Prevalence and risk factors for extended-spectrum ß-lactamase producing antimicrobial-resistant E. coli in urinary tract infections among inpatients in the tertiary hospitals in Zanzibar (Tanzania): a prospective cross-sectional study.

Introduction: Extended-spectrum ß-lactamase (ESBL) production among Enterobacteriaceae, such as E. coli, has been increasing worldwide, which causes treatment failure for urinary tract infections. Therefore, this study aimed to determine the prevalence and risk factors for the production of ESBL in E. coli from patients with urinary tract infections (UTI) in Zanzibar. Methods: a prospective cross-sectional study was conducted from January 2018 to December 2021 in Zanzibar. Data were retrieved from a routine bacteriological laboratory culture report from urine samples of 4306 patients at the Lancet Laboratory. In addition, the patient's social demographics and clinical data were retrieved by examining the medical records in the respective hospitals. All inpatients older than fifteen years diagnosed with urinary tract infections (UTI) and requested urine culture and sensitivity were included. The Chi-square and Fischer's exact tests were used to compare antibiotic resistance. In addition, a binary logistic regression analysis was used to predict ESBL production risk factors. Results: the prevalence of E. coli-producing ESBL was 13.4% (578/4030). Infection of ESBL. E. coli was prevalent in females 52.6% (n=304) compared to male patients, 47.4% (n=274), and the majority 38.8% (n=224), were people of young age, between 16-30 years. The average age of patients was 31.5±10.2 years, with minimum age of 16 years and a maximum age of 72 years. In multivariate analysis, results shown that previously hospitalised patients aOR: 6.35, 95% Cl 3.37-11.92; p=0.001, long hospital stays aOR: 10.34, 95% Cl 3.03-22.29; p <0.001, prior use of penicillin aOR: 7.78, 95% Cl 2.99-29.11; p < 0.001, and prior use of cephalosporin drugs aOR: 4.64, 95% Cl 2.99-9.96; p=0.001, were strongly associated with the emergence of ESBL-producing E. coli in urinary tract infection patients. ESBL E. coli showed high resistance to amoxicillin 99.5% (n=575), ampicillin 97.8.% (n=570), cotrimazaxole 86.2% (n=344), ceftriaxone 73.7% (n=344), ciprofloxacin 73.2% (n=423), and ceftaxime 59.5% (n=426). There was a less resistance to ampicillin -cloxacillin 44.3% (n=256), gentamicin 22.5% (n=22.5), and norfloxacin 18.9% (n=109) respectively. Isolates were shown to be more susceptible to meropenem at 1.6% (n=9). Conclusion: the overall prevalence of ESBL-producing E. coli is 13.4%. The risk of emergence ESBL was higher in patients with previous history of hospitalisation, long hospital stay, prior use of penicillin and cephalosporin drugs. High level of antimicrobial resistance observed against most commonly used antibiotics in treatment of urinary tract infections. The clinicians should rely on microbiological diagnosis in treatment of UTIs to reduce risk of treatment failure. Further study should be carried out to assess the prevalence and resistance pattern of other uropathogens and other risk factors.

Extended-Spectrum ß-Lactamase and carbapenemase-producing Escherichia coli O157:H7 among diarrheic patients in Shashemene, Ethiopia.

BACKGROUND: The worldwide increase in multidrug resistance is a major threat to public health. One particular concern is the presence of Escherichia coli strains that carry Extended-Spectrum ß-Lactamase (ESBL) and Carbapenemase enzymes, which can make multiple antibiotics ineffective. This complicates treatment strategies and raises the risk of illness and death. The aim of this study was to isolate E. coli O157:H7, assess its susceptibility against antimicrobial agents, and determine the presence of ESBL and Carbapenemase production in stool samples collected from diarrheic patients in Shashemene, west Arsi, Ethiopia from July to November 2022. METHODS: The samples were cultured McConkey Agar and E. coli were isolated and identified by standard biochemical tests using API 20E. E. coli O157:H7 was further identified using sorbitol McConkey Agar and antisera for O157 antigen test. The antimicrobial susceptibility test was performed using the Kirby-Bauer disc diffusion method using different antibiotics. Each identified isolate was screened and tested for phenotypical ESBL and Carbapenemase production using combined disc method and modified carbapenem inactivation method, respectively. Bivariant and multivariant analyses were employed using a logistic regression model for further analysis and were interpreted based on the odds ratio and level of statistical significance at a p-value <0.05 with 95% confidence interval. RESULTS: E. coli O157:H7 strain was found from 9% (38/423) study participants. The majority of the participants [61.9% (262/423)] were males; and 19.1% (81/ 423) of the participants were under five children. Living in urban areas, having domestic animals, and ≥5 family size in the household were identified as statistically significant factors associated with E. coli O157:H7. Twenty-seven (71.1%) and 12 (31.6%) of the 38 E. coli O157:H7 isolates were phenotypically confirmed to be ESBL and carbapenemase producers, respectively. All isolates were resistant against Ampicillin, but sensitive to ciprofloxacin. High resistance to Ampicillin and Amoxicillin/Clavulanic acid was observed among the ESBL and carbapenemase producing isolates also. The extent of detection of multidrug resistant E. coli O157:H7 isolates against three or more classes of antimicrobial agents tested was alarmingly very high (84%). CONCLUSION: The E. coli O157:H7 isolates in this study showed a significant resistance to certain antimicrobials that were tested. The level of ESBL and Carbapenemase production among these isolates was found to be quite high. We observed a high resistance to Ampicillin and Amoxicillin/Clavulanic acid among the ESBL and carbapenemase producing isolates. Ciprofloxacin was found to be the most effective drug against both the ESBL producers and nonproducers.

