Inactivation Method Research Articles

Extended spectrum betalactamase and Carbapenemase producing gram negative bacteria from healthcare workers gowns at Debre Berhan Comprehensive Specialized Hospital, Ethiopia

Extended-spectrum beta-lactamase (ESBL) and carbapenemase-producing (CP) gram-negative bacteria are the major public health concerns. Gowns used by healthcare workers (HCWs) in daily practice are a source of hospital-acquired infections in hospital settings. The study aimed to determine the prevalence of extended-spectrum beta-lactamase and carbapenemase-producing gram-negative bacteria from gowns of healthcare workers at Debre Berhan Comprehensive Specialized Hospital, Amhara Regional State, Ethiopia. A hospital-based cross-sectional study was conducted from August to October 2022. A total of 226 swab samples were collected from gowns of HCWs and cultured on MacConkey agar. Bacterial identifications were done by standard biochemical tests. Screening for ESBL and carbapenemase production was done using CHROME agar. Confirmation of ESBL and carbapenemase production was done by the combination disk method and modified carbapenem inactivation method, respectively. Data were analyzed by using SPSS version 25. The overall contamination rate of gowns was 46.9% (106/226). Among 226 swab samples, a total of 117 (51.7%) gram-negative bacteria were isolated. Among these, the most frequent isolates were E. coli, accounted 36 (30.8%) followed by K. pneumoniae, 26(22.2%). The overall multi-drug resistance (MDR) rate was 65 (55.6%). Of the 117 isolates, 17(14.5%) and 12(10.3%) were ESBL and carbapenemase producers respectively. Gown type (p = 0.041), laundering practice (p = 0.045), number of gowns (p = 0.002), and gown washing frequency per week (p = 0.017).were significantly associated with bacterial contamination of gowns. In this study, the prevalence of ESBL and CP Gram-negative bacteria from gowns of healthcare workers was found to be alarming. Therefore, strict infection prevention and control practices, as well as good hygienic practices, should be implemented to reduce and prevent cross-contamination and the spread of antimicrobial-resistant strains in hospital settings.

Assessment of the impact of different SARS-CoV-2 inactivation operations on colistin sulfate plasma concentration results

ObjectiveTo evaluate the effects of different SARS-CoV-2 inactivation methods on the blood concentration of colistin sulfate.MethodsA colistin sulfate reference substance, a quality control plasma sample, and a clinically measured sample were transferred and heated in a 56 °C water batch for 30 min or irradiated under an ultraviolet (UV) lamp for 60 min to examine the stability of the reference solution and quality control plasma sample. Statistical analysis was conducted for the concentration of the clinically measured sample before and after inactivation with the intraclass correlation coefficient (ICC) method, the Passing–Bablok regression, and the Bland–Altman analysis.ResultsThe reference substance of colistin sulfate was unstable under UV inactivation. Under moist heat inactivation, the reference substance and quality control plasma sample of colistin sulfate were stable. The ICC of the concentration results of 27 clinical blood samples with and without moist heat inactivation was 0.995 (P < 0.001). The Passing–Bablok regression equation was Y = 0.01347 + 0.9861X (R2 = 0.994, n = 27). Finally, 96.30% (26/27) of test data was within the 95% limit of agreement. The results obtained with the three statistical analysis methods all showed a high correlation and agreement between the plasma concentration results of samples not inactivated and inactivated with moist heat inactivation.ConclusionThe clinical plasma concentration test samples for colistin sulfate can be processed with the moist heat virus inactivation method to reduce the risk of virus infection of medical staff.

