API 20NE System Research Articles

Phenotypic Characterization and Prevalence of Carbapenemase-Producing Pseudomonas aeruginosa Isolates in Six Health Facilities in Cameroon

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with a great ability to adapt to stress, in particular, to the selective pressure of antibiotics in the hospital environment. This pathogen constitutes a real public health concern, especially in low- and middle-income countries. In Cameroon, little is known about the drug resistance patterns of Pseudomonas aeruginosa. This study sought to determine the prevalence of Pseudomonas aeruginosa strains producing carbapenemases in six health facilities in the center, littoral, and west regions of Cameroon. An analytical cross-sectional study was conducted over a four-month period from July to October 2021. All Pseudomonas aeruginosa or suspected strains isolated from pathological products at the bacteriology laboratory of different health facilities were systematically collected and underwent a re-identification. After growing on cetrimide agar and successfully subculturing on nutrient agar, an oxidase test was performed on pure colonies, followed by biochemical identification (API 20NE system) of the bacterial suspension (0.5McFarland standard). Drug susceptibility testing for the detection of extended-spectrum beta-lactamases of overproduced inducible cephalosporinases and carbapenemases was performed according to adequate standard procedures. Of the 468 isolates collected, 347 (74.14%) were confirmed Pseudomonas aeruginosa after re-identification, of which 34.49% (120/347) produced inducible cephalosporinases (CAZR and C/TS) and 32.26% (112/347) extended-spectrum beta-lactamases. The prevalence of carbapenemase-producing P. aeruginosa (IMPR and C/TR) was 25.07% (87/347), with 17.24% (15/87) class A and 82.76% (72/87) class B. A high rate of resistance to penicillin (piperacillin: 70.58% and ticarcillin: 60.24%) was observed. We also noted a 34.49% resistance to ceftazidime, 30.22% to imipenem against 37.02% to meropenem, and 25.1% to ceftolozane/tazobactam (C/T). These strains also exhibited 79.57% resistance to quinolones and about 26% to aminoglycoside families. Multivariate analysis revealed that carbapenemase-producing Pseudomonas aeruginosa-related infections were significantly associated with hospitalization (p = 0.04), maternity (p = 0.03), surgery (p = 0.04), and intensive care wards (p = 0.04). This study highlighted a high burden of resistant strains of carbapenemase-producing Pseudomonas aeruginosa. Surveillance should be intensified to prevent the dissemination and spread of these strains.

Multilocus Sequence Typing of Carbapenem-Resistant Acinetobacter baumannii Isolates Harboring blaOXA-23 and blaIMP in Cattle from Punjab, Pakistan.

Acinetobacter baumannii is a notorious bacterial pathogen that can cause an array of nosocomial infections in clinical settings. However, the data from the veterinary settings is limited and especially in Pakistan, no such study is conducted so far. To investigate the prevalence, antimicrobial resistance, and distribution of specific sequence types of A. baumannii in cattle, a total of 1,960 samples were collected from cattle over 18 months from Punjab, Pakistan. The isolates obtained were identified using the API20NE system and confirmed through PCR. The isolated A. baumannii isolates were further screened for antimicrobial susceptibility and the presence of resistance genes. Multilocus sequence typing was carried out to characterize the carbapenem-resistant A. baumannii (CRAB) isolates. Results revealed an overall prevalence of A. baumannii at 3.31% (65/1,960) with a higher prevalence of 7.38% (54/731) in dairy cattle compared to beef cattle at 4.41% (11/249). Among 65 A. baumannii isolates, 27.7% (18/65) were CRAB. All CRAB isolates harbor class D β-lactamases genes blaOXA-23 and blaOXA-51, whereas 94.4% (17/18) CRAB isolates carried class B β-lactamases gene blaIMP, and only one isolate had blaNDM-1 gene. The commonly found sequence types for CRAB isolates were ST2 and ST642 corresponding to 10 and 05 isolates, respectively. The presence of CRAB in cattle indicates an alarming situation that necessitates an urgent and efficient surveillance system to limit the transmission of CRAB among the cattle population and its possible transmission to humans and the environment.

