Pathogenicity assessment of Arcobacter butzleri isolated from Canadian agricultural surface water.

BACKGROUND: Water is considered a source for the transmission of Arcobacter species to both humans and animals. This study was conducted to assess the prevalence, distribution, and pathogenicity of A. butzleri strains, which can potentially pose health risks to humans and animals. Cultures were isolated from surface waters of a mixed-use but predominately agricultural watershed in eastern Ontario, Canada. The detection of antimicrobial resistance (AMR) and virulence-associated genes (VAGs), as well as enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) assays were performed on 913 A. butzleri strains isolated from 11 agricultural sampling sites. RESULTS: All strains were resistant to one or more antimicrobial agents, with a high rate of resistance to clindamycin (99%) and chloramphenicol (77%), followed by azithromycin (48%) and nalidixic acid (49%). However, isolates showed a significantly (p < 0.05) high rate of susceptibility to tetracycline (1%), gentamycin (2%), ciprofloxacin (4%), and erythromycin (5%). Of the eight VAGs tested, ciaB, mviN, tlyA, and pldA were detected at high frequency (> 85%) compared to irgA (25%), hecB (19%), hecA (15%), and cj1349 (12%) genes. Co-occurrence analysis showed A. butzleri strains resistant to clindamycin, chloramphenicol, nalidixic acid, and azithromycin were positive for ciaB, tlyA, mviN and pldA VAGs. ERIC-PCR fingerprint analysis revealed high genetic similarity among strains isolated from three sites, and the genotypes were significantly associated with AMR and VAGs results, which highlight their potential environmental ubiquity and potential as pathogenic. CONCLUSIONS: The study results show that agricultural activities likely contribute to the contamination of A. butzleri in surface water. The findings underscore the importance of farm management practices in controlling the potential spread of A. butzleri and its associated health risks to humans and animals through contaminated water.

Characterization of a colistin resistant, hypervirulent hospital isolate of Acinetobacter courvalinii from Canada.

Non-baumannii Acinetobacter spp. are becoming more prevalent in clinical settings including those that present resistance to last-resort antibiotics such as colistin. AB222-IK40 is an Acinetobacter courvalinii strain isolated from the Ottawa Hospital Research Institute located in Ottawa, Canada. To our knowledge, it is the first report of clinical A. courvalinii in Canada. Based on the susceptibility profile, AB222-IK40 is resistant to colistin and non-susceptible to ertapenem. Whole-genome sequencing allowed for genomic investigation into colistin resistance mechanisms. No previously identified mechanism(s) were observed, but a mobile colistin resistance (mcr)-like gene and a UDP-glucose dehydrogenase gene were identified. Based on phylogenomic analyses, the mcr-like gene is an intrinsic phosphoethanolamine transferase. This gene family is implicated in one of the many mechanisms responsible for colistin resistance in Acinetobacter baumannii as well as Acinetobacter modestus. UDP-glucose dehydrogenase is involved in colistin resistance in Enterobacterales and has been shown to be involved in capsule formation in A. baumannii. Global lipidomics revealed greater abundance of phosphatidyl-myo-inositol and lyso-phosphatidyl ethanolamine moieties in the membrane of A. courvalinii than in A. baumannii. Lipidomic profiles showed differences that were probably responsible for the colistin resistance phenotype in AB222-IK40. This isolate was also hypervirulent based on survival assays in Galleria mellonella. As this is the first report of A. courvalinii from a hospital in Canada, this species may be an emerging clinical pathogen, and therefore, it is important to understand this mechanism of its colistin resistance and hypervirulence.

Three novel multiplex PCR assays for rapid detection of virulence, antimicrobial resistance, and toxin genes in Acinetobacter calcoaceticus-baumannii complex species.

The Acinetobacter calcoaceticus-baumannii (ACB) complex is an often-overlooked group of nosocomial pathogens with a significant environmental presence. Rapid molecular screening methods for virulence, antimicrobial resistance, and toxin (VAT) genes are required to investigate the potential pathogenicity of environmental isolates. This study aimed to develop and apply novel ACB complex-specific multiplex PCR (mPCR) primers and protocols for the rapid detection of eight VAT genes. We optimized three single-tube mPCR assays using reference DNA from ACB complex and other Acinetobacter species. These assays were then applied to detect VAT genes in cultured ACB complex isolates recovered from clinical and environmental sources. Widespread detection of VAT genes in environmental isolates confirmed the validity, functionality, and applicability of these novel assays. Overall, the three newly developed ACB complex species-specific mPCR assays are rapid and simple tools that can be adopted in diagnostic and clinical lab settings. The detection of VAT genes in environmental isolates suggests that environmental niches could serve as a reservoir for potentially pathogenic ACB complex and warrants further investigation. The newly developed mPCR assays are specific, sensitive, and efficient, making them well-suited for high-throughput screening in epidemiological studies and evaluating the potential pathogenicity of ACB complex recovered from various sources.

