Microbial Communities in Changing Aquatic Environments.

The quality of aquatic ecosystems is an important public health concern [...].

The Efficacy of Selected Probiotic Strains and Their Combination to Inhibit the Interaction of Adherent-Invasive Escherichia coli (AIEC) with a Co-Culture of Caco-2:HT29-MTX Cells.

The gastrointestinal tract's microbiota plays a crucial role in human health, with dysbiosis linked to the development of diseases such as inflammatory bowel disease (IBD). Whilst the pathogenic mechanisms underlying IBD remain poorly characterised, adherent-invasive Escherichia coli (AIEC) has been implicated as a microbiological factor in disease pathogenesis. These strains show an enhanced ability to diffusely adhere to and invade intestinal epithelial cells, along with the ability to survive and replicate within macrophages. Probiotics, such as Lactobacillus strains, have been identified as potential treatment options due to their abilities to compete with pathogens for binding sites and regulate the host immune response. In this study, we used four well-characterised Lactobacillus strains and their combination to test their ability to inhibit the adhesion, invasion, and translocation of a well-characterized AIEC strain, F44A-1, in a co-culture of Caco-2 and HT29-MTX cell lines representing the gut epithelium. The results demonstrated that the pre-inoculation of the probiotic candidates 90 min prior to the introduction of the AIEC was more effective in inhibiting AIEC interaction than the co-inoculation of the strains. While the individual probiotic strains greatly reduced AIEC colonisation and invasion of the co-cultured cells, their combination was only more effective in reducing the translocation of the AIEC. These results suggest that probiotics are more effective when used prophylactically against pathogens and that the combination of strains may enhance their efficacy against AIEC translocation once used as a prophylactic measure.

Nocardia australiensis sp. nov. and Nocardia spumae sp. nov., isolated from sea foam in Queensland, Australia.

Strains USC-21046T and USC-21048T were isolated from foaming coastal marine waters on the Sunshine Coast, Queensland, Australia. Both strains displayed growth and morphological characteristics typical for members belonging to the genus Nocardia. The major polar lipids were diphosphatidylglycerol and phosphatidylethanolamine, and the major fatty acids were C16 : 0, C18 : 1 ω9c, C18 : 0 and C18 : 0 10-methyl. The mycolic acids of strains USC-21046T and USC-21048T consisted of chain lengths between 50-64 and 56-68, respectively. Moreover, both of those strains contained meso-diaminopimelic acid and ribose, arabinose, glucose and galactose as whole cell sugars. Based on the phylogenomic results, both strains belonged to the genus Nocardia with strain USC-21046T showing an 80.4 % genome similarity to N. vinacea NBRC 16497T and N. pseudovaccinii NBRC 100343T, whereas USC-21048T strain showed an 83.6 % genome similarity to N. aobensis NBRC 100429T. Both strains were delineated from their closely related relatives based on physiological (e.g. growth on sole carbon source) and chemotaxonomic (e.g. cellular fatty composition) differences. The digital DNA-DNA hybridization (dDDH) values between USC-21046T and USC-21048T and their closely related relatives were below the dDDH threshold value of ≤70 % used for the taxonomic classification of novel species status. The genome length of strains USC-21046T and USC-21048T were 6 878 863 and 7 066 978 bp, with G+C contents of 65.2 and 67.8 mol%, respectively. For the novel isolates, we propose the names Nocardia australiensis sp. nov. with the type strain USC-21046T (=DSM 111727T=NCCB 100867T) and Nocardia spumae sp. nov. with the type strain USC-21048T (=DSM 111726T=NCCB 100868T).

Cardiac rehabilitation in heart failure with severely reduced ejection fraction: effects on mortality.

Thirty years ago, patients with low ejection fraction (EF) have often been excluded from rehabilitation programs due to concern about possibility of sudden death or other adverse cardiovascular events during exercise sessions. Recent studies have highlighted the fact that cardiac rehabilitation could improve exercise capacity, cardiac function, and health-related quality of life in congestive heart failure patients. This encouraged us to write a review article and update our latest knowledge about the outcome of rehabilitation program in patients with severely depressed cardiac function. We were particularly interested in effect of cardiac rehabilitation on exercise capacity, quality of life, vascular effects, neuro-hormonal changes, and mortality. We also conducted a mini-systematic review and meta-analysis on randomized controlled trials comparing exercise training with usual care in patients with severely reduced left ventricular ejection fraction, for the mortality subsection to obtain precise estimates of overall treatment benefit on mortality. It is our privilege to submit our manuscript for possible publication in your prestigious journal.

