Mutations In Penicillin-binding Proteins Research Articles

In silico exploration of phenolics as modulators of penicillin binding protein (PBP) 2× of Streptococcus pneumoniae

Infections caused by multidrug-resistant Streptococcus pneumoniae remain the leading cause of pneumonia-related deaths in children < 5 years globally, and mutations in penicillin-binding protein (PBP) 2 × have been identified as the major cause of resistance in the organism to beta-lactams. Thus, the development of new modulators with enhanced binding of PBP2x is highly encouraged. In this study, phenolics, due to their reported antibacterial activities, were screened against the active site of PBP2x using structure-based pharmacophore and molecular docking techniques, and the ability of the top-hit phenolics to inhibit the active and allosteric sites of PBP2x was refined through 120 ns molecular dynamic simulation. Except for gallocatechin gallate and lysidicichin, respectively, at the active and allosteric sites of PBP2x, the top-hit phenolics had higher negative binding free energy (ΔGbind) than amoxicillin [active site (− 19.23 kcal/mol), allosteric site (− 33.75 kcal/mol)]. Although silicristin had the best broad-spectrum effects at the active (− 38.41 kcal/mol) and allosteric (− 50.54 kcal/mol) sites of PBP2x, the high thermodynamic entropy (4.90 Å) of the resulting complex might suggest the need for its possible structural refinement for enhanced potency. Interestingly, silicristin had a predicted synthetic feasibility score of < 5 and quantum calculations using the DFT B3LYP/6-31G+ (dp) revealed that silicristin is less stable and more reactive than amoxicillin. These findings point to the possible benefits of the top-hit phenolics, and most especially silicristin, in the direct and synergistic treatment of infections caused by S. pneumoniae. Accordingly, silicristin is currently the subject of further confirmatory in vitro research.

Affinity of β-Lactam Antibiotics for Neisseria gonorrhoeae Penicillin-Binding Protein 2 Having Wild, Cefixime-Reduced-Susceptible, and Cephalosporin (Ceftriaxone)-Resistant penA Alleles.

Multidrug-resistant Neisseria gonorrhoeae is a serious concern worldwide. Resistance to β-lactam antibiotics occurs through mutations in penicillin-binding proteins (PBPs), acquisition of β-lactamases, and alteration of antibiotic penetration. Mosaic structures of penA, which encodes PBP2, play a major role in resistance to β-lactams, especially cephalosporins. Ceftriaxone (CRO) is recognized as the only satisfiable antibiotic for the treatment of gonococcal infections; however, CRO-resistant isolates have emerged in the community. Here, we examined the affinity of β-lactam antibiotics for recombinant PBP2 in a competition assay using fluorescence-labeled penicillin. We found no or little difference in the affinities of penicillins and meropenem (MEM) for PBP2 from cefixime (CFM)-reduced-susceptible strain and cephalosporin-resistant strain. However, the affinity of cephalosporins, including CRO, for PBP2 from the cephalosporin-resistant strain was markedly lower than that for PBP2 from the CFM-reduced-susceptible-resistant strain. Notably, piperacillin (PIP) showed almost the same affinity for PBP2 from penicillin-susceptible, CFM-reduced-susceptible, and cephalosporin (including CRO)-resistant strains. Thus, PIP/tazobactam and MEM are candidate antibiotics for the treatment of CRO-resistant/multidrug-resistant N. gonorrhoeae.

Role of transcriptomic and genomic analyses in improving the comprehension of cefiderocol activity in Acinetobacter baumannii.

