Mycolyltransferase is important for biofilm formation and pathogenesis of Tsukamurella keratitis.

Tsukamurella, a group of multi-drug resistant, Gram-positive, aerobic, and partially acid-fast bacteria, are emerging causes of bacterial conjunctivitis and keratitis. However, the pathogenesis of Tsukamurella keratitis is largely unknown. To address this, we used New Zealand White rabbits to develop the first eye infection model and conducted in vitro tests to study the pathogenesis mechanisms of Tsukamurella. There is increasing evidence that biofilms play a significant role in ocular infections, leading us to hypothesize that biofilm formation is crucial for effective Tsukamurella infection. In order to look for potential candidate genes which are important in biofilm formation and Tsukamurella keratitis. We performed genome sequencing of two ocular isolates, T. pulmonis-PW1004 and T. tyrosinosolvens-PW899, to identify potential virulence factors. Through in vitro and in vivo studies, we characterized their biological roles in mediating Tsukamurella keratitis. Our findings confirmed that Tsukamurella is an ocular pathogen by fulfilling Koch's postulates, and using genome sequence data, we identified tmytC, encoding a mycolyltransferase, as a crucial gene in biofilm formation and causing Tsukamurella keratitis in the rabbit model. This is the first report demonstrating the novel role of mycolyltransferase in causing ocular infections. Overall, our findings contribute to a better understanding of Tsukamurella pathogenesis and provide a potential target for treatment. Specific inhibitors targeting TmytC could serve as an effective treatment option for Tsukamurella infections.

Genetic mechanisms of co-emergence of INH-resistant Mycobacterium tuberculosis strains during the standard course of antituberculosis therapy.

The incidence of isoniazid (INH) resistant Mycobacterium tuberculosis is increasing globally. This study aimed to identify the molecular mechanisms behind the development of INH resistance in M. tuberculosis strains collected from the same patients during the standard course of treatment. Three M. tuberculosis strains were collected from a patient before and during antituberculosis (anti-TB) therapy. The strains were characterized using phenotypic drug susceptibility tests, Mycobacterial Interspersed Repeated Unit-Variable-Number Tandem Repeats (MIRU-VNTR), and whole-genome sequencing (WGS) to identify mutations associated with INH resistance. To validate the role of the novel mutations in INH resistance, the mutated katG genes were electroporated into a KatG-deleted M. tuberculosis strain (GA03). Three-dimensional structures of mutated KatG were modeled to predict their impact on INH binding. The pre-treatment strain was susceptible to INH. However, two INH-resistant strains were isolated from the patient after anti-TB therapy. MIRU-VNTR and WGS revealed that the three strains were clonally identical. A missense mutation (P232L) and a nonsense mutation (Q461Stop) were identified in the katG of the two post-treatment strains, respectively. Transformation experiments showed that katG of the pre-treatment strain restored INH susceptibility in GA03, whereas the mutated katG genes from the post-treatment strains rendered negative catalase activity and INH resistance. The protein model indicated that P232L reduced INH-KatG binding affinity while Q461Stop truncated gene transcription. Our results showed that the two katG mutations, P232L and Q461Stop, accounted for the co-emergence of INH-resistant clones during anti-TB therapy. The inclusion of these mutations in the design of molecular assays could increase the diagnostic performance.IMPORTANCEThe evolution of drug-resistant strains of Mycobacterium tuberculosis within the lung lesions of a patient has a detrimental impact on treatment outcomes. This is particularly concerning for isoniazid (INH), which is the most potent first-line antimycobacterial drug. However, the precise genetic factors responsible for drug resistance in patients have not been fully elucidated, with approximately 15% of INH-resistant strains harboring unknown genetic factors. This raises concerns about the emergence of drug-resistant clones within patients, further contributing to the global epidemic of resistance. In this study, we revealed the presence of two novel katG mutations, which emerged independently due to the stress exerted by antituberculosis (anti-TB) treatment on a parental strain. Importantly, we experimentally demonstrated the functional significance of both mutations in conferring resistance to INH. Overall, this research sheds light on the genetic mechanisms underlying the evolution of INH resistance within patients and provides valuable insights for improving diagnostic performance by targeting specific mutations.

Spatial Relevance: Teaching History to Medical Students at a Medical Museum in Hong Kong.

