Efflux-Related Carbapenem Resistance in Acinetobacter baumannii Is Associated with Two-Component Regulatory Efflux Systems' Alteration and Insertion of ΔAbaR25-Type Island Fragment.

The efflux pumps, beside the class D carbapenem-hydrolysing enzymes (CHLDs), are being increasingly investigated as a mechanism of carbapenem resistance in Acinetobacter baumannii. This study investigates the contribution of efflux mechanism to carbapenem resistance in 61 acquired blaCHDL-genes-carrying A. baumannii clinical strains isolated in Warsaw, Poland. Studies were conducted using phenotypic (susceptibility testing to carbapenems ± efflux pump inhibitors (EPIs)) and molecular (determining expression levels of efflux operon with regulatory-gene and whole genome sequencing (WGS)) methods. EPIs reduced carbapenem resistance of 14/61 isolates. Upregulation (5-67-fold) of adeB was observed together with mutations in the sequences of AdeRS local and of BaeS global regulators in all 15 selected isolates. Long-read WGS of isolate no. AB96 revealed the presence of AbaR25 resistance island and its two disrupted elements: the first contained a duplicate ISAba1-blaOXA-23, and the second was located between adeR and adeA in the efflux operon. This insert was flanked by two copies of ISAba1, and one of them provides a strong promoter for adeABC, elevating the adeB expression levels. Our study for the first time reports the involvement of the insertion of the ΔAbaR25-type resistance island fragment with ISAba1 element upstream the efflux operon in the carbapenem resistance of A. baumannii.

Sustainable access of quality seeds of genetically engineered crops in Eastern Africa - Case study of Bt Cotton.

The genetically engineered bollworm-resistant Bt cotton hybrid varieties offer opportunities for reducing crop losses and enhancing productivity. In Eastern Africa region, Sudan, Ethiopia, and Kenya have approved and released Bt cotton in 2012, in 2018, and in 2019, respectively. The region has potential to grow cotton in over 5 million hectares. For commercial plantings in Ethiopia, Sudan and Kenya, hybrid Bt cotton seeds have been imported from India. Due to the COVID-19 pandemic-induced supply chain disruptions, high shipment costs, bureaucratic procedures for importing seeds, and foreign exchange shortages, farmers have not been able to access Bt cotton seeds. Stakeholders are seeking local production of seeds to provide sustainable access by farmers at affordable cost. Country case studies reveal the importance of enhancing capacity for local seed production and extension advisory services. Revival of the cotton sector needs enhanced public-private partnerships to pave the way for sustainable seeds access in the region.

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.

Acquisition of T6SS Effector TseL Contributes to the Emerging of Novel Epidemic Strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen with multiple strategies to interact with other microbes and host cells, gaining fitness in complicated infection sites. The contact-dependent type VI secretion system (T6SS) is one critical secretion apparatus involved in both interbacterial competition and pathogenesis. To date, only limited numbers of T6SS-effectors have been clearly characterized in P. aeruginosa laboratory strains, and the importance of T6SS diversity in the evolution of clinical P. aeruginosa remains unclear. Recently, we characterized a P. aeruginosa clinical strain LYSZa7 from a COVID-19 patient, which adopted complex genetic adaptations toward chronic infections. Bioinformatic analysis has revealed a putative type VI secretion system (T6SS) dependent lipase effector in LYSZa7, which is a homologue of TseL in Vibrio cholerae and is widely distributed in pathogens. We experimentally validated that this TseL homologue belongs to the Tle2, a subfamily of T6SS-lipase effectors; thereby, we name this effector TseL (TseLPA in this work). Further, we showed the lipase-dependent bacterial toxicity of TseLPA, which primarily targets bacterial periplasm. The toxicity of TseLPA can be neutralized by two immunity proteins, TsiP1 and TsiP2, which are encoded upstream of tseL. In addition, we proved this TseLPA contributes to bacterial pathogenesis by promoting bacterial internalization into host cells. Our study suggests that clinical bacterial strains employ a diversified group of T6SS effectors for interbacterial competition and might contribute to emerging of new epidemic clonal lineages. IMPORTANCE Pseudomonas aeruginosa is one predominant pathogen that causes hospital-acquired infections and is one of the commonest coinfecting bacteria in immunocompromised patients and chronic wounds. This bacterium harbors a diverse accessory genome with a high frequency of gene recombination, rendering its population highly heterogeneous. Numerous Pa lineages coexist in the biofilm, where successful epidemic clonal lineage or strain-specific type commonly acquires genes to increase its fitness over the other organisms. Current studies of Pa genomic diversity commonly focused on antibiotic resistant genes and novel phages, overlooking the contribution of type VI secretion system (T6SS). We characterized a Pa clinical strain LYSZa7 from a COVID-19 patient, which adopted complex genetic adaptations toward chronic infections. We report, in this study, a novel T6SS-lipase effector that is broadly distributed in Pa clinical isolates and other predominant pathogens. The study suggests that hospital transmission may raise the emergence of new epidemic clonal lineages with specified T6SS effectors.

