Antimicrobial resistance and genomic relationships of Salmonella enterica from Australian cattle.

The aim of this study was to evaluate phenotypic and genotypic AMR characteristics of Salmonella enterica isolates from Australian cattle collected through a structured national survey utilizing 1001 faecal samples collected from healthy cattle at slaughter. A total of 184 Salmonella isolates were subsequently derived and subjected to microbroth dilution to 16 drugs from 11 classes with interpretation of minimum inhibitory concentrations (MICs) using epidemiological cut off (ECOFF) values to distinguish between wild-type and non-wild-type populations. Most isolates were susceptible (wild type) to all antimicrobials tested, with no resistance (non-wild type) detected for colistin, nalidixic acid, meropenem, gentamicin, florfenicol or chloramphenicol. Low rates of resistance were detected for ampicillin (2.2%), cefoxitin (2.2%), ceftiofur (2.2%), ceftriaxone (2.2%), ciprofloxacin (0.5%), streptomycin (3.3%) and tetracycline (0.5%). Isolates resistant to ceftriaxone (a critically important antimicrobial, CIA) carried the extended spectrum cephalosporin gene blaCMY-2 while no known mutation in the QRDR region or qnrS genes were detected for the CIA ciprofloxacin-resistant isolate. Thirty-six serovars were detected among the 184 Salmonella isolates using whole genome sequencing, dominated by Typhimurium (n = 36), Saintpaul (n = 22) and Anatum (n = 16). Genomic analysis clustered the cattle isolates based on serovar, with the majority of serovars containing a single sequence type (ST). Further analysis of the bovine Typhimurium isolates (ST19) and comparison with publicly available data from human Typhimurium isolates from Australia, revealed the majority of cattle isolates were unrelated to human isolates. In conclusion, this study demonstrates the continued low prevalence of AMR among Salmonella within the beef, dairy and veal industries in Australia. Salmonella Typhimurium from cattle were genetically distinct from isolates sourced from human infections. Further investigations are warranted to expand on the potential clinical and public health relevance of these findings to inform risk-management of this key pathogen.

Phenotypic and Genotypic Assessment of Antimicrobial Resistance in Escherichia coli from Australian Cattle Populations at Slaughter.

ABSTRACT: Australia relies on periodic antimicrobial resistance (AMR) surveys to determine trends and changes in AMR in animal production systems. This study is a follow-up to a survey of Escherichia coli from healthy cattle at slaughter conducted in 2013, which provided baseline data on AMR prevalence across cattle groups and production practices. In this study, 591 beef cattle, 194 dairy cattle, and 216 veal calf fecal samples were collected from 25 beef and veal processing establishments in Australia, representing approximately 77% of total export volume. A total of 969 matrix-assisted laser desorption-ionization results confirmed commensal E. coli isolates from 574 beef cattle, 186 dairy cattle, and 209 veal calves were recovered, and antimicrobial susceptibility testing was carried out by microbroth dilution to 16 drugs from 10 classes interpreted against epidemiological cutoff breakpoints. Overall, a high proportion of E. coli isolates (83.8%) were wild type for all antimicrobials assessed. In addition, isolates that were non-wild type (NWT) for three or more classes of antimicrobial did not exceed 4% for any of the cattle groups. The prevalence of E. coli that were NWT for antimicrobials that are critical or of high importance to human health was very low, with 1.4% of all isolates tested determined to be NWT for fluoroquinolones, third-generation cephalosporins, or polymyxins. Genomic analysis of NWT isolates identified one beef cattle isolate (ST-10) harboring blaCMY-2 and a dairy isolate (ST-58) and two veal calf isolates (ST-58 and ST-394) that had qnrS1, which confer resistance to extended-spectrum cephalosporins and fluoroquinolones, respectively. The low levels of AMR reported in this study confirm previous Australian studies, which indicated that there is minimal evidence that specific production practices lead to widespread disproportionate development of NWT isolates.

