Genetic diversity of Escherichia coli recovered from the oral cavity of beef cattle and their relatedness to faecal E. coli

AIMS: To determine the genetic diversity of generic Escherichia coli recovered from the oral cavities of beef cattle and their relatedness to E. coli isolated from the faeces of cattle during pasture grazing and feedlot finishing. METHODS AND RESULTS: A total of 484 E. coli (248 oral and 236 faecal isolates) were obtained from eight beef cattle after 1 and 5 months of grazing on pasture and after 1 and 5 months in a feedlot. The random amplification of polymorphic DNA (RAPD) method was used to genetically characterize these isolates. The RAPD patterns showed that ca 60% of E. coli recovered from the oral cavities and faeces during pasture and feedlot shared a close genetic relatedness. A number of E. coli with unique RAPD types were also found either in the oral cavities or faeces. Most of the E. coli RAPD types recovered from the oral cavities were shared among animals, but there were also RAPD types which were unique to individual animals. The E. coli populations of the oral cavities were genetically diverse and changed over time. CONCLUSIONS: This study indicates that there are large numbers of E. coli carried in the oral cavities of beef cattle and those E. coli are closely related to strains found in the faeces. The oral cavities of cattle harbour a genetically diverse E. coli population. SIGNIFICANCE AND IMPACT OF THE STUDY: The oral cavity may be an important reservoir of enteric pathogens which may transfer to meat during carcass dressing. A better understanding of the molecular ecology of E. coli in cattle would assist the design of approaches to control pathogenic strains during beef production and processing.     

Genotypic analysis of Escherichia coli recovered from product and equipment at a beef-packing plant

AIMS: To identify sources of Escherichia coli on beef by characterizing strains of the organism on animals, equipment and product at beef-packing plant. METHODS AND RESULTS: Generic E. coli were recovered from hides, carcasses, beef trimmings, conveyers and ground beef during the summer of 2001 (750 isolates) and winter of 2002 (500 isolates). The isolates were characterized by Random Amplification of Polymorphic DNA (RAPD). The numbers of E. coli recovered from dressed carcasses were less than the numbers recovered from hides. The numbers recovered from chilled carcasses were too few for meaningful analysis of the strains present on them but the numbers recovered from trimmings and ground beef were larger. The RAPD patterns showed that the majority of isolates from hides, carcasses, beef trimmings, conveyers and ground beef were of similar RAPD types, but a few unique RAPD types were recovered from only one of those sources. The E. coli populations present on the hides of incoming animals and in the beef-processing environment were highly diverse. Randomly selected E. coli isolates from each of the five sources were further characterized by pulsed-field gel electrophoresis (PFGE). Most genotypes of E. coli defined by PFGE corresponded to the E. coli types defined by RAPD. CONCLUSIONS: The hides of the incoming animals appeared to be only one of the sources of the E. coli on trimmings and in ground beef, as additional sources were apparently present in equipment used for carcass breaking. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that hazardous microbiological contamination of meat may occur after the dressing of carcasses at commercial beef-packing plants, which suggests that attention should be given to the control of the contamination of meat during carcass breaking as well as during the dressing of carcasses.     

Delineation of Pseudomonas savastanoi pv. savastanoi strains isolated in Tunisia by random-amplified polymorphic DNA analysis

Aims:  To investigate the genetic diversity of Pseudomonas savastanoi pv. savastanoi strains and to look whether these strains were distributed to geographical location.
Methods and Results:  Random amplification of polymorphic DNA (RAPD) was used to discriminate between 58 Tunisian strains and 21 strains from various other countries of P. savastanoi pv. savastanoi , the causal agent of olive knot disease. Isolates were separated into three groups by cluster analysis and principal coordinate analysis of RAPD fingerprint data obtained with three primers (OPR-12, OPX-7 and OPX-14). Group 1 contained isolates from the southeast of Tunisia and European strains. Group 2 comprised strains isolated from the north of Tunisia exclusively while group 3 encompassed the majority of isolates obtained from five orchards located in the centre of Tunisia.
Conclusions:  The results indicated that isolates of P. savastanoi pv. savastanoi were genetically distinct according to geographic regions. RAPD grouped isolates derived from the same orchard as identical.
Significance and Impact of the Study:  This is the first application of RAPD in the delineation of P. savastanoi pv. savastanoi strains.     