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.

Increased Detection of Carbapenemase-Producing Enterobacterales Bacteria in Latin America and the Caribbean during the COVID-19 Pandemic.

During 2020-2021, countries in Latin America and the Caribbean reported clinical emergence of carbapenemase-producing Enterobacterales that had not been previously characterized locally, increased prevalence of carbapenemases that had previously been detected, and co-production of multiple carbapenemases in some isolates. These increases were likely fueled by changes related to the COVID-19 pandemic, including empirical antibiotic use for potential COVID-19-related bacterial infections and healthcare limitations resulting from the rapid rise in COVID-19 cases. Strengthening antimicrobial resistance surveillance, epidemiologic research, and infection prevention and control programs and antimicrobial stewardship in clinical settings can help prevent emergence and transmission of carbapenemase-producing Enterobacterales.

A protein-protein interaction map reveals that the Coxiella burnetii effector CirB inhibits host proteasome activity.

Coxiella burnetii is the etiological agent of the zoonotic disease Q fever, which is featured by its ability to replicate in acid vacuoles resembling the lysosomal network. One key virulence determinant of C. burnetii is the Dot/Icm system that transfers more than 150 effector proteins into host cells. These effectors function to construct the lysosome-like compartment permissive for bacterial replication, but the functions of most of these effectors remain elusive. In this study, we used an affinity tag purification mass spectrometry (AP-MS) approach to generate a C. burnetii-human protein-protein interaction (PPI) map involving 53 C. burnetii effectors and 3480 host proteins. This PPI map revealed that the C. burnetii effector CBU0425 (designated CirB) interacts with most subunits of the 20S core proteasome. We found that ectopically expressed CirB inhibits hydrolytic activity of the proteasome. In addition, overexpression of CirB in C. burnetii caused dramatic inhibition of proteasome activity in host cells, while knocking down CirB expression alleviated such inhibitory effects. Moreover, we showed that a region of CirB that spans residues 91-120 binds to the proteasome subunit PSMB5 (beta 5). Finally, PSMB5 knockdown promotes C. burnetii virulence, highlighting the importance of proteasome activity modulation during the course of C. burnetii infection.

Multidrug-resistant OXA-48/CTX-M-15 Klebsiella pneumoniae cluster in a COVID-19 intensive care unit: salient lessons for infection prevention and control during the COVID-19 pandemic.

