石和田 稔彦

Introduction. Certain nontypeable Haemophilus influenzae cannot be assigned a sequence type (ST) by Multilocus Sequence Typing (MLST) due to the lack of the fucK gene, one of seven MLST loci in H. influenzae, which encodes a fucose-operon enzyme.Aims. To confirm whether the loss of fucK is also found in the encapsulated strains, we analysed clinical isolates of H. influenzae serotype e (Hie).Methodology. We conducted MLST, PFGE, and antimicrobial susceptibility tests of 45 Hie strains; the majority (n=43) were derived from respiratory samples of pediatric patients at Chiba Children's Hospital between 2000 and 2016. The two remaining strains were obtained from the blood of elderly patients with invasive H. influenzae diseases (IHiDs) between 2015 and 2016 at general hospitals. For the fucK-negative strains, PCR analysis for fucose operon was also performed.Results. Four STs (ST18, 122, 621 and 1758) were assigned to 13 strains, and remaining 32 (including one associated with IHiD) were fucK-negative, completely missing the fucose operon. The allelic profiles of six other loci were identical among 31 strains and to that of ST18, 122 and 621, and these strains were genetically closely related. Forty of 45 isolates were ampicillin-sensitive.Conclusions. The loss of fucK was frequently observed in clinical isolates of Hie from children. Moreover, fucK-negative Hie may be the cause of IHiD in adult patients. The majority of Hie, including fucK-negative strains, were shown to be clonally related and were ampicillin sensitive. This represents the first report examining fucK losses in encapsulated H. influenzae.

A novel stereoisomer of eushearilide, 23-demethyleushearilide, was synthesized, and the structure-activity relationships of this compound along with known eushearilide stereoisomers were investigated in order to design novel lead compounds for the treatment of fungal infections. It was discovered that all of these congeners, together with the natural product, exhibited a wide range of antimicrobial activity against not only fungi but also against bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).

Introduction. In 2016-2017, there was an increase in the number of paediatric invasive pneumococcal disease (IPD) cases caused by Streptococcus pneumoniae serotype 12F in Chiba Prefecture, Japan. Serotype 12F is one of the major causative serotypes of IPD following the introduction of pneumococcal conjugate vaccine 13 (PCV13), and outbreaks of IPD caused by serotype 12F have recently been reported in several countries.Aim. Our goal here was to clarify the relationship among local outbreak strains and the outbreak strains in other countries, and for this we analysed clinical isolates of S. pneumoniae serotype 12F using several genetic identification methods.Methodology. All reported IPD cases caused by serotype 12F were reviewed and bacterial strains were collected and analysed. We also analysed S. pneumoniae serotype 12F strains isolated from other time periods, geographical areas, cases of adult IPD and respiratory specimens as control strains. Multi-locus sequence typing, PFGE and multi-locus variable number tandem repeat analysis (MLVA) were conducted on all isolates.Results. All 26 S. pneumoniae serotype 12F isolates, including control strains, belonged to a single sequence type (ST4846) that was the specific ST in Japan. All tested strains demonstrated five MLVA patterns and two PFGE patterns.Conclusion. We determined that the 2016-2017 outbreak of IPD in Chiba Prefecture was caused by clonally related isolates of serotype 12F. The continuous monitoring of IPD caused by serotype 12F is important for evaluating the impact of re-emerging pneumococcal serotypes following the PCV13 introduction era, and MLVA could be a useful tool for identification of outbreak strains.

Panton Valentine Leukocidin (PVL) is one of the many toxins produced by Staphylococcus aureus. In Japan, PVL-positive S. aureus strains are mainly methicillin-resistant S. aureus (MRSA). Data regarding PVL-positive methicillin-sensitive S. aureus (MSSA) are scarce. In this report, we describe a case of severe infection by PVL-positive MSSA. A 12-year-old healthy girl was admitted with high fever and pain in the lower back. Computed tomography revealed a diagnosis of psoitis and multiple venous thromboses. Blood cultures obtained after admission revealed infection with MSSA. Her fever continued despite adequate antibiotic therapy. On the fifth hospitalization day, she developed bladder dysfunction, and an abscess was noted near the third lumbar vertebra. She underwent an emergency operation and recovered. Bacterial analyses revealed that the causative MSSA was a PVL-producing single variant of ST8 (related to USA300clone), of sequence type 2149. PVL is known to cause platelet activation. This case demonstrates the need for detailed analyses of the causative strain of bacteria in cases of S. aureus infection with deep vein thrombosis, even in cases of known MSSA infection.

