Genomic Analysis of Enterobacter Species Isolated from Patients in United States Hospitals.

We analyzed the whole genome sequences (WGS) and antibiograms of 35 Enterobacter isolates, including E. hormaechei and E. asburiae, and the recently described E. bugandensis, E. kobei, E. ludwigii, and E. roggenkampii species. Isolates were obtained from human blood and urinary tract infections in patients in the United States. Our goal was to understand the genetic diversity of antimicrobial resistance genes and virulence factors among the various species. Thirty-four of 35 isolates contained an AmpC class blaACT allele; however, the E. roggenkampii isolate contained blaMIR-5. Of the six Enterobacter isolates resistant to ertapenem, imipenem, and meropenem, four harbored a carbapenemase gene, including blaKPC or blaNDM. All four isolates were mCIM-positive. The remaining two isolates had alterations in ompC genes that may have contributed to the resistance phenotype. Interpretations of cefepime test results were variable when disk diffusion and automated broth microdilution results were compared due to the Clinical Laboratory and Standards Institute use of the "susceptible dose-dependent" classification. The diversity of the blaACT alleles paralleled species identifications, as did the presence of various virulence genes. The classification of recently described Enterobacter species is consistent with their resistance gene and virulence gene profiles.

Emergence of multidrug-resistant Providencia rettgeri clone in food-producing animals: A public health threat.

The occurrence of carbapenemases encoding genes in Providencia rettgeri is a critical public health concern since this species has intrinsic resistance to several antimicrobials, including polymyxins. The identification of this multidrug-resistant (MDR) pathogen outside the hospital setting has become increasingly frequent, and raises an alert for the global health agencies, as they indicate a possible spread of such pathogens. Herein, we described three MDR P. rettgeri isolates carrying a diversity of antimicrobial resistance genes (ARGs) isolated from stool samples of swine and bovine in Brazil. Molecular analysis revealed that all isolates belonged to the same clone. The whole genome sequencing (WGS) of a representative isolate (PVR-188) was performed by MiSeq Illumina® platform, while the assembling and annotation was achieved using SPAdes and Prooka, respectively. The WGS analyses indicated the presence of ARGs that confer resistance to ß-lactams (bla NDM-1, bla CTX-M-2), quinolones (qnrD1), aminoglycosides (aadA2, aadA1, aph(3')-Via), phenicol (catB2), sulfonamides (sul1, sul2), and trimethoprim (dfrA12, dfrA1). The presence of three plasmid replicons (Col3M, IncQ1, and IncT) was detected, but no phage sequences were found. The phylogenetic analyses confirmed the genomic relationship of the PVR-188 with P. rettgeri isolates recovered from animals and humans in the USA and Malaysia. In conclusion, we report the occurrence of MDR P. rettgeri clone colonizing the gut microbiota of food-producing animals in Brazil, revealing the spread of this pathogen beyond hospital boundaries.

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.

An outbreak of severe or lethal infections by a multidrug-resistant Acinetobacter baumannii ST126 strain carrying a plasmid with blaNDM-1 and blaOXA-58 carbapenemases.

Acinetobacter baumannii poses a significant health threat because of its frequent implications in hospital outbreaks and multidrug resistance (MDR). Here, we studied four A. baumannii isolates recovered during a hospital outbreak of severe or fatal cases to elucidate their diversity and factors contributing to their increased virulence and antibiotic resistance. The isolates were identified using MALDI-ToF and characterized using comparative genomics, PCR, and antimicrobial susceptibility tests. They were classified as ST126 and exhibited fewer than five chromosomal single-nucleotide variants and the same extrachromosomal content, indicating that they are a single strain (A. baumannii AB01). A. baumannii AB01 showed an MDR phenotype that could be linked to the carriage of parC and gyrA mutations, efflux transporters, aminoglycoside resistance genes, a class C beta-lactamase, and three carbapenemases, some of which are encoded on a 72 kb plasmid. ST126 is infrequent and has not been reported in Latin America, and our genomic data indicate a plausible origin for A. baumannii AB01 within the Pan Pacific region.

