Rapid identification of melioidosis agent by an insulated isothermal PCR on a field–deployable device

Background. Burkholderia pseudomallei causes melioidosis, a serious illness that can be fatal if untreated or misdiagnosed. Culture from clinical specimens remains the gold standard but has low diagnostic sensitivity. Method. In this study, we developed a rapid, sensitive and specific insulated isothermal Polymerase Chain Reaction (iiPCR) targeting bimA gene (Burkholderia Intracellular Motility A; BPSS1492) for the identification of B. pseudomallei. A pair of novel primers: BimA(F) and BimA(R) together with a probe were designed and 121 clinical B. pseudomallei strains obtained from numerous clinical sources and 10 ATCC nontargeted strains were tested with iiPCR and qPCR in parallel. Results. All 121 B. pseudomallei isolates were positive for qPCR while 118 isolates were positive for iiPCR, demonstrating satisfactory agreement (97.71%; 95% CI [93.45– 99.53%]; k = 0.87). Sensitivity of the bimA iiPCR/POCKIT assay was 97.52% with the lower detection limit of 14 ng/µL of B. pseudomallei DNA. The developed iiPCR assay did not cross-react with 10 types of non-targeted strains, indicating good specificity. Conclusion. This bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen

Isolation, molecular characterization and antimicrobial susceptibility of Aeromonas spp. obtained from Tiger Grouper (Epinephelus fuscoguttatus) and Marble Goby (Oxyeleotris marmoratus) fish in Sabah, Malaysia

Aeromonads are ubiquitous in aquatic environments and have been implicated in fish and human infections. In this study, we isolated, studied antimicrobial susceptibility patterns and screened the existence of 15 virulence genes in aeromonads from two famously consumed fish species—seven marine Tiger Grouper (Epinephelus fuscoguttatus) and eight freshwater Marble Goby (Oxyeleotris marmoratus) from the aquaculture hatchery in Sabah, Malaysia. A total of 30 aeromonads (17 A. caviae, 9 A. rivuli, 4 A. dhakensis) were identified using PCR targeting GCAT gene, rpoD‐restriction fragment length polymorphism and multi‐locus phylogenetic analysis. All 30 strains were resistant to amoxicillin and cephalothin and five strains were multidrug‐resistant. Nine virulence genes (lip, ela, eno, fla, aerA, hylA, dam, alt and ser) present in A. dhakensis, suggesting the virulence potential of this species as a fish pathogen. This study offers as a baseline for future studies in monitoring and managing these two fish in aquaculture industry

Molecular Characterization of Clinical Isolates of Aeromonas Species from Malaysia

Background: Aeromonas species are common inhabitants of aquatic environments giving rise to infections in both fish and humans. Identification of aeromonads to the species level is problematic and complex due to their phenotypic and genotypic heterogeneity. Methodology/Principal Findings: Aeromonas hydrophila or Aeromonas sp were genetically re-identified using a combination of previously published methods targeting GCAT, 16S rDNA and rpoD genes. Characterization based on the genus specific GCAT-PCR showed that 94 (96%) of the 98 strains belonged to the genus Aeromonas. Considering the patterns obtained for the 94 isolates with the 16S rDNA-RFLP identification method, 3 clusters were recognised, i.e. A. caviae (61%), A. hydrophila (17%) and an unknown group (22%) with atypical RFLP restriction patterns. However, the phylogenetic tree constructed with the obtained rpoD sequences showed that 47 strains (50%) clustered with the sequence of the type strain of A. aquariorum, 18 (19%) with A. caviae, 16 (17%) with A. hydrophila, 12 (13%) with A. veronii and one strain (1%) with the type strain of A. trota. PCR investigation revealed the presence of 10 virulence genes in the 94 isolates as: lip (91%), exu (87%), ela (86%), alt (79%), ser (77%), fla (74%), aer (72%), act (43%), aexT (24%) and ast (23%). Conclusions/Significance: This study emphasizes the importance of using more than one method for the correct identification of Aeromonas strains. The sequences of the rpoD gene enabled the unambiguous identication of the 9

Molecular characterization of putative virulence determinants and identification of specific immunogenic polypeptides for the serological diagnosis of burkholderia pseudomallei infections / Puah Suat Moi

