Differential impact of environmental factors on airborne live bacteria and inorganic particles in an underground walkway.

We previously reported that variations in the number and type of bacteria found in public spaces are influenced by environmental factors. However, based on field survey data alone, whether the dynamics of bacteria in the air change as a result of a single environmental factor or multiple factors working together remains unclear. To address this, mathematical modeling may be applied. We therefore conducted a reanalysis of the previously acquired data using principal component analysis (PCA) in conjunction with a generalized linear model (Glm2) and a statistical analysis of variance (ANOVA) test employing the χ2 distribution. The data used for the analysis were reused from a previous public environmental survey conducted at 8:00-20:00 on May 2, June 1, and July 5, 2016 (regular sampling) and at 5:50-7:50 and 20:15-24:15 on July 17, 2017 (baseline sampling) in the Sapporo underground walking space, a 520-meter-long underground walkway. The dataset consisted of 60 samples (22 samples for "bacterial flora"), including variables such as "temperature (T)," "humidity (H)," "atmospheric pressure (A)," "traffic pedestrians (TP)," "number of inorganic particles (Δ5: 1-5 µm)," "number of live airborne bacteria," and "bacterial flora." Our PCA with these environmental factors (T, H, A, and TP) revealed that the 60 samples could be categorized into four groups (G1 to G4), primarily based on variations in PC1 [Loadings: T(-0.62), H(-0.647), TP(0.399), A(0.196)] and PC2 [Loadings: A(-0.825), TP(0.501), H(0.209), T(-0.155)]. Notably, the number of inorganic particles significantly increased from G4 to G1, but the count of live bacteria was highest in G2, with no other clear pattern. Further analysis with Glm2 indicated that changes in inorganic particles could largely be explained by two variables (H/TP), while live bacteria levels were influenced by all explanatory variables (TP/A/H/T). ANOVA tests confirmed that inorganic particles and live bacteria were influenced by different factors. Moreover, there were minimal changes in bacterial flora observed among the groups (G1-G4). In conclusion, our findings suggest that the dynamics of live bacteria in the underground walkway differ from those of inorganic particles and are regulated in a complex manner by multiple environmental factors. This discovery may contribute to improving public health in urban settings.

Human pathogenic bacteria on high-touch dry surfaces can be controlled by warming to human-skin temperature under moderate humidity.

Healthcare-associated infections have become a major health issue worldwide. One route of transmission of pathogenic bacteria is through contact with "high-touch" dry surfaces, such as handrails. Regular cleaning of surfaces with disinfectant chemicals is insufficient against pathogenic bacteria and alternative control methods are therefore required. We previously showed that warming to human-skin temperature affected the survival of pathogenic bacteria on dry surfaces, but humidity was not considered in that study. Here, we investigated environmental factors that affect the number of live bacteria on dry surfaces in hospitals by principal component analysis of previously-collected data (n = 576, for CFU counts), and experimentally verified the effect of warming to human-skin temperature on the survival of pathogenic bacteria on dry surfaces under humidity control. The results revealed that PCA divided hospital dry surfaces into four groups (Group 1~4) and hospital dry surfaces at low temperature and low humidity (Group 3) had much higher bacterial counts as compared to the others (Group 1 and 4) (p<0.05). Experimentally, warming to human-skin temperature (37°C with 90% humidity) for 18~72h significantly suppressed the survival of pathogenic bacteria on dry surfaces, such as plastic surfaces [p<0.05 vs. 15°C (Escherichia coli DH5α, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and blaNDM-5 E. coli)] or handrails [p<0.05 vs. 15~25°C (E. coli DH5α, S. aureus, P. aeruginosa, A. baumannii)], under moderate 55% humidity. Furthermore, intermittent heating to human-skin temperature reduced the survival of spore-forming bacteria (Bacillus subtilis) (p<0.01 vs. continuous heating to human-skin temperature). NhaA, an Na+/H+ antiporter, was found to regulate the survival of bacteria on dry surfaces, and the inhibitor 2-aminoperimidine enhanced the effect of warming at human-skin temperature on the survival of pathogenic bacteria (E. coli DH5α, S. aureus, A. baumannii) on dry surfaces. Thus, warming to human-skin temperature under moderate humidity is a useful method for impairing live pathogenic bacteria on high-touch surfaces, thereby helping to prevent the spread of healthcare-associated infections.

