海氷および南極アイスコアにおける微生物叢の解析と抗菌薬耐性遺伝子の検出に関する研究

抗菌薬が人医療および獣医療において広く用いられるのに伴い、薬剤耐性菌の出現と増加が公衆衛生学上の問題として世界的に懸念されている。また、薬剤耐性菌がもつ耐性遺伝子は、その多くが非病原性の環境細菌に由来するとみられていることから、現在蔓延している耐性遺伝子は、環境細菌から病原性細菌へと伝達されてから拡散したものと考えられている。これに加えて、合成抗菌薬に対する耐性遺伝子を染色体上にもつ細菌が報告されている点や、永久凍土層や洞窟などの隔離条件下からも耐性菌と耐性遺伝子が検出されている点から、耐性菌と耐性遺伝子は人為的な抗菌薬の使用による選択圧の有無に関わらず、自然環境中に存在しているものと予見されている。本研究では、上記の仮説を補完しうる知見を収集するため、絶対的抗菌薬非存在環境として南極のアイスコアを選択し、その中に含まれる耐性遺伝子の検出を目標とした。まず第１章では、氷サンプルの安全かつ十分なクリーニング方法を立ち上げるため、南氷洋およびオホーツク海で採材した海氷をサンプルとして、氷の洗浄および氷からのDNA抽出方法を検討した。また、海氷中の細菌叢を16S rDNAに基づく分類で特定し、その構成を両サンプル間で比較した。続いて第２章では、上記のクリーニング方法に基づいて実際の南極アイスコアを処理するとともに、含まれる微生物叢をメタゲノム解析によって網羅的に特定した。第3章では、海氷および南極アイスコア中から薬剤耐性遺伝子の検出を試み、検出した遺伝子について既知の遺伝子との比較を行なった。第1章における検討の結果、氷サンプルの洗浄方法が開発された。また、海氷に含まれる細菌叢を南氷洋とオホーツク海とで比較したところ、門レベルあるいは網レベルの広い分類ではおおむね似た傾向を示したが、科レベルや属レベルの詳細な解析で明確に菌叢が異なることがわかった。また、オホーツク海の海氷はバクテロイデス門の細菌を多く含むことが特徴であった。海氷を対象とする研究は数が少なく、また本実験で用いた採材地域でのサンプリングは過去に行なわれていないため、本研究の結果は海氷や低温環境中での細菌叢の構成に関する新たな知見を加えるものとなった。第2章では、1,670年前および2,860年前の南極アイスコアについて、細菌および真菌の構成をメタゲノム解析で特定した。グリーンランドなどのアイスコアを対象としたメタゲノム解析の既報と比べ、南極アイスコアのメタゲノム解析では遺伝子のヒット数が大幅に少ないことが特徴であったが、これは南極（ドームふじ基地）が周囲から隔絶された環境にあることが影響したものと考えられた。一方、2,860年前の氷については、デイノコッカス‐テルムス門の細菌が極端に多く検出された。本菌は極限環境微生物として知られており、ドームふじ基地における南極アイスコア中の微粒子の起源であるオーストラリアの砂漠環境にも分布しうる細菌であることから、これは細菌の付着した微粒子が当該アイスコア中に多量に含まれていた結果であると考えられた。アイスコアを対象としたメタゲノム解析は本研究が初報告であることから、今回得られた南極アイスコア固有の特性が今後の他のアイスコア研究の参考となることが期待される。第3章においては、南極アイスコアのうち約1,200～1,400年前の氷（サンプル名DF-63.5）から、sul2-strA-strBの耐性遺伝子クラスターを検出することに成功した。この遺伝子群は現代でも非常に広範な種類の細菌が保有していることから、この遺伝子は古くから環境細菌が保有しており、その細菌が南極の氷にトラップされていたことが予想された。また、DF-63.5から得られた遺伝子の塩基配列が現代の当該遺伝子と相同的であったこと、およびこの遺伝子群を組み込んだ形質転換株（大腸菌）が抗菌薬耐性を発現したことから、今回得られた耐性遺伝子群は現代の相同遺伝子と極めて近縁であることが確認された。さらに、耐性遺伝子が単体ではなくクラスターとして検出されたことから、人為的な抗菌薬使用が無い環境においても、薬剤耐性遺伝子がまとまって細菌間を移動していることが考えられた。これらに加え、sul2遺伝子が合成抗菌薬であるサルファ剤に対する耐性遺伝子であることから、合成抗菌薬の発明以前にもその耐性遺伝子が存在しうることが改めて示された。以上の成績から、抗菌薬の非存在下においても薬剤耐性遺伝子が存在することが証明された。これまでに永久凍土層や南極表層の雪などからの耐性菌および耐性遺伝子の検出報告はあるが、南極アイスコアという完全に外界から隔絶された環境からの報告は今回が初めてである。抗菌薬耐性菌の出現と蔓延を予見するためには、それらの起源や環境中における動態を明らかにすることが必要であることから、本研究の結果は抗菌薬の選択圧非存在下における耐性遺伝子の存在を証明したものであり、新たな薬剤耐性菌対策の構築に有用な知見を提供した。酪農学園大

