Molecular Epidemiology of Streptococcus pneumoniae Detected in Hospitalized Pediatric Acute Respiratory Infection Cases in Central Vietnam.

Streptococcus pneumoniae is the major bacterial pathogen causing high pneumonia morbidity and mortality in children <5 years of age. This study aimed to determine the molecular epidemiology of S. pneumoniae detected among hospitalized pediatric ARI cases at Khanh Hoa General Hospital, Nha Trang, Vietnam, from October 2015 to September 2016 (pre-PCV). We performed semi-quantitative culture to isolate S. pneumoniae. Serotyping, antimicrobial susceptibility testing, resistance gene detection and multi-locus sequence typing were also performed. During the study period, 1300 cases were enrolled and 413 (31.8%) S. pneumoniae were isolated. School attendance, age <3 years old and prior antibiotic use before admission were positively associated with S. pneumoniae isolation. Major serotypes were 6A/B (35.9%), 19F (23.7%) and 23F (12.7%), which accounted for 80.3% of vaccine-type pneumococci. High resistance to Clarithromycin, Erythromycin and Clindamycin (86.7%, 85%, 78.2%) and the mutant drug-resistant genes pbp1A (98.1%), pbp2b (98.8%), pbp2x (99.6%) ermB (96.6%) and mefA (30.3%) were detected. MLST data showed high genetic diversity among the isolates with dominant ST 320 (21.2%) and ST 13223 (19.3%), which were mainly found in Vietnam. Non-typeables accounted for most of the new STs found in the study. Vaccine-type pneumococcus and macrolide resistance were commonly detected among hospitalized pediatric ARI cases.

Antibiotic-Resistant Vibrio cholerae O1 and Its SXT Elements Associated with Two Cholera Epidemics in Kenya in 2007 to 2010 and 2015 to 2016.

Multidrug-resistant Vibrio cholerae O1 strains have long been observed in Africa, and strains exhibiting new resistance phenotypes have emerged during recent epidemics in Kenya. This study aimed to determine the epidemiological aspects, drug resistance patterns, and genetic elements of V. cholerae O1 strains isolated from two cholera epidemics in Kenya between 2007 and 2010 and between 2015 and 2016. A total of 228 V. cholerae O1 strains, including 226 clinical strains isolated from 13 counties in Kenya during the 2007-2010 and 2015-2016 cholera epidemics and two environmental isolates (from shallow well water and spring water isolates) isolated from Pokot and Kwale Counties, respectively, in 2010 were subjected to biotyping, serotyping, and antimicrobial susceptibility testing, including the detection of antibiotic resistance genes and mobile genetic elements. All V. cholerae isolates were identified as El Tor biotypes and susceptible to ceftriaxone, gentamicin, and ciprofloxacin. The majority of isolates were resistant to trimethoprim-sulfamethoxazole (94.6%), streptomycin (92.8%), and nalidixic acid (64.5%), while lower resistance was observed against ampicillin (3.6%), amoxicillin (4.2%), chloramphenicol (3.0%), and doxycycline (1.8%). Concurrently, the integrating conjugative (SXT) element was found in 95.5% of the V. cholerae isolates; conversely, class 1, 2, and 3 integrons were absent. Additionally, 64.5% of the isolates exhibited multidrug resistance patterns. Antibiotic-resistant gene clusters suggest that environmental bacteria may act as cassette reservoirs that favor resistant pathogens. On the other hand, the 2015-2016 epidemic strains were found susceptible to most antibiotics except nalidixic acid. This revealed the replacement of multidrug-resistant strains exhibiting new resistance phenotypes that emerged after Kenya's 2007-2010 epidemic. IMPORTANCE Kenya is a country where cholera is endemic; it has experienced three substantial epidemics over the past few decades, but there are limited data on the drug resistance patterns of V. cholerae at the national level. To the best of our knowledge, this is the first study to investigate the antimicrobial susceptibility profiles of V. cholerae O1 strains isolated from two consecutive epidemics and to examine their associated antimicrobial genetic determinants. Our study results revealed two distinct antibiotic resistance trends in two separate epidemics, particularly trends for multidrug-associated mobile genetic elements and chromosomal mutation-oriented resistant strains from the 2007-2010 epidemic. In contrast, only nalidixic acid-associated chromosomal mutated strains were isolated from the 2015-2016 epidemic. This study also found similar patterns of antibiotic resistance in environmental and clinical strains. Continuous monitoring is needed to control emerging multidrug-resistant isolates in the future.

