Eco-alternative treatments for Vibrio parahaemolyticus and V. cholerae biofilms from shrimp industry through Eucalyptus (Eucalyptus globulus) and Guava (Psidium guajava) extracts: A road for an Ecuadorian sustainable economy.

Understanding how environmental variables influence biofilm formation becomes relevant for managing Vibrio biofilm-related infections in shrimp production. Therefore, we evaluated the impact of temperature, time, and initial inoculum in the biofilm development of these two Vibrio species using a multifactorial experimental design. Planktonic growth inhibition and inhibition/eradication of Vibrio biofilms, more exactly V. parahaemolyticus (VP87 and VP275) and V. cholerae (VC112) isolated from shrimp farms were evaluated by Eucalyptus and Guava aqueous leaf extracts and compared to tetracycline and ceftriaxone. Preliminary results showed that the best growth conditions of biofilm development for V. parahaemolyticus were 24 h and 24°C (p <0.001), while V. cholerae biofilms were 72 h and 30°C (p <0.001). Multivariate linear regression ANOVA was applied using colony-forming unit (CFU) counting assays as a reference, and R-squared values were applied as goodness-of-fit measurements for biofilm analysis. Then, both plant extracts were analyzed with HPLC using double online detection by diode array detector (DAD) and mass spectrometry (MS) for the evaluation of their chemical composition, where the main identified compounds for Eucalyptus extract were cypellogin A, cypellogin B, and cypellocarpin C, while guavinoside A, B, and C compounds were the main compounds for Guava extract. For planktonic growth inhibition, Eucalyptus extract showed its maximum effect at 200 µg/mL with an inhibition of 75% (p < 0.0001) against all Vibrio strains, while Guava extract exhibited its maximum inhibition at 1600 µg/mL with an inhibition of 70% (p < 0.0001). Both biofilm inhibition and eradication assays were performed by the two conditions (24 h at 24°C and 72 h at 30°C) on Vibrio strains according to desirability analysis. Regarding 24 h at 24°C, differences were observed in the CFU counting between antibiotics and plant extracts, where both plant extracts demonstrated a higher reduction of viable cells when compared with both antibiotics at 8x, 16x, and 32x MIC values (Eucalyptus extract: 1600, 3200, and 6400 µg/mL; while Guava extract: 12800, 25600, and 52000 µg/mL). Concerning 72 h at 30°C, results showed a less notorious biomass inhibition by Guava leaf extract and tetracycline. However, Eucalyptus extract significantly reduced the total number of viable cells within Vibrio biofilms from 2x to 32x MIC values (400-6400 µg/mL) when compared to the same MIC values of ceftriaxone (5-80 µg/mL), which was not able to reduce viable cells. Eucalyptus extract demonstrated similar results at both growth conditions, showing an average inhibition of approximately 80% at 400 µg/mL concentration for all Vibrio isolates (p < 0.0001). Moreover, eradication biofilm assays demonstrated significant eradication against all Vibrio strains at both growth conditions, but biofilm eradication values were substantially lower. Both extract plants demonstrated a higher reduction of viable cells when compared with both antibiotics at 8x, 16x, and 32x MIC values at both growth sets, where Eucalyptus extract at 800 µg/mL reduced 70% of biomass and 90% of viable cells for all Vibrio strains (p < 0.0001). Overall results suggested a viable alternative against vibriosis in the shrimp industry in Ecuador.

Exploring extended-spectrum beta lactamase resistance in Vibrio parahaemolyticus and Vibrio cholerae within the tropical mangrove ecosystem of southwest India.

