Phenotypic and genotypic characterization of colistin-resistant Escherichia Coli with mcr-4, mcr-5, mcr-6, and mcr-9 genes from broiler chicken and farm environment.

BACKGROUND: Colistin is an antibiotic used as a last-resort to treat multidrug-resistant Gram-negative bacterial infections. Colistin had been used for a long time in veterinary medicine for disease control and as a growth promoter in food-producing animals. This excessive use of colistin in food animals causes an increase in colistin resistance. This study aimed to determine molecular characteristics of colistin-resistant Escherichia coli in broiler chicken and chicken farm environments. RESULTS: Four hundred fifty-three cloacal and farm environment samples were collected from six different commercial chicken farms in Kelantan, Malaysia. E. coli was isolated using standard bacteriological methods, and the isolates were tested for antimicrobial susceptibility using disc diffusion and colistin minimum inhibitory concentration (MIC) by broth microdilution. Multiplex PCR was used to detect mcr genes, and DNA sequencing was used to confirm the resistance genes. Virulence gene detection, phylogroup, and multilocus sequence typing (MLST) were done to further characterize the E. coli isolates. Out of the 425 (94%; 425/453) E. coli isolated from the chicken and farm environment samples, 10.8% (48/425) isolates were carrying one or more colistin-resistance encoding genes. Of the 48 colistin-resistant isolates, 54.2% (26/48) of the mcr positive isolates were genotypically and phenotypically resistant to colistin with MIC of colistin ≥ 4 µg/ml. The most prominent mcr gene detected was mcr-1 (47.9%; 23/48), followed by mcr-8 (18.8%; 9/48), mcr-7 (14.5%; 7/48), mcr-6 (12.5%; 6/48), mcr-4 (2.1%; 1/48), mcr-5 (2.1%; 1/48), and mcr-9 (2.1%; 1/48) genes. One E. coli isolate originating from the fecal sample was found to harbor both mcr-4 and mcr-6 genes and another isolate from the drinking water sample was carrying mcr-1 and mcr-8 genes. The majority of the mcr positive isolates were categorized under phylogroup A followed by phylogroup B1. The most prevalent sequence typing (ST) was ST1771 (n = 4) followed by ST206 (n = 3). 100% of the mcr positive E. coli isolates were multidrug resistant. The most frequently detected virulence genes among mcr positive E. coli isolates were ast (38%; 18/48) followed by iss (23%; 11/48). This is the first research to report the prevalence of mcr-4, mcr-5, mcr-6, mcr-7, and mcr-8 genes in E. coli from broiler chickens and farm environments in Malaysia. CONCLUSION: Our findings suggest that broiler chickens and broiler farm environments could be reservoirs of colistin-resistant E. coli, posing a risk to public health and food safety.

A multicentre study to determine the in vitro efficacy of flomoxef against extended-spectrum beta-lactamase producing Escherichia coli in Malaysia.

Background: The high burden of extended-spectrum beta-lactamase-producing (ESBL)-producing Enterobacterales worldwide, especially in the densely populated South East Asia poses a significant threat to the global transmission of antibiotic resistance. Molecular surveillance of ESBL-producing pathogens in this region is vital for understanding the local epidemiology, informing treatment choices, and addressing the regional and global implications of antibiotic resistance. Methods: Therefore, an inventory surveillance of the ESBL-Escherichia coli (ESBL-EC) isolates responsible for infections in Malaysian hospitals was conducted. Additionally, the in vitro efficacy of flomoxef and other established antibiotics against ESBL-EC was evaluated. Results: A total of 127 non-repetitive ESBL-EC strains isolated from clinical samples were collected during a multicentre study performed in five representative Malaysian hospitals. Of all the isolates, 33.9% were isolated from surgical site infections and 85.8% were hospital-acquired infections. High rates of resistance to cefotaxime (100%), cefepime (100%), aztreonam (100%) and trimethoprim-sulfamethoxazole (100%) were observed based on the broth microdilution test. Carbapenems remained the most effective antibiotics against the ESBL-EC, followed by flomoxef. Antibiotic resistance genes were identified by PCR. The blaCTX-M-1 was the most prevalent ESBL gene, with 28 isolates (22%) harbouring blaCTX-M-1 only, 27 isolates (21.3%) co-harbouring blaCTX-M-1 and blaTEM, and ten isolates (7.9%) co-harbouring blaCTX-M-1, blaTEM and blaSHV. A generalised linear model showed significant antibacterial activity of imipenem against different types of infection. Besides carbapenems, this study also demonstrated a satisfactory antibacterial activity of flomoxef (81.9%) on ESBL-EC, regardless of the types of ESBL genes.

