Targeted next-generation sequencing of pathogens reveals the profile of secondary infections in COVID-19 patients (preprint)

PURPOSE: To use targeted next-generation sequencing (tNGS) of pathogens for analysing the etiological distribution of secondary infections in patients with severe and critical novel coronavirus pneumonia (COVID-19), to obtain microbial epidemiological data on secondary infections in patients with COVID-19, and to provide a reference for early empirical antibiotic treatment of such patients. METHODS: Patients with infections secondary to severe and critical COVID-19 and hospitalised at the First Affiliated Hospital of Shandong First Medical University between 1 December 2022 and 30 June 2023 were included in the study. The characteristics and etiological distribution of secondary infections in these patients were analysed using tNGS. RESULTS: A total of 95 patients with COVID-19 secondary infections were included in the study, of whom 87.37% had one or more underlying diseases. Forty-eight pathogens were detected, the most common being HSV-4, Candida albicans, Klebsiella pneumoniae, Enterococcus faecium, HSV-1, Staphylococcus aureus, Aspergillus fumigatus, Acinetobacter baumannii, HSV-5, and Stenotrophomonas maltophilia, with Pneumocystis jirovecii being detected in 14.29% of cases. The majority (76.84%) of COVID-19 secondary infections were mixed infections, with mixed viral-bacterial-fungal infections being the most common (28.42%). CONCLUSION: Most secondary infections in severe and critical COVID-19 patients are mixed, with high rates of viral and fungal infections. In clinical settings, monitoring for reactivation or secondary infections by Herpesviridae viruses is crucial; additionally, these patients have a significantly higher rate of P. jirovecii infection. tNGS testing on bronchoalveolar lavage fluid can help determine the aetiology of secondary infections early in COVID-19 patients and assist in choosing appropriate antibiotics.

Changes of respiratory pathogen and clinical features of in acute exacerbation of chronic obstructive pulmonary disease after COVID-19 pandemic (preprint)

Respiratory infections are common causes of acute exacerbation of chronic obstructive lung disease (AECOPD). We explored whether the pathogens causing AECOPD and clinical features changed from before to after the coronavirus disease 2019 (COVID-19) outbreak. We reviewed the medical records of patients hospitalized with AECOPD at four university hospitals between January 2017 and December 2018 and between January 2021 and December. We evaluated 1,180 patients with AECOPD for whom medication histories were available. After the outbreak, the number of patients hospitalized with AECOPD was almost 44% lower compared with before the outbreak. Patients hospitalized with AECOPD after the outbreak were younger (75 vs. 77 years, p = 0.003) and more often stayed at home (96.6% vs. 88.6%, p < 0.001) than patients of AECOPD before the outbreak. Hospital stay was longer after the outbreak than before the outbreak (10 vs. 8 days. p < 0.001). After the COVID-19 outbreak, the identification rates of S. pneumoniae (15.3 vs. 6.2%, p < 0.001) and Hemophilus influenzae (6.4 vs. 2.4%, p = 0.002) decreased, whereas the identification rates of P. aeruginosa (9.4 vs. 13.7%, p = 0.023), Klebsiella pneumoniae (5.3 vs. 9.8%, p = 0.004), and methicillin-resistant Staphylococcus aureus (1.0 vs. 2.8%, p = 0.023) increased. After the outbreak, the identification rate of influenza A decreased (10.4 vs. 1.0%, p = 0.023). After the outbreak, the number of patients hospitalized with AECOPD was lower and the identification rates of community-transmitted pathogens tended to decrease, whereas the rates of pathogens capable of chronic colonization tended to increase. During the period of large-scale viral outbreaks that require quarantine, patients with AECOPD should consider treatment against strains that can colonize chronic respiratory disease rather than community acquired pathogens.

