Multidrug-resistant Enterobacterales Research Articles

High prevalence of colonization with extended-spectrum β-lactamase-producing and multidrug-resistant Enterobacterales in the community in Addis Ababa Ethiopia: risk factors, carbapenem resistance, and molecular characterization

BackgroundGlobally, extended-spectrum beta-lactamase-producing and carbapenem-resistant Enterobacterales are major causes of hospital-acquired infections and there are increasing concerns about their role in community-acquired infections.ObjectiveWe aimed to investigate the prevalence of extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-PE) and Carbapenemase-producing-Carbapenemresistant-Enterobacterales (CP-CRE) and associated factors in community settings in Gulele sub city, Addis Ababa, Ethiopia.MethodsA cross-sectional study was conducted among 261 healthy individuals. Stool samples were collected and processed using standard microbiological methods. Antimicrobial susceptibility and phenotypic ESBL and carbapenemase tests were performed. Antibiotic resistance genes were detected by Polymerase Chain Reaction (PCR).ResultsThe colonization rate of ESBL-PE and CP-CRE were 31.4% (82/261, 95% CI: 25.91–37.48) and 0.8% (2/261, 95% CI: 0.13–3.1), respectively by phenotypic method. Molecular detection of genes for ESBL-PE was 27.9% (73/261, 95% CI:22.7–33.9), and for CP-CRE was 0.8% (2/261, 95% CI: 0.13–3.1). The most prevalent genes were blaTEM [76.7% (56/73)] and blaCTX-M [45.2% (33/73)]. Previous antibiotic use (AOR:2.04, 95%CI: 1.35–4.41, P:0.041) and age between 42 and 53 years old (AOR:3.00, 95%CI:1.12–7.48, P:0.019) were significantly associated with ESBL-PE colonization.ConclusionIntestinal colonization by ESBL-PE harboring the associated antibiotic resistance genes was substantially high but with low CP-CRE. Continued surveillance of community-level carriage of antimicrobial resistance Enterobacterales is warranted.

In vitro activity of cefepime-enmetazobactam on carbapenem-resistant gram negatives

ObjectivesCefepime-enmetazobactam is a new β-lactam/βlactamase inhibitor combination with broad-spectrum activity against multidrug-resistant Enterobacterales, including extended-spectrum β-lactamase producers. This study evaluated the in vitro activity of cefepime-enmetazobactam towards a collection of carbapenem-resistant Enterobacterales (CRE), Pseudomonas aeruginosa and Acinetobacter baumannii compared to the other β-lactam/β-lactamase inhibitor combinations. MethodsThe MIC of cefepime, cefepime-enmetazobactam, ceftazidime, ceftazidime-avibactam, meropenem, meropenem-vaborbactam, imipenem, imipenem-relebactam, and ertapenem were determined by broth microdilution on 2212 CRE, including 2089 carbapenemase producers (1000 OXA-48-like, 49 KPC, 697 NDM, 180 VIM, 1 IMP, 9 IMI, and 158 multiple carbapenemases) and 123 CRE that do not produce carbapenemase received at the French National Reference Centre (from March 1, 2023 to August 31, 2023), 50 P. aeruginosa, and 30 A. baumannii. All strains were fully sequenced. ResultsWe confirmed the absence of inhibitory activity of enmetazobactam towards metallo-β-lactamases. Cefepime-enmetazobactam and ceftazidime-avibactam exhibited a similar susceptibility (96.7% vs. 99.5%, respectively) on OXA-48-producers. Cefepime-enmetazobactam exhibited 66.9% and 63.3% susceptibility for CRE non-EPC and KPC, whereas those rates rose to 96.7%/95.9%, 93.4%/95.9%, and 95.9%/98.0% for ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam, respectively. Low MICs (≤0.25 mg/L) were obtained for ceftazidime-avibactam-resistant KPC variants.Cefepime-enmetazobactam did not display a significant added value when compared with cefepime alone on Pseudomonas aeruginosa and Acinetobacter baumannii. DiscussionOXA-48 producers displayed high susceptibility to cefepime-enmetazobactam, which is similar to ceftazidime-avibactam, including for OXA-48 producers that coproduce a ceftazidime hydrolyzing enzyme (extended-spectrum β-lactamases or AmpC). In vivo experiments have to be implemented to confirm if cefepime-enmetazobactam might be a relevant alternative to ceftazidime-avibactam for the treatment of infections caused by OXA-48 producers.

