Multi-Locus Sequence Typing (MLST) is a key method for allocation of Sequence Types (STs) for bacterial isolates. Traditionally, this is performed by the Sanger sequencing method, which can be highly time-consuming and laborious. In this study, we present NanoMLST, a high-throughput MLST workflow using multiplex PCR, Oxford Nanopore Technologies Next-Generation Sequencing, and the Krocus program for typing ESKAPE + E pathogens (Enterococcus faecium [E. faecium], Staphylococcus aureus, Klebsiella pneumoniae [K. pneumoniae], Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli). Bacterial isolates were obtained from the Hospital Universitario La Paz's Microbiology Department and the Centro Nacional de Microbiología. Primers that can be multiplexed in a single PCR reaction were designed for the seven housekeeping genes for each species. DNA was extracted from single colonies by heating at 95°C for 10 min, mechanical lysis at 4.20 m/s for 2 min, and then by the MagCore extraction system. Multiplex PCRs were then performed with the respective primer mixes for each species, and libraries were prepared for sequencing by ONT Flongle cells. The Krocus program was then used to determine the STs from the raw FastQ reads. STs for 221 isolates were obtained through this workflow with an average time of 12 h per 24 isolates. In line with local data, the K. pneumoniae and E. faecium isolates were relatively oligoclonal, while the rest were polyclonal. STs from representative isolates showed 100% concordance between Sanger sequencing and the proposed workflow. NanoMLST offers a fast, cheaper, and less labor-intensive alternative for large-scale MLST applications targeting clinically important pathogens.

Patients in burn intensive care units (BICUs) are at high risk of infections caused by multidrug-resistant pathogens, which can lead to hospital outbreaks. To investigate an outbreak caused by carbapenem-resistant Acinetobacter baumannii (CRAB) in a BICU at a tertiary care hospital in Madrid, Spain, using whole-genome sequencing (WGS) and antibiotic susceptibility testing. CRAB isolates were obtained from rectal swabs collected during routine epidemiological screening of BICU patients, as well as clinical and environmental samples collected throughout the outbreak. The genomes of the isolates were sequenced using Ion Torrent™ and MinION™ technologies. Antibiotic susceptibility testing was conducted by microdilution using the MicroScan NF50 panel. Susceptibility to cefiderocol was tested using disk diffusion and broth microdilution (ComASP®). The outbreak occurred between October 2022 and August 2023, affecting 26 patients (median age: 56 years, IQR 34–70). Genomic analysis identified the isolates as belonging to ST2/ST451 (Pasteur/Oxford schemes), carrying blaOXA−23 carbapenemase gene. Environmental sampling detected CRAB on 20 of 105 tested surfaces. The outbreak’s genomic evolution included the emergence of hypermucoid isolates and blaADC−73 mutants with increased cefiderocol minimum inhibitory concentrations (MICs). Novel mutations and combinations in blaADC−73 were observed, likely selected under cefiderocol pressure. Antibiotic susceptibility testing using the microdilution method was more sensitive in detecting these mutations. The outbreak ultimately necessitated the closure of the BICU for disinfection with high-concentration H2O2. WGS enabled detailed tracking of CRAB’s genomic evolution during the outbreak, identifying mutations associated with increased cefiderocol MICs. This information supported effective infection control measures, highlighting the utility of WGS in managing hospital outbreaks.

Over 60 years ago, researchers started the genetic analysis of bacterial cell division by isolating conditional, temperature-sensitive mutants of essential Escherichia coli cell division genes. These early mutants were obtained by mutagenesis with chemical agents that introduced dozens to hundreds of mutations in the bacterial genomes. In this work, we present the complete genome sequences of six of these original mutants on ftsA, ftsZ and ftsQ genes, along with two of the strains used to generate them. The genomes of mutants obtained by exposure to nitrosoguanidine had 100 to 400 mutations. Transducing target alleles into a new strain effectively reduced the number of mutations, but those near the target gene were co-transduced with it. In contrast, a mutant generated by site-directed mutagenesis maintained the genomic background intact. The genomic analysis improves our understanding of these foundational strains, offering insights into the effects of historical mutagenesis techniques. These findings underscore the importance of genomic characterization in ensuring accurate interpretations of experimental results in microbiological research.

