Metagenome and Resistome Analysis of Beta-Lactam-Resistant Bacteria Isolated from River Waters in Surabaya, Indonesia.

Antimicrobial agents are administered to humans and livestock, and bacterial antimicrobial resistance (AMR) and antimicrobial agents are released into the environment. In this study, to investigate the trend of AMR in humans, livestock, and the environment, we performed a metagenomic analysis of multidrug-resistant bacteria with CHROMagar ESBL in environmental river water samples, which were collected using syringe filter units from waters near hospitals, downtown areas, residential areas, and water treatment plants in Surabaya, Indonesia. Our results showed that Acinetobacter, Pseudomonas, Aeromonas, Enterobacter, Escherichia, and Klebsiella grew in CHROMagar ESBL; they were most frequently detected in water samples from rivers surrounding hospitals contaminated with various AMR genes (ARGs) in high levels. These results identified bacteria as ARG reservoirs and revealed that hospitals could be sources for various ARGs disseminated into the environment. In conclusion, this study details a novel metagenomic analysis of collected bacteria in environmental water samples using a syringe filter unit for an AMR epidemiological study based on the One Health approach.

An oral cancer vaccine using Bifidobacterium vector augments combination of anti-PD-1 and anti-CTLA-4 antibodies in mouse renal cell carcinoma model.

Recently, immune checkpoint inhibitor (ICI) based combination therapies, including anti-PD-1 antibody, nivolumab with anti-CTLA-4 antibody, and ipilimumab have become the primary treatment option for metastatic or unresectable renal cell carcinoma (RCC). However, despite the combination of two ICIs, 60-70% of patients are still resistant to first-line cancer immunotherapy. In the present study, undertook combination immunotherapy for RCC using an oral cancer vaccine (Bifidobacterium longum displaying WT1 tumor associated antigen (B. longum 420)) with anti-PD-1 and anti-CTLA-4 antibodies in a mouse syngeneic model of RCC to explore possible synergistic effects. We found that B. longum 420 significantly improved the survival of mice bearing RCC tumors treated by anti-PD-1 and anti-CTLA-4 antibodies compared to the mice treated by the antibodies alone. This result suggests that B. longum 420 oral cancer vaccine as an adjunct to ICIs could provide a novel treatment option for RCC patients. Our microbiome analysis revealed that the proportion of Lactobacilli was significantly increased by B. longum 420. Although the detailed mechanism of action is unknown, it is possible that microbiome alteration by B. longum 420 enhances the efficacy of the ICIs.

A recombinant adenovirus vector containing the synNotch receptor gene for the treatment of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is known as the most difficult molecular subtype of breast cancer to treat. Recent studies revealed that cancer stem cells (CSCs) play a critical role in TNBC recurrence and metastasis. In this study, we developed a recombinant replication-deficient adenoviral vector (Ad-CD44-N-HIF-3α4), which contains a gene encoding a synthetic Notch (synNotch) receptor composed of the extracellular domain of CD44 (CD44-ECD) and the hypoxia-inducible factor (HIF)-3α4 connected by the Notch core regulatory region. CD44 is a transmembrane glycoprotein and known as a CSC marker in breast cancer and other malignancies. HIF-3α4 is a dominant-negative regulator of HIF-1α and HIF-2α and inhibits hypoxia-inducing effect. Both CD44 and HIF signals contribute cancer stemness and maintaining CSCs in breast cancer. The CD44-ECD in the synNotch receptor acts as the CD44 decoy receptor, and after a ligand such as a hyaluronic acid binds to the CD44-ECD, HIF-3α4 is released from the Notch core domain. We performed an in vivo study using a mouse xenograft model of MDA-MB-231, a highly invasive TNBC cell, and confirmed the significant antitumor activity of the intratumoral injections of Ad-CD44-N-HIF3α4. Our findings in this study warrant the further development of Ad-CD44-N-HIF3α4 for the treatment of patients with TNBC.

