Probiotic candidates for controlling Paenibacillus larvae, a causative agent of American foulbrood disease in honey bee.

BACKGROUND: American foulbrood (AFB) disease caused by Paenibacillus larvae is dangerous, and threatens beekeeping. The eco-friendly treatment method using probiotics is expected to be the prospective method for controlling this pathogen in honey bees. Therefore, this study investigated the bacterial species that have antimicrobial activity against P. larvae. RESULTS: Overall, 67 strains of the gut microbiome were isolated and identified in three phyla; the isolates had the following prevalence rates: Firmicutes 41/67 (61.19%), Actinobacteria 24/67 (35.82%), and Proteobacteria 2/67 (2.99%). Antimicrobial properties against P. larvae on agar plates were seen in 20 isolates of the genus Lactobacillus, Firmicutes phylum. Six representative strains from each species (L. apis HSY8_B25, L. panisapium PKH2_L3, L. melliventris HSY3_B5, L. kimbladii AHS3_B36, L. kullabergensis OMG2_B25, and L. mellis OMG2_B33) with the largest inhibition zones on agar plates were selected for in vitro larvae rearing challenges. The results showed that three isolates (L. apis HSY8_B25, L. panisapium PKH2_L3, and L. melliventris HSY3_B5) had the potential to be probiotic candidates with the properties of safety to larvae, inhibition against P. larvae in infected larvae, and high adhesion ability. CONCLUSIONS: Overall, 20 strains of the genus Lactobacillus with antimicrobial properties against P. larvae were identified in this study. Three representative strains from different species (L. apis HSY8_B25, L. panisapium PKH2_L3, and L. melliventris HSY3_B5) were evaluated to be potential probiotic candidates and were selected for probiotic development for the prevention of AFB. Importantly, the species L. panisapium isolated from larvae was identified with antimicrobial activity for the first time in this study.

Current Status of Q Fever and the Challenge of Outbreak Preparedness in Korea: One Health Approach to Zoonoses.

Human Q fever, a zoonosis caused by Coxiella burnetii, presents with diverse clinical manifestations ranging from mild self-limited febrile illnesses to life-threatening complications such as endocarditis or vascular infection. Although acute Q fever is a benign illness with a low mortality rate, a large-scale outbreak of Q fever in the Netherlands led to concerns about the possibility of blood transfusion-related transmission or obstetric complications in pregnant women. Furthermore, a small minority (< 5%) of patients with asymptomatic or symptomatic infection progress to chronic Q fever. Chronic Q fever is fatal in 5-50% of patients if left untreated. In South Korea, Q fever in humans was designated as a notifiable infectious disease in 2006, and the number of Q fever cases has increased sharply since 2015. Nonetheless, it is still considered a neglected and under-recognized infectious disease. In this review, recent trends of human and animal Q fever in South Korea, and public health concerns regarding Q fever outbreaks are reviewed, and we consider how a One Health approach could be applied as a preventive measure to prepare for zoonotic Q fever outbreaks.

Prevalence of Campylobacter species in wild birds of South Korea.

Campylobacter species cause human gastrointestinal infections worldwide. They commonly inhabit intestines of avian species including wild birds. They might play a role in the spread of infections to humans and other bird species. The prevalence of Campylobacter species in 2164 faecal samples of wild birds (representing 71 species and 28 families) captured across the Korean peninsula was evaluated in this study. The overall prevalence was 15.3% (332/2164). Bird species belonging to the family Charadriidae had the highest isolation rate (30.0%), followed by those belonging to the families Ardeidae (26.4%), Turdidae (21.9%), and Anatidae (15.3%). The prevalence of Campylobacter spp. differed significantly according to migratory habit. Stopover birds were the most commonly infected (19.0%), followed by winter migratory (16.7%) and summer migratory birds (12.3%). However, indigenous birds showed very low prevalence (2.7%). Antimicrobial susceptibility tests were performed for 213 isolates. Results showed that Campylobacter jejuni isolates (n = 169) exhibited resistance to nalidixic acid (5.3%), ciprofloxacin (3.0%), and tetracycline (1.8%), while Campylobacter lari (n = 1) displayed resistance to nalidixic acid and ciprofloxacin. However, all Campylobacter coli isolates (n = 20) were susceptible to all antimicrobials tested. This is the first report on the prevalence of Campylobacter species in wild birds that seasonally or indigenously inhabit the Korean peninsula. Our results indicate that the overall prevalence of Campylobacter in wild birds is moderate. Therefore, birds might serve as significant reservoirs for Campylobacter pathogens.

