Molecular epidemiology of carbapenem-resistant Enterobacteriaceae isolated from patients in COVID-19 wards and ICUs in a Bulgarian University Hospital.

Many studies report an increase in antimicrobial resistance of Gram - negative bacteria during the COVID-19 pandemic. Our aim was to evaluate the epidemiological relationship between carbapenem-resistant (CR) Enterobacteriaceae isolates from patients in COVID-19 wards and to investigate the main mechanisms of carbapenem resistance in these isolates during the period April 2020-July 2021. A total of 45 isolates were studied: Klebsiella pneumoniae (n = 37), Klebsiella oxytoca (n = 2), Enterobacter cloacae complex (n = 4) and Escherichia coli (n = 2). Multiplex PCR was used for detection of genes encoding carbapenemases from different classes (blaKPC, blaIMP, blaVIM, blaNDM, blaOXA-48). For epidemiological typing and analysis, ERIC PCR was performed. Two clinical isolates of E. cloacae, previously identified as representatives of two dominant hospital clones from the period 2014-2017, were included in the study for comparison. In the CR K. pneumoniae group, 23 (62.2%) carried blaKPC, 13 (35.1%) blaNDM, 10 (27.0%) blaVIM, and 9 (24.3%) were positive for both blaKPC and blaVIM. The blaKPC was identified also in the two isolates of K. oxytoca and blaVIM in all E. cloacae complex isolates. The two CR isolates of E. coli possessed blaKPC and blaOXA-48 genes. Epidemiological typing identified 18 ERIC profiles among K. pneumoniae, some presented as clusters of identical and/or closely related isolates. The carbapenem resistance in the studied collection of isolates is mediated mainly by blaKPC. During the COVID-19 pandemic intrahospital dissemination of CR K. pneumoniae, producing carbapenemases of different molecular classes, as well as continuing circulation of dominant hospital clones of multidrug-resistant E. cloacae complex was documented.

Isolation of Carbapenem and Colistin Resistant Gram-Negative Bacteria Colonizing Immunocompromised SARS-CoV-2 Patients Admitted to Some Libyan Hospitals.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a devastating effect, globally. We describe, for the first time, the occurrence of carbapenem-resistant bacteria colonizing SARS-CoV-2 patients who developed hospital-associated infections with carbapenemase-producing, Gram-negative bacteria at some isolation centers of SARS-CoV-2 in the eastern part of Libya. In total, at first, 109 samples were collected from 43 patients, with the samples being recovered from oral (n = 35), nasal (n = 45), and rectal (n = 29) cavities. Strain identification was performed via matrix assisted laser desorption ionization-time of flight (MALDI-TOF). Antibiotic susceptibility testing was carried out on Mueller-Hinton agar, using the standard disk diffusion method. MIC determination was confirmed via E-TEST and microdilution standard methods. A molecular study was carried out to characterize the carbapenem and colistin resistance in Gram-negative bacterial strains. All of the positive results were confirmed via sequencing. Klebsiella pneumoniae (n = 32), Citrobacter freundii (n = 21), Escherichia coli (n = 7), and Acinetobacter baumannii (n = 21) were the predominant isolated bacteria. Gram-negative isolates were multidrug-resistant and carried different carbapenem resistance-associated genes, including NDM-1 (56/119; 47.05%), OXA-48 (15/119; 12.60%), OXA-23 (19/119; 15.96%), VIM (10/119; 8.40%), and the colistin resistance mobile gene mcr-1 (4/119; 3.36%). The overuse of antimicrobials, particularly carbapenem antibiotics, during the SARS-CoV-2 pandemic has led to the emergence of multidrug-resistant bacteria, mainly K. pneumoniae, A. baumannii, and colistin-resistant E. coli strains. Increased surveillance as well as the rational use of carbapenem antibiotics and, recently, colistin are required to reduce the propagation of multidrug-resistant strains and to optimally maintain the efficacy of these antibiotics. IMPORTANCE In this work, we describe, for the first time, the occurrence of carbapenem-resistant bacteria colonizing COVID-19 patients who developed hospital-associated infections with carbapenemase-producing, Gram-negative bacteria at some isolation centers of COVID-19 in the eastern part of Libya. Our results confirmed that the overuse of antimicrobials, such as carbapenem antibiotics, during the COVID-19 pandemic has led to the emergence of multidrug-resistant bacteria, mainly K. pneumoniae and A. baumannii, as well as colistin resistance.