Enhanced antitumor activity of a novel, oral, helper epitope-containing WT1 protein vaccine in a model of murine leukemia.

BACKGROUND: A Wilms' tumor 1 (WT1) oral vaccine, Bifidobacterium longum (B. longum) 420, in which the bacterium is used as a vector for WT1 protein, triggers immune responses through cellular immunity consisting of cytotoxic T lymphocytes (CTLs) and other immunocompetent cells (e.g., helper T cells). We developed a novel, oral, helper epitope-containing WT1 protein vaccine (B. longum 2656) to examine whether or not B. longum 420/2656 combination further accelerates the CD4+ T cell help-enhanced antitumor activity in a model of murine leukemia. METHODS: C1498-murine WT1-a genetically-engineered, murine leukemia cell line to express murine WT1-was used as tumor cell. Female C57BL/6 J mice were allocated to the B. longum 420, 2656, and 420/2656 combination groups. The day of subcutaneous inoculation of tumor cells was considered as day 0, and successful engraftment was verified on day 7. The oral administration of the vaccine by gavage was initiated on day 8. Tumor volume, the frequency and phenotypes of WT1-specific CTLs in CD8+ T cells in peripheral blood (PB) and tumor-infiltrating lymphocytes (TILs), as well as the proportion of interferon-gamma (INF-γ)-producing CD3+CD4+ T cells pulsed with WT135-52 peptide in splenocytes and TILs were determined. RESULTS: Tumor volume was significantly smaller (p < 0.01) in the B. longum 420/2656 combination group than in the B. longum 420 group on day 24. WT1-specific CTL frequency in CD8+ T cells in PB was significantly greater in the B. longum 420/2656 combination group than in the B. longum 420 group at weeks 4 (p < 0.05) and 6 (p < 0.01). The proportion of WT1-specific, effector memory CTLs in PB increased significantly in the B. longum 420/2656 combination group than in the B. longum 420 group at weeks 4 and 6 (p < 0.05 each). WT1-specific CTL frequency in intratumoral CD8+ T cells and the proportion of IFN-γ-producing CD3+CD4+ T cells in intratumoral CD4+ T cells increased significantly (p < 0.05 each) in the B. longum 420/2656 combination group than in the 420 group. CONCLUSIONS: B. longum 420/2656 combination further accelerated antitumor activity that relies on WT1-specific CTLs in the tumor compared with B. longum 420.

An oral WT1 protein vaccine composed of WT1-anchored, genetically engineered Bifidobacterium longum allows for intestinal immunity in mice with acute myeloid leukemia.

Wilms' tumor 1 (WT1) is a promising tumor-associated antigen for cancer immunotherapy. We developed an oral protein vaccine platform composed of WT1-anchored, genetically engineered Bifidobacterium longum (B. longum) and conducted an in vivo study in mice to examine its anticancer activity. Mice were orally treated with phosphate-buffered saline, wild-type B. longum105-A, B. longum 2012 displaying only galacto-N-biose/lacto-N-biose I-binding protein (GLBP), and WT1 protein- and GLBP-expressing B. longum 420. Tumor size reduced significantly in the B. longum 420 group than in the B. longum 105-A and 2012 groups (P < 0.00 l each), indicating B. longum 420's antitumor activity via WT1-specific immune responses. CD8+ T cells played a major role in the antitumor activity of B. longum 420. The proportion of CD103+CD11b+CD11c+ dendritic cells (DCs) increased in the Peyer's patches (PPs) from mice in the B. longum 420 group, indicating the definite activation of DCs. In the PPs, the number and proportion of CD8+ T cells capable of producing interferon-gamma were significantly greater in the B. longum 420 group than in the B. longum 2012 group (P < 0.05 or < 0.01). The production of WT1-specific IgG antibody was significantly higher in the B. longum 420 group than in the 2012 group (P < 0.05). The B. longum 420 group showed the most intense intratumoral infiltration of CD4+ and CD8+ T cells primed by activated DCs in the PPs of mice in the B. longum 420 group. Our findings provide insights into a novel, intestinal bacterium-based, cancer immunotherapy through intestinal immunity.