This article examines the pedagogical significance of history workshops as part of the mandatory medical curriculum in Hong Kong. At the University of Hong Kong, year one medical students must take a three-hour long history workshop at the Hong Kong Museum of Medical Sciences. We argue that by immersing experiential museum learning into the official medical curriculum, students can grow interest in Hong Kong's local medical history and discover its spatial relevance to their future practice. Moreover, students are equipped with analytical skills to tackle important agendas, such as historical contingency, multicausality of diseases, and perspectivism in dealing with conflicting narratives. However, we also notice that the way histories are curated in the museum and through the heritage trail could potentially constrain students to develop a limited historiography. The museum exhibitions and the trail walk mostly curated by medical professionals emphasize too much of the comparison of the bubonic plague and SARS that took place in 1894 and 2003. Students might assume the linear progression of medical sciences and the oversimplified dichotomy between traditional and modern medicine. In addition, disproportionate narratives of infectious and non-infectious disease in Hong Kong might result in oversight of the chronicity of general ill health conditions that have long been suffered by local people. Workshop conveners, therefore, need to constantly modify discussion questions to balance demands between the advancement of contemporary medicine emphasized in medical education and the critical thinking process offered by history.

Risk Factors for Slow Viral Decline in COVID-19 Patients during the 2022 Omicron Wave.

Formulating termination of isolation (de-isolation) policies requires up-to-date knowledge about viral shedding dynamics. However, current de-isolation policies are largely based on viral load data obtained before the emergence of Omicron variant. In this retrospective cohort study involving adult patients hospitalised for COVID-19 between January and February 2022, we sought to determine SARS-CoV-2 viral shedding kinetics and to investigate the risk factors associated with slow viral decline during the 2022 Omicron wave. A total of 104 patients were included. The viral load was highest (Ct value was lowest) on days 1 post-symptom-onset (PSO) and gradually declined. Older age, hypertension, hyperlipidaemia and chronic kidney disease were associated with slow viral decline in the univariate analysis on both day 7 and day 10 PSO, while incomplete or no vaccination was associated with slow viral decline on day 7 PSO only. However, older age was the only risk factor that remained statistically significant in the multivariate analysis. In conclusion, older age is an independent risk factor associated with slow viral decline in this study conducted during the Omicron-dominant 2022 COVID-19 wave. Transmission-based precaution guidelines should take age into consideration when determining the timing of de-isolation.

SARS-CoV-2 IgG seropositivity after the severe Omicron wave of COVID-19 in Hong Kong.

The SARS-CoV-2 Omicron variant has led to a major wave of COVID-19 in Hong Kong between January and May 2022. Here, we used seroprevalence to estimate the combined incidence of vaccination and SARS-CoV-2 infection, including subclinical infection which were not diagnosed at the acute stage. The overall seropositive rate of IgG against receptor binding domain (anti-RBD IgG) increased from 52.2% in December 2021 to 89.3% in May 2022. The level of anti-RBD IgG was lowest in the 0-9 and ≥80 year-old age groups in May 2022. The seropositive rate of antibody against ORF8, which reflects the rate of prior infection, was 23.4% in May 2022. Our data suggest that although most individuals were either vaccinated or infected after the fifth wave, children and older adults remain most vulnerable. Public health measures should target these age groups in order to ameliorate the healthcare consequences of upcoming waves.

Streptococcus oriscaviae sp. nov. Infection Associated with Guinea Pigs.

