Quantitative Measurement of Enteric Bacteria Load From Public Surfaces in Restaurants in Nnewi-Town

Background: Bio-contamination of surfaces of various items and equipment used by the public is the main cause of disease epidemic. Most outbreaks of infection associated with inanimate objects are caused by items that should be sterile but have been contaminated and are generally known as fomites. Objective: This study is to evaluate the presence of enteric bacteria from different public surfaces in restaurants in Nnewi town. Methodology: Selected public surfaces in eight (8) randomly selected restaurants were analyzed for the presence of enteric bacteria via culture and biochemical tests following a standard criterion. The sample sites included; tables, chairs, counters, sinks and walls. Also, Disc susceptibility test were carried out on the isolates using conventional antibiotics. Results: Eighty-eight (88) bacteria isolates were isolated and identified, and S. aureus showed the highest prevlence 29(33%), followed by E. coli 15(17%), Proteus spp 13(15%), Klebsiella spp 12(14%), Pseudomonas spp 10(11%), and Coagulase-negative Staphylococci 9(10%). Tables and sinks were the most contaminated fomites each constituting of 19(22%) of the total bacteria isolated in this study. Escherichia coli, Pseudomonas spp, Proteus spp and Staphylococcus aureus, were the most resistant isolates to the antibiotics, Coagulase-negative Staphylococci was the most sensitive isolate. Out of the ten antibiotics tested ceporex was found to be the least effective with about 100% resistance by the isolates while ciprofloxacin, ceftriaxone, and levofloxacin were the most effective antibiotics with 0% resistance by the bacteria isolates. Conclusion: the study has shown that public surfaces in restaurant can easily be contaminated with a variety of bacterial contaminants that may be multi-drug resistant bacteria strains posing as a possible public health issue.

Distribution of copper resistance gene variants of Xanthomonas citri subsp. citri and Xanthomonas euvesicatoria pv. perforans

In Taiwan, numerous crops are threatened by Xanthomonas diseases such as citrus bacterial canker caused by X. citri subsp. citri and tomato bacterial spot mainly caused by X. euvesicatoria pv. perforans. Foliar sprays of copper-based bactericides have been frequently used for control of plant bacterial diseases. However, in Taiwan not much attention was paid on copper-resistant (Cu^R) Xanthomonas spp. and their impact on disease control efficacy of copper-based bactericides. In this study, Cu^R Xanthomonas isolates were collected from citrus and tomato in Taiwan. Compared with the pronounced effect on the copper sensitive isolate, spraying of copper hydroxide at the recommended rate of 0.5 kg/ha could not protect tomato plants against bacterial spot caused by the Cu^R isolate. Phylogenetic analysis of concatenated copper resistance genes, copL, copA, and copB, indicate that the Taiwanese Cu^R isolates belong to the worldwide clade. In addition to the three previously reported variants of the copB gene, analysis of complete copB sequences from xanthomonads associated with citrus and solanaceous hosts revealed the other three variants of copB and their global distribution. Copper-resistant Xanthomonas isolates from Taiwan have the two unreported variants of copB genes which differ from the other three previously reported types in the sizes and structures. The information provided here reveals the necessity to develop and include alternative measures rather than relying on foliar sprays of copper bactericides for sustainable control of tomato bacterial spot in Taiwan.

Evaluating fungicide selections to manage Pythium root rot on poinsettia cultivars with varying levels of partial resistance

Pythium aphanidermatum is the predominant species causing Pythium root rot of commercially grown poinsettia (Euphorbia pulcherrima Willd. ex Kotzch) in North Carolina. Pythium root rot is managed primarily through a combination of sanitation practices and preventative fungicide applications of mefenoxam or etridiazole. Insensitivity to mefenoxam is common but growers continue to rely on it due to lack of inexpensive and efficacious alternatives. This research was conducted to identify alternative fungicides for Pythium root rot control and evaluate their efficacy on poinsettia cultivars with varying levels of partial resistance. Greenhouse studies were conducted to assess efficacy of fungicide treatments in seven poinsettia cultivars inoculated with a mefenoxam-sensitive isolate of P. aphanidermatum. One study examined control with a single fungicide drench made at transplant and a second study examined repeat fungicide applications made throughout the experiment. Treatments containing etridiazole, mefenoxam, fenamidone, and cyazofamid provided control of Pythium root rot across all cultivars in both experiments whereas Fosetyl-al, potassium phosphite, and Trichoderma spp. failed to offer satisfactory control. Azoxystrobin, pyraclostrobin, and propamocarb reduced disease on some cultivars but failed to control Pythium root rot on highly susceptible cultivars. Four isolates of P. aphanidermatum cultured from plants growing in commercial greenhouses were evaluated for in vitro sensitivity to fungicides labeled for Pythium root rot control at four rates. Etridiazole, fosetyl-al, and potassium phosphite completely inhibited mycelial growth, whereas isolates varied in response to mefenoxam, cyazofamid, propamocarb, fenamidone, azoxystrobin, and pyraclostrobin in vitro. Twenty-one additional isolates then were evaluated at label rates of these fungicides. Seven isolates were insensitive to label rates of all three quinone outside inhibitors (QoIs) and one isolate was insensitive to the QoIs and mefenoxam. These results provide guidelines for selecting fungicides to maximize control of Pythium root rot on poinsettia cultivars.