Core and conditionally rare taxa as indicators of agricultural drainage ditch and stream health and function.

BACKGROUND: The freshwater microbiome regulates aquatic ecological functionality, nutrient cycling, pathogenicity, and has the capacity to dissipate and regulate pollutants. Agricultural drainage ditches are ubiquitous in regions where field drainage is necessary for crop productivity, and as such, are first-line receptors of agricultural drainage and runoff. How bacterial communities in these systems respond to environmental and anthropogenic stressors are not well understood. In this study, we carried out a three year study in an agriculturally dominated river basin in eastern Ontario, Canada to explore the spatial and temporal dynamics of the core and conditionally rare taxa (CRT) of the instream bacterial communities using a 16S rRNA gene amplicon sequencing approach. Water samples were collected from nine stream and drainage ditch sites that represented the influence of a range of upstream land uses. RESULTS: The cross-site core and CRT accounted for 5.6% of the total number of amplicon sequence variants (ASVs), yet represented, on average, over 60% of the heterogeneity of the overall bacterial community; hence, well reflected the spatial and temporal microbial dynamics in the water courses. The contribution of core microbiome to the overall community heterogeneity represented the community stability across all sampling sites. CRT was primarily composed of functional taxa involved in nitrogen (N) cycling and was linked to nutrient loading, water levels, and flow, particularly in the smaller agricultural drainage ditches. Both the core and the CRT were sensitive responders to changes in hydrological conditions. CONCLUSIONS: We demonstrate that core and CRT can be considered as holistic tools to explore the temporal and spatial variations of the aquatic microbial community and can be used as sensitive indicators of the health and function of agriculturally dominated water courses. This approach also reduces computational complexity in relation to analyzing the entire microbial community for such purposes.

Comparative genomics analysis and virulence-related factors in novel Aliarcobacter faecis and Aliarcobacter lanthieri species identified as potential opportunistic pathogens.

BACKGROUND: Emerging pathogenic bacteria are an increasing threat to public health. Two recently described species of the genus Aliarcobacter, A. faecis and A. lanthieri, isolated from human or livestock feces, are closely related to Aliarcobacter zoonotic pathogens (A. cryaerophilus, A. skirrowii, and A. butzleri). In this study, comparative genomics analysis was carried out to examine the virulence-related, including virulence, antibiotic, and toxin (VAT) factors in the reference strains of A. faecis and A. lanthieri that may enable them to become potentially opportunistic zoonotic pathogens. RESULTS: Our results showed that the genomes of the reference strains of both species have flagella genes (flaA, flaB, flgG, flhA, flhB, fliI, fliP, motA and cheY1) as motility and export apparatus, as well as genes encoding the Twin-arginine translocation (Tat) (tatA, tatB and tatC), type II (pulE and pulF) and III (fliF, fliN and ylqH) secretory pathways, allowing them to secrete proteins into the periplasm and host cells. Invasion and immune evasion genes (ciaB, iamA, mviN, pldA, irgA and fur2) are found in both species, while adherence genes (cadF and cj1349) are only found in A. lanthieri. Acid (clpB), heat (clpA and clpB), osmotic (mviN), and low-iron (irgA and fur2) stress resistance genes were observed in both species, although urease genes were not found in them. In addition, arcB, gyrA and gyrB were found in both species, mutations of which may mediate the resistance to quaternary ammonium compounds (QACs). Furthermore, 11 VAT genes including six virulence (cadF, ciaB, irgA, mviN, pldA, and tlyA), two antibiotic resistance [tet(O) and tet(W)] and three cytolethal distending toxin (cdtA, cdtB, and cdtC) genes were validated with the PCR assays. A. lanthieri tested positive for all 11 VAT genes. By contrast, A. faecis showed positive for ten genes except for cdtB because no PCR assay for this gene was available for this species. CONCLUSIONS: The identification of the virulence, antibiotic-resistance, and toxin genes in the genomes of A. faecis and A. lanthieri reference strains through comparative genomics analysis and PCR assays highlighted the potential zoonotic pathogenicity of these two species. However, it is necessary to extend this study to include more clinical and environmental strains to explore inter-species and strain-level genetic variations in virulence-related genes and assess their potential to be opportunistic pathogens for animals and humans.

