In Vitro Antibacterial Activity of Ceftobiprole and Comparator Compounds against Nation-Wide Bloodstream Isolates and Different Sequence Types of MRSA

Bloodstream infections by bacteria, especially multidrug-resistant bacteria, remain a worldwide public health concern. We evaluated the antibacterial activity of ceftobiprole and comparable drugs against different bloodstream isolates and different sequence types of methicillin-resistant Staphylococcus aureus (MRSA) in China. We found that MRSA, methicillin-susceptible Staphylococcus aureus (MSSA), and methicillin-susceptible coagulase-negative Staphylococcus (MSCNS) displayed ceftobiprole sensitivity rates of >95%, which are similar to the rates for linezolid, daptomycin, and vancomycin. Of the tested MRCNS strains, 90.4% were sensitive to ceftobiprole. The sensitivities of ST59, ST398, and ST22 MRSA to ceftobiprole were higher than that of ST239. Ceftobiprole’s MIC50/90 value against Enterococcus faecalis was 0.25/2 mg/L, whereas Enterococcus faecium was completely resistant to this drug. Ceftobiprole exhibited no activity against ESBL-positive Enterobacterales, with resistance rates between 78.6% and 100%. For ESBL-negative Enterobacterales, excluding Klebsiella oxytoca, the sensitivity to ceftobiprole was comparable to that of ceftazidime, ceftriaxone, and cefepime. The MIC50/90 value of ceftobiprole against Pseudomonas aeruginosa was 2/16 mg/L, and for Acinetobacter baumannii, it was 32/>32 mg/L. Thus, ceftobiprole shows excellent antimicrobial activity against ESBL-negative Enterobacterales and Pseudomonas aeruginosa (comparable to that of ceftazidime, ceftriaxone, and cefepime); however, it is not effective against ESBL-positive Enterobacterales and Acinetobacter baumannii. These results provide important information to clinicians.


Introduction
Bacterial bloodstream infections remain a major cause of the life-threatening condition of sepsis.Studies have demonstrated that one-third of hospital-associated deaths were related to sepsis, and bacterial infections causing sepsis account for 87% of hospitalizations [1].Xu et al. found that among 1318 patients with positive blood cultures in China, the associated mortality rate was 48.9% [2].The GBD 2019 Antimicrobial Resistance Collaborators estimated that deaths caused by 33 bacterial pathogens accounted for 13.6% of all global deaths and 56.2% of all sepsis-related deaths in 2019 [3].Reducing the mortality burden from infections is an urgent global public health priority.Bloodstream infections have arguably been encompassed within the scope of more recent global sepsis initiatives [4].Appropriate selection of antimicrobial agents is crucial to enhancing the treatment efficacy for bloodstream infections.In the United States, methicillinresistant Staphylococcus aureus (MRSA) accounted for 11% of bloodstream infections, with extended-spectrum β-lactamases (ESBL)-positive Enterobacterales and carbapenem-resistant Enterobacterales accounting for 7% and 1.3%, respectively [5].The use of broad-spectrum antibiotics, particularly those with activity against multidrug-resistant bacteria, remains the major treatment strategy for bloodstream infections.Ceftobiprole, a fifth-generation cephalosporin, differs from most β-lactam antibiotics in that it can bind to penicillin-binding protein 2a (PBP2a) in MRSA and displays associated antibacterial activity.Ceftobiprole medocari, the prodrug of ceftobiprole, has been approved by the Food and Drug Administration (FDA) for the treatment of complicated skin and soft-tissue infections, communityacquired pneumonia, and hospital-acquired bacterial pneumonia (excluding ventilatorassociated pneumonia).The monitoring of ceftobiprole activity following its introduction in Europe, the United States, Canada, and other countries has indicated its antimicrobial activity against various bacteria including MRSA, Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, ESBL-negative Enterobacterales, and Pseudomonas aeruginosa [6][7][8][9].However, studies on clinical isolates from bloodstream infections in China are limited because ceftobiprole medocari has not yet been employed in this region.We, therefore, conducted a large-sample antimicrobial activity study on bloodstream isolates collected from more than 50 hospitals in China, comparing the activity of ceftobiprole and comparator drugs.We subsequently investigated the antimicrobial activity of these drugs against different sequence types (STs) of MRSA to provide guidance for clinical drug use.
Ceftobiprole also exhibited strong activity against Enterococcus faecalis with MIC 50/90 values of 0.25/2 mg/L, while the MIC 50 and MIC 90 values against E. faecium strains were >32 mg/L.All Enterococci were sensitive to vancomycin and teicoplanin.All E. faecalis strains were sensitive to tigecycline, whereas 1.5% of E. faecium strains were not.The sensitivity rates of E. faecium strains to daptomycin and linezolid were higher than those of E. faecalis (99.5% vs. 92.9%,and 98.2% vs. 95.3%,respectively) (Table 1).