Multidrug-resistant pathogenic vibrios are a crisis of concern as they cause multiple illnesses, including gastroenteritis in humans and acute hepatopancreatic necrosis in aquaculture. In the current study, we investigated the prevalence of the beta-lactamase gene CTX-M-group 1 in Vibrio spp. (Vibrio cholerae and Vibrio parahaemolyticus) from the water and sediment of urban tropical mangrove ecosystems of Kerala, southwest India. A total of 120 isolates of Vibrio spp. were tested for antibiotic susceptibility to 14 antibiotics. In water, ampicillin resistance was very high in isolates of V. cholerae (94.1%, n = 17) and V. parahaemolyticus (89.1%, n = 46). 26.9% of V. parahaemolyticus and 14.2% of V. cholerae harbored the CTX-M-group 1 gene in water samples. Compared to V. cholerae, the CTX-M-group 1 gene was exclusively hosted by V. parahaemolyticus (49%) in sediment samples. A significant difference in the prevalence of the CTX-M-group 1 gene was observed among Vibrio spp. in both water and sediment samples (p < 0.05). The results revealed the presence of multidrug-resistant and beta-lactamase harboring Vibrio spp. in mangrove ecosystems, which may have evolved as a consequence of the misuse and abuse of broad-spectrum antibiotics as prophylaxis in human health care and aquaculture.

Antibiotic Resistance in Vibrio cholerae: Mechanistic Insights from IncC Plasmid-Mediated Dissemination of a Novel Family of Genomic Islands Inserted at trmE.

Cholera remains a formidable disease, and reports of multidrug-resistant strains of the causative agent Vibrio cholerae have become common during the last 3 decades. The pervasiveness of resistance determinants has largely been ascribed to mobile genetic elements, including SXT/R391 integrative conjugative elements, IncC plasmids, and genomic islands (GIs). Conjugative transfer of IncC plasmids is activated by the master activator AcaCD whose regulatory network extends to chromosomally integrated GIs. MGIVchHai6 is a multidrug resistance GI integrated at the 3' end of trmE (mnmE or thdF) in chromosome 1 of non-O1/non-O139 V. cholerae clinical isolates from the 2010 Haitian cholera outbreak. In the presence of an IncC plasmid expressing AcaCD, MGIVchHai6 excises from the chromosome and transfers at high frequency. Herein, the mechanism of mobilization of MGIVchHai6 GIs by IncC plasmids was dissected. Our results show that AcaCD drives expression of GI-borne genes, including xis and mobIM , involved in excision and mobilization. A 49-bp fragment upstream of mobIM was found to serve as the minimal origin of transfer (oriT) of MGIVchHai6. The direction of transfer initiated at oriT was determined using IncC plasmid-driven mobilization of chromosomal markers via MGIVchHai6. In addition, IncC plasmid-encoded factors, including the relaxase TraI, were found to be required for GI transfer. Finally, in silico exploration of Gammaproteobacteria genomes identified 47 novel related and potentially AcaCD-responsive GIs in 13 different genera. Despite sharing conserved features, these GIs integrate at trmE, yicC, or dusA and carry a diverse cargo of genes involved in phage resistance.IMPORTANCE The increasing association of the etiological agent of cholera, Vibrio cholerae serogroup O1 and O139, with multiple antibiotic resistance threatens to deprive health practitioners of this effective tool. Drug resistance in cholera results mainly from acquisition of mobile genetic elements. Genomic islands conferring multidrug resistance and mobilizable by IncC conjugative plasmids were reported to circulate in non-O1/non-O139 V. cholerae clinical strains isolated from the 2010 Haitian cholera outbreak. As these genomic islands can be transmitted to pandemic V. cholerae serogroups, their mechanism of transmission needed to be investigated. Our research revealed plasmid- and genomic island-encoded factors required for the resistance island excision, mobilization, and integration, as well as regulation of these functions. The discovery of related genomic islands carrying diverse phage resistance genes but lacking antibiotic resistance-conferring genes in a wide range of marine dwelling bacteria suggests that these elements are ancient and recently acquired drug resistance genes.

A comprehensive review of therapeutic approaches available for the treatment of cholera.

OBJECTIVES: The oral rehydration solution is the most efficient method to treat cholera; however, it does not interfere in the action mechanism of the main virulence factor produced by Vibrio cholerae, the cholera toxin (CT), and this disease still stands out as a problem for human health worldwide. This review aimed to describe therapeutic alternatives available in the literature, especially those related to the search for molecules acting upon the physiopathology of cholera. KEY FINDINGS: New molecules have offered a protection effect against diarrhoea induced by CT or even by infection from V. cholerae. The receptor regulator cystic fibrosis channel transmembrane (CFTR), monosialoganglioside (GM1), enkephalinase, AMP-activated protein kinase (AMPK), inhibitors of expression of virulence factors and activators of ADP-ribosylarginine hydrolase are the main therapeutic targets studied. Many of these molecules or extracts still present unclear action mechanisms. CONCLUSIONS: Knowing therapeutic alternatives and their molecular mechanisms for the treatment of cholera could guide us to develop a new drug that could be used in combination with the rehydration solution.

