Common Enteric Pathogens Research Articles

Assessment of Biofilm Formation and Anti-Inflammatory Response of a Probiotic Blend in a Cultured Canine Cell Model

Gut dysbiosis and an inflamed bowel are growing concerns in mammals, including dogs. Probiotic supplements have been used to restore the natural microbial community and improve gastrointestinal health. Biofilm formation, antimicrobial activities, and immunological responses of probiotics are crucial to improving gut health. Thus, we tested a commercial probiotic blend (LabMAX-3), a canine kibble additive comprising Lactobacillus acidophilus, Lacticaseibacillus casei, and Enterococcus faecium for their ability to inactivate common enteric pathogens; their ability to form biofilms; epithelial cell adhesion; and their anti-inflammatory response in the Madin-Darby Canine Kidney (MDCK) cell line. Probiotic LabMAX-3 blend or individual isolates showed a strong inhibitory effect against Salmonella enterica, Listeria monocytogenes, enterotoxigenic Escherichia coli, and Campylobacter jejuni. LabMAX-3 formed biofilms comparable to Staphylococcus aureus. LabMAX-3 adhesion to the MDCK cell line (with or without lipopolysaccharide (LPS) pretreatment) showed comparable adhesion and biofilm formation (p < 0.05) to L. casei ATCC 334 used as a control. LabMAX-3 had no cytotoxic effects on the MDCK cell line during 1 h exposure. The interleukin-10 (IL-10) and tumor necrosis factor alpha (TNFα) ratio of LabMAX-3, compared to the L. casei control, showed a significant increase (p < 0.05), indicating a more pronounced anti-inflammatory response. The data show that LabMAX-3, a canine kibble supplement, can improve gastrointestinal health.

Herd-level occurrence and risk factors associated with respiratory and enteric pathogens from dairy calves in Ontario: A cross-sectional study

This cross-sectional herd-level study aimed to determine the occurrence of and risk factors for pathogens associated with neonatal calf diarrhea and bovine respiratory disease on Ontario dairy farms. From April to August 2022, a convenience sample of 100 dairy farms were visited once. A questionnaire covering farm biosecurity, calving and colostrum management, preweaning nutrition, and housing was administered on-farm. At each farm visit, approximately 5 calves between 2 and 35 d old were randomly selected for fecal sampling. Furthermore, approximately 5 calves between 21 to 122 d old were randomly selected for nasopharyngeal sampling. In total, 363 fecal samples (from 83 dairy farms) and 390 nasopharyngeal swab samples (from 80 dairy farms) were collected. Fecal samples were analyzed individually using a multiplex PCR to identify bacterial and parasitic enteric pathogens. Nasopharyngeal swabs were analyzed as one pooled sample per farm using bacterial culture and real-time PCR. The most common enteric pathogens detected at herd-level were Cryptosporidium parvum (67.4%) and Escherichia coli K99+ (13.2%). The most common respiratory pathogens detected at herd-level were Pasteurella multocida (62.5%), bovine coronavirus (42.5%), and Mycoplasma bovis (21.2%). Multivariable logistic models were built to explore associations between the most common pathogens and herd-level predictors selected from the questionnaire. Herd positivity for C. parvum was positively associated with having more than 61 preweaned calves per year and feeding mainly whole milk to calves. The presence of M. bovis was positively associated with herds that combined manual and automatic milk-feeding systems, and the presence of bovine coronavirus was positively associated with having more than 98 preweaned calves during the year. Univariable Poisson regression models were built to explore the association between the most common pathogens and preweaning calf mortality. Herds that were positive for C. parvum, M. bovis, or bovine coronavirus had a greater risk of preweaning calf mortality. These results provide insights for future research on pathogens associated with NCD and BRD and offer guidance for veterinarians and dairy farmers in implementing disease control measures in dairy calf herds.