Modification of carbapenemase inhibition test and comparison of its performance with NG-Test CARBA 5 for detection of carbapenemase-producing Enterobacterales.

AIMS: Adequately and accurately identifying carbapenemase-producing Enterobacterales (CPE) is vital for selecting appropriate antimicrobial therapy and implementing effective infection control measures. This study aims to optimize the phenotypic detection method of carbapenemase for routine diagnostics in clinical microbiology laboratories. METHODS AND RESULTS: Carbapenemase genes in 2665 non-duplicate CRE clinical strains collected from various regions of China were confirmed through whole-genome sequencing (WGS). The carbapenemase inhibition test (CIT) was conducted and interpreted using different methods and breakpoints, then compared with the NG-Test CARBA 5 for carbapenemase detection. The diagnostic performance of the CIT method was optimal when the carbapenemase types were determined by comparing the inhibition zone diameters of the imipenem disc with 3-aminophenylboronic acid (APB) plus ethylenediaminetetraacetic acid (EDTA) to those of the imipenem disc with either APB or EDTA alone, with a breakpoint of 4 mm. The overall sensitivities of the current CIT, the modified CIT, and NG-Test CARBA 5 were 91.4%, 94.9%, and 99.9%, respectively. For detecting isolates co-producing Klebsiella pneumoniae carbapenemase (KPC) and metallo-ß-lactamases (MBLs), the modified CIT method had higher sensitivity than the current method (70.0% vs. 53.3%), though this difference was not statistically significant (P = 0.063). The NG-Test CARBA 5 showed excellent performance for multi-carbapenemases diagnosis, with sensitivity and specificity of 97.1% and 100%, respectively. CONCLUSIONS: Optimizing and standardizing the CIT method for clinical use is necessary. It has certain advantages in diagnosing multi-carbapenemase and rare carbapenemase production. However, for identifying common carbapenemase types, the NG-Test CARBA 5 demonstrated superior performance.

Characterization of GQA as a novel ß-lactamase inhibitor of CTX-M-15 and KPC-2 enzymes.

ß-lactam resistance is a significant global public health issue. Outbreaks of bacteria resistant to extended-spectrum ß-lactams and carbapenems are serious health concerns that not only complicate medical care but also impact patient outcomes. The primary objective of this work was to express and purify two soluble recombinant representative serine ß­lactamases using Escherichia coli strain as an expression host and pET101/D as a cloning vector. Furthermore, a second objective was to evaluate the potential, innovative, and safe use of galloylquinic acid (GQA) from Copaifera lucens as a potential ß-lactamase inhibitor.In the present study, blaCTX-M-15 and blaKPC-2 represented genes encoding for serine ß-lactamases that were cloned from parent isolates of E. coli and K. pneumoniae, respectively, and expression as well as purification were performed. Moreover, susceptibility results demonstrated that recombinant cells became resistant to all test carbapenems (MICs; 64-128 µg/mL) and cephalosporins (MICs; 128-512 µg/mL). The MICs of the tested ß-lactam antibiotics were determined in combination with 4 µg/mL of GQA, clavulanic acid, or tazobactam against E. coli strains expressing CTX-M-15 or KPC-2-ß-lactamases. Interestingly, the combination with GQA resulted in an important reduction in the MIC values by 64-512-fold to the susceptible range with comparable results for other reference inhibitors. Additionally, the half-maximal inhibitory concentration of GQA was determined using nitrocefin as a ß-lactamase substrate. Data showed that the test agent was similar to tazobactam as an efficient inhibitors of the test enzymes, recording smaller IC50 values (CTX-M-15; 17.51 for tazobactam, 28.16 µg/mL for GQA however, KPC-2; 20.91 for tazobactam, 24.76 µg/mL for GQA) compared to clavulanic acid. Our work introduces GQA as a novel non-ß-lactam inhibitor, which interacts with the crucial residues involved in ß-lactam recognition and hydrolysis by non-covalent interactions, complementing the enzyme's active site. GQA markedly enhanced the potency of ß-lactams against carbapenemase and extended-spectrum ß-lactamase-producing strains, reducing the MICs of ß-lactams to the susceptible range. The ß-lactamase inhibitory activity of GQA makes it a promising lead molecule for the development of more potent ß-lactamase inhibitors.

