A novel metagenomic approach uncovers phage genes as markers for increased disinfectant tolerance in mixed Listeria monocytogenes communities.

Listeria monocytogenes is an important human pathogen with a high mortality rate. Consumption of contaminated ready-to-eat food is the main mode of transmission to humans. Disinfectant-tolerant L. monocytogenes have emerged, which are believed to have increased persistence potential. Elucidating the mechanisms of L. monocytogenes disinfectant tolerance has been the focus of previous studies using pure cultures. A limitation of such approach is the difficulty to identify strains with reduced susceptibility due to inter-strain variation and the need to screen large numbers of strains and genes. In this study, we applied a novel metagenomic approach to detect genes associated with disinfectant tolerance in mixed L. monocytogenes planktonic communities. Two communities, consisting of 71 and 80 isolates each, were treated with the food industry disinfectants benzalkonium chloride (BC, 1.75 mg/L) or peracetic acid (PAA, 38 mg/L). The communities were subjected to metagenomic sequencing and differences in individual gene abundances between biocide-free control communities and biocide-treated communities were determined. A significant increase in the abundance of Listeria phage-associated genes was observed in both communities after treatment, suggesting that prophage carriage could lead to an increased disinfectant tolerance in mixed L. monocytogenes planktonic communities. In contrast, a significant decrease in the abundance of a high-copy emrC-harbouring plasmid pLmN12-0935 was observed in both communities after treatment. In PAA-treated community, a putative ABC transporter previously found to be necessary for L. monocytogenes resistance to antimicrobial agents and virulence, was among the genes with the highest weight for differentiating treated from control samples. The undertaken metagenomic approach in this study can be applied to identify genes associated with increased tolerance to other antimicrobials in mixed bacterial communities.

Mur ligase F as a new target for the flavonoids quercitrin, myricetin, and (-)-epicatechin.

MurC, D, E, and F are ATP-dependent ligases involved in the stepwise assembly of the tetrapeptide stem of forming peptidoglycan. As highly conserved targets found exclusively in bacterial cells, they are of significant interest for antibacterial drug discovery. In this study, we employed a computer-aided molecular design approach to identify potential inhibitors of MurF. A biochemical inhibition assay was conducted, screening twenty-four flavonoids and related compounds against MurC-F, resulting in the identification of quercitrin, myricetin, and (-)-epicatechin as MurF inhibitors with IC50 values of 143 µM, 139 µM, and 92 µM, respectively. Notably, (-)-epicatechin demonstrated mixed type inhibition with ATP and uncompetitive inhibition with D-Ala-D-Ala dipeptide and UM3DAP substrates. Furthermore, in silico analysis using Sitemap and subsequent docking analysis using Glide revealed two plausible binding sites for (-)-epicatechin. The study also investigated the crucial structural features required for activity, with a particular focus on the substitution pattern and hydroxyl group positions, which were found to be important for the activity. The study highlights the significance of computational approaches in targeting essential enzymes involved in bacterial peptidoglycan synthesis.

Exploring Alternative Pathways to Target Bacterial Type II Topoisomerases Using NBTI Antibacterials: Beyond Halogen-Bonding Interactions.

Novel bacterial topoisomerase inhibitors (NBTIs) are a new class of antibacterial agents that target bacterial type II topoisomerases (DNA gyrase and topoisomerase IV). Our recently disclosed crystal structure of an NBTI ligand in complex with DNA gyrase and DNA revealed that the halogen atom in the para position of the phenyl right hand side (RHS) moiety is able to establish strong symmetrical bifurcated halogen bonds with the enzyme; these are responsible for the excellent enzyme inhibitory potency and antibacterial activity of these NBTIs. To further assess the possibility of any alternative interactions (e.g., hydrogen-bonding and/or hydrophobic interactions), we introduced various non-halogen groups at the p-position of the phenyl RHS moiety. Considering the hydrophobic nature of amino acid residues delineating the NBTI's binding pocket in bacterial topoisomerases, we demonstrated that designed NBTIs cannot establish any hydrogen-bonding interactions with the enzyme; hydrophobic interactions are feasible in all respects, while halogen-bonding interactions are apparently the most preferred.