Potential Usefulness of Bacteriophages for the Treatment of Multidrug-Resistant Acinetobacter Infection.

Bacteriophages were discovered in early 20th century. However, the interest in bacteriophage research was reduced with the discovery of antibiotics. With the increasing number of infections due to multidrug-resistant (MDR) organisms, the potential usefulness of bacteriophages as therapeutic agents has been re-evaluated. In this review, we found that more than 30 lytic bacteriophages that infect Acinetobacter species have been characterised. These are mainly members of Caudovirales, with genome sizes ranging from 31 kb to 234 kb and G+C contents ranging from 33.5% to 45.5%. The host range can be as low as < 10% of all tested Acinetobacter strains. Fourteen published murine trials indicated positive outcomes in bacteriophage-treated groups. Only two case reports were pertaining to the use of bacteriophages in the treatment of Acinetobacter infections in humans; in both cases, the infections were resolved with bacteriophage therapy. The use of bacteriophages has been associated with reduced Acinetobacter burden in the environment, as shown in two studies. The major limitation of bacteriophage therapy is its highly selective host strain. In conclusion, the potential usefulness of bacteriophage therapy for the treatment of MDR Acinetobacter species has been documented only in limited studies and more research is needed prior to its extensive use in clinical practice.

Molecular detection and antimicrobial resistance profiles of Extended-Spectrum Beta-Lactamase (ESBL) producing Escherichia coli in broiler chicken farms in Malaysia.

Antimicrobial resistance is one of the major public health threats globally. This challenge has been aggravated with the overuse and misuse of antibiotics in food animals and humans. The present study aimed to investigate the prevalence of Extended-Spectrum ß-lactamase (ESBL) genes in Escherichia coli (E. coli) isolated from broiler chickens in Kelantan, Malaysia. A total of 320 cloacal swabs were collected from farms in different districts of Kelantan and were analyzed using routine bacteriology, antimicrobial susceptibility test, and molecular techniques for further identification and characterization of ESBL encoding genes. Based on PCR detection for the E. coli species-specific Pho gene, 30.3% (97/320) of isolates were confirmed as E. coli, and 84.5% (82/97) of the isolates were positive for at least one ESBL gene. Majority of the isolates, 62.9% (61/97) were harboring blaCTX-M followed by 45.4% (44/97) of blaTEM genes, while 16.5% (16/97) of the isolates were positive for both mcr-1 and ESBL genes. Overall, 93.8% (90/97) of the E. coli were resistant to three or more antimicrobials; indicating that the isolates were multi-drug resistance. 90.7% of multiple antibiotic resistance (MAR) index value greater than 0.2, would also suggest the isolates were from high-risk sources of contamination. The MLST result shows that the isolates are widely diverse. Our findings provide insight into the alarmingly high distribution of antimicrobial resistant bacteria, mainly ESBL producing E. coli in apparently healthy chickens indicating the role of food animals in the emergence and spread of antimicrobial resistance, and the potential public health threats it may pose.

A Nanoplex PCR Assay for the Simultaneous Detection of Vancomycin- and Linezolid-Resistant Genes in Enterococcus.

BACKGROUND: Enterococci are Gram-positive cocci found in the guts of humans and animals. The goal of this research is to develop a multiplex PCR assay that can detect the Enterococcus genus, four VRE genes, and three LZRE genes simultaneously. METHODS: Primers used in this study were specifically designed for the detection of 16S rRNA of Enterococcus genus, vanA-vanB-vanC-vanD for vancomycin, cfr methyltransferase, and optrA, and poxtA, as well as an adenosine triphosphate-binding cassette (ABC) transporter for linezolid. A Vibrio cholerae ctxA (internal amplification control) was included. Optimization of primer concentrations and PCR components was also done. This was followed by evaluating the sensitivity and specificity of the optimized multiplex PCR. RESULTS: Final Primer concentrations were optimized as follows: 16S rRNA is 1.0 pmol/µL, vanA is 1.0 pmol/µL, optrA is 1.0 pmol/µL, cfr is 1.0 pmol/µL, poxtA is 0.1 pmol/µL, vanB is 0.08 pmol/µL, ctxA is 0.07 pmol/µL, vanC is 0.8 pmol/µL, and vanD is 0.1 pmol/µL. Further, the optimized concentrations for MgCl2, dNTPs and Taq DNA polymerase were 2.5 mM, 0.16 mM, and 0.75 units respectively, and an annealing temperature of 64.5 °C. CONCLUSIONS: The developed multiplex PCR is sensitive and species-specific. The development of a multiplex PCR assay that will take into account all known VRE genes and linezolid mutation is highly recommended.

