Prevalence of antibiotic resistance and virulent factors in nosocomial clinical isolates of Pseudomonas aeruginosa from Panamá

ABSTRACT Background: Pseudomonas aeruginosa is an important causative agent of nosocomial infections. As pathogen, P. aeruginosa is of increasing clinical importance due to its ability to develop high-level multidrug resistance (MDR). Methods: The aim of the present study was to better understand the intrinsic virulence of circulating strains of Pseudomonas aeruginosa, by surveying and characterizing the antibiotic resistance profiles and prevalence of virulence factors in 51 clinical isolates of P. aeruginosa obtained from children admitted to Hospital del Niño-Panamá during the period of October 2016 until March 2017. Antimicrobial susceptibilities were assessed by determining the minimum inhibitory concentration for 12 antibiotics against P. aeruginosa clinical isolates using the VITEK system (https://www.biomerieux.com). Additionally, all isolates were examined by Polymerase Chain Reaction (PCR) for the presence of components of the MexAB-OprM efflux pump system (mexABR) and pyoverdine receptor genes and betalactamases resistance genes (ESBL) using gene-specific primers. Results: A total of 51 pyoverdine producing clinical isolates were analyzed, all of which expressed resistance genes such as genes of the MexAB-OprM efflux pump system (mexABR) and pyoverdine receptor genes (fpvA). Out of 51 MDR isolates, 22 were ESBL producers. The most common ESBL gene was blaTEM expressed by 43% of the isolates. The isolates tested in this study showed increased resistance to antibiotics in the following categories: (i) penicillins (ampicillin (69%), piperacillin (22%); (ii) pyrimethamines (trimethoprim, 65%); (iii) nitrofurans (nitrofurantoin, 63%), and (iv) third-generation cephalosporin cefotaxime (53%). These results underscore a high prevalence of MDR amongst clinical isolates from Panama. Conclusions: The present study indicates that prevalence of BlaTEM-carrying strains is increasing with subsequent multidrug resistance in Panamá and as well reported worldwide. The virulent factors identified in this study provide valuable information regarding the prevalence of resistance genes and their potential impact on treatments that exploit the unique physiology of the pathogen. To prevent further spread of MDR, the proportions of resistant strains of Pseudomonas aeruginosa should be constantly evaluated on healthcare institutions of Panamá. More importantly, this information can be used to better understand the evolution and dissemination of strains hoping to prevent the development of resistance in Pseudomonas aeruginosa. Future studies quantifying the expression of these virulent genes will emphasize on the acquisition of multidrug resistance.

Genotypic profiles of virulent genes detected among the Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa isolated from swiftlets in Borneo

Aims@#The occurrence of multiple pathogenic Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa are important nosocomial and hazardous infection clinically challenge worldwide. Thus, the aim of this study was to screen for the virulent genes profiles to ascertain their prevalence in swiftlets in Borneo. @*Methodology and results@#The Enterococci, E. coli and P. aeruginosa bacteria were isolated from the swiftlets’ faeces and air inside swiftlet houses, which located in the Southern, Central and Northern regions of Borneo. The isolates were identified to the species level by 16S rRNA sequencing assay. Specific primers were designed for detection of the potential virulence genes in E. faecalis (ace, AS, efaA and gelE), E. coli (stx) and P. aeruginosa (oprL) by PCR assay. A total of 38 Enterococci, 26 of E. coli and 2 of P. aeruginosa fecal and airborne bacteria were identified. Sixty-seven percent of E. faecalis isolates were detected positive for four virulence genes, 27% possessed three (AS, efaA, gelE) genes and 6% possessed two (ace, AS) genes. There were no stx genes detected among all the E. coli isolates. The oprL gene was detected in all the P. aeruginosa isolates. @*Conclusion, significance and impact of study@#Virulence genes are important in the pathogenesis of both clinical and avian infections which considered to be a serious public health threat. The high incidence of virulence genes detection in E. faecalis and P. aeruginosa indicates these genes were widely disseminated among the bacteria found in swiftlet houses, suggesting the important issues in the pathogenesis of infections and diseases which may cause potential health risks to humans.

