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Wastewater-based epidemiology provides temporal and spatial information about the health status of a population. The objective of this study was to analyze and report the epidemiological dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the province of Tucumán, Argentina during the second and third waves of coronavirus disease 2019 (COVID-19) between April 2021 and March 2022. The study aimed to quantify SARS-CoV-2 RNA in wastewater, correlating it with clinically reported COVID-19 cases. Wastewater samples (n = 72) were collected from 16 sampling points located in three cities of Tucumán (San Miguel de Tucumán, Yerba Buena y Banda del Río Salí). Detection of viral nucleocapsid markers (N1 gene) was carried out using one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Viral loads were determined for each positive sample using a standard curve. A positive correlation (p < 0.05) was observed between viral load (copies/mL) and the clinically confirmed COVID-19 cases reported at specific sampling points in San Miguel de Tucumán (SP4, SP7, and SP8) in both months, May and June. Indeed, the high viral load concurred with the peaks of COVID-19 cases. This method allowed us to follow the behavior of SARS-CoV-2 infection during epidemic outbreaks. Thus, wastewater monitoring is a valuable epidemiological indicator that enables the anticipation of increases in COVID-19 cases and tracking the progress of the pandemic. SARS-CoV-2 genome-based surveillance should be implemented as a routine practice to prepare for any future surge in infections.

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Lactic acid bacteria can be considered as natural biopreservative and good biotechnological alternative to food safety. In this study, the antilisterial compounds produced by Enterococcus isolates from the Patagonian environment and their effectiveness for the control of Listeria monocytogenes in a food model were studied. Enterococcus isolates whose cell-free supernatant presented activity against Listeria monocytogenes were identified and evaluated for their virulence factors. The activity of the antimicrobial compounds produced by Enterococcus sp. against Listeria monocytogenes Scott A in meat gravy and ground beef during refrigerated storage was tested. The results indicated that ten Enterococcus isolates presented activity against Listeria monocytogenes and none of the selected strains presented virulence factors. L. monocytogenes in the food models containing the antilisterial compounds produced by Enterococcus sp. has decreased over the days, indicating that these compounds and cultures are an alternative to control the growth of L. monocytogenes in foods.

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Microcin J25 (MccJ25), an antimicrobial peptide, targets the respiratory chain but the exact mechanism by which it does so remains unclear. Here, we reveal that MccJ25 is able to inhibit the enzymatic activity of the isolated cytochrome bd-I from E. coli and induces at the same time production of reactive oxygen species. MccJ25 behaves as a dose-dependent weak inhibitor. Intriguingly, MccJ25 is capable of producing a change in the oxidation state of cytochrome bd-I causing its partial reduction in the presence of cyanide. These effects are specific for cytochrome bd-I, since the peptide is not able to act on purified cytochrome bo3.

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This review attempts to analyze the mechanism of action and immunity of class IIa bacteriocins. These peptides are promising alternative food preservatives and they have a great potential application in medical sciences. Class IIa bacteriocins act on the cytoplasmic membrane of Gram-positive cells dissipating the transmembrane electrical potential by forming pores. However, their toxicity and immunity mechanism remains elusive. Here we discuss the role of the mannose phosphotransferase system (man-PTS) as the receptor for class IIa bacteriocins and the influence of the membrane composition on the activity of these antimicrobial peptides. A model that is consistent with experimental results obtained by different researchers involves the non-specific binding of the bacteriocin to the negatively charged membrane of target bacteria. This step would facilitate a specific binding to the receptor protein, altering its functionality and forming an independent pore in which the bacteriocin is inserted in the membrane. An immunity protein could specifically recognize and block the pore. Bacteriocins function in bacterial ecosystems and energetic costs associated with their production are also discussed. Theoretical models based on solid experimental evidence are vital to understand bacteriocins mechanism of action and to promote new technological developments.

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The role of the class IIa bacteriocin membrane receptor protein remains unclear, and the following two different mechanisms have been proposed: the bacteriocin could interact with the receptor changing it to an open conformation or the receptor might act as an anchor allowing subsequent bacteriocin insertion and membrane disruption. Bacteriocin-producing cells synthesize an immunity protein that forms an inactive bacteriocin-receptor-immunity complex. To better understand the molecular mechanism of enterocin CRL35, the peptide was expressed as the suicidal probe EtpM-enterocin CRL35 in Escherichia coli, a naturally insensitive microorganism since it does not express the receptor. When the bacteriocin is anchored to the periplasmic face of the plasma membrane through the bitopic membrane protein, EtpM, E. coli cells depolarize and die. Moreover, co-expression of the immunity protein prevents the deleterious effect of EtpM-enterocin CRL35. The binding and anchoring of the bacteriocin to the membrane has demonstrated to be a sufficient condition for its membrane insertion. The final step of membrane disruption by EtpM-enterocin CRL35 is independent from the receptor, which means that the mannose PTS might not be involved in the pore structure. In addition, the immunity protein can protect even in the absence of the receptor.