Phenotypic and molecular characterization of multidrug-resistant Enterobacterales isolated from clinical samples in Palestine: a focus on extended-spectrum β-lactamase- and carbapenemase-producing isolates

BackgroundInfections resulting from multidrug-resistant Enterobacterales (MDR-E) pose a growing global threat, presenting challenges in treatment and contributing significantly to morbidity and mortality rates. The main objective of this study was to characterize phenotypically and genetically extended-spectrum β-lactamase- and carbapenemase- producing Enterobacterales (ESBLE and CPE respectively) isolated from clinical samples in the West Bank, Palestine.MethodsA cross sectional study was conducted in October 2023 on clinical bacterial isolates collected from five governmental hospitals in the West Bank, Palestine. The isolates obtained from the microbiology laboratories of the participating hospitals, underwent identification and antibiotic susceptibility testing (AST) using the VITEK® 2 Compact system. ESBL production was determined by the Vitek2 Compact system. A modified carbapenem inactivation method (mCIM) was employed to identify carbapenemase-producing Enterobacterales (CPE). Resistance genes were detected by real-time PCR.ResultsOut of the total 1380 collected isolates, we randomly selected 600 isolates for analysis. Our analysis indicated that 287 (47.83%) were extended-spectrum beta-lactamase producers (ESBLE), and 102 (17%) as carbapenem-resistant Enterobacterales (CRE) isolates. A total of 424 isolates (70.67%) were identified as multidrug-resistant Enterobacterales (MDRE). The most prevalent ESBL species were K. pneumoniae (n = 124; 43.2%), E. coli (n = 119; 41.5%) and E. cloacae (n = 31; 10.8%). Among the CRE isolates, 85 (83.33%) were carbapenemase-producing Enterobacterales (CPE). The most frequent CRE species were K. pneumoniae (n = 63; 61.7%), E. coli (n = 25; 24.5%) and E. cloacae (n = 13; 12.8%). Additionally, 47 (7.83%) isolates exhibited resistance to colistin (CT), with 38 (37.62%) being CT-resistant CRE and 9 (3.14%) being CT-resistant ESBLE while sensitive to carbapenems. We noticed that 11 isolates (6 Klebsiella pneumoniae and 5 Enterobacter cloacae complex) demonstrated sensitivity to carbapenems by phenotype but carried silent CPE genes (1 blaOXA48, and 6 blaNDM, 4 blaOXA48, blaNDM). ESBL-producing Enterobacterales strains exhibited varied resistance patterns across different antibiotic classes. E. coli isolates showed notable 48% resistance to trimethoprim/sulfamethoxazole. K. pneumoniae isolates displayed a significant resistance to trimethoprim/sulfamethoxazole, nitrofurantoin, and fosfomycin (54%, 90%, and 70% respectively). E. cloacae isolates showed complete resistance to nitrofurantoin and fosfomycin. P. mirabilis isolates exhibited high resistance against fluoroquinolones (83%), and complete resistance to trimethoprim/sulfamethoxazole, nitrofurantoin and fosfomycin.ConclusionThis study showed the high burden of the ESBLE and CRE among the samples collected from the participating hospitals. The most common species were K. pneumoniae and E. coli. There was a high prevalence of blaCTXm. Adopting both conventional and molecular techniques is essential for better surveillance of the emergence and spread of antimicrobial-resistant Enterobacterales infections in Palestine.

FosA3 emerging in clinical carbapenemase-producing C. freundii.

Fosfomycin (FOS) is an effective antibiotic against multidrug-resistant Enterobacterales, but its effectiveness is reducing. Little is known on the current prevalence of FosA enzymes in low-risk pathogens, such as Citrobacter freundii. The aim of the study was the molecular characterization of a carbapenemase- and FosA-producing C. freundii collected in Italy. AK867, collected in 2023, showed an XDR profile, retaining susceptibility only to colistin. AK867 showed a FOS MIC >128 mg/L by ADM. Based on WGS, AK867 belonged to ST116 and owned a wide resistome, including fosA3, blaKPC-2, and blaVIM-1. fosA3 was carried by a conjugative pKPC-CAV1312 plasmid of 320,480 bp, on a novel composite transposon (12,907 bp). FosA3 transposon shared similarities with other fosA3-harboring pKPC-CAV1312 plasmids among Citrobacter spp. We report the first case of FosA3 production in clinical carbapenemase-producing C.freundii ST116. The incidence of FosA3 enzymes is increasing among Enterobacterales, affecting even low-virulence pathogens, as C. freundii.