The first case of Ochrobactrum intermedium bacteremia in a pediatric patient with malignant tumor

BackgroundOchrobactrum spp. are non-fermenting, Gram-negative bacilli that are regarded as emerging human pathogens of low virulence that can cause infections. The first identified case of Ochrobactrum intermedium was reported in 1998 in a liver transplantation patient with liver abcess. There are no reports of infections in pediatric patients. Here, we report the first case of O. intermedium bacteremia in a pediatric patient.Case presentationA two and a half years old male was admitted with fever, chills and nausea. He had been diagnosed as pineoblastoma and underwent surgical resection and chemotherapy. O. intermedium was isolated from his blood cultures and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), however, the Vitek II automated system failed to identify the organism. Then the pathogen was confirmed by 16S rDNA sequencing and average nucleotide identity result (ANI) confirmed the precise identification of O. intermedium at genomic level. In addition, the patient recovered well after antibiotic combined therapy.ConclusionsThis, to our knowledge, is the first case of O. intermedium bacteremia in a pediatric patient with malignant tumor. Traditional biochemical identification methods such as API 20NE or VITEK2 system cannot differentiate O. anthropi and O. intermedium. MALDI-TOF may be a promising tool for rapid identification of microorganisms such as O. intermedium.

Characterization of a novel shrimp pathogen, Vibrio brasiliensis, isolated from Pacific white shrimp, Penaeus vannamei.

A novel pathogenic strain Vibrio 20190611023 was isolated from the hepatopancreas of moribund cultured Penaeus vannamei suffering from black gill disease. This strain was identified as V.brasiliensis based on the phylogenetic analyses of 16S rDNA gene and five other housekeeping genes (i.e., gapA, ftsZ, mreB, topA and gyrB). Some biochemical features of this strain were determined with an API 20NE system, and its haemolytic activity was determined using a sheep blood agar plate. The pathogenicity of this isolate 20190611023 was confirmed by the experimental challenge tests and histopathological examinations. P.vannamei were challenged via reverse gavage with different doses of bacterial suspensions. The calculated median lethal dose (LD50 ) was (3.16±1.78)×105 CFU/g (body weight). Moreover, antibiotic susceptibility tests were performed, the results of which showed that the strain 20190611023 was sensitive to chloramphenicol, compound sulphamethoxazole, ciprofloxacin, doxycycline and oxacillin, but resistant to erythromycin, kanamycin, gentamicin, cefoperazone, ceftriaxone, cefamezin and piperacillin. To our knowledge, this is the first report for demonstrating V.brasiliensis as a shrimp pathogen, which expands the host range of V.brasiliensis infection. The present study highlights that more attention should be paid to this novel pathogen in intensive shrimp aquaculture.

Misidentification of Burkholderia pseudomallei, China.

We report a case of melioidosis in China and offer a comparison of 5 commercial detection systems for Burkholderia pseudomallei. The organism was misidentified by the VITEK 2 Compact, Phoenix, VITEK mass spectrometry, and API 20NE systems but was eventually identified by the Bruker Biotyper system and 16S rRNA sequencing.

Emergence of plasmid-mediated mcr genes from Gram-negative bacteria at the human-animal interface

