ScanGrow: Deep Learning-Based Live Tracking of Bacterial Growth in Broth.

Monitoring the growth of bacterial cultures is one of the most common techniques in microbiology. This is usually achieved by using expensive and bulky spectrophotometric plate readers which periodically measure the optical density of bacterial cultures during the incubation period. In this study, we present a completely novel way of obtaining bacterial growth curves based on the classification of scanned images of cultures rather than using spectrophotometric measurements. We trained a deep learning model with images of bacterial broths contained in microplates, and we integrated it into a custom-made software application that triggers a flatbed scanner to timely capture images, automatically processes the images, and represents all growth curves. The developed tool, ScanGrow, is presented as a low-cost and high-throughput alternative to plate readers, and it only requires a computer connected to a flatbed scanner and equipped with our open-source ScanGrow application. In addition, this application also assists in the pre-processing of data to create and evaluate new models, having the potential to facilitate many routine microbiological techniques.

Imitation of ß-lactam binding enables broad-spectrum metallo-ß-lactamase inhibitors.

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-ß-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential ß-lactamase stable ß-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.

An approach to increase the success rate of cultivation of soil bacteria based on fluorescence-activated cell sorting.

Soil microbiota are considered a source of undiscovered bioactive compounds, yet cultivation of most bacteria within a sample remains generally unsuccessful. Two main reasons behind the unculturability of bacteria are the presence of cells in a viable but not culturable state (such as dormant cells) and the failure to provide the necessary growth requirements in vitro (leading to the classification of some bacterial taxa as yet-to-be-cultured). The present work focuses on the development of a single procedure that helps distinguish between both phenomena of unculturability based on viability staining coupled with flow cytometry and fluorescence-activated cell sorting. In the selected soil sample, the success rate of cultured bacteria was doubled by selecting viable and metabolically active bacteria. It was determined that most of the uncultured fraction was not dormant or dead but likely required different growth conditions. It was also determined that the staining process introduced changes in the taxonomic composition of the outgrown bacterial biomass, which should be considered for further developments. This research shows the potential of flow cytometry and fluorescence-activated cell sorting applied to soil samples to improve the success rate of bacterial cultivation by estimating the proportion of dormant and yet-to-be-cultured bacteria and by directly excluding dormant cells from being inoculated into growth media.

Emergence of Mobile Colistin Resistance (mcr-8) in a Highly Successful Klebsiella pneumoniae Sequence Type 15 Clone from Clinical Infections in Bangladesh.

The emergence of mobilized colistin resistance genes (mcr) has become a serious concern in clinical practice, compromising treatment options for life-threatening infections. In this study, colistin-resistant Klebsiella pneumoniae harboring mcr-8.1 was recovered from infected patients in the largest public hospital of Bangladesh, with a prevalence of 0.3% (3/1,097). We found mcr-8.1 in an identical highly stable multidrug-resistant IncFIB(pQil) plasmid of ∼113 kb, which belonged to an epidemiologically successful K. pneumoniae clone, ST15. The resistance mechanism was proven to be horizontally transferable, which incurred a fitness cost to the host. The core genome phylogeny suggested the clonal spread of mcr-8.1 in a Bangladeshi hospital. Core genome single-nucleotide polymorphisms among the mcr-8.1-positive K. pneumoniae isolates ranged from 23 to 110. It has been hypothesized that mcr-8.1 was inserted into IncFIB(pQil) with preexisting resistance loci, blaTEM-1b and blaCTX-M-15, by IS903B Coincidentally, all resistance determinants in the plasmid [mcr-8.1, ampC, sul2, 1d-APH(6), APH(3'')-Ib, blaTEM-1b, blaCTX-M-15] were bracketed by IS903B, demonstrating the possibility of intra- and interspecies and intra- and intergenus transposition of entire resistance loci. This is the first report of an mcr-like mechanism from human infections in Bangladesh. However, given the acquisition of mcr-8.1 by a sable conjugative plasmid in a successful high-risk clone of K. pneumoniae ST15, there is a serious risk of dissemination of mcr-8.1 in Bangladesh from 2017 onwards.IMPORTANCE There is a marked paucity in our understanding of the epidemiology of colistin-resistant bacterial pathogens in South Asia. A report by Davies and Walsh (Lancet Infect Dis 18:256-257, https://doi.org/10.1016/S1473-3099(18)30072-0, 2018) suggests the export of colistin from China to India, Vietnam, and South Korea in 2016 was approximately 1,000 tons and mainly used as a poultry feed additive. A few reports forecast that the prevalence of mcr in humans and livestock will increase in South Asia. Given the high prevalence of blaCTX-M-15 and blaNDM in India, Bangladesh, and Pakistan, colistin has become the invariable option for the management of serious infections, leading to the emergence of mcr-like mechanisms in South Asia. Systematic scrutiny of the prevalence and transmission of mcr variants in South Asia is vital to understanding the drivers of mcr genes and to initiate interventions to overcome colistin resistance.