Genetic plurality of blaKPC-2-harboring plasmids in high-risk clones of Klebsiella pneumoniae of environmental origin.

International high-risk clones of Klebsiella pneumoniae are important human pathogens that are spreading to the environment. In the COVID-19 pandemic scenario, the frequency of carbapenemase-producing strains increased, which can contribute to the contamination of the environment, impacting the surrounding and associated ecosystems. In this regard, KPC-producing strains were recovered from aquatic ecosystems located in commercial, industrial, or agricultural areas and were submitted to whole-genome characterization. K. pneumoniae and Klebsiella quasipneumoniae subsp. quasipneumoniae strains were assigned to high-risk clones (ST11, ST340, ST307) and the new ST6325. Virulome analysis showed genes related to putative hypervirulence. Strains were resistant to almost all antimicrobials tested, being classified as extensively drug-resistant or multidrug-resistant. In this context, a broad resistome (clinically important antimicrobials and hazardous metal) was detected. Single replicon (IncX5, IncN-pST15, IncU) and multireplicon [IncFII(K1)/IncFIB(pQil), IncFIA(HI1)/IncR] plasmids were identified carrying the blaKPC-2 gene with Tn4401 and non-Tn4401 elements. An unusual association of blaKPC-2 and qnrVC1 and the coexistence of blaKPC-2 and mer operon (mercury tolerance) was found. Comparative analysis revealed that blaKPC-2-bearing plasmids were most similar to plasmids from Enterobacterales of Brazil, China, and the United States, evidencing the long persistence of plasmids at the human-animal-environmental interface. Furthermore, the presence of uncommon plasmids, displaying the interspecies, intraspecies, and clonal transmission, was highlighted. These findings alert for the spread of high-risk clones producing blaKPC-2 in the environmental sector and call attention to rapid dispersion in a post-pandemic world.

Ceftazidime-Avibactam (C/A) Resistant, Meropenem Sensitive KPC-Producing Klebsiella pneumoniae in ICU Setting: We Are What We Are Treated with?

The continuous spread of carbapenem-resistant Klebsiella pneumoniae (CP-Kp) strains presents a severe challenge to the healthcare system due to limited therapeutic options and high mortality. Since its availability, ceftazidime/avibactam (C/A) has become a first-line option against KPC-Kp, but C/A-resistant strains have been reported increasingly, especially with pneumonia or prior suboptimal blood exposure to C/A treatment. A retrospective, observational study was conducted with all patients admitted to the Intensive Care Unit (ICU) dedicated to COVID-19 patients at the City of Health & Sciences in Turin, between 1 May 2021 and 31 January 2022, with the primary endpoint to study strains with resistance to C/A, and secondly to describe the characteristics of this population, with or without previous exposure to C/A. Seventeen patients with colonization or invasive infection due to Klebsiella pneumoniae, C/A resistance, and susceptibility to meropenem (MIC = 2 µg/L) were included; the blaKPC genotype was detected in all isolates revealing D179Y mutation in the blaKPC-2 (blaKPC-33) gene. Cluster analysis showed that 16 out of the 17 C/A-resistant KPC-Kp isolates belonged to a single clone. Thirteen strains (76.5%) were isolated in a 60-day period. Only some patients had a previous infection with non-mutant KPC at other sites (5; 29.4%). Eight patients (47.1%) underwent previous large-spectrum antibiotic treatment, and four patients (23.5%) had prior treatment with C/A. The secondary spread of the D179Y mutation in the blaKPC-2 during the COVID-19 pandemic needs to be addressed constantly by an interdisciplinary interaction between microbiologists, infection control personnel, clinicians, and infectious diseases consultants to properly diagnose and treat patients.