The mechanisms of action and resistance of cefiderocol (FDC) in Acinetobacter baumannii are still not fully elucidated, but iron transport systems have been evoked in its entry into the cell to reach the penicillin-binding proteins (PBPs). To capture the dynamics of gene expression related to FDC action in various conditions, we report on the genomic and transcriptomic features of seven A. baumannii strains with different FDC susceptibility, focusing on the variants in genes associated with β-lactam resistance and the expression of the siderophore biosynthesis and transport systems acinetobactin and baumannoferrin. We also investigated the expression of the TonB energy transduction system (ETS) and siderophore receptors piuA and pirA. The four clinical samples belonged to the same clonal complex (CC2), and the two strains with the highest FDC MICs showed peculiar variants in PBP2 and ampC. Similarly, the two clinical strains with the lowest MICs shared variants in an outer membrane protein as well as ampC. Gene expression analyses highlighted the up-regulation of the acinetobactin and baumannoferrin genes in response to iron depletion and a down-regulation in the presence of high iron concentrations. In response to FDC, gene expression seemed strain-dependent, probably due to the different metabolic features of each strain. Overall, FDC activates the ETS, confirming the active import of the drug; baumannoferrin, more than acinetobactin, appeared stimulated by FDC in an iron-depleted medium. In conclusion, iron transport systems play a clear role in the FDC uptake, and their expression likely contributes to MIC variation together with β-lactam resistance determinants.IMPORTANCEAcinetobacter baumannii poses a threat to healthcare due to its ability to give difficult-to-treat infections as a consequence of our shortage of antibiotic molecules active on this multidrug-resistant bacterium. Cefiderocol (FDC) represents one of the few drugs active on A. baumannii, and to preserve its activity, this study explored the transcriptomic and genomic features of seven strains with varying susceptibility to FDC. Transcriptomic analyses revealed the different effects of FDC on iron transport systems, promoting mainly baumannoferrin expression-thus more likely related to FDC entry-and the energy transduction systems. These findings suggest that not all iron transport systems are equally involved in FDC entry into A. baumannii cells. Finally, mutations in PBPs and β-lactamases may contribute to the resistance onset. Overall, the study sheds light on the importance of iron availability and metabolic differences in FDC resistance, offering insights into understanding the evolution of resistance in A. baumannii strains.

The morphogenic protein CopD controls the spatio-temporal dynamics of PBP1a and PBP2b in Streptococcus pneumoniae.

Penicillin-binding proteins (PBPs) are essential for proper bacterial cell division and morphogenesis. The genome of Streptococcus pneumoniae encodes for two class B PBPs (PBP2x and 2b), which are required for the assembly of the peptidoglycan framework and three class A PBPs (PBP1a, 1b and 2a), which remodel the peptidoglycan mesh during cell division. Therefore, their activities should be finely regulated in space and time to generate the pneumococcal ovoid cell shape. To date, two proteins, CozE and MacP, are known to regulate the function of PBP1a and PBP2a, respectively. In this study, we describe a novel regulator (CopD) that acts on both PBP1a and PBP2b. These findings provide valuable information for understanding bacterial cell division. Furthermore, knowing that ß-lactam antibiotic resistance often arises from PBP mutations, the characterization of such a regulator represents a promising opportunity to develop new strategies to resensitize resistant strains.

Methycillin-Resistant Staphylococcus Aureus (Mrsa) Pada Ulserasi Septum Nasi

Staphylococcus aureus is a gram-positive, which belongs to the Staphylococcus aureus family forming grape-like cluster. Staphylococcus aureus can be found in parts of the human body such as the skin, skin glands, mucous membranes, nose and intestines. Methycillin-resistant Staphylococcus aureus (MRSA) is a Staphylococcus aureus that has mutations in penicillin-binding protein (PBP), namely the mecA gene with decreased affinity for most penicillins. Reported one case of a 59-year-old man with MRSA in nasal septal ulceration. Diagnosis is based on anamnesis, physical examination and laboratory test like complete blood count and antibiotic culture test. The therapy in this case was giving tetracycline 4 x 500mg for 14 days.

Genomic Epidemiology of Streptococcus pneumoniae Isolated in a Tertiary Hospital in Beijing, China, from 2018 to 2022.

Streptococcus pneumoniae is one of the most common bacterial pathogens of a wide range of community-acquired infections. It has been more and more recognized that this bacterium could also play a role as a cause of nosocomial infections. In this study, by retrospective analysis of the phenotypic resistance characteristics and genomic characteristics of 52 S. pneumoniae isolates in a hospital in Beijing, China, from 2018 to 2022, we explored the carriage of resistance genes and mutations in penicillin-binding proteins corresponding to the resistances, and identified the population diversity based on the prediction of serotypes and identification of sequence types (STs). The isolates displayed resistances to erythromycin (98%), tetracycline (96%), sulfonamide (72%) and penicillin G (42%). Among the 52 isolates, 41 displayed multiple-drug resistance. In total, 37 STs and 21 serotypes were identified, and the clonal complex 271 serogroup 19 was the most prevalent subtype. Only 24 isolates (46.2%) of 7 serotypes were covered by the 13-valent pneumococcal conjugate vaccination. The isolates showed high carriages of resistance genes, including tet(M) (100%) and erm(B) (98.1%); additionally, 32 isolates (61.5%) had mutations in penicillin-binding proteins. We also observed 11 healthcare-associated infections and 3 cases infected by different subtypes of isolates. We did not find nosocomial transmissions between the patients, and these cases might be associated with the asymptomatic colonization of S. pneumoniae in the human population. Our results called for further active surveillance of these subtypes, as well as the continuous optimization of the treatment protocols.

