Phenotypic Virulence Factors Research Articles

Antibiotics resistance and phenotypic virulence factors of Vibrio species isolated from poultry litters at Obafemi Awolowo University, research farm.

The study investigates the antimicrobial resistance and phenotypic virulence factors of Vibrio isolates in poultry litter. Samples were enriched in 5 mL alkaline peptone water 1:10 (w/v) for 6 hours and cultured on Thiosulphate Citrate Bile salt (TCBS) agar. Vibrio isolates were identified through biochemical tests assay and were subjected to antibiotic susceptibility tests using standards disc diffusion technique. The phenotypic virulence factors, including hemolytic and proteolytic activity were determined. A total of 100 Vibrio isolates were identified as Vibrio parahaemolyticus (49%), V. cholerae (26%), V. vulnificus (11%), V. alginolyticus (9%), and V. damsela (5%). The Vibrio isolates showed a significant resistance to the selected antibiotics tested with 99% resistant to Gentamycin, 95% Cefixime, 96% Ofloxacin, 98% Augmentin, 96% Nitrofurantoin, 100% Ciprofloxacin, and 100% Cefuroxime. The Vibrio isolates showed varying levels of haemolysis with 17% alpha haemolysis, 61% beta haemolysis, 22% gamma haemolysis, and 29% proteolytic activity. The study reveals that poultry litter contains Vibrio isolates with antibiotic resistance and virulence factors, increasing their risk of severe infections. The high beta haemolysis prevalence highlights the interconnected relationship between animal, environmental, and human health, underscoring the need for effective management.

Characterization of Virulence Genotypes, Antimicrobial Resistance Patterns, and Biofilm Synthesis in Salmonella spp Isolated from Foodborne Outbreaks.

Salmonella is the main bacterial pathogen that causes foodborne disease, particularly in developing countries. Nontyphoidal Salmonella (NTS) include Enteritidis and Typhimurium as the most prevalent strains which are one of the significant causes of acute gastroenteritis in children. Therefore, identifying the most predominant serovars, types of common contaminated food, and paying attention to their antibiotic resistance are the main factors in the prevention and control strategy of salmonellosis. This study was undertaken to evaluate the prevalence rate of serovars, the biofilm formation, antimicrobial resistance (AMR) status, and phenotypic virulence factors of Salmonella strains isolated from diarrhea samples in some cities of Iran. A total of 40 (10.41%) Salmonella isolates were recovered from 384 diarrhea samples processed and the most common serovar was Salmonella serovar Typhimurium (82.5). Also, all isolates belonging to serovar Typhimurium showed more virulence factors compared to other serovars. The isolates showed a high resistance rate to ampicillin (95%) and nalidixic acid (87.5%), while a low resistance rate was found for chloramphenicol (2.5%). Moreover, significant variances in the capacity of biofilm formation were found between different Salmonella serotypes. The resistance of NTS to extant choice drugs is a potential public health problem. Constant monitoring of AMR pattern and virulence profile of NTS serovars is suggested for the prevention of salmonellosis in humans.

Colistin-resistant <i>Escherichia coli</i> from hospital and community nexus: public and environmental health concern

Antimicrobial resistance (AMR) is one of the greatest encounters for human health globally, which is associated with elevated morbidity and increased economic burdens. Colistin represents an important resort in treating severe infections caused by multidrug-resistant difficult-to-treat Gram-negative bacteria, including Escherichia coli. The study aimed to investigate the prevalence of colistin-resistant E. coli (CREC) in community and hospital sources and their associated risk factors. Eighty-five samples from hospital and community sources were screened using cultural and biochemical techniques and confirmed via Analytical Profile Index 20E. The antimicrobial susceptibility profile was determined using disc diffusion procedure and the phenotypic virulence factors were determined using standard culture protocols. The occurrence of CREC based on sample types includes cow carcass (26.7%), abattoir effluents (20%) and hospital effluents (8.6%). Pumpkin leaf, carrots and smoked fish were negative for CREC. The percentage distribution of CREC positive samples based on sample sources were slaughterhouses (77%) and hospital environment (33%) with open markets showing no case of CREC. Overall, CREC positive samples were 9/85 (10.6%). CREC demonstrated 100% ampicillin and trimethoprim/sulfamethoxazole resistance. The multiple antibiotics resistance profile of the Escherichia coli in this study ranged from 0.83 – 0.46. The phenotypic virulence distribution of CREC were lipase activity (33.3%), β-hemolytic activity (22.2%) and gelatinase activity (22.2%).The detection of CREC in both community and hospital environment allays a notable threat to human and animal health. Public enlightenment campaign and proper education against indiscriminate antibiotics usage as animal feed and growth promoters coupled with routine antibiotic resistance monitoring is crucial to curtail the dissemination of superbugs