Detection of Thermal Sublethal Injury in Escherichia coli via the Selective Medium Plating Technique: Mechanisms and Improvements.

In food preservation, the synergistic combination of different technologies aims to maximize the total lethality of the process and minimize the intensity of each hurdle. This is especially the case when at least one of the treatments can cause sublethal (reparable) injury in a great proportion of the population, so that sublethally injured cells can end up being entirely inactivated by the other hurdle(s). The selective medium plating technique (SMPT) is extensively used to enumerate bacterial sublethal injury after inimical treatments, being sodium chloride added to the recovery medium to detect damaged bacterial envelopes. However, little work has been done to explain the reasons for the inability of sublethally injured cells to outgrow in selective agar media, whereas they are able to grow in non-selective agar. In the present paper, the performance of SMPT on Escherichia coli cells after heat treatments is explored by applying different selective agents in the recovery media, using mutants lacking factors involved in osmoregulation, and also by examining the integrity of the cytoplasmic membrane. In view of the results, the possibility of a specific toxic effect of Na(+) as the main mechanism under SMPT was discarded, since the same level of sublethal injury was detected using KCl instead of NaCl. The synthesis of the osmoprotectant trehalose determined the maximum osmotolerance of intact cells to the selective agents, but was not crucial in the quantification of sublethal injury. Moreover, for the first time, the extent of sublethal injury detected via SMPT was directly correlated with the physical loss of integrity of the cell membrane in 99.999% of the initial population. This was achieved through statistical analysis of flow cytometry data using propidium iodide-exclusion technique when that dye was added before thermal treatments. The present work confirms the adequacy of SMPT as a tool for detecting the occurrence and quantity of sublethally injured cells after thermal treatments and thus, for efficiently designing the combination of heat with other preservation techniques. We also propose the study of statistical analysis from flow cytometry data for a more rapid quantification of bacterial sublethal injury in a broad detection range.

Individual Constituents from Essential Oils Inhibit Biofilm Mass Production by Multi-Drug Resistant Staphylococcus aureus.

Biofilm formation by Staphylococcus aureus represents a problem in both the medical field and the food industry, because the biofilm structure provides protection to embedded cells and it strongly attaches to surfaces. This circumstance is leading to many research programs seeking new alternatives to control biofilm formation by this pathogen. In this study we show that a potent inhibition of biofilm mass production can be achieved in community-associated methicillin-resistant S. aureus (CA-MRSA) and methicillin-sensitive strains using plant compounds, such as individual constituents (ICs) of essential oils (carvacrol, citral, and (+)-limonene). The Crystal Violet staining technique was used to evaluate biofilm mass formation during 40 h of incubation. Carvacrol is the most effective IC, abrogating biofilm formation in all strains tested, while CA-MRSA was the most sensitive phenotype to any of the ICs tested. Inhibition of planktonic cells by ICs during initial growth stages could partially explain the inhibition of biofilm formation. Overall, our results show the potential of EOs to prevent biofilm formation, especially in strains that exhibit resistance to other antimicrobials. As these compounds are food additives generally recognized as safe, their anti-biofilm properties may lead to important new applications, such as sanitizers, in the food industry or in clinical settings.

Bioactive properties of a propolis-based dietary supplement and its use in combination with mild heat for apple juice preservation.

This study characterizes the antioxidant and antibacterial properties of a propolis-based dietary supplement (PDS) and investigates its incorporation into apple juice to decrease the intensity of the heat treatment required to inactivate 5 log10 cycles of Escherichia coli O157:H7. As the source of propolis, we used a PDS containing 0.2 mg/µL of propylene glycol-extracted propolis (propolis). The total phenolic content and antioxidant activity (IC50) of the PDS were 82.15±3.53 mg/g and 0.055±0.003 mg/mL, respectively. Regarding antimicrobial activity, propolis (0.2 mg/mL) was very effective under acidic pH against Listeria monocytogenes EGD-e, inactivating more than 5 log10 cell cycles in 1h, but hardly inactivated or sub-lethally injured E. coli O157:H7 Sakai. However, incorporating propolis (0.2 mg/mL) into acidic buffer decreased the time needed to inactivate 5 log10 cycles of E. coli O157:H7 Sakai at 51 °C by more than 40 times. Moreover, when combined with heat in apple juice, propolis (0.1mg/mL) reduced the thermal treatment time and temperature needed to inactivate 5 log10 cycles of E. coli by 75% and 3 °C, respectively. The corresponding PDS concentration did not decrease the organoleptic properties of the apple juice, which implies the possibility of obtaining a sensorially appealing, low-pasteurized apple juice with the functional properties provided by propolis.