Burden and Risk Factors of Melioidosis in Southeast Asia: A Scoping Review.

This scoping review aims to provide a comprehensive overview of human melioidosis in Southeast Asia as well as to highlight knowledge gaps in the prevalence and risk factors of this life-threatening disease using available evidence-based data for better diagnosis and treatment. Preferred Reporting Items for Systematic Review and Meta-Analyses Extension for Scoping Reviews (PRISMA-ScR) was used as the guideline for this review. The literature search was conducted on 23 March 2022 through two electronic databases (PubMed and Scopus) using lists of keywords referring to the Medical Subject Headings (MeSH) thesaurus. A total of 38 articles related to human melioidosis were included from 645 screened articles. These studies were carried out between 1986 and 2019 in six Southeast Asian countries: Thailand, Cambodia, Malaysia, Myanmar, Singapore, and Vietnam. Melioidosis has been reported with a high disease prevalence among high-risk populations. Studies in Thailand (48.0%) and Cambodia (74.4%) revealed disease prevalence in patients with septic arthritis and children with suppurative parotitis, respectively. Other studies in Thailand (63.5%) and Malaysia (54.4% and 65.7%) showed a high seroprevalence of melioidosis among Tsunami survivors and military personnel, respectively. Additionally, this review documented soil and water exposure, diabetes mellitus, chronic renal failure, thalassemia, and children under the age of 15 as the main risk factors for melioidosis. Human melioidosis is currently under-reported in Southeast Asia and its true prevalence is unknown.

Draft Genome Sequence of Clinical Isolate USM039K of the Pathogenic Yeast Candida parapsilosis.

Here, we announce the draft genome sequence of a Candida parapsilosis clinical isolate (USM039K) recovered from a patient with catheter-related bloodstream infection (CRBSI). The genome size is 12,860,016 bp long, with 188 scaffolds, a G+C content of 38.65%, and 5,467 genes.

Draft Genome Sequence of Clinical Isolate USM026 of the Pathogenic Yeast Candida parapsilosis.

Here, we report the draft genome sequence of a Candida parapsilosis clinical isolate (USM026) that was recovered from a blood sample from a patient who was treated for a catheter-related bloodstream infection (CRBSI). The draft genome is 12,839,916 bp in length, with 22,076,712 reads, 249 scaffolds, and 5,537 genes.

Distribution of sasX, qacA/B and mupA genes and determination of genetic relatedness of methicillin-resistant Staphylococcus aureus among clinical isolates and nasal swab samples from the same patients in a hospital in Malaysia.

INTRODUCTION: This study determined the distribution of sasX, qacA/B and mupA genes from methicillin-resistant Staphylococcus aureus (MRSA) isolated from clinical samples and nasal swab samples of the same patients and analysed their genetic relatedness. METHODS: Polymerase chain reaction was used to detect the presence of sasX, qacA/B and mupA genes from 47 paired MRSA isolates. A paired isolate was defined as one nasal swab (colonising) isolate and clinical isolate that caused infection in the same patient. 22 selected paired isolates were subjected to multilocus sequence typing (MLST). The genetic relatedness among the isolates and association between the putative genes with epidemic sequence types (STs) were investigated. RESULTS: 7 (14.9%, n = 14) paired isolates were positive for the sasX gene. qacA/B genes were positive in 7.4% (n = 7) of the isolates, from three paired isolates and one clinical isolate whose paired colonising isolate was negative. The paired sample of three patients were positive for both genes. The mupA gene was not detected in all the isolates. MLST revealed two epidemic STs, ST22 and ST239, and a novel ST4649. sasX and qacA/B genes were found in ST239 in 29.5% (n = 13) and 13.6% (n = 6) of cases, respectively. Gene co-existence occurred in 13.6% (n = 6) of MRSA ST239 and 2.3% (n = 1) of MRSA ST4649. CONCLUSION: sasX and qacA/B genes were present in the MRSA isolates, while the mupA gene was undetected. ST22 and ST239 were the major MRSA clones. The circulating MRSA genotypes conferred different virulence and resistance determinants in our healthcare settings.

