Detection of OXA-181 Carbapenemase in Shigella flexneri.

We report the detection of OXA-181 carbapenemase in an azithromycin-resistant Shigella spp. bacteria in an immunocompromised patient. The emergence of OXA-181 in Shigella spp. bacteria raises concerns about the global dissemination of carbapenem resistance in Enterobacterales and its implications for the treatment of infections caused by Shigella bacteria.

In vitro and in vivo activities of KSP-1007, a broad-spectrum inhibitor of serine- and metallo-ß-lactamases, in combination with meropenem against carbapenem-resistant Gram-negative bacteria.

KSP-1007 is a novel bicyclic boronate-based broad-spectrum ß-lactamase inhibitor and is being developed in combination with meropenem (MEM) for the treatment of infections caused by carbapenem-resistant Gram-negative bacteria, a global health concern, and here, we describe its characteristics. KSP-1007 exhibited low apparent inhibition constant (Ki app) values against all classes of ß-lactamase, including imipenemase types and oxacillinase types from Acinetobacter baumannii. Against 207 Enterobacterales and 55 A. baumannii, including carbapenemase producers, KSP-1007 at fixed concentrations of 4, 8, and 16 µg/mL dose-dependently potentiated the in vitro activity of MEM in broth microdilution MIC testing. The MIC90 of MEM/KSP-1007 at 8 µg/mL against Enterobacterales was lower than those of MEM/vaborbactam, ceftazidime/avibactam, imipenem/relebactam, and colistin and similar to those of aztreonam/avibactam, cefiderocol, and tigecycline. The in vitro activity of MEM/KSP-1007 at ≥4 µg/mL against Enterobacterales harboring metallo-ß-lactamase was superior to that of cefepime/taniborbactam. MEM/KSP-1007 showed excellent activity against Escherichia coli with PBP3 mutations and New Delhi metallo-ß-lactamase compared to aztreonam/avibactam, cefepime/taniborbactam, and cefiderocol. MEM/KSP-1007 at 8 µg/mL showed greater efficacy against A. baumannii than these comparators except for cefiderocol, tigecycline, and colistin. A 2-fold reduction in MEM MIC against 96 Pseudomonas aeruginosa was observed in combination with KSP-1007. MEM/KSP-1007 demonstrated bactericidal activity against carbapenemase-producing Enterobacterales, A. baumannii, and P. aeruginosa based on minimum bactericidal concentration/MIC ratios of ≤4. KSP-1007 enhanced the in vivo activity of MEM against carbapenemase-producing Enterobacterales, A. baumannii, and P. aeruginosa in murine systemic, complicated urinary tract, and thigh infection models. Collectively, MEM/KSP-1007 has a good profile for treating carbapenem-resistant Gram-negative bacterial infections.

Structural basis of metallo-ß-lactamase resistance to taniborbactam.

The design of inhibitors against metallo-ß-lactamases (MBLs), the largest family of carbapenemases, has been a strategic goal in designing novel antimicrobial therapies. In this regard, the development of bicyclic boronates, such as taniborbactam (TAN) and xeruborbactam, is a major achievement that may help in overcoming the threat of MBL-producing and carbapenem-resistant Gram-negative pathogens. Of concern, a recent report has shown that New Delhi MBL-9 (NDM-9) escapes the inhibitory action of TAN by a single amino acid substitution with respect to New Delhi MBL-1 (NDM-1), the most widely disseminated MBL. Here, we report a docking and computational analysis that identifies that "escape variants" against TAN can arise by disruption of the electrostatic interaction of negative charges in the active site loops of MBLs with the N-(2-aminoethyl)cyclohexylamine side chain of TAN. These changes result in non-productive binding modes of TAN that preclude reaction with the MBLs, a phenomenon that is not restricted to NDM-9. This analysis demonstrates that single amino acid substitutions in non-essential residues in MBL loops can unexpectedly elicit resistance to TAN.

Balancing the scales: achieving the optimal beta-lactam to beta-lactamase inhibitor ratio with continuous infusion piperacillin/tazobactam against extended spectrum beta-lactamase producing Enterobacterales.