Rapid Evaporative Ionization Mass Spectrometry: A Review on Its Application to the Red Meat Industry with an Australian Context.

The red meat supply chain is a complex network transferring product from producers to consumers in a safe and secure way. There can be times when fragmentation can arise within the supply chain, which could be exploited. This risk needs reduction so that meat products enter the market with the desired attributes. Rapid Evaporative Ionisation Mass Spectrometry (REIMS) is a novel ambient mass spectrometry technique originally developed for rapid and accurate classification of biological tissue which is now being considered for use in a range of additional applications. It has subsequently shown promise for a range of food provenance, quality and safety applications with its ability to conduct ex vivo and in situ analysis. These are regarded as critical characteristics for technologies which can enable real-time decision making in meat processing plants and more broadly throughout the sector. This review presents an overview of the REIMS technology, and its application to the areas of provenance, quality and safety to the red meat industry, particularly in an Australian context.

Utilizing the Food-Pathogen Metabolome to Putatively Identify Biomarkers for the Detection of Shiga Toxin-Producing E. coli (STEC) from Spinach.

Shiga toxigenic E. coli (STEC) are an important cause of foodborne disease globally with many outbreaks linked to the consumption of contaminated foods such as leafy greens. Existing methods for STEC detection and isolation are time-consuming. Rapid methods may assist in preventing contaminated products from reaching consumers. This proof-of-concept study aimed to determine if a metabolomics approach could be used to detect STEC contamination in spinach. Using untargeted metabolic profiling, the bacterial pellets and supernatants arising from bacterial and inoculated spinach enrichments were investigated for the presence of unique metabolites that enabled categorization of three E. coli risk groups. A total of 109 and 471 metabolite features were identified in bacterial and inoculated spinach enrichments, respectively. Supervised OPLS-DA analysis demonstrated clear discrimination between bacterial enrichments containing different risk groups. Further analysis of the spinach enrichments determined that pathogen risk groups 1 and 2 could be easily discriminated from the other groups, though some clustering of risk groups 1 and 2 was observed, likely representing their genomic similarity. Biomarker discovery identified metabolites that were significantly associated with risk groups and may be appropriate targets for potential biosensor development. This study has confirmed that metabolomics can be used to identify the presence of pathogenic E. coli likely to be implicated in human disease.

Changes in STEC and bacterial communities during enrichment of manufacturing beef in selective and non-selective media.

Detection and isolation of Shiga toxin-producing Escherichia coli (STEC) from manufacturing beef is challenging and it may be affected by microbial changes during enrichment. This study was designed to understand population changes during enrichment of beef from an integrated (Samples A and B) and a fragmented (Samples C and D) abattoir. The samples were enriched in buffered peptone water (BPW), Assurance GDS MPX top 7 STEC mEHEC®, BAX® E. coli O157:H7 MP and PDX-STEC media then were processed for 16 S rRNA sequencing. Escherichia dominated Sample B enrichment broths regardless of the media used (71.6-97.9%) but only in mEHEC broth (79.6%) of Sample A. Escherichia was dominant in Sample C in mEHEC (95.2%) and PDX-STEC (99.2%) broths but less in BPW (58.5%) and MP (64.9%) broths. In Sample D, Clostridium dominated in mEHEC (65.5%), MP (80.2%) and PDX-STEC (90.6%) broths. O157 STEC was isolated from Sample C only. The study suggested that MP may not be as effective as mEHEC and PDX-STEC and that Clostridium could interfere with enrichment of Escherichia. Understanding the ecological changes during enrichment provides meaningful insight to optimising the enrichment protocol for STEC and subsequently enhance the efficiency of STEC detection.

A low-density SNP genotyping panel for the accurate prediction of cattle breeds.