Identification and pathogen detection of a Neocypholaelaps species (Acari: Mesostigmata: Ameroseiidae) from beehives in the Republic of Korea.

In this study, we identified a new strain of the genus Neocypholaelaps from the beehives of Apis mellifera colonies in the Republic of Korea (ROK). The Neocypholaelap sp. KOR23 mites were collected from the hives of honeybee apiaries in Wonju, Gangwon-do, in May 2023. Morphological and molecular analyses based on 18S and 28S rRNA gene regions conclusively identified that these mites belong to the genus Neocypholaelaps, closely resembling Neocypholaelaps sp. APGD-2010 that was first isolated from the United States. The presence of 9 of 25 honeybee pathogens in these mite samples suggests that Neocypholaelaps sp. KOR23 mite may act as an intermediate vector and carrier of honeybee diseases. The identification of various honeybee pathogens within this mite highlights their significance in disease transmission among honeybee colonies. This comprehensive study provides valuable insights into the taxonomy and implications of these mites for bee health management and pathogen dissemination.

First detection and prevalence of Apis mellifera filamentous virus in Apis mellifera and Varroa destructor in the Republic of Korea.

Apis mellifera filamentous virus (AmFV) is a double-stranded DNA virus that infects Apis mellifera bees. To our knowledge, this is the first comprehensive study aiming to detect and analyse the genetic diversity and prevalence of AmFV in Korean honeybee colonies. Phylogenetic analysis based on baculovirus repeat open reading frame-N gene (Bro-N) sequences revealed that AmFV isolates from the Republic of Korea (ROK) fell into two distinct lineages, with genetic origins in Switzerland and China, with nucleotide similarities of 98.3% and 98.2%, respectively. Our prevalence analysis demonstrated a noteworthy infection rate of AmFV in 545 honeybee colonies, reaching 33.09% in 2022 and increasing to 44.90% by 2023. Intriguingly, we also detected AmFV in Varroa destructor mites, highlighting their potential role as vectors and carriers of AmFV. The presence of AmFV was correlated with an increased infection rate of sacbrood virus, deformed wing virus, Lake Sinai virus 2, black queen cell virus, and Nosema ceranae in honeybee colonies. These findings provide valuable insight into the prevalence and potential transmission mechanisms of AmFV in honeybee colonies in the ROK. The results of this study may be instrumental in the effective management of viral infections in honeybee apiaries.

Molecular Identification and Prevalence of the Mite Carpoglyphus lactis (Acarina: Carpoglyphidae) in Apis mellifera in the Republic of Korea.

Apis mellifera, especially weak ones, are highly vulnerable to Carpoglyphus lactis mites, which can rapidly infest and consume stored pollen, leading to weakened colonies and potential colony collapse. This study aimed to ascertain and investigate the prevalence of this mite in honeybee colonies across nine provinces in the Republic of Korea (ROK). A total of 615 honeybee colony samples were collected from 66 apiaries during the spring and 58 apiaries during the summer of 2023. A 1242 bp segment of the Cytochrome c oxidase subunit 1 (COI) gene was amplified using the polymerase chain reaction method. The detection levels of C. lactis in the honeybees were compared between winter and summer. Based on the COI sequence analysis, the nucleotide sequence similarity of C. lactis mites isolated in the ROK with those from China (NC048990.1) was found to be 99.5%, and with those from the United Kingdom (KY922482.1) was 99.3%. This study is the first report of C. lactis in Korean apiaries. The findings of this study demonstrate a significantly higher detection rate in winter, which is 4.1 times greater than that in summer (p < 0.001). Furthermore, the results underscore the usefulness of molecular diagnostic techniques for detecting C. lactis mites.

Prevalence and genome features of lake sinai virus isolated from Apis mellifera in the Republic of Korea.

