Biofilm Formation by Listeria monocytogenes 15G01, a Persistent Isolate from a Seafood-Processing Plant, Is Influenced by Inactivation of Multiple Genes Belonging to Different Functional Groups.

Listeria monocytogenes is a ubiquitous foodborne pathogen that results in a high rate of mortality in sensitive and immunocompromised people. Contamination of food with L. monocytogenes is thought to occur during food processing, most often as a result of the pathogen producing a biofilm that persists in the environment and acting as the source for subsequent dispersal of cells onto food. A survey of seafood-processing plants in New Zealand identified the persistent strain 15G01, which has a high capacity to form biofilms. In this study, a transposon library of L. monocytogenes 15G01 was screened for mutants with altered biofilm formation, assessed by a crystal violet assay, to identify genes involved in biofilm formation. This screen identified 36 transposants that showed a significant change in biofilm formation compared to the wild type. The insertion sites were in 27 genes, 20 of which led to decreased biofilm formation and seven to an increase. Two insertions were in intergenic regions. Annotation of the genes suggested that they are involved in diverse cellular processes, including stress response, autolysis, transporter systems, and cell wall/membrane synthesis. Analysis of the biofilms produced by the transposants using scanning electron microscopy and fluorescence microscopy showed notable differences in the structure of the biofilms compared to the wild type. In particular, inactivation of uvrB and mltD produced coccoid-shaped cells and elongated cells in long chains, respectively, and the mgtB mutant produced a unique biofilm with a sandwich structure which was reversed to the wild-type level upon magnesium addition. The mltD transposant was successfully complemented with the wild-type gene, whereas the phenotypes were not or only partially restored for the remaining mutants.IMPORTANCE The major source of contamination of food with Listeria monocytogenes is thought to be due to biofilm formation and/or persistence in food-processing plants. By establishing as a biofilm, L. monocytogenes cells become harder to eradicate due to their increased resistance to environmental threats. Understanding the genes involved in biofilm formation and their influence on biofilm structure will help identify new ways to eliminate harmful biofilms in food processing environments. To date, multiple genes have been identified as being involved in biofilm formation by L. monocytogenes; however, the exact mechanism remains unclear. This study identified four genes associated with biofilm formation by a persistent strain. Extensive microscopic analysis illustrated the effect of the disruption of mgtB, clsA, uvrB, and mltD and the influence of magnesium on the biofilm structure. The results strongly suggest an involvement in biofilm formation for the four genes and provide a basis for further studies to analyze gene regulation to assess the specific role of these biofilm-associated genes.

Design, synthesis and biological evaluation of novel DNA gyrase inhibitors and their siderophore mimic conjugates.

Bacterial DNA gyrase is an important target for the development of novel antibacterial drugs, which are urgently needed because of high level of antibiotic resistance worldwide. We designed and synthesized new 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA gyrase B inhibitors and their conjugates with siderophore mimics, which were introduced to increase the uptake of inhibitors into the bacterial cytoplasm. The most potent conjugate 34 had an IC50 of 58 nM against Escherichia coli DNA gyrase and displayed MIC of 14 µg/mL against E. coli ΔtolC strain. Only minor improvements in the antibacterial activities against wild-type E. coli in low-iron conditions were seen for DNA gyrase inhibitor - siderophore mimic conjugates.

Defining conditions for biofilm inhibition and eradication assays for Gram-positive clinical reference strains.