A case report of fatal COVID-19 complicated by rapidly progressive sepsis caused by Klebsiella variicola.

BACKGROUND: There is a growing interest in Klebsiella variicola as a causative pathogen in humans, though its clinical features and the impact of co-infection or secondary infection with COVID-19 remain unknown. CASE PRESENTATION: A 71-year-old man presented with fever, altered mental status and generalized weakness and was admitted to ICU due to severe COVID-19 pneumonia. He was newly diagnosed with type II diabetes mellitus upon admission. On hospital day 3, his respiratory status deteriorated, requiring invasive mechanical ventilation. On hospital day 10, superimposed bacterial pneumonia was suspected and subsequently, broad-spectrum antibiotics were administered for the associated bloodstream infection. On hospital day 13, despite administration of active antibiotics and appropriate source control, he decompensated and died. The causative organism isolated from blood cultures was initially reported as K. pneumoniae, but it was identified as K. variicola by a genetic analysis. A representative isolate (FUJ01370) had a novel multilocus sequence typing allelic profile (gapA-infB-mdh-pgi-phoE-rpoB-tonB: 16-24-21-27-52-17-152), to which sequence type 5794 was assigned (GenBank assembly accession: GCA_019042755.1). CONCLUSIONS: We report a fatal case of respiratory and bloodstream infection due to K. variicola complicating severe COVID-19. Co-infection or secondary infection of K. variicola in COVID-19 is likely under-recognized and can be fulminant as in this case.

Isolation, characterization, therapeutic potency, and genomic analysis of a novel bacteriophage vB_KshKPC-M against carbapenemase-producing Klebsiella pneumoniae strains (CRKP) isolated from Ventilator-associated pneumoniae (VAP) infection of COVID-19 patients.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant clinical problem, given the lack of therapeutic options. The CRKP strains have emerged as an essential worldwide healthcare issue during the last 10 years. Global expansion of the CRKP has made it a significant public health hazard. We must consider to novel therapeutic techniques. Bacteriophages are potent restorative cases against infections with multiple drug-resistant bacteria. The Phages offer promising prospects for the treatment of CRKP infections. OBJECTIVE: In this study, a novel K. pneumoniae phage vB_KshKPC-M was isolated, characterized, and sequenced, which was able to infect and lyse Carbapenem-resistant K. pneumoniae host specifically. METHODS: One hundred clinical isolates of K. pneumoniae were collected from patients with COVID-19 associated with ventilator-associated acute pneumonia hospitalized at Shahid Beheshti Hospital, Kashan, Iran, from 2020 to 2021. Initially, all samples were cultured, and bacterial isolates identified by conventional biochemical tests, and then the ureD gene was used by PCR to confirm the isolates. The Antibiotic susceptibility test in the disc diffusion method and Minimum inhibitory concentrations for Colistin was done and interpreted according to guidelines. Phenotypic and molecular methods determined the Carbapenem resistance of isolates. The blaKPC, blaNDM, and blaOXA-23 genes were amplified for this detection. Biofilm determination of CRKP isolates was performed using a quantitative microtiter plate (MTP) method. The phage was isolated from wastewater during the summer season at a specific position from Beheshti Hospital (Kashan, Iran). The sample was processed and purified against the bacterial host, a CRKP strain isolated from a patient suffering from COVID-19 pneumoniae and resistance to Colistin with high potency for biofilm production. This isolate is called Kp100. The separated phages were diluted and titration by the double overlay agar plaque assay. The separate Phage is concentrated with 10% PEG and stored at -80 °C until use. The phage host range was identified by the spot test method. The purified phage morphology was determined using a transmission electron microscope. The phage stability tests (pH and temperature) were analyzed. The effect of cationic ions on phage adsorption was evaluated. The optimal titer of bacteriophage was determined to reduce the concentration of the CRKP strain. One-step growth assays were performed to identify the purified phage burst's latent cycle and size. The SDS-PAGE was used for phage proteins analysis. Phage DNA was extracted by chloroform technique, and the whole genome of lytic phage was sequenced using Illumina HiSeq technology (Illumina, San Diego, CA). For quality assurance and preprocessing, such as trimming, Geneious Prime 2021.2.2 and Spades 3.9.0. The whole genome sequence of the lytic phage is linked to the GenBank database accession number. RASTtk-v1.073 was used to predict and annotate the ORFs. Prediction of ORF was performed using PHASTER software. ResFinder is used to assess the presence of antimicrobial resistance and virulence genes in the genome. The tRNAs can-SE v2.0.6 is used to determine the presence of tRNA in the genome. Linear genome comparisons of phages and visualization of coding regions were performed using Easyfig 2.2.3 and Mauve 2.4.0. Phage lifestyles were predicted using the program PHACTS. Phylogenetic analysis and amino acid sequences of phage core proteins, such as the major capsid protein. Phylogenies were reconstructed using the Neighbor-Joining method with 1000 bootstrap repeat. HHpred software was used to predict depolymerase. In this study, GraphPad Prism version 9.1 was used for the statistical analysis. Student's t-test was used to compare the sets and the control sets, and the significance level was set at P ≤ 0.05. RESULTS: Phage vB_KshKPC-M is assigned to the Siphoviridae, order Caudovirales. It was identified as a linear double-stranded DNA phage of 54,378 bp with 50.08% G + C content, had a relatively broad host range (97.7%), a short latency of 20 min, and a high burst size of 260 PFU/cell, and was maintained stable at different pH (3-11) and temperature (45-65 °C). The vB_KshKPC-M genome contains 91 open-reading frames. No tRNA, antibiotic resistance, toxin, virulence-related genes, or lysogen-forming gene clusters were detected in the phage genome. Comparative genomic analysis revealed that phage vB_KshKPC-M has sequence similarity to the Klebsiella phages, phage 13 (NC_049844.1), phage Sushi (NC_028774.1), phage vB_KpnD_PeteCarol (OL539448.1) and phage PWKp14 (MZ634345.1). CONCLUSION: The broad host range and antibacterial activity make it a promising candidate for future phage therapy applications. The isolated phage was able to lyse most of the antibiotic-resistant clinical isolates. Therefore, this phage can be used alone or as a phage mixture in future studies to control and inhibit respiratory infections caused by these bacteria, especially in treating respiratory infections caused by resistant strains in sick patients.