Antimicrobial Resistance in Bacteria Isolated from Foods in Cuba.

INTRODUCTION Antimicrobial drug resistance constitutes a health risk of increasing concern worldwide. One of the most common av-enues for the acquisition of clinically-relevant antimicrobial resistance can be traced back to the food supply, where resistance is acquired through the ingestion of antimicrobial resistant microorganisms pres-ent in food. Antimicrobial resistance constitutes a health risk, leading to production losses and negative consequences for livelihood and food safety. OBJECTIVE Determine whether resistant bacteria are present in foods in Cuba. METHODS A descriptive observational study was conducted in theMicrobiology Laboratory of Cuba's National Institute of Hygiene, Epi-demiology and Microbiology from September 2004 through Decem-ber 2018. Researchers analyzed 1178 bacterial isolates from food samples. The isolates were identifi ed as Escherichia coli, Salmonella, Vibrio cholerae and coagulase-positive Staphylococcus. The antimi-crobial susceptibility study was performed using the Bauer-Kirby disk diffusion method, following procedures outlined by the Clinical and Laboratory Standards Institute. The data were analyzed using WHO-NET version 5.6. RESULTS Of the total isolates, 62.1% were resistant to at least one antibiotic. Within each group, >50% of isolates showed some type of resistance. E. coli and V. cholerae exceeded 50% resistance to tetracycline and ampicillin, respectively. Staphylococcus showed the highest resistance to penicillin, and Salmonella to tetracycline, nali-dixic acid and ampicillin. The highest percentages of non-susceptible microorganisms were identifi ed in meats and meat products. CONCLUSIONS These results serve as an alert to the dangers of acquiring antibiotic-resistant bacteria from food and demonstrate the need to establish a surveillance system and institute measures bacte-rial control in food products.KEYWORDS Microbial drug resistance, bacteria, food, foodborne disease, Cuba.

Antimicrobial resistance not related to 1,2,3 integrons and Superintegron in Vibrio spp. isolated from seawater sample of Lima (Peru).

Antimicrobial resistance (AMR) in microorganisms has been attributed to integrons, which have the ability to capture antimicrobial resistance gene cassettes and express them in their hosts. 170 strains of Vibrio spp. were isolated from Lima (Peru) seawater samples and identified by biochemical tests and PCR. AMR profiles were generated using 15 standard antibiotics. The presence of class 1, 2 and 3 integrons and Superintegron in these strains were also investigated by PCR. Ten species of Vibrio were identified with V. alginolyticus the most frequent. All strains were resistant to antibiotics, especially to penicillin group. No resistance to norfloxacin or tetracycline was observed. Class 1, 2 and 3 integrons were not found, only one Superintegron containing the mutT gene was identified in V. cholerae L22 strain. This indicated that AMR is not related to integrons as mentioned previously and that these strains can be reservoirs of resistance genes in marine environments.

Antimicrobial Resistance Risks of Cholera Prophylaxis for United Nations Peacekeepers.

More than 5 years after a United Nations peacekeeping battalion introduced cholera to Haiti, over 150,000 peacekeepers continue to be deployed annually from countries where cholera is endemic. The United Nations has thus far declined to provide antimicrobial chemoprophylaxis to peacekeepers, a policy based largely on concerns that the risks of drug resistance generation and spread would outweigh the potential benefits of preventing future cholera importations. In this study, we sought to better understand the relative benefits and risks of cholera chemoprophylaxis for peacekeepers in terms of antibiotic resistance. Using a stochastic model to quantify the potential impact of chemoprophylaxis on importation and transmission of drug-resistant and drug-sensitive Vibrio cholerae, we found that chemoprophylaxis would decrease the probability of cholera importation but would increase the expected number of drug-resistant infections if an importation event were to occur. Despite this potential increase, we found that at least 10 drug-sensitive infections would likely be averted per excess drug-resistant infection under a wide range of assumptions about the underlying prevalence of drug resistance and risk of acquired resistance. Given these findings, policymakers should reconsider whether the potential resistance risks of providing antimicrobial chemoprophylaxis to peacekeepers are sufficient to outweigh the anticipated benefits.