Amide containing NBTI antibacterials with reduced hERG inhibition, retained antimicrobial activity against gram-positive bacteria and in vivo efficacy.

Novel bacterial topoisomerase inhibitors (NBTIs) are new promising antimicrobials for the treatment of multidrug-resistant bacterial infections. In recent years, many new NBTIs have been discovered, however most of them struggle with the same issue - the balance between antibacterial activity and hERG-related toxicity. We started a new campaign by optimizing the previous series of NBTIs, followed by the design and synthesis of a new, amide-containing focused NBTI library to reduce hERG inhibition and maintain antibacterial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). This optimization strategy yielded the lead compound 12 that exhibits potent antibacterial activity against Gram-positive bacteria, reduced hERG inhibition, no cardiotoxicity in zebrafish model, and a favorable in vivo efficacy in a neutropenic murine thigh infection model of MRSA infection.

Antimicrobial Resistance and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus from Two Pig Farms: Longitudinal Study of LA-MRSA.

Pigs were identified as the most important reservoir of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), mostly belonging to the emergent zoonotic clonal complex (CC) 398. Here, we investigated the presence of MRSA in sows and piglets over a period of several months in two pig farms (intensive farm A and family-run farm B). Isolates underwent antimicrobial susceptibility testing, PCR characterization and spa typing. We collected 280 samples, namely 206 nasal swabs from pigs and 74 environmental samples from pig housings at 12 consecutive time points. A total of 120/161 (74.5%) and 75/119 (63.0%) samples were MRSA-positive in farms A and B, respectively. All isolates harbored mecA but lacked mecC and PVL-encoding genes. The identified spa types (t571, t034, t1250 and t898 in farm A, t1451 and t011 in farm B) were indicative of CC398. Antimicrobial resistance patterns (all multidrug resistant in farm A, 57.2% in farm B) depended on the farm, suggesting the impact of farm size and management practices on the prevalence and characteristics of MRSA. Due to the intermittent colonization of pigs and the high contamination of their immediate environment, MRSA status should be determined at the farm level when considering preventive measures or animal trade between farms.

Covalent inhibitors of bacterial peptidoglycan biosynthesis enzyme MurA with chloroacetamide warhead.

MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) catalyzes the first committed step in the cytoplasmic part of peptidoglycan biosynthesis and is a validated target enzyme for antibacterial drug discovery; the inhibitor fosfomycin has been used clinically for decades. Like fosfomycin, most MurA inhibitors are small heterocyclic compounds that inhibit the enzyme by forming a covalent bond with the active site cysteine. The reactive chloroacetamide group was selected from a series of suitable electrophilic thiol-reactive warheads. The predominantly one-step synthesis led to the construction of the final library of 47 fragment-sized chloroacetamide compounds. Several new E. coli MurA inhibitors were identified, with the most potent compound having an IC50 value in the low micromolar range. The electrophilic reactivity of all chloroacetamide fragments in our library was evaluated by a high-throughput spectrophotometric assay using the reduced Ellman reagent as a surrogate for the cysteine thiol. LC-MS/MS experiments confirmed the covalent binding of the most potent inhibitor to Cys115 of the digested MurA enzyme. The covalent binding was further investigated by a biochemical time-dependent assay and a dilution assay, which confirmed the irreversible and time-dependent mode of action. The efficacy of chloroacetamide derivatives against MurA does not correlate with their thiol reactivity, making the active fragments valuable starting points for fragment-based development of new antibacterial agents targeting MurA.

Lactic acid bacteria and bifidobacteria deliberately introduced into the agro-food chain do not significantly increase the antimicrobial resistance gene pool.