Detection of putative virulence traits in Aeromonas spp. from brazilian food chain through phenotypic tests and polymerase chain reaction

Aeromonads are natural inhabitants of aquatic environments and may be associated with numerous infections in humans and animals. The human disease may range from self-limiting diarrhea to a more severe form. The pathogenesis of infections are multifactorial, because of their wide variety of virulence factors. This study aimed to evaluate virulence markers in Aeromonas isolates and determinate their virulence profiles. There were analyzed 120 strains of A. caviae (n = 57) and A. hydrophila (n = 63) from human, animal and environmental sources between 2008 and 2012. All isolates were examined to detect extracellular virulence enzymes by phenotypic activity and the presence of virulence genes hlyA, aerA, lip, gcat, ser, act, alt and exu by PCR. We observed more than 90% of positivity for at least one phenotypic virulence factors and all of them had at least two of the virulence genes measured. Among the virulence enzymes detected, the DNase was present in 93.33% of the isolates and hemolytic activity was detected in 62.5%. Collagenase and elastase were found in 13.33% and 10.83% of the strains, respectively. We found exu and gcat in 100% of the isolates, lip in 40.83%, aerA in 40.83%, hlyA in 40%, alt in 19.16%, act in 17.5% and ser in 11.66%. It was possible to observe different combinations of virulence factors between the isolates showing the multifactorial virulence among the isolates. The diversity of virulence profiles found in this study hint heterogeneity in clones circulating in our environment.

Relationship between Biofilm-Formation, Phenotypic Virulence Factors and Antibiotic Resistance in Environmental Pseudomonas aeruginosa

The formation of a protective biofilm by Pseudomonas aeruginosa (PA) is one of the hallmarks of their survival both in vivo and in harsh environmental conditions, thus, biofilm-eradication has relevance from therapeutic perspectives and for infection control. The aim of our study was to investigate the possible relationship between antibiotic resistance, biofilm-forming capacity and virulence factors in n = 166 PA isolates of environmental origin. Antimicrobial susceptibility testing and the phenotypic detection of resistance determinants were carried out using standard protocols. The biofilm-forming capacity of PA was tested using a standardized crystal violet microtiter plate-based method. Motility (swimming, swarming, and twitching) and siderophore production of the isolates were also assessed. Resistance rates were highest for ciprofloxacin (46.98%), levofloxacin (45.18%), ceftazidime (31.92%) and cefepime (30.12%); 19.28% of isolates met the criteria to be classified as multidrug-resistant (MDR). Efflux pump overexpression, AmpC overexpression, and modified Hodge-test positivity were noted in 28.31%, 18.07% and 3.61%, respectively. 22.89% of isolates were weak/non-biofilm producers, while 27.71% and 49.40% were moderate and strong biofilm producers, respectively. Based on MDR status of the isolates, no significant differences in biofilm-production were shown among environmental PA (non-MDR OD570 [mean ± SD]: 0.416 ± 0.167 vs. MDR OD570: 0.399 ± 0.192; p > 0.05). No significant association was observed between either motility types in the context of drug resistance or biofilm-forming capacity (p > 0.05). 83.13% of isolates tested were positive for siderophore production. The importance of PA as a pathogen in chronic and healthcare-associated infections has been described extensively, while there is increasing awareness of PA as an environmental agent in agriculture and aquaculture. Additional studies in this field would be an important undertaking to understand the interrelated nature of biofilm production and antimicrobial resistance, as these insights may become relevant bases for developing novel therapeutics and eradication strategies against PA.

Selection of Debaryomyces hansenii isolates as starters in meat products based on phenotypic virulence factors, tolerance to abiotic stress conditions and aroma generation.