Klebsiella pneumoniae Co-infection Leads to Fatal Pneumonia in SARS-CoV-2-infected Mice (preprint)

SARS-CoV-2 patients have been reported to have high rates of secondary Klebsiella pneumoniae infections. Klebsiella pneumoniae is a commensal that is typically found in the respiratory and gastrointestinal tracts. However, it can cause severe disease when a person's immune system is compromised. Despite a high number of K. pneumoniae cases reported in SARS-CoV-2 patients, a co-infection animal model evaluating the pathogenesis is not available. We describe a mouse model to study disease pathogenesis of SARS-CoV-2 and K. pneumoniae co-infection. BALB/cJ mice were inoculated with mouse-adapted SARS-CoV-2 followed by a challenge with K. pneumoniae. Mice were monitored for body weight change, clinical signs, and survival during infection. The bacterial load, viral titers, immune cell accumulation and phenotype, and histopathology were evaluated in the lungs. The co-infected mice showed severe clinical disease and a higher mortality rate within 48 h of K. pneumoniae infection. The co-infected mice had significantly elevated bacterial load in the lungs, however, viral loads were similar between co-infected and single-infected mice. Histopathology of co-infected mice showed severe bronchointerstitial pneumonia with copious intralesional bacteria. Flow cytometry analysis showed significantly higher numbers of neutrophils and macrophages in the lungs. Collectively, our results demonstrated that co-infection of SARS-CoV-2 with K. pneumoniae causes severe disease with increased mortality in mice.

Identification of Potential Public Health Associated Viruses, Bacteria, and Antimicrobial Resistance Genes in Upper Respiratory Tract of Domestic Cats in China: A Nationwide Genomic Surveillance Study (preprint)

Background: As a highly popular choice for a household pet worldwide, domestic cats may carry pathogens and antimicrobial resistance genes (ARGs) that pose severe risks to human health. However, our knowledge about pathogens and ARGs carried by cats remains limited. We aimed to identify public health-associated pathogens and ARGs in the respiratory tracts of domestic cats.Methods: In this genomic surveillance study, zoonotic viruses were identified through meta-transcriptome sequencing of 1454 oropharyngeal-nasal swabs collected from domestic cats in 22 Chinese provinces; while bacterial species and ARGs were identified using metagenomic sequencing. Potential role of domestic cats in transmitting specific virus such as Zika virus (ZIKV) was assessed in laboratory.Findings: We identified DNAs or RNAs of 85 viral species that can infect humans. Of particular note are SARS-related coronavirus, monkeypox virus, and ZIKV. For the first time, we demonstrated that domestic cats can be infected with and transmit ZIKV via mosquito bites. We also identified a substantial number of bacterial species associated with public health, including the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) which showed a broad detection range. Additionally, a plentiful amount of ARGs, including those conferring resistance to the last resort antibiotics (carbapenems [blaNDM, blaOXA, blaVIM], colistin [mcr], and high-level tigecycline [tetX3, tetX4, tetX5, tetX6]), were also detected broadly.Interpretation: Although additional studies to isolate specific viruses (e.g., ZIKV) are needed, this study still provides comprehensive knowledge about the possible pathogens and ARGs carried by domestic cats from large geographic regions in China. It is important to monitoring domestic cats within the One Health approach to control these critical global public health issues.Funding: This work was supported by National Key Research and Development Program of China (2022YFD1800105, 2022YFD1801500, 2021YFC2600204), National Natural Science Foundation of China (32022082, 31972721), Natural Science Foundation of Hubei Province (2021CFA056), and Fundamental Research Funds for the Central Universities (Project 2662023PY005).Declaration of Interest: All authors declare no competing interests.Ethical Approval: This study was approved by the Animal Management and Ethics Committee of Huazhong Agricultural University. The reference ID number is 202303010001.

A Sequent of Gram-Negative Co-Infectome Induced Severe Res-Piratory Distress Syndrome Are Potential Subtle Aggravators As-Sociated to SARS-CoV-2 Evolution of Virulence (preprint)