Pulmonary abscess secondary to epididymitis caused by extended spectrum ß-lactamase-producing hypervirulent Klebsiella pneumoniae: a case report.

BACKGROUND: Pulmonary abscesses resulting from epididymitis caused by extended spectrum ß-lactamase-producing hypervirulent Klebsiella pneumoniae (ESBL-hvKp) in a nondiabetic patient are extremely uncommon. The infection caused by this disseminated drug-resistant bacteria, which is generally considered an intractable case, poses a potential challenge in clinical practice. CASE PRESENTATION: In this case report, we present the clinical course of a 71-year-old male patient with epididymitis, who subsequently developed cough and dyspnea following anti-infection treatment. Imaging examinations revealed severe pneumonia and pulmonary abscess. The infection of ESBL-hvKp in the epididymis led to bacteremia and subsequent lung lesions. Due to poor response to anti-infection therapy, the patient required an extended duration of anti-infection treatment and ultimately chosed to discontinue treatment. CONCLUSIONS: Acute epididymitis caused by ESBL-hvKP infection can result in the spread of the infection through the bloodstream, leading to severe pneumonia and lung abscess. Given the critical condition of the patient, even with active anti-infection treatment, there is a risk of treatment failure or potentially fatal outcomes.

Molecular characterization of NDM and OXA-48-like-producing Klebsiella pneumoniae ST16 and hypervirulent ST337 clone among two patients; a case report.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a major public health problem, requiring the use of last-resort antibiotics such as colistin. However, there is concern regarding the emergence of isolates resistant to this agent. The report describes two patients with urinary tract infection (UTI) and ventilator-associated pneumonia (VAP) infection caused by CRKP strains. The first case was a 23-year-old male with UTI caused by a strain of ST16 co-harboring blaCTX-M, blaTEM, blaSHV, blaNDM, blaOXA-48-like genes. The second case was a 39-year-old woman with VAP due to hypervirulent ST337-K2 co-harboring blaSHV, blaNDM, blaOXA-48-like, iucA, rmpA2 and rmpA. The patients' general condition improved after combination therapy with colistin (plus meropenem and rifampin, respectively) and both of them recovered and were discharged from the hospital. This study highlights the necessary prevention and control steps to prevent the further spread of CRKP strains should be a priority in our hospital.

Antimicrobial Susceptibility and Genetic Epidemiology of Extended-Spectrum ß-Lactamase-Positive Enterobacterales Clinical Isolates in Central Poland.

The extended-spectrum ß-lactamases (ESßLs) are bacterial enzymes capable of hydrolyzing penicillins, cephalosporins, and aztreonam. The prevalence of ESßL is increasing among clinically significant microorganisms worldwide, drastically reducing the therapeutic management of infectious diseases. The study aimed to determine the drug susceptibility of ESßL-positive clinical isolates acquired from patients hospitalized in Lodz, central Poland, and analyze the prevalence of specific genes, determining acquired resistance in these bacteria. The samples of ESßL-positive clinical isolates were gathered in 2022 from medical microbiological laboratories in the city of Lodz, central Poland. The strains were subjected to biochemical identification and antimicrobial susceptibility testing following EUCAST guidelines. The presence of studied genes (blaCTX-M, blaSHV, blaTEM, blaPER, blaVEB) was confirmed by PCR. Over 50% of studied isolates were resistant to gentamicin, cefepime, ceftazidime and ciprofloxacin. The most common ESßL gene was blaCTX-M. In most isolates, the resistance genes occurred simultaneously. The blaPER was not detected in any of the tested strains. ESßL-producing strains are largely susceptible to the currently available antibiotics. The observation of the coexistence of different genes in most clinical isolates is alarming.

Tracing the origin of NDM-1-producing and extensively drug-resistant Pseudomonas aeruginosa ST357 in the Netherlands.