Analysis of Environmental and Pathogenic Bacteria Attached to Aerosol Particles Size-Separated with a Metal Mesh Device.

Metal mesh devices (MMDs) are novel materials that enable the precise separation of particles by size. Structurally, MMDs consist of a periodic arrangement of square apertures of characteristic shapes and sizes on a thin nickel membrane. The present study describes the separation of aerosol particles using palm-top-size collection devices equipped with three types of MMDs differing in pore size. Aerosols were collected at a farm located in the suburbs of Nairobi, Kenya; aerosol particles were isolated, and pathogenic bacteria were identified in this microflora by next-generation sequencing analysis. The composition of the microflora in aerosol particles was found to depend on particle size. Gene fragments were obtained from the collected aerosols by PCR using primers specific for the genus Mycobacterium. This analysis showed that Mycobacterium obuense, a non-tuberculous species of mycobacteria that causes lung diseases, was present in these aerosols. These findings showed that application of this MMD analytical protocol to aerosol particles can facilitate the investigation of airborne pathogenic bacteria.

Characterization of Vibrio cholerae O1 isolates responsible for cholera outbreaks in Kenya between 1975 and 2017.

Kenya is endemic for cholera with different waves of outbreaks having been documented since 1971. In recent years, new variants of Vibrio cholerae O1 have emerged and have replaced most of the traditional El Tor biotype globally. These strains also appear to have increased virulence, and it is important to describe and document their phenotypic and genotypic traits. This study characterized 146 V. cholerae O1 isolates from cholera outbreaks that occurred in Kenya between 1975 and 2017. Our study reports that the 1975-1984 strains had typical classical or El Tor biotype characters. New variants of V. cholerae O1 having traits of both classical and El Tor biotypes were observed from 2007 with all strains isolated between 2015 and 2017 being sensitive to polymyxin B and carrying both classical and El Tor type ctxB. All strains were resistant to Phage IV and harbored rstR, rtxC, hlyA, rtxA and tcpA genes specific for El Tor biotype indicating that the strains had an El Tor backbone. Pulsed field gel electrophoresis (PFGE) genotyping differentiated the isolates into 14 pulsotypes. The clustering also corresponded with the year of isolation signifying that the cholera outbreaks occurred as separate waves of different genetic fingerprints exhibiting different genotypic and phenotypic characteristics. The emergence and prevalence of V. cholerae O1 strains carrying El Tor type and classical type ctxB in Kenya are reported. These strains have replaced the typical El Tor biotype in Kenya and are potentially more virulent and easily transmitted within the population.

Epidemiology and Pathogenesis of Providencia alcalifaciens Infections.

Providencia alcalifaciens is a member of the family Enterobacteriaceae that has been commonly implicated as a causative agent of diarrheal infection in humans and animals. Recent outbreaks of P. alcalifaciens in both developing and developed countries have raised public health concerns. Several studies have suggested that P. alcalifaciens can cause diarrhea by invading the intestinal mucosa, although its pathogenicity has not been well established. Often routine laboratory investigations that seek etiological agents of diarrhea do not actively pursue P. alcalifaciens detection. Therefore, routine laboratory diagnosis should be given more attention for better understanding the epidemiology and pathogenicity of P. alcalifaciens.

Case Report: Bacillus pumilus-Caused Bacteremia in a Patient with Food Poisoning.

Bacillus pumilus has rarely been reported as a cause of human infections. We report a case of a B. pumilus causing food poisoning in an adult male. A 51-year-old Japanese man complained of severe abdominal cramps, fever with chills, diarrhea, dizziness, and loss of appetite after eating reheated rice with stewed minced meat purchased from a Kenyan restaurant. Bacillus pumilus was isolated from blood culture and was identified using a biochemical test and 16S rRNA gene sequencing analysis. The patient was treated with probiotics and ciprofloxacin and recovered after 3 days. To our knowledge, this is the first report describing the potential role of B. pumilus as a foodborne pathogen in Kenya and highlights the importance of good hygiene and food preparation practices.

