New method to study bacterial adhesion to meat

A new method was developed for the study of bacterial adhesion to meat surfaces. Thin slices of meat (40 microns thick) were inserted into a specially designed observation chamber. The meat slices were then exposed to a bacterial suspension (ca. 10(6) CFU.ml-1) to initiate adhesion (20 min of contact time) and subsequently rinsed to eliminate nonadherent bacteria. Because of the special chamber design, the disruptive force exerted on the bacteria during rinsing (shear stress) was uniform over the whole surface of the meat slices, was constant, and could be varied from 0 to 0.08 N.m-2. After being rinsed, the meat slices were stained with basic fuschin and observed under light microscopy to determine the number and distribution of adherent bacteria. This new method was used to study the adhesion of Acinetobacter strain LD2, a Lactobacillus sp., and Pseudomonas fluorescens to slices of beef fat and tendon. At 25 degrees C, most (greater than or equal to 99.9%) of the cells of the Lactobacillus sp. deposited on the meat were washed off the surface during rinsing (0.05 N.m-2), whereas a large number (ca. 10(5) CFU.cm-2) of Acinetobacter strain LD2 and P. fluorescens cells remained adherent. The extent of adhesion was similar on fat and tendon, and adherent bacteria were distributed evenly over the whole surface of the slices. This preliminary study indicates that the combined use of thin slices of meat and of the observation chamber provides us with the means to more accurately study bacterial adhesion to meat surfaces.     

New method to study bacterial adhesion to meat.

A new method was developed for the study of bacterial adhesion to meat surfaces. Thin slices of meat (40 microns thick) were inserted into a specially designed observation chamber. The meat slices were then exposed to a bacterial suspension (ca. 10(6) CFU.ml-1) to initiate adhesion (20 min of contact time) and subsequently rinsed to eliminate nonadherent bacteria. Because of the special chamber design, the disruptive force exerted on the bacteria during rinsing (shear stress) was uniform over the whole surface of the meat slices, was constant, and could be varied from 0 to 0.08 N.m-2. After being rinsed, the meat slices were stained with basic fuschin and observed under light microscopy to determine the number and distribution of adherent bacteria. This new method was used to study the adhesion of Acinetobacter strain LD2, a Lactobacillus sp., and Pseudomonas fluorescens to slices of beef fat and tendon. At 25 degrees C, most (greater than or equal to 99.9%) of the cells of the Lactobacillus sp. deposited on the meat were washed off the surface during rinsing (0.05 N.m-2), whereas a large number (ca. 10(5) CFU.cm-2) of Acinetobacter strain LD2 and P. fluorescens cells remained adherent. The extent of adhesion was similar on fat and tendon, and adherent bacteria were distributed evenly over the whole surface of the slices. This preliminary study indicates that the combined use of thin slices of meat and of the observation chamber provides us with the means to more accurately study bacterial adhesion to meat surfaces.     

Bacterial communities of brown and red algae from Peter the Great Bay, the Sea of Japan

The structure of microbial communities of brown algae, red algae, and of the red alga Gracilaria verrucosa, healthy and affected with rotten thallus, were comparatively investigated; 61 strains of heterotrophic bacteria were isolated and characterized. Most of them were identified to the genus level, some Vibrio spp., to the species level according to their phenotypic properties and the fatty acid composition of cellular lipids. The composition of the microflora of two species of brown algae was different. In Chordaria flagelliformis, Pseudomonas spp. prevailed, and in Desmarestia viridis, Bacillus spp. The composition of the microflora of two red algae, G. verrucosa and Camphylaephora hyphaeoides, differed mainly in the ratio of prevailing groups of bacteria. The most abundant were bacteria of the CFB cluster and pseudoalteromonads. In addition, the following bacteria were found on the surface of the algae: Sulfitobacter spp., Halomonas spp., Acinetobacter sp., Planococcus sp., Arthrobacter sp., and Agromyces sp. From tissues of the affected G. verrucosa, only vibrios were isolated, both agarolytic and nonagarolytic. The existence of specific bacterial communities characteristic of different species of algae is suggested and the relation of Vibrio sp. to the pathological process in the tissues of G. verrucosa is supposed.     