Piperacillin/tazobactam (TZP) is administered intravenously in a fixed ratio (8:1) with the potential for inadequate tazobactam exposure to ensure piperacillin activity against Enterobacterales. Adult patients receiving continuous infusion (CI) of TZP and therapeutic drug monitoring (TDM) of both agents were evaluated. Demographic variables and other pertinent laboratory data were collected retrospectively. A population pharmacokinetic approach was used to select the best kidney function model predictive of TZP clearance (CL). The probability of target attainment (PTA), cumulative fraction of response (CFR) and the ratio between piperacillin and tazobactam were computed to identify optimal dosage regimens by continuous infusion across kidney function. This study included 257 critically ill patients (79.3% male) with intra-abdominal, bloodstream, and hospital-acquired pneumonia infections in 89.5% as the primary indication. The median (min-max range) age, body weight, and estimated glomerular filtration rate (eGFR) were 66 (23-93) years, 75 (39-310) kg, and 79.2 (6.4-234) mL/min, respectively. Doses of up to 22.5 g/day were used to optimize TZP based on TDM. The 2021 chronic kidney disease epidemiology equation in mL/min best modeled TZP CL. The ratio of piperacillin:tazobactam increased from 6:1 to 10:1 between an eGFR of <20 mL/min and >120 mL/min. At conventional doses, the PTA is below 90% when eGFR is ≥100 mL/min. Daily doses of 18 g/day and 22.5 g/day by CI are expected to achieve a >80% CFR when eGFR is 100-120 mL/min and >120-160 mL/min, respectively. Inadequate piperacillin and tazobactam exposure is likely in patients with eGFR ≥ 100 mL/min. Dose regimen adjustments informed by TDM should be evaluated in this specific population.

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

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

IncC plasmid genome rearrangements influence the vertical and horizontal transmission tradeoff in Escherichia coli.

It has been shown that an evolutionary tradeoff between vertical (host growth rate) and horizontal (plasmid conjugation) transmissions contributes to global plasmid fitness. As conjugative IncC plasmids are important for the spread of multidrug resistance (MDR), in a broad range of bacterial hosts, we investigated vertical and horizontal transmissions of two multidrug-resistant IncC plasmids according to their backbones and MDR-region rearrangements, upon plasmid entry into a new host. We observed plasmid genome deletions after conjugation in three diverse natural Escherichia coli clinical strains, varying from null to high number depending on the plasmid, all occurring in the MDR region. The plasmid burden on bacterial fitness depended more on the strain background than on the structure of the MDR region, with deletions appearing to have no impact. Besides, we observed an increase in plasmid transfer rate, from ancestral host to new clinical recipient strains, when the IncC plasmid was rearranged. Finally, using a second set of conjugation experiments, we investigated the evolutionary tradeoff of the IncC plasmid during the critical period of plasmid establishment in E. coli K-12, by correlating the transfer rates of deleted or non-deleted IncC plasmids and their costs on the recipient strain. Plasmid deletions strongly improved conjugation efficiency with no negative growth effect. Our findings indicate that the flexibility of the MDR-region of the IncC plasmids can promote their dissemination, and provide diverse opportunities to capture new resistance genes. In a broader view, they suggest that the vertical-horizontal transmission tradeoff can be manipulated by the plasmid to improve its fitness.

Cefiderocol heteroresistance associated with mutations in TonB-dependent receptor genes in Pseudomonas aeruginosa of clinical origin.

The siderophore-cephalosporin cefiderocol (FDC) presents a promising treatment option for carbapenem-resistant (CR) P. aeruginosa (PA). FDC circumvents traditional porin and efflux-mediated resistance by utilizing TonB-dependent receptors (TBDRs) to access the periplasmic space. Emerging FDC resistance has been associated with loss of function mutations within TBDR genes or the regulatory genes controlling TBDR expression. Further, difficulties with antimicrobial susceptibility testing (AST) and unexpected negative clinical treatment outcomes have prompted concerns for heteroresistance, where a single lineage isolate contains resistant subpopulations not detectable by standard AST. This study aimed to evaluate the prevalence of TBDR mutations among clinical isolates of P. aeruginosa and the phenotypic effect on FDC susceptibility and heteroresistance. We evaluated the sequence of pirR, pirS, pirA, piuA, or piuD from 498 unique isolates collected before the introduction of FDC from four clinical sites in Portland, OR (1), Houston, TX (2), and Santiago, Chile (1). At some clinical sites, TBDR mutations were seen in up to 25% of isolates, and insertion, deletion, or frameshift mutations were predicted to impair protein function were seen in 3% of all isolates (n = 15). Using population analysis profile testing, we found that P. aeruginosa with major TBDR mutations were enriched for a heteroresistant phenotype and undergo a shift in the susceptibility distribution of the population as compared to susceptible strains with wild-type TBDR genes. Our results indicate that mutations in TBDR genes predate the clinical introduction of FDC, and these mutations may predispose to the emergence of FDC resistance.

