Joanna Koort

Role of Broiler Carcasses and Processing Plant Air in Contamination of Modified-Atmosphere-Packaged Broiler Products with Psychrotrophic Lactic Acid Bacteria

ABSTRACT Some psychrotrophic lactic acid bacteria (LAB) are specific meat spoilage organisms in modified-atmosphere-packaged (MAP), cold-stored meat products. To determine if incoming broilers or the production plant environment is a source of spoilage LAB, a total of 86, 122, and 447 LAB isolates from broiler carcasses, production plant air, and MAP broiler products, respectively, were characterized using a library of HindIII restriction fragment length polymorphism (RFLP) patterns of the 16 and 23S rRNA genes as operational taxonomic units in numerical analyses. Six hundred thirteen LAB isolates from the total of 655 clustered in 29 groups considered to be species specific. Sixty-four percent of product isolates clustered either with Carnobacterium divergens or with Carnobacterium maltaromaticum type strains. The third major product-associated cluster (17% of isolates) was formed by unknown LAB. Representative strains from these three clusters were analyzed for the phylogeny of their 16S rRNA genes. This analysis verified that the two largest RFLP clusters consisted of carnobacteria and showed that the unknown LAB group consisted of Lactococcus spp. No product-associated LAB were detected in broiler carcasses sampled at the beginning of slaughter, whereas carnobacteria and lactococci, along with some other specific meat spoilage LAB, were recovered from processing plant air at many sites. This study reveals that incoming broiler chickens are not major sources of psychrotrophic spoilage LAB, whereas the detection of these organisms from the air of the processing environment highlights the role of processing facilities as sources of LAB contamination.

Leuconostoc gelidum and Leuconostoc gasicomitatum strains dominated the lactic acid bacterium population associated with strong slime formation in an acetic-acid herring preserve.

Spoilage characterised by strong slime and gas formation affected some manufacture lots of an acetic-acid Baltic herring (Culpea haerengus membras) preserve after few weeks of storage at 0-6 degrees C. The product consisted of herring filets in acetic acid marinade containing sugar, salt, allspice and carrot slices. Microbiological analyses of the spoiled product showed high lactic acid bacterium (LAB) levels ranging from 4.5x10(8) to 2.4x10(9) CFU/g. Yeasts were not detected in any of the herring samples. Since LAB contaminants are seldom associated with fresh fish, LAB populations associated with marinade ingredients (carrots, allspice) were also analyzed. The highest LAB levels exceeding 10(7) CFU/g were detected in equilibrium modified atmosphere packaged baby carrots whereas the levels detected in the allspice samples did not exceed 4.3x10(5). A total of 176 randomly selected LAB isolates originating from herring, carrot and allspice samples were further identified to species level using a 16 and 23S rRNA gene RFLP (ribotyping) database. Leuconostoc gelidum and Leuconostoc gasicomitatum strains dominated both in the spoiled herring and carrot samples. These species are heterofermentative-producing CO(2) from glucose and they also produce dextran from sucrose. Inoculation of some commercial-herring products with spoilage-associated L. gelidum and L. gasicomitatum strains verified that these strains have the capability of producing slime and gas in herring preserves although slime formation was not as strong as in the original samples. Since L. gelidum and L. gasicomitatum strains were commonly detected in carrots, carrot slices used for the fish marinade were considered to be the probable source of these specific spoilage organisms.

Probiotic properties of Lactobacillus isolates originating from porcine intestine and feces.

In this study, a total of 94 lactic acid bacterial (LAB) isolates of porcine small intestinal and fecal origin were screened for their probiotic properties. The aim was to evaluate whether their isolation site and putative species identity play a role in these characteristics and whether either of these can be used as a predictive factor for the probiotic potential of bacterial isolates. The isolates were preliminarily identified by partial 16S rRNA gene sequencing and characterized in vitro for their pH and bile tolerance, adhesion capacity towards porcine enterocytes isolated from five intestinal sites and for antimicrobial activity towards five indicator pathogens. The interdependence of these characteristics was statistically evaluated. The isolates tolerated low pH and bile well. Adherence to the enterocytes of different origins did not correlate with the strain isolation site. In general, higher adherence was observed to colon cells in comparison to the small intestinal enterocytes. Culture filtrates of the isolates caused a decrease of up to three orders of magnitude in the intestinal pathogen cell numbers. The inhibition was mostly due to lactic and other organic acids. The predominating phylotypes identified were Lactobacillus reuteri and Lactobacillus salivarius, of which the former generally had the best adhesion capacity, whereas the latter appeared to be the best inhibitor. Based on the results, several strains of the pig Lactobacillus isolates tested may function as promising candidates for use in probiotic products. However, it was not possible to use the isolation site or the species identity of the isolates as reliable preliminary screening factors.

