Metallo-B-lactamase (MBL)-producing Enterobacteriaceae are of grave clinical concern particularly as there are no MBL inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available B-lactams is thus imperative. We investigated a zinc-chelating moiety, 1, 4, 7-triazacyclononane (TACN) for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae (CRE). Minimum inhibitory concentrations, minimum bactericidal concentrations, serum effect, fractional inhibitory concentrations index and time-kill kinetics were performed using broth micro-dilution techniques according to the CLSI guidelines. Enzyme kinetic parameters and cytotoxicity effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem (MEM) regained its activity against carbapenemase-producing Enterobacteriaceae, with the MIC decreasing to 0.03 mg/L in the presence of TACN. TACN-MEM combinations showed bactericidal effect with MIC/MBC ratio of less than or equal to 4 and synergistic activity was observed. Human serum effects on the MICs were insignificant and TACN was found to be non-cytotoxic at concentrations above the MIC values. Computational studies predicted that TACN inhibit MBLs by targeting their catalytic active site pockets. This was supported by its inhibition constant Ki = 0.044 uM and inactivation constant kinact = 0.0406 (min -1 ) demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor. Importance of study: Carbapenem-resistant Enterobacteriaceae (CRE)-mediated infections remain a huge public health concern and have been reported as critical in the World Health Organization Priority Pathogens List for the Research and Development of New Antibiotics. CRE produce enzymes such as Metallo-B-lactamases (MBLs), which inactivate B-lactam antibiotics. Combination therapies involving a B-lactam antibiotic and a B-lactamase inhibitor remain a major treatment option for infections caused by B-lactamase-producing organisms. Currently, no MBL inhibitor-B-lactam combination therapy is clinically available for MBL-positive bacterial infections. Hence, developing efficient molecules capable of inhibiting these enzymes remain a way forward to overcome this phenomenon. TACN played a significant role in the inhibitory activity of the tested molecules against CREs by potentiating the carbapenem. This study demonstrated that TACN inhibits MBLs efficiently and holds promise as a potential MBLs inhibitor to help curb the global health threat posed by MBL-producing CREs.
Antibiotics are now “endangered species” facing extinction due to the worldwide emergence of antibiotic resistance (ABR). Food animals are considered as key reservoirs of antibiotic-resistant bacteria with the use of antibiotics in the food production industry having contributed to the actual global challenge of ABR. There are no geographic boundaries to impede the worldwide spread of ABR. If preventive and containment measures are not applied locally, nationally and regionally, the limited interventions in one country, continent and for instance, in the developing world, could compromise the efficacy and endanger ABR containment policies implemented in other parts of the world, the best-managed high-resource countries included. Multifaceted, comprehensive and integrated measures complying with the One Health approach are imperative to ensure food safety and security, effectively combat infectious diseases, curb the emergence and spread of ABR, and preserve the efficacy of antibiotics for future generations. Countries should follow the World Health Organization, World Organization for Animal Health, and the Food and Agriculture Organization of the United Nations recommendations to implement national action plans encompassing human, (food) animal, and environmental sectors to improve policies, interventions and activities that address the prevention and containment of ABR from farm-to-fork. This review covers (i) the origin of antibiotic resistance, (ii) pathways by which bacteria spread to humans from farm-to-fork, (iii) differences in levels of antibiotic resistance between developed and developing countries, and (iv) prevention and containment measures of antibiotic resistance in the food chain.
Herein, we highlight the genome sequence of a novel Enterococcus faecalis sequence type 922 (ST922) strain isolated in South Africa. The 3,564,442-bp genome harbored defense systems, a resistome, a virulome, and genetic support, which is of importance to the control of hospital-acquired infections. The genomics of Enterococcus faecalis yields greater understanding into its pathogenesis.