Burden of Multidrug-Resistant Gram-Negative Bacterial Infections in a Tertiary Care Hospital

Multidrug-resistant (MDR) Gram-negative bacterial infections have emerged as a major public health concern. The aim of the present study was to detect the rate of infections due to MDR Gram-negative bacteria (GNB) in a tertiary care hospital, the rate of Carbapenemases and AmpC-β-lactamases production and the Antimicrobial susceptibility test pattern (AST) among MDR GNB. The rate of MDR GNB during the study period was 25.70%. Urine samples showed the highest contribution to the total MDR GNB. Among the total MDR GNB isolates, 166 were randomly selected and included in the present study. A higher rate of MDR GNB was reported among male patients (61.5%) compared to the females (38.5%) and most of them were from the patients aged between 61-70 years (30.7%). The most prevalent MDR GNB was Klebsiella pneumoniae 80 (48.12%), followed by Escherichia coli 43 (25.9%). AST of MDR GNB revealed their significant resistance to β-lactamases/β-lactamases inhibitors, cephalosporins, fluoroquinolones and carbapenem drugs (98%). Of 123 MDR Enterobacterales, 83% of them were found to be Metallo β-lactamase (MBLs) producers by mCIM and eCIM methods. Of 43 MDR non-fermenters, 29 (67.4%) of them were found to be carbapenemase producers by MHT. About 29.51% of MDR GNB isolates were found to be AmpC producers by AmpC disk test. A reliable and rapid phenotypic method to detect carbapenemases and AmpC β-lactamases among MDR GNB in a routine microbiology laboratory method is clinically important to guide antibiotic therapy and implementation of effective infection control practices.

Typical pneumonia among human immunodeficiency virus-infected patients in public hospitals in southern Ethiopia.

Typical pneumonia is a pressing issue in the treatment of human immunodeficiency virus (HIV) patients, especially in Sub-Saharan Africa, where it remains a significant menace. Addressing this problem is crucial in improving health outcomes and the reduction of the burden of diseases in this vulnerable category of patients. To determine the prevalence of community-acquired typical pneumonia among HIV patients in Public Hospitals in southern Ethiopia. A cross-sectional study was done among 386 HIV patients clinically suspected of typical pneumonia attending the anti-retroviral therapy (ART) clinics of two hospitals from March to September 2022. A pretested structured questionnaire was employed to collect the demographic, clinical, and behavioral data. Sputum samples were collected and inspected for bacteria following standard procedures, and antimicrobial susceptibility testing was performed employing the Kirby-Bauer disk diffusion method. Besides, extended-spectrum β-lactamase (ESβL) and carbapenemase-producing Gram-negative bacteria were inspected by the double disk synergy test and modified carbapenem inactivation method. Descriptive and inferential statistical analyses were also done. Overall, 39.1% (151/386) of sputum cultures (95% Confidence Interval: 32.4-44) were bacteriologically positive. A total of 151 bacteria were identified, comprising 72.8% (n = 110) of Gram-negative bacteria. The predominant isolate was Klebsiella pneumoniae (25.8%, n = 39), followed by Staphylococcus aureus (17.9%, n = 27); 59.6% (n = 90) of the entire isolates were multidrug-resistant (MDR). Forty percent (11/27) of S. aureus were methicillin-resistant S. aureus (MRSA), and 28.1% (n = 31) and 20.9% (n = 23) of Gram-negative bacteria were extended-spectrum beta-lactamases (ESBL) and carbapenemase producers, respectively. Occupational status, alcohol consumption, cluster of differentiation4 (CD4) Thymocyte cell count < 350, interruption of trimethoprim-sulfamethoxazole prophylaxis and antiretroviral treatment, and recent viral load ≥ 150 were found statistically significant. The higher rates of MDR, MRSA, ESBL, and carbapenem-resistant Enterobacterales (CRE) indicate that bacterial pneumonia is a vexing problem among HIV patients and therefore it is advisable to implement an antimicrobial stewardship program in the study area.

Small RNA-regulated expression of efflux pump affects tigecycline resistance and heteroresistance in clinical isolates of Klebsiella pneumoniae

Tigecycline and the newly Food and Drug Administration-approved tetracyclines, including eravacycline and omadacycline, are regarded as last-resort treatments for multidrug-resistant Enterobacterales. However, tigecycline resistance in Klebsiella pneumoniae has increased, especially the underlying mechanism of heteroresistance is unclear. This study aimed to elucidate the mechanisms underlying tigecycline resistance and heteroresistance in clinical K. pneumoniae isolates. A total of 153 clinical K. pneumoniae isolates were collected, and identified 15 tigecycline-resistant and three tigecycline-heteroresistant isolates using broth microdilution and population analysis profile methods, respectively. Total RNAs from K. pneumoniae ATCC13883 and the laboratory-induced tigecycline-resistant strain were extracted and sequenced on an Illumina platform. Differentially expressed genes and regulatory small RNAs (sRNAs) were analyzed and validated in clinical isolates of K. pneumoniae using quantitative real-time PCR. RNA sequencing results showed that mdtABC efflux pump genes were significantly upregulated in the tigecycline-resistant strains. Overexpression of mdtABC was observed in a clinical K. pneumoniae isolate, which increased tigecycline minimum inhibitory concentrations (MICs) and was involved in tigecycline heteroresistance. Sequencing analysis of sRNA demonstrated that candidate sRNA-120 directly interacted with the mdtABC operon and was downregulated in tigecycline-resistant strains. We generated an sRNA-120 deletion mutation strain and a complemented strain of K. pneumoniae. The sRNA-120 deletion strain displayed increased mRNA levels of mdtA, mdtB, and mdtC and an increase in MICs of tigecycline. The complemented strain of sRNA-120 restored the mRNA levels of these genes and the susceptibility to tigecycline. RNA antisense purification and parallel reaction monitoring mass spectrometry were performed to verify the interactions between sRNA-120 and mdtABC. Collectively, our study highlights that the post-transcriptional repression of mdtABC through sRNA-120 may provide an additional layer of efflux pump gene expression control, which is important for resistance and heteroresistance in clinical K. pneumoniae isolates.