A comparative study of the clonal diversity and virulence characteristics of uropathogenic Escherichia coli isolated from Australian and Turkish (Turkey) children and adults with urinary tract infections.

Introduction: The virulence-associated gene (VAG) repertoire and clonal organization of uropathogenic Escherichia coli (UPEC) strains is influenced by host demographic, geographic locale, and the setting of urinary tract infection (UTI). Nevertheless, a direct comparison of these features among Australian and Turkish UPEC remains unexplored. Accordingly, this study investigated the clonal composition and virulence characteristics of a collection of UPEC isolated from Australian and Turkish UTI patients. Methods: A total of 715 UPEC strains isolated from Australian (n=361) and Turkish (n=354) children and adults with hospital (HA)- and community-acquired (CA)-UTIs were included in this study. Typing of the strains using RAPD-PCR and PhPlate fingerprinting grouped all strains into 25 clonal groups (CGs). CG representatives were phylogrouped and screened for the presence of 18 VAGs associated with extraintestinal pathogenic E. coli. Results: Turkish UPEC strains were characterized by high clonal diversity and predominance of the phylogroup D, while few distinct clonal groups with phylogenetic group B2 backgrounds dominated among the Australian strains. Twelve identical CGs were shared between ≥1 patient group from either country. Australian strains, particularly those isolated from children with HA-UTI, showed higher virulence potential than their Turkish counterparts, carrying significantly more genes associated with adhesion, iron acquisition and capsule biosynthesis. Conclusions: This study identified identical CGs of UPEC causing HA- and CA-UTIs among Australian and Turkish UTI patients. These CGs frequently carried VAGs associated with adhesion, iron acquisition, immune evasion, and toxin production, which may contribute to their ability to disseminate internationally and to cause UTI.

Efficacy of Selected Live Biotherapeutic Candidates to Inhibit the Interaction of an Adhesive-Invasive Escherichia coli Strain with Caco-2, HT29-MTX Cells and Their Co-Culture.

Adherent-invasive Escherichia coli (AIEC) has been implicated as a microbiological factor in the pathogenesis of inflammatory bowel disease (IBD). We evaluated the ability of six live biotherapeutic products (LBPs) to inhibit the interaction of an AIEC strain to three cell lines representing human gut epithelium. Co-inoculation of LBPs with AIEC showed a reduction in adhesion (up to 73%) and invasion of AIEC (up to 89%). Pre-inoculation of LBPs in HT-29-MTX and Caco-2 cells before challenging with AIEC further reduced the adhesion and invasion of the AIEC, with three LBPs showing significantly (p < 0.0001) higher efficiency in reducing the adhesion of AIEC. In co-inoculation experiments, the highest reduction in adhesion (73%) of AIEC was observed in HT-29-MTX cells, whereas the highest reduction in invasion (89%) was seen in HT-29-MTX and the co-culture of cells. Pre-inoculation of LBPs further reduced the invasion of AIEC with highest reduction (97%) observed in co-culture of cells. Our results indicated that whilst there were differences in the efficacy of LBPs, they all reduced interaction of AIEC with cell lines representing gut epithelium. Their efficiency was higher when they were pre-inoculated onto the cells, suggesting their potential as candidates for alleviating pathogenesis of AIEC in patients with IBD.

Comparative genomics of Nocardia seriolae reveals recent importation and subsequent widespread dissemination in mariculture farms in the South Central Coast region, Vietnam.

