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Methicillin-resistant Staphylococcus aureus (MRSA) mastitis poses a significant threat to dairy herds worldwide, given its resistance to methicillin and other β-lactam antibiotics, which often leads to treatment failure. Consequently, there is an urgent need for safe and effective alternative therapeutic approaches. Recent investigations have highlighted the potential of baicalein, a natural flavonoid known for its potent anti-inflammatory and antibacterial properties, especially its synergistic effects with β-lactam antibiotics against MRSA. However, the limited solubility and bioavailability of baicalein hinder its biomedical utility. The present study assessed the therapeutic efficacy of encapsulated baicalein in chitosan, forming a tricomplex with a β-lactam antibiotic, using a murine model of MRSA-induced mastitis. The experimental design comprised seven groups, each consisting of six mice. We evaluated the ability of various treatment regimens to mitigate histopathological alterations and bacterial burden induced by MRSA infection, aiming to elucidate underlying mechanisms. Our results revealed that tricomplex treatment significantly reduced bacterial load in mammary tissue and preserved tissue integrity, resulting in decreased inflammatory responses post-MRSA inoculation. In addition, tricomplex treatment markedly reduced mean leukocyte and neutrophil counts in blood and suppressed the matrix metalloproteinase-9 (MMP-9) concentration and C-reactive protein (CRP) response. Notably, the synergistic interaction between baicalein and amoxicillin was particularly pronounced. Our findings suggest that chitosan-encapsulated baicalein combined with a β-lactam antibiotic holds promise as a therapeutic option for MRSA-induced mastitis. Further investigations, particularly in target animal species, are warranted to comprehensively evaluate its clinical feasibility.

ABSTRACT: β-Lactam antibiotics are one of the most clinical importance in human and veterinary medicine because they are used for both preventive and therapeutic purposes against several gram-positive, gram-negative, and anaerobic organisms. In this study, it was confirmed that β-lactams (81.1%) were found to be significantly prescribed the most among 74 farms in 5 integrated broiler operations, and single prescription (84.6%), 2-day (41.5%) or 3-day (40.0%) administration, and 15 to 22 d of age (67.7%) administration was significantly higher in the farms (P < 0.05). Among the E. coli isolated from 74 farms in 5 integrated broiler operations, β-lactam-resistant E. coli isolates were detected more frequently in fecal sample (94.6%) than in dust sample (60.8%) (P < 0.05). The prevalence of MDR in β-lactam-resistant isolates, ranging from 88.1 to 96.5%, was significantly higher than that in non–β-lactam-resistant isolates (P < 0.05), without significant differences among operations. Of 466 β-lactam-resistant isolates, 432 (92.7%) isolates harbored β-lactamase genes. The non–extended-spectrum β-lactamase (ESBL) gene blaTEM-1 (81.8%) showed the highest prevalence among isolates, followed by the non-ESBL gene blaTEM-135 (6.4%) (P < 0.05). Five ESBL genes, SHV-12, OXA-1, CTX-M-27, CTX-M-55, and CTX-M-65, were found in 0.9 to 6.0% of the isolates. The pAmpC gene blaCMY-2 was detected in 17 isolates (3.6%). These results suggest that feces and dust are important reservoirs of antimicrobial-resistant bacteria, highlighting the need to strengthen farm management regulations, such as cleaning, disinfection, and litter disposal and to reduce the use of antibiotics in broiler operations.

β-lactamase, a kind of hydrolase in multi-drug resistant pathogens, can hydrolyze β-lactam antibiotics and make these kinds of antibiotics invalid. Small-molecular inhibitors about the enzyme and their mechanism are widely investigated but they may result in unavoidable adverse reactions and drug-resistance. Herein, we propose a new therapeutic strategy of Chinese materioherbology, in which herbal medicine or traditional Chinese medicinal herbs can be employed as biological functional materials or refreshed/excited by means of materialogy. Natural tea nanoclusters (TNCs) were extracted from tea to inhibit β-lactamase. Different from the mechanism of small-molecular inhibitors inhibiting enzymes by binding to the corresponding active sites, the TNCs as a cap cover the protein pocket and create a spatial barrier between the active sites and antibiotics, which was named “capping-pocket” effect. TNCs were combined with amoxicillin sodium (Amo) to treat the methicillin-resistant Staphylococcus aureus (MRSA) pneumonia in mice. This combinatorial therapy remarkably outperforms antibiotic monotherapy in reducing MRSA infections and the associated inflammation in mice. The therapeutic strategy exhibited excellent biosafety, without any side effects, even in piglets. Hence, TNCs have great clinical value in potentiating β-lactam antibiotic activity for combatting multi-drug resistant pathogen infections and the ''pocket capping'' effect can guide the design of new enzyme inhibitors in near future.

