Genomic diversity of antimicrobial resistance in non-typhoidal Salmonella in Victoria, Australia

Non-typhoidal Salmonella (NTS) is the second most common cause of foodborne bacterial gastroenteritis in Australia with antimicrobial resistance (AMR) increasing in recent years. Whole-genome sequencing (WGS) provides opportunities for in silico detection of AMR determinants. The objectives of this study were two-fold: (1) establish the utility of WGS analyses for inferring phenotypic resistance in NTS, and (2) explore clinically relevant genotypic AMR profiles to third generation cephalosporins (3GC) in NTS lineages. The concordance of 2490 NTS isolates with matched WGS and phenotypic susceptibility data against 13 clinically relevant antimicrobials was explored. In silico serovar prediction and typing was performed on assembled reads and interrogated for known AMR determinants. The surrounding genomic context, plasmid determinants and co-occurring AMR patterns were further investigated for multidrug resistant serovars harbouring bla CMY-2, bla CTX-M-55 or bla CTX-M-65. Our data demonstrated a high correlation between WGS and phenotypic susceptibility testing. Phenotypic-genotypic concordance was observed between 2440/2490 (98.0 %) isolates, with overall sensitivity and specificity rates >98 % and positive and negative predictive values >97 %. The most common AMR determinants were bla TEM-1, sul2, tet(A), strA-strB and floR. Phenotypic resistance to cefotaxime and azithromycin was low and observed in 6.2 % (151/2486) and 0.9 % (16/1834) of the isolates, respectively. Several multi-drug resistant NTS lineages were resistant to 3GC due to different genetic mechanisms including bla CMY-2, bla CTX-M-55 or bla CTX-M-65. This study shows WGS can enhance existing AMR surveillance in NTS datasets routinely produced in public health laboratories to identify emerging AMR in NTS. These approaches will be critical for developing capacity to detect emerging public health threats such as resistance to 3GC.

Prediction of antimicrobial resistance in clinical Enterococcus faecium isolates using a rules-based analysis of whole genome sequences

Background: Enterococcus faecium is a major cause of clinical infections, often due to multidrug-resistant (MDR) strains. Whole genome sequencing (WGS) is a powerful tool to study MDR bacteria and their antimicrobial resistance (AMR) mechanisms. Here we use WGS to characterize E. faecium clinical isolates and test the feasibility of rules-based genotypic prediction of AMR. Methods: Clinical isolates were divided into derivation and validation sets. Phenotypic susceptibility testing for ampicillin, vancomycin, high-level gentamicin, ciprofloxacin, levofloxacin, doxycycline, tetracycline, and linezolid was performed using the VITEK 2 automated system, with confirmation and discrepancy resolution by broth microdilution, disk diffusion, or gradient diffusion when needed. WGS was performed to identify isolate lineage and AMR genotype. AMR prediction rules were derived by analyzing the genotypic-phenotypic relationship in the derivation set. Results: Phylogenetic analysis demonstrated that 88% of isolates in the collection belonged to hospital-associated clonal complex 17. Additionally, 12% of isolates had novel sequence types. When applied to the validation set, the derived prediction rules demonstrated an overall positive predictive value of 98% and negative predictive value of 99% compared to standard phenotypic methods. Most errors were falsely resistant predictions for tetracycline and doxycycline. Further analysis of genotypic-phenotypic discrepancies revealed potentially novel pbp5 and tet (M) alleles that provide insight into ampicillin and tetracycline class resistance mechanisms. The prediction rules demonstrated generalizability when tested on an external dataset. Conclusions: Known AMR genes and mutations can predict E. faecium phenotypic susceptibility with high accuracy for most routinely tested antibiotics, providing opportunities for advancing molecular diagnostics.