Penicillin and Cefotaxime Resistance of Quinolone-Resistant Neisseria meningitidis Clonal Complex 4821, Shanghai, China, 1965-2020.

Clonal complex 4821 (CC4821) Neisseria meningitidis, usually resistant to quinolones but susceptible to penicillin and third-generation cephalosporins, is increasing worldwide. To characterize the penicillin-nonsusceptible (PenNS) meningococci, we analyzed 491 meningococci and 724 commensal Neisseria isolates in Shanghai, China, during 1965-2020. The PenNS proportion increased from 0.3% in 1965-1985 to 7.0% in 2005-2014 and to 33.3% in 2015-2020. Of the 26 PenNS meningococci, 11 (42.3%) belonged to the CC4821 cluster; all possessed mutations in penicillin-binding protein 2, mostly from commensal Neisseria. Genetic analyses and transformation identified potential donors of 6 penA alleles. Three PenNS meningococci were resistant to cefotaxime, 2 within the CC4821 cluster. With 96% of the PenNS meningococci beyond the coverage of scheduled vaccination and the cefotaxime-resistant isolates all from toddlers, quinolone-resistant CC4821 has acquired penicillin and cefotaxime resistance closely related to the internationally disseminated ceftriaxone-resistant gonococcal FC428 clone, posing a greater threat especially to young children.

Molecular characterisation of a meropenem-resistant Haemophilus influenzae

Background: Haemophilus influenzae β-lactam resistance is mediated by production of β-lactamase and mutations in penicillin-binding protein 3 (PBP3, encoded by ftsI). Only one of the 646 patient-unique H. influenzae isolated in our laboratory between 2012–July 2022 was meropenem (amoxicillin, co-amoxiclav and ceftriaxone) resistant. Methods: Identification and susceptibility of this meropenem-resistant H. influenzae (MRHI) was confirmed by disk diffusion and E-test (bioMérieux) (CLSI M100). WGS was performed on GridION (Nanopore) and reference mapped with Geneious Prime 2022.2. PCR and Sanger sequencing was used to confirm results of WGS. Results: The MRHI isolate was β-lactamase negative and non-typeable. ftsI subgroup IIc1 mutations were present, signifying previously described low level PBP3 alterations that should not solely account for carbapenem resistance. Group VII2 acrR (repressor of AcrAB efflux pump) substitutions were observed but without the reported carbapenem resistance associated frameshift mutation. There were also multiple substitutions and insertions in ompP2 (outer membrane protein P2) porin, associated with carbapenem resistance.2 The 100% concordance between Sanger sequencing and Nanopore increases our confidence for application of this technology for future investigations. Conclusion: Meropenem resistance in this isolate is likely multifactorial, with PBP3 alterations and decreased influx as contributing factors.

Resistance to Cefiderocol Involved Expression of PER-1 β-Lactamase and Downregulation of Iron Transporter System in Carbapenem-Resistant Acinetobacter baumannii.