Gram-negative ESKAPE bacteria bloodstream infections in patients during the COVID-19 pandemic.

Bloodstream infections due to bacteria are a highly consequential nosocomial occurrences and the organisms responsible for them are usually multidrug-resistant. The aims of this study were to describe the incidence of bacteremia caused by Gram-negative ESKAPE bacilli during the COVID-19 pandemic and characterize the clinical and microbiological findings including antimicrobial resistance. A total of 115 Gram-negative ESKAPE isolates were collected from patients with nosocomial bacteremia (18% of the total bacteremias) in a tertiary care center in Mexico City from February 2020 to January 2021. These isolates were more frequently derived from the Respiratory Diseases Ward (27), followed by the Neurosurgery (12), Intensive Care Unit (11), Internal Medicine (11), and Infectious Diseases Unit (7). The most frequently isolated bacteria were Acinetobacter baumannii (34%), followed by Klebsiella pneumoniae (28%), Pseudomonas aeruginosa (23%) and Enterobacter spp (16%). A. baumannii showed the highest levels of multidrug-resistance (100%), followed by K. pneumoniae (87%), Enterobacter spp (34%) and P. aeruginosa (20%). The bla CTX-M-15 and bla TEM-1 genes were identified in all beta-lactam-resistant K. pneumoniae (27), while bla TEM-1 was found in 84.6% (33/39) of A. baumannii isolates. The carbapenemase gene bla OXA-398 was predominant among carbapenem-resistant A. baumannii (74%, 29/39) and bla OXA-24was detected in four isolates. One P. aeruginosa isolate was bla VIM-2 gene carrier, while two K. pneumoniae and one Enterobacter spp were bla NDM gene carriers. Among colistin-resistant isolates mcr-1 gene was not detected. Clonal diversity was observed in K. pneumoniae, P. aeruginosa and Enterobacter spp. Two outbreaks caused by A. baumannii ST208 and ST369 were detected, both belonging to the clonal complex CC92 and IC2. A. baumannii was associated with a death rate of 72% (28/32), most of them (86%, 24/28) extensively drug-resistant or pandrug-resistant isolates, mainly in patients with COVID-19 (86%, 24/28) in the Respiratory Diseases Ward. A. baumannii isolates had a higher mortality rate (72%), which was higher in patients with COVID-19. There was no statistically significant association between the multidrug-resistant profile in Gram-negative ESKAPE bacilli and COVID-19 disease. The results point to the important role of multidrug-resistant Gram-negative ESKAPE bacteria causing bacteremia in nosocomial settings before and during the COVID-19 epidemic. Additionally, we were unable to identify a local impact of the COVID-19 pandemic on antimicrobial resistance rates, at least in the short term.

