VIRULENCE GENE PROFILING AND PATHOGENICITY OF Streptococcus agalactiae ISOLATED FROM TILAPIA, Oreochromis niloticus FARMS IN INDONESIA

Streptococcosis caused by Streptococcus agalactiae has become a major disease problem in tilapia culture in Indonesia. This study aimed to detect virulence genes of S. agalactiae during streptococcosis disease outbreaks in several tilapia farms in Indonesia and evaluate the correlation between biotype and virulence genes to bacterial virulence. The presence of virulence genes was determined in 10 strains of S. agalactiae isolated from farm-raised tilapia. Polymerase chain reaction (PCR) protocol was used to determine genes for cylE, hylB, lmb, bib A, PI-2b, fbs A, fbs B, gap, PI-1, and cfb in the template DNA. Pathogenicity test was carried out by intraperitoneal injection of 24 hour-cultured S. agalactiae to tilapia with 108 CFU/fish. Four isolates have seven of virulence genes (cylE, hylB, bibA, PI-2b, fbs A, fbs B, and gap genes), three isolates have six virulence genes (hylB, bib A, fbs A, fbs B, gap, cfb genes), one isolate has four virulence gene (hyl B, bib A, fbs, and cfb genes), and one isolate has one virulence gene (PI-2b gene). None of the isolates has lmb or PI-1 genes. Bacteria with more virulence genes showed higher pathogenicity post injection. Mortality of tilapia injected with b-hemolytic bacteria was 100% within the period of 14-19 hours, while non-hemolytic bacteria was 53.3%-86.6% on 14 days post-injection. Pathological changes associated with the infection by either isolate included melanosis, slow response, anorexia, ocular opacity, gasping, erratic, C-shape, and whirling. It can be concluded that S. agalactiae with more virulence genes show a higher level of pathogenicity. The presence of a virulent gene has the potential to be used as a basis for selecting candidate isolates and designing vaccine compositions as an effort to prevent streptococcosis infection in tilapia in Indonesia.

Prevalence of Methicillin-Resistant Staphylococcus Species Among Filarial Lymphedema Patients in Ahanta West District of Ghana

BackgroundFilarial pathologies such as lymphedema may be associated with complications such as chronic non-healing wounds. Nonetheless, the role of bacterial population colonizing the lymphedematous legs has been posited to worsen the conditions of those living with the infection. These bacteria are usually composed of staphylococcal species partly because they are commensals. Thus, this present study sought to type the methicillin-resistant Staphylococcus aureus (MRSA) prevalence among individuals presenting with filarial lymphedema, particularly as MRSA tends to affect treatments options.MethodsWe recruited individuals (n = 321) with stages I–VII of lymphedema in a cross-sectional study in the Ahanta West district of the Western Region of Ghana. Swabs from lymphedematous limb ulcers, pus, and cutaneous surfaces were cultured using standard culture-based techniques. The culture isolates were later identified using Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry.ResultsA total of 192 Staphylococci species were isolated, with an overall prevalence of 39.7% (95% CI: 35%–44%; N = 483). S. hominis was the most prevalent species (23.95%), followed by S. haemolyticus (20.83%), S. epidermidis (15.10%), S. aureus (10.41%), and S. saprophyticus (9.32%). The remaining 20.34% were distributed among S. wanneri, S. sciuri, S. pasteuri, S. xylosus, S. simulans, S. cohnii, S. caprae, S. lugdunensis, and S. capitis. MRSA, containing mecA gene, was detected in 21 out of 31 Staphylococci isolates tested, with an overall prevalence of 68% (95% CI: 51%–84%). In addition, a virulent gene, Panton–Valentine leukocidin (PVL), which is usually associated with S. aureus, was detected in 20/31 (64.5%) S. aureus in the study.ConclusionThese results suggest that MRSA species may pose a challenge to the treatment of filarial lymphedema with antibiotics particularly, as doxycycline is currently being piloted in some endemic areas to treat the infection. Thus, intensive antimicrobial resistance surveillance should be conducted in endemic areas by health authorities to forestall the dilemma of multidrug resistance not only against lymphatic filariasis (LF) infection but other diseases.