BackgroundThe global emergence of plasmid-mediated colistin resistance (Col-R) conferred by mcr genes in gram-negative rods (GNRs) has jeopardized the last treatment option for multidrug-resistant bacterial infections in humans. This study aimed to assess the emergence of mcr gene-mediated Col-R in GNRs isolated from humans and animals in Pakistan.MethodsAnimal and clinical specimens collected from various sources were prospectively analysed using standard microbiological procedures. Pathogens were identified using the API 20E and API 20NE systems (bioMerieux). Minimum inhibitory concentration (MIC) against colistin was determined using the MIC detection methods, and multiplex polymerase chain reaction (PCR) was used to amplify the mcr-1 to mcr-5 genes.ResultsWe isolated 126 (88.1%) animal and 17 (11.9%) human Col-R phenotypes, among which there was a significant association (P < 0.01) of Escherichia coli and Proteus mirabilis with animals and of Acinetobacter baumannii with humans. Animal strains exhibited statistically significant (P < 0.05) resistance to co-trimoxazole, chloramphenicol, and moxifloxacin, and the human pathogens exhibited statistically significant (P < 0.05) antibiotic resistance to cephalosporins, carbapenems, and piperacillin-tazobactam. For Col-R strains, MIC50 values were > 6 µg/mL and > 12 µg/mL for human and animal isolates, respectively. mcr genes were detected in 110 (76.9%) bacterial strains, of which 108 (98.2%) were mcr-1 and 2 (1.8%) were mcr-2.ConclusionsThe detection of a considerable number of mcr-1 and mcr-2 genes in animals is worrisome, as they are now being detected in clinical pathogens. The acquisition of mcr genes by colistin-susceptible bacteria could leave us in a post-antibiotic era.

DIVERSITY OF NODD GENE OF SOME RHIZOBIUM ISOLATES FROM DIFFERENT LEGUMES.

This study was conducted to 1) isolate and characterize some rhizobia from some legumes, 2) determine the phylogenetic relationships among them; and 3) investigate their potential biological control agent. 24 Rhizobial isolates were obtained from five legumes namely; faba bean, lentil, pea, clover, and soybean. Morphological characterization classified isolates into fast grower and slow grower. Also, their biochemical profiles using API 20E and API 20NE system showed large diversity which may reflect their adaptation in different environments. Moreover, molecular detection by 16S rDNA gene was able to characterize 19 of them to species level. Isolates from pea, faba bean, clover and lentil were Rhizobium leguminosarum and isolates from soybean were Bradyrhizobium japonicum. This data supported the idea that there is a narrow diversity of species of rhizobia in Egypt. Phylogenetic analysis of the 19 isolates confirmed that the Bradyrhizobium japonicum isolates were phylogenetically divergent from all other isolates. Also, the phylogram revealed that each group of isolates originated from root nodule of a certain legume formed a separate sub-cluster. Besides a narrow range of interspecies variations was found which is consistent with the idea of biovars presence among species. In addition, the 24 isolates showed strong inhibition of growth of Fusarium and Rhizoctonia phytopathogens. However, results indicated that 25% of isolates were not able to solubilize tricalcium phosphate. On the other hand, the IAA production by the 24 isolates varied greatly among isolates. Therefore, these results demonstrated that they may have the potential to act as biocontrol agents. Moreover, we characterize Nod D gene on the molecular level. Upon its isolation its size came to be 820 bp of nucleotides. This came to be true for all bacterial isolates except four isolates we did not succeed to isolate NodD gene. Moreover we used Quantitative Real Time PCR to study NodD gene expression. Seven nod D gene were down regulated and five were up regulated than 16S housekeeping gene.

A novel research on isolation and characterization of Photobacterium damselae subsp. damselae from Pacific white shrimp, Penaeus vannamei, displaying black gill disease cultured in China.

In June 2019, massive mortalities of cultured Penaeus vannamei occurred in a local farm in Hainan Province, China. The diseased shrimp displayed evident black gills. Three bacterial strains 20190611001, 20190611007 and 20190611022 were isolated from hepatopancreas and gills of the diseased shrimp and identified as Photobacterium damselae subsp. damselae based on the sequence analysis of 16S rRNA and toxR genes. These three isolates showed haemolytic activities. Of them, strain 20190611022 isolated from hepatopancreas was selected and processed for pathogenic analysis. The calculated median lethal dose (LD50 ) was 9.75±4.29×105 CFU/g (body weight) by challenging P.vannameivia reverse gavage. The diseased shrimp displayed enlarged hepatopancreatic tubules and sloughing of epithelial cells in tubular lumens. The strain 20190611022 was also characterized by the testing of API 20NE systems and antibiotic susceptibility. The results of disc diffusion test showed that strain 20190611022 was sensitive to chloramphenicol, compound sulfamethoxazole, cefoperazone, ceftriaxone, ceftazidime and cefuroxime. To our knowledge, this is the first report of isolation and characterization of Photobacterium damselae subsp. damselae from natural diseased P.vannamei. Our findings can serve as a basis for further studies of its pathogenicity and provide technological support for disease controlling in shrimp aquaculture.

