Phenotypic and molecular characterization of β-lactamase and plasmid-mediated quinolone resistance genes in Klebsiella oxytoca isolated from slaughtered pigs in Thailand

Research (Published online: 13-02-2022)

10. Phenotypic and molecular characterization of β-lactamase and plasmid-mediated quinolone resistance genes in Klebsiella oxytoca isolated from slaughtered pigs in Thailand

Nattamol Phetburom, Parichart Boueroy, Peechanika Chopjitt, Rujirat Hatrongjit, Suphachai Nuanualsuwan and Anusak Kerdsin

Veterinary World, 15(2): 309-315

ABSTRACT

Background and Aim: Over recent years, antimicrobial-resistant Klebsiella species in humans, animals, food animals, food products, and agricultural environments have been the center of attention due to its role in the evolution of antimicrobial resistance. The emergence of resistance to fluoroquinolones and cephalosporins of third and higher generations in Klebsiella oxytoca has not received much attention in animal husbandry compared to that in Klebsiella pneumoniae. Reports on K. oxytoca are limited in the study area. Therefore, we investigated the antimicrobial susceptibility and resistance genes in K. oxytoca isolated from slaughtered pigs in Thailand.

Materials and Methods: Microbiological examination was conducted on 384 Klebsiella spp. isolates recovered from slaughtered pigs in ten provinces of Thailand. Seventy-two K. oxytoca isolates (18.75%) were examined for antimicrobial-resistant genes (β-lactamase [blaTEM, blaCTX-M, and blaSHV]) and fluoroquinolone-resistant genes (qnrA, qnrB, qnrC, qnrD, qnrS, oqxAB, aac(6')-Ib-cr, and qepA).

Results: The most common genotype was blaCTX-M (58/72, 80.55%), followed by blaTEM with blaCTX-M (7/72, 9.72%) and blaTEM (6/72, 8.33%). The most common blaCTX-M group was blaCTX-M-1 (19/58, 32.76%), followed by blaCTX-M-9 (1/58, 1.72%). Plasmid-mediated quinolone resistance genes were identified in 13 (18.05%) isolates: qnrS (16.70%) and qnrB (1.4%). All 13 isolates had qnrS transferable to an Escherichia coli recipient, whereas qnrB was not detected in any transconjugants. Either blaCTX-M or blaTEM harbored by one K. oxytoca strain was transferable to an E. coli recipient. Analysis of antimicrobial susceptibility revealed that more than 90% of the blaCTX-M-carrying K. oxytoca isolates were susceptible to chloramphenicol, trimethoprim, ceftazidime, cefepime, cefotaxime, amoxicillin-clavulanic acid, piperacillin–tazobactam, and fosfomycin. All K. oxytoca isolates (13) harboring qnr were susceptible to carbapenem and ceftriaxone; however, 43 (74.13%) of the K. oxytoca isolates harboring blaCTX-M exhibited extended-spectrum β-lactamase activity. Most of the K. oxytoca isolates from pigs were highly resistant to ampicillin, azithromycin, and gentamicin.

Conclusion: To prevent further transmission of Klebsiella spp. between food animals and humans, strict control of antibiotic use in clinical and livestock settings is necessary along with routine disinfection of the livestock environment and efforts to increase awareness of antimicrobial resistance transmission.

Keywords: Klebsiella oxytoca, plasmid-mediated quinolone resistance, slaughtered pigs, Thailand, β-lactamase.

Lacticaseibacillus spp.; Probiotic candidates from Palmyra palm sugar, possess antimicrobial, and anti-biofilm activities against methicillin-resistant Staphylococcus aureus

Research (Published online: 12-02-2022)

9. Lacticaseibacillus spp.; Probiotic candidates from Palmyra palm sugar, possess antimicrobial, and anti-biofilm activities against methicillin-resistant Staphylococcus aureus

Watcharapong Mitsuwan, Phoomjai Sornsenee and Chonticha Romyasamit

Veterinary World, 15(2): 299-308

ABSTRACT

Background and Aim: Probiotics are beneficial microorganisms that play important roles by adhering to the gut and producing antimicrobial substances to inhibit pathogens. The objective of this study was to isolate and characterize the probiotic lactic acid bacteria (LAB) from Palmyra palm sugar, which can produce antimicrobial compounds against methicillin-resistant Staphylococcus aureus (MRSA), a new zoonotic and food-borne pathogens.

