Cefotaxime-non-susceptible Haemophilus influenzae has rarely been isolated from clinical specimens. Although several reports have shown that amino acid (AA) alteration in penicillin-binding protein 3 ...(PBP3), encoded by the ftsI gene, reduces activity of cefotaxime, precise mechanisms conferring the non-susceptibility have been unclear. We analyzed the ftsI gene of two clinically isolated cefotaxime-non-susceptible H. influenzae strains, 16-11 and 20-07 (minimum inhibitory concentrations MICs: 16 and 8 μg/mL, respectively), and found that their deduced AA sequences of PBP3 included two AA substitutions of G555E and Y557H in addition to previously described AA alterations. To clarify whether the two additional substitutions are requisite for cefotaxime non-susceptibility, we produced transformants of Rd KW20 (cefotaxime MIC: ≤0.06 μg/mL) with the ftsI gene of 16-11. Cefotaxime MICs against transformants M1 and M2, of which deduced PBP3s were altered with that of 16-11 entirely and partially (only the N-terminal side up to the AA position 519), were 8 and 0.25 μg/mL, respectively. We also produced M2-555/7 through site-directed mutagenesis inducing additional substitutions of G555E and Y557H into the PBP3 of M2, against which cefotaxime MIC was 8 μg/mL. These findings show that the additional substitutions of G555E and Y557H in PBP3 with previously described alterations cause cefotaxime non-susceptibility. An additional substitution of either G555E or Y557H alone in altered PBP3 reduced cefotaxime activity but the elevation of MICs were within the category of susceptibility. To our knowledge, this is the first study clarifying a genetic factor in the PBP3 causing cefotaxime non-susceptibility among H. influenzae strains.
Mesoporous graphitic carbon nitride (MCN) photocatalyst was blended in polyvinylidene fluoride (PVDF) membrane through immersion-precipitation phase transformation. Various techniques were adopted to ...characterize the structure and morphology of MCN-PVDF hybrid membrane, which indicated that MCN was successfully integrated in PVDF. The MCN80-PVDF membrane exhibited enhanced hydrophilicity and antifouling properties. As the amount of MCN addition increased, the contact angle of membranes decreased from 68.33° to 57.12°, whereas the flux recovery rate increased by 28% as compared to the pristine PVDF. Meanwhile, MCN-PVDF membrane possessed photocatalytic, self-cleaning and antimicrobial activities. The MCN80-PVDF membrane was used to degrade cefotaxime (CFX) under sunlight irradiation. After five cycles of experiments, the degradation rate of CFX remained 97.4%. Additionally, MCN80-PVDF membrane achieved 3 log of E. coli inactivation under visible light irradiation for 4 h. The photogenerated h+ and reactive oxidative species were the primary reason for organics’ degradation and bacterial inactivation. In summary, the prepared hybrid MCN80-PVDF membrane with sunlight irradiation exhibited great potential for the treatment of real wastewater.
•MCN was blended in PVDF via immersion-precipitation phase transformation.•MCN enhanced the permeability, hydrophilicity and antifouling of membrane.•The degradation of CFX under sunlight irradiation by MCN-PVDF achieved over 97%.•MCN-PVDF was stable and exhibited great antimicrobial and self-cleaning properties.
CTX-M β-lactamases are a widespread source of resistance to β-lactam antibiotics in Gram-negative bacteria. These enzymes readily hydrolyze penicillins and cephalosporins, including ...oxyimino-cephalosporins such as cefotaxime. To investigate the preference of CTX-M enzymes for cephalosporins, we examined eleven active-site residues in the CTX-M-14 β-lactamase model system by alanine mutagenesis to assess the contribution of the residues to catalysis and specificity for the hydrolysis of the penicillin, ampicillin, and the cephalosporins cephalothin and cefotaxime. Key active site residues for class A β-lactamases, including Lys73, Ser130, Asn132, Lys234, Thr216, and Thr235, contribute significantly to substrate binding and catalysis of penicillin and cephalosporin substrates in that alanine substitutions decrease both kcat and kcat/KM. A second group of residues, including Asn104, Tyr105, Asn106, Thr215, and Thr216, contribute only to substrate binding, with the substitutions decreasing only kcat/KM. Importantly, calculating the average effect of a substitution across the 11 active-site residues shows that the most significant impact is on cefotaxime hydrolysis while ampicillin hydrolysis is least affected, suggesting the active site is highly optimized for cefotaxime catalysis. Furthermore, we determined X-ray crystal structures for the apo-enzymes of the mutants N106A, S130A, N132A, N170A, T215A, and T235A. Surprisingly, in the structures of some mutants, particularly N106A and T235A, the changes in structure propagate from the site of substitution to other regions of the active site, suggesting that the impact of substitutions is due to more widespread changes in structure and illustrating the interconnected nature of the active site.