Molecular typing of Acinetobacter baumannii clinical strains by enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR)

Acinetobacter baumannii is one of the most important causes of nosocomial infections. Because of the prevalence and recurrence of infections associated with A. baumannii that occur in different regions of hospitals, such as the ICU ward, is important to find the source of the infection by different techniques including molecular methods. This study aimed to determine A. baumannii isolates by ERIC-PCR.In this cross-sectional study, 80 A. baumannii isolates were retrieved from patients admitted to ICU, CCU, and post CCU wards of Ghaem hospital, Karaj, Iran. After detecting the isolates by phenotypic methods, standard biochemical tests were first used to identify the isolates and then API 20NE System kits (Biomerieux, France). The final confirmation was done by tracking the blaOXA-51 gene through PCR. Then, isolates were typed by the ERIC-PCR technique. The gel electrophoresis image was then analyzed by GEL compareII software and the relating dendrogram was depicted.The dendrogram obtained from the results of the study showed that the isolates were divided into 14 clusters using ERIC-PCR method.The present study showed that the ERIC-PCR technique is a useful tool for studying the genetic diversity of A. baumannii isolates. Also, A. baumannii isolates were rotating between ICU, CCU, and post CCU of Ghaem hospital, Karaj, Iran. And also, no specific pattern was observed in the distribution of the isolates from one ward to another.

PHENOTYPIC AND GENOTYPIC DETECTION OF CARBAPENEM RESISTANT ACINETOBACTER BAUMANNII IN SURGICAL AND INTENSIVE CARE UNITS IN AL AZHAR UNIVERSITY HOSPITAL – NEW DAMIETTA

Background: Acinetobacter baumannii (A. baumannii) has emerged as a healthcare-associated pathogen worldwide. Several epidemiological studies have reported the occurrence of multi-drug resistant A. baumannii infections in different regions of the world. The spread of carbapenem-resistant A. baumannii is of a global concern. Objectives: This work was carried out to detect carbapenem-resistant A. baumannii in Surgical Departments and intensive care units (ICUs), Al-Azhar University hospital, New Damietta. It was also aimed to determine the occurrence of bla OXA -51-like and bla OXA-23 like genes among the isolated strains. Patients and methods: The current study was conducted on 500 patients attending the Surgical Departments and ICUs during the period between May 2016 and September 2018. The choice of samples differs according to the clinical manifestations. The isolates were obtained from various clinical specimens, i.e. sputum, endotracheal aspirate (ETA), pus, urine and blood samples. All clinical specimens were collected under complete aseptic conditions. The clinical specimens were traced to the species level using API 20NE system followed by an assesment of the different phenotypic assays for detection of carbapenemase production using multiplex polymerase chain reaction (PCR). Results: Post-operative infections were detected in 217 (43.4%) out of 500 of patients. A. baumannii was considered the third common isolated Gram-negative organisms (27, 12.3 %). A. baumannii isolates were predominant in ICUs (14, 51.9%). Carbapenemase production was detected in A. baumannii isolates using the modified Carbapenem Inactivation Method (mCIM), the modified Hodge test (MHT) and the Carba NP test. Using multiplex PCR analysis, most isolates (12; 44.4%) carried bla OXA-51-like gene, followed by ten (37%) isolates that carried both bla OXA-51-like and bla OXA-23-like genes. Only one (3.7%) isolate carried bla OXA-23-like gene. Conclusion: The current study suggested that A. baumannii is one of the most commonly detected isolates in our hospital. The mCIM is the most useful phenotypic method for detection of carbapenemase production. Detection of carbapenem resistance genes is alarming a serious healthcare problem in our hospital.