Colonization Mediated by T6SS-ClpV Disrupts Host Gut Microbiota and Enhances Virulence of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common foodborne enteric pathogen that infects humans or mammals and colonizes the intestinal tract primarily by invading the host following ingestion. Meanwhile, ClpV is a core secreted protein of the bacterial type VI secretion system (T6SS). Because elucidating ClpV's role in the pathogenesis of T6SS is pivotal for revealing the virulence mechanism of Salmonella, in our study, clpV gene deletion mutants were constructed using a λ-red-based recombination system, and the effect of clpV mutation on SL1344's pathogenicity was examined in terms of stress resistance, motility, cytokine secretion, gut microbiota, and a BALB/c mouse model. Among the results, ClpV affected SL1344's motility and was also involved in cell invasion, adhesion, and intracellular survival in the MDBK cell model but did not affect invasion or intracellular survival in the RAW264.7 cell model. Moreover, clpV gene deletion significantly reduced the transcription levels of GBP2b, IFNB1, IL-6, NLRP3, NOS2, and TNF-α proinflammatory factor levels but significantly increased transcription levels of IL-4 and IL-10 anti-inflammatory factors. Last, ClpV appeared to closely relate to the pathogenicity of S. Typhimurium in vivo, which can change the gut environment and cause dysbiosis of gut microbiota. Our findings elucidate the functions of ClpV in S. Typhimurium and illustrating interactions between T6SS and gut microbiota help to clarify the mechanisms of the pathogenesis of foodborne diseases.

Occurrence, Sources and Virulence Potential of Arcobacter butzleri in Urban Municipal Stormwater Systems.

A. butzleri is an underappreciated emerging global pathogen, despite growing evidence that it is a major contributor of diarrheal illness. Few studies have investigated the occurrence and public health risks that this organism possesses from waterborne exposure routes including through stormwater use. In this study, we assessed the prevalence, virulence potential, and primary sources of stormwater-isolated A. butzleri in fecally contaminated urban stormwater systems. Based on qPCR, A. butzleri was the most common enteric bacterial pathogen [25%] found in stormwater among a panel of pathogens surveyed, including Shiga-toxin producing Escherichia coli (STEC) [6%], Campylobacter spp. [4%], and Salmonella spp. [<1%]. Concentrations of the bacteria, based on qPCR amplification of the single copy gene hsp60, were as high as 6.2 log10 copies/100 mL, suggesting significant loading of this pathogen in some stormwater systems. Importantly, out of 73 unique stormwater culture isolates, 90% were positive for the putative virulence genes cadF, ciaB, tlyA, cjl349, pldA, and mviN, while 50-75% of isolates also possessed the virulence genes irgA, hecA, and hecB. Occurrence of A. butzleri was most often associated with the human fecal pollution marker HF183 in stormwater samples. These results suggest that A. butzleri may be an important bacterial pathogen in stormwater, warranting further study on the risks it represents to public health during stormwater use.

Epistasis, core-genome disharmony, and adaptation in recombining bacteria.

Recombination of short DNA fragments via horizontal gene transfer (HGT) can introduce beneficial alleles, create genomic disharmony through negative epistasis, and create adaptive gene combinations through positive epistasis. For non-core (accessory) genes, the negative epistatic cost is likely to be minimal because the incoming genes have not co-evolved with the recipient genome and are frequently observed as tightly linked cassettes with major effects. By contrast, interspecific recombination in the core genome is expected to be rare because disruptive allelic replacement is likely to introduce negative epistasis. Why then is homologous recombination common in the core of bacterial genomes? To understand this enigma, we take advantage of an exceptional model system, the common enteric pathogens Campylobacter jejuni and C. coli that are known for very high magnitude interspecies gene flow in the core genome. As expected, HGT does indeed disrupt co-adapted allele pairings, indirect evidence of negative epistasis. However, multiple HGT events enable recovery of the genome's co-adaption between introgressing alleles, even in core metabolism genes (e.g., formate dehydrogenase). These findings demonstrate that, even for complex traits, genetic coalitions can be decoupled, transferred, and independently reinstated in a new genetic background-facilitating transition between fitness peaks. In this example, the two-step recombinational process is associated with C. coli that are adapted to the agricultural niche.IMPORTANCEGenetic exchange among bacteria shapes the microbial world. From the acquisition of antimicrobial resistance genes to fundamental questions about the nature of bacterial species, this powerful evolutionary force has preoccupied scientists for decades. However, the mixing of genes between species rests on a paradox: 0n one hand, promoting adaptation by conferring novel functionality; on the other, potentially introducing disharmonious gene combinations (negative epistasis) that will be selected against. Taking an interdisciplinary approach to analyze natural populations of the enteric bacteria Campylobacter, an ideal example of long-range admixture, we demonstrate that genes can independently transfer across species boundaries and rejoin in functional networks in a recipient genome. The positive impact of two-gene interactions appears to be adaptive by expanding metabolic capacity and facilitating niche shifts through interspecific hybridization. This challenges conventional ideas and highlights the possibility of multiple-step evolution of multi-gene traits by interspecific introgression.