Phenotypic and molecular characterization of ß-lactamase-producing Klebsiella species among children discharged from hospital in Western Kenya.

BACKGROUND: The emergence and spread of ß-lactamase-producing Klebsiella spp. has been associated with a substantial healthcare burden resulting in therapeutic failures. We sought to describe the proportion of phenotypic resistance to commonly used antibiotics, characterize ß-lactamase genes among isolates with antimicrobial resistance (AMR), and assess the correlates of phenotypic AMR in Klebsiella spp. isolated from stool or rectal swab samples collected from children being discharged from hospital. METHODS: We conducted a cross-sectional study involving 245 children aged 1-59 months who were being discharged from hospitals in western Kenya between June 2016 and November 2019. Whole stool or rectal swab samples were collected and Klebsiella spp. isolated by standard microbiological culture. ß-lactamase genes were detected by PCR whilst phenotypic antimicrobial susceptibility was determined using the disc diffusion technique following standard microbiology protocols. Descriptive analyses were used to characterize phenotypic AMR and carriage of ß-lactamase-producing genes. The modified Poisson regression models were used to assess correlates of phenotypic beta-lactam resistance. RESULTS: The prevalence of ß-lactamase carriage among Klebsiella spp. isolates at hospital discharge was 62.9% (154/245). Antibiotic use during hospitalization (adjusted prevalence ratio [aPR] = 4.51; 95%CI: 1.79-11.4, p < 0.001), longer duration of hospitalization (aPR = 1.42; 95%CI: 1.14-1.77, p < 0.002), and access to treated water (aPR = 1.38; 95%CI: 1.12-1.71, p < 0.003), were significant predictors of phenotypically determined ß-lactamase. All the 154 ß-lactamase-producing Klebsiella spp. isolates had at least one genetic marker of ß-lactam/third-generation cephalosporin resistance. The most prevalent genes were blaCTX-M 142/154 (92.2%,) and blaSHV 142/154 (92.2%,) followed by blaTEM 88/154 (57.1%,) and blaOXA 48/154 (31.2%,) respectively. CONCLUSION: Carriage of ß-lactamase producing Klebsiella spp. in stool is common among children discharged from hospital in western Kenya and is associated with longer duration of hospitalization, antibiotic use, and access to treated water. The findings emphasize the need for continued monitoring of antimicrobial susceptibility patterns to inform the development and implementation of appropriate treatment guidelines. In addition, we recommend measures beyond antimicrobial stewardship and infection control within hospitals, improved sanitation, and access to safe drinking water to mitigate the spread of ß-lactamase-producing Klebsiella pathogens in these and similar settings.

Outcomes of 23 patients diagnosed with New Delhi metallo-beta-lactamase (NDM)-producing Klebsiella pneumoniae infection treated with ceftazidime/avibactam and aztreonam at a single center in Poland.