Detecting Pneumocystis jirovecii by bronchoalveolar lavage or lung biopsy is the gold standard for diagnosis of P. jirovecii pneumonia (PJP); however, these techniques are not always applicable in children because of their high invasiveness. We report two pediatric cases of PJP diagnosed by polymerase chain reaction (PCR) of gastric lavage that were successfully treated. To date, there are no reported cases of using PCR of gastric lavage to diagnose PJP. On the day of PJP onset, both the infants required respiratory support and infiltrative shadows were observed in both lung fields on chest radiography. Furthermore, their (1 → 3)-β-D glucan levels were elevated. P. jirovecii was detected by PCR of gastric lavage and trimethoprim-sulfamethoxazole was administered for 3 weeks, following which their condition improved. They were long-term steroid users, but without any prophylaxis. PCR of gastric lavage in cases of suspected PJP may help in confirming the diagnosis in children who have mild to moderate airway symptoms, or have difficulty with invasive examination like bronchoscopy.

症例は新生児期に2度の遅発型B群溶連菌(Streptococcus agalactiae、Group B Streptococcus;GBS)菌血症を発症した女児。免疫学的素因のない正期産児であった。母体の産前GBSスクリーニングは陰性で、乳腺炎症状もなかったが、経過より経母乳感染が否定できないと考え、母乳培養を行った。児血液および母乳より発育したGBSの解析を行ったところ、血清型およびMLST(multi locus sequence typing)による遺伝子型が完全に一致した。経母乳感染の場合、通常の遅発型感染症よりも再発が多いことが知られているが、加えて本症例の血清型はIII型、遺伝子型はST17で、ともに高い再発率や侵襲性との関与が指摘されている血清型、遺伝子型であったことから、それも感染を繰り返した一因となったと考えている。本症例では、母乳栄養の中止と菌血症として通常の抗菌薬投与を行い、後遺症なく治癒し、再発もなく経過しているが、母体への抗菌薬投与を含めた発症時対応の標準化や、感染予防としての母体向けワクチンの導入など、今後の課題は多く、類似症例の蓄積が必須である。(著者抄録)

Chronic granulomatous disease (CGD) is a type of primary immunodeficiency disease, which increases susceptibility to recurrent bacterial and fungal infections. Sputum and bronchoalveolar lavage fluid are often obtained using bronchoscopy from adult patients for pathogenic diagnosis, although this approach is much more invasive for infants. We report the case of a 2-month-old boy with CGD, in which gastric aspirate culture was used to diagnose fungal pneumonia. Rasamsonia piperina was isolated from the gastric aspirate, and the patient was successfully treated with micafungin based on the drug susceptibility test results for the fungal isolate. The acid tolerance test revealed that R. piperina could grow at pH 2, indicating high acid resistance. Although we can only report our experience with a single case, gastric aspirate culture may be a useful tool for detecting fungal respiratory pathogens in children with primary immunodeficiency. Detecting these pathogens may help improve outcomes, as early diagnosis and appropriate treatment are extremely important for immunocompromised patients with respiratory infections.

肺炎球菌による尿路感染症の2歳女児例の経験を契機に、当院での過去の尿検体から同菌を検出した症例を抽出した。2010年4月から2017年9月までの過去7年6ヵ月間の尿培養結果から、肺炎球菌が検出された症例を後方視的に抽出した。肺炎球菌検出率は過去7年6ヵ月間で4/5390件(0.07%)であった。尿路感染症と診断された症例は1例のみで、他は汚染菌とみなされた。汚染菌とみなされた3検体の2症例はいずれも尿路奇形を伴っていなかった。また、全例が複雑な腎尿路疾患を有しており、免疫抑制状態が示唆されたのは腎移植後の1例であった。全例、βラクタム系抗菌薬7〜14日間の投与で軽快し、外科的介入を要するなどの治療に難渋した例はなかった。本症例では複数菌が同時検出されており、菌量も最も少なく汚染菌との鑑別がより重要であった。