Microbiological Quality Assessment and Identification of Antibiotic Resistant Bacteria at Different Stages of the Milk Supply Chain in Dhaka City of Bangladesh

Aims: Milk works as an excellent medium for bacterial growth and can turn into a fatal source of food borne diseases when consumed without pasteurization. This study was carried out to examine the microbiological quality of milk from three different points of milk supply chain to investigate whether the dairy stakeholders are maintaining the consumer safety or not. Study Design: A cross sectional study Place and Duration: The study took place at the Food Microbiology lab of Institute of Nutrition and Food Science, University of Dhaka from November 2019 to February 2020. Methodology: A total of 60 samples were studied including raw milk from collection centers, unpackaged pasteurized milk from processing plants and packaged pasteurized milks from retail shops. After carrying out the microbiological analysis the samples were examined for determining the total bacterial count (TBC) and total coliform count (TCC). Antibiotic susceptibility test was done using disk diffusion assay and detection of virulent gene in Salmonella spp. was done by Polymerase Chain Reaction (PCR) using specific invA primer. Results: The results revealed that all raw milk samples were substandard in terms of TBC and TCC and pasteurized milks from processing plants maintained the standard quality. Importantly, packaged pasteurized milk samples from retail shops had high TBC (>4.0× 104 CFU/mL) and TCC (1.2×103 CFU/mL) containing Pseudomonas, Micrococcus, Streptococcus, Salmonella, Proteus, Staphylococcus, Bacillus and E. coli. Bacteria like Salmonella (75%), Proteus (62.5%) and Vibrio (62.5%) possessed high Multiple Antibiotic Resistance (MAR) index and showed resistance towards antibiotics namely Ampicillin, Amoxicillin, Erythromycin and Colistin. Through further molecular analysis we detected invA virulent gene one of the Salmonella isolates which was collected from the pasteurized milk samples of the retail shops. Conclusion: High bacterial load in raw milk and packaged pasteurized milk indicate that the milk we consume is substandard in microbiological quality. Precautionary measurements and careful processing of milk may reduce the prevalence of microbiological contamination in the milk supply chain.

Glucose 6-phosphate transport regulatory protein UhpA regulates the virulent genes in Edwardsiella piscicida

Edwardsiella piscicida (E. piscicida) is an important zoonotic pathogen, which infects animals by colonizing the intestine. Glucose 6-phosphate (Glu6P) was an important carbohydrate in intestine and could be used as a regulate signal. Here we identify a virulence-regulating pathway named Glu6P transport regulatory protein UhpA, which affects the virulent genes of hemolysins, flagellar, T3SS, T6SS and metabolism related genes how to promote E. piscicida infect the host. The results showed that the metabolism related gene expression of cysteine synthase (orf 1134) and sulfate transporter (ychM) in the uhpA mutant strain ΔuhpA was 0.76-fold and 0.68-fold lower than the ones in the wild strains (P < 0.05), the gene expression of ethA and ethB in the ΔuhpA strain was 0.80-fold and 0.72-fold lower than the ones in the wild strains (P < 0.05). However, the gene expression of fliC and flgN in the ΔuhpA was 1.51-fold and 1.21-fold higher than the ones in the wild strains (P < 0.05), the gene expression of T3SS (esrB and esrC) and T6SS (evpB and evpC) in the ΔuhpA was 1.27-fold, 1.13-fold 1.28-fold and 1.23-fold higher than the ones in the wild strains (P < 0.05). Besides, the survival rate of fish challenged with E. piscicida EIB202 and ΔuhpA was 50% and 30% respectively. These suggested that although the uhpA gene deletion decreased the metabolic level and the hemolysins related gene expression in E. piscicida, the uhpA gene could down regulate the key virulent gene expression to decrease the pathogenicity of E. piscicida in fish.

DIFFERENCES IN GENOTYPE OF E. FAECALIS AND E. FAECIUM CLINICAL ISOLATES REGARDING CYTOLYSIN AND GELATINASE PRODUCTION IN BULGARIAN PATIENTS

Purpose - compare the phenotype and genotype correlation of cytolysin and gelatinase production in clinical isolates Enterococcus spp. Materials and methods - 100 Enterococcus strains collected over a period of one year from inpatients of two Bulgarian university hospitals, were tested for phenotype production of cytolysin and gelatinase. Multiplex PCR was performed to screen the presence of gelE and cylA virulence genes. Results – 17% of the enterococcal isolates demonstrated only cytolysin production phenotypically. Gelatinase activity was found in 21% of the isolates. Only E. faecalis showed combined phenotypic production of cytolysin plus gelatinase (21%). Forty-five percent of the tested enterococci were identified negative for both hemolysin and gelatinase activity. GelE was the most prevalent virulent gene (48% of the isolates). CylA gene was present alone only in four non-invasive E. faecalis isolates. Twenty-six percent of the isolates possessed both cylA and gelE genes and 21% did not harbor any of the virulence factors genotypically. Conclusion - our results prove that it is appropriate to perform both phenotypic and genotypic analysis of the enterococci virulence profile in parallel in order to better characterize the strains, which in turn may serve to develop more effective methods to limit the spread of infections caused by these microorganisms.