Assessment Of Selected Cardiac Enzymes To Find Out Cardiac Damage In Admitted COVID 19 Patients In Tertiary Care Hospital, Kolkata

Introduction: The systemic inflammatory response of SARS COV2 virus affects cardiovascular system too along with the respiratory system. The prediction of prognosis &amp; though unclear still it can be assessed by CRP level. The cardioselective biomarkers of LDH &amp; CPK (CKMB &amp; LDH1) has been seen to be elevated in covid infection which is more profound in severity of infection The study was done to find any association of cardioselective isoenzyemes of LDH &amp; CPK with the inflammatory marker (CRP) in admitted covid patients Materials &amp; methods: 88 admitted covid patients in CMSDH having LDH 663 U/L to 955 U/L (as 95% CI) were assessed for LDH1 by heat inactivation method at 650C &amp; total CPK, CKMB &amp; CRP were measured using standardized kits. Result: LDH1: LDH is significantly correlated with total CPK (r=0.387 &amp; P&lt;0.05) &amp; CKMB (r=0.55 &amp; p&lt;0.05) in covid patients. By Chi- square test significant association has been found between CRP with LDH1: total LDH ratio 2 value= 5.934 &amp; p=0.03) and between CRP with CK-MB ( 2 value= 11.37 &amp; p&lt;0.001). Conclusion: in our study there was significant myocardial involvement indicated by increased cardioselective biomarkers (LDH1 &amp; CKMB) which threaten the possibility of adverse cardiovascular complications in patients suffering &amp; recovered from covid infection.

Evaluation of simplified carbapenem inactivation method (sCIM) as a phenotypic method for rapid detection of carbapenemase-producing Enterobacterales: Study from a tertiary care hospital in North India

Evaluation of simplified carbapenem inactivation method (sCIM) as a phenotypic method for rapid detection of carbapenemase-producing Enterobacterales: Study from a tertiary care hospital in North India

Carbapenem resistant Enterobacteriaceae: Detection by modified carbapenem inactivation method (mCIM) and their association with clinical outcome at a teaching hospital in southern India.

Carbapenem resistant Enterobacteriaceae: Detection by modified carbapenem inactivation method (mCIM) and their association with clinical outcome at a teaching hospital in southern India.

A highly immunogenic UVC inactivated Sabin based polio vaccine

Despite their efficacy, the currently available polio vaccines, oral polio vaccine (OPV) and inactivated polio vaccine (IPV), possess inherent flaws posing significant challenges in the global eradication of polio. OPV, which uses live Sabin attenuated strains, carries the risk of reversion to pathogenic forms and causing vaccine-associated paralytic poliomyelitis (VAPP) and vaccine-derived polio disease (VDPD) in incompletely vaccinated or immune-compromised individuals. Conventional IPVs, which are non-replicative, are more expensive to manufacture and introduce biohazard and biosecurity risks due to the use of neuropathogenic strains in production. These types of limitations have led to a call by the Global Polio Eradication Initiative and others for the development of updated polio vaccines. We are developing a novel Ultraviolet-C radiation (UVC) inactivation method that preserves immunogenicity and is compatible with attenuated strains of polio. The method incorporates an antioxidant complex, manganese-decapeptide-phosphate (MDP), derived from the radioresistant bacterium Deinococcus radiodurans. The inclusion of MDP protects the immunogenic neutralizing epitopes from damage during UVC inactivation. The novel vaccine candidate, ultraIPVTM, produced using these methods demonstrates three crucial attributes: complete inactivation, which precludes the risk of vaccine-associated disease; use of non-pathogenic strains to reduce production risks; and significantly enhanced yield of doses per milligram of input virus, which could increase vaccine supply while reducing costs. Additionally, ultraIPVTM retains antigenicity post-freeze–thaw cycles, a testament to its robustness.

How Can Restoration Improve the Environmental Conditions of a Meromictic Urban Lake?

This study was conducted on Klasztorne Małe Lake located in the city of Kartuzy. For about 100 years, the lake served as a receiver of domestic, industrial, and rain sewage. The lake was completely degraded. In addition, excessive sewage inflow caused the accumulation of dense pollution in the area of the lake’s deepest part, which caused the formation of a monimolimnion layer. The bradymictic KML became a meromictic lake. After cutting off the inflow of pollutants, the lake was restored by the P inactivation method using coagulants PIX 111 and PAX 18. Before the restoration process of this lake, concentrations of nutrients in the bottom water layers achieved very high values of TN (total N) 65 mg/L and TP (total P) 30 mg/L. An average concentration of chlorophyll was 65 µg/L and visibility did not exceed 1 m. The method used brought very good results. In the entire volume of water, over 90% reduction in TP and a several-fold decrease in the content of TN were noted. The withdrawal of mineral P from the water limited the production processes in the lake, which was reflected in the reduction in chlorophyll amounts (19 µg/L) and the increase in water visibility (2 m). In addition, oxygen conditions in the lake improved. The application of coagulants caused a decrease in the density of the monimolimnion water, which was indicated by a significant decrease in water conductivity. However, the restoration method did not improve the water dynamics.

