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We investigated the prevalence, persistence and virulence determinants of enterococci populations in water samples collected over three rounds following an extreme flood event in a metropolitan river. Enterococci (n = 482) were typed using the high resolution biochemical fingerprinting method (PhP typing) and grouped into common (C) or single (S) biochemical phenotypes (BPTs). In all, 23 C-BPTs (72.6% of isolates) were found across the sites. A representative isolate of each C-BPT was identified to the species level and tested for the presence of seven virulence genes (VGs), biofilm formation and resistance to 14 antibiotics. The enterococci concentrations in samples collected during the first two rounds were above national recreational water guidelines. By round three, enterococci concentrations decreased significantly (P < 0.05). However, 11 C-BPTs (55.5% of isolates) persisted across all sampling rounds. E. casseliflavus and E. mundtii were the most common enterococci populations comprising of >57% of all isolates. Ten of the 11 most dominant C-BPTs were resistant to multiple antibiotics and harboured one or more VGs. The high prevalence of antibiotic resistance and VGs among enterococci isolates in this catchment not only provides them with niche advantages but also poses a risk to public health.

Stingless bees accumulate deposits of plant resins that are mixed with beeswax to produce propolis. Previous studies have reported anti-microbial constituents of stingless bee (Tetragonula carbonaria) propolis from East Australia, but several components remained to be characterized. In the search of natural products yet unreported for Australian propolis, four bee deposit-resins of T. carbonaria bees were analysed by gas and liquid chromatography mass spectrometry with accurate mass measurements. Ethanolic extracts of the deposit-resins were tested in vitro against Staphylococcus aureus ATCC 25983 and Pseudomonas aeruginosa ATCC 27853 by the agar diffusion method. Phloroglucinols, flavonoids and isoprenoids were identified in samples. The crude extracts showed strong anti-staphylococcal effects but were less active against the Gram-negative bacterium. The diagnostic data enabled the identification of markers that can be used for profiling other Australian propolis sources and to target the isolation of bioactive phloroglucinols in future studies against antibiotic resistant S. aureus strains. Copyright © 2014 John Wiley & Sons, Ltd.

Propolis of Australian stingless bees (Tetragonula carbonaria, Meliponini) originating from Corymbia torelliana (Myrtaceae) fruit resins was tested for its antimicrobial activities as well as its flavonoid contents. This study aimed at the isolation, structural elucidation and antibacterial testing of flavanones of C. torelliana fruit resins that are incorporated into stingless bee propolis. Flavanones of this study were elucidated by spectroscopic and spectrometric methods including UV, 1D and 2D NMR, EI-MS, ESI-MS and HR-MS. The results indicated known C-methylated flavanones namely, 1 (2S)-cryptostrobin, its regioisomer 2 (2S)- stroboponin, 3 (2S)- cryptostrobin 7-methyl ether, and 6 (2S)- desmethoxymatteucinol, and known flavanones 4 (2S)- pinostrobin and 5 (2S)- pinocembrin as markers for C. torelliana fruit resins and one propolis type. Ethanolic preparations of propolis were shown to be active against Staphylococcus aureus (ATCC 25923) and to a lesser extent against Pseudomonas aeruginosa (ATCC 27853). C. torelliana flavanones inhibited the growth of S. aureus therefore contributing to the antibacterial effects observed for Australian stingless bee propolis extracts.

Farmed Tasmanian Atlantic salmon in Australia may experience water temperatures as high as 20°C during summer, which may impact on health and mariculture productivity. In this study we investigated the impact of two commercial feed on the major bacterial population in the gut of Atlantic salmon using an anaerobic semi-continuous fermenter model set at 20°C. Fermentation was conducted in a 5L culture vessel with 100 rpm agitation under CO2. For each diet the hindgut contents of three farmed Tasmanian Atlantic salmon were collected, mixed and used as fermenter inocula. Samples were collected at day 0, 1, 6 and 12 and used for bacterial enumeration and measurement of the functional status of the gut microbiota as well as their metabolic capacity (MC) values. With diet A, Vibrio spp. and lactic acid bacteria (LAB) increased over the course of fermentation. In contrast, diet B did not support the growth of LAB and instead promoted the growth of Plesiomonasshigelloides. MC values of gut microbiota receiving either diet also increased over the course of fermentation, reaching the highest level on day 12. This was independent of the type of diet used as the functional status of the microbiota for both diets was highly similar at each sampling round. Our results indicate that at the temperature experienced by Tasmanian Atlantic salmon during warm season i.e. 20°C, the type of diet may select for the growth of specific species of bacteria.

