Synergistic Activity of Temocillin and Fosfomycin Combination against KPC-Producing Klebsiella pneumoniae Clinical Isolates

Infections caused by KPC-producing K. pneumoniae continue to pose a significant clinical challenge due to their emerging resistance to new antimicrobials. We investigated the association between two drugs whose roles have been repurposed against multidrug-resistant bacteria: fosfomycin and temocillin. Temocillin exhibits unusual stability against KPC enzymes, while fosfomycin acts as a potent “synergizer”. We conducted in vitro antimicrobial activity studies on 100 clinical isolates of KPC-producing K. pneumoniae using a combination of fosfomycin and temocillin. The results demonstrated synergistic activity in 91% of the isolates. Subsequently, we assessed the effect on Galleria mellonella larvae using five genetically different KPC-Kp isolates. The addition of fosfomycin to temocillin increased larvae survival from 73 to 97% (+Δ 32%; isolate 1), from 93 to 100% (+Δ 7%; isolate 2), from 63 to 86% (+Δ 36%; isolate 3), from 63 to 90% (+Δ 42%; isolate 4), and from 93 to 97% (+Δ 4%; isolate 10). Among the temocillin-resistant KPC-producing K. pneumoniae isolates (24 isolates), the addition of fosfomycin reduced temocillin MIC values below the resistance breakpoint in all isolates except one. Temocillin combined with fosfomycin emerges as a promising combination against KPC-producing K. pneumoniae, warranting further clinical evaluation.


Introduction
Infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae are associated with high mortality rates [1].In Europe, from 2007 to 2015, the burden of carbapenem-resistant K. pneumoniae increased by 6.16 times in terms of both the number of infections and number of deaths [2].In patients with bloodstream infections (BSI), the resistance to carbapenems is associated with excess mortality [3].It has been demonstrated that initiating the appropriate antibiotic therapy as early as possible for KPC-producing K. pneumoniae infections reduces mortality [4].Currently, apart from colistin and eravacycline, we have four new β-lactam drugs with activity against KPCproducing K. pneumoniae: cefiderocol, ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam.However, the microbial landscape is dynamic, with resistances emerging easily and increasingly reported [5].According to the Infectious Diseases Society of America (IDSA), the preferred antibiotics for the treatment of infections outside of the urinary tract caused by carbapenem-resistant Enterobacterales (CRE) if KPC production is present are the following: meropenem-vaborbactam, followed by ceftazidime-avibactam, and then imipenem cilastatin-relebactam.Cefiderocol is listed as an alternative option [6].The European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guidelines recommend meropenem-vaborbactam or ceftazidime-avibactam for patients with severe CRE infections [7].IDSA and the ESCMID does not recommend combination therapies for CRE infections, if susceptible to the new beta-lactams/beta-lactamase inhibitors.Moreover, antimicrobial stewardship principles advocate for the use of an antibiotic with the narrowest spectrum possible, at least for monomicrobial infections.In this context, temocillin, an old antibiotic, emerges as an interesting option.Temocillin is a semisynthetic derivative of ticarcillin, a carboxypenicillin commonly used in the 1980s and 1990s as an agent against Gram-negative bacteria [8].Temocillin is used mainly for urinary tract infections (UTI), pneumonia, abdominal infections, and BSI.Generally, it is well tolerated and is associated with a low rate of C. difficile infections [8].Temocillin exhibits unusual stability against extended-spectrum β-lactamases (ESBL), AmpC and KPC enzymes [8], raising interest in drug repurposing.Indeed, currently, there are two ongoing clinical trials whose aim is to compare between temocillin and carbapenems in complicated UTI [9] and BSI [10] due to Gram-negative bacteria resistant to third-generation cephalosporins.From an antimicrobial stewardship perspective, the inactivity of temocillin against bacteria producing metallobeta-lactamase and OXA enzymes may be seen as an advantage, making it a strict anti-KPC agent.Fosfomycin is another old antibiotic with unique properties.It is a small molecule capable of synergizing with several other antibiotics, especially beta-lactams.It is a broadspectrum antibiotic active against antimicrobial-resistant and multidrug-resistant (MDR) Gram-positive and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), Enterobacterales, and Pseudomonas aeruginosa [11].Moreover, both fosfomycin and temocillin have proven to be stable for at least 24 h in elastomeric pumps, making them excellent options for outpatient parenteral antimicrobial therapy [12,13].Temocillin offers the pharmacokinetic/pharmacodynamic advantages of a β-lactam drug, while fosfomycin provides the advantages of a small molecule, with good tissue penetration and anti-biofilm activity.The combination of the two molecules theoretically helps to reduce the bacterial minimum inhibitory concentration (MIC) against antibiotics without requiring a higher dose of the single drugs [14], potentially increasing bacterial killing and limiting drug-related toxicity.In the setting of MDR infections, the number of in vitro and in vivo studies on different antimicrobial combination treatments are increasing [11,15].To the best of our knowledge, currently there are only a few studies on the synergistic activity in vitro and in vivo of temocillin and fosfomycin against Enterobacterales.Only one of these is focused on a KPC-producing E. coli infection in a murine model of peritonitis [16,17].In light of this premise, we aimed to test the in vitro and in vivo potential of temocillin and fosfomycin combinations against clinical isolates of KPC-producing K. pneumoniae.