Clinical distribution, virulence factors, and molecular epidemiology of hypervirulent Klebsiella pneumoniae in Hainan Province in 2016 / 中国感染控制杂志

Objective To investigate clinical distribution,capsular serotyping,molecular typing,virulence gene carriage,and antimicrobial susceptibility of hypervirulent Klebsiella pneumoniae (hvKP) strains isolated from a hospital in Hainan Province in 2016.Methods Klebsiella pneumoniae(K.pneumoniae) isolated from the hospital between January and December 2016 were analyzed retrospectively,hvKP strains were selected through string test,antimicrobial susceptibility testing was performed and compared with classic K.pneumoniae(cKP);capsular serotyping,virulence genes,and drug resistance genes of hvKP strains were detected with polymerase chain reaction,molecular typing was performed with pulsed-field gel electrophoresis (PFGE) and multiloeus sequence typing.Results A total of 84 hvKP strains were isolated,the main specimen source was sputum(45 strains);K1 and K2 were the major capsular serotypes of hvKP,while ST23,ST65,and ST86 were the main sequence types of hvKP.The carriage rates of rmpA,aerobatin,allS,kfuBC,and cf29a in hvKP were 90.48％,96.43％,42.86％,66.67％,and 53.57％ respectively,all of them were statistically higher than those of cKP strains,PFGE found that allS was positive only among K1 strains;most antimicrobial resistance rates of hvKP were lower than those of the cKP.Conclusion Sputum is the main specimen source of hvKP,especially K1 serotype;more than 90％ of hvKP strains carry rmpA and aerobatin genes,allS gene only exists in K1 type hvKP.

Molecular epidemiological characteristics of human derived Brucella isolated in Hohhot / 中华地方病学杂志

Objective To get knowledge of the molecular epidemiological characteristics of human derived Brucella isolated in Hohhot,and to provide experimental basis in guiding prevention and treatment of Brucella infection.Methods Twenty-seven Brucella isolates derived from patients in Affiliated Hospital of Inner Mongolian Medical University from 2013 to 2015 were identified by routine bacteriological methods and molecular methods.Multiple-locus variable number tandem repeats analysis (MLVA-16) was used to detect molecular typing and do cluster analysis.Sixteen virulent genes were detected and analyzed by polymerase chain reaction (PCR).Results Twenty-seven Brucella isolates were identified as Brucella melitensis (B.melitensis) by routine bacteriological methods and PCR.Out of them,six isolates were B.melitensis biovar 1,and twenty-one isolates were B.melitensis biovar 3.MLVA-16 analysis showed that seven genotypes were obtained from nine Brucella isolates,which showed significant difference in variable number of tandem repeats,which suggested that they originated from sporadic outbreak.Moreover,two isolates were clustered into the same clade,which suggested they were epidemiologically correlated and may be derived from the same origin.Sixteen virulent genes were detected in all of the twenty-seven isolates.Conclusions Brucella isolates from patients in Hohhot are mainly B.melitensis biovar 3 and B.melitensis biovar 1,and the distribution profile of multiple virulence genes is similar.Some isolates have showed epidemic correlation,and the epidemic mechanism should be further explored.

Progress in pathogenic mechanism of hypervirulent Klebsiella pneumoniae / 中华微生物学和免疫学杂志

Hypervirulent Klebsiella pneumoniae ( hvKP) mainly infects healthy people and causes serious infections, such as liver abscess, meningitis, necrotizing fasciitis, endophthalmitis and severe pneu-monia. Studies have shown that hvKP is more virulent than classic Klebsiella pneumoniae characterized by ex-pressing more capsular polysaccharide and carrying the virulence factors including magA, rmpA and iron ac-quisition molecules. The greater survival and anti-phagocytosis abilities of hvKP strains contribute to the spread and metastasis of hvKP infection. This review describes the virulence factors, colonization and infec-tion of hvKP as well as the host immunity to hvKP.

Establishment of An Indirect ELISA Method with Helicobacter Pylori Recombinated Antigene OipA6 as Coating Antigen / 现代检验医学杂志

Objective To establish an indirect ELISA method with Helicobacter pylori (HP)recombinated antigene OipA6 as coating antigen after prokaryotic expression and purification.Methods The ELISA plate was coated with Hp OipA6 recom-binant protein as antigen.Serum specimens and enzyme labeled anti human IgG were added.The reaction conditions was op-timized.The results of indirect ELISA were compared with the detection of oipA gene.Results When Hp OipA6 recombi-nant protein was 1.0 mg/ml,the condition of coating was 2~8℃ one night,the dilution of serum specimens was 1∶100 and enzyme labeled anti human IgG was 1∶4 000,the incubation time of enzyme labeled anti human IgG was 40 min,the chro-mogenic time was 15 min,the result of ELISA was the best.The sensitivity and specificity of the indirect ELISA were both higher than the detection of oipA gene.Conclusion It suggested that the indirect ELISA,in which Hp OipA6 recombinant protein were coated,may be an appropriate assisted method in detection of high virulent Hp infection.

