A new phenothiazine derivate is active against Clostridioides difficile and shows low cytotoxicity.

The rapid evolution of antibiotic resistance in Clostridioides difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are matters of concern for public health. Thioridazine, a compound belonging to the phenothiazine group, has previous shown antimicrobial activity against C. difficile. The purpose of this present study was to investigate the potential of a novel phenothiazine derivative, JBC 1847, as an oral antimicrobial for treatment of intestinal pathogens and CDIs. The minimal inhibition concentration and the minimum bactericidal concentration of JBC 1847 against C. difficile ATCC 43255 were determined 4 µg/mL and high tolerance after oral administration in mice was observed (up to 100 mg/kg bodyweight). Pharmacokinetic modeling was conducted in silico using GastroPlusTM, predicting low (< 10%) systemic uptake after oral exposure and corresponding low Cmax in plasma. Impact on the intestinal bacterial composition after four days of treatment was determined by 16s rRNA MiSeq sequencing and revealed only minor impact on the microbiota in non-clinically affected mice, and there was no difference between colony-forming unit (CFU)/gram fecal material between JBC 1847 and placebo treated mice. The cytotoxicity of the compound was assessed in Caco-2 cell-line assays, in which indication of toxicity was not observed in concentrations up to seven times the minimal bactericidal concentration. In conclusion, the novel phenothiazine derivative demonstrated high antimicrobial activity against C. difficile, had low predicted gastrointestinal absorption, low intestinal (in vitro) cytotoxicity, and only induced minor changes of the healthy microbiota, altogether supporting that JBC 1847 could represent a novel antimicrobial candidate. The clinical importance hereof calls for future experimental studies in CDI models.

Efficacy of a novel antimicrobial hydrogel for eradication of Staphylococcus epidermidis, Staphylococcus aureus and Cutibacterium acnes from preformed biofilm and treatment performance in an in vivo MRSA wound model.

BACKGROUND: Bacterial biofilm formation is a complicating factor in the antimicrobial treatment of bacterial infections. OBJECTIVES: In this study, we assessed the impact of a novel hydrogel with the active antimicrobial compound JBC 1847 on eradication of preformed biofilms of Staphylococcus epidermidis, Cutibacterium acnes and MRSA in vitro, and evaluated the in vivo efficacy of MRSA wound treatment. METHODS: Biofilms were exposed to JBC 1847 for 24 h and subsequently the treatments were neutralized and surviving biofilm-associated bacteria recovered and enumerated. The efficacy of the hydrogel on post-treatment load of MRSA was determined in a murine model of MRSA wound infection, and skin samples of the infected mice were examined histologically to evaluate the degree of healing. RESULTS: A concentration-dependent eradication of biofilm-embedded bacteria by JBC 1847 was observed for all three pathogens, and the hydrogel caused a greater than four log reduction of cfu in all cases. In the mouse model, treatment with the hydrogel significantly reduced the cfu/mL of MRSA compared with treatment of MRSA-infected wounds with pure hydrogel. Histopathological analysis of the wounds showed that the JBC 1847 treatment group had a lower grade of inflammation, a higher mean score of re-epithelization and higher mean scores of parameters assessing the maturity of the newly formed epidermis, compared with both the fusidic acid 2% and vehicle treatment groups. CONCLUSIONS: The novel hydrogel shows promising results as a candidate for future wound treatment, likely to be highly effective even in the case of biofilm-complicating infected wounds.

Insight Into the Anti-staphylococcal Activity of JBC 1847 at Sub-Inhibitory Concentration.

Multidrug-resistant pathogens constitute a serious global issue and, therefore, novel antimicrobials with new modes of action are urgently needed. Here, we investigated the effect of a phenothiazine derivative (JBC 1847) with high antimicrobial activity on Staphylococcus aureus, using a wide range of in vitro assays, flow cytometry, and RNA transcriptomics. The flow cytometry results showed that JBC 1847 rapidly caused depolarization of the cell membrane, while the macromolecule synthesis inhibition assay showed that the synthesis rates of DNA, RNA, cell wall, and proteins, respectively, were strongly decreased. Transcriptome analysis of S. aureus exposed to sub-inhibitory concentrations of JBC 1847 identified a total of 78 downregulated genes, whereas not a single gene was found to be significantly upregulated. Most importantly, there was downregulation of genes involved in adenosintrifosfat (ATP)-dependent pathways, including histidine biosynthesis, which is likely to correlate with the observed lower level of intracellular ATP in JBC 1847-treated cells. Furthermore, we showed that JBC 1847 is bactericidal against both exponentially growing cells and cells in a stationary growth phase. In conclusion, our results showed that the antimicrobial properties of JBC 1847 were primarily caused by depolarization of the cell membrane resulting in dissipation of the proton motive force (PMF), whereby many essential bacterial processes are affected. JBC 1847 resulted in lowered intracellular levels of ATP followed by decreased macromolecule synthesis rate and downregulation of genes essential for the amino acid metabolism in S. aureus. Bacterial compensatory mechanisms for this proposed multi-target activity of JBC 1847 seem to be limited based on the observed very low frequency of resistance toward the compound.