PURPOSE: Amongst all etiologic hospital-acquired infection factors, K. pneumoniae strains producing New Delhi metallo-ß-lactamase (KP-NDM) belong to pathogens with the most effective antibiotic resistance mechanisms. Clinical guidelines recommend using ceftazidime/avibactam with aztreonam (CZA + AT) as the preferred option for NDM-producing Enterobacterales. However, the number of observations on such treatment regimen is limited. This retrospective study reports the clinical and microbiological outcomes of 23 patients with KP-NDM hospital-acquired infection treated with CZA + AT at a single center in Poland. METHODS: The isolates were derived from the urine, lungs, blood, peritoneal cavity, wounds, and peritonsillar abscess. In microbiological analysis, mass spectrometry for pathogen identification, polymerase chain reaction, or an immunochromatographic assay for detection of carbapenemase, as well as VITEK-2 system, broth microdilution, and microdilution in agar method for antimicrobial susceptibility tests were used, depending of the pathogens' nature. CZA was administered intravenously (IV) at 2.5 g every eight hours in patients with normal kidney function, and aztreonam was administered at 2 g every eight hours IV. Such dosage was modified when renal function was reduced. RESULTS: KP-NDM was eradicated in all cases. Four patients (17.4%) died: three of them had a neoplastic disease, and one - a COVID-19 infection. CONCLUSION: The combination of CZA + AT is a safe and effective therapy for infections caused by KP-NDM, both at the clinical and microbiological levels. The synergistic action of all compounds resulted in a good agreement between the clinical efficacy of CZA + AT and the results of in vitro susceptibility testing.

Prevalence and risk factors of extended-spectrum beta-lactamase producing E. coli causing urinary tract infections in Iceland during 2012-2021.

PURPOSE: To investigate the association of potential risk factors for urinary tract infections (UTI) caused by E. coli producing ESBL vs. not producing ESBL in Iceland. METHODS: Observational, case-control study including a cohort of 27,747 patients (22,800 females, 4,947 males; 1207 cases, 26,540 controls) of all ages with UTI caused by E. coli in 2012 to 2021 at the clinical microbiology laboratory covering about 2/3 of the Icelandic population. Clinical patient data was obtained from three national databases. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) as a measure of association between ESBL and exposure variables. RESULTS: The proportion of samples with ESBL-producing E. coli increased during the study period, from 2.6% in 2012 to 7.6% in 2021 (p < 0.001). ESBL-positive strains were detected in 1207 individuals (4.4%), 905 females (4.0%) and 302 males (6.1%). The following risk factors were identified: Male sex, higher age, institution type (hospital, nursing home), hospital-associated UTI, Charlson comorbidity index score ≥ 3, history of cystitis or hospitalization in the past year, and prescriptions for certain antibiotics or proton pump inhibitors (PPIs: OR 1.51) in the past half year. The antibiotic associated with the highest risk was ciprofloxacin (OR 2.45). CONCLUSION: The prevalence of UTIs caused by ESBL-producing E. coli has been increasing in Iceland. The strongest risk factors for ESBL production were previous antibiotic use, especially ciprofloxacin, and previous PPI use, both considered to be overprescribed. It is important to promote the prudent use of these drugs.

New Antibiotics Against Multidrug-Resistant Gram-Negative Bacteria in Liver Transplantation: Clinical Perspectives, Toxicity, and PK/PD Properties.

Antimicrobial resistance is a growing global health problem, and it is especially relevant among liver transplant recipients where infections, particularly when caused by microorganisms with a difficult-to-treat profile, are a significant cause of morbidity and mortality. We provide here a complete dissection of the antibiotics active against multidrug-resistant Gram-negative bacteria approved over the last years, focusing on their activity spectrum, toxicity profile and PK/PD properties, including therapeutic drug monitoring, in the setting of liver transplantation. Specifically, the following drugs are presented: ceftolozane/tazobactam, ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam, cefiderocol, and eravacycline. Overall, studies on the safety and optimal employment of these drugs in liver transplant recipients are limited and especially needed. Nevertheless, these pharmaceuticals have undeniably enhanced therapeutic options for infected liver transplant recipients.

Identification of Phytochemicals with Inhibitory Potential Against Beta-lactamase Enzymes via Computer-aided Approach.