Lake Sinai Virus (LSV) is an emerging pathogen known to affect the honeybee (Apis mellifera). However, its prevalence and genomic characteristics in the Republic of Korea (ROK) remain unexplored. This study aimed to assess the prevalence of and analyze the LSVs by examining 266 honeybee samples from the ROK. Our findings revealed that LSV exhibited the highest infection rate among the pathogens observed in Korean apiaries, particularly during the reported period of severe winter loss (SWL) in A. mellifera apiaries in 2022. Three LSV genotypes- 2, 3, and 4 -were identified using RNA-dependent RNA polymerase gene analysis. Importantly, the infection rates of LSV2 (65.2%) and LSV3 (73.3%) were significantly higher in colonies experiencing SWL than in those experiencing normal winter loss (NWL) (p < 0.03). Furthermore, this study provides the first near-complete genome sequences of the Korean LSV2, LSV3, and LSV4 strains, comprising 5,759, 6,040, and 5,985 nt, respectively. Phylogenetic analysis based on these near-complete genome sequences demonstrated a close relationship between LSVs in the ROK and China. The high LSV infection rate in colonies experiencing a heightened mortality rate during winter suggests that this pathogen might contribute to SWL in ROK. Moreover, the genomic characteristic information on LSVs in this study holds immense potential for epidemiological information and the selection of specific genes suitable for preventing and treating LSV, including the promising utilization of RNA interference medicine in the future.

Evaluation of acaricidal activity in entomopathogenic fungi for poultry red mite (Dermanyssus gallinae) control.

The poultry red mite (PRM), Dermanyssus gallinae, significantly impacts the health of egg-laying hens. Mites feed on the blood of infested chickens and have a great economic impact on the poultry industry. Chemical treatment of mites raises concerns about their resistance to miticides and residues in eggs and poultry. Biocontrol using entomopathogenic fungi is expected to be a chemical-free strategy for reducing PRM infestations. Therefore, the present study aimed to investigate the effects of various entomopathogenic fungal species collected in South Korea on the inhibition of PRM. Seventeen strains of six fungal species collected from various sources were used to evaluate acaricidal activity against PRM. The results showed that 16/17 strains had acaricidal properties against PRM, of which strains of Metarhizium anisopliae had the highest acaricidal activity. Mites treated with M. anisopliae CBNU 4-2 showed 100 % mortality 5 d after inoculation, followed by M. flavoviride var. pemphigi. The M. flavoviride var. pemphigi CBNU 1-1-1 showed 97.78 % mortality after 10 d of exposure to fungi. The mortality rate of PRM treated with other strains slowly increased and reached its highest value on the 14th day of inoculation. The results of this study provide information on the acaricidal activity of different entomopathogenic fungi against PRM. This information is important for the selection of fungal species for developing biocontrol methods for PRM treatment. These strains could be used for further evaluation of PRM treatment on chicken farms, or in combination with other methods, to increase PRM treatment efficiency.

16S rRNA metabarcoding for the identification of tick-borne bacteria in ticks in the Republic of Korea.

Ticks are blood-sucking ectoparasites that act as vectors for transmission of various pathogens. The purpose of this study was to assess tick-borne bacteria, whether pathogenic or not, in ticks distributed in Korea using 16S rRNA metabarcoding and to confirm the results by PCR. Questing ticks were collected from four provinces in Korea in 2021 using the flagging method. After pooling the DNAs from the 61 tick pools (including 372 ticks), the bacterial 16S rRNA V3-V4 hypervariable region was amplified and sequenced using the MiSeq platform. Rickettsia, Ehrlichia, and the endosymbiont Wolbachia were confirmed by conventional PCR and molecular analysis. In total, 6907 ticks (534 pools) were collected and identified as belonging to five species (Haemaphysalis spp., H. longicornis, H. flava, I. nipponensis, and A. testudinarium). Through 16S rRNA metabarcoding, 240 amplicon sequence variants were identified. The dominant taxa were Rickettsiella and Coxiella. Additionally, pathogenic bacteria such as Rickettsia and Ehrlichia, endosymbiotic bacteria such as Wolbachia and Spiroplasma were identified. Polymerase chain reaction (PCR) was performed to confirm the presence of Rickettsia, Ehrlichia, Bartonella, and Wolbachia in individual ticks. Overall, 352 (65.92%) of 534 pools tested positive for at least one of the screened tick-borne bacteria. Rickettsia was the most prevalent (61.42%), followed by Wolbachia (5.05%). Ehrlichia was detected in 4.86% of tested samples, whereas Bartonella was not detected. In this study, 16S rRNA metabarcoding revealed the presence of Rickettsia, Wolbachia, and Ehrlichia, in that order of abundance, while showing absence of Bartonella. These results were confirmed to exhibit the same trend as that of the conventional PCR. Therefore, large-scale screening studies based on pooling, as applied in this study, will be useful for examining novel or rare pathogens present in various hosts and vectors.