Pet bite-related infections are commonly caused by the pet's oral flora transmitted to the animal handlers through the bite wounds. In this study, we isolated a streptococcus, HKU75T, in pure culture from the purulent discharge collected from a guinea pig bite wound in a previously healthy young patient. HKU75T was alpha-hemolytic on sheep blood agar and agglutinated with Lancefield group D and group G antisera. API 20 STREP showed that the most likely identity for HKU75T was S. suis I with 85.4% confidence while Vitek 2 showed that HKU75T was unidentifiable. MALDI-TOF MS identified HKU75T as Streptococcus suis (score of 1.86 only). 16S rRNA gene sequencing showed that HKU75T was most closely related to S. parasuis (98.3% nucleotide identity), whereas partial groEL and rpoB gene sequencing showed that it was most closely related to S. suis (81.8% and 89.8% nucleotide identity respectively). Whole genome sequencing and intergenomic distance determined by ANI revealed that there was <85% identity between the genome of HKU75T and those of all other known Streptococcus species. Genome classification using concatenated sequences of 92 bacterial core genes showed that HKU75T belonged to the Suis group. groEL gene sequences identical to that of HKU75T could be directly amplified from the oral cavities of the two guinea pigs owned by the patient. HKU75T is a novel Streptococcus species, which we propose to be named S. oriscaviae. The oral cavity of guinea pigs is presumably a reservoir of S. oriscaviae. Some of the reported S. suis strains isolated from clinical specimens may be S. oriscaviae. IMPORTANCE We reported the discovery of a novel Streptococcus species, propose to be named Streptococcus oriscaviae, from the pus collected from a guinea pig bite wound in a healthy young patient. The bacterium was initially misidentified as S. suis/S. parasuis by biochemical tests, mass spectrometry. and housekeeping genes sequencing. Its novelty was confirmed by whole genome sequencing. Comparative genomic studies showed that S. oriscaviae belongs to the Suis group. S. oriscaviae sequences were detected in the oral cavities of the two guinea pigs owned by the patient, suggesting that the oral cavity of guinea pigs could be a reservoir of S. oriscaviae. Some of the reported S. suis strains may be S. oriscaviae. Further studies are warranted to refine our knowledge on this novel Streptococcus species.

IFN-α blockade during ART-treated SIV infection lowers tissue vDNA, rescues immune function, and improves overall health.

Type I IFNs (TI-IFNs) drive immune effector functions during acute viral infections and regulate cell cycling and systemic metabolism. That said, chronic TI-IFN signaling in the context of HIV infection treated with antiretroviral therapy (ART) also facilitates viral persistence, in part by promoting immunosuppressive responses and CD8+ T cell exhaustion. To determine whether inhibition of IFN-α might provide benefit in the setting of chronic, ART-treated SIV infection of rhesus macaques, we administered an anti-IFN-α antibody followed by an analytical treatment interruption (ATI). IFN-α blockade was well-tolerated and associated with lower expression of TI-IFN-inducible genes (including those that are antiviral) and reduced tissue viral DNA (vDNA). The reduction in vDNA was further accompanied by higher innate proinflammatory plasma cytokines, expression of monocyte activation genes, IL-12-induced effector CD8+ T cell genes, increased heme/metabolic activity, and lower plasma TGF-ß levels. Upon ATI, SIV-infected, ART-suppressed nonhuman primates treated with anti-IFN-α displayed lower levels of weight loss and improved erythroid function relative to untreated controls. Overall, these data demonstrated that IFN-α blockade during ART-treated SIV infection was safe and associated with the induction of immune/erythroid pathways that reduced viral persistence during ART while mitigating the weight loss and anemia that typically ensue after ART interruption.

COVID-19-Related Deaths And Excess Deaths Among Medicare Fee-For-Service Beneficiaries.

Millions of Americans have been infected with SARS-CoV-2, and more than 575,000 had died as of early May 2021. Understanding who are the most vulnerable populations for COVID-19 mortality and excess deaths is critical, especially as the US prioritizes vaccine distribution. Using Medicare administrative data, we found that beneficiaries residing in nursing homes, the oldest beneficiaries, members of racial/ethnic minority groups, beneficiaries with multiple comorbid conditions, and beneficiaries who are dually eligible for Medicare and Medicaid were disproportionately likely to die after infection with SARS-CoV-2. As the pandemic developed, Medicare data were quickly adapted to provide analyses and inform the nation's response to COVID-19. Similar data for the rest of the population, however, are not readily available. Developing policies and methods around data collection and access will be important to address the consequences of future pandemics and other health emergencies.

Tsukamurella asaccharolytica sp. nov., Tsukamurella conjunctivitidis sp. nov. and Tsukamurella sputi sp. nov., isolated from patients with bacteraemia, conjunctivitis and respiratory infection in Hong Kong.