Impact of essential oils on the taste acceptance of tomato juice, vegetable soup, or poultry burgers.

Despite the vast body of available literature on the possibilities of essential oils (EOs) as food preservatives or functional ingredients, the sensory impact of their addition to foods has barely been approached. This work focuses on the hedonic taste acceptance of 3 food products (tomato juice, vegetable soup, and poultry burgers) when they are incorporated with potentially antimicrobial concentrations (20 to 200 µL/L) of 6 selected EOs (lemon, pennyroyal mint, thyme, and rosemary) and individual compounds (carvacrol, p-cymene). Although addition of 20 µL/L of pennyroyal mint or lemon EO did not change the taste acceptance of tomato juice, higher concentrations of these compounds or any concentration of the other 4 compounds did. In vegetable soup, the tolerance limit for rosemary EO, thyme EO, carvacrol, or p-cymene was 20 µL/L, while the addition of 200 µL/L of lemon EO was accepted. Tolerance limits in poultry burgers were established in 20 µL/L for carvacrol and thyme EOs, 100 µL/L for pennyroyal mint EO and p-cymene, and 200 µL/L for lemon and rosemary EOs. Moreover, incorporation of pennyroyal mint EO to tomato juice or poultry burgers, and enrichment of vegetable soup with lemon EO, could contribute to the development of food products with an improved sensory appeal.

Combination of pulsed electric fields, mild heat and essential oils as an alternative to the ultrapasteurization of liquid whole egg.

The production of microbiologically safe liquid whole egg (LWE) by industrial ultrapasteurization is restricted by the high thermal sensitivity of LWE components. This research proposes an alternative treatment based on the application of pulsed electric fields (PEF) and mild heat, in the presence of natural essential oils (EOs) or their individual components (ICs). The obtained results indicate that the successive application of PEF (25kV/ and 100kJ/kg) followed by heat (60°C during 3.5') to LWE added with 200µL/L of lemon EO would reach 4log10cycles of inactivation of Salmonella Senftenberg 775W and Listeria monocytogenes, when any of these barriers acting alone inactivated less than 1.5log10cycles of either bacteria. Therefore, the synergism between lemon EO and the successive application of PEF and heat would provide a safety level similar to that of ultrapasteurization treatment for Salmonella Senftenberg 775W and L. monocytogenes, but at a lower temperature. To a lesser extent, synergism with the successive application of PEF and heat was also observed in the presence of 200µL/L of carvacrol, citral, (+)-limonene, or mandarin EO, reaching about 3.5log10cycles of inactivation in Salmonella Senftenberg and 3.0log10cycles in L. monocytogenes, respectively. A sensory test on LWE containing 200µL/L of each additive in the form of omelets and sponge cakes revealed that this concentration of mandarin EO, lemon EO, or (+)-limonene did not decrease the sensory acceptability of the LWE-containing products, and lemon EO and mandarin EO even increased the hedonic acceptability of sponge cakes. In conclusion, this process could be applied in the food industry to obtain microbiologically safe LWE, which could be used to produce egg-based products without decreasing (and even increasing) their sensory appeal.

Chemical composition and antioxidant properties of Laurus nobilis L. and Myrtus communis L. essential oils from Morocco and evaluation of their antimicrobial activity acting alone or in combined processes for food preservation.

BACKGROUND: This study describes the antioxidant and antimicrobial activity of Laurus nobilis L. and Myrtus communis L. essential oils (EOs). This is the first report of the synergistic antimicrobial effect of these EOs in combination with physical food preservation treatments. RESULTS: EOs obtained by steam distillation from aerial parts of Laurus nobilis and Myrtus communis were analysed by using gas chromatography-mass spectrometry. The main compounds were 1,8-cineole and 2-carene (L. nobilis EO); and myrtenyl acetate, 1,8-cineole and α-pinene (M. communis EO). L. nobilis EO showed higher antioxidant activity than M. communis EO in three complementary antioxidant tests. Although antimicrobial activity tests demonstrated the effectiveness of L. nobilis EO and the lack of bactericidal effect of M. communis EO, synergistic lethal effects were observed when combining each EO (0.2 µL mL(-1)) with mild heat (54°C for 10 min) or high hydrostatic pressure (175-400 MPa for 20 min). In contrast, combination of EOs with pulsed electric fields (30 kV cm(-1) for 25 pulses) showed no additional effects. CONCLUSION: This study shows the great potential of these EOs in combined treatments with mild heat and high hydrostatic pressure to obtain a higher inactivation of foodborne pathogens, which might help in the design of safe processes applied at low intensity.