Molecular epidemiology of Theileria species in ticks and its potential threat to livestock in the Republic of Korea.

The purpose of this study was to investigate molecular epidemiology of Theileria spp. in ticks in Korea and assess their potential threat from wildlife animals to domestic animals. A total of 21152 hard ticks were collected from Chungcheong and Jeolla provinces of Korea from March to October 2021. Tick species were identified by microscopy and Theileria spp. were screened by nested PCR targeting 18S rRNA. Haemaphysalis spp. were the most abundant tick species, followed by H. longicornis, H. flava, Amblyomma testudinarium, and Ixodes nipponensis. Of the collected ticks, 6914 ticks (541 pools) were screened, and PCR showed 211 positive pools (39.0%; MIR: 3.05). The PCR and phylogenetic analysis identified two Theileria species, T. luwenshuni and Theileria sp., with T. luwenshuni (162/211, 76.78%; MIR: 2.34) being more abundant than Theileria sp. (36/211, 17.06%; MIR: 0.52); co-infection of the two species were noted (13/211, 6.16%; MIR: 0.19). Among the tick species, H. longicornis, especially nymphs, showed the highest prevalence. Regarding season, the highest prevalence was observed in May. Considering the tick and Theileria species identified in this study, H. longicornis nymph and cervine play a critical role in maintaining Theileria spp. in Korea and could be a potential threat to domestic animals, including deer and goats. In addition, there are significant correlations among tick distribution, region, season, and prevalence of Theileria.

Large-Scale Application of Double-Stranded RNA Shows Potential for Reduction of Sacbrood Virus Disease in Apis cerana Apiaries.

Sacbrood virus (SBV) infection has emerged as a remarkable threat to Apis cerana colonies in South Korea, necessitating prompt control measures. In this study, RNA interference (RNAi) targeting the VP3 gene was developed to assess its safety and efficacy in protecting and treating SBV in vitro and in infected colonies in South Korean apiaries. The efficacy of VP3 double-stranded RNA (dsRNA) was demonstrated in laboratory-based experiments, wherein infected larvae treated with VP3 dsRNA exhibited a 32.7% increase in survival rate compared to untreated larvae. Data from a large-scale field trial indicate the efficacy of dsRNA treatment since none of the treated colonies had symptomatic SBV infections, whereas disease was observed in 43% (3/7) of the control colonies. In the 102 colonies exhibiting symptoms of SBV disease, RNAi treatment provided partial protection with weekly treatment, prolonging the survival period of colonies to 8 months compared to 2 months in colonies treated at 2- and 4-week intervals. Therefore, this study demonstrated that RNAi is a valuable tool for preventing SBV disease outbreaks in healthy and low-level SBV-infected colonies.

First identification of Tyrophagus curvipenis (Acari: Acaridae) and pathogen detection in Apis mellifera colonies in the Republic of Korea.

Mites of the genus Tyrophagus (Acari: Acaridae) are among the most widely distributed mites. The species in this genus cause damage to stored products and crops, and pose a threat to human health. However, the influence of Tyrophagus spp. in apiculture remains unknown. In 2022, a study focusing on the identification of Tyrophagus species within five apiaries was conducted in Chungcheongnam Province, Republic of Korea. Its specific objective was to investigate the presence of Tyrophagus mites in response to the reported high mortality of honey bee colonies in this area. Morphological identification and phylogenetic analysis using the mitochondrial gene cytochrome-c oxidase subunit 1 (CO1) confirmed for the first time the presence of the mite species Tyrophagus curvipenis in a honey bee colony in the Republic of Korea. Two honey bee pathogens were detected in the mite, a viral pathogen (deformed wing virus, DWV) and a protozoal pathogen (Trypanosoma spp.). The presence of the two honey bee pathogens in the mite suggests that this mite could contribute to the spread of related honey bee diseases. However, the direct influence of the mite T. curvipenis on honey bee health remains unknown and should be further investigated.