Clinical performance evaluation of TAQPATH Enteric Bacterial Select Panel for the detection of common enteric bacterial pathogens in comparison to routine stool culture and other qPCR-based diagnostic tests.

Enteric bacterial infections caused by Salmonella, Shigella, pathogenic Escherichia coli, and Campylobacter represent one of the most common causes of infectious enteritis worldwide. The timely and accurate diagnosis of pathogens causing gastroenteritis is crucial for patient care, public health, and disease surveillance. While stool culture has long been the standard and highly specific method for detecting enteric pathogens, it is labor-intensive and time-consuming with limited sensitivity. To improve patient outcomes, there is a need to implement new cost-effective approaches for the detection of bacterial enteric pathogens with higher sensitivity and faster time to result. This study shows that multiplex real-time polymerase chain reaction-based tests, such as the TAQPATH Enteric Bacterial Select Panel, are accurate and cost-effective diagnostic alternatives for the detection and differentiation of the most common enteric bacterial pathogens, offering quicker time to result and higher sensitivity compared to routine stool culture.

Comparative assessment of essential oils for antioxidant and anti-microbial potential against common enteric pathogens

Comparative assessment of essential oils for antioxidant and anti-microbial potential against common enteric pathogens

Demystifying the global outbreak of severe acute hepatitis of unknown aetiology in children: A systematic review and meta-analysis

The sudden outbreak of severe acute hepatitis of unknown aetiology (SAHUA) in the first half of 2022 affected more than 1010 children in 35 countries worldwide. Dire clinical outcomes, such as acute liver failure necessitating transplantation, neurological symptoms, long-term sequelae, and death, highlight the need to determine the pathogenesis of this condition. Hypotheses on the aetiology include adenovirus and SARS-CoV-2 infections and an aberrant immune response to multiple pathogen exposure following lifting of lockdown measures but further investigation is required to reach an informed consensus. A literature search was performed on MEDLINE and EMBASE in accordance with PRISMA guidelines for systematic reviews. Primary studies reporting data on severe acute hepatitis of unknown aetiology in children from the COVID-19 era were selected for inclusion in our review. Data on patient demographics, clinical presentation and outcomes, and diagnostic testing for coinfection were extracted. Meta-analysis used a random-effects model. The 33 included studies (30 case series and 3 case-control studies) described a total of 3636 cases of SAHUA (reported 1 January, 2019-31 December, 2022), with a median age of 3.5years. Of these, 214 children (5.9%) received a liver transplant and 66 (1.8%) died. Whilst data on diagnostic testing was incomplete, the most frequently detected coinfections were with adenovirus and/or adeno-associated virus 2 (AAV2). Other common childhood respiratory and enteric pathogens, such as enterovirus, rhinovirus, and herpesviruses (EBV and HHV-6), were also identified. Coinfection with AAV2 and other common childhood pathogens may predispose children to develop this novel severe hepatitis. Altered susceptibility and response to such pathogens may be a consequence of immunological naivety following pandemic restrictions. Further investigations are needed to generate high-quality evidence on aetiology for different patient demographics and geographical areas.

Gasdermin D is the only Gasdermin that provides protection against acute Salmonella gut infection in mice.

Gasdermins (GSDMs) share a common functional domain structure and are best known for their capacity to form membrane pores. These pores are hallmarks of a specific form of cell death called pyroptosis and mediate the secretion of pro-inflammatory cytokines such as interleukin 1β (IL1β) and interleukin 18 (IL18). Thereby, Gasdermins have been implicated in various immune responses against cancer and infectious diseases such as acute Salmonella Typhimurium (S.Tm) gut infection. However, to date, we lack a comprehensive functional assessment of the different Gasdermins (GSDMA-E) during S.Tm infection in vivo. Here, we used epithelium-specific ablation, bone marrow chimeras, and mouse lines lacking individual Gasdermins, combinations of Gasdermins or even all Gasdermins (GSDMA1-3C1-4DE) at once and performed littermate-controlled oral S.Tm infections in streptomycin-pretreated mice to investigate the impact of all murine Gasdermins. While GSDMA, C, and E appear dispensable, we show that GSDMD i) restricts S.Tm loads in the gut tissue and systemic organs, ii) controls gut inflammation kinetics, and iii) prevents epithelium disruption by 72 h of the infection. Full protection requires GSDMD expression by both bone-marrow-derived lamina propria cells and intestinal epithelial cells (IECs). In vivo experiments as well as 3D-, 2D-, and chimeric enteroid infections further show that infected IEC extrusion proceeds also without GSDMD, but that GSDMD controls the permeabilization and morphology of the extruding IECs, affects extrusion kinetics, and promotes overall mucosal barrier capacity. As such, this work identifies a unique multipronged role of GSDMD among the Gasdermins for mucosal tissue defense against a common enteric pathogen.