Antibiotic Resistance in Non-Typhoidal Salmonella enterica Strains Isolated from Chicken Meat in Indonesia.

The increase in antibiotic resistance in non-typhoidal Salmonella enterica (NTS) has been confirmed in Indonesia by this study. We confirmed the virulence genes and antimicrobial susceptibilities of clinical NTS (n = 50) isolated from chicken meat in Indonesia and also detected antimicrobial resistance genes. Of 50 strains, 30 (60%) were non-susceptible to nalidixic acid (NA) and all of them had amino acid mutations in gyrA. Among 27 tetracycline (TC) non-susceptible strains, 22 (81.5%) had tetA and/or tetB. The non-susceptibility rates to ampicillin, gentamicin or kanamycin were lower than that of NA or TC, but the prevalence of blaTEM or aadA was high. Non-susceptible strains showed a high prevalence of virulence genes compared with the susceptible strains (tcfA, p = 0.014; cdtB, p < 0.001; sfbA, p < 0.001; fimA, p = 0.002). S. Schwarzengrund was the most prevalent serotype (23 strains, 46%) and the most frequently detected as multi-antimicrobial resistant. The prevalence of virulence genes in S. Schwarzengrund was significantly higher than other serotypes in hlyE (p = 0.011) and phoP/Q (p = 0.011) in addition to the genes above. In conclusion, NTS strains isolated from Indonesian chicken had a high resistance to antibiotics and many virulence factors. In particular, S. Schwarzengrund strains were most frequently detected as multi-antimicrobial resistant and had a high prevalence of virulence genes.

Occurrence of Carriage of Multidrug Resistant Enterobacteriaceae among Pregnant Women in the Primary Health Center and Hospital Setting in Surabaya, Indonesia.

Objectives: The incidence of healthy individuals carrying multidrug resistant Enterobacteriaceae, including extended-spectrum ß-lactamase producing Enterobacteriaceae (ESBL-E), especially extended-spectrum ß-lactamase producing Escherichia coli (ESBL-EC) and extended-spectrum ß-lactamase producing Klebsiella pneumoniae (ESBL-KP), is increasing worldwide. Although ESBL-E causes early or late onset of neonatal sepsis, the prevalence of ESBL-E carriage among pregnant women in Indonesia is not clear. In the present study, we compared the occurrence of carriage of ESBL-E among pregnant women in a primary health center (PHC) versus two hospitals. Materials and Methods: We collected rectal swab samples from 200 pregnant women who visited a PHC or were admitted to two hospitals in Surabaya, Indonesia from July to October 2018. The ESBL-E strains were isolated from the samples and phenotypically and genotypically analyzed. Results: ESBL-E strains were isolated from 25 (24.8%) pregnant women who visited the PHC and 49 (49.5%) pregnant women who were admitted to the hospitals. The rate of ESBL-E carriage of pregnant women in the hospitals was significantly higher than that in the PHC. Among the 74 isolated ESBL-E strains, ESBL-EC was most frequently isolated (62 strains), followed by ESBL-KP (12 strains). In addition, blaCTX-M-15 was the most frequent ESBL gene type of the isolated ESBL-E strains. Conclusions: Our results revealed the high occurrence of ESBL-E carriage in pregnant women, especially those who were admitted to the hospitals.