INTRODUCTION: Antibacterial drugs have been widely used for the past century to treat diseases, but their efficacy has been limited by multi-resistant pathogens, particularly those that utilize beta-lactamase enzymes. The inhibition of beta-lactamase enzymes holds great promise for reducing the influence of such pathogens. OBJECTIVE: This study aims to evaluate the mechanism of inhibition of phytochemicals with antibacterial activity against two classes of beta-lactamases using computational methods. METHODS: To achieve this objective, a total of thirty phytochemicals were docked against SHV-1 beta-lactamase and AmpC beta-lactamase after procurement from Protein Data Bank. The pharmacokinetics (ADMET) and density functional theory (DFT) analysis study were also conducted to unravel the nature of the top six most promising compounds on each protein. RESULTS: The results showed that a significant percentage of the compounds had binding affinities greater than that of avibactam, the positive control. Quercetin-3-O-rutinoside showed the most promising results against SHV-1 beta-lactamase with an affinity of -9.4 kcal/mol, while luteolin was found to be the most promising candidate against AmpC beta-lactamase with an affinity of -8.5 kcal/mol. DFT analysis demonstrated the reactivity of these compounds, and the ADMET study indicated that they were relatively safe. CONCLUSION: In conclusion, the study's findings suggest that the selected compounds have significant potential to inhibit beta-lactamase and may be used in combination with antibiotics against organisms that produce beta-lactamase. This study provides a basis for further research in a wet-lab setting to validate the results.

Blue Light Compromises Bacterial ß-Lactamases Activity to Overcome ß-Lactam Resistance.

OBJECTIVE: In this study, we evaluated the effectiveness of antimicrobial blue light (aBL; 410 nm wavelength) against ß-lactamase-carrying bacteria and the effect of aBL on the activity of ß-lactamases. METHODS: Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae strains carrying ß-lactamases as well as a purified ß-lactamase enzymes were studied. ß-lactamase activity was assessed using a chromogenic cephalosporin hydrolysis assay. Additionally, we evaluated the role of porphyrins in the photoreaction, as well as protein degradation by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Finally, we investigated the bactericidal effect of combined aBL-ceftazidime exposure against a metallo-ß-lactamase expressing P. aeruginosa strain. RESULTS: Our study demonstrated that aBL effectively killed ß-lactamase-producing bacteria and reduced ß-lactamase activity. After an aBL exposure of 1.52 J/cm2, a 50% reduction in enzymatic activity was observed in P. aeruginosa. Additionally, we found a 40% decrease in the photoreaction activity of porphyrins following an aBL exposure of 64.8 J/cm2. We also revealed that aBL reduced ß-lactamase activity via protein degradation (after 136.4 J/cm2). Additionally, aBL markedly improved the bactericidal effect of ceftazidime (by >4-log10) in the metallo-ß-lactamase P. aeruginosa strain. CONCLUSION: Our results provide evidence that aBL compromises bacterial ß-lactamase activity, offering a potential approach to overcome ß-lactam resistance in bacteria.

Antibiotic Resistance and Virulence Gene Patterns Associated with Multi Drug Resistant Avian Pathogenic Escherichia coli (APEC) Isolated from Broiler Chickens in India.

In the present study, a total of 102 samples were collected from chickens of different flocks, died due to suspected colibacillosis. Bacteriological and PCR methods were applied to detect avian pathogenic Escherichia coli (APEC). Phenotypic antimicrobial resistance (AMR) was determined by disk diffusion method. Extended spectrum beta lactamases (ESBL) detection was carried out via PCR by targeting blaTEM, blaSHV, blaOXA, and blaCTX-M groups 1, 2, and 9. Genes of eight virulence factors and class I integrons were also detected by PCR using gene specific primers. Culture, microscopic, biochemical tests and PCR recognised 69/102 (67.64%) samples as E. coli. Phenotypic AST revealed higher resistance against fluoroquinolone antibiotics, i.e., enrofloxacin (72.46%), levofloxacin (69.56%) & ciprofloxacin (66.66%), followed by amoxyclav (63.77%) and tetracycline (59.42%). Six isolates were found as pan-drug-resistant E. coli. A total of 48 (69.56%) and 7 (10.14%) isolates were positive for the presence of blaTEM and blaCTX-M-G9 genes, respectively, whereas 2 (2.90%) isolates each were found positive for blaSHV, blaOXA, and blaCTX-M-G1 genes. Among APEC associated virulence genes, iss (79.71%) was the most predominant, followed by tsh (50.72%), ast (30.43%), cvaf (26.08%), pap (23.18%), vat (8.69%) and stx-1 (1.44%). Thirty-two isolates harboured class I integrons, either with or without ESBL genes. Conclusively, the isolates under study showed pan and multiple-drug resistance, specifically against fluoroquinolone drugs. ESBL production was mediated principally through bla TEM and blaCTX-M-G9. Multiple virulence factors, toxins, and carriage & spread factor render these as zoonotically potential pathogens for humans. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01132-2.

