Cy3-Based Nanoviscosity Determination of Mucus: Effect of Mucus Collection Methods and Antibiotics Treatment.

The integrity of the protective mucus layer as a primary defense against pathogen invasion and microbial leakage into the intestinal epithelium can be compromised by the effects of antibiotics on the commensal microbiome. Changes in mucus integrity directly affect the solvent viscosity in the immediate vicinity of the mucin network, that is, the nanoviscosity, which in turn affects both biochemical reactions and selective transport. To assess mucus nanoviscosity, a reliable readout via the viscosity-dependent fluorescence lifetime of the molecular rotor dye cyanine 3 is established and nanoviscosities from porcine and murine ex vivo mucus are determined. To account for different mucin concentrations due to the removal of digestive residues during mucus collection, the power law dependence of mucin concentration on viscosity is used. The impact of antibiotics combinations (meropenem/vancomycin, gentamycin/ampicillin) on ex vivo intestinal mucus nanoviscosity is presented. The significant increase in viscosity of murine intestinal mucus after treatment suggests an effect of antibiotics on the microbiota that affects mucus integrity. This method will be a useful tool to assess how drugs, directly or indirectly, affect mucus integrity. Additionally, the method can be utilized to analyze the role of mucus nanoviscosity in health and disease, as well as in drug development.

Gene amplifications cause high-level resistance against albicidin in gram-negative bacteria.

Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. The peptide antibiotic albicidin is such a promising candidate that, as a gyrase poison, shows bactericidal activity against a wide range of gram-positive and gram-negative bacteria. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against albicidin. RNA sequencing and proteomics data show that this novel mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of STM3175 (YgiV), a transcription regulator with a GyrI-like small molecule binding domain that traps albicidin with high affinity. X-ray crystallography and molecular docking reveal a new conserved motif in the binding groove of the GyrI-like domain that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in gram-negative bacteria, and our experiments confirm that STM3175 homologs can confer resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.

A virulence factor as a therapeutic: the probiotic Enterococcus faecium SF68 arginine deiminase inhibits innate immune signaling pathways.

The probiotic bacterial strain Enterococcus faecium SF68 has been shown to alleviate symptoms of intestinal inflammation in human clinical trials and animal feed supplementation studies. To identify factors involved in immunomodulatory effects on host cells, E. faecium SF68 and other commensal and clinical Enterococcus isolates were screened using intestinal epithelial cell lines harboring reporter fusions for NF-κB and JNK(AP-1) activation to determine the responses of host cell innate immune signaling pathways when challenged with bacterial protein and cell components. Cell-free, whole-cell lysates of E. faecium SF68 showed a reversible, inhibitory effect on both NF-κB and JNK(AP-1) signaling pathway activation in intestinal epithelial cells and abrogated the response to bacterial and other Toll-like receptor (TLR) ligands. The inhibitory effect was species-specific, and was not observed for E. avium, E. gallinarum, or E. casseliflavus. Screening of protein fractions of E. faecium SF68 lysates yielded an active fraction containing a prominent protein identified as arginine deiminase (ADI). The E. faecium SF68 arcA gene encoding arginine deiminase was cloned and introduced into E. avium where it conferred the same NF-κB inhibitory effects on intestinal epithelial cells as seen for E. faecium SF68. Our results indicate that the arginine deiminase of E. faecium SF68 is responsible for inhibition of host cell NF-κB and JNK(AP-1) pathway activation, and is likely to be responsible for the anti-inflammatory and immunomodulatory effects observed in prior clinical human and animal trials. The implications for the use of this probiotic strain for preventive and therapeutic purposes are discussed.

Frequency, Local Dynamics, and Genomic Characteristics of ESBL-Producing Escherichia coli Isolated From Specimens of Hospitalized Horses.