Detection of rotavirus in clinical specimens using an immunosensor prototype based on the photon burst counting technique.

In this study, a sensitive fluorescence sensor was developed for the detection of small, fluorescence-labeled particles dispersed in a solution. The prototype system comprises of a laser confocal optical system and a mechanical sample stage to detect photon bursting of fluorescence-labeled small particles in sample volumes less than 5 µL within 3 minutes. To examine the feasibility of the prototype system as a diagnostic tool, assemblages of rotavirus and fluorescence-labeled antibody were analyzed. The detection sensitivity for rotavirus was 1 × 104 pfu/mL. Rotavirus in stool samples from patients with acute gastroenteritis was also detected. The advantages and disadvantages of this immunosensor with respect to ELISA and RT-PCR, the current gold standards for virus detection, are discussed.

Synthesis and Antimicrobial Activity of Nitrobenzyl-oxy-phenol Derivatives.

Two hydroquinone derivatives were prepared and their antimicrobial activity evaluated. Their minimum inhibitory concentrations (MICs) were determined using a broth dilution method. Gentamycin and ciprofloxacin were used as reference antibiotics. The antimicrobial activity of 4-(benzyloxy)phenol (monobenzone) was also evaluated based on its structural similarity to the new compounds; activity was comparable to that of 3,5-dimethyl-4-((4-nitrobenzyl)oxy)phenol (4a). 2,3,5-Trimethyl-4-((4-nitrobenzyl)oxy)phenol (4b) exhibited the best antibacterial activity against both clinical isolates and type strain of Moraxella catarrhalis (M. catarrhalis), with a MIC value of 11 µM, comparable to ciprofloxacin 9 µM.

First Report of a Foodborne Providencia alcalifaciens Outbreak in Kenya.

Providencia alcalifaciens is an emerging bacterial pathogen known to cause acute gastroenteritis in children and travelers. In July 2013, P. alcalifaciens was isolated from four children appearing for diarrhea at Kiambu District Hospital (KDH) in Kenya. This study describes the outbreak investigation, which aimed to identify the source and mechanisms of infection. We identified seven primary and four secondary cases. Among primary cases were four mothers who had children and experienced mild diarrhea after eating mashed potatoes. The mothers reported feeding children after visiting the toilet and washing their hands without soap. P. alcalifaciens was detected from all secondary cases, and the isolates were found to be clonal by random amplified polymorphic DNA (RAPD) fingerprinting. Our study suggests that the outbreak was caused by P. alcalifaciens, although no fluid accumulation was observed in rabbit ileal loops. The vehicle of the outbreak was believed to be the mashed potato dish, but the source of P. alcalifaciens could not be confirmed. We found that lack of hygiene, inadequate food storage, and improper hand washing before food preparation was the likely cause of the current outbreak. This is the first report of a foodborne infection caused by P. alcalifaciens in Kenya.

Activity of Aristolochia bracteolata against Moraxella catarrhalis.

A bioassay-guided fractionation of methanol extract of Aristolochia bracteolata whole plant was carried out in order to evaluate its antimicrobial activity and to identify the active compounds in this extract. Antibacterial and antifungal activities of methanol extract against gram-positive, gram-negative, and fungal strains were investigated by the agar disk diffusion method. Among the strains tested, Moraxella catarrhalis and sea urchin-derived Bacillus sp. showed the highest sensitivity towards the methanol extract and hence they are used as test organisms for the bioassay-guided fractionation. From this extract, aristolochic acid 1 (AA-1) has been isolated and has showed the greatest antibacterial activity against both standard strain and clinical isolates of Moraxella catarrhalis with equal minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 25 and 50 µg/mL. Modification of the AA-1 to AA-1 methyl ester completely abolished the antibacterial activity of the compound and the piperonylic acid moiety of AA-1 which suggested that the coexistence of phenanthrene ring and free carboxylic acid is essential for AA-1 antibacterial activity.

Lactobacillus acidophilus strain L-92 induces CD4(+)CD25(+)Foxp3(+) regulatory T cells and suppresses allergic contact dermatitis.