Debaryomyces hansenii is a yeast widely used in meat fermentations as starter for the purpose of improving the aromatic quality of the final product. However, it has not been the subject of an extensive study regarding phenotypic characteristics important for starter selection, such as the capacity to grow at abiotic stress conditions occurring during fermentation, the ability to generate desirable aromas and the absence of virulence traits in yeasts. The aim of this study was to screen 60 strains of D. hansenii isolated from assorted foods for their potential application as starters in dry-cured fermented sausages manufacture. The abiotic stress factors tested were low aw and pH and high concentration of salt, acetic acid and lactic acid. The phenotypic virulence traits explored were growth at 37°C, pseudohyphal and biofilm generation, invasiveness and enzymatic activities present in virulent yeasts. The generation of desirable meat aromas was tested in models containing aroma precursors applying an olfactory analysis. A quantitative profiling of stress tolerance was used to test the potential performance of selected strains in meat fermentations. The results demonstrated that most strains displayed no virulence trait or were only positive for biofilm production. Moreover, the strains showed large heterogeneity regarding their tolerance to abiotic stress factors, although most of them could grow at intermediate to high levels of the traits. The sensory analysis was the criteria determining the selection of starter strains. The evaluation of the phenotypic traits demonstrates that D. hansenii is a safe yeast, it is able to tolerate the stress in meat fermentation and it is able to generate desirable aromas. The results of this study confirm the adequacy of selected D. hansenii strains to be applied as starters in meat products.

Novel lytic bacteriophages (AhFM4 & AhFM5) as bio-control measures against multidrug resistant biofilm producing Aeromonas hydrophila (AhZ1K)

Motile Aeromonas Septicemia (Aeromonas hydrophila) has caused severe economic loss in several aquaculture fish species such as carps, tilapia, salmon, and catfishes. In the present study, mass mortality of zebrafish (Danio rerio) was observed in fish maintained in the hatchery cum rearing center, along with clinical signs including enlarged abdomen and haemorrhages on the body surface. The etiological agent was identified as Aeromonas hydrophila through biochemical and molecular methods. The isolated A. hydrophila, AhZ1K was positive for all the phenotypic virulence factors and cytopathic effect was observed in D. rerio gill (DRG) cell line. Further, A. hydrophila, AhZ1K was found to produce biofilm and was positive for eight virulence genes and five antibiotic resistance genes with a Multiple Antibiotic Resistance (MAR) index of 0.6 and 0.825 for free and biofilm cells respectively, indicating the property of multidrug resistance. Therefore, as a preliminary approach to develop bio-control measure, novel lytic bacteriophages (phages namely AhFM4 & AhFM5) were isolated from Western Ghats riverine water systems. Transmission electron microscopy (TEM) confirms that both the lytic phages (AhFM4 & AhFM5) belong to Myoviridae. Interestingly, AhFM4 & AhFM5 displayed 96% and 70%; 15 and 25 min; 152 ± 27 PFU/host cell and 112 ± 13 PFU/host cell of adsorption, latency and burst size respectively. The phages were stable over a broad array of physicochemical parameters (temperatures, salinity, and pH), indicating that AhFM4 and AhFM5 have an immense capability to be developed as a substitute for antibiotics to control motile Aeromonas septicemia in aquaculture.

Virulence Factors of Pseudomonas aeruginosa Isolated from ICU

Background: P.aeruginosa has many virulence factors which are the main reason for infection and the emergence of antibiotic resistance leading to an increase of morbidity and mortality. Currently, multidrug resistance is the hardest problem, which made it imperative to search for alternative treatment strategies. Objective: detection of some phenotypic virulence factors of P.aeruginosa isolated from ICU patients and the possibility of any antibiotic resistance related to certain virulence factors released by P. aeruginosa. Methodology: Our study was carried out on patients admitted to ICU Department in Benha University Hospital and infected with P.aeruginosa, the isolates subjected to phenotypic detection of the virulence factors: phospholipase, alkaline protease, lipase, gelatinase, esculin hydrolysis, biofilm formation, hemolysin and DNase production using specific media for each and evaluation of the antibiotic susceptability pattern using Kirby Bauer disk diffusion assay. Results: P.aeruginosa virulence factors were recorded as follow: hemolysin (70%) followed by alkaline protease (68%), phospholipase (62%), gelatinase & biofilm formation (60%) for each, lipase & bile esculin hydrolysis (54%) for each and DNase (2%).High antibiotic resistance was detected to mostly all of the used antibiotic discs. Also, presence of invasive device, prolonged hospital stay, ICU stay and higher number of virulence factors were associated with poor outcome. Conclusions: Production of different phenotypic virulence factors in high amount reflects their important role in spread of infection and pathogenicity with increased antibiotic resistance. Therefore finding anti-virulence factors as adjuvant therapy has an important role in treatment of P. aeruginosa especially MDR isolates.