Materials and Methods: Twenty-six LAB isolates were isolated from 30 Palmyra palm sugar samples. Three selected LAB were further characterized as probiotics. In addition, the antibacterial and anti-biofilm-forming activities of the probiotics' culture supernatants against MRSA and food-borne pathogens were investigated. Finally, the selected probiotics were identified by aligning 16S rRNA sequences.

Results: The three confirmed probiotics, WU 0904, WU 2302, and WU 2503, showed strong antibacterial activities against S. aureus, MRSA, Escherichia coli O157:H7, and Listeria monocytogenes, as measured by a broth microdilution assay. Among the LAB isolates, 82.22-86.58%, 91.83-96.06%, and 64.35-74.93% exhibited resistance to low pH, pancreatin treatment, and bile salts, respectively. It was found that 59.46% and 83.33% auto-aggregation was observed in 2 and 24 h, respectively. Moreover, 50.25-57.24% adhesion was detected after the incubation of the bacterial cells to Caco-2 cells. . Biofilm inhibition (82.81-87.24%) was detected after the treatment of MRSA with the culture supernatants, when compared with that to the control. By the alignment of 16S rRNA sequences, the isolate WU 2302 was identified as Lacticaseibacillus spp. with 98.82% homology when compared to the GenBank database.

Conclusion: This study indicates that isolated probiotics can produce antimicrobial compounds against MRSA and food-borne pathogens. The obtained results strongly suggest that these probiotics are promising candidates for pharmaceutical products.

Keywords: antibacterial activity, Lacticaseibacillus spp., methicillin-resistant Staphylococcus aureus, Palmyra palm sugar, probiotics.

Immunosuppression by piperine as a regulator of the NLRP3 inflammasome through MAPK/NF-κB in monosodium urate-induced rat gouty arthritis

Research (Published online: 11-02-2022)

8. Immunosuppression by piperine as a regulator of the NLRP3 inflammasome through MAPK/NF-κB in monosodium urate-induced rat gouty arthritis

Galih Aji Kuncoro Jati, Nazzun Assihhah, Anas Ardiana Wati and Siti Isrina Oktavia Salasia

Veterinary World, 15(2): 288-298

ABSTRACT

Background and Aim: Gouty arthritis is a metabolic disorder involving monosodium urate (MSU) crystal deposition as a key initiator of acute inflammation. Dysregulation of the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome is associated with the pathogenesis of gout through the maturation of interleukin-1β. Piperine (PIP) is a phytochemical with an anti-inflammatory activity that has the potential as an alternative treatment for gout. In this study, we examined the effect of PIP in immunosuppression of gout inflammation through the regulation of the NLRP3 inflammasome.

Materials and Methods: An in silico study was done by pharmacodynamic modeling of PIP in suppressing MSU-induced inflammation through disruption of the NLRP3 inflammasome. In vivo tests, including inflammatory assessment, histopathology, cytology, estimation of lipid peroxidation index, and detection of systemic inflammatory reactants, were performed on two groups using preventive and curative protocols.

Results: In silico studies of molecular docking demonstrated the activity of PIP as a competitive inhibitor of the mitogen-activated protein kinases/nuclear factor-kappaB axis, upstream of the NLRP3 inflammasome. Analysis of gout models with curative and preventive protocols revealed the immunosuppression activity of PIP by reducing inflammatory symptoms, inhibiting tophus formation resulting from NETosis, reducing cartilage erosion, inhibiting leukocyte exudation, suppressing lipid peroxidation index, and inhibiting the production of C-reactive protein.

Conclusion: The results demonstrate the activity of PIP as an immunosuppressant in gout flare. These findings indicate the potential of PIP as a candidate for prophylactic and therapeutic agent for the treatment of gouty arthritis.

Keywords: gout, immunosuppression, leucine-rich repeat, monosodium urate, nucleotide-binding oligomerization domain, piperine, pyrin domain-containing protein 3 inflammasome, piperine.