Mastitis on selected farms in Wakiso district, Uganda: Burden, pathogens and predictors of infectivity of antimicrobial resistant bacteria in dairy herds.

BACKGROUND: Mastitis and associated antimicrobial resistance (AMR) are major challenges to the dairy industry worldwide. OBJECTIVE: This study aimed to expose the mastitis burden, causative bacteria and drivers for mastitis-causing multi-drug-resistant (MDR) Staphylococci infectivity in cows on dairy farms in Wakiso district, Uganda. METHODS: On 22 farms, practices were documented using questionnaires, and 175 cows were screened by the California mastitis test. Composite milk samples from the positive reactors were submitted to the laboratory for bacterial culture testing. Antimicrobial sensitivity testing by the Kirby Bauer disc diffusion method was done only on Staphylococci with a panel of 10 antimicrobials of clinical relevance. RESULTS: Mastitis was detected in 80.6% (n = 141) of the 175 sampled cows, of which sub-clinical mastitis (76.0%: n = 133) was predominant. The Chi-squared analysis hypothesized that cow age (p = 0.017), sub-county (p = 0.013), parity (p < 0.0001), sex of farm owner (p = 0.003), farm duration in dairy production (p = 0.048) and the use of milking salve (p = 0.006) were associated with mastitis. Coagulase-negative Staphylococci were the most prevalent (71.4%; n = 95), followed by Staphylococcus aureus (30.1%, n = 40). Staphylococci (76.3%; n = 135) were majorly resistant to penicillin and tetracycline. Only one isolate was phenotyped as a methicillin-resistant Staphylococcus specie (MRSS). The prevalences of MDR strains at cow and isolate level were 6.3% and 8.3%. The major MDR phenotype identified was penicillin-tetracycline-trimethoprim-sulphamethoxazole. The isolate detected as an MRSS exhibited the broadest MDR pattern. Cow parity was identified as a predictor of infectivity of mastitis-causing MDR Staphylococci in dairy herds. CONCLUSION: The high prevalence of mastitis and associated pathogen AMR found exposes possibilities of economic losses for the dairy sector warranting the need for farmer sensitization on the institution of proper mastitis prevention and control programs, with emphasis on milking hygiene practices and routine disease monitoring.

The fecal microbiomes analysis of Marabou storks (Leptoptilos crumenifer) reveals their acclimatization to the feeding environment in the Kampala urban areas, Uganda.

The Marabou stork (Leptoptilos crumenifer) is a typical scavenging bird and adapted to the Savannah environment, where they show a carnivorous feeding style. However, Marabou stork recently penetrated into the city areas and acclimatized to the urban environment, where they modified their feeding habits to an omnivorous type toward more carbohydrate. To reveal their adaptation to the variable feeding customs, this study compared the gut microbiomes and chemical compositions of feces of Marabou storks inhabiting two different locations in peri urban Kampala: one is a slaughter house floc that predicted their original carnivorous feeding, and the other is a landfill floc that adapted more to the omnivorous feeding. 16S rRNA gene sequencing analysis revealed more diverse gut microbiome, more enriched Lactobacilli, and less abundant Peptostreptococci in the landfill flock comparing to the slaughter house flock. Isolation work and predicted metagenome analysis confirmed more diverse Lactobacilli and more enriched functions for carbohydrate metabolism in the landfill flock. In addition, chemical composition of feces revealed higher ammonia in the former, which is consisting with higher Peptostreptococci and their practice of carnivorous feeding. These results highlighted their adaptation to the variable feeding environment, which presumably protects their health and ensure survival of species.

Chlamydia trachomatis relies on the scavenger role of aryl hydrocarbon receptor with detyrosinated tubulin for its intracellular growth, but this is impaired by excess indole.