Identification and Characterization of Salmonella Serovars Isolated from Pig Farms in Benin City, Edo State, Nigeria: One Health Perspective.

The present study was carried out to characterize Salmonella serovars from commercial pig farms in Benin City, Nigeria. A total of 81 samples were collected from the Agricultural Development Program farms between January and June 2017. Standard culture-based and polymerase chain reaction procedures were adopted in the isolation and identification of Salmonella serovars. Antibiotic susceptibility profiles of the isolates were conducted using the Kirby-Bauer disc diffusion method with prominent resistance determinants screened for using specific primer sets. The 84 identified Salmonella serovars include 15 Salmonella ser. Enteritidis, 11 Salmonella ser. Typhimurium, and 58 other Salmonella serovars. Phenotypic virulence factors include: hemolytic activity (51.7-100%), lipase activity (48.3-81.8%), protease activity (60.3-100%), gelatinase production (50-90.9%), DNA degrading activity (55.2-90.9%), and S-layer formation (63.8-100%). The biofilm formation profiles include nonbiofilm producers (0-12.1%), weak biofilm producers (0-20%), moderate biofilm producers (24.1-27.3%), and strong biofilm producers (48.3-72.7%). Salmonella serovars in this study harbored resistant determinants, such as tetA, tetC, ampC, sul1, sul2, sul3, floR, ermA, and ermC. The occurrence of resistance phenotype and determinants in pathogenic Salmonella serovars from pig farms is a significant public health concern, which could result in the dissemination of resistant elements within the environment.

Biofilm formation and transfer of a streptomycin resistance gene in enterococci from fermented pork

ObjectivesMultidrug-resistant (MDR) enterococci are found extensively in food samples. This study characterized the phenotypic virulence factors and the ability of horizontal gene transfer of a streptomycin resistance gene among enterococci isolated from fermented pork. MethodsThirty-six MDR enterococci were subjected to screening of gelatinase, biofilm formation at various temperatures (4 °C, 25 °C and 37 °C), clumping ability and conjugation. ResultsAll gelatinase-positive and clumping-positive strains were Enterococcus faecalis (41.7% and 38.9%, respectively). None of Enterococcus faecium and Enterococcus hirae demonstrated both phenotypes. Moderate and strong biofilm formations were found mostly at optimal temperatures in all the three species tested. However, moderate and weak biofilm formations could be found in 52.8% at 4 °C. No association was observed between biofilm formation and asa1, efaA, gelE and esp genes. Surprisingly, our data revealed evidence of the streptomycin resistance gene (aadE) being transferred among meat E. faecalis isolates as characterized by the pheromone-clumping response. ConclusionsHere we report the co-existence of some virulence factors and MDR enterococci from fermented pork. Our data demonstrated for the first time that the aadE gene could be transferred via conjugation among enterococci isolated from meat, contributing to streptomycin resistance. This study highlights the importance of horizontal gene transfer within the food chain reservoir and that transfer to humans might be possible, causing harm or untreatable diseases.

Genetic diversity and virulence characteristics of Staphylococcus aureus isolates from cases of bovine mastitis