The Clinical Pattern and Prevalence of Streptococcus mutans and Streptococcus sobrinus among Adult and Children Patients with Dental Caries

Abstract Objective: To explore the clinical pattern, host factors, and presentation of Streptococcus mutans related to caries incidence among children and adults visiting Universitas Airlangga dental clinic. Material and Methods: This was an observational study with a cross-sectional approach with 50 patients in each group of carious children (6-12 years) and adults (18-35 years). Dental decay samples were taken by sterile excavator, put in a BHI's transport medium, and directly incubated overnight at 37 ºC. The next day, they were sub-cultured microbiologically in Tryptone Yeast Cystine Sucrose Bacitracin (TYCSB) selective medium. Bacterial species and serogroups were examined by PCR. All patient's data were collected from medical records and direct observation. Results: Caries were mostly media type in both children and adults. Oral hygiene (OHIS) in children was higher than in adults but not significantly different according to their DMFT. The highest scores for decay, missed and filled teeth were 16, 8 and 7, with an average of 6.82, 1.22 and 0.63, considered quite high. Conclusion: The prevalence of S. mutans was higher in children's caries than in adults, but among the adult patients the co-incidence of S. mutans and S. sobrinus was associated with higher DMFT. The mutans serotypes e, f, and d were more prevalent among children than adults.

An oral cancer vaccine using a Bifidobacterium vector suppresses tumor growth in a syngeneic mouse bladder cancer model.

Cancer immunotherapy using immune-checkpoint inhibitors (ICIs) such as PD-1/PD-L1 inhibitors has been well established for various types of cancer. Monotherapy with ICIs, however, can achieve a durable response in only a subset of patients. There is a great unmet need for the ICI-resistant-tumors. Since patients who respond to ICIs should have preexisting antitumor T cell response, combining ICIs with cancer vaccines that forcibly induce an antitumor T cell response is a reasonable strategy. However, the preferred administration sequence of the combination of ICIs and cancer vaccines is unknown. In this study, we demonstrated that combining an oral WT1 cancer vaccine using a Bifidobacterium vector and following anti-PD-1 antibody treatment eliminated tumor growth in a syngeneic mouse model of bladder cancer. This vaccine induced T cell responses specific to multiple WT1 epitopes through the gut immune system. Moreover, in a tumor model poorly responsive to an initial anti-PD-1 antibody, this vaccine alone significantly inhibited the tumor growth, whereas combination with continuous anti-PD-1 antibody could not inhibit the tumor growth. These results suggest that this oral cancer vaccine alone or as an adjunct to anti-PD-1 antibody could provide a novel treatment option for patients with advanced urothelial cancer including bladder cancer.

Analysis of the upstream genetic structures of the ISEcp1-blaCTX-M transposition units in Escherichia coli isolates carrying blaCTX-M obtained from the Indonesian and Vietnamese communities.

Extended spectrum ß-lactamase (ESBL)-producing Escherichia coli have been found in healthy individuals in Indonesia and Vietnam. The ISEcp1-blaCTX-M transposition unit of ESBL-producing bacterial isolates has been considered responsible for the production of CTX-M type ESBL and it is important for the dissemination of blaCTX-M . This study aimed to characterize the upstream genetic structure (UGS) of E. coli isolates possessing blaCTX-M-1 group and/or blaCTX-M-9 group genes obtained from healthy individuals in Indonesia and Vietnam. A total of 501 CTX-M type ESBL-producing E. coli isolates possessing blaCTX-M-1 group and/or blaCTX-M-9 group genes were obtained from healthy individuals of the two countries in 2018. The UGSs of the ISEcp1-blaCTX-M transposition unit of the 501 ESBL-producing E. coli isolates were amplified by barcode-adaptor-ligation-mediated PCR and analyzed using the Nanopore sequencer. The obtained sequence information was used to classify the UGSs of the ISEcp1-blaCTX-M transposition unit. From the 501 ESBL-producing E. coli isolates, 502 UGSs were obtained, which were classified into 85 UGS types based on the sequence. ISEcp1 of 359 (71.5%) of the 502 UGSs was disrupted by gene insertion, and ISEcp1-blaCTX-M transposition unit of most (87.1%) of the determined UGSs was confirmed as plasmidic. Only 6 (7.1%) of the 85 UGS types were common to both countries. Our results indicated that many different UGSs of ISEcp1-blaCTX-M transposition units were detected in Indonesia and Vietnam; hence, we suggest that structurally different kinds of plasmids harboring blaCTX-M were separately distributed in the two countries.