The increased use of antibiotics in food animals has resulted in the selection of drug-resistant bacteria across the farm-to-fork continuum. This study aimed to investigate the molecular epidemiology of antibiotic-resistant Escherichia coli from intensively produced poultry in the uMgungundlovu District, KwaZulu-Natal, South Africa. Samples were collected weekly between August and September 2017 from hatching to final retail products. E. coli was isolated on eosin methylene blue agar, identified biochemically, and confirmed using polymerase chain reaction (PCR). Susceptibility to 19 antibiotics was ascertained by the Kirby–Bauer disc diffusion method. PCR was used to test for resistance genes. The clonal similarity was investigated using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). In total, 266 E. coli isolates were obtained from all the samples, with 67.3% being non-susceptible to at least one antibiotic tested and 6.7% multidrug resistant. The highest non-susceptibility was to ampicillin (48.1%) and the lowest non-susceptibility to ceftriaxone and azithromycin (0.8%). Significant non-susceptibility was observed to tetracycline (27.4%), nalidixic acid (20.3%), trimethoprim-sulfamethoxazole (13.9%), and chloramphenicol (11.7%) which have homologues used in the poultry industry. The most frequently observed resistance genes were blaCTX-M (100%), sul1 (80%), tetA (77%), and tetB (71%). ERIC-PCR grouped isolates into 27 clusters suggesting the spread of diverse clones across the farm-to-fork continuum. This reiterates the role of intensive poultry farming as a reservoir and a potential vehicle for the transmission of antibiotic resistance, with potentially severe public health implications, thus, requiring prompt and careful mitigation measures to protect human and environmental health.
Extended-spectrum beta-lactamaseproducing Escherichia coli harbouring mcr-1 gene isolated from pigs in South AfricaTo the Editor: Extended-spectrum betalactamase-producing Enterobacteriaceae (ESBL-PE) produce enzymes that confer resistance to penicillins, cephalosporins and monobactams. [1]ESBL-PE represent a global concern in humans, in animals and in the environment and have therefore been recognised as pathogens of critical priority. [2]f additional concern is the emergence of the plasmid-borne colistin resistance (mcr-1) genes that encode for resistance to polymyxins, the antibiotics of last resort for treatment of serious difficult-to-treat infections. [3]They have both been detected in developed and developing countries, and food animals have been recognised as their principal reservoir. [4]asal and rectal swabs were collected from 432 pigs during a multicentre study carried out from March to October 2016 in five abattoirs in Cameroon (n=3) and South Africa (SA) (n=2).Samples were screened on a selective medium and puta tive ESBL-PE were tested for their antimicrobial susceptibility using the Vitek 2 System and Vitek 2 Gram Negative Susceptibility card (AST-N255) (bioMérieux, France).The results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guideline, [5] with the exception of colistin, amoxicillin plus clavulanic acid, piperacillin plus tazobactam and ami kacin, which were based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. [6]Closely related isolates underwent whole-genome sequencing (WGS) analysis on an Illumina MiSeq platform (Illumina Inc., USA) with 100 × coverage.ResFinder, [7] VirulenceFinder [8] and PlasmidFinder [9] were used to identify resistance genes, virulence factors and plasmids, respectively.The multi-locus sequence type was also determined from WGS data.One of the ESBL-producing Escherichia coli isolated, PN256E8 (GenBank accession no.QJRZ00000000), showed resistance to several beta-lactam and non-beta-lactam antibiotics, including colistin.This resistance phenotype was corroborated by the identification of the bla TEM-1B and bla CTX-M-55 genes through WGS, which also evidenced the colistin (mcr-1)-resistant gene (Table 1).The isolate PN256E8 was ascribed to the sequence type (ST) 446 and phylogenetic group A. It further harboured two virulence factors
Background and objectives: Extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae is a serious public health issue globally. In this study, the antibiotic resistance genes, virulence factors, mobile genetic elements, and genetic lineages of circulating ESBL-producing K. pneumoniae strains isolated from pigs and humans in Cameroonian abattoirs were investigated using whole genome sequencing (WGS), in order to ascertain zoonotic transmission (viz. from animals to humans and/or vice-versa) in the food chain. Methods: During March-October 2016, 288 nasal and rectal pooled samples from 432 pigs as well as nasal and hand swabs from 82 humans were collected from Cameroon and South Africa. Seven ESBL-producing K. pneumoniae circulating in Cameroonian pig abattoirs were selected and their genomic DNA sequenced using an Illumina MiSeq platform. Generated reads were de novo assembled