was a statistically significant (p < 0.001) increase in minocycline susceptibility among MRSA over the study duration (Table 2). Methicillin resistance had no impact on the activity of linezolid, minocycline, tigecycline or vancomycin.
S. agalactiae S. agalactiae (N = 859) were highly susceptible to most agents on the TEST panel where breakpoints exist, the notable exception being minocycline (against which only 15.4% of isolates were susceptible) ( Table 1).

S. marcescens
The most active antimicrobial agents in this study against S.  (Table 3).

Discussion
This report updates data previously presented by Rodloff et al. [6] for France (as well as Germany, Italy, Spain and the U.K.) between 2004 and 2006 and Nørskov-Lauritsen et al. [7] for data collected between 2004 and 2007. The data described in their reports are included in the dataset described in this manuscript. Susceptibility results are difficult to compare between these two earlier reports and the current study as CLSI interpretive breakpoints were used in Rodloff et al. [6] and Nørskov-Lauritsen et al. [7] while EUCAST breakpoints have been used in the current manuscript. No vancomycin-resistant enterococci were reported in either earlier study in France; however, small percentages of vancomycin-resistant E. faecalis (0.7%) and E. faecium (5.4%) were collected in the current study. As the data show, the majority of vancomycin-resistant enterococci were collected during or after 2008 (three isolates were collected in 2006 and 2007 but were not reported by Rodloff et al. [6] and Nørskov-Lauritsen et al. [7] as they were entered into the database after the data cut-offs for these publications). Rates of MRSA were comparable between the three reports (28.3% in the current study, 28.3% in Rodloff et al. [6], and 31.5% in Nørskov-Lauritsen et al. [7]); however, the rate of penicillinresistant S. pneumoniae was lower in the current study when compared with Nørskov-Lauritsen et al. [7] (3.2% and 16.8%, respectively). No S. pneumoniae data was presented by Rodloff et al. [6]. This difference is likely due in part to the use of CLSI breakpoints by Nørskov-Lauritsen et al. (resistance breakpoint ≥2 mg/L, compared to ≥4 mg/L used by EUCAST); the removal of 236 S. pneumoniae isolates from the T.E.S.T. database whose MICs could not be verified (i.e., isolates which could not be revived for retesting or which died on transport from the contributing centre to IHMA) may have also influenced this PRSP difference.
ESBL production among E. coli and K. pneumoniae was higher in the current study; 12.0% and 18.0% compared with 4.9% and 9.5% and 5.1% and 9.8% in Rodloff et al. [6] and Nørskov-Lauritsen et al. [7], respectively. As rates of ESBLs were higher in the later years of this study (2008 onwards) this difference is not unexpected. Rates of multidrug-resistant A. baumannii and β-lactamase producing H. influenzae were similar between the current report and Nørskov-Lauritsen et al. [7]. (approximately 5% and 22%, respectively), although the definition of MDR A. baumannii in Nørskov-Lauritsen et al. [7]. also included cephalosporins. Data on multidrug-resistant A. baumannii and H. influenzae were not reported by Rodloff et al. [6]. As the isolates presented by Rodloff et al. [6] and Nørskov-Lauritsen et al. [7] are also included in this report comparisons between these three reported must be treated with some caution. However, the increases in rates of vancomycin-resistant enterococci, and ESBL-producing E. coli and K. pneumoniae are cause for concern and warrant further monitoring.
One factor that could influence the difference in resistance rates between the reports is the presence of centre specific outbreaks. Outbreaks of resistant pathogens have been described in several medical centres in France in recent years, caused by carbapenemase-producing [11] or metallo-β-lactamase-producing K. pneumoniae [12], MDR A. baumannii [13], glycopeptide-intermediate S. aureus Table 3 Percentages of resistant phenotypes among Gram-positive and Gram-negative isolates by year, 2004-2012   Pathogen  2004  2005  2006  2007  2008  2009  2010  2011   [14] and vancomycin-resistant enterococci [4,15]. These outbreaks were controlled with infection control measures, including strict enforcement of hygiene precautions, limiting transfer of patients to other wards, isolating infected patients with dedicated staff and the closure of infected wards. These outbreaks of highly resistant pathogens reinforce the clinical importance of antimicrobial agents such as tigecycline, daptomycin, linezolid, and vancomycin, which