Differential effects of chromosome and plasmid blaCTX-M-15 genes on antibiotic susceptibilities in extended-spectrum beta-lactamase-producing Escherichia coli isolates from patients with urinary tract infection.

OBJECTIVES: To compare antibiotic susceptibilities between chromosomal and plasmid blaCTX-M-15 locations in urinary tract infection-causing extended-spectrum ß-lactamases-producing Escherichia coli blaCTX-M-15 isolated in Indonesia. METHODS: A total of 84 strains identified as extended-spectrum ß-lactamases-producing E. coli were isolated from patients with urinary tract infection in Indonesia in 2015. Antimicrobial susceptibility tests were performed on these strains using 18 antibiotics, and extended-spectrum ß-lactamase bla genes were detected by polymerase chain reaction. Gene localization of blaCTX-M-15 -positive strains was confirmed by Southern blot hybridization, and epidemiological typing was conducted using multilocus sequence typing. RESULTS: Of 54 strains harboring the blaCTX-M-15 gene, 27 showed localization on chromosome, 20 on plasmid, and seven on chromosome and plasmid. Most multilocus sequence typing sequence types of the 27 strains with chromosomal blaCTX-M-15 were ST405 (25.9%) and ST131 (22.2%) strains, whereas the 20 strains with plasmid-blaCTX-M-15 were mostly ST410 (55.0%). CONCLUSIONS: Extended-spectrum ß-lactamases-producing E. coli blaCTX-M-15 with plasmid genes show significantly higher resistant rates against piperacillin-tazobactam but lower resistant rates against chloramphenicol compared to chromosomal strains in Indonesian patients with urinary tract infection. Mechanistic investigations will be necessary to advance our knowledge of antimicrobial resistance in urinary tract infection.

Cross-Resistance and the Mechanisms of Cephalosporin-Resistant Bacteria in Urinary Tract Infections Isolated in Indonesia.

Urinary tract infection (UTI) by antibiotic-resistant strains has become increasingly problematic, with trends that differ from country to country. This study examined cross-resistance and the mechanisms of cephalosporin resistance in UTI-causative bacteria isolated in Indonesia. Antibiotic susceptibility tests based on Clinical Laboratory Standards Institute (CLSI) standards were done for UTI-causative strains (n = 50) isolated from patients in Indonesia in 2015-2016 and showed resistance against the third-generation cephalosporin. Mechanistic studies were carried out to confirm the presence of extended-spectrum ß-lactamase (ESBL) genes, carbapenemase-related genes, the fosA3 gene related to fosfomycin resistance, and mutations of quinolone-resistance-related genes. Isolated UTI-causative bacteria included Escherichia coli (64.0%), Pseudomonas aeruginosa (16.0%), Klebsiella pneumoniae (10.0%), and others (10.0%). These strains showed 96.0% susceptibility to amikacin, 76.0% to fosfomycin, 90.0% to imipenem, 28.0% to levofloxacin, 92.0% to meropenem, and 74.0% to tazobactam/piperacillin. ESBL was produced by 68.0% of these strains. Mechanistic studies found no strains with carbapenemase genes but 6.0% of strains had the fosA3 gene. Seventy-two % of the strains had mutations in the gyrA gene and 74.0% in the parC gene. Most E. coli strains (87.5%) had Ser-83 → Leu and Asp-87 → Asn in gyrA and 93.8% of E. coli had Ser-80 → Ile in parC. There were significant correlations among mutations in gyrA and parC, and fosA3 gene detection (P < 0.05), respectively. To our knowledge, this is the first mechanistic study of antibiotic-cross-resistant UTI-causative bacteria in Indonesia. Further studies with a longer period of observation are necessary, especially for changes in carbapenem resistance without carbapenemase-related genes.

