Pheno- and Genotypic Epidemiological Characterization of Vancomycin-Resistant Enterococcus faecium Isolates from Intensive Care Unit Patients in Central Türkiye.

Vancomycin-resistant Enterococcus faecium (VRE) has been detected in Türkiye. Only limited information is available on its dissemination in the central regions of the country. This study describes the first epidemiological characterization of VRE clinical isolates detected in patients in a hospital in the province of Aksaray. In this one-year study conducted between 2021 and 2022, stool samples from intensive care unit patients were screened for VRE using the phenotypic E-test method, and the antibiotic sensitivity test was analyzed by using the VITEK® 2 system. A molecular assay for confirmation of species level was carried out by 16S rRNA gene-based sequencing and testing for antibiotic resistance (vanA or vanB) and virulence factor-encoding genes (esp, asa1, and hyl). Further, genotypic characterization was determined by macro-restriction fragment pattern analysis (MRFPA) of genomic DNA digested with SmaI restriction enzyme. Of the total 350 Enterococcus positive patients from different hospital intensive care units, 22 (6.3%) were positive for VRE using the phenotypic E-test method. All isolates showed resistance to ampicillin, ciprofloxacin, vancomycin, and teicoplanin and positive amplification for the vanA gene. However, none of the isolates was positive for the vanB gene. The most prevalent virulence gene was esp. The results indicate that the isolates are persistent in the hospital environment and subsequently transmitted to hospitalized patients, thus representing challenges to an outbreak and infection control. These study results would also help formulate more effective strategies to reduce the transmission and propagation of VRE contamination in various hospital settings.

Complete genome sequence of Arcanobacterium wilhelmae strain DSM 102162 isolated from the genital tract of a Rhinoceros unicornis.

Previous case reports indicate Arcanobacterium's opportunistic pathogenic potential. However, the true diversity of the genus remains understudied. Here, we present the complete genome of Arcanobacterium wilhelmae isolated from a diseased rhinoceros, suspected to play a role in its condition. These genomic data may enable future advancements in understanding Arcanobacterium pathogenicity.

Changes in Hepatitis E Virus Contamination during the Production of Liver Sausage from Naturally Contaminated Pig Liver and the Potential of Individual Production Parameters to Reduce Hepatitis E Virus Contamination in the Processing Chain.

In this study, changes in hepatitis E virus (HEV) contamination in the production of liver sausage from naturally contaminated pork liver were investigated. Furthermore, the potential effectiveness of individual production parameters in reducing viral loads was measured. When processing moderately contaminated liver (initial Cq-value 29), HEV RNA persisted in the finished sausages, even after heating for 90 min at 75 °C. A matrix-specific standard curve was created using a spiking experiment to accurately quantify HEV RNA in a particularly challenging matrix like liver sausage. Variations in product-specific production parameters, including mincing and heating times, showed some reduction in contamination levels, but even prolonged heating did not render all finished products HEV negative. The persistence of HEV contamination underscores the importance of ongoing monitoring in the pig population and raw materials to enhance food safety measures and reduce the likelihood of transmission through pork consumption. The detection of HEV RNA within all processing stages of pork liver in the production of liver sausage suggests that further research into the risk of infection posed by this detection and vigilance in managing HEV risks in the food chain, particularly in pork products, are required to protect public health.

Assessment and Assay Comparison for Detection of Antimicrobial Residues in Freshwater Aquaculture Fish in Erbil Governorate, Iraq.

The excessive and uncontrolled application of antibiotics in the fish farming industry, coupled with a lack of health monitoring and medication practices, is a driving force behind the escalating development of antimicrobial resistance. The present study assessed and compared qualitative field diffusion (QFD) and disk diffusion (DD) assays for the detection of antimicrobial residues (ARs) in diverse freshwater aquaculture fish. A total of 380 freshwater aquaculture fish (160 fresh and 180 frozen) samples were systematically collected between January and June 2021 from various retail stores located in Erbil Governorate, Iraq. Based on QFDA results, overall, ARs were detected (52; 15.3%) at a relatively lower frequency with comparatively higher frequency (21; 31.1%) in fresh than (31; 17.2%) frozen fish samples. On the other hand, DDA also revealed a comparable (45; 13.2%) prevalence rate of ARs. However, a low detection was observed more in fresh (17; 10.6%) than frozen (28; 15.6%) fish samples. Moreover, no statistically significant disparity (χ2 = 0.069; p = 0.79) between two assays and types of fish was recorded. In conclusion, the results of the present study showed that detecting a considerable frequency of ARs in these fish samples raises concerns about potential threats to public health. This underscores the necessity for understanding antibiotic application in aquaculture and its potential connection to antibiotic resistance in bacterial pathogens. Such comprehension is pivotal for formulating and implementing effective control and farm management strategies to address this pressing issue.