The severe respiratory distress syndrome (SRDS) has been a significant clinical issue in modern respiratory medicine. Its role in COVID-19 pandemic is not well defined. Outside-the-hospital SRDS is usually community-acquired septic pneumonia; however, in-hospitals incidences are complicated by co-infections. While SRDS is genetically selected in European and African origins, these are not clear in Middle East, particularly in COVID-19 backgrounds. There is a severe paucity in high quality data on correlations between COVID-19, ARDS, co-infectome, and patient demographics. We have conducted a comprehensive investigation on 298 patients for associations of SRDS, coinfections, and patient demographics on COVID-19 patients’ outcomes. Of these, 9.4% (n=28) had SRDS, and the rest (90.6%) had not. 54% of those with SRDS died while 84% survived; SRDS fatality was highly significant (Chi-square test P-value = 0.00000246). Irrespective of gender, the age of patients was significantly associated with SRDS (72.9 +/-8.9) compared to those without it (56.2 +/-15.1). However, there was no significant difference neither in the age of admitted patients before COVID-19 (58.5 +/-15.3) and during COVID-19 (57.2 +/-15.5) nor in the gender and COVID-19 fatality (Fisher Exact test 2-sided 1.000, 1-sided .546) ruling out that SRDS age-specificity were selected by virus susceptibility. A 100% of SRDS patients without bacterial co-infections survived while only 25% of those with co- infectome did not; this association was highly significant (P value= 0.00041). Co-infections alone without underlying SRDS was also associated with high fatality among patients (P value= 0.00000000076). Almost all COVID-19 patients without co-infectome (99.2%) survived while 28% of those with bacterial co-infection died. The major bacterial pathogens that potentially predisposed to SRDS, were Acinetobacter baumannii, and Escherichia coli either alone or in a mixed infection with Klebsiella pneumoniae were predominant species identified during SRDS attack. Thus, Gram-negative co-infectome potentially induced fatal SRDS aggravating COVID-19 outcome. These findings have significant clinical implications in specific differential diagnosis of SRDS syndromes for subsequent empiric therapy and patient management strategies. Future vertical investigation for similar mechanisms of cytokine-induced SRDS by Gram negative pathogens is imperative since hypervirulent strains are rapidly circulating in the region. The study is limited by a single center study confined to Ha’il hospitals; large scale investigation in major national hospitals would gain more insights.

Green synthesis of biocompatible Fe 3 O 4 magnetic nanoparticles using Citrus Sinensis peels extract for their biological activities and magnetic- hyperthermia applications (preprint)

Plants include active chemicals known as phytochemicals and biomolecules that serve as decreasing and biostability factors for nanoparticle (NP) creation. Citrus Sinensis peels are rich in phenolics, flavonoids, antioxidants, and biophysical benefits. Herein, we prepared superparamagnetic iron oxide nanoparticles (SPIONs) by co-precipitation using Citrus Sinensis peel extract as a novel green synthesis method. The antioxidant, anti-inflammatory, dye degradation activities, and antimicrobial activities of Fe3O4 MNPs were investigated. Furthermore, the produced materials were characterized using FTIR, UV, TEM, VSM, and XRD analysis. The Fe3O4 MNPs showed higher antibacterial activities against multi antibiotic resistant bacterial strains: Escherichia coli, Streptococcus mutans, Candida albicans, Staphylococcus aureus, Bacillus subtilis, and Klebsiella pneumonia. The sample has generated a lot of attention in the scientific community for magnetic hyperthermia (MHT) applications. The maximum value of the specific absorption rate (SAR) was evaluated at sample concentrations of 10mg under the magnetic field condition. Additionally, these newly fabricated SPIONs virtually achieve significant execution under the alternating magnetic field (AMF) in fluid HT and are suitable for biomedical applications.

Molecular Characterization of the Convergent Carbapenem-Resistant and Hypervirulent Klebsiella pneumoniae Strain K1-ST23, Collected in Chile during the COVID-19 Pandemic.

