Activity of temocillin against ESBL-, AmpC-, and/or KPC-producing Enterobacterales isolated in Poland

We evaluated the in vitro effectiveness of temocillin and several commonly used antimicrobials against Enterobacterales bacteria in isolates from Polish patients. We tested 400 isolates: 260 extended-spectrum β-lactamase (ESBL)- and/or ampC β-lactamase (AmpC)-producing isolates; 40 Klebsiella pneumoniae carbapenemase (KPC)-producing isolates; and 100 ESBL-, AmpC-, and KPC-negative isolates. The minimal inhibitory concentrations (MICs) of temocillin and 16 other antimicrobials were determined by reference microdilution. We also determined the activities of fosfomycin and ceftazidime/avibactam in KPC-producing isolates. The antibiotic sensitivities were interpreted according to EUCAST, BSAC, and CLSI criteria. Overall, 91% of the isolates were susceptible to temocillin using the urinary tract infection breakpoint (≤ 32 mg/L), and 61.8% were susceptible using the systemic infection breakpoint (≤ 8 mg/L). Meropenem and imipenem were the most active drugs (MIC50 values of 0.06 and 0.5 mg/L, respectively). Colistin and ertapenem (both MIC50 = 0.12 mg/L) were less active than meropenem or imipenem, but some strains were 77% susceptible to each of them. Among the KPC-producing isolates, 42.5% had MIC values of ≤ 32 mg/L (urinary tract infection breakpoint), but 100% were resistant to temocillin (systemic infection breakpoint). Ceftazidime/avibactam was active against 100% of the KPC-producing isolates, and fosfomycin was active against 40%. The empirical susceptibility rate observed among the urinary isolates suggests that temocillin may be considered as an alternative to carbapenems in the absence of KPC-producing bacteria. With regard to isolates from other sources, temocillin might be useful as a documented therapy agent or an empirical treatment in hospitals with a low prevalence of ESBL/AmpC-producing strains.


Introduction
Multidrug-resistant Enterobacterales bacteria have become a serious global concern, with limited therapeutic options for their control [1]. To meet the current challenges, there is an urgent need to discover new antimicrobials, or to re-examine known compounds such as fosfomycin, polymyxins, and temocillin [2].
Temocillin is a β-lactamase-resistant penicillin. It is the 6-α-methoxy derivative of ticarcillin, and is resilient to all classical and extended-spectrum TEM, SHV, and CTX-M enzymes and AmpC β-lactamases. Temocillin is used in few Western Europe countries [3], and only limited data are available on temocillin susceptibility in Eastern Europe, where microbial resistance to many antimicrobials is prevalent. According to the most recent report from the European Centre for Disease Prevention and Control in Poland over 65% of Klebsiella pneumoniae isolates reported to the European Antimicrobial Resistance Surveillance Network (EARS-Net) in 2018 were resistant to extended-spectrum cephalosporins; this represents an increase compared to previous years [4].
The aim of the present study was to evaluate the in vitro activity of temocillin and compare it to the activities of commonly used antimicrobials in respect of a large collection of Enterobacterales bacteria, especially ESBL-and/or AmpCproducing strains isolated from Polish patients with various infections.

Materials and methods
The non -du plic ate , wel l -ch ara cter ized , c linic al Enterobacterales isolates used in the present study were collected during laboratory surveillance conducted by the National Reference Centre for Susceptibility Testing between January 2000 and 2017 (Table 1). All the isolates were subcultured from storage (at -70°C) and reidentified prior to testing. ESBL production and AmpC expression were verified using a double-disk synergy test, as previously described [5]. Klebsiella pneumoniae carbapenemase (KPC) production was verified using the disk test combined with phenylboronic acid [6] followed by polymerase chain reaction (PCR) [7]. βlactamase production was controlled by PCR using specific primers for bla CTX-M-1 -, bla SHV -, bla TEM -, and bla KPC -like genes [8]. The bla KPC gene amplicons were all digested using a restriction enzyme (RsaI; Thermo Scientific, Vilnius, Lithuania), which allowed bla KPC-2 -and bla KPC-3 -like variants to be distinguished [9].
The minimal inhibitory concentrations (MICs) of temocillin and 16 other antibiotics listed in Table 2 were evaluated using the microdilution method according to standard ISO 20776-1. The MIC of ceftazidime/avibactam was determined only for KPC-producing strains using a MIC Test Strip (Liofilchem®, Roseto degli Abruzzi, Italy). The quality control strains used in the study were: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli mcr-1-producing strain. The data were interpreted using EUCAST guidelines, except for temocillin and cefoxitin, where BSAC and CLSI breakpoints were used, respectively [10][11][12].
According to the urinary breakpoint, among the ESBLand/or AmpC-producing isolates, the temocillin susceptibility rates were between 94% and 97%. According to the systemic breakpoint, the susceptibility rates were 22.7% for ESBL-and AmpC-producers, 61.6% for ESBL-producers, and 74.4% for AmpC-producers ( Table 2).
The KPC producers were resistant to several antimicrobials. These isolates were all resistant to temocillin according to the systemic infection breakpoint, but only 42.5% were susceptible according to the urinary tract infection breakpoint. Ceftazidime/avibactam (100% susceptibility) and colistin (95% susceptibility) were the most effective against KPCproducing isolates.