Trends in carbapenem resistance in Pre-COVID and COVID times in a tertiary care hospital in North India.

BACKGROUND: Carbapenem resistance is endemic in the Indian sub-continent. In this study, carbapenem resistance rates and the prevalence of different carbapenemases were determined in Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa during two periods; Pre-COVID (August to October 2019) and COVID (January to February 2021) in a north-Indian tertiary care hospital. METHODS: Details of patient demographics and clinical condition was collated from the Hospital Information System and detection of carbapenemases NDM, OXA-48, VIM, IMP and KPC was done by Polymerase chain reaction (PCR) in 152 and 138 non-consecutive carbapenem resistant isolates during the two study periods respectively. Conjugation assay and sequencing of NDM and OXA-48 gene was done on a few selected isolates. RESULTS: As compared to Pre-COVID period, co-morbidities and the mortality rates were higher in patients harbouring carbapenem resistant organisms during the COVID period. The overall carbapenem resistance rate for all the four organisms increased from 23 to 41% between the two periods of study; with Pseudomonas aeruginosa and Klebsiella pneumoniae showing significant increase (p < 0.05). OXA-48, NDM and co-expression of NDM and OXA-48 were the most common genotypes detected. NDM-5 and OXA-232 were most common variants of NDM and OXA-48 family respectively during both the study periods. CONCLUSION: Higher rate of carbapenem resistance in COVID times could be attributed to increase in number of patients with co-morbidities. However, genetic elements of carbapenem resistance largely remained the same in the two time periods.

Implementation of Chromatic Super CAZ/AVI® medium for active surveillance of ceftazidime-avibactam resistance: preventing the loop from becoming a spiral.

Acquired resistance towards ceftazidime-avibactam (CAZ-AVI) is increasingly reported. Several mechanisms can be involved, but mutations in the Ω-loop region of ß-lactamases are the most described. Herein, we assessed the implementation of Chromatic Super CAZ/AVI® medium in rectal swab surveillance cultures in a geographic area with endemic distribution of KPC-producing Klebsiella pneumoniae. Routine rectal swabs collected from the intensive care unit (ICU) and non-ICU patients were screened for carbapenemase-producing Enterobacterales (CPE), carbapenem-resistant Gram-negative organisms (CR-GN) and CAZ-AVI-resistant organisms by Chromatic CRE and Super CAZ/AVI® media. Among the 1839 patients screened, 146 (7.9%) were found to be colonized by one or more CPE and/or CR-GN isolates during hospitalization. Overall, among colonized patients the most common bacteria encountered were KPC-producing Enterobacterales (n = 60; 41.1%), carbapenem-resistant Pseudomonas aeruginosa (n = 41; 28.1%) and carbapenem-resistant A. baumannii (n = 34; 23.3%). Among patients colonized by KPC-producing Enterobacterales, thirty-five (58.3%) had CAZ-AVI-resistant strains. A 30.5% rate of faecal carriage of CAZ-AVI-resistant KPC-producing K. pneumoniae, substantially higher than that of susceptible isolates (2.8%), was observed in the COVID-19 ICU. Prevalence of faecal carriage of metallo-ß-lactamase-producing organisms was low (0.5% and 0.2% for Enterobacterales and P. aeruginosa, respectively). Chromatic Super CAZ/AVI® medium showed 100% sensitivity in detecting CPE or CR-GN isolates resistant to CAZ-AVI regardless of both MIC values and carbapenemase content. Specificity was 86.8%. The Chromatic Super CAZ/AVI® medium might be implemented in rectal swab surveillance cultures for identification of patients carrying CAZ-AVI-resistant organisms to contain the spread of these difficult-to-treat pathogens.