often retain excellent in vitro activity against even highly resistant pathogens [16,17]. As a result of a resistance control programme started in 2003 in 38 French teaching hospitals, vancomycinresistant enterococci and carbapenemase-producing Enterobacteriaceae cases were controlled while MRSA incidence declined by two thirds; however, a dramatic increase in the percentage of ESBL-positive Enterobacteriaceae was noted [4]. Similarly, a long-term study involving 933 health care facilities carried out by the French national healthcare-associated infection early-warning, investigation and surveillance network (RAISIN) led to a 43% decrease in MRSA while ESBL-positive Enterobacteriaceae increased by 182% [18]. The epidemiology of ESBLproducing pathogens can be very complex [19], and ESBL-positive Enterobacteriaceae are increasing in prevalence so rapidly that they may soon become the most widespread MDR pathogens in French hospitals [20]. ESBL levels among E. coli and K. pneumoniae increased markedly over the course of the T.E.S.T. study; however, MRSA levels in the current study decreased between 2004 (34.3%) and 2009 (20.0%) but increased from 2011 (23.4%) to 2012 (34.7%). This increase in MRSA levels was unexpected and may have been due to regional factors such as localised outbreak(s) of resistant isolates.
In a review of data collected by the Pneumococcus Surveillance Network (PSN) in France in 2007, Kempf et al. [21] reported a PRSP percentage of 6.6% among S. pneumoniae isolates collected from adults and children. This PRSP occurrence is twice that recorded in the current manuscript for France between 2004 and 2012, and three times higher than the value reported in T.E.S.T. for 2007 alone. This difference is due in part to Kempf et al. [21] using a resistance breakpoint of >1 mg/L for penicillin, compared with ≥4 mg/L used in this T.E.S.T. study. Sizeable (>20%) regional variations in the prevalence of penicillin-non-susceptible S. pneumoniae and a high number of isolates collected from children (27.9%) were also reported by Kempf et al. [21].
Tigecycline and linezolid demonstrated good activity against the Gram-positive isolates in this study. In the case of enterococci the activity of tigecycline and linezolid has also been demonstrated by others [15,22,23]. Bourdon et al. [15] performed susceptibility testing on 602 E. faecium and 30 E. faecalis isolates, all VRE, collected from 112 French hospitals between 2006 and 2008 and observed 100% susceptibility to tigecycline and linezolid. Similarly, Marcadé et al. [22] described seven glycopeptideresistant E. faecium isolates from a single hospital in Paris which possessed both vanA and vanB resistance genes; all were susceptible to tigecycline and linezolid, as well as daptomycin. Bérenger et al. [23] examined 60 glycopeptide-resistant, epidemiologically unrelated clinical isolates of E. faecium collected in France between 2006 and 2008; all were susceptible to linezolid while 59 were tigecycline-susceptible (the remaining isolate had intermediate susceptibility for tigecycline).
In the current T.E.S.T. report, the levels of β-lactamase positive isolates of H. influenzae fluctuated year-on-year (between 18.1% and 27.7%) between 2004 and 2012, with no discernible pattern over time. A statistically significant decrease in the occurrence of β-lactamase-positive, ampicillin-resistant isolates among non-typeable H. influenzae was reported in France between 2001 and 2008 [24], with the rate decreasing from 35.6% in 2001-02 to 13.5% in 2007-08; however, this study only included isolates collected from patients ≤5 years in age.

Conclusions
Programmes aimed at controlling and/or reducing the prevalence of drug-resistant pathogens in France have been successful against some important pathogens, such as MRSA and VRE, but other resistant pathogens continue to increase in prevalence across the country, most notably ESBL-positive Enterobacteriaceae. These trends highlight the importance of surveillance studies such as T.E.S.T., which monitor pathogen resistance rates against key antimicrobial agents both nationally and globally. Tigecycline possesses good in vitro activity against many resistant pathogens, including ESBL producers, and thus could be a useful tool in the treatment of resistant infections in France in the future.
No authors were paid for their contributions to this manuscript. Medical writing support was provided by Dr. Rod Taylor at Micron Research Ltd, Ely, UK and was funded by Pfizer Inc. Micron Research Ltd also provided data management services which were funded by Pfizer Inc.