Pheno- and genotypic characterization and identification of novel subtypes of Peste des Petits Ruminants virus in domestic and captive wild goats in Northern Iraq.

BACKGROUND: Peste des Petits Ruminants (PPR) is an acute or peracute contagious transboundary viral disease that mainly affects caprine and ovine and causes significant economic impact in developing countries. After two PPR virus outbreaks in 2011 and 2014, an investigation, from August 2015 to September 2016, was carried out in Northern Iraq when an increased morbidity and mortality rates were reported in the domestic and captive wild goats. In the present study, ten domestic goat farms and seven captive wild goat herds located in seven geographical areas of Northern Iraq were clinically, pathologically, serologically and genotypically characterized to determine the prevalence and potential cause of PPR virus outbreak. RESULTS: The outbreak occurred with rate of morbidity (26.1%) and mortality (11.1%) in domestic goat farms as compared to captive wild goat herds where relatively high mortality (42.9%) and low morbidity (10.9%) rates were recorded. Based on the clinical symptoms (mucopurulent nasal discharges, ulceration and erosion of oral mucosa, profuse watery diarrhea) and necropsy (hemorrhage and congestion on mucous membranes of the colon and rectum with zebra stripes lesions) results, overall, the serological test findings revealed a high frequency (47.9%) of positive samples for anti-PPRV nucleoprotein antibodies. Furthermore, the nucleoprotein (N) gene was detected in 63.2 and 89.1% of samples using conventional and reverse transcription real-time quantitative PCR assays. A phylogenetic analysis of N gene amino acid sequences clustered with the reference strain revealed lineage IV similar to the strains isolated in 2011 and 2014, respectively. However, two sub-types of lineage IV (I and II), significantly distinct from the previous strains, were also observed. CONCLUSION: The phylogenetic analysis suggests that movements of goats are possible cause and one of the important factors responsible for the spread of virus across the region. The study results would help in improving farm management practices by establishing a PPR virus eradication program using regular monitoring and vaccination program to control and mitigate the risk of re-emergence of PPR virus infection in domestic and captive wild goats in Iraq.

Arcobacter Identification and Species Determination Using Raman Spectroscopy Combined with Neural Networks.

Rapid and accurate identification of Arcobacter is of great importance because it is considered an emerging food- and waterborne pathogen and potential zoonotic agent. Raman spectroscopy can differentiate bacteria based on Raman scattering spectral patterns of whole cells in a fast, reagentless, and easy-to-use manner. We aimed to detect and discriminate Arcobacter bacteria at the species level using confocal micro-Raman spectroscopy (785 nm) coupled with neural networks. A total of 82 reference and field isolates of 18 Arcobacter species from clinical, environmental, and agri-food sources were included. We determined that the bacterial cultivation time and growth temperature did not significantly influence the Raman spectral reproducibility and discrimination capability. The genus Arcobacter could be successfully differentiated from the closely related genera Campylobacter and Helicobacter using principal-component analysis. For the identification of Arcobacter to the species level, an accuracy of 97.2% was achieved for all 18 Arcobacter species using Raman spectroscopy combined with a convolutional neural network (CNN). The predictive capability of Raman-CNN was further validated using an independent data set of 12 Arcobacter strains. Furthermore, a Raman spectroscopy-based fully connected artificial neural network (ANN) was constructed to determine the actual ratio of a specific Arcobacter species in a bacterial mixture ranging from 5% to 100% by biomass (regression coefficient >0.99). The application of both CNN and fully connected ANN improved the accuracy of Raman spectroscopy for bacterial species determination compared to the conventional chemometrics. This newly developed approach enables rapid identification and species determination of Arcobacter within an hour following cultivation.IMPORTANCE Rapid identification of bacterial pathogens is critical for developing an early warning system and performing epidemiological investigation. Arcobacter is an emerging foodborne pathogen and has become more important in recent decades. The incidence of Arcobacter species in the agro-ecosystem is probably underestimated mainly due to the limitation in the available detection and characterization techniques. Raman spectroscopy combined with machine learning can accurately identify Arcobacter at the species level in a rapid and reliable manner, providing a promising tool for epidemiological surveillance of this microbe in the agri-food chain. The knowledge elicited from this study has the potential to be used for routine bacterial screening and diagnostics by the government, food industry, and clinics.