Previous research identified veterinary clinics as hotspots with respect to accumulation and spread of multidrug resistant extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (EC). Therefore, promoting the prudent use of antibiotics to decrease selective pressure in that particular clinical environment is preferable to enhance biosecurity for animal patients and hospital staff. Accordingly, this study comparatively investigated the impact of two distinct perioperative antibiotic prophylaxis (PAP) regimens (short-term versus prolonged) on ESBL-EC carriage of horses subjected to colic surgery. While all horses received a combination of penicillin/gentamicin (P/G) as PAP, they were assigned to either the "single-shot group" (SSG) or the conventional "5-day group" (5DG). Fecal samples collected on arrival (t0), on the 3rd (t1) and on the 10th day after surgery (t2) were screened for ESBL-EC. All isolates were further investigated using whole genome sequences. In total, 81 of 98 horses met the inclusion criteria for this study. ESBL-EC identified in samples available at t0, t1 and t2 were 4.8% (SSG) and 9.7% (5DG), 37% (SSG) and 47.2% (5DG) as well as 55.6% (SSG) and 56.8% (5DG), respectively. Regardless of the P/G PAP regimen, horses were 9.12 times (95% CI 2.79-29.7) more likely to carry ESBL-EC at t1 compared to t0 (p < 0.001) and 15.64 times (95% CI 4.57-53.55) more likely to carry ESBL-EC at t2 compared to t0 (p < 0.001). ESBL-EC belonging to sequence type (ST) 10, ST86, ST641, and ST410 were the most prevalent lineages, with bla CTX - M - 1 (60%) being the dominant ESBL gene. A close spatio-temporal relationship between isolates sharing a particular ST was revealed by genome analysis, strongly indicating local spread. Consequently, hospitalization itself has a strong impact on ESBL-EC isolation rates in horses, possibly masking differences between distinct PAP regimens. The results of this study reveal accumulation and spread of multi-drug resistant ESBL-EC among horses subjected to colic surgery with different P/G PAP regimens, challenging the local hygiene management system and work-place safety of veterinary staff. Moreover, the predominance of particular ESBL-EC lineages in clinics providing health care for horses needs further investigation.

Phenotypic zinc resistance does not correlate with antimicrobial multi-resistance in fecal E. coli isolates of piglets.

BACKGROUND: Following the ban on antimicrobial usage for growth promotion in animal husbandry in the EU, non-antimicrobial agents including heavy metal ions (e.g. zinc and copper), prebiotics or probiotics have been suggested as alternatives. Zinc has extensively been used in pig farming, particularly during weaning of piglets to improve animal health and growth rates. Recent studies, however, have suggested that high dietary zinc feeding during weaning of piglets increases the proportion of multi-drug resistant E. coli in the gut, contraindicating the appropriateness of zinc as an alternative. The underlying mechanisms of zinc effects on resistant bacteria remains unclear, but co-selection processes could be involved. In this study, we determined whether E. coli isolates from intestinal contents of piglets that had been supplemented with high concentrations of zinc acquired a higher tolerance towards zinc, and whether multi-drug resistant isolates tolerated higher zinc concentrations. In addition, we compared phenotypic zinc and copper resistance of E. coli isolates for possible correlation between phenotypic resistance/tolerance to different bivalent ionic metals. RESULTS: We screened phenotypic zinc/copper tolerance of 210 isolates (including antimicrobial resistant, multi-drug resistant, and non-resistant E. coli) selected from two, independent zinc-feeding animal trials by determining a zinc/copper minimal inhibitory concentration (Merlin, Bornheim-Hersel, Germany). In both trials, groups of piglets were supplemented either with high dietary zinc (> 2000 ppm) or control (50-70 ppm, background) concentrations. Our observations showed that high concentration zinc exposure did not have an effect on either zinc or copper phenotypic tolerance of E. coli isolates from the animals. No significant association was found between antimicrobial resistance and phenotypic zinc/copper tolerance of the same isolates. CONCLUSION: Our findings argue against a co-selection mechanism of antimicrobial drug-resistance and zinc tolerance after dietary zinc supplementation in weaning piglets. An explanation for an increase in multi-drug resistant isolates from piglets with high zinc dietary feeding could be that resistant bacteria to antimicrobial agents are more persistent to stresses such as zinc or copper exposure.

High-Zinc Supplementation of Weaned Piglets Affects Frequencies of Virulence and Bacteriocin Associated Genes Among Intestinal Escherichia coli Populations.