Inactivated quadrivalent influenza vaccine (IIV4) has been used as seasonal influenza vaccine since 2016 in Japan. This study examined the safety of IIV4 in comparison with the AH1pdm monovalent vaccine used for novel influenza in 2009. Questionnaire surveillance associated with adverse events (AEs) was conducted at Chiba University Hospital, Japan. After being vaccinated, all health care workers (HCWs) were given a daily AEs check sheet on which they recorded solicited events, the same surveillance program used after AH1pdm vaccination in 2009. The frequency of injection site AEs with IIV4 was significantly higher than with the monovalent vaccine, but there was no significant difference with systemic AEs. Injection site and systemic AEs were reported as 83.7% and 25.5%, respectively, with IIV4. The grades of AE, mild, moderate and severe, were 67.2%, 16.4% and 0.1% with IIV4, respectively, indicating that almost all of the AEs reported with IIV4 were mild or moderate. Systemic AEs with IIV4 and monovalent vaccine were reported to be 25.5% and 23.1%, respectively, with the difference not being significant. The grade of AEs with IIV4, mild, moderate and severe, was 19.1%, 5.6% and 0.9%, respectively. The ratio of HCWs reporting AEs peaked at around 80% on day 1, then decreasing to less than 5% by day 7. AEs with IIV4 were reported more frequently compared with the AH1pdm monovalent vaccine. However, in consideration of the grade and duration of AEs, IIV4 was a well-tolerated, safe vaccine.

煩雑であった核酸精製から核酸増幅・検出を自動化したTRCReadyシステムを利用し、開発したマイコプラズマ・リボゾームRNA試薬(TRCReady MP)を用い、本試薬の臨床的有用性を評価した。臨床的に肺炎マイコプラズマ感染症が疑われた症例を含む急性呼吸器感染症と診断した患者290例を対象とした。PCR法を基にした本キットの感度は98.2%、特異度は95.7%、全体一致率は96.2%、LAMP法を基にした本キットの感度は100%、特異度は95.3%、全体一致率は96.2%であった。小児例を対象とした本キットの感度は97.7%、特異度93.0%、全体一致率95.0%、成人例を対象とした感度は100%、特異度96.6%、全体一致率96.8%であり、いずれも成人の方が高い感度・特異度・全体一致率を示した。また、上咽頭ぬぐい液でのキットとPCRの比較では、感度97.6%、特異度88.7%、全体一致率92.0%、喀痰でのキットとPCR法の比較の感度100%、特異度98.8%、全体一致率98.9%であり、感度・特異度・全体一致率いずれも喀痰の方が咽頭ぬぐい液に比べ高い値を示した。

症例は6歳女児。短腸症候群のため10ヵ月時より中心静脈で栄養管理していた。肺炎球菌による菌血症で入院し、抗菌薬開始後解熱したが、肉眼的血尿と蛋白尿に加え、乏尿と顔面・四肢浮腫、高血圧、低補体血症を認めた。カテーテルを抜去したところ、速やかに解熱、蛋白尿は消失し、顕微鏡学的血尿のみ残存した。中心静脈カテーテル感染症に関連した腎炎では抗菌薬治療に加え、早期のカテーテル抜去が望ましいと考えられた。(著者抄録)

BACKGROUND: Haemophilus influenzae type b (Hib) vaccine was introduced as a voluntary vaccine in December 2008 and was included in the national routine immunization program in April 2013 in Japan. Currently, no nationwide data are available to evaluate the effectiveness of Hib vaccine in Japan. METHODS: To evaluate the effectiveness of Hib vaccine in Japan, nationwide active population-based surveillance of culture-proven invasive infections caused by H. influenzae in children was performed in 2008-2017 in 10 prefectures in Japan (covering approximately 23% of the total Japanese population). Clinical data were recorded on a standardized case report form. Capsular type and antimicrobial susceptibility of the H. influenzae isolates were examined. The incidence rate ratio (IRR) and its confidence interval (CI) were calculated to compare data from 5 years before and that from after the introduction of the national routine Hib vaccine immunization program. RESULTS: During the 10-year study period, 566 invasive H. influenzae disease cases including 336 meningitis cases were identified. The average number of invasive H. influenzae disease cases among children <5 years of age during 2013-2017 decreased by 93% (IRR: 0.07, 95%CI 0.05-0.10, p < 0.001) compared with those occurring during 2008-2012. Hib strains have not been isolated from invasive H. influenzae disease cases since 2014; however, non-typeable H. influenzae and H. influenzae type f isolates have been noted as causes of invasive H. influenzae diseases among children <5 years in the post-Hib vaccine era. CONCLUSIONS: After the governmental subsidization of the Hib vaccine, invasive Hib disease cases decreased dramatically in the study population, as per our surveillance. Continuous surveillance is necessary to monitor the effectiveness of Hib vaccine and for detecting any emerging invasive capsular types.