Real-time quantitative PCR assay development and application for assessment of agricultural surface water and various fecal matter for prevalence of Aliarcobacter faecis and Aliarcobacter lanthieri.

BACKGROUND: Aliarcobacter faecis and Aliarcobacter lanthieri are recently identified as emerging human and animal pathogens. In this paper, we demonstrate the development and optimization of two direct DNA-based quantitative real-time PCR assays using species-specific oligonucleotide primer pairs derived from rpoB and gyrA genes for A. faecis and A. lanthieri, respectively. Initially, the specificity of primers and amplicon size of each target reference strain was verified and confirmed by melt curve analysis. Standard curves were developed with a minimum quantification limit of 100 cells mL- 1 or g- 1 obtained using known quantities of spiked A. faecis and A. lanthieri reference strains in autoclaved agricultural surface water and dairy cow manure samples. RESULTS: Each species-specific qPCR assay was validated and applied to determine the rate of prevalence and quantify the total number of cells of each target species in natural surface waters of an agriculturally-dominant and non-agricultural reference watershed. In addition, the prevalence and densities were determined for human and various animal (e.g., dogs, cats, dairy cow, and poultry) fecal samples. Overall, the prevalence of A. faecis for surface water and feces was 21 and 28%, respectively. The maximum A. faecis concentration for water and feces was 2.3 × 107 cells 100 mL- 1 and 1.2 × 107 cells g- 1, respectively. A. lanthieri was detected at a lower frequency (2%) with a maximum concentration in surface water of 4.2 × 105 cells 100 mL- 1; fecal samples had a prevalence and maximum density of 10% and 2.0 × 106 cells g- 1, respectively. CONCLUSIONS: The results indicate that the occurrence of these species in agricultural surface water is potentially due to fecal contamination of water from livestock, human, or wildlife as both species were detected in fecal samples. The new real-time qPCR assays can facilitate rapid and accurate detection in < 3 h to quantify total numbers of A. faecis and A. lanthieri cells present in various complex environmental samples.

Novel virulence, antibiotic resistance and toxin gene-specific PCR-based assays for rapid pathogenicity assessment of Arcobacter faecis and Arcobacter lanthieri.

BACKGROUND: Arcobacter faecis and A. lanthieri are two newly classified species of genus Arcobacter. The prevalence and distribution of virulence, antibiotic resistance and toxin (VAT) genes in these species are required to assess their potential pathogenic health impacts to humans and animals. This study (i) developed species- and gene-specific primer pairs for the detection of six virulence, two antibiotic resistance, and three toxin genes in two target species; (ii) optimized eight single-tube multiplex and three monoplex PCR protocols using the newly developed species- and gene-specific primers; and (iii) conducted specificity and sensitivity evaluations as well as validation of eleven mono- and multiplex PCR assays by testing A. faecis (n= 29) and A. lanthieri (n= 10) strains isolated from various fecal and agricultural water sources to determine the prevalence and distribution of VAT genes and assess the degree of pathogenicity within the two species. RESULTS: Detection of all ten and eleven target VAT genes, and expression of cytolethal distending toxin (cdtA, cdtB and cdtC) genes in A. faecis and A. lanthieri reference strains with high frequency in field isolates suggest that they are potentially pathogenic strains. These findings indicate that these two species can pose a health risk to humans and animals. CONCLUSIONS: The study results show that the developed mono- and multiplex PCR (mPCR) assays are simple, rapid, reliable and sensitive for the simultaneous assessment of the potential pathogenicity and antibiotic resistance profiling of tet(O) and tet(W) genes in these two newly discovered species. Also, these assays can be useful in diagnostic and analytical laboratories to determine the pathotypes and assessment of the virulence and toxin factors associated to human and animal infections.

Arcobacter lanthieri sp. nov., isolated from pig and dairy cattle manure.

