Beta-lactam Antibiotics Research Articles

Multidrug Resistance of Escherichia coli in cats and the Level of Understanding of Cat Owners on Antimicrobial Resistance

Multidrug resistance poses a global health risk to humans, animals, and the environment.The impact of this issue is so great that it has gained international attention, ranging from the rising morbidity to the mortality rate of infectious diseases in humans and animals. Because of their peculiar eliminative behavior habit of burying their waste in the ground, cats may contribute to the spread of microorganisms resistant to antibiotics. The purpose of this study was to map the prevalence of antibiotic-resistant E. coli bacteria in cats and to determine the degree of cat owners' comprehension as a risk factor for the development of antimicrobial resistance. In this investigation, 353 cat anal swab samples were found to contain E. coli. The study was carried out in 2023 between March and May. The Mueller Hilton Agar medium was used to test for multidrug resistance to beta-lactam, aminoglycoside, and tetracycline class antibiotics using the diffusion method. Using a questionnaire, the degree of knowledge on antibiotic resistance among 373 cat owners was assessed. Respondent variables include identification, knowledge, demeanor, attitude, and cat care. After that, data were descriptively examined. E. coli was detected in 87.82% (310/353) of the cat anal swab samples (n = 353) in this investigation. Additionally, multidrug resistance was shown in 7.4% (23/310) of the isolates; this frequency was higher in stray cats (8.75%) than in domestic cats (6%). For ampicillin, tetracycline, and streptomycin, the rates of antimicrobial resistance were 28.39% (88/310), 15.81% (49/310), and 14.84% (46/310), in that order. According to an analysis of the questionnaires used to gauge cat owners' comprehension of antimicrobial-resistant events, 64.3% (240/373) of the owners did not know what antimicrobial resistance was. In addition, 72.7% (271/373) of the cat owners were aware of the medications their cats were given, and 66% (246/373) of them knew how to give antibiotics to their pets. These findings are consistent with the findings of another survey that showed 70% (261/373) of cat owners took their sick cats to the veterinarian. Compared to domestic cats, stray cats in this study had a higher rate of multidrug resistance.This is consistent with the relatively positive findings of a survey on cat owners' comprehension of cat maintenance and care; yet, the poor awareness of antimicrobial resistance among cat owners may contribute to its emergence. To raise awareness regarding antimicrobial resistance in cats, cat owners need to get education.

Severe septicemia and electrolyte imbalance in a 2-month-old infant caused by<i> Brevundimonas diminuta</i>

Brevundimonas diminuta, an opportunistic Gram-negative bacterium, is increasingly recognized as a potential pathogen in clinical settings, particularly among immunocompromised individuals. We present a case of B. diminuta bacteremia in a 2-month-old male infant presenting with high-grade fever and hypocalcemic seizures. Despite initial treatment, the patient experienced recurrent seizures, leading to hospital readmission. Microbiological analysis revealed B. diminuta, sensitive to beta-lactam antibiotics and carbapenems. Prompt initiation of targeted antibiotic therapy resulted in significant clinical improvement, highlighting the importance of timely diagnosis and tailored treatment. This case underscores the evolving clinical significance of B. diminuta and emphasizes the need for heightened awareness among health-care providers for effective management of such infections, especially in pediatric patients. Further research collaboration is warranted to better understand the epidemiology and optimal treatment strategies for B. diminuta infections.

Structural shifts in TolC facilitate Efflux-Mediated β-lactam resistance

Efflux-mediated β-lactam resistance is a major public health concern, reducing the effectiveness of β-lactam antibiotics against many bacteria. Structural analyses show the efflux protein TolC in Gram-negative bacteria acts as a channel for antibiotics, impacting bacterial susceptibility and virulence. This study examines β-lactam drug efflux mediated by TolC using experimental and computational methods. Molecular dynamics simulations of drug-free TolC reveal essential movements and key residues involved in TolC opening. A whole-gene-saturation mutagenesis assay, mutating each TolC residue and measuring fitness effects under β-lactam selection, is performed. Here we show the TolC-mediated efflux of three antibiotics: oxacillin, piperacillin, and carbenicillin. Steered molecular dynamics simulations identify general and drug-specific efflux mechanisms, revealing key positions at TolC’s periplasmic entry affecting efflux motions. Our findings provide insights into TolC’s structural dynamics, aiding the design of new antibiotics to overcome bacterial efflux mechanisms.