BACKGROUND: In the hospital environment, carbapenemase-producing Pseudomonas aeruginosa (CPPA) may lead to fatal patient infections. However, the transmission routes of CPPA often remain unknown. Therefore, this case study aimed to trace the origin of CPPA ST357, which caused a hospital-acquired pneumonia in a repatriated critically ill patient suffering from Guillain-Barré Syndrome in 2023. METHODS: Antimicrobial susceptibility of the CPPA isolate for 30 single and combination therapies was determined by disk-diffusion, Etest or broth microdilution. Whole-genome sequencing was performed for three case CPPA isolates (one patient and two sinks) and four distinct CPPA ST357 patient isolates received in the Dutch CPPA surveillance program. Furthermore, 193 international P. aeruginosa ST357 assemblies were collected via three genome repositories and analyzed using whole-genome multi-locus sequence typing in combination with antimicrobial resistance gene (ARG) characterization. RESULTS: A Dutch patient who carried NDM-1-producing CPPA was transferred from Kenya to the Netherlands, with subsequent dissemination of CPPA isolates to the local sinks within a month after admission. The CPPA case isolates presented an extensively drug-resistant phenotype, with susceptibility only for colistin and cefiderocol-fosfomycin. Phylogenetic analysis showed considerable variation in allelic distances (mean = 150, max = 527 alleles) among the ST357 isolates from Asia (n = 92), Europe (n = 58), Africa (n = 21), America (n = 16), Oceania (n = 2) and unregistered regions (n = 4). However, the case isolates (n = 3) and additional Dutch patient surveillance program isolates (n = 2) were located in a sub-clade of isolates from Kenya (n = 17; varying 15-49 alleles), the United States (n = 7; 21-115 alleles) and other countries (n = 6; 14-121 alleles). This was consistent with previous hospitalization in Kenya of 2/3 Dutch patients. Additionally, over half of the isolates (20/35) in this sub-clade presented an identical resistome with 9/17 Kenyan, 5/5 Dutch, 4/7 United States and 2/6 other countries, which were characterized by the blaNDM-1, aph(3')-VI, ARR-3 and cmlA1 ARGs. CONCLUSION: This study presents an extensively-drug resistant subclone of NDM-producing P. aeruginosa ST357 with a unique resistome which was introduced to the Netherlands via repatriation of critically ill patients from Kenya. Therefore, the monitoring of repatriated patients for CPPA in conjunction with vigilance for the risk of environmental contamination is advisable to detect and prevent further dissemination.

Extended spectrum and metalo beta lactamase producing gram negative bacterial pathogens from cockroaches collected at hospital, Southern Ethiopia.

BACKGROUND: Cockroaches can pose a significant health risk in hospital environments because they may serve as reservoirs and vectors for nosocomial pathogens. Cockroaches harbor epidemiologically significant extended spectrum and metalo beta lactamase producing Gram negative bacterial pathogens, which complicate nosocomial infections. OBJECTIVES: The main aim of this study is to determine aetiology and phenotypic extended spectrum and metalo beta lactamase producing Gram negative bacteria pathogens from cockroaches collected in hospitals. METHODS: A cross-sectional study was employed from February to May 2022 to determine the antibiotic resistance producing bacterial isolates from cockroaches by giving special emphasis to metalo beta lactamase and extended spectrum beta lactamase production from different wards of WSUCSH. Cockroaches were collected with hands wearing sterile gloves. External homogenate was prepared and incubated microbiologically by using different culture media and differentiated biochemically. Antimicrobial susceptibility testing was performed by disk diffusion method. ESBL production was conducted using double disc synergy method and double disk method was used to detect MBL enzyme detection. Descriptive statistics was used to determine prevalence and percentage. RESULT: Out of 245 cockroaches, 108 Gram negative bacteria were isolated. K. pneumoniae 29(26.9%) was the most predominant bacteria and Enetrobacter spp. 8(7.4%), was the least. All, K. pneumoniae, P. mirabilis, and Enterobacter isolates were pan-resistant to Ampicillin. P.aeruginosa and P.mirabilis antibiotics showed ≥ 80% resistant for amoxicillin/clavulanic acid antibiotics. Cefotaxime, ceftazidime, ceftriaxone and imipenem showed relative efficacy compared with other antibiotics. Out of 78 amoxicillin-clavulanic acid resistant isolates, 42(34.7%) were ESBL producers. ESBL production is more depicted by P. aeruginosa, A. baumannii, K. pneumoniae and E. coli. The overall prevalence of MBL production is 29(23.1%). K. pneumoniae P. aeruginosa, E.coli, A. baumannii, Enterobacter spp and K.oxytoca revealed MBL production. CONCLUSION: The overall prevalence of ESBL and MBL producing nosocomial agents from hospital cockroaches was 34.7% and 23.1% respectively. P.aeruginosa, A.baumannii, K.pneumoniae and E.coli showed pronounced ESBL production. All bacterial isolates except P. mirabilis and C. freundii showed MBL production. The needed to evaluate our antibiotic stewardship program and antibiotic resistance detection for treatment is mandatory. The impact of cockroach as a source of AMR should be sought.

Solid waste dumpsite leachate and contiguous surface water contain multidrug-resistant ESBL-producing Escherichia coli carrying Extended Spectrum ß-Lactamase (ESBL) genes.