Genotyping of Coxiella burnetii from Cattle by Multispacer Sequence Typing and Multiple Locus Variable Number of Tandem Repeat Analysis in the Republic of Korea.

Genotyping of Coxiella burnetii using multispacer sequence typing (MST) and multiple locus variable number tandem repeat analysis (MLVA) was conducted from infected animals for the first time in the Republic of Korea. C. burnetii was detected by real-time PCR, and followed by MST and MLVA genotyping. The result showed that detected C. burnetii all had the same MLVA genotype, 6-13-2-7-9-10 for markers MS23-MS24-MS27-MS28-MS33-MS34, respectively, and genotype group 61 for MST. The same genotypes were previously identified in Poland. Importantly, this MLVA type was detected in humans in France, suggesting that the Korean strain can also potentially cause Q fever in humans. MST and MLVA were very useful tools for analyzing the molecular epidemiology of C. burnetii and helpful for interpreting the epidemiological relationship between isolates from domestic and international resources.

Genetic and Pathogenic Characterization of QX(GI-19)-Recombinant Infectious Bronchitis Viruses in South Korea.

Infectious bronchitis viruses (IBVs) are evolving continuously via genetic drift and genetic recombination, making disease prevention and control difficult. In this study, we undertook genetic and pathogenic characterization of recombinant IBVs isolated from chickens in South Korea between 2003 and 2019. Phylogenetic analysis showed that 46 IBV isolates belonged to GI-19, which includes nephropathogenic IBVs. Ten isolates formed a new cluster, the genomic sequences of which were different from those of reference sequences. Recombination events in the S1 gene were identified, with putative parental strains identified as QX-like, KM91-like, and GI-15. Recombination detection methods identified three patterns (rGI-19-I, rGI-19-II, and rGI-19-III). To better understand the pathogenicity of recombinant IBVs, we compared the pathogenicity of GI-19 with that of the rGI-19s. The results suggest that rGI-19s may be more likely to cause trachea infections than GI-19, whereas rGI-19s were less pathogenic in the kidney. Additionally, the pathogenicity of rGI-19s varied according to the genotype of the major parent. These results indicate that genetic recombination between heterologous strains belonging to different genotypes has occurred, resulting in the emergence of new recombinant IBVs in South Korea.

Impact of Dermanyssus gallinae infestation on persistent outbreaks of fowl typhoid in commercial layer chicken farms.

Although it has rapidly decreased since the early 2000s, fowl typhoid still occurs in commercial layer chickens, causing a significant economic loss in Korea. There is growing concern about the emergence of new pathogenic strains of the causative agent, Salmonella Gallinarum, which is able to overcome vaccine immunity. It has also been suspected that the poultry red mite, Dermanyssus gallinae, which is commonly found in layer chicken farms, may be an important cause of the recurrence of fowl typhoid in the farms. This study was conducted to examine changes in the virulence of recent isolates of S. Gallinarum obtained from layer farms and estimate the potential of the disease transmission of D. gallinae in the farms. Clinical and environmental samples and mites collected from layer farms affected by fowl typhoid between 2013 and 2018 were tested for S. Gallinarum. The isolates were characterized by genotypic analyses and in vitro virulence assays with chicken-derived cell lines. Vaccine protection against recent isolates was examined in the chickens. A total of 45 isolates of S. Gallinarum were collected and there was no evidence of changes in their virulence. It has also been demonstrated that the S. Gallinarum 9R vaccine strain widely used in Korea is still effective in controlling fowl typhoid if the susceptibility of birds to the disease is not increased by stress. Salmonella Gallinarum isolated from the outer and inner parts of D. gallinae, environmental dust, and dead birds of the same farm showed the same or closely related genotypes. Consequently, the present study indicated that the horizontal transmission and environmental persistence of S. Gallinarum and the increased disease susceptibility of chickens in layer farms could be mediated by D. gallinae, causing persistent outbreaks of fowl typhoid.