PHENOTYPIC AND GENOTYPIC DETECTION OF CARBAPENEM RESISTANT ACINETOBACTER BAUMANNII IN SURGICAL AND INTENSIVE CARE UNITS IN AL AZHAR UNIVERSITY HOSPITAL – NEW DAMIETTA

Background: Acinetobacter baumannii (A. baumannii) has emerged as a healthcare-associated pathogen worldwide. Several epidemiological studies have reported the occurrence of multi-drug resistant A. baumannii infections in different regions of the world. The spread of carbapenem-resistant A. baumannii is of a global concern. Objectives: This work was carried out to detect carbapenem-resistant A. baumannii in Surgical Departments and intensive care units (ICUs), Al-Azhar University hospital, New Damietta. It was also aimed to determine the occurrence of bla OXA -51-like and bla OXA-23 like genes among the isolated strains. Patients and methods: The current study was conducted on 500 patients attending the Surgical Departments and ICUs during the period between May 2016 and September 2018. The choice of samples differs according to the clinical manifestations. The isolates were obtained from various clinical specimens, i.e. sputum, endotracheal aspirate (ETA), pus, urine and blood samples. All clinical specimens were collected under complete aseptic conditions. The clinical specimens were traced to the species level using API 20NE system followed by an assesment of the different phenotypic assays for detection of carbapenemase production using multiplex polymerase chain reaction (PCR). Results: Post-operative infections were detected in 217 (43.4%) out of 500 of patients. A. baumannii was considered the third common isolated Gram-negative organisms (27, 12.3 %). A. baumannii isolates were predominant in ICUs (14, 51.9%). Carbapenemase production was detected in A. baumannii isolates using the modified Carbapenem Inactivation Method (mCIM), the modified Hodge test (MHT) and the Carba NP test. Using multiplex PCR analysis, most isolates (12; 44.4%) carried bla OXA-51-like gene, followed by ten (37%) isolates that carried both bla OXA-51-like and bla OXA-23-like genes. Only one (3.7%) isolate carried bla OXA-23-like gene. Conclusion: The current study suggested that A. baumannii is one of the most commonly detected isolates in our hospital. The mCIM is the most useful phenotypic method for detection of carbapenemase production. Detection of carbapenem resistance genes is alarming a serious healthcare problem in our hospital.

Molecular feature of lasB gene of Pseudomonas aeruginosa isolated from different clinical sources

The study was carried out through a period from June to November 2017 from out-patients and in-patients of Al-Hussein Teaching Hospital in Thi-Qar province South of Iraq. A sum of 132 specimens from the hospital facility condition and different clinical samples of various patients were randomly collected and analyzed for diagnosis of Pseudomonas aeruginosa. These clinical specimens were included wound, burn, ear swabs, urine and sputum specimens. Every single specimen was screened to detect of P. aeruginosa by culturing on suitable media, and 36 isolates of P. aeruginosa were distinguished through biochemical tests and using API 20NE system for confirmation the isolates. All P. aeruginosa isolates were tested against nine antimicrobial discs in vitro. They revealed that having different classes and the results demonstrated that variable resistance to the anti-microbial agents. In attempting to the recognizable proof of P. aeruginosa having the DNA, Polymerase chain reaction (PCR) was utilized in light of particular groundwork for 16S rRNA genetic marker. The results demonstrated that PCR has observed to be quick and sensitive and particular for recognizable proof of P. aeruginosa. Furthermore, 16S rRNA was utilized as confirmation feature, while lasB detected as virulence gene also to utilize as pathogenesis quality of the bacteria.