Sensitivity of Different Sequence Types of MRSA to Ceftobiprole and Comparator Compounds
Among the 294 MRSA strains isolated from bloodstream infections in 2019-2020, the main MLST types were ST59 (33.0%),ST5 (15.0%),ST398 (13.0%),ST22 (5.4%), and ST239 (5.4%).All MRSA strains were susceptible to linezolid, vancomycin, and daptomycin.Ceftobiprole's MIC 50 values for all STs, except ST5 and ST239, were 0.5 mg/L.Meanwhile, ST5 and ST239 displayed a higher ceftobiprole MIC 50 of 2 mg/L.Three ST239 MRSA strains were resistant to ceftobiprole, and resistance to ceftobiprole and cotrimoxazole was only found in ST239 MRSA, with resistance rates of 18.8% (3/16).ST239 MRSA also showed the highest resistance rates to rifampicin (56.3%) and levofloxacin (87.5%) among all MLST types.The antimicrobial activity of all agents against MRSA types other than ST239 was comparable, with the exception of detected resistance to rifampicin and levofloxacin.ST5 MRSA showed higher resistance rates to levofloxacin, but no resistance was observed for the other drugs.The ST22 MRSA strains were sensitive to all the seven drugs tested (Table 2 and Figure 1).

The Antibacterial Activity of Ceftobiprole and Comparator Compounds against Gram-Negative Bacteria
The sensitivity rates of ESBL-negative Escherichia coli, Enterobacter aerogenes, and Klebsiella pneumoniae to ceftobiprole were 92.1%, 95.9%, and 87.2%, respectively, and these rates were similar to those observed for ceftazidime, ceftriaxone, and cefepime, and slightly lower than that for ertapenem.However, the sensitivity rates of ESBL-negative Klebsiella oxytoca were 63.7%, significantly lower than those for the comparator compounds.More than 50.0% of Enterobacter cloacae, Salmonella spp., and Serratia marcescens isolates were inhibited by ceftobiprole at concentrations of 0.06-0.125mg/L, with sensitivity rates of 74.0%, 88.8%, and 86.0%, respectively, similar to those of ceftriaxone, cefepime, and ceftazidime.For ceftobiprole, almost no activity was observed against ESBL-positive Enterobacterales, with resistance rates ranging from 78.6% to 100.0% (Table 3).   2 MIC 50 : minimum inhibitory concentration that inhibits 50% of bacterial isolates tested. 3MIC 90 : minimum inhibitory concentration that inhibits 90% of bacterial isolates tested. 4R: resistance rate (%). 5 S: sensitivity rate (%).

The Antibacterial Activity of Ceftobiprole and Comparator Compounds against Gram-Negative Bacteria
The sensitivity rates of ESBL-negative Escherichia coli, Enterobacter aerogenes, and Klebsiella pneumoniae to ceftobiprole were 92.1%, 95.9%, and 87.2%, respectively, and these rates were similar to those observed for ceftazidime, ceftriaxone, and cefepime, and slightly lower than that for ertapenem.However, the sensitivity rates of ESBL-negative    2 MIC 50 : minimum inhibitory concentration that inhibits 50% of bacterial isolates tested. 3MIC 90 : minimum inhibitory concentration that inhibits 90% of bacterial isolates tested. 4R: resistance rate (%). 5 S: sensitivity rate (%). 6"-": no relevant breakpoint data were available for ceftobiprole against the corresponding bacteria.