The aim of this study was to investigate the genomic features of a carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) isolate (K-2157) collected in Chile. Antibiotic susceptibility was determined using the disk diffusion and broth microdilution methods. Whole-genome sequencing (WGS) and hybrid assembly were performed, using data generated on the Illumina and Nanopore platforms. The mucoid phenotype was analyzed using both the string test and sedimentation profile. The genomic features of K-2157 (e.g., sequence type, K locus, and mobile genetic elements) were retrieved using different bioinformatic tools. Strain K-2157 exhibited resistance to carbapenems and was identified as a high-risk virulent clone belonging to capsular serotype K1 and sequence type 23 (ST23). Strikingly, K-2157 displayed a resistome composed of ß-lactam resistance genes (blaSHV-190, blaTEM-1, blaOXA-9, and blaKPC-2), the fosfomycin resistance gene fosA, and the fluoroquinolones resistance genes oqxA and oqxB. Moreover, several genes involved in siderophore biosynthesis (ybt, iro, and iuc), bacteriocins (clb), and capsule hyperproduction (plasmid-borne rmpA [prmpA] and prmpA2) were found, which is congruent with the positive string test displayed by K-2157. In addition, K-2157 harbored two plasmids: one of 113,644 bp (KPC+) and another of 230,602 bp, containing virulence genes, in addition to an integrative and conjugative element (ICE) embedded on its chromosome, revealing that the presence of these mobile genetic elements mediates the convergence between virulence and antibiotic resistance. Our report is the first genomic characterization of a hypervirulent and highly resistant K. pneumoniae isolate in Chile, which was collected during the coronavirus disease 2019 (COVID-19) pandemic. Due to their global dissemination and public health impact, genomic surveillance of the spread of convergent high-risk K1-ST23 K. pneumoniae clones should be highly prioritized. IMPORTANCE Klebsiella pneumoniae is a resistant pathogen involved primarily in hospital-acquired infections. This pathogen is characterized by its notorious resistance to last-line antibiotics, such as carbapenems. Moreover, hypervirulent K. pneumoniae (hvKp) isolates, first identified in Southeast Asia, have emerged globally and are able to cause infections in healthy people. Alarmingly, isolates displaying a convergence phenotype of carbapenem resistance and hypervirulence have been detected in several countries, representing a serious threat to public health. In this work, we analyzed the genomic characteristics of a carbapenem-resistant hvKp isolate recovered in 2022 from a patient with COVID-19 in Chile, representing the first analysis of this type in the country. Our results will provide a baseline for the study of these isolates in Chile, which will support the adoption of local measures aimed at controlling their dissemination.

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.

Cryo-EM Structures of the Klebsiella pneumoniae AcrB Multidrug Efflux Pump.

The continued challenges of the COVID-19 pandemic combined with the growing problem of antimicrobial-resistant bacterial infections has severely impacted global health. Specifically, the Gram-negative pathogen Klebsiella pneumoniae is one of the most prevalent causes of secondary bacterial infection in COVID-19 patients, with approximately an 83% mortality rate observed among COVID-19 patients with these bacterial coinfections. K. pneumoniae belongs to the ESKAPE group of pathogens, a group that commonly gives rise to severe infections that are often life-threatening. Recently, K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae has drawn wide public attention, as the mortality rate for this infection can be as high as 71%. The most predominant and clinically important multidrug efflux system in K. pneumoniae is the acriflavine resistance B (AcrB) multidrug efflux pump. This pump mediates resistance to different classes of structurally diverse antimicrobial agents, including quinolones, ß-lactams, tetracyclines, macrolides, aminoglycosides, and chloramphenicol. We here report single-particle cryo-electron microscopy (cryo-EM) structures of K. pneumoniae AcrB, in both the absence and the presence of the antibiotic erythromycin. These structures allow us to elucidate specific pump-drug interactions and pinpoint exactly how this pump recognizes antibiotics. IMPORTANCE Klebsiella pneumoniae has emerged as one of the most problematic and highly antibiotic-resistant pathogens worldwide. It is the second most common causative agent involved in secondary bacterial infection in COVID-19 patients. K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae is a major concern in global public health because of the high mortality rate of this infection. Its drug resistance is due, in a significant part, to active efflux of these bactericides, a major mechanism that K. pneumoniae uses to resist to the action of multiple classes of antibiotics. Here, we report cryo-electron microscopy (cryo-EM) structures of the prevalent and clinically important K. pneumoniae AcrB multidrug efflux pump, in both the absence and the presence of the erythromycin antibiotic. These structures allow us to understand the action mechanism for drug recognition in this pump. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

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.