Microbiological Findings
One hundred non-repetitive K. pneumoniae clinical isolates were investigated.All of the isolates were recovered from different patients (n = 100) and were identified at the species level via MALDI-TOF mass spectrometry.All K. pneumoniae were KPC producers resistant to carbapenems (MICs > 1 mg/L).The isolates were from rectal swabs (n = 62), urine (n = 29), blood culture (n = 7) and bronchial aspirate (n = 2).Isolates were recovered from patients hospitalized in the medical department (n = 68), surgical department (n = 9), intensive care unit (n = 22) and emergency department (n = 1).In particular, among the medical department patients, about 40% were from the infectious diseases unit (n = 27).Thirty-four isolates were resistant to temocillin (MICs > 16 mg/L), while no resistant isolate was recovered for fosfomycin (MICs ≤ 32 mg/L).The fosfomycin-temocillin combination showed a prevalent synergistic activity against 91% of KPC-producing K. pneumoniae isolates, while additive activity was obtained against 8% of them.Only in one case was indifferent activity revealed.No antagonism was detected (Table 1).In particular, synergistic activity lowered temocillin MIC values under the resistance breakpoint (MICs ≤ 16 mg/L) for all isolates except one (99%), with an FIC index ranging from 0.21 to 1 considering all combinations (Table 1).

In Vivo Findings
Temocillin was determined to not be toxic to G. mellonella larvae, either alone or in combination with fosfomycin.A variable degree of synergism was observed between temocillin and fosfomycin in time-kill assays of K. pneumoniae.This synergism was recapitulated in vivo using the G. mellonella infection model, using select K. pneumoniae isolates.We observed clear increases in survival rates of larvae treated with combination therapy compared to either agent alone (Figure 1).
Temocillin was determined to not be toxic to G. mellonella larvae, either alone or in combination with fosfomycin.A variable degree of synergism was observed between temocillin and fosfomycin in time-kill assays of K. pneumoniae.This synergism was recapitulated in vivo using the G. mellonella infection model, using select K. pneumoniae isolates.We observed clear increases in survival rates of larvae treated with combination therapy compared to either agent alone (Figure 1).Larvae infected with K. pneumoniae isolate 1 and treated with a combination of temocillin and fosfomycin exhibited a survival rate of 96.6% compared to 90% for fosfomycin alone, and 73.3% for temocillin alone (p = 0.0252, n = 30).In larvae infected with K. pneumoniae isolate 2, a combination of both antimicrobials resulted in a 100% survival rate, relative to an 86% survival rate for temocillin monotherapy.Fosfomycin monotherapy led to a 93% survival rate, with only a single larva succumbing to challenge at 72 h post-infection.K. pneumoniae isolate 3 elicited 90% mortality; however, the combination of temocillin at 24 and fosfomycin at 8 significantly improved the survival rate relative to 86% relative to temocillin alone 63% (p = 0.0768, n = 30), or fosfomycin alone 53% (p = 0.0205, n = 30).Combination treatment of larvae infected with K. pneumoniae isolate 4 resulted in a survival rate of 90% compared to a survival rate of 46% without antibiotic therapy, and 63% for temocillin alone (p = 0.0344, n = 30) and 73% for fosfomycin alone (p = 0.1915, n = 30).The survival rate of larvae infected with K. pneumoniae isolate 10 was 76.6%.Treatment of larvae with temocillin increased survival to 93.3%, and treatment with fosfomycin alone increased survival to 96.6%.Combination therapy with both of these agents resulted in a survival rate of 96.6% (Figure 1).