The Cumulative MIC Distribution of Ceftobiprole and Comparator Compounds against Major Bacteria
Ceftobiprole was highly potent against Staphylococci, with an activity curve shifted to the left of vancomycin, linezolid, cotrimoxazole, and levofloxacin.Its antibacterial activity was also superior to that of daptomycin against MSSA and MSCNS.Daptomycin displayed stronger antibacterial activity than the comparator compounds against MRCNS (Figure 2).The bacteriostatic curves of ceftobiprole, cefepime, ceftriaxone, and ceftazidime against ESBLpositive E. coli and K. pneumoniae were similar, inhibiting over 90% of bacteria at concentrations of ≥32 mg/L (Figure 3a,c).Ertapenem displayed the greatest activity against ESBL-negative E. coli and K. pneumoniae, followed by ceftobiprole, cefepime, ceftriaxone, and ceftazidime.Ceftobiprole inhibited over 80% of P. aeruginosa at concentrations of ≤4 mg/L.Furthermore, the bacteriostatic curve of ceftobiprole was shifted to the left of cefepime and ceftazidime, with the highest MIC 90 (32 mg/L) observed for ceftazidime (Figure 3e).
against ESBL-positive E. coli and K. pneumoniae were similar, inhibiting over 90% of bacteria at concentrations of ≥32 mg/L (Figure 3a,c).Ertapenem displayed the greatest activity against ESBL-negative E. coli and K. pneumoniae, followed by ceftobiprole, cefepime, ceftriaxone, and ceftazidime.Ceftobiprole inhibited over 80% of P. aeruginosa at concentrations of ≤ 4 mg/L.Furthermore, the bacteriostatic curve of ceftobiprole was shifted to the left of cefepime and ceftazidime, with the highest MIC90 (32 mg/L) observed for ceftazidime (Figure 3e).

Discussion
Antimicrobial resistance (AMR) is firmly lodged in the medical community's collective mind as a challenge to be reckoned with in the decades to come [10].This study determined the antibacterial activity of ceftobiprole and comparator compounds against 9860 bacterial strains isolated from blood and 294 MRSA strains collected in China.In the period from January 2021 to December 2021, this research involved gathering 9860 strains