Invasive fungal infection (IFI) is a life-threating infectious disease in high-risk neonates. Strategies for the treatment and prevention of IFI in neonates in Japan remain unclear. We conducted a nationwide retrospective survey to determine IFI incidence between January 2014 and October 2015. Primary survey questionnaires were submitted to 309 medical facilities that regularly treat high-risk neonates. The questionnaire assessed IFI incidence during the study period, methods for preventing fungal infection in early delivery neonates, and methods for preventing mother-to-child fungal transmission. The secondary questionnaire was for facilities that had IFI cases and replied to the primary questionnaire. In total, 128 medical facilities (41.4%) completed the primary questionnaire, 17/128 facilities recorded 23 proven or probable IFI cases. Estimated annual IFI incidence was 0.33/1000 live births of hospitalized neonates. Patient data at IFI onset were available for all 23 patients. Birth weight was < 1000 g in 18 patients. Causative microorganisms were identified in 22 patients. Candida species (n = 21) were the most common pathogens, and one patient had mucormycosis. The mortality rate was 17.4%. Regarding neonatal fungal prophylaxis, 55/128 facilities (43.0%) reported administering therapy. The most frequently used prophylactic drugs were fluconazole, then micafungin. Fungal prophylaxis for mothers who showed fungal colonization was performed in 30/128 facilities (23.4%). Oxiconazole vaginal tablets were most commonly used as prophylaxis for high-risk mothers. In Japan, the diagnosis, treatment, and prevention of neonatal IFI varied. Continuous surveillance and treatment regimen for neonatal IFI are required to improve outcomes in high-risk neonates.

Haemophilus influenzae is a major pathogenic bacteria causing invasive disease, which is classified into six capsular serotypes (a-f) and non-typeable (NT) strains. Capsular serotyping of H. influenzae is traditionally determined by serological methods and more recently by PCR methods. However, these methods are time-consuming and expensive. In the present study, matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated as an alternative method for capsular serotyping of H. influenzae clinical strains. We created an in-house database of all six serotypes and NT H. influenzae strains using the main spectrum creation standard method set to the default parameters in MADI-TOF MS. We evaluated the performance of the in-house database using 79 clinical strains already identified by PCR and 58 prospectively collected clinical strains. Measurements were performed using the Bruker MALDI BioTyper system. The peak list was matched against the reference library using the integrated pattern algorithm of the software. The best-matched spectrum was considered the serotyping result. All 137 test strains were correctly identified as H. influenzae using MALDI-TOF MS. The sensitivity and specificity for identification for type b, type e, and type f capsular serotypes and NT H. influenzae using MALDI-TOF MS were 100%/94.3%, 94.7%/97.9%, 97.4%/97.9%, and 85.5%/99.2%, respectively. Our findings indicate that MALDI-TOF MS is a useful alternative method for capsular serotyping of H. influenzae strains. This method is faster and more cost-effective than traditional methods and will therefore be useful for routine applications in clinical laboratories.