Increase of multidrug-resistant bacteria after the COVID-19 pandemic in a major teaching Hospital in Sicily (2018-2021) (preprint)

Introduction The COVID-19 pandemic has further highlighted the continuing threat of antimicrobial resistance (AMR) to global health and economic development. In the last two decades, AMR has raised increasing concern with an estimated 4.95 million deaths globally due to bacterial AMR in 2019 alone. This study aims to analyze the impact of the pandemic on the spread of multi-drug resistant organisms (MDROs) using data from the Hospital «P. Giaccone» in Palermo, comparing pre-pandemic and pandemic periods. Methods This observational study involved adult patients discharged from the hospital between 01/01/2018-31/12/2021. Hospital Discharge Cards were linked with microbiological laboratory reports to assess MDRO isolations. SARS-CoV-2 positivity during hospitalization was evaluated using the National Institute of Health's surveillance system. Results This study evaluated 58,427 hospitalizations, where half of the patients were over 65 years old (N=26,984) and most admissions were in the medical area (N=31,716). During the hospitalization period, the isolated MDRO were 2,681 (5%), whereas there were 946 subjects (2%) positive for SARS-CoV-2. In the multivariable analyses, during 2020 and 2021 there was a significant increased risk of isolation of Staphylococcus aureus, Acinetobacter baumannii, and Klebsiella pneumoniae. Age, DRG-weight, and length-of-stay were associated to a higher risk of MDRO isolation. Conclusion Our study provides new insights into the impact of the COVID-19 pandemic on MDRO isolation and has important implications for infection control and prevention efforts in healthcare facilities. Age, DRG-weight, and longer hospital stays further increase the risk of MDRO isolation. Thus, it is imperative to improve and follow hospital protocols to prevent healthcare-associated infections.

Changes and Comparison of drug resistance surveillance in lower respiratory tract specimens from adult isolates before and during the COVID-19 epidemic (preprint)

The aim of this paper is to study the changes in the resistance and distribution of pathogenic bacteria isolated from adult lower respiratory tract specimens from our hospital during the novel coronavirus (COVID-19) epidemic.Antimicrobial susceptibility testing is used for adult lower respiratory tract isolates from 2017 to 2022 using automated micro-broth dilution and Kirby-Bauer methods.The top six pathogens isolated in our hospital from 2017 to 2022 were A.baumannii, K.pneumoniae, P.aeruginosa and S.aureus,E.coli and S.maltophilia. The numbers of strains increased from 1041 in 2017 to 1799 in 2022, antibacterial drug resistance rates also changed. There is decline in the number of ESBLs-producing strains. The rate of isolation of carbapenem-resistant Acinetobacter baumannii (CRAB)decreased to75%、52.1%、61.5%、61.8%、36.8% and 51.6% respectively. Although the number of detected strains of K.pneumoniae increased, the isolation rate of carbapenem-resistant Klebsiella pneumoniae（CRKP）showed a decreasing trend with 12.6%、20.3%、28.3%、17.7%、9.8% and 13.8%, respectively. The Carbapenem-resistant Pseudomonas aeruginosa（CRPA）also increased from 32 strains in 2017 to 67 strains in 2022，but did not increase isolation rates, The separation rate for 2019 is 26.4% and 17.7% in 2020. The isolation rates of methicillin-resistant Staphylococcus aureus(MRSA) showed a decreasing trend during the COVID-19 epidemic, with 58.1%、41.3%、44.0%、34.1%、32.2% and 31.3%, respectively.During the COVID-19 epidemic, the spread of the pathogenic bacteria within the hospital was interrupted by measures such as reducing outings, wearing masks, nucleic acid testing , sanitary environment disinfection and blocking control. The detection rate of ESBLs, CRKP, CRAB and CRPA in adult lower respiratory tract specimens has been generally decreasing, and the detection rate of MRSA has been gradually decreasing.The rate of CRPA did not increase.