Lactobacillus oligofermentans sp. nov., Associated with Spoilage of Modified-Atmosphere-Packaged Poultry Products

ABSTRACT Unidentified lactic acid bacterium (LAB) isolates which had mainly been detected in spoiled, marinated, modified atmosphere packaged (MAP) broiler meat products during two previous studies, were identified and analyzed for their phenotypic properties and the capability to produce biogenic amines. To establish the taxonomic position of these isolates, 16S rRNA gene sequence analysis, numerical analysis of ribopatterns, and DNA-DNA hybridization experiments were done. Unexpectedly for a meat-spoilage-associated LAB, the strains utilized glucose very weakly. According to the API 50 CHL test, arabinose and xylose were the only carbohydrates strongly fermented. None of the six strains tested for production of histamine, tyramine, tryptamine, phenylethylamine, putrescine, and cadaverine were able to produce these main meat-associated biogenic amines in vitro. The polyphasic taxonomy approach showed that these strains represent a new Lactobacillus species. The six isolates sequenced for the 16S rRNA encoding genes shared the highest similarity (95.0 to 96.3%) with the sequence of the Lactobacillus durianis type strain. In the phylogenetic tree, these isolates formed a distinct cluster within the Lactobacillus reuteri group, which also includes L. durianis. Numerical analyses of HindIII-EcoRI ribotypes placed all isolates together in a cluster with seven subclusters well separated from the L. reuteri group reference strains. The DNA-DNA hybridization levels between Lactobacillus sp. nov. isolates varied from 67 to 96%, and low hybridization levels (3 to 15%) were obtained with the L. durianis type strain confirming that these isolates belong to the same species different from L. durianis. The name Lactobacillus oligofermentans sp. nov. is proposed, with strain LMG 22743T (also known as DSM 15707T or AMKR18T) as the type strain.

Staphylococcus agnetis sp nov., a coagulase-variable species from bovine subclinical and mild clinical mastitis

Thirteen Gram-positive-staining coagulase-variable staphylococci were isolated from subclinical and mild clinical mastitic bovine milk (n=12) and a teat apex (n=1). The results of sequence analysis of the 16S rRNA gene and two housekeeping genes, rpoB and tuf, and DNA fingerprinting with amplified fragment length polymorphism (AFLP) analysis showed that the isolates formed a separate branch within the genus Staphylococcus. The phylogenetically most closely related species were Staphylococcus hyicus and Staphylococcus chromogenes. DNA-DNA hybridization with S. hyicus DSM 20459(T) and S. chromogenes DSM 20674(T) confirmed that the isolates belonged to a separate species. The predominant fatty acids were i-C(15:0), ai-C(15:0), i-C(17:0) and C(20:0) and the peptidoglycan type was A3α L-Lys-Gly(5). Based on the results of genotypic and phenotypic analyses, it is proposed that the thirteen isolates represent a novel species, for which the name Staphylococcus agnetis sp. nov. is proposed. Strain 6-4(T) (=DSM 23656(T)=CCUG 59809(T)) is the type strain.

Streptococcus alactolyticus is the dominating culturable lactic acid bacterium species in canine jejunum and feces of four fistulated dogs.

Canine intestinal lactic acid bacterium (LAB) population in four fistulated dogs was cultured and enumerated using MRS agar. LAB levels ranging from 1.4x10(6) to 1.5x10(7) CFU ml(-1) were obtained in jejunal chyme. In the fecal samples 7.0x10(7) and 2.0x10(8) CFU g(-1) were detected. Thirty randomly selected isolates growing in the highest sample dilutions were identified to species level using numerical analysis of 16S and 23S rDNA restriction fragment length polymorphism patterns (ribotyping) and 16S rDNA sequence analysis. According to these results, Streptococcus alactolyticus was the dominant culturable LAB species in both feces and jejunal chyme. In addition, Lactobacillus murinus and Lactobacillus reuteri were detected.

Microbial changes and growth of Listeria monocytogenes during chilled storage of brined shrimp (Pandalus borealis)