Transformation of fluoroquinolone-resistance Neisseria gonorrhoeae gyrA and parC genes

Introduction: N. gonorrhoeae is one of the etiological causative agents of one of the most common sexually transmitted diseases. Gonococci has created many resistance mechanisms, which is associated with bacterial evolution. Natural transformation is the basic method of horizontal gene transfer in bacteria of the genus Neisseria, which can lead to a mutation in the gyrA gene encoding DNA gyrase. The aim of the study was to verify the view on the significance of mutations at positions 91 and 95 of the gyrA protein on the sensitivity of N. gonorrhoeae to antibiotics of the quinolone type. Methods: GyrA gene was introduced into an sensitive isolate of N. gonorrhoeae using genetic transformation. Resistance gene donor, recipient and transform strains were tested for susceptibility and the gyrA gene was sequenced. Results: It has been shown that double mutation in amino acid acid sequence of the GyrA protein at positions 91 and 95 increase the value of MIC from 0,003 mg / L to 0,125 mg / L at CIP sensitive N. gonorrhoeae strain. Conclusions: Mutations in the amino acid sequence at positions 91 and 95 affet the strain’s sensitivity to ciprofloxacin, but it is not the only mechanism which could alter the MIC value of quinolones.

Molecular Analysis of Aquaglyceroporin 1 Gene in Non-Healing Clinical Iso¬lates Obtained from Patients with Cutaneous Leishmaniasis from Central of Iran

Background: Regarding the antimonial-resistant of Leishmania spp., understanding of related mechanism is neces­sary. One of the most important involved molecules is aquaglyceropin1 (AQP1). The aim of this study was molecu­lar analysis of AQP1 gene from antimonial-resistant clinical isolates and its expression. Methods: Overall, 150 patients with cutaneous leishmaniasis referring to the reference laboratories of Yazd and Varzaneh,, located 105km southeast of Isfahan and 240km away from Yazd, were assessed from Jun 2015 to Dec 2017. After sampling, staining was done and evaluated for Leishman by microscope. Samples were collected in RNAlater solution for gene expression analysis in non-healing isolates. DNA extraction was performed from each slide with Leishman body. All patients with L. major isolates detected by ITS1-PCR-RFLP were followed for find­ing the resistant isolates, consequence of molecular characterization of AQP1 using PCR-RFLP. Gene expression of AQP1 from all resistant isolates was assessed in comparison with the one in a sensitive isolate. Statistical analysis was done using SPSS. The significance level was considered ≤0.05. Results: Five isolates were detected as antimonial resistant. Molecular detection and identification were appeared that all were L. major. The molecular characterization of AQP1 showed G562A mutation. Gene expression of AQP1 in resistant isolates showed 1.67 fold higher than the sensitive isolate. Conclusion: We reported a new point mutation of G562A in AQP1 gene involved in molecular mechanism in re­sistant isolates.

Resistance to Fluopicolide and Propamocarb and Baseline Sensitivity to Ethaboxam Among Isolates of Pseudoperonospora cubensis From the Eastern United States

Chemical control is currently the most effective method for controlling cucurbit downy mildew (CDM) caused by Pseudoperonospora cubensis. Most commercial cucurbit cultivars, with the exception of a few new cucumber cultivars, lack adequate disease resistance. Fluopicolide and propamocarb were among the most effective fungicides against CDM in the United States between 2006 and 2009. Since then, reduced efficacy of these two fungicides under field conditions was reported starting around 2013 but occurrence of resistance to fluopicolide and propamocarb in field isolates of P. cubensis had not been established. Thirty-one isolates collected from cucurbits in the eastern United States were tested for their sensitivity to fluopicolide and propamocarb using a leaf disc assay. This same set of isolates and four additional isolates (i.e., 35 isolates) were also used to establish the baseline sensitivity of P. cubensis to ethaboxam, an ethylamino-thiazole-carboxamide fungicide, which was recently granted registration to control CDM in the United States. About 65% of the isolates tested were resistant to fluopicolide with at least one resistant isolate being present in samples collected from 12 of the 13 states in the eastern United States. About 74% of the isolates tested were sensitive to propamocarb with at least one resistant isolate being among samples collected from 8 of the 12 states in the study. The frequency of resistance to fluopicolide and propamocarb was high among isolates collected from cucumber, while the frequency was low among isolates collected from other cucurbit host types. All isolates tested were found to be sensitive to ethaboxam and EC50 values ranged from 0.18 to 3.08 mg a.i./liter with a median of 1.55 mg a.i./liter. The ratio of EC50 values for the least sensitive and the most sensitive isolate was 17.1, indicating that P. cubensis isolates were highly sensitive to ethaboxam. The most sensitive isolates to ethaboxam were collected from New York, North Carolina, and Ohio, while the least sensitive isolates were collected from Georgia, Michigan, and New Jersey. These results show that ethaboxam could be a viable addition to fungicide programs used to control CDM in the United States.