BACKGROUND: Biofilms are formed by a complex bacterial community encapsulated by a polymeric matrix, with strong adherent properties and persistent phenotype. Biofilms are considered one of the most challenging areas of modern medicine. Existing antibiotics have been developed against free-floating bacterial cells, and thus, many treatments of biofilm-related infection fail. In this study, we compared the effects of different media on biofilm growth of clinical reference strains of Staphylococci and Enterococci, including multi-drug resistant representatives. Further, we optimized the resazurin-based assay for determining the minimal biofilm inhibitory concentration (MBIC) of standard antibiotics, and evaluated its use for the determination of minimal biofilm eradication concentration (MBEC). RESULTS: We showed that tryptic soy broth supplemented with 1% glucose was an optimal media for maximum biofilm growth of all strains tested, with an extended incubation time for Enterococci. A range of parameters were tested for the resazurin assay, including concentration, temperature and time of incubation. Using quality parameters to analyze the assay's performance, the conditions for the resazurin assay were set as follows: 4 µg/mL and 8 µg/mL, with incubation at 25 °C for 20 min and 40 min for Staphylococci and Enterococci, respectively. CONCLUSIONS: In summary, we defined conditions for optimal biofilm growth and for standardized resazurin assay for MBIC determination against six Gram-positive clinical reference strains. We also observed that MBEC determination by the resazurin-based assay is limited due to the poor detection limit of the assay. Complementary cell counting data is needed for precise determination of MBEC.

Listeria monocytogenes associated with New Zealand seafood production and clinical cases: unique sequence types, truncated InlA, and attenuated invasiveness.

Listeriosis is caused by the food-borne pathogen Listeria monocytogenes, which can be found in seafood and processing plants. To evaluate the risk to human health associated with seafood production in New Zealand, multi-virulence-locus sequence typing (MVLST) was used to define the sequence types (STs) of 31 L. monocytogenes isolates collected from seafood-processing plants, 15 from processed foods, and 6 from human listeriosis cases. The STs of these isolates were then compared with those from a collection of seafood isolates and epidemic strains from overseas. A total of 17 STs from New Zealand clustered into two lineages: seafood-related isolates in lineages I and II and all human isolates in lineage II. None of the New Zealand STs matched previously described STs from other countries. Isolates (belonging to ST01-N and ST03-N) from mussels and their processing environments, however, were identical to those of sporadic listeriosis cases in New Zealand. ST03-N isolates (16 from mussel-processing environments, 2 from humans, and 1 from a mussel) contained an inlA premature stop codon (PMSC) mutation. Therefore, the levels of invasiveness of 22 isolates from ST03-N and the three other common STs were compared using human intestinal epithelial Caco-2 cell lines. STs carrying inlA PMSCs, including ST03-N isolates associated with clinical cases, had a low invasion phenotype. The close relatedness of some clinical and environmental strains, as revealed by identical MVLST profiles, suggests that local and persistent environmental strains in seafood-processing environments pose a potential health risk. Furthermore, a PMSC in inlA does not appear to give L. monocytogenes a noninvasive profile.

Host range and in vitro lysis of Listeria monocytogenes seafood isolates by bacteriophages.

Listeria-infecting bacteriophages (listeriaphages) can be used to control Listeria monocytogenes in the food industry. However, the sensitivity of many of seafood-borne Listeria strains to phages has not been reported. This research investigated the host ranges of three listeriaphages (FWLLm1, FWLLm3 and FWLLm5) by the formation of lytic zones and plaques on host lawns and in vitro lysis kinetics of listeriaphage FWLLm3. The study also predicted the phage titres required to lyse host cells. The host ranges of the phages were determined using 50 L. monocytogenes strains, of which 48 were isolated from the seafood industry and two from clinical cases. Of the 50 strains, 36 were tested at 25 and 30 ℃ and the remainder (14) at 15 and 25 ℃. Based on the formation of either discrete plaques or lytic zones (host kill zones), the host ranges of FWLLm1, FWLLm3 and FWLLm5 were about 87%, 81% and 87%, respectively, at 25 ℃. Six L. monocytogenes strains from the seafood environment were insensitive to all three phages, while the other seafood strains (42) were phage-sensitive. The adsorption rate constant (k value) of listeriaphage FWLLm3 was between 1.2 × 10(-9) and 1.6 × 10(-9 )ml/min across four host strains in tryptic soy broth at 25 ℃. The cultures (at 3-4 log colony-forming unit (CFU/ml) were completely lysed (<1 log CFU/ml) when cultures were infected with FWLLm3 at > 8.7 log phage-forming units (PFU/ml) for 30 min. Re-growth of phage-infected cultures was not detected after 24 h. The effective empirical phage titre was similar to the calculated titre using a kinetic model. Results indicate the potential use of the three phages for controlling L. monocytogenes strains in seafood processing environments.