Cefiderocol (CFDC) is a promising antimicrobial agent against multidrug resistant Gram-negative bacteria. However, CFDC resistance has emerged in carbapenem-resistant Acinetobacter baumannii (CR-AB) but the underlying mechanisms remain unclear. Whole-genome sequencing and transcriptome sequencing were performed on CFDC-non-susceptible and CFDC-susceptible isolates. Two different recombinant plasmids was electro-transformed into the E. coli BL21 strain to determine the impact of blaPER and the combined impact of blaPER-1 and blaOXA-23 on CFDC resistance. Fifty-five CR-AB isolates with minimum inhibitory concentrations (MICs) ranged from 0.06 mg/L to >256 mg/L were sequenced, including 47 CFDC-non-susceptible and eight CFDC-susceptible isolates. Two CFDC-non-susceptible isolates belonged to ST104 whereas the remaining isolates belonged to ST2, and blaPER-1 was present only in CFDC-non-susceptible isolates. Amino acid substitutions were noted in penicillin-binding proteins (PBPs) in four CFDC-susceptible isolates, with slightly elevated MICs. The MICs of recombinant E. coli BL21 carrying the blaPER-1 gene increased 64-fold and recombinant E. coli BL21 carrying both the blaPER-1 and blaOXA-23 genes increased 8-fold but both remained within the susceptibility range. Transcriptome sequencing of 17 CFDC-non-susceptible isolates and eight CFDC-susceptible isolates revealed that transcriptional levels of various iron transport proteins, such as fiu, feoA, and feoB, and the energy transduction system, TonB-ExbB-ExbD, were relatively downregulated in CFDC-non-susceptible isolates. GO enrichment analysis revealed that the upregulated genes in CFDC-non-susceptible isolates were mainly associated with redox homeostasis and stress response. Besides, the expression levels of the blaOXA-23 and exbD genes were negatively correlated with the MICs. PER-1 production, iron transport system downregulation, and mutations in PBPs may synergistically impart high-level resistance to CFDC in CR-AB.

Acinetobacter baumannii and Cefiderocol, between Cidality and Adaptability.

ABSTRACTAmong the bacterial species included in the ESKAPE group, Acinetobacter baumannii is of great interest due to its intrinsic and acquired resistance to many antibiotics and its ability to infect different body regions. Cefiderocol (FDC) is a novel cephalosporin that is active against Gram-negative bacteria, with promising efficacy for A. baumannii infections, but some studies have reported therapeutic failures even in the presence of susceptible strains. This study aims to investigate the interactions between FDC and 10 A. baumannii strains with different susceptibilities to this drug. We confirmed diverse susceptibility profiles, with resistance values close to the EUCAST-proposed breakpoints. The minimal bactericidal concentration (MBC)/MIC ratios demonstrated bactericidal activity of the drug, with ratio values of ≤4 for all of the strains except ATCC 19606; however, bacterial regrowth was evident after exposure to FDC, as were changes in the shapes of colonies and bacterial cells. A switch to a nonsusceptible phenotype in the presence of high FDC concentrations was found in 1 strain as an adaptation mechanism implemented to overcome the cidal activity of this antibiotic, which was confirmed by the presence of heteroresistant, unstable subpopulations in 8/10 samples. Genomic analyses revealed the presence of mutations in penicillin-binding protein 3 (PBP3) and TonB3 that were shared by all of the strains regardless of their resistance phenotype. Because our isolates harbored β-lactamase genes, β-lactamase inhibitors showed the ability to restore the antimicrobial activity of FDC despite the different nonsusceptibility levels of the tested strains. These in vitro results support the concept of using combination therapy to eliminate drug-adapted subpopulations and regain full FDC activity in this difficult-to-treat species.IMPORTANCE This work demonstrates the underrated presence of Acinetobacter baumannii heteroresistant subpopulations after exposure of A. baumannii strains to FDC and its instability. Both A. baumannii and FDC are of great interest for the scientific community, as well as for clinicians; the former represents a major threat to public health due to its resistance to antibiotics, with related costs of prolonged hospitalization, and the latter is a novel, promising cephalosporin currently under the magnifying glass.

Mutations in PBP2 from ceftriaxone-resistant Neisseria gonorrhoeae alter the dynamics of the β3–β4 loop to favor a low-affinity drug-binding state

Resistance to the extended-spectrum cephalosporin ceftriaxone in the pathogenic bacteria Neisseria gonorrhoeae is conferred by mutations in penicillin-binding protein 2 (PBP2), the lethal target of the antibiotic, but how these mutations exert their effect at the molecular level is unclear. Using solution NMR, X-ray crystallography, and isothermal titration calorimetry, we report that WT PBP2 exchanges dynamically between a low-affinity state with an extended β3–β4 loop conformation and a high-affinity state with an inward β3–β4 loop conformation. Histidine-514, which is located at the boundary of the β4 strand, plays an important role during the exchange between these two conformational states. We also find that mutations present in PBP2 from H041, a ceftriaxone-resistant strain of N. gonorrhoeae, increase resistance to ceftriaxone by destabilizing the inward β3–β4 loop conformation or stabilizing the extended β3–β4 loop conformation to favor the low-affinity drug-binding state. These observations reveal a unique mechanism for ceftriaxone resistance, whereby mutations in PBP2 lower the proportion of target molecules in the high-affinity drug-binding state and thus reduce inhibition at lower drug concentrations.

