In Vivo Cross-Linking Sheds Light on the Salmonella Divisome in Which PBP3 and PBP3SAL Compete for Occupancy.

Bacterial cell division is orchestrated by proteins that assemble in dynamic complexes collectively known as the divisome. Essential monofunctional enzymes with glycosyltransferase or transpeptidase (TPase) activities, FtsW and FtsI respectively, engage in the synthesis of septal peptidoglycan (sPG). Enigmatically, Salmonella has two TPases that can promote cell division independently: FtsI (PBP3) and the pathogen-specific paralogue PBP3SAL. How Salmonella regulates the assembly of the sPG synthase complex with these two TPases, is unknown. Here, we characterized Salmonella division complexes in wild-type cells and isogenic mutants lacking PBP3 or PBP3SAL. The complexes were cross-linked in vivo and pulled down with antibodies recognizing each enzyme. Proteomics of the immunoprecipitates showed that PBP3 and PBP3SAL do not extensively cross-link in wild type cells, supporting the presence of independent complexes. More than 40 proteins cross-link in complexes in which these two TPases are present. Those identified with high scores include FtsA, FtsK, FtsQLB, FtsW, PBP1B, SPOR domain-containing proteins (FtsN, DedD, RlpA, DamX), amidase activators (FtsX, EnvC, NlpD) and Tol-Pal proteins. Other cross-linked proteins are the protease Prc, the elongasome TPase PBP2 and, D,D-endo- and D,D-carboxypeptidases. PBP3 and PBP3SAL localize at midcell and compete for occupying the division complex in response to environmental cues. Thus, a catalytic-dead PBP3SAL-S300A variant impairs cell division in a high osmolarity and acidic condition in which it is produced at levels exceeding those of PBP3. Salmonella may therefore exploit an 'adjustable' divisome to exchange TPases for ensuring cell division in distinct environments and, in this manner, expand its colonization capacities.

Effect of modification of penicillin-binding protein 3 on susceptibility to ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and cefiderocol of Escherichia coli strains producing broad-spectrum ß-lactamases.

The impact of penicillin-binding protein 3 (PBP3) modifications that may be identified in Escherichia coli was evaluated with respect to susceptibility to ß-lactam/ß-lactamase inhibitor combinations including ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and to cefiderocol. A large series of E. coli recombinant strains producing broad-spectrum ß-lactamases was evaluated. While imipenem-relebactam showed a similar activity regardless of the PBP3 background, susceptibility to other molecules tested was affected at various levels. This was particularly the case for ceftazidime-avibactam, aztreonam-avibactam, and cefepime-taniborbactam.

Evolution of mutations in the ftsI gene leading to amino acid substitutions in PBP3 in Haemophilus influenzae strains under the selective pressure of ampicillin and cefuroxime.

BACKGROUND: Aminopenicillins are recommended agents for non-invasive Haemophilus influenzae infections. One of the mechanisms of resistance to ß-lactams is the alteration of the transpeptidase region of penicillin binding protein 3 (PBP3) which is caused by mutations in the ftsI gene. It was shown that exposure to beta-lactams has a stimulating effect on increase of prevalence of H. influenzae strains with the non-enzymatic mechanism of resistance. OBJECTIVES: The aim of our study was to compare the mutational potential of ampicillin and cefuroxime in H. influenzae strains, determination of minimum inhibitory concentration and the evolution of mutations over time, focusing on amino acid substitutions in PBP3. METHODS: 30 days of serial passaging of strains in liquid broth containing increasing concentrations of ampicillin or cefuroxime was followed by whole-genome sequencing. RESULTS: On average, cefuroxime increased the minimum inhibitory concentration more than ampicillin. The minimum inhibitory concentration was increased by a maximum of 32 fold. Substitutions in the PBP3 started to appear after 15 days of passaging. In PBP3, cefuroxime caused different substitutions than ampicillin. CONCLUSIONS: Our experiment observed differences in mutation selection by ampicillin and cefuroxime. Selection pressure of antibiotics in vitro generated substitutions that do not occur in clinical strains in the Czech Republic.