Prevalence and antimicrobial resistance of enterococcus species isolated from retail meats

From March 2001 to June 2002, a total of 981 samples of retail raw meats (chicken, turkey, pork, and beef) were randomly obtained from 263 grocery stores in Iowa and cultured for the presence of Enterococcus spp. A total of 1,357 enterococcal isolates were recovered from the samples, with contamination rates ranging from 97% of pork samples to 100% of ground beef samples. Enterococcus faecium was the predominant species recovered (61%), followed by E. faecalis (29%), and E. hirae (5.7%). E. faecium was the predominant species recovered from ground turkey (60%), ground beef (65%), and chicken breast (79%), while E. faecalis was the predominant species recovered from pork chops (54%). The incidence of resistance to many production and therapeutic antimicrobials differed among enterococci recovered from retail meat samples. Resistance to quinupristin-dalfopristin, a human analogue of the production drug virginiamycin, was observed in 54, 27, 9, and 18% of E. faecium isolates from turkey, chicken, pork, and beef samples, respectively. No resistance to linezolid or vancomycin was observed, but high-level gentamicin resistance was observed in 4% of enterococci, the majority of which were recovered from poultry retail meats. Results indicate that Enterococcus spp. commonly contaminate retail meats and that dissimilarities in antimicrobial resistance patterns among enterococci recovered from different meat types may reflect the use of approved antimicrobial agents in each food animal production class.     

Quantitative isolation efficiency of O26, O103, O111, O145 and O157 STEC serotypes from artificially contaminated food and cattle faeces samples using a new isolation protocol

Aims:  A range of new differential and confirmation plating media for some non-O157 Shiga toxin producing Escherichia coli (STEC) serotypes (O26, O103, O111, O145) and both sorbitol-positive and -negative O157 were evaluated using artificially contaminated samples.
Methods and Results:  Dairy products (raw milk, cheese made from pasteurized milk and raw milk), meat (ground beef, fermented meat) and cattle faeces were artificially contaminated using clinical STEC strains. Isolation efficiency was 100%, 82·3%, 88·5%, 65·9%, 64·3% and 15·8%, respectively, for an inoculum size of ≤100 CFU 25 g⁻¹. The consecutive use of differential and confirmation media limited the incidence of false positive isolates from 0% for raw milk samples, cheese made from pasteurized milk and for fermented meat to 2·1% for cheese made from raw milk, and to 8·9% for ground beef.
Conclusions:  Data presented in this paper indicated that the efficiency of the applied isolation method was dependent on sample-to-sample variation but not on the inoculum size.
Significance and Impact of Study:  Data in this paper indicated that isolation of low levels of non-O157 and sorbitol-positive O157 STEC from food samples is possible.     

Isolation and sources of 'blown pack' spoilage clostridia in beef abattoirs

Aims:  (i) To evaluate methods for isolation and molecular detection of blown pack spoilage (BPS) clostridia and (ii) to survey beef abattoirs for sources and distributions of Clostridium estertheticum and Cl. gasigenes .
Methods and Results:  Molecular detection and conventional isolation methods were used to detect and recover BPS associated clostridia ( Cl. estertheticum and Cl. gasigenes ), from four commercial Irish beef abattoirs and their environments, during a one year study. DNA-based methods detected 218 Cl. estertheticum and 300 Cl. gasigenes , from 1680 samples, whereas culture-methods only yielded 17 Cl. estertheticum and 176 Cl. gasigenes isolates. BPS Clostridia were frequently detected in beef abattoirs and their environments, especially at areas prior to hide removal. The study noted a higher percentage of positive samples during the month of May (38·6%).
Conclusions:  (i) DNA-based techniques are the most reliable ways to determine the presence of these organisms in various samples and (ii) hides and faeces are the main reservoirs of BPS clostridia in the abattoirs.
Significance and Impact of the Study:  This paper provides useful information to detect BPS organisms, as well as to develop a science-based control strategy of the problem.     

Changes in the Microbiology of Vacuum-packaged Beef

The development of the microbial flora on meat stored in vacuum-bags at 0–2° for up to 9 weeks was studied. Although the proportion of lactic acid bacteria increased relative to the aerobic spoilage organisms, the numbers of the latter continued to increase throughout storage. The initial contamination of the meat before vacuum-packaging was important; meat with a very low initial number had lower numbers of bacteria throughout storage for up to 9 weeks and steaks cut from such meat which had been stored always had 1–2 days' additional aerobic shelf life at 4°. Spoilage of these steaks was due either to slime formation and off-odour associated with high counts of presumptive Pseudomonas spp., or by discoloration and souring (lactic acid bacteria). Extract release volume and pH measurements performed on the vacuum-packaged primal joints were only of value in determining the onset of aerobic spoilage when large numbers of Gram negative organisms were present, whereas the titrimetric method of spoilage evaluation of the vacuum-packaged meat showed a correlation with spoilage due to lactic organisms.     