Application of Baculovirus Expression Vector system (BEV) for COVID-19 diagnostics and therapeutics: a review.

BACKGROUND: The baculovirus expression vector system has been developed for expressing a wide range of proteins, including enzymes, glycoproteins, recombinant viruses, and vaccines. The availability of the SARS-CoV-2 genome sequence has enabled the synthesis of SARS-CoV2 proteins in a baculovirus-insect cell platform for various applications. The most cloned SARS-CoV-2 protein is the spike protein, which plays a critical role in SARS-CoV-2 infection. It is available in its whole length or as subunits like S1 or the receptor-binding domain (RBD). Non-structural proteins (Nsps), another recombinant SARS-CoV-2 protein generated by the baculovirus expression vector system (BEV), are used in the identification of new medications or the repurposing of existing therapies for the treatment of COVID-19. Non-SARS-CoV-2 proteins generated by BEV for SARS-CoV-2 diagnosis or treatment include moloney murine leukemia virus reverse transcriptase (MMLVRT), angiotensin converting enzyme 2 (ACE2), therapeutic proteins, and recombinant antibodies. The recombinant proteins were modified to boost the yield or to stabilize the protein. CONCLUSION: This review covers the wide application of the recombinant protein produced using the baculovirus expression technology for COVID-19 research. A lot of improvements have been made to produce functional proteins with high yields. However, there is still room for improvement and there are parts of this field of research that have not been investigated yet.

Molecular characterization of blaNDM, blaOXA-48, mcr-1 and blaTEM-52 positive and concurrently carbapenem and colistin resistant and extended spectrum beta-lactamase producing Escherichia coli in chicken in Malaysia.

BACKGROUND: Antimicrobial resistance (AMR) is a global public health threat and the use of antibiotics growth promoters in food animals has been implicated as a potential contributing factor in the emergence and spread of AMR. This study was conducted to investigate colistin and carbapenem resistance and extended spectrum beta-lactamase producing E. coli from live broiler chicken and chicken meat in Kelantan, Malaysia. RESULTS: Among the E. coli isolates, 37.5% (27/72 were positive for at least one of the resistance genes and one isolate was positive for mcr-1, blaTEM-52, blaNDM and blaOXA-48 whereas 4.17% (3/72) and 2.78% (2/72) were positive for mcr-1, blaTEM-52 and blaOXA-48, and mcr-1, blaTEM-52 and blaIMP. Multilocus sequence typing (MLST) results revealed the presence of widespread E. coli strains belonging to the sequence types ST410 and ST155 and other extra-intestinal E. coli (ExPEC) strains. Phylogroup A made up the majority 51.85% (14/27) followed by phylogroup B1 22.22% (6/27). CONCLUSIONS: The findings imply the potential threats of colistin, extended-spectrum beta-lactamase producing and carbapenem resistant E. coli in food animals to the public health and underscores the need for judicious use of antibiotics in animal production and good hygiene practices to curb the rising risks of AMR.

Development of Multiplex PCR Assay for Screening of T6SS-5 Gene Cluster: The Burkholderia pseudomallei Virulence Factor.