Five point mutations in a particular {szligbeta}-lactamase allele jointly increase bacterial resistance to a clinically important antibiotic by a factor of approximately100,000. In principle, ...evolution to this high-resistance {szligbeta}-lactamase might follow any of the 120 mutational trajectories linking these alleles. However, we demonstrate that 102 trajectories are inaccessible to Darwinian selection and that many of the remaining trajectories have negligible probabilities of realization, because four of these five mutations fail to increase drug resistance in some combinations. Pervasive biophysical pleiotropy within the {szligbeta}-lactamase seems to be responsible, and because such pleiotropy appears to be a general property of missense mutations, we conclude that much protein evolution will be similarly constrained. This implies that the protein tape of life may be largely reproducible and even predictable.
Ser/Thr protein kinase (STK1) plays a critical role in cell wall biosynthesis of and drug resistance in methicillin-resistant Staphylococcus aureus (MRSA). MRSA strains lacking STK1 become ...susceptible to failing cephalosporins, such as Ceftriaxone and Cefotaxime. STK1, despite being nonessential protein for MRSA survival, it can serve as an important therapeutic agent for combination therapy. Here, we report a novel small molecule quinazoline compound, Inh2-B1, which specifically inhibits STK1 activity by directly binding to its ATP-binding catalytic domain. Functional analyses encompassing in vitro growth inhibition of MRSA, and in vivo protection studies in mice against the lethal MRSA challenge indicated that at high concentration neither Inh2-B1 nor Ceftriaxone or Cefotaxime alone was able to inhibit the growth of bacteria or protect the challenged mice. However, the growth of MRSA was inhibited, and a significant protection in mice against the bacterial challenge was observed at a micromolar concentration of Ceftriaxone or Cefotaxime in the presence of Inh2-B1. Cell-dependent minimal to no toxicity of Inh2-B1, and its abilities to down-regulate cell wall hydrolase genes and disrupt the biofilm formation of MRSA clearly indicated that Inh2-B1 serves as a therapeutically important "antibiotic-resistance-breaker," which enhances the bactericidal activity of Ceftriaxone/Cefotaxime against highly pathogenic MRSA infection.
ABO blood type incompatibility hemolytic disease of newborn (ABO-HDN) and drug-induced immune hemolytic anemia (DIIHA) due to non-immunologic protein adsorption (NIPA) mainly cause extravascular ...hemolysis. All the reported severe DIIHA were caused by drug-induced antibodies, and rare report of acute intravascular hemolysis was caused by the NIPA mechanism or ABO-HDN.
We report the first case of acute intravascular hemolysis induced by cefotaxime sodium - sulbactam sodium (CTX - SBT) in a case of ABO-HDN which resulted in death at 55 h after birth. The mother's blood type was O and RhD-positive, and the newborn's blood type was B and RhD-positive. No irregular red blood cell (RBC) antibodies or drug-dependent antibodies related to CTX or SBT was detected in the mother's plasma and the plasma or the RBC acid eluent of the newborn. Before the newborn received CTX - SBT treatment, the result of direct antiglobulin test (DAT) was negative while anti-B was positive (2 +) in both plasma and acid eluent. After the newborn received CTX - SBT treatment, the results of DAT for anti-IgG and anti-C3d were both positive, while anti-B was not detected in plasma, but stronger anti-B (3 +) was detected in acid eluent.
experiments confirmed that NIPA of SBT promoted the specific binding of maternal-derived IgG anti-B to B antigen on RBCs of the newborn, thereby inducing acute intravascular hemolysis.
The NIPA effect of SBT promoted the specific binding of mother-derived IgG anti-B in newborn's plasma to the newborn's RBC B antigens and formed an immune complex, and then activated complement, which led to acute intravascular hemolysis. Drugs such as SBT with NIPA effect should not be used for newborns with HDN.
Adaptive evolutionary processes are constrained by the availability of mutations which cause a fitness benefit and together make up the fitness landscape, which maps genotype space onto fitness under ...specified conditions. Experimentally derived fitness landscapes have demonstrated a predictability to evolution by identifying limited "mutational routes" that evolution by natural selection may take between low and high-fitness genotypes. However, such studies often utilize indirect measures to determine fitness. We estimated the competitive fitness of mutants relative to all single-mutation neighbors to describe the fitness landscape of three mutations in a β-lactamase enzyme. Fitness assays were performed at sublethal concentrations of the antibiotic cefotaxime in a structured and unstructured environment. In the unstructured environment, the antibiotic selected for higher-resistance types-but with an equivalent fitness for a subset of mutants, despite substantial variation in resistance-resulting in a stratified fitness landscape. In contrast, in a structured environment with a low antibiotic concentration, antibiotic-susceptible genotypes had a relative fitness advantage, which was associated with antibiotic-induced filamentation. These results cast doubt that highly resistant genotypes have a unique selective advantage in environments with subinhibitory concentrations of antibiotics and demonstrate that direct fitness measures are required for meaningful predictions of the accessibility of evolutionary routes.