Clinical manifestations of scarlet fever in children in the megalopolis at the present stage.

BACKGROUND: according to the literature, an increase in severe forms of scarlet fever among children was noted in a number of foreign countries in 2022-2023. AIMS: to identify the current clinical features of scarlet fever in children in a large metropolis of Russia. MATERIALS AND METHODS: an observational descriptive retrospective continuous study of the clinical features of the course of scarlet fever in children was conducted according to 295 case histories of patients diagnosed with Scarlet fever from 2020 to 2022 on the basis of the GBUZ "DGKB named after Z.A. Bashlyaeva DZM", GBUZ "DGKB No. 9 named after G.N. Speransky DZM" in Moscow. The statistical processing of the material uses traditional methods for calculating extensive and intensive indicators (p1 and p2), errors of extensive indicators (m1 and m2) and standard deviations (rmax↔rmip), taking into account the confidence interval (t) and confidence probability (P=95%), a method for comparing indicators using a well-known formula, where at t ≥ 2.0 P ≥ 95% p ≤ 0.05, the definition of standard deviations, taking into account the confidence interval (t) and the confidence probability P = 95%. The comparison of percentages in the analysis of four-field conjugacy tables was performed using the exact Fisher criterion. The differences were considered statistically significant at p 0.05. RESULTS: as a result of the study, it was revealed that the abolition of strict anti-epidemic restrictions on COVID-19 in 2022 led not only to an increase in the incidence of scarlet fever among children from 0 to 18 years of age by 3.6 times in 2022 compared with 2021, but, as a result, to an increase in hospitalization of children in hospitals in Moscow. Thus, the number of hospitalized children with scarlet fever in 2022 increased by 18.0 times compared to 2020. The majority of those hospitalized (83.4%) were children from organized groups, including those aged 3 to 7 years (52.9%) and from 7 to 14 years (39.3%), with an average severity of the disease (93.6%). It was noted that that among the examined children, 53 children (18%) had a history of COVID-19, and among this group of children, one in five children had severe scarlet fever, while among children who did not have a history of COVID-19, these were significantly (p 0.001) 8.4 times less (respectively, 20.7% versus 2.5%). It was revealed that every 8-9 patients (12.2%) had various complications, mainly of a septic nature and those that arose in the first week of the disease, including otitis media, sinusitis, community-acquired pneumonia, parathonsillar and lateral pharyngeal abscesses and toxic shock syndrome. In the second week and later, complications of an infectious and allergic nature were reported, including tubulointerstitial nephritis, acute renal injury, post-infectious cardiomyopathy, acute post-streptococcal glomerulonephritis, toxic allergic vasculitis, post-streptococcal arthritis. Beta-lactam antibiotics remain the etiotropic drugs of choice. Antibiotics of the cephalosporin 53.6% and penicillin 46.4% series were used to treat scarlet fever. CONCLUSIONS: in modern conditions of the megalopolis, scarlet fever occurs with a typical clinical picture. The majority of those hospitalized were children of preschool and school age, only 12.2% of children were diagnosed with various complications of scarlet fever and the vast majority of children (93.6%) had a moderate severity of scarlet fever. It was noted that against the background of an increase in the number of hospitalized patients, the number of patients with severe scarlet fever decreases, while the incidence of complications in patients does not change (12.3% - 12.8%). It was shown that among patients who had suffered from COVID-19, scarlet fever was 8.3 times more likely to occur in severe form compared with children who had no history of COVID-19 disease (respectively, 20.7% vs. 2.5, p 0.0001).

Bacteriophage Therapy on an In Vitro Wound Model and Synergistic Effects in Combination with Beta-Lactam Antibiotics.