BACKGROUND: Wards caring for COVID-19 patients, including intensive care units (ICUs), have an important focus on preventing transmission of SARS-CoV-2 to other patients and healthcare workers. AIM: To describe an outbreak of carbapenemase-producing Enterobacterales (CPE) in a COVID-19 ICU and to discuss key infection control measures enabling prompt termination of the cluster. METHODS: CPE were isolated from clinical specimens and screening swabs from intensive care patients with COVID-19 disease and from environmental screening. Whole-genome sequencing analysis was instrumental in informing phylogenetic relationships. FINDINGS: Seven clinical isolates and one environmental carbapenemase-producing Klebsiella pneumoniae isolate - all carrying OXA-48, CTX-M-15 and outer membrane porin mutations in ompK35/ompK36 - were identified with ≤1 single nucleotide polymorphism difference, indicative of clonality. A bundle of infection control interventions including careful adherence with contact precautions and hand hygiene, twice weekly screening for multidrug-resistant organisms, strict antimicrobial stewardship, and enhanced cleaning protocols promptly terminated the outbreak. CONCLUSION: Prolonged use of personal protective equipment is common with donning and doffing stations at the ward entrance, leaving healthcare workers prone to reduced hand hygiene practices between patients. Minimizing transmission of pathogens other than SARS-CoV-2 by careful adherence to normal contact precautions including hand hygiene, even during high patient contact manoeuvres, is critical to prevent outbreaks of multidrug-resistant organisms. Appropriate antimicrobial stewardship and screening for multidrug-resistant organisms must also be maintained throughout surge periods to prevent medium-term escalation in antimicrobial resistance rates. Whole-genome sequencing is highly informative for multidrug-resistant Enterobacterales surveillance strategies.

Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2.

Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/µL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.

Clonal spread of ArmA- and OXA-23-coproducing Acinetobacter baumannii International Clone 2 in Brazil during the first wave of the COVID-19 pandemic.

Introduction. Carbapenem-resistant Acinetobacter baumannii (CRAB) is the primary pathogen causing hospital-acquired infections. The spread of CRAB is mainly driven by the dissemination of resistant clones, and in Latin America, International Clones IC-1 (also known as clonal complex CC1), IC-4 (CC15) and IC-5 (CC79) are the most prevalent.Gap Statement. There are no documented outbreaks of CRAB International Clone 2 (IC-2) reported in Brazil.Aim. To describe a large outbreak of CRAB caused by the uncommon IC-2 in a Brazilian COVID-19 hospital.Methodology. From May 2020 to May 2021, 224 patients infected or colonized with CRAB were identified in a single hospital; 92 % of them were also infected with SARS-CoV-2. From these patients, 137 isolates were recovered and subjected to antimicrobial susceptibility testing, PCR analysis and molecular typing. Whole-genome sequencing and downstream analysis were carried out on a representative isolate (the first available isolate).Results. In 76 % of the patients, a single OXA-23-producing CRAB IC-2 was identified. All the isolates were susceptible to polymyxin B, but highly resistant (>95 %) to aminoglycosides, fluoroquinolones and beta-lactams. Genomic analysis revealed that the representative isolate also carried the 16S rRNA Methylase ArmA, which was detected for the first time in this species in Brazil.Conclusion. We report the rapid spread of an emerging CRAB clone responsible for causing a large outbreak in a hospital in Brazil, a country with predominance of other CRAB clones. Continuous and prospective surveillance is warranted to evaluate the impact of this clone in Brazilian hospital settings.

Characterization of a Programmable Argonaute Nuclease from the Mesophilic Bacterium Rummeliibacillus suwonensis.

Prokaryotic Argonautes (pAgos) from mesophilic bacteria are attracting increasing attention for their genome editing potential. So far, it has been reported that KmAgo from Kurthia massiliensis can utilize DNA and RNA guide of any sequence to effectively cleave DNA and RNA targets. Here we find that three active pAgos, which have about 50% sequence identity with KmAgo, possess typical DNA-guided DNA target cleavage ability. Among them, RsuAgo from Rummeliibacillus suwonensis is mainly explored for which can cleave both DNA and RNA targets. Interestingly, RsuAgo-mediated RNA target cleavage occurs only with short guide DNAs in a narrow length range (16-20 nt), and mismatches between the guide and target sequence greatly affect the efficiency of RNA target cleavage. RsuAgo-mediated target cleavage shows a preference for a guide strand with a 5'-terminal A residue. Furthermore, we have found that RsuAgo can cleave double-stranded DNA in a low-salt buffer at 37 °C. These properties of RsuAgo provide a new tool for DNA and RNA manipulation at moderate temperatures.