Combined Use of Phenotypic Screening and of a Novel Commercial Assay (REALQUALITY Carba-Screen) for the Rapid Molecular Detection of Carbapenemases: A Single-Center Experience.

Carbapenem resistance is a serious public health threat, causing numerous deaths annually primarily due to healthcare-associated infections. To face this menace, surveillance programs in high-risk patients are becoming a widespread practice. Here we report the performance of the combined use of a recently approved commercial multiplex real-time PCR assay (REALQUALITY Carba-Screen kit) with conventional phenotypic screening. In this three-month study, 479 rectal swabs from 309 patients across high-risk units were evaluated by combining the two approaches. Although the molecular assay showed a higher positivity rate than phenotypic screening (7.1% vs. 5%), it should be noted that the molecular method alone would have missed eight carbapenem-resistant isolates, while using only phenotypic screening would not have detected sixteen isolates. This demonstrates the complementary strengths of each method. Our study confirms the need for a combined approach to maximize the possible clinical impact of this kind of screening, ensuring a more comprehensive detection of resistant strains.

Development and Validation of a Novel Multiplex Real-Time PCR Assay for Rapid Detection of Carbapenemase Genes in Carbapenem-Resistant Enterobacterales Isolates and Clinical Samples.

Purpose: Carbapenem-resistant Enterobacterales (CRE) infection is an urgent threat to human health. This study aimed to develop and validate a novel multiplex real-time PCR (multi-qPCR) assay for the detection of the blaKPC, blaNDM, blaIMP, blaOXA-48-like, and blaVIM genes in CRE isolates and clinical samples, as well as to compare it with three phenotypic methods. Methods: The reliability and limit of detection (LOD) of the multi-qPCR assay were evaluated. PCR and DNA sequencing were used as the reference methods to identify carbapenemase genes in CRE isolates and clinical samples. The accuracy of the multi-qPCR assay, modified carbapenem inactivation and EDTA-modified carbapenem inactivation method (mCIMandeCIM), carbapenemase inhibitor-based combined disk test (CDT), and colloidal gold-based immunochromatographic test was compared with the reference methods with 182 isolates of CRE. Furthermore, 112 clinical samples were collected to validate the efficacy of this multi-qPCR assay. Results: The standard deviations (CVs) of intra-assay and inter-assay of the multi-qPCR assay were ≤ 0.53% and ≤ 2.04% for detecting the five major carbapenemase genes, respectively; while the LOD ranged from 2×102 copies/mL to 8×102 copies/mL. PCR and DNA sequencing confirmed 168 out of 182 CRE isolates producing carbapenemase(s): KPC (n = 93), NDM (n = 46), IMP (n = 8), OXA-48-like (n = 14), VIM (n = 1), KPC&NDM (n = 5), and KPC&NDM&IMP (n = 1). The accuracy of mCIMandeCIM, CDT, Colloidal Gold, and the multi-qPCR assay was 96.2%, 89.6%, 100%, and 100% respectively for detecting carbapenemase(s) producers. Moreover, the sensitivity and specificity of the multi-qPCR assay were all 100% for the detection of each carbapenemase gene in clinical samples, compared with PCR and sequencing. Conclusion: For clinical isolate detection, the multi-qPCR assay is comparable to Colloidal Gold, and superior to mCIMandeCIM and CDT; while for clinical samples detection, it also shows excellent performance. Therefore, the multi-qPCR assay has great potential for clinical diagnosis.

RESIST ACINETO test for the rapid detection of NDM and OXA acquired carbapenemases directly from blood culture in Acinetobacter species.