To prevent economic losses due to post-weaning diarrhea (PWD) in industrial pig production, zinc (Zn) feed additives have been widely used, especially since awareness has risen that the regular application of antibiotics promotes buildup of antimicrobial resistance in both commensal and pathogenic bacteria. In a previous study on 179 Escherichia coli collected from piglets sacrificed at the end of a Zn feeding trial, including isolates obtained from animals of a high-zinc fed group (HZG) and a corresponding control group (CG), we found that the isolate collection exhibited three different levels of tolerance toward zinc, i.e., the minimal inhibitory concentration (MIC) detected was 128, followed by 256 and 512 µg/ml ZnCl2. We further provided evidence that enhanced zinc tolerance in porcine intestinal E. coli populations is clearly linked to excessive zinc feeding. Here we provide insights about the genomic make-up and phylogenetic background of these 179 E. coli genomes. Bayesian analysis of the population structure (BAPS) revealed a lack of association between the actual zinc tolerance level and a particular phylogenetic E. coli cluster or even branch for both, isolates belonging to the HZG and CG. In addition, detection rates for genes and operons associated with virulence (VAG) and bacteriocins (BAG) were lower in isolates originating from the HZG (41 vs. 65% and 22 vs. 35%, p < 0.001 and p = 0.002, resp.). Strikingly, E. coli harboring genes defining distinct pathotypes associated with intestinal disease, i.e., enterotoxigenic, enteropathogenic, and Shiga toxin-producing E. coli (ETEC, EPEC, and STEC) constituted 1% of the isolates belonging to the HZG but 14% of those from the CG. Notably, these pathotypes were positively associated with enhanced zinc tolerance (512 µg/ml ZnCl2 MIC, p < 0.001). Taken together, zinc excess seems to influence carriage rates of VAGs and BAGs in porcine intestinal E. coli populations, and high-zinc feeding is negatively correlated with enteral pathotype occurrences, which might explain earlier observations concerning the relative increase of Enterobacterales considering the overall intestinal microbiota of piglets during zinc feeding trials while PWD rates have decreased.

Effects of a Four-Week High-Dosage Zinc Oxide Supplemented Diet on Commensal Escherichia coli of Weaned Pigs.

Strategies to reduce economic losses associated with post-weaning diarrhea in pig farming include high-level dietary zinc oxide supplementation. However, excessive usage of zinc oxide in the pig production sector was found to be associated with accumulation of multidrug resistant bacteria in these animals, presenting an environmental burden through contaminated manure. Here we report on zinc tolerance among a random selection of intestinal Escherichia coli comprising of different antibiotic resistance phenotypes and sampling sites isolated during a controlled feeding trial from 16 weaned piglets: In total, 179 isolates from "pigs fed with high zinc concentrations" (high zinc group, [HZG]: n = 99) and a corresponding "control group" ([CG]: n = 80) were investigated with regard to zinc tolerance, antimicrobial- and biocide susceptibilities by determining minimum inhibitory concentrations (MICs). In addition, in silico whole genome screening (WGSc) for antibiotic resistance genes (ARGs) as well as biocide- and heavy metal tolerance genes was performed using an in-house BLAST-based pipeline. Overall, porcine E. coli isolates showed three different ZnCl2 MICs: 128 µg/ml (HZG, 2%; CG, 6%), 256 µg/ml (HZG, 64%; CG, 91%) and 512 µg/ml ZnCl2 (HZG, 34%, CG, 3%), a unimodal distribution most likely reflecting natural differences in zinc tolerance associated with different genetic lineages. However, a selective impact of the zinc-rich supplemented diet seems to be reasonable, since the linear mixed regression model revealed a statistically significant association between "higher" ZnCl2 MICs and isolates representing the HZG as well as "lower ZnCl2 MICs" with isolates of the CG (p = 0.005). None of the zinc chloride MICs was associated with a particular antibiotic-, heavy metal- or biocide- tolerance/resistance phenotype. Isolates expressing the 512 µg/ml MIC were either positive for ARGs conferring resistance to aminoglycosides, tetracycline and sulfamethoxazole-trimethoprim, or harbored no ARGs at all. Moreover, WGSc revealed a ubiquitous presence of zinc homeostasis and - detoxification genes, including zitB, zntA, and pit. In conclusion, we provide evidence that zinc-rich supplementation of pig feed selects for more zinc tolerant E. coli, including isolates harboring ARGs and biocide- and heavy metal tolerance genes - a putative selective advantage considering substances and antibiotics currently used in industrial pork production systems.

