Genomic epidemiology of multidrug‐resistant Gram‐negative organisms

The emergence and spread of antibiotic‐resistant Gram‐negative bacteria (rGNB) across global healthcare networks presents a significant threat to public health. As the number of effective antibiotics available to treat these resistant organisms dwindles, it is essential that we devise more effective strategies for controlling their proliferation. Recently, whole‐genome sequencing has emerged as a disruptive technology that has transformed our understanding of the evolution and epidemiology of diverse rGNB species, and it has the potential to guide strategies for controlling the evolution and spread of resistance. Here, we review specific areas in which genomics has already made a significant impact, including outbreak investigations, regional epidemiology, clinical diagnostics, resistance evolution, and the study of epidemic lineages. While highlighting early successes, we also point to the next steps needed to translate this technology into strategies to improve public health and clinical medicine.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147016/1/nyas13672.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147016/2/nyas13672_am.pd

Evaluation of risk based microbiological criteria for Campylobacter in broiler carcasses in Belgium using TRiMiCri

Campylobacteriosis is the most frequently reported foodborne zoonosis worldwide. Consumer´s exposure to Campylobacter might be reduced by establishing a microbiological criterion (MC) for Campylobacter on broiler meat. In the present study two possible approaches were evaluated, using the freely available software tool for risk based microbiological criteria TRiMiCri (http://tools.food.dtu.dk/trimicri). The first approach was the traditional one that implies a microbiological limit (ML-MC) and the second one which is based on the relative risk estimate (RRL-MC). The analyses were based on Campylobacter quantitative data collected from 28 Campylobacter positive bathes processed in 6 Belgian broiler slaughterhouses. To evaluate the performance of ML-MC, n=6, different c (0,1,2) and m (100,1 000,10 000) were used. Results showed that more than 90% of Campylobacter positive batches were not complying with strict ML criteria based on the m=100 for all applied combination of c. The RRL approach requires a baseline risk which was estimated based on the Campylobacter baseline data collected in Belgium in 2008. Approximately 60% of evaluated Campylobacter positive batches account for higher risk than the baseline risk. For both approaches, application of less stringent criteria results in lower percentage of NC and higher minimum relative residual risks (MRRR; it refers to the change in risk when all batches are sampled and all NC batches undergo treatment that effectively eliminates Campylobacter so they are replaced by zero risk batches). It was also observed that the number of samples (n) had little effect on risk estimates. Additionally, the results from ML-MC and RRL-MC follow the same curve when plotting percentage of NC against MRRR. However, for RRL-MC the percentage of NC batches and MRRR was lower and higher, respectively. To conclude, obtained results indicate that TRiMiCri is a useful and user friendly tool to make a risk based decision on the choice of the MC

Resistance mechanisms and population structure of highly drug resistant Klebsiella in Pakistan during the introduction of the carbapenemase NDM-1.

Klebsiella pneumoniae is a major threat to public health with the emergence of isolates resistant to most, if not all, useful antibiotics. We present an in-depth analysis of 178 extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae collected from patients resident in a region of Pakistan, during the period 2010-2012, when the now globally-distributed carbapenemase bla-NDM-1 was being acquired by Klebsiella. We observed two dominant lineages, but neither the overall resistance profile nor virulence-associated factors, explain their evolutionary success. Phenotypic analysis of resistance shows few differences between the acquisition of resistance genes and the phenotypic resistance profile, including beta-lactam antibiotics that were used to treat ESBL-positive strains. Resistance against these drugs could be explained by inhibitor-resistant beta-lactamase enzymes, carbapenemases or ampC type beta-lactamases, at least one of which was detected in most, but not all relevant strains analysed. Complete genomes for six selected strains are reported, these provide detailed insights into the mobile elements present in these isolates during the initial spread of NDM-1. The unexplained success of some lineages within this pool of highly resistant strains, and the discontinuity between phenotypic resistance and genotype at the macro level, indicate that intrinsic mechanisms contribute to competitive advantage and/or resistance

Genomic methods for enhanced surveillance and persistence investigations of foodborne pathogens