Efficacy of dihydroartemisinin/piperaquine in patients with non-complicated Plasmodium falciparum malaria in Yaoundé, Cameroon

Background Dihydroartemisinin/piperaquine is increasingly used for the treatment of uncomplicated Plasmodium falciparum malaria in Africa. The efficacy of this combination in Cameroon is poorly documented, while resistance to dihydroartemisinin/piperaquine readily spreads in Southeast Asia. Objectives This study evaluated the clinical efficacy of dihydroartemisinin/piperaquine in Cameroon, as well as the molecular profile and phenotypic susceptibility of collected isolates to dihydroartemisinin and piperaquine. Patients and methods Dihydroartemisinin/piperaquine efficacy in 42 days was followed-up for 138 patients presenting non-complicated falciparum malaria. Piperaquine concentration was determined at day 7 for 124 patients. kelch13 gene polymorphisms (n = 150) and plasmepsin2 gene amplification (n = 148) were determined as molecular markers of resistance to dihydroartemisinin and piperaquine, respectively. Parasite susceptibility to dihydroartemisinin and piperaquine was determined using validated in vitro survival assays. Results The efficacy of dihydroartemisinin/piperaquine treatment was 100% after PCR correction. The reinfections were not associated with a variation of piperaquine concentration at day 7. Ninety-six percent (144/150) of the samples presented a WT allele of the kelch13 gene. Two percent (3/150) presented the non-synonymous mutation A578S, which is not associated with resistance to dihydroartemisinin. No duplication of the plasmepsin2 gene was observed (0/148). All the samples tested in vitro by survival assays (n = 87) were susceptible to dihydroartemisinin and piperaquine. Conclusions Dihydroartemisinin/piperaquine has demonstrated excellent therapeutic efficacy with no evidence of emerging artemisinin or piperaquine resistance in Yaoundé, Cameroon. This observation suggests that dihydroartemisinin/piperaquine could be a sustainable therapeutic solution for P. falciparum malaria if implemented in areas previously free of artemisinin- and piperaquine-resistant parasites, unlike Southeast Asia.

Core Genome Multi-Locus Sequence Typing and Prediction of Antimicrobial Susceptibility Using Whole Genome Sequences of Escherichia coli Bloodstream Infection Isolates

50 Escherichia coli bloodstream isolates from the clinical laboratory and 12 E. coli isolates referred for PFGE were sequenced, assessed for clonality using cgMLST and evaluated for genomic susceptibility predictions using ARESdb. Sequence typing using WGS-based MLST and ST-specific PCR were identical. Overall categorical agreement between genotypic (ARESdb) and phenotypic susceptibility testing for 62 isolates and 11 antimicrobial agents was 91%. Among the referred isolates, high ceftazidime, cefepime, and piperacillin-tazobactam major error rates were found.

Overcoming the Challenges of Pyrazinamide Susceptibility Testing in Clinical Mycobacterium tuberculosis isolates

Pyrazinamide (PZA) is one of the first-line agents used for the treatment of tuberculosis. However, current phenotypic PZA susceptibility testing in the BACTEC MGIT 960 system is unreliable and false resistance is well documented. Rapid identification of resistance-associated mutations can confirm the phenotypic result. This study aimed to investigate the use of genotypic methods in combination with phenotypic susceptibility testing for confirmation of PZA resistant M. tuberculosis isolates. Sanger sequencing and/or whole genome sequencing were performed to detect mutations in pncA, rpsA, panD and clpC1. Isolates were screened for heteroresistance, and PZA susceptibility testing was performed in the BACTEC MGIT 960 system using a reduced inoculum to investigate false resistance. Overall, 40 phenotypically PZA resistant isolates were identified. Of these, PZA resistance was confirmed in 22/40 (55%) isolates by detecting mutations in pncA, rpsA and panD genes. 16/40 (40%) isolates were found to be susceptible using the reduced inoculum method (i.e. false resistance). No mutations were detected in two PZA resistant isolates. False resistance was observed in isolates with MICs close to the critical concentration. In particular, EAI strains (lineage 1) appeared to have an elevated MIC that is close to the critical concentration. While this study illustrates the complexity and challenges associated with PZA susceptibility testing of M. tuberculosis, we conclude that a combination of genotypic and phenotypic drug susceptibility testing methods is required for accurate detection of PZA resistance.