IntroductionCarbapenemase-Producing Escherichia coli (CP-Eco) isolates, though less prevalent than other CP-Enterobacterales, have the capacity to rapidly disseminate antibiotic resistance genes (ARGs) and cause serious difficult-to-treat infections. The aim of this study is phenotypically and genotypically characterizing CP-Eco isolates collected from Spain to better understand their resistance mechanisms and population structure.MethodsNinety representative isolates received from 2015 to 2020 from 25 provinces and 59 hospitals Spanish hospitals were included. Antibiotic susceptibility was determined according to EUCAST guidelines and whole-genome sequencing was performed. Antibiotic resistance and virulence-associated genes, phylogeny and population structure, and carbapenemase genes-carrying plasmids were analyzed.Results and discussionThe 90 CP-Eco isolates were highly polyclonal, where the most prevalent was ST131, detected in 14 (15.6%) of the isolates. The carbapenemase genes detected were blaOXA-48 (45.6%), blaVIM-1 (23.3%), blaNDM-1 (7.8%), blaKPC-3 (6.7%), and blaNDM-5 (6.7%). Forty (44.4%) were resistant to 6 or more antibiotic groups and the most active antibiotics were colistin (98.9%), plazomicin (92.2%) and cefiderocol (92.2%). Four of the seven cefiderocol-resistant isolates belonged to ST167 and six harbored blaNDM. Five of the plazomicin-resistant isolates harbored rmt. IncL plasmids were the most frequent (45.7%) and eight of these harbored blaVIM-1. blaOXA-48 was found in IncF plasmids in eight isolates. Metallo-β-lactamases were more frequent in isolates with resistance to six or more antibiotic groups, with their genes often present on the same plasmid/integron. ST131 isolates were associated with sat and pap virulence genes. This study highlights the genetic versatility of CP-Eco and its potential to disseminate ARGs and cause community and nosocomial infections.

Nanoparticles and nanocomposites with low concentration in the body are non-toxic and can be suitable alternative to antibiotics. Gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), and silver-gold nanocomposite (Ag-Au NCs) were synthesized for the first time using galangin (GA) from Suaeda maritima (SM). A simple method was studied and optimized for the biosynthesis of AuNPs, AgNPs, and Ag-Au NCs using GA of SM. A variety of phytochemical compounds found in SM, such as phenols, convert metal into ions and serve as capping agents. The SM phytochemical analysis indicates a high concentration of GA in leaf shell extract, which is a flavonoid with anti-tumor properties. Characterization of GASMAuNPs, GASMAgNPs, and GASMAg-Au NCs was performed using a variety of spectroscopy and microscopy techniques. Based on transmission electron microscopy and field emission scanning electron microscopy, the size of the GASM-Ag-AuNCs (45 nm), GASM-AgNPs (50 nm) and GASM-AuNPs (7 nm) and was spherical. The cytotoxicity effects of these nanoparticles and nanocomposites on Michigan Cancer Foundation-7 (MCF-7) breast cancer cell lines were studied. GA-SM-Ag-AuNCs reduced the cell viability of MCF-7 breast cancer cell lines. Antibacterial activities were evaluated against Staphylococcus aureus, Klebsiella pneumoniae, Proteus mirabilis, and Escherichia coli. GA-SM-Ag-Au NCs had maximum inhibition zones for Escherichia coli and Staphylococcus aureus. The maximum inhibition zones observed with GA-SM-Ag-AuNCs were higher than clindamycin, gentamycin, and tetracycline. The minimum inhibitory concentration (MIC) of GA-SM-Ag-AuNCs against Escherichia coli and Staphylococcus aureus was 15.6 μgmL−1. GA-SM-Ag-AuNCs have powerful antimicrobial potential, while having less cytotoxicity at low concentrations. The GA-SM-Ag-AuNCs reduced the cytotoxicity effects on MCF-7. This study showed that GASM-Ag-AuNCs synthesized using GA of SM has antibacterial and anticancer properties with fewer side effects and significant efficacy, which can be effective as a potential therapy in the future.