Dumpsites generate leachates containing bacteria that may carry antibiotic resistance genes, such as extended spectrum ß-lactamase (ESBL). However, the contribution of dumpsite leachates in the environmental spread of ESBL genes has not been investigated in greater detail. This study aimed to quantify the impact of Ajakanga dumpsite leachate on the spread of ESBL genes through surface water. The susceptibility of Escherichia coli isolated from dumpsite leachate and the accompanying surface water to selected antibiotics was assessed by the standardized disc diffusion method. The isolates were evaluated for phenotypic ESBL production using the double disc synergy test (DDST). The detection of ESBL genes in the isolates was carried out using a primer-specific polymerase chain reaction (PCR). Escherichia coli isolates from leachate (n = 26/32) and surface water (n = 9/12) expressed ESBL phenotype. The ESBL-producing isolates showed the highest level of resistance to the 3rd generation cephalosporin antibiotics: cefotaxime (100%), cefpodoxime (97%), ceftazidime (97%), with low resistance observed to imipenem (6%) and azithromycin (3%). All the isolates were multidrug-resistant, showing resistance to three or more classes of antibiotics. All the ESBL-producing E. coli obtained carried blaCTX-M, 21/35 (60%) carried blaTEM while none of the isolates bore blaSHV. This study found that ESBL-producing Escherichia coli from dumpsite leachate and nearby surface water had identical resistance signatures indicating the relatedness of the isolates, and that dumpsite leachate could contribute to the transfer of ESBL-producing bacteria and their genes to receiving surface water. This study has necessitated the need for a review of the guidelines and operational procedures of dumpsites to forestall a potential public health challenge.

Proteomic analysis of carbapenem-resistant Klebsiella pneumoniae outer membrane vesicles under the action of phages combined with tigecycline.

BACKGROUND: Klebsiella pneumoniae is the most commonly encountered pathogen in clinical practice. Widespread use of broad-spectrum antibiotics has led to the current global dissemination of carbapenem-resistant K. pneumoniae, which poses a significant threat to antibacterial treatment efficacy and public health. Outer membrane vesicles (OMVs) have been identified as carriers capable of facilitating the transfer of virulence and resistance genes. However, the role of OMVs in carbapenem-resistant K. pneumoniae under external pressures such as antibiotic and phage treatments remains unclear. METHODS: To isolate and purify OMVs under the pressure of phages and tigecycline, we subjected K. pneumoniae 0692 harboring plasmid-mediated blaNDM-1 and blaKPC-2 genes to density gradient separation. The double-layer plate method was used to isolate MJ1, which efficiently lysed K. pneumoniae 0692 cells. Transmission electron microscopy (TEM) was used to characterize the isolated phages and extract OMV groups for relevant morphological identification. Determination of protein content of each OMV group was conducted through bicinchoninic acid assay (BCA) and proteomic analysis. RESULTS: K. pneumoniae 0692 released OMVs in response to different environmental stimuli, which were characterized through TEM as having the typical structure and particle size of OMVs. Phage or tigecycline treatment alone resulted in a slight increase in the mean protein concentration of OMVs secreted by K. pneumoniae 0692 compared to that in the untreated group. However, when phage treatment was combined with tigecycline, there was a significant reduction in the average protein concentration of OMVs compared to tigecycline treatment alone. Proteomics showed that OMVs encapsulated numerous functional proteins and that under different external stresses of phages and tigecycline, the proteins carried by K. pneumoniae 0692-derived OMVs were significantly upregulated or downregulated compared with those in the untreated group. CONCLUSIONS: This study confirmed the ability of OMVs to carry abundant proteins and highlighted the important role of OMV-associated proteins in bacterial responses to phages and tigecycline, representing an important advancement in microbial resistance research.

Antibiofilm activity of promethazine against ESBL-producing strains of Escherichia coli in urinary catheters.

The bacterium Escherichia coli is one of the main causes of urinary tract infections. The formation of bacterial biofilms, especially associated with the use of urinary catheters, contributes to the establishment of recurrent infections and the development of resistance to treatment. Strains of E. coli that produce extended-spectrum beta-lactamases (ESBL) have a greater ability to form biofilms. In addition, there is a lack of drugs available in the market with antibiofilm activity. Promethazine (PMZ) is an antihistamine known to have antimicrobial activity against different pathogens, including in the form of biofilms, but there are still few studies of its activity against ESBL E. coli biofilms. The aim of this study was to evaluate the antimicrobial activity of PMZ against ESBL E. coli biofilms, as well as to assess the application of this drug as a biofilm prevention agent in urinary catheters. To this end, the minimum inhibitory concentration and minimum bactericidal concentration of PMZ in ESBL E. coli strains were determined using the broth microdilution assay and tolerance level measurement. The activity of PMZ against the cell viability of the in vitro biofilm formation of ESBL E. coli was analyzed by the MTT colorimetric assay and its ability to prevent biofilm formation when impregnated in a urinary catheter was investigated by counting colony-forming units (CFU) and confirmed by scanning electron microscopy (SEM). PMZ showed bactericidal activity and significantly reduced (p < 0.05) the viability of the biofilm being formed by ESBL E. coli at concentrations of 256 and 512 µg/ml, as well as preventing the formation of biofilm on urinary catheters at concentrations starting at 512 µg/ml by reducing the number of CFUs, as also observed by SEM. Thus, PMZ is a promising candidate to prevent the formation of ESBL E. coli biofilms on abiotic surfaces.