using the Qiagen CLC Genomics Workbench and SPAdes. The assembled contigs were annotated using RAST and antibiotic resistance genes, virulence factors, plasmids, and bacteriophages were identified with ResFinder, Virulence Finder, PlasmidFinder, and PHAST, respectively. Results: ESBL-producing K. pneumoniae were detected in pigs (34/158; 21.52%) and exposed workers (8/71; 11.26%) in Cameroon only. The circulating K. pneumoniae strains were dominated principally by the sequence type (ST) 14 and 39. In addition, the "high-risk" ST307 clone and two novel STs assigned ST2958 and ST2959 were detected. Genomic analysis identified various antibiotic resistance genes associated with resistance to β-lactams, aminoglycosides, fluoroquinolones, macrolide, lincosamide and streptogramins, rifampicin, sulfonamides, trimethoprim, phenicols and tetracycline. None of the ESBL-producing K. pneumoniae harbored virulence genes. Intermingled K. pneumoniae populations were observed between pig- and human-source within and across abattoirs in the country. Conclusion: Our study shows that ESBL-producing K. pneumoniae is actively disseminating in pigs and occupationally exposed workers in Cameroonian pig abattoirs and is probably underestimated in the absence of molecular epidemiological studies. It suggests pigs, abattoir workers and food products as potential reservoirs and sources of zoonotic transmission in Cameroon. Our findings underline the existence of a potential unheeded food safety and public health threat associated with these resistant strains and reinforce the crucial importance of implementing appropriate food safety measures and promoting rational antibiotic use.
Companion animals are globally documented to harbour antibiotic-resistant E. coli . We investigated the prevalence and phylotyping of antibiotic-resistant E. coli from companion animals (dogs and cats) at selected veterinary practices in Durban, KwaZulu-Natal, South Africa. E. coli was isolated from rectal swab samples on Eosin methylene blue agar and confirmed using real-time PCR. Antibiotic susceptibility testing against 20 antibiotics was done using the Kirby-Bauer method. Selected antibiotic-resistance genes (ARGs), conferring third-generation cephalosporins ( bla TEM , bla SHV , and bla CTX-M ), tetracycline ( tet A, and tet B), and tigecycline ( tet X/X2, tet X3, and tet X4) resistance, were detected using PCR and amplicon sequencing. Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction was used to determine the clonality of antibiotic-resistant E. coli . Three hundred and thirty E. coli (234 dog and 96 cat) isolates were obtained. Isolates were most resistant to tetracycline (dogs - 25.2%; cats - 22.4%) and least to amikacin and piperacillin-tazobactam (Dogs - 0.4%) and piperacillin-tazobactam and ceftazidime (cats - 1.0%). Thirty-five (10.6%) isolates were multidrug-resistant, displaying twenty-two different phenotypic patterns. The bla CTX-M-15 (8%) and tet A (24%) were the most prevalent resistance genes; bla SHV, tet X/X2, tet X3, and tet X4 were undetected. Clonality revealed 48 clusters using a 75% similarity cut-off, suggesting highly diverse E. coli circulating in Durban, South Africa, potentially no evidence of antibiotic-resistant E. coli transmission among these animals in Durban. Resistance of E. coli from companion animals to medically important antimicrobials for humans is of particular concern, requiring measures to control the spread of antibiotic-resistant bacteria and ARGs between companion animals, veterinary practice personnel, and owners.
Bacteria’s ability to withstand the detrimental effects of antimicrobials may take the form of resistance or tolerance with the minimum inhibitory concentration (MIC), the mutant prevention concentration (MPC), and the mutant selection window as salient concepts. Isolates exposed for 30 days to different concentrations of selected antibiotics, biocides, and heavy metals, singly and in combination, were additionally exposed to very high (15 × MIC) concentrations of ampicillin., and their tolerance was calculated the time to kill 99.9% of the isolate population (MDK99.9). These values were increased by 30 to 50% in test isolates compared to the untreated control. BAC 10-exposed isolates had the highest tolerance increase, and the tolerance appeared to originate from two sources, i.e., stochastic persistence and genetic-induced persistence, involving multiple genes using diverse mechanisms. The mutant selection window of the isolates to ampicillin, amoxicillin, and oxytetracycline also slightly increased compared to the control, indicating the amplification of persister cells during the 30-day exposure. These findings indicate that bacterial exposure to sub-inhibitory concentrations of environmental chemical stressors may not always result in the development of antimicrobial resistance but could initiate this process by selecting for persisters that could evolve into resistant isolates.
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