Despite the advanced understanding of the disease, melioidosis, an infection caused by Burkholderia pseudomallei, continues to be of global interest. The bacterial virulence factor, type six secretion system-5 (T6SS-5), in particular, is an essential factor for B. pseudomallei that is associated with internalization and intracellular survival of the pathogen. To detect the virulence gene cluster, this study has successfully developed a novel seven-gene (tssC-5, tagD-5, tssA-5, hcp-5, tssB-5, tssF-5, and vgrG-5) multiplex PCR assay. The optimum annealing temperature for this assay ranged between 59 and 62 °C. The limit of detection for this assay was 103 CFU/mL for all genes, excluding tssF-5, which was found at 105 CFU/mL of the bacterial concentration. In sensitivity and specificity tests, this multiplex assay was able to amplify all of the seven target genes from 93.8% (n = 33/35) clinical and 100% (n = 2/2) environmental isolates of B. pseudomallei. Whereas only four genes (tssC-5, tagD-5, tssF-5, and vgrG-5) were amplified from Bukholderia thailandesis, two genes (tagD-5 and tssB-5) were amplified from Bukholderia stagnalis, and zero target genes were amplified from Bukholderia ubonensis. No amplification of any genes was obtained when tested against isolated DNA from non-Bukholderia species (n = 20), which include Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, and others. In conclusion, this multiplex PCR assay is sensitive, species-specific, rapid, and reliable to detect the virulent gene cluster T6SS-5 of B. pseudomallei.

Prevalence of Vancomycin-Resistant Enterococcus (VRE) in Poultry in Malaysia: The First Meta-Analysis and Systematic Review.

Databases such as PubMed, Scopus and Google Scholar were searched. Data extraction and assessment of study protocol was done by two independent reviewers and the results were reviewed by a third. OpenMeta analyst and comprehensive meta-analysis (CMA) were used for the meta-analysis. The random effect model was used, publication bias and between-study heterogeneity was assessed. Seventeen studies were added to the final meta-analysis. Studies were sampled from 2000-2018 and of the 8684 isolates tested, 2824 were VRE. The pooled prevalence of VRE among poultry in Malaysia was estimated at 24.0% (95% CI; 16.7-33.1%; I2 = 98.14%; p < 0.001). Between-study variability was high (t2 = 0.788; heterogeneity I2 = 98.14% with heterogeneity chi-square (Q) = 858.379, degrees of freedom (df) = 16, and p < 0.001). The funnel plot showed bias which was confirmed by Egger's test and estimates from the leave-one-out forest plot did not affect the pooled prevalence. Pooled prevalence of VRE in chickens and ducks were 29.2% (CI = 18.8-42.5%) and 11.2%, CI = 9.0-14.0%) respectively. Enterococcus faecalis was reported most with more studies being reported in Peninsular Malaysia Central region and used antibiotic disc diffusion as detection method. Increased surveillance of VRE in poultry in Malaysia is required.

Vancomycin and Linezolid dosing in Obese and Overweight Patients: Is There a Universally Accepted Dosing Protocol to Improve their Efficacy?

@#Vancomycin is used to manage methicillin-resistant Staphylococcus aureus (MRSA) and other bacterial infections that are Gram-positive in nature. Linezolid belongs to the oxazolidinone class of antibiotics, which is primarily used to treat vancomycin-resistant Enterococcus (VRE), MRSA, diabetic foot, soft tissue, and skin infections. Here, we discuss vancomycin and linezolid dosing in obese patients, their mechanism of actions, pharmacokinetics, problems with dosing and evaluation of several dosing protocols in the obese patient population. There is no generally accepted dosing protocol for linezolid and vancomycin. Evidence suggests that using trough concentrations alone is insufficient for estimating vancomycin and linezolid exposure accurately as many researchers have revised protocol guidelines, developed more rigorous dosing and monitoring guidelines, or developed novel dosage strategies to meet the needs of overweight patients. Peaks and troughs measurement should be considered because it improves precision and reduces the area under the curve (AUC) estimate bias. To provide better dosing guidelines in this vulnerable group, obese patients must be included in all phases of drug design.

Phylogenetically Diverse Escherichia coli Strains from Chicken Coharbor Multiple Carbapenemase-Encoding Genes (bla NDM -bla OXA-blaIMP).

Carbapenem-resistant Enterobacteriaceae (CRE) has been a public health risk in several countries, and recent reports indicate the emergence of CRE in food animals. This study was conducted to investigate the occurrence, resistance patterns, and phylogenetic diversity of carbapenem-resistant E. coli (CREC) from chicken. Routine bacteriology, PCR detection of E. coli species, multiplex PCR to detect carbapenemase-encoding genes, and phylogeny of CRE E. coli were conducted. The results show that 24.36% (19/78) were identified as CREC based on the phenotypic identifications of which 17 were positive for the tested carbapenemases genes. The majority, 57.99% (11/19), of the isolates harbored multiple carbapenemase genes. Four isolates harbored all bla NDM, bla OXA, and bla IMP, and five and two different isolates harbored bla NDM and bla OXA and bla OXA and bla IMP, respectively. The meropenem, imipenem, and ertapenem MIC values for the isolates ranged from 2 µg/mL to ≥256 µg/mL. Phylogenetic grouping showed that the CREC isolates belonged to five different groups: groups A, B1, C, D, and unknown. The detection of CREC in this study shows that it has become an emerging problem in farm animals, particularly, in poultry farms. This also implies the potential public health risks posed by CRE from chicken to the consumers.