Carbapenem-resistant Acinetobacter baumannii (CRAB) are increasingly reported worldwide and a leading cause of mortality associated with antimicrobial resistance. Their early detection, particularly in the cases of bloodstream infections, is crucial in attempting to initiate effective antibiotic treatment. The immunochromatographic assay RESIST ACINETO (Coris BioConcept) is a new test developed for the detection of OXA-23, OXA-40/58, and New-Delhi Metallo-beta-lactamase (NDM) carbapenemases in Acinetobacter spp. We evaluated this test on a collection of 121 Acinetobacter spp. clinical isolates, including 104 carbapenemase producers (97 carbapenemases targeted by the test) and 17 non-carbapenemase producers. The performance of the RESIST ACINETO test was evaluated according to the manufacturer's recommendations from bacterial and blood cultures. The strains producing the carbapenemases OXA-23, -40, -58, or/and NDM were accurately detected from bacterial cultures and directly from blood cultures, with the exception of one OXA-23/NDM-1-positive Acinetobacter radioresistens isolate (only detected through standard culture). None of the non-carbapenemase producers tested positive. The RESIST ACINETO test demonstrated sensitivity/specificity of 100%/100% and 99%/100% on bacterial and blood cultures, respectively. IMPORTANCE: The incidence of bloodstream infections with carbapenem-resistant Acinetobacter baumannii (CRAB) could be very high in some countries such as the Balkans or Southeast Asia. In case of positive blood cultures with Gram-negative bacteria, the use of the RESIST ACINETO test could prove highly beneficial for the rapid identification of these imipenem-resistant bacteria and their antibiotic resistance mechanisms. In addition, it is now well established that New-Delhi Metallo-beta-lactamase (NDM) carbapenemase-producing isolates can have increased MICs of cefiderocol, which is an alternative treatment for these infections. This test may also allow the optimization of treatment based on the type of carbapenemase present. Finally, the RESIST ACINETO test is a rapid, easy-to-use, and cost-effective assay that demonstrates excellent performance in detecting the major acquired carbapenemases present in the Acinetobacter species.

Rapid Reversal of Carbapenemase-Producing Pseudomonas aeruginosa Epidemiology from blaVIM- to blaNDM-harbouring Isolates in a Greek Tertiary Care Hospital.

Carbapenemase-producing Pseudomonas aeruginosa strains present a specific geographical distribution regarding the type of carbapenemase-encoding genes that they harbor. For more than twenty years, VIM-type enzymes were the only major carbapenemases that were detected among P. aeruginosa isolates in Greece until the emergence of NDM-1-encoding P. aeruginosa in early 2023. In the present study, we present the rapid reversal of the carbapenemase-producing P. aeruginosa epidemiology from blaVIM- to blaNDM-harbouring isolates that occurred in our hospital since then. Between January 2023 and February 2024, 139 isolates tested positive for carbapenemase production with the NG-Test CARBA 5 immunochromatographic assay. Eight isolates were processed with the Hybrispot antimicrobial resistance direct flow chip molecular assay, and the first NDM-producing isolate was further analyzed through whole genome sequencing and bioinformatics analysis. Multiple resistance genes were detected by molecular techniques in accordance with the extensively drug-resistant phenotype. The isolate that was subjected to whole-genome sequencing belonged to the P. aeruginosa high-risk clone ST308, and the blaNDM was located in the chromosome in accordance with previously reported data. During the study period, NDM-producing isolates were increasingly detected, and only five months after their emergence, they overcame VIM producers. Our results indicate the potential of this new clone to spread rapidly and predominate within healthcare institutions, further restricting the already limited treatment options.

Identificación de enterobacterias productoras de carbapenemasas en el Hospital Universitario Católico de Cuenca / Identification of carbapenemase producing enterobacteriaceae at the Catholic University Hospital of Cuenca / Identificação de enterobacteriaceae produtoras de carbapenemase no Hospital Universitário Católico de Cuenca