Characterization and Clonal Diffusion of Ceftaroline Non-Susceptible MRSA in Two Hospitals in Central Italy.

Background: Ceftaroline represents a novel fifth-generation cephalosporin to treat infections caused by methicillin-resistant Staphylococcus aureus (MRSA). Methods: Ceftaroline susceptibility of 239 MRSA isolates was assessed by disk diffusion and a MIC test strip following both EUCAST and CLSI guidelines. Non-susceptible isolates were epidemiologically characterized by pulsed-field gel electrophoresis, spa typing, and multilocus sequence typing, and further investigated by PCR and whole genome sequencing to detect penicillin-binding protein (PBP) mutations as well as antibiotic resistance and virulence genes. Results: Fourteen isolates out of two hundred and thirty-nine (5.8%) were non-susceptible to ceftaroline (MIC > 1 mg/L), with differences between the EUCAST and CLSI interpretations. The characterized isolates belonged to seven different pulsotypes and three different clones (ST228/CC5-t041-SCCmecI, ST22/CC22-t18014-SCCmecIV, and ST22/CC22-t022-SCCmecIV), confirming a clonal diffusion of ceftaroline non-susceptible strains. Mutations in PBPs involved PBP2a for ST228-t041-SCCmecI strains and all the other PBPs for ST22-t18014-SCCmecIV and ST22-t022-SCCmecIV clones. All isolates harbored antibiotic resistance and virulence genes with a clonal distribution. Conclusion: Our study demonstrated that ceftaroline non-susceptibile isolates belonged not only to ST228 strains (the most widespread clone in Italy) but also to ST22, confirming the increasing role of these clones in hospital infections.

Evolution of Sequence Type 4821 Clonal Complex Hyperinvasive and Quinolone-Resistant Meningococci.

Expansion of quinolone-resistant Neisseria meningitidis clone ChinaCC4821-R1-C/B from sequence type (ST) 4821 clonal complex (CC4821) caused a serogroup shift from serogroup A to serogroup C invasive meningococcal disease (IMD) in China. To determine the relationship among globally distributed CC4821 meningococci, we analyzed whole-genome sequence data from 173 CC4821 meningococci isolated from 4 continents during 1972–2019. These meningococci clustered into 4 sublineages (1–4); sublineage 1 primarily comprised of IMD isolates (41/50, 82%). Most isolates from outside China (40/49, 81.6%) formed a distinct sublineage, the Europe–USA cluster, with the typical strain designation B:P1.17-6,23:F3-36:ST-3200(CC4821), harboring mutations in penicillin-binding protein 2. These data show that the quinolone-resistant clone ChinaCC4821-R1-C/B has expanded to other countries. The increasing distribution worldwide of serogroup B CC4821 raises the concern that CC4821 has the potential to cause a pandemic that would be challenging to control, despite indirect evidence that the Trumenba vaccine might afford some protection.

Penicillin-Binding Proteins and Associated Protein Mutations Confer Oxacillin/Cefoxitin Tolerance in Borderline Oxacillin-Resistant Staphylococcus aureus.

Among clinical isolates of Staphylococcus aureus, borderline oxacillin-resistant S. aureus (BORSA), which is mildly resistant to oxacillin (OXA) without harboring the mecA or mecC gene, is considered a risk factor for further resistance against multiple antibiotics. In this study, BORSA isolates and their derivatives were characterized through antibiotic susceptibility testing and mutation analysis of the genes encoding penicillin-binding proteins (PBPs) and their related proteins, including the promoter region. Eight BORSA isolates were confirmed to harbor the blaZ gene, and hyperproduction of blaZ-encoded penicillinase was predicted based on the minimum inhibitory concentrations (MICs). Of these, four derivative strains that were spontaneously selected based on viability on media containing high concentrations of OXA showed higher MICs than the parent isolates. The minimum bactericidal concentrations, MIC ratios, and TDtest results identified many strains with cefoxitin tolerance. Sequencing of pbp1, pbp2, pbp3, pbp4, gdpP, and yjbH, and the promoter of pbp4 revealed mutations in BORSA isolates and derivatives, despite their absence in parent isolates, suggesting that mutations in PBPs confer OXA/cefoxitin tolerance in BORSA strains.