One of the primary opportunistic pathogens that can cause a wide range of diseases is Pseudomonas aeruginosa. This microorganism can become resistant to practically every antibacterial currently in use, including beta-lactam antibiotics. Its ability to proliferate as biofilm has been linked to, among other things, the failure of antimicrobial therapies. Due to a variety of virulence factors and host immune system modifications, P. aeruginosa is one of the most significant and common bacteria that colonize wounds and burns. A novel therapeutic option for treating these multidrug-resistant (MDR) bacterial infections is the combination of antibiotics and bacteriophages. This approach has been linked to improved biofilm penetration, a decreased selection of antibiotic and bacteriophage resistance, and an enhanced antibacterial impact. Combining the F1Pa bacteriophage and beta-lactam antibiotics reduced the viability of the mature biofilm of MDR P. aeruginosa strains and suppressed bacterial growth in vitro. F1Pa critically reduced the amount of biofilm that MDR P. aeruginosa clinical strains formed in the in vitro wound model. These findings highlight the bacteriophage F1Pa's therapeutic potential as a prophylactic topical treatment against MDR pseudomonal infections in wounds and burns.

Australian Group on Antimicrobial Research surveillance outcome programs - bloodstream infections and antimicrobial resistance patterns from patients less than 18 years of age, January 2020 - December 2021.

From 1 January 2020 to 31 December 2021, thirty-eight institutions across Australia submitted data to the Australian Group on Antimicrobial Resistance (AGAR) from patients aged < 18 years (AGAR-Kids). Over the two years, 1,679 isolates were reported from 1,611 patients. This AGAR-Kids report aims to describe the population of children and adolescents with bacteraemia reported to AGAR and the proportion of resistant isolates. Overall, there were 902 gram-negative isolates reported: 800 Enterobacterales, 61 Pseudomonas aeruginosa and 41 Acinetobacter spp. Among the Enterobacterales, 12.9% were resistant to third generation cephalosporins; 11.6% to gentamicin/tobramycin; and 11.2% to piperacillin-tazobactam. In total, 14.5% of Enterobacterales were multi-drug resistant (MDR). Only 3.3% of P. aeruginosa were resistant to carbapenems and 4.9% were MDR. Resistance in Acinetobacter spp was uncommon. Of 607 Staphylococcus aureus isolates, 12.9% were methicillin-resistant (MRSA). Almost half of S. aureus isolates from the Northern Territory were MRSA. In S. aureus, resistance to erythromycin was 13.2%; 12.4% to clindamycin; and 5.3% to ciprofloxacin. Resistance to all antibiotics tested was higher in MRSA. Overall, 6.5% of S. aureus were MDR, of which 65% were MRSA. Almost three-quarters of the 170 Enterococcus spp. reported were E. faecalis, and half were from patients < 1 year old. Ampicillin resistance in enterococci was 19.6%. Eight isolates were vancomycin resistant and three isolates were teicoplanin resistant. Five E. faecium isolates were classified as MDR. This AGAR-Kids report highlights clear differences in the geographic distribution of pathogens and resistance profiles across Australia.

Reassessing the management of uncomplicated urinary tract infection: A retrospective analysis using machine learning causal inference.