A study was undertaken to determine the prevalence and diversity of species of the genus Arcobacter in pig and dairy cattle manure, which led to the identification of strains AF1440T, AF1430 and AF1581. Initially identified as Arcobacter butzleri based on colony morphology and initial PCR-confirmation tests, analyses of 16S rRNA gene sequences of these strains confirmed that they belonged to the genus Arcobacter and were different from all known species of the genus. The isolates formed a distinct group within the genus Arcobacter based on their 16S rRNA, gyrB, rpoB, cpn60, gyrA and atpA gene sequences and fatty acid profiles. Their unique species status was further supported by physiological properties and DNA-DNA hybridization that allowed phenotypic and genotypic differentiation of the strains from other species of the genus Arcobacter. The isolates were found to be oxidase, catalase and esterase positive and urease negative; they grew well at 30 °C under microaerophilic conditions and produced nitrite and acetoin. Based on their common origin and various physiological properties, it is proposed that the isolates are classified as members of a novel species with the name Arcobacter lanthieri sp. nov. The type strain is AF1440T ( = LMG 28516T = CCUG 66485T); strains AF1430 ( = LMG 28515 = CCUG 66486) and AF1581 ( = LMG 28517 = CCUG 66487) are reference strains.

Long-term monitoring of waterborne pathogens and microbial source tracking markers in paired agricultural watersheds under controlled and conventional tile drainage management.

Surface waters from paired agricultural watersheds under controlled tile drainage (CTD) and uncontrolled tile drainage (UCTD) were monitored over 7 years in order to determine if there was an effect of CTD (imposed during the growing season) on occurrences and loadings of bacterial and viral pathogens, coliphages, and microbial source tracking markers. There were significantly lower occurrences of human, ruminant, and livestock (ruminant plus pig) Bacteroidales markers in the CTD watershed in relation to the UCTD watershed. As for pathogens, there were significantly lower occurrences of Salmonella spp. and Arcobacter spp. in the CTD watershed. There were no instances where there were significantly higher quantitative loadings of any microbial target in the CTD watershed, except for F-specific DNA (F-DNA) and F-RNA coliphages, perhaps as a result of fecal inputs from a hobby farm independent of the drainage practice treatments. There was lower loading of the ruminant marker in the CTD watershed in relation to the UCTD system, and results were significant at the level P = 0.06. The odds of Salmonella spp. occurring increased when a ruminant marker was present relative to when the ruminant marker was absent, yet for Arcobacter spp., the odds of this pathogen occurring significantly decreased when a ruminant marker was present relative to when the ruminant marker was absent (but increased when a wildlife marker was present relative to when the wildlife marker was absent). Interestingly, the odds of norovirus GII (associated with human and swine) occurring in water increased significantly when a ruminant marker was present relative to when a ruminant marker was absent. Overall, this study suggests that fecal pollution from tile-drained fields to stream could be reduced by CTD utilization.

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.

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.

Acinetobacter calcoaceticus-baumannii complex prevalence, spatial-temporal distribution, and contamination sources in Canadian aquatic environments.

Acinetobacter calcoaceticus-baumannii (ACB) complex has been identified as a group of emerging opportunistic pathogens that cause nosocomial infections. The current study investigates the prevalence, distribution, and diversity of pathogenic ACB complex in various aquatic systems with different uses. Of the total 157 agricultural, raw drinking water intake, recreational beach, and wastewater treatment plant (WWTP) effluent samples, acinetobacters were isolated, quantified, and confirmed by genus- and ACB complex-specific PCR assays. Of all agricultural surface water samples, A. calcoaceticus (65%) was more frequently detected than A. pittii (14%), A. nosocomialis (9%), and A. baumannii (3%). In WWTP effluent samples, A. baumannii was more prevalent in de-chlorinated (60%) samples compared to both A. pittii and A. nosocomialis (40%). Interestingly, A. nosocomialis (43%), A. calcoaceticus (29%), and A. baumannii (14%) were detected in raw drinking water intake samples, whereas A. pittii (50%) and A. nosocomialis (25%) were detected in beach samples. Although no sampling location-specific differences were recorded, significant (P < 0.05) seasonal differences were observed when agricultural surface water samples collected in spring were compared with the summer and fall. Whereas effluent chlorination significantly impacted the degree of prevalence of Acinetobacter in WWTP effluent samples, overall, the prevalence of ACB complex in all sampling locations and seasons indicates that these water sources, containing human-associated ACB complex, may pose potential health risks as community-acquired opportunistic infections.IMPORTANCEAcinetobacter calcoaceticus-baumannii (ACB) complex is a group of organisms known to cause problematic nosocomial opportunistic infections. A member of the species complex, A. baumannii, is becoming a global threat to infection treatment as strains are increasingly develop resistance to antibiotics. The prevalence and distribution of potentially pathogenic Acinetobacter calcoaceticus-baumannii complex species remain poorly understood, and there is a need to better understand the occurrence of A. baumannii in non-nosocomial environments. Our research details the spatial-temporal distribution of ACB complex species in a regional watershed and highlights the presence of ACB complex in wastewater effluent that is discharged into a river. These findings deepen our understanding of this group of species in non-nosocomial environments and encourage the development of monitoring programs for these species in regional waters.