Shigella flexneri vaccine development: Oral administration of peptides derived from the 49.8 kDa pili protein subunit activates the intestinal immune response in mice

Research (Published online: 11-02-2022)

7. Shigella flexneri vaccine development: Oral administration of peptides derived from the 49.8 kDa pili protein subunit activates the intestinal immune response in mice

Khoirul Anam, Agustina Tri Endharti, Sri Poeranto, Hidayat Sujuti, Dwi Yuni Nur Hidayati and Sumarno Reto Prawiro

Veterinary World, 15(2): 281-287

ABSTRACT

Background and Aim: The morbidity and mortality of Shigella infections remain a global challenge. Epitope-based vaccine development is an emerging strategy to prevent bacterial invasion. This study aimed to identify the ability of the 49.8 kDa pili subunit adhesin protein epitope of Shigella flexneri to induce an intestinal immune response in mice.

Materials and Methods: Thirty adult male Balb/c mice were divided into a control group, cholera toxin B subunit (CTB) group, CTB+QSSTGTNSQSDLDS (pep_1) group, CTB+DTTITKAETKTVTKNQVVDTPVTTDAAK (pep_2) group, and CTB+ ATLGATLNRLDFNVNNK (pep_3). We performed immunization by orally administering 50 μg of antigen and 50 μl of adjuvant once a week over 4 weeks. We assessed the cellular immune response by quantifying T helper 2 (Th2) and Th17 using flow cytometry. In addition, we assessed the humoral immune response by quantifying interleukin (IL-4), IL-17, secretory immunoglobulin A (sIgA), and β-defensin using enzyme-linked immunoassay. Statistical analysis was performed using one-way analysis of variance and Kruskal–Wallis test.

Results: Peptide oral immunization increases the cellular immune response as reflected by the increase of Th2 (p=0.019) and Th17 (p=0.004) cell counts, particularly in the CTB_pep_1 group. Humoral immune response activation was demonstrated by increased IL-4 levels, especially in the CTB+pep_3 group (p=0.000). The IL-17 level was increased significantly in the CTB+pep_1 group (p=0.042). The mucosal immune response was demonstrated by the sIgA levels increase in the CTB+pep_3 group (p=0.042) and the β-defensin protein levels (p=0.000).

Conclusion: All selected peptides activated the cellular and humoral immune responses in the intestine of mice. Further studies are necessary to optimize antigen delivery and evaluate whether the neutralizing properties of these peptides allow them to prevent bacterial infection.

Keywords: epitope, immune response, oral, Shigella flexneri, shigellosis.

Phagocytosis and intracellular killing of Pasteurella multocida B:2 by macrophages: A comparative study between buffalo and cattle

Research (Published online: 10-02-2022)

6. Phagocytosis and intracellular killing of Pasteurella multocida B:2 by macrophages: A comparative study between buffalo and cattle

Qistina Hasnan, Yulianna Puspitasari, Sarah Othman, Mohd Zamri-Saad and Annas Salleh

Veterinary World, 15(2): 275-280

ABSTRACT

Background and Aim: Pasteurella multocida B:2 is the causative agent of hemorrhagic septicemia (HS) in buffalo and cattle. Buffaloes are known to be more susceptible to HS than cattle, but the reason for this remains unknown. This study aimed to compare the in vitro efficiency with which buffalo and cattle macrophages can kill P. multocida B:2.

Materials and Methods: Monocyte-derived macrophages of buffalo and cattle were used in this study. They were exposed to 1×106 colony-forming unit/mL of live P. multocida B:2 before the cells were harvested at 0, 30, 60, and 120 min post-exposure and viewed under a fluorescence microscope to count viable and non-viable macrophages and the macrophages with phagocytosing P. multocida B:2 cells. The phagocytosis, intracellular bacterial killing, and macrophage death rates were calculated and compared between the two species and sampling points.

Results: In general, the rates of phagocytosis, intracellular killing, and macrophage death increased with time of exposure for both animal species. No significant (p>0.05) differences were noted between the phagocytosis rates by the macrophages of buffalo and cattle throughout the experiment. However, the rates of intracellular killing were significantly (p<0.05) higher in cattle macrophages at 30 min and 120 min post-exposure than those of buffalo. The death rates of buffalo macrophages were significantly (p<0.05) higher than those of cattle at 60 min and 120 min post-exposure.

Conclusion: With higher bacteria killing ability and lower macrophage death, cattle appeared to be more efficient at handling P. multocida B:2 infection than buffalo.

Keywords: buffalo, cattle, in vitro efficiency, macrophages, Pasteurella multocida B:2.