The evolution of antibiotic-resistant bacterial populations underpins the ongoing antibiotic resistance crisis. We aim to understand how antibiotic-degrading enzymes can evolve to cause increased resistance, how this process is constrained, and whether it can be predictable. To this end, competition experiments were performed with a combinatorially complete set of mutants of a β-lactamase gene subject to subinhibitory concentrations of the antibiotic cefotaxime. While some mutations confer on their hosts high resistance to cefotaxime, in competition these mutations do not always confer a selective advantage. Specifically, high-resistance mutants had equivalent fitnesses despite different resistance levels and even had selective disadvantages under conditions involving spatial structure. Together, our findings suggest that the relationship between resistance level and fitness at subinhibitory concentrations is complex; predicting the evolution of antibiotic resistance requires knowledge of the conditions that select for resistant genotypes and the selective advantage evolved types have over their predecessors.
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•Water disinfection was realized through magnetic CFO/CFS/MBO photocatalyst.•CFO/CFS/MBO exhibited excellent photocatalytic activity for the removal of CTX.•The water disinfection ...efficiency of CFO/CFS/MBO (0.5 mg/mL) was up to 99.8%.•The mechanism of charge transfer and separation in p-n-p heterojunction was explored.
Although advanced photocatalysts have been developed for water disinfection, photocatalytic activity is still limited owing to the difficulty of recycling nanoparticles and the high recombination rate of photogenerated electron-hole pairs. In this paper, efficient and recyclable CoFe2O4/CoFe2S4/MgBi2O6 (CFO/CFS/MBO) magnetic fibers (MNFs) photocatalyst with p-n-p type heterojunction are obtained by a simple hydrothermal method. As expected, when the optimal concentration of CFO/CFS/MBO was 0.5 mg/mL, the antibacterial rate reached 99.8% toward Methicillin-resistant Staphylococcus aureus (MRSA) after 60 min visible light irradiation. The optimal sample 30% CFO/CFS/MBO composite can degrade 82.7% Cefotaxime sodium (CTX) in 60 min. The mechanism was shown to be the severe damage of bacterial cell membranes and decomposition of antibiotic molecules caused by ROS generated by CFO/CFS/MBO photocatalyst under visible light irradiation. Due to the p-n-p heterojunction structural characteristics and abundant oxygen vacancies, CFO/CFS/MBO possessed a stronger absorption capacity of visible light and enhanced photo-generated carrier separation efficiency, which has a remarkably positive impact on the ability of photo-generated ROS. PL and EIS revealed the improvement of the separation efficiency of photogenerated carriers. LC-MS analysis, EPR and scavenger experiments verified the intermediate product, active species and photodegradation pathway in the photocatalytic process. Such recyclable and highly efficient CFO/CFS/MBO MNFs photocatalysts have promising applications in wastewater treatment.
•Most βRGs’ abundance decreased firstly and then increased induced by cefotaxime.•Environment-dose cefotaxime altered composition patterns of βRGs.•Cefotaxime enhanced the potential mobility of βRGs ...among bacteria in fish guts.•Shifts of bacterial community and MGEs were key drivers to the variation of βRGs.•Cefotaxime increased β-lactam resistance in aeromonas strains isolated from fish guts.
With large amounts of cephalosporin end up in natural ecosystems, water has been acknowledged as the large reservoir of β-lactam resistance over the past decades. However, there is still insufficient knowledge available on the function of the living organisms to the transmission of antibiotic resistance. For this reason, in this study, using adult zebrafish (Danio rerio) as animal model, exposing them to environmentally relevant dose of cefotaxime for 150 days, we asked whether cefotaxime contamination accelerated β-lactam resistance in gut microbiota as well as its potential transmission. Results showed that some of β-lactam resistance genes (βRGs) were intrinsic embedded in intestinal microbiome of zebrafish even without antibiotic stressor. Across cefotaxime treatment, the abundance of most βRGs in fish gut microbiome decreased apparently in the short term firstly, and then increased with the prolonged exposure, forming distinctly divergent βRG profiles with antibiotic-untreated zebrafish. Meanwhile, with the rising concentration of cefotaxime, the range of βRGs’ host-taxa expanded and the co-occurrence relationships of mobile genetics elements (MGEs) with βRGs intensified, indicating the enhancement of βRGs’ mobility in gut microbiome when the fish suffered from cefotaxime contamination. Furthermore, the path of partial least squares path modeling (PLS-PM) gave an integral assessment on the specific causality of cefotaxime treatment to βRG profiles, showing that cefotaxime-mediated βRGs variation was most ascribed to the alteration of MGEs under cefotaxime stress, followed by bacterial community, functioning both direct influence as βRG-hosts and indirect effects via affecting MGEs. Finally, pathogenic bacteria Aeromonas was identified as the critical host for multiple βRGs in fish guts, and its β-lactam resistance increased over the duration time of cefotaxime exposure, suggesting the potential spreading risks for the antibiotic-resistant pathogens from environmental ecosystems to clinic. Overall, our finding emphasized cefotaxime contamination in aquatic surroundings could enhance the β-lactam resistance and its transmission mobility in fish bodies.
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