Uncomplicated urinary tract infection (UTI) is a common indication for outpatient antimicrobial therapy. National guidelines for the management of uncomplicated UTI were published by the Infectious Diseases Society of America in 2011, however it is not fully known the extent to which they align with current practices, patient diversity, and pathogen biology, all of which have evolved significantly in the time since their publication. We aimed to re-evaluate efficacy and adverse events for first-line antibiotics (nitrofurantoin, and trimethoprim-sulfamethoxazole), versus second-line antibiotics (fluoroquinolones) and versus alternative agents (oral β-lactams) for uncomplicated UTI in contemporary clinical practice by applying machine learning algorithms to a large claims database formatted into the Observational Medical Outcomes Partnership (OMOP) common data model. Our primary outcome was a composite endpoint for treatment failure, defined as outpatient or inpatient re-visit within 30 days for UTI, pyelonephritis or sepsis. Secondary outcomes were the risk of 4 common antibiotic-associated adverse events: gastrointestinal symptoms, rash, kidney injury and C. difficile infection. We adjusted for covariate-dependent censoring and treatment indication using a broad set of domain-expert derived features. Sensitivity analyses were conducted using OMOP-learn, an automated feature engineering package for OMOP datasets. Our study included 57,585 episodes of UTI from 49,037 patients. First-line antibiotics were prescribed in 35,018 (61%) episodes, second-line antibiotics were prescribed in 21,140 (37%) episodes and alternative antibiotics were prescribed in 1,427 (2%) episodes. After adjustment, patients receiving first-line therapies had an absolute risk difference of -2.1% [95% CI -2.9% to -1.6%] for having a revisit for UTI within 30 days of diagnosis relative to second-line antibiotics. First-line therapies had an absolute risk difference of -6.6% [95% CI -9.4% to -3.8%] for 30-day revisit compared to alternative β-lactam antibiotics. Differences in adverse events were clinically similar between first and second line agents, but lower for first-line agents relative to alternative antibiotics (-3.5% [95% CI -5.9% to -1.2%]). Results were similar for models built with OMOPlearn. Our study provides support for the continued use of first-line antibiotics for the management of uncomplicated UTI. Our results also provide proof-of-principle that automated feature extraction methods for OMOP formatted data can emulate manually curated models, thereby promoting reproducibility and generalizability.

Surface enhanced Raman spectroscopy and machine learning for identification of beta-lactam antibiotics resistance gene fragment in bacterial plasmid

BackgroundAntibiotic resistance stands as a critical medical concern, notably evident in commonly prescribed beta-lactam antibiotics. The imperative need for expeditious and precise early detection methods underscores their role in facilitating timely intervention, curbing the propagation of antibiotic resistance, and enhancing patient outcomes. ResultsThis study introduces the utilization of surface-enhanced Raman spectroscopy (SERS) in tandem with machine learning (ML) for the sensitive detection of characteristic gene fragments responsible for antibiotic resistance appearance and spreading. To make the detection procedure close to the real case, we used bacterial plasmids as starting biological objects, containing or not the characteristic gene fragment (up to 1:10 ratio), encoding beta-lactam antibiotics resistance. The plasmids were subjected to enzymatic digestion and without preliminary purification or isolation the created fragments were captured by functional SERS substrates. Based on subsequent SERS measurements, a database was created for the training and validation of ML. Method validation was performed using separately measured spectra, which did not overlap with the database used for ML training. To check the efficiency of recognising the target fragment, control experiments involved bacterial plasmids containing different resistance genes, the use of inappropriate enzymes, or the absence of plasmid. SignificanceSERS-ML allowed express detection of bacterial plasmids containing a characteristic gene fragment up to the 10−7 concentration of the initial plasmid, despite the complex composition of the biological sample, including the presence of interfering plasmids. Our approach offers a promising alternative to existing methods for monitoring antibiotic-resistant bacteria, characterized by its simplicity, low detection limit, and the potential for rapid and straightforward analysis.

Methods for degrading dicloxacillin in water using inorganic peroxides and their combination with UVC- experimental and theoretical aspects

Antibiotics are currently recognized as environmental pollutants. In this work, the methods involved in the degradation of a β-lactam antibiotic (i.e., DXC) by treatments based on inorganic peroxides and UVC (e.g., UVC alone, UV-C/H2O2, UVC/peroxymonosulfate, and UVC/peroxydisulfate) are presented. The methodology of computational calculations to obtain frontier orbitals and Fukui indices for DXC, and elucidate the reactive moieties on the target substance is also shown. Finally, the direct oxidation by peroxides and UV-C/H2O2 action to treat DXC in simulated pharmaceutical wastewater are depicted. The chromatographic and theoretical analyses allowed for determining the degrading performance of inorganic peroxides and UVC-based treatments toward the target pollutant in aqueous samples.•Treatments based on inorganic peroxides and UVC as useful methods for degrading the β-lactam antibiotic dicloxacillin.•Persulfates and UV-C/H2O2 showed high degrading action on the target pharmaceutical.•Methodologies based on theoretical calculations for the identification of reactive moieties on the DXC susceptible to radical attacks are presented.