Reduced susceptibility to aztreonam-avibactam conferred by acquired AmpC-type ß-lactamases in PBP3-modified Escherichia coli.

PURPOSE: Carbapenemase-producing Enterobacterales are a growing threat, and very few therapeutic options remain active against those multidrug resistant bacteria. Aztreonam is the molecule of choice against metallo-beta-lactamases (MBL) producers since it is not hydrolyzed by those enzymes, but the co-production of acquired plasmidic cephalosporinases or extended-spectrum ß-lactamases leading to aztreonam resistance may reduce the efficacy of this molecule. Hence, the development of the aztreonam-avibactam (AZA) combination provides an interesting therapeutic alternative since avibactam inhibits the activity of both cephalosporinases and extended-spectrum ß-lactamases. However, structural modifications of penicillin binding protein PBP3, the target of aztreonam, may lead to reduced susceptibility to aztreonam-avibactam. METHODS: Here the impact of various plasmid-encoded AmpC-type ß-lactamases (ACC-1, ACT-7, ACT-17, CMY-2, CMY-42, DHA-1, FOX-1, and FOX-5) on susceptibility to aztreonam-avibactam was evaluated using isogenic E. coli MG1655 strains harboring insertions in PBP3 (YRIN and YRIK). The inhibitory activity of various ß-lactamase inhibitors (clavulanic acid, tazobactam, avibactam, relebactam, and vaborbactam) were also compared against these enzymes. RESULTS: Hence, we showed that reduced susceptibility to AZA was due to the combined effect of both AmpC production and amino acid insertions in PBP3. The highest resistance level was achieved in strains possessing the insertions in PBP3 in association with the production of ACT-7, ACC-1, or CMY-42. CONCLUSION: Although none of the recombinant strains tested displayed clinical resistance to aztreonam-avibactam, our data emphasize that the occurrence of such profile might be of clinical relevance for MBL-producing strains.

Use of trans-complementation method to determine the effects of various ftsI mutations on ß-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae strains.

Haemophilus influenzae is a causative agent of serious infections, especially among children. ß-lactam antibiotics are commonly used for the treatment of these infections. Among H. influenzae isolates, ß-lactam resistance is due to the presence of ß-lactamase, or to mutations in the ftsI gene that generate altered PBP3 (penicillin-binding protein 3) with reduced affinity for ß-lactams (BLNAR-ß-lactamase-negative, ampicillin-resistant). Wild-type ftsI gene encoding for PBP3 was amplified in whole from ß-lactam susceptible H. influenzae Rd and cloned in pLS88 plasmid to obtain pADUTAS17, which was then used to transform known BLNAR strains, susceptible strains, and a strain (CF55) with wild-type ftsI but unexplained reduced ß-lactam susceptibility. Ampicillin and cefotaxime MICs (minimum inhibitory concentration) were determined after transformation with pLS88 and pADUTAS17 plasmids. The results showed that antibiotic susceptibilities were not affected by trans-complementation for isolates carrying wild-type ftsI gene. However, trans-complementation for all BLNAR strains showed decreases between - 0.957 and 0.5-fold for ampicillin and cefotaxime, confirming the role of the PBP3 substitutions in the BLNAR phenotype of these isolates. The first article showed that trans-complementation might be a useful tool in the investigation of decreased ß-lactam susceptibility in H. influenzae.

Modifiable Risk Factors for the Emergence of Ceftolozane-tazobactam Resistance.

