Characterization and genomic analysis of the lytic bacteriophage vB_EclM_HK6 as a potential approach to biocontrol the spread of Enterobacter cloacae contaminating food

Background: Increased prevalence of Enterobacter cloacae within food products underscores food as an underexplored reservoir for antibiotic resistance, thus requiring particular intervention. Bacteriophages have been explored as a promising approach for controlling bacterial growth in different matrices. Moreover, their specific interaction and self-replication, put them apart from traditional methods for controlling bacteria in different matrices. Methods: Sixteen Enterobacter cloacae strains were recovered from raw chicken. These strains were used to isolate bacteriophages using enrichment protocol. The broad-spectrum bacteriophage was evaluated in terms of thermal, pH, shearing stress and storge. Moreover, its infection kinetics, in vitro antibacterial activity, cytotoxicity were also assessed. Genomic sequencing was performed to exclude any potential virulence or resistance genes. Finally, the capability of the isolated phages to control bacterial growth in different chicken samples was assessed alone and in combination with sodium nitrite. Results: The lytic bacteriophage vB_EclM_HK6 was isolated and showed the broadest spectrum being able to infect 8/16 E. cloacae strains with a lytic activity against its host strain, E. cloacae EC21, as low as MOI of 10–6. The phage displays a latent period of 10 min and burst size of 115 ± 44 and resistance frequency of 5.7 × 10–4 ± 3.0 × 10–4. Stability assessment revealed a thermal tolerance up to 60 ˚C, wide range pH stability (3–10) and the ability to withstand shearing stress up to 250 rpm. HK6 shows no cytotoxicity against oral epithelial cells up to 1012 PFU/ml. Genomic analysis revealed a Strabovirus with total size of 177,845 bp that is free from known resistance and virulence genes. Finally, HK6 pretreatment of raw chicken, chicken nuggets and ready-made cheese salad shows a reduced bacterial count up to 4.6, 2.96 and 2.81 log-units, respectively. Moreover, combing HK6 with sodium nitrite further improved the antibacterial activity in both raw chicken and chicken nuggets without significant enhancement in case of cheese salad. Conclusion: Enterobacter bacteriophage vB_EclM_HK6 presents a safe and effective approach for controlling E. cloacae contaminating stored chicken food samples. Moreover, they could be combined with a reduced concentrations of sodium nitrite to improve the killing capacity

Phage-host co-evolution has led to distinct generalized transduction strategies

Generalized transduction is pivotal in bacterial evolution but lacks comprehensive understanding regarding the facilitating features and variations among phages. We addressed this gap by sequencing and comparing the transducing particle content of three different Salmonella Typhimurium phages (i.e. Det7, ES18 and P22) that share a headful packaging mechanism that is typically initiated from a cognate pac site within the phage chromosome. This revealed substantial disparities in both the extent and content of transducing particles among these phages. While Det7 outperformed ES18 in terms of relative number of transducing particles, both phages contrasted with P22 in terms of content. In fact, we found evidence for the presence of conserved P22 pac-like sequences in the host chromosome that direct tremendously increased packaging and transduction frequencies of downstream regions by P22. More specifically, a ca. 561 kb host region between oppositely oriented pac-like sequences in the purF and minE loci was identified as highly packaged and transduced during both P22 prophage induction and lytic infection. Our findings underscore the evolution of phage transducing capacity towards attenuation, promiscuity or directionality, and suggest that pac-like sequences in the host chromosome could become selected as sites directing high frequency of transduction

AN OUTBREAK OF EXTENSIVELY DRUG-RESISTANT <i>ACINETOBACTER BAUMANNII</i> IN A BELGIAN TERTIARY BURN WOUND CENTER

The burn intensive care unit (ICU) of the Queen Astrid Military Hospital experienced an outbreak with an extensively drug-resistant Acinetobacter baumannii (XDR-Ab) strain, which began when all burn wound patients from all over Belgium were sent there as part of the national COVID-19 action plan. The purpose of this study is to report on the investigation and strategies that were implemented to contain the outbreak. Between October 2020 and May 2021, five of the 72 patients admitted to the ICU met the acute outbreak case definition (attack rate 7%). Their median age was 46 years and their median total body surface area burned was 39%. All patients developed at least one XDR-Ab infection, with in total three pulmonary, three bloodstream and five burn wound infections. One patient died. All XDR-Ab isolates were only susceptible to colistin. Whole genome sequencing of the isolates from the first two patients revealed an identical A. baumannii ST2 genotype, suggesting an outbreak. XDR-Ab-positive patients were cohorted with dedicated staff. The infection control team intensified its training on hand hygiene, excreta management and bio-cleaning procedures. Concurrently, 30 environmental samples were collected, which proved negative for XDR-Ab. Spatio-temporal associations were found for all XDR-Ab-positive patients, suggesting cross-transmission via staff’s hands. We describe an XDR-Ab outbreak in a burn ICU over a seven-month period, in a context of increased workload. This series underlines the importance of a correct staff-to-patient ratio, especially in outbreak situations.</p