Genetic Characterization, Antibiotic Resistance, and Virulence Genes Profiling of Bacillus cereus Strains from Various Foods in Japan.

Bacillus cereus sensu stricto is a foodborne pathogen that causes food poisoning. Their spore and biofilm-forming abilities persist in various environments and foods. This study investigated the prevalence, virulence, antibiotic resistance, and genetic diversity of B. cereus s. s. strains isolated from various food samples. Of 179 samples, 22.34% were positive for B. cereus s. s., with significantly high detection rates in milk products and raw chicken meat. Forty strains were isolated from positive samples. Matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis revealed nine distinct clusters and multi-locus sequence typing revealed 34 sequence types including 23 novel sequences, demonstrating high genetic diversity among the isolates. PCR analysis revealed that all the strains contained at least one toxin gene, but none contained the cytK gene. Antibiotic resistance tests revealed that all isolates were classified as multidrug-resistant, with high resistance levels, particularly to β-lactam antibiotics and vancomycin, but were susceptible to gentamicin. All isolates showed variations in biofilm formation. This study highlights the significant public health risk due to B. cereus s. s. and underscores the need for stringent monitoring and control measures in food production to manage antimicrobial resistance and ensure food safety.

A MRSA mystery: how PBP4 and cyclic-di-AMP join forces against β-lactam antibiotics.

The high-level resistance to next-generation β-lactams frequently found in Staphylococcus aureus isolates lacking mec, which encodes the transpeptidase PBP2a traditionally associated with methicillin-resistant Staphylococcus aureus (MRSA), has remained incompletely understood for decades. A new study by Lai et al. found that the co-occurrence of mutations in pbp4 and gdpP, which respectively cause increased PBP4-mediated cell wall crosslinking and elevated cyclic-di-AMP levels, produces synergistic β-lactam resistance rivaling that of PBP2a-producing MRSA (L.-Y. Lai, N. Satishkumar, S. Cardozo, V. Hemmadi, et al., mBio 15:e02889-23. 2024, https://doi.org/10.1128/mbio.02889-23). The combined mutations are sufficient to explain the high-level β-lactam resistance of some mec-lacking strains, but the mechanism of synergy remains elusive and an avenue for further research. Importantly, the authors establish that co-occurrence of these mutations leads to antibiotic therapy failure in a Caenorhabditis elegans infection model. These results underscore the need to consider this unique and novel β-lactam resistance mechanism during the clinical diagnosis of MRSA, rather than relying on mec as a diagnostic.

Occurrence and Antibiotic Resistance Profiles of Metallo Beta Lactamase Producing Pseudomonas aeruginosa among Clinical Isolates in Enugu Metropolis, Nigeria

Introduction: Antibiotic resistance is rising worldwide, posing a challenge for contemporary medicine. Metallo-beta-lactamases (MBLs) confer resistance to beta-lactam antibiotics other than monbactams. It threatens public health because of its vast scope of action and quick spread. The study was undertaken to assess the occurrence of MBL in clinical isolates of Pseudomonas aeruginosa in the Enugu metropolis and to determine their resistance profile. Materials and Methods: The work was conducted from October 2020 to July 2021 in the microbiology laboratory of the University of Nigeria Teaching Hospital, Ituku-Ozalla. A total of 127 non-duplicate bacterial isolates recovered from clinical samples including wounds, urine, sputum, ear discharge, and catheter tip processed in the microbiology laboratory of four referral hospitals within the Enugu metropolis was used for the study. Isolates were identified and characterized using standard microbiology protocols. Antimicrobial susceptibility was done using the Kirby-Bauer disc diffusion method. Phenotypic detection of Metallo-beta-lactamase production was done using the Combined Disk diffusion Test (CDDT). Results: Of the 127 isolates, 68 (53.5%) were resistant to imipenem. Among these, 35 strains were positive for MBL production while 33 isolates were non-MBL producers. The highest percentage occurrence of MBL producers was recorded from catheter tips 33% followed by urine 30.6%. Both MBL producers and non-MBL producers displayed high resistance to most of the antibiotics used except Aztreonam. Conclusion: The overall occurrence of MBL among Pseudomonas aeruginosa in our study was found to be 27.6%. MBL-producing strains showed higher resistance than the non-MBL-producing strains. Aztreonam was the most potent antibiotic. The most effective approaches to combating this organism include early detection, stringent antibiotic regimens, and adherence to infection control measures.