Lactic acid bacteria (LAB) and bifidobacteria may serve as reservoirs of antimicrobial resistance, but the risk posed by strains intentionally introduced into the agro-food chain has not yet been thoroughly investigated. The aim of our study was to evaluate whether probiotics, starter and protective cultures, and feed additives represent a risk to human health. In addition to commercial strains of LAB and bifidobacteria, isolates from human milk or colostrum, intestinal mucosa or feces, and fermented products were analyzed. Phenotypic susceptibility data of 474 strains showed that antimicrobial resistance was more common in intestinal isolates than in commercial strains. Antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) were characterized in the whole genome sequences of 1114 strains using comparative genomics. Intrinsic ARGs were abundant in enterococci, bifidobacteria, and lactococci but were considered non-risky due to the absence of MGEs. The results revealed that 13.8% of commercial strains contained acquired ARGs, most frequently for tetracycline. We associated 75.5% of the acquired ARGs with known or novel MGEs, and their potential for transmission was assessed by examining metagenomic sequences. We confirmed that ARGs and MGEs were not as abundant or diverse in commercial strains as in human intestinal isolates or isolates from human milk, suggesting that strains intentionally introduced into the agro-food chain do not pose a significant threat. However, attention should be paid especially to individual probiotic strains containing elements that have been shown to have high potential for transferability in the gut microbiota.Abbreviations: ARG, antimicrobial resistance gene; ICE, integrative and conjugative element; IME, integrative and mobilizable element; LAB, lactic acid bacteria; MDR, multidrug resistance; MIC, minimum inhibitory concentration; MGE, mobile genetic element; TRRPP, tetracycline-resistant ribosomal protection protein; WGS, whole genome sequences.

Diminishing hERG inhibitory activity of aminopiperidine-naphthyridine linked NBTI antibacterials by structural and physicochemical optimizations.

Novel bacterial topoisomerase inhibitors (NBTIs) are an important new class of antibacterials targeting bacterial type II topoisomerases (DNA gyrase and topoisomerase IV). Notwithstanding their potent antibacterial activity, they suffer from a detrimental class-related hERG blockage. In this study, we designed and synthesized an optimized library of NBTIs comprising different linker moieties that exhibit reduced hERG inhibition and retain inhibitory potencies on DNA gyrase and topoisomerase IV of Staphylococcus aureus and Escherichia coli, respectively, as well as potent antibacterial activities. Substitution of the linker's tertiary amine with polar groups outcome in diminished hERG inhibition. Compound 17 expresses nanomolar enzyme inhibitory potency and antibacterial activity against both Gram-positive and Gram-negative bacteria as well as reduced hERG inhibition relative to our previously published NBTI analogs. Here, we point to some important NBTI's structural features that influence their hERG inhibitory activity.

Novel TaqMan PCR Assay for the Quantification of Paenibacillus larvae Spores in Bee-Related Samples.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybees. P. larvae spore counts in bee-related samples correlate with the presence of AFB symptoms and may, therefore, be used to identify at-risk colonies. Here, we constructed a TaqMan-based real-time PCR (qPCR) assay targeting a single-copy chromosomal metalloproteinase gene for reliable quantification of P. larvae. The assay was calibrated using digital PCR (dPCR) to allow absolute quantification of P. larvae spores in honey and hive debris samples. The limits of detection and quantification were 8 and 58 spores/g for honey and 188 and 707 spores/mL for hive debris, respectively. To assess the association between AFB clinical symptoms and spore counts, we quantified spores in honey and hive debris samples originating from honeybee colonies with known severity of clinical symptoms. Spore counts in AFB-positive colonies were significantly higher than those in asymptomatic colonies but did not differ significantly with regard to the severity of clinical symptoms. For honey, the average spore germination rate was 0.52% (range = 0.04-6.05%), indicating poor and inconsistent in vitro germination. The newly developed qPCR assay allows reliable detection and quantification of P. larvae in honey and hive debris samples but can also be extended to other sample types.

How Accurate Are Veterinary Clinicians Employing Flexicult Vet for Identification and Antimicrobial Susceptibility Testing of Urinary Bacteria?