Changes in the spoilage-related microbiota of beef during refrigerated storage under different packaging conditions

The microbial spoilage of beef was monitored during storage at 5 degrees C under three different conditions of modified-atmosphere packaging (MAP): (i) air (MAP1), (ii) 60% O2 and 40% CO2 (MAP2), and (iii) 20% O2 and 40% CO2 (MAP3). Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta, and lactic acid bacteria were monitored by viable counts and PCR-denaturing gradient gel electrophoresis (DGGE) analysis during 14 days of storage. Moreover, headspace gas composition, weight loss, and beef color change were also determined at each sampling time. Overall, MAP2 was shown to have the best protective effect, keeping the microbial loads and color change to acceptable levels in the first 7 days of refrigerated storage. The microbial colonies from the plate counts of each microbial group were identified by PCR-DGGE of the variable V6-V8 region of the 16S rRNA gene. Thirteen different genera and at least 17 different species were identified after sequencing of DGGE fragments that showed a wide diversity of spoilage-related bacteria taking turns during beef storage in the function of the packaging conditions. The countable species for each spoilage-related microbial group were different according to packaging conditions and times of storage. In fact, the DGGE profiles displayed significant changes during time and depending on the initial atmosphere used. The spoilage occurred between 7 and 14 days of storage, and the microbial species found in the spoiled meat varied according to the packaging conditions. Rahnella aquatilis, Rahnella spp., Pseudomonas spp., and Carnobacterium divergens were identified as acting during beef storage in air (MAP1). Pseudomonas spp. and Lactobacillus sakei were found in beef stored under MAP conditions with high oxygen content (MAP2), while Rahnella spp. and L. sakei were the main species found during storage using MAP3. The identification of the spoilage-related microbiota by molecular methods can help in the effective establishment of storage conditions for fresh meat.     

Effect of Packaging under Carbon Dioxide, Nitrogen or Air on the Microbial Flora of Pork Stored at 4°C

Changes in the microbial flora of pork packed in laminated plastic bags and stored at 4 °C were studied in an initial atmosphere of carbon dioxide, nitrogen or air. The time needed for the total aerobic count at 28 °C to reach 5 × 10⁶ organisms/cm² was about 7 times longer in carbon dioxide than in air, whilst in nitrogen it was about twice as long.
The predominant organisms on fresh pork taken directly from the processing line were: Acinetobacter calcoaceticus , non-fluorescent Pseudomonas spp. and Flavobacterium spp. After storage in air for 7 d, more than 90% of the flora consisted of non-fluorescent Pseudomonas spp. After storage in nitrogen for 10 d, 70% of the flora consisted of non-fluorescent Pseudomonas spp. with lower levels of fluorescent Pseudomonas spp., Kurthia zopfii, Aeromonas hydrophila and Lactobacillus plantarum. The non-fluorescent Pseudomonas spp. could be divided into three different groups, on proteolytic and lipolytic ability; the distribution of the groups was markedly different between pork loins stored in air and nitrogen.
On pork stored in carbon dioxide for 21 d the flora consisted of L. plantarum together with lower levels of heterofermentative lactic acid bacteria. When the storage time in carbon doxide was prolonged to 35 d, the proportion of heterofermentative lactic acid bacteria increased to about 50% of the flora.     

Effect of Hyperbaric Carbon Dioxide Pressure on the Microbial Flora of Pork Stored at 4 or 14°C

Changes in the microbial flora of pork stored at 4 or 14°C were studied in 5 atm CO₂, 1 atm CO₂ or 1 atm air. The time needed for the total aerobic count at 4°C to reach 5 × 10⁶ organisms/cm² was about three times longer in 5 atm CO₂ than in 1 atm CO₂, and about 15 times longer in 5 atm CO₂ than in air. At 14°C there was no difference in growth rate between 5 atm CO₂ and 1 atm CO₂. No off-odour was detected after storage in 5 atm CO₂ for 14 d, but the pork in 1 atm CO₂ (6 d) was organoleptically unacceptable.
The predominant organisms on the pork from the processing line were: Flavobacterium spp., Acinetobacter calcoaceticus, Pseudomonas spp., Micrococcus spp. and Moraxella spp. After aerobic storage at 4°C (8 d) or 14°C (3 d) more than 90% of the flora consisted of Pseudomonas spp. At 4°C all Pseudomonas spp. were of the non-fluorescent type, whilst at 14°C 32% were Ps. putida and Ps. fluorescens. After storage in 1 atm CO₂ Lactobacillus spp. represented 66% of the flora at 14°C (6 d) and 100% at 4°C (40 d), with L. xylosus dominating. After storage in 5 atm CO₂ Lactobacillus spp. constituted the total flora at both temperatures with L. lactis (14°C) and L. xylosus (4°C) dominating.
It was concluded that high partial pressures of CO₂ have a considerable shelf-life prolonging effect by (i) selecting the microflora towards Lactobacillus spp. and (ii) reducing the growth rate of these Lactobacillus spp. The controlling and growth inhibitory effect of CO₂ was promoted by reduced temperatures.     