Staphylococcus aureus is one of the major bacterial mastitis pathogens with significant effects on animal and human health. Some studies showed that S. aureus strains that infect different host species are genetically distinct, although most strains can infect a wide range of host species. However, there are no clearly defined clonal patterns of S. aureus strains that are known to infect a specific host. The objectives of this study were to evaluate the clonal diversity and virulence characteristics of S. aureus isolates from cases of bovine mastitis. Bacteriological tests were conducted on milk samples from cases of bovine mastitis from 11 dairy farms including some milk samples from unknown farms in Eastern Tennessee. Overall, a total of 111 S. aureus were isolated and identified, and further evaluated for their genetic diversity by pulsed-field gel electrophoresis (PFGE) and virulence characteristics by PCR. Genotypic virulence factors including staphylococcal enterotoxins, and toxic shock syndrome toxin 1 (tsst-1) were tested by PCR. In addition, the association among several known virulence factors of these isolates based on our current and previous studies in our lab were evaluated. Previously generated data that were included in the analysis of association among virulence factors were the presence of biofilm production associated genes in the ica operon such as icaA, icaD and icaAB, and phenotypic virulence characteristics such as hemolysis on blood agar, slime production and resistance or susceptibility to ten commonly used antimicrobials in dairy farms. The PFGE results showed the presence of 16 PFGE types (designated A – P) throughout farms, of which three pulsotypes, I, M and O were the most frequently isolated PFGE types from most farms. The PFGE type M was the most prevalent of all 16 PFGE types, with 64 isolates being present among nine farms. The PCR results of enterotoxin genes showed that out of the total 111 tested 84 (75.7%) were negative whereas 13 (11.7%), 2 (1.8%), 3 (2.7%), 1 (0.9%) and 8 (7.2%) were positive for seb, seb and sec, sec, see, and tsst-1, respectively. All 111 isolates were negative for sea and sej.Results of the evaluation of I, M and O strains adhesion to and invasion into mammary epithelial cells showed that the total count of each strain of bacteria adhered to and invaded into mammary epithelial cell line (MAC-T cells) was not significantly different (P > 0.05). This may be an indication that there is no significant difference in their ability to establish early host-pathogen interaction and colonization of the host.There were no statistically significant associations among PFGE types and other known virulence factors of these strains. However, PFGE types O and M tend to cluster with β-hemolysin, absence of enterotoxins and susceptibility to antimicrobials. In conclusion, there was not any association between pulsotype and genotypic and phenotypic virulence factors. S. aureus isolates from cases of bovine mastitis had diverse genotypes that possessed variable virulence factors.

Suppressing the phenotypic virulence factors of Uropathogenic Escherichia coli using marine polysaccharide

Uropathogenic Escherichia coli (UPEC) is one of the keystone pathogen that cause 80–90% of community acquired urinary tract infections (UTIs) and Catheter associated urinary tract infections (CAUTIs). Pathogenicity and ability of UPEC to colonize the bladder majorly relies on the expression of phenotypic virulence factors like flagella, pili, curli, and non pilus adhesion. Pathogens that colonize on the indwelling medical devices are able to communicate using quorum sensing (QS) signals. QS Plays a vital role in coordinating biofilm formation which results in the bacterial cells encased inside an extracellular polymeric substance (EPS). Chitosan is a marine polysaccharide which is known for its antibacterial activity. In the present study we investigated the ability of chitosan extracted from marine biowaste to mitigate the QS mediated biofilm formation in UPEC. Extracted chitosan (EC) and Commercial chitosan (CC) showed percentage inhibition of 80–85% and 60–75% respectively on young biofilm inhibition and preformed biofilm disruption. EC and CC were assessed for its ability to suppress QS mediated virulence in UPEC. Hemolysis assay showed a percentage inhibition of 79% against EC. Both chitosan showed profound activity to suppress the phenotypic virulence factors like swarming motility which is mediated by type I pili and colony morphology assay showed repression in cellulose production in UPEC. Furthermore, Real-Time PCR confirmed the ability of EC to down regulate the virulent genes which are responsible for invasion in UPEC. Accordingly, the current study foresees the quorum sensing inhibiting (QSI) potential of chitosan extracted from marine biowaste which offers an antibiotic free approach to combat UTI caused by UPEC.

Phylogenetic characteristics, virulence properties and antibiogram profile of motile Aeromonas spp. isolated from ornamental guppy (Poecilia reticulata).