IntroductionIntestinal colonization by Multi-Drug Resistant Organisms (MDROs) can pose a threat on the health of critically ill patients. The extent of colonization by these organisms is related to previous antibiotic treatments and their ability to cause infections among adult patients. The aim of this study is to determine the relationship between the intestinal Relative Loads (RLs) of selected antibiotic resistance genes, antibiotic consumption and extra-intestinal spread among critically ill pediatric patients.MethodsRLs of blaCTX-M-1-Family, blaOXA-1, blaOXA-48 and blaVIM were determined in 382 rectal swabs obtained from 90 pediatric critically ill patients using qPCRs. The RLs were compared to the patients’ demographics, antibiotic consumption, and detection of MDROs from extra-intestinal sites. 16SrDNA metagenomic sequencing was performed for 40 samples and clonality analyses were done for representative isolates.Results and discussion76 (74.45%) patients from which 340 (89.01%) rectal swabs were collected had at least one swab that was positive for one of the tested genes. Routine cultures did not identify carbapenemases in 32 (45.1%) and 78 (58.2%) swabs that were positive by PCR for blaOXA-48 and blaVIM, respectively. RLs of above 6.5% were associated with extra-intestinal spread of blaOXA-48-harboring MDROs. Consumption of carbapenems, non-carbapenem β-lactams, and glycopeptides were statistically associated with testing negative for blaCTX-M-1-Family and blaOXA-1 while the consumption of trimethoprim/sulfamethoxazole and aminoglycosides was associated with testing negative for blaOXA-48 (P<0.05). In conclusion, targeted qPCRs can be used to determine the extent of intestinal dominance by antibiotic resistant opportunistic pathogens and their potential to cause extra-intestinal infections among a critically ill pediatric population.

Objectives To evaluate the performance of oral saliva swab (OSS) reverse transcription PCR (RT-PCR) compared with RT-PCR and antigen rapid diagnostic test (Ag-RDT) on nasopharyngeal swabs (NPS) for SARS-CoV-2 in children. Design Cross-sectional multicentre diagnostic study. Setting Study nested in a prospective, observational cohort (EPICO-AEP) performed between February and March 2021 including 10 hospitals in Spain. Patients Children from 0 to 18 years with symptoms compatible with Covid-19 of ≤5 days of duration were included. Two NPS samples (Ag-RDT and RT-PCR) and one OSS sample for RT-PCR were collected. Main outcome Performance of Ag-RDT and RT-PCR on NPS and RT-PCR on OSS sample for SARS-CoV-2. Results 1174 children were included, aged 3.8 years (IQR 1.7–9.0); 73/1174 (6.2%) patients tested positive by at least one of the techniques. Sensitivity and specificity of OSS RT-PCR were 72.1% (95% CI 59.7 to 81.9) and 99.6% (95% CI 99 to 99.9), respectively, versus 61.8% (95% CI 49.1 to 73) and 99.9% (95% CI 99.4 to 100) for the Ag-RDT. Kappa index was 0.79 (95% CI 0.72 to 0.88) for OSS RT-PCR and 0.74 (95% CI 0.65 to 0.84) for Ag-RDT versus NPS RT-PCR. Conclusions RT-PCR on the OSS sample is an accurate option for SARS-CoV-2 testing in children. A less intrusive technique for younger patients, who usually are tested frequently, might increase the number of patients tested. The results from this multi-centre prospective diagnostic test accuracy study suggest that molecular testing of oral saliva samples for SARS-CoV-2 in children is an accurate and less invasive alternative to molecular testing of nasopharyngeal/oropharyngeal swabs.