Pneumonia due to Mycoplasma hominis in a Full-Term Newborn - A Case Report with Literature Review

Mycoplasma ssp can colonize various human tissues and can cause infections. Their lack of a cell wall makes them difficult to cultivate and to treat as they are resistant to beta-lactam antibiotics. Mycoplasma hominis and ureaplasma urealyticum can colonize the genital tract. While colonization in healthy adults is usually asymptomatic, they can cause neonatal infections during pregnancy through transmission to the fetus or during birth and lead to increased morbidity and mortality, especially in premature infants. However, in full-term neonates with high colonization rates, the pathogenic role and its treatment is controversial. In the following, we present a case of neonatal pneumonia caused by M. hominis in a full-term neonate, which was successfully treated with clindamycin. Mycoplasma spp. can cause symptomatic infections in neonates in individual cases and should be considered as potential pathogens, especially in the case of a protracted course.

A Hunt for the Resistance of Haemophilus influnezae to Beta-Lactams.

Infections due to Haemophilus influnezae require prompt treatment using beta-lactam antibiotics. We used a collection of 81 isolates obtained between 1940 and 2001 from several countries. Whole genome sequencing showed the high heterogeneity of these isolates but allowed us to track the acquisition of beta-lactamase, which was first detected in 1980. Modifications of the ftsI gene encoding the penicillin-binding protein 3, PBP3, also involved in resistance to beta-lactams, appeared in 1991. These modifications (G490E, A502V, R517H, and N526K) were associated with resistance to amoxicillin that was not relieved by a beta-lactamase inhibitor (clavulanic acid), but the isolates retained susceptibility to third-generation cephalosporins (3GC). The modeling of the PBP3 structure suggested that these modifications may reduce the accessibility to the PBP3 active site. Other modifications appeared in 1998 and were associated with resistance to 3GC (S357N, M377I, S385T, and L389F). Modeling of the PBP3 structure suggested that they lie near the S379xN motif of the active site of PBP3. Overall resistance to amoxicillin was detected among 25 isolates (30.8%) of this collection. Resistance to sulfonamides was predicted by a genomic approach from the sequences of the folP gene (encoding the dihydropteroate synthase) due to difficulties in interpreting phenotypic anti-microbial testing and found in 13 isolates (16.0%). Our data suggest a slower spread of resistance to sulfonamides, which may be used for the treatment of H. influnezae infections. Genomic analysis may help in the prediction of antibiotic resistance, inform structure-function analysis, and guide the optimal use of antibiotics.

Efficacy of aspergillomarasmine A/meropenem combinations with and without avibactam against bacterial strains producing multiple β-lactamases.