Specific length and structure rather than high thermodynamic stability enable regulatory mRNA stem-loops to pause translation.

Translating ribosomes unwind mRNA secondary structures by three basepairs each elongation cycle. Despite the ribosome helicase, certain mRNA stem-loops stimulate programmed ribosomal frameshift by inhibiting translation elongation. Here, using mutagenesis, biochemical and single-molecule experiments, we examine whether high stability of three basepairs, which are unwound by the translating ribosome, is critical for inducing ribosome pauses. We find that encountering frameshift-inducing mRNA stem-loops from the E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) hinders A-site tRNA binding and slows down ribosome translocation by 15-20 folds. By contrast, unwinding of first three basepairs adjacent to the mRNA entry channel slows down the translating ribosome by only 2-3 folds. Rather than high thermodynamic stability, specific length and structure enable regulatory mRNA stem-loops to stall translation by forming inhibitory interactions with the ribosome. Our data provide the basis for rationalizing transcriptome-wide studies of translation and searching for novel regulatory mRNA stem-loops.

Listeria toxin promotes phosphorylation of the inflammasome adaptor ASC through Lyn and Syk to exacerbate pathogen expansion.

Inflammasome activation exacerbates infectious disease caused by pathogens such as Listeria monocytogenes, Staphylococcus aureus, and severe acute respiratory syndrome coronavirus 2. Although these pathogens activate host inflammasomes to regulate pathogen expansion, the mechanisms by which pathogen toxins contribute to inflammasome activation remain poorly understood. Here we show that activation of inflammasomes by Listeria infection is promoted by amino acid residue T223 of listeriolysin O (LLO) independently of its pore-forming activity. LLO T223 is critical for phosphorylation of the inflammasome adaptor ASC at amino acid residue Y144 through Lyn-Syk signaling, which is essential for ASC oligomerization. Notably, a Listeria mutant expressing LLO T223A is impaired in inducing ASC phosphorylation and inflammasome activation. Furthermore, the virulence of LLO T223A mutant is markedly attenuated in vivo due to impaired ability to activate the inflammasome. Our results reveal a function of a pathogen toxin that exacerbates infection by promoting phosphorylation of ASC.

Cryptococcal Protease(s) and the Activation of SARS-CoV-2 Spike (S) Protein.

In this contribution, we report on the possibility that cryptococcal protease(s) could activate the SARS-CoV-2 spike (S) protein. The S protein is documented to have a unique four-amino-acid sequence (underlined, SPRRAR↓S) at the interface between the S1 and S2 sites, that serves as a cleavage site for the human protease, furin. We compared the biochemical efficiency of cryptococcal protease(s) and furin to mediate the proteolytic cleavage of the S1/S2 site in a fluorogenic peptide. We show that cryptococcal protease(s) processes this site in a manner comparable to the efficiency of furin (p > 0.581). We conclude the paper by discussing the impact of these findings in the context of a SARS-CoV-2 disease manifesting while there is an underlying cryptococcal infection.

Occurrence of multi-carbapenemases producers among carbapenemase-producing Enterobacterales and in vitro activity of combinations including cefiderocol, ceftazidime-avibactam, meropenem-vaborbactam, and aztreonam in the COVID-19 era.