Seven-day antibiotic therapy for Enterobacterales bacteremia in high-risk neutropenic patients: toward a new paradigm.

Data on short courses of antibiotic therapy for Enterobacterales bacteremia in high-risk neutropenic patients are limited. The aim of the study was to describe and compare the frequency of bacteremia relapse, 30-day overall and infection-related mortality, Clostridiodes difficile infection and length of hospital stay since bacteremia among those who received antibiotic therapy for 7 or 14days. This is a multicenter, prospective, observational cohort study in adult high-risk neutropenic patients with hematologic malignancies or hematopoietic stem cell transplant and monomicrobial Enterobacterales bacteremia. They received appropriate empirical antibiotic therapy, had a clinical response within 7days, and infection source control. Clinical, epidemiological and outcomes variables were compared based on 7 or 14days of AT. Two hundred patients were included (100, 7-day antibiotic therapy; 100, 14-day antibiotic therapy). Escherichia coli was the pathogen most frequently isolated (47.5%), followed by Klebsiella sp. (40.5%). Among those patients that received 7-day vs. 14-day antibiotic course, a clinical source of bacteremia was found in 54% vs. 57% (p = 0.66), multidrug-resistant Enterobacterales isolates in 28% vs. 30% (p = 0.75), and 40% vs. 47% (p = 0.31) received combined empirical antibiotic therapy. Overall mortality was 3% vs. 1% (p = 0.62), in no case related to infection; bacteremia relapse was 7% vs. 2% (p = 0.17), and length of hospital stay since bacteremia had a median of 9days (IQR: 7-15) vs. 14days (IQR: 13-22) (p = < 0.001). These data suggest that seven-day antibiotic therapy might be adequate for patients with high-risk neutropenia and Enterobacterales bacteremia, who receive appropriate empirical therapy, with clinical response and infection source control.

In Vitro Antimicrobial Activity of Taurolidine against Isolates Associated with Catheter-Related Bloodstream Infections

Background: Taurolidine exhibits broad antimicrobial activity and is a component of a recently FDA approved catheter lock solution (DefenCath®, taurolidine 13,500 μg/mL and heparin 1000 Units/mL) indicated for reducing the risk of catheter-related bloodstream infections (CRBSI) in adult patients receiving chronic hemodialysis through a central venous catheter (HD-CVC). FDA approval was based on a Phase 3 randomized trial (LOCK-IT-100) in which DefenCath showed a 71% reduction in CRBSI risk among HD-CVC patients as compared with heparin alone. Although individual isolates from the clinical program were not available for testing, this study evaluated the in vitro antimicrobial activity of taurolidine against a set of recent clinical isolates representative of those recovered from the LOCK-IT-100 trial and/or those commonly associated with CRBSI. Methods: 420 bacterial and 50 yeast isolates were selected from the SENTRY Antimicrobial Surveillance Program. All isolates were collected from the bloodstream of patients in the U.S. between 2018-2023. Isolates were tested for susceptibility to taurolidine and comparators using Clinical and Laboratory Standards Institute (CLSI) broth microdilution guidelines. JMI Laboratories produced susceptibility test panels for testing. CLSI-recommended quality control strains were also tested concurrently. MIC values were determined after 24 hours. Results: Taurolidine exhibited broad antimicrobial activity against all isolates tested (see table). Against gram-positive bacteria, taurolidine MIC50/90 values ranged from 256-512/512-1,024 μg/mL for S. aureus, Coagulase-negative Staphylococcus, Enterococcus species, and Viridans group streptococci. This activity was maintained regardless of methicillin susceptibility for Staphylococcal isolates or vancomycin resistance among Enterococcal species. Against gram-negative bacteria, taurolidine MIC50/90 values ranged from 256-1,024/512-2,048 μg/mL for Enterobacterales, P. aeruginosa, S. maltophilia, A. baumannii-calcoaceticus, and B. cepacia. This activity was maintained in both multidrug resistant Enterobacterales and P. aeruginosa isolates. Among Candida isolates, taurolidine MIC50/90 values ranged from 256-512/512 μg/mL for C. glabrata and C. parapsilosis while taurolidine MIC50/90 values of 4,096/4,096 μg/mL were observed for C. albicans. Conclusions: Taurolidine activity was very similar among a large collection of gram-positive, gram-negative, and yeast organisms. MIC90 values for all species/groups were ≤2,048 μg/mL, except C. albicans where an MIC90 of 4,096 μg/mL was observed. The activity of taurolidine was unaffected by resistance to antibiotics (i.e. methicillin, vancomycin, or multi-drug resistance) among gram-positive or gram-negative organisms. Based on these data, catheter lock solutions containing the broad-spectrum antimicrobial taurolidine at 13,500 μg/mL have the potential to prevent CRBSI caused by a variety of species, including those observed in the recent LOCK-IT-100 clinical trial and other common bloodstream pathogens