Genomic tools to better define breed composition in agriculturally important species have sparked scientific and commercial industry interest. Knowledge of breed composition can inform multiple scientifically important decisions of industry application including DNA marker-assisted selection, identification of signatures of selection, and inference of product provenance to improve supply chain integrity. Genomic tools are expensive but can be economized by deploying a relatively small number of highly informative single-nucleotide polymorphisms (SNP) scattered evenly across the genome. Using resources from the 1000 Bull Genomes Project we established calibration (more stringent quality criteria; N = 1,243 cattle) and validation (less stringent; N = 864) data sets representing 17 breeds derived from both taurine and indicine bovine subspecies. Fifteen successively smaller panels (from 500,000 to 50 SNP) were built from those SNP in the calibration data that increasingly satisfied 2 criteria, high differential allele frequencies across the breeds as measured by average Euclidean distance (AED) and high uniformity (even spacing) across the physical genome. Those SNP awarded the highest AED were in or near genes previously identified as important signatures of selection in cattle such as LCORL, NCAPG, KITLG, and PLAG1. For each panel, the genomic breed composition (GBC) of each animal in the validation dataset was estimated using a linear regression model. A systematic exploration of the predictive accuracy of the various sized panels was then undertaken on the validation population using 3 benchmarking approaches: (1) % error (expressed relative to the estimated GBC made from over 1 million SNP), (2) % breed misassignment (expressed relative to each individual's breed recorded), and (3) Shannon's entropy of estimated GBC across the 17 target breeds. Our analyses suggest that a panel of just 250 SNP represents an adequate balance between accuracy and cost-only modest gains in accuracy are made as one increases panel density beyond this point.

Bacterial community analysis using 16S rRNA amplicon sequencing in the boning room of Australian beef export abattoirs.

Microbial contamination associated with beef slaughter and boning has been investigated using traditional culture dependent approaches. However, conventional counting methods have disadvantages of detecting only cultivable bacterial groups that may be a small subset of the true microbial population. This study investigated the microbiology in the boning room of an integrated (abattoir A) and a fragmented (abattoir B) Australian beef export abattoirs using culture independent 16S rRNA gene amplicon sequencing coupled with total viable count (TVC). Transmission of microbial populations during processing of carcases onto beef trim was monitored and compared between the two abattoirs. The results showed that the abattoirs produced beef trim with a mean TVC of 2.64-2.70 log10 CFU/cm2. Initial counts of microbes on the chilled carcases entering the boning room were <1.5 log10 CFU/cm2 and the environmental surfaces had ≤2.0 log10 CFU/cm2 throughout the boning room. Profiling of 16S gene sequences demonstrated that the contamination of boned products (beef trim) may be a result of contamination accumulating from environmental surfaces that are regularly in contact with beef trim. The 16S data also showed that the bacterial communities on the carcases and trim shared similar community composition with microbiota on environmental surfaces at varying proportions depending on the day of processing. Bacteroidales, Clostridiales, Enterobacteriales, Lactobacillales and Pseudomonadales were predominantly present in the bacterial communities in both abattoirs. However, the changes in relative abundance of these bacteria through the boning process varied between the abattoirs. The findings from this study suggested that the transfer of bacterial contaminants in the beef cattle boning room can be dynamic, and a 16 s rRNA gene sequencing-based approach can improve our understanding of the sources of contamination in the boning environment.

Analysis of Bacterial Diversity in Relation to the Presence of the Top 7 Shiga Toxin-Producing Escherichia coli throughout Australian Beef Abattoirs.