Mechanism of bacterial inactivation by (+)-limonene and its potential use in food preservation combined processes.

This work explores the bactericidal effect of (+)-limonene, the major constituent of citrus fruits' essential oils, against E. coli. The degree of E. coli BJ4 inactivation achieved by (+)-limonene was influenced by the pH of the treatment medium, being more bactericidal at pH 4.0 than at pH 7.0. Deletion of rpoS and exposure to a sub-lethal heat or an acid shock did not modify E. coli BJ4 resistance to (+)-limonene. However, exposure to a sub-lethal cold shock decreased its resistance to (+)-limonene. Although no sub-lethal injury was detected in the cell envelopes after exposure to (+)-limonene by the selective-plating technique, the uptake of propidium iodide by inactivated E. coli BJ4 cells pointed out these structures as important targets in the mechanism of action. Attenuated Total Reflectance Infrared Microspectroscopy (ATR-IRMS) allowed identification of altered E. coli BJ4 structures after (+)-limonene treatments as a function of the treatment pH: ß-sheet proteins at pH 4.0 and phosphodiester bonds at pH 7.0. The increased sensitivity to (+)-limonene observed at pH 4.0 in an E. coli MC4100 lptD4213 mutant with an increased outer membrane permeability along with the identification of altered ß-sheet proteins by ATR-IRMS indicated the importance of this structure in the mechanism of action of (+)-limonene. The study of mechanism of inactivation by (+)-limonene led to the design of a synergistic combined process with heat for the inactivation of the pathogen E. coli O157:H7 in fruit juices. These results show the potential of (+)-limonene in food preservation, either acting alone or in combination with lethal heat treatments.

Synergistic combinations of high hydrostatic pressure and essential oils or their constituents and their use in preservation of fruit juices.

This work addresses the inactivation achieved with Escherichia coli O157:H7 and Listeria monocytogenes EGD-e by combined processes of high hydrostatic pressure (HHP) and essential oils (EOs) or their chemical constituents (CCs). HHP treatments (175-400 MPa for 20 min) were combined with 200 µL/L of each EO (Citrus sinensis L., Citrus lemon L., Citrus reticulata L., Thymus algeriensis L., Eucalyptus globulus L., Rosmarinus officinalis L., Mentha pulegium L., Juniperus phoenicea L., and Cyperus longus L.) or each CC ((+)-limonene, α-pinene, ß-pinene, p-cymene, thymol, carvacrol, borneol, linalool, terpinen-4-ol, 1,8-cineole, α-terpinyl acetate, camphor, and (+)-pulegone) in buffer of pH 4.0 or 7.0. The tested combinations achieved different degrees of inactivation, the most effective being (+)-limonene, carvacrol, C. reticulata L. EO, T. algeriensis L. EO and C. sinensis L. EO which were capable of inactivating about 4-5 log(10) cycles of the initial cell populations in combination with HHP, and therefore showed outstanding synergistic effects. (+)-Limonene was also capable of inactivating 5 log(10) cycles of the initial E. coli O157:H7 population in combination with HHP (300 MPa for 20 min) in orange and apple juices, and a direct relationship was established between the inactivation degree caused by the combined process with (+)-limonene and the occurrence of sublethal injury after the HHP treatment. This work shows the potential of EOs and CCs in the inactivation of foodborne pathogens in combined treatments with HHP, and proposes their possible use in liquid food such as fruit juices.

Inactivation of Escherichia coli O157:H7 in fruit juices by combined treatments of citrus fruit essential oils and heat.

This work approaches the possibility of combining mild heat treatments with citrus fruit essential oils (EOs) to improve the effectiveness of heat treatments and thus to reduce treatment intensity. Concentrations between 10 and 200 µL/L of lemon, mandarin, or orange EO were tested at 54 °C for 10 min in laboratory media, determining that 200 µL/L of each EO was necessary to achieve a 5 log(10) reduction of the initial Escherichia coli O157:H7 concentration. A relationship could be established between sublethally injured cells after the heat treatment and inactivated cells after the combined process. In apple juice, the synergism in the inactivation of E. coli O157:H7 when adding 200 µL/L of lemon EO might suppose a reduction in the treatment temperature (of 4.5 °C) or in the treatment time (by 5.7 times) within the range of temperature assayed (54-60 °C). Addition of 75 µL/L of lemon EO was determined to achieve the same synergistic effect of the combined treatment when the initial inoculum was reduced from 3×10(7) to 3×10(4) CFU/mL. Since the addition of lemon EO did not decrease the hedonic acceptability of apple juice, the proposed combined treatment could be further studied and optimized for the production of new minimally processed juices.