Antibiotics are frequently used for treating urinary tract infections (UTI) in dogs and cats. UTI often requires time-consuming and expensive antimicrobial susceptibility testing (AST). Alternatively, clinicians can employ Flexicult Vet, an affordable chromogenic agar with added antibiotics for in-clinic AST. We investigated how well veterinary microbiologists and clinicians, without any prior experience, employ Flexicult Vet for the identification and AST of the most common canine and feline urinary pathogenic bacteria. We prepared 47 monoculture plates containing 10 bacterial species. The test's mean accuracy was 75.1% for bacteria identification (84.6% and 68.7% for microbiologists and clinicians, respectively) and 79.2% for AST (80.7% and 78.2%). All evaluators employed Flexicult Vet with the accuracies over 90% for the distinctively colored bacteria like Escherichia coli (red), Enterococcus faecalis (turquoise), and Proteus spp. (pale brown). However, the evaluators' experience proved important in recognizing lightly colored bacteria like Staphylococcus pseudintermedius (accuracies of 82.6% and 40.3%). Misidentifications of E. faecium additionally worsened AST performance since bacterial intrinsic resistance could not be considered. Finally, only 33.3% (3/9) of methicillin-resistant S. pseudintermedius (MRSP) were correctly detected. To conclude, Flexicult Vet proved reliable for certain urinary pathogens. In contrast, light-colored bacteria (e.g., Staphylococcus), often misidentified, require a standard AST.

Structurally Optimized Potent Dual-Targeting NBTI Antibacterials with an Enhanced Bifurcated Halogen-Bonding Propensity.

We designed and synthesized an optimized library of novel bacterial topoisomerase inhibitors with p-halogenated phenyl right-hand side fragments and significantly enhanced and balanced dual-targeted DNA gyrase and topoisomerase IV activities of Staphylococcus aureus and Escherichia coli. By increasing the electron-withdrawing properties of the p-halogenated phenyl right-hand side fragment and maintaining a similar lipophilicity and size, an increased potency was achieved, indicating that the antibacterial activities of this series of novel bacterial topoisomerase inhibitors against all target enzymes are determined by halogen-bonding rather than van der Waals interactions. They show nanomolar enzyme inhibitory and whole-cell antibacterial activities against S. aureus and methicillin-resistant S. aureus (MRSA) strains. However, due to the relatively high substrate specificity for the bacterial efflux pumps, they tend to be less potent against E. coli and other Gram-negative pathogens.

A Fine-Tuned Lipophilicity/Hydrophilicity Ratio Governs Antibacterial Potency and Selectivity of Bifurcated Halogen Bond-Forming NBTIs.

Herein, we report the design of a focused library of novel bacterial topoisomerase inhibitors (NBTIs) based on innovative mainly monocyclic right-hand side fragments active against DNA gyrase and Topo IV. They exhibit a very potent and wide range of antibacterial activity, even against some of the most concerning hard-to-treat pathogens for which new antibacterials are urgently needed, as reported by the WHO and CDC. NBTIs enzyme activity and whole cell potency seems to depend on the fine-tuned lipophilicity/hydrophilicity ratio that governs the permeability of those compounds through the bacterial membranes. Lipophilicity of NBTIs is apparently optimal for passing through the membrane of Gram-positive bacteria, but the higher, although not excessive lipophilicity and suitable hydrophilicity seems to determine the passage through Gram-negative bacterial membranes. However, due to the considerable hERG inhibition, which is still at least two orders of magnitude away from MICs, continued optimization is required to realize their full potential.

Impact of Type of Parturition on Colostrum Microbiota Composition and Puppy Survival.

The objective of our study was to determine whether the type of parturition affects the microbiota of the colostrum and the growth and survival of the puppies. Seventy-nine newborn puppies were divided into three groups regarding the type of parturition: vaginal delivery (VD), elective caesarean section (EL-CS), and emergency caesarean section (EM-CS). After the birth of the puppies, swabs of meconium were collected from the puppies and colostrum was obtained from the dam. Many aerobic and anaerobic bacteria were isolated and identified by mass spectrometry (MALDI-TOF MS). The colostrum microbiota of VD and EL-CS puppies contained a significantly higher abundance of bacteria belonging to the genera Staphylococcus, Kocuria and Enterococcus compared with EM-CS colostrum samples. The composition of the meconium microbiota of the puppies present at birth was similar to the colostrum microbiota of their mothers. It was also found that puppies without a meconium microbiota at birth gained weight more slowly compared with puppies with a meconium microbiota at birth. The type of parturition influenced the bacterial composition of the microbiota in the colostrum. Future studies are necessary to further define the significance of the observed differences in microbiota composition between EM-CS compared with EL-CS and VD colostrum microbiota.