ABSTRACT: There is increasing evidence that diversity changes in bacterial communities of beef cattle correlate to the presence of Shiga toxin-producing Escherichia coli (STEC). However, studies that found an association between STEC and bacterial diversity have been focused on preslaughter stages in the beef supply chain. This study was designed to test a hypothesis that there are no differences in bacterial diversity between samples with and those without the presence of the top 7 STEC (O26, O45, O103, O111, O121, O145, and O157) throughout processing in an integrated (abattoir A) and a fragmented (abattoir B) Australian beef abattoir. Slaughter and boning room surface samples from each abattoir were analyzed using 16S rRNA amplicon sequencing and tested for the top 7 STEC following the Food Safety and Inspection Service protocol. Potential positives through slaughter were similar between the abattoirs (64 to 81%). However, abattoir B had substantially reduced potential positives in the boning room compared with abattoir A (abattoir A: 23 and 48%; abattoir B: 2 and 7%). Alpha diversity between the sample groups was not significantly different (P > 0.05) regardless of different STEC markers. Nonmetric multidimensional scaling of slaughter samples showed that the bacterial composition in fecal and hide samples shared the least similarity with the communities in carcass and environmental samples. Surface samples from slaughter (carcass and environmental) and boning (carcass, beef trim, and environmental) all appeared randomly plotted on the scale. This indicated that the STEC presence also did not have a significant effect (P > 0.05) on beta diversity. Although presence of STEC appeared to correlate with changes in diversity of fecal and hide bacterial communities in previous studies, it did not appear to have the same effect on other samples throughout processing.

Using a noninfectious MVM surrogate for assessing viral clearance during downstream process development.

Viral contamination is an inherent risk during the manufacture of biopharmaceuticals. As such, biopharmaceutical companies must demonstrate the viral clearance efficacy of their downstream process steps prior to clinical trials and commercial approval. This is accomplished through expensive and logistically challenging spiking studies, which utilize live mammalian viruses. These hurdles deter companies from analyzing viral clearance during process development and characterization. We utilized a noninfectious minute virus of mice-mock virus particle (MVM-MVP) as a surrogate spiking agent during small scale viral filtration (VF) and anion exchange chromatography (AEX) studies. For VF experiments, in-process mAb material was spiked and processed through Asahi Kasei P15, P20, P35, and BioEX nanofilters. Across each filter type, flux decay profiles and log reduction values (LRVs) were nearly identical for either particle. For AEX experiments, loads were conditioned with various amounts of sodium chloride (9, 20, 23, and 41 mS/cm), spiked with either particle and processed through a Q-SFF packed column. LRV results met our expectations of predicting MVM removal.

A Comparison of 16S rRNA Profiles Through Slaughter in Australian Export Beef Abattoirs.

Microbial contamination of beef cattle carcases and subsequent cross-contamination during processing is inevitable and virtually impossible to prevent. The understanding of microbial contamination in the beef industry is currently limited to hypotheses based on traditional microbiological tools. Additionally, the complex structural and functional responses of beef cattle microbial communities to the fragmentation in the supply chain remain unknown. This study used 16S rRNA gene sequencing in combination with traditional microbiology to monitor and compare changes in the microbiota throughout slaughter in an integrated (abattoir A) and a fragmented (abattoir B) beef abattoir in Australia. Briefly, the primary difference between an integrated and a fragmented abattoir is that fragmented abattoirs receive cattle from multiple sources, whereas integrated abattoirs typically receive cattle that has been produced using the same production system and from a limited number of sources. The composition in the bacterial communities varied between the abattoirs, though the presence of the most predominant bacterial species within the microbiota at each abattoir was similar. Lactobacillales (2.4-56.2%) and Pseudomonadales (2.4-59.4%) most notably dominated hides, carcases, and the environment in abattoir B. In abattoir A, Bacteroidales (3.9-43.8%), Lactobacillales (0.0-61.9%), and Pseudomonadales (0.5-72.1%) fluctuated but generally shared the dominance over the rest. Combined results of total viable count (TVC) and 16S rRNA gene profiling indicated that an upward hide pulling system adopted by abattoir B may lead to increased transmission of hide contaminants to post-hide pull carcases. Abattoir B had 3.2 log10CFU/cm2 reduction from hide to carcase, where abattoir A had 4.5 log10CFU/cm2 reduction. The findings from this study indicated that common beef-associated microbiota exist in varying composition in Australian abattoirs, and 16S rRNA amplicon sequencing is a powerful tool to understand in-depth movement of microbial contaminants.

Correction to: Characterization of biofilm-forming capacity and resistance to sanitizers of a range of E.coli O26 pathotypes from clinical cases and cattle in Australia.