Between 2010 and 2015, nocardiosis outbreaks caused by Nocardia seriolae affected many permit farms throughout Vietnam, causing mass fish mortalities. To understand the biology, origin and epidemiology of these outbreaks, 20 N. seriolae strains collected from farms in four provinces in the South Central Coast region of Vietnam, along with two Taiwanese strains, were analysed using genetics and genomics. PFGE identified a single cluster amongst all Vietnamese strains that was distinct from the Taiwanese strains. Like the PFGE findings, phylogenomic and SNP genotyping analyses revealed that all Vietnamese N. seriolae strains belonged to a single, unique clade. Strains fell into two subclades that differed by 103 SNPs, with almost no diversity within clades (0-5 SNPs). There was no association between geographical origin and subclade placement, suggesting frequent N. seriolae transmission between Vietnamese mariculture facilities during the outbreaks. The Vietnamese strains shared a common ancestor with strains from Japan and China, with the closest strain, UTF1 from Japan, differing by just 220 SNPs from the Vietnamese ancestral node. Draft Vietnamese genomes range from 7.55 to 7.96 Mbp in size, have an average G+C content of 68.2 % and encode 7 602-7958 predicted genes. Several putative virulence factors were identified, including genes associated with host cell adhesion, invasion, intracellular survival, antibiotic and toxic compound resistance, and haemolysin biosynthesis. Our findings provide important new insights into the epidemiology and pathogenicity of N. seriolae and will aid future vaccine development and disease management strategies, with the ultimate goal of nocardiosis-free aquaculture.

Genetic diversity and virulence characteristics of biofilm-producing uropathogenic Escherichia coli / Diversidad genética y características de virulencia de Escherichia coli uropatógena productora de biopelículas

Uropathogenic E. coli (UPEC) strains exhibit different levels of biofilm formation that help adhesion of the bacteria to uroepithelial cells. We investigated the genetic diversity and virulence-associated genes (VAGs) of biofilm-producing UPEC. A collection of 107 biofilm-producing (BFP) UPEC strains isolated from patients with UTI in Iran were divided into three groups of strong, moderate, and weak BFPs after a quantitative microtiter plate assay, and the involvement of curli and cellulose in adhesion of the strains to T24 cell line was confirmed by the construction of csgD and yedQ mutants of two representative UPEC strains. BFP strains were tested for their genetic diversity, phylogenetic groups, and the presence of 15 VAGs. A significant decrease in adhesion of csgD and yedQ mutant strains confirmed the role of biofilm production in adhesion to uroepithelial cells. A high diversity was found among all three groups of strong (Di = 0.998), moderate (Di = 0.998), and weak (Di = 0.988) BFPs with majority of the strains belonging to phylogroups B2 (44.9%) and A (24.3%). Strong BFP strains carried significantly higher level papEF, hlyA, and iutA than other BFP groups. In contrast, the presence of fimH, focG, sfaS, set-1, and cvaC was more pronounced among weak BFP strains. There exists a high genetic diversity among the BFP strains with different VGA profiles. However, the high prevalence of phylogroup A among BFP strains suggests the fitness of commensal E. coli strains to cause UTI in this country.(AU)

Genetic diversity and virulence characteristics of biofilm-producing uropathogenic Escherichia coli.

Uropathogenic E. coli (UPEC) strains exhibit different levels of biofilm formation that help adhesion of the bacteria to uroepithelial cells. We investigated the genetic diversity and virulence-associated genes (VAGs) of biofilm-producing UPEC. A collection of 107 biofilm-producing (BFP) UPEC strains isolated from patients with UTI in Iran were divided into three groups of strong, moderate, and weak BFPs after a quantitative microtiter plate assay, and the involvement of curli and cellulose in adhesion of the strains to T24 cell line was confirmed by the construction of csgD and yedQ mutants of two representative UPEC strains. BFP strains were tested for their genetic diversity, phylogenetic groups, and the presence of 15 VAGs. A significant decrease in adhesion of csgD and yedQ mutant strains confirmed the role of biofilm production in adhesion to uroepithelial cells. A high diversity was found among all three groups of strong (Di = 0.998), moderate (Di = 0.998), and weak (Di = 0.988) BFPs with majority of the strains belonging to phylogroups B2 (44.9%) and A (24.3%). Strong BFP strains carried significantly higher level papEF, hlyA, and iutA than other BFP groups. In contrast, the presence of fimH, focG, sfaS, set-1, and cvaC was more pronounced among weak BFP strains. There exists a high genetic diversity among the BFP strains with different VGA profiles. However, the high prevalence of phylogroup A among BFP strains suggests the fitness of commensal E. coli strains to cause UTI in this country.