Feline Otitis Externa Caused by Methicillin-Resistant Staphylococcus aureus with Mixed Hemolytic Phenotype and Overview of Possible Genetic Backgrounds.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of nosocomial infections in humans, but its importance in small animal practice is increasing. Here, we present a case of feline otitis externa (OE) caused by MRSA; both hemolytic and nonhemolytic variants with a stable phenotype were recovered from the external auditory canal after infection was detected by routine otoscopy. One isolate per variant underwent antimicrobial susceptibility testing (AST) by broth microdilution method, conventional spa typing and whole-genome sequencing (WGS). The results showed that both variants were genetically related and were of sequence type (ST) 1327, SCCmec type IV and spa type t005. AST and WGS showed that both isolates were resistant to ß-lactams and sensitive to all tested non-ß-lactam antibiotics. Both isolates were pvl-negative, but encoded several other virulence genes (aur, hlgABC, sak, scn, seg, sei, sem, sen, seo and seu). Genetic background of the mixed hemolytic phenotype was not identified; no differences in the agr locus or other regulatory regions were detected. Three single-nucleotide polymorphisms were identified but could not be associated with hemolysis. This well-documented case of MRSA infection in companion animals adds to the reports of MRSA infections with a mixed hemolytic phenotype.

Genomic insights into the emergence and spread of methicillin-resistant Staphylococcus pseudintermedius in veterinary clinics.

Staphylococcus pseudintermedius is a common cause of skin and soft tissue infections in dogs but can also cause infections in cats and humans. The frequency of methicillin-resistant S. pseudintermedius (MRSP) strains is increasing worldwide. Here, we obtained 43 MRSP isolates from dogs (n = 41), one cat (n = 1) and the small animal clinic environment (n = 1) in Slovenia from the period 2008-2018, which underwent whole-genome sequencing (WGS) and antimicrobial susceptibility testing. Five sequence types (STs) were identified, with ST71 (32/43) and ST551 (8/43) being the predominant. In Slovenia, ST551 was first detected in 2016, whereas a decrease in the frequency of ST71 was observed after 2015. All isolates were multidrug-resistant and most antimicrobial-resistant phenotypes could be linked to acquisition of the corresponding resistance genes or gene mutations. Core-genome multilocus sequence typing (cgMLST) revealed several potential MRSP transmission routes: (i) between two veterinary clinics by a single MRSP-positive dog, (ii) between the environment of a veterinary clinic and a dog, and (iii) between a canine and a feline patient through the contaminated environment of a veterinary clinic. Of the six dogs that were additionally sampled from 14 days to five months after the initial sampling, each harbored the same MRSP strain, suggesting a limited within-host diversity of MRSP in symptomatic dogs. The present results highlight the importance of MRSP-positive dogs in the spread of veterinary care-associated MRSP infections and call for the implementation of strict control measures to reduce MRSP contamination in veterinary clinic environments originating from animal-contact surfaces.

Computational Design and Development of Benzodioxane-Benzamides as Potent Inhibitors of FtsZ by Exploring the Hydrophobic Subpocket.

Multidrug resistant Staphylococcus aureus is a severe threat, responsible for most of the nosocomial infections globally. This resistant strain is associated with a 64% increase in death compared to the antibiotic-susceptible strain. The prokaryotic protein FtsZ and the cell division cycle have been validated as potential targets to exploit in the general battle against antibiotic resistance. Despite the discovery and development of several anti-FtsZ compounds, no FtsZ inhibitors are currently used in therapy. This work further develops benzodioxane-benzamide FtsZ inhibitors. We seek to find more potent compounds using computational studies, with encouraging predicted drug-like profiles. We report the synthesis and the characterization of novel promising derivatives that exhibit very low MICs towards both methicillin-susceptible and -resistant S. aureus, as well as another Gram positive species, Bacillus subtilis, while possessing good predicted physical-chemical properties in terms of solubility, permeability, and chemical and physical stability. In addition, we demonstrate by fluorescence microscopy that Z ring formation and FtsZ localization are strongly perturbed by our derivatives, thus validating the target.