Development of a microbial model for the combined effect of temperature and pH on spoilage of ground meat, and validation of the model under dynamic temperature conditions

The changes in microbial flora and sensory characteristics of fresh ground meat (beef and pork) with pH values ranging from 5.34 to 6.13 were monitored at different isothermal storage temperatures (0 to 20 degrees C) under aerobic conditions. At all conditions tested, pseudomonads were the predominant bacteria, followed by Brochothrix thermosphacta, while the other members of the microbial association (e.g., lactic acid bacteria and Enterobacteriaceae) remained at lower levels. The results from microbiological and sensory analysis showed that changes in pseudomonad populations followed closely sensory changes during storage and could be used as a good index for spoilage of aerobically stored ground meat. The kinetic parameters (maximum specific growth rate [mu(max)] and the duration of lag phase [lambda]) of the spoilage bacteria were modeled by using a modified Arrhenius equation for the combined effect of temperature and pH. Meat pH affected growth of all spoilage bacteria except that of lactic acid bacteria. The "adaptation work," characterized by the product of mu(max) and lambda(mu(max) x lambda) was found to be unaffected by temperature for all tested bacteria but was affected by pH for pseudomonads and B. thermosphacta. For the latter bacteria, a negative linear correlation between ln(mu(max) x lambda) and meat pH was observed. The developed models were further validated under dynamic temperature conditions using different fluctuating temperatures. Graphical comparison between predicted and observed growth and the examination of the relative errors of predictions showed that the model predicted satisfactorily growth under dynamic conditions. Predicted shelf life based on pseudomonads growth was slightly shorter than shelf life observed by sensory analysis with a mean difference of 13.1%. The present study provides a "ready-to-use," well-validated model for predicting spoilage of aerobically stored ground meat. The use of the model by the meat industry can lead to effective management systems for the optimization of meat quality.     

The Aerobic Flora of Stored Meat with Particular Reference to the Use of Selective Media

The changes in the aerobic flora of meats stored at 15, 9 and 4° have been compared by means of selective media and by screening representative isolates. By both methods the predominant spoilage flora at the three temperatures belonged to the genus Pseudomonas . Using the medium of Masurvosky, Goldblith & Voss (1963) preliminary investigations have been made into the numbers of pseudomonads in meats.     

Identification and tracing of Enterococcus spp. by RAPD-PCR in traditional fermented sausages and meat environment

Aims:  Four local small-scale factories were studied to determine the sources of enterococci in traditional fermented sausages.
Methods and Results:  Different points during the production of a traditional fermented sausage type ( fuet ) were evaluated. Randomly amplified polymorphic DNA (RAPD)-PCR was used to type 596 Enterococcus isolates from the final products, the initial meat batter, the casing, the workers' hands and the equipment. Species-specific PCR-multiplex and the partial sequencing of atpA gene and 16S rRNA gene sequencing allowed the identification of the isolates: Enterococcus faecalis (31·4%), Enterococcus faecium (30·7%), Enterococcus sanguinicola (14·9%), Enterococcus devriesei (9·7%), Enterococcus malodoratus (7·2%), Enterococcus gilvus (1·0%), Enterococcus gallinarum (1·3%), Enterococcus casseliflavus (3·4%), Enterococcus hermanniensis (0·2%), and Enterococcus durans (0·2%) . A total of 92 different RAPD-PCR profiles were distributed among the different factories and samples evaluated. Most of the genotypes found in fuet samples were traced back to their source.
Conclusions:  The major sources of enterococci in the traditional fermented sausages studied were mainly the equipment followed by the raw ingredients, although a low proportion was traced back to human origin.
Significance and Impact of the Study:  This work contributes to determine the source of enterococcal contamination in fermented sausages and also to the knowledge of the meat environment.     

A numerical taxonomic study of Pseudomonas strains from spoiled meat

A numerical taxonomic study using 160 unit characters has been performed on 110 isolates of pseudomonads from aerobically spoiled beef and pork and on 13 named strains from the genus Pseudomonas. Four clusters of meat strains were detected at 87% S. Non-fluorescent strains were contained in two closely related clusters (1 and 2) which were identified with P. fragi. Clusters 3 and 4 contained fluorescent strains which were distinct from P. fluorescens, P. putida and the other named strains examined. An identification scheme based on 11 carbon source tests is presented for the recognition of cluster members.     