Molecular Characterization by WGS of 1, 2, 3, 4 and 10 K. pneumoniae Isolates
The whole genome sequence (WGS) of five K. pneumoniae clinical isolates was performed, and the antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) are reported in Tables 2 and 3.The isolates 2 and 4 belonged to ST307, isolate 1 belonged to ST512 and isolate 3 belonged to ST101.The ST of isolate 10 is not present in the MLST database of the Institut Pasteur; for this reason, we have inserted it as an unknown sequence type.As shown in Table 2, all five clinical isolates harbored KPC-βlactamases, specifically KPC-2 (isolates 2, 4 and 10) and KPC-3 (isolates 1 and 3).Larvae infected with K. pneumoniae isolate 1 and treated with a combination of temocillin and fosfomycin exhibited a survival rate of 96.6% compared to 90% for fosfomycin alone, and 73.3% for temocillin alone (p = 0.0252, n = 30).In larvae infected with K. pneumoniae isolate 2, a combination of both antimicrobials resulted in a 100% survival rate, relative to an 86% survival rate for temocillin monotherapy.Fosfomycin monotherapy led to a 93% survival rate, with only a single larva succumbing to challenge at 72 h post-infection.K. pneumoniae isolate 3 elicited 90% mortality; however, the combination of temocillin at 24 and fosfomycin at 8 significantly improved the survival rate relative to 86% relative to temocillin alone 63% (p = 0.0768, n = 30), or fosfomycin alone 53% (p = 0.0205, n = 30).Combination treatment of larvae infected with K. pneumoniae isolate 4 resulted in a survival rate of 90% compared to a survival rate of 46% without antibiotic therapy, and 63% for temocillin alone (p = 0.0344, n = 30) and 73% for fosfomycin alone (p = 0.1915, n = 30).The survival rate of larvae infected with K. pneumoniae isolate 10 was 76.6%.Treatment of larvae with temocillin increased survival to 93.3%, and treatment with fosfomycin alone increased survival to 96.6%.Combination therapy with both of these agents resulted in a survival rate of 96.6% (Figure 1).

Materials and Methods
Clinical bacterial isolates from various sample types (rectal swabs, urine, blood, bronchial aspirate) collected from January 2021 and August 2022 at the microbiology unit of the University Hospital of Trieste (Azienda Sanitaria Universitaria Giuliano Isontina) underwent screening for carbapenemase production using selective chromogenic agar medium (bioMérieux, Marcy-l'Etoile, France) as part of routine laboratory activities.Only one isolate per patient was studied.Species identification on bacterial isolates on selective medium was performed using MALDI-TOF mass spectrometry (bioMérieux, Marcy-l'Etoile, France), while antimicrobial susceptibility was assessed with the Vitek2 system (bioMérieux, Marcy-l'Etoile, France).The susceptibility profile of temocillin and fosfomycin was assessed using the Etest (bioMérieux, Marcy-l'Etoile, France).Isolates with meropenem MIC values greater than 0.125 mg/L underwent further investigation for carbapenemase production via PCR (Cepheid, Sunnyvale, CA, USA), according to the EUCAST criteria for detection of resistance mechanisms (https://www.eucast.org,accessed on 1 March 2024).Synergy tests between temocillin and fosfomycin were conducted for KPC-producing K. pneumoniae isolates.Although the recommended testing for fosfomycin is agar dilution, we used the Etest also for fosfomycin since it allowed us to perform synergy testing for the standard MIC of drugs A and B before creating the synergy.Synergy testing methods have been used to assess the interactions of antibiotic combinations in vitro.The goal of synergy testing is to assess the in vitro interactions of antimicrobial combinations, in order to determine whether the effect of the two antimicrobials is greater than the sum of their individual activities.Conventional, single-drug testing with the MIC test strip (gradient diffusion method) relies on the diffusion of a continuous concentration gradient of antimicrobial from an impregnated strip into solid agar.MIC test strips are placed on agar medium that has been