Strain-resolved metagenomic analysis of the gut as a reservoir for bloodstream infection pathogens among premature infants in Singapore

BackgroundGut dysbiosis contributes to the high risk of bloodstream infection (BSI) among premature infants. Most prior studies of the premature infant gut microbiota were conducted in Western countries and prior to development of current tools for strain-resolved analysis.MethodsWe performed metagenomic sequencing of weekly fecal samples from 75 premature infants at a single hospital in Singapore. We evaluated associations between clinical factors and gut microbiota composition using PERMANOVA and mixed effects linear regression. We used inStrain to perform strain-level analyses evaluating for gut colonization by BSI-causing strains.ResultsMedian (interquartile range) gestation was 27 (25, 29) weeks, and 63% of infants were born via Cesarean section. Antibiotic exposures (PERMANOVA; R2 = 0.017, p = 0.001) and postnatal age (R2 = 0.015, p = 0.001) accounted for the largest amount of variability in gut microbiota composition. Increasing postnatal age was associated with higher relative abundances of several common pathogens (Enterococcus faecalis: p < 0.0001; Escherichia coli: p < 0.0001; Klebsiella aerogenes: p < 0.0001; Klebsiella pneumoniae: p < 0.0001). Antibiotic exposures were generally associated with lower relative abundances of both frequently beneficial bacteria (e.g., Bifidobacterium species) and common enteric pathogens (e.g., Enterobacter, Klebsiella species). We identified strains identical to the blood culture isolate in fecal samples from 12 of 16 (75%) infants who developed BSI, including all infections caused by typical enteric bacteria.ConclusionsAntibiotic exposures were the dominant modifiable factor affecting gut microbiota composition in a large cohort of premature infants from South-East Asia. Strain-resolved analyses indicate that the gut is an important reservoir for organisms causing BSI among premature infants.

First identification and coinfection detection of Enterocytozoon bieneusi, Encephalitozoon spp., Cryptosporidium spp. and Giardia duodenalis in diarrheic pigs in Southwest China

BackgroundEnterocytozoon bieneusi, Encephalitozoon spp., Cryptosporidium spp., and Giardia duodenalis (G. intestinalis) are enteric pathogens that cause diarrhea in pigs. This study aimed to determine the prevalence of these enteric parasites and their coinfection with E. bieneusi in diarrheic pigs in Southwest China (Chongqing and Sichuan) using nested polymerase chain reaction (nPCR) based methods.ResultsA total of 514 fecal samples were collected from diarrheic pigs from 14 pig farms in Chongqing (five farms) and Sichuan (nine farms) Provinces. The prevalence of Encephalitozoon spp., Cryptosporidium spp. and G. duodenalis was 16.14% (83/514), 0% (0/514), and 8.95% (46/514), respectively. Nested PCR revealed 305 mono-infections of E. bieneusi, six of E. cuniculi, two of E. hellem, and nine of G. duodenalis and 106 concurrent infections of E. bieneusi with the other enteric pathogens. No infections of E. intestinalis and Cryptosporidium species were detected. The highest coinfection was detected between E. bieneusi and E. cuniculi (10.5%, 54/514), followed by E. bieneusi and G. duodenalis (5.8%, 30/514) and E. bieneusi and E. hellem (2.9%, 15/514). E. bieneusi was the most frequently detected enteric pathogen, followed by E. cuniculi, G. duodenalis and E. hellem. There was a significant age-related difference in the prevalence of E. cuniculi in fattening pigs (χ2 = 15.266, df = 3, P = 0.002) and G. duodenalis in suckling pigs (χ2 = 11.92, df = 3, P = 0.008) compared with the other age groups. Sequence analysis of the ITS region of Encephalitozoon species showed two genotypes (II and III) for E. cuniculi and one (TURK1B) for E. hellem. Only G. duodenalis assemblage A was identified in all nested PCR-positive samples. E. bieneusi was found more often than other enteric pathogens.ConclusionsThis study showed that E. bieneusi, Encephalitozoon spp. [E. cuniculi and E. hellem] and G. duodenalis were common enteric parasites in diarrheic pigs in Chongqing and Sichuan Provinces. In case of both mono-infection and coinfection, E. bieneusi was the most common enteric pathogen in diarrheic pigs. Thus, it may be a significant cause of diarrhea in pigs. Precautions should be taken to prevent the spread of these enteric parasites.