Although IFN-γ depletes tryptophan (Trp) as a defense against intracellular Chlamydia trachomatis (Ct) infected to hypoxic vagina, the presence of indole, a precursor of Trp, enables Ct to infect IFN-γ-exposed culture cells. Meanwhile, Trp-derived indole derivatives interact the aryl hydrocarbon receptor (AhR), which is a ligand-dependent transcription factor involved in the cellular homeostasis with tubulin dynamics. Here, the amounts of IFN-γ and indole in cervical swabs with known Ct infection status were measured, and Ct growth in the presence of indole was determined from the perspective of the AhR axis under hypoxia. A positive correlation between the amounts of IFN-γ and indole was found, and both of these amounts were lower in Ct-positive swabs than in Ct-negative ones. Indole as well as other AhR ligands inhibited Ct growth, especially under normoxia. Ct prompted the expression of detyrosinated tubulin (dTTub), but indole inhibited it. Indole did not stimulate the translocation of AhR to nucleus, and it blocked AhR activation in AhR-reporter cells. Ct growth was reduced more effectively under normoxia in AhR-knockdown cells, an effect that was enhanced by indole, which in turn diminished dTTub. Thus, Ct growth relies on the scavenger role of cytosolic AhR responsible for promoting dTTub expression.

Chlamydia trachomatis L2/434/Bu Favors Hypoxia for its Growth in Human Lymphoid Jurkat Cells While Maintaining Production of Proinflammatory Cytokines.

The role of lymphocytes as a cornerstone of the inflammatory response in the invasive pathogenesis of Chlamydia trachomatis (Ct) LGV (L1-3) infection is unclear. Therefore, we assessed whether the adaptation of CtL2 to immortal lymphoid Jurkat cells under hypoxic conditions occurred through proinflammatory cytokine profile modification. The quantities of inclusion-forming units with chlamydial 16S rDNA confirmed that CtL2 grew well under hypoxic rather than normoxic conditions in the cells. Confocal microscopic imaging and transmission electron microscopy revealed the presence of bacterial progeny in the inclusions and showed that the inclusions were larger under hypoxic rather than normoxic conditions; this was supported by the results of 3D image construction. Furthermore, PCR-based analysis of proinflammatory cytokines revealed that the gene expression levels under hypoxic conditions were significantly higher than those under normoxic conditions. In particular, the expression of two genes (CXCL8 and CXCR3) was significantly diminished under normoxic conditions. Taken together, the results indicated that hypoxia promoted CtL2 growth in Jurkat cells while maintaining the levels of proinflammatory cytokines. Thus, Ct LGV infection in lymphocytes under hypoxic conditions might be crucial to a complete understanding of the invasive pathogenesis.

Chlamydia trachomatis Requires Functional Host-Cell Mitochondria and NADPH Oxidase 4/p38MAPK Signaling for Growth in Normoxia.

Chlamydia trachomatis (Ct) is an intracellular energy-parasitic bacterium that requires ATP derived from infected cells for its growth. Meanwhile, depending on the O2 concentration, the host cells change their mode of ATP production between oxidative phosphorylation in mitochondria (Mt) and glycolysis; this change depends on signaling via reactive oxygen species (ROS) produced by NADPH oxidases (NOXs) as well as Mt. It has been proposed that Ct correspondingly switches its source of acquisition of ATP between host-cell Mt and glycolysis, but this has not been verified experimentally. In the present study, we assessed the roles of host-cell NOXs and Mt in the intracellular growth of CtL2 (L2 434/Bu) under normoxia (21% O2) and hypoxia (2% O2) by using several inhibitors of NOXs (or the downstream molecule) and Mt-dysfunctional (Mtd) HEp-2 cells. Under normoxia, diphenyleneiodonium, an inhibitor of ROS diffusion, abolished the growth of CtL2 and other Chlamydiae (CtD and C. pneumoniae). Both ML171 (a pan-NOX inhibitor) and GLX351322 (a NOX4-specific inhibitor) impaired the growth of CtL2 under normoxia, but not hypoxia. NOX4-knockdown cells diminished the bacterial growth. SB203580, an inhibitor of the NOX4-downstream molecule p38MAPK, also inhibited the growth of CtL2 under normoxia but not hypoxia. Furthermore, CtL2 failed to grow in Mtd cells under normoxia, but no effect was observed under hypoxia. We conclude that under normoxia, Ct requires functional Mt in its host cells as an ATP source, and that this process requires NOX4/p38MAPK signaling in the host cells. In contrast to hypoxia, crosstalk between NOX4 and Mt via p38MAPK may be crucial for the growth of Ct under normoxia.

Wild ciliates differ in susceptibility to Legionella pneumophila JR32.

