Polyphasic Taxonomic Studies of Lactic Acid Bacteria Associated with Non-fermented Meats

Systematiikka on tieteenala, joka tutkii eliöiden, esimerkiksi bakteereiden, sukulaisuussuhteita, määrittelyä ja luokittelua. Taksonomian voidaan ajatella olevan tämän tieteenalan käytännön sovellus: taksonomian tavoitteena on jakaa eliöt niiden sukulaisuussuhteita kuvaaviin, järkeviin ryhmiin, antaa jokaiselle ryhmälle sitä kuvaava nimi sekä löytää ne ryhmän ominaispiirteet, joiden avulla kyseinen ryhmä voidaan jatkossakin vaikeuksitta tunnistaa uudelleen. Taksonomia siis tarjoaa tutkijoille yhteisen kielen, jotta kaikki puhuisivat asioista niiden "oikeilla nimillä" ja vältyttäisiin väärinkäsityksiltä. Taksonomisten joukkojen perusyksikkö on laji. Suvullisesti lisääntyvillä eliöillä voidaan ajatella, että samaan lajiin kuuluvat ne eliöt, jotka pystyisivät keskenään tuottamaan lisääntymiskykyisiä jälkeläisiä. Asia tosin ei läheskään aina ole näin yksinkertainen. Suvullisen lisääntymisen puuttuessa bakteereiden perhesuhteita on vieläkin vaikeampi määritellä, ja siksi tiedon lisääntyessä aiempaan luokitukseen tulee jatkuvasti muutoksia. Jotta luokittelusta saataisiin mahdollisimman pysyvä ja pitkäkestoinen, on bakteereiden luokittelussa ryhdytty käyttämään niin sanottua polyfaasista (monivaiheista) taksonomiaa, menetelmää, jossa bakteerijoukon ominaisuuksia tutkitaan mahdollisimman monella tavalla ja lopuksi yhdistetään kaikki saatu tieto. Tässä tutkimuksessa polyfaasista taksonomiaa käytettiin hyväksi viiden broilerinlihavalmistesiin liittyvän maitohappobakteeriryhmän tunnistuksessa. Samalla arvioitiin polyfaasisen taksonomian soveltuvuutta maitohappobakteereiden luokittelussa. Tutkimuksessa käytettiin sekä fenotyyppisä (ilmiasuun perustuvia), genotyyppisiä (perimään perustuvia) että fylogeneettisiä (kehityshistoriaan eli evoluutioon perustuvia) menetelmiä. Lopuksi kunkin bakteeriryhmän tulokset koottiin yhteen. Useissa aiemmissa tutkimuksissa oli saatu viitteitä siitä, että Lactobacillus curvatus ja Lactobacillus sakei -lajien taksonomiassa on epäselvyyttä. Tämän tutkimuksen tulosten perusteella luokittelua korjattiin siirtämällä Lactobacillus curvatus- bakteerin melibiosus- alalaji osaksi Lactobacillus sakei- lajia, sen alalajiin carnosus. Polyfaasista lähestymistapaa sovellettiin myös neljän tuntemattoman bakteeriryhmän tunnistamisessa. Tulosten perusteella havaittiin että kaksi näistä, kokkimainen koiran nielurisoista ja suojakaasupakatusta broilerista eristetty ryhmä ja sauvamainen, pääosin pilaantuneesta, marinoimattomasta, suojakaasupakatusta broilerinlihavalmisteesta eristetty ryhmä, oli aiemmin kuvaamattomia lajeja. Molemmat uudet lajit kuvattiin, eli niille annettiin nimet ja niiden ominaisuudet listattiin. Kokkibakteeri sai nimekseen Enterococcus hermanniensis, eläinlääketieteellisen tiedekunnan Helsingin Hermannin kaupunginosassa sijainneen vanhan kampuksen mukaan, ja sauvabakteeri Lactobacillus oligofermentans, sen energiankäytön mukaan (bakteeri pystyy käyttämään eli fermentoimaan vain harvoja = oligo sokereita). Kaksi muuta ryhmää sen sijaan tunnistettiin jo olemassa oleviksi lajeiksi. Kokkimainen bakteeri, joka oli eristetty suojakaasupakatusta, marinoidusta ja marinoimattomasta broilerin koipireisivalmisteesta ja broilerinlihanleikkaamon ilmasta tunnistettiin Streptococcus parauberis- bakteeriksi. Osalla näistä bakteereista oli kuitenkin kaksi ominaisuutta, joiden perusteella ne erosivat muista samaan lajiin kuuluvista kannoista: ne eivät kyenneet hyödyntämään laktoosia energianlähteenään, ja galaktoosiakin vain huonosti. Poikkeuksellisiksi nämä ominaisuudet tekee se, että tätä lajia on pidetty lähinnä lehmän utaretulehdusta aiheuttavana bakteerina, ja siten olisi ollut odotettavissa että kaikki sen jäsenet pystyisivät käyttämään laktoosia ja galaktoosia, maidon yleisimpiä energianlähteitä. Toinen, sauvamainen, espanjalaisesta, vakuumi- ja suojakaasupakatusta "Morcilla de Burgos" verimakkarasta eristetty ryhmä tunnistettiin Weissella viridescens- bakteeriksi. Polyfaasinen lähestymistapa sopi hyvin näille maitohappobakteereille. Yksittäisten testien perusteella ei kestävää lajimääritystä olisi saatu aikaan. Vaikka fenotyyppisten testien tulokset voivat ollakin kirjavia, ne kuuluvat kuitenkin uuden lajin kuvaukseen uusien genotyyppisten menetelmien rinnalle. Tutkimuksessa havaittiin myös, että erilaisten testien tuloksista luodut kirjastot ovat luotettavia apuvälineitä bakteereiden tunnistuksessa, kunhan niitä päivitetään jatkuvasti