ANTIMICROBIAL ACTIVITY OF BURSERA MORELENSIS RAMÍREZ ESSENTIAL OIL.

BACKGROUND: Bursera morelensis, known as "Aceitillo", is an endemic tree of Mexico. Infusions made from the bark of this species have been used for the treatment of skin infections and for their wound healing properties. In this work, we present the results of a phytochemical and antimicrobial investigation of the essential oil of B. morelensis. MATERIALS AND METHODS: The essential oil was obtained by a steam distillation method and analyzed using GC-MS. The antibacterial and antifungal activities were evaluated. RESULTS: GC-MS of the essential oil demonstrated the presence of 28 compounds. The principal compound of the essential oil was a-Phellandrene (32.69%). The essential oil had antibacterial activity against Gram positive and negative strains. The most sensitive strains were S. pneumoniae, V. cholerae (cc) and E. coli (MIC 0.125 mg/mL, MBC 0.25 mg/mL). The essential oil was bactericidal for V. cholera (cc). The essential oil inhibited all the filamentous fungi. F. monilifome (IC50 = 2.27 mg/mL) was the most sensitive fungal strain. CONCLUSIONS: This work provides evidence that confirms the antimicrobial activity of the B. morelensis essential oil and this is a scientific support about of traditional uses of this species.

BIOFILM FORMATION OF Vibrio cholerae ON STAINLESS STEEL USED IN FOOD PROCESSING.

Vibrio cholerae represents a significant threat to human health in developing countries. This pathogen forms biofilms which favors its attachment to surfaces and its survival and transmission by water or food. This work evaluated the in vitro biofilm formation of V. cholerae isolated from clinical and environmental sources on stainless steel of the type used in food processing by using the environmental scanning electron microscopy (ESEM). Results showed no cell adhesion at 4 h and scarce surface colonization at 24 h. Biofilms from the environmental strain were observed at 48 h with high cellular aggregations embedded in Vibrio exopolysaccharide (VPS), while less confluence and VPS production with microcolonies of elongated cells were observed in biofilms produced by the clinical strain. At 96 h the biofilms of the environmental strain were released from the surface leaving coccoid cells and residual structures, whereas biofilms of the clinical strain formed highly organized structures such as channels, mushroom-like and pillars. This is the first study that has shown the in vitro ability of V. cholerae to colonize and form biofilms on stainless steel used in food processing.

Prevalence and Antimicrobial Resistance of Vibrio spp. in Retail and Farm Shrimps in Ecuador.

The aim of this study was to investigate the prevalence of Vibrio spp. in shrimp at retail and in shrimp farms in Ecuador and to determine the antimicrobial agent resistance patterns of farm isolates. The presence of genes linked to early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) also was evaluated. Vibrio spp. were isolated from retail shrimps in Cuenca, Ecuador, and farm shrimps originating from provinces El Oro and Guayas, Ecuador. A total of 229 shrimp samples were collected, of which 71 originated from retail markets in Cuenca and 158 came from shrimp farms. Overall, 219 (95.6%) samples tested positive for Vibrio spp. Vibrio parahaemolyticus (80.8%) was the most common species detected, followed by Vibrio alginolyticus (50.2%), Vibrio cholerae (11.3%), and Vibrio vulnificus (3.5%). None of the V. parahaemolyticus isolates carried the virulence-associated tdh and trh genes. In V. parahaemolyticus shrimp farm isolates, high resistance was found to ampicillin (92.2%), and intermediate resistance was found to tetracycline (51.3%) and amikacin (22.1%). Of the V. parahaemolyticus strains, 68 were resistant to at least three antimicrobial agents, and 2 were resistant to seven antimicrobial agents simultaneously. Up to 18 resistant isolates were found for V. alginolyticus, whereas V. vulnificus and V. cholerae isolates were more susceptible. None of the V. parahaemolyticus isolates carried the EMS-AHPND plasmid. The results of this study revealed the ubiquitous occurrence of Vibrio spp. in shrimps at retail and on shrimp farms in Ecuador.