BACKGROUND: Ceftolozane-tazobactam (TOL-TAZ) affords broad coverage against Pseudomonas aeruginosa. Regrettably, TOL-TAZ resistance has been reported. We sought to identify modifiable risk factors that may reduce the emergence of TOL-TAZ resistance. METHODS: Twenty-eight consecutive patients infected with carbapenem-resistant P. aeruginosa isolates susceptible to TOL-TAZ, treated with ≥72 hours of TOL-TAZ , and with P. aeruginosa isolates available both before and after TOL-TAZ exposure between January 2018 and December 2019 in Baltimore, Maryland, were included. Cases were defined as patients with at least a 4-fold increase in P. aeruginosa TOL-TAZ MICs after exposure to TOL-TAZ. Independent risk factors for the emergence of TOL-TAZ resistance comparing cases and controls were investigated using logistic regression. Whole genome sequencing of paired isolates was used to identify mechanisms of resistance that emerged during TOL-TAZ therapy. RESULTS: Fourteen patients (50%) had P. aeruginosa isolates which developed at least a 4-fold increase in TOL-TAZ MICs(ie, cases). Cases were more likely to have inadequate source control (29% vs 0%, P = .04) and were less likely to receive TOL-TAZ as an extended 3-hour infusion (0% vs 29%; P = .04). Eighty-six percent of index isolates susceptible to ceftazidime-avibactam (CAZ-AVI) had subsequent P. aeruginosa isolates with high-level resistance to CAZ-AVI, after TOL-TAZ exposure and without any CAZ-AVI exposure. Common mutations identified in TOL-TAZ resistant isolates involved AmpC, a known binding site for both ceftolozane and ceftazidime, and DNA polymerase. CONCLUSIONS: Due to our small sample size, our results remain exploratory but forewarn of the potential emergence of TOL-TAZ resistance during therapy and suggest extending TOL-TAZ infusions may be protective. Larger studies are needed to investigate this association.

Genetic Features Leading to Reduced Susceptibility to Aztreonam-Avibactam among Metallo-ß-Lactamase-Producing Escherichia coli Isolates.

Metallo-ß-lactamase (MBL)-producing Escherichia coli isolates resistant to the newly developed ß-lactam/ß-lactamase inhibitor drug combination aztreonam-avibactam (ATM-AVI) have been reported. Here, we analyzed a series of 118 clinical MBL-producing E. coli isolates of various geographical origins for susceptibility to ATM-AVI. The nature of the PBP3 protein sequence and the occurrence of blaCMY genes for susceptibility to ATM-AVI were investigated. We showed here that elevated MICs of ATM-AVI among MBL-producing E. coli isolates resulted from a combination of different features, including modification of PBP3 protein sequence through specific amino acid insertions and production of CMY-type enzymes, particularly, CMY-42. We showed here that those insertions identified in the PBP3 sequence are not considered the unique basis of resistance to ATM-AVI, but they significantly contribute to it.

Influence of the α-Methoxy Group on the Reaction of Temocillin with Pseudomonas aeruginosa PBP3 and CTX-M-14 ß-Lactamase.

The prevalence of multidrug-resistant Pseudomonas aeruginosa has led to the reexamination of older "forgotten" drugs, such as temocillin, for their ability to combat resistant microbes. Temocillin is the 6-α-methoxy analogue of ticarcillin, a carboxypenicillin with well-characterized antipseudomonal properties. The α-methoxy modification confers resistance to serine ß-lactamases, yet temocillin is ineffective against P. aeruginosa growth. The origins of temocillin's inferior antibacterial properties against P. aeruginosa have remained relatively unexplored. Here, we analyze the reaction kinetics, protein stability, and binding conformations of temocillin and ticarcillin with penicillin-binding protein 3 (PBP3), an essential PBP in P. aeruginosa We show that the 6-α-methoxy group perturbs the stability of the PBP3 acyl-enzyme, which manifests in an elevated off-rate constant (koff) in biochemical assays comparing temocillin with ticarcillin. Complex crystal structures with PBP3 reveal similar binding modes of the two drugs but with important differences. Most notably, the 6-α-methoxy group disrupts a high-quality hydrogen bond with a conserved residue important for ligand binding while also being inserted into a crowded active site, possibly destabilizing the active site and enabling water molecule from bulk solvent to access and cleave the acyl-enzyme bond. This hypothesis is supported by the observation that the acyl-enzyme complex of temocillin has reduced thermal stability compared with ticarcillin. Furthermore, we explore temocillin's mechanism of ß-lactamase inhibition with a high-resolution complex structure of CTX-M-14 class A serine ß-lactamase. The results suggest that the α-methoxy group prevents hydrolysis by locking the compound into an unexpected conformation that impedes access of the catalytic water to the acyl-enzyme adduct.

Stationary phase persister formation in Escherichia coli can be suppressed by piperacillin and PBP3 inhibition.