Incidence of Acinetobacter spp. and other gram-negative, oxidase-negative bacteria in fresh and spoiled ground beef

A total of 1,409 gram-negative bacterial colonies were randomly selected from 19 samples of fresh and spoiled ground beef plated on six media. Only 137 (9.7%) were oxidase negative, and 20 (14.6%) of these were Acinetobacter spp., all of which were recovered from fresh meat samples. The importance of this group in both fresh and spoiled beef is less than is generally believed.     

Incidence of Acinetobacter spp. and other gram-negative, oxidase-negative bacteria in fresh and spoiled ground beef.

A total of 1,409 gram-negative bacterial colonies were randomly selected from 19 samples of fresh and spoiled ground beef plated on six media. Only 137 (9.7%) were oxidase negative, and 20 (14.6%) of these were Acinetobacter spp., all of which were recovered from fresh meat samples. The importance of this group in both fresh and spoiled beef is less than is generally believed.     

Ecophysiological attributes of a Lactobacillus sp. and a Pseudomonas sp. on sterile beef fillets in relation to storage temperature and film permeability

AIMS: To determine the combined effect of packaging film and temperature on the rate and type of end-products caused by the growth of two main contrasting prevailing organisms in air and 100% CO2, Pseudomonas sp. and Lactobacillus sp., respectively. METHODS AND RESULTS: Pseudomonas sp. and Lactobacillus sp. were inoculated individually on sterile meat fillets. The samples were packed in air or 100% CO2, using a high and a low permeable film, and stored at 0 and 10 degrees C. Pseudomonas sp. grew aerobically and in 100% CO2 using high permeable film at both storage temperatures, while film permeability significantly affected the growth of Lactobacillus sp. only at 10 degrees C. Enzymatic kits and HPLC and GC analysis were used to determine the chemical changes of the samples throughout storage. Pseudomonas sp. presented a greater rate of consumption of glucose and lactate than Lactobacillus sp. in samples stored aerobically or with high permeable film. Propanol-1 and two unidentified organic acids were present only in samples inoculated with Pseudomonas sp., while acetaldehyde, ethanol, diacetyl and acetoin were detected in samples inoculated with Lactobacillus sp. CONCLUSION: Since different microbial species and introduction of new packaging methods affect spoilage reactions of meat either qualitatively or quantitatively, a combination of several chemical indicators should be thoroughly investigated. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides information on how and when such potential indicators can be exploited for the benefit of the industry and consumer.     

The microbial flora of smoked pork loin and frankfurter sausage stored in different gas atmospheres at 4 degrees C

The development of the microflora of smoked pork loin and frankfurter sausage was followed during storage in vacuum, N2 and CO2 atmospheres at 4 degrees C. The total aerobic count on the smoked pork loin reached 10(7) organisms/g after 37 d in vacuum, 43 d in N2 and 49 d in CO2. The corresponding value for the sausage was 77 d in vacuum, while the growth stopped at 6 x 10(40 organisms/g after 98 d in N2, and at 4 x 10(2) organisms/g after 48 d in CO2. The predominant organisms on the fresh products were Bacillus spp., coryneform bacteria, Flavobacterium spp. and Pseudomonas spp. At the end of the storage time the microflora on both products in the three gas atmospheres, consisted mainly of Lactobacillus spp. and two large groups of organisms that could not be identified as any described genus. Some of the unidentified strains could be classified as a Lactobacillus sp. after subsequent subculturing on laboratory media. The numbers of Lactobacillus spp. at the end of storage decreased in the order, CO2 greater than N2 greater than vacuum. Lactobacillus viridescens generally constituted a substantial part of the Lactobacillus flora (5-72%). On the sausages two large uniform groups of unidentifiable homofermentative Lactobacillus spp. were also found.     