Benzothiazole DNA gyrase inhibitors and their conjugates with siderophore mimics: design, synthesis and evaluation.

Benzothiazole-based bacterial DNA gyrase and topoisomerase IV inhibitors are promising new antibacterial agents with potent activity against Gram-positive and Gram-negative bacterial strains. The aim of this study was to improve the uptake of these inhibitors into the cytoplasm of Gram-negative bacteria by conjugating them to the small siderophore mimics. The best conjugate 18b displayed potent Escherichia coli DNA gyrase and topoisomerase IV inhibition. The interaction analysis of molecular dynamics simulation trajectory showed the important contribution of the siderophore mimic moiety to binding affinity. By NMR spectroscopy, we demonstrated that the hydroxypyridinone moiety alone was responsible for the chelation of iron(iii). Moreover, 18b showed an enhancement of antibacterial activity against E. coli JW5503 in an iron-depleted medium, clearly indicating an increased uptake of 18b in this bacterial strain.

Effectiveness of phages in the decontamination of Listeria monocytogenes adhered to clean stainless steel, stainless steel coated with fish protein, and as a biofilm.

Listeria monocytogenes is a food-borne pathogen which causes listeriosis and is difficult to eradicate from seafood processing environments; therefore, more effective control methods need to be developed. This study investigated the effectiveness of three bacteriophages (LiMN4L, LiMN4p and LiMN17), individually or as a three-phage cocktail at ≈9 log10 PFU/ml, in the lysis of three seafood-borne L. monocytogenes strains (19CO9, 19DO3 and 19EO3) adhered to a fish broth layer on stainless steel coupon (FBSSC) and clean stainless steel coupon (SSC), in 7-day biofilm, and dislodged biofilm cells at 15 ± 1 °C. Single phage treatments (LiMN4L, LiMN4p or LiMN17) decreased bacterial cells adhered to FBSSC and SSC by ≈3-4.5 log units. Phage cocktail reduced the cells on both surfaces (≈3.8-4.5 and 4.6-5.4 log10 CFU/cm², respectively), to less than detectable levels after ≈75 min (detection limit = 0.9 log10 CFU/cm²). The phage cocktail at ≈5.8, 6.5 and 7.5 log10 PFU/cm² eliminated Listeria contamination (≈1.5-1.7 log10 CFU/cm²) on SSC in ≈15 min. One-hour phage treatments (LiMN4p, LiMN4L and cocktail) in three consecutive applications resulted in a decrease of 7-day L. monocytogenes biofilms (≈4 log10 CFU/cm²) by ≈2-3 log units. Single phage treatments reduced dislodged biofilm cells of each L. monocytogenes strain by ≈5 log10 CFU/ml in 1 h. The three phages were effective in controlling L. monocytogenes on stainless steel either clean or soiled with fish proteins which is likely to occur in seafood processing environments. Phages were more effective on biofilm cells dislodged from the surface compared with undisturbed biofilm cells. Therefore, for short-term phage treatments of biofilm it should be considered that some disruption of the biofilm cells from the surface prior to phage application will be required.

Biochemometry identifies ostruthin as pluripotent antimicrobial and anthelmintic agent from masterwort.

The root extract of Peucedanum ostruthium (PO-E) was identified as a promising antibacterial source from a screening of 158 extracts against Staphylococcus aureus. It has also recently been shown to significantly decrease the survival of the nematode Caenorhabditis elegans. We used the biochemometric approach ELINA to investigate the phytochemical characteristics of the multicomponent mixture PO-E to identify the anti-infective constituent(s) targeting S. aureus and C. elegans.1H NMR spectra of PO-E-derived microfractions were correlated with their respective bioactivity data. Heterocovariance analyses unambiguously identified ostruthin as an anti-staphylococcal constituent, which potently also inhibited Enterococcus spp.. ELINA demonstrated that anthelmintic activity was due to a combinatorial effect of ostruthin and isoimperatorin. A C. elegans-based survival and motility assay confirmed that isoimperatorin, imperatorin, and verapamil modulated the susceptibility of ostruthin. The combinatorial effect of these natural products was shown in larvae studies to be related to the function of the nematodes' efflux pump.