Thirteen storage trials and ten challenge tests were carried out to examine microbial changes, spoilage and the potential growth of Listeria monocytogenes in brined shrimp (Pandalus borealis). Shrimp in brine as well as brined and drained shrimp in modified atmosphere packaging (MAP) were produced and studied. Different recipes were used to study the effect of preserving parameters (organic acids, pH and NaCl) on growth of microorganisms and shelf life at 7-8 degrees C or 12 degrees C. Particularly, brines with different concentrations of (i) benzoic, citric and sorbic acids or (ii) acetic, citric and lactic acids were studied. Furthermore, the effect of adding diacetate to brined shrimp was evaluated. A single batch of cooked and peeled shrimp was used to study both industrially and manually processed brined shrimp with respect to the effect of process hygiene on microbial changes and the shelf life of products. Concentrations of microorganisms on newly produced brined shrimp from an industrial scale processing line were 1.0-2.3 log (CFU g(-1)) higher than comparable concentrations in manually processed samples. This resulted in a substantially shorter shelf life and a more diverse spoilage microflora of the industrially processed brined shrimp. In addition, shelf life of brined shrimp was affected by the types and concentrations of organic acids and by the storage temperature as expected. The effect of MAP was less pronounced. Eighty-two isolates from the spoilage microflora of brined shrimp were identified and they included 53 lactic acid bacteria, 6 coagulase negative Staphylococcus spp., 18 Pseudomonas fluorescens and 5 yeast isolates. After storage at 7 degrees C, P. fluorescens, Enterococcus-like isolates, E. malodoratus, Carnobacterium maltaromaticum, coagulase negative Staphylococcus spp. and Lactobacillus sakei constituted the dominating microflora of shrimp in brines that contained benzoic, citric and sorbic acids as preservatives. L. sakei dominated the spoilage microflora of brined and drained MAP shrimp, and of brined shrimp preserved using acetic, citric and lactic acids, irrespective of packaging conditions. Shrimp in brine with benzoic, citric and sorbic acids prevented growth of L. monocytogenes during more than 40 days at 7 degrees C when the preserving parameters resembled those of commercial products. However, small changes in the preserving parameters and, particularly, reduced concentrations of benzoic acid led to growth of L. monocytogenes in brined shrimp. The present study provides significant new information on microbial changes, shelf life and growth of L. monocytogenes in brined shrimp. This information can facilitate development of new and safe brined shrimp products.

Identification and Antimicrobial Resistance of Streptococcus uberis and Streptococcus parauberis Isolated from Bovine Milk Samples

The conventional identification of Streptococcus uberis/parauberis group (n = 137) in clinical and subclinical bovine mastitis samples originating from 111 different farms was compared with identification based on 16 and 23S rRNA gene HindIII RFLP patterns used as operational taxonomic units in numerical analyses. On the basis of ribopattern analysis only 2 isolates belonged to S. parauberis, which is thus not a frequent cause of bovine intramammary infections in Finland. According to in vitro antimicrobial susceptibility testing, Streptococcus uberis is susceptible to beta-lactam antibiotics. The prevalence of erythromycin (15.6%) and oxytetracycline (40.6%) resistance of clinical S. uberis isolates was higher than reported previously among subclinical isolates. The 2 subclinical S. parauberis isolates were susceptible to all the antimicrobials tested.

Bovine-associated CNS species resist phagocytosis differently

BackgroundCoagulase-negative staphylococci (CNS) cause usually subclinical or mild clinical bovine mastitis, which often remains persistent. Symptoms are usually mild, mostly only comprising slight changes in the appearance of milk and possibly slight swelling. However, clinical mastitis with severe signs has also been reported. The reasons for the differences in clinical expression are largely unknown. Macrophages play an important role in the innate immunity of the udder. This study examined phagocytosis and killing by mouse macrophage cells of three CNS species: Staphylococcus chromogenes (15 isolates), Staphylococcus agnetis (6 isolates) and Staphylococcus simulans (15 isolates). Staphylococcus aureus (7 isolates) was also included as a control.ResultsAll the studied CNS species were phagocytosed by macrophages, but S. simulans resisted phagocytosis more effectively than the other CNS species. Only S. chromogenes was substantially killed by macrophages. Significant variations between isolates were seen in both phagocytosis and killing by macrophages and were more common in the killing assays. Significant differences between single CNS species and S. aureus were observed in both assays.ConclusionThis study demonstrated that differences in the phagocytosis and killing of mastitis-causing staphylococci by macrophages exist at both the species and isolate level.

Genetic basis of penicillin resistance of S. aureus isolated in bovine mastitis

BackgroundThe blaZ gene encoding penicillin resistance can be located either chromosomally or on plasmids. The aim of this study was to investigate the genetic relationships and to determine the location of the blaZ gene in S. aureus isolated in bovine mastitis in Finland and Sweden.MethodsSeventy-eight β-lactamase positive S. aureus isolates from bovine mastitis (34 from Finland and 44 from Sweden) were included in the study. The localization of blaZ gene was determined by Southern blotting. The blaZ genes of the isolates were sequenced and the sequences were translated to beta-lactamase proteins and further grouped as different protein signatures. The isolates and, as control, 33 Swedish and 36 Finnish beta-lactamase negative isolates were typed with pulsed-field gel electrophoresis (PFGE).ResultsIn 26 out of 34 Finnish isolates (76.5%) and in 25 out of 44 Swedish isolates (56.8%) the blaZ gene was localized on a plasmid. Six different protein signatures were found. One signature was found only in four Swedish isolates, but all other signatures were found both in Finnish and Swedish isolates. The PFGE results revealed a diversity of S. aureus clones. The protein signatures were not clearly associated with certain pulsotypes.ConclusionsThe plasmid location of the blaZ gene was not statistically significantly more common in Finland than in Sweden, and hence does not explain the higher proportion of penicillin-resistant isolates of S. aureus causing bovine mastitis in Finland compared to Sweden.