Genetic analysis of ESBL-producing Klebsiella pneumoniae isolated from UTI patients in Indonesia.

INTRODUCTION: Extended spectrum beta-lactamase (ESBL)-producing Klebsiellapneumoniae is a serious concern for nosocomial infection and the emergence rate in Indonesia is higher than that in developed countries. The purpose of this study was to investigate the genetic characteristics of ESBL-producing K. pneumoniae isolated from UTI patients in Indonesia. MATERIALS AND METHODS: We collected K. pneumoniae resistant to ceftazidime or cefotaxime isolated from UTI patients in Dr. Soetomo's Academic Hospital in Surabaya, Indonesia in 2015. Ninety-four strains were identified as ESBL-producing bacteria by confirmation tests. The isolates were investigated by antimicrobial susceptibility testing with 20 drugs and ESBL gene detection, plasmid replicon typing and virulence genes as hypermucoviscous (HMV) strains were tested by the string test. RESULTS: High rates of resistance to ciprofloxacin (86.2%), tetracycline (80.9%) and nalidixic acid (78.7%) were observed. CTX-M-15 was the most common ESBL gene (89.4%), 33 of which also carried SHV-type ESBL. IncF was the most prevalent plasmid replicon typing (47.6%). Sixteen (17.0%) strains were judged as HMV, all of which had rmpA and more than half of which had fimH, uge, and wab. IncL/M was the most common replicon plasmid in the HMV strains, and the difference in the positive rate was statistically significant (p = 0.0024). CONCLUSION: This study showed the high prevalence of multiple-drug resistant and predominately CTX-M-15-positive ESBL-producing K. pneumoniae in Indonesia. There was a correlation between IncL/M and the HMV phenotype in this study. As such hypervirulent strains continue to emerge, studying their dissemination with resistance determinants is an urgent priority.

Combination of rAd-p53 in situ gene therapy and anti-PD-1 antibody immunotherapy induced anti-tumor activity in mouse syngeneic urogenital cancer models.

In this study we undertook a novel combination therapy using rAd-p53 in situ gene therapy and immunotherapy with immune checkpoint inhibitor (ICI) anti-PD-1 antibody for urogenital cancers. Three mouse syngeneic tumor cell lines, TRAMP-C2 (prostate cancer derived from C57BL/6 mice), MBT-2 (bladder cancer derived from C3H mice) and Renca (kidney cancer derived from BALB/c mice) were used in this study. The highest coxsackie and adenovirus receptor (CAR) mRNA expression was observed in TRAMP-C2 cells, followed by Renca and then MBT-2 cells. Consistent with the CAR expressions, rAd-p53 at 160 multiplicity of infection (MOI) significantly inhibited the cell proliferation of TRAMP-C2 and Renca cells, but not MBT-2 cells. In in vivo experiments, the combination of intratumoral injections of rAd-p53 (1 × 109 plaque-forming units) every other day and intraperitoneal injections of anti-mouse PD-1 antibody (200 µg) twice a week suppressed tumor growth and prolonged survival compared to rAd-p53 or anti-PD-1 antibody monotherapy in both the TRAMP-C2 and Renca models. Our results encourage the clinical development of combination therapy comprised of in situ gene therapy with rAd-p53 and immunotherapy with an ICI anti-PD-1 antibody for urogenital cancers.

Molecular detection of a new pathotype enteroaggregative haemorrhagic Escherichia coli (EAHEC) in Indonesia, 2015.