In vitro efficacy of humanized regimen of flomoxef against extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae.

This study compared the efficacy of flomoxef with other ß-lactam antibiotics against extended-spectrum ß-lactamases (ESBL)-producing bacteria of clinical relevance. First, the prevalence and ß-lactamase genotypes of ESBL-producing strains among Escherichia coli and Klebsiella pneumoniae isolates collected in Japan from 2004 to 2018 were investigated. High MIC90 values (>64 µg/mL) of ceftriaxone, cefepime, and ceftazidime and low MIC90 values (≤0.06-2 µg/mL) of flomoxef, cefmetazole, and meropenem against both species were observed. Second, a chemostat model was used to analyze the efficacy of humanized regimens of three oxacephem/cephamycin antibiotics (flomoxef, cefmetazole, cefoxitin) and two other antibiotics (meropenem and piperacillin/tazobactam) in suppressing the growth of five ESBL-producing E. coli and two K. pneumoniae strains. Flomoxef, piperacillin/tazobactam, and meropenem showed good bactericidal effects with >4 log10 CFU/mL reduction without bacterial regrowth at 24 h even when the MIC of test isolates was >MIC90. Cefmetazole and cefoxitin resulted in regrowth of test isolates with MIC ≥MIC90 at 24 h. Cefmetazole, cefoxitin, flomoxef, and meropenem showed increased MICs for regrown samples. A clear relationship between the proportion of time that the free drug concentration exceeded the MIC (%fT>MIC) and antibiotic efficacy was found for flomoxef, cefoxitin, and cefmetazole, and flomoxef had the highest %fT>MIC, whereas discrepancies between Clinical and Laboratory Standards Institute breakpoint and bactericidal activity were observed for cefmetazole. Flomoxef was effective in preventing the growth of all ESBL-producing strains, even those with an MIC eight times the MIC90. Thus, flomoxef may be a good alternative to meropenem in context of carbapenems sparing stewardship.

The directed evolution of NDM-1.

ß-Lactam antibiotics are among the most frequently prescribed therapeutic agents. A common mechanism of resistance toward ß-lactam antibiotics is the production of ß-lactamases. These enzymes are capable of hydrolyzing the ß-lactam bond, rendering the drug inactive. Among the four described classes, the metallo- ß-lactamases (MBLs, class B) employ one or two zinc ions in the active site for catalysis. One of the three most clinically relevant MBLs is New Delhi Metallo- ß-Lactamase (NDM-1). The current study sought to investigate the in vitro protein evolution of NDM-1 ß-lactamase using error-prone polymerase chain reaction. Evaluation revealed that variants were not found to confer higher levels of resistance toward meropenem based on amino acid substitutions. Thus, we postulate that increases in transcription or changes in zinc transport may be clinically more relevant to meropenem resistance than amino acid substitutions.

Natural protein engineering in the Ω-loop: the role of Y221 in ceftazidime and ceftolozane resistance in Pseudomonas-derived cephalosporinase.

A wide variety of clinically observed single amino acid substitutions in the Ω-loop region have been associated with increased minimum inhibitory concentrations and resistance to ceftazidime (CAZ) and ceftolozane (TOL) in Pseudomonas-derived cephalosporinase and other class C ß-lactamases. Herein, we demonstrate the naturally occurring tyrosine to histidine substitution of amino acid 221 (Y221H) in Pseudomonas-derived cephalosporinase (PDC) enables CAZ and TOL hydrolysis, leading to similar kinetic profiles (k cat = 2.3 ± 0.2 µM and 2.6 ± 0.1 µM, respectively). Mass spectrometry of PDC-3 establishes the formation of stable adducts consistent with the formation of an acyl enzyme complex, while spectra of E219K (a well-characterized, CAZ- and TOL-resistant comparator) and Y221H are consistent with more rapid turnover. Thermal denaturation experiments reveal decreased stability of the variants. Importantly, PDC-3, E219K, and Y221H are all inhibited by avibactam and the boronic acid transition state inhibitors (BATSIs) LP06 and S02030 with nanomolar IC50 values and the BATSIs stabilize all three enzymes. Crystal structures of PDC-3 and Y221H as apo enzymes and complexed with LP06 and S02030 (1.35-2.10 Å resolution) demonstrate ligand-induced conformational changes, including a significant shift in the position of the sidechain of residue 221 in Y221H (as predicted by enhanced sampling well-tempered metadynamics simulations) and extensive hydrogen bonding between the enzymes and BATSIs. The shift of residue 221 leads to the expansion of the active site pocket, and molecular docking suggests substrates orientate differently and make different intermolecular interactions in the enlarged active site compared to the wild-type enzyme.