PSXIV-28 Post-Confluence Cell Age Affects Lipopolysaccharide-Induced Interleukin-8 Secretion in Ipec-J2 Cells

Abstract Porcine intestinal epithelial cell line IPEC-J2 is routinely used to investigate epithelial cell interactions with common enteric porcine antigens and pathogens. Lipopolysaccharides (LPS) are bacterial antigens metabolized from Gram negative bacteria that can induce a cellular inflammatory cascade, resulting in changes to cell structure and integrity, and are often used to challenge IPEC-J2 cells. Although numerous investigators employ the IPEC-J2 LPS model, consistent methodology regarding cell culture conditions and treatment times is not well defined. The objective of the current study was to determine the optimal post-confluence cell age for LPS induction of Interleukin-8 (IL-8) secretion and related cell integrity changes. For this study, cells were seeded at 2x104/cm2 in 24 well plates on day zero and incubated at 37°C, 5% CO2 for 3 days (confluence) prior to treatment in complete growth media (CGM) containing 5% fetal bovine serum. One day prior to treatment, CGM was removed and replaced with serum free (SF), phenol red-free media. On days 0, 5, 7, 12, 14 and 18 post confluence, cells were treated with LPS (0 or 10μg/mL) in SF and phenol red-free media for 24 hours. There were 5 replicates per treatment per timepoint. Lactate dehydrogenase (LDH) release, trans-epithelial electrical resistance (TEER), and IL-8 secretion were measured at each experimental timepoint. Data were statistically analyzed for the effect of time, treatment, and the subsequent interactive effect. There was no effect of treatment, time or their interaction on LDH release (P &amp;gt; 0.05), indicating cellular health was maintained over time and 10μg/ml LPS did not influence cellular toxicity. There was an effect of treatment, time and the respective interaction that showed IL-8 secretory responsiveness was increased with LPS challenge (P &amp;lt; 0.01), was increased on d 7 regardless of LPS challenge (P &amp;lt; 0.01) and peaked on d 5 post-confluence (P &amp;lt; 0.01). Furthermore, TEER was maximized at d 5 post-confluence and remained consistently greater relative to the d 0 baseline throughout the experiment (P &amp;lt; 0.01). These data indicate that LPS-induced IL-8 secretion and related cellular integrity are optimized at d 5 post-confluence in IPEC-J2 cells. In conclusion, in-vitro research utilizing the IPEC-J2 LPS model should allow cells to age 5 days post-confluence to maximize responses.

Total and pathogen-specific serum Immunoglobulin G concentrations in neonatal beef calves, Part 1: Risk factors.

Maternal antibodies, delivered to the calf via colostrum, are crucial to prevent calfhood diseases and death. However, knowledge regarding the factors influencing this transfer of total and specific Immunoglobulin G (IgG) against common enteric and respiratory disease pathogens under current production conditions is sparse. The objectives of this study were to determine risk factors influencing total and pathogen-specific immunoglobulin G (IgG) concentrations against Escherichia coli (E. coli), bovine Rotavirus (BRoV), Cryptosporidium parvum (C. parvum), Bovine Viral Diarrhea Virus type 1 and 2 (BVDV), Parainfluenza Virus Type 3 (PI-3), Bovine Respiratory Syncytial Virus (BRSV), and Bovine Herpesvirus type 1 (BHV-1) in the serum of newborn beef calves. A total of 420 serum samples were collected from 1- to 7-day-old beef calves born on 6 farms in Alberta, Canada. Samples were analyzed by radial immunodiffusion for total IgG concentration and by enzyme-linked immunosorbent assays for pathogen-specific IgG concentrations against E. coli, BRoV, C. parvum, BVDV, PI-3, BRSV, and BHV-1. Multivariable multilevel linear and logistic regression models were built to evaluate dam- and calf-level risk factors associated with total and pathogen-specific IgG concentrations, failed transfer of passive immunity (FTPI; serum IgG < 10g/L), and inadequate transfer of passive immunity (ITPI; serum IgG < 24g/L). Farm was included as a random effect in all models to account for clustering at the herd level. Of the 420 calves included in this study, 5% (n=20) and 18% (n=75) of calves had FTPI and ITPI, respectively. Receiving colostrum intervention (i.e., being fed colostrum or colostrum product by either bottle or tube) was the most consistent risk factor for low total IgG concentration and significantly increased the odds of FTPI (Odds ratio (OR): 6.1, 95% CI: 2.0-18.9) and ITPI (OR: 4.8, 95% CI: 2.1-10.8). Calves born to cows consistently had higher pathogen-specific IgG concentrations (P<0.0001), compared to calves born from heifers, and calves born to vaccinated dams had significantly higher BRoV, BVDV, and BHV-1-specific IgG concentrations. Interestingly, E.coli-specific IgG concentrations were associated with dam vaccination only in cows but not in heifers, which was likely due to differing vaccination strategies used. This study highlights the need to review and refine protocols with respect to dam vaccination and colostrum intervention on cow-calf operations.