Extended-spectrum β-lactamase- producing gram-negative bacterial infections in severely ill COVID-19 patients admitted in a national referral hospital, Kenya (preprint)

Background: Bacterial infections in COVID-19 patients, especially those caused by multidrug-resistant gram-negative strains, are associated with increased morbidity, hospital stay and mortality. However, there is limited data on the epidemiology of extended-spectrum β-lactamase (ESBL)-producing bacteria in COVID-19 patients. Here, we assessed the prevalence and the factors associated with ESBL-producing gram-negative bacteria (GNB) infections among severely ill laboratory-confirmed COVID-19 patients admitted at Kenyatta National Hospital (KNH), Kenya. Methods: We adopted a descriptive cross-sectional study design for patients admitted between October 2021 and February 2022, purposively recruiting 120 participants based on clinical presentation. Demographics and clinical characteristics data were collected using structured questionnaires and case report forms. Clinical samples were collected and analyzed by standard microbiological methods in the KNH Microbiology laboratory and the Centre for Microbiology, Kenya Medical Research Institute. Results: GNB infections prevalence was 40.8%, with the majority caused by ESBL – producers (67.3%) predominated by Klebsiella pneumoniae (45.5%). Generally, 73% of the ESBL producers harboured our target ESBL genes, mainly CTX-M-type (59%, 17/29) in K. pneumoniae (76.9%, 20/26). GNB harbouring TEM-type (83%, 10/12) and SHV-type (100%, 7/7) genes showed ESBLs phenotypes and inhibitor resistance, mainly involving clavulanate, but most of them remained susceptible to tazobactam (60%, 6/10). SHV-type genes carrying ESBL producers showed resistance to both cefotaxime CTX) and ceftazidime (CAZ) (K. pneumoniae), CAZ (E. coli) or CTX (E. cloacae complex and K. pneumoniae). About 87% (20/23) of isolates encoding CTX-M-type β-lactamases displayed the typical CTX/ceftriaxone (CRO) resistance phenotype. About 42% of isolates with CTX-M-type β-lactamases only hydrolyzed ceftazidime (CAZ). Isolates with OXA-type β-lactamases were resistant to CTX, CAZ, CRO, cefepime and aztreonam. Patients with comorbidities were about ten (10) times more likely to have an ESB-producing GNB infection (aOR =9.86, 95%CI: 1.30 – 74.63, p =0.003). Conclusion: We report a high prevalence of ESBL-GNB infections in severely ill COVID-19 patients, predominantly due to Klebsiella pneumoniae harbouring CTX-M type ESBL genes. The patient’s underlying comorbidities increased the risk of ESBL-producing GNB infection. Enhanced systematic and continuous surveillance of ESBL-producing GNB, strict adherence to infection control measures and antimicrobial stewardship policies in the current study setting are warranted.

Trends in the Rates of Extended-Spectrum-ß-Lactamase-Producing Enterobacterales Isolated from Urine Cultures during the COVID-19 Pandemic in Ontario, Canada.

Worldwide, extended-spectrum ß-lactamase (ESBL) rates are increasing at an alarming level with increasing rates of health care exposures, international travel, and antibiotic usage. In this study, we investigated whether enhanced social isolation, travel restrictions, and the reduced use of antibiotics in Ontario, Canada during coronavirus disease 2019 (COVID-19) pandemic had an impact on ESBL rates in urine cultures collected from the community and long-term-care (LTC) facilities across the province. Data from a total of 8.6 million urine cultures performed at LifeLabs Ontario from 2016 to 2021 were utilized for analysis. ESBL-producing Escherichia coli (ESBL Escherichia coli) and ESBL Klebsiella pneumoniae were identified using standard operating procedures. Data trends were estimated by interrupted time series (ITS) regression analysis. Among 2.3 million positive urine cultures, 48.9% and 7.2% grew E. coli and K. pneumoniae, of which 5.8% and 3.3% produced ESBLs, respectively. While the overall rate of ESBL isolation was higher in the pandemic period than in the prepandemic period, by ITS regression analysis of the monthly rates of ESBL isolation, decreasing trends were noted for ESBL E. coli in both the community and LTC facilities and for ESBL K. pneumoniae in the community. The ESBL K. pneumoniae rates in LTC facilities continued to increase throughout the COVID-19 period. By subgroup analysis for different genders, age groups, and local health integration network (LHIN) units, similar trends were seen in most cases (P < 0.05), except for a few densely populated LHINs where rate changes were not statistically significant. IMPORTANCE Community-onset urinary tract infections (UTIs) caused by ESBL-producing Enterobacterales, particularly E. coli and K. pneumoniae, are a major public health concern. In this study, we assessed the impact of COVID-19 on ESBL rates in urine cultures in Ontario, Canada. Our results show the recent epidemiology of ESBL-producing Enterobacterales in urine cultures from both the community and LTC facilities in Ontario, Canada, and the impact of COVID-19 restrictions on ESBL trends for the entire province as well as different subgroups of the population based on demographic and geographic characteristics. Our results may have important public health implications in the context of the gradual easing of COVID-19 restrictions.