The effectiveness of β-lactam antibiotics is increasingly threatened by resistant bacteria that harbor hydrolytic β-lactamase enzymes. Depending on the class of β-lactamase present, β-lactam hydrolysis can occur through one of two general molecular mechanisms. Metallo-β-lactamases (MBLs) require active site Zn2+ ions, whereas serine-β-lactamases (SBLs) deploy a catalytic serine residue. The result in both cases is drug inactivation via the opening of the β-lactam warhead of the antibiotic. MBLs confer resistance to most β-lactams and are non-susceptible to SBL inhibitors, including recently approved diazabicyclooctanes, such as avibactam; consequently, these enzymes represent a growing threat to public health. Aspergillomarasmine A (AMA), a fungal natural product, can rescue the activity of the β-lactam antibiotic meropenem against MBL-expressing bacterial strains. However, the effectiveness of this β-lactam/β-lactamase inhibitor combination against bacteria producing multiple β-lactamases remains unknown. We systematically investigated the efficacy of AMA/meropenem combination therapy with and without avibactam against 10 Escherichia coli and 10 Klebsiella pneumoniae laboratory strains tandemly expressing single MBL and SBL enzymes. Cell-based assays demonstrated that laboratory strains producing NDM-1 and KPC-2 carbapenemases were resistant to the AMA/meropenem combination but became drug-susceptible upon adding avibactam. We also probed these combinations against 30 clinical isolates expressing multiple β-lactamases. E. coli, Enterobacter cloacae, and K. pneumoniae clinical isolates were more susceptible to AMA, avibactam, and meropenem than Pseudomonas aeruginosa and Acinetobacter baumannii isolates. Overall, the results demonstrate that a triple combination of AMA/avibactam/meropenem has potential for empirical treatment of infections caused by multiple β-lactamase-producing bacteria, especially Enterobacterales.

Covalent penicillin-protein conjugates elicit anti-drug antibodies that are clonally and functionally restricted

Many archetypal and emerging classes of small-molecule therapeutics form covalent protein adducts. In vivo, both the resulting conjugates and their off-target side-conjugates have the potential to elicit antibodies, with implications for allergy and drug sequestration. Although β-lactam antibiotics are a drug class long associated with these immunological phenomena, the molecular underpinnings of off-target drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, using the classical β-lactam penicillin G (PenG), we probe the B and T cell determinants of drug-specific IgG responses to such conjugates in mice. Deep B cell clonotyping reveals a dominant murine clonal antibody class encompassing phylogenetically-related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Protein NMR and x-ray structural analyses reveal that these drive structurally convergent binding modes in adduct-specific antibody clones. Their common primary recognition mechanisms of the penicillin side-chain moiety (phenylacetamide in PenG)—regardless of CDRH3 length—limits cross-reactivity against other β-lactam antibiotics. This immunogenetics-guided discovery of the limited binding solutions available to antibodies against side products of an archetypal covalent inhibitor now suggests future potential strategies for the ‘germline-guided reverse engineering’ of such drugs away from unwanted immune responses.

A low-barrier proton shared between two aspartates acts as a conformational switch that changes the substrate specificity of the β-lactamase BlaC

Serine β-lactamases inactivate β-lactam antibiotics in a two-step mechanism comprising acylation and deacylation. For the deacylation step, a water molecule is activated by a conserved glutamate residue to release the adduct from the enzyme. The third-generation cephalosporin ceftazidime is a poor substrate for the class A β-lactamase BlaC from Mycobacterium tuberculosis but it can be hydrolyzed faster when the active site pocket is enlarged, as was reported for mutant BlaC P167S. The conformational change in the Ω-loop of the P167S mutant displaces the conserved glutamate (Glu166), suggesting it is not required for deacylation of the ceftazidime adduct. Here, we report the characterization of wild type BlaC and BlaC E166A at various pH values. The presence of Glu166 strongly enhances activity against nitrocefin but not ceftazidime, indicating it is indeed not required for deacylation of the adduct of the latter substrate. At high pH wild type BlaC was found to exist in two states, one of which converts ceftazidime much faster, resembling the open state previously reported for the BlaC mutant P167S. The pH-dependent switch between the closed and open states is caused by the loss at high pH of a low-barrier hydrogen bond, a proton shared between Asp172 and Asp179. These results illustrate how readily shifts in substrate specificity can occur as a consequence of subtle changes in protein structure.