ABSTRACT The emergence of antibiotic resistance has led to a global crisis for the physician to handle infection control issues. All antibiotics, including colistin, have lost efficiency against emerging drug-resistant bacterial strains due to the production of metallo-β-lactamases (MBLs) and serine-β-lactamases (SBLs). Therefore, it is of the utmost importance to design inhibitors against these enzymes to block the hydrolytic action against antibiotics being used. Although various novel β-lactamase inhibitors are being authorized or are under clinical studies, the coverage of their activity spectrum does not include MDR organisms expressing multiple classes of β-lactamases at a single time. This study reports three novel broad-spectrum inhibitors effective against both SBLs and MBLs. Virtual screening, molecular docking, molecular dynamics simulations, and an in silico pharmacokinetic study were performed to identify the lead molecules with broad-spectrum ability to inhibit the hydrolysis of β-lactam. The selected compounds were further assessed by in vitro cell assays (MIC, 50% inhibitory concentration [IC50], kinetics, and fluorescence against class A, B, and C type β-lactamases) to confirm their efficacies. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to check the toxicity of screened lead molecules. All three selected inhibitors were found to reduce MIC and showed good affinity against all the SBLs and MBLs produced by class A, B, and C type β-lactamases. These nontoxic novel non-β-lactam broad-spectrum inhibitors bind to the active site residues of selected β-lactamases, which are crucial for β-lactam antibiotic hydrolysis. These inhibitors may be proposed as a future drug candidate in combination with antibiotics as a single formulation to control infection caused by resistant strains. Hence, this study plays a significant role in the cure of infections caused by antibiotic-resistant bacteria. IMPORTANCE Several inhibitors for usage in conjunction with antibiotics have been developed. However, to date, there is no commercially available broad-spectrum β-lactamase inhibitor that targets both MBLs and SBLs. Here, we showed three novel broad-spectrum inhibitors with promising results through computational techniques and in vitro studies. These inhibitors are effective against both SBLs and MBLs and hence could be used as future drug candidates to treat infections caused by multidrug-resistant bacteria producing both types of enzymes (SBLs and MBLs).

In this study, a multi-residue determination method of QuECHERS combined with UHPLC-MS/MS was developed to determine nine β-lactam antibiotics (penicillin G, amoxicillin, ampicillin, cephalexin, cefquinome, ceftiofur, cefazolin, cephapirin, cefuroxime) in aquaculture products. All the nine β-lactam antibiotics exhibited excellent linearity within the range of 2.0–200 ng/mL (cefuroxime: 5.0–200 ng/mL, r greater than 0.999), while the limits of detection (LODs) and quantification (LOQs) were 2.0–5.0 μg/kg and 5.0–10.0 μg/kg, respectively. The recoveries of this method in five aquaculture products (Penaeus Orientalis, Cyprinus Carpio, Channa Argus, Aristichthys Nobilis, and Ctenopharyngodon Idella) ranged from 85.4% to 113.3% and the intra-day and inter-day precisions (%RSDs) were less than 15%. The results suggested the feasibility of this method as a rapid, simple, and accurate approach for determining the residues of the β-lactam antibiotics in aquaculture products.