Enteroaggregative haemorrhagic Escherichia coli (E. Coli, EAHEC) has been identified as the agent responsible for one of the largest outbreaks of gastroenteritis and Haemolytic-uremic syndrome (HUS) that is transmitted through food in Germany in 2011. The hypervirulent pathotype has a unique combination of two pathogens namely enterohemorrhagic E.coli strain (EHEC) which produces shiga/verotoxin and enteroaggregative E.coli toxins (EAEC) which produces toxins similar to ST and hemolysin. The toxin produced by the EAHEC strain is a hybrid pathotype that combines the virulence potential of the EAEC and EHEC strains that will damage the microcirculation, cause vasculitis and other toxic effects. The purpose of this study was to determine the percentage of samples infected with enteroaggregative hemorrhagic E. coli bacteria (EAHEC) in pediatric diarrhea patients at DR. Soetomo Hospital, Surabaya, Indonesia, 2015. This study used PCR (Polymerase Chain Reaction) method to detect enteroaggregative E. coli strains (CVD432 and aaic genes) and enterohemorrhagic E.coli (eae gene).The results showed that 33 out of 40 (82,5%) stool samples examined were detected enteroaggregative E. coli (EAEC), 4 out of 40 (10%) enterohemorrhagic E. coli (EHEC) and 3 out of 40 (7,5%) enteroaggregative haemorrhagic E. coli bacteria (EAHEC), which caused diarrhea in pediatric diarrhea patients at Dr. Soetomo General Hospital. The unique combination of genomic features of the Surabaya outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype enteroaggregative haemorrhagic E. coli (EAHEC). It is expected that development of specific primer design and sequencing are needed to continue in this research.

Oncolytic effect of Midkine promoter-based conditionally replicating adenoviruses expressing EGFR siRNA in head and neck squamous cancer cell line T891.

BACKGROUND: Epidermal growth factor receptor (EGFR) is overexpressed in head and neck squamous cell carcinomas (HNSCCs). Midkine expression is restricted in adult tissues but is increased in several malignant tumors, including HNSCCs. AIM: Here, we evaluated the antitumor effect of Midkine promoter-based conditionally replicative adenovirus expressing siRNA against EGFR for targeting HNSCCs expressing Midkine. METHODS AND RESULTS: A conditionally replicative adenovirus vector controlled by the Midkine promoter, Ad-MK-siEGFR, was generated by integrating gene-expressing siRNA against EGFR. Antitumor effect of Ad-MK-siEGFR was tested in vitro using established HNSCC cell line, T891 with strong Midkine expression. Expression of EGFR in T891 infected with Ad-MK-siEGFR was significantly lower than that of T891 infected with control. Cytotoxicity assays showed significant growth suppression of Ad-MK-siEGFR in T891 cells. CONCLUSIONS: This study demonstrated the possibility of oncolytic therapy using the Midkine promoter-based conditional replication-selective adenovirus containing siRNA against EGFR in HNSCC cell line T891. Further validation of the findings in more cell lines and in vivo should be performed to clarify the potential clinical application.

Can ultrasound irradiation be a therapeutic option for prostate cancer?

BACKGROUND: Focal therapies for prostate cancer (PC) can reduce adverse events and do not lead to androgen-independent progression. Ultrasound could be used for cancer treatments if the repetition frequency is fitted to the purpose. We investigated the possible therapeutic effect of ultrasound irradiation on PC cells. MATERIALS AND METHODS: We irradiated two PC cell lines, androgen-dependent LNCaP and -independent PC-3 with ultrasound (3.0 W/cm2 , 3 MHz, irradiation time rate: 20%) for 2 minutes for 1 day or 3 consecutive days at a repetition frequency of 1, 10, or 100 Hz in vitro. Cell proliferation and apoptosis were determined after irradiation. RESULTS: Cell proliferation of PC-3 was significantly inhibited after 1 day (P < .0001) and 3 days (P < .0001) of 10 Hz ultrasound irradiation, and that of LNCaP after 1 day (P < .0001) and 3 days (P < .0001) of irradiation. LNCaP was more sensitive to ultrasound at both lower and higher cell density but PC-3 was only sensitive at a lower cell density (P < .01). Irradiation with 10 Hz ultrasound-induced significantly more PC-3 apoptotic cells than control (1 day, P = .0137; 3 days, P = .0386) rather than irradiation with 1 Hz. Apoptosis via caspase-3 was induced at 10 Hz in 1-day (P < .05) irradiation in both cell lines. CONCLUSIONS: Ultrasound irradiation with even 1 day of 10 Hz significantly inhibited cell proliferation in both LNCaP and PC-3, especially by the remarkable induction of apoptosis in vitro. Our study indicated that ultrasound irradiation can be a therapeutic option for PC and further studies in vivo will be undertaken.