Enteric Aeromonas Infection: a Common Enteric Bacterial Infection with a Novel Infection Pattern Detected in an Australian Population with Gastroenteritis.

Aeromonas species are emerging human enteric pathogens. However, they are currently not routinely detected in many diagnostic laboratories, and information regarding Aeromonas enteric infections detected using molecular methods is lacking. Here, we investigated the detection of Aeromonas species and four other enteric bacterial pathogens in 341,330 fecal samples from patients with gastroenteritis processed in a large Australian diagnostic laboratory between 2015 and 2019. These enteric pathogens were detected using quantitative real-time PCR (qPCR) methods. Furthermore, we compared the qPCR cycle threshold (CT) values obtained from fecal samples that tested positive for Aeromonas only by molecular detection with those of samples that tested positive by both molecular detection and bacterial isolation methods. Aeromonas species were found to be the second most common bacterial enteric pathogens among patients with gastroenteritis. We observed a unique pattern of three infection peaks for Aeromonas, which correlated with the age of the patients. Aeromonas species were the most common enteric bacterial pathogens in children younger than 18 months. Fecal samples that tested positive for Aeromonas only by molecular detection had significantly higher CT values than fecal samples that tested positive by both molecular detection and bacterial culture. In conclusion, our findings reveal that Aeromonas enteric pathogens exhibit an age-related three-peak infection pattern, distinguishing them from other enteric bacterial pathogens. Moreover, the high rate of Aeromonas enteric infection discovered in this study suggests that Aeromonas species should be routinely tested in diagnostic laboratories. Our data also show that combining qPCR with bacterial culture can enhance the detection of enteric pathogens. IMPORTANCE Aeromonas species are emerging human enteric pathogens. However, these species are currently not routinely detected in many diagnostic laboratories, and no studies have reported the detection of Aeromonas enteric infection using molecular methods. We investigated the presence of Aeromonas species and four other enteric bacterial pathogens in 341,330 fecal samples from patients with gastroenteritis using quantitative real-time PCR (qPCR) methods. Interestingly, we discovered that Aeromonas species were the second most common bacterial enteric pathogens in patients with gastroenteritis, exhibiting a novel infection pattern compared to those of other enteric pathogens. Furthermore, we found that Aeromonas species were the most prevalent enteric bacterial pathogens in children aged 6 to 18 months. Our data also revealed that qPCR methods exhibit higher sensitivity in detecting enteric pathogens compared to that of bacterial culture alone. Moreover, combining qPCR with bacterial culture enhances the detection of enteric pathogens. These findings emphasize the importance of Aeromonas species in public health.

Detection of Antimicrobial Resistance Genes from Shiga Toxin Producing Escherichia coli by Multiplex PCR

Background: Shiga toxin-producing Escherichia coli (STEC) strains are considered to be most common food-borne enteric zoonotic pathogen, causing various disease conditions in both animals and humans and are highly pathogenic to human in low infectious doses. Resistance against antibiotics by STEC is also a big concern now a days. Hence in view of the public health significance of STEC, present work was planned to know the combination of phenotypic and genotypic resistance patterns against certain most commonly used antibiotics by using disk diffusion method and multiplex PCR respectively. Methods: A total of 426 PCR confirmed STEC isolates isolated from pooled samples (animal faecal(179), farm water(122) and human faecal samples (125) of different livestock farms in and around Proddatur andhra Pradesh, were subjected to antibiotic sensitivity test by using disc diffusion method. The isolates that showed resistance for tetracycline, streptomycin, sulphonamides and ampicillin were selected and subjected to multiplex PCR for the detection of resistance genes. Result: Disk diffusion assay revealed highest phenotypic resistance for STEC isolates against Cepahlothin (100%), followed by Ampicillin (99.06%), Tetracycline (97.2%), Streptomycin (94.3%), Sulphonamides (90.8%) and Trimethoprim (84.5%). Pooled samples also revealed the presence of antimicrobial resistance genes like tetA(59.2%), tetB(43.5%), tetC (9.2%), strA (39.3%), strB(54.1%), sul1 (40.8%), sul2 (58.7%), sul3 (3.8%) and blaTEM(83.4%). These findings indicate the highest prevalence of antimicrobial resistance among the STEC isolates, which alarms indiscriminate use of antibiotics both for therapeutic purpose and as growth promoters. Strict hygienic, sanitation and HACCP programmes should be applied to counter STEC prevalence.