Exploring the Sources of Bacterial Spoilers in Beefsteaks by Culture-Independent High-Throughput Sequencing

Microbial growth on meat to unacceptable levels contributes significantly to change meat structure, color and flavor and to cause meat spoilage. The types of microorganisms initially present in meat depend on several factors and multiple sources of contamination can be identified. The aims of this study were to evaluate the microbial diversity in beefsteaks before and after aerobic storage at 4°C and to investigate the sources of microbial contamination by examining the microbiota of carcasses wherefrom the steaks originated and of the processing environment where the beef was handled. Carcass, environmental (processing plant) and meat samples were analyzed by culture-independent high-throughput sequencing of 16S rRNA gene amplicons. The microbiota of carcass swabs was very complex, including more than 600 operational taxonomic units (OTUs) belonging to 15 different phyla. A significant association was found between beef microbiota and specific beef cuts (P<0.01) indicating that different cuts of the same carcass can influence the microbial contamination of beef. Despite the initially high complexity of the carcass microbiota, the steaks after aerobic storage at 4°C showed a dramatic decrease in microbial complexity. Pseudomonas sp. and Brochothrix thermosphacta were the main contaminants, and Acinetobacter, Psychrobacter and Enterobacteriaceae were also found. Comparing the relative abundance of OTUs in the different samples it was shown that abundant OTUs in beefsteaks after storage occurred in the corresponding carcass. However, the abundance of these same OTUs clearly increased in environmental samples taken in the processing plant suggesting that spoilage-associated microbial species originate from carcasses, they are carried to the processing environment where the meat is handled and there they become a resident microbiota. Such microbiota is then further spread on meat when it is handled and it represents the starting microbial association wherefrom the most efficiently growing microbial species take over during storage and can cause spoilage.     

Electrochemical checking of aerobic isolates from electrochemically active biofilms formed in compost

Aims:  To design a cyclic voltammetry (CV) procedure to check the electrochemical activity of bacterial isolates that may explain the electrochemical properties of biofilms formed in compost.
Methods and Results:  Bacteria catalysing acetate oxidation in garden compost were able to form electrochemically active biofilms by transferring electrons to an electrode under chronoamperometry. They were recovered from the electrode surface and identification of the isolates using 16S rRNA sequencing showed that most of them were Gammaproteobacteria, mainly related to Enterobacter and Pseudomonas spp. A CV procedure was designed to check the electrochemical activity of both groups of isolates. Preliminary CVs suggested that the bacteria were not responsible for the catalysis of acetate oxidation. In contrast, both groups of isolates were found to catalyse the electrochemical reduction of oxygen under experimental conditions that favoured adsorption of the microbial cells on the electrode surface.
Conclusions:  Members of the genera Enterobacter and Pseudomonas were found to be able to catalyse the electrochemical reduction of oxygen.
Significance and Impact of the Study:  This study has shown the unexpected efficiency of Enterobacter and Pseudomonas spp. in catalysing the reduction of oxygen, suggesting a possible involvement of these species in biocorrosion, or possible application of these strains in designing bio-cathode for microbial fuel cells.     

Proof of concept: predicting the onset of meat spoilage by an integrated oxygen sensor spot in MAP packages

During storage of modified atmosphere packaged (MAP) meat, the initial microbiota grows to high cell numbers, resulting in perceptible spoilage after exceeding a specific threshold level. This study analyses, whether elevated oxygen consumption in the headspace of MA-packages would enable a prediction method for meat spoilage. We monitored the growth of single spoiling species inoculated on high-oxygen MAP beef and poultry, performed sensorial analysis and determined oxygen concentrations of the headspace via a non-invasive sensor spot technology. We detected microbial headspace oxygen consumption occurring prior to perceptible meat spoilage for certain species inoculated on beef steaks. However, headspace oxygen consumption and cell counts at the onset of spoilage were highly species-dependent, which resulted in a strong (Brochothrix thermosphacta) and moderate (Leuconostoc gelidum subspecies) decrease of the headspace oxygen content. No linear decrease of the headspace oxygen could be observed for Carnobacterium divergens and Carnobacterium maltaromaticum inoculated on poultry meat. We demonstrate the applicability of an incorporated oxygen sensor spot technology in MAP meat packages for detection of spoilage in individual packages prior to its perceptible onset. This enables individual package evaluation and sorting within retail, and consequently reduces meat disposal as waste.     