Willow Bark-Derived Material with Antibacterial and Antibiofilm Properties for Potential Wound Dressing Applications.

Tree stems contain wood in addition to 10-20% bark, which remains one of the largest underutilized biomasses on earth. Unique macromolecules (like lignin, suberin, pectin, and tannin), extractives, and sclerenchyma fibers form the main part of the bark. Here, we perform detailed investigation of antibacterial and antibiofilm properties of bark-derived fiber bundles and discuss their potential application as wound dressing for treatment of infected chronic wounds. We show that the yarns containing at least 50% of willow bark fiber bundles significantly inhibit biofilm formation by wound-isolated Staphylococcus aureus strains. We then correlate antibacterial effects of the material to its chemical composition. Lignin plays the major role in antibacterial activity against planktonic bacteria [i.e., minimum inhibitory concentration (MIC) 1.25 mg/mL]. Acetone extract (unsaturated fatty acid-enriched) and tannin-like (dicarboxylic acid-enriched) substances inhibit both bacterial planktonic growth [MIC 1 and 3 mg/mL, respectively] and biofilm formation. The yarn lost its antibacterial activity once its surface lignin reached 20.1%, based on X-ray photoelectron spectroscopy. The proportion of fiber bundles at the fabricated yarn correlates positively with its surface lignin. Overall, this study paves the way to the use of bark-derived fiber bundles as a natural-based material for active (antibacterial and antibiofilm) wound dressings, upgrading this underappreciated bark residue from an energy source into high-value pharmaceutical use.

Antibacterial and antibiofilm activity of permanently ionized quaternary ammonium fluoroquinolones.

A series of quaternary ammonium fluoroquinolones was obtained by exhaustive methylation of the amine groups present at the 7-position of fluoroquinolones, including ciprofloxacin, enoxacin, gatifloxacin, lomefloxacin, and norfloxacin. The synthesized molecules were tested for their antibacterial and antibiofilm activities against Gram-positive and Gram-negative human pathogens, i.e. Staphylococcus aureus and Pseudomonas aeruginosa. The study showed that the synthesized compounds are potent antibacterial agents (MIC values at the lowest 6.25 µM) with low cytotoxicity in vitro as assessed on the BALB 3T3 mouse embryo cell line. Further experiments proved that the tested derivatives are able to bind to the DNA gyrase and topoisomerase IV active sites in a fluoroquinolone-characteristic manner. The most active quaternary ammonium fluoroquinolones, in contrast to ciprofloxacin, reduce the total biomass of P. aeruginosa ATCC 15442 biofilm in post-exposure experiments. The latter effect may be due to the dual mechanism of action of the quaternary fluoroquinolones, which also involves disruption of bacterial cell membranes. IAM-HPLC chromatographic experiments with immobilized artificial membranes (phospholipids) showed that the most active compounds were those with moderate lipophilicity and containing a cyclopropyl group at the N1 nitrogen atom in the fluoroquinolone core.

Prevalence and biofilm-forming ability of Listeria monocytogenes in New Zealand mussel (Perna canaliculus) processing plants.