Biochemical fingerprinting (BF) databases of 524 enterococci and 571 Escherichia coli isolates and an antibiotic resistance analysis (ARA) database comprising of 380 E. coli isolates from four suspected sources (i.e. dogs, chickens, waterfowls, and human sewage) were developed to predict the sources of faecal pollution in a recreational coastal lake. Twenty water samples representing four sampling episodes were collected from five sites and the enterococci and E. coli population from each site were compared with those of the databases. The degree of similarity between bacterial populations was measured as population similarity (Sp) coefficient. Using the BF-database, bacterial populations of waterfowls showed the highest similarity with the water samples followed by a sewage treatment plant (STP). Higher population similarities were found between samples from STP and water samples especially at two sites (T2 and T3) which were located near the sewerage pipes collecting wastewater from the study area. When using the ARA-database, the highest similarity was found between E. coli populations from STP and water samples at sites T2 and T4. Both faecal indicators and as well as methods predicted human faecal pollution, possibly through leakage from submerged sewerage pipes. The results indicated that the Sp-analysis of faecal indicator bacterial populations from suspected sources and water samples can be used as a simple tool to predict the source(s) of faecal pollution in surface waters.

Aims: The aim of this study was to identify the prevalence of environmentally adapted enterococci strains by analysing biochemical fingerprinting (BF) data of 3952 enterococci isolates collected over 5 years from the six catchments in Southeast Queensland, Australia. Methods and Results: A BF method was used to type 3952 enterococci isolates from six catchments. The environmental isolates were compared with a large existing BF library comprised of 5803 enterococci isolates from 10 host groups. Environmental isolates belonged to 801 biochemical phenotypes (BPTs), of which, an average of 29.2% was specific to each catchment. When compared with the BF library, an average of 79·5% BPTs from each catchment was identical to those in the library (i.e. host-origin BPTs). The remaining 20·5% was regarded as non-host origin BPTs, as they were not in the library and constituted only 5·3% of the total isolates tested for each catchment. Conclusions: Our data suggest that less than 5% of studied environmental strains was not identical to those in the library and seemed to be of environmental origin. From a microbial source tracking context, such low level of environmentally adapted strains can have a minimal impact on the performance of the library-based methods if a large number of isolates were tested from both the host groups and environmental waters. Significance and Impact of the Study: These data shed light on the importance of the size and representativeness of library–based source-tracking methods and their implications for the identification of faecal pollution in environmental waters.

A collection of 366 Escherichia coli strains from 10 host groups and surface waters were tested for the presence of 15 virulence genes associated with strains causing intestinal and extra-intestinal infections. The virulence genes included eaeA, VT1, 2 and 2e, LT1, ST1 and 2, Einv gene, EAgg gene, CNF1 and 2, papC, O111 and O157 side chain LPS. Of the 262 strains obtained from nine different hosts, 39 (15%) carried one or more of these virulence genes. These included six strains from humans, two from horses, eight from dogs, two from ducks, five from cattle, seven from chickens, four from pigs, two from sheep and three from deer. Of the remaining 104 strains obtained from water samples, 10 (10%) also carried one or more of the tested virulence genes. Of these, six had identical biochemical phenotypes (BPTs) to strains isolated from humans (two strains), dogs (two strains), chickens (one strain) and sheep (one strain) with 4 BPTs also carrying same virulence genes. Our results indicate that the sources of clinically important E. coli strains found in surface waters due to faecal contamination can be predicted by using a combination of biochemical fingerprinting method and the detection of virulence genes. From the public health point of view this information will be of great importance for evaluating the risk associated with public use of the catchment.

A comparison of the efficacy of an existing large metabolic fingerprint database of enterococci and Escherichia coli with a locally developed database was undertaken to identify the sources of faecal contamination in a coastal lake, in southeast Qld., Australia. The local database comprised of 776 enterococci and 780 E. coli isolates from six host groups. In all, 189 enterococci and 245 E. coli biochemical phenotypes (BPTs) were found, of which 118 and 137 BPTs were unique (UQ) to host groups. The existing database comprised of 295 enterococci UQ-BPTs and 273 E. coli UQ-BPTs from 10 host groups. The representativeness and the stability of the existing database were assessed by comparing with isolates that were external to the database. In all, 197 enterococci BPTs and 179 E. coli BPTs were found in water samples. The existing database was able to identify 62.4% of enterococci BPTs and 64.8% of E. coli BPTs as human and animal sources. The results indicated that a representative database developed from a catchment can be used to predict the sources of faecal contamination in another catchment with similar landuse features within the same geographical area. However, the representativeness and the stability of the database should be evaluated prior to its application in such investigation

A biochemical fingerprinting method (the PhPlate system) was used to compare similarities between Escherichia coli and enterococci populations from surface water samples with those found in different animal species during the wet and the dry seasons in order to predict the dominant source(s) of fecal contamination in a local creek. A significant increase in the number and diversity of enterococci was observed in the creek during the wet season. Enterococci population from water samples also showed a higher population similarity with animal species than did E. coli. A higher population similarity was found between both indicator bacteria and animal species during the wet season with highest population similarities found in dogs, horses, cows and kangaroos. In contrast, a low population similarity was found for both fecal indicator bacteria from humans with water samples during the wet and the dry seasons, indicating that humans are not a major source of contamination in the studied creek. The results also indicate that the population similarity analysis of enterococci population has an advantage over E. coli in tracing the possible source(s) of contamination in the studied creek and that population similarity analysis as used in this study can be used to predict the source(s) of fecal contamination in surface waters.