Aeromonas spp. are opportunistic pathogenic bacteria related to an assembly of infectious diseases in ornamental fish. In the present study, virulence properties and antibiotic susceptibility of 52 guppy-borne Aeromonas spp. were investigated. The isolates were identified as A. veronii (n = 34), A. dhakensis (n = 10), A. hydrophila (n = 3), A. caviae (n = 3) and A. enteropelogenes (n = 2) by gyrB gene sequencing. The gyrB sequence deviation within and among the species ranged from 0 to 2.6% and 2.7-9.2%. Each species formed a distinct group in the unrooted neighbor-joining phylogenetic tree. The phenotypic virulence factors such as β-hemolysis, slime, caseinase, DNase, gelatinase and lipase production were observed in 28 (53.9%), 33 (63.5%), 28 (53.9%), 42 (80.8%), 37 (71.2%) and 42 (80.8%) isolates, respectively. The virulence genes were detected by PCR assay in the following proportions- act (84.6%), hly (80.8%), aer (73.1%), lip (73.1%), gcaT (73.1%), ascV (53.8%), ahyB (53.8%) fla (51.9%), alt (48.1%), ast (36.5%) and ser (34.6%), respectively. The amoxicillin, ampicillin, imipenem, nalidixic acid, oxytetracycline and rifampicin were resistant to more than 70.0% of the isolates in antibiotic susceptibility test. Our study suggests that the ornamental guppy can be a potential reservoir of virulent and multi-drug resistant Aeromonas spp.

Biofilm formation and potential virulence factors of Salmonella strains isolated from ready-to-eat shrimps.

Salmonella species is an important foodborne pathogen with the non-typhoidal serovars such as Enteritidis and Typhimurium as the most predominant strains. This study examines the biofilm formation, phenotypic virulence factors and cell surface characteristics of Salmonella strains from ready-to-eat shrimps. The ready-to-eat shrimps were obtained from open markets between November 2016 and October 2017 in Edo and Delta States, Nigeria. The occurrence of Salmonella strains in this study was 210/1440 (14.58%) of the ready-to-eat shrimp’s samples. The identified strains comprise of Salmonella Enteritidis 11, Salmonella Typhimurium 14 and other Salmonella spp. 20. The 45 identified Salmonella strains revealed the following virulence properties: swimming and swarming motility 45(100%); S-layer 39(86.67%); haemolytic activity 40(88.89%); lipase activity 43(95.56%); protease activity 43(95.56%); gelatinase production 43(95.56%); and DNA degrading activity 41(91.11%). The variation in the formation of biofilm-based on the diversity of Salmonella species was observed with higher percentage of Salmonella Typhimurium strains as strong biofilms producers under different environmental conditions. For surface hydrophobicity using bacterial adherence to hydrocarbons, 25(55.56%) were hydrophilic while 20(44.44%) were moderately hydrophobic from the 45 Salmonella isolates. Using salting aggregation test for surface hydrophobicity, all selected isolates 45(100%) was hydrophilic. Autoaggregation index for the 12 selected Salmonella isolates ranged from 15.2–47.2%, while the autoaggragation index for the 12 selected test bacteria ranged from 26.2–71.3%. Coaggragation between the 12 selected test bacteria and 12 Salmonella isolates ranged from 12.5–81.0%. The occurrence of pathogenic species of Salmonella from ready-to-eat shrimps could be detrimental to the consumers. Findings on the physiological conditions of biofilms formed by the foodborne pathogenic Salmonella and the cell surface characteristics therein are crucial for the advancement of methods for controlling Salmonella from ready-to-eat foods.

Phenotypic Assay of Virulence Factors, Biofilms and Antibiotic Resistance among Enterobacterial Uropathogens from Cancer Patients

Background: Virulence factors are associated with Enterobacterial uropathogens in cancer patients. Antimicrobial resistance in association with biofilm formation has intensified the pathogenicity of these uropathogens leading to high morbidity and therapeutic failures. Objective: This study determined and correlated the phenotypic virulence factors, antimicrobial resistance and biofilm forming Enterobacterial uropathogens in cancer patients. Materials and Methods: One hundred and seven (107) samples were collected randomly and processed between April and October, 2018. Twenty-five (25) isolates were recovered and characterized. Antimicrobial susceptibility testing against ten antimicrobials was performed using Kirby Bauer method. Extended spectrum beta-lactamase (ESBL) was determined with double disk synergy test. Virulence markers haemagglutinin was detected with haemagglutination test, haemolysin was detected by red cell haemolysis and biofilm formation was determined with the Congo red agar on all the isolates. Results: Out of the twenty-five (25) isolates, E. coli (72%), Klebsiella pneumoniae (24%) and Proteus mirabilis (4%) were isolated. 92% isolates were resistant to cefotaxime and 80% resistance to amoxicillin-clavulanic acid. 24% were positive for, ESBL, 4% for haemolysin test, 60% for haemagglutination and 40% for biofilm formation. From this study virulence markers were distributed among the isolates, however antibiotic resistance did not determine the presence or type of virulence markers. Conclusion: The present results may assist in developing strategic management of antibiotic therapy in cancer patients with urinary tract infections.