Clonal groups of extended-spectrum ß-lactamase and biofilm producing uropathogenic Escherichia coli in Iran.

Pathogenicity of a bacterium is affected by the social characteristics of the population and environmental factors. The ability of biofilm formation among ß-lactamase-producing uropathogenic Escherichia coli (UPEC) could facilitate the exchange of antibiotic-resistance genes, which resulted in widespread dissemination of antibacterial drug resistance. We investigated the prevalence of biofilm and ß-lactamase producing UPECs among patients with urinary tract infection (UTI) in two cities with different demographics and climates in Iran. A total of 265 E. coli was isolated from patients with UTIs from two referral hospitals (n = 191) and two outpatient clinics (n = 74) in Isfahan and Zahedan, Iran. Production of curli and cellulose, and, biofilm formation was investigated using Congo red agar and microtiter plate methods, respectively. Biofilm producing (BFP) isolates (n = 107) were further characterized using rep-PCR, antimicrobial susceptibility testing and extended-spectrum ß-lactamase (ESBL)/AmpC phenotypic production. Isolates were also screened for the presence of carbapenemase, ESBL and AmpC genes using multiplex PCR.   High diversity was found among BFP strains in both cities, with 58% strains producing ESBL and 21% producing AmpC. ESBL (98%), AmpC (50%) and carbapenemase genes (40%) were identified in BFP strains with ESBL-positive phenotype, respectively. The prevalence of BFP strains, antibiotic resistance and ß-lactamase genes in Zahedan, a low socioeconomic city with a warm climate, was significantly higher than that of Isfahan. High prevalence of biofilm and ß-lactamase producing UPEC strains among strains from Zahedan suggests that socioeconomic status and environmental factors might have a role in pathogenicity of the strains.

Microbial Diversity Profiling of Gut Microbiota of Macropus giganteus Using Three Hypervariable Regions of the Bacterial 16S rRNA.

Animal faecal contamination of surface waters poses a human health risk, as they may contain pathogenic bacteria or viruses. Of the numerous animal species residing along surface waterways in Australia, macropod species are a top contributor to wild animals' faecal pollution load. We characterised the gut microbiota of 30 native Australian Eastern Grey Kangaroos from six geographical regions (five kangaroos from each region) within South East Queensland in order to establish their bacterial diversity and identify potential novel species-specific bacteria for the rapid detection of faecal contamination of surface waters by these animals. Using three hypervariable regions (HVRs) of the 16S rRNA gene (i.e., V1-V3, V3-V4, and V5-V6), for their effectiveness in delineating the gut microbial diversity, faecal samples from each region were pooled and microbial genomic DNA was extracted, sequenced, and analysed. Results indicated that V1-V3 yielded a higher taxa richness due to its larger target region (~480 bp); however, higher levels of unassigned taxa were observed using the V1-V3 region. In contrast, the V3-V4 HVR (~569 bp) attained a higher likelihood of a taxonomic hit identity to the bacterial species level, with a 5-fold decrease in unassigned taxa. There were distinct dissimilarities in beta diversity between the regions, with the V1-V3 region displaying the highest number of unique taxa (n = 42), followed by V3-V4 (n = 11) and V5-V6 (n = 8). Variations in the gut microbial diversity profiles of kangaroos from different regions were also observed, which indicates that environmental factors may impact the microbial development and, thus, the composition of the gut microbiome of these animals.

Isolation and Characterization of Nocardiae Associated with Foaming Coastal Marine Waters.