A Novel Promazine Derivative Shows High in vitro and in vivo Antimicrobial Activity Against Staphylococcus aureus.

The emergence of multidrug-resistant bacteria constitutes a significant public health issue worldwide. Consequently, there is an urgent clinical need for novel treatment solutions. It has been shown in vitro that phenothiazines can act as adjuvants to antibiotics whereby the minimum inhibitory concentration (MIC) of the antibiotic is decreased. However, phenothiazines do not perform well in vivo, most likely because they can permeate the blood-brain (BBB) barrier and cause severe side-effects to the central nervous system. Therefore, the aim of this study was to synthesize a promazine derivate that would not cross the BBB but retain its properties as antimicrobial helper compound. Surprisingly, in vitro studies showed that the novel compound, JBC 1847 exhibited highly increased antimicrobial activity against eight Gram-positive pathogens (MIC, 0.5-2 mg/L), whereas a disc diffusion assay indicated that the properties as an adjuvant were lost. JBC 1847 showed significant (P < 0.0001) activity against a Staphylococcus aureus strain compared with the vehicle, in an in vivo wound infection model. However, both in vitro and in silico analyses showed that JBC 1847 possesses strong affinity for human plasma proteins and an Ames test showed that generally, it is a non-mutagenic compound. Finally, in silico predictions suggested that the compound was not prone to pass the BBB and had a suitable permeability to the skin. In conclusion, JBC 1847 is therefore suggested to hold potential as a novel topical agent for the clinical treatment of S. aureus skin and soft tissue infections, but pharmacokinetics and pharmacodynamics need to be further investigated.

A Novel Derivative of Thioridazine Shows Low Toxicity and Efficient Activity against Gram-Positive Pathogens.

Thioridazine hydrochloride (HCl) has been suggested as a promising antimicrobial helper compound for the treatment of infections with antimicrobial-resistant bacteria. Unfortunately, the therapeutic concentration of thioridazine HCl is generally higher than what can be tolerated clinically, in part due to its toxic side effects on the central nervous system. Therefore, we aimed to synthesize a less toxic thioridazine derivative that would still retain its properties as a helper compound. This resulted in a compound designated 1-methyl-2-(2-(2-(methylthio)-10H-phenothiazin-10-yl)ethyl)-1-pentylpiperidin-1-ium bromide (abbreviated T5), which exhibited low blood-brain barrier permeability. The lowest minimal inhibitory concentration (MIC) against Staphylococcus aureus exposed to the novel compound was reduced 32-fold compared to thioridazine HCl (from 32 µg/mL to 1 µg/mL). The MIC values for T5 against five Gram-positive pathogens ranged from 1 µg/mL to 8 µg/mL. In contrast to thioridazine HCl, T5 does not act synergistically with oxacillin. In silico predictive structure analysis of T5 suggests that an acceptably low toxicity and lack of induced cytotoxicity was demonstrated by a lactate dehydrogenase assay. Conclusively, T5 is suggested as a novel antimicrobial agent against Gram-positive bacteria. However, future pharmacokinetic and pharmacodynamic studies are needed to clarify the clinical potential of this novel discovery.

GtxA is a virulence factor that promotes a Th2-like response during Gallibacterium anatis infection in laying hens.