inoculated with a lawn of the test organism.The "MTS Synergy Applicator System" was used for the synergy test.In particular, the MIC test strip (MTS) of the antibiotic (A) was positioned on the MTS synergy applicator platform.The MTS (antibiotic A) was adjusted such that the MIC value of the first antibiotic (MICA) was placed at the intersection of the base.An MTS of the second antibiotic (B) was placed on the second base.The second STD (antibiotic B) was adjusted such that the MIC value of the second antibiotic (MICB) was positioned at the base intersection and intersects MTS-antibiotic A at its MIC value.Finally, the plates were incubated according to the standard MTS procedure for the specific microorganism [18].A four-fold reduction in the MIC values of the antibiotics in combinations, in comparison with MIC values alone, was considered synergistic (FIC index ≤ 0.5).An FIC index (FICI) between >0.5 and ≤1 was considered additive interaction; an FICI between >1 and ≤4 was considered indifferent interaction; an FICI > 4 was considered antagonistic.EUCAST breakpoints were used to value susceptibility to temocillin and fosfomycin [19].

In Vivo Toxicity of Antibiotic Compounds
We have previously determined that fosfomycin is not toxic to Galleria mellonella larvae at doses of up to 512 mg/L [20].The in vivo toxicity of temocillin was determined according to the approach described previously for zidovudine and fosfomycin [21].G. mellonella were purchased from LiveFoods UK Ltd. (Axbridge, UK) and stored at 15 • C prior to use.Only larvae weighing between 0.7 g and 1.3 g, showing no discoloration or injury, were used in the assays.Groups of 10 larvae were injected into the proleg using a 26S gauge sterile syringe with either temocillin (48 mg/L) and separately, a combination of temocillin (48 mg/L) and fosfomycin (32 mg/L).These concentrations are equivalent to the highest MIC observed for each agent against K. pneumoniae isolates used in this study.Larvae mock-infected with phosphate-buffered saline (PBS) were used as injection controls.Following inoculation, all larvae were incubated aerobically at 37 • C for 72 h and were assessed for mortality at 24 h intervals.Larvae were classed as dead when movement was not detected when applying stimulus.This assay was performed in triplicate.

In Vivo Challenge and Antibiotic Therapy
A single colony of the test K. pneumoniae isolates was harvested from a freshly streaked Luria-Bertani (LB) agar plate.Then, the colony was transferred to 10 mL of lysogeny broth and incubated at 37 • C, with gentle agitation (150 rpm) under aerobic conditions for 16 h.Following incubation, the cultures were serially diluted 10-fold in PBS and enumerated on LB agar in triplicate.Groups of 10 G. mellonella larvae were placed in Petri dishes, and challenged with 5000 CFU/mL of either K. pneumoniae isolate 1, 2, 3, 4 and 10-reference, using the top-left proleg (Table 1).Additionally, a group of 10 larvae were mock-infected with 10 µL of sterile PBS.Within 15 min of infection, a second injection into the top right proleg was performed to administer fosfomycin, temocillin, a combination of both agents or PBS control.The concentration of each drug was equal to that used in combination with time-kill assays.Larvae were incubated aerobically at 37 • C and scored for mortality at 0, 24, 48 and 72 h post-infection.All assays were performed in triplicate, and the data were plotted using GraphPad Prism 8.4.3 software (GraphPad Software Inc., San Diego, CA, USA).ria efflux pump systems, in particular, AcrAB-TolC are common resistance mechanisms against multiple antibiotic classes.Overexpression of acrAB in association with inactivated OmpK35 and OmpK36 porins increased the MIC of meropenem/vaborbactam [28].Previous studies demonstrated that imipenem/relebactam resistance was associated with mutations, resulting in a non-functional OmpK35 and OmpK36 porins in KPC-producing K. pneumoniae strains, or AmpC overexpression in combination with porins loss [29].Similarly, resistance to cefiderocol, although uncommon, is