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      Occurrence, Antibiotic Susceptibility and Genes Encoding Antibacterial Resistance of Salmonella spp. and Escherichia coli From Milk and Meat Sold in Markets of Bushenyi District, Uganda

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          Abstract

          The bacteriological safety of food/food products and the menace of antimicrobial resistance amongst enteropathogenic bacteria raise therapeutic management concerns within the public health system. Recently consumers of food/food products purchased from the public market of Bushenyi District presents with Enterobacteriaceae infection-associated symptoms and clinical conditions. We determine the molecular characterization and antibiotic signatures of some enteric bacterial recovered from foods/food products in markets of Bushenyi District, Uganda. Standard molecular biology techniques (Polymerase chain reaction PCR) and microbiological procedures were applied. Meat (MT) and milk (MK) samples were collected from 4 communities/town markets (Kizinda, Ishaka, Bushenyi, kashenyi) between April and September 2020 and analyzed. Our result reveals high differential counts of Salmonella species (175.33 ± 59.71 Log 10 CFU/100 ml) and Escherichia coli (53.33 ± 26.03 Log 10 CFU/100 ml) within the 4 markets with the count of Salmonella species higher than that of E. coli in each sampled market. The PCR further confirmed the detected strains (22.72% of E. coli and 54.29% of Salmonella species) and diverse multiple antibiotic-resistant determinants {TEM: (12 (23.1%) blaTEM-2 gene, 3 (5.8%) blaTEM gene}, 5 (9.6%) blaSHV gene, 3 (5.8%) bla-CTX-M-2, 1 (1.9%) bla-CTX-M-9 }. Other resistance genes detected were {10 (21.7%) strA gene} and 8 (17.4%) aadA gene} indicating a potential antibiotic failure. The need for alternative medicine and therapeutic measure is suggestive. Astute and routine surveillance/monitoring of potential pathogens and food products in the public market remains a core for maintaining future consumer safety.

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          Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae.

          To develop a rapid and reliable tool to detect by multiplex PCR assays the most frequently widespread beta-lactamase genes encoding the OXA-1-like broad-spectrum beta-lactamases, extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC beta-lactamases and class A, B and D carbapenemases. Following the design of a specific group of primers and optimization using control strains, a set of six multiplex PCRs and one simplex PCR was created. An evaluation of the set was performed using a collection of 31 Enterobacteriaceae strains isolated from clinical specimens showing a resistance phenotype towards broad-spectrum cephalosporins and/or cephamycins and/or carbapenems. Direct sequencing from PCR products was subsequently carried out to identify beta-lactamase genes. Under optimized conditions, all positive controls confirmed the specificity of group-specific PCR primers. Except for the detection of carbapenemase genes, multiplex and simplex PCR assays were carried out using the same PCR conditions, allowing assays to be performed in a single run. Out of 31 isolates selected, 22 strains produced an ESBL, mostly CTX-M-15 but also CTX-M-1 and CTX-M-9, SHV-12, SHV-5, SHV-2, TEM-21, TEM-52 and a VEB-type ESBL, 6 strains produced a plasmid-mediated AmpC beta-lactamase (five DHA-1 and one CMY-2) and 3 strains produced both an ESBL (two SHV-12, one CTX-M-15) and a plasmid-mediated AmpC beta-lactamase (DHA-1). We report here the development of a useful method composed of a set of six multiplex PCRs and one simplex PCR for the rapid screening of the most frequently encountered beta-lactamases. This method allowed direct sequencing from the PCR products.
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            Commensal Escherichia coli of healthy humans: a reservoir for antibiotic-resistance determinants.

            This study examined in detail the population structure of Escherichia coli from healthy adults with respect to the prevalence of antibiotic resistance and specific resistance determinants. E. coli isolated from the faeces of 20 healthy adults not recently exposed to antibiotics was tested for resistance to ten antibiotics and for carriage of integrons and resistance determinants using PCR. Strain diversity was assessed using biochemical and molecular criteria. E. coli was present in 19 subjects at levels ranging from 2.0×10(4) to 1.7×10(8) c.f.u. (g faeces)(-1). Strains resistant to one to six antibiotics were found at high levels (>30 %) in only ten individuals, but at significant levels (>0.5 %) in 14. Resistant isolates with the same phenotype from the same individual were indistinguishable, but more than one susceptible strain was sometimes found. Overall, individuals harboured one to four E. coli strains, although in 17 samples one strain was dominant (>70 % of isolates). Eighteen strains resistant to ampicillin, sulfamethoxazole, tetracycline and trimethoprim in 15 different combinations were observed. One resistant strain was carried by two unrelated individuals and a susceptible strain was shared by two cohabiting subjects. Two minority strains were derivatives of a more abundant resistant strain in the same sample, showing that continuous evolution is occurring in vivo. The trimethoprim-resistance genes dfrA1, dfrA5, dfrA7, dfrA12 or dfrA17 were in cassettes in a class 1 or class 2 integron. Ampicillin resistance was conferred by the bla(TEM) gene, sulfamethoxazole resistance by sul1, sul2 or sul3 and tetracycline resistance by tetA(A) or tetA(B). Chloramphenicol resistance (cmlA1 gene) was detected only once. Phylogenetic groups A and B2 were more common than B1 and D. Commensal E. coli of healthy humans represent an important reservoir for numerous antibiotic-resistance genes in many combinations. However, measuring the true extent of resistance carriage in commensal E. coli requires in-depth analysis.
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              Aseptic Laboratory Techniques: Plating Methods

              Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: ● Perform plating procedures without contaminating media. ● Isolate single bacterial colonies by the streak-plating method. ● Use pour-plating and spread-plating methods to determine the concentration of bacteria. ● Perform soft agar overlays when working with phage. ● Transfer bacterial cells from one plate to another using the replica-plating procedure. ● Given an experimental task, select the appropriate plating method.
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                Author and article information

                Journal
                Microbiol Insights
                Microbiol Insights
                MBI
                spmbi
                Microbiology Insights
                SAGE Publications (Sage UK: London, England )
                1178-6361
                11 April 2022
                2022
                : 15
                : 11786361221088992
                Affiliations
                [1 ]Biomolecules, Metagenomics, Endocrine and Tropical Disease Research Group (BMETDREG), Kampala International University, Western Campus, Ishaka-Bushenyi, Uganda
                [2 ]Biopharmaceutics unit, Department of Pharmacology and Toxicology, School of Pharmacy, Kampala International University Uganda, Ishaka, Uganda
                [3 ]Department of Microbiology and Biotechnology, Western Delta University Oghara, Delta State, Nigeria
                Author notes
                [*]Hope Onohuean, Biopharmaceutics unit, Department of Pharmacology and Toxicology, School of Pharmacy, Kampala International University, Western Campus, Ishaka, Bushenyi 256, Uganda. Email: onohuean@ 123456gmail.com
                Author information
                https://orcid.org/0000-0002-1890-6324
                Article
                10.1177_11786361221088992
                10.1177/11786361221088992
                9008818
                de728a1f-3a48-4367-b927-3b6eca80c8a0
                © The Author(s) 2022

                This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License ( https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages ( https://us.sagepub.com/en-us/nam/open-access-at-sage).

                History
                : 13 July 2021
                : 25 February 2022
                Categories
                Original Research
                Custom metadata
                January-December 2022
                ts1

                antimicrobial agents,food/food product,enteric pathogens,antibiotic-resistant genes

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