We investigated how Legionella pneumophila (Lp) JR32 interacts with Anteglaucoma CS11A and Colpoda E6, two ciliates that we isolated from sewage and sink trap sludge, respectively, using a handmade maze device containing a 96-well crafting plate. Our 18S rDNA-based phylogenetic analysis showed that Anteglaucoma CS11A and Colpoda E6 formed distinct clades. Scanning electron microscopy showed that Anteglaucoma CS11A had a bigger-sized body than Colpoda E6 and, unlike Tetrahymena IB (the reference strain), neither ciliate produced pellets, which are extracellular vacuoles. Fluorescence microscopic observations revealed that although the intake amounts differed, all three ciliates rapidly ingested LpJR32 regardless of the presence or absence of the icm/dot virulence genes, indicating that they all interacted with LpJR32. In co-cultures with Anteglaucoma CS11A, the LpJR32 levels were maintained but fell dramatically when the co-culture contained the LpJR32 icm/dot deletion mutant instead. Anteglaucoma CS11A died within 2 days of co-culture with LpJR32, but survived co-culture with the deletion mutant. In co-cultures with Colpoda E6, LpJR32 levels were maintained but temporarily decreased independently of the virulence gene. Concurrently, the Colpoda E6 ciliates survived by forming cysts, which may enable them to resist harsh environments, and by diminishing the sensitivity of trophozoites to Lp. In the Tetrahymena IB co-cultures with LpJR32 or Δicm/dot, the Lp levels were maintained, albeit with temporal decreases, and the Tetrahymena IB levels were also maintained. We conclude that unlike Tetrahymena IB with pellet production, Anteglaucoma CS11A can be killed by LpJR32 infection, and Colpoda E6 can resist LpJR32 infection through cyst formation and the low sensitivity of trophozoites to Lp. Thus, the two ciliates that we isolated had different susceptibilities to LpJR32 infection.

Usefulness of a 3D-printing air sampler for capturing live airborne bacteria and exploring the environmental factors that can influence bacterial dynamics.

We created a handmade 3D-printed air sampler to effectively collect live airborne bacteria, and determined which environmental factors influenced the bacteria. Bacterial colony forming units (CFUs) in the air samples (n = 37) were monitored by recording the environmental changes occurring over time, then determining the presence/absence of correlations among such changes. The bacterial CFUs changed sharply and were significantly correlated with the DNA concentrations, indicating that the captured bacteria made up most of the airborne bacteria. Spearman's rank correlation analysis revealed significant correlations between the bacterial CFU values and some environmental factors (humidity, wind speed, insolation, and 24-h rainfall). Similarly the significant associations of CFU with humidity and wind speed were also found by multiple regression analysis with box-cox transformation. Among our panel of airborne bacteria (952 strains), 70 strains were identified as soil-derived Bacillus via the production of Escherichia coli- and Staphylococcus aureus-growth inhibiting antibiotics and by 16S rDNA typing. Soil-derived protozoa were also isolated from the air samples. We conclude that the airborne bacteria mainly derived from soil can alter in number according to environmental changes. Our sampler, which was created by easy-to-customize 3D printing, is a useful device for understanding the dynamics of live airborne bacteria.

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

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

Distribution of amoebal endosymbiotic environmental chlamydia Neochlamydia S13 via amoebal cytokinesis.

We previously isolated a symbiotic environmental amoeba, harboring an environmental chlamydia, Neochlamydia S13. Interestingly, this bacterium failed to survive outside of host cells and was immediately digested inside other amoebae, indicating bacterial distribution via cytokinesis. This may provide a model for understanding organelle development and chlamydial pathogenesis and evolution; therefore, we assessed our hypothesis of Neochlamydia S13 distribution via cytokinesis by comparative analysis with other environmental Chlamydiae (Protochlamydia R18 and Parachlamydia Bn9 ). Dual staining with 4',6-diamidino-2-phenylindole and phalloidin revealed that the progeny of Neochlamydia S13 and Protochlamydia R18 existed in both daughter cells with a contractile ring on the verge of separation. However, in contrast to other environmental Chlamydiae, little Neochlamydia S13 16S ribosomal DNA was amplified from the culture supernatant. Interestingly, Neochlamydia S13 failed to infect aposymbiotic amoebae, indicating an intimate interaction with the host cells. Furthermore, its infectious rates in cultures expanded from a single amoeba were always maintained at 100%, indicating distribution via cytokinesis. We concluded that unlike other environmental Chlamydiae, Neochlamydia S13 has a unique ability to divide its progeny only via host amoebal cytokinesis. This may be a suitable model to elucidate the mechanism of cell organelle distribution and of chlamydial pathogenesis and evolution.