Saccharomyces boulardii and bismuth subsalicylate as low-cost interventions to reduce the duration and severity of cholera.

We conducted a randomised single-blinded clinical trial of 100 cholera patients in Port-au-Prince, Haiti to determine if the probiotic Saccharomyces cerevisiae var. boulardii and the anti-diarrhoeal drug bismuth subsalicylate (BS) were able to reduce the duration and severity of cholera. Subjects received either: S. boulardii 250 mg, S. boulardii 250 mg capsule plus BS 524 mg tablet, BS 524 mg, or two placebo capsules every 6 hours alongside standard treatment for cholera. The length of hospitalisation plus the number and volume of emesis, stool and urine were recorded every 6 hours until the study subject was discharged (n = 83), left against medical advice (n = 11), or requested removal from the study (n = 6). There were no reported deaths or adverse study-related events. There were no statistically significant differences between the study arms and the outcomes of interest.

Evaluation of the medicinal properties of Cyrtocarpa procera Kunth fruit extracts.

BACKGROUND: The fruit of Cyrtocarpa procera is used to treat stomach diseases by people living in San Rafael, Coxcatlan, Puebla. This work investigated the antibacterial, antioxidant, cytotoxic and anti-inflammatory activities of the fruit produced by this species. METHODS: Methanol extract was obtained by maceration. After obtaining the methanol extract (MeOH1), methanol subextract (MeOH2) and hexane (H) were obtained. The antibacterial activities of MeOH1, MeOH2 and H were evaluated through disc-diffusion. The quenching of free radicals was evaluated by decolorizing a methanolic DPPH solution. The cytotoxic activity of MeOH2 was evaluated by in vitro assay system of growth inhibition of human cervical carcinoma cell line (CasKi). The IL-1ß and TNF-α were determined through ELISA in the supernatants of the macrophage cell line (RAW 264.7). The MeOH2 subextract was separated by column chromatography, seventy-three fractions were collected. RESULTS: The Gram-positive and -negative bacteria examined were sensitive to MeOH1 and MeOH2; the MeOH2 was bactericidal toward Staphyloccocus aureus (MIC = 4 mg/mL) and Vibrio cholera (MIC = 4 mg/mL). The MeOH2 inhibited the DPPH radical (SC50 = 69.7 µg/mL), but a cytotoxicity assay revealed that the extract is not toxic according to the National Cancer Institute (LD50 = 22.03 µg/mL). The production of proinflammatory cytokines (IL- 1ß and TNF- α) by LPS- stimulated macrophages was reduced after the treatments. The methanol extract contained various organic acids, such as citric acid, palmitic acid and α- linoleic acid. CONCLUSIONS: The fruits of Cyrtocarpa procera are employed to treat ailments such as diarrhea, in this study were demonstrated some biological activities involved in a bacterial infection. This is the first research about of the medicinal properties of C. procera fruit.

In vivo antipyretic, antiemetic, in vitro membrane stabilization, antimicrobial, and cytotoxic activities of different extracts from Spilanthes paniculata leaves.