Acanthamoeba containing endosymbiotic chlamydia isolated from hospital environments and its potential role in inflammatory exacerbation

Background: Environmental chlamydiae belonging to the Parachlamydiaceae are obligate intracellular bacteria that infect Acanthamoeba, a free-living amoeba, and are a risk for hospital-acquired pneumonia. However, whether amoebae harboring environmental chlamydiae actually survive in hospital environments is unknown. We therefore isolated living amoebae with symbiotic chlamydiae from hospital environments. Results: One hundred smear samples were collected from Hokkaido University Hospital, Sapporo, Japan; 50 in winter (February to March, 2012) and 50 in summer (August, 2012), and used for the study. Acanthamoebae were isolated from the smear samples, and endosymbiotic chlamydial traits were assessed by infectivity, cytokine induction, and draft genomic analysis. From these, 23 amoebae were enriched on agar plates spread with heatkilled Escherichia coli. Amoeba prevalence was greater in the summer-collected samples (15/30, 50%) than those of the winter season (8/30, 26.7%), possibly indicating a seasonal variation (p = 0.096). Morphological assessment of cysts revealed 21 amoebae (21/23, 91%) to be Acanthamoeba, and cultures in PYG medium were established for 11 of these amoebae. Three amoebae contained environmental chlamydiae; however, only one amoeba (Acanthamoeba T4) with an environmental chlamydia (Protochlamydia W-9) was shown the infectious ability to Acanthamoeba C3 (reference amoebae). While Protochlamydia W-9 could infect C3 amoeba, it failed to replicate in immortal human epithelial, although exposure of HEp-2 cells to living bacteria induced the proinflammatory cytokine, IL-8. Comparative genome analysis with KEGG revealed similar genomic features compared with other Protochlamydia genomes (UWE25 and R18), except for a lack of genes encoding the type IV secretion system. Interestingly, resistance genes associated with several antibiotics and toxic compounds were dentified. Conclusion: These findings are the first demonstration of the distribution in a hospital of a living Acanthamoeba carrying an endosymbiotic chlamydial pathogen

A retrospective analysis of antimicrobial resistance in pathogenic Escherichia coli and Salmonella spp. isolates from poultry in Uganda

There are increasing reports of antimicrobial treatment failures for bacterial diseases of poultry in Uganda. The paucity of data on antimicrobial resistance (AMR) of pathogenic bacteria in Uganda is a major setback to AMR control. This study investigated the occurrence of fowl typhoid, colibacillosis, and AMR in associated pathogens from 2012 to 2018. Laboratory records from the Central Diagnostic Laboratory (CDL), a National Veterinary Diagnostic Facility located at Makerere University, were reviewed. Archived isolates of the causative bacteria for the two diseases were also evaluated for AMR. The frequencies of the two disease conditions, their clinical and necropsy presentations and the demographic data of the diagnostic samples were summarized from the records. Archived bacterial isolates were revived before antimicrobial susceptibility testing. This was done on Mueller Hinton agar using the disk diffusion method, against 16 antimicrobials of medical and veterinary importance according to the Clinical Laboratory Standards Institute guidelines. A total of 697 poultry cases were presented for bacteriological investigations in the review period. Colibacillosis and salmonellosis had prevalence rates of 39.7% (277/697) and 16.2% (113/697), respectively. A total of 63 and 92 isolates of Escherichia coli and Salmonella spp., respectively, were archived but 43 (68.3%) E. coli and 47 (51.1%) Salmonella spp. isolates were recovered and evaluated for AMR. Multidrug resistance was more frequent in E. coli (38; 88.4%) than salmonellae (25; 53.2%), (p < 0.001). The high prevalence of colibacillosis, salmonellosis and the AMR of associated pathogens warrants immediate institution of appropriate disease control measures

Impact of capsaicin, an active component of chili pepper, on pathogenic chlamydial growth (Chlamydia trachomatis and Chlamydia pneumoniae) in immortal human epithelial HeLa cells