Nocardiosis is an infectious disease caused by Nocardia species that occurs worldwide, albeit more prevalently in tropical/subtropical regions. It can appear as either acute, subacute or as a chronic infection mostly with those with a compromised/weakened immune system. Inhalation of spores and or mycelium fragments is the main transmission route for developing pulmonary nocardiosis. In contrast, cutaneous nocardiosis usually occurs via direct contact. In the subtropical region of the Sunshine Coast in Australia foaming events with thick and persistent and orange-brown color foam have been observed during summer seasons in the near shore marine environments. This study reports the existence of nocardiae in these near shore marine environments by the use of a novel isolation method which used the gas requirements of nocardiae as a selective battery. A total of 32 nocardiae were isolated with the use of this novel method and subsequently conducted molecular identification methods confirmed that the isolates belonged to the genus Nocardia. Twenty-one isolates out of the 32 were closely related to N. nova strains MGA115 and one was related to CBU 09/875, in addition when compared with human pathogenic nocardiae twenty of the isolates were found to be related to N. nova strain JCM 6044. Isolates displayed varied resistance against some of the antibiotics tested when interpretation threshold recommended the Comite de L'Antibiogramme de la Societe Francaise de Microbiologie were used. The highest level of resistance against cefotaxime (n = 27) and ceftriaxone (n = 24). Some of the isolates (n = 6) that displayed resistance to selected antibiotics also possessed potential human pathogenic characteristics such as adherence and translocation through human long epithelial cells as well as displaying phage resistance (n = 26). They might thus present a potential public health risk if frequently encountered through exposure to aerosols generated by the foam as well as direct contact through a wound. Preventative measures to control the growth of nocardiae in such environments such as the control of pollutants, might prevent potential infections that might be caused by these bacteria in humans as well as in marine animals.

Characterization of methicillin-resistant Staphylococcus aureus strains in sewage treatment plants in Tehran, Iran.

Multidrug-resistant Staphylococcus aureus strains have been commonly found in hospitals and communities causing wide ranges of infections among humans and animals. Typing of these strains is a key factor to reveal their clonal dissemination in different regions. We investigated the prevalence and dissemination of different clonal groups of S. aureus with resistance phenotype to multiple antibiotics in two sewage treatment plants (STPs) in Tehran, Iran over four sampling occasions. A total of 576 S. aureus were isolated from the inlet, sludge and outlet. Of these, 80 were identified as methicillin-resistant S. aureus (MRSA) and were further characterized using a combination of Phene Plate (PhP) typing, staphylococcal cassette chromosome mec (SCCmec), ccr types, prophage and antibiotic-resistant profiling. In all, eight common type (CT) and 13 single PhP type were identified in both STPs, with one major CT accounting for 38.8% of the MRSA strains. These strains belonged to three prophage patterns and five prophage types with SCCmec type III being the predominant type. Resistance to 11 out of the 17 antibiotics tested was significantly (P < 0.0059) higher among the MRSA isolates than methicillin-sensitive S. aureus (MSSA) strains. The persistence of the strains in samples collected from the outlet of both STPs was 31.9% for MRSA and 23.1% for MSSA. These data indicated that while the sewage treatment process, in general, is still useful for removing most MRSA populations, some strains with SCCmec type III may have a better ability to survive the STP process.

A Comparative Study of the Adherent-Invasive Escherichia coli Population and Gut Microbiota of Healthy Vegans versus Omnivores.

Adherent-invasive Escherichia coli (AIEC) strains carry virulence genes (VGs) which are rarely found in strains other than E. coli. These strains are abundantly found in gut mucosa of patients with inflammatory bowel disease (IBD); however, it is not clear whether their prevalence in the gut is affected by the diet of the individual. Therefore, in this study, we compared the population structure of E. coli and the prevalence of AIEC as well as the composition of gut microbiota in fecal samples of healthy participants (n = 61) on either a vegan (n = 34) or omnivore (n = 27) diet to determine whether diet is associated with the presence of AIEC. From each participant, 28 colonies of E. coli were typed using Random Amplified Polymorphic DNA (RAPD)-PCR. A representative of each common type within an individual was tested for the presence of six AIEC-associated VGs. Whole genomic DNA of the gut microbiota was also analyzed for its diversity profiles, utilizing the V5-V6 region of the16S rRNA gene sequence. There were no significant differences in the abundance and diversity of E. coli between the two diet groups. The occurrence of AIEC-associated VGs was also similar among the two groups. However, the diversity of fecal microbiota in vegans was generally higher than omnivores, with Prevotella and Bacteroides dominant in both groups. Whilst 88 microbial taxa were present in both diet groups, 28 taxa were unique to vegans, compared to seven unique taxa in the omnivores. Our results indicate that a vegan diet may not affect the number and diversity of E. coli populations and AIEC prevalence compared to omnivores. The dominance of Prevotella and Bacteroides among omnivores might be accounted for the effect of diet in these groups.