PURPOSE: To evaluate the prevalence of multi-carbapenemase-producing Enterobacterales (EB) and the activity of cefiderocol (CFDC), meropenem-vaborbactam (MEV), ceftazidime-avibactam (CZA), and combinations of CZA plus aztreonam (ATM), MEV plus ATM and CFDC plus CZA against them. METHODS: A collection of carbapenemase-producing EB clinical isolates (n = 1242) was investigated by lateral flow immunoassay NG-Test CARBA-5 and molecular testing. Cefiderocol MICs were determined using broth microdilution SensititreTM panel. MICs of CZA and MEV were determined by the gradient diffusion method. Antimicrobial synergy testing was performed using gradient diffusion strip crossing. RESULTS: KPC were the most frequent carbapenemases (83.2%), followed by VIM (9.2 %), OXA-48-like (4.3 %) and NDM enzymes (4.1%). Multi-carbapenemase producers were found in 10 (0.8%) isolates. Three combinations of two different carbapenemases were observed: KPC+VIM (n = 4), NDM+OXA-48-like (n = 4), and VIM+OXA-48-like (n = 2). CFDC showed potent activity against eight out of ten dual-carbapenemases producers, while resistance or reduced susceptibility was shown towards CZA and MEV. CFDC in combination with CZA showed no synergistic effects and only two additive effects on seven (87.5%) of the CFDC-susceptible strains. Conversely, CZA plus ATM and MEV plus ATM combinations were synergistic against all ATM-resistant strains regardless of dual-carbapenemases phenotype. CONCLUSIONS: The occurrence of multi-carbapenemase producers is not uncommon in Northern Italy area. MEV in combination with ATM might be considered as a potential therapeutic option, alternative to CZA plus ATM. CFDC susceptibility testing and synergy evaluation of ATM-based combinations should be performed in the lab routine to evaluate the most in vitro active antimicrobial regimen.

Spread of hypervirulent multidrug-resistant ST147 Klebsiella pneumoniae in patients with severe COVID-19: an observational study from Italy, 2020-21.

OBJECTIVES: To report an outbreak of hypervirulent Klebsiella pneumoniae (hvKp) in COVID-19 patients. METHODS: Prospective, observational study including consecutive COVID-19 patients with hvKp infections admitted to the University Hospital of Pisa (Italy). Clinical data and outcome of patients were collected. All patients were followed-up to 30 days from the diagnosis of infection. Mortality within 30 days of the diagnosis of hvKp infection was reported. The hypermucoviscous phenotype was determined by the 'string test'. Molecular typing was performed on three strains collected during different periods of the outbreak. The strains underwent whole genome sequencing using the Illumina MiSeq instrument. The complete circular assemblies were also obtained for the chromosome and a large plasmid using the Unicycler tool. RESULTS: From November 2020 to March 2021, hvKp has been isolated from 36 COVID-19 patients: 29/36 (80.6%) had infections (15 bloodstream infections, 8 ventilator-associated pneumonias and 6 complicated urinary tract infections), while 7/36 (19.4%) had colonization (3 urine, 2 rectal and 2 skin). The isolates belonged to ST147 and their plasmid carried three replicons of the IncFIB (Mar), IncR and IncHI1B types and several resistance genes, including the rmpADC genes encoding enhancers of capsular synthesis. The hvKp isolates displayed an ESBL phenotype, with resistance to piperacillin/tazobactam and ceftolozane/tazobactam and susceptibility only to meropenem and ceftazidime/avibactam. The majority of patients were treated with meropenem alone or in combination with fosfomycin. Thirty-day mortality was 48.3% (14/29). CONCLUSIONS: ST147 ESBL-producing hvKp is associated with high mortality in COVID-19 patients. Strict microbiological surveillance and infection control measures are needed in this population.

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.

Equivalence of the GeneXpert System and GeneXpert Omni System for tuberculosis and rifampicin resistance detection.

A laboratory validation study was conducted to assess the equivalence of Xpert MTB/RIF Ultra testing on the GeneXpert System and the GeneXpert Omni System ('Omni') for tuberculosis and rifampicin resistance. High concordance of the two devices was demonstrated for well-characterized clinical samples as well as control materials, with controls tested on Omni at normal and challenging environmental conditions (i.e. 35°C, 90% relative humidity). Equivalence of the Cts for all probes was also shown. Equivalence was demonstrated for the Omni and GeneXpert devices for tuberculosis and rifampicin resistance detection for a diverse range of clinical specimens and environmental conditions.