Increasing prevalence of resistance to second-line drugs among multidrug-resistant Mycobacterium tuberculosis isolates in Kuwait

Molecular methods detect genetic mutations associated with drug resistance. This study detected resistance-conferring mutations in gyrA/gyrB for fluoroquinolones and rrs/eis genes for second-line injectable drugs (SLIDs) among multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates in Kuwait. Fifty pansusceptible M. tuberculosis and 102 MDR-TB strains were tested. Phenotypic susceptibility testing was performed by MGIT 960 system using SIRE drug kit. GenoType MTBDRsl version 1 (gMTBDRslv1) and GenoType MTBDRsl version 2 (gMTBDRslv2) tests were used for mutation detection. Results were validated by PCR-sequencing of respective genes. Fingerprinting was performed by spoligotyping. No mutations were detected in pansusceptible isolates. gMTBDRslv1 detected gyrA mutations in 12 and rrs mutations in 8 MDR-TB isolates. gMTBDRsl2 additionally detected gyrB mutations in 2 and eis mutation in 1 isolate. Mutations in both gyrA/gyrB and rrs/eis were not detected. gMTBDRslv1 also detected ethambutol resistance-conferring embB mutations in 59 isolates. Although XDR-TB was not detected, frequency of resistance-conferring mutations for fluoroquinolones or SLIDs was significantly higher among isolates collected during 2013–2019 versus 2006–2012. Application of both tests is warranted for proper management of MDR-TB patients in Kuwait as gMTBDRslv2 detected resistance to fluoroquinolones and/or SLIDs in 3 additional isolates while gMTBDRslv1 additionally detected resistance to ethambutol in 58% of MDR-TB isolates.

Reduced Susceptibility to Chlorhexidine among Staphylococcus aureus Isolates in Israel: Phenotypic and Genotypic Tolerance

Antiseptic use for body decolonization is the main activity applied to prevent healthcare-associated infections, including those caused by S. aureus. Consequentially, tolerance to several antiseptics such as chlorhexidine gluconate (CHG) has developed. This study aimed to estimate the prevalence of CHG tolerance among S. aureus strains in Israel and to evaluate factors that may affect this tolerance. Furthermore, it tested the associations between phenotypic and genotypic CHG tolerance. S. aureus strains (n = 190) were isolated from clinical samples of patients admitted to various medical institutions in Israel. Phenotypic susceptibility to CHG was assessed by determining minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Genotypic tolerance was detected using real-time PCR for detection of qac A/B genes. MIC for the antibiotic mupirocin was determined using the Etest method. Presence of the Panton–Valentine Leucocidin (pvl) toxin, mecA and mecC genes was detected using an eazyplex® MRSAplus kit (AmplexDiagnostics GmbH, Gars, Germany). CHG tolerance was observed in 13.15% of the isolates. An association between phenotypic and genotypic tolerance to CHG was observed. Phenotypic tolerance to CHG was associated with methicillin resistance but not with mupirocin resistance. Additionally, most of the CHG-tolerant strains were isolated from blood cultures. In conclusion, this work shed light on the prevalence of reduced susceptibility to CHG among S. aureus strains in Israel and on the characteristics of tolerant strains. CHG-tolerant strains were more common than methicillin-resistant ones in samples from invasive infections. Further research should be performed to evaluate risk factors for the development of CHG tolerance.

EUCAST rapid antimicrobial susceptibility testing (RAST): analytical performance and impact on patient management