Molecular feature of lasB gene of Pseudomonas aeruginosa isolated from different clinical sources

The study was carried out through a period from June to November 2017 from out-patients and in-patients of Al-Hussein Teaching Hospital in Thi-Qar province South of Iraq. A sum of 132 specimens from the hospital facility condition and different clinical samples of various patients were randomly collected and analyzed for diagnosis of Pseudomonas aeruginosa. These clinical specimens were included wound, burn, ear swabs, urine and sputum specimens. Every single specimen was screened to detect of P. aeruginosa by culturing on suitable media, and 36 isolates of P. aeruginosa were distinguished through biochemical tests and using API 20NE system for confirmation the isolates. All P. aeruginosa isolates were tested against nine antimicrobial discs in vitro. They revealed that having different classes and the results demonstrated that variable resistance to the anti-microbial agents. In attempting to the recognizable proof of P. aeruginosa having the DNA, Polymerase chain reaction (PCR) was utilized in light of particular groundwork for 16S rRNA genetic marker. The results demonstrated that PCR has observed to be quick and sensitive and particular for recognizable proof of P. aeruginosa. Furthermore, 16S rRNA was utilized as confirmation feature, while lasB detected as virulence gene also to utilize as pathogenesis quality of the bacteria.

Biodegradability of Disinfectants in Surface Waters from Buenos Aires: Isolation of an Indigenous Strain Able to Degrade and Detoxify Benzalkonium Chloride

Biodegradability of chlorhexidine (CH), triclosan (TC), and benzalkonium chloride (CBA) has been tested in 18 surface water sampling points in the urban area of Buenos Aires. Sampling points were located in both the Reconquista and the Matanza-Riachuelo basins as well as in the La Plata River. High tolerance to the three disinfectants was found and indigenous strains capable of degrading CBA and TC were isolated. Neither tolerance nor biodegradation were correlated with sewage pollution. A strain that degrades CBA was identified as belonging to the genus Pseudomonas using the API20NE system and 16SRNA sequencing. In batch assays, the strain was capable of degrading 100, 200, and up to 500 mg L−1 of CBA in 10, 25, and 46 h respectively with specific growth rates (μ) of 0.56, 0.30, and 0.14 h−1. The efficiency of the process was between 99.5–98.0% in terms of compound removal and between 93.8–89.1% in terms of chemical oxygen demand (COD). The detoxification of the compound as a result of the biodegradation was assessed using Pseudokirchneriella subcapitata, Vibrio fischeri, and Lactuca sativa as test organisms.

Morphological and Molecular Identification of Hospital Pseudomonas spp

The study was designed for isolation and identification of Pseudomonas spp. from neonatal nosocomial infections, from Three Teaching Hospitals of Maternity and Pediatrics in Al-Mosul city. The total collected samples were 288,which included clinical(134samples collected from preterm and full-term neonates who less one year of age ) and environmental samples(154)from anuary 2011 till end July 2011, in order to evaluate the classical laboratory diagnostic procedures for diagnosis of Pseudomonas isolates and compared them with molecular technique on the basis Polymerase Chain Reaction (PCR). Pseudomonas spp. has been isolated and identified by using cultural method, biochemical tests and Mini API 20NE system as confirmative test. On the other hand, PCR technique was utilized to detected 16SrRNA gene as a house keeping gene, specific for diagnosis of Pseudomonas spp. The present study showed that the isolation percent of Pseudomonas spp. from clinical samples was 17/30 (18.8%)and 30/111(27%) from environmental samples, whereas the PCR results showed that there were high specificity (100%) in detection of Pseudomonas spp. versus each of cultural, biochemical and Mini API 20NE system

A bacterium with close genetic identity to Pseudomonas mandelii associated with spring fish kills in wild bluegill Lepomis macrochirus Rafinesque and pumpkinseed sunfish Lepomis gibbosus (Linnaeus).