Las enterobacterias productoras de carbapenemasas desarrollan infecciones resistentes a los medicamentos en neumonía, infección del tracto urinario e infecciones relacionadas con dispositivos. Klebsiella pneumoniae, Escherichia coli y Enterobacter cloacae son amenazas de resistencia emergentes importantes a nivel mundial, lo que representa alta mortalidad y limitadas opciones de tratamiento. Objetivo: detectar la presencia de EPC de clase A, mediante la aplicación del test fenotípico de sinergia con ácido borónico en cepas de enterobacterias aisladas de superficies inertes en el Hospital Universitario Católico de Cuenca, Ecuador. Materiales y Métodos: estudio cuali-cuantitativo de tipo experimento puro de corte transversal y alcance exploratorio - descriptivo. Las enterobacterias se identificaron mediante test bioquímicos del sistema estandarizado API 20 E. Para la detección fenotípica de carbapenamasas de clase A se utilizó el método de sinergia de discos con ácido borónico y discos imipenem, meropenem y ertapenem. Resultados: se identificaron 25 géneros de enterobacterias, el 24 % fue Pseudomonas aeruginosam, el 20 % de enterobacterias fue productoras de carpapenemasas clase Am mientras que el 32 % fue resistente para los tres carbapenémicos en estudio, el 68 % mostró sensibilidad para imipenem, el 56 % para meropenem y 44 % para ertapenem. El 48 % de enterobacterias fueron resistentes a ertapenem, el 44 % a meropenem y 32 % a imipenem. Conclusiones: Enterobacterias como P. aureginosa, E. cloacae, Cronobacter spp. y E. coli presentan mecanismos de resistencia asociados a carbapenemasas clase A tipo KPC por lo que se recomienda vigilancia continua y estrategias de manejo para abordar la resistencia a carbapenémicos en entornos hospitalarios

Carbapenemase-producing Enterobacteriaceae develop drug-resistant infections in pneumonia, urinary tract infection, and device-related infections. Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae are important emerging resistance threats globally, representing high mortality and limited treatment options. Objective: detect the presence of class A EPC, by applying the phenotypic synergy test with boronic acid in strains of enterobacteria isolated from inert surfaces at the Catholic University Hospital of Cuenca, Ecuador. Materials and Methods: Qualitative-quantitative study of pure cross-sectional experiment type and exploratorydescriptive scope. Enterobacteriaceae were identified using biochemical tests of the standardized API 20 E system. For the phenotypic detection of class A carbapenamases, the synergy method of disks with boronic acid and imipenem, meropenem and ertapenem disks was used. Results: 25 genera of enterobacteria were identified, 24 % were Pseudomonas aeruginosam, 20 % of enterobacteria were producers of class Am carbapenemases while 32 % were resistant to the three carbapenems under study, 68 % showed sensitivity to imipenem, 56 % for meropenem and 44 % for ertapenem. 48 % of enterobacteria were resistant to ertapenem, 44 % to meropenem and 32 % to imipenem. Conclusions: Enterobacteriaceae such as P. aureginosa, E. cloacae, Cronobacter spp. and E. coli present resistance mechanisms associated with class A carbapenemases type KPC, so continuous surveillance and management strategies are recommended to address resistance to carbapenems in hospital environments

Enterobacteriaceae produtoras de carbapenemases desenvolvem infecções resistentes a medicamentos em pneumonia, infecção do trato urinário e infecções relacionadas a dispositivos. Klebsiella pneumoniae, Escherichia coli e Enterobacter cloacae são importantes ameaças emergentes de resistência em todo o mundo, representando alta mortalidade e opções de tratamento limitadas. Objetivo: detectar a presença de CPE classe A, aplicando o teste de sinergia fenotípica com ácido borônico em cepas de enterobactérias isoladas de superfícies inertes no Hospital Universitário Católico de Cuenca, Equador. Materiais e Métodos: estudo cualitativo quantitativo, do tipo experimento transversal puro e escopo exploratório-descritivo. As enterobactérias foram identificadas por meio de testes bioquímicos do sistema padronizado API 20 E. Para a detecção fenotípica das carbapenamases classe A foi utilizado o método de sinergia de discos com ácido borônico e discos de imipenem, meropenem e ertapenem. Resultados: foram identificados 25 gêneros de enterobactérias, 24 % eram Pseudomonas aeruginosam, 20 % das enterobactérias eram produtoras de carbapenemases da classe Am enquanto 32 % eram resistentes aos três carbapenêmicos em estudo, 68 % apresentaram sensibilidade ao imipenem, 56 % ao meropenem e 44. % para ertapenem. 48 % das enterobactérias eram resistentes ao ertapenem, 44 % ao meropenem e 32 % ao imipenem. Conclusões: Enterobacteriaceae como P. aureginosa, E. cloacae, Cronobacter spp. e. coli apresentam mecanismos de resistência associados às carbapenemases classe A tipo KPC, portanto estratégias contínuas de vigilância e manejo são recomendadas para abordar a resistência aos carbapenêmicos em ambientes hospitalares.