ERIC and WGS Typing of Paenibacillus larvae in Slovenia: Investigation of ERIC I Outbreaks.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a fatal disease of honeybee brood. Here, we obtained 506 P. larvae isolates originating from honey or brood samples and from different geographic regions of Slovenia in the period 2017-2019. In the first part of the study, we conducted ERIC-PCR typing to assess the frequency of ERIC types in Slovenia. Capillary electrophoresis was used for the analysis of ERIC patterns, revealing good separation efficiency and enabling easy lane-to-lane comparisons. ERIC II was the predominant type (70.2%), followed by ERIC I (29.8%); two slightly altered ERIC I banding patterns were observed but were not considered relevant for the discrimination of ERIC types. No evident spatiotemporal clustering of ERIC types was observed. To assess the clonality of the outbreak-related P. larvae ERIC I isolates, 59 isolates of this type underwent whole-genome sequencing (WGS). Whole-genome multilocus sequence typing (wgMLST) revealed seven ERIC I-ST2 outbreak clusters (≤35 allele differences) with the median intra-outbreak diversity ranging from 7 to 27 allele differences. In all seven clusters, the transmission of P. larvae outbreak clone within a 3-km radius (AFB zone) was observed, which could be explained by the activity of honeybees. In three clusters, the transmission of the outbreak clone between geographically distant apiaries was revealed, which could be explained by the activities of beekeepers such as migratory beekeeping and trading of bee colonies. The present findings reinforce the importance of beekeeping activities in the transmission of P. larvae. WGS should be used as a reference typing method for the detection of P. larvae transmission clusters.

Mur ligases inhibitors with azastilbene scaffold: Expanding the structure-activity relationship.

Antibiotic resistance represents one of the biggest public health challenges in the last few years. Mur ligases (MurC-MurF) are involved in the synthesis of UDP-N-acetylmuramyl-pentapeptide, the main building block of bacterial peptidoglycan polymer. They are essential for the survival of bacteria and therefore important antibacterial targets. We report herein the synthesis and structure-activity relationships of Mur ligases inhibitors with an azastilbene scaffold. Several compounds showed promising inhibitory potencies against multiple ligases and one compound also possessed moderate antibacterial activity. These results represent a solid ground for further development and optimization of structurally novel antimicrobial agents to combat the rising bacterial resistance.

Livestock-associated methicillin-resistant Staphylococcus aureus: Establishing links between animals and humans on livestock holdings.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) represents a concern in both human and veterinary medicine. The aim of this study was to investigate potential LA-MRSA transmission between animals and humans in rural settings. To this aim, a study was designed to include 14 farms in Slovenia, which were selected on the basis of a farmer (initial patient) with confirmed LA-MRSA infection and regular animal contacts. On all farms, the initial patients, their household members, animals and barn environment were analysed for the presence of LA-MRSA. In addition, the epidemiologically linked hospital-related LA-MRSA isolates were included to investigate possible nosocomial transmissions. On five farms, LA-MRSA was discovered both in animals and in humans. In total, 49 LA-MRSA isolates of different origins underwent whole-genome sequencing, antimicrobial susceptibility testing and spa typing. All 49 isolates belonged to the sequence type 398 (ST398), spa types t011 and t034, and harboured staphylococcal chromosomal cassette mec Vc. High levels of concordance between resistance phenotypes and genotypes were observed. No transmission pairs between animals and initial patients were discovered. However, several isolates originating from farm animals and other household members formed clusters with pairwise distances of ≤14 single nucleotide polymorphisms (SNPs), indicating recent transmission events. In addition, three closely related isolates (0 SNP) form hospitalized patients were observed, indicating a possible nosocomial transmission. Two hospital-related isolates harboured the immune evasion cluster genes, which are associated with adaptation to the human host; however, these two isolates differed in >30 SNPs from the remaining isolates. Characteristics of LA-MRSA from Slovenia reflect those observed previously in other European studies. Immune evasion cluster-positive LA-MRSA ST398 suggests its re-adaptation to the human host and calls for a closer monitoring of such emerging LA-MRSA lineages, in addition to monitoring and preventing the introduction of LA-MRSA from farms to hospitals where transmission is highly plausible.