Haemophilus influenzae type b (Hib) conjugate vaccines have led to dramatic reductions in Hib disease among young children worldwide. Nontypeable H. influenzae (NTHi) is now the major cause of invasive H. influenzae infections. We investigated the clinical characteristics of invasive NTHi diseases among children in Japan, to clarify the pathogenicity of isolated NTHi strains. The mortality rate was 10.7%, with deaths occurring mainly among children with underlying comorbidities. Biotypes II and III were the most common, and most strains (64.3%) had multiple amino acid substitutions at the Asp-350, Ser-357, Ser-385, and/or Met-377 sites of penicillin-binding protein 3. Two strains were β-lactamase positive and ampicillin-clavulanate resistant. Biofilm indices varied widely, and IS1016 was detected in 10.7% of the strains tested. Moreover, there was wide variation in the characteristics of invasive NTHi strains. NTHi strains, showing great genetic diversity, are responsible for most invasive H. influenzae infections in children in the postvaccine era. Continuous monitoring of NTHi strains responsible for invasive diseases in children is important to detect changes in the epidemiology of invasive H. influenzae infections in the postvaccine era.

We elucidated a unique feature of sulfur metabolism in Cryptococcus neoformans. C. neoformans produces cysteine solely by the O-acetylserine pathway that consists of serine-O-acetyl transferase and cysteine synthase. We designated the gene encoding the former enzyme CYS2 (locus tag CNE02740) and the latter enzyme CYS1 (locus tag CNL05880). The cys1Δmutant strain was found to be avirulent in a murine infection model. Methionine practically does not support growth of the cys1Δ strain, and cysteine does not serve as a methionine source, indicating that the transsulfuration pathway does not contribute to sulfur amino acid synthesis in C. neoformans. Among the genes encoding enzymes catalyzing the reactions from homoserine to methionine, the gene corresponding to the Saccharomyces cerevisiae MET17 encoding O-acetylhomoserine sulfhydrylase (Met17p) had remained to be identified in C. neoformans. By genetic analysis of Met- mutants obtained by Agrobacterium tumefaciens-mediated mutagenesis, we concluded that Cnc01220, most similar to Str2p (36% identity), cystathionine-γ-synthase, in the Saccharomyces genome, is the C. neoformans version of O-acetylhomoserine sulfhydrylase. We designated CNC01220 as MET17. The C. neoformans met3Δ mutant defective in the first step of the sulfate assimilation pathway, sulfate adenylyltransferase, barely uses methionine as a sulfur source, whereas it uses cysteine efficiently. The poor utilization of methionine by the met3Δ mutant is most probably due to the absence of the transsulfuration pathway, causing an incapability of C. neoformans to produce cysteine and hydrogen sulfide from methionine. When cysteine is used as a sulfur source, methionine is likely produced de novo by using hydrogen sulfide derived from cysteine via an unidentified pathway. Altogether, the unique features of sulfur amino acid metabolism in C. neoformans will make this fungus a valuable experimental system to develop anti-fungal agents and to investigate physiology of hydrogen sulfide.

Although invasive meningococcal disease is rare in Japan (0.028 cases per 100,000 population), its incidence is 10 times greater in many other countries. Colonization is a prerequisite for invasive meningococcal disease. However, no study in Japan has involved specifically analyzing the carriage rate of Neisseria meningitidis in children. During 5 months in 2015, the respiratory tract specimens of patients who presented to 3 hospitals with respiratory symptoms were cultured. The bacteria were identified in selective media using a meningococcal detection kit and the serogroup was identified using polymerase chain reaction analysis. In 389 patients aged ≤15 years with respiratory symptoms, the N. meningitidis isolation rate was 0.26% (1/389). The serogroup of the only child who tested positive was Y. In this study, we detected a low meningococcal isolation rate in pediatric patients. Due to increasing globalization, the risk of invasive meningococcal disease is likely increasing in Japan. Accordingly, invasive meningococcal diseases should be continuously monitored in Japan. Future large-scale studies should assess meningococcal isolation rates and corresponding serogroups.

We report a case of mediastinal subcutaneous and multiple muscular abscesses caused by group B streptococcus serotype VIII in a type 2 diabetes mellitus patient. The patient arrived at the hospital with the chief complaint of immobility, and blood examination results suggested an acute infection and poorly controlled diabetes mellitus. Group B streptococcal bacteria were cultured from the patient's blood, and identified as serotype VIII upon further analysis. The patient recovered without any sequelae after percutaneous drainage, antibiotic therapy, and intensive glycemic control. Although the incidence of group B streptococcal infection in non-pregnant adults has recently increased in many developed countries, information on serotype VIII infection is quite limited. The reason is that serotype VIII group B streptococci are a Japan-specific serotype, and rarely cause invasive infections, even in Japan. Therefore, further surveillance and case reports should be documented in the future.