Redesigning and teaching veterinary microbiology laboratory exercises with combined on-site and online participation during the COVID-19 pandemic

The COVID-19 (coronavirus disease 2019) pandemic has forced universities to find new ways to conduct learning and teaching, as traditional face-to-face teaching has been prevented or restricted to an absolute minimum in many instances. Therefore, we redesigned and taught second-year veterinary student microbiology laboratory exercises (labs) with a hybrid learning approach. For this, a novel 'remote partner' model was implemented in which students present on-site in the laboratory worked synchronously pairwise with their remote partner present online. A student feedback survey revealed that in this remote partner model, both on-site and online participation in the labs were experienced as being useful in improving their laboratory skills. The students' overall performance in hands-on microbiological laboratory skills and safe working practices was similar in the hybrid learning approach (the 2021 class) and in the traditional on-site participation approach (the 2018-20 classes). This study suggests that the remote partner model is an effective way to acquire microbiological laboratory skills. This learning approach can be used in the non-pandemic future and/or also be applied to other fields.Microbiological laboratory skills can be developed using a hybrid model combining synchronous on-site and online learning for students working in pairs.Peer reviewe

Genomic Analysis of Staphylococcus aureus Isolates Associated With Peracute Non-gangrenous or Gangrenous Mastitis and Comparison With Other Mastitis-Associated Staphylococcus aureus Isolates

Staphylococcus aureus is a highly prevalent cause of mastitis in dairy herds worldwide, capable of causing outcomes that vary from subclinical to peracute gangrenous mastitis. We performed a comparative genomic analysis between 14 isolates of S. aureus, originating from peracute bovine mastitis with very severe signs (9 gangrenous, 5 non-gangrenous) and six isolates originating from subclinical or clinical mastitis with mild to moderate signs, to find differences that could be associated with the clinical outcome of mastitis. Of the 296 virulence factors studied, 219 were detected in all isolates. No difference in the presence of virulence genes was detected between the peracute and control groups. None of the virulence factors were significantly associated with only a single study group. Most of the variation in virulence gene profiles existed between the clonal complexes. Our isolates belonged to five clonal complexes (CC97, CC133, CC151, CC479, and CC522), of which CC522 has previously been detected only in isolates originating from caprine and ovine mastitis, but not from bovine mastitis. For statistical analysis, we sorted the CCs into two groups. The group of CCs including CC133, CC479, and CC522 was associated with gangrenous mastitis, in contrast to the group of CCs including CC97 and CC151. The presence of virulence genes does not explain the clinical outcome of mastitis, but may be affected by allelic variation, and especially different regulation and thus expression in the virulence genes.Peer reviewe