Iron Restriction to Clinical Isolates ofCandida albicansby the Novel Chelator DIBI Inhibits Growth and Increases Sensitivity to AzolesIn VitroandIn Vivoin a Murine Model of Experimental Vaginitis

ABSTRACTCandida albicansis an important opportunistic pathogen causing various human infections that are often treated with azole antifungals. The U.S. CDC now regards developing candidal antifungal resistance as a threat, creating a need for new and more effective antifungal treatments. Iron is an essential nutrient for all living cells, and there is growing evidence that interference with iron homeostasis ofC. albicanscan improve its response to antifungals. This study was aimed at establishing whether withholding iron by currently used medical iron chelators and the novel chelator DIBI could restrict growth and also enhance the activity of azoles against clinical isolates ofC. albicans. DIBI, but not deferoxamine or deferiprone, inhibited the growth ofC. albicansat relatively low concentrationsin vitro, and this inhibition was reversed by iron addition. DIBI in combination with various azoles demonstrated stronger growth inhibition than the azoles alone and greatly prolonged the inhibition of cell multiplication. In addition, the administration of DIBI along with fluconazole (FLC) to mice inoculated with an FLC-sensitive isolate in a model of experimentalC. albicansvaginitis showed a markedly improved clearance of infection. These results suggest that iron chelation by DIBI has the potential to enhance azole efficacy for the treatment of candidiasis.

Frequency and Mechanism of Spontaneous Resistance to Sulbactam Combined with the Novel β-Lactamase Inhibitor ETX2514 in Clinical Isolates of Acinetobacter baumannii

ABSTRACTThe novel diazabicyclooctenone ETX2514 is a potent, broad-spectrum serine β-lactamase inhibitor that restores sulbactam activity against resistantAcinetobacter baumannii. The frequency of spontaneous resistance to sulbactam-ETX2514 in clinical isolates was found to be 7.6 × 10−10to <9.0 × 10−10at 4× MIC and mapped to residues near the active site of penicillin binding protein 3 (PBP3). Purified mutant PBP3 proteins demonstrated reduced affinity for sulbactam. In a sulbactam-sensitive isolate, resistance also mapped to stringent response genes associated with resistance to PBP2 inhibitors, suggesting that in addition to β-lactamase inhibition, ETX2514 may enhance sulbactam activity inA. baumanniivia inhibition of PBP2.

Molecular Mechanisms Involved in Qualitative and Quantitative Resistance to the Dicarboximide Fungicide Iprodione in Sclerotinia homoeocarpa Field Isolates

The dicarboximide fungicide class is commonly used to control Sclerotinia homoeocarpa, the causal agent of dollar spot on turfgrass. Despite frequent occurrences of S. homoeocarpa field resistance to iprodione (dicarboximide active ingredient), the genetic mechanisms of iprodione resistance have not been elucidated. In this study, 15 field isolates (seven suspected dicarboximide resistant, three multidrug resistance (MDR)-like, and five dicarboximide sensitive) were used for sequence comparison of a histidine kinase gene, Shos1, of S. homoeocarpa. The suspected dicarboximide-resistant isolates displayed nonsynonymous polymorphisms in codon 366 (isoleucine to asparagine) in Shos1, while the MDR-like and sensitive isolates did not. Further elucidation of the Shos1 function, using polyethylene glycol–mediated protoplast transformation indicated that S. homoeocarpa mutants (Shos1I366N) from a sensitive isolate gained resistance to dicarboximides but not phenylpyrrole and polyols. The deletion of Shos1 resulted in higher resistance to dicarboximide and phenylpyrrole and higher sensitivity to polyols than Shos1I366N. Levels of dicarboximide sensitivity in the sensitive isolate, Shos1I366N, and Shos1 deletion mutants were negatively correlated to values of iprodione-induced expression of ShHog1, the last kinase in the high-osmolarity glycerol pathway. Increased constitutive and induced expression of the ATP-binding cassette multidrug efflux transporter ShPDR1 was observed in six of seven dicarboximide-resistant isolates. In conclusion, S. homoeocarpa field isolates gained dicarboximide resistance through the polymorphism in Shos1 and the overexpression of ShPDR1.