A Two Amino Acid Duplication, L167E168, in the Ω-Loop Drastically Decreases Carbapenemase Activity of KPC-53, a Natural Class A ß-Lactamase.

KPC-53 enzyme is a natural KPC variant which showed a duplication of L167E168 residues in the Ω-loop structure. The blaKPC-53 gene was cloned both into pBC-SK and pET-24a vectors, and the recombinant plasmids were transferred by transformation in Escherichia coli competent cells to evaluate the antimicrobial susceptibility and to produce the enzyme. Compared to KPC-3, the KPC-53 was less stable and showed a dramatic reduction of kcat and kcat/Km versus several ß-lactams, in particular carbapenems. Indeed, a 2,000-fold reduction was observed in the kcat values of KPC-53 for imipenem and meropenem. Concerning inhibitors, KPC-53 was susceptible to tazobactam and clavulanic acid but maintained resistance to avibactam. The molecular modeling indicates that the L167E168 duplication in KPC-53 modifies the interactions between residues involved in the catalytic pocket, changing the flexibility of the Ω-loop, which is directly coupled with the catalytic properties of the KPC enzymes.

Laboratory Variants GESG170L, GESG170K, and GESG170H Increase Carbapenem Hydrolysis and Confer Resistance to Clavulanic Acid.

The Guiana extended-spectrum (GES) ß-lactamase GESG170H, GESG170L, and GESG170K mutants showed kcat, Km , and kcat/Km values very dissimilar to those of GES-1 and GES-5. The enhancement of the hydrolytic activity against carbapenems is potentially due to a shift of the substrate in the active site that provides better positioning of the deacylating water molecule caused by the presence of the imidazole ring of H170 and of the long side chain of K170 and L170.

Identification of New Natural CphA Metallo-ß-Lactamases CphA4 and CphA5 in Aeromonas veronii and Aeromonas hydrophila Isolates from Municipal Sewage in Central Italy.

Two new natural CphA metallo-ß-lactamases, the CphA4 and CphA5 enzymes, were identified in water samples from municipal sewage in central Italy. Compared to CphA, the CphA4 and CphA5 enzymes showed numerous point mutations. These enzymes have a narrow spectrum of substrates focused on carbapenems only. CphA5 showed kcat values about 40-, 12-, and 97-fold higher than those observed for CphA4 versus imipenem, ertapenem, and biapenem, respectively.

Kinetic study of the effect of histidines 240 and 164 on TEM-149 enzyme probed by ß-lactam inhibitors.

In the present study, we performed a detailed kinetic analysis of the enzymes TEM-149, TEM-149(H240), and TEM-149(H164-H240) versus a large panel of inhibitors/inactivators, including penicillins, penems, carbapenems, monobactams, cephamycin, and carbacephem. These compounds behaved as poor substrates versus TEM-149, TEM-149(H240), and TEM-149(H164-H240) ß-lactamases, and the Ki (inhibition constant), K (dissociation constant of the Henri-Michaelis complex), k+2 and k+3 (first-order acylation and deacylation constants, respectively), and k+2/K values were calculated.

Coexistence of blaNDM-5, blaCTX-M-15, blaOXA-232, blaSHV-182 genes in multidrug-resistant K. pneumoniae ST437-carrying OmpK36 and OmpK37 porin mutations: First report in Italy.

OBJECTIVES: K. pneumoniae is a common cause of severe hospital-acquired infections. In the present study, we have characterised the whole-genome of two K. pneumoniae ST437 belonging to the clonal complex CC258. METHODS: The whole-genome sequencing was performed by MiSeq Illumina, with a 2 × 300bp paired-end run. ResFinder 4.4.2 was used to detect acquired antimicrobial resistance genes (ARGs) and chromosomal mutations. Mobile genetic elements (plasmids and ISs) were identified by MobileElementFinder v1.0.3. The genome was also assigned to ST using MLST 2.0.9. Virulence factors were detected using the Virulence Factor Database (VFDB). RESULTS: K. pneumoniae KPNAQ_1/23 and KPNAQ_2/23 strains, isolated from urine samples of hospitalised patients, showed resistance to most antibiotics, including ceftazidime-avibactam, ceftolozane-tazobactam, and meropenem-vaborbactam combinations. Both strains were susceptible only to cefiderocol. Multiple mechanisms of resistance were identified. Resistance to ß-lactams was due to the presence of NDM-5, OXA-232, CTX-M-15, SHV-182 ß-lactamases, and OmpK36 and OmpK37 porin mutations. Resistance to fluoroquinolones was mediated by chromosomal mutations in acrR, oqxAB efflux pumps, and the bifunctional gene aac(6')-Ib-cr. CONCLUSION: The presence of different virulence genes makes these KPNAQ_1/23 and KPNAQ_2/23 high-risk clones.