On page 4of the original publication [1], the correct sentence should read.

Characterization of biofilm-forming capacity and resistance to sanitizers of a range of E. coli O26 pathotypes from clinical cases and cattle in Australia.

BACKGROUND: The formation of biofilms and subsequent encasement of bacterial cells in a complex matrix can enhance resistance to antimicrobials and sterilizing agents making these organisms difficult to eradicate and control. The aim of this study was to evaluate and compare the capacity of 40 E. coli O26 isolates of enterohemorrhagic E. coli (EHEC, n = 27), potential EHEC (pEHEC, n = 3), atypical enteropathogenic E. coli (aEPEC, n = 8) and non-toxigenic E. coli (NTEC, n = 2) from human and cattle sources to form biofilms on different surfaces, and determine whether extracellular matrix (ECM) components (cellulose, curli), motility, prophage insertion in mlrA and cell surface hydrophobicity could influence biofilm formation. Finally, the influence of biofilm formation on the sensitivity of isolates to quaternary ammonium compounds (QACs; Profoam, Kwiksan 22) and peracetic acid-based sanitizer (Topactive Des.) for 2 min on polystyrene plate were also evaluated. RESULTS: Biofilm production on one surface may not indicate biofilm formation on a different surface. Biofilm was formed by different pathotypes on polystyrene (70%), stainless steel (87.5%) and glass slides (95%), however only 50% demonstrated pellicle formation. EHEC isolates were significantly more likely to form a pellicle at the air-liquid interface and biofilms on polystyrene surface at 48 h than aEPEC. Strains that don't produce ECM (curli or cellulose), harbor a prophage insertion in mlrA, and are non-motile have lower biofilm forming capacities than those isolates possessing combinations of these attributes. Hydrophobicity had no impact on biofilm formation. After 2 min exposure, none of the disinfectants tested were able to completely inactivate all cells within a biofilm regardless of pathotypes and the amount of biofilm formed. CONCLUSION: Pathotypes of E. coli O26 showed varying capacities to form biofilms, however, most EHEC strains had the capacity to form biofilm on all surfaces and at the air-liquid interface under the conditions used in this study. Biofilms provided a protective effect to E. coli O26 strains against the three sanitizers, previously shown to successfully control the growth of their planktonic counterparts. Whether the characteristics of biofilm forming and non-biofilm forming strains observed in this study reflect their attributes within the food and meat-processing environments is unknown. Further studies that represent the food and meat-processing environments are required.

Antimicrobial resistance status of Enterococcus from Australian cattle populations at slaughter.

Antimicrobial agents are used in cattle production systems for the prevention and control of bacterial associated diseases. A consequence of their use is the potential development of antimicrobial resistance (AMR). Enterococcus faecium and Enterococcus faecalis that are resistant to antimicrobials are of increased concern to public health officials throughout the world as they may compromise the ability of various treatment regimens to control disease and infection in human medicine. Australia is a major exporter of beef; however it does not have an ongoing surveillance system for AMR in cattle or foods derived from these animals. This study examined 910 beef cattle, 290 dairy cattle and 300 veal calf faecal samples collected at slaughter for the presence of enterococci. Enterococcus were isolated from 805 (88.5%) beef cattle faeces, 244 (84.1%) dairy cattle faeces and 247 (82.3%) veal calf faeces with a total of 800 enterococci subsequently selected for AMR testing. The results of AMR testing identified high levels of resistance to antimicrobials that are not critically or highly important to human medicine with resistance to flavomycin (80.2%) and lincomycin (85.4-94.2%) routinely observed. Conversely, resistance to antibiotics considered critically or highly important to human medicine such as tigecycline, daptomycin, vancomycin and linezolid was not present in this study. There is minimal evidence that Australian cattle production practices are responsible for disproportionate contributions to AMR development and in general resistance to antimicrobials of critical and high importance in human medicine was low regardless of the isolate source. The low level of antimicrobial resistance in Enterococcus from Australian cattle is likely to result from comprehensive controls around the use of antimicrobials in food-production animals in Australia. Nevertheless, continued monitoring of the effects of all antimicrobial use is required to support Australia's reputation as a supplier of safe and healthy food.