Multidrug-Resistant Streptococcus agalactiae Strains Found in Human and Fish with High Penicillin and Cefotaxime Non-Susceptibilities.

Penicillin non-susceptible Streptococcus agalactiae (PEN-NS GBS) has been increasingly reported, with multidrug-resistant (MDR) GBS documented in Japan. Here we identified two PEN-NS GBS strains during our surveillance studies: one from a patient’s wound and the other from a tilapia. The patient’s GBS (H21) and fish GBS (F49) were serotyped and tested for antibiotic susceptibility. Whole-genome sequencing was performed to find the sequence type, antimicrobial resistance genes, and mutations in penicillin-binding proteins (PBPs) and fluoroquinolone (FQ) resistance genes. H21 and F49 belonged to ST651, serotype Ib, and ST7, serotype Ia, respectively. H21 showed PEN and cefotaxime minimum inhibitory concentrations (MICs) of 2.0 mg/L. F49 showed PEN MIC 0.5 mg/L. H21 was MDR with ermB, lnuB, tetS, ant6-Ia, sat4a, and aph3-III antimicrobial resistance genes observed. Alignment of PBPs showed the combination of PBP1B (A95D) and 2B mutations (V80A, S147A, S160A) in H21 and a novel mutation in F49 at N192S in PBP2B. Alignment of FQ-resistant determinants revealed mutation sites on gyrA, gyrB, and parC and E in H21. To our knowledge, this is the first report of GBS isolates with such high penicillin and cefotaxime MICs. This raises the concern of emergence of MDR and PEN-NS GBS in and beyond healthcare facilities.

Mutations in penicillin-binding protein 2 from cephalosporin-resistant Neisseria gonorrhoeae hinder ceftriaxone acylation by restricting protein dynamics

The global incidence of the sexually transmitted disease gonorrhea is expected to rise due to the spread of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins (ESCs). ESC resistance is conferred by mosaic variants of penicillin-binding protein 2 (PBP2) that have diminished capacity to form acylated adducts with cephalosporins. To elucidate the molecular mechanisms of ESC resistance, we conducted a biochemical and high-resolution structural analysis of PBP2 variants derived from the decreased-susceptibility N. gonorrhoeae strain 35/02 and ESC-resistant strain H041. Our data reveal that mutations both lower affinity of PBP2 for ceftriaxone and restrict conformational changes that normally accompany acylation. Specifically, we observe that a G545S substitution hinders rotation of the β3 strand necessary to form the oxyanion hole for acylation and also traps ceftriaxone in a noncanonical configuration. In addition, F504L and N512Y substitutions appear to prevent bending of the β3-β4 loop that is required to contact the R1 group of ceftriaxone in the active site. Other mutations also appear to act by reducing flexibility in the protein. Overall, our findings reveal that restriction of protein dynamics in PBP2 underpins the ESC resistance of N. gonorrhoeae.

Diversification and Evolution of Vancomycin-Resistant Enterococcus faecium during Intestinal Domination.

Vancomycin-resistant Enterococcus faecium (VRE) is a leading cause of hospital-acquired infections. This is particularly true in immunocompromised patients, where the damage to the microbiota caused by antibiotics can lead to VRE domination of the intestine, increasing a patient's risk for bloodstream infection. In previous studies we observed that the intestinal domination by VRE of patients hospitalized to receive allogeneic bone marrow transplantation can persist for weeks, but little is known about subspecies diversification and evolution during prolonged domination. Here we combined a longitudinal analysis of patient data and in vivo experiments to reveal previously unappreciated subspecies dynamics during VRE domination that appeared to be stable from 16S rRNA microbiota analyses. Whole-genome sequencing of isolates obtained from sequential stool samples provided by VRE-dominated patients revealed an unanticipated level of VRE population complexity that evolved over time. In experiments with ampicillin-treated mice colonized with a single CFU, VRE rapidly diversified and expanded into distinct lineages that competed for dominance. Mathematical modeling shows that in vivo evolution follows mostly a parabolic fitness landscape, where each new mutation provides diminishing returns and, in the setting of continuous ampicillin treatment, reveals a fitness advantage for mutations in penicillin-binding protein 5 (pbp5) that increase resistance to ampicillin. Our results reveal the rapid diversification of host-colonizing VRE populations, with implications for epidemiologic tracking of in-hospital VRE transmission and susceptibility to antibiotic treatment.