Preliminary snapshot reveals a relationship between multidrug-resistance and biofilm production among enterobacteriaceae isolated from fecal samples of farm-raised poultry in ceará, Brazil.

Antimicrobial resistance and biofilm formation by microbial pathogens pose a significant challenge to poultry production systems due to the persistent risk of dissemination and compromise of bird health and productivity. In this context, the study aimed to investigate the occurrence of different multiresistance phenotypes and the biofilm-forming ability of Enterobacteriaceae isolated from broiler chicken excreta in poultry production units in Ceará, Brazil. Samples were collected from three distinct broiler breeding facilities and subjected to isolation, identification, antibiotic susceptibility testing, phenotypic screening for ß-lactamases enzymes, and biofilm formation evaluation. Seventy-one strains were identified, being Escherichia coli (37 %) and Proteus mirabilis (32 %), followed by Klebsiella pneumoniae (11 %), Providencia stuartii (9 %), Klebsiella aerogenes (6 %), Alcaligenes faecalis (4 %), and Salmonella sp. (1 %). A significant proportion (87 %) of multiresistant strains were detected. For the phenotypic evaluation of ß-lactamases production, strains with resistance to second and third-generation cephalosporins and carbapenems were tested. About 4 of 6 and 10 of 26 were positive for inducible chromosomal AmpC ß-lactamase and extended-spectrum ß-lactamase (ESBL), respectively. Regarding biofilm formation, it was observed that all MDR strains were capable of forming biofilm. In this sense the potential of these MDR bacteria to develop biofilms becomes a significant concern, representing a real threat to both human and animal health, as biofilms offer stability, antimicrobial protection, and facilitate genetic transfer.

Antimicrobial resistance genes harbored in invasive Acinetobacter calcoaceticus-baumannii complex isolated from Korean children during the pre-COVID-19 pandemic periods, 2015-2020.

Background: Acinetobacter baumannii (AB) has emerged as one of the most challenging pathogens worldwide, causing invasive infections in the critically ill patients due to their ability to rapidly acquire resistance to antibiotics. This study aimed to analyze antibiotic resistance genes harbored in AB and non-baumannii Acinetobacter calcoaceticus-baumannii (NB-ACB) complex causing invasive diseases in Korean children. Methods: ACB complexes isolated from sterile body fluid of children in three referral hospitals were prospectively collected. Colistin susceptibility was additionally tested via broth microdilution. Whole genome sequencing was performed and antibiotic resistance genes were analyzed. Results: During January 2015 to December 2020, a total of 67 ACB complexes were isolated from sterile body fluid of children in three referral hospitals. The median age of the patients was 0.6 (interquartile range, 0.1-7.2) years old. Among all the isolates, 73.1% (n=49) were confirmed as AB and others as NB-ACB complex by whole genome sequencing. Among the AB isolates, only 22.4% susceptible to carbapenem. In particular, all clonal complex (CC) 92 AB (n=33) showed multi-drug resistance, whereas 31.3% in non-CC92 AB (n=16) (P<0.001). NB-ACB showed 100% susceptibility to all classes of antibiotics except 3rd generation cephalosporin (72.2%). The main mechanism of carbapenem resistance in AB was the bla oxa23 gene with ISAba1 insertion sequence upstream. Presence of pmr gene and/or mutation of lpxA/C gene were not correlated with the phenotype of colistin resistance of ACB. All AB and NB-ACB isolates carried the abe and ade multidrug efflux pumps. Conclusions: In conclusion, monitoring and research for resistome in ACB complex is needed to identify and manage drug-resistant AB, particularly CC92 AB carrying the bla oxa23 gene.

Nordalbergin Synergizes with Novel ß-Lactam Antibiotics against MRSA Infection.

The synergetic strategy has created tremendous advantages in drug-resistance bacterial infection treatment, whereas challenges related to novel compound discovery and identifying drug-binding targets still remain. The mechanisms of antimicrobial resistance involving ß-lactamase catalysis and the degradation of ß-lactam antibiotics are being revealed, with relevant therapies promising to improve the efficacy of existing major classes of antibiotics in the foreseeable future. In this study, it is demonstrated that nordalbergin, a coumarin isolated from the wood bark of Dalbergia sissoo, efficiently potentiated the activities of ß-lactam antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) by suppressing ß-lactamase performance and improving the bacterial biofilm susceptibility to antibiotics. Nordalbergin was found to destabilize the cell membrane and promote its permeabilization. Moreover, nordalbergin efficiently improved the therapeutic efficacy of amoxicillin against MRSA pneumonia in mice, as supported by the lower bacterial load, attenuated pathological damage, and decreased inflammation level. These results demonstrate that nordalbergin might be a promising synergist of amoxicillin against MRSA infections. This study provided a new approach for developing potentiators for ß-lactam antibiotics against MRSA infections.