Mingjia Peng,1,2 Renru Han,1,2 Yan Guo,1,2 Yonggui Zheng,1,2 Feifei Yang,1,2 Xiaogang Xu,1,2 Fupin Hu1,2 1Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, People’s Republic of China; 2Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, Shanghai, People’s Republic of China; 3National Clinical Research Centre for Aging and Medicine, Huashan Hospital, Fudan University, Shanghai, People’s Republic of ChinaCorrespondence: Fupin Hu; Xiaogang Xu Email hufupin@fudan.edu.cn; xuxiaogang@fudan.edu.cnBackground: The spread of KPC-producing Enterobacteriaceae has triggered a global public health concern, with KPC-2-positive strains being the most prevalent in China. We hereby studied the in vitro combined inhibitory activities of three kinds of β-lactam antibiotics and clavulanic acid at different concentrations against blaKPC-2-positive Klebsiella pneumoniae to explore the antimicrobial characteristics of these combinations and alternative therapeutic regimens for infections caused by blaKPC-2-positive K. pneumoniae strains.Materials and Methods: In this study, 153 clinically isolated blaKPC-2-positive K. pneumoniae strains from 19 provinces in China were collected from 2016 to 2018. Antimicrobial susceptibility testing of imipenem/clavulanic acid, meropenem/clavulanic acid, ceftazidime/clavulanic acid, and each antimicrobial agent alone was performed by broth microdilution technique according to the CLSI guidelines. The concentration ratios of β-lactam antibiotics to clavulanic acid were as follows: 1:1, 1:2, 1:4, 1:8, 1:16, 1:32. The antimicrobial susceptibility of the combinations was determined according to the breakpoints of Imipenem, meropenem, and ceftazidime established by the CLSI directives for Enterobacteriaceae.Results: The MICs of all three combinations gradually declined with increments in the proportion of clavulanic acid in the regimens, and the most significant decline in the MIC50 and MIC90 was seen in combinations at the concentration ratio of 1:1 (also 1:2 for meropenem/clavulanic acid). When the concentration of clavulanic acid was restricted to 4 mg/L, the susceptibility of more than 70% of the isolates to the regimens could be restored with imipenem MIC 2– 4 mg/L, meropenem MIC 2– 8 mg/L or ceftazidime MIC 8mg/L. However, the percentage decreased to 30 to 40% when the initial MIC level was higher.Conclusion: The highest combined inhibitory activity of β-lactam antibiotics/clavulanic acid at low concentration ratios against blaKPC-2-positive K. pneumoniae may offer a new way to optimize the effects of these antimicrobial regimens.Keywords: KPC, Enterobacteriaceae, combined inhibitory activity, clavulanic acid, β-lactam antibiotic

Abstract Methicillin-resistant Staphylococcus aureus (MRSA) strains are distinct from general Staphylococcus strains with respect to the composition of the membrane, ability to form a thicker biofilm, and, importantly, ability to modify the target of antibiotics to evade their activity. The agr gene is an accessory global regulator of gram-positive bacteria that governs virulence or resistant mechanisms and therefore an important target for the control of resistant strains. However, the mechanism by which agr impacts resistance to β-lactam antibiotics remains unclear. In the present study, we found the Δagr mutant strain having higher resistance to high concentrations of β-lactam antibiotics such as oxacillin and ampicillin. To determine the influence of variation in the microenvironment of cells between the parental and mutant strains, fatty acid analysis of the supernatant, total lipids, and phospholipid fatty acids were compared. The Δagr mutant strain tended to produce fewer fatty acids and retained lower amounts of C16, C18 fatty acids in the supernatant. Phospholipid analysis showed a dramatic increase in the hydrophobic longer-chain fatty acids in the membrane. To target membrane, we applied several surfactants and found that sorbitan monolaurate (Span20) had a synergistic effect with oxacillin by decreasing biofilm formation and growth. These findings indicate that agr deletion allows for MRSA to resist antibiotics via several changes including constant expression of mecA, fatty acid metabolism, and biofilm thickening.

