$\beta$-Lactams are the most widely used and effective antibiotics for the treatment of infectious diseases. Unfortunately, bacteria have developed several mechanisms to combat these therapeutic agents. One of the major resistance mechanisms involves the production of $\beta$-lactamase that hydrolyzes the $\beta$-lactam ring thereby inactivating the drug. To overcome this threat, the small molecule $\beta$-lactamase inhibitors (e.g., clavulanic acid, sulbactam and tazobactam) have been used in combination with $\beta$-lactams for treatment. However, the bacterial resistance to this kind of combination therapy has evolved recently. Therefore, multiple attempts have been made to discover and develop novel broad-spectrum $\beta$-lactamase inhibitors that sufficiently work against $\beta$-lactamase producing bacteria. $\beta$-lactamase inhibitory proteins (BLIPs) (e.g., BLIP, BLIP-I and BLIP-II) are potential inhibitors that have been found from soil bacterium Streptomyces spp. BLIPs bind and inhibit a wide range of class A $\beta$-lactamases from a diverse set of Gram-positive and Gram-negative bacteria, including TEM-1, PC1, SME-1, SHV-1 and KPC-2. To the best of our knowledge, this article represents the first systematic review on $\beta$-lactamase inhibitors with a particular focus on BLIPs and their inherent properties that favorably position them as a source of biologically-inspired drugs to combat antimicrobial resistance. Furthermore, an extensive compilation of binding data from $\beta$-lactamase–BLIP interaction studies is presented herein. Such information help to provide key insights into the origin of interaction that may be useful for rationally guiding future drug design efforts.

Introduction: Infections caused by drug-resistant Klebsiella pneumoniae are now a serious problem for public health, associated with high morbidity and mortality due to limited treatment options. Therefore, new antibacterial agents or a combination of agents as the first line of treatment are urgently needed. K11 is a novel antimicrobial peptide (AMP) that has demonstrated in vitro antimicrobial activity against several types of bacteria. Additionally, K11 has previously shown no hemolytic activity. Herein, the antibacterial activity, the synergistic action of K11 in combination with different conventional antibiotics and the antibiofilm activity of K11 against multidrug-resistant (MDR) and extensively drug-resistant (XDR) K. pneumoniae were investigated. Meanwhile, the stability and ability to induce the bacterial resistance of K11 were also tested.Methods: Fifteen clinical isolates of MDR/XDR K. pneumoniae were used in this study. The minimum inhibitory concentration (MIC) of K11 against these isolates was determined by the broth microdilution method. In vitro synergy between K11 and antibiotics was evaluated using the checkerboard methodology. The antibiofilm activity of K11 against K. pneumoniae strong biofilm producers were explored by the crystal violet staining. The stability in different environments and resistance induction of K11 were evaluated by MIC determination.Results: The MIC values of K11 against MDR/XDR K. pneumoniae isolates were 8-512 μg/mL. Intriguingly, the synergistic effects were clearly observed for K11 in combination with chloramphenicol, meropenem, rifampicin, or ceftazidime, whereas no synergy was observed when K11 was combined with colistin. Besides, K11 effectively prevented biofilm formation against K. pneumoniae strong biofilm producers in a concentration-dependent manner starting at 0.25×MIC and exerted an enhancing effect when administered in combination with meropenem, chloramphenicol, or rifampicin. Additionally, K11 demonstrated high thermal and wide pH stability along with good stability in serum and physiological salts. Significantly, K. pneumoniae showed no induction of resistance even after prolonged exposure to a sub-inhibitory concentration of K11.Conclusion: These findings indicate that K11 is a promising candidate with potent antibacterial and antibiofilm activities without inducing resistance and acts synergistically with conventional antibiotics against drug-resistant K. pneumoniae.

Drug-resistant Enterobacterales infections are a great health concern due to the lack of effective treatments. Consequently, finding novel antimicrobials or combining therapies becomes a crucial approach in addressing this problem. BP203 and MAP-0403 J-2, novel antimicrobial peptides, have exhibited effectiveness against Gram-negative bacteria. In this study, we assessed the in vitro antibacterial activity of BP203 and MAP-0403 J-2, along with their synergistic interaction with conventional antibiotics including colistin, rifampicin, chloramphenicol, ceftazidime, meropenem, and ciprofloxacin against colistin-resistant Escherichia coli and Klebsiella pneumoniae clinical isolates. The minimal inhibitory concentrations (MIC) of BP203 and MAP-0403 J-2 against tested E. coli isolates were 2–16 and 8–32 μg/mL, respectively. However, for the majority of K. pneumoniae isolates, the MIC of BP203 and MAP-0403 J-2 were >128 μg/mL. Notably, our results demonstrated a synergistic effect when combining BP203 with rifampicin, meropenem, or chloramphenicol, primarily observed in most K. pneumoniae isolates. In contrast, no synergism was evident between BP203 and colistin, chloramphenicol, ceftazidime, rifampicin, or ciprofloxacin when tested against all E. coli isolates. Furthermore, synergistic effects between MAP-0403 J-2 and rifampicin, ceftazidime or colistin were observed against the majority of E. coli isolates. Similarly, the combined effect of MAP-0403 J-2 with rifampicin or chloramphenicol was synergistic in the majority of K. pneumoniae isolates. Importantly, these peptides displayed the stability at high temperatures, across a wide range of pH values, in specific serum concentrations and under physiological salt conditions. Both peptides also showed no significant hemolysis and cytotoxicity against mammalian cells. Our findings suggested that BP203 and MAP-0403 J-2 are promising candidates against colistin-resistant E. coli. Meanwhile, the synergism of these peptides and certain antibiotics could be of great therapeutic value as antimicrobial drugs against infections caused by colistin-resistant E. coli and K. pneumoniae.

The growing prevalence of antibiotic resistance in multidrug-resistant Gram-negative bacteria (MDR-GNB), exacerbated by the misuse of antibiotics, presents a critical global health challenge. Colistin, a last-resort antibiotic for severe MDR-GNB infections, has faced diminishing efficacy due to the emergence of colistin-resistant (COL-R) strains. This study evaluates the potential of penfluridol (PF), an antipsychotic drug with notable antibacterial and antibiofilm properties, to restore colistin activity against COL-R GNB in vitro. PF alone exhibited limited antibacterial activity against COL-R GNB; however, its combination with colistin demonstrated strong synergistic effects, significantly reducing colistin’s minimum inhibitory concentrations (MICs) by 4-128 times. Time-kill assays confirmed the combination’s superior bactericidal activity compared to either agent alone. Membrane permeability assays revealed that PF enhanced colistin’s ability to disrupt bacterial membranes, likely by facilitating colistin binding to lipopolysaccharide. Furthermore, PF significantly inhibited the development of colistin resistance over a 30-day resistance development assay. In addition to its antibacterial effects, PF exhibited notable antibiofilm activity. The combination of PF and colistin effectively inhibited biofilm formation and eradicated mature biofilms in most of the tested COL-R GNB strains. These findings mark the first report of PF’s synergistic interaction with colistin against GNB biofilms, offering a promising strategy to combat biofilm-associated infections. Overall, the colistin/PF combination holds potential as an effective therapeutic strategy to enhance colistin efficacy, delay resistance development, and manage biofilm-associated infections in MDR-GNB.