Streptococcus parauberis associated with modified atmosphere packaged broiler meat products and air samples from a poultry meat processing plant

http://www.elsevier.com/locate/issn/01681605Lactic acid bacteria (LAB) isolated from marinated or non-marinated, modified atmosphere packaged (MAP) broiler leg products and air samples of a large-scale broiler meat processing plant were identified and analyzed for their phenotypic properties. Previously, these strains had been found to be coccal LAB. However, the use of a 16 and 23S rRNA gene RFLP database had not resulted in species identification because none of the typically meat-associated LAB type strains had clustered together with these strains in the numerical analysis of the RFLP patterns. To establish the taxonomic position of these isolates, 16S rRNA gene sequence analysis, numerical analysis of ribopatterns, and DNA–DNA hybridization experiments were done. The 16S rRNA gene sequences of three isolates possessed the highest similarities (over 99%) with the sequence of S. parauberis type strain. However, in the numerical analysis of HindIII ribopatterns, the type strain did not cluster together with these isolates. Reassociation values between S. parauberis type or reference strain and the strains studied varied from 82 to 97%, confirming that these strains belong to S. parauberis. Unexpectedly, most of the broiler meat-originating strains studied for their phenotypical properties did not utilize lactose at all and the same strains fermented also galactose very weakly, properties considered atypical for S. parauberis. This is, to our knowledge, the first report of lactose negative S. parauberis strains and also the first report associating S. parauberis with broiler slaughter and meat products

Developing microbial spoilage population in vacuum-packaged charcoal-broiled European river lamprey (Lampetra fluviatilis)

http://www.elsevier.com/locate/issn/01681605Microbiological and sensory changes in vacuum-packaged charcoal-broiled river lampreys from three lamprey processing plants were monitored as a function of time at 8°C. The lampreys were examined every 7 days up to 8 weeks for aerobic plate count (APC) and lactic acid bacteria (LAB). The highest mean APC and LAB were 6.01 log CFU/g and 4.86 log CFU/g, respectively. Only 6 out of 15 lots reached an APC value of 7.0 log CFU/g during storage. The sensory scores remained at the baseline levels after 8 weeks´ storage. Twenty-seven isolates were randomly picked from MRS agar and identified to species level using a 16S and 23S rDNA HindIII RFLP (ribotyping) database and sequencing of the 16S rRNA gene if no database match was obtained. Twelve of the 27 isolates were identified as Lactobacillus curvatus subsp. curvatus, and two Leuconostoc mesenteroides and one Weissella halotolerans strain were also detected. Twelve isolates were not identified by the LAB database. However, they possessed very high (99.9%) 16S gene sequence similarity with either Staphylococcus warneri or Staphylococcus pasteuri type strains. The LAB detected, with the exception of W. halotolerans, have commonly been associated with spoilage of fishery products, but in these vacuum-packaged lampreys they were not the dominant organisms within the developing spoilage population

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

http://www.elsevier.com/locate/issn/03781097Canine intestinal lactic acid bacterium (LAB) population in four fistulated dogs was cultured and enumerated using MRS agar. LAB levels ranging from 1.4×106 to 1.5×107 CFU ml-1 were obtained in jejunal chyme. In the fecal samples 7.0×107 and 2.0×108 CFU g-1 were detected. Thirty randomly selected isolates growing in the highest sample dilutions were identified to species level using numerical analysis of 16 and 23 S rDNA RFLP patterns (ribotyping) and 16S rDNA sequence analysis. According to these results, Streptococcus alactolyticus was the dominant culturable LAB species in both faeces and jejunal chyme. In addition, Lactobacillus murinus and Lactobacillus reuteri were detected