Interpreting the results of rapid molecular diagnostic tests for carbapenem-resistant Enterobacterales infection: current clinical perspective while waiting for further evidence.

INTRODUCTION: Carbapenem-resistant Enterobacterales (CRE) causing severe infections in humans have represented an important challenge for clinicians worldwide during the past two decades. AREAS COVERED: Novel ß-lactams and ß-lactam/ß-lactamase inhibitor combinations have led to a shift in the first-line approach to the treatment of severe CRE infections from polymyxin-based regimens to treatment with less toxic agents. This new scenario offers the opportunity to apply rapid molecular diagnostic tests for CRE infection to identify different types of carbapenemases. Herein, the authors provide an overview of this subject and follow it with their expert perspectives. EXPERT OPINION: When considering studies actually measuring the clinical impact of rapid molecular tests in real-life scenarios, high certainty evidence from randomized controlled trials is still limited and not focused on CRE infections. Nonetheless, it is indisputable that rapid molecular tests have been shown to impact early therapeutic choices (in terms of both escalation and de-escalation) when used in real-life settings, thus issues in the clinical interpretation of their results are already relevant. Overall, increased expertise is required for the appropriate interpretation of rapid molecular tests for personalized antibiotic selection by understanding their strengths and limitations.

Assessment of Posttransplant Bacteremia Caused by Extended-Spectrum Beta-Lactamase-Producing Gram-Negative Bacteria Among Kidney Transplant Recipients.

BACKGROUND: Extended-spectrum beta-lactamase-producing gram-negative rods (ESBL-GNR) are a rising cause of bacteremia in kidney transplant recipients (KT). The study purpose was to examine patient mortality, allograft survival, estimated glomerular filtration rate (eGFR) at the end of 1 year, and readmission rates while looking at treatment strategies among KTs with ESBL-GNR and non-ESBL-GNR bacteremia at our institution. METHODS: This study was a retrospective, cohort analysis of KTs with gram-negative bacteremia from January 1, 2020, to December 31, 2021. The primary outcome of the study was mortality. Patient outcomes were assessed for 365 days after positive blood cultures. RESULTS: The study included 63 patients. Of these, 18 (29%) patients had bacteremia caused by an ESBL-GNR and 45 (71%) patients had bacteremia caused by a non-ESBL-GNR. Patient survival at 90 days was 94% in the ESBL-GNR group and 96% in the non-ESBL-GNR group. Ciprofloxacin was the most common antimicrobial therapy at discharge (68.9%) in the non-ESBL-GNR group whereas ertapenem was the most common in the ESBL-GNR group (44.5%). Median eGFR at discharge was 41 mL/min/1.73 m2 in the ESBL-GNR group and 48 mL/min/1.73 m2 in the non-ESBL-GNR group. Ninety-day readmission occurred in 9 (50%) ESBL-GNR patients and 14 (32%) non-ESBL-GNR patients. None of the above comparisons are statistically significant (p > 0.05). Eleven (61%) ESBL-GNR and 2 (4%) non-ESBL-GNR patients used outpatient parenteral antimicrobial therapy (p < 0.001). CONCLUSIONS: Among KTs with ESBL-GNR bacteremia, no significant difference was detected in mortality or allograft function compared to non-ESBL-GNR bacteremia.

Impact of the COVID-19 pandemic on extended-spectrum ß-lactamase producing Escherichia coli in urinary tract and blood stream infections: results from a nationwide surveillance network, Finland, 2018 to 2022.

BACKGROUND: Before the COVID-19 pandemic there has been a constant increase in antimicrobial resistance (AMR) of Escherichia coli, the most common cause of urinary tract infections and bloodstream infections. The aim of this study was to investigate the impact of the COVID-19 pandemic on extended-spectrum ß-lactamase (ESBL) production in urine and blood E. coli isolates in Finland to improve our understanding on the source attribution of this major multidrug-resistant pathogen. METHODS: Susceptibility test results of 564,233 urine (88.3% from females) and 23,860 blood E. coli isolates (58.8% from females) were obtained from the nationwide surveillance database of Finnish clinical microbiology laboratories. Susceptibility testing was performed according to EUCAST guidelines. We compared ESBL-producing E. coli proportions and incidence before (2018-2019), during (2020-2021), and after (2022) the pandemic and stratified these by age groups and sex. RESULTS: The annual number of urine E. coli isolates tested for antimicrobial susceptibility decreased 23.3% during 2018-2022 whereas the number of blood E. coli isolates increased 1.1%. The annual proportion of ESBL-producing E. coli in urine E. coli isolates decreased 28.7% among males, from 6.9% (average during 2018-2019) to 4.9% in 2022, and 28.7% among females, from 3.0 to 2.1%. In blood E. coli isolates, the proportion decreased 32.9% among males, from 9.3 to 6.2%, and 26.6% among females, from 6.2 to 4.6%. A significant decreasing trend was also observed in most age groups, but risk remained highest among persons aged ≥ 60 years. CONCLUSIONS: The reduction in the proportions of ESBL-producing E. coli was comprehensive, covering both specimen types, both sexes, and all age groups, showing that the continuously increasing trends could be reversed. Decrease in international travel and antimicrobial use were likely behind this reduction, suggesting that informing travellers about the risk of multidrug-resistant bacteria, hygiene measures, and appropriate antimicrobial use is crucial in prevention. Evaluation of infection control measures in healthcare settings could be beneficial, especially in long-term care.