Three bacterial strains, HKU70T, HKU71T and HKU72T, were isolated from the conjunctival swab, blood and sputum samples of three patients with conjunctivitis, bacteraemia and respiratory infection, respectively, in Hong Kong. The three strains were aerobic, Gram-stain positive, catalase-positive, non-sporulating and non-motile bacilli and exhibited unique biochemical profiles distinguishable from currently recognized Tsukamurella species. 16S rRNA, secA, rpoB and groEL gene sequence analyses revealed that the three strains shared 99.6-99.9, 94.5-96.8, 95.7-97.8 and 97.7-98.9 % nucleotide identities with their corresponding closest Tsukamurella species respectively. DNA-DNA hybridization confirmed that they were distinct from other known species of the genus Tsukamurella (26.2±2.4 to 36.8±1.2 % DNA-DNA relatedness), in line with results of in silico genome-to-genome comparison (32.2-40.9 % Genome-to-Genome Distance Calculator and 86.3-88.9 % average nucleotide identity values]. Fatty acids, mycolic acids, cell-wall sugars and peptidoglycan analyses showed that they were typical of members of Tsukamurella. The G+C content determined based on the genome sequence of strains HKU70T, HKU71T and HKU72T were 69.9, 70.2 and 70.5 mol%, respectively. Taken together, our results supported the proposition and description of three new species, i.e. Tsukamurella sputi HKU70T (=JCM 33387T=DSM 109106T) sp. nov., Tsukamurella asaccharolytica HKU71T (=JCM 33388T=DSM 109107T) sp. nov. and Tsukamurella conjunctivitidis HKU72T (=JCM 33389T=DSM 109108T) sp. nov.

Direct Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis by Use of pncA PCR Sequencing.

An in-house-developed pncA sequencing assay for analysis of pyrazinamide (PZA) resistance was evaluated using 162 archived Mycobacterium tuberculosis complex (MTBC) isolates with phenotypic PZA susceptibility profiles that were well defined by analysis of Bactec MGIT 960 PZA kit and PZase activity data. Preliminary results showed 100% concordance between pncA sequencing and phenotypic PZA drug susceptibility test (DST) results among archived isolates. Also, 637 respiratory specimens were prospectively collected, and 158 were reported as MTBC positive by the Abbott Realtime MTB assay (96.3% sensitivity [95% confidence interval {CI}: 92.2% to 98.7%]; 100% specificity [95% CI: 99.2% to 100.0%]). Genotypic and phenotypic PZA resistance profiles of these 158 MTBC-positive specimens were analyzed by pncA sequencing and Bactec MGIT 960 PZA kit, respectively. For analysis of PZA resistance, pncA sequencing detected pncA mutations in 5/5 (100%) phenotypic PZA-resistant respiratory specimens within 4 working days. No pncA mutations were detected among PZA-susceptible specimens. Combining archived isolates with prospective specimens, 27 were identified as phenotypic PZA resistant with pncA mutation. Among these 27 samples, 6/27 (22.2%) phenotypic PZA-resistant strains carried novel pncA mutations without rpsA and panD mutations. These included 5 with mutations (a deletion [Del] at 383T [Del383T], Del 380 to 390, insertion of A [A Ins] at position 127, A Ins at position 407, and G Ins at position 508) in pncA structural genes and 1 with a mutation (T-12C) at the pncA promoter region. All six of these strains had no or reduced PZase activities, indicating that the novel mutations might confer PZA resistance. Additionally, 25/27 phenotypic PZA-resistant strains were confirmed multidrug-resistant tuberculosis (MDR-TB) strains. As PZA is commonly used in MDR-TB treatment regimens, direct pncA sequencing will rapidly detect PZA resistance and facilitate judicious use of PZA in treating PZA-susceptible MDR-TB.

Improving the specific diagnosis of trematode, cestode and nematode infections by a multiplex single-tube real-time PCR assay.

AIMS: Helminth infections are becoming uncommon in high-income countries and laboratory staff may lose expertise in their morphological identification, especially in histological sections where speciation of helminths is challenging. Commercially available molecular diagnostic panels for faecal specimens only offer tests for protozoa but not helminths. We aim to improve the identification accuracy of helminths using a multiplex PCR assay. METHODS: We designed three pairs of PCR primers and probes targeting multicopy genes for a multiplex single-tube real-time PCR assay which covers 16 trematode (28S rRNA gene), 24 cestode (cox1 gene) and 33 nematode (cox1 gene) species. Helminths (n=27) from faecal samples (n=10), fresh parasites (n=11), formalin-fixed specimens (n=4), cerebrospinal fluid (n=1) and bile (n=1) were examined morphologically and tested by PCR. Fifty stool samples negative for parasites by microscopy were also tested. RESULTS: The PCR assay correctly identified the genera of all tested helminths. Agarose gel electrophoresis and sequencing of the purified PCR amplicons confirmed that the PCR products were of correct sizes with 100% correlation with the respective species. Sequencing of the cox1 gene failed to identify Capillaria spp. in one sample owing to the lack of corresponding sequences in GenBank. PCR and sequencing of the nematode 18S rRNA gene using consensus primers showed 100% homology with Capillaria spp. sequence. No positive PCR products were found in the negative stool samples. CONCLUSIONS: The highly specific test correctly identified all helminths in our cohort. It is a useful adjunct to helminth identification in difficult situations such as histological sections.