Decellularised whole ovine testis as a potential bio-scaffold for tissue engineering.

The aim of this study was to determine an efficient whole-organ decellularisation protocol of a human-sized testis by perfusion through the testicular arteries. In the first step of this study, we determined the most efficient detergent agent, whereas the second phase delineated the optimal time required for the decellularisation process. Initially sheep testes were decellularised by one of three different detergent agents: sodium dodecyl sulphate (SDS), Triton X-100 and trypsin-ethylenediamine tetraacetic acid (EDTA) solutions, each perfused for 6h. In the second phase, the selected detergent agent was applied for different time periods. A total number of 20 organs were processed during this investigation. The efficacy of the decellularisation process and the preservation of the extracellular matrix components and structure were evaluated by histopathological examinations, 4',6'-diamidino-2-phenylindole (DAPI) staining, DNA quantification, hydroxyproline measurement, magnetic resonance imaging and scanning electron microscopy. Organ perfusion with 1% SDS solution for 6 to 8h demonstrated the most desirable outcomes regarding decellularisation and extracellular matrix preservation. The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay was used to determine the toxicity of the scaffold and its potential for further application in tissue-engineering investigations. This investigation introduces an efficient method to produce a three-dimensional testicular bio-scaffold resembling the properties of the native organ that could be employed in tissue-engineering studies.

Genetic characterisation of methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in pets and veterinary personnel in Iran: new insights into emerging methicillin-resistant S. pseudintermedius (MRSP).

OBJECTIVES: Methicillin-resistant staphylococci, including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP), pose a threat to animal and human health worldwide. Veterinary staff and pets may play a role in the spread of resistant clones. METHODS: A total of 125 samples from veterinary staff (n=50), dogs (n=49) and cats (n=26) were investigated. Obtained isolates were tested for the methicillin resistance gene mecA and were subjected to multiplex PCR to differentiate coagulase-positive species. Following SCCmec and spa typing, isolates were tested for the presence of various toxin and virulence genes and phenotypic resistance to common antimicrobials. RESULTS: Overall, 4 MRSA were isolated from two veterinarians and two dogs and 19 MRSP were found in eleven dogs (12 isolates) and five cats (7 isolates). The MRSA isolates possessed sea (2) and eta (3) virulence genes and the MRSP isolates possessed sea (6), expA (15), expB (1) and siet (19) genes. SCCmec type II and three spa types (t186, t1816 and t10897) were identified in the MRSA isolates. Most of the MRSP isolates belonged to SCCmec types II (2 isolates) and V (10 isolates); however, the remaining 7 isolates were untypeable and contained class C1 mec. The majority of isolates were multidrug-resistant (MDR). CONCLUSION: These findings show that pets and veterinarians could be potential sources of MDR-MRSA and MDR-MRSP in Iran. Taken together, these findings warrant future investigations on the epidemiology and public-health significance of MDR-MRSA and MDR-MRSP both in veterinarians and companion animals in Iran.

Detection and molecular characterization of feline hemoplasmas in wild felid species in Iran in the Middle East.

Three feline hemoplasma species exist in felids: Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum', and 'Candidatus Mycoplasma turicensis'. The aims of the study were to determine the presence of, and molecularly characterize, any hemoplasmas in wild felids, including the endangered Persian leopard in Iran, the Middle East. Blood samples were collected from 19 wild felids, including three Persian leopards. Using species-specific hemoplasma PCRs and ELISA serological testing for feline leukaemia virus and feline immunodeficiency virus (FIV), two Persian leopards were found to be infected with 'Ca. M. haemominutum' and were seropositive for FIV. Partial 16S rRNA gene sequences were generated for these 'Ca. M. haemominutum' species and subsequent phylogenetic analysis revealed 97.70% to 99.45% sequence identity with those found in domestic cats from Iran and other countries. This study confirms the presence of 'Ca. M. haemominutum' and concurrent FIV antibody in wild felids in Iran. This represents the first report of hemoplasma in wild felids in the Middle East as well as the first report of infection in Persian leopards.

Molecular characterization and phylogenetic analysis of feline hemoplasmas in domestic cats in Iran.