The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Six genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131 and BURPS1710A_1419 were identified earlier in a screen by PCR-based subtractive hybridization, using a virulent clinical isolate of B. pseudomallei and a laboratory-acquired attenuated strain of the same isolate of B. pseudomallei. Therefore, the first objective of this study was to extensively characterize these genes at the molecular level, as well as one additional gene BPSL3147 identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and confirmed by PCR. Likewise, complemented strains were successfully obtained by reintroducing an intact copy of the defective gene into the corresponding mutant strains. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced survival inside macrophage RAW 264.7 cells and also low attenuation level in the virulence of nematode infection model. However, BPSL2033::Km only demonstrated a weak statistical significance (p=0.049) of intracellular survival compared to the wild type at 8 hour post infection in macrophage infection study but BURPS1710A_1419::Km showed a p-value of 0.165. Nevertheless, complemented strains of both genes were able to partially restore the gene defect both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei. Lack of a universally acceptable antigen for serodiagnosis of infections caused by B. pseudomallei is another clinical challenge. The second objective of this study was to search for an immunogen via a shotgun expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening with sera from melioidosis patients, 6 sero-positive clones expressing immunogenic polypeptides were sequenced and their identities were: BPSS1904 (benzoate 1,2-dioxygenase beta subunit), BURPS1710b_0454 (a putative 200 kDa antigen p200), BPSS1856 (phosphotransferase enzyme family protein), BPSS0897 (short chain dehydrogenase), BPSL3130 and BPSS1757 (hypothetical proteins). These immunogenic polypeptides were then purified and transferred to an ELISA platform for further large scale screening. Experimental screening using 60 melioidosis positive and 123 non-melioidosis sera allowed the identification of 2 immunogenic polypeptides BPSS1904 and BPSL3130 with diagnostic potential, which demonstrated sensitivities of 75% and 90%, and specificities of 90.24% and 88.62%, respectively. The results suggest that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei. In summary, the present study suggests that BPSL2033 and BURPS1710A_1419 genes to be associated with virulence of B. pseudomallei, while immunogenic polypeptides BPSS1904 and BPSL3130 were shown to be potential antigens for the serological diagnosis of its infection

Molecular Characterization of Putative Virulence Determinants in Burkholderia pseudomallei

The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033:: Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei

First report of extended-spectrum β-lactamases TEM-116 and OXA-10 in clinical isolates of alcaligenes species from Kuala Lumpur, Malaysia

There is an alarming increase in the prevalence of extended-spectrum β-lactamases (ESBLs) present mainly in Enterobacteriaceae and other nonfermenting gram-negative bacteria, such as Alcaligenes faecalis, which is the only species in that genus that is clinically relevant. We investigated Alcaligenes species from 7 cases (6 inpatients and one outpatient) at our tertiary-care hospital. Four patients had urinary tract infections, and one each had systemic lupus erythematosus, pulmonary stenosis, and diabetic ulcer. All 7 isolates were identified as Alcaligenes spp. based on their 16S rRNA gene sequences, and antibiotic susceptibility was determined using a Vitek 2 system with AST-GN87 cards. All the strains were resistant to cefazolin; 6 were resistant to trimethoprim/sulfamethoxazole; 5 manifested resistance to ampicillin/sulbactam, cefepime, tobramycin, ciprofloxacin, and nitrofurantoin; whereas 5 had multidrug resistance profiles. All the strains (7/7) expressed ESBL activity; PCR screening and sequencing showed evidence of genes blaTEM-116 (7/7) and blaOXA-10 (4/7), and we believe that this is the first report on the presence of TEM-116 and OXA-10 in an Alcaligenes spp. A combination of the 2 genes was present in 4 strains. All 7 strains were found to harbor at least one ESBL gene probably contributing to the drug resistance. © 2019, National Institute of Health. All rights reserved

Virulence Factors and Antibiotic Susceptibility of Staphylococcus aureus Isolates in Ready-to-Eat Foods: Detection of S. aureus Contamination and a High Prevalence of Virulence Genes

Staphylococcus aureus is one of the leading causes of food poisoning. Its pathogenicity results from the possession of virulence genes that produce different toxins which result in self-limiting to severe illness often requiring hospitalization. In this study of 200 sushi and sashimi samples, S. aureus contamination was confirmed in 26% of the food samples. The S. aureus isolates were further characterized for virulence genes and antibiotic susceptibility. A high incidence of virulence genes was identified in 96.2% of the isolates and 20 different virulence gene profiles were confirmed. DNA amplification showed that 30.8% (16/52) of the S. aureus carried at least one SE gene which causes staphylococcal food poisoning. The most common enterotoxin gene was seg (11.5%) and the egc cluster was detected in 5.8% of the isolates. A combination of hla and hld was the most prevalent coexistence virulence genes and accounted for 59.6% of all isolates. Antibiotic resistance studies showed tetracycline resistance to be the most common at 28.8% while multi-drug resistance was found to be low at 3.8%. In conclusion, the high rate of S. aureus in the sampled sushi and sashimi indicates the need for food safety guidelines

Identification and speciation of 98 <i>Aeromonas</i> clinical isolates using <i>GCAT</i>, 16S rDNA-RFLP and <i>rpoD</i> genes.

<p>Identification and speciation of 98 <i>Aeromonas</i> clinical isolates using <i>GCAT</i>, 16S rDNA-RFLP and <i>rpoD</i> genes.</p

Source and distribution of 94 clinical isolates of <i>Aeromonas</i> species.

a<p>Pus/Pus swab from wounds , hand injury, cellulitis, abscess and unknown source.</p>b<p>Two <i>A. aquariorum</i> from tracheal secretion and urine; one <i>A. veronii</i> from biliary tract secretion.</p

Phylogenetic relationship of the <i>rpoD</i> sequences between 94 <i>Aeromonas</i> isolates and 9 references strains using neighbor-joining method.

<p>Numbers next to nodes indicate percentage bootstrap values of 5000 replicates.</p