BackgroundIt remains largely unknown how physicochemical properties of hydrolysed infant formulas influence their allergy preventive capacity, and results from clinical and animal studies comparing the preventive capacity of hydrolysed infant formula with conventional infant formula are inconclusive. Thus, the use of hydrolysed infant formula for allergy prevention in atopy-prone infants is highly debated. Furthermore, knowledge on how gut microbiota influences allergy prevention remains scarce.ObjectiveTo gain knowledge on (1) how physicochemical properties of hydrolysed whey products influence the allergy preventive capacity, (2) whether host microbiota disturbance influences allergy prevention, and (3) to what extent hydrolysed whey products influence gut microbiota composition.MethodsThe preventive capacity of four different ad libitum administered whey products was investigated in Brown Norway rats with either a conventional or an amoxicillin-disturbed gut microbiota. The preventive capacity of products was evaluated as the capacity to reduce whey-specific sensitisation and allergic reactions to intact whey after intraperitoneal post-immunisations with intact whey. Additionally, the direct effect of the whey products on the growth of gut bacteria derived from healthy human infant donors was evaluated by in vitro incubation.ResultsTwo partially hydrolysed whey products with different physicochemical characteristics were found to be superior in preventing whey-specific sensitisation compared to intact and extensively hydrolysed whey products. Daily oral amoxicillin administration, initiated one week prior to intervention with whey products, disturbed the gut microbiota but did not impair the prevention of whey-specific sensitisation. The in vitro incubation of infant faecal samples with whey products indicated that partially hydrolysed whey products might confer a selective advantage to enterococci.ConclusionsOur results support the use of partially hydrolysed whey products for prevention of cow’s milk allergy in atopy-predisposed infants regardless of their microbiota status. However, possible direct effects of partially hydrolysed whey products on gut microbiota composition warrants further investigation.

Chien-Chih Wu,1,2 Chih-Hsun Tai,1 Wen-You Liao,2 Chi-Chuan Wang,2 Ching-Hua Kuo,2 Shu-Wen Lin,1,3 Shih-Chi Ku41Department of Pharmacy, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan; 2School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan; 3Graduate Institute of Clinical Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan; 4Division of Chest Medicine, Department of Internal Medicine, National Taiwan University Hospital, Taipei, TaiwanCorrespondence: Shih-Chi KuDivision of Chest Medicine, Department of Internal Medicine, National Taiwan University Hospital, 7 Chung Shan S. Road, Taipei, TaiwanTel +886 22 356 2905Fax +886 22 358 2867Email scku1015@gmail.comBackground: Augmented renal clearance (ARC) is common in critically ill patients and could result in subtherapeutic antibiotic concentration. However, data in the Asian population are still lacking. The aim of this study was to explore the incidence and risk factors of ARC and its effect on β-lactam pharmacokinetics/pharmacodynamics (PK/PD) in Asian populations admitted to a medical ICU. In addition, we evaluated the appropriateness of using three estimated glomerular filtration (eGFR) formulas [Cockcroft–Gault (CG), Modification of Diet in Renal Disease (MDRD), and the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI)] as screening tools.Methods: We measured 2-, 8-, and 24-hr creatinine clearance (CLCr) and calculated eGFR by using three formulas for each. ARC was defined as CLCr24hr >130 mL/min/1.73 m2. Concentrations at the mid-dosing interval and prior to the next dose were collected if patients received the β-lactam antibiotic of piperacillin/tazobactam, cefepime, and meropenem, to determine the PK/PD index of fT > MIC. Multiple logistic regression analysis was conducted to identify the risk factors for ARC. Pearson correlation coefficient and the Bland and Altman method were applied to assess the accuracy of CLCr2hr, CLCr8hr, and eGFR for predicting ARC.Results: Of 100 patients, 46 (46%) manifested ARC. Younger age (4MIC (33% vs 75%, p MIC (23% vs 69%, p4MIC (3% vs 25%, p

Staphylococcus aureus is among the pathogens capable of developing a broad spectrum of infections in human beings. In addition to the hospital, the bacterium is present in the community and has a high resistance to antibiotics, which is also increasing on an ongoing basis. Resistance to β-lactam antibiotic family is one of the concerns about the bacterium that has encountered the treatment of such infections with difficulty. Due to the increased resistance and importance of this bacterium, new strategies are needed to control this pathogen. One of these approaches is the use of medicinal plants, which has attracted many researchers in the last decade. Several studies have been carried out or are being designed using various herbs to find active ingredients to deal with this bacterium. The aim of this study was to present the antibacterial activity of different medicinal plants and the effects of their active ingredients on methicillin-resistant and methicillin-sensitive S. aureus and to clarify the pathway to further studies in this regard.