BACKGROUND: Persisters are rare phenotypic variants within a bacterial population that are capable of tolerating lethal antibiotic concentrations. Passage through stationary phase is associated with the formation of persisters (type I), and a major physiological response of Escherichia coli during stationary phase is cell wall restructuring. Given the concurrence of these processes, we sought to assess whether perturbation to cell wall synthesis during stationary phase impacts type I persister formation. RESULTS: We tested a panel of cell wall inhibitors and found that piperacillin, which primarily targets penicillin binding protein 3 (PBP3 encoded by ftsI), resulted in a significant reduction in both ß-lactam (ampicillin, carbenicillin) and fluoroquinolone (ofloxacin, ciprofloxacin) persister levels. Further analyses showed that piperacillin exposure through stationary phase resulted in cells with more ATP, DNA, RNA, and protein (including PBPs) than untreated controls; and that their physiology led to more rapid resumption of DNA gyrase supercoiling activity, translation, and cell division upon introduction into fresh media. Previously, PBP3 inhibition had been linked to antibiotic efficacy through the DpiBA two component system; however, piperacillin suppressed persister formation in ΔdpiA to the same extent as it did in wild-type, suggesting that DpiBA is not required for the phenomenon reported here. To test the generality of PBP3 inhibition on persister formation, we expressed FtsI Ser307Ala to genetically inhibit PBP3, and suppression of persister formation was also observed, although not to the same magnitude as that seen for piperacillin treatment. CONCLUSIONS: From these data we conclude that stationary phase PBP3 activity is important to type I persister formation in E. coli.

A novel PBP3 substitution in Haemophilus influenzae confers reduced aminopenicillin susceptibility.

BACKGROUND: Identification and characterization of non-typeable Haemophilus influenzae (NTHi) with reduced susceptibility to ß-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. RESULTS: Site-directed mutagenesis of a ß-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 ß-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 ß-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. CONCLUSIONS: In parallel to the previously well-described PBP3-substitutions R517H and N526K, we demonstrate that Y528H confers reduced aminopenicillin susceptibility.

Optimization of novel monobactams with activity against carbapenem-resistant Enterobacteriaceae - Identification of LYS228.

Metallo-ß-lactamases (MBLs), such as New Delhi metallo-ß-lactamase (NDM-1) have spread world-wide and present a serious threat. Expression of MBLs confers resistance in Gram-negative bacteria to all classes of ß-lactam antibiotics, with the exception of monobactams, which are intrinsically stable to MBLs. However, existing first generation monobactam drugs like aztreonam have limited clinical utility against MBL-expressing strains because they are impacted by serine ß-lactamases (SBLs), which are often co-expressed in clinical isolates. Here, we optimized novel monobactams for stability against SBLs, which led to the identification of LYS228 (compound 31). LYS228 is potent in the presence of all classes of ß-lactamases and shows potent activity against carbapenem-resistant isolates of Enterobacteriaceae (CRE).

Unusual Escherichia coli PBP 3 Insertion Sequence Identified from a Collection of Carbapenem-Resistant Enterobacteriaceae Tested In Vitro with a Combination of Ceftazidime-, Ceftaroline-, or Aztreonam-Avibactam.

Carbapenemase-producing Enterobacteriaceae isolates (n = 110) from health care centers in central Indiana (from 2010 to 2013) were tested for susceptibility to combinations of avibactam (4 µg/ml) with ceftazidime, ceftaroline, or aztreonam. MIC50/MIC90 values were 1/2 µg/ml (ceftazidime-avibactam), 0.5/2 µg/ml (ceftaroline-avibactam), and 0.25/0.5 µg/ml (aztreonam-avibactam.) A ß-lactam MIC of 8 µg/ml was reported for the three combinations against one Escherichia coli isolate with an unusual TIPY insertion following Tyr344 in penicillin-binding protein 3 (PBP 3) as the result of gene duplication.

Bypass of genetic constraints during mutator evolution to antibiotic resistance.