Stress tolerant virulent strains of Cronobacter sakazakii from food

BACKGROUND: Cronobacter sakazakii is considered as an emerging foodborne pathogen. The aim of this study was to isolate and characterize virulent strains of Cronobacter sakazakii from food samples of Bangladesh. RESULT: Six (6) Cronobacter sakazakii was isolated and identified from 54 food samples on the basis of biochemical characteristics, sugar fermentation, SDS-PAGE of whole cell protein, plasmid profile and PCR of Cronobacter spp. specific genes (esak, gluA, zpx, ompA, ERIC, BOX-AIR) and sequencing. These strains were found to have moderately high antibiotic resistance against common antibiotics and some are ESBL producer. Most of the C. sakazakii isolates were capable of producing biofilm (strong biofilm producer), extracellular protease and siderophores, curli expression, haemolysin, haemagglutinin, mannose resistant haemagglutinin, had high cell surface hydrophobicity, significant resistance to human serum, can tolerate high concentration of salt, bile and DNase production. Most of them produced enterotoxins of different molecular weight. The isolates pose significant serological cross-reactivity with other gram negative pathogens such as serotypes of Salmonella spp., Shigella boydii, Shigella sonnei, Shigella flexneri and Vibrio cholerae. They had significant tolerance to high temperature, low pH, dryness and osmotic stress. CONCLUSION: Special attention should be given in ensuring hygiene in production and post-processing to prevent contamination of food with such stress-tolerant virulent Cronobacter sakazakii.

Characterization of a virulent Leptospira interrogans strain isolated from an abandoned swimming pool

Pathogenic Leptospira spp. are the etiological agents of leptospirosis, an important disease of both humans and animals. In urban settings, L. interrogans serovars are the predominant cause of disease in humans. The purpose of this study was to characterize a novel Leptospira isolate recovered from an abandoned swimming pool. Molecular characterization through sequencing of the rpoB gene revealed 100% identity with L. interrogans and variable-number tandem-repeat (VNTR) analysis resulted in a banding pattern identical to L. interrogans serogroup Icterohaemorrhagiae, serovar Copenhageni or Icterohaemorrhagiae. The virulence of the strain was determined in a hamster model of lethal leptospirosis. The lethal dose 50% (LD50) was calculated to be two leptospires in female hamsters and a histopathological examination of infected animals found typical lesions associated with severe leptospirosis, including renal epithelium degeneration, hepatic karyomegaly, liver-plate disarray and lymphocyte infiltration. This highly virulent strain is now available for use in further studies, especially evaluation of vaccine candidates.

A Virulent Salmonella enterica Serovar Enteritidis Phage SE2 with a Strong Bacteriolytic Activity of Planktonic and Biofilmed Cells

Salmonella enterica serovar Enteritidis is one of the major food borne pathogens. Utilizing lytic bacteriophages against this pathogen can be a new and effective approach for the prevention of food-contamination and food-borne infection. In this study, we isolated and characterized a Salmonella Enteritidis specific lytic bacteriophage (phage SE2). The bacteriolytic activity of planktonic and biofilmed cells against an antibiotic resistant strain of Salmonella Enteritidis was also evaluated. Phage SE2 revealed an efficient bacteriolytic effect with biofilm dispersing ability and could maintain its virulence even at extreme pH and temperature. It can be a potential biotherapeutic agent against Salmonella Enteritidis.

Partial VP1 sequencing of Brazilian infectious bursal disease virus strains

Infectious bursal disease virus (IBDV) is classified according to the antigenicity and virulence into classical virulent (cv), very virulent (vv), and antigenic variant strains. The molecular basis for the IBDV antigenic variation is well established and is associated to the capsid protein, VP2 (gene VP2 of segment A), whereas both VP2 and the RNA-dependent RNA polymerase, VP1 (gene VP1 of segment B), have been correlated with the virulence. In this study, seventeen Brazilian IBDV samples previously characterized by the VP2 gene as cv (three) and vv (fourteen) strains were genetically and molecularly analyzed for their VP1 gene. All of the strains kept with the same cv or vv classification except one sample, Br/03/DR. This sample was classified as vv by its VP2 gene, but it was most closely related to the cv strains by its VP1 partial sequence and phylogeny. Studies on the phylogeny of VP1 have suggested a possible reassortment event that originated the vvVP1. In this case, the sample carrying vvVP2 and cvVP1 could be a descendant of IBDV ancestors prior to the reassortment of vvVP1; alternatively, it could be the result of a genetic exchange between the segments of different strains or with a live attenuated vaccine. Nevertheless, this is the first report of natural genetic reassortment of IBDV in Brazil.