Detection of Carbapenemase-Producing Enterobacterales: A Comparative Analysis of Available Phenotypic Methods

Background: Carbapenems serve as a critical last-line defence against infections from multidrug-resistant Enterobacterales. The increasing prevalence of CRE (carbapenem-resistant Enterobacterales) poses significant challenges in clinical settings. This greatly complicates the handling of gram-negative bacilli (GNB) infections and represents a significant global health threat. Aim: This study evaluates and contrasts different phenotypic techniques used to identify carbapenemase-producing Enterobacterales isolated from various clinical samples. Objectives: To compare the accuracy and reliability of Combined Disc Test (CDT) and Modified Carbapenem Inactivation Method/EDTA - Carbapenem Inactivation Method (mCIM/eCIM) in identifying Metallo-beta-lactamase (MBL)-producing Enterobacterales. Methods: All clinical specimens submitted for culture and sensitivity testing at the Department of Microbiology were analysed to isolate and identify Enterobacterales. Carbapenemase production was assessed through using the phenotypic CDT Confirmation was carried out using the mCIM and eCIM in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: During a two-year period, 254 strains of Enterobacterales were isolated. Among those, 80/254 (31.49%) isolates exhibited resistance to the Imipenem (IMP) disc. The CDT identified 30/80 (37.5%) isolates as serine carbapenemase producers and 50/80 (62.5%) isolates as MBL producers. In comparison, the mCIM/eCIM methods detected 26/80 (32.5%) isolates as serine carbapenemase producers, 48/80 (60.0%) isolates as MBL producers, and 6/80 (7.5%) isolates were tested negative for carbapenemase production. Conclusion: This study evaluates the precision and effectiveness of the mCIM/eCIM and CDT methods for identifying CRE. The combination of the mCIM and eCIM tests may be a more reliable phenotypic method for detecting carbapenemase compared to the CDT.

Evaluation of biofilm formation and carbapenem resistance in Klebsiella pneumoniae isolated from clinical samples at a rural hospital in western Uttar Pradesh.

Klebsiella pneumoniae commonly causes healthcare-associated infections and shows multidrug resistance. K. pneumoniae can produce biofilm. Carbapenem resistance in K. pneumoniae is due to the production of carbapenemases mainly. This study was done to evaluate the formation of biofilm and carbapenemase resistance in K. pneumoniae isolates. A total of 110 K. pneumoniae isolated from various clinical samples were taken, the antibiotic susceptibility test was done by the Kirby disk diffusion method, and biofilm detection was done by the tissue culture plate method. All the carbapenem-resistant isolates were confirmed by multiplex real-time PCR (mPCR). Those found positive for any of the carbapenemase genes were tested by the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), and ethylenediamine tetraacetic acid (EDTA)-modified carbapenem inactivation method (eCIM). Out of 110 isolates, 66% (72/110) were carbapenem-resistant (suggestive of carbapenemase producers) by Kirby-Bauer disk diffusion but 58% (42/72) of Klebsiella isolates were confirmed for carbapenemase production by mPCR. Maximum number of carbapenemase gene were New Delhi metallo-β-lactamase (NDM) 52% (N = 22), 29% (N = 12) coproducers (NDM+OXA-48), and lowest in oxacillinase (OXA-48), 19% (N = 8). The overall sensitivity of MHT and mCIM+eCIM was 62% and 93%, and specificity was 88% and 97%, respectively. Our study showed that moderate biofilm producers were 51% (N = 56) K. pneumoniae isolates, strong biofilm producers 27% (N = 30), and 22% (N = 30) were weak/non-biofilm producers. We also found the correlation between biofilm formation and carbapenem-resistant K. pneumoniae (CR-KP) genes was statistically significant with a P value of 0.01*<0.05. Most isolates of K. pneumoniae demonstrated a wide range of antibiotic resistance and were biofilm producers. Our results indicated that the combination of mCIM with eCIM showed high sensitivity and specificity to detect CR-KP compared with MHT.