Genetic constraints can block many mutational pathways to optimal genotypes in real fitness landscapes, yet the extent to which this can limit evolution remains to be determined. Interestingly, mutator bacteria elevate only specific types of mutations, and therefore could be very sensitive to genetic constraints. Testing this possibility is not only clinically relevant, but can also inform about the general impact of genetic constraints in adaptation. Here, we evolved 576 populations of two mutator and one wild-type Escherichia coli to doubling concentrations of the antibiotic cefotaxime. All strains carried TEM-1, a ß-lactamase enzyme well known by its low availability of mutational pathways. Crucially, one of the mutators does not elevate any of the relevant first-step mutations known to improve cefatoximase activity. Despite this, both mutators displayed a similar ability to evolve more than 1000-fold resistance. Initial adaptation proceeded in parallel through general multi-drug resistance mechanisms. High-level resistance, in contrast, was achieved through divergent paths; with the a priori inferior mutator exploiting alternative mutational pathways in PBP3, the target of the antibiotic. These results have implications for mutator management in clinical infections and, more generally, illustrate that limits to natural selection in real organisms are alleviated by the existence of multiple loci contributing to fitness.

Isolates of ß-lactamase-negative ampicillin-resistant Haemophilus influenzae causing invasive infections in Spain remain susceptible to cefotaxime and imipenem.

OBJECTIVES: The epidemiology of invasive Haemophilus influenzae has changed in recent years. ß-Lactamase-negative ampicillin-resistant (BLNAR) invasive isolates have recently been described in Europe but their clinical significance is unclear. Our main goal was to determine whether invasive H. influenzae remains susceptible to ß-lactam antibiotics indicated in the treatment of invasive infections. METHODS: The antibiotic susceptibility of 307 invasive H. influenzae isolates to seven ß-lactam antibiotics was determined by microdilution and interpreted by EUCAST and CLSI breakpoints. We also identified the bla genes, the amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3), the molecular epidemiology of invasive BLNAR isolates by PFGE and MLST, and the time-kill curves of two isolates with PBP3 mutations conferring reduced susceptibility to aminopenicillins and cephalosporins. RESULTS: Of the invasive isolates, 86.6% were non-typeable and 62% were isolated from adults. Decreased susceptibility to ß-lactams was due to the BLNAR genotype (gBLNAR; 19.2%) and to ß-lactamase production (16.9%). Susceptibility rates to amoxicillin/clavulanic acid, cefotaxime, cefixime and imipenem were greater than 98%. Of 18 gBLNAR non-typeable isolates studied by MLST, 15 different STs were obtained. Amoxicillin and cefotaxime were bactericidal after 2 and 4 h of incubation, respectively. CONCLUSIONS: Invasive H. influenzae disease was mainly due to non-typeable isolates infecting adults, and the most common mechanism of ß-lactam resistance was mutations in the transpeptidase domain of PBP3. The gBLNAR non-typeable isolates were genetically diverse. The majority of invasive H. influenzae remained susceptible to third-generation cephalosporins; amoxicillin and cefotaxime were bactericidal in two gBLNAR isolates.

Development and validation of a pentaplex PCR assay for rapid detection of blaCTX-M, blaOXA-1, blaCMY, blaNDM and the PBP3 insert in Enterobacterales.

BACKGROUND: There is a high diversity of beta-lactamases in gram negative pathogens, making them difficult to treat. In the presence of OXA-1 and ampC, PTZ is no longer clinically relevant when treating Enterobacterales expressing ESBLs. Further, MBL infections are often treated with the combination of ceftazidime/avibactam with aztreonam. . It has recently been reported that NDM-expressing E. coli isolates co-harboring PBP3 insert develops resistance to this triple combination. METHODS: A pentaplex PCR is developed and validated to simultaneously detect blaCTX-M, blaOXA-1, blaCMY, blaNDM, and the PBP3 insert in whole genome sequenced E. coli and K. pneumoniae isolates. In addition, the isolates chosen for pentaplex PCR evaluation were tested for their minimum inhibitory concentrations (MICs) against piperacillin/tazobactam, cefoperazone/sulbactam (C/S), ertapenem, imipenem, meropenem, ceftazidime/avibactam, aztreonam/avibactam, cefepime/taniborbactam, and cefiderocol. RESULTS: The developed pentaplex PCR showed 100 % reproducibility with the antimicrobial resistance profile generated from whole genome sequenced data. PTZ and C/S are not effective against ESBL and/or OXA-1 expressing E. coli and K. pneumoniae isolates and do not offer any activity against CMY co-producers. Further, the combined effect of CMY, NDM and PBP3 inserts impacts aztreonam/avibactam activity and reduces the susceptibility to 40 % in E. coli isolates. While, aztreonam/avibactam showed potent activity against NDM-expressing K. pneumoniae isolates. Importantly, cefepime/taniborbactam and cefiderocol showed limited activity against NDM-expressing E. coli and K. pneumoniae isolates. CONCLUSION: The pentaplex PCR was effective in detecting four beta-lactamases (blaCTX-M, blaOXA-1, blaCMY, blaNDM) as well as PBP3 inserts. It is expected that using pentaplex PCR as a diagnostic test for resistance detection in clinical practice will improve patient outcomes by providing prompt and targeted treatment.