Species Distribution and Prevalence of Putative Virulence Factors in Mesophilic Aeromonas spp. Isolated from Fresh Retail Sushi.

Aeromonas spp. are ubiquitous bacteria that have received increasing attention as human pathogens because of their widespread occurrence in food, especially seafood and vegetables. The aim of this work was to assess the species identity and phylogenetic relationship of 118 Aeromonas strains isolated from fresh retail sushi from three producers, and to characterize the isolates with respect to genetic and phenotypic virulence factors. We also evaluate the potential hazard associated with their presence in ready-to-eat seafood not subjected to heat treatment. Mesophilic Aeromonas salmonicida was most prevalent (74%), followed by A. bestiarum (9%), A. dhakensis (5%), A. caviae (5%), A. media (4%), A. hydrophila (2%), and A. piscicola (1%). All isolates were considered potentially pathogenic due to the high prevalence of genes encoding hemolysin (hlyA) (99%), aerolysin (aerA) (98%), cytotoxic enterotoxin (act) (86%), heat-labile cytotonic enterotoxin (alt) (99%), and heat-stable cytotonic enterotoxin (ast) (31%). The shiga-like toxins 1 and 2 (stx-1 and stx-2) were not detected. Moreover, there was heterogeneity in toxin gene distribution among the isolates, and the combination of act/alt/hlyA/aerA was most commonly detected (63%). β-hemolysis was species-dependent and observed in 91% of the isolates. All A. media and A. caviae strains were non-hemolytic. For isolates belonging to this group, lack of hemolysis was possibly related to the absence of the act gene. Swimming motility, linked to adhesion and host invasion, occurred in 65% of the isolates. Partial sequencing of the gyrB gene demonstrated its suitability as a genetic marker for Aeromonas species identification and for assessment of the phylogenetic relationship between the isolates. The gyrB sequence divergence within a given species ranged from 1.3 to 2.9%. A. bestiarum, A. salmonicida, and A. piscicola were the most closely related species; their sequences differed by 2.7–3.4%. The average gyrB sequence similarity between all species was 93%, demonstrating its acceptable taxonomic resolution. The presence of multiple species of potential pathogenic Aeromonas in fresh retail sushi raises new food safety issues related to the increased consumption of ready-to-eat food composed of raw ingredients.

An alternative approach for evaluating the phenotypic virulence factors of pathogenic Escherichia coli

Escherichia coli is a recognized zoonotic food-borne pathogen; however, the use of polymerase chain reaction (PCR) in the underdeveloped countries to differentiate pathogenic from non-pathogenic E. coli is a problematic issue. Our grail was to assess the phenotypic virulence markers motility, hemolysin, congo red agar, embryo lethality assay and serum resistance for pathogenic E. coli (PEC) correlated to PCR tests which is currently used world-wide to evaluate the PEC. The 448 strains of Escherichia coli that were isolated from different sources, were characterized for phenotypic virulence factors such as motility, hemolysin, Congo red binding, Embryo Lethality assay (ELA) and serum resistance, as well as antibiotic susceptibility using disc diffusion method to 23 antibiotics. Results exhibited 100% motility and Congo red binding, 97.1% for hemolysin production and 90.2% in the ELA. As a result, we were able to hypothetically conclude that the aforementioned virulence markers are plain, straightforward, economical, rapid, more dynamic, uncomplicated methodology, duplicatable and cost next to nothing when compared to the molecular PCR. Their implementation in a diagnostic microbiology laboratory for vetting is a rewarding task in the underdeveloped countries. It augments endeavors to minimize the use of PCR in our investigations especially during epidemiological and outbreak investigations of PEC.

Molecular characterization and detection of enterotoxins, methicillin resistance genes and antimicrobial resistance of Staphylococcus aureus from fish and ground beef.