The globalized food supply chain became a vast and complex network leading to an increased risk of spread of known and emerging foodborne pathogens, Listeria monocytogenes and Salmonella enterica serovar Typhimurium variant 4,[5],12:i:-. Based on a previous study of persistence of L. monocytogenes, 27 ST14 and 6 ST121 newly sequenced genomes collected over one year on the same rabbit meat processing plant were investigated in comparison to a selection of public genomes. cgMLST analysis of sequenced genomes showed higher discriminatory power in comparison to conventional typing methods. wgSNPs phylogeny inferred on 273 newly sequenced and publicly available ST121 and ST14 genomes confirmed that a persistent clone was circulating in the Italian rabbit-meat plant along with not persistent strains. Mass screening of a novel dataset of genes involved in physiological adaptation to food-processing environment showed a significant enrichment in ST121 genomes concerned genetic features related to sanitizing procedures adaptation, whereas genes related to biofilm forming enhanced ability and cadmium resistance was associated to ST14, as confirmed by phenotypic tests. Genomic data of 148 Salmonella enterica ser. Typhimurium monophasic variant 4,[5],12:i:- (MVSTm) isolates circulating in human and swine in Italy have been investigated in comparison to publicly available S. Typhimurium/ MVSTm strains collected in Italy and worldwide. The innovative genome-wide approach applied in this study allowed to mine population structure of a large Salmonella serovars dataset (~4,000 genomes) including Italian MVST strains belonging to a large population of ~1,300 clonal S. Typhimurium/ MVSTm isolates (2.5% of allele differences) isolated from a wide-range of countries in last two decades. cgSNPs phylogeny revealed and Genome-Wide Association Study suggested that discrete geographical segregation has had a strong impact on the accessory gene content with particular significance for a large SopE-containing prophage associated to Italian population and constituting specific biomarkers relevant in course of large epidemics

Molecular profile of gram-negative ESKAPE pathogens from Komfo Anokye Teaching Hospital in Ghana.

Doctoral Degree. University of KwaZulu-Natal, Durban.Gram-negative ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp) pathogens are a major healthcare concern globally due to their increasing multidrug resistance and ability to cause debilitating infections. Phenotypic and genotypic characteristics of multidrug resistant Gram-negative ESKAPE pathogens from Komfo Anokye Teaching Hospital in Ghana were investigated. Two hundred (200) clinical, non-duplicate Gram-negative bacterial pathogens were randomly selected from various human specimens routinely processed by the diagnostic microbiological laboratory in the hospital. Multidrug resistant (isolates resistant to at least one agent in three or more antibiotic class) isolates selected from each group of Gram-negative ESKAPE pathogens constituted the final sample. Identification and antibiotic susceptibility profiles were carried out using Vitek-2. Identity of isolates for whole genome sequencing was further confirmed by MALDI-TOF MS. Four P. aeruginosa and 10 K. pneumoniae were subjected to whole genome sequencing based on their extensively drug resistant profiles and resistance to third-generation cephalosporins respectively using Illumina MiSeq, after genomic DNA extraction using the NucliSens easyMAG®. Antibiotic resistance genes and plasmids were identified by mapping the sequence data to an online database using ResFinder and plasmidFinder respectively. MLST was also determined from the WGS data. The raw read sequences and assembled whole genome contigs have been deposited in GenBank under project number PRJNA411997. An average multidrug resistance of 89.5% was observed, ranging from 53.8% in Enterobacter spp to 100.0% in Acinetobacter spp and P. aeruginosa. Gram-negative ESKAPE bacteria constituted 48.5% (97) of the 200 isolates. P. aeruginosa (n=4) belonging to ST234 harboured blaDIM-1, blaIMP-34, blaOXA-10, blaOXA-129, blaOXA-50, blaPAO aadA1, aac4 aph(3’)-IIb, fosA, sul1, dfrB5, catB7, arr-2 conferring resistance to β-lactams, aminoglycosides, fosfomycin, sulphonamides, trimethoprim phenicols and rifampin respectively. qnrVC was detected in two of the four isolates . Both blaDIM-1 and blaIMP-34-like positive contigs showed identical DNA sequences and were linked to type 1 integron structures. BlaDIM-1 was 100% identical to the blaDIM-1 prototype gene, while blaIMP-34-like had two base pair (bp) differences T190C and C314G respectively compared to blaIMP-34, leading to one amino acid substitution in IMP-34-like indicating that, the gene may have independently evolved, perhaps due to selection pressure. Blast analysis did not reveal identical genetic structures deposited in NCBI, neither among the nucleotide collection, completed genomes nor among the completed plasmids. β-lactamases (blaCTX-M-15, blaSHV-11, blaTEM-1B) and resistance genes for aminoglycosides (aac(3)-IIa-like,aph(3')-Ia) quinolones/fluoroquinolones (oqxA-like,oqxB-like,qnrB10-like,qnrB2) and others including fosfomycin (fosA), trimethoprim (dfrA14), and sulphonamide (sul2) were found in the K. pneumoniae (n=10). Multiple and diverse mutations of the quinolone resistance-determining regions gyrA, gyrB and parC genes were detected in the K. pneumoniae (n=4), which were clonally distinct. The diversity of resistance genes expressed by Gram-negative ESKAPE pathogens conferring resistance to multiple antibiotics is problematic in a resource-constrained country like Ghana, necessitating urgent antibiotic stewardship and infection prevention and control interventions