Influence of the T to S mutation at the STMK motif on antibiotic resistance of penicillin binding protein 1A: A comprehensive computational study

The emergence of antibiotic resistance has attracted the attention of scientists and scientific circles over the decades. β-Lactam antibiotics resistance is a worldwide therapeutic challenge in bacterial infections, mediated through several mechanisms of which mutations in Penicillin Binding Proteins (PBPs) are an important issue, making critical therapeutic problems in the human population. Accordingly, investigating the dynamic structures of mutant variants could result in a profound understanding of such a specific resistance. Therefore, this work investigated structural properties sampled by all-atom molecular dynamics (MD) simulations, umbrella sampling, and binding free energy calculations for both a wild-type and a cefotaxime-resistant T to S mutant of PBP1A. The T to S mutation significantly reduces the binding affinity of cefotaxime (a frequently clinically-administrated β-lactam antibiotic) as the PBP1A inhibitor. In the conventional MD simulations presented here, more fluctuations of the mutant's active site cleft margins were detected. The cleft of the mutant protein also opened remarkably more than the wild-type's cleft and displayed more flexibility. Thus, our findings have shown that flexibility of cleft margins of the active site in the mutant PBP1A immediately results in the catalytic cleft opening. In addition, binding free energy calculation suggests that reducing hydrophobic contacts and increasing the polar contribution in the binding energy may play an important role in cefotaxime resistance.

A novel PBP3 substitution in Haemophilus influenzae confers reduced aminopenicillin susceptibility

BackgroundIdentification and characterization of non-typeable Haemophilus influenzae (NTHi) with reduced susceptibility to β-lactam antibiotics due to mutations in penicillin binding protein 3 (PBP3) is a clinical challenge. We analyzed a blood isolate, NTHi93–57485, that was categorized as aminopenicillin resistant but lacked key amino acid substitutions in PBP3 that have previously been associated with reduced aminopenicillin susceptibility. The significance of an alternative amino acid substitution (Y528H) in this isolate was examined.ResultsSite-directed mutagenesis of a β-lactam susceptible H. influenzae (NTHi3655) was performed to introduce the Y528H substitution into wild-type ftsI (encoding for PBP3). Disc diffusion screening and broth microdilution determination of MICs for β-lactam agents were done with the NTHi3655-PBP3Y528H mutant and were compared with the NTHi3655 wild-type as well as the original clinical isolate NTHi93–57485. Introduction of the Y528H substitution in NTHi3655 resulted in positive screening for β-lactam resistance. MICs for aminopenicillins were increased in the mutant compared to the wild-type. However, the mutant remained susceptible to aminopenicillins according to EUCAST clinical breakpoints (assuming intravenous treatment) and the introduction of Y528H alone did not increase the resistance to the same level as NTHi93–57485. None of the isolates had frame shift insertions in the acrR gene regulating the AcrAB efflux pump.ConclusionsIn parallel to the previously well-described PBP3-substitutions R517H and N526K, we demonstrate that Y528H confers reduced aminopenicillin susceptibility.

New Mutations of Penicillin-Binding Proteins inStreptococcus agalactiaeIsolates from Cattle with Decreased Susceptibility to Penicillin

Streptococcus agalactiae is a causal agent of bovine mastitis and is treated by β-lactam antibiotics (BLAs). Compared to penicillin-resistant S. agalactiae from humans, resistant strains in bovine are rarely reported. In this study, we aimed to investigate BLA resistance and mutations in penicillin-binding proteins (PBPs) of S. agalactiae in central and northeast China. The minimum inhibitory concentrations (MICs) of 129 penicillin-resistant S. agalactiae isolates from cows with mastitis were determined, and the related PBP genes were detected and sequenced. All strains were unsusceptible to penicillin G and mostly resistant to ampicillin, cefalexin, and ceftiofur sodium. One hundred twenty-nine strains were divided into 4 clonal groups and 8 sequence types by multilocus sequence typing analysis. We found a set of new substitutions in PBP1B, PBP2B, and PBP2X from most strains isolated from three provinces. The strains with high PBP mutations showed a broader unsusceptible spectrum and higher MICs than those with few or single mutation. Our research indicates unpredicted mutations in the PBP genes of S. agalactiae isolated from cows with mastitis treated by BLAs. This screening is the first of S. agalactiae from cattle.