Diagnostic evaluation of an in-house developed single-tube, duplex, nested IS6110 real-time PCR assay for rapid pulmonary tuberculosis diagnosis.

OBJECTIVE: To perform a prospective evaluation on the diagnostic performance of an in-house developed, duplex nested IS6110 real-time Polymerase-Chain-Reaction (PCR) assay (IS6110-qPCR assay) for rapid pulmonary TB diagnosis. METHODS: A total of 503 sputum specimens were prospectively collected from July 2016 to November 2016. Diagnostic accuracy and optimal cut-off Cycle-threshold (Ct) value for IS6110-qPCR assay was determined by Receiver Operating Characteristic (ROC) curve. Using the optimal cut-off Ct, diagnostic performance of IS6110-qPCR assay was assessed with reference to both bacteriological and clinical information. Meanwhile, limit of detection (LOD) was calculated using Mycobacterium tuberculosis H37Rv as reference strain. RESULT: ROC curve analysis of IS6110-qPCR assay showed a high Area Under the Curve (AUC) value (0.948) with optimal Ct value at 24.140. Prospective analysis of IS6110-qPCR assay with cut-off Ct = 24.140 showed a high overall sensitivity and specificity of 97.2% and 99.7%, respectively. No cross reactivity was observed among all non-tuberculous mycobacteria specimens in this study. LOD analysis on MTB-spiked sputum showed an average detection limit of 5.0 CFU/mL at Ct = 23.18 (±SD, 0.57). CONCLUSION: IS6110-qPCR assay is a highly accurate and cost-effective assay developed for primary screening of suspected TB cases, which is particularly suitable for regions with limited resources but high TB burden.

Comparative evaluation of a laboratory-developed real-time PCR assay and RealStar® Adenovirus PCR Kit for quantitative detection of human adenovirus.

BACKGROUND: Human adenoviruses are common causes of community-acquired respiratory tract and enteric infections. Severe disseminated infections with high mortality rates may be seen in immunocompromised individuals. An accurate and cost-effective quantitative assay is essential not only for laboratory diagnosis of adenoviral infections, but also for monitoring of response to antiviral treatment. The diagnostic performance of an in-house quantitative polymerase chain reaction assay was compared to a commercial system. METHODS: The analytical sensitivity, specificity, linearity, precision and accuracy of an in-house adenovirus quantitative polymerase chain reaction assay were evaluated against the RealStar® Adenovirus PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany), using 122 clinical specimens and 18 proficiency testing samples. RESULTS: Linear regression analysis of the quantitative results by the in-house assay showed the dynamic range from 2.60 to 9 log10 (plasma) and 2.94 to 9 log10 (viral transport medium) copies/mL, with the coefficient of determination (R2) of 0.996 and 0.998, respectively. A dilution series demonstrated the limits of detection and lower limits of quantification for plasma were 2.06 log10 and 2.60 log10 copies/mL and those for viral transport medium were 2.31 log10 and 2.94 log10 copies/mL respectively. The precision of the in-house assay was highly reproducible among runs with coefficients of variance ranging from 0.07 to 3.21% for plasma and 0.17% to 2.11% for viral transport medium. A comparison of 52 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house assay and the RealStar® Adenovirus PCR Kit (R2 = 0.984), with an average bias of - 0.16 log10 copies/mL. CONCLUSIONS: The in-house adenovirus assay is a sensitive and reliable assay with lower cost for the detection and quantification of adenoviral DNA when compared to the RealStar® Adenovirus PCR Kit.

Performance in the Medicare Shared Savings Program by Accountable Care Organizations Disproportionately Serving Dual and Disabled Populations.