increasingly being reported [30,31].In this context, some old antibiotics such as temocillin and fosfomycin could represent betalactams/beta-lactamase inhibitors and cefiderocol-sparing options, but also may provide pharmacokinetic/pharmacodynamic and biochemical advantages (e.g., central nervous system penetration, anti-biofilm activity, etc.), hence improving the antimicrobial armamentarium against KPC-producing isolates.Fosfomycin, in combination with beta-lactams, represents a classical (and historical) synergistic option.Beta-lactam antibiotics target the bacterial cell wall's penicillin-binding proteins, while fosfomycin inhibits the peptidoglycan precursor UDP N-acetylmuramic acid involved in peptidoglycan biosynthesis, hence providing a greater chance of synergistic/additive effects against Gram-negative isolates.Among the beta-lactams, the best synergistic activity against KPC-producing K. pneumoniae isolates was found in ceftazidime/avibactam.Fosfomycin demonstrated synergistic activity against KPC-producing K. pneumoniae isolates with tigecycline, colistin and aminoglycosides [11].Moreover, recently, synergistic activity was also reported between fosfomycin and meropenem/vaborbactam [32,33], as well as between fosfomycin and cefiderocol [34].In the present study, we assessed the antimicrobial interactions among these drugs against KPC-producing K. pneumoniae clinical isolates, demonstrating a significant in vitro and in vivo synergistic activity.In particular, in vitro synergistic activity between temocillin and fosfomycin was demonstrated against 91% of KPC-producing K. pneumoniae isolates, lowering the temocillin MIC values under the resistance breakpoint in all cases.In the animal study, we tested the temocillin and fosfomycin combination on five different KPC-producing K. pneumoniae isolates.The addition of fosfomycin to temocillin increased larvae survival from 73 to 97% (+∆ 32%; isolate 1), from 93 to 100% (+∆ 7%; isolate 2), from 63 to 86% (+∆ 36%; isolate 3), from 63 to 90% (+∆ 42%; isolate 4) and from 93 to 97% (+∆ 4%; isolate 10).To our knowledge, another study evaluated the in vitro and in vivo synergism of fosfomycin and temocillin against Enterobacterales KPC-producers, in particular an E. coli KPC-3 producer strain.In vitro, the addition of fosfomycin reduced the temocillin MIC 16-fold for this isolate of E. coli KPC-3, with an FICI of 0.562, showing an additive effect.This was eventually demonstrated in vivo in murine models of peritonitis, with a survival rate of 93% in mice infected with E. coli KPC-3 and treated with temocillin plus fosfomycin [16].In our study, there were no strains resistant to fosfomycin.In cases of temocillin resistance, the co-administration of fosfomycin resulted in lowered temocillin MIC values under the resistance breakpoint (MICs ≤ 16 mg/L) for all isolates except one (99%).In our infected larvae, the combination therapy with temocillin and fosfomycin resulted in a survival rate of 96.6%, showing results similar to those already reported in the study of Berleur et al. [16].The combination of fosfomycin and temocillin was also found to be synergistic in 27% of Gram-negative invasive isolates (E. coli or K. pneumoniae with a relatively low percentage of multiple drug-resistant organisms) of an observational study in the UK.However, no KPC-producing Enterobacterales were reported in this study [17]; thus, a comparison is not possible with our findings.
Our study has limitations.Firstly, it is monocentric.The epidemiology, especially for Gram-negative infections, could vary significantly among different areas within the same country.Thus, our study reflects only the local epidemiology.Secondly, we did not include many invasive clinical isolates.
In conclusion, this study represents the first in vitro and in vivo description of temocillinfosfomycin synergistic interactions against KPC-producing K. pneumoniae isolates.Temocillin combined with fosfomycin emerges as a promising combination against KPC-producing K. pneumoniae, warranting further clinical investigations.