Difference of Phenotype and Genotype Between Human and Environmental: Isolated Vibrio cholerae in Surabaya, Indonesia.

Cholera due to Vibrio cholerae has been spreading worldwide, although the reports focusing on Indonesian V. cholerae are few. In this study, in order to investigate how V. cholerae transmitted to human from environment. We extended an epidemiological report that had investigated the genotype of V. cholerae isolated from human pediatric samples and environmental samples. We examined 44 strains of V. cholerae isolated from pediatric diarrhea patients and the environment such as shrimps or oysters collected in three adjacent towns in Surabaya, Indonesia. Susceptibilities were examined for 11 antibiotics. Serotype O1 or O139 genes and pathogenic genes including cholera toxin were detected. Multi-locus sequence typing (MLST) and enterobacterial repetitive intergenic consensus (ERIC)-PCR were also performed to determine genetic diversity of those isolates. Serotype O1 was seen in 17 strains (38.6%) with all pathogenic genes among 44 isolates. Other isolates were non-O1/non-O139 V. cholerae. Regarding antibiotic susceptibilities, those isolates from environmental samples showed resistance to ampicillin (11.4%), streptomycin (9.1%) and nalidixic acid (2.3%) but those isolates from pediatric stools showed no resistance to those 3 kinds of antibiotics. MLST revealed sequence type (ST) 69 in 17 strains (38.6%), ST198 in 3 strains (6.8%) and non-types in 24 strains (54.5%). All the ST69 strains were classified to O1 type with more than 95% similarity by ERIC-PCR, including all 6 (13.6%) isolates from environmental samples with resistance to streptomycin. In conclusion, V. cholerae O1 ST69 strains has been clonally spreading in Surabaya, exhibiting pathogenic factors and antibiotic resistance to streptomycin, especially in the isolates from environment.

Characterization of CTX-M-type-extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae isolated from Indonesian undergraduate medical students of a university in Surabaya, Indonesia.

Enterobacteriaceae isolates producing CTX-M-type extended-spectrum ß-lactamase (ESBL) has been found in hospitalized patients and healthy individuals in communities of the Southeast Asian countries. Medical students might have more risk of ESBL-producing Enterobacteriaceae contagion, because medical students who belong to communities have direct and indirect contacts with workers and patients in healthcare facilities. The aim of this study was to collect information for evaluation of the potential risk of ESBL-producing Enterobacteriaceae contagion in Indonesian undergraduate medical students by characterizing genotypic properties of Escherichia coli isolates-producing CTX-M-type ESBL. A total 141 fecal samples collected from 207 medical students of a university in Surabaya, Indonesia were subjected to PCR, XbaI and S1 nuclease-pulsed-field gel electrophoresis (PFGE), Southern blotting, and sequencing analysis. Eighty-two ESBL-producing Enterobacteriaceae, including 75 E. coli and 7 Klebsiella pneumoniae were isolated from 79 (56.0%) students. Among 75 ESBL-producing E. coli, blaCTX-M-15 was the most prevalent type (44.0%). Although XbaI-PFGE results showed genetic background of the E. coli isolates producing CTX-M-type ESBL were diverse, five clonal spread cases of certain E. coli producing CTX-M-type ESBL isolates were observed among the medical students. Our results suggested that ESBL-producing Enterobacteriaceae might be circulating among the medical students through contaminated environment such as in a university or communities they belonged.