Investigation of Carbapenemase-Producing Pseudomonas aeruginosa at Secondary Care Hospital in Bolu, Turkey.

The global increase in carbapenem resistance poses a significant public health threat due to the potential emergence of multidrug-resistant pathogens and limited treatment options. To learn more about this issue and offer potential solutions, we conducted a study of carbapenem-resistant Pseudomonas aeruginosa (CRPA) infections in a secondary care hospital setting. The study utilized the carbapenem inactivation method (CIM), a leading phenotypic analysis, to determine carbapenemase activity in 63 CRPA isolates. Additionally, polymerase chain reaction (PCR) analysis was conducted to test for the presence of carbapenemase genes associated with the production or expression of various carbapenemase enzymes, including blaKPC, blaNDM, blaVIM, blaOXA-48, blaIMP, and blaGES. Arbitrary primed PCR (AP-PCR) was performed to assess the clonal relationship between different isolates. The isolates were also classified as either health care-associated infections or community-acquired infections, and their clonal relationship and gene positivity were evaluated. A total of 63 CRPA samples underwent evaluation, with 14 isolates determined to be carbapenemase producers via CIM tests. PCR assays revealed that 14 isolates carried carbapenemase genes, with 9 carrying blaNDM, 2 carrying blaGES, 2 carrying blaVIM, and 1 carrying blaIMP. CRPA exhibited a 22% prevalence of carbapenemase genes, of which 64% were attributed to the NDM gene responsible for multidrug resistance. AP-PCR revealed high clonal diversity among the isolates. Molecular epidemiological evaluation also showed no dominant outbreak strain among PA isolates. This study presents significant data on the prevalence and distribution of carbapenemase-producing CRPA strains isolated from secondary health care facilities. Typically, the literature focuses on resistance rates in tertiary care public hospitals. These findings may aid in understanding resistance and its mechanisms, as well as in developing effective treatment strategies and infection control measures.

Nosocomial cluster of patients infected with imipenemase-1-producing Enterobacter ludwigii.

Introduction. Imipenemase (IMI) enzymes are an uncommon class A carbapenemases that have been isolated from aquatic environments and, occasionally, from clinical isolates of Enterobacterales.Aim. We describe a cluster of three patients infected by IMI-1 carbapenemase-producing Enterobacter ludwigii (IMI-1-Elud) in a tertiary university hospital in Gran Canaria, Spain.Methodology. Antimicrobial susceptibility was determined using the Vitek2 AST-N355 card and antibiotic gradient strips. The modified carbapenem inactivation method (CIM) test was performed in cases where the ertapenem MIC value was higher than 0.125 mg l-1. The carbapenemase was identified by PCR and DNA microarray and later characterized by whole-genome next-generation sequencing (NGS) with Illumina.Results. Three patients presented thoracic or abdominal infections caused by IMI-1-Elud ST1677 from 14 June 2022 to 14 July 2022. All patients underwent at least one gastroscopy during their admission, and two of them were located in adjoining rooms. Isolates were resistant to carbapenems, colistin and fosfomycin but susceptible to ciprofloxacin. IMI/NMC-A carbapenemase was detected by PCR and hybridization test and confirmed by NGS as IMI-1. All patients underwent at least one gastroscopy, and two of them were in nearby rooms. Patients showed microbiological and clinical improvement following focus drainage and targeted antibiotic treatment with a fluoroquinolone.Conclusions. This study reports the first documented global outbreak of patients infected with IMI-1-Elud. The source appeared to be related to endoscopes. Contact transmission may also have played a role. A screening method such as the modified CIM test is crucial for detecting less common carbapenemases that might not be identified by rapid molecular or immunochromatographic tests, as these often do not include bla IMI genes, which could lead to the undetected dissemination of carbapenemase-producing Enterobacterales. Effective infection source control and targeted treatment are essential for achieving a favourable clinical outcome.