Validation method for determining enrofloxacin and tylosin levels in broiler liver, kidney, and muscle using high-performance liquid chromatography

Research (Published online: 08-02-2022)

5. Validation method for determining enrofloxacin and tylosin levels in broiler liver, kidney, and muscle using high-performance liquid chromatography

Agustina Dwi Wijayanti, Rahmad Dwi Ardiansyah, Anggi Muhtar Pratama, Aris Haryanto and Ida Fitriana

Veterinary World, 15(2): 268-274

ABSTRACT

Background and Aim: Enrofloxacin and tylosin can be combined into an antibiotic formulation which is expected to have a broader range of antibacterial activity against various infections in broilers. Validation method analysis of the levels of these two active compounds needs to be done for future use in pharmacokinetic or residual studies. The present study aims to determine a suitable validation method of isocratic high-performance liquid chromatography (HPLC) to measure the concentration of antibiotic combinations in the broiler liver, kidney, and muscles.

Materials and Methods: The combination of enrofloxacin and tylosin in the liver, kidney, and muscle was validated by HPLC method to find the procedures, processes, equipment, and systems used, consistently provides the appropriate results. The chromatography system consisted of an Octadecyl-silica column of 5 μm in diameter and 150 mm in length with a mobile phase of a mixture of 0.05 M monobasic sodium phosphate (pH 2.5) and acetonitrile (65:35 v/v). The solution was detected at a wavelength of 280 nm, 30°C, a flow rate of 1 mL/min, and an injection volume of 20 μL. The combination antibiotics powder was produced from PT Tekad Mandiri Citra, Bandung, Indonesia, and broiler tissues obtained from day-old chick broilers maintained for 30 days with free antibiotic feed.

Results: Validation of a combination solution of enrofloxacin and tylosin shows the linearity values of enrofloxacin and tylosin in the liver, kidney, and muscles as r2=0.9988, r2=0.9999, r2=0.9997, r2=0.9989, r2=0.9978, and r2=0.9962. The accuracy and precision values of enrofloxacin in the liver, kidney, and muscles were 5.53, 6.23, and 6.93, respectively. The values of accuracy and precision of tylosin in the liver, kidney, and muscles were 10.43, 4.63, and 7.16%, respectively. The retention times for enrofloxacin and tylosin were 1.945-2.000 min and 4.175-4.342 min. The limit of detection (LOD) and limit of quantity (LOQ) values for enrofloxacin were 3.03 and 10.1 μg/g, respectively. In contrast, the LOD and LOQ values for tylosin were 9.05 and 30.17 μg/g, respectively.

Conclusion: The value of linearity, accuracy, precision, specificity, and sensitivity of the combined solution of enrofloxacin and tylosin showed promising results.

Keywords: enrofloxacin-tylosin combination, high-performance liquid chromatography, validation method.

The first study on seroprevalence and risk factors for zoonotic transmission of ovine and caprine brucellosis in the Province of Bam, Burkina Faso

Research (Published online: 05-02-2022)

4. The first study on seroprevalence and risk factors for zoonotic transmission of ovine and caprine brucellosis in the Province of Bam, Burkina Faso

Dieudonné Tialla

Veterinary World, 15(2): 262-267

ABSTRACT

Background and Aim: Brucellosis is a bacterial disease notorious for its ability to infect a wide range of domestic and wildlife animals, as well as humans. This study aimed to determine the seroprevalence of ovine and caprine brucellosis and the associated risk factors in the Province of Bam in Burkina Faso.

Materials and Methods: The individual serological status of 300 unvaccinated sheep and 300 unvaccinated goats was determined by Rose Bengal and indirect enzyme-linked immunosorbent assay (iELISA) serological tests used in parallel. The frequency of behaviors conferring risk of developing this zoonotic disease was determined through two epidemiological questionnaires, which identified known risk factors for the transmission of brucellosis between animals and humans.

Results: Individual seroprevalence was estimated at 6.0% (18/300) in sheep and 4.3% (13/300) in goats. The "herd" prevalence of brucellosis was estimated at 60% in sheep while 40% in goats. Positivity in the iELISA serological test was significantly associated with age, sex, and husbandry system in sheep and goats.

Conclusion: These results indicate that Brucella melitensis circulates in sheep and goat farms in the Province of Bam in Burkina Faso. As B. melitensis is highly pathogenic to humans, adequate measures must be taken to protect the population against this zoonotic disease.

Keywords: brucellosis ovine and caprine, Burkina Faso, Province of Bam, seroprevalence, zoonosis.