Current State of COVID-19 Pandemic in Africa: Lessons for Today and the Future.

This study is a cross-sectional, observational analysis of the COVID-19 pandemic in Africa, to understand the progression of the disease across the continent. Published data on COVID-19 from 20 January 2020 to 21 June 2021 were obtained and analyzed. Case fatality ratios, as well as case growth rates and other indices were computed. On 21 June 2021, a total of 178,210,532 confirmed cases and 3,865,978 deaths had been recorded worldwide. While the Americas recorded the highest number of cases, Southern Africa recorded the majority of African cases. Fatality rate since from 20 February 2020 to 21 June 2021 was highest in the Americas (2.63%) and low in the South Eastern Asia region (1.39%), globally increasing from 2.17% at the end of January to 6.36% in May 2020 and decreasing to a range between 2.14% to 2.30% since January 2021. In Africa, the infection rate per 100,000 persons was up to 3090.18, while deaths per 100,000 and case fatality ratio were as high as 119.64 and 5.72%, respectively, among the 20 most-affected countries. The testing rate per million population was highest in Botswana (512,547.08). Fatality appears to be increasing in some regions of Africa. The rate of infection and fatality in Africa could still likely take an upward turn. Strict control measures are required, considering the continent's weak healthcare systems.

Risk Factors of Candida parapsilosis Catheter-Related Bloodstream Infection.

Catheter-related bloodstream infection (CRBSI) is an important healthcare-associated infection caused by various nosocomial pathogens. Candida parapsilosis has emerged as a crucial causative agent for the CRBSI in the last two decades. Many factors have been associated with the development of CRBSI including, demography, pre-maturity, comorbidities (diabetes mellitus, hypertension, heart diseases, neuropathy, respiratory diseases, renal dysfunction, hematological and solid organ malignancies, and intestinal dysfunction), intensive care unit (ICU) admission, mechanical ventilation (MV), total parenteral nutrition (TPN), prior antibiotic and/or antifungal therapy, neutropenia, prior surgery, immunosuppressant, and type, site, number, and duration of catheters. This study aims to determine C. parapsilosis CRBSI risk factors. A retrospective study has been performed in an 853-bedded tertiary-care hospital in north-eastern Malaysia. All inpatients with C. parapsilosis positive blood cultures from January 2006 to December 2018 were included, and their medical records were reviewed using a standardized checklist. Out of 208 candidemia episodes, 177 had at least one catheter during admission, and 31 cases had not been catheterized and were excluded. Among the 177 cases, 30 CRBSI cases were compared to 147 non-CRBSI cases [81 bloodstream infections (BSIs), 66 catheter colonizers]. The significance of different risk factors was calculated using multivariate analysis. Multivariate analysis of potential risk factors shows that ICU admission was significantly associated with non-CRBSI as compared to CRBSI [OR, 0.242; 95% CI (0.080-0.734); p = 0.012], and TPN was significantly positively associated with CRBSI than non-CRBSI [OR, 3.079; 95%CI (1.125-8.429); p = 0.029], while other risk factors were not associated significantly. Patients admitted in ICU were less likely to develop C. parapsilosis CRBSI while patients receiving TPN were more likely to have C. parapsilosis CRBSI when compared to the non-CRBSI group.

One-step, multiplex, dual-function oligonucleotide of loop-mediated isothermal amplification assay for the detection of pathogenic Burkholderia pseudomallei.