GtxA, a leukotoxic RTX-toxin, has been proposed a main virulence factor of Gallibacterium anatis. To evaluate the impact of GtxA during infection, we experimentally infected laying hens with a G. anatis wild-type (WT) strain and its isogenic gtxA deletion mutant (ΔgtxA), respectively, and monitored the birds during a 6 day period. Birds inoculated with ΔgtxA had significantly reduced gross lesions and microscopic changes compared to the birds inoculated with the WT strain. To assess the host response further, we quantified the expression of pro-inflammatory cytokines and apoptosis genes by RT-qPCR. In the ovarian tissue, the expression levels of IL-4 and TNF-α were significantly lower in the ΔgtxA group compared to the WT group, while IL-6 and IL-10 levels appeared similar in the two groups. In the spleen tissue of ΔgtxA infected chickens, IL-4 expression was also lower compared to the WT infected chickens. The results indicated that GtxA plays a key role in an acute cytokine-mediated Th2-like response against G. anatis infection in the ovary tissue. The pro-inflammatory response in the ovary tissue of birds inoculated with ΔgtxA mutant was thus significantly lower than the wild-type response. This was, at least partly, supported by the apoptosis gene expression levels, which were significantly higher in the ΔgtxA mutant compared to the wild-type infected chickens. In conclusion, GtxA clearly plays an important role in the pathogenesis of G. anatis infections in laying hens. Further investigations into the specific factors regulating the host response is however needed to provide a more complete understanding of the bacteria-host interaction.

Draft Genome Sequences of Three Human Pathogenic Acinetobacter baumannii Strains.

Acinetobacter baumannii is an opportunistic human pathogen with the ability to develop multiple resistances against the main antibiotic classes. It causes nosocomial infections, especially in intensive care units. Here, we present the draft genome sequences of three isolates (AB1, AB2, and AB3) from humans, collected from two hospitals in Tabriz, Iran.

Diversity and Population Overlap between Avian and Human Escherichia coli Belonging to Sequence Type 95.

Avian-pathogenic Escherichia coli (APEC) is a subgroup of extraintestinal pathogenic E.coli (ExPEC) presumed to be zoonotic and to represent an external reservoir for extraintestinal infections in humans, including uropathogenic E. coli (UPEC) causing urinary tract infections. Comparative genomics has previously been applied to investigate whether APEC and human ExPEC are distinct entities. Even so, whole-genome-based studies are limited, and large-scale comparisons focused on single sequence types (STs) are not available yet. In this study, comparative genomic analysis was performed on 323 APEC and human ExPEC genomes belonging to sequence type 95 (ST95) to investigate whether APEC and human ExPEC are distinct entities. Our study showed that APEC of ST95 did not constitute a unique ExPEC branch and was genetically diverse. A large genetic overlap between APEC and certain human ExPEC was observed, with APEC located on multiple branches together with closely related human ExPEC, including nearly identical APEC and human ExPEC. These results illustrate that certain ExPEC clones may indeed have the potential to cause infection in both poultry and humans. Previously described ExPEC-associated genes were found to be encoded on ColV plasmids. These virulence-associated plasmids seem to be crucial for ExPEC strains to cause avian colibacillosis and are strongly associated with strains of the mixed APEC/human ExPEC clusters. The phylogenetic analysis revealed two distinct branches consisting of exclusively closely related human ExPEC which did not carry the virulence-associated plasmids, emphasizing a lower avian virulence potential of human ExPEC in relation to an avian host.IMPORTANCE APEC causes a range of infections in poultry, collectively called colibacillosis, and is the leading cause of mortality and is associated with major economic significance in the poultry industry. A growing number of studies have suggested APEC as an external reservoir of human ExPEC, including UPEC, which is a reservoir. ExPEC belonging to ST95 is considered one of the most important pathogens in both poultry and humans. This study is the first in-depth whole-genome-based comparison of ST95 E. coli which investigates both the core genomes as well as the accessory genomes of avian and human ExPEC. We demonstrated that multiple lineages of ExPEC belonging to ST95 exist, of which the majority may cause infection in humans, while only part of the ST95 cluster seem to be avian pathogenic. These findings further support the idea that urinary tract infections may be a zoonotic infection.

Reclassification of Bisgaard taxon 37 and taxon 44 as Psittacicella melopsittaci gen. nov., sp. nov., Psittacicella hinzii sp. nov. and Psittacicella gerlachiana sp. nov. within Psittacicellaceae fam. nov. of the order Pasteurellales.