BACKGROUND: The study was conducted to evaluate the in vitro antimicrobial activity, cytotoxic, and membrane stabilization activities, and in vivo antiemetic and antipyretic potentials of ethanolic extract, n-hexane and ethyl acetate soluble fractions of Spilanthes paniculata leaves for the first time widely used in the traditional treatments in Bangladesh. RESULTS: In antipyretic activity assay, a significant reduction (P < 0.05) was observed in the temperature in the mice tested. At dose 400 mg/kg-body weight, the n-hexane soluble fraction showed the effect (36.7 ± 0.63°C ) as like as the standard (dose 150 mg/kg-body weight) after 5 h of administration. Extracts showed significant (P < 0.001) potential when tested for the antiemetic activity compared to the standard, metoclopramide. At dose 50 mg/kg-body weight, the standard showed 67.23% inhibition, whereas n-hexane and ethyl acetate soluble fractions showed 37.53% and 24.93% inhibition of emesis respectively at dose 400 mg/kg-body weight. In antimicrobial activity assay, the n-hexane soluble fraction (400 µg/disc) showed salient activity against the tested organisms. It exerts highest activity against Salmonella typhi (16.9 mm zone of inhibition); besides, crude, and ethyl acetate extracts showed resistance to Bacillus cereus and Bacillus subtilis, and Vibrio cholera respectively. All the extracts were tested for lysis of the erythrocytes. At the concentration of 1mg/ml, ethanol extract, and n-hexane and ethyl acetate soluble fractions significantly inhibited hypotonic solution induced lysis of the human red blood cell (HRBC) (27.406 ± 3.57, 46.034 ± 3.251, and 30.72 ± 5.679% respectively); where standard drug acetylsalicylic acid (concentration 0.1 mg/ml) showed 77.276 ± 0.321% inhibition. In case of heat induced HRBC hemolysis, the plant extracts also showed significant activity (34.21 ± 4.72, 21.81 ± 3.08, and 27.62 ± 8.79% inhibition respectively). In the brine shrimp lethality bioassay, the n-hexane fraction showed potent (LC50 value 48.978 µg/ml) activity, whereas ethyl acetate fraction showed mild (LC50 value 216.77 µg/ml) cytotoxic activity. CONCLUSIONS: Our results showed that the n-hexane extract has better effects than the other in all trials. In the context, it can be said that the leaves of S. paniculata possess remarkable pharmacological effects, and justify its folkloric use as antimicrobial, antipyretic, anti-inflammatory, and antiemetic agent. Therefore, further research may be suggested to find possible mode of action of the plant part.

In vivo antipyretic, antiemetic, in vitro membrane stabilization, antimicrobial, and cytotoxic activities of different extracts from Spilanthes paniculata leaves

BACKGROUND: The study was conducted to evaluate the in vitro antimicrobial activity, cytotoxic, and membrane stabilization activities, and in vivo antiemetic and antipyretic potentials of ethanolic extract, n-hexane and ethyl acetate soluble fractions of Spilanthes paniculata leaves for the first time widely used in the traditional treatments in Bangladesh. RESULTS: In antipyretic activity assay, a significant reduction (P < 0.05) was observed in the temperature in the mice tested. At dose 400 mg/kg-body weight, the n-hexane soluble fraction showed the effect (36.7 ± 0.63°C ) as like as the standard (dose 150 mg/kg-body weight) after 5 h of administration. Extracts showed significant (P < 0.001) potential when tested for the antiemetic activity compared to the standard, metoclopramide. At dose 50 mg/kg-body weight, the standard showed 67.23% inhibition, whereas n-hexane and ethyl acetate soluble fractions showed 37.53% and 24.93% inhibition of emesis respectively at dose 400 mg/kg-body weight. In antimicrobial activity assay, the n-hexane soluble fraction (400 µg/disc) showed salient activity against the tested organisms. It exerts highest activity against Salmonella typhi (16.9 mm zone of inhibition); besides, crude, and ethyl acetate extracts showed resistance to Bacillus cereus and Bacillus subtilis, and Vibrio cholera respectively. All the extracts were tested for lysis of the erythrocytes. At the concentration of 1mg/ml, ethanol extract, and n-hexane and ethyl acetate soluble fractions significantly inhibited hypotonic solution induced lysis of the human red blood cell (HRBC) (27.406 ± 3.57, 46.034 ± 3.251, and 30.72 ± 5.679% respectively); where standard drug acetylsalicylic acid (concentration 0.1 mg/ml) showed 77.276 ± 0.321% inhibition. In case of heat induced HRBC hemolysis, the plant extracts also showed significant activity (34.21 ± 4.72, 21.81 ± 3.08, and 27.62 ± 8.79% inhibition respectively). In the brine shrimp lethality bioassay, the n-hexane fraction showed potent (LC50 value 48.978 µg/ml) activity, whereas ethyl acetate fraction showed mild (LC50 value 216.77 µg/ml) cytotoxic activity. CONCLUSIONS: Our results showed that the n-hexane extract has better effects than the other in all trials. In the context, it can be said that the leaves of S. paniculata possess remarkable pharmacological effects, and justify its folkloric use as antimicrobial, antipyretic, anti-inflammatory, and antiemetic agent. Therefore, further research may be suggested to find possible mode of action of the plant part.