Survival capabilities of Escherichia coli O26 isolated from cattle and clinical sources in Australia to disinfectants, acids and antimicrobials.

BACKGROUND: After E. coli O157, E. coli O26 is the second most prevalent enterohaemorrhagic E. coli (EHEC) serotype identified in cases of foodborne illness in Australia and throughout the world. E. coli O26 associated foodborne outbreaks have drawn attention to the survival capabilities of this organism in a range of environments. The aim of the present study was to assess the ability of E. coli O26 to survive the effects of disinfectants, acids and antimicrobials and investigate the possible influence of virulence genes in survival and persistence of E. coli O26 from human and cattle sources from Australia. RESULTS: Initial characterization indicated that E. coli O26 are a genetically diverse group that were shown to belong to a number of pathotypes. Overall, 86.4% of isolates were susceptible to all antimicrobials tested with no significant differences in resistance observed between pathotypes. A representative subset of isolates (n = 40) were selected to determine their ability to survive disinfectants at proposed industry working concentrations and acid stress. Profoam, Kwiksan 22, and Topactive DES. were able to inhibit the growth of 100% of isolates. The remaining three disinfectants (Dairy Chlor 12.5%, Envirosan and Maxifoam) were not effective against the subset of 40 E. coli O26. Finally, elevated MICs (1,024 to 4,096 µg/ml) of acetic, propionic, lactic, and citric acids were determined for the majority of the isolates (85%). CONCLUSIONS: Australian E. coli O26 isolates belong to a range of pathotypes that harbor differing virulence markers. Despite this, their response to antimicrobials, disinfectants and acids is similar confirming that stress response appears unrelated to the presence of EHEC virulence markers. Notwithstanding, the tolerance to disinfectants and the elevated acid MICs for EHEC and the other E. coli O26 pathotypes examined in this study may contribute to bacterial colonization on food contact surfaces and subsequent foodborne illness caused by this pathogen.

National Survey of Shiga Toxin-Producing Escherichia coli Serotypes O26, O45, O103, O111, O121, O145, and O157 in Australian Beef Cattle Feces.

Escherichia coli O157 and six non-O157 Shiga toxin-producing E. coli (STEC) serotypes (O26, O45, O103, O111, O121, and O145, colloquially referred to as the "big 6") have been classified as adulterants of raw nonintact beef products in the United States. While beef cattle are a known reservoir for the prototype STEC serotype, E. coli O157, less is known about the dissemination of non-O157 STEC serotypes in Australian cattle. In the present study, 1,500 fecal samples were collected at slaughter from adult (n =628) and young (n =286) beef cattle, adult (n =128) and young (n =143) dairy cattle, and veal calves (n = 315) across 31 Australian export-registered processing establishments. Fecal samples were enriched and tested for E. coli O157 and the big 6 STEC serotypes using BAX System PCR and immunomagnetic separation methods. Pathogenic STEC (pSTEC; isolates that possess stx, eae, and an O antigen marker for O157 or a big 6 serotype) were isolated from 115 samples (7.7%), of which 100 (6.7%) contained E. coli O157 and 19 (1.3%) contained a big 6 serotype. Four of the 115 samples contained multiple pSTEC serotypes. Among samples confirmed for big 6 pSTEC, 15 (1%) contained E. coli O26 and 4 (0.3%) contained E. coli O111. pSTEC of serotypes O45, O103, O121, and O145 were not isolated from any sample, even though genes indicative of E. coli belonging to these serotypes were detected by PCR. Analysis of animal classes revealed a higher pSTEC prevalence in younger animals, including veal (12.7%), young beef (9.8%), and young dairy (7.0%), than in adult animals, including adult beef (5.1%) and adult dairy (3.9%). This study is the largest of its kind undertaken in Australia. In contrast to E. coli O157 and consistent with previous findings, this study reports a relatively low prevalence of big 6 pSTEC serotypes in Australian cattle populations.