BACKGROUND: The growth of accountable care organizations (ACOs) and other alternative payment models has prompted concern about whether these models will disadvantage providers who serve vulnerable populations, particularly those living in poverty or with a disability. OBJECTIVE: To examine performance by ACOs in the top quintile of their proportion of beneficiaries dually enrolled in Medicare and Medicaid (high-dual) and the top quintile of disabled beneficiaries (high-disabled). RESEARCH DESIGN: This is a retrospective cohort study. SUBJECTS: The 333 ACOs in the Medicare Shared Savings Program in 2014, followed through 2016. MEASURES: Quality scores, savings per beneficiary, whether or not the ACO shared savings, and amount of shared savings. RESULTS: High-dual and high-disabled ACOs had slightly lower quality and similar or higher baseline spending than other ACOs, but achieved greater savings per beneficiary than other ACOs ($212 vs. $51 for high-dual ACOs, P=0.04; $241 vs. $44 for high-disabled ACOs, P=0.012). Further, these ACOs were equally or more likely to earn shared savings; just over 30% of high-dual ACOs earned shared savings compared with 25% of non-high-dual ACOs (P=0.35) and 38% of high-disabled ACOs earned shared savings compared with 23% of non-high-disabled ACOs (P=0.013). In longitudinal analyses, we found a decrease in the differences in quality between high-social risk and other ACOs over time. Savings remained higher for high-dual and high-disabled ACOs relative to other ACOs over 2014-2016 though the gap narrowed over time. CONCLUSIONS: High-dual and high-disabled ACOs had similar or higher spending than other ACOs at baseline, but achieved greater savings and were equally or more likely to earn shared savings, suggesting that alternative payment models can have positive financial outcomes for providers who serve vulnerable populations.

Metagenomic analysis of Sichuan takin fecal sample viromes reveals novel enterovirus and astrovirus.

The Sichuan takin inhabits the bamboo forests in the Eastern Himalayas and is considered as a national treasure of China with the highest legal protection and conservation status considered as vulnerable according to The IUCN Red List of Threatened Species. In this study, fecal samples of 71 Sichuan takins were pooled and deep sequenced. Among the 103,553 viral sequences, 21,961 were assigned to mammalian viruses. De novo assembly revealed genomes of an enterovirus and an astrovirus and contigs of circoviruses and genogroup I picobirnaviruses. Complete genome sequencing and phylogenetic analysis showed that Sichuan takin enterovirus is a novel serotype/genotype of the species Enterovirus G, with evidence of recombination. Sichuan takin astrovirus is a new subtype of bovine astrovirus, probably belonging to a new genogroup in the genus Mamastrovirus. Further studies will reveal whether these viruses can also be found in Mishmi takin and Shaanxi takin and their pathogenic potentials.

MALDI-TOF MS for identification of Tsukamurella species: Tsukamurella tyrosinosolvens as the predominant species associated with ocular infections.

Although Tsukamurella infections have been increasingly reported in Europe, Asia, America, and Africa, indicating that diseases caused by this group of bacteria are emerging in a global scale, species identification within this genus is difficult in most clinical microbiology laboratories. Recently, we showed that groEL gene sequencing is useful for identification of all existing Tsukamurella species. Nevertheless, PCR sequencing is still considered expensive, time-consuming, and technically demanding, and therefore is yet to be incorporated as a routine identification method in clinical laboratories. Using groEL gene sequencing as the reference method, 60 Tsukamurella isolates were identified as five different Tsukamurella species [T. tyrosinosolvens (n = 31), T. pulmonis (n = 25), T. hongkongensis (n = 2), T. strandjordii (n = 1), and T. sinensis (n = 1)]. The most common source of the patient isolates were the eye (n = 18), sputum (n = 6), and blood (n = 6). None of the 60 isolates were identified correctly to species level by MALDI-TOF MS with the original Bruker database V.6.0.0.0. Using the Bruker database extended with 15 type and reference strains which covered all the currently recognized 11 Tsukamurella species, 59 of the 60 isolates were correctly identified to the species level with score ≥2.0. MALDI-TOF MS should be useful for routine species identification of Tsukamurella in clinical microbiology laboratories after optimization of the database. T. tyrosinosolvens was the most common species observed in patients with Tsukamurella infections and the predominant species associated with ocular infections.

Tsukamurella ocularis sp. nov. and Tsukamurella hominis sp. nov., isolated from patients with conjunctivitis in Hong Kong.