Sequential Transmission of Salmonella in the Slaughtering Process of Chicken in Korea.

Salmonella is one of the most common foodborne pathogens worldwide. Salmonella infections in humans are mainly associated with consumption of poultry products contaminated with this foodborne pathogen. Therefore, strict sanitary measures are necessary to control Salmonella contamination during the slaughtering process of poultry. The objective of this study was to determine the occurrence and transmission of Salmonella at a series of steps in the slaughtering process of chicken. A total of 601 samples were collected from a series of slaughtering steps (10 sampling sites) of 26 chicken slaughterhouses throughout Korea. Salmonella was isolated from samples and its distribution was analyzed along the slaughtering process. Isolates from each sampling site were tested for susceptibility to 15 antibiotics by the broth microdilution method. They were also genotypically characterized by pulsed-field gel electrophoresis (PFGE). Salmonela was isolated from 168 out of 601 samples. Sixteen serotypes were identified while six isolates were untypable. Salmonella enterica serovars Montevideo (n = 29) and Virchow (n = 27) were the most common serotypes out of 119 nonredundant isolates. Relatively high contamination rates of Salmonella were found in shackles (75.0%), feathers near plucking machine (68.5%), and feces from crates (44.0%). Twenty-three antibiotic resistance patterns were recognized and 40 (33.6%) isolates were resistant to five or more antibiotics. The same serotypes of Salmonella were distributed along the slaughtering process of each Salmonella-positive slaughterhouse. Most of those isolates belonging to the same serotype had identical or closely related PFGE profiles. They also shared common antibiotic resistance patterns. Overall findings of this study indicated that Salmonella were sequentially transmitted through the chicken slaughtering process. PRACTICAL APPLICATION: This study provides useful information on the distribution and transmission of Salmonella serotypes through the chicken slaughtering process. Overall findings indicated the need for routine microbiological monitoring along the slaughtering process. This study also showed that on-farm control of Salmonella is needed to obtain Salmonella-free chicken carcasses.

Synthesis of Stachyobifiose Using Bifidobacterial α-Galactosidase Purified from Recombinant Escherichia coli.

The prebiotic effects of GOS (galactooligosaccharides) are known to depend on the glycosidic linkages, degree of polymerization (DP), and the monosaccharide composition. In this study, a novel form of α-GOS with a potentially improved prebiotic effect was synthesized using bifidobacterial α-galactosidase (α-Gal) purified from recombinant Escherichia coli. The carbohydrate produced was identified as α-d-galactopyranosyl-(1→6)-O-α-d-glucopyranosyl-(1→2)-[α-d-galactopyranosyl-(1→6)-O-ß-d-fructofuranoside] and was termed stachyobifiose. Among 17 nonprobiotics, 16 nonprobiotics showed lower growth on stachyobifiose than ß-GOS. In contrast, among the 16 probiotics, 6 probiotics showed higher growth on stachyobifiose than ß-GOS. When compared with raffinose, stachyobifiose was used less by nonprobiotics than raffinose. Moreover, compared with stachyose, stachyobifiose was used less by Escherichia coli, Enterobacter cloacae, and Clostridium butyricum. The average amounts of total short-chain fatty acids (SCFA) produced were in the order of stachyobifiose > stachyose > raffinose > ß-GOS. Taken together, stachyobifiose is expected to contribute to beneficial changes of gut microbiota.

Development of Strain-Specific Primers for Identification of Bifidobacterium bifidum BGN4.

Bifidobacterium bifidum BGN4 (BGN4) has many proven beneficial effects, including antiallergy and anticancer properties. It has been commercialized and used in several probiotic products, and thus strain-specific identification of this strain is very valuable for further strain-dependent physiological study. For this purpose, we developed novel multiplex polymerase chain reaction (PCR) primer sets for strain-specific detection of BGN4 in commercial products and fecal samples of animal models. The primer set was tested on seven strains of B. bifidum and 75 strains of the other Bifidobacterium species. The BGN4-specific regions were derived using megaBLAST against genome sequences of various B. bifidum databases and four sets of primers were designed. As a result, only BGN4 produced four PCR products simultaneously whereas the other strains did not. The PCR detection limit using BGN4-specific primer sets was 2.8 × 101 CFU/ml of BGN4. Those primer sets also detected and identified BGN4 in the probiotic products containing BNG4 and fecal samples from a BGN4-fed animal model with high specificity. Our results indicate that the PCR assay from this study is an efficient tool for the simple, rapid, and reliable identification of BGN4, for which probiotic strains are known.