Characterization and technological functions of different lactic acid bacteria from traditionally produced Kirklareli white brined cheese during the ripening period.

In the present study, the evolution of the physicochemical and microbiological characteristics of lactic acid bacteria (LAB) in traditional Kirklareli white brined cheese collected from 14 different cheese manufacturing facilities were investigated on different days of the 90-day ripening period. The obtained LAB within the species Lactococcus (Lc.) lactis, Latilactobacillus (Lt.) curvatus, Lactobacillus (Lb.) casei and Lb. plantarum, Enterococcus (E.) durans, E. faecium, E. faecalis, Streptococcus macedonicus, and Weissella paramesenteroides were characterized in terms of their influence on technological properties and their potential as starter cultures for traditional white brined cheese production. The results of the microbiological and physicochemical investigations showed that a few selected isolates of Lc. lactis, Lb. casei, and Lb. plantarum had certain functions as starter germs. Moderate acidification capacity, antibacterial activity and proteolytic activity, which are characteristic of their use as starter lactic acid bacteria, were found. Importantly, antibiotic resistance among selected Lc. lactis, Lb. casei, and Lb. plantarum isolates was extremely low, whereas some of these isolates demonstrated antibacterial activity against major foodborne pathogenic bacteria. Based on the results obtained in this study, selected Lc. and Lb. isolates can also be considered as starter culture in traditional cheese production.

Development of a Sensitive and Specific Quantitative RT-qPCR Method for the Detection of Hepatitis E Virus Genotype 3 in Porcine Liver and Foodstuff.

As an international and zoonotic cause of hepatitis, hepatitis E virus (HEV) poses a significant risk to public health. However, the frequency of occurrence and the degree of contamination of food of animal origin require further research. The aim of this study was to develop and validate a highly sensitive quantitative RT-qPCR assay for the detection and quantification of HEV contamination in porcine liver and food. The focus was on genotype 3, which is most common as a food contaminant in developed countries and Europe. The selected assay has its target sequence in the open reading frame 1 (ORF1) of the HEV genome and showed good results in inclusivity testing, especially for HEV genotype 3. The developed assay seems to show high efficiency and a low intercept when compared to other assays, while having a comparable limit of detection (LOD). In addition, a standard curve was generated using artificially spiked liver to provide more accurate quantitative results for contamination assessment and tracking in this matrix. Application of the assay to test 67 pig livers from different origins resulted in a positivity rate of 7.5%, which is consistent with the results of numerous other prevalence studies. Quantitative detection of the viral genome in the food chain, particularly in pig livers, is essential for understanding the presence and evolution of HEV contamination and thus ensures consumer safety.

Arcanobacterium canis strain DSM 25104 isolated from an English bulldog suffering from otitis externa: complete genome sequence.

Many species of the genus Arcanobacterium are known as opportunistic pathogens and have been isolated in association with infectious diseases in humans and animals. Here, we present the complete genome sequence of another opportunistic pathogenic representative, namely Arcanobacterium canis, isolated from the otitis externa of an English bulldog.

Detection of Clostridium difficile among diarrheic children using cultural and polymerase chain reaction technique.