Isolation and Characterization of Antimicrobial-Resistant Escherichia coli from Retail Meats from Roadside Butcheries in Uganda.

Retail meats are one of the main routes for spreading antimicrobial-resistant bacteria (ARB) from livestock to humans through the food chain. In African countries, retail meats are often sold at roadside butcheries without chilling or refrigeration. Retail meats in those butcheries are suspected to be contaminated by ARB, but it was not clear. In this study, we tested for the presence of antimicrobial-resistant Escherichia coli from retail meats (n = 64) from roadside butcheries in Kampala, Uganda. The meat surfaces were swabbed and inoculated on PetriFilm SEC agar to isolate E. coli. We successfully isolated E. coli from 90.6% of these retail meat samples. We identified the phylogenetic type, antimicrobial susceptibility, and antimicrobial resistance genes prevalence between retail meat isolates (n = 89). Phylogenetic type B1 was identified from 70.8% of the retail meat isolates, suggesting that the isolates originated primarily from fecal contamination during meat processing. Tetracycline (TET)-resistant isolates with tetA and/or tetB gene(s) were the most frequently detected (28.1%), followed by ampicillin (AMP) resistance genes with blaTEM (15.7%,) and sulfamethoxazole-trimethoprim (SXT) resistance genes with sul2 (15.7%). No extended-spectrum beta-lactamase-producing isolates were detected. A conjugation assay showed that resistance to AMP, TET, and SXT could be simultaneously transferred to recipients. These findings suggest that antimicrobial-resistant E. coli can easily be transferred from farms to tables from retail meats obtained from roadside butcheries.

Hypoxia promotes Chlamydia trachomatis L2/434/Bu growth in immortal human epithelial cells via activation of the PI3K-AKT pathway and maintenance of a balanced NAD+/NADH ratio.

Chlamydia trachomatis LGV (CtL2) causes systemic infection and proliferates in lymph nodes as well as genital tract or rectum producing a robust inflammatory response, presumably leading to a low oxygen environment. We therefore assessed how CtL2 growth in immortal human epithelial cells adapts to hypoxic conditions. Assessment of inclusion forming units, the quantity of chlamydial 16S rDNA, and inclusion size showed that hypoxia promotes CtL2 growth. Under hypoxia, HIF-1α was stabilized and p53 was degraded in infected cells. Moreover, AKT was strongly phosphorylated at S473 by CtL2 infection. This activation was significantly diminished by LY-294002, a PI3K-AKT inhibitor, which decreased the number of CtL2 progeny. HIF-1α stabilizers (CoCl2, desferrioxamine) had no effect on increasing CtL2 growth, indicating no autocrine impact of growth factors produced by HIF-1α stabilization. Furthermore, in normoxia, CtL2 infection changed the NAD+/NADH ratio of cells with increased gapdh expression; in contrast, under hypoxia, the NAD+/NADH ratio was the same in infected and uninfected cells with high and stable expression of gapdh, suggesting that CtL2-infected cells adapted better to hypoxia. Together, these data indicate that hypoxia promotes CtL2 growth in immortal human epithelial cells by activating the PI3K-AKT pathway and maintaining the NAD+/NADH ratio with stably activated glycolysis.

Chlamydia trachomatis isolated from cervicovaginal samples in Sapporo, Japan, reveals the circulation of genetically diverse strains.