Phylogenomic and phenotypic analyses highlight the diversity of antibiotic resistance and virulence in both human and non-human Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative, opportunistic pathogen that causes infections in the immunocompromised. With a high incidence of muti-drug resistance, carbapenem-resistant A. baumannii is designated as a priority 1 pathogen by the WHO. The current literature has expertly characterized clinical isolates of A. baumannii. As the challenge of these infections has recently been classified as a One Health issue, we set out to explore the diversity of isolates from human and non-clinical sources, such as agricultural surface water, urban streams, various effluents from wastewater treatment plants, and food (tank milk); and, importantly, these isolates came from a wide geographic distribution. Phylogenomic analysis considering almost 200 isolates showed that our diverse set is well-differentiated from the main international clones of A. baumannii. We discovered novel sequence types in both hospital and non-clinical settings and five strains that overexpress the resistance-nodulation-division efflux pump adeIJK without changes in susceptibility reflected by this overexpression. Furthermore, we detected a bla ADC-79 in a non-human isolate despite its sensitivity to all antibiotics. There was no significant differentiation between the virulence profiles of clinical and non-clinical isolates in the Galleria mellonella insect model of virulence, suggesting that virulence is neither dependent on geographic origin nor isolation source. The detection of antibiotic resistance and virulence genes in non-human strains suggests that these isolates may act as a genetic reservoir for clinical strains. This endorses the notion that in order to combat multi-drug-resistant infection caused by A. baumannii, a One Health approach is required, and a deeper understanding of non-clinical strains must be achieved.IMPORTANCEThe global crisis of antibiotic resistance is a silent one. More and more bacteria are becoming resistant to all antibiotics available for treatment, leaving no options remaining. This includes Acinetobacter baumannii. This Gram-negative, opportunistic pathogen shows a high frequency of multi-drug resistance, and many strains are resistant to the last-resort drugs carbapenem and colistin. Research has focused on strains of clinical origin, but there is a knowledge gap regarding virulence traits, particularly how A. baumannii became the notorious pathogen of today. Antibiotic resistance and virulence genes have been detected in strains from animals and environmental locations such as grass and soil. As such, A. baumannii is a One Health concern, which includes the health of humans, animals, and the environment. Thus, in order to truly combat the antibiotic resistance crisis, we need to understand the antibiotic resistance and virulence gene reservoirs of this pathogen under the One Health continuum.

Effect of incubation temperature on the detection of thermophilic campylobacter species from freshwater beaches, nearby wastewater effluents, and bird fecal droppings.

This large-scale study compared incubation temperatures (37°C versus 42°C) to study the detection of thermophilic Campylobacter species, including Campylobacter jejuni, C. coli, and C. lari, in various surface water samples and bird fecal droppings around Hamilton Harbor, Lake Ontario. The putative culture isolates obtained from incubation temperatures of 37 and 42°C were confirmed by Campylobacter genus- and species-specific triplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA gene internal transcribed spacer (ITS) region. A total of 759 water, wastewater, and bird fecal dropping samples were tested. Positive amplification reactions for the genus Campylobacter were found for 454 (60%) samples incubated at 37°C, compared to 258 (34%) samples incubated at 42°C. C. jejuni (16%) and C. lari (12%) were detected significantly more frequently at the 42°C incubation temperature than at 37°C (8% and 5%, respectively). In contrast, significantly higher rates of C. coli (14%) and other Campylobacter spp. (36%) were detected at the 37°C incubation temperature than at 42°C (8% and 7%, respectively). These results were consistent across surface water, wastewater, and bird fecal dropping samples. At times, Campylobacter spp. were recovered and detected at 37°C (3% for C. jejuni, 10% for C. coli, and 3% for C. lari) when the same samples incubated at 42°C were negative. A significantly higher rate of other Campylobacter spp. was detected only at 37°C (32%) than only at 42°C (3%). These results indicate that incubation temperature can significantly influence the culturability and detection of thermophilic and other fastidious Campylobacter spp. and that a comprehensive characterization of the Campylobacter spp. in surface water, wastewaters, or bird fecal droppings will require incubation at both 37 and 42°C.