Allogenic succession of marine bacterial communities in response to pharmaceutical waste

Vibrio spp. predominated in the culturable bacterial community of surface waters of the Puerto Rico Trench at the site of disposal for nearly ten years of pharmaceutical wastes. In this area and surrounding waters as far as 1000 km north of the dumpsite and south into the Caribbean Sea, Vibrio spp. comprised up to 100% of the culturable bacteria, with Acinetobacter spp. being the second most prevalent group. Pseudomonas spp., reported to be common in these waters a decade earlier, were virtually absent from all samples examined during a three year study involving 9 cruises. Staphylococcus spp. were also found in water samples collected within the dumpsite. Using cultures isolated from surface water samples collected at the dumpsite, laboratory experiments confirmed that pharmaceutical waste can enrich for Vibrio spp., in preference to Pseudomonas spp., with growth of the strains proportional to the amount of waste added.     

Allogenic succession of marine bacterial communities in response to pharmaceutical waste

Vibrio spp. predominated in the culturable bacterial community of surface waters of the Puerto Rico Trench at the site of disposal for nearly ten years of pharmaceutical wastes. In this area and surrounding waters as far as 1000 km north of the dumpsite and south into the Caribbean Sea, Vibrio spp. comprised up to 100% of the culturable bacteria, with Acinetobacter spp. being the second most prevalent group. Pseudomonas spp., reported to be common in these waters a decade earlier, were virtually absent from all samples examined during a three year study involving 9 cruises. Staphylococcus spp. were also found in water samples collected within the dumpsite. Using cultures isolated from surface water samples collected at the dumpsite, laboratory experiments confirmed that pharmaceutical waste can enrich for Vibrio spp., in preference to Pseudomonas spp., with growth of the strains proportional to the amount of waste added.     

Natural transformation of Acinetobacter sp. strain BD413 with cell lysates of Acinetobacter sp., Pseudomonas fluorescens, and Burkholderia cepacia in soil microcosms

To elucidate the biological significance of dead bacterial cells in soil to the intra- and interspecies transfer of gene fragments by natural transformation, we have exposed the kanamycin-sensitive recipient Acinetobacter sp. strain BD413(pFG4) to lysates of the kanamycin-resistant donor bacteria Acinetobacter spp., Pseudomonas fluorescens, and Burkholderia cepacia. Detection of gene transfer was facilitated by the recombinational repair of a partially (317 bp) deleted kanamycin resistance gene in the recipient bacterium. The investigation revealed a significant potential of these DNA sources to transform Acinetobacter spp. residing both in sterile and in nonsterile silt loam soil. Heat-treated (80 degrees C, 15 min) cell lysates were capable of transforming strain BD413 after 4 days of incubation in sterile soil and for up to 8 h in nonsterile soil. Transformation efficiencies obtained in vitro and in situ with the various lysates were similar to or exceeded those obtained with conventionally purified DNA. The presence of cell debris did not inhibit transformation in soil, and the debris may protect DNA from rapid biological inactivation. Natural transformation thus provides Acinetobacter spp. with an efficient mechanism to access genetic information from different bacterial species in soil. The relatively short-term biological activity (e.g., transforming activity) of chromosomal DNA in soil contrasts the earlier reported long-term physical stability of DNA, where fractions have been found to persist for several weeks in soil. Thus, there seems to be a clear difference between the physical and the functional significance of chromosomal DNA in soil.     

Taxonomic composition of bacteria associated with cultivated mollusks Crassostrea lugubris and Perna viridis and with the water of the Gulf of Nha Trang Lagoon, Vietnam

One hundred and four strains of heterotrophic bacteria have been isolated and characterized from two species of bivalve mollusks cultivated in the Gulf of Nha Trang (Vietnam) and from the water of a mariculture farm. The isolates have been identified on the basis of morphological, physiological, biochemical, and chemotaxonomic properties, as well as by the content of G+C bases in DNA. In the microflora of mollusks, Vibrio alginolyticus was predominant; the pathogenic species V. harveyi and V. splendidus were found as well. Staphylococci and bacilli occupied the second place in abundance after vibrios. In addition, coryneforms and enterobacteria, as well as Pseudomonas spp. and Pseudoalteromonas spp., were revealed. The composition of the water microflora was more diverse as compared with the microflora of mollusks. In the water, Bacillus spp., Vibrio spp., and Pseudomonas spp. were predominant. Brevibacterium spp. and other coryneform bacteria, as well as enterobacteria, occurred in significant amounts. In addition, Pseudoalteromonas spp., Marinococcus sp., Halobacillus sp., Shewanella sp., Sulfitobacter sp., and bacteria of the CFB cluster were noticed. The presence of pathogenic and conditionally pathogenic bacterial species in the water and mollusks is probably the reason for the high death rate of cultivated animals at the mariculture farm.     