Evaluation of blaOXA-48-like point mutation carbapenemase-producing Enterobacterales in Prapokklao Hospital, Thailand.

In this study, we aimed to investigate genetic variation and CPE among blaOXA-48-like carrying isolates recovered from Prapokklao Hospital, Chanthaburi Province, Thailand, during 2016-2017. A total of 122 carbapenem-resistant Enterobacterales (CRE) were recovered from clinical samples in Prapokklao Hospital. All CRE samples were confirmed by standard biochemical tests and minimum inhibitory concentration (MIC) test strips (E-test). The carbapenemase production was determined using the modified Hodge test (MHT), the modified carbapenem inactivation method (mCIM), and EDTA-CIM (eCIM). Three single mutations (E168Q, S171A, and R214S) were characterized in this study. This mutation might reflect the hydrolysis of the modified β-lactam spectrum, especially carbapenem, by OXA-48-like. Our report provides evidence of the blaOXA-48-like point mutation and carbapenemase-producing phenotype of CRE detected in this healthcare setting. Effective control measures and active surveillance of drug resistance in nosocomial pathogens are crucial for controlling diseases associated with difficult-to-treat bacteria.

Blood-rsCDM: a new rapid and simplified carbapenemase detection method for detecting carbapenemases in Enterobacterales directly from positive blood cultures

ObjectiveWe aim to validate and evaluate a new rapid and simplified method, called Blood-rsCDM, for the detection and characterization of carbapenemase using 3-aminophenylboronic acid (APBA) and ethylenediaminetetraacetic acid (EDTA) β-lactamase inhibitors from positive blood cultures.MethodWe utilized a panel of 172 Enterobacterales strains, including blaKPC (77), blaNDM (48), blaIMP (9), blaVIM (2), blaOXA-181 (2), blaKPC and blaNDM (6), as well as 28 carbapenem-susceptible Enterobacterales isolates, to assess the performance of Blood-rsCDM and the EDTA-carbapenem inactivation method (eCIM). Carbapenemase class was determined using specific inhibitors at 4 h and 6 h by Blood-rsCDM.ResultsBlood-rsCDM exhibited a sensitivity of 97.9% at both time points, with a specificity of 100%, regardless of the culture duration. The sensitivity of eCIM was 94.4%, with a specificity of 100%. Blood-rsCDM accurately characterized KPC-producing isolates as 77/77, metallo-β-lactamases (MBLs) as 58/59, and KPC and NDM carbapenemases as 6/6 at 4 h. There was no difference in results between the 4 h and 6 h time points. However, Blood-rsCDM could not differentiate OXA-181-producing strains. For eCIM, the characterization numbers for KPC-, OXA-181-, and MBLs-producing strains were 77/77, 2/2, and 57/59, respectively, but it failed to detect the coproduction of KPC and NDM isolates.ConclusionBlood-rsCDM accurately discriminates carbapenemase within 4 h and is capable of directly differentiating multi-enzyme (KPC and NDM) presence from positive blood culture broths. Therefore, Blood-rsCDM represents a rapid, simple, easy-to-read, and accurate tool that can be utilized in resource-limited settings.

Combination of a novel bacteriophage and d-serine effectively controls Vibrio parahaemolyticus growth in seafood