AN OUTBREAK OF EXTENSIVELY DRUG-RESISTANT <i>ACINETOBACTER BAUMANNII</i> IN A BELGIAN TERTIARY BURN WOUND CENTER

The burn intensive care unit (ICU) of the Queen Astrid Military Hospital experienced an outbreak with an extensively drug-resistant Acinetobacter baumannii (XDR-Ab) strain, which began when all burn wound patients from all over Belgium were sent there as part of the national COVID-19 action plan. The purpose of this study is to report on the investigation and strategies that were implemented to contain the outbreak. Between October 2020 and May 2021, five of the 72 patients admitted to the ICU met the acute outbreak case definition (attack rate 7%). Their median age was 46 years and their median total body surface area burned was 39%. All patients developed at least one XDR-Ab infection, with in total three pulmonary, three bloodstream and five burn wound infections. One patient died. All XDR-Ab isolates were only susceptible to colistin. Whole genome sequencing of the isolates from the first two patients revealed an identical A. baumannii ST2 genotype, suggesting an outbreak. XDR-Ab-positive patients were cohorted with dedicated staff. The infection control team intensified its training on hand hygiene, excreta management and bio-cleaning procedures. Concurrently, 30 environmental samples were collected, which proved negative for XDR-Ab. Spatio-temporal associations were found for all XDR-Ab-positive patients, suggesting cross-transmission via staff’s hands. We describe an XDR-Ab outbreak in a burn ICU over a seven-month period, in a context of increased workload. This series underlines the importance of a correct staff-to-patient ratio, especially in outbreak situations.</p

Combining sequencing approaches to fully resolve a carbapenemase-encoding megaplasmid in a Pseudomonas shirazica clinical strain

ABSTRACTHorizontal transfer of plasmids plays a pivotal role in dissemination of antibiotic resistance genes and emergence of multidrug-resistant bacteria. Plasmid sequencing is thus paramount for accurate epidemiological tracking in hospitals and routine surveillance. Combining Nanopore and Illumina sequencing allowed full assembly of a carbapenemase-encoding megaplasmid carried by multidrug-resistant clinical isolate FFUP\_PS\_41. Average nucleotide identity analyses revealed that FFUP\_PS\_41 belongs to the recently proposed new species Pseudomonas shirazica, related to the P. putida phylogenetic group. FFUP\_PS\_41 harbours a 498,516-bp megaplasmid (pJBCL41) with limited similarity to publicly-available plasmids. pJBCL41 contains genes predicted to encode replication, conjugation, partitioning and maintenance functions and heavy metal resistance. The |aacA7|blaVIM-2|aacA4| cassette array (resistance to carbapenems and aminoglycosides) is located within a class 1 integron that is a defective Tn402 derivative. This transposon lies within a 50,273-bp region bound by Tn3-family 38-bp inverted repeats and flanked by 5-bp direct repeats (DR) that composes additional transposon fragments, five insertion sequences and a Tn3-Derived Inverted-Repeat Miniature Element. The hybrid Nanopore/Illumina approach allowed full resolution of a carbapenemase-encoding megaplasmid from P. shirazica. Identification of novel megaplasmids sheds new light on the evolutionary effects of gene transfer and the selective forces driving antibiotic resistance

Direct Comparison of the Hinge-Cleaving Proteases IgdE and BdpK for LC-MS-Based IgG1 Clonal Profiling

Human antibodies are heterogeneous molecules primarily due to clonal sequence variations. Analytical techniques to assess antibody levels quantitatively, such as ELISA, lack the power to resolve abundances at the clonal level. Recently, we introduced an LC-MS-based approach that can distinguish and quantify antibody clones using the mass and retention time of their corresponding Fab-fragments. We used specific hinge-cleaving protease IgdE (FabALACTICA) to release the Fab-fragments from the constant Fc region of the antibody. Here, we explore an alternative IgG1 hinge-cleaving protease, BdpK (FabDELLO), and compare it directly to IgdE for use in IgG1 repertoire profiling. We used IgdE and BdpK in parallel to digest all IgG1s from the same set of plasma samples. Both proteases cleave IgG1 specifically in the hinge, albeit via different mechanisms and at two distinct cleavage sites. Notwithstanding these differences, the Fab fragments generated by IgdE or BdpK produced highly similar clonal repertoires. However, IgdE required ∼16 h of incubation to digest plasma IgG1s, while BdpK required ∼2 h. We authenticated the similarity of the clones by top-down proteomics using electron transfer dissociation. We conclude that BdpK performs very well in digesting polyclonal plasma IgG1s and that neither BdpK nor IgdE displays detectable biases in cleaving IgG1s. We anticipate that BdpK may emerge as the preferred protease for IgG1 hinge-digestion because it offers a shorter digestion time compared to IgdE, an equally specific digestion site, and no bias against any IgG1 present in plasma

Evaluation of mountain pine beetle infestations, Gallatin Ranger District, Gallatin National Forest, Montana, 1975

Mountain pine beetle has occurred at epidemic level in lodgepole pine stands in the west Gallatin River drainage since 1969. Infestation now encompasses about 5,500 acres. Since 1969, approximately 463,212 trees, with an estimated volume of 20,529,244 board feet have been killed. Approximately 69 percent of the stands on the Gallatin District are classed as susceptible. It is predicted that about 927,781 trees will be killed in 1976. Selective logging to remove infested and susceptible trees is recommended to reduce the potential for a continued epidemic

Combination of pre-adapted bacteriophage therapy and antibiotics for treatment of fracture-related infection due to pandrug-resistant Klebsiella pneumoniae

Immunogenetics and cellular immunology of bacterial infectious disease