R164H and V240H replacements by site-directed mutagenesis of TEM-149 extended-spectrum ß-lactamase: kinetic analysis of TEM-149H240 and TEM-149H164-H240 laboratory mutants.

Two laboratory mutant forms, TEM-149(H240) and TEM-149(H164-H240), of the TEM-149 extended-spectrum ß-lactamase enzyme were constructed by site-directed mutagenesis. TEM-149(H240) and TEM-149(H164-H240) were similar in kinetic behavior, except with respect to benzylpenicillin and ceftazidime. Molecular modeling of the two mutant enzymes demonstrated the role of histidine at position 240 in the reduction of the affinity of the enzyme for ceftazidime.

Whole-Genome Sequencing of ST2 A. baumannii Causing Bloodstream Infections in COVID-19 Patients.

A total of 43 A. baumannii strains, isolated from 43 patients affected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and by bacterial sepsis, were analyzed by antimicrobial susceptibility testing. All strains were resistant to almost three different classes of antibiotics, including carbapenems and colistin. The whole-genome sequencing (WGS) of eight selected A. baumannii isolates showed the presence of different insertion sequences (ISs), such as ISAba13, ISAba26, IS26, ISVsa3, ISEc29, IS6100 and IS17, giving to A. baumannii a high ability to capture and mobilize antibiotic resistance genes. Resistance to carbapenems is mainly mediated by the presence of OXA-23, OXA-66 and OXA-82 oxacillinases belonging to OXA-51-like enzymes. The presence of AmpC cephalosporinase, ADC-25, was identified in all A. baumannii. The pathogenicity of A. baumannii was exacerbated by the presence of several virulence factors. The multi-locus sequence typing (MLST) analysis showed that all strains belong to sequence type 2 (ST) international clone.

In Vitro Activity of Sulbactam-Durlobactam against Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates: A Multicentre Report from Italy.

In the present study, the in vitro activity of the sulbactam-durlobactam (SUL-DUR) combination was evaluated against 141 carbapenem-resistant A. baumannii (CRAb) clinical strains collected from six Italian laboratories. Over half (54.6%) of these isolates were resistant to colistin. The SUL-DUR combination was active against these CRAb isolates with MIC50 and MIC90 values of 0.5 mg/L and 4 mg/L, respectively. Only eleven isolates were resistant to SUL-DUR with MIC values ranging from 8 to 128 mg/L. The SUL-DUR resistant A. baumannii exhibited several antimicrobial resistance genes (ARGs) such as blaOXA-20, blaOXA-58, blaOXA-66, blaADC-25, aac(6')-Ib3 and aac(6')-Ib-cr and mutations in gyrA (S81L) and parC (V104I, D105E). However, in these isolates, mutations Q488K and Y528H were found in PBP3. Different determinants were also identified in these CRAb isolates, including adeABC, adeFGH, adeIJK, abeS, abaQ and abaR, which encode multidrug efflux pumps associated with resistance to multiple antibacterial agents. This is the first report on the antimicrobial activity of SUL-DUR against carbapenem-resistant A. baumannii isolates selected from multiple regions in Italy.

Potent inhibitory activity of taniborbactam towards NDM-1 and NDM-1Q119X mutants, and in vitro activity of cefepime/taniborbactam against MBLs producing Enterobacterales.

OBJECTIVE: This study aimed to investigate the in vitro activity of taniborbactam (VNRX-5133), a novel broad-spectrum bicyclic boronate, against NDM-1 and Q119E, Q119K, Q119C, Q119F, Q119V, and Q119Y NDM-1 variants, which showed an increased activity towards some ß-lactams, including cefepime. METHODS: Inhibition kinetic assays were spectrophotometrically performed using cefepime (50 µM) as the reporter substrate and 80 nM of each enzyme. Taniborbactam behaves as a competitive inhibitor towards NDM-1 and NDM-1 Q119 variants with lower Ki values (range 3-16 nM). The phenotypic profile was assessed in both Enterobacterales clinical isolates and engineered Escherichia coli BL21(DE3) strains by conventional broth microdilution procedures according to the Clinical and Laboratory Standards Institute (CLSI). RESULTS: Taniborbactam at a fixed concentration of 4 mg/L was able to restore activity of cefepime in 24 of 26 Enterobacterales clinical isolates harbouring metallo-ß-lactamases with MIC50/MIC90 values of 14 mg/L. Cefepime MICs were drastically reduced in all clinical isolates and in NDM-1 and Q119X producing Escherichia coli BL21(DE3). Taniborbactam was unable to restore susceptibility to cefepime in two IMP variants producing clinical isolates. CONCLUSION: The inhibition level of NDM enzymes provided by taniborbactam protects the antibacterial activity of cefepime from this important metallo-ß-lactamase.