Evolution of blaKPC Under the Pressure of Carbapenems and Ceftazidime/Avibactam in a Patient With Persistent Bacteremia Caused by Klebsiella pneumoniae.

A 30-year-old Korean man with myelodysplastic syndrome admitted hospital due to undifferentiated fever and recurrent skin lesions. He received combination therapy with high doses of meropenem, tigecycline and amikacin, yielding carbapenem resistant Klebsiella pneumoniae (CRKP) harboring K. pneumoniae carbapenemase (KPC)-2 from blood cultures on hospital day (HD) 23. Ceftazidime/avibactam was started at HD 37 and CRKP was eradicated from blood cultures after 5 days. However, ceftazidime/avibactam-resistant CRKP carrying KPC-44 emerged after 26 days of ceftazidime/avibactam treatment and then ceftazidime/avibactam-resistant, carbapenem-susceptible K. pneumoniae carrying KPC-135 was isolated on HD 65. The 3-D homology of KPC protein showed that hot spot changes in the omega loop could be attributed to ceftazidime/avibactam resistance and loss of carbapenem resistance. Whole genome sequencing of serial isolates supported that phenotypic variation was due to clonal evolution than clonal replacement. The treatment regimen was changed from CAZ/AVI to meropenem-based therapy (meropenem 1 g iv q 8 hours and amikacin 600 mg iv per day) starting with HD 72. CAZ/AVI-susceptible CRKP was presented again from blood cultures on HD 84, and the patient expired on HD 85. This is the first Korean report on the acquisition of ceftazidime/avibactam resistance through the emergence of blaKPC variants.

Genetic background of aminoglycoside-modifying enzymes in various genetic lineages of clinical aminoglycosides-resistant E. coli and K. pneumoniae isolates in Tunisia.

AIMS: This study was conducted to evaluate the in vitro activity of clinically relevant aminoglycosides and to determine the prevalence of genes encoding aminoglycoside modifying enzymes (AMEs) and 16S ribosomal RNA (rRNA) methyltransferases among aminoglycoside-resistant E. coli (n = 61) and K. pneumoniae (n = 44) clinical isolates. Associated resistances to beta-lactams and their bla genes as well as the genetic relatedness of isolates were also investigated. MATERIALS AND METHODS: A total of 105 aminoglycoside-resistant E. coli (n = 61) and K. pneumoniae (n = 44) isolates recovered between March and May 2017 from 100 patients hospitalized in different wards of Charles Nicolle Hospital of Tunis, Tunisia, were studied. Minimal inhibitory concentrations of aminoglycoside compounds were determined by broth microdilution method. Aminoglycosides resistance encoding genes [aph(3´)-Ia, aph(3') IIa, aph(3´)-VIa, ant(2″)-Ia, aac(3)-IIa, aac(3)-IVa, aac(6')-Ib, rmtA, rmtB, rmtC, armA, and npmA] and bla genes were investigated by PCR and sequencing. Genetic relatedness was examined by multilocus sequence typing (MLST) for representative isolates. RESULTS: High rates of aminoglycoside resistance were found: gentamicin (85.7%), tobramycin (87.6%), kanamycin (78.0%), netilmincin (74.3%), and amikcin (18.0%). Most common AME gene was aac(3)-IIa (42%), followed by aac(6')-Ib (36.2%) and aph(3')-VIa (32.4%). The majority of isolates were resistant to beta-lactams and blaCTX-M-15 was the most common ESBL. The blaNDM-1 and blaOXA-48 were also produced by 1 and 23 isolates, respectively. Novel sequence types have been reported among our isolates and high-risk clonal lineages have been detected, such as E. coli ST43 (ST131 in Achtman MLST scheme) and K. pneumoniae (ST11/ST13). CONCLUSIONS: The high prevalence of aminoglycoside resistance rates and the diversity of corresponding genes, with diverse ß-lactamase enzymes among genetically heterogeneous clinical isolates present a matter of concern.