INTRODUCTION: Clostridium difficile is the most common cause of antibiotic-associated diarrhea and colitis. Several methods are available for the detection of C. difficile in stool samples. This study aimed to use glutamate dehydrogenase (GDH), toxin detection, culture and polymerase chain reaction (PCR) techniques for the diagnosis of this pathogen. METHODOLOGY: A total of 300 stool samples were collected from children with hospital acquired diarrhea (HA-D), community acquired diarrhea (CA-D), and hospitalized non-diarrheic children as control with ages ranging from 6 months to 6 years (mean 3.7 ± 1.7). Each stool sample was divided into two parts; one part was tested for the enzyme GDH, toxin A and B and then cultured on selective media; and the other part for direct DNA extraction. RESULTS: From a total of 300 stool samples, 9 (3.0%) were positive for C. difficile by the PCR technique, 7 (7%) samples of which were from HA-D cases and 2 (2.0%) from CA-D cases; the control group samples were negative. The enzyme GDH was detected in 12 (12%) samples and toxins A and B in 8 (8%) samples from HA-D cases compared to 5 (5%) and 2 (2%), respectively from CA-D cases. Both GDH and the toxins were negative in control samples. Only 19 (19.0%) samples from HA-D cases gave suspected growth and all of these were negative by PCR. CONCLUSIONS: Based on the results of this study, we conclude that the PCR technique is the only reliable method for the diagnosis of this pathogen.

Rapid Diagnostic of Streptococcus suis in Necropsy Samples of Pigs by thrA-Based Loop-Mediated Isothermal Amplification Assay.

Streptococcus (S.) suis presents a serious threat to the pig industry as well as food safety and public health. Although several LAMP assays have been developed for the identification of S. suis, no universal assay is so far available for the field-suitable examination of clinical pig specimens. Based on the thrA housekeeping gene, a new loop-mediated isothermal amplification (LAMP) assay was developed and validated for the detection of S. suis in the brain and joints of pigs. For this LAMP assay, two different methods for the extraction of DNA from brain and joint swabs were compared. Using the LPTV boiling method, the detection limit of LAMP was 1.08 CFU/reaction, while the detection limit was 53.8 CFU/reaction using a commercial DNA extraction kit. The detection limits of thrA-LAMP in combination with the LPTV boiling method were 104-105 CFU/swab in the presence of brain tissue and 103-104 CFU/swab in the presence of joint tissue. The diagnostic quality criteria of LAMP were determined by the examination of 49 brain swabs and 34 joint swabs obtained during routine diagnostic necropsies. Applying the LPTV boiling method to brain swabs, the sensitivity, specificity, and positive and negative predictive values of thrA-LAMP were 88.0, 95.8, 95.7, and 88.5% using cultural investigation as a reference method, and 76.7, 100, 100, and 73.1% using real-time PCR as a reference method. Based on these results, the thrA-LAMP assay combined with the LPTV boiling method is suitable for rapid detection of S. suis from brain swabs.

Identification of the novel potential pathogen Trueperella pecoris with interspecies significance by LAMP diagnostics.

Trueperella pecoris was described as a new species of the genus Trueperella in 2021 and might be pathogenic to various animal species. However, the lack of a suitable diagnostic test system stands in the way of epidemiological surveys to clarify possible causalities. In this study, a Loop-mediated Isothermal Amplification (LAMP) assay was developed and validated that was highly specific for T. pecoris. The assay provided an analytical sensitivity of 0.5 pg/25 µL and showed 100% inclusivity and exclusivity for 11 target and 33 non-target strains, respectively. Three different DNA extraction methods were evaluated to select the most LAMP-compatible method for cell disruption in pure and complex samples. Using an on-site applicable single-buffer DNA extraction with additional heating, the cell-based detection limit was 2.3 CFU/reaction. Finally, the LAMP assay was validated by means of artificially contaminated porcine lung tissue samples in which minimal microbial loads between 6.54 and 8.37 × 103 CFU per swab sample were detectable. The LAMP assay established in this study represents a suitable diagnostic procedure for identifying T. pecoris in clinical specimens and will help to collect epidemiological data on the pathogenicity of this species.

Arcanobacterium pinnipediorum Strain DSM 28752 Isolated from a Harbour Seal: Complete Genome Sequence.

The genus Arcanobacterium is constantly growing as novel species are identified. In particular, harbor seals have proven to be a common reservoir for bacteria of this genus. Here, we announce the complete genome sequence of another Arcanobacterium species-namely, Arcanobacterium pinnipediorum strain DSM 28752, isolated from a harbor seal.

First report of paratuberculosis (Johne's disease) in livestock farms of river buffaloes (Bubalus Bubalis) in Nineveh, Iraq.