Molecular characterization of carbapenem-resistant Klebsiella pneumoniae ST14 and ST512 causing bloodstream infections in ICU and surgery wards of a tertiary university hospital of Verona (northern Italy): co-production of KPC-3, OXA-48, and CTX-M-15 ß-lactamases.

Klebsiella pneumoniae strain is an important opportunistic pathogen that causes severe nosocomial infections. In the present study a molecular characterization of carbapenem resistant K. pneumoniae, isolated from blood samples of hospitalized patients of Verona University Hospital, was performed. The simultaneous presence of SHV-1/CTX-M-15/KPC-3 and SHV-1/CTX-M-15/OXA-48 serin-ß-lactamases was ascertained in the 89% and 11% of K. pneumoniae ST512 and K. pneumoniae ST14, respectively. Molecular characterization of bla genes showed that blaKPC-3 was found in Tn4401a transposon with the tnpR, tnpA, ISKpn6, and ISKpn7 mobile elements whereas blaCTX-M-15 was detected downstream ISEcp1 genetic element. A class 1 integron with a gene cassette of 780 bp corresponding to aadA2 gene was identified in 33 K. pneumoniae ST512 isolates.

Antimycotic Activity of Ozonized Oil in Liposome Eye Drops against Candida spp.

Purpose: This study aims to investigate the antifungal activity and mechanism of action of ozonized oil eye drops in liposomes (Ozodrop), commercialized as eye lubricant for the treatment of dry eye syndrome and eye inflammation. The activity was tested against four clinical Candida species: Calbicans,Cglabrata,Ckrusei, and Corthopsilosis. Methods: The antifungal activity of the eye drop solution was ascertained by microdilution method in accordance with EUCAST obtaining the minimum inhibitory concentration for Ozodrop. The mechanism of action was further investigated in Calbicans by measuring cell vitality, intracellular reactive oxygen species production, levels of cellular and mitochondrial (∆Ψm) membrane potential, and the extent of membrane lipid peroxidation. Results: All Candida isolates were susceptible to Ozodrop with minimum inhibitory concentration values ranging from 0.195% (v/v) for Cglabrata to 6.25% (v/v) for Corthopsilosis. After 1 hour of exposure at the minimum inhibitory concentration value about 30% of cells were killed, reaching about 70% at the highest Ozodrop value. After Ozodrop exposure, Calbicans showed cell membrane depolarization, increased levels of lipid peroxidation, depolarized ∆Ψm, and increased reactive oxygen species generation. Conclusions: The significant increases in reactive oxygen species production cause the accumulation of reactive oxygen species-associated damages leading to progressive Candida cell dysfunction. Translational Relevance: The antifungal activity of Ozodrop was demonstrated at concentrations several times lower than the concentration that can be retrieved in ocular surface after its application. The antifungal activity of the eye drops Ozodrop would represent an interesting off-label indication for a product basically conceived as an eye lubricant.

First Identification of ß-Lactamases in Antibiotic-Resistant Escherichia coli, Citrobacter freundii, and Aeromonas spp. Isolated in Stream Macroinvertebrates in a Central Italian Region.

Antibiotic-resistant bacteria (ARB) are widespread in nature and represent a serious public and environmental problem. In the present study, we report for the first time the presence of bacterial ß-lactamases in two macroinvertebrate species with different feeding traits. The class A ß-lactamases, SHV-1 and TEM-1, were found in Citrobacter freundii isolated from Gammarus elvirae and Escherichia coli from water samples, respectively. The metallo-ß-lactamase CphA was found in Aeromonas veronii and Aeromonas hydrophila strains isolated from the predator Dina lineata. The presence of a large plasmid was ascertained only in E. coli strains isolated from water. In all strains studied, an integrase I typical of class I integrin was found. In contaminated freshwater habitats, ARB and antibiotic resistance genes could be disseminated through trophic links with important ecological implications. Transmission through the food chain may contribute to spreading and transferring antibiotic resistance not only in freshwater ecosystems but also outside the aquatic habitat.

Identification of bla(IMP-22) in Pseudomonas spp. in urban wastewater and nosocomial environments: biochemical characterization of a new IMP metallo-enzyme variant and its genetic location.