Rapid Detection of Methicillin Resistant Staphylococcus Aureus (MRSA) Using Electric-Double-Layer (EDL) Field-Effect Transistor (FET) Biosensors

According to the latest statistics and analysis by American researchers based on the data from 195 countries worldwide, it has been found that one person in every five people died due to sepsis. The annual death caused by sepsis reaches up to eleven million people, and the number of deaths from sepsis is even higher than those from cancer. We believe that the detection of MRSA is necessary.Staphylococcus aureus is the most common pathogen of sepsis. Methicillin-resistant Staphylococcus aureus (MRSA) or Multiple-resistant Staphylococcus aureus is a distinct strain of Staphylococcus aureus. It has mecA gene which codes for penicillin binding protein 2a (PBP 2A) that will remain active under beta lactams antibiotics. This gives bacterial the ability to grow under concentrations of beta-lactam antibiotics which leads to MRSA's resistance to almost all the different penicillin-type antibiotics including methicillin and other beta-lactamase-resistant penicillins.To detect MRSA in the shortest possible time, we adopt field-effect transistor (FET) biosensor for the real-time detection of binding reactions between DNA probe and cDNA. We select DNA probe sequence of mecA to be the aptamer and immobilize probe DNA which are complementary to the target cDNA on the sensor. This process allows DNA strands to attach to the gold surface. After that, we detect the binding reaction of DNA probe and cDNA by analyzing electrical response of field-effect transistor (FET). We can observe that the Id current changes after cDNA hybridizes with DNA probe.In conclusion, in our research results, Field-effect transistor (FET) biosensor can detect concentration ranging from 1fM to 1pM. In addition, we can achieve other advantages such as more portable, higher sensitivity, real-time monitoring capability, and lower detection limits. With this rapid detection procedure, we can do early diagnosis and treatment, thus improving the quality of human life. Keywords: FET, EDL, Aptamer, DNA, MRSA Figure 1

A Dual-Channel Biosensor Enables Concurrent Detection of Pathogens and Antibiotic Resistance

Diarrhoeagenic infections, commonly treated with β-lactam antibiotics, contribute to antibiotic resistance - a pressing public health concern. Rapid monitoring of pathogen antibiotic resistance is vital to combat antimicrobial spread. Current bacterial diagnosis methods identify pathogens or determine antibiotic resistance separately, necessitating multiple assays. There is an urgent need for tools that simultaneously identify infectious agents and their antibiotic resistance at the point of care (POC). We developed an integrated electrochemical chip-based biosensor for detecting enteropathogenic E. coli. Leveraging an antibody against the virulence marker EspB, the dual-channel chip facilitated electrochemical impedance spectroscopy-based detection of EspB antigen (sensitivity of 10 ng/mL) and EspB-expressing bacteria. For β-lactam resistance profiling, a second channel enabled differential-pulse voltammetric measurement of hydrolyzed nitrocefin, a β-lactam analogue, demonstrating sensitive detection of the antibiotic resistance marker, β-lactamase at concentrations as low as 1 ng/mL. The integrated electrochemical biosensor successfully achieved simultaneous, rapid detection of double positive EspB- and β-lactamase-expressing bacteria, promoting advancements in POC clinical diagnosis.

A β-Lactamase Responsive Peptide Inhibits MRSA Infection through Self-Assembled Nanonet.

Gram-positive S. aureus is one of the leading pathogens for death associated with antimicrobial resistance. The β-lactamase (Bla) secreted by methicillin-resistant S. aureus (MRSA) hydrolyzes nearly all β-lactam antibiotics, leaving only a few antibiotics available for the clinical treatment of MRSA infections. Thereby, a Bla-responsive peptide (BLAP) is designed here with the capacity of inhibiting MRSA infection through mimicking the host defense mechanism of human defensin-6. The BLAP comprising a self-assembling peptide sequence can respond specifically to the secreted Bla and assemble in situ surrounding MRSA. The assembled nanofibrous network is able to trap MRSA, preventing its invasion into the host cells effectively. As a consequence, the intramuscular injection of BLAP significantly restricted bacterial infection and abscess formation in mice. The biomimetic BLAP holds great potential for the efficient treatment of drug-resistant gram-positive bacterial infections.