Discussion
Antimicrobial resistance (AMR) is firmly lodged in the medical community's collective mind as a challenge to be reckoned with in the decades to come [10].This study determined the antibacterial activity of ceftobiprole and comparator compounds against 9860 bacterial strains isolated from blood and 294 MRSA strains collected in China.In the period from January 2021 to December 2021, this research involved gathering 9860 strains to assess the in vitro antibacterial properties of ceftobiprole in comparison with other compounds against a range of Gram-positive and Gram-negative bacteria.Moreover, for a detailed investigation into the sensitivity of different MRSA sequence types, 294 MRSA isolates collected between January 2019 and December 2020 were analyzed to determine their reactivity to ceftobiprole and various comparator compounds.We found that ceftobiprole exhibited antibacterial activity against most Gram-positive and Gram-negative bacteria, with most showing activity comparable to or greater than that of commonly used antibacterial drugs.
S. aureus was identified as the primary bacterial cause of death in 135 countries and additionally held the highest age-standardized mortality rate in 16 of the 21 GBD regions [3].This is largely attributed to the emergence of resistant strains, particularly the widespread prevalence of MRSA.The resistance mechanism of MRSA to antibiotics is primarily characterized by alterations in the target site or a reduction in the affinity between the target and the antibiotic.Moreover, changes in cell membrane proteins and plasmid-mediated efflux pumps also play a significant role in the antibiotic resistance observed in MRSA.In this study, ceftobiprole exhibited potent antibacterial activity against MRSA, MSSA, and MSCNS, comparable to or better than that of linezolid, daptomycin, and vancomycin.Despite this, we identified some MRSA, MSCNS, and MRCNS strains that were resistant to ceftobiprole.These findings are consistent with studies conducted in Europe and China [11][12][13].For example, Amsler et al. reported the in vitro antibacterial activity of ceftobiprole in the United States, with MIC 50/90 values of 0.5/2 mg/L, 0.25/0.5 mg/L, 0.5/2 mg/L, and 0.25/0.5 mg/L against MRSA, MSSA, MRCNS, and MSCNS, respectively, and Yin et al. identified values 1/2 mg/L, 0.5/1 mg/L, 1/2 mg/L, and 0.25/0.5 mg/L in a study conducted in China, respectively.Holland et al. conducted a double-blind, non-inferiority trial in 387 patients with Staphylococcus aureus bacteremia and found that the efficacy of ceftobiprole was noninferior to that of daptomycin [14], consistent with the in vitro susceptibility data reported in our study.In this study, we found that the ST59, ST398, and ST22 MRSA strains were more sensitive to ceftobiprole, levofloxacin, rifampin, and cotrimoxazole than ST239 MRSA strains and that ceftobiprole-resistant MRSA strains were uncommon.Three resistant strains were found for ST239 MRSA, with MIC values of 4 mg/L.Zhu et al. previously identified a correlation between ceftobiprole's MIC and MRSA's genetic background, with stable ceftobiprole resistance mutations in mecA and other genes only occurring in ST5 MRSA and ST239 MRSA.SCCmec amplification may also occur following ceftobiprole exposure in ST239 MRSA isolates, although this has not been observed in the other lineages [15].Since 2010, the prevalence of ST59 MRSA in China has been increasing, gradually replacing ST239 and becoming the dominant clone in most Chinese hospitals [16,17], supporting the clinical use of ceftobiprole.In an Italian study, three ST228 strains of ceftobiprole-resistant MRSA were detected, with MIC values of 4 mg/L, before the clonal replacement of ST228 with ST22 [18].Hawser et al. analyzed the whole-genome sequences of three resistant MRSA strains from European countries and identified two as clonal complex 8 (CC8) and one as CC5, with different mutations in the gene encoding the penicillin-binding protein MecA [19].Therefore, research on different parts of the genotyping of MRSA and the analysis of MRSA whole-genome sequence data can aid in drug resistance monitoring.The antibacterial activity of ceftobiprole against MRCNS in our study (with a sensitivity rate of 90.4% and an MIC 50/90 of 1/2 mg/L) was slightly lower than that against other Staphylococci, which is consistent with the results reported by Pfaller et al. (with a sensitivity rate of 85.7% and an MIC 50/90 of 1/4 mg/L) [20], but the mechanism of resistance in MRCNS remains unclear.We also identified 13 strains of Staphylococcus resistant to linezolid, including two MRSA, ten MRCNS, and one MSCNS, all of which were sensitive to ceftobiprole.These results are consistent with the findings of Rossolini et al. [6].Despite the use of ceftobiprole in Europe and other countries over the years, the current resistance rate of MRSA to ceftobiprole remains very low (<2%) [11,[21][22][23].