Greenshell™ mussels are New Zealand's largest seafood export species. Some export markets require compliance with 'zero' tolerance legislation for Listeria monocytogenes in 25 g of product. Even though individually quick frozen (IQF) mussel products are labeled 'to be cooked', and are not classified as ready-to-eat, some markets still require them to comply with the strict policy. Three mussel processing plants were assessed for the pattern of L. monocytogenes contamination on raw material, environment, food contact surfaces, and in the final product. Cultures (n = 101) obtained from an industrial Listeria monitoring program from August 2007 to June 2009 were characterized by serotyping and pulsed field gel electrophoresis. Using the crystal violet method, isolates were assessed for their ability to form biofilms. This work confirmed the presence of L. monocytogenes in raw and processed product, and the importance of cross-contamination from external and internal environments. Processing plants had L. monocytogenes pulsotypes that were detected more than once over 6 months. No correlation was found between biofilm-forming ability and persistent isolates. Two pulsotypes (including a persistent one), were previously isolated in human cases of listeriosis in New Zealand, but none of the pulsotypes matched those involved in international outbreaks.

Compounding Parenteral Products in Pediatric Wards-Effect of Environment and Aseptic Technique on Product Sterility.

Parenteral products must be compounded using an aseptic technique to ensure sterility of the medicine. We compared the effect of three clinical environments as compounding areas as well as different aseptic techniques on the sterility of the compounded parenteral product. Clinical pharmacists and pediatric nurses compounded 220 samples in total in three clinical environments: a patient room, a medicine room and biological safety cabinet. The study combined four methods: observation, environmental monitoring (settle plates), monitoring of personnel (finger dab plates) and sterility testing (membrane filtration). Of the compounded samples, 99% were sterile and no significant differences emerged between the clinical environments. Based on the settle plates, the biological safety cabinet was the only area that fulfilled the requirements for eliminating microbial contamination. Most of the steps on the observation form for aseptic techniques were followed. All participants disinfected their hands, wore gloves and disinfected the septum of the vial. Non-contaminated finger dab plates were mostly detected after compounding in the biological safety cabinet. Aseptic techniques were followed relatively well in all environments. However, these results emphasize the importance of good aseptic techniques and support the recommendation of compounding parenteral products in biological safety cabinets in clinical environments.

Installation of an aryl boronic acid function into the external section of N-aryl-oxazolidinones: Synthesis and antimicrobial evaluation.

N-aryl-oxazolidinones is a prominent family of antimicrobials used for treating infections caused by clinically prevalent Gram-positive bacteria. Recently, boron-containing compounds have displayed intriguing potential in the antibiotic discovery setting. Herein, we report the unprecedented introduction of a boron-containing moiety such as an aryl boronic acid in the external region of the oxazolidinone structure via a chemoselective acyl coupling reaction. As a result, we accessed a series of analogues with a distal aryl boronic pharmacophore on the oxazolidinone scaffold. We identified that a peripheric linear conformation coupled with freedom of rotation and no further substitution on the external aryl boronic ring, an amido linkage with hydrogen bonding character, in addition to a para-relative disposition between boronic group and linker, are the optimal combination of structural features in this series for antimicrobial activity. In comparison to linezolid, the analogue comprising all those features, compound 20b, displayed levels of antimicrobial activity augmented by an eight-fold to a thirty-two-fold against a panel of Gram-positive strains, and a near one hundred-fold against Escherichia coli JW5503, a Gram-negative mutant strain with a defective efflux capability.

Genomic diversity of Listeria monocytogenes isolates from seafood, horticulture and factory environments in New Zealand.

Listeria monocytogenes is a foodborne human pathogen that causes systemic infection, fetal-placental infection in pregnant women causing abortion and stillbirth and meningoencephalitis in elderly and immunocompromised individuals. This study aimed to analyse L. monocytogenes from different sources from New Zealand (NZ) and to compare them with international strains. We used pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and whole-genome single nucleotide polymorphisms (SNP) to study the population structure of the NZ L. monocytogenes isolates and their relationship with the international strains. The NZ isolates formed unique clusters in PFGE, MLST and whole-genome SNP comparisons compared to the international isolates for which data were available. PFGE identified 31 AscI and 29 ApaI PFGE patterns with indistinguishable pulsotypes being present in seafood, horticultural products and environmental samples. Apart from the Asc0002:Apa0002 pulsotype which was distributed across different sources, other pulsotypes were site or factory associated. Whole-genome analysis of 200 randomly selected L. monocytogenes isolates revealed that lineage II dominated the NZ L. monocytogenes populations. MLST comparison of international and NZ isolates with lineage II accounted for 89% (177 of 200) of the total L. monocytogenes population, while the international representation was 45.3% (1674 of 3473). Rarefaction analysis showed that sequence type richness was greater in NZ isolates compared to international trend, however, it should be noted that NZ isolates predominantly came from seafood, horticulture and their respective processing environments or factories, unlike international isolates where there was a good mixture of clinical, food and environmental isolates.