Novel Modified Piperacillin Inhibitors of Penicillin-Binding Protein 3 (PBP3) and Their Intermolecular Interactions.

<b>Background and Objective:</b> Despite the rise of antibiotic resistance, penicillin and the broader group of ß-lactams have continued to be the most crucial class of antibiotics. Penicillin-Binding Protein 3 (PBP3) in <i>Pseudomonas aeruginosa</i> is the specific molecule that ß-lactam-based medicines target. The objective is to design and study several piperacillin derivatives to create novel antibacterial agents. <b>Materials and Methods:</b> Piperacillin derivatives were drawn using chem sketch and prepared using AutoDock 4.2.6 Tools. Molecular docking simulations were conducted on novel piperacillin derivatives and piperacillin (Control) against the 6r3x.PDB protein. The AutoDock log files were analyzed to determine the lowest energy of binding (LEB) values for each ligand. Consequently, the conformer with the most favorable binding energy may be identified. <b>Results:</b> All of the proposed piperacillin derivatives displayed improved binding energies when compared to the reference chemical piperacillin. This suggests the potential for stronger interactions between derivatives and proteins, resulting in an enhanced likelihood of biological effects. Compounds b, e and j, when used alongside piperacillin, showed similar binding sites inside the active site and have the potential for additional characterization. <b>Conclusion:</b> Compounds b, e and j are highly likely to exhibit inhibitory activity, indicating that they should be synthesized and tested for biological activity.

Resistance to aztreonam-avibactam among clinical isolates of Escherichia coli is primarily mediated by altered penicillin-binding protein 3 and impermeability.

This study was conducted to investigate decreased susceptibility (minimum inhibitory concentrations [MICs] 0.25-4 mg/L) and resistance (MICs > 4 mg/L) to aztreonam-avibactam (ATM-AVI). Contemporary non-replicate clinical isolates of carbapenemase-producing Escherichia coli (CP-EC) (n=90) and ESBL-producing E. coli (EP-EC) (n=12) were used. CP-EC belonged to 25 distinct sequence types (STs) and all EP-EC belonged to ST405. All strains were isolated from 2019 to 2022 at the Karolinska University Laboratory, Stockholm, Sweden. ATM-AVI MICs were determined using broth microdilution. The EUCAST epidemiological cut-off value of 0.125 mg/L was used to define the wild type (WT). Whole-genome sequences (Illumina) were analysed for detecting resistance determinants among WT vs. non-WT isolates. Among 102 isolates, 40 (39%) and 62 (61%) were WT and non-WT, respectively. Among non-WT isolates, resistance was noted for 20 and decreased susceptibility for 42. Resistance was observed among 14/47 New Delhi metallo-ß-lactamase (NDM)-producers, 5/43 OXA-48 group producers, and 1/12 EP-EC. Decreased susceptibility was observed among 29/47 NDM, 13/43 OXA-48 group, and 3/12 EP-EC. Resistant isolates predominantly belonged to ST405, followed by STs 410, 361, 167, 617, and 1284. Penicillin-binding protein 3 (PBP3) inserts (YRIK/YRIN) were observed in 20/20 and CMY-42 in 5/20 resistant isolates. Several mutations in the ftsI (encoding PBP3) and regulatory genes of outer membrane proteins (OmpC and OmpF) and efflux pumps (AcrAB-TolC) were detected. A ≥ 2-fold reduction in MICs was observed among 20/20 vs. 7/20 isolates tested in the presence of the membrane permeabiliser, polymyxin B nanopeptide (PMBN) and efflux inhibitor, phenylalanine arginine ß-naphthylamide (PAßN), respectively. In conclusion, resistance to ATM-AVI is a result of interplay of various determinants, including target alterations, deactivating enzymes, and decreased permeability.