Study of the coumarate decarboxylase and vinylphenol reductase activities of Dekkera bruxellensis (anamorph Brettanomyces bruxellensis) isolates

Aim:  To evaluate the coumarate descarboxylase (CD) and vinylphenol reductase (VR) activities in Dekkera bruxellensis isolates and study their relationship to the growth rate, protein profile and random amplified polymorphic DNA (RAPD) molecular pattern.
Methods and Results:  CD and VR activities were quantified, as well, the growth rate, intracellular protein profile and molecular analysis (RAPD) were determined in 12 isolates of D. bruxellensis . All the isolates studied showed CD activity, but only some showed VR activity. Those isolates with the greatest growth rate did not present a different protein profile from the others. The FASC showed a relationship between RAPD molecular patterns and VR activity.
Conclusion:  CD activity is common to all of the D. bruxellensis isolates. This was not the case with VR activity, which was detected at a low percentage in the analysed micro-organisms. A correlation was observed between VR activity and the RAPD patterns.
Significance and Impact of the Study:  This is the first study that quantifies the CD and VR enzyme activities in D. bruxellensis, demonstrating that these activities are not present in all isolates of this yeast.     

Diversity of staphylococcal cassette chromosome in coagulase-negative staphylococci from animal sources

Aims:  To type the staphylococcal cassette chromosome (SCC) in coagulase-negative staphylococci (CoNS) from animal sources.
Methods and Results:  A total of 92 CoNS isolates recovered from farm animals was analysed. The top three staphylococcal species were Staphylococcus lentus (34), S. sciuri (31), and S. xylosus (13). The presence of the cassette chromosome recombinase (ccr) genes ccrA1 , ccrB1 , ccrA2 , ccrB2 , ccrA3 , ccrB3 and ccrC , the mec regulatory genes mecI and mecR1 , and Tn 554 was used to differentiate the SCC. A total of 60 of the 92 isolates were methicillin resistant. Among the 60 methicillin-resistant Staphylococcus spp. isolates, SCC mec ( mecA -carrying SCC) types I, III, IV and V were identified in 24 isolates based on the combinations of the ccr genes and the mec regulatory genes, with type III being predominant. The single S. epidermidis carried SCC mec type IV. SCC type III was also identified in two of 32 methicillin-susceptible isolates. Identical SCC mec types were present in different species of CoNS. Pulsed-field gel electrophoresis (PFGE) generated 64 patterns out of 81 PFGE typeable isolates. Indistinguishable clones were detected in animals from different farms.
Conclusions:  Heterogeneous SCC existed in CoNS of diverse genetic background. Both clonal transmission of methicillin-resistant CoNS and horizontal transfer of SCC mec occurred in the animal production environment.
Significance and Impact of the Study:  This study adds to our knowledge of SCC mec type and the diversity of SCC in CoNS.     

Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing

Aims:  To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), Sau -PCR and amplified fragment length polymorphism (AFLP).
Methods and Results:  Eighty-one isolates from bovine and caprine dairy products ( n  = 53) and from diseased rainbow trouts and other fishes ( n  = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84·4% to 97·5% and discriminatory powers from 0·798 to 0·986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau -PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes.
Conclusion:  L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks.
Significance and Impact of the Study:  L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau -PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.     

Characterization of an extremely heat-resistant Escherichia coli obtained from a beef processing facility

Aims:  This study aimed to determine the survival of Escherichia coli strains during steam and lactic acid decontamination interventions currently used by the beef‐processing industry, and to determine their heat resistance. Methods and Results:  Strains were grouped into cocktails of five strains each differing in their RAPD patterns for subsequent identification. Steam and lactic acid treatments on meat reduced cell counts of E. coli strain cocktails by 90–99%. The 20 slaughter plant isolates exhibited only minor variation in their resistance to steam and lactic acid treatments but were more resistant than reference strains (three strains) or isolates from live cattle (seven strains). D₆₀ values of strains from live cattle, and reference strains ranged from 0·1 to 0·5 min, in keeping with literature data. However, D₆₀ values of current slaughter plant isolates ranged between 15 for E. coli DM18.3 and 71 min AW 1.7. Cell counts of E. coli AW 1.7 were reduced by <5 log₁₀ CFU g^?1 in ground beef patties cooked to an internal temperature of 71°C. Conclusions:  Strains of E. coli that survive cooking of ground beef to the recommended internal temperature of 71°C can be isolated from beef‐processing facilities. Significance and Impact of the Study:  Pathogen interventions in current commercial beef slaughter may select for extremely heat‐resistant strains of E. coli.