Plasmid genomic epidemiology of carbapenem-hydrolysing class D β-lactamase (CDHL)-producing Enterobacterales in Canada, 2010-2021.

Carbapenems are last-resort antibiotics for treatment of infections caused by multidrug-resistant Enterobacterales, but carbapenem resistance is a rising global threat due to the acquisition of carbapenemase genes. Oxacillinase-48 (bla OXA-48)-type carbapenemases are increasing in abundance in Canada and elsewhere; these genes are frequently found on mobile genetic elements and are associated with specific transposons. This means that alongside clonal dissemination, bla OXA-48-type genes can spread through plasmid-mediated horizontal gene transfer. We applied whole genome sequencing to characterize 249 bla OXA-48-type-producing Enterobacterales isolates collected by the Canadian Nosocomial Infection Surveillance Program from 2010 to 2021. Using a combination of short- and long-read sequencing, we obtained 70 complete and circular bla OXA-48-type-encoding plasmids. Using MOB-suite, four major plasmids clustered were identified, and we further estimated a plasmid cluster for 91.9 % (147/160) of incomplete bla OXA-48-type-encoding contigs. We identified different patterns of carbapenemase mobilization across Canada, including horizontal transmission of bla OXA-181/IncX3 plasmids (75/249, 30.1 %) and bla OXA-48/IncL/M plasmids (47/249, 18.9 %), and both horizontal transmission and clonal transmission of bla OXA-232 for Klebsiella pneumoniae ST231 on ColE2-type/ColKP3 plasmids (25/249, 10.0 %). Our findings highlight the diversity of OXA-48-type plasmids and indicate that multiple plasmid clusters and clonal transmission have contributed to bla OXA-48-type spread and persistence in Canada.

MultiRapid ATB NP test for detecting concomitant susceptibility and resistance of last-resort novel antibiotics available to treat multidrug-resistant Enterobacterales infections

BackgroundRecently developed therapeutics against Gram-negative bacteria include the β-lactam-β-lactamase inhibitor combinations ceftazidime–avibactam (CZA), meropenem–vaborbactam (MEV), and imipenem–relebatam (IPR), and the siderophore cephalosporin cefiderocol (FDC). The aim of this study was to develop a test for rapid identification of susceptibility/resistance to CZA, MEV, IPR, and FDC for Enterobacterales in a single test for rapid clinical decision making. MethodsThe MultiRapid ATB NP test is based on the detection of glucose metabolism occurring after bacterial growth in the presence of defined concentrations of CZA, MEV, IPR, and FDC, followed by visual detection of colour change of the pH indicator red phenol (red to yellow) generated by the acidification of the medium upon bacterial growth. This test is performed in 96-well microplates. The MultiRapid ATB NP test was evaluated using 78 Enterobacterales isolates and compared to the reference method broth microdilution. ResultsThe MultiRapid ATB NP test displayed 97.0% (confidence interval [CI] 92.6–98.8) sensitivity, 97.7% (CI 94.3–99.1) specificity, and 97.4% (CI 95.0–98.7) accuracy. The results were obtained after 3 h of incubation at 35 °C ± 2 °C, representing at least a 15-h gain-of-time compared with currently used antimicrobial susceptibility testing methods. ConclusionThe MultiRapid ATB NP test provided accurate results for the concomitant detection of susceptibility/resistance to CZA, MEV, IPR, and FDC in Enterobacterales, independent of the resistance mechanism. This test may be suitable for implementation in any microbiology routine laboratory.