Cholera outbreaks in Nigeria are associated with multidrug resistant atypical El Tor and non-O1/non-O139 Vibrio cholerae.

BACKGROUND: The current millennium has seen a steep rise in the number, size and case-fatalities of cholera outbreaks in many African countries. Over 40,000 cases of cholera were reported from Nigeria in 2010. Variants of Vibrio cholerae O1 El Tor biotype have emerged but very little is known about strains causing cholera outbreaks in West Africa, which is crucial for the implementation of interventions to control epidemic cholera. METHODOLOGY/PRINCIPAL FINDINGS: V. cholerae isolates from outbreaks of acute watery diarrhea in Nigeria from December, 2009 to October, 2010 were identified by standard culture methods. Fifteen O1 and five non-O1/non-O139 strains were analyzed; PCR and sequencing targeted regions associated with virulence, resistance and biotype were performed. We also studied genetic interrelatedness among the strains by multilocus sequence analysis and pulsed-field gel electrophoresis. The antibiotic susceptibility was tested by the disk diffusion method and E-test. We found that multidrug resistant atypical El Tor strains, with reduced susceptibility to ciprofloxacin and chloramphenicol, characterized by the presence of the SXT element, and gyrA(Ser83Ile)/parC(Ser85Leu) alleles as well CTX phage and TCP cluster characterized by rstR(ElTor), ctxB-7 and tcpA(CIRS) alleles, respectively, were largely responsible for cholera outbreaks in 2009 and 2010. We also identified and characterized a V. cholerae non-O1/non-O139 lineage from cholera-like diarrhea cases in Nigeria. CONCLUSIONS/SIGNIFICANCE: The recent Nigeria outbreaks have been determined by multidrug resistant atypical El Tor and non-O1/non-O139 V. cholerae strains, and it seems that the typical El Tor, from the beginning of seventh cholera pandemic, is no longer epidemic/endemic in this country. This scenario is similar to the East Africa, Asia and Caribbean countries. The detection of a highly virulent, antimicrobial resistant lineage in Nigeria is worrisome and points to a need for vaccine-based control of the disease. This study has also revealed the putative importance of non-O1/non-O139 V. cholerae in diarrheal disease in Nigeria.

Population genetics of Vibrio cholerae from Nepal in 2010: evidence on the origin of the Haitian outbreak.

Cholera continues to be an important cause of human infections, and outbreaks are often observed after natural disasters, such as the one following the 2010 earthquake in Haiti. Once the cholera outbreak was confirmed, rumors spread that the disease was brought to Haiti by a battalion of Nepalese soldiers serving as United Nations peacekeepers. This possible connection has never been confirmed. We used whole-genome sequence typing (WGST), pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing to characterize 24 recent Vibrio cholerae isolates from Nepal and evaluate the suggested epidemiological link with the Haitian outbreak. The isolates were obtained from 30 July to 1 November 2010 from five different districts in Nepal. We compared the 24 genomes to 10 previously sequenced V. cholerae isolates, including 3 from the Haitian outbreak (began July 2010). Antimicrobial susceptibility and PFGE patterns were consistent with an epidemiological link between the isolates from Nepal and Haiti. WGST showed that all 24 V. cholerae isolates from Nepal belonged to a single monophyletic group that also contained isolates from Bangladesh and Haiti. The Nepalese isolates were divided into four closely related clusters. One cluster contained three Nepalese isolates and three Haitian isolates that were almost identical, with only 1- or 2-bp differences. Results in this study are consistent with Nepal as the origin of the Haitian outbreak. This highlights how rapidly infectious diseases might be transmitted globally through international travel and how public health officials need advanced molecular tools along with standard epidemiological analyses to quickly determine the sources of outbreaks.