Carbapenem-resistant Klebsiella pneumoniae outbreak in a COVID-19 intensive care unit; a case-control study.

We analysed a carbapenem-resistant Klebsiella pneumoniae (CRKP) outbreak in the coronavirus disease (COVID) ICU. We retrospectively collected data from ICU records. We identified 25 cases between 12 November 2020 and 19 December 2020, and compared them to 42 controls present in the ICU during the same period. The presence of a femoral haemodialysis catheter was strongly associated with invasive CRKP infections (cases, 9 [36%]; controls, 0 [0%]; odds ratio [OR] 95% confidence intervals [CIs], 21 (5; 89)). We found a significant association between old age and CRKP infection with adverse outcomes. Sequence analysis revealed three distinct carbapenemase genes: blaNDM-1, blaOXA-48 and blaKPC-2. We launched rectal swab sampling upon admission to the ICU, cohorted colonized patients and cases and conducted an intensive training programme for newly employed staff. This study revealed that the emergence and dissemination of CRKP in COVID ICUs were associated with increased adverse outcomes. The presence of a femoral haemodialysis catheter was a significant risk factor for CRKP infections.

Interplay between Klebsiella pneumoniae producing KPC-31 and KPC-3 under treatment with high dosage meropenem: a case report.

The objective was to study ceftazidime-avibactam resistant and susceptible Klebsiella pneumoniae isolated from a patient admitted to the Policlinico Umberto I of Rome for SARS-CoV2. Data on the evolution of patient's conditions, antimicrobial therapies, and microbiological data were collected. Whole-genome sequencing performed by Illumina and Nanopore sequencing methods were used to type the strains. During the hospitalization, a SARS-CoV2-infected patient was colonized by a KPC-producing K. pneumoniae strain and empirically treated with ceftazidime-avibactam (CZA) when presenting spiking fever symptoms. Successively, ST2502 CZA-resistant strain producing the KPC-31 variant gave a pulmonary infection to the patient. The infection was treated with high doses of meropenem. The KPC-31-producing strain disappeared but the patient remained colonized by a KPC-3-producing K. pneumoniae strain. An interplay between highly conserved KPC-31- and KPC-3-producing ST2502 strains occurred in the SARS-CoV2 patient during the hospitalization, selected by CZA and carbapenem treatments, respectively.

Genetic Diversity, Antimicrobial Resistance Pattern, and Biofilm Formation in Klebsiella pneumoniae Isolated from Patients with Coronavirus Disease 2019 (COVID-19) and Ventilator-Associated Pneumonia.