Norovirus acute gastroenteritis amongst US and European travellers to areas of moderate to high risk of travellers' diarrhoea: a prospective cohort study.

Acute gastroenteritis (AGE) is a major medical condition for travellers worldwide, particularly travellers to low- and middle-income countries. Norovirus (NoV) is the most common cause of viral AGE in older children and adults, but data on prevalence and impact amongst travellers is limited. Prospective, multi-site, observational cohort study conducted 2015-2017, amongst adult international travellers from the US and Europe to areas of moderate to high risk of travel-acquired AGE. Participants provided self-collected pre-travel stool samples and self-reported AGE symptoms whilst travelling. Post-travel stool samples were requested from symptomatic subjects and a sample of asymptomatic travellers within 14days of return. Samples were tested for NoV by RT-qPCR, genotyped if positive and tested for other common enteric pathogens by Luminex xTAG GPP. Of the 1109 participants included, 437 (39.4%) developed AGE symptoms resulting in an overall AGE incidence of 24.7 per 100 person-weeks [95% confidence interval (CI): 22.4; 27.1]. In total, 20 NoV-positive AGE cases (5.2% of those tested) were identified at an incidence of 1.1 per 100 person-weeks (95% CI: 0.7; 1.7). NoV-positive samples belonged mostly to genogroup GII (18, 85.7%); None of the 13 samples sequenced belonged to genotype GII.4. Clinical severity of AGE was higher for NoV-positive than for NoV-negative cases (mean modified Vesikari Score 6.8 vs 4.9) with more cases classified as severe or moderate (25% vs 6.8%). In total, 80% of NoV-positive participants (vs 38.9% in NoV-negative) reported at least moderate impact on travel plans. AGE is a prevalent disease amongst travellers with a small proportion associated with NoV. Post-travel stool sample collection timing might have influenced the low number of NoV cases detected; however, NoV infections resulted in high clinical severity and impact on travel plans. These results may contribute to targeted vaccine development and the design of future studies on NoV epidemiology.

Association between histo-blood group antigens and Pseudomonas aeruginosa-associated diarrheal diseases.

Pseudomonas aeruginosa is not a common enteric pathogen. The association between human histo-blood group antigens (HBGAs) and P. aeruginosa enteric infection has not yet been studied. We collected stool samples from healthy children under 2 years of age for P. aeruginosa gut colonization rate. Saliva samples were collected from patients with P. aeruginosa-associated diarrheal diseases and normal healthy children. Genomic DNA was extracted from saliva samples for ABO blood group typing and FUT2 genotyping. Lewis phenotype was detected using ELISA assay. A total of 85 patients with P. aeruginosa-associated diarrheal diseases and 105 healthy children were enrolled for collecting saliva specimens. The stool colonization rate was 5/101 (5%) in healthy children, 4/58 (6.9%) in infants, and 1/43 (2.3%) in children 1-2 years old, respectively. Blood group A was more frequent in patients with P. aeruginosa-associated diarrheal diseases 24/77 (31.2%) than in healthy children 18/102 (17.6%) (P=0.035). All patients and healthy children were secretor positive. The distribution of weak-secretor genotype Se385/Se385 was 23/84 (27.4%) in patients with P. aeruginosa-associated diarrheal diseases and 17/104 (16.3%) in healthy children, respectively (P=0.06). Patients with P. aeruginosa-associated diarrheal diseases had a higher percentage of Lea+b+phenotype 25/81 (30.9%) than healthy children 17/105 (16.2%) (P=0.018). There was no association between ABO or secretor or Lewis status with the clinical severity of P. aeruginosa-associated diarrheal diseases. Infants had a higher gut P. aeruginosa colonization rate than children. Children with blood group A and Lea+b+phenotype are prone to P. aeruginosa-associated diarrheal diseases.