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BACKGROUND: Enterocin CRL35 is a class IIa bacteriocin with anti-Listeria activity. Resistance to these peptides has been associated with either the downregulation of the receptor expression or changes in the membrane and cell walls. The scope of the present work was to characterize enterocin CRL35 resistant Listeria strains with MICs more than 10,000 times higher than the MIC of the WT sensitive strain. METHODS: Listeria monocytogenes INS7 resistant isolates R2 and R3 were characterized by 16S RNA gene sequencing and rep-PCR. Bacterial growth kinetic was studied in different culture media. Plasma membranes of sensitive and resistant bacteria were characterized by FTIR and Langmuir monolayer techniques. RESULTS: The growth kinetic of the resistant isolates was slower as compared to the parental strain in TSB medium. Moreover, the resistant isolates barely grew in a glucose-based synthetic medium, suggesting that these cells had a major alteration in glucose transport. Resistant bacteria also had alterations in their cell wall and, most importantly, membrane lipids. In fact, even though enterocin CRL35 was able to bind to the membrane-water interface of both resistant and parental sensitive strains, this peptide was only able to get inserted into the latter membranes. CONCLUSIONS: These results indicate that bacteriocin receptor is altered in combination with membrane structural modifications in enterocin CRL35-resistant L. monocytogenes strains. GENERAL SIGNIFICANCE: Highly enterocin CRL35-resistant isolates derived from Listeria monocytogenes INS7 have not only an impaired glucose transport but also display structural changes in the hydrophobic core of their plasma membranes.

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Bacteriocins and microcins are ribosomally synthesized antimicrobial peptides that are usually active against phylogenetically related bacteria. Thus, bacteriocins are active against Gram-positive while microcins are active against Gram-negative bacteria. The narrow spectrum of action generally displayed by bacteriocins from lactic acid bacteria represents an important limitation for the application of these peptides as clinical drugs or as food biopreservatives. The present study describes the design and expression of a novel recombinant hybrid peptide combining enterocin CRL35 and microcin V named Ent35-MccV. The chimerical bacteriocin displayed antimicrobial activity against enterohemorrhagic Escherichia coli and Listeria monocytogenes clinical isolates, among other pathogenic bacteria. Therefore, Ent35-MccV may find important applications in food or pharmaceutical industries.

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We previously showed that the antimicrobial peptide microcin J25 induced the over-production of reactive oxygen species with the concomitant release of cytochrome c from rat heart mitochondria via the opening of the mitochondrial permeability transition pore. Here, we were able to demonstrate that indeed, as a consequence of the oxidative burst, MccJ25 induces carbonylation of mitochondrial proteins, which may explain the irreversible inhibition of complex III and the partial inhibition of superoxide dismutase and catalase. Moreover, the peptide raised the levels of oxidized membrane lipids, which triggers the release of cytochrome c. From in silico analysis, we hypothesize that microcin would elicit these effects through interaction with heme c1 at mitochondrial complex III. On the other hand, under an excess of l-arginine, MccJ25 caused nitric oxide overproduction with no oxidative damage and a marked inhibition in oxygen consumption. Therefore, a beneficial anti-oxidative activity could be favored by the addition of l-arginine. Conversely, MccJ25 pro-oxidative-apoptotic effect can be unleashed in either an arginine-free medium or by suppressing the nitric oxide synthase activity.

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Escherichia coli microcin J25 (MccJ25) is a lasso-peptide antibiotic comprising 21 L-amino acid residues (G(1)-G-A-G-H(5)-V-P-E-Y-F(10)-V-G-I-G-T(15)-P-I-S-F-Y(20)-G). MccJ25 has two independent substrates: RNA-polymerase (RNAP) and the membrane respiratory chain. The latter is mediated by oxygen consumption inhibition together with an increase of superoxide production. In the present paper, the antibiotic MccJ25 was engineered by substituting Tyr(9) or Tyr(20) with phenylalanine. Both mutants were well transported into the cells and remained active on RNAP. Only the Y9F mutant lost the ability to overproduce superoxide and inhibit oxygen consumption. The last results confirm that the Tyr(9), and not Tyr(20), is involved in the MccJ25 action on the respiratory chain target.

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Microcin J25, an antimicrobial lasso-structure peptide, induces the opening of mitochondrial permeability transition pores and the subsequent loss of cytochrome c. The microcin J25 effect is mediated by the stimulation of superoxide anion overproduction. An increased uptake of calcium is also involved in this process. Additional studies with superoxide dismutase, ascorbic acid and different specific inhibitors, such as ruthenium red, cyclosporin A and Mn(2+), allowed us to establish a time sequence of events starting with the binding of microcin J25, followed by superoxide anion overproduction, opening of mitochondrial permeability transition pores, mitochondrial swelling and the concomitant leakage of cytochrome c.

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