BACKGROUND: This study was conducted to understand the molecular epidemiology of circulating Chlamydia trachomatis (Ct) strains in Sapporo, Japan. METHODS: A total of 713 endocervical samples collected from April 2016 to March 2019 were screened for Ct. The obtained Ct positive samples were analyzed by ompA genotyping and multilocus sequence analysis (MLSA). RESULTS: Eighty-three (11.6%) samples were positive for Ct plasmid DNA. Sequence analysis of the ompA gene from the 61 positive cases revealed eight genotypes: F (40.9%), E (19.6%), D (14.7%), G (9.8%), H (6.5%), I (3.2%), K (3.2%), and J (1.6%). The globally dominant genotype E and F strains were highly conserved with 13 ompA genetic variants being detected, whereas genotype D strains were the most diverse. Genetic characterization of D strains revealed that D1 genetic variants may be potentially specific to Sapporo. MLSA revealed 13 unique sequence types (STs) including four novel STs from 53 positive samples, with the globally dominant STs 39 and 19 being predominant. STs 39, 34, and 21 were exclusively associated with genotypes E and F indicating their global dominance. Novel ST70 and ST30 were specifically associated with genotype D. CONCLUSION: Our study has revealed the circulation of genetically diverse Ct strains in the women population of Sapporo, Japan. We suggest identifying a transmission network of those successful strains and implementing public health prevention strategies to control the spread of Ct in Sapporo.

Complete genome and bimodal genomic structure of the amoebal symbiont Neochlamydia strain S13 revealed by ultra-long reads obtained from MinION.

Neochlamydia strain S13 is an amoebal symbiont of an Acanthamoeba sp. The symbiont confers resistance to Legionella pneumophila on its host; however, the molecular mechanism underlying this resistance is not completely understood. Genome analyses have been crucial for understanding the complicated host-symbiont relationship but segregating the host's genome DNA from the symbiont's DNA is often challenging. In this study, we successfully identified a bimodal genomic structure in Neochlamydia strain S13 using PacBio RS II supported by ultra-long reads derived from MinION. One mode consisted of circular sequences of 2,586,667 and 231,307 bp; the other was an integrated sequence of the two via long homologous regions. They encoded 2175 protein-coding regions, some of which were implied to be acquired via horizontal gene transfer. They were specifically conserved in the genus Neochlamydia and formed a cluster in the genome, presumably by multiplication through genome replication. Moreover, it was notable that the sequenced DNA was obtained without segregating the symbiont DNA from the host. This is an easy and versatile technique that facilitates the characterization of diverse hosts and symbionts in nature.

Effect of thermal control of dry fomites on regulating the survival of human pathogenic bacteria responsible for nosocomial infections.

We monitored the survival of human pathogenic bacteria [Escherichia coli (ATCC), extended-spectrum ß-lactamase-producing E. coli (Clinical isolate), New Delhi metallo-ß-lactamase-producing E. coli (clinical isolate), Staphylococcus aureus (ATCC)] on dry materials (vinyl chloride, aluminum, plastic, stainless steel) at distinct temperatures (room temperature or 15°C-37°C). These bacteria favored a lower temperature for their prolonged survival on the dry fomites, regardless of the material type. Interestingly, when mixed with S. aureus, E. coli survived for a longer time at a lower temperature. Cardiolipin, which can promote the survival of S. aureus in harsh environments, had no effect on maintaining the survival of E. coli. Although the trends remained unchanged, adjusting the humidity from 40% to 60% affected the survival of bacteria on dry surfaces. Scanning electron microscopic analysis revealed no morphological differences in these bacteria immediately before or after one day of dry conditions. In addition, ATP assessment, a method used to visualize high-touch surfaces in hospitals, was not effective at monitoring bacterial dynamics. A specialized handrail device fitted with a heater, which was maintained at normal human body core temperature, successfully prohibited the prolonged survival of bacteria [Enterococcus faecalis (ATCC), E. coli (ATCC), Pseudomonas aeruginosa (ATCC), S. aureus (ATCC), Acinetobacter baumannii (clinical isolate), and Serratia marcescens (clinical isolate)], with the exception of spore-forming Bacillus subtilis (from our laboratory collection) and the yeast-like fungus Candida albicans (from our laboratory collection)] on dry surfaces. Taken together, we concluded that the tested bacteria favor lower temperatures for their survival in dry environments. Therefore, the thermal control of dry fomites has the potential to control bacterial survival on high-touch surfaces in hospitals.

Chlamydia pneumoniae enhances Interleukin 8 (IL-8) production with reduced azithromycin sensitivity under hypoxia.