This study highlights the development of a multiplex real-time loop-mediated isothermal amplification assay. The developed assay employed a dual-function oligonucleotide (DFO) which simultaneously monitors the emitted amplification signals and accelerates the amplification process. The DFO was a modification of loop primer (LP); the 5'-end and 3'-end of the LP was tagged with fluorophore and quencher, respectively. The DFO was quenched in its unbound state and fluoresces only when it anneals to the specific target during the amplification process. With the same working mechanism as LP, DFO allowed the detection of target genes in less than 1 h in a real time monitoring system. We demonstrated this detection platform with Burkholderia pseudomallei, the causative agent of melioidosis. An internal amplification control (IAC) was incorporated in the assay to rule out false negative result and to demonstrate that the assay was successfully developed in a multiplex system. The assay was 100% specific when it was evaluated against 96 B. pseudomallei clinical isolates and 48 other bacteria species. The detection limit (sensitivity) of the developed assay was 1 fg/µl of B. pseudomallei genomic DNA and 18.2 CFU/ml at the bacterial cell level. In spiked blood samples, the assay's detection limit was 14 CFU/ml. The assay's diagnostic evaluation showed 100% diagnostic sensitivity, diagnostic specificity, positive predictive value, and negative predictive value. An integrated multiplex LAMP and real-time monitoring system was successfully developed, simplifying the workflow for the rapid and specific nucleic acid diagnostic test.

BipD of Burkholderia pseudomallei: Structure, Functions, and Detection Methods.

Melioidosis is a severe disease caused by Burkholderia pseudomallei (B. pseudomallei), a Gram-negative environmental bacterium. It is endemic in Southeast Asia and Northern Australia, but it is underreported in many other countries. The principal routes of entry for B. pseudomallei are skin penetration, inhalation, and ingestion. It mainly affects immunocompromised populations, especially patients with type 2 diabetes mellitus. The laboratory diagnosis of melioidosis is challenging due to its non-specific clinical manifestations, which mimic other severe infections. The culture method is considered an imperfect gold standard for the diagnosis of melioidosis due to its low sensitivity. Antibody detection has low sensitivity and specificity due to the high seropositivity among healthy people in endemic regions. Antigen detection using various proteins has been tested for the rapid determination of B. pseudomallei; however, it presents certain limitations in terms of its sensitivity and specificity. Therefore, this review aims to frame the present knowledge of a potential target known as the Burkholderia invasion protein D (BipD), including future directions for its detection using an aptamer-based sensor (aptasensor).

Prevalence of Vancomycin-Resistant Enterococcus (VRE) in Companion Animals: The First Meta-Analysis and Systematic Review.

Antimicrobial resistance in companion animals is a major public health concern worldwide due to the animals' zoonotic potential and ability to act as a reservoir for resistant genes. We report on the first use of meta-analysis and a systematic review to analyze the prevalence of vancomycin-resistant Enterococcus (VRE) in companion animals. Databases such as MedLib, PubMed, Web of Science, Scopus, and Google Scholar were searched. The information was extracted by two independent reviewers and the results were reviewed by a third. Two reviewers independently assessed the study protocol using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) checklist and the study quality using the Joanna Briggs Institute (JBI) critical appraisal checklist for prevalence data. OpenMeta analyst and comprehensive meta-analysis (CMA) were used for the meta-analysis. The random effect model was used, and publication bias was assessed using the Eggers test and funnel plot. Between-study heterogeneity was assessed, and the sources were analyzed using the leave-one-out meta-analysis, subgroup analysis and meta-regression. Twenty-two studies met the eligibility criteria, but because some studies reported the prevalence of VRE in more than one companion animal, they were considered as individual studies, and 35 studies were therefore added to the final meta-analysis. Sampling period of the included studies was from 1995-2018. Of the 4288 isolates tested in the included studies, 1241 were VRE. The pooled prevalence of VRE in companion animals was estimated at 14.6% (95% CI; 8.7-23.5%; I2 = 97.10%; p < 0.001). Between-study variability was high (t2 = 2.859; heterogeneity I2 = 97.10% with heterogeneity chi-square (Q) = 1173.346, degrees of freedom (df) = 34, and p < 0.001). The funnel plot showed bias, which was confirmed by Eggers test (t-value = 3.97165; p = 0.00036), and estimates from the leave-one-out forest plot did not affect the pooled prevalence. Pooled prevalence of VRE in dogs and cats were 18.2% (CI = 9.4-32.5%) and 12.3%, CI = 3.8-33.1%), respectively. More studies were reported in Europe than in any other continent, with most studies using feces as the sample type and disc diffusion as the detection method. With the emergence of resistant strains, new antimicrobials are required in veterinary medicine.