Pseudomonas fluorescens are known bacterial pathogens in fish. The P. fluorescens group contains at least nine different bacterial species, although species from fish have rarely been differentiated. Two isolated fish kills affecting wild bluegills, Lepomis macrochirus Rafinesque, and pumpkinseed sunfish, Lepomis gibbosus (Linnaeus), occurred in the spring of 2015 during cool water temperatures (12.5°C-15.5°C). Disease signs included severe bacteraemia with rare gross external signs. Pure bacterial cultures isolated from kidneys of all affected fish were identified as P. fluorescens using the API 20NE system, while no bacteria were isolated from asymptomatic fish. To further identify the species of bacterium within the P. fluorescens complex, genetic analysis of the 16S rRNA, rpoD and gyrB genes was conducted. DNA sequences of bacterial isolates from both mortality events were identical and had close identity (≥99.7%) to Pseudomonas mandelii. Although likely widespread in the aquatic environment, this is the first report of a bacterium closely resembling P. mandelii infecting and causing disease in fish. The bacterium grew at temperatures between 5°C and 30°C, but not at 37°C. It is possible that infections in fish were a result of immunosuppression associated with spring conditions combined with the psychrotrophic nature of the bacterium.

First recording of Shewanella putrefaciens in cultured Oreochromis niloticus and its identification by 16Sr RNA in Egypt

A survey to determine the pathogenic bacterial strains of an important commercial capture fish, Oreochromis niloticus in an Egyptian fish farm was conducted. One strain presumptively identified as Shewanella putrefaciens was isolated from the fish and identified by a biochemical test, API20NE system and 16S rRNA gene. Intraperitoneal (I.P) challenge by S. putrefaciens revealed its pathogenicity with 50% mortality. The infected fish showed skin hemorrhage, fin rot and shallow necrotizing ulcers on the skin with congestion and enlargement of the liver, spleen and kidneys. The antibiogram of S. putrefaciens revealed its sensitivity to ciprofloxacin, norfloxacin, nalidixic acid, streptomycin, gentamycin, oxytetracyclin and sulfamethoxazole. Complete resistance to cephazolin and fusidic acid was recorded. Histological changes in the liver of infected O. niloticus included hepatocyte degeneration, fatty vacuolation in liver cells, Kupffer cell increase, hemolysis with infiltration in blood vessels, deposition of hemosiderin associated with the liver blood vessels and necrosis hepatocytes in the fatty liver tissue. The results concluded that the S. putrefaciens should be considered as an opportunistic pathogen for fish, which had caused fish infections, diseases with septicemia and mortality in O. niloticus.

Isolation and identification of pseudomonas aeruginosa from infected sheep and detection of phosolipase C (lecithinase

The current study dealt with the isolation and identification of 19 isolates (14.07%) for the period (October 2013 to January 2014) from different samples (135 samples) number of isolates belonging to Pseudomonas aeruginosa were 8 isolated from urine samples, their percentage were 33.33 % and 5 isolates from nasal swabs, (11.11%) and 4 isolated from milk samples (9.30%), 2 isolated from wound swabs (24%) and isolates from eye infection (9.09%). These isolates were identified by morphological examination and biochemical tests and API-20 NE system. The second part was the study of the antibiotic susceptibility that was carried out on 19 isolates of P. aeruginosa for 12 types of antibiotic. The results showed that isolates were resistant to 9 out of 12 antibiotics with percentage (Penicillin 100%, nalidixic acid100%, pipracillin 100%, erythromycin 68,4%, Chloramphenicol 63%, 78%, trimthoprim 73%, ceftacizidem 89%, cefitriaxon 84% and cefotaxime 89%) on the other hand most of isolates were sensitive to (epipenime, azythromycin, and polymixin). Detection of phospholipase C (Lecithinase) by agar well diffusion method on different selective media as (egg yolk agar, briliant green crystal violates lecithnase agar BCL), 12 isolates out of 19 showed a positive reaction on these media. The result depended on measurement the diameter of opacity zone produced and the blush green zone on the BCL media, the diameter of zone ranged from 8mm to 32mm. both media (EYA and BCL media is the best media for detection of phospholipase by P. aeruginosa.

First Report of Pantoea beijingensis Induced Soft Rot Disease of Pleurotus nebrodensis in China.