Bacteria isolated from lesions as well as apparently normal tissues of psittacine birds have previously been reported as taxon 37 and taxon 44 of Bisgaard. 16S rRNA gene sequence comparisons revealed a distant relationship to members of Pasteurellaceae at the species, genus and family levels. The polar lipid profile consisted of the major components phosphatidylethanolamine and phosphatidylglycerol. A new family Psittacicellaceae fam. nov. is proposed with the type genus Psittacicella gen. nov. The new genus Psittacicella includes the type species Psittacicella melopsittaci sp. nov. with type strain B96/4T (=CCUG 70858T=DSM 105476T), Psittacicella hinzii sp. nov. with type strain 111T (=CCUG 52861T=CCM 8842T) and Psittacicella gerlachiana sp. nov. with type strain EEAB3T1T (=CCUG 70857T=DSM 105477T). In addition to the major polar lipids, strain 111T possessed the non-identified aminophospholipids APL1 and APL2 and trace amounts of four lipids (L1-L4) whereas strain B94/4T showed the minor unidentified aminophospholipids APL3 and APL2 and trace amounts of unidentified lipid L3. These results demonstrate that strain B96/4T can be distinguished from 111T based on presence/absence of the unidentified lipids APL1 and APL3. The total polar lipid profile of strain EEAB3T1T differed from B96/4Tonly in one minor lipid. Strain B96/4T can further be distinguished from 111T by acid formation from trehalose and raffinose and the α-glucosidase test. Strains 111T and EEAB3T1T can be separated based on acid formation from trehalose and the α-glucosidase test. Strains B96/4T and EEAB3T1T can be separated by acid formation from raffinose and eight signature indels in the RpoB protein.

Chromosomal features of Escherichia coli serotype O2:K2, an avian pathogenic E. coli.

Escherichia coli causing infection outside the gastrointestinal system are referred to as extra-intestinal pathogenic E. coli. Avian pathogenic E. coli is a subgroup of extra-intestinal pathogenic E. coli and infections due to avian pathogenic E. coli have major impact on poultry production economy and welfare worldwide. An almost defining characteristic of avian pathogenic E. coli is the carriage of plasmids, which may encode virulence factors and antibiotic resistance determinates. For the same reason, plasmids of avian pathogenic E. coli have been intensively studied. However, genes encoded by the chromosome may also be important for disease manifestation and antimicrobial resistance. For the E. coli strain APEC_O2 the plasmids have been sequenced and analyzed in several studies, and E. coli APEC_O2 may therefore serve as a reference strain in future studies. Here we describe the chromosomal features of E. coli APEC_O2. E. coli APEC_O2 is a sequence type ST135, has a chromosome of 4,908,820 bp (plasmid removed), comprising 4672 protein-coding genes, 110 RNA genes, and 156 pseudogenes, with an average G + C content of 50.69%. We identified 82 insertion sequences as well as 4672 protein coding sequences, 12 predicated genomic islands, three prophage-related sequences, and two clustered regularly interspaced short palindromic repeats regions on the chromosome, suggesting the possible occurrence of horizontal gene transfer in this strain. The wildtype strain of E. coli APEC_O2 is resistant towards multiple antimicrobials, however, no (complete) antibiotic resistance genes were present on the chromosome, but a number of genes associated with extra-intestinal disease were identified. Together, the information provided here on E. coli APEC_O2 will assist in future studies of avian pathogenic E. coli strains, in particular regarding strain of E. coli APEC_O2, and aid in the general understanding of the pathogenesis of avian pathogenic E. coli.

Evaluation of the efficacy of an autogenous Escherichia coli vaccine in broiler breeders.

In poultry production Escherichia coli autogenous vaccines are often used. However, the efficacy of autogenous E. coli vaccinations has not been evaluated experimentally in chickens after start of lay. The aim of the present study was to evaluate the protective effect of an autogenous E. coli vaccine in broiler breeders. Three groups of 28-week-old broiler breeders (unvaccinated, vaccinated once and twice, respectively) were challenged with a homologous E. coli strain (same strain as included in the vaccine) or a heterologous challenge strain in an experimental ascending model. The clinical outcome was most pronounced in the unvaccinated group; however, the vast majority of chickens in the vaccinated groups had severe pathological manifestations similar to findings in the unvaccinated group after challenge with a homologous as well as a heterologous E. coli strain. Although significant titre rises in IgY antibodies were observed in the twice vaccinated group, antibodies did not confer significant protection in terms of pathological impact. Neither could transfer of maternal-derived antibodies to offspring be demonstrated. In conclusion, with the use of the present model for ascending infection, significant protection of an autogenous E. coli vaccine against neither a homologous nor a heterologous E. coli challenge could not be documented.

Draft Genome Sequences of Two Avian Pathogenic Escherichia coli Strains of Clinical Importance, E44 and E51.

Avian pathogenic Escherichia coli strains have remarkable impacts on animal welfare and the production economy in the poultry industry worldwide. Here, we present the draft genomes of two isolates from chickens (E44 and E51) obtained from field outbreaks and subsequently investigated for their potential for use in autogenous vaccines for broiler breeders.