Core-Shell Beads as Microreactors for Phylogrouping of E. coli Strains.

Multiplex polymerase chain reaction (PCR) is an effective tool for simultaneous detection of target genes. Nevertheless, their use has been restricted due to the intrinsic interference between primer pairs. Performing several single PCRs in an array format instead of a multiplex PCR is a simple way to overcome this obstacle. However, there are still major technical challenges in designing a new generation of single PCR microreactors with a small sample volume, rapid thermal cycling, and no evaporation during amplification. We report a simple and robust core-shell bead array for a series of single amplifications. Four core-shell beads with a polymer coating and PCR mixture were synthesized using liquid marble formation and subsequent photo polymerization. Each bead can detect one target gene. We constructed a customised system for thermal cycling of these core-shell beads. Phylogrouping of the E. coli strains was carried out based on the fluorescent signal of the core-shell beads. This platform can be a promising alternative for multiplex nucleic acid analyses due to its simplicity and high throughput. The platform reported here also reduces the cycling time and avoids evaporation as well as contamination of the sample during the amplification process.

Field assessment of horse-associated genetic markers HoF597 and mtCytb for detecting the source of contamination in surface waters.

We investigated the specificity and sensitivity of two horse-associated markers, HoF597 and Horse mtCytb, and 12 mitochondrial and bacterial markers of six animal species (human, cow, pig, bird, dog, chicken) in the faecal samples of 50 individual horses. Both horse markers were detected in 48 (96%) faecal samples. Cross-reactivity with dog (BacCan545) and pig (P23-2) occurred in 88% and 72% of horse faecal samples, respectively. Several other bacterial and mitochondrial markers of non-target hosts were also detected; however, their specificities were >80%. Analyses of samples from surface waters (n = 11) on or adjacent to properties from which horse faecal samples had been collected showed only the presence of HoF597 but not horse mitochondrial marker. Our data suggest that while bacterial and (or) mitochondrial markers of other animal species may be present in horse faeces, dog and pig markers may predominantly be present in horse faecal samples, which points to their nonspecificity as markers for microbial source tracking. Although HoF597 and Horse mtCytb are highly sensitive and specific for the detection of horse faecal pollution, because of their low numbers, mitochondrial (mtDNA) markers may not be robust for screening surface waters.

Acinetobacter baumannii detected on modified charcoal-cefoperazone-deoxycholate agar in a waste stabilization pond.

Campylobacter is a recommended reference pathogen for the verification and validation of water recycling schemes in Australia and globally. In a larger study investigating the efficacy of pathogen removal in waste stabilization ponds (WSP), we cultivated bacteria from wastewater samples on modified charcoal-cefoperazone-deoxycholate agar (mCCDA) targeting the growth of Campylobacter. A high number of colonies characteristic of Campylobacter grew on this selective medium, but this did not correlate with qPCR data. Using primers targeting the 16S rRNA gene, and additional confirmatory tests to detect VS1, ompA, blaOXA-51-like, blaOXA-23-like genes, we tested 80 random colonies from 10 WSP samples. All 80 were identified as Acinetobacter baumannii. Wastewater grab samples taken three times over 6 months throughout the WSP system showed removal of A. baumannii in the WSP at rates similar to that of Escherichia coli. Our study suggests that mCCDA agar is not a suitable medium for isolating Campylobacter from environmental samples and that A. baumannii can be used as an indicator for removal of pathogens in WSPs.

Compositional and temporal stability of fecal taxon libraries for use with SourceTracker in sub-tropical catchments.