Pleurotus nebrodensis is a popular edible fungus in China. In December 2009, soft rot disease appeared on fruiting bodies of P. neberodensis in greenhouses located at Haidian District of Beijing, China. Early symptoms were water-soaked lesions on the pilei of the fruiting bodies. Lesions then spread and purulent tissues were formed. Severe soft rot induced production of deformed fruiting bodies and offensive odor. Diseased fruiting bodies stopped growth and commercial losses were estimated at 5 to 10%. Internal sections of the diseased pilei about 0.5 × 0.5 cm were suspended in 0.85% NaCl and the suspension was spread on trypticase soy agar (TSA) media. After incubation at 30°C for 2 days, dominant bacterial colonies were yellow, smooth, round, and convex. Cells were gram-negative, short rods, non-capsulated, motile, and non-spore forming. The 16S rRNA gene (1,408 bp, GenBank Accession No. KF849293) was amplified by using the universal forward primer P1 (5'-AGAGTTTGATCCTGGTCAGAACGCT-3') and the universal reverse primer P6 (5'-TACGGCTACCTTGTTACGACTTCACCCC-3'). Neighbor-joining tree analysis based on 16S rRNA gene sequences demonstrated the isolates belonged to the genus Pantoea and showed highest similarity with Pantoea beijingensis LMG 27579T (99.9% similarity, 2 base pair differences), which was isolated from the diseased fruiting bodies of P. eryngii in China. Pantoea sp. PA4, reported to be the pathogen of soft rot disease of P. eryngii in Korea (1), showed 99.8% similarity with P. ananatis ATCC 33244T, which suggested our isolates belonged to a different species with Pantoea sp. PA4 (supported by 97.9% similarity between the present isolates and Pantoea sp. PA4) (2). Results of physiological properties and enzyme activities determined by the API 20E, API 20NE, API 50CH, API ZYM (bioMérieux), and GN2 MicroPlate (Biolog) system showed that there was no difference between the present isolates and P. beijingensis LMG 27579T. Pathogenicity tests for these isolates were performed with bacterial suspensions (approximately 1 × 106 CFU/ml) that were grown for 24 h in trypticase soy broth (TSB). Fruiting bodies of P. nebrodensis were induced after spawn was run for 30 days in plastic bags. When the young fruiting bodies were forming, the prepared bacterial suspension was sprayed onto the surface of the pilei. Sterilized TSB media was used as a negative control. All inoculated fruiting bodies were then incubated at 16°C with 80 to 95% relative humidity. Assays were conducted twice and five fruiting bodies were used each time. Results were observed after 5 to 10 days of incubation. The symptoms that developed were similar to those observed in the original samples. The negative control remained symptomless. Koch's postulates were fulfilled by re-isolating bacteria, which were identical to the original isolates based on 16S rRNA gene sequence analysis, physiological properties and enzyme activities. Pantoea ananatis was first reported as a pathogen of Pleurotus eryngii, causing soft rot disease (1), but to the best of our knowledge, this is the first report of P. beijingensis- induced soft rot disease of P. nebrodensis in China. This disease may be an emerging disease problem in the near future and information on this pathogen will be useful in the development of management practices.

Pseudomonas aeruginosa inhibits the growth of pathogenic fungi: In vitro and in vivo studies.

The aim of the present study was to investigate the inhibitory effect of Pseudomonas aeruginosa (PA) on pathogenic fungi, including Candida albicans (CA), Candida tropicalis (CT), Candida glabrata (CG), Candida parapsilosis (CP) and Candida krusei (CK), in vitro and in vivo. In total, 24 PA strains were collected from clinical specimens and identified by Gram staining, oxidase production and the API 20NE system. Cross-streak, disk diffusion and co-culture methods were used to observe the inhibitory effect of PA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze differences in the bacterial proteins of PA. A blood infection model in mice was used to evaluate the effect of PA on fungi in vivo. The in vitro and in vivo results demonstrated that a number of PA isolates exhibited a marked inhibitory effect on pathogenic fungi, including CA, CT, CP, CG and CK, while other PA strains exhibited no effect. Therefore, PA exhibits an inhibitory effect on pathogenic fungi and this activity may be important in the treatment of patients. It was hypothesized that PA secretes various types of proteins to suppress the growth of fungal filaments, which subsequently inhibits pathogenic fungi.