In this study, we track the relative intestinal loads of antibiotic resistance genes among pediatric liver transplant patients and determine the relationship between this load, antibiotic consumption, and infections caused by antibiotic-resistant organisms. We demonstrate that the consumption of broad spectrum antibiotics increase this load and decrease the gut microbial diversity among these patients.

The population structure of a set of OXA- 48-producing Klebsiella pneumoniae isolates belonging to sequence type 11 (ST11 KpOXA) and obtained from two hospitals in Madrid in the period from 2012 to 2015 was studied by genome sequencing. Overall, 97 ST11 Kp- OXA isolates were sequenced and their population structure and demography were studied by Bayesian phylodynamic analysis using core-genome SNVs. In total, 92 isolates were from Hospital La Paz, 57 of them from two selected units. The remaining five isolates were from different units of Hospital Doce de Octubre. Altogether, 96 out of the 97 ST11 Kp- OXA isolates could be ascribed to a single lineage that evolved into three sublineages. Demographic inference showed an expansion of the ST11 Kp- OXA in the first half of 2013 in agreement with the registered incidences. Dated phylogeny showed transmission clusters within hospital wards, between wards and between hospitals. The ST11 Kp- OXA outbreak in Hospital La Paz was largely due to the expansion of a single clone that was transmitted between different units and to Hospital Doce de Octubre. This clone diverged into three sub-lineages and spread out following a mixed mode of neutral core-genome evolution with some features of antibiotic selection, frequent large deletions and plasmid loss and gain events.

Vaborbactam is a novel boron-based β-lactamase inhibitor with high activity against Ambler class A and C enzymes, including KPC carbapenemases [ [1] Lomovskaya O. Sun D. Rubio-Aparicio D. Nelson K. Tsivkovski R. Griffith D.C. et al. Vaborbactam: spectrum of beta-lactamase inhibition and impact of resistance mechanisms on activity in Enterobacteriaceae. Antimicrob Agents Chemother. 2017; 61e01443https://doi.org/10.1128/AAC.01443-17 Crossref Scopus (241) Google Scholar , [2] Sun D. Rubio-Aparicio D. Nelson K. Dudley M.N. Lomovskaya O. Meropenem-vaborbactam resistance selection, resistance prevention, and molecular mechanisms in mutants of KPC-producing Klebsiella pneumoniae. Antimicrob Agents Chemother. 2017; 61: 1-14https://doi.org/10.1128/AAC.01694-17 Crossref Scopus (121) Google Scholar ]. The combination of vaborbactam with meropenem restores susceptibility to KPC-producing, carbapenem-resistant Enterobacteriaceae. Resistance to meropenem-vaborbactam (M/V) is uncommon but may occur because of loss of porins in KPC-producing Klebsiella pneumoniae [ [3] Dulyayangkul P. Wan Nur Ismah W.A. Douglas E.J. Avison M.B. Mutation of kvrA causes OmpK35 and OmpK36 porin downregulation and reduced meropenem-vaborbactam susceptibility in KPC-producing Klebsiella pneumoniae. Antimicrob Agents Chemother. 2020; 64: e02208-e02219https://doi.org/10.1128/AAC.02208-19 Crossref PubMed Scopus (18) Google Scholar ].