Use of Cefiderocol in Adult Patients: Descriptive Analysis from a Prospective, Multicenter, Cohort Study.

INTRODUCTION: Cefiderocol is a siderophore cephalosporin showing activity against various carbapenem-resistant Gram-negative bacteria (CR-GNB). No data currently exist about real-world use of cefiderocol in terms of types of therapy (e.g., empirical or targeted, monotherapy or combined regimens), indications, and patient characteristics. METHODS: In this multicenter, prospective study, we aimed at describing the use of cefiderocol in terms of types of therapy, indications, and patient characteristics. RESULTS: Cefiderocol was administered as empirical and targeted therapy in 27.5% (55/200) and 72.5% (145/200) of cases, respectively. Overall, it was administered as monotherapy in 101/200 cases (50.5%) and as part of a combined regimen for CR-GNB infections in the remaining 99/200 cases (49.5%). In multivariable analysis, previous isolation of carbapenem-resistant Acinetobacter baumannii odds ratio (OR) 2.56, with 95% confidence interval (95% CI) 1.01-6.46, p = 0.047] and previous hematopoietic stem cell transplantation (OR 8.73, 95% CI 1.05-72.54, p = 0.045) were associated with administration of cefiderocol as part of a combined regimen, whereas chronic kidney disease was associated with cefiderocol monotherapy (OR 0.38 for combined regimen, 95% CI 0.16-0.91, p = 0.029). Cumulative 30-day mortality was 19.8%, 45.0%, 20.7%, and 22.7% in patients receiving targeted cefiderocol for infections by Enterobacterales, A. baumannii, Pseudomonas aeruginosa, and any metallo-ß-lactamase producers, respectively. CONCLUSIONS: Cefiderocol is mainly used for targeted treatment, although empirical therapies account for more than 25% of prescriptions, thus requiring dedicated standardization and guidance. The almost equal distribution of cefiderocol monotherapy and cefiderocol-based combination therapies underlines the need for further study to ascertain possible differences in efficacy between the two approaches.

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

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

Performance of Flow Cytometry-Based Rapid Assay in Detection of Carbapenemase-Producing Enterobacterales.

Carbapenemase-producing Enterobacterales are increasingly being recognized in nosocomial infections. The performance of a flow cytometry-based rapid assay for their detection and differentiation was evaluated. This is a disruptive phenotypic technology, phenotypic and growth-independent, that searches for the lesions produced by drugs acting on cells after a short incubation time. Overall, 180 Gram-negative bacteria were studied, and results were compared with those obtained molecularly by PCR and phenotypically by 'KPC, MBL and OXA-48 Confirm Kit'. This phenotypic method was used as reference for comparison purposes. Susceptibility to carbapenems (imipenem, meropenem, and ertapenem) was determined by standard broth microdilution. Overall, 112 isolates (62.2%) were carbapenemase producers, 41 KPCs, 36 MßLs, and 31 OXA-48, and 4 strains were KPC + MßL co-producers. Sixty-eight isolates were carbapenemase-negative. The percentage of agreement, sensitivity, and specificity were calculated according to ISO 20776-2:2021. The FASTinov assay showed 97.7% agreement with the reference method for carbapenemase detection. Discrepant flow cytometry results were obtained in four isolates compared with both reference and PCR results. The sensitivity and specificity of this new technology were 95.3% and 98.5%, respectively, for KPCs, 97.6% and 99.3% for MßLs, and 96.9% and 98% for OXA-48 detection. In conclusion, we describe a rapid flow cytometry assay with high accuracy for carbapenemase detection and the differentiation of various carbapenemases, which should impact clinical microbiology laboratories and patient management.