In Europe and the United States, E. faecalis is predominantly responsible for enterococcal bloodstream infections [24], whereas in China, E. faecium is more prevalent [25].Concurrently, data from the 20-year global SENTRY antimicrobial surveillance program revealed that the resistance rate of Enterococcus isolates to vancomycin was 16% in 1997, and this rate has been steadily increasing [26].In this study, we demonstrated that ceftobiprole displays potent in vitro activity against E. faecalis, with MIC 50/90 values of 0.25/2 mg/L, but not against E. faecium.These data are consistent with a study by Yin et al., who reported ceftobiprole MIC 50/90 values against E. faecalis and E. faecium of 0.5/1 mg/L and 32/>32 mg/L, respectively [12].The antibacterial activity of ceftobiprole against E. faecalis is mediated by the inhibition of peptidoglycan cross-linking by the acylation of serine at the active sites of penicillin-binding proteins (PBPs) with a greater affinity for PBP4 [27].Both the overexpression and substitution of amino acids in low-affinity PBP4 can induce resistance [28].The weaker activity of ceftobiprole against E. faecium correlates with the lower affinity of ceftobiprole to PBP5 [29].
According to the study conducted by Swiss ANRESIS, E. coli has been identified as the primary resistant pathogen causing bloodstream infections.In terms of treatment, infections caused by third-generation cephalosporin-resistant E. coli pose a significant challenge [30].Like other cephalosporins, the activity of ceftobiprole against Enterobacterales largely depends on the expression of β-lactamases.Ceftobiprole is degraded by extendedspectrum β-lactamases, explaining the lack of antibacterial activity against ESBL-positive Enterobacterales in our study, whilst carbapenems retain activity [20,31,32].Ceftobiprole possesses antibacterial activity against ESBL-negative Enterobacterales (except for K. oxytoca), E. cloacae, Salmonella spp., and S. marcescens, similar to that of ceftriaxone, ceftazidime, and cefepime, but slightly inferior to carbapenems, such as ertapenem.These findings are consistent with other studies conducted in China and Europe [12,20].A large-sample study by Pfaller et al. demonstrated sensitivity rates of Enterobacterales to ceftobiprole, ceftriaxone, ceftazidime, cefepime, and imipenem of 73.8%, 73.3%, 74.2%, 78.2%, and 96.3%, respectively.The ceftobiprole MIC 50/90 values against ESBL-negative E. coli and K. pneumoniae were 0.03/0.06mg/L, similar to those reported by Yin et al. in China (≤0.06/0.25 mg/L).Many Enterobacterales and P. aeruginosa encode an inducible AmpC β-lactamase, which is usually expressed at low levels but can be up-regulated in response to cell wall damage or exposure to β-lactam antibiotics [33].Ceftobiprole and cefepime are stable against AmpC enzymes.In this study, most ESBL-negative Enterobacterales and P. aeruginosa had lower ceftobiprole and cefepime MIC values than ceftazidime, and an analysis of the bacteriostatic curves indicated greater antibacterial activity than ceftazidime.These two drugs may rapidly penetrate the cytoplasm of Gram-negative bacteria [34].P. aeruginosa, a major cause of hospital-acquired infection, is resistant to multiple antibiotics [35].When the sensitivity breakpoints were set to 4 mg/L, most P. aeruginosa isolates were classified as sensitive to ceftobiprole (>80%, with MIC 50/90 values of 2/16 mg/L).The sensitivity rates were higher than that in Europe (63.2%)[23], but were consistent with previous data from China, with the same MIC 50/90 values (2/16 mg/mL) [36], observations which are likely indicative of earlier applications of the drug in European countries.The response of P. aeruginosa exposed to ceftobiprole differs from that of bacteria exposed to the other two drugs, being characterized by increased efflux rather than overexpression of AmpC [37].Ceftobiprole and other β-lactam antibiotics have poor antibacterial activity against A. baumannii.
The results of the above studies are based on in vitro susceptibility to address clinical needs and guide clinical treatment.However, it is important to note that discrepancies still exist between in vitro drug susceptibility testing and clinical applications in practice.This gap may be attributed to the incorrect identification of pathogenic bacteria, or a failure to consider pharmacokinetic factors in the treatment process.Monte Carlo simulation (MCS), a statistical modeling method, integrates in vitro antimicrobial susceptibility with pharmacodynamic principles, offering substantial reference value for clinical evaluation and the rational use of antibiotics.By employing the MCS method, clinicians can simulate various scenarios for different patient populations to compare the efficacy of different drugs, dosages, and dosing intervals [38].