Antibacterial effect (in vitro) of Moringa oleifera and Annona muricata against Gram positive and Gram negative bacteria.

Antibacterial effects of aqueous and ethanolic extracts of seeds of moringa (Moringa oleifera) and pods of soursop (Annona muricata) in the concentration of 1:5 and 1:10 in volumes 50, 100, 150 and 200 microL were examined against Staphylococcus aureus, Vibrio cholerae, Escherichia coli (isolated from the organism and the aquatic environment) and Salmonella Enteritidis. Antibacterial activity (inhibition halo > 13 mm) against S. aureus, V. cholerae and E. coli isolated from the whiteleg shrimp, Litopenaeus vannmaei, was detected in aqueous and ethanolic extracts of moringa. E. coli isolated from tilapiafish, Oreochromis niloticus, was sensitive to the ethanolic extract of moringa. The aqueous extracts of soursop showed an antibacterial effect against S. aureus and V. cholerae, but the antibacterial activity by the ethanol extracts of this plant was not demonstrated.

Antibacterial effect (in vitro) of Moringa oleifera and Annona muricata against Gram positive and Gram negative bacteria / Efeito antibacteriano (in vitro) de Moringa oleifera (moringa) e Annona muricata (graviola) frente a bactérias Gram-negativas e Gram-positiva

Antibacterial effects of aqueous and ethanolic extracts of seeds of moringa (Moringa oleifera) and pods of soursop (Annona muricata) in the concentration of 1:5 and 1:10 in volumes 50, 100, 150 and 200 µL were examined against Staphylococcus aureus, Vibrio cholerae, Escherichia coli (isolated from the organism and the aquatic environment) and Salmonella Enteritidis. Antibacterial activity (inhibition halo > 13 mm) against S. aureus, V. cholerae and E. coli isolated from the whiteleg shrimp, Litopenaeus vannmaei, was detected in aqueous and ethanolic extracts of moringa. E. coli isolated from tilapiafish, Oreochromis niloticus, was sensitive to the ethanolic extract of moringa. The aqueous extracts of soursop showed an antibacterial effect against S. aureus and V. cholerae, but the antibacterial activity by the ethanol extracts of this plant was not demonstrated.

Para avaliação do efeito bactericida frente à Staphylococcus aureus, Vibrio cholerae, Escherichia coli (isolada de pescados e ambiente aquático) e Salmonella Enteretidis, foram testados extratos aquosos e etanólicos de sementes de moringa (Moringa oleifera) e casca de graviola (Annona muricata) na concentração de 1:5 e 1:10, nos volumes de 50, 100, 150 e 200 µL. Os resultados mostraram efeito antibacteriano (halo de inibição > 13mm) dos extratos aquosos e etanólicos de moringa frente a S. aureus, V. cholerae e E. coli isoladas de camarão cinza Litopenaeus vannmaei. A cepa de E. coli isolada do pescado Oreochromis niloticus apresentou sensibilidade frente ao extrato etanólico de moringa. Os extratos aquosos de graviola apresentaram efeito bactericida frente a S. aureus e V. cholerae, entretanto, os extratos etanólicos dessa planta não mostraram atividade antibacteriana.

Occurrence and composition of class 1 and class 2 integrons in clinical and environmental O1 and non-O1/non-O139 Vibrio cholerae strains from the Brazilian Amazon.

This study identified and characterised class 1 and 2 integrons in clinical and environmental Vibrio cholerae O1 and non-O1/non-O139 strains isolated from the Brazilian Amazon. The aadA2 and aadA7 gene cassettes were found in class 1 integrons in two genotypes of environmental V. cholerae non-O1/non-O139. Empty integrons were found in strains from the Brazilian cholera epidemic. A class 2 integron was detected in one strain from the V. cholerae Amazonia lineage harbouring sat1 and aadA1 genes. All isolates were resistant to aminoglycosides, indicating aadA functionality. These findings suggest that environmental bacteria act as cassette reservoirs that favour the emergence of resistant pathogens.