Seafood is an important high-protein and low-fat food source prone to spoilage and may harbor food poisoning bacteria. Vibrio parahaemolyticus is a gram-negative halophilic bacterium that causes food poisoning. It is heat labile and can be eliminated through heating; however, V. parahaemolyticus may be present in seafood for raw consumption, such as sashimi. Recently, the use of bacteriophages or phages has been proposed as a non-thermal inactivation method for microbial control. Here, we characterized VF16, a novel phage that infects V. parahaemolyticus, and evaluated the effectiveness of the combination of VF16 with the antimicrobial amino acid d‑serine in controlling V. parahaemolyticus through antimicrobial testing in broth cultures and raw scallops. The morphology of VF16 was examined using transmission electron microscopy, which demonstrated that it had a long, flexible tail characteristic of a siphovirus. Genome sequencing revealed that VF16 is a novel phage belonging to the family Demerecviridae, subfamily Ermolyevavirinae, and genus Vipunavirus. Analysis of adsorption and one-step growth curve in a liquid medium indicated that Ca2+ and Mg2+ are necessary for one or more subsequent replication processes of VF16 but are not essential for its adsorption. The combination of VF16 and d‑serine completely inhibited the regrowth of V. parahaemolyticus in the liquid medium and effectively suppressed its growth in the scallop samples. No VF16-resistant mutants were isolated from the scallop samples treated with VF16 alone or in combination with d‑serine. The combination of phages and d-amino acids is an effective non-thermal inactivation method for controlling foodborne pathogens.

Colistin Insusceptibility in Carbapenem-Resistant Enterobacteriaceae Isolates From a Tertiary Referral Centre.

Background Antimicrobial resistance has developed significant importance as a worldwide health concern more so in the 21st century. This is more specifically observed among the Enterobacteriaceae family, the major group of Gram-negative bacteria. Resistance to carbapenems and colistin antimicrobials from the reserve group, in critical infection treatment poses a substantial therapeutic challenge. This study aims to detect resistance to both carbapenems and colistin in Enterobacteriaceae isolates, emphasizing the importance of monitoring. Materials and methods A laboratory-based study investigated 82 Carbapenem-Resistant Enterobacteriaceae (CRE) isolates from hospitalized patients at Krishna Hospital & Medical Research Centre, Karad, India, for two years (November 2021-November 2023). The Kirby-Bauer disc diffusion method was used to determine the antimicrobial susceptibility following Clinical Laboratory Standards Institute 2022 recommendations. To validate Carbapenem-Resistant Enterobacteriaceae isolates, the Modified Carbapenem Inactivation Method (mCIM) was used, and Colistin resistance was ascertained using Broth Microdilution Susceptibility Testing (BMD). Results Among 309 Enterobacteriaceae isolates, 82 (26.5%) exhibited carbapenem resistance, with 75 confirmed by Modified Carbapenem Inactivation Method (mCIM) testing. For patients in the age group 51-60 years (20%), with men being the most prevalent, the predominance of CRE isolates was from the Intensive Care Unit (44%), mostly from urine samples (34.6%). The most dominant CRE was Klebsiella pneumoniae (62.7%), followed by Escherichia coli (32%). Colistin resistance was found in 14 (18.7%) of CRE isolates, with the highest minimum inhibitory concentration (MIC) at 4 µg/mL (n=10) and 16 µg/mL (n=4). Colistin-resistant isolates (n=10) were sensitive to amikacin (71.4%) and resistant to many antibiotics. Conclusion The extreme rise of colistin-resistant cases among CRE in healthcare settings is a solemn alarm. Among the most effective treatments, aminoglycosides amikacin and netilmicin show the greatest sensitivity against these colistin-resistant CRE infections. The findings stress the difficulties in treating such infections and the dire need for solid antimicrobial stewardship and novel therapeutic strategies.

Prevalence of Carbapenemase Production Among Klebsiella and Escherichia coli Isolated From Urinary Tract Infections.