Three known feline hemoplasmas are Mycoplsama haemofelis, 'CandidatusMycoplasma haemominutum' and 'CandidatusMycoplasma turicensis'. They are described as cause of feline infectious anemia in domestic and wild felids. Other blood parasites or blood-related pathogens like concurrent retroviral infections may deteriorate the clinical condition and severity of anemia. The aims of this study were molecular characterization and phylogenetic analysis of hemoplasmas in domestic cats in Iran for the first time. Blood samples were collected from 185 healthy and diseased domestic cats. Blood smears were prepared and hematological parameters were measured to determine possible anemia. Using 16S rRNA gene universal and species specific polymerase chain reactions with the following sequencing, 47 (25.40%) of cats were hemoplasma positive. Also, 17.02%, 72.50% and 40.40% of total positive samples were M.haemofelis, 'Ca. M. haemominutum' and 'Ca. M. turicensis' infected, respectively. 10 (21.20%) of hemoplasma positive cats had anemic blood profiles (HCT < 24.00%). All M. haemofelis infected cases were included. Partial 16S rRNA gene phylogenetic analysis revealed a high identity between the hemoplasma species found in this study and domestic cat sequences existing in GenBank. Phylogenetic analysis revealed 94.00% to 100% sequence identity between sequences of this study and existing sequences in Genbank. All hemoplasma isolates in this study were grouped within a single clade and additionally subdivided into two groups; haemofelis group including M.haemofelis and 'Ca. M. turicensis' and haemominutum group including 'Ca. M. haemominutum'.

A molecular study of hemotropic mycoplasmas (hemoplasmas) in cats in Iran.

BACKGROUND: Three feline hemoplasma species are recognized: Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum', and 'Candidatus Mycoplasma turicensis'. These species can cause anemia in cats and have a worldwide distribution. OBJECTIVES: There was no previous information on hemotropic mycoplasma spp in cats in Iran and the Middle East. Accordingly, we investigated the molecular presence, and clinical signs and hematological profile in cats infected with these microorganisms in Iranian cats. METHODS: Polymerase chain reaction (PCR) assays and cytology were performed on 100 blood samples collected from Iranian Shorthair cats. ACBC and case history were also collected for each sample. RESULTS: By PCR, 22 (22%; 14-30%, 95% CI) samples were positive. The prevalence of M haemofelis, 'Ca M haemominutum', and 'Ca M turicensis' was 63.63% (14/22), 54.54% (12/22), and 18.18% (4/22), respectively. Some double and triple co-infections were also found. Using PCR as the reference method, cytology had poor sensitivity (27%) and reasonable specificity (89.74%). Male cats were at a higher risk of infection (P = .001). Cats older than 8 years were more frequently infected than the younger cats (P = .0018). Lower HCT (P = .018), RBC count (P = .028) and HGB concentration (P = .003) were also associated with hemoplasma PCR-positive status. CONCLUSIONS: Based on this study, the most prevalent feline hemoplasma species in Iranian cats was M haemofelis, but double and triple co-infections are also documented. Age and sex, as well as reduced RBC parameters, were predisposing factors for hemoplasma infection.

Evaluation of Effects of Intraperitoneal CO2 Pressure in Laparoscopic Operations on Kidney, Pancreas, Liver and Spleen in Dogs.

BACKGROUND: During laparoscopy, insufflation of an inert gas in the peritoneal cavity creates a working space to facilitate surgery. The space should be large enough to facilitate surgery without increasing intra-abdominal pressure (IAP) over a threshold limit (usually 15 mm Hg). OBJECTIVES: This experimental study was performed to evaluate the effects of increasing in intra-abdominal pressure on internal organs. MATERIALS AND METHODS: Twenty female mixed breed dogs (20 ± 3 kg, 18 ± 1.2 months) were selected. They were randomly divided to two groups (n = 10). The intra-abdominal pressure was maintained 12 mm Hg and 20 mm Hg during the operation in control group and in test group respectively. RESULTS: Histopathologic evaluations revealed more pathological changes at the kidney of all the dogs in test group in comparison to control group. CONCLUSIONS: Our findings revealed that organs that their blood supplies are related to one single or two arteries and their blood drainage are related to one or two veins are more sensitive to increased intra-abdominal pressure.