Extended-spectrum ß-lactamase-producing Escherichia coli isolated from poultry: a review of current problems, illustrated with some laboratory findings.

Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli has been documented in humans as well as in food-producing birds, including chickens, and for unknown reasons the prevalence has increased significantly during the last decade. With E. coli as a major opportunistic pathogen in chickens and with a potential for zoonotic transfer to human beings, ESBL-producing E. coli represents a major risk both to poultry production and to human health. This review presents some of the current problems with ESBL-producing E. coli in relation to poultry production, with a focus on chickens. To illustrate issues in relation to screening and typing, two case studies are included where one collection of ESBL-producing E. coli isolates was obtained from asymptomatic carrier chickens while the other was obtained from lesions in chickens. Pulsed-field gel electrophoresis and multi-locus sequence typing revealed a highly heterogeneous population of ESBL-producing E. coli. All isolates harboured between one and three large plasmids (>100 kb). Among isolates associated with asymptomatic chickens, the ESBL types SHV and TEM dominated, while CTX-M-1 dominated in disease-associated isolates. The isolates from diseased birds were occasionally of sequence types often associated with human infections, such as ST131. With improved tools to trace and screen for ESBL-producing E. coli at farm level, strategies can be selected that aim to reduce or eliminate the presence of ESBL-producing E. coli in poultry and poultry products meant for human consumption.

In vitro and in vivo investigation on genomic stability of Salmonella enterica Typhimurium DT41 obtained from broiler breeders in Denmark.

Salmonella enterica serovar Typhimurium phage type DT41 has previously been identified from salmonella-positive broiler breeder flocks in Denmark and isolates obtained from different flocks have demonstrated major diversity by multiple-locus variable-number tandem-repeats analysis (MLVA) typing. To elucidate whether the high diversity observed by MLVA was related to multiple independent introductions at farm level or genetic instability of markers, we investigated the genomic stability of different clones of S. Typhimurium DT41. In the in vitro genomic stability experiment, feed pellet- and dust samples inoculated with four strains of DT41 were kept at three different temperatures. The in vitro genomic stability was also assessed by conducting a serial passage experiment. In a subsequent in vivo experiment, broiler breeders of three different age groups were challenged with a strain of poultry and human origin, respectively. The in vitro experiment demonstrated that DT41 survived more than 6 months in feed-pellets at 20 °C whereas the survival in dust was less than 4 weeks. Infection pattern and excretion varied for the poultry and human strain and birds of different age groups as revealed by the in vivo experiment. Genetic stability of cultures obtained from the in vitro and in vivo survival/passage was investigated by plasmid profiling, pulsed-field gel electrophoresis (PFGE) and MLVA. The results of plasmid profiling and PFGE demonstrated genomic stability of all but one strain kept in dust at 20 °C for 3 weeks. Minor genetic changes were observed in isolates from the in vitro experiment as revealed by MLVA. The epidemiological impact of these findings is briefly discussed.

Transmission and genetic diversity of Enterococcus faecalis among layer chickens during hatch.

BACKGROUND: Studies on transmission of Enterococcus faecalis among chickens during hatch have not been carried out so far. Information about vertical transmission and subsequent spreading and colonization of the cloacal mucosa through cloacal 'drinking' during hatch are important to understand the epidemiology of E. faecalis infections. In the present investigation vertical transmission and subsequent spreading and colonization of the cloacal mucosa of chickens by E. faecalis through cloacal 'drinking' were examined. METHODS: Two different batches of layer chickens originating from 45 weeks old Brown and White Lohmann parents, respectively from the same farm were sampled in the hatcher. Isolates were confirmed to be E. faecalis by polymerase chain reaction (PCR) and further by multilocus sequence typing (MLST) to state their population structure and comparison made to sequence types previously obtained from chicken. RESULTS: A total of 480 chickens were swabbed from the cloacae just after hatch and after 24 hours. A total of 101 isolates were confirmed as E. faecalis by a species specific PCR. The prevalence of E. faecalis increased from 14% at 0 h to 97% after 24 h for the Brown Lohmann chickens and from 0.5% to 23% for the White Lohmann flock. The 84 isolates analysed by MLST were distributed on 14 sequence types (ST). Three ST (401, 82 and 249) accounted for 64% of all isolates analysed by MLST after 24 h. ST 82 has previously been reported from amyloid arthropathy and other lesions in poultry. CONCLUSIONS: The present findings demonstrated a high potential of a few contaminated eggs or embryos to rapidly facilitate the spread of E. faecalis to almost all chickens during hatch.