Characterization of microbial communities using high-throughput amplicon sequencing is an emerging approach for microbial source tracking of fecal pollution. This study used SourceTracker software to examine temporal and geographical variability of fecal bacterial community profiles to identify pollutant sources in three freshwater catchments in sub-tropical Australia. Fecal bacterial communities from 10 animal species, humans, and composite wastewater samples from six sewage treatment plants were characterized and compared to freshwater samples using Illumina amplicon sequencing of the V5-V6 regions of the 16S rRNA gene. Source contributions were calculated in SourceTracker using new fecal taxon libraries as well as previously generated libraries to determine the effects of geographic and temporal variability on source assignments. SourceTracker determined 16S rRNA bacterial communites within freshwater samples, shared taxonomic similarities to that of wastewater at low levels (typically <3%). SourceTraker also predicted occasional fecal detection of deer and flying fox sources in the water samples. No significant differences in source contributions were observed within sequences from current and previously characterized fecal samples (P ≥ 0.107). However, significant differences were observed between previously characterized and newly characterized source communities (ANOSIM P ≤ 0.001), which shared <15% community composition. Results suggest temporal instability of fecal taxon libraries among tested sources and highlight continual evaluation of community-based MST using confirmatory qPCR analyses of marker genes.

Marker genes of fecal indicator bacteria and potential pathogens in animal feces in subtropical catchments.

We investigated the abundance of marker genes of two fecal indicator bacteria (FIB) and eight potential pathogens in fecal samples of humans (n = 14) and 10 domestic and native wild animals (n = 134). For each target animal, between 10 and 14 individual fecal samples were collected (n = 148 individual fecal samples in total). The abundance of FIB and potential pathogens within each sample was determined using quantitative PCR (qPCR) assays. All animals tested were positive for Escherichia coli (EC) and the concentrations ranged from 6.13 (flying fox) to 8.87 (chicken) log10 GC/g of feces. These values for Enterococcus spp. (ENT) were 5.25 log10 GC/g for flying fox and 8.12 log10 GC/g of feces for chicken. Moderate correlations were observed between EC with P. aeruginosa, EC O157 and Cryptosporidium parvum, whereas weak correlations were observed between EC and Salmonella spp. and Giardia lamblia, Mycobacterium avium complex (MAC) and Campylobacter spp. The prevalence of MAC and P. aeruginosa were low in dog (14.3% each) and moderate (57.2%, MAC; 42.9% P. aeruginosa) in Eastern grey kangaroo fecal samples. Cryptosporidium parvum was detected in one cattle and one human fecal sample, while G. lamblia was detected in one dog, one flying fox, and one pig fecal samples. Among the eight potential pathogens tested, five pathogens were detected in chicken and dog fecal samples. The remaining animal species contained up to three potential pathogens in their feces. The data generated in this study may aid in the calculation of pathogen loads in the environment, and hence to assess the risks from human and animal fecal contamination of source waters.

The relationship between phylogenetic groups and antibiotic susceptibility patterns of Escherichia coli strains isolated from feces and urine of patients with acute or recurrent urinary tract infection.

BACKGROUND AND OBJECTIVES: B2 and D have been mentioned as the most common phylogenetic groups among uropathogenic Escherichia coli. However, there is still controversy about the importance of these phylo-groups. This study was conducted to investigate the probable relation between these groups and antibiotic resistance patterns of E. coli isolates derived from urine and feces of the patients with acute or recurrent UTI. MATERIALS AND METHODS: 10 isolates were recovered from urine and feces samples of patients with different phases of UTI in whom E. coli was causative pathogen. Biochemical fingerprinting was performed to classify the isolates and select their appropriate representatives. Phylogenetic grouping was performed using multiplex PCR, and antibiotic resistance was determined by disk diffusion method. RESULTS: Five-hundred-sixty E. coli isolates were derived from 56 UTI patients (27 acute, 29 recurrent). Among them, 261 isolates were selected using biochemical fingerprinting. All the isolates were sensitive to imipenem and nitrofurantoin. Compared to other phylo-groups, the isolates in group D showed considerably different frequencies in acute vs. recurrent phase of UTI, in urine vs. stool samples, in males vs. females, and in- vs. out-patients. They were more resistant to the antibiotics (except norfloxacin), and in contrast to others, this was seen more in acute UTI, especially in urine samples. Multi-drug resistance pattern was also meaningfully higher in group D. CONCLUSION: Although phylo-groups B2 and D of E. coli bacteria are more responsible for UTI, group D isolates seem to be more resistant and probably more virulent, even than the ones from group B2.