The emergence of transposon-driven multidrug resistance in invasive nontypeable Haemophilus influenzae over the last decade.

Nontypeable Haemophilus influenzae (NTHi), once considered a harmless commensal, has emerged as a significant concern due to the increased prevalence of multidrug-resistant (MDR) strains and their association with invasive infections. This study aimed to explore the epidemiology and molecular resistance mechanisms of 51 NTHi isolates collected from patients with invasive infections in northern Taiwan between 2011 and 2020. This investigation revealed substantial genetic diversity, encompassing 29 distinct sequence types and 18 clonal complexes. Notably, 68.6% of the isolates exhibited ampicillin resistance, with 28 categorised as MDR and four isolates were even resistant to up to six antibiotic classes. Among the MDR isolates, 18 pulsotypes were identified, indicating diverse genetic lineages. Elucidation of their resistance mechanisms revealed 18 ß-lactamase-producing amoxicillin-clavulanate-resistant (BLPACR) isolates, 12 ß-lactamase-producing ampicillin-resistant (BLPAR) isolates, and 5 ß-lactamase-nonproducing ampicillin-resistant (BLNAR) isolates. PBP3 analysis revealed 22 unique substitutions in BLPACR and BLNAR, potentially contributing to cephem resistance. Notably, novel transposons, Tn7736-Tn7739, which contain critical resistance genes, were discovered. Three strains harboured Tn7739, containing seven resistance genes [aph(3')-Ia, blaTEM-1, catA, sul2, strA, strB, and tet(B)], while four other strains carried Tn7736, Tn7737, and Tn7738, each containing three resistance genes [blaTEM-1, catA, and tet(B)]. The emergence of these novel transposons underscores the alarming threat posed by highly resistant NTHi strains. Our findings indicated that robust surveillance and comprehensive genomic studies are needed to address this growing public health challenge.

Ampicillin susceptibility testing of Haemophilus influenzae in the routine clinical laboratory by the EUCAST methodology compared to broth microdilution and the presence of ftsI gene mutations.

OBJECTIVES: To investigate the prevalence of ampicillin resistance in Haemophilus influenzae and the diagnostic accuracy of the EUCAST recommended disc diffusion method to detect the increasingly prevalent ampicillin resistance due to the presence of PBP3 alterations based on mutations in the ftsI gene. METHODS: During a 6-month period all consecutive non-duplicate H. influenzae isolates were prospectively collected and stored. MICs of ampicillin were determined by broth microdilution (BMD). PCR was performed to detect mutations in the ftsI gene. Results of routine disc diffusion susceptibility testing, including the penicillin screening test in accordance with the current EUCAST methodology, as well as additional Etest results, were compared to the BMD as the reference method. RESULTS: In 102 isolates, the prevalence of ampicillin resistance was 28% (29/102) by BMD. There was a good correlation between MICs of ampicillin and the presence of a ß-lactamase and/or an ftsI gene mutation. The prevalence of ampicillin resistance was overestimated using the EUCAST method (33% (34/102)) and underestimated when an additional Etest was used (24% (24/102)) (not significant). The sensitivity and specificity of the EUCAST methodology for the detection of ampicillin resistance were 97% ((28/29); 95% CI, 82-100%) and 92% ((67/73); 95% CI, 83-97%), respectively. CONCLUSIONS: The prevalence of ampicillin resistance was 28%, as determined by BMD. Although the overall diagnostic accuracy of the EUCAST ampicillin disc diffusion was high, misclassification of ampicillin susceptibility may still occur.