Secondary bacterial infections & extensively drug-resistant bacteria among COVID-19 hospitalized patients at the University Hospital in Kraków (preprint)

Introduction: Healthcare-associated infections (HAI) and bacterial antimicrobial resistance posed a therapeutic risk during the coronavirus disease 2019 (COVID-19) pandemic. The aim of this study was to analyze the HAIs in COVID-19 patients in the Intensive Care Unit (ICU) and non-ICU at the University Hospital in Krakow (UHK) with an emphasis on the susceptibility of the most frequently isolated pathogens and the prevalence of extensively drug resistant (XDR) microorganisms. Methods: This laboratory-based study was carried out at the University Hospital in Krakow in the ICU and non-ICUs dedicated to COVID-19 patients between May 2021 and January 2022. All isolates of Klebsiella pneumoniae were analyzed using PFGE protocol. Results: 288 independent HAI cases were identified, with the predominance of urinary tract infections (UTI), especially in the non-ICU setting. The most common ICU syndrome was pneumonia (PNA). The prevalence of XDR organisms was 29.1% in the ICU and 26.4% in non-ICUs among all isolates. The incidence of carbapenem-resistant Enterobacteriaceae infection was 24.8 cases per 10,000 hospitalizations and the carbapenem-resistant A. baumannii infection incidence was 208.8 cases per 10,000 hospitalizations. The prevalence of XDR strains was highest in Acinetobacter spp, in PNA cases. The PFGE typing demonstrated that almost all XDR strains varied widely from each other. Conclusions: In this study, there was a high incidence of HAI in COVID-19 patients. Similarly, the prevalence of XDR microorganisms, especially XDR-A.baumannii, was also high. PFGE did not confirm the horizontal spread of any organism strains.

Assessment of the activity of tigecycline and colistin on biofilm producer Klebsiella pneumoniae isolated from COVID-19 patients, Iran (preprint)

Background Pneumonia caused by β-lactamase-producing Klebsiella pneumoniae is encountered in hospitals. We aimed to investigate the activity of tigecycline, colistin and other selected antibiotics against 27 β-lactamase-producing Klebsiella pneumonia isolated from bronchoalveolar lavage (BAL) in COVID-19 patients.Methods In the current survey phenotypic screening of ESBL, AmpC, and carbapenemase enzymes was carried out. Detection of ESBL, AmpC, and carbapenemase genes was also performed by the PCR method. Biofilm formation was assessed by staining with 1% crystal violet. Finally, a set of the virulence-encoding genes were detected by the PCR method.Results This study demonstrated the high percentage of ESBL and carbapenemase-producing Klebsiella pneumoniae among COVID-19 patients. Tigecycline and colistin were more effective against these isolates. ESBL associated genes were detected in (18 (66.7%) blaTEM, 27 (100%) blaSHV, and 18 (66.7%) blaCTX−M) isolates. Also, carbapenemase-related genes were detected in 16 (59.3%) isolates. The prevalence of these genes were blaOXA−48−like 14(51.9%), and blaNDM1 2 (7.4%). Twenty-seven isolates exhibited biofilm formation. Molecular distribution of virulence genes revealed that 92.59%, 92.5%, 88.88%, 11.11% and 18.5% of the isolates carried entB, mrkD, Irp2, fimH and magA genes, respectively. MLST results for four colistin-resistant isolates showed three different sequence types-ST: ST3500, ST273, and 2 cases of ST2558.Conclusion The results of this study demonstrated the prevalence of infections caused by β-lactamase-producing Klebsiella pneumoniae, which are biofilm producers among respiratory hospitalized Patients. The effective antimicrobial activity of tigecycline to the bacteria that produce these enzymes may be efficient in faster and better treating COVID-19 patients which are hospitalized.