A Note on the Microflora of Beef Muscle Stored in Nitrogen at 0°

Fresh beef stored in nitrogen at 0° was spoiled by Gram positive rods. Almost all the organisms isolated were Microbacterium spp., although in one experiment about 10% of the organisms isolated were Lactobacillus spp. The numbers and types of organisms isolated were similar on each of two media incubated under aerobic and anaerobic conditions.     

Isolation and characterization of acyl homoserine lactone–producing bacteria during an urban river biofilm formation

The presence and diversity of acyl homoserine lactone (AHL)-producers in an urban river biofilm were investigated during 60-day biofilm formation. AHL biosensors detected the presence of AHL-producers in 1–60-day river biofilms. Screening for AHL-producers resulted in 17 Aeromonas spp., 3 Pseudomonas spp., 3 Ensifer spp., and 1 Acinetobacter sp. Among these isolates, six of them were closely related to Acinetobacter tjernbergiae, Aeromonas allosaccharophila, Aeromonas aquariorum, Aeromonas jandaei, Pseudomonas panipatensis, and Ensifer adhaerens and represented novel AHL-producing species. Thin layer chromatography revealed that C4-homoserine lactone was prevailing in Aeromonas spp., whereas C6- and C8-homoserine lactones and their derivatives were prevailing in other strains. Using degenerate primers, novel AHL synthetase genes from the three Ensifer spp. were successfully amplified. This study reports for the first time the diversity of AHL-producers from a river biofilm and the variety of novel AHL synthetase genes in Ensifer group.     

Genes involved in aniline degradation by Delftia acidovorans strain 7N and its distribution in the natural environment

Aniline-degraders were isolated from activated sludge and environmental samples and classified into eight phylogenetic groups. Seven groups were classified into Gram-negative bacteria, such as Acidovorax sp., Acinetobacter sp., Delftia sp., Comamonas sp., and Pseudomonas sp., suggesting the possible dominance of Gram-negative aniline-degraders in the environment. Aniline degradative genes were cloned from D. acidovorans strain 7N, and the nucleotide sequence of the 8,039-bp fragment containing eight open reading frames was determined. Their deduced amino acid sequences showed homologies to glutamine synthetase (GS)-like protein, glutamine amidotransferase (GA)-like protein, large and small subunits of aniline dioxygenase, reductase, LysR-type regulator, small ferredoxin-like protein, and catechol 2,3-dioxygenase, suggesting a high similarity of this gene cluster to those in P. putida strain UCC22 and Acinetobacter sp. strain YAA. Polymerase chain reaction (PCR) and sequencing analyses of GS-like protein gene segments of other Gram-negative bacteria suggested that Gram-negative bacteria have aniline degradative gene that can be divided into two distinctive groups.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coli-form bacteria on three occasions during 1983. Eighty percent or more of the coli-form bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherchia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 μg/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.     

The microbial flora of smoked pork loin and frankfurter sausage stored in different gas atmospheres at 4°C

The development of the microflora of smoked pork loin and frankfurter sausage was followed during storage in vacuum, N₂ and CO₂ atmospheres at 4°C. The total aerobic count on the smoked pork loin reached 10⁷ organisms/g after 37 d in vacuum, 43 d in N₂ and 49 d in CO₂. The corresponding value for the sausage was 77 d in vacuum, while the growth stopped at 6 times 10⁴ organisms/g after 98 d in N₂, and at 4 times 10² organisms/g after 48 d in CO².
The predominant organisms on the fresh products were Bacillus spp., coryneform bacteria, Flavobacterium spp. and Pseudomonas spp.
At the end of the storage time the microflora on both products in the three gas atmospheres, consisted mainly of Lactobacillus spp. and two large groups of organisms that could not be identified as any described genus. Some of the unidentified strains could be classified as a Lactobacillus sp. after subsequent subculturing on laboratory media.
The numbers of Lactobacillus spp. at the end of storage decreased in the order, CO₂ > N₂ > vacuum. Lactobacillus viridescens generally constituted a substantial part of the Lactobacillus flora (5–72%). On the sausages two large uniform groups of unidentifiable homofermentative Lactobacillus spp. were also found.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coliform bacteria on three occasions during 1983. Eighty percent or more of the coliform bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherichia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 micrograms/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.