Sequence analysis of segment A gene of a very virulent infectious bursal disease virus recently isolated in Korea / 대한수의학회지

Infectious bursal disease virus (IBDV) is a member of the Avibirnavirus genus of the Birnaviridae family which genome consists of two segments (A and B) of double stranded RNA. Segment A gene of KNU08010 isolate, which was isolated from a 15-day-old chicken flock in 2008, was sequenced and compared with other IBDV isolates including SH/92 strain, the first Korean very virulent (vv) IBDV isolate. The amino acid sequences of segment A gene showed that KNU08010 had 99.2% homology with SH92 strain. KNU08010 isolate had specific amino acids A222, I242, I256, I294 and S299 which are highly conserved among vvIBDV strains. Phylogenetic analysis based on the nucleotide sequences of variable region of the VP2 gene of 18 IBDV strains revealed that KNU08010 was grouped with vvIBDVs and was closely related to Korean vvIBDVs isolated from wild birds.

Growth responses of in vitro Fusarium oxysporum f. sp. niveum to external supply of tannic acid.

Allelochemicals released from root exudates or decaying residues of plants play diversified roles in ecological interactions of plant-pathogen. The objective of this work was to evaluate the allelopathic effect of an externally supplied tannic acid on soil-borne in vitro Fusarium oxysporum f.sp.niveum. Results showed that the tannic acid decreased the growth of the fungus up to 9.5% at 800 mg l-1. Conidial germination was reduced by 52.3% in comparison with the control. However, sporulation and mycotoxin production by the fungus were stimulated. The activity of pectinase and proteinase were initially increased and finally decreased with increase in concentrations of tannic acid. Tannic acid served as an ecological allelochemical, repressing the growth of the pathogen.

PCR-based detection of enterotoxigenic isolates of Bacillus cereus from tropical seafood.

Background & objectives: Bacillus cereus is an important enterotoxigenic food borne pathogen. The present study was undertaken to assess the occurrence of B. cereus in tropical fish and evaluation of virulent gene specific PCR for differentiation of diarrhoeal enterotoxin producing isolates of B. cereus from non enterotoxigenic isolates. Methods: Selective plating on polymixin-pyruvate-egg yolk-mannitol-bromocresol purple agar (PEMPA) was used for isolation of B. cereus from finfish, prawn and clams. Enterotoxin producing ability of all 42 isolates obtained from the samples was judged by reverse passive latex agglutination (RPLA) test and the presence of different virulent genes i.e. hbla, bceT and entFM was screened by PCR. Results: B. cereus and enterotoxigenic B. cereus were found to be in 36.7 and 29.41 per cent of fish samples, respectively. All the diarrhoeal enterotoxin producing isolates showed the presence of hbla gene, but hbla gene was not present in any of the non-enterotoxigenic isolates tested in this study. Interpretation & conclusions: Our findings indicated that hbla gene specific PCR can be employed for differentiation of enterotoxigenic B. cereus isolates from non-enterotoxigenic isolates.

Growth of in vitro Fusarium oxysporum f. sp. niveum in chemically defined media amended with gallic acid

Gallic acid was artificially added to the media to grow Fusarium oxysporum f.sp.niveum to investigate its effect on the pathogenic fungus. Results indicate that gallic acid inhibited the growth of F. oxysporum f.sp.niveum. The colony diameter, the conidia germinating rate and the conidia yield were reduced by 5.7-22.9 percent percent, 35.8-55.6 percent and 38.9-62.2 percent respectively. However, the virulence factors by the fungus were stimulated. The activity of pectinase, proteinase and cellulase increased by 12.3-627.8 percent, 11.8-41.2 percent and 0.5-325.0 percent respectively, while the activity of amylase increased slightly. The results suggest that gallic acid repressed growth but facilitated the relative pathogenicity of invading pathogens.