Population- and Gender-Based Investigation for Prevalence of Helicobacter pylori in Dhamar, Yemen.

Among 35 species of genus Helicobacter, H. pylori is the most common causative agent of human gastritis, peptic ulcer, and gastric cancer. The infection can spread through direct human-to-human contact, fecal-oral route, and contaminated water. The study was designed to investigate the rate of prevalence of H. pylori in the population of Dhamar, Yemen. In this one-year study, 460 including 250 male and 210 female stool specimens were collected between January to December 2020 in Dhamar Governorate, Yemen. Of the total 460, 215 rural (male: n = 120 and female: n = 95) and 245 urban (male: n = 130 and female: n = 115) specimens were investigated for identification of H. pylori by serological test using Helicobacter pylori stool antigen (HpSA) test. In addition, for comparing an improved recovery of H. pylori, conventional culture-based isolation was also carried out using three selective media. Modified Campy-blood Agar (MCA), Belo Horizonte Agar (BHA), and Egg yolk Emulsion (EYE) medium supplemented with antimicrobial agents including vancomycin (10 mg/L), cefsulodin (5 mg/L), trimethoprim (5 mg/L), and amphotericin B (5 mg/L) and isolates were phenotypically characterized. The HpSA test results revealed that of the total 460 specimens, 89 (19.3%) were positive for H. pylori with relatively low in male (n = 43; 17.2%) as compared to the female (n = 46; 21.9%) specimens. After 3-10 days of incubation, H. pylori was recovered at a variable rate on each selective (MCA: 16.5%; BHA: 15.0%; EYE: 13.0%) media. However, culture-based assay results showed less recovery (n = 81; 17.6%) with no significant difference among all selective media tested and between genders (male: n = 39; 15.6%; female: n = 42; 20.0%). The infection rate was comparatively higher in rural (n = 45; 20.9%) as compared to urban (n = 36; 14.7%) population. Overall, the study data showed the prevalence of infection in both genders of all age groups. The present study showed a relatively high rate of infection of H. pylori in the Dhamar population. The serological identification and culture-based methods are important for rapid detection, aid in treatment, and developing policies for the control and eradication of H. pylori infection and to prevent the disease in different age groups in Yemen.

Comparison of gull feces-specific assays targeting the 16S rRNA genes of Catellicoccus marimammalium and Streptococcus spp.

Two novel gull-specific quantitative PCR (qPCR) assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR green assay targeting Streptococcus spp. (gull3) and a hydrolysis TaqMan assay targeting Catellicoccus marimammalium (gull4). The objectives of this study were to compare the host specificity of a previous C. marimammalium qPCR assay (gull2) with that of the new markers and to examine the presence of the three gull markers in environmental water samples from different geographic locations. Most of the gull fecal samples tested (n = 255) generated positive signals with the gull2 and gull4 assays (i.e., >86%), whereas only 28% were positive with gull3. Low prevalence and abundance of tested gull markers (0.6 to 15%) were observed in fecal samples from six nonavian species (n = 180 fecal samples), whereas the assays cross-reacted to some extent (13 to 31%) with other (nongull) avian fecal samples. The gull3 assay was positive against fecal samples from 11 of 15 avian species, including gull. Of the presumed gull-impacted water samples (n = 349), 86%, 59%, and 91% were positive with the gull2, the gull3, and the gull4 assays, respectively. Approximately 5% of 239 non-gull-impacted water samples were positive with the gull2 and the gull4 assays, whereas 21% were positive witg the gull3 assay. While the relatively high occurrence of gull2 and gull4 markers in waters impacted by gull feces suggests that these assays could be used in environmental monitoring studies, the data also suggest that multiple avian-specific assays will be needed to accurately assess the contribution of different avian sources in recreational waters.

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.

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.