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

http://www.elsevier.com/locate/issn/01681605Spoilage characterised by strong slime and gas formation affected some manufacture lots of 2 an acetic-acid Baltic herring (Culpea haerengus membras) preserve after few weeks’ storage 3 at 0-6°C. The product consisted of herring filets in acetic acid marinade containing sugar, 4 salt, allspice and carrot slices. Microbiological analyses of the spoiled product showed high 5 lactic acid bacterium (LAB) levels ranging from 4.5 ×108 to 2.4 × 109 CFU/g. Yeasts were 6 not detected in any of the herring samples. Since LAB contaminants seldom are associated 7 with fresh fish, LAB populations associated with marinade ingredients (carrots, allspice) 8 were also analysed. The highest LAB levels exceeding 107 CFU/g were detected in 9 equilibrium modified atmosphere packaged baby carrots whereas the levels detected in the 10 allspice samples did nor exceed 4.3 × 105. A total of 176 randomly selected LAB isolates 11 originating from herring, carrot and allspice samples were further identified to species level 12 using a 16 and 23S rRNA gene RFLP (ribotyping) database. Leuconostoc gelidum and 13 Leuconostoc gasicomitatum strains dominated both in the spoiled herring and carrot samples. 14 These species are heterofermentative producing CO2 from glucose and they also produce 15 dextran from sucrose. Inoculation of some commercial herring products with spoilage- 16 associated L. gelidum and L. gasicomitatum strains verified that these strains have the 17 capability of producing slime and gas in herring preserves although slime formation was not 18 as strong as in the original samples. Since L. gelidum and L. gasicomitatum strains were 19 commonly detected in carrots, carrot slices used for the fish marinade were considered to be 20 the probable source of these specific spoilage organisms. 2

Enterococcus hermanniensis sp. nov., from modified-atmosphere-packaged broiler meat and canine tonsils

Isolates 302, 334, 356, 377 and 379, detected in modified atmosphere packaged (MAP) broiler meat, together with LMG 12317T and LMG 13617, detected in dog tonsils, were analysed in a polyphasic taxonomy study including numerical analysis of ribopatterns and whole cell protein patterns, 16S rDNA sequence analysis, DNA-DNA hybridisation and determination of some phenotypic properties. The results indicated that these isolates represent a new species in the genus Enterococcus. The isolates showed classical phenotypic reactions for the genus Enterococcus with the exception of not possessing the Lancefield group D antigen. Isolates 334, LMG 12317T, and LMG 13617 showed the highest 16S rDNA sequence similarity (98.3-99.0%) with the Enterococcus pallens type strain. In the distance matrix tree based on 16S rDNA sequences, the three isolates were located in the Enterococcus avium –group with E. pallens as their closest phylogenic neighbour. Numerical analyses of whole cell protein patterns and HindIII/EcoRI ribotypes placed all seven isolates together in a single cluster separated from the E. avium group reference strains. The DNA-DNA hybridisation level between strains 334 and LMG 12317T was 93.5% confirming that they represent the same species. Low hybridisation levels (12-30%) were, on the other hand, obtained with the E. pallens and Enterococcus raffinosus type strains. The name Enterococcus hermanniensis sp. nov. is proposed, with strain LMG 12317T (=CCUG 48100 T) as the type strain

Lactobacillus curvatus subsp. melibiosus is a later synonym of Lactobacillus sakei subsp. carnosus

On the basis of phenotypic and DNA-DNA reassociation studies, strain CCUG 34545T has been considered to represent a distinct Lactobacillus curvatus subspecies, Lactobacillus curvatus subsp. melibiosus. However, in several independent studies dealing with Lactobacillus sakei and L. curvatus strains, the subspecies division of L. curvatus has been found to be controversial. The original study distinguishing the two subspecies within both L. curvatus and L. sakei also lacked 16S rRNA gene sequence analyses. Therefore, the taxonomic position of L. curvatus subsp. melibiosus CCUG 34545T was re-evaluated in a polyphasic taxonomy study that included 16S rRNA gene sequence analysis, DNA-DNA reassociation, DNA G+C content deternmination, numerical analysis of ribotypes and whole-cell protein patterns and the examination of some fundamental phenotypic properties. The results obtained indicate that strain CCUG 34545T and its duplicate, CCUG 41580T, are Lactobacillus sakei subsp. carnosus strains and that L. curvatus subsp. melibiosus is a later synonym of L. sakei subsp. carnosus