Development and Clinical Application of RT-PCR Differential Diagnosis Method for High Virulent Porcine Reproductive and Respiratory Syndrome / 微生物学通报

Based on the deletion information of high virulent PRRSV genome, 3 oligonucleotide primer were designed and synthesized. Specific and sensitive reverse transcription-PCR (RT-PCR) assays were de-veloped for the detection of high virulent PRRSV. The sensitivity and specificity of RT-PCR assays were evaluated, the results showing that the detection limit of the assay was found to be 0.265 pg of tissue total RNA, and the protocol have no cross-reaction with classical swine fever virus, porcine circovirus type 2,pseudorabies virus, streptococcus, haemophilus parasuis and Escherichia coli. Then 36 cell cultures, two PRRSV live vaccine strains and 184 clinical specimens from 52 farms were tested. Five PRRSV field iso-lates were the high virulent PRRSV; two PRRSV live vaccine strains from normal PRRSV, and 123 speci-mens from 42 farmer were positive (only 1 specimen was normal PRRSV). This RT-PCR method proved to be accurate differential diagnosis of the high virulent PRRSV and normal PRRSV with the characteristics of rapidity, sensitivity and specificity, and has a strong clinical relevance.

Carriage prevalence and distribution of virulence genes of diarrheagenic Escherichia coli isolated from healthy children under 5 years of age in community

Introduction: Diarrheagenic Escherichia Coli (DEC) is getting more and more important as a cause of diarrhea in children under 5 years of age. Detection of DEC prevalence and distribution of their virulent genes plays an important role in prevention and treatment for E.coli-related diseases and vaccine development. Objectives: This study was conducted with the aim to detect DEC prevalence and the distribution of virulent genes of DEC isolated from healthy children under 5 who were living in the community. Subjects and method: 826 children under-5 living in Ba Vi District, Ha Tay Province were selected. Polymerase chain reactions using specific primers to virulent genes of DEC were used. Results. The study found that the prevalence of DEC was 9.8%, among this EAEC accounted for 3.1%, EHEC 1.8%, EIEC 0.1%, EPEC 1.1%, ETEC 0.1% and two DECs 3.5%. Combinations of virulent genes of EHEC and EHEC+ETEC accounted for 50% of total virulent genes. Conclusion: Five types of DEC were isolated from subjects with the prevalence of 9.8%. The most common virulent genes were combinations of EHEC and ETEC. Further studies are needed to investigate the transmission pathway of DEC in children living in the community.

Detection of Virulent Gene Distribution of Diarrheagenic Escherichia Coli (DEC)

Introduction: There are 5 identified DEC including EAEC, EHEC, EIEC, EPEC and ETEe. Virulent genes (for adherrnee, toxin, antibiotic resistance ...) play important roles in pathogenesis of DEe. Detection of DEC is very important in diagnosis, epidemiology survey and vaccine development. \r\n', u'Objectives: Detection of virulent gene distribution of DEC and non - DEe.\r\n', u'Object and methods: 161 strains of DEC (EAEC, EIEC, EPEC, TEC) and 100 strains of non - DEC were subjected to this study. PCR with specific primers were used to test these genes. \r\n', u'Results: EAEC that accounted for 50% of DEC, was identified and isolated. Aap gene was the highest prevalence in EAEC (96.5%), followed by aggR (79.1 %) and astA (60.5%). 37.2% of the strains harbor all three genes. None of strains had PCR results negative for these 3 genes. ETEC, EPEC and EIEC had aap, and astA gene at the prevalence from 7% to 72.7%. The highest prevalence of aap was seen in EIEC 72.7%), aggR in EIEC (45.5%), and astA in ETEC (50%). 14% of non - DEC had aggR and more than 30% of E. coli had aap and astA gene. \r\n', u'Conclusion: EAEC is prevalent at 50% among Diarreagenic E. coli. Aap is the most prevalent and the most commonly seen among EAEC isolates. The other three genes are at different prevalence. The findings contribute towards the vaccine development against diarrhea caused by E. coli. \r\n', u'

BIOLOGICAL CHARACTERIZATION OF FOWL INTESTINAL BACTERIOPHAGE / 微生物学通报

More than ten bacteriophage of E.coli were isolated from the soil and the dung of the fowl-run, then three of named bacteriophage A, C, D which lysis E.coli virulently were selected to investigate biological characterizations. The results showed that high activities were obtained after the phages incubated at 50℃ for 1 h or 60℃ for 30 min. The phages could be alive at the range of pH from 4 to 12, Ca 2+ or Mg 2+ added to the medium could stimulate the lysis of phages. However, the formation of the plaque could be inhibited obviously by adding sodium citrate to the medium.