Urinary Tract Infections with Carbapenem-Resistant Klebsiella pneumoniae in a Urology Clinic-A Case-Control Study.

BACKGROUND: The aim of our study was to analyze the factors associated with the increased risk of urinary tract infection (UTI) with carbapenem-resistant (CR) Klebsiella pneumoniae (Kpn) and the antibiotic resistance spectrum of the strains in patients. As secondary objectives, we elaborated the profile of these patients and the incidence of different types of carbapenemases. METHODS: We conducted a retrospective case-control study in which we compared a group of 62 patients with urinary tract infections with CR Kpn with a control group consisting of 136 patients with urinary tract infections with multidrug-resistant (MDR), but carbapenem-sensitive (CS), Kpn, who were hospitalized between 1 January 2022 and 31 March 2024. RESULTS: Compared to patients with urinary tract infections with CS Kpn, patients with urinary tract infections with CR Kpn were preponderant in rural areas (62.9% vs. 47.1%, p = 0.038) and more frequently had an upper urinary tract infection (69.4% vs. 36.8%, p < 0.01). Among the risk factors examined, patients in the study group had a higher presence of urinary catheters inserted for up to one month (50% vs. 34.6%, p = 0.03), rate of hospitalization in the last 180 days (96.8% vs. 69.9%, p < 0.01) and incidence of antibiotic therapy in the last 180 days (100% vs. 64.7%, p < 0.01). They also had a higher rate of carbapenem treatment in the last 180 days (8.1% vs. 0%, p < 0.01). Patients in the study group had a broader spectrum of resistance to all antibiotics tested (p < 0.01), with the exception of sulfamethoxazole-trimethoprim, where the resistance rate was similar in both groups (80.6% vs. 67.6%, p = 0.059). In the multivariate analysis, transfer from other hospitals (OR = 3.51, 95% and CI: 1.430-8.629) and treatment with carbapenems in the last 180 days (OR = 11.779 and 95% CI: 1.274-108.952) were factors associated with an increased risk of disease compared to the control group. The presence of carbapenemases was observed in all patients with CR Kpn, in the order of frequency New Delhi metallo-ß-lactamase (NDM) (52.2%), Klebsiella pneumoniae carbapenemase (KPC) (32.6%), and carbapenem-hydrolyzing oxacillinase (Oxa-48) (15.2%). CONCLUSIONS: The environment of origin and previous treatment with carbapenems appear to be the factors associated with an increased risk of urinary tract infection with CR Kpn compared to patients with urinary tract infections with CS Kpn. CR Kpn exhibits a broad spectrum of antibiotic resistance, among which is resistance to carbapenem antibiotics.

Coexistence of ß-lactamase genes and biofilm forming potential among carbapenem-resistant Acinetobacter baumannii in Lahore, Pakistan.