Global epidemiology of campylobacteriosis and the impact of COVID-19.

Campylobacteriosis is a gastroenteritis caused by pathogenic Campylobacter species and an important topic in public health. Here we review the global epidemiology of campylobacteriosis in the last eight years between 2014-2021, providing comprehensive and updated information on the reported incidence and outbreaks of Campylobacter infections. The government public health website of each of the 195 countries and publications from 2014 to September 2022 in public databases were searched. The reported incidence of campylobacteriosis in pre-COVID-19 years was compared to that during the COVID-19 pandemic in countries where data were available. Czech Republic had the highest reported incidence of campylobacteriosis worldwide (215 per 100,000 in 2019), followed by Australia (146.8 per 100,000 in 2016) and New Zealand (126.1 per 100,000 in 2019). Campylobacter was one of the most common human enteric pathogens in both developed and developing countries. About 90% of cases of campylobacteriosis were caused by Campylobacter jejuni, whereas less than 10% of cases were caused by Campylobacter coli. Other Campylobacter species were also isolated. The reported incidence and case numbers of campylobacteriosis in developed nations have remained steadily high prior to the COVID-19 pandemic, whilst some countries reported an increasing trend such as France and Japan. While outbreaks were more frequently reported in some countries, Campylobacter infections were mainly sporadic cases in most of the developed countries. Campylobacter infection was more common in summer in some but not all countries. Campylobacter infection was more common in males than females. The COVID-19 pandemic has reduced the reported incidence of campylobacteriosis in most countries where 2020 epidemiology data were available. In conclusion, Campylobacter infection remains a global health concern. Increased research and improved strategies are needed for prevention and reduction of Campylobacter infection.

Improved molecular detection of Neorickettsia risticii with a duplex real-time PCR assay in the diagnosis of Potomac horse fever.

Neorickettsia risticii, an obligate intracellular bacterium, is the causative agent of Potomac horse fever (PHF). Diagnosis of PHF is based on demonstration of serum antibodies, isolation of N. risticii, and/or detection of nucleic acid by a PCR assay. An existing real-time PCR assay targeting the N. risticii 16S rRNA has been validated using blood samples from horses with colitis, and snails; to our knowledge, the performance of the assay for other sample types has not been reported. We describe here a modification of the 16S rRNA gene assay by the addition of a set of primers and probe targeting the N. risticii p51 gene to form a duplex assay. We validated the new assay using diagnostic specimens from 56 horses with suspected PHF. The assay consistently detected down to 5 copies of synthetic targets, and did not show any cross-reaction with common equine enteric pathogens. Although we did not establish the diagnostic sensitivity and specificity of the duplex assay, results for both gene targets were in complete agreement, with the exception of 4 fecal samples that tested positive for the 16S rRNA gene only. Further analysis indicated that testing of fecal samples using our 16S rRNA gene assay alone can produce a false-positive result.

Iron-enriched Aspergillus oryzae as an alternative to iron sulphate to limit iron accumulation, growth and motility of the enteric pathogen S. Typhimurium.

Excess unabsorbed iron in the gastrointestinal tract may select for enteric pathogens and increase the incidence and severity of infectious disease. Aspergillus oryzae (Ao) is a filamentous fungus that has the ability to accumulate and store large amounts of iron, and when used as a supplement or fortificant, has similar absorption to ferrous sulphate (FeSO4) in humans. The objective of this study was to determine the effect of iron-enriched Ao (Ao iron) compared with FeSO4 on iron accumulation, growth and motility of the Gram-negative enteric pathogen, S. Typhimurium. S. Typhimurium was cultured in media containing no added iron or 1 μM elemental iron as either Ao iron or FeSO4. S. Typhimurium cultured with FeSO4 accumulated more iron than those cultured with Ao iron. Genes regulated by the iron-activated transcriptional repressor, Fur, did not differ between control and Ao iron, but decreased in S. Typhimurium cultured with FeSO4 compared with both groups. Growth of S. Typhimurium was greater when cultured with FeSO4 compared with Ao iron and control. S. Typhimurium swam faster, had greater acceleration and travelled further when cultured with FeSO4 compared with Ao iron and control; swim speed, acceleration and distance travelled did not differ between Ao iron and control. These findings provide evidence that Ao iron reduces the virulence of a common enteric pathogen in vitro. Further research is required to determine whether iron-enriched Ao is a suitable iron supplement to improve iron delivery in areas with a high infection burden.