Efficacy of an inulin-based treatment on intestinal colonization by multidrug-resistant E. coli: insight into the mechanism of action

ABSTRACT Inulin, an increasingly studied dietary fiber, alters intestinal microbiota. The aim of this study was to assess whether inulin decreases intestinal colonization by multidrug resistant E. coli and to investigate its potential mechanisms of action. Mice with amoxicillin-induced intestinal dysbiosis mice were inoculated with extended spectrum beta-lactamase producing E. coli (ESBL-E. coli). The combination of inulin and pantoprazole (IP) significantly reduced ESBL-E. coli fecal titers, whereas pantoprazole alone did not and inulin had a delayed and limited effect. Fecal microbiome was assessed using shotgun metagenomic sequencing and qPCR. The efficacy of IP was predicted by increased abundance of 74 taxa, including two species of Adlercreutzia. Preventive treatments with A. caecimuris or A. muris also reduced ESBL-E. coli fecal titers. Fecal microbiota of mice effectively treated by IP was enriched in genes involved in inulin catabolism, production of propionate and expression of beta-lactamases. They also had increased beta-lactamase activity and decreased amoxicillin concentration. These results suggest that IP act through production of propionate and degradation of amoxicillin by the microbiota. The combination of pantoprazole and inulin is a potential treatment of intestinal colonization by multidrug-resistant E. coli. The ability of prebiotics to promote propionate and/or beta-lactamase producing bacteria may be used as a screening tool to identify potential treatments of intestinal colonization by multidrug resistant Enterobacterales.

High prevalence of multidrug-resistant Enterobacterales carrying extended-spectrum beta-lactamase and AmpC genes isolated from neonatal sepsis in Ahvaz, Iran

ObjectivesIn the recent years, multidrug resistant (MDR) neonatal septicemia-causing Enterobacterales has been dramatically increased due to the extended-spectrum beta-lactamases (ESBLs) and AmpC enzymes. This study aimed to assess the antibiotic resistance pattern, prevalence of ESBLs/AmpC beta-lactamase genes, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) fingerprints in Enterobacterales isolated from neonatal sepsis.ResultsIn total, 59 Enterobacterales isolates including 41 (69.5%) Enterobacter species, 15 (25.4%) Klebsiella pneumoniae and 3 (5.1%) Escherichia coli were isolated respectively. Resistance to ceftazidime and cefotaxime was seen in all of isolates. Furthermore, all of them were multidrug-resistant (resistant to three different antibiotic categories). The phenotypic tests showed that 100% of isolates were ESBL-positive. Moreover, AmpC production was observed in 84.7% (n = 50/59) of isolates. Among 59 ESBL-positive isolates, the highest percentage belonged to blaCTX−M−15 gene (66.1%) followed by blaCTX−M (45.8%), blaCTX−M−14 (30.5%), blaSHV (28.8%), and blaTEM (13.6%). The frequency of blaDHA, blaEBC, blaMOX and blaCIT genes were 24%, 24%, 4%, and 2% respectively. ERIC-PCR analysis revealed that Enterobacterales isolates were genetically diverse. The remarkable prevalence of MDR Enterobacterales isolates carrying ESBL and AmpC beta-lactamase genes emphasizes that efficient surveillance measures are essential to avoid the more expansion of drug resistance amongst isolates.

Spread and evolution of blaKPC-plasmid between Serratia marcescens and Klebsiella pneumoniae

ObjectivesblaKPC-carrying Enterobacterales have post great challenges to global healthcare systems. In this study, we reported the evolution and spread of blaKPC between Serratia marcescens and Klebsiella pneumoniae. MethodsFour S. marcescens and one K. pneumoniae strains were isolated from the sputum samples of the patient. Antimicrobial susceptibility tests and whole genome sequencing were performed to investigate the phenotype & genotype of strains. Conjugation assays, cloning experiment and kinetic parameters measuring were performed to explore the spread and antimicrobial resistance mechanisms. ResultsThe evolution and transmission of blaKPC-2 occurred during the treatment of ceftazidime-avibactam and trimethoprim-sulfamethoxazole. Analysis of the antimicrobial susceptibility and genetic profiles of the clinical strains showed that blaKPC-2 evolved into blaKPC-71 and blaKPC-44, together with resistance to ceftazidime-avibactam and carbapenems susceptibility recovery under antimicrobial pressure. Cloning and expression of blaKPC-44 & blaKPC-71 in E. coli DH5α showed that KPC-44 and KPC-71 resulted in a 64∼128-fold increase in the MIC value for ceftazidime-avibactam. Meanwhile, the kinetic assays also showed that the enzyme activity of KPC-44 and KPC-71 towards carbapenems was destroyed and couldn't be inhibited by avibactam. Based on the conjugation assay and whole genome sequence analyses, we provided evolutionary insights into the transmission pathway trace of blaKPC-bearing plasmids between S. marcescens and K. pneumoniae. ConclusionsMixed-species co-infection is one of the risk factors leading to the spread of plasmids carrying carbapenem-resistant genes, and increased surveillance of multidrug-resistant Enterobacterales is urgently needed.