A total of 120 samples including 40 freshwaterfish(Oncorhynchus mykiss), 40 seawater fish (Sparus aurata) and 40 ground beef samples were examined for the presence of Staphylococcus aureus. The isolates were identified using biochemical tests and a PCR for the species-specific fragment (Sa442) and thermonuclease gene (nucA). The presence of staphylococcal enterotoxin genes (sea, seb, sec, sed and see), toxin genes (eta, etb, tsst), methicillin resistance gene (mecA) and some phenotypic virulence factors was also tested. Genotypic characterization of the isolates was analyzed by PCR-RFLP of the coa gene. Overall, 36 (30%) meat samples were contaminated with S. aureus. Of the 36 isolates, 3 (8.3%) were found to be positive for enterotoxin genes. Only 1 isolate (5.9%) from ground beef had the sea gene. In addition, 1 (12.5%) of the freshwater fish and 1 (9.1%) of the seawater fish carried both the sea and sed genes. The presence of seb, sec, see, eta, etb and tsst was not detected among the isolates of S. aureus. The amplified coa gene revealed five different clusters. Seven and six distinct RFLP patterns were obtained with AluI and HaeIII digestion, respectively. All isolates were found to be positive for slime, hemolytic and DNase activity while 41.7% of them were beta-lactamase positive. The presence of methicillin resistance was neither detected by PCR nor the disk diffusion method. A total of 94.4% of the isolates were resistant to at least one antimicrobial while 44.4% of them were resistant to at least two or more antimicrobials.

Detection of AmpC β-lactamase and adherence factors in uropathogenic Escherichia coli isolated from aged patients

Escherichia coli mediated urinary tract infection has been reported to be most prevalent among patients of different class, gender and ages. Currently, multidrug resistant E. coli harboring several virulence factors are most perilous threats for patients especially for elders. The aim of this study was to determine the antibiotic resistance pattern, co-resistance and phenotypic virulence factors present in uropathogenic E. coli isolated from aged patients. Thirty-nine E. coli isolates were collected during May–June 2014 from patients between 50 to 80 years of age. Experiments have been carried out to determine the antibiotic resistance, co-resistances and phenotypic adherent factors present in each isolate. Clonal relatedness was also determined in the AmpC positive uropathogenic E. coli (UPEC). 97.43% isolates were found to be multidrug resistant and 41.02% of them were AmpC producer. AmpC producer group showed higher multiple antibiotic resistance index than AmpC non-producer (p value < 0.01) group. Interestingly, adherence factor Type 1 fimbriae were found among 84.61% of total isolates which were more prevalent in elderly female patients than males. Biofilm production studies revealed that 84.61% of total isolates are more common in elderly males. This study adds value for the proper empiric selection of antibiotic therapy as well as calls for continuous monitoring of the incidence of drug resistance virulent uropathogenic E. coli mediated urinary tract infection in elderly patients.

Fluconazole Susceptibility in Cryptococcus gattii Is Dependent on the ABC Transporter Pdr11

Cryptococcus gattii isolates from the Pacific Northwest have exhibited higher fluconazole MICs than isolates from other sites. The mechanism of fluconazole resistance in C. gattii is unknown. We sought to determine the role of the efflux pumps Mdr1 and Pdr11 in fluconazole susceptibility. Using biolistic transformation of the parent isolate, we created a strain lacking Mdr1 (mdr1Δ) and another strain lacking Pdr11 (pdr11Δ). Phenotypic virulence factors were assessed by standard methods (capsule size, melanin production, growth at 30 and 37 °C). Survival was assessed in an intranasal murine model of cryptococcosis. Antifungal MICs were determined by the M27-A3 methodology. No differences in key virulence phenotypic components were identified. Fluconazole susceptibility was unchanged in the Mdr1 knockout or reconstituted isolates. However, fluconazole MICs decreased from 32 μg/ml for the wild-type isolate to <0.03 μg/ml for the pdr11Δ strain and reverted to 32 μg/ml for the reconstituted strain. In murine models, no difference in virulence was observed between wild-type, knockout, or reconstituted isolates. We conclude that Pdr11 plays an essential role in fluconazole susceptibility in C. gattii. Genomic and expression differences between resistant and susceptible C. gattii clinical isolates should be assessed further in order to identify other potential mechanisms of resistance.