Prevalence and Antimicrobial Resistance of Salmonella and Escherichia coli from Australian Cattle Populations at Slaughter.

Antimicrobial agents are used in cattle production systems for the prevention and control of bacteria associated with diseases. Australia is the world's third largest exporter of beef; however, this country does not have an ongoing surveillance system for antimicrobial resistance (AMR) in cattle or in foods derived from these animals. In this study, 910 beef cattle, 290 dairy cattle, and 300 veal calf fecal samples collected at slaughter were examined for the presence of Escherichia coli and Salmonella, and the phenotypic AMR of 800 E. coli and 217 Salmonella isolates was determined. E. coli was readily isolated from all types of samples (92.3% of total samples), whereas Salmonella was recovered from only 14.4% of samples and was more likely to be isolated from dairy cattle samples than from beef cattle or veal calf samples. The results of AMR testing corroborate previous Australian animal and retail food surveys, which have indicated a low level of AMR. Multidrug resistance in Salmonella isolates from beef cattle was detected infrequently; however, the resistance was to antimicrobials of low importance in human medicine. Although some differences in AMR between isolates from the different types of animals were observed, there is minimal evidence that specific production practices are responsible for disproportionate contributions to AMR development. In general, resistance to antimicrobials of critical and high importance in human medicine was low regardless of the isolate source. The low level of AMR in bacteria from Australian cattle is likely a result of strict regulation of antimicrobials in food animals in Australia and animal management systems that do not favor bacterial disease.

Role of capsular polysaccharides and lipooligosaccharides in Campylobacter surface properties, autoagglutination, and attachment to abiotic surfaces.

The role of capsular polysaccharides and lipooligosaccharides in cell surface hydrophobicity, surface charge, autoagglutination (AAG), and attachment to abiotic surfaces of three strains of Campylobacter jejuni and one strain of C. coli were investigated. This was achieved by removal of capsular polysaccharides and truncation of lipooligosaccharides core oligosaccharides by inactivation of the kpsE and waaF genes, respectively. The mutants and the wild-type strains were compared after growth under planktonic (broth) and sessile (agar) conditions. Cells grown as planktonic cultures showed a significantly (p<0.05) higher degree of hydrophobicity and AAG activity but differed from their sessile counterparts with respect to surface charge and attachment counts, depending on the strain. These results suggest that prior mode of growth affects the surface properties and attachment of Campylobacter in a strain-dependent manner. There were no significant (p>0.05) differences between the three C. jejuni strains and their ΔkpsE and ΔwaaF mutants with respect to all traits tested. Inactivation of the kpsE gene significantly (p<0.05) reduced the surface charge of the C. coli strain from ∼-10 to ∼-6 mV and increased its AAG activity, while disruption of the waaF gene significantly (p<0.05) increased its surface hydrophobicity by >8° and decreased the numbers of cells attaching to stainless steel and glass by ∼0.5 log/cm². These results suggest that surface polysaccharides may influence the surface properties and attachment to abiotic surfaces of C. coli but not C. jejuni. This suggestion, however, requires further investigation using a larger number of strains of both species.

The relationship between slow photoresponse recovery rate and temporal resolution of vision.

The rate at which photoreceptors recover from excitation is thought to be critical for setting the temporal resolution of vision. Indeed, mutations in RGS9 (regulator of G-protein signaling 9) and R9AP (RGS9 anchor protein) proteins mediating rapid photoresponse recovery impair patients' ability to see moving objects. In this study, we analyzed temporal properties of retinal sensitivity and spatiotemporal aspects of visual behavior in R9AP knock-out mice. Surprisingly, we have found that this knock-out does not affect dim-light vision mediated by rods acting as single-photon counters. Under these conditions, vision was also unaffected in mice overexpressing R9AP in rods, which causes accelerated photoresponse recovery. However, in brighter light, slow photoresponse recovery in rods and cones impaired visual responses to high temporal frequency stimuli, as reported for the daylight vision of human patients. Therefore, the speed of photoresponse recovery can affect temporal resolution and motion detection when photoreceptors integrate signals from multiple photons but not when they act as single-photon counters.