Discussion
Temocillin has been used for several years in some European countries, and is approved for the treatment of septicaemia, and urinary tract and lower respiratory tract infections [13][14][15][16]. However, it is still not available in Poland. Therefore, in the present analysis we assessed the prevalence of susceptibility to temocillin in Enterobacterales bacteria before it becomes available in our country.
There are currently no EUCAST breakpoints for temocillin [10]. Therefore, Enterobacterales bacteria are categorized as susceptible at MIC values of 8, 16, or 32 mg/L, depending on the country [14]. In the present study, we used BSAC [11] clinical breakpoints with criteria established separately for systemic (8 mg/L) and urinary tract (32 mg/L) infections. The temocillin MIC 50 and MIC 90 values for the total set of isolates calculated according to the urinary breakpoint were higher than those reported by Alexandre et al. [14] for urinary tract infection (UTI) isolates (8 and 32 mg/L versus 3 and 6 mg/L, respectively). However, our set of isolates comprised both UTI cases and isolates from other sites of infection.
When MICs were calculated only for isolates from urine, the MIC 50 and MIC 90 values were similar (data not shown), suggesting a possible role for temocillin in the treatment of urinary tract infections in Poland, regardless of the clinical breakpoint used. As expected, based on the literature non-ESBL/AmpC/KPC-producing isolates were very susceptible (100% and 99%), regardless of the clinical breakpoint used [15][16][17][18].
With regard to bacterial species tested and according to the urinary breakpoint, temocillin retained a high level of activity against all bacterial species producing ESBL and/or AmpC enzymes. However, when the systemic infection breakpoint was applied, only E. coli and Proteus spp. species remained susceptible, suggesting a possible role for temocillin in the treatment of urinary tract infections due to those two species [19]. More generally, temocillin was highly effective against most Enterobacterales bacteria, especially when the urinary tract infection breakpoint was used, and was least active against K. pneumoniae. The second most temocillin-resistant species was S. marcescens, with 77.8% of resistant isolates harbouring CTX-M enzymes (data not shown).
The majority of ESBL-or ESBL/AmpC-producers were susceptible using the urinary breakpoint but the resistance rate increased significantly using the systemic breakpoint. Previously, Rodriguez-Villalobos et al. [18] and Kresken et al. [20] pointed out that CTX-M-15 producers were less frequently susceptible to temocillin than other CTX-M-typeproducing isolates. In Poland, as in other European countries, the population of Enterobacterales bacteria has been dominated by CTX-M-15 producers [21].
With regard to K. pneumoniae, the temocillin-resistant strains were dominated by CTX-M-and KPC-producing isolates, regardless of the sample origin (data not shown). Contrary to the previous reports by Adams-Haduch et al. [22] and Woodford et al. [23], we were unable to confirm the susceptibility to temocillin among KPC-producing isolates. In the present study, 100% and over 50% of the KPCproducers were resistant to temocillin according to the systemic and urinary breakpoints, respectively. Our data are however in line with a report from Greece describing low temocillin activity against KPC-producers: 97.3% and 42% were resistant according to systemic and urinary breakpoints, respectively [24]. The low number of KPC-producing isolates is a limitation of the study, but the set of clinical isolates tested was representative of the population of KPC-producing isolates cultured from clinical specimens between 2010 and 2017 in Poland. However, among our KPC-producers, 20% had CTX-M enzymes and this could already be a reason why they are more resistant to temocillin. It would be of interest to further investigate the presence of other resistance mechanisms such as upregulated efflux or permeability for instance.
The present study is the first Polish evaluation of the in vitro susceptibility of Enterobacterales isolates to  temocillin. It demonstrated the effectiveness of the antibiotic against a collection of tested microbes, especially when the urinary breakpoint was used. In contrast with previously published data, our study did not confirm the susceptibility of KPC-producing isolates to temocillin.