Strain Sources
From January 2021 to December 2021, a total of 9860 strains of Gram-positive and Gram-negative bacteria causing bloodstream infections were collected from 51 hospitals (16 provinces) participating in the Blood Bacterial Resistance Investigation Collaborative System (BRICS) (Figure 4).various scenarios for different patient populations to compare the efficacy of different drugs, dosages, and dosing intervals [38].

Statistical Analysis
WHONET5.6 software was used to statistically analyze the drug susceptibility results.

Conclusions
Ceftobiprole displayed strong antibacterial activity against Staphylococci and E. faecalis in vitro, at levels comparable to vancomycin, daptomycin, and linezolid.The clinically dominant MRSA types in China, including ST59, ST398, and ST22, showed higher sensitivity to ceftobiprole, levofloxacin, and rifampicin compared with ST239.Ceftobiprole displayed antibacterial activity against ESBL-negative Enterobacterales (except for K. oxytoca), E. cloacae, Salmonella spp., S. marcescens, and P. aeruginosa in vitro at levels comparable to ceftazidime, ceftriaxone, and cefepime.However, ceftobiprole exhibited weaker antibacterial effects against E. faecium, ESBL-positive Enterobacterales, and A. baumannii.Therefore, for bloodstream infection by Staphylococci, E. faecalis, ESBL-negative Enterobacterales, P. aeruginosa, and other gram-negative bacteria, ceftobiprole can be an alternative agent in clinical practice.
However, our study does have certain limitations that need to be acknowledged.Firstly, although the dataset of bloodstream infections was extensive, it was sourced from only about half of the provinces in China, with less coverage in the northwest and southern regions.This limitation might have impacted the comprehensive understanding of drug susceptibility patterns across the country.Secondly, research from other countries has indicated resistance to ceftobiprole in various STs of MRSA, such as ST22, ST5, and ST8 [40].Hence, owing to the diverse genetic backgrounds of MRSA in different countries, the patterns of drug resistance also vary.In our study, ceftobiprole resistance was observed exclusively in the ST239 strain of MRSA, necessitating further investigations to elucidate its resistance mechanisms.
The following bacteria were excluded: single-bottle-culturepositive coagulase-negative Staphylococcus, Bacillus, Streptococcus viridans, Corynebacterium, Propionibacterium, Aerococcus, Micrococcus, Brucella, duplicate strains from the same patient, and contaminated bacteria.All strains were stored in Microbank tubes and stored at −80 • C. BRICS members transmitted clinical isolates quarterly to the central lab at Zhejiang University.After receiving the strains, the central lab re-identified and stored them for sensitivity tests.Two-hundred and ninety-four MRSA strains causing bloodstream infections from BRICS's 54 hospitals from January 2019 to December 2020 were subjected to ST typing and drug susceptibility testing.Quality control strains included Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and ATCC35218, Pseudomonas aeruginosa ATCC 27853, and Klebsiella pneumoniae ATCC 700603.Antibiotics 2024, 13, x FOR PEER REVIEW 12 of 16 The following bacteria were excluded: single-bottle-culturepositive coagulase-negative Staphylococcus, Bacillus, Streptococcus viridans, Corynebacterium, Propionibacterium, Aerococcus, Micrococcus, Brucella, duplicate strains from the same patient, and contaminated bacteria.All strains were stored in Microbank tubes and stored at −80 °C.BRICS members transmitted clinical isolates quarterly to the central lab at Zhejiang University.After receiving the strains, the central lab re-identified and stored them for sensitivity tests.Two-hundred and ninety-four MRSA strains causing bloodstream infections from BRICS's 54 hospitals from January 2019 to December 2020 were subjected to ST typing and drug susceptibility testing.Quality control strains included Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and ATCC35218, Pseudomonas aeruginosa ATCC 27853, and Klebsiella pneumoniae ATCC 700603.

Figure 4 .
Figure 4. Chinese provinces distribution of 9860 bacterial strains collected from January 2021 to December 2021.

Figure 4 .
Figure 4. Chinese provinces distribution of 9860 bacterial strains collected from January 2021 to December 2021.

Table 1 .
Activities of ceftobiprole and comparator compounds against Gram-positive bacteria isolated from blood.

Table 2 .
Activities of ceftobiprole and comparator compounds against different sequence types (STs) of MRSA isolated from blood.

Table 3 .
Activities of ceftobiprole and comparator compounds against Gram-negative bacilli isolated from blood.