LSPR-Based Biosensing Enables the Detection of Antimicrobial Resistance Genes.

The development of rapid, simple, and accurate bioassays for the detection of nucleic acids has received increasing demand in recent years. Here, localized surface plasmon resonance (LSPR) spectroscopy for the detection of an antimicrobial resistance gene, sulfhydryl variable ß-lactamase (blaSHV), which confers resistance against a broad spectrum of ß-lactam antibiotics is used. By performing limit of detection experiments, a 23 nucleotide (nt) long deoxyribonucleic acid (DNA) sequence down to 25 nm was detected, whereby the signal intensity is inversely correlated with sequence length (23, 43, 63, and 100 nt). In addition to endpoint measurements of hybridization events, the setup also allowed to monitor the hybridization events in real-time, and consequently enabled to extract kinetic parameters of the studied binding reaction. Performing LSPR measurements using single nucleotide polymorphism (SNP) variants of blaSHV revealed that these sequences can be distinguished from the fully complementary sequence. The possibility to distinguish such sequences is of utmost importance in clinical environments, as it allows to identify mutations essential for enzyme function and thus, is crucial for the correct treatment with antibiotics. Taken together, this system provides a robust, label-free, and cost-efficient analytical tool for the detection of nucleic acids and will enable the surveillance of antimicrobial resistance determinants.

Characterisation of ESBL/AmpC-Producing Enterobacteriaceae isolated from poultry farms in Peninsular Malaysia.

Extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases (AmpCs)-producing Enterobacteriaceae have been increasingly reported and imposing significant threat to public. Livestock production industry might be the important source for clinically important ESBL-producing Enterobacteriaceae. This study aims to investigate the resistance profile, phenotypic ESBL production, beta-lactamase genes, virulence factors, and plasmid replicon types among 59 Enterobacteriaceae strains isolated from poultry faecal samples in Malaysia's commercial poultry farm. There were 38.7% and 32.3% of Escherichia coli resistant to cefotaxime and cefoxitin, respectively, while Klebsiellaspp. demonstrated resistance rate of 52.6% to both mentioned antimicrobials. Majority of the E. coli isolates carried blaTEM and blaCMY-2 group. blaSHV was the most prevalent gene detected in Klebsiellaspp., followed by blaDHA and blaTEM. Resistance to extended spectrum cephalosporin in our isolates was primarily mediated by plasmid mediated AmpC beta-lactamase such as CMY-2 group and DHA enzyme. The CTX-M genes were found in two ESBL-producing E. coli. IncF, IncI1, and IncN plasmids were most frequently detected in E. coli and Klebsiellaspp. The virulence factor, including EAST1 and pAA were identified at low frequency. This study highlights the poultry as a reservoir of resistance and virulence determinants and prevalence of plasmids in Enterobacteriaceae might drive their dissemination.

CRISPR/cas9 cassette targeting Escherichia coli blaCTX-M specific gene of mastitis cow milk origin can alter the antibiotic resistant phenotype for cefotaxime.

CTX-M beta-lactamases are one of the most important extended spectrum beta-lactamase (ESBL) resistance enzymes found in E. coli. In the present study, 59% of E. coli isolates from mastitis cow milk were reported to be positive for ESBL types. The prevalence of beta-lactam (ß-lactam) antibiotic resistance was reported to be 84%, 72.7%, 52.27%, 50%, and 45.4% for cefotaxime, cefepime, cefuroxime, oxacillin, and cephalexine, respectively. The blaCTX-M gene was found in 65% (n = 17) of the E. coli isolates when they were genotyped. Further, the use of a CRISPR/cas9 cassette to target the E. coli blaCTX-M gene revealed changes in antibiotic phenotypes for cefotaxime.