Chlamydia trachomatis is the leading cause of sexually transmitted infections worldwide. Capsaicin, a component of chili pepper, which can stimulate actin remodeling via capsaicin receptor TRPV1 (transient receptor potential vanilloid 1) and anti-inflammatory effects via PPAR gamma (peroxisome proliferator-activated receptor-gamma) and LXR alpha (liver X receptor alpha), is a potential candidate to control chlamydial growth in host cells. We examined whether capsaicin could inhibit C. trachomatis growth in immortal human epithelial HeLa cells. Inclusion forming unit and quantitative PCR assays showed that capsaicin significantly inhibited bacterial growth in cells in a dose-dependent manner, even in the presence of cycloheximide, a eukaryotic protein synthesis inhibitor. Confocal microscopic and transmission electron microscopic observations revealed an obvious decrease in bacterial numbers to inclusions bodies formed in the cells. Although capsaicin can stimulate the apoptosis of cells, no increase in cleaved PARP (poly (ADP-ribose) polymerase), an apoptotic indicator, was observed at a working concentration. All of the drugs tested (capsazepine, a TRPV1 antagonist; 5CPPSS-50, an LXR alpha inhibitor; and T0070907, a PPAR gamma inhibitor) had no effect on chlamydial inhibition in the presence of capsaicin. In addition, we also confirmed that capsaicin inhibited Chlamydia pneumoniae growth, indicating a phenomena not specific to C. trachomatis. Thus, we conclude that capsaicin can block chlamydial growth without the requirement of host cell protein synthesis, but by another, yet to be defined, mechanism

Long-term survival of Naegleria polaris from Antarctica after 10 years of storage at 4 A degrees C

A free-living amoeba, Naegleria is ubiquitously distributed in various natural environments. Since some Naegleria spp. are exclusively distributed in the Arctic and sub-Antarctic regions, we hypothesized that the amoeba may be useful to determine long-term survival of Naegleria in laboratory conditions at 4 A degrees C. The main objective of the study is to determine that a species of an environmental amoebal isolated can live at low temperatures after a long time. Here, we therefore show long-term survival of an amoeba, Naegleria polaris isolated from a sediment sample, which was collected from Antarctica 10 years ago, and since stored at 4 A degrees C. The sample was put on non-nutrient agar plates with heat-killed Escherichia coli, and then the plate was incubated at 4, 15, or 30 A degrees C. Motile amoebae were seen only when the plate was incubated at 15 A degrees C. The sequencing of ribosomal DNA including internal transcribed spacers (ITS) 1, 5.8S rDNA, and ITS2 region revealed the amoebae to be N. polaris, which is exclusively distributed in the Arctic and sub-Antarctic regions. Scanning electron microscopic observation showed that no typical sucker-like structure was seen on the surface of N. polaris, but the cysts were similar to those of Naegleria fowleri. Thus, our result shows, for the first time, that N. polaris can survive after 10 years of storage at 4 A degrees C. This finding may help us understand the still undescribed effects of environmental samples on viability of amoebae

Subtle changes in host cell density cause a serious error in monitoring of the intracellular growth of Chlamydia trachomatis in a low-oxygen environment: Proposal for a standardized culture method

We monitored Chlamydia trachomatis growth in HeLa cells cultured with either DMEM or RPMI medium containing 10% FCS under 2% or 21% O-2 conditions for 2 days. Bacterial numbers, host cell numbers, and fibrosis related gene expression in the host cells were estimated by an inclusion forming unit assay, a cell counting assay, and a PCR array, respectively. In contrast to RPMI, bacterial growth under low oxygen conditions in DMEM rapidly decreased with increasing host cell density. The addition of supplements (glucose, glutamine, vitamin B12, D-biotin, non-essential amino acids, glutathione) to the media had no effect. The growth of host cells in DMEM under low oxygen conditions rapidly decreased, although the cells remained healthy morphologically. Furthermore, the downregulation of 17 genes was observed under low oxygen in DMEM. Whereas no effect on bacterial growth was observed when culturing in RPMI medium at low oxygen, and the downregulation of three genes (CTGF, SERPINEI, JUN) was observed following bacterial infection compared with the uninfected control cells. Thus, our findings indicate the need for carefully selected culture conditions when performing experiments with C. trachomatis under low-oxygen environments, and RPMI (rather than DMEM) is recommended when a low host cell density is to be used, proposing the major modification of cell culturing method of C. trachomatis in a low-oxygen environment