The EPIC consortium brings together experts from a wide range of fields that include clinical, molecular and basic microbiology, infectious diseases, computational biology and chemistry, drug discovery and design, bioinformatics, biochemistry, biophysics, pharmacology, toxicology, veterinary sciences, environmental sciences, and epidemiology. The main question to be answered by the EPIC alliance is the following: “What is the best approach for data mining on carbapenemase inhibitors and how to translate this data into experiments?” From this forum, we propose that the scientific community think up new strategies to be followed for the discovery of new carbapenemase inhibitors, so that this process is efficient and capable of providing results in the shortest possible time and within acceptable time and economic costs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Madrid, Spain, on 25 February 2020. It increased in frequency very fast and by the end of May more than 70,000 cases had been confirmed by reverse transcription-polymerase chain reaction (RT-PCR). To study the lineages and the diversity of the viral population during this first epidemic wave in Madrid we sequenced 224 SARS-CoV-2 viral genomes collected from three hospitals from February to May 2020. All the known major lineages were found in this set of samples, though B.1 and B.1.5 were the most frequent ones, accounting for more than 60% of the sequences. In parallel with the B lineages and sublineages, the D614G mutation in the Spike protein sequence was detected soon after the detection of the first coronavirus disease 19 (COVID-19) case in Madrid and in two weeks became dominant, being found in 80% of the samples and remaining at this level during all the study periods. The lineage composition of the viral population found in Madrid was more similar to the European population than to the publicly available Spanish data, underlining the role of Madrid as a national and international transport hub. In agreement with this, phylodynamic analysis suggested multiple independent entries before the national lockdown and air transportation restrictions.

(1) Background: We determined the relevance of intestinal dominance by Serratia spp. during a neonatal outbreak over 13 weeks. (2) Methods: Rectal swabs (n = 110) were obtained from 42 neonates. Serratia spp. was cultured from swabs obtained from 13 neonates (Group 1), while the other 29 neonates were culture-negative (Group 2). Total DNA was extracted from rectal swabs, and quantitative PCRs (qPCRs) using Serratia- and 16SrRNA-gene-specific primers were performed. relative intestinal loads (RLs) were determined using ΔΔCt. Clonality was investigated by random amplified polymorphic DNA analysis and whole-genome sequencing. (3) Results: The outbreak was caused by Serratia marcescens during the first eight weeks and Serratia ureilytica during the remaining five weeks. Serratia spp. were detected by qPCR in all Group 1 neonates and eleven Group 2 neonates. RLs of Serratia spp. were higher in Group 1 as compared to Group 2 (6.31% vs. 0.09%, p < 0.05) and in the first swab compared to the last (26.9% vs. 4.37%, p < 0.05). Nine neonates had extraintestinal detection of Serratia spp.; eight of them were infected. RLs of the patients with extraintestinal spread were higher than the rest (2.79% vs. 0.29%, p < 0.05). (4) Conclusions: Intestinal dominance by Serratia spp. plays a role in outbreaks and extraintestinal spread.

We report a case of osteomyelitis due to methicillin-resistant Staphylococcus aureus (MRSA) that is also non-susceptible to vancomycin, dalbavancin, ceftaroline, and ceftobiprole, in the absence of exposure to the latter three antibiotics. It was isolated from a patient with a 26-year history of cranial surgeries and episodes of osteomyelitis. Whole-genome sequencing was performed. It was found to belong to ST247 and the mecA gene was detected within the SSCmec type I (1B) gene cassette that lacked the E447K mutation known to produce resistance to ceftobiprole and ceftaroline. However, mutations in other genes related to resistance to these antibiotics were found.

This protocol is for the preparation of libraries for 96 samples of SARS-CoV-2 using the New England Biolabs NEBNext®Fast DNA Library Prep Set for Ion Torrent™ kit, ARTIC primer pools, the Ion Express Barcodes, and sequencing through Ion Torrent. The library preparation is semi-automated with the open source Opentrons robot. The volumes, concentrations, cycling conditions, and Opentrons protocols have been optimized through a series of experiments and are currently being used at our lab on a weekly basis. The samples used for this protocol were extracted after being obtained from nasopharyngeal swabs, placed in viral transport medium, and mixed 1:1 with AVL buffer (Qiagen). It is advisable to perform a screening for the samples before starting the library preparation by RT-qPCR and select the samples that have Ct values below 30.

•The amount of material collected by nasopharyngeal swabs is imprecise.•The determinations of SARS-CoV-2 viral loads from Cts ignore this error source.•SARS-CoV-2 Cts should be normalized with an internal marker to correct this errors.