INTRODUCTION: Patients with acute respiratory distress syndrome caused by coronavirus disease 2019 (COVID-19) are at risk for superadded infections, especially infections caused by multidrug resistant (MDR) pathogens. Before the COVID-19 pandemic, the prevalence of MDR infections, including infections caused by MDR Klebsiella pneumoniae (K. pneumoniae), was very high in Iran. This study is aimed at assessing the genetic diversity, antimicrobial resistance pattern, and biofilm formation in K. pneumoniae isolates obtained from patients with COVID-19 and ventilator-associated pneumonia (VAP) hospitalized in an intensive care unit (ICU) in Iran. METHODS: In this cross-sectional study, seventy K. pneumoniae isolates were obtained from seventy patients with COVID-19 hospitalized in the ICU of Shahid Beheshti hospital, Kashan, Iran, from May to September, 2020. K. pneumoniae was detected through the ureD gene. Antimicrobial susceptibility testing was done using the Kirby-Bauer disc diffusion method, and biofilm was detected using the microtiter plate assay method. Genetic diversity was also analyzed through polymerase chain reaction based on enterobacterial repetitive intergenic consensus (ERIC-PCR). The BioNumerics software (v. 8.0, Applied Maths, Belgium) was used for analyzing the data and drawing dendrogram and minimum spanning tree. Findings. K. pneumoniae isolates had varying levels of resistance to antibiotics meropenem (80.4%), cefepime-aztreonam-piperacillin/tazobactam (70%), tobramycin (61.4%), ciprofloxacin (57.7%), gentamicin (55.7%), and imipenem (50%). Around 77.14% of isolates were MDR, and 42.8% of them formed biofilm. Genetic diversity analysis revealed 28 genotypes (E1-E28) and 74.28% of isolates were grouped into ten clusters (i.e., clusters A-J). Clusters were further categorized into three major clusters, i.e., clusters E, H, and J. Antimicrobial resistance to meropenem, tobramycin, gentamicin, and ciprofloxacin in cluster J was significantly higher than cluster H, denoting significant relationship between ERIC clusters and antimicrobial resistance. However, there was no significant difference among major clusters E, H, and J respecting biofilm formation. CONCLUSION: K. pneumoniae isolates obtained from patients with COVID-19 have high antimicrobial resistance, and 44.2% of them have genetic similarity and can be clustered in three major clusters. There is a significant difference among clusters respecting antimicrobial resistance.

Evaluating the potential for respiratory metagenomics to improve treatment of secondary infection and detection of nosocomial transmission on expanded COVID-19 intensive care units.

BACKGROUND: Clinical metagenomics (CMg) has the potential to be translated from a research tool into routine service to improve antimicrobial treatment and infection control decisions. The SARS-CoV-2 pandemic provides added impetus to realise these benefits, given the increased risk of secondary infection and nosocomial transmission of multi-drug-resistant (MDR) pathogens linked with the expansion of critical care capacity. METHODS: CMg using nanopore sequencing was evaluated in a proof-of-concept study on 43 respiratory samples from 34 intubated patients across seven intensive care units (ICUs) over a 9-week period during the first COVID-19 pandemic wave. RESULTS: An 8-h CMg workflow was 92% sensitive (95% CI, 75-99%) and 82% specific (95% CI, 57-96%) for bacterial identification based on culture-positive and culture-negative samples, respectively. CMg sequencing reported the presence or absence of ß-lactam-resistant genes carried by Enterobacterales that would modify the initial guideline-recommended antibiotics in every case. CMg was also 100% concordant with quantitative PCR for detecting Aspergillus fumigatus from 4 positive and 39 negative samples. Molecular typing using 24-h sequencing data identified an MDR-K. pneumoniae ST307 outbreak involving 4 patients and an MDR-C. striatum outbreak involving 14 patients across three ICUs. CONCLUSION: CMg testing provides accurate pathogen detection and antibiotic resistance prediction in a same-day laboratory workflow, with assembled genomes available the next day for genomic surveillance. The provision of this technology in a service setting could fundamentally change the multi-disciplinary team approach to managing ICU infections. The potential to improve the initial targeted treatment and rapidly detect unsuspected outbreaks of MDR-pathogens justifies further expedited clinical assessment of CMg.

Fecal Carriage and Molecular Characterization of Carbapenemase-Producing Enterobacterales in the Pediatric Population in Qatar.