OBJECTIVES: The aim of the study was the biochemical characterization of a new variant of the metallo-beta-lactamase, IMP-22. Moreover, the genetic environment of the bla(IMP-22) gene was investigated in Pseudomonas fluorescens and Pseudomonas aeruginosa collected from urban wastewater and a teaching hospital in L'Aquila, Italy. METHODS: Molecular characterization of genetic elements was carried out by PCR and DNA sequencing methods. The new enzyme was purified from recombinant Escherichia coli BL21(DE)Rosetta/pBC-SK/IMP-22. Steady-state kinetic parameters (K(m) and V(max)) were determined for a large pattern of substrates. RESULTS: A new IMP metallo-beta-lactamase gene was found in a class 1 integron and in one case, in a plasmid of Pseudomonas spp. The bla(IMP-22) encodes for a pre-protein of 246 amino acids and the N-terminus of the mature beta-lactamase (NH(2)-PDLK) was also determined. The molecular mass and pI were 24 930 Da and 6.2, respectively. On the basis of the kinetic parameters calculated (K(m) and V(max)), IMP-22 was found to hydrolyse narrow- and extended-spectrum beta-lactams. Enzyme activity was found to be inhibited by metal chelators such as EDTA, 1,10-o-phenathroline and dipicolinic acid with an IC(50) of 800, 750 and 300 microM, respectively. CONCLUSIONS: The finding of the bla(IMP-22) gene in P. fluorescens environmental strains and P. aeruginosa clinical isolate suggests the ongoing spread of bla(MBL) genes in several bacterial species and in different environments.

Identification of CTX-M-15 and CTX-M-27 in Antibiotic-Resistant Gram-Negative Bacteria Isolated from Three Rivers Running in Central Italy.

The main goal of this study was to identify Gram-negative bacteria resistant to antibiotics, in particular ß-lactams, in stream waters and effluents from urban wastewater treatment plants draining into Fino, Tavo, and Saline rivers of the Abruzzo region, Italy. Eight sampling sites were selected because they were the most contaminated by coliforms during previous sampling campaign. One sample for each site was collected for the detection of total and fecal coliforms, Escherichia coli and Enterococcus species by Colilert-18 and Enterolert-E Quanti-Tray/2000. Antibiotic-resistant bacteria, selected on ampicillin and cefotaxime-supplemented agar plates, were identified by EnteroPluri test systems and then confirmed by MALDI-TOF. The resistant determinants were identified and characterized by PCR and sequencing. The microbiological analysis allowed to detect E. coli, total coliforms, fecal coliforms, and enterococci with a coefficient of variation of 215.7%, 212.8%, 242.5%, and 188.5%, respectively. Several Gram-negative bacteria were identified: Serratia liquefaciens, E. coli, Enterobacter cloacae, Citrobacter freundii, Raoultella ornithinolytica, Acinetobacter johnsonii, Aeromonas veronii, Aeromonas hydrophila, and Pseudomonas koreensis. All strains possessed class 1 integrons, insertion sequences, and genes encoding for serin- and metallo-ß-lactamases. Extended-spectrum ß-lactamases, such as CTX-M-15 and CTX-M-27, were found in Enterobacteriaceae, whereas CphA metallo-ß-lactamase was found in A. veronii and A. hydrophila. The main resistance's mechanism to ß-lactams observed among the analyzed strains is represented by the production of serin ß-lactamases (CTX-M-15, CTX-M-27, and SHV-1) and metallo ß-lactamase (CphA).

Natural D240G Toho-1 mutant conferring resistance to ceftazidime: biochemical characterization of CTX-M-43.

OBJECTIVES: The aim of this article is biochemical and kinetic characterization of CTX-M-43, a natural Asp-240-->Gly mutant of CTX-M-44 (ex Toho-1), from a clinical isolate of Acinetobacter baumannii isolated in a Bolivian hospital. METHODS: Steady-state kinetic parameters (K(m) and k(cat)) were determined for a large pattern of substrates. Analysis of inactivators and transient inactivators was performed to determine the efficiency of acylation (k(+2)/K) and the deacylation constant (k(+3)). Molecular modelling of Michaelis complex of ceftazidime, cefotaxime and ceftibuten, obtained from molecular mechanics calculations, was carried out. RESULTS: CTX-M-43 showed a general increase in affinity towards all cephalosporins tested, with respect to CTX-M-44. Carbapenems acted as inactivators with a good acylation efficiency for meropenem and ertapenem and significant deacylation constant for imipenem. MICs of imipenem obtained at a higher bacterial inoculum of recombinant Escherichia coli were increased. CONCLUSIONS: Kinetic data and molecular modelling of Michaelis complex confirmed that Asp-240-->Gly allows a better accommodation of the bulky C7beta aminothiazol-oxyimino substituent, resulting in a general increase in the enzyme affinity towards oxyimino cephalosporins. The ascertained potentialities of CTX-M-type enzymes, supported by the kinetic data and the behaviour of the recombinant E. coli at different bacterial inocula towards carbapenems, make a possible evolution of those enzymes towards a carbapenemase activity plausible.