＜文献概要＞RSウイルスは,小児呼吸器感染症の代表的原因ウイルスであり,2歳までにほぼ100%の児が感染する.なかには重症化する例もあり,世界的にみると死亡者数も多い感染症である.しかし,いまだ確立された特異的治療はなく,認可されたRSウイルスワクチンもない.現在開発中のものとしては,生後早期の乳児の感染予防目的の妊婦ワクチン,乳幼児や高齢者対象のワクチンの臨床試験が進められている.また,予防接種ではないが,半減期が長く接種回数が少なくて済むモノクローナル抗体製剤の開発も進められている.治療薬に関してもいくつか臨床試験が進められている.今後さらに研究が進み,早期にこれらの臨床使用が可能となることが望まれる.

日本において、侵襲性インフルエンザ菌b型(Hib)感染症、侵襲性肺炎球菌感染症に易感染性を示す基礎疾患を有する定期接種対象年齢外のハイリスク小児に対するHibワクチン、肺炎球菌ワクチンが、臨床現場でどの程度接種されているか明確になっていない。そこで、定期接種対象年齢外の無脾症・摘脾・脾機能不全、血液腫瘍疾患、原発性免疫不全症、臓器移植後の小児に対するHibワクチン、13価肺炎球菌結合型ワクチン(PCV13)、23価肺炎球菌莢膜多糖体ワクチン(PPSV23)接種状況調査を日本小児感染症学会のメーリングリストを用いて行った。91施設から回答があり、48%が上記ハイリスク小児の診療を行っていた。約50%の施設が定期接種年齢対象外のハイリスク小児にHibワクチン、PCV13を推奨していたが、その推定接種率には幅が認められた。PPSV23に関しては、無脾症・摘脾・脾機能不全群では、92%の施設で最低1回の接種が推奨されていたが、他のハイリスク小児に対しては、積極的な接種は行われていなかった。今後、ハイリスク小児に対する具体的な接種勧奨指針の策定と接種環境の整備が必要である。(著者抄録)

造血器腫瘍または固型腫瘍にて経過観察中の小児41名(5〜18歳)から血清試料を採取し、肺炎球菌PCV13(非PCV7)血清型に対する特異的抗体を測定した。患児はいずれもPCV13接種を受けていなかった。6つの血清型すべてについて患児のIgGレベルは同年齢の健常対照と比べて低かった。採血前6ヵ月以内に化学療法を受けた患児では他の患児と比べて血清型特異的IgGレベルが低かった。以上より、年齢を理由に定期的ワクチン接種を推奨されていない免疫不全状態の小児の多くにおいて、侵襲性肺炎球菌感染症の予防に十分なPCV13血清型特異的IgG値に達していないことが判明した。

&lt;p&gt;小児がん患者では，真菌感染症が疑われる場合でも，真菌の分離同定はしばしば困難であり臨床経過により診断治療が行われることが多い．2004年1月から2014年12月までに当科で治療を受けた小児血液がん患者6人より分離同定された糸状菌2株，酵母5株に関し，薬剤感受性試験を行い，分離同定することの意義について後方視的に検討した．糸状菌はいずれも耳漏より検出された．1例では好中球抑制期間に外耳炎を繰り返し，&lt;i&gt;Aspergillus terreus&lt;/i&gt;が同定された．薬剤感受性試験の結果よりミカファンギン（MCFG），ボリコナゾール（VRCZ）を併用し造血幹細胞移植を行った．酵母はすべてカンジダで，血液より分離同定された．&lt;i&gt;Candida tropicalis&lt;/i&gt;分離例は治療開始後にβ-Dグルカンの上昇，脾膿瘍の悪化を認めたが感受性試験にてMCFG感受性良好であることを確認し治療遂行できた．&lt;i&gt;C. parapsilosis&lt;/i&gt;，&lt;i&gt;C. glabrata&lt;/i&gt;分離例はいずれもMCFG投与下のブレイクスルー感染であった．MCFG感受性良好として知られている&lt;i&gt;C. glabrata&lt;/i&gt;に関しては薬剤感受性試験の結果，キャンディン系薬剤に対するMICの上昇が確認された．近年米国でもキャンディン系耐性カンジダが問題となっており，今後小児がん患者においても，治療効果が思わしくない際には薬剤感受試験を行うことが必要だろう．&lt;/p&gt;