Phylogenetically related Argentinean and Australian Escherichia coli O157 isolates are distinguished by virulence clades and alternative Shiga toxin 1 and 2 prophages.

Shiga toxigenic Escherichia coli O157 is the leading cause of hemolytic uremic syndrome (HUS) worldwide. The frequencies of stx genotypes and the incidences of O157-related illness and HUS vary significantly between Argentina and Australia. Locus-specific polymorphism analysis revealed that lineage I/II (LI/II) E. coli O157 isolates were most prevalent in Argentina (90%) and Australia (88%). Argentinean LI/II isolates were shown to belong to clades 4 (28%) and 8 (72%), while Australian LI/II isolates were identified as clades 6 (15%), 7 (83%), and 8 (2%). Clade 8 was significantly associated with Shiga toxin bacteriophage insertion (SBI) type stx(2) (locus of insertion, argW) in Argentinean isolates (P < 0.0001). In Argentinean LI/II strains, stx(2) is carried by a prophage inserted at argW, whereas in Australian LI/II strains the argW locus is occupied by the novel stx(1) prophage. In both Argentinean and Australian LI/II strains, stx(2c) is almost exclusively carried by a prophage inserted at sbcB. However, alternative q(933)- or q(21)-related alleles were identified in the Australian stx(2c) prophage. Argentinean LI/II isolates were also distinguished from Australian isolates by the presence of the putative virulence determinant ECSP_3286 and the predominance of motile O157:H7 strains. Characteristics common to both Argentinean and Australian LI/II O157 strains included the presence of putative virulence determinants (ECSP_3620, ECSP_0242, ECSP_2687, ECSP_2870, and ECSP_2872) and the predominance of the tir255T allele. These data support further understanding of O157 phylogeny and may foster greater insight into the differential virulence of O157 lineages.

Partial rescue of retinal function in chronically hypoglycemic mice.

PURPOSE: Mice rendered hypoglycemic by a null mutation in the glucagon receptor gene Gcgr display late-onset retinal degeneration and loss of retinal sensitivity. Acute hyperglycemia induced by dextrose ingestion does not restore their retinal function, which is consistent with irreversible loss of vision. The goal of this study was to establish whether long-term administration of high dietary glucose rescues retinal function and circuit connectivity in aged Gcgr-/- mice. METHODS: Gcgr-/- mice were administered a carbohydrate-rich diet starting at 12 months of age. After 1 month of treatment, retinal function and structure were evaluated using electroretinographic (ERG) recordings and immunohistochemistry. RESULTS: Treatment with a carbohydrate-rich diet raised blood glucose levels and improved retinal function in Gcgr-/- mice. Blood glucose increased from moderate hypoglycemia to euglycemic levels, whereas ERG b-wave sensitivity improved approximately 10-fold. Because the b-wave reflects the electrical activity of second-order cells, we examined for changes in rod-to-bipolar cell synapses. Gcgr-/- retinas have 20% fewer synaptic pairings than Gcgr+/- retinas. Remarkably, most of the lost synapses were located farthest from the bipolar cell body, near the distal boundary of the outer plexiform layer (OPL), suggesting that apical synapses are most vulnerable to chronic hypoglycemia. Although treatment with the carbohydrate-rich diet restored retinal function, it did not restore these synaptic contacts. CONCLUSIONS: Prolonged exposure to diet-induced euglycemia improves retinal function but does not reestablish synaptic contacts lost by chronic hypoglycemia. These results suggest that retinal neurons have a homeostatic mechanism that integrates energetic status over prolonged periods of time and allows them to recover functionality despite synaptic loss.