Background and aim Urinary tract infections represent a substantial portion of healthcare-associated infections due to E. coli and Klebsiella. Carbapenems are broad-spectrum antibiotics considered highly effective in treating infections caused by multidrug-resistant bacteria. Carbapenem-resistantEnterobacteriaceae (CRE), including carbapenem-producing E. coli and Klebsiella isolates, have become a major concern as they limit treatment options. The study aims to determine the prevalence of carbapenemase-producing E. coli and Klebsiella while also comparing the effectiveness of two detection methods, namely the modified carbapenem inactivation method (mCIM) and modified Hodge test (MHT). Materials and methods A cross-sectional study was conducted from July 2022 to June 2023 in a tertiary care hospital, in Karad, Satara, India. Three hundred urinary isolates of E. coli (150) and Klebsiella (150) were studied. These isolates were tested for antimicrobial susceptibility testing. Two phenotypic methods, the modified carbapenem inactivation method (mCIM) and the modified Hodge test (MHT), were used to study carbapenemase production. Results Out of three hundred isolates, carbapenemase production was detected in 72 isolates (24%) by the modified Hodge test (MHT) and in 111 isolates (37%) by the modified carbapenem inactivation method (mCIM). Among the MHT-positive isolates, 46 (63.8%) were identified as Klebsiella and 26 (36.1%) as E. coli. In contrast, of the mCIM-positive isolates, 68 (61.2%) were Klebsiella, and 43 (38.7%) were E. coli. A total of 41 Klebsiella (27.33%) and 25 E. coli (16.66%) isolates tested positive by both methods, highlighting variability in detection rates between the two methods. Conclusion This study observed MHT and mCIM to be accurate for the detection of carbapenemase among carbapenem-resistant isolates. However, the mCIM demonstrated greater sensitivity compared to the MHT.

PMCA to demonstrate the efficacy of prion inactivation methods on reusable medical devices: a relevant alternative to animal bioassays

Validation of prion inactivation processes for medical devices relies on in-vivo experimental protocols. However, bioassays are costly, long (1-2 years) and ethically disputable. Additionally, results obtained with one prion strain - for example, 263K (hamster-adapted strain originating from sheep scrapie) - cannot be easily extrapolated to relevant human prion strains, further questioning the utility of bioassays. Over the past two decades, cell-free prion amplification assays have emerged as potential alternatives to bioassays. Rather than measuring prion infectivity, they quantify prion seeding activity (i.e. the capacity to convert the normal prion protein into the disease-associated isoform). The results obtained from an optimized cell-free assay termed 'miniaturized-bead protein misfolding cyclic amplification' (mb-PMCA) with four processes using three different prion strains - 263K and two human prions derived from variant and sporadic Creutzfeldt-Jakob disease - were compared with published bioassays using the same three strains and processes, when available. Tests performed on reference processes (steam, sodium hydroxide, sodium hypochlorite) and low temperature H2O2 sterilization (STERRAD NXTM Advanced cycle) showed perfect alignment between mb-PMCA and available bioassays. STERRAD NXTM Advanced cycle was efficacious against all three prion strains. These data confirm that PMCA, particularly mb-PMCA, is a relevant alternative to animal bioassays for the assessment of prion inactivation processes, and highlight the interest of some low temperature H2O2 sterilization cycles.

Survival of infection with TEM β-lactamase-producing Escherichia coli with Pan-β-lactam resistance

Antimicrobial resistance is a critical global health issue, significantly contributing to patient mortality. Recent antibiotic developments have aimed to counteract carbapenemase-producing Enterobacterales; however, the impact of their use on the emergence of antibiotic resistance is unknown. This study investigates the first case of a non-carbapenemase-producing, pan-β-lactam-resistant Escherichia coli strain from a patient previously treated with ceftolozane-tazobactam and cefiderocol. This study describes the clinical progression of a 39-year-old ICU patient who developed multiple infections, culminating in the isolation of a pan-β-lactam-resistant E. coli strain (EC554). The resistance profile was characterised through MIC determination, whole-genome sequencing, the use of the β-lactam inactivation method, RT-qPCR, efflux pump inhibition assays, outer membrane protein analysis, and blaTEM transformation. The EC554 isolate displayed resistance to all tested β-lactams and β-lactam-β-lactamase inhibitor combinations. Whole-genome sequencing revealed four plasmids in EC554, with the only β-lactamase gene being blaTEM-252 on the pEC554-PBR-X1-X1 plasmid. We found that the extremely resistant phenotype was attributable to a combination of different mechanisms: a high expression of TEM-252, efflux pump activity, porin loss, and PBP3 mutations. The findings illustrate the complex interplay of multiple resistance mechanisms in E. coli, highlighting the potential for high-level resistance even without carbapenemase production. This study underscores the importance of comprehensively characterising resistance mechanisms in order to inform effective treatment strategies and mitigate the spread of resistant strains.