The present study was designed to investigate Mycobacterium avium subsp. paratuberculosis (MAP) in dairy buffalo herds from six different geographical areas in Nineveh, Iraq. A total of 87 individual faecal samples from river buffaloes, representing 12 dairy herds, were investigated for detection of MAP using cultural, Ziehl­Neelsen and MAP­specific PCR­based methods. Overall, MAP was detected at a higher frequency at herd­level (4/12; 33%) compared to the total individual faecal samples (14/87; 16%) with a cell density ranging from 101 to 103 CFU g­1. A significantly (p < 0.05) higher frequency (9/17; 53%) of MAP was observed in faecal samples collected from clinically diseased as compared to healthy (5/70; 7%) buffaloes selected for the study. However, no statistically significant difference (p ≥ 0.05) was observed in the frequency of MAP occurrence between clinical (9; 64%) and apparently healthy (5; 36%) cases. This report, which is the first MAP study based on data from Iraqi dairy buffalo herds suggests that MAP transmission is a significant health risk for grazing livestock. In conclusion, this study would help farm owners and regulatory authorities to realise the importance of developing and applying best farm management practices in order to prevent transmission of MAP to healthy animals and the environment. In addition, effective diagnostic tests should be taken into account when carrying out the screening tests.

Development of loop-mediated isothermal amplification (LAMP) assay for rapid and direct screening of yellowfin tuna (Thunnus albacares) in commercial fish products.

Tuna is one of the most widely consumed fish on the European market, being available in various consumable options. Among them, Thunnus albacares, also called yellowfin tuna, is a delicacy and is consumed by millions of people around the world. Due to its comparatively high cost and demand, it is more vulnerable to fraud, where low-cost tuna or other fish varieties might be replaced for economic gain. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for targeting the mitochondrial cytochrome b gene for fast and direct detection of Thunnus albacares, which is a valuable tuna species. The analytical specificity was confirmed using 18 target samples (Thunnus albacares) and 18 samples of non-target fish species. The analytical sensitivity of the LAMP assay was 540 fg DNA per reaction. In addition, a simple and direct swab method without time-consuming nucleic acid extraction procedures and the necessity for cost-intensive laboratory equipment was performed that allowed LAMP detection of Thunnus albacares samples within 13 minutes. Due to its high specificity and sensitivity, the LAMP assay can be used as a rapid and on-site screening method for identifying Thunnus albacares, potentially providing a valuable monitoring tool for food authenticity control by the authorities.

Arcanobacterium buesumense sp. nov., isolated from an anal swab of a male harbour seal (Phoca vitulina).

A polyphasic taxonomic study was performed on an unidentified previously described Arcanobacterium-like Gram-positive strain 2701T isolated from an anal swab of a dead male harbour seal. Comparative 16S rRNA sequencing showed that the bacterium belonged to the genus Arcanobacterium in the family Arcanobacteriaceae. The genome sequence of the strain was obtained by Borowiak et al. [1]. The genome had a G+C content of 49 mol% and a total length of 1.94 Mb. The presence of the major menaquinone MK-9(H4) supported the affiliation of the isolate with the genus Arcanobacterium. The polar lipid profile consisted of diphosphatidylglycerol and an unidentified phospholipid as major components and two unidentified lipids, a further unidentified phospholipid, two unidentified phosphoglycolipids as well as phosphatidylglycerol. The major fatty acids were C16 : 0, C18 : 1 and C18 : 0. Biochemical and phylogenetic analyses clearly distinguished the isolate from other members of the genus Arcanobacterium and closely related other species. Based on these results, it is proposed that the unknown Arcanobacterium sp. strain 2701T should be classified as representing a novel species with the name Arcanobacterium buesumense sp. nov. The type strain is 2701T (=DSM 112952T=LMG 32446T).

Establishment and Validation of a Two-Step LAMP Assay for Detection of Bacillus cereus-Group Isolates in Food and Their Possibility of Non-haemolytic Enterotoxin Production.