The introduction of the Haemophilus influenzae type b (Hib) vaccine and the 7-valent pneumococcal conjugate vaccine (PCV7) has led to dramatic reductions in cases of invasive H. influenzae disease and invasive pneumococcal disease (IPD). After the introduction of the PCV7 and the 13-valent pneumococcal conjugate vaccine (PCV13), the number of children with IPD markedly decreased in our hospital. However, since 2015, three children with IPD have been admitted to our hospital. We analyzed the serotype, multilocus sequence type, and antimicrobial susceptibility of Streptococcus pneumoniae strains isolated in these newly diagnosed cases. The strains were serotypes 7F and 12F. In addition, we analyzed the incidence of invasive bacterial disease before and after the introduction of conjugate vaccines and found no change in the incidences. We found that cases of IPD and invasive H. influenzae disease clearly decreased following the introduction of the PCV7, the PCV13, and the Hib vaccine, as well as disease caused by antibiotic-resistant strains.

We constructed deletion mutants of Cryptococcus neoformans var neoformans (serotype D) genes encoding late ergosterol biosynthetic pathway enzymes and found that the mutations enhanced susceptibility to various drugs including micafungin, one of the echinocandins, to which wild-type Cryptococcus strains show no susceptibility. Furthermore, through isolation of a mutant resistant to micafungin from a micafungin-sensitive erg mutant and genetic analysis of it, we found that the responsible mutation occurred in the hotspot 2 of FKS1 encoding β-1, 3-glucan synthase, indicating that micafungin inhibited the growth of the erg mutant via inhibiting Fks1 activity. Addition of ergosterol to the culture of the erg mutants recovered the resistance to micafungin, suggesting that the presence of ergosterol in membrane inhibits the accession of micafungin to its target. We found that a loss of one of genes encoding subunits of v-ATPase, VPH1, made Cryptococcus cells sensitive to micafungin. Our observation that the erg2 vph1 double mutant was more sensitive to micafungin than either single mutant suggests that these two genes act differently in becoming resistant to micafungin. The erg mutants allowed us to study the physiological significance of β-1, 3-glucan synthesis in C. neoformans; the inhibition of β-1, 3-glucan synthesis induced cell death and changes in cellular morphology. By observing the erg mutant cells recovering from the growth inhibition imposed by micafungin, we recognized β-1, 3-glucan synthesis would suppress filamentous growth in C. neoformans.

It is well known that 5-fluoroorotic acid (5-FOA)-resistant mutants isolated from wild-type Cryptococcus neoformans are exclusively either ura3 or ura5 mutants. Unexpectedly, many of the 5-FOA-resistant mutants isolated in our selective regime were Ura+. We identified CNM00460 as the gene responsible for these mutations. Cnm00460 belongs to the nucleobase cation symporter 1/purine-related transporter (NCS1/PRT) super family of fungal transporters, representative members of which are uracil transporter, uridine transporter and allantoin transporter of Saccharomyces cerevisiae. Since the CNM00460 gene turned out to be involved in utilization of orotic acid, most probably as transporter, we designated this gene Orotic Acid Transporter 1 (OAT1). This is the first report of orotic acid transporter in this family. C. neoformans has four members of the NCS1/PRT family, including Cnm00460, Cnm02550, Cnj00690, and Cnn02280. Since the cnm02550∆ strain showed resistance to 5-fluorouridine, we concluded that CNM02550 encodes uridine permease and designated it URidine Permease 1 (URP1). We found that oat1 mutants were sensitive to 5-FOA in the medium containing proline as nitrogen source. A mutation in the GAT1 gene, a positive transcriptional regulator of genes under the control of nitrogen metabolite repression, in the genetic background of oat1 conferred the phenotype of weak resistance to 5-FOA even in the medium using proline as nitrogen source. Thus, we proposed the existence of another orotic acid utilization system (tentatively designated OAT2) whose expression is under the control of nitrogen metabolite repression at least in part. We found that the OAT1 gene is necessary for full pathogenic activity of C. neoformans var. neoformans.