The anti-allergic mechanism of heat-killed Lactobacillus acidophilus strain L-92 has not been fully investigated. Recent studies have reported that CD4(+)CD25(+)Foxp3(+) (forkhead box P3) T regulatory (Treg) cells play important roles in controlling allergic diseases. Hence, we examined the effect of orally administered L-92 on CD4(+)CD25(+)Foxp3(+) cell populations. BALB/c mice were supplemented daily with L-92 by gavage for 5 weeks. 2,4-Dinitrofluorobenzene (DNFB) was used to induce allergic contact dermatitis (ACD) in mice. Fluorescent-activated cell sorter (FACS) analysis was used to determine CD4(+)CD25(+)Foxp3(+) T cell populations in spleen and cervical lymph nodes (CLN). Interleukin-10 (IL-10), transforming growth factor-ß (TGF-ß), and Foxp3 mRNA expressions in mouse ear skin were investigated by real-time reverse transcription-polymerase chain reaction (RT-PCR). The percentage of CD4(+)CD25(+)Foxp3(+) T cell populations were significantly increased in both spleen and CLN of L-92-fed group than vehicle and control. In addition, L-92 produced higher levels of Foxp3, IL-10 and TGF-ß compared to control mice. These results suggest that L-92 can up-regulate the number of Treg cells to suppress the progression of DNFB-induced contact dermatitis in mice.

Orally supplemented Lactobacillus acidophilus strain L-92 inhibits passive and active cutaneous anaphylaxis as well as 2,4-dinitroflurobenzene and mite fecal antigen induced atopic dermatitis-like skin lesions in mice.

Oral supplementation of lactic acid bacteria is a potential approach to the prevention and manipulation of allergic diseases such as atopic dermatitis. Our previous report showed that heat-killed Lactobacillus acidophilus strain L-92 (L-92) possessed anti-allergic properties, although its physiological function in atopic dermatitis has largely remained undefined. To evaluate the anti-allergic efficacy of L-92, we used four experimental animal models with the major features of atopic dermatitis and compared the results to those of clinically active drugs. ICR mice were passively sensitized by anti-dinitrophenyl mouse monoclonal IgE for passive cutaneous anaphylaxis (PCA), and BALB/c mice were actively sensitized by ovalbumin for active cutaneous anaphylaxis (ACA). Allergic reaction was induced by repeated exposure to 2,4-dinitroflurobenzene (DNFB) and mite (Dermatophagoides farinae) fecal allergen, in BALB/c and NC/Nga mice, respectively. Orally administrated L-92 significantly inhibited the vascular permeability increase in both PCA and ACA, and the elevation of ovalbumin-specific IgE titer in ACA. Moreover, repeated applications of DNFB and mite fecal antigen onto the BALB/c and NC/Nga mouse ear, respectively, caused clinical symptoms similar to atopic dermatitis such as ear swelling, scratching behavior and elevation of total serum IgE levels that were also moderately suppressed by L-92. In addition, L-92 treated mice exhibited lower levels of mast cells, eosinophil infiltration and Th1/Th2 cytokine expression. Our results, therefore, suggest that oral administration of L-92 might be useful for alleviating allergic symptoms.

Rapid multiplex immunofluorescent assay to detect antibodies against Burkholderia pseudomallei and taxonomically closely related nonfermenters.

An indirect immunofluorescent assay to detect antibodies against the lipopolysaccharide (LPS) of Burkholderia pseudomallei and taxonomically closely related species was developed with the Luminex system. LPSs of Pseudomonas aeruginosa, Burkholderia cepacia, Burkholderia thailandensis, Burkholderia vietnamiensis, B. pseudomallei, and Burkholderia mallei were successfully conjugated to Luminex microspheres. Antibodies measured against the LPS of B. pseudomallei-conjugated Luminex beads only cross-reacted with those of two genetically closely related species, B. mallei and B. thailandensis (previously classified as non-pathogenic arabinose-negative B. pseudomallei). However, this system could distinguish other closely related species from B. pseudomallei. This assay is able to detect significantly high levels of anti-LPS antibodies of B. pseudomallei in serum from patients with culture-proven melioidosis.

Mycobacterium species identification--a new approach via dnaJ gene sequencing.