Optimizing cell permeation of an antibiotic resistance inhibitor for improved efficacy.

Benzo[b]thiophene-2-ylboronic acid, 1, is a 27 nM inhibitor of the class C beta-lactamase AmpC and potentiates the activity of beta-lactam antibiotics in bacteria that express this and related enzymes. As is often true, the potency of compound 1 against the enzymes is much attenuated in cell culture against Gram negative bacteria, where the minimum inhibitor concentration of compound 1 is in the mid-micromolar range. Here, we modulated the properties of this lead to enhance its ability to cross the membrane, using a combination of X-ray crystallography, structure-based design, and application of physical models of outer membrane crossing. This strategy led us to derivatives with substantially improved permeability. Also, the greater solubility of these compounds allowed us to measure their efficacy at higher concentrations than with the lead 1, leading to higher maximum potentiation of the antibiotic effect of ceftazidime on resistant bacteria.

BlaB-15, a new BlaB metallo-ß-lactamase variant found in an Elizabethkingia miricola clinical isolate.

A multidrug-resistant strain of Elizabethkingia miricola was isolated from the urine of a 2-year-old boy hospitalized for severe clinical conditions. The strain produces 2 metallo-ß-lactamases belonging to subclasses B1 and B3: a new BlaB variant (BlaB-15) and a GOB-7-like enzyme.

Interaction between lichen secondary metabolites and antibiotics against clinical isolates methicillin-resistant Staphylococcus aureus strains.

The in vitro antimicrobial activities of five compounds isolated from lichens, collected in several Southern regions of Chile (including the Chilean Antarctic Territory), were evaluated alone and in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC90, MIC50, as well as MBC90 and MBC50, for the lichen compounds were evaluated. The MIC90 was ranging from 32 µg/ml for perlatolic acid to 128 µg/ml for α-collatolic acid. MBC90 was ranging from onefold up to twofold the MIC90 for each compound. A synergistic action was observed in combination with gentamicin, whilst antagonism was observed for some lichen compounds in combination with levofloxacin. All combinations with erythromycin were indifferent, whilst variability was observed for clindamycin and oxacillin combinations. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. These could mainly be explained by the failure of FIC approach, being too much subjective and sensitive to experimental errors. These findings suggest, however, that the natural compounds from lichens are good candidates for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

OXA-23 carbapenemase in multidrug-resistant Acinetobacter baumannii ST2 type: first identification in L'Aquila Hospital (Italy).

In this study 114 extensively drug-resistant Acinetobacter baumannii clinical isolates were characterized. The strains were collected at L'Aquila Hospital after the earthquake in L'Aquila city (central Italy) on the 6th of April 2009. The genes blaOXA-23 and blaOXA-51 were detected in all clinical isolates analyzed, whereas blaTEM-1 allele was detected in 56/114 isolates. The blaOXA-23 gene is located downstream the ISAba region and is under control of a strong promoter. On 42/80 A. baumannii the presence of two class 1 integrons was ascertained on chromosomal DNA. Variable regions show different gene array: (1) aadB and aadA2, (2) aacA4, aac(6')-Ib-cr, and aadA1. Macrorestriction analysis using ApaI restriction endonuclease identifies three clusters (A, B, and C) according to pulsed-field gel electrophoresis profiles. All isolates analyzed belong to the clone A. baumannii sequence type 2.

Overexpression system and biochemical profile of CTX-M-3 extended-spectrum beta-lactamase expressed in Escherichia coli.

An efficient over-expression system was developed for CTX-M-3 extended-spectrum-beta-lactamase. The recombinant enzyme was purified from 1 l of culture to yield 22 mg of pure enzyme. The N-terminal amino acid sequence was determined to be NH2-QTADVQ... Determination of kinetic parameters with the purified CTX-M-3 revealed efficient hydrolysis of penicillins and cephalosporins, while ceftazidime and aztreonam were very poor substrates. Clavulanic acid, sulbactam and especially tazobactam inhibited the CTX-M-3 enzyme.