Occurrence and persistence of multidrug-resistant Enterobacterales isolated from urban, industrial and surface water in Monastir, Tunisia

The One Health approach of antimicrobial resistance highlighted the role of the aquatic environment as a reservoir and dissemination source of resistance genes and resistant bacteria, especially due to anthropogenic activities. Resistance to extended-spectrum cephalosporins (ESC) conferred by extended-spectrum beta-lactamases (ESBLs) in E. coli has been proposed as the major marker of the AMR burden in cross-sectoral approaches. In this study, we investigated wastewater, surface water and seawater that are subjected to official water quality monitoring in Monastir, Tunisia. While all but one sample were declared compliant according to the official tests, ESC-resistant bacteria were detected in 31 (19.1 %) samples. Thirty-nine isolates, coming from urban, industrial and surface water in Monastir, were collected and characterized using antibiograms and whole-genome sequencing. These isolates were identified as 27 Escherichia coli (69.3 %) belonging to 13 STs, 10 Klebsiella pneumoniae (25.6 %) belonging to six STs, and two Citrobacter freundii (5.1 %). We observed the persistence and dissemination of clones over time and in different sampling sites, and no typically human-associated pathogens could be identified apart from one ST131. All isolates presented a blaCTX-M gene – blaCTX-M-15 (n = 22) and blaCTX-M-55 (n = 8) being the most frequent variants – which were identified on plasmids (n = 20) or on the chromosome (n = 19). In conclusion, we observed ESC resistance in rather ubiquitous bacteria that are capable of surviving in the water environment. This suggests that including the total coliform count and the ESBL count as determined by bacterial growth on selective plates in the official monitoring would greatly improve water quality control in Tunisia.

Antibiotic Susceptibility Profiles of Bacterial Isolates Recovered from Abscesses in Cattle and Sheep at a Slaughterhouse in Algeria.

Abscesses represent the most prominent emerging problem in the red meat industry, leading to great economic constraints and public health hazards. Data on etiological agents present in these purulent lesions in Algeria are very scarce. The aim of this study was to identify the bacteria responsible for these abscesses and to determine their antibiotic susceptibility profiles. A total of 123 samples of abscesses from 100 slaughtered sheep and 23 slaughtered cattle were cultured in several media. A total of 114 bacterial isolates were cultured from 103 abscesses. Bacteria were identified using MALDI-TOF, and antibiotic susceptibility was determined by the disk diffusion method on Mueller-Hinton agar. A total of 73.6% (n = 84) corresponded to Enterobacterales, of which four were multidrug-resistant (MDR). These isolates, together with Staphylococcus aureus, coagulase negative Staphylococci, and seven randomly chosen susceptible Escherichia coli isolates, were further characterized using WGS. Resistome analysis of the four MDR Enterobacterales isolates revealed the presence of OXA-48 carbapenemase in two Klebsiella pneumoniae ST985 and one E. coli ST10 isolates and a CTX-M-15 ESBL in one E. coli isolate ST1706. Two coagulase-negative Staphylococci isolates were found to carry the mecA gene. WGS showed the presence of different resistance genes and virulence genes. Our study revealed 5% of MDR Enterobacterales (including ESBLs and carbapenemases) identified from abscesses, thus urging the need for abscess monitoring in slaughterhouses.

First Detection of International High-Risk blaKPC-2-Harbouring Escherichia coli Pandemic Lineage ST648 in Pet Food Packages

The continued worldwide increase in pet ownership has significantly boosted the growth of the pet food industry accompanied by new food safety risks and challenges. This study was designed to determine the occurrence and molecularly characterize multidrug-resistant (MDR) Enterobacterales in pet food. Eighty-six (86) packages of dry and wet pet food purchased in different retail stores were screened for carbapenem-resistant Enterobacterales (CRE). Antimicrobial susceptibility testing was performed by agar dilution technique using EUCAST/BrCAST recommendations. Blue-Carba test was further used to screen for carbapenemase-producing isolates. Isolated CRE strains were identified at the species level using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Detection of carbapenemase-encoding genes was carried out by PCR, Sanger sequencing, and whole genome sequencing (WGS). A total of 15 (17.4%) MDR-CRE (Escherichia coli (n = 2), Enterobacter cloacae (n = 10), Leclercia adecarboxylata (n = 2), and Cronobacter spp. (n = 1)) were isolated from 86 pet food samples. In addition to being resistant to beta-lactams, the Gram-negative bacterial isolates were also resistant to aminoglycosides, fluoroquinolones, and tigecycline. Interestingly, two carbapenem-resistant E. coli isolates harboured blaKPC-2 gene. WGS analysis of the two blaKPC-2-producing E. coli isolates revealed that they both belong to ST648 and serotype O153:H2 group. The genetic context of the blaKPC-2 showed that they were carried by an IncN plasmid on a Tn4401b transposon element. To the best of our knowledge, this is the first description of blaKPC-2-harbouring E. coli ST648 pathogens in pet food. The detection of blaKPC-2-harbouring E. coli ST648 pandemic high-risk lineage in pet food is worrisome and a serious “One Health” issue. Therefore, pet food should be considered as a potential vehicle for the transmission of MDR pathogens to companion animals, and a risk factor for the dissemination of these bacterial pathogens to pet animals and their human guardians.