The closely related members of the Bacillus cereus-group can mainly only be differentiated by whole genome sequencing. Among them, there are potentially toxin-producing bacteria. When consumed with food, these can cause vomiting or diarrhea and abdominal cramps. To date, although no EU-wide threshold exists, a bacterial count of 105 CFU/g can be regarded as critical. Specific and rapid detection of the bacteria is difficult due to their close relationship, and no loop-mediated isothermal amplification (LAMP) assay has been developed so far to detect potentially toxin-producing members of the B. cereus-group. Aim of this study was to develop a LAMP method to detect critical cell counts specifically and rapidly of potentially non-haemolytic enterotoxin (NHE)-producing cells of this group. A two-step LAMP assay was developed. First, the target sequence groEL was used to determine the representatives of the B. cereus-group. Second, since bacteria in which nheB is present are basically capable of producing enterotoxins, this gene was chosen for detection. The specificity of the developed assay was 100% for B. cereus-group isolates and 93.7% for the detection of nheB. The analytical sensitivity was 0.1 pg DNA/µl. Using simplified DNA extraction by boiling, cell-based sensitivity was determined. Targeting groEL and nheB, 11.35-27.05 CFU/reaction and 11.35-270.5 CFU/reaction were detectable, respectively. Artificially contaminated samples were investigated to prove the application in foods. Direct detection of the critical value of B. cereus-group cells was possible in 83.3% of samples and detecting the toxin-gene 50% thereof. After a 6-h incubation period, the detection rate increased to 100 and 91.7%, respectively. Additionally, 100 natively contaminated food samples were tested, also quantitatively and culturally. Samples with relevant contamination levels were reliably detected using groEL-LAMP. After a 6-h incubation period, isolates bearing the toxin gene nheB could also be reliably detected. In addition, colony material was boiled and used as a LAMP template for simple detection. Specificity for the B. cereus-group was 100 and 93.22% detecting nheB. The study demonstrated that screening of food samples with the groEL/nheB-LAMP assay can be performed within 1 day, making it possible to detect critical levels of potentially NHE-toxin-producing cells of the B. cereus-group.

Assessing synergistic effect of Jerusalem Artichoke juice and antioxidant compounds on enhanced viability and persistence of Bifidobacterium species, palatability, and shelf life.

Commercial vegetable and fruit juices with probiotics are new functional type of beverages; however, limitations including persistence and impact of probiotic bacteria on palatability and shelf life may prevent their industrial development. This study evaluated the effect of antioxidant compounds (ascorbic acid, astaxanthin, and ginseng) on viability and persistence of Bifidobacterium spp. in Jerusalem Artichoke (JA) juice; and determine the impact of these antioxidants on the sensory (color, texture, flavor, acidity) properties, free reducing sugar (inulin and fructose), and shelf life in the fortified JA juice. Overall, the JA juice fortified with ascorbic acid showed a significant impact on the rate of persistence of two targeted bifidobacterial strains from 1 to 28 days at 5°C. Both strains produced slight acidity in ascorbic acid fortified JA juice as compared to other tested samples. Similarly, the JA juice fortified with ascorbic acid showed a significantly high increase in the total number of bifidobacterial cells of both species, enhanced palatability, and shelf life as compared to astaxanthin and ginseng extract. The quadratic model indicated a strong association between ascorbic acid, ginseng extract, and astaxanthin with a bifidobacterial cell concentration in the fortified JA juices. The Box-Behnken design was considered a feasible analysis for describing fortified JA juice and the rate of viability and persistence of bifidobacteria during 28 days of storage at 5°C in all trials. In conclusion, JA juice fortified with ascorbic acid showed a significant impact on improving the cell viability and persistence of probiotic bacteria, enhanced palatability, and shelf life as compared to other compounds tested.

Development of a loop-mediated isothermal amplification (LAMP) assay for molecular identification of Trueperella abortisuis isolated from pigs.