Comparison of the Antimicrobial and Sanitizer Resistance of Salmonella Isolates from Chicken Slaughter Processes in Korea.

Salmonella is a foodborne pathogen worldwide. Outbreaks of Salmonella are commonly associated with consumption of contaminated foods such as poultry products. Therefore, the objective of this study was to determine the occurrence, biofilm formation, antibiotic resistance, and sanitizer resistance of Salmonella enterica isolated from chicken carcasses. A total of 318 samples were collected from 15 chicken slaughterhouses in 8 provinces of Korea. They were then examined for Salmonella contamination. S. enterica isolates were tested for their susceptibilities to 15 antimicrobials by broth microdilution method. Their biofilm formation ability and resistance to sanitizers were also evaluated. Eighty-two isolates of S. enterica were obtained from the 318 samples. There were 14 serotypes and 2 untypable isolates. Fifty-seven (69.5%) isolates were resistant to at least one antibiotic while 30 (36.6%) isolates were resistant to 5 or more antibiotics. Two S. Senftenberg and 3 S. Montevideo isolates exhibited considerable biofilm formation ability (A600 >0.2) following incubation in Luria-Bertani (LB) broth for 48 h. Biofilm cell survival and recovery growth assay after sanitization showed that most isolates were highly susceptible to 2.5% lactic acid and 0.1% cetylpyridinium chloride. Therefore, lactic acid and cetylpyridinium chloride might be alternatively or additionally used in addition to chlorine-based sanitizers that are frequently used to reduce Salmonella contamination of chicken carcasses. Our results provide basic information on the distribution of Salmonella serotypes in chicken slaughterhouses. This study also highlights the necessity to improve farming practices and use antimicrobial agents cautiously. This study also suggests that sanitization during the slaughtering process might be necessary to reduce Salmonella contamination of chicken carcasses.

Synthesis of ß-Galactooligosaccharide Using Bifidobacterial ß-Galactosidase Purified from Recombinant Escherichia coli.

Galactooligosaccharides (GOSs) are known to be selectively utilized by Bifidobacterium, which can bring about healthy changes of the composition of intestinal microflora. In this study, ß-GOS were synthesized using bifidobacterial ß-galactosidase (G1) purified from recombinant E. coli with a high GOS yield and with high productivity and enhanced bifidogenic activity. The purified recombinant G1 showed maximum production of ß-GOSs at pH 8.5 and 45°C. A matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the major peaks of the produced ß-GOSs showed MW of 527 and 689, indicating the synthesis of ß-GOSs at degrees of polymerization (DP) of 3 and DP4, respectively. The trisaccharides were identified as ß-D-galactopyranosyl-(1→4)-O-ß-D-galactopyranosyl-(1→4)-O-ß-D-glucopyranose, and the tetrasaccharides were identified as ß-D-galactopyranosyl-(1→4)-O-ß-D-galactopyranosyl- (1→4)-O-ß-D-galactopyranosyl-(1→4)-O-ß-D-glucopyranose. The maximal production yield of GOSs was as high as 25.3% (w/v) using purified recombinant ß-galactosidase and 36% (w/v) of lactose as a substrate at pH 8.5 and 45°C. After 140 min of the reaction under this condition, 268.3 g/l of GOSs was obtained. With regard to the prebiotic effect, all of the tested Bifidobacterium except for B. breve grew well in BHI medium containing ß-GOS as a sole carbon source, whereas lactobacilli and Streptococcus thermophilus scarcely grew in the same medium. Only Bacteroides fragilis, Clostridium ramosum, and Enterobacter cloacae among the 17 pathogens tested grew in BHI medium containing ß-GOS as a sole carbon source; the remaining pathogens did not grow in the same medium. Consequently, the ß-GOS are expected to contribute to the beneficial change of intestinal microbial flora.