3D-printed devices for optimized generation of cold atmospheric plasma to improve decontamination of surfaces from respiratory pathogens

Three-dimensional (3D)-printing technology is instrumental in creating devices for biological applications, including the exploitation of cold atmospheric plasma (CAP). CAP, a partially ionized gas that functions at ambient temperatures, serves as a safe, inexpensive, and effective tool for the inactivation of various pathogens on different surfaces. In this study, we compared three different 3D-printed devices with respect to their ability to provide optimized CAP compositions effective against select respiratory viruses (SARS-CoV-2, influenza virus, adenovirus, and rhinovirus) and the bacterium Pseudomonas aeruginosa, which is associated with serious lung diseases. The transmission of respiratory pathogens via surface contamination may pose a serious health threat, thus highlighting the biological importance of the current study. The properties of a prototype 3D-printed CAP-generating device and two optimized versions were characterized by detecting reactive oxygen and nitrogen species (RONS) in a gaseous environment via infrared spectroscopy and analyzing the composition of the reactive compounds. The virucidal effects of CAP were examined by determining virus infectivity and particle integrity. The bactericidal effect was documented by viability testing and visualization via transmission electron microscopy. The findings indicate that optimization of the 3D-printed devices for CAP production yielded an environment with relatively high amounts of RONS (O3, N2O, NO2, and H2O2), reducing the exposure time required for inactivation of respiratory pathogens by approximately 50%. In addition to reducing infectivity and viability, CAP treatment led to the destruction of viral nucleic acids and physical damage to bacterial cells. Owing to its flexibility and easy implementation, optimized CAP generated by 3D-printed devices provides an attractive inactivation method adaptable for different biological applications, including surface decontamination from viral and bacterial pathogens. &amp;nbsp;&amp;nbsp;

Molecular characterization of superbugs K. pneumoniae harboring extended-spectrum β-lactamase (ESBL) and carbapenemase resistance genes among hospitalized patients in southwestern Iran, Western Asia

BackgroundDetection of K. pneumoniae superbugs carrying Extended-spectrum β-lactamase (ESBL) and Carbapenemase resistance genes among hospitalized patients is crucial for infection control and prevention. The aim of this molecular study was to investigate the spread of ESBL and Carbapenemase-producing K. pneumoniae in two hospitals located in Southwest Iran. MethodsOne hundred clinical isolates of K. pneumoniae were randomly collected from two hospitals over a period of five months, from November 2023. The isolates were confirmed using biochemical and genotypic tests. According to the CLSI 2022 guidelines, K. pneumoniae isolates that exhibited resistance to at least one of the three indicator cephalosporins or carbapenems were selected for evaluation of ESBL and carbapenemase production. This was done using a combination disk confirmatory test and the modified carbapenem inactivation method (mCIM). Finally, the presence of ESBLs and carbapenemase resistance encoding genes was assessed using PCR and specific primers. ResultsOut of the 100 isolates, the percentage of antibiotic resistance was cefoxitin (29 %), cefixime (28 %), ceftazidime (26 %), cefotaxime (24 %), cefepime (22 %), ceftriaxone (21 %), imipenem (20 %), and meropenem (17 %). Additionally, thirty isolated strains were found to be multidrug-resistant. Out of these, twenty-seven strains demonstrated a potential for ESBLs, twenty strains for Carbapenemase, and seventeen strains for both ESBLs and Carbapenemase production. Moreover, the occurrence of ESBLs and carbapenemase genes was as follows: blaSHV (25 %), blaTEM (23 %), blaCTX-M (20 %), blaOXA-48 (17 %), and blaVIM (13 %). It is important to mention that we did not detect the blaIMP and blaKPC. resistant genes among clinical isolates. ConclusionBased on the results, the existence of this type of resistance in hospital centers needs to be reevaluated in terms of empirical antibiotic prescribing. Additionally, it is recommended that infection control measures should be taken for public health. Also, it's suggested that hospital-acquired infections caused by superbug K. pneumoniae resistant strains should be addressed.