Wild ciliates differ in susceptibility to Legionella pneumophila JR32

We investigated how Legionella pneumophila (Lp) JR32 interacts with Anteglaucoma CS11A and Colpoda E6, two ciliates that we isolated from sewage and sink trap sludge, respectively, using a handmade maze device containing a 96-well crafting plate. Our 185 rDNA-based phylogenetic analysis showed that Anteglaucoma CS11A and Colpoda E6 formed distinct clades. Scanning electron microscopy showed that Anteglaucoma CS11A had a bigger-sized body than Colpoda E6 and, unlike Tetrahymena IB (the reference strain), neither ciliate produced pellets, which are extracellular vacuoles. Fluorescence microscopic observations revealed that although the intake amounts differed, all three ciliates rapidly ingested LpJR32 regardless of the presence or absence of the icm/dot virulence genes, indicating that they all interacted with LpJR32. In co-cultures with Anteglaucoma CS11A, the LpJR32 levels were maintained but fell dramatically when the co-culture contained the LpJR32 icm/dot deletion mutant instead. Anteglaucoma CS11 A died within 2days of co-culture with LpJR32, but survived co-culture with the deletion mutant. In co-cultures with Colpoda E6, LpJR32 levels were maintained but temporarily decreased independently of the virulence gene. Concurrently, the Colpoda E6 ciliates survived by forming cysts, which may enable them to resist harsh environments, and by diminishing the sensitivity of trophozoites to Lp. In the Tetrahymena IB co-cultures with LpJR32 or Delta icm/dot, the Lp levels were maintained, albeit with temporal decreases, and the Tetrahymena IB levels were also maintained. We conclude that unlike Tetrahymena IB with pellet production, Anteglaucoma CS11A can be killed by LpJR32 infection, and Colpoda E6 can resist LpJR32 infection through cyst formation and the low sensitivity of trophozoites to Lp. Thus, the two ciliates that we isolated had different susceptibilities to LpJR32 infection

Occurrence of Selected Bacterial Antibiotic Resistance Genes in Soils of Animal Farms in Uganda

The antibiotic resistance genes (ARG) of bacteria can be found in diverse environments such as the gut of mammals, soil, air, and water. In this research, we examined soils (for ARG) from 27 animal farms in Uganda (Mbarara, Wakiso and Mpigi districts) rearing poultry, pigs, dairy, or beef animals. Among these 27 places, there were antibiotic-free farms and those, which routinely used antibiotics to control diseases. DNA was extracted from soil samples using a commercial kit. A polymerase chain reaction (PCR) was performed to detect baScterial genes of resistance to sulphonamide (3 genes), beta lactam (1 gene), and Tetracycline (8 genes) antibiotics. The highest number of soils contaminated with these genes were from Mpigi district, whereas in Mbarara we found contamination of farm soils to a lesser extent. In all districts, the ARG were detected in farm soils regardless of the evidence of antibiotic usage; however, ARG were predominant in severe antibiotic consuming farms than the less consuming ones. Sulphonamide resistance genes were predominant in most district samples. In particular, the sul2 (Sulphonamide) and tetW (Tetracycline) genes were the most prevalent in all samples, suggesting that fecal disposal or use of animal manure could drive the accumulation of ARG in these soils, making it a deadly reservoir, especially in areas with vast consumption of antibiotics

Amino Acid Substitutions in GyrA and ParC are Associated with Fluoroquinolone Resistance in Mycoplasma bovis Isolates from Japanese Dairy Calves

We investigated the contribution of quinolone resistance-determining region (QRDR) mutations to fluoroquinolone (enrofloxacin, orbifloxacin and danofloxacin) susceptibility in 58 Mycoplasma bovis isolates from dairy cattle in Japan. Fluoroquinolone non-resistant isolates (fluoroquinolone-MICs≤2μg/ml) possessed no QRDR mutations (19 isolates) or Ser83Leu in GyrA (32 isolates). Fluoroquinoloneresistant isolates (fluoroquinolone-MICs≥4μg/ml) possessed Ser83Leu in GyrA and Ser81Pro in ParC (3 isolates) or Ser83Phe in GyrA and Ser80Ile in ParC (4 isolates). Laboratory-derived fluoroquinolone-resistant mutants selected from 2 isolates (possessing Ser83Leu in GyrA) had an amino acid substitution in ParC at the same position (Ser80Ile or Ser81Tyr) as fluoroquinolone-resistant isolates, suggesting a concurrent amino acid substitution in ParC (Ser80 or Ser81) is important in fluoroquinolone resistance in M. bovis isolates