New dual ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV active against ESKAPE pathogens.

The rise in multidrug-resistant bacteria defines the need for identification of new antibacterial agents that are less prone to resistance acquisition. Compounds that simultaneously inhibit multiple bacterial targets are more likely to suppress the evolution of target-based resistance than monotargeting compounds. The structurally similar ATP binding sites of DNA gyrase and topoisomerase Ⅳ offer an opportunity to accomplish this goal. Here we present the design and structure-activity relationship analysis of balanced, low nanomolar inhibitors of bacterial DNA gyrase and topoisomerase IV that show potent antibacterial activities against the ESKAPE pathogens. For inhibitor 31c, a crystal structure in complex with Staphylococcus aureus DNA gyrase B was obtained that confirms the mode of action of these compounds. The best inhibitor, 31h, does not show any in vitro cytotoxicity and has excellent potency against Gram-positive (MICs: range, 0.0078-0.0625 µg/mL) and Gram-negative pathogens (MICs: range, 1-2 µg/mL). Furthermore, 31h inhibits GyrB mutants that can develop resistance to other drugs. Based on these data, we expect that structural derivatives of 31h will represent a step toward clinically efficacious multitargeting antimicrobials that are not impacted by existing antimicrobial resistance.

Influence of farming methods and seawater depth on Vibrio species in New Zealand Pacific oysters.

Studies conducted in seawaters around New Zealand have shown the numbers of human pathogenic Vibrio spp. are usually low, but high numbers sometimes occur during warmer summer/autumn months (January - April). In this study, Pacific oysters (Crassostrea gigas) were grown at Kaipara Harbour and Mahurangi Harbour in New Zealand at different heights from the seafloor in different ways: fixed positons intertidally and subtidally, and as floating long lines over the 2013 and 2014 summer periods. Two geographically distinct commercial harvest areas: Coromandel Harbour (North Island) and Croisilles Harbour (South Island) in New Zealand were also compared in 2015 where oysters are grown under different methods. Detection and enumeration of Vibrio spp. was performed according to the Bacteriological Analytical Manual using the Most Probable Number approach and real-time polymerase chain reaction technique. The only significant growing method effect was observed in Mahurangi Harbour, where intertidal oysters at 1.5 m from the seafloor had higher numbers of trh + Vibrio parahaemolyticus than other intertidal samples from Kaipara Harbour and Coromandel Harbour. All other samples showed a relationship with surface seawater temperature, but not with distance from seafloor or farming method. Overall, there is no clear evidence that different oyster farming methods (floating, subtidal or intertidal at different depths) affect Vibrio spp. population sizes, which were dominated by seasonal changes and environmental parameters.

Second-generation 4,5,6,7-tetrahydrobenzo[d]thiazoles as novel DNA gyrase inhibitors.

Aim: DNA gyrase and topoisomerase IV are essential bacterial enzymes, and in the fight against bacterial resistance, they are important targets for the development of novel antibacterial drugs. Results: Building from our first generation of 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA gyrase inhibitors, we designed and prepared an optimized series of analogs that show improved inhibition of DNA gyrase and topoisomerase IV from Staphylococcus aureus and Escherichia coli, with IC50 values in the nanomolar range. Importantly, these inhibitors also show improved antibacterial activity against Gram-positive strains. Conclusion: The most promising inhibitor, 29, is active against Enterococcus faecalis, Enterococcus faecium and S. aureus wild-type and resistant strains, with minimum inhibitory concentrations between 4 and 8 µg/ml, which represents good starting point for development of novel antibacterials.