The emergence of multi-drug-resistant bacteria, coupled with the lack of new antibiotics in development, is fast evolving into a global crisis. New strategies utilizing existing antibacterial agents are urgently needed. We propose one such strategy in which four outmoded β-lactam antibiotics (ampicillin, carbenicillin, cephalothin and oxacillin) and a well-known antiseptic (chlorhexidine di-acetate) were fashioned into a group of uniform materials based on organic salts (GUMBOS) as an alternative to conventional combination drug dosing strategies. The antibacterial activity of precursor ions (e.g., chlorhexidine diacetate and β-lactam antibiotics), GUMBOS and their unreacted mixtures were studied with 25 clinical isolates with varying antibiotic resistance using a micro-broth dilution method. Acute cytotoxicity and therapeutic indices were determined using fibroblasts, endothelial and cervical cell lines. Intestinal permeability was predicted using a parallel artificial membrane permeability assay. GUMBOS formed from ineffective β-lactam antibiotics and cytotoxic chlorhexidine diacetate exhibited unique pharmacological properties and profound antibacterial activity at lower concentrations than the unreacted mixture of precursor ions at equivalent stoichiometry. Reduced cytotoxicity to invasive cell types commonly found in superficial and chronic wounds was also observed using GUMBOS. GUMBOS show promise as an alternative combination drug strategy for treating wound infections caused by drug-resistant bacteria.

The problem of determining the formation of complexes of β-lactam antibiotics with cyclodextrins (CDs) and the interactions involved in this process were addressed by machine learning on multispectral images. Complexes of β-lactam antibiotics, including cefuroxime axetil, cefetamet pivoxil, and pivampicillin, as well as CDs, including αCD, βCD, γCD, hydroxypropyl-αCD, methyl-βCD, hydroxypropyl-βCD, and hydroxypropyl-γCD, were prepared in all combinations. Thermograms confirming the formation of cyclodextrin complexes were obtained using differential scanning calorimetry. Transmission Fourier-transform infrared (tFTIR) and complementary attenuated total reflectance FTIR (ATR) coupled with machine learning were techniques chosen as a nondestructive alternative. The machine learning algorithm was used to determine the formation of complexes in samples using solely their tFTIR and ATR spectra at the prediction stage. Parameterized method 7 (PM7) was used to support the analysis by molecular modeling of the complexes. The model developed through machine learning properly distinguished samples with formed complexes form noncomplexed samples with a cross-validation accuracy of 90.4%. Analysis of the contribution of spectral bands to the model indicated interactions of ester groups of β-lactam antibiotics with CDs, as well as some interactions of cephem ring in cefetamet pivoxil and penam moiety in pivampicillin. Molecular modeling with PM7 helped to explain experimental results and allowed to propose possible binding modes.

Artemisinin (ART) and its derivatives artesunate (AS), dihydroartemisinin (DHA) are a group of drugs containing a sesquiterpene lactone used to treat malaria. Previously, AS was shown to not have antibacterial activity but to significantly increase the antibacterial activities of β-lactam antibiotics against E. coli. Herein, molecular docking experiments showed that ART, AS and DHA could dock into AcrB very well, especially DHA and AS; both DHA and AS had the same docking pose. The affinity between AS and AcrB seemed weaker than that of DHA, while the succinate tail of AS, which was like a “bug”, could extend in the binding pocket very well. Imitating the parent nucleus of DHA and the succinate tail of AS, twenty-one DHA derivatives 4a–u were designed and synthesized. Among them, seventeen were new compounds. The synergistic effects against E. coli AG100A/pET28a-AcrB showed among the new structures 4k, 4l, 4m, 4n, and 4r exhibited significant synergism with β-lactam antibiotics although they had no direct antibacterial activities themelves. The bacterial growth assay showed that only 4k in combination with ampicillin or cefuroxime could totally inhibit bacterial growth from 0 to 12 h, demonstrating that 4k had the best antibacterial enhancement effect. In conclusion, our results provided a new idea and several candidate compounds for antibacterial activity enhancers against multidrug resistant E. coli.

The aim of this work was to compare the composition of a complex and soluble culture medium to eight other media described in the literature through the batch cultivation in a conventional bench-scale bioreactor for the production of cephamycin C by a wild strain of Streptomyces clavuligerus. The proposed medium resulted in an antibiotic production 1.5 to 7.5 times higher than the other culture media.