The availability of the dnaJ1 gene for identifying Mycobacterium species was examined by analyzing the complete dnaJ1 sequences (approximately 1200 bp) of 56 species (54 of them were type strains) and comparing sequence homologies with those of the 16S rRNA gene and other housekeeping genes (rpoB, hsp65). Among the 56 Mycobacterium species, the mean sequence similarity of the dnaJ1 gene (80.4%) was significantly less than that of the 16S rRNA, rpoB and hsp65 genes (96.6%, 91.3% and 91.1%, respectively), indicating a high discriminatory power of the dnaJ1 gene. Seventy-one clinical isolates were correctly clustered to the corresponding type strains, showing isolates belonging to the same species. In order to propose a method for strain identification, we identified an area with a high degree of polymorphism, bordered by conserved sequences, that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (approximately 350 bp) allows accurate species identification and may be used as a new tool for the identification of Mycobacterium species.

Use of the novel phylogenetic marker dnaJ and DNA-DNA hybridization to clarify interrelationships within the genus Aeromonas.

The interrelationships of 27 Aeromonas strains were investigated using dnaJ sequences and DNA-DNA hybridization. dnaJ sequence similarities showed a stronger relationship with DNA-DNA relatedness values than did 16S rRNA gene sequence similarities. Additionally, dnaJ sequence analysis, with interspecies divergence over 5.2 % in most cases, gave better resolution than 16S rRNA gene sequences for the differentiation of strains at the species level. Relationships among Aeromonas species were therefore elucidated on the basis of dnaJ sequences and DNA-DNA reassociation. Strains of Aeromonas encheleia and Aeromonas sp. HG11 were unquestionably grouped in the same genetic species, since they shared 98.7 % dnaJ sequence similarity and 82-85 % genomic relatedness. The phylogenetically close relationships obtained from dnaJ sequence analysis (1.7-3.3 % genetic distance) were corroborated by high DNA-DNA relatedness (73-97 %) to support the previous suggestion that Aeromonas culicicola and Aeromonas allosaccharophila are later heterotypic synonyms of Aeromonas veronii. Our findings will contribute to the clarification of controversial relationships in the genus Aeromonas and also demonstrate that analysis of dnaJ sequences can be a powerful tool for interspecies study of the genus.

dnaJ gene sequence-based assay for species identification and phylogenetic grouping in the genus Staphylococcus.

In the last few years, many attempts have been made to use conserved gene sequences for identification and for phylogenetic studies of Staphylococcus species. In an effort to identify a more reliable approach, a dnaJ gene sequence-based database was created. In this study, an approximately 883 bp portion of the dnaJ gene sequence from 45 staphylococcal type strains was compared with 16S rRNA and other conserved gene (hsp60, sodA and rpoB) sequences available in public databases. Nucleotide sequence comparisons revealed that the staphylococcal dnaJ gene showed higher discrimination (mean similarity 77.6 %) than the 16S rRNA (mean similarity 97.4 %), rpoB (mean similarity 86 %), hsp60 (mean similarity 82 %) and sodA (mean similarity 81.5 %) genes. Analysis of the dnaJ gene sequence from 20 Staphylococcus isolates representing two clinically important species showed <1 % sequence divergence. Phylogenetic data obtained from the dnaJ gene sequence were in general agreement with those of 16S rRNA gene sequence analysis and DNA-DNA reassociation studies. In conclusion, the dnaJ gene sequence-based assay is an effective alternative to currently used methods, including 16S rRNA gene sequencing, for identification and taxonomical analysis of Staphylococcus species.

The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species.

The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.

dnaJ and gyrB gene sequence relationship among species and strains of genus Streptococcus.

The dnaJ and gyrB nucleotide sequences were determined for members of the genus Streptococcus. The average similarity between the species tested was 76.4% (69.7-100%) for dnaJ and 75.9 (70.1-98.7%) for gyrB. These data indicated that the dnaJ and gyrB genes are more divergent and more discriminatory than the 16S rDNA gene. Furthermore, the variation in the dnaJ nucleotide sequences among the mitis group was greater than that of the gyrB nucleotide sequences, especially between Streptococcus pneumoniae and Streptococcus mitis. Subsequently, the high discrimination power of dnaJ within the mitis group was confirmed. Thus, we conclude that the dnaJ and gyrB genes are efficient alternative targets for the classification of the genus Streptococcus, and that dnaJ is suitable for phylogenetic analysis of closely related Streptococcus strains.