An optimised series of substituted N-phenylpyrrolamides as DNA gyrase B inhibitors.

ATP competitive inhibitors of DNA gyrase and topoisomerase IV have great therapeutic potential, but none of the described synthetic compounds has so far reached the market. To optimise the activities and physicochemical properties of our previously reported N-phenylpyrrolamide inhibitors, we have synthesized an improved, chemically variegated selection of compounds and evaluated them against DNA gyrase and topoisomerase IV enzymes, and against selected Gram-positive and Gram-negative bacteria. The most potent compound displayed IC50 values of 6.9 nM against Escherichia coli DNA gyrase and 960 nM against Staphylococcus aureus topoisomerase IV. Several compounds displayed minimum inhibitory concentrations (MICs) against Gram-positive strains in the 1-50 µM range, one of which inhibited the growth of Enterococcus faecalis, Enterococcus faecium, S. aureus and Streptococcus pyogenes with MIC values of 1.56 µM, 1.56 µM, 0.78 µM and 0.72 µM, respectively. This compound has been investigated further on methicillin-resistant S. aureus (MRSA) and on ciprofloxacin non-susceptible and extremely drug resistant strain of S. aureus (MRSA VISA). It exhibited the MIC value of 2.5 µM on both strains, and MIC value of 32 µM against MRSA in the presence of inactivated human blood serum. Further studies are needed to confirm its mode of action.

α-Amino Diphenyl Phosphonates as Novel Inhibitors of Escherichia coli ClpP Protease.

Increased Gram-negative bacteria resistance to antibiotics is becoming a global problem, and new classes of antibiotics with novel mechanisms of action are required. The caseinolytic protease subunit P (ClpP) is a serine protease conserved among bacteria that is considered as an interesting drug target. ClpP function is involved in protein turnover and homeostasis, stress response, and virulence among other processes. The focus of this study was to identify new inhibitors of Escherichia coli ClpP and to understand their mode of action. A focused library of serine protease inhibitors based on diaryl phosphonate warheads was tested for ClpP inhibition, and a chemical exploration around the hit compounds was conducted. Altogether, 14 new potent inhibitors of E. coli ClpP were identified. Compounds 85 and 92 emerged as most interesting compounds from this study due to their potency and, respectively, to its moderate but consistent antibacterial properties as well as the favorable cytotoxicity profile.

Persistent Listeria monocytogenes strains isolated from mussel production facilities form more biofilm but are not linked to specific genetic markers.

Contamination of mussels with the human pathogen Listeria monocytogenes occurs during processing in the factory, possibly from bacteria persisting in the factory's indoor and outdoor areas. In this study, a selection of persistent (n=8) and sporadic (n=8) L. monocytogenes isolates associated with mussel-processing premises in New Zealand were investigated for their phenotypic and genomic characteristics. To identify traits that favour or contribute to bacterial persistence, biofilm formation, heat resistance, motility and recovery from dry surfaces were compared between persistent and sporadic isolates. All isolates exhibited low biofilm formation at 20°C, however, at 30°C persistent isolates showed significantly higher biofilm formation after 48h using cell enumeration and near significant difference using the crystal violet assay. All 16 isolates were motile at 20°C and 30°C and motility was fractionally higher for sporadic isolates, but no significant difference was observed. We found persistent isolates tend to exhibit greater recovery after incubation on dry surfaces compared to sporadic isolates. Two of the three most heat-resistant isolates were persistent, while four of five isolates lacking heat resistance were sporadic isolates. Comparison of genome sequences of persistent and sporadic isolates showed that there was no overall clustering of persistent or sporadic isolates, and that differences in prophages and plasmids were not associated with persistence. Our results suggest a link between persistence and biofilm formation, which is most likely multifactorial, combining subtle phenotypic and genotypic differences between isolates.