A metabolic fingerprint database of enterococci and Escherichia coli from 10 host groups of animals was developed to trace the sources of fecal contamination in surface waters. In all, 526 biochemical phenotypes (BPTs) of enterococci and 530 E. coli BPTs were obtained from 4,057 enterococci and 3,728 E. coli isolates tested. Of these, 231 Enterococcus BPTs and 257 E. coli BPTs were found in multiple host groups. The remaining 295 Enterococcus BPTs and 273 E. coli BPTs were unique to individual host groups. The database was used to trace the sources of fecal contamination in a local creek. The mean diversities (Di) of enterococci (Di = 0.76 +/- 0.05) and E. coli (Di = 0.88 +/- 0.04) were high (maximum 1) in water samples, indicating diverse sources of fecal contamination. Overall, 71% of BPTs of enterococci and 67% of E. coli BPTs from water samples were identified as human and animal sources. Altogether, 248 Enterococcus BPTs and 282 E. coli BPTs were found in water samples. Among enterococci, 26 (10%) BPTs were identical to those of humans and 152 BPTs (61%) were identical to those of animals (animal BPTs). Among E. coli isolates, 36 (13%) BPTs were identical to those of humans and 151 (54%) BPTs were identical to those of animals. Of the animal BPTs, 101 (66%) Enterococcus BPTs and 93 (62%) E. coli BPTs were also unique to individual animal groups. On the basis of these unique Enterococcus BPTs, chickens contributed 14% of contamination, followed by humans (10%), dogs (7%), and horses (6%). For E. coli, humans contributed 13% of contamination, followed by ducks (9%), cattle (7%), and chickens (6%). The developed metabolic fingerprint database was able to distinguish between human and animal sources as well as among animal species in the studied catchment.

Faecal indicator microorganisms have long been used by water quality industries to assess the quality of surface, ground and shellfish-harvesting waters. However, none of these indicators are universally accepted as they lack of desired characteristics of a suitable indicator. Furthermore, they do not provide sufficient information regarding their source of origin. Over the past ten years, researchers have developed many phenotypic, genotypic and chemical methods to identify the faecal contamination in waterways by typing various indicator microorganisms. Ironically, there is no "gold standard" method for faecal source tracking. This paper is a critical evaluation of faecal indicators along with the advantages and limitations of current methods for tracking the sources of faecal contamination.

Coliforms are the members of the indigenous gut flora that most often translocate to mesenteric lymph nodes. Very few strains of Escherichia coli found in cecal contents of rats are able to translocate. The present study investigated the role of the composition of the gut flora for the occurrence of bacterial translocation. Two strains of E. coli (KI-C1 and KI-C2), previously shown to translocate in rats subjected to stress, were given by oral inoculation to rats lacking these strains. A biochemical fingerprinting method was used to identify bacteria in cecal contents, on cecal epithelium, and in mesenteric lymph nodes. In a challenge study, the inoculated E. coli strains were shown to colonize the rats and persist for up to 75 days in cecum. Subsequently, one group was starved for 24 h and a second group was subjected to experimental hemorrhage and then starved for 24 h before sampling for bacteriological analyses from blood, cecum, and mesenteric lymph nodes. Two parallel groups of rats served as controls and were not inoculated but otherwise received the same treatment before sampling. In the inoculated group, starved for 24 h, seven out of 11 rats showed translocation, whereas in the noninoculated group one of 11 rats showed translocation (P < 0.05). In groups subjected to hemorrhage and then starved for 24 h, 15/22 rats in the inoculated and 5/20 rats in the noninoculated group showed translocation (P < 0.01). These findings show that orally inoculated KI-C1 and KI-C2 strains can colonize the gut and can substantially increase bacterial translocation in rats subjected to mild and severe stress. The composition of the gut flora seems to be an underestimated factor in the pathophysiology of bacterial translocation.

Background: starvation for 24 h prior to experimental haemorrhage increases bacterial translocation in rats. Forty-eight hours starvation alone causes pronounced microbiological changes in caecal contents and a marked increase in bacterial adherence to caecal epithelium. The aim of the present study was to examine whether bulking fibre prevents these microbiological changes induced by starvation, i.e. mucosal adherence and/or bacterial translocationwith and without haemorrhage in rats. Methods: 32 rats were inoculated with the translocating Escherichia cell strain KI-C1. Groups of these rats were then starved for 48 h with or without access to bulking fibre. An additional group of rats was given bulking fibre and subjected to haemorrhage. A control group was untreated and given regular food. Samples were taken from caecal contents, caecal epithelium, mesenteric lymph nodes and blood. A biochemical fingerprinting method was used to characterize and compare E. coil strains in all samples. Results: ingestion of bulking fibre alone for 48 h significantly reduced the frequency of KI-C1 both in caecal contents and on caecal epithelium and completely prevented translocation of the strain, compared to starvation without bulking fibre for 48 h. Enforced stress (haemorrhage) increased bacterial translocation to the same level as starvation for 48 h. E. coli phenotypes found in mesenteric lymph nodes were also found adhering to the caecal epithelium. Conclusions: the presence of bulking fibre in gut lumen, by unknown mechanisms, reduces the frequency of an inoculated translocating strain of E. coil in caecal contents and on caecal epithelium and prevents its translocationto mesenteric lymph nodes.