The first description of Trueperella (T.) abortisuis was presented in Japan in 2009 by Azuma and colleagues. In the current study, eight T. abortisuis strains were identified by a newly developed loop-mediated isothermal amplification (LAMP) assay based on the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) encoding gene gap. Two T. abortisuis strains were obtained from prepuce of a seven-month-old boar and pooled foetal stomach contents in the United Kingdom, while the other six T. abortisuis strains were recovered from aborted foetal material of six pigs from a single farm in Germany. The developed LAMP assay showed an analytical sensitivity of 22 pg µL-1T. abortisuis DNA. T. abortisuis DSM 19515T and field strain T. abortisuis P504054/19/1 were directly detectable in artificially contaminated vaginal swabs up to concentrations of 980 CFU and 770 CFU per swab, respectively. There was no cross reactivity with control strains representing six species of genus Trueperella and six species of the closely related genus Arcanobacterium and Schaalia (Actinomyces) hyovaginalis. Further field research is required to determine the usefulness of the designed LAMP assay for identifying T. abortisuis isolated from pigs of various origins and from test samples directly obtained at farm level.

Mpl-Gene-Based Loop-Mediated Isothermal Amplification Assay for Specific and Rapid Detection of Listeria monocytogenes in Various Food Samples.

Listeria monocytogenes represents a high risk in food and can trigger potentially fatal listeriosis. The objective of this study was to detect L. monocytogenes in food using the LAMP method in a fast, specific, sensitive manner and thus to preventively test food for the presence of the target species. The reaction was performed and established using the portable real-time fluorometer Genie® II (OptiGene Ltd., Horsham, United Kingdom). In this new assay, six LAMP primers targeted the mpl-gene sequence of L. monocytogenes. A total of 148 different isolates, including 105 L. monocytogenes and 43 non-L. monocytogenes strains, were tested. Analytical sensitivity was determined based on different DNA- and cell concentrations. The detection limit with a detection rate of 100% was 5 pg of DNA or 275 colony-forming units (CFU) per reaction. Artificially contaminated minced beef and grated mozzarella were also tested. The assay was 100% successful to detect an initial bacterial contamination of 0.4-4 CFU g-1 food after 24 h enrichment in half-Fraser broth. Finally, natively contaminated samples were tested in comparison to the microbiological reference method and real-time polymerase chain reaction. Native sample testing revealed 100% consistent findings between LAMP and the standard culture method after first enrichment for 24 h. In addition, a rapid colony-confirmation method was established that enabled reliable identification of L. monocytogenes isolates on different selective culture media using a simplified DNA extraction by boiling. This study showed that the developed assay was able to determine whether a food is safe with respect to the food-safety criteria of 100 CFU per gram, according to standards of the European Union, for L. monocytogenes and provided faster results than the cultural reference method.

First isolation of Arcanobacterium pinnipediorum from a grey seal pup (Halichoerus grypus) in the UK.

In the present study, a single Arcanobacterium (A.) pinnipediorum strain isolated from discharge of a jaw swelling of a grey seal pup (Halichoerus grypus) in England, UK, was identified. This strain was further characterized by phenotypical investigations, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), by Fourier transform infrared spectroscopy (FT-IR), and genotypically by sequencing the 16S rRNA gene and the genes gap encoding glyceraldehyde 3-phosphate dehydrogenase, tuf encoding elongation factor tu, and rpoB encoding the ß subunit of bacterial RNA polymerase. The present study gives a first detailed characterization of the species A. pinnipediorum from a grey seal in the UK. However, the route of infection of the grey seal with the bacterial pathogen remains unclear.

Identification of Trueperella bernardiae isolated from peking ducks (Anas platyrhynchos domesticus) by phenotypical and genotypical investigations and by a newly developed loop-mediated isothermal amplification (LAMP) assay.

Trueperella (T.) bernardiae is a well-known bacterial pathogen in infections of humans, rarely in animals. In the present study, five T. bernardiae isolates, isolated from five Peking ducks of four different farms, were identified by phenotypic properties, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and genotypically by sequencing the 16S ribosomal RNA (rRNA) gene, the superoxide dismutase A encoding gene sodA, and the glyceraldehyde-3-phosphate dehydrogenase encoding gene gap. In addition, the T. bernardiae isolates could be identified with a newly developed loop-mediated isothermal amplification (LAMP) assay based on the gyrase encoding housekeeping gene gyrA. All these tests clearly identified the T. bernardiae isolates to the species level. However, the detection of the specific gene gyrA with the newly designed LAMP assay appeared with a high sensitivity and specificity, and could help to identify this bacterial species in human and animal infections in future. The importance of the T. bernardiae isolates for the clinical condition of the ducks and for the problems at farm level remains unclear.