In the present study, a fast and straightforward method was presented for the preparation of silver nanoparticles (AgNPs), zinc oxide nanoparticles (ZnNPs), and silver-zinc oxide nanocomposites (Ag-ZnNPs). Biosynthesis was carried out using resin extraction from Pistacia Atlantica as a reductant and capping agent. In-vitro antimicrobial activities of these particles, silver nitrate (AgNO3), and zinc nitrate (Zn (NO3)2) were also evaluated in comparison with resin extracts. Synthesis of the nanoparticles and nanocomposites was characterized and confirmed by ultraviolet-visible spectroscopy (UV–vis), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FESEM) equipped with an energy dispersive spectroscopy (EDS) and transmission electron microscope (TEM). Then, antimicrobial activity was tested against Klebsiella pneumonia, Escherichia coli, Staphylococcus aureus, and Proteus mirabilis using the agar well diffusion method. Maximum inhibition zones were observed for resin-AgZnNPs and resin-ZnNPs (21.1 ± 0.002 mm), demonstrating their antibacterial activity, while Zn (NO3)2 had the smallest inhibition zone (5.4 ± 0.06 mm). These results are promising in terms of their potential ability to be used against pathogenic bacteria.

We used metagenomics to analyze one sputum sample from a patient with symptoms of a respiratory infection that yielded negative results for all pathogens tested. We detected two viral genomes that could be assembled and showed sequence similarity to redondoviruses, a recently described group within the CRESS-DNA viruses. One hundred sputum samples were screened for the presence of these viruses using specific primers. One sample was positive for the same two viruses, and another was positive for one of them. These findings raise questions about a possible role of redondoviruses in respiratory infections in humans.

In this study, two of the polycyclic aromatic hydrocarbons (PAHs) were investigated in water samples of the Arvand and Bahmanshir rivers in Iran. High performance liquid chromatography with Solid phase extraction (HPLC-SPE) was used to analyze the PAHs in warwe. Our results show that the average concentration of benzo (α) pyrene and anthracene in the outlet of the rivers is lower than the permissible amount as compared to the WHO standard.

Currently, antimicrobial resistance is one of the most prominent public health issues. In fact, there is increasing evidence that animals constitute a reservoir of antimicrobial resistance. In collaboration with the Lebanese Ministry of Agriculture, the aim of this study was to determine the prevalence of intestinal carriage of multi-drug-resistant Gram-negative Bacilli in poultry farms at the national level. Between August and December 2015, 981 fecal swabs were obtained from 49 poultry farms distributed across Lebanon. The swabs were subcultured on MacConkey agar supplemented with cefotaxime (2 μg/ml). Isolated strains were identified using MALDI-TOF mass spectrometry. Multilocus sequence typing analysis was performed for Escherichia coli. Phenotypic detection of extended spectrum β-lactamases (ESBL) and AmpC production was performed using double disk synergy and the ampC disk test, respectively. β-lactamase encoding genes blaCTX-M, blaTEM, blaSHV, blaFOX, blaMOX, blaEBC, blaACC, blaDHA, and blaCMY using PCR amplification. Out of 981 fecal swabs obtained, 203 (20.6%) showed bacterial growth on the selective medium. Of the 235 strains isolated, 217 were identified as E. coli (92%), eight as Klebsiella pneumoniae (3%), three as Proteus mirabilis (1%) and three as Enterobacter cloacae (1%). MLST analysis of E. coli isolates showed the presence of ST156, ST5470, ST354, ST155, and ST3224. The phenotypic tests revealed that 43.5, 28.5, and 20.5% of the strains were ampC, ESBL, and ampC/ESBL producers, respectively. The putative TEM gene was detected in 83% of the isolates, SHV in 20%, CTX-M in 53% and CMY ampC β-lactamase gene in 65%. Our study showed that chicken farms in Lebanon are reservoirs of ESBL and AmpC producing Gram-negative bacilli. The level of antibiotic consumption in the Lebanese veterinary medicine should be evaluated. Future studies should focus on the risk factors associated with the acquisition of multi-drug-resistant organisms in farm animals in Lebanon.