INTRODUCTION: Our goal was to investigate the antimicrobial resistance due to beta-lactamase genes and virulent determinants (biofilm-forming ability) expressed by Acinetobacter collected from health settings in Pakistan. A cross-sectional study was conducted for the molecular characterization of carbapenemases and biofilm-producing strains of Acinetobacter spp. METHODOLOGY: Two twenty-three imipenem-resistant Acinetobacter isolates were analyzed from 2020 to 2023.The combination disk test and modified hodge test were performed. Biofilm forming ability was determined by polystyrene tube assay. Multiplex polymerase chain reaction (PCR) for virulent and biofilm-forming genes, and 16S rRNA sequencing were performed. RESULTS: 118 (52.9%) carbapenem-resistant Acinetobacter (CR-AB) were isolated from wounds and pus, 121 (54.2%) from males, and 92 (41.2%) from 26-50-years-olds. More than 80% of strains produced ß-lactamases and carbapenemases. Based on the PCR amplification of the ITS gene, 174 (78.0%) CR-AB strains were identified from CR-Acinetobacter non-baumannii (ANB). Most CR-AB were strong and moderate biofilm producers. Genetic analysis revealed the blaOXA-23, blaTEM, blaCTX-M blaNDM-1 and blaVIM were prevalent in CR-AB with frequencies 91 (94.8%), 68 (70.8%), 19 (19.7%), 53 (55.2%), 2 (2.0%) respectively. Among virulence genes, OmpA was dominant in CR-AB isolates from wound (83, 86.4%), csuE 63 (80.7%) from non-wound specimens and significantly correlated with blaNDM and blaOXA genes. Phylogenetic analysis revealed three different clades for strains based on specimens. CONCLUSIONS: CR-AB was highly prevalent in Pakistan and associated with wound infections. The genes, blaOXA-23, blaTEM, blaCTX-M, and blaNDM-1 were detected in CR-AB. Most CR-AB were strong biofilm producers with virulent genes OmpA and csuE.

Phytobacter spp: the emergence of a new genus of healthcare-associated Enterobacterales encoding carbapenemases in Argentina: a case series.

Members of the genus Phytobacter (order Enterobacterales) are isolated from the natural environment and clinical settings. Identification of Phytobacter strains based on biochemical characteristics is complicated due to taxonomic confusion, and they are often misidentified by automated identification systems in laboratories. In this study we describe the first three clinical cases associated with Phytobacter spp. reported in Argentina. We describe the identification, the molecular analysis using whole genome sequencing and the potential clinical relevance.

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.

Molecular characteristics and evaluation of the phenotypic detection of carbapenemases among Enterobacterales and Pseudomonas via whole genome sequencing.

Background/purposes: The continuously increasing carbapenem resistance within Enterobacterales and Pseudomonas poses a threat to public health, nevertheless, the molecular characteristics of which in southern China still remain limited. And carbapenemase identification is a key factor in effective early therapy of carbapenem-resistant bacteria infections. We aimed to determine the molecular characteristics of these pathogens and compare commercial combined disc tests (CDTs) with the modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) in detecting and distinguishing carbapenemases using whole genome sequencing (WGS). Methods: A total of 78 Enterobacterales, 30 Pseudomonas were obtained from two tertiary hospitals in southern China. Susceptibility tests were conducted using an automated VITEK2 compact system with confirmation via the Kirby-Bauer method. The WGS was conducted on all clinical isolates and the molecular characteristics were analyzed by screening the whole genome sequences. CDTs with or without cloxacillin, mCIM, and eCIM, were performed and compared by taking WGS results as the benchmark. Results: A total of 103 carbapenem non-susceptible and 5 carbapenem susceptible bacteria were determined, with Klebsiella pneumoniae (42.7%), Pseudomonas aeruginosa (23.3%) and Escherichia coli (18.4%) being most prevalent. Carbapenemase genes were detected in 58 (56.3%) of the 103 carbapenem-non-susceptible clinical isolates, including 46 NDM, 6 KPC, 3 IMP, 1 IPM+VIM,1NDM+KPC, and 1 OXA-181. Carbapenemase-producing isolates were detected more frequently in Enterobacterales (76.3%). Among K. pneumoniae, the major sequence types were st307 and st11, while among E. coli and P. aeruginosa, the most prevalent ones were st410 and st242 respectively. For carbapenemase detection in Enterobacterales, the mCIM method achieved 100.00% (95% CI, 92.13-100.00%) sensitivity and 94.44% (70.63-99.71%) specificity (kappa, 0.96); for Pseudomonas, detection sensitivity was 100% (5.46-100.00%), and 100% (84.50-100.00%) specificity (kappa, 0.65). Commercial CDT carbapenemase detection sensitivity for Enterobacterales was 96.49% (86.84-99.39%), and 95.24% (74.13-99.75%) specificity (kappa, 0.90); for Pseudomonas, carbapenemase detection sensitivity was 100.00% (5.46-100.00%) and 37.93% (21.30-57.64%) specificity (kappa, 0.04). When cloxacillin testing was added, CDT specificity reached 84.61% (64.27-94.95%). Conclusion: The molecular epidemiology of carbapenem-non-susceptible isolates from pediatric patients in Southern China exhibited distinctive characteristics. Both the mCIM-eCIM combination and CDT methods effectively detected and differentiated carbapenemases among Enterobacterales isolates, and the former performed better than CDT among Pseudomonas.