Obligate intracellular bacterium Chlamydia pneumoniae causes respiratory tract infections such as community-acquired pneumonia. During infection, C. pneumoniae induces inflammatory responses in host cells and the oxygen concentration at the infection sites subsequently decreases. Because hypoxic conditions influence further inflammatory responses and reduced antibacterial effects, this may exacerbate the C. pneumoniae infection. Here, we show inflammatory responses and drug sensitivity in C. pneumoniae-infected cells under hypoxic conditions. First, we confirmed the enhanced growth of C. pneumoniae under hypoxia, which indicates that the hypoxic condition we used could adequately reproduce past reports. We then demonstrated a significant increase in production of the pro-inflammatory cytokine Interleukin 8 (IL-8) in C. pneumoniae-infected cells under hypoxic conditions. Furthermore, hypoxia decreased the antibacterial effects of azithromycin against C. pneumoniae compared with normoxic conditions. Together, our data suggest that inflammatory responses and drug sensitivity may have been underestimated in C. pneumoniae infection in previous studies. Thus, to accurately understand the Chlamydia infection, it may be necessary to perform in vitro experiments under hypoxic conditions.

A simple and short microbiology practical improves undergraduate nursing students' awareness of bacterial traits and ability to avoid spreading infections.

BACKGROUND: Nurses are responsible for implementing appropriate measures to reduce hospital infections, especially with multidrug resistant bacteria, so nursing students should learn about microbiology. This helps them to understand bacterial dissemination and infectious disease control. Because of tight schedules, however, its teaching is limited in undergraduate nursing classes in Japan. We therefore tested whether a simple short practical session in a microbiology class could help to improve undergraduate nursing students' awareness of bacterial traits and how to prevent infections. METHODS: This study involved second-grade nursing students (n = 76). Two short practical sessions (a total of 3 h, across 2 days) were used to assess the effectiveness of washing or disinfection on hand bacteria in a 16-class microbiology course (total class time was 24 h, plus an exam). Hand bacteria were sampled on LB agar plates with orientation during the first half-day, and the plates examined for colonies with distinct color or morphological traits, and discussed, in the second session, a week later. Questionnaires before and after the exercise were used to assess changes in awareness of unseen bacteria inhabiting around us connecting bacterial traits and how to prevent infections. RESULTS: The results showed that the practical increased the nursing students' awareness of fomites (utensils) (p = 0.0115), fomites (contact-based) (p = 0.0016), habitats (body surface) (p = 0.0127), action facilitating hospital infection (p = 0.0166), and changes in physical condition caused by bacterial infections (p = 0.0136). There were no changes in word associations (p = 0.627) or habitats (inside body) (p = 0.308). Difficulty score, which is an element in questionnaire psychometric properties, tended to be close to the expected score through the practical, but not statistical significant. In addition, regardless of before or after practical, Cronbach α score, which is an indicator of the reliability among items of multi-choice questions, showed > 0.8, indicating validity of evaluation items. Thus, the student's awareness of unseen bacteria inhabiting around us was significantly increased as compared to those before practical in microbiology class. CONCLUSIONS: The simple short practical effectively improved nursing students' awareness of unseen bacteria inhabiting around us in microbiology course, useful for even tight teaching schedules.

Phenotypic and genotypic analyses of antimicrobial resistant bacteria in livestock in Uganda.

Antimicrobial resistant bacteria (ARB) in livestock are a global public health concern, not only because they prolong infectious diseases but also they can be transferred from animals to humans via the food chain. Here, we studied ARB in livestock at commercial and subsistence farms (n = 13) in Wakiso and Mpigi districts, Uganda. We enquired from the farmers about the type and the purpose of antimicrobial agents they have used to treat their livestock. After collecting faeces, we isolated antimicrobial resistant Escherichia coli from livestock faeces (n = 134) as an indicator bacterium. These strains showed resistance to ampicillin (44.8%), tetracycline (97.0%), and sulfamethoxazole-trimethoprim (56.7%). The frequency of ampicillin-resistance was significantly correlated with the usage of penicillins to livestock in the farms (p = 0.04). The metagenomics data detected 911 antimicrobial resistant genes that were classified into 16 categories. Genes for multidrug efflux pumps were the most prevalent category in all except in one sample. Interestingly, the genes encoding third-generation cephalosporins (blaCTX-M ), carbapenems (blaACT ), and colistin (arnA) were detected by metagenomics analysis although these phenotypes were not detected in our E. coli strains. Our results suggest that the emergence and transmission of cephalosporin, carbapenem, and/or colistin-resistant bacteria among livestock can occur in future if these antimicrobial agents are used.