Three bacterial strains, HKU63T, HKU64 and HKU65T, were isolated from the conjunctival swabs of three patients with conjunctivitis in Hong Kong. The three strains were aerobic, Gram-stain-positive, catalase-positive, non-sporulating and non-motile bacilli and exhibited unique biochemical profiles distinguishable from closely related Tsukamurella species. 16S rRNA gene sequence analysis revealed that the three strains shared identical sequences with each other, being most closely related to Tsukamurella tyrosinosolvens and Tsukamurella pulmonis, sharing 99.9 % sequence identity. Sequence analysis of three additional housekeeping genes, groEL, secA and rpoB, revealed 100 % nucleotide sequence identity between HKU63T and HKU64, 94.2-97.0 % nucleotide sequence identities between HKU63T/HKU64 and HKU65T and the three strains shared 82.9-98.9 % sequence identities with other currently recognized Tsukamurella species. DNA-DNA hybridization confirmed that they were distinct from other known species of the genus Tsukamurella(23.0±4.2 to 50.7±3.7 % DNA-DNA relatedness), of which HKU63T and HKU64 represented the same species (≥95.2±4.8 % DNA-DNA relatedness) while HKU65T represented another species. Fatty acid, mycolic acid, cell-wall sugar and peptidoglycan analyses showed that they were typical of members of Tsukamurella. The G+C content of strains HKU63T, HKU64 and HKU65T were 71.3±1.9, 71.3±2.0 and 71.2±2.3 mol% (mean±sd; n=3), respectively. A novel species, Tsukamurella ocularis sp. nov. is proposed to accommodate strains HKU63T and HKU64, with HKU63T (=JCM 31969T=DSM 105034T) designated as the type strain whilst another novel species, Tsukamurella hominis sp. nov., is proposed to accommodate the third strain, HKU65T, which is designated as the type strain (=JCM 31971T=DSM 105036T).

Comparative Genomic Analysis of Two Clonally Related Multidrug Resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing.

Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB under in vivo challenge of anti-TB drugs. Result: Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations at rpoB S531L, inhA C-15T, and embB M306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 of rv0888 in pre-treatment strain, and a missense mutation at rv3303c (lpdA) V44I and a 6-bp inframe deletion at codon 67-68 in rv2071c (cobM) in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function. Conclusion: This is the first study that compared the full genomes of two clonally-related MDR-TB clinical isolates during the course of anti-TB treatment. Our work has demonstrated the robustness of SMRT Sequencing in identifying mutations among MDR-TB clinical isolates. Comparative genome analysis also suggested novel mutations at rv0888, lpdA, and cobM that might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.

Bats host diverse parvoviruses as possible origin of mammalian dependoparvoviruses and source for bat-swine interspecies transmission.

Compared to the enormous species diversity of bats, relatively few parvoviruses have been reported. We detected diverse and potentially novel parvoviruses from bats in Hong Kong and mainland China. Parvoviruses belonging to Amdoparvovirus, Bocaparvovirus and Dependoparvovirus were detected in alimentary, liver and spleen samples from 16 different chiropteran species of five families by PCR. Phylogenetic analysis of partial helicase sequences showed that they potentially belonged to 25 bocaparvovirus, three dependoparvovirus and one amdoparvovirus species. Nearly complete genome sequencing confirmed the existence of at least four novel bat bocaparvovirus species (Rp-BtBoV1 and Rp-BtBoV2 from Rhinolophus pusillus, Rs-BtBoV2 from Rhinolophus sinicus and Rol-BtBoV1 from Rousettus leschenaultii) and two novel bat dependoparvovirus species (Rp-BtAAV1 from Rhinolophus pusillus and Rs-BtAAV1 from Rhinolophus sinicus). Rs-BtBoV2 was closely related to Ungulate bocaparvovirus 5 with 93, 72.1 and 78.7 % amino acid identities in the NS1, NP1 and VP1/VP2 genes, respectively. The detection of bat bocaparvoviruses, including Rs-BtBoV2, closely related to porcine bocaparvoviruses, suggests recent interspecies transmission of bocaparvoviruses between bats and swine. Moreover, Rp-BtAAV1 and Rs-BtAAV1 were most closely related to human AAV1 with 48.7 and 57.5 % amino acid identities in the rep gene. The phylogenetic relationship between BtAAVs and other mammalian AAVs suggests bats as the ancestral origin of mammalian AAVs. Furthermore, parvoviruses of the same species were detected from multiple bat species or families, supporting the ability of bat parvoviruses to cross species barriers. The results extend our knowledge on the diversity of bat parvoviruses and the role of bats in parvovirus evolution and emergence in humans and animals.