Epidemiological and clinical characteristics of patients with carbapenem-resistant Enterobacterales in a university hospital of Colombia: Enzyme coproductions in rise.

The distribution of carbapenemases in Carbapenem-Resistant Enterobacterales (CRE) has recently undergone a change in our region. According to the Colombian National Institute of Health, there is an increasing prevalence of NDM and NDM-KPC co-producing strains. We carried-out an ambispective cohort study of adult inpatients from Hospital Universitario San Ignacio (2021-2023), infected or colonized with CRE, in which carbapenemases immunochromatographic assay was performed. Out of the 150 patients included in the study, 71.3 % presented with an infection, and carbapenemases were detected in 92.7 % of these cases. Among them, KPC predominated (54 %), while 16.7 % demonstrated enzyme coproductions, mainly KPC-NDM. CRE infected patients had an 18.7 % 30-days mortality, but we could not demonstrate an association between type of carbapenemase and mortality rate (p = 0.82). Logistic regression analysis suggested that ICU admission was independently correlated to fatality (OR 5.08; CI 1.68-16.01). NDM and KPC-NDM presence in CRE poses a public health threat and a therapeutic challenge, with unknown mortality differences according to the carbapenemases pattern. Nevertheless, there was not an association between enzyme type and mortality.

Study of the Epidemiological and Mechanistic Differences Between Carbapenem-Resistant Klebsiella pneumoniae Infections in Children and Adults.

Background: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has garnered international concern due to its significant antibiotic resistance. Notably, children exhibit distinct resistance mechanisms compared to adults, necessitating a differential approach to antibiotic selection. A thorough analysis of CRKP's epidemiology and drug resistance mechanisms is essential for establishing a robust foundation for clinical anti-infection strategies and precise prevention and control measures. Methods: This study involved the collection of 31 non-repetitive strains from pediatric and adult patients at a tertiary hospital in China, spanning from July 2016 to July 2022, testing for resistance genes, antimicrobial susceptibility, and homology analysis. Results: Infants (0-1 year) were the largest pediatric CRKP group, with 61.3% of cases. The neonatal intensive care unit (NICU) and pediatrics were the main departments affected. Adults with CRKP had a mean age of 67 years, with the highest prevalence in neurology and emergency ICU. Antimicrobial susceptibility testing revealed that adult CRKP strains exhibited higher resistance to amikacin, ciprofloxacin, cotrimoxazole, and aztreonam compared to pediatric strains. Conversely, pediatric strains showed a higher rate of resistance to ceftazidime/avibactam. The predominant resistance genes identified were bla NDM-5 in children (58.1%) and bla KPC-2 in adults (87.1%), with over 93% of both groups testing positive for extended-spectrum beta-lactamase (ESBL) genes. Multilocus Sequence Typing (MLST) indicated ST2735 and ST11 as the predominant types in children and adults, respectively. Pulsed-field gel electrophoresis (PFGE) identified clonal transmission patterns of ST11 bla KPC-2 and ST15 bla OXA-232 across both age groups. Notably, this study reports the first instance of ST1114-type CRKP co-producing bla NDM-5 and bla OXA-181 in the NICU. Conclusion: This study reveals distinct resistance mechanisms and epidemiology in CRKP from children and adults. The identified clonal transmission patterns emphasize the need for improved infection control to prevent the spread of resistant strains.