Background The emergence of antibiotic-resistant species calls for fast and reliable phenotypic susceptibility testing to adapt clinical management as fast as possible. Objectives We assessed the real-life performance of EUCAST rapid antimicrobial susceptibility testing (RAST) and analysed its impact on patient management. Methods RAST was performed on clinical blood cultures containing Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa or Acinetobacter baumannii complex. Categorical agreement with VITEK2 was analysed. A pre-post quasi-experimental observational study was designed to compare antibiotic treatment in sepsis patients in the RAST patient group (n = 51) and a historical control cohort (n = 54). Results In total, 436 isolates, corresponding to 2314 disc diameters, were measured; 18.4% of these measurements were in the area of technical uncertainty. For the 81.6% categorical results, which could be compared, 94.7% were in agreement, whereas 5.3% of the results were not. In the RAST group, optimal therapy was initiated on the same day as blood culture positivity, while this was the case in the historical group after 24 h. In six cases, RAST allowed for rapid antibiotic escalation. The 30 day mortality rate was lower in the RAST group, although this was not statistically significant. Conclusions RAST provides a reliable tool to improve clinical management of sepsis patients by providing rapid phenotypic susceptibility data. While not necessarily being an instrument for de-escalation, especially in areas of low prevalence, early detection allows for timely coverage of resistant isolates. Thus, RAST significantly adds to successful antibiotic stewardship programmes.

The Genotype to Phenotype Dilemma: How Should Laboratories Approach Discordant Susceptibility Results?

Traditional culture-based methods for identification and antimicrobial susceptibility testing (AST) of bacteria on average takes 2-3 days. Syndromic molecular diagnostic panels have revolutionized clinical microbiology laboratories as they can simultaneously identify an organism and detect some of the most significant antimicrobial resistance (AMR) genes directly from positive blood culture broth or from various specimen types (e.g., whole blood, cerebrospinal fluid, respiratory specimens). The presence or absence of an AMR marker associated with a particular organism can be used to predict the phenotypic AST results to more rapidly guide therapy. Numerous studies have shown that genotypic susceptibility predictions by syndromic panels can improve patient outcomes. However, an important limitation of AMR marker detection to predict phenotype is the potential discrepancies that may arise upon performing phenotypic AST of the recovered organism in culture. The focus of this minireview is to address how clinical laboratories should interpret rapid molecular results from commercial platforms in relation to phenotypic AST. Stepwise approaches and solutions are provided to resolve discordant results between genotypic and phenotypic susceptibility results.

Occurrence and associated characteristics of a mutated ant(6’)-Ia gene among Enterococcus faecium strains expressing phenotypic susceptibility to high levels of streptomycin

ABSTRACTEnterococcal high-level resistance to streptomycin (HLR-S) (MIC ≥ 2000 µg/ml), conferred by production of a variety of aminoglycoside modifying enzymes (AMES), has been reported worldwide and a nucleotidyltransferase (ANT) enzyme encoded by the ant(6’)-Ia gene is frequently associated with this phenotype. However, during a study conducted by our group on whole genome sequencing (WGS) analyses of Enterococcus faecium isolates, we observed that 32 E. faecium strains identified as susceptible to high-levels of streptomycin by the disk diffusion method had the of ant(6’)-Ia gene annotated in their genomes. Antimicrobial susceptibility to streptomycin was reassessed by phenotypic testing and the presence of the ant(6’)-Ia gene was confirmed by PCR in all the isolates. Alignment of the ant(6’)-Ia gene with a reference sequence revealed a deletion of the first 48 nucleotides and four nonsynonymous mutations, leading to the substitution of a Glutamine to Methionine and an Aspartic Acid to Asparagine in the amino acid sequence. The protein structure was modelled by using the Phyre2 platform and the results indicated alterations in the N-terminus region leading to changes in the predicted binding site. Also, by searching the NCBI database we identified the genomes of 71 strains carrying the mutated gene. MLST analysis revealed that most strains carrying the mutated gene, including those described in this study belonged to hospital-adapted lineages, suggesting the occurrence of clonal dissemination of a subset of mutated isolates.HIGHLIGHTSThe presence of a mutated ant(6’)-Ia gene was identified among Enterococcus faecium isolates expressing phenotypic susceptibility to high levels of streptomycin.Nonsynonymous mutations and inactivating changes in the ant(6’)-Ia gene led to incongruities between phenotypes and genotypes.Alterations in the amino acid sequence had impacts on protein structure, with changes in the N-terminus region and the binding site.