Whole-genome sequencing was used to characterize carbapenemase-producing Enterobacterales (CPE) strains recovered from rectal screening swab samples obtained from children at a tertiary-care pediatric hospital in Qatar during a 3-year period. A total of 72 CPE isolates recovered from 61 fecal carriers were characterized. Escherichia coli (47 isolates [65.3%]) and Klebsiella pneumoniae (22 isolates [30.6%]) were the most common species identified. High levels of genetic diversity were observed for both species. These 72 isolates produced 78 carbapenemases, characterized as either NDM-type (41 enzymes [52.6%]) or OXA-48-type (37 enzymes [47.4%]). NDM-5 (24 enzymes [30.8%]), NDM-1 (15 enzymes [19.2%]), and OXA-181 (15 enzymes [19.2%]) were the most common variants detected within each type. Twenty-three NDM producers exhibited difficult-to-treat resistance, compared with only 2 of the OXA-48 producers. Multiple comorbidities were identified in 88.5% of the patients, whereas recent travel history to countries in which CPE are endemic was documented for 57.4% of the patients. All 9 blaOXA-48-type-gene-containing E. coli sequence type 38 (ST38) strains were isolated from patients without international travel history. The mean quarterly incidence of fecal carriage decreased more than 6-fold after the implementation of coronavirus disease 2019 (COVID-19)-related international travel restrictions in Qatar in mid-March 2020. Our data suggest that NDM-type and OXA-48-type carbapenemases expressed by a large diversity of E. coli and K. pneumoniae genotypes are largely dominant in the pediatric population of Qatar. Although our data indicate successful local expansion of E. coli ST38 strains harboring blaOXA-244 genes, at least within health care settings, blaOXA-48-type and blaNDM-type genes appear to have been mainly introduced sporadically by asymptomatic carriers who visited or received health care in some nearby countries in which the genes are endemic. IMPORTANCE To the best of our knowledge, this is the first study addressing the molecular characteristics of CPE in a pediatric population in Qatar using whole-genome sequencing. Since several countries in the Arabian Peninsula share relatively similar demographic patterns and international links, it is plausible that the molecular characteristics of CPE in children, at least in the middle and eastern parts of the region, are similar to those observed in our study.

Successful Treatment of Klebsiella pneumoniae NDM Sepsis and Intestinal Decolonization with Ceftazidime/Avibactam Plus Aztreonam Combination in a Patient with TTP Complicated by SARSCoV-2 Nosocomial Infection.

Carbapenem-resistant Enterobacteriaceae (CRE) are a serious public health threat. Infections due to these organisms are associated with significant morbidity and mortality. Among them, metallo-ß-lactamases (MBLs)-producing Klebsiella pneumoniae are of global concern today. The ceftazidime/avibactam combination and the ceftazidime/avibactam + aztreonam combination currently represent the most promising antibiotic strategies to stave off these kinds of infections. We describe the case of a patient affected by thrombotic thrombocytopenic purpura (TTP) admitted in our ICU after developing a hospital-acquired SarsCoV2 interstitial pneumonia during his stay in the hematology department. His medical conditions during his ICU stay were further complicated by a K. Pneumoniae NDM sepsis. To our knowledge, the patient had no risk factors for multidrug-resistant bacteria exposure or contamination during his stay in the hematology department. During his stay in the ICU, we treated the sepsis with a combination therapy of ceftazidime/avibactam + aztreonam. The therapy solved his septic state, allowing for a progressive improvement in his general condition. Moreover, we noticed that the negativization of the hemocultures was also associated to a decontamination of his known rectal colonization. The ceftazidime/avibactam + aztreonam treatment could not only be a valid therapeutic option for these kinds of infections, but it could also be considered as a useful tool in selected patients' intestinal decolonizations.

Outbreak of NDM-1-producing Klebsiella pneumoniae in the intensive care unit during the COVID-19 pandemic: Another nightmare.

An outbreak of Klebsiella pneumoniae producing the carbapenemase NDM-1 occurred in our ICU during the last COVID-19 wave. Twelve patients were tested positive, seven remained asymptomatic whereas 5 developed an infection. Resistome and in silico multilocus sequence typing confirmed the clonal origin of the strains. The identification of a possible environmental reservoir suggested that difficulties in observing optimal bio-cleaning procedures due to workload and exhaustion contributed to the outbreak besides the inappropriate excessive glove use.