Enhancing colistin efficacy against Salmonella infections with a quinazoline-based dual therapeutic strategy

Colistin remains one of the last-resort therapies for combating infections caused by multidrug-resistant (MDR) Enterobacterales, despite its adverse nephro- and neuro-toxic effects. This study elucidates the mechanism of action of a non-antibiotic 4-anilinoquinazoline-based compound that synergistically enhances the effectiveness of colistin against Salmonella enterica. The quinazoline sensitizes Salmonella by deactivating intrinsic, mutational, and transferable resistance mechanisms that enable Salmonella to counteract the antibiotic impact colistin, together with an induced disruption to the electrochemical balance of the bacterial membrane. The attenuation of colistin resistance via the combined treatment approach also proves efficacious against E. coli, Klebsiella, and Acinetobacter strains. The dual therapy reduces the mortality of Galleria mellonella larvae undergoing a systemic Salmonella infection when compared to individual drug treatments. Overall, our findings unveil the potential of the quinazoline-colistin combined therapy as an innovative strategy against MDR bacteria.

In vitro activity of ceftazidime-avibactam against gram-negative bacteria in patients with bacteremia and skin and soft-tissue infections in Colombia 2019–2021

ObjectivesCeftazidime-avibactam (CAZ-AVI) is an option for infections caused by MDR gram-negative bacilli. In this study, we aimed to analyze the in vitro antimicrobial activity of CAZ-AVI and other antimicrobial agents against gram-negative bacilli that were collected in Colombia between 2019 and 2021 from patients with bacteremia and skin and soft-tissue infections (SSTIs). MethodsA total of 600 Enterobacterales and 259 P. aeruginosa strains were analyzed. The phenotypic resistance of isolates, particularly non-susceptibility to meropenem, multidrug-resistant (MDR) isolates, and difficult-to-treat (DTR) P. aeruginosa, was evaluated according to CLSI breakpoints. ResultsEnterobacterales had the most susceptibility to CAZ-AVI (96.5 %) and tigecycline (95 %). Tigecycline and CAZ-AVI were the antimicrobial agents with the most in vitro activity against carbapenem-resistant Enterobacterales (CRE). CAZ-AVI was the antimicrobial treatment with the most activity against P. aeruginosa. ConclusionsTigecycline and CAZ-AVI were the antimicrobial agents with the most activity against CRE and MDR Enterobacterales. For P. aeruginosa, CAZ-AVI was the antimicrobial treatment with the most in vitro activity.

Effects of breastfeeding on children’s gut colonization with multidrug-resistant Enterobacterales in peri-urban Lima, Peru

ABSTRACT Children living in low-resource settings are frequently gut-colonized with multidrug-resistant bacteria. We explored whether breastfeeding may protect against children’s incident gut colonization with extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-Ec) and Klebsiella, Enterobacter, or Citrobacter spp. (ESBL-KEC). We screened 937 monthly stool samples collected from 112 children aged 1–16 months during a 2016–19 prospective cohort study of enteric infections in peri-urban Lima. We used 52,816 daily surveys to examine how exposures to breastfeeding in the 30 days prior to a stool sample were associated with children’s risks of incident gut-colonization, controlling for antibiotic use and other covariates. We sequenced 78 ESBL-Ec from 47 children to explore their diversity. Gut-colonization with ESBL-Ec was increasingly prevalent as children aged, approaching 75% by 16 months, while ESBL-KEC prevalence fluctuated between 18% and 36%. Through 6 months of age, exclusively providing human milk in the 30 days prior to a stool sample did not reduce children’s risk of incident gut-colonization with ESBL-Ec or ESBL-KEC. From 6 to 16 months of age, every 3 additional days of breastfeeding in the prior 30 days was associated with 6% lower risk of incident ESBL-Ec gut-colonization (95% CI: 0.90, 0.98, p = .003). No effects were observed on incident ESBL-KEC colonization. We detected highly diverse ESBL-Ec among children and few differences between children who were predominantly breastfed (mean age: 4.1 months) versus older children (10.8 months). Continued breastfeeding after 6 months conferred protection against children’s incident gut colonization with ESBL-Ec in this setting. Policies supporting continued breastfeeding should be considered in efforts to combat antibiotic resistance.