Rapid polymyxin NP test for the detection of polymyxin resistance mediated by the MCR-1/MCR-2 genes

The Rapid Polymyxin NP test has been recently developed to rapidly detect polymyxin resistance in Enterobacteriaceae. Here we evaluated this test for detecting MCR- 1/MCR-2-producing Enterobacteriaceae using a collection of 70 non-redundant strains either recovered from the environment, animals, or humans. Sensitivity and specificity were found to be 100%

Antibiotic resistance in the environment, with particular reference to MRSA

The introduction of β-lactam antibiotics (penicillins and cephalosporins) in the 1940s and 1950s probably represents the most dramatic event in the battle against infection in human medicine. Even before widespread global use of penicillin, resistance was already recorded. E. coli producing a penicillinase was reported in Nature in 1940 (Abraham, 1940) and soon after a similar penicillinase was discovered in Staphylococcus aureus (Kirby, 1944). The appearance of these genes, so quickly after the discovery and before the widespread introduction of penicillin, clearly shows that the resistance genes pre-dated clinical use of the antibiotic itself

An improved PKPD modeling approach to characterize the pharmacodynamic interaction over time between ceftazidime/avibactam and colistin from in vitro time-kill experiments against multidrug-resistant Klebsiella pneumoniae isolates.

In contrast to the checkerboard method, bactericidal experiments [time-kill curves (TKCs)] allow an assessment of pharmacodynamic (PD) interactions over time. However, TKCs in combination pose interpretation problems. The objective of this study was to characterize the PD interaction over time between ceftazidime/avibactam (CZA) and colistin (CST) using TKC against four multidrug-resistant Klebsiella pneumoniae susceptible to both antibiotics and expressing a widespread carbapenemase determinant KPC-3. In vitro TKCs were performed and analyzed using pharmacokinetic/pharmacodynamic (PKPD) modeling. The general pharmacodynamic interaction model was used to characterize PD interactions between drugs. The 95% confidence intervals (95%CIs) of the expected additivity and of the observed interaction were built using parametric bootstraps and compared to evaluate the in vitro PD interaction over time. Further simulations were conducted to investigate the effect of the combination at varying concentrations typically observed in patients. Regrowth was observed in TKCs at high concentrations of drugs alone [from 4 to 32× minimum inhibitory concentrations (MIC)], while the combination systematically prevented the regrowth at concentrations close to the MIC. Significant synergy or antagonism were observed under specific conditions but overall 95%CIs overlapped widely over time indicating an additive interaction between antibiotics. Moreover, simulations of typical PK profile at standard dosages indicated that the interaction should be additive in clinical conditions. The nature of the PD interaction varied with time and concentration in TKC. Against the four K. pneumoniae isolates, the bactericidal effect of CZA + CST combination was predicted to be additive and to prevent the emergence of resistance at clinical concentrations

PKPD modeling of the inoculum effect of combined ceftazidime/avibactam and colistin against KPC-3 Klebsiella pneumoniae isolate.

The inoculum effect (IE) characterizes a decrease in the antimicrobial effect of antibiotics with increasing inoculum. To face antimicrobial resistance, antibiotic combinations are progressively used. In this context, the effect of combination may be affected by IE, especially drugs for which an IE has been described. The objective was to characterize the IE of a carbapenemase (KPC-3) Klebsiella pneumoniae isolate on the combination of ceftazidime/avibactam (CZA) and colistin (CST). In vitro time-kill curves with single and combined drugs were performed at four different inocula. The IE of each drug was described using pharmacokinetic/pharmacodynamic modeling, and interactions on IE were investigated with the general pharmacodynamic interaction model when drugs were combined. The IE was assessed by evaluating the significance of the parameters associated with the IE model compared to the no IE model and by comparing the CFU counts over time predicted with the IE model vs the no IE model. Rapid bacterial killing was observed at 10 <sup>4</sup> CFU/mL. For both 5·10 <sup>5</sup> and 10 <sup>7</sup> CFU/mL inocula, initial decays followed by re-growth were observed with drugs alone, while the combination prevented the emergence of resistance. Eradication was never achieved at 10 <sup>8</sup> CFU/mL. The IE was best modeled as a reduction of CZA maximum bactericidal effect and as an increase in CST EC <sub>50</sub> with increasing inoculum. However, no interaction between IEs was significant, meaning that CST did not modify the IE of CZA and inversely. IE may be important at least as demonstrated by in vitro antibiotic combination studies

Acquisition of broad-spectrum cephalosporin resistance leading to colistin resistance in Klebsiella pneumoniae

An extended-spectrum β-lactamase (ESBL)-producing and colistin-resistant Klebsiella pneumoniae clinical isolate was recovered from a patient who was treated with cefotaxime. This isolate harbored a blaCTX-M-15 ESBL gene that was associated with an ISEcp1 insertion sequence. Transposition of that tandem occurred within the chromosomal mgrB gene, leading to inactivation of the mgrB gene and consequently to acquired resistance to colistin. We showed here a coselection of colistin resistance as a result of a broad- spectrum cephalosporin selective pressure

CTX-M and Plasmid-mediated AmpC-Producing Enterobacteriaceae, Singapore

10.3201/eid1006.030726Emerging Infectious Diseases1061172-117

Interactions of Aspergillus fumigatus and Stenotrophomonas maltophilia in an in vitro Mixed Biofilm Model: Does the Strain Matter?

Introduction:Aspergillus fumigatus (Af) and Stenotrophomonas maltophilia (Sm) are pathogenic microorganisms, which coexist in the respiratory tract of cystic fibrosis (CF) patients. We recently developed an in vitro model of mixed biofilm associating Af ATCC 13073-GFP (Af13073) and Sm ATCC 13637 (Sm13637) and described an antibiosis effect. The present study aim was to assess the antibiosis of Sm on Af using different strains and to analyze the potential synergistic virulence of these strains in an in vivo Galleria mellonella model.Methods: The effect of Sm13637 was evaluated on eight Af strains and the effect of nine Sm strains was evaluated on Af13073. The strains originated from clinical cases (human and animal) and from environment. Fungal and bacterial inocula were simultaneously inoculated to initiate mixed biofilm formation. Fungal growth inhibition was analyzed by qPCR and CLSM and the fungal cell wall modifications by TEM analysis. The virulence of different Sm strains was assessed in association with Af in G. mellonella larvae.Results: All strains of Af and Sm were able to produce single and mixed biofilms. The antibiosis effect of Sm13637 was similar whatever the Af strain tested. On the other hand, the antibiosis effect of Sm strains was bacterial-fitness and strain dependent. One strain (1/9) originated from animal clinical case was never able to induce an antibiosis, even with high bacterial concentration. In the G. mellonella model, co-inoculation with Sm13637 and Af13073 showed synergism since the mortality was 50%, i.e., more than the summed virulence of both.Conclusion: Human clinical strains of Sm yielded in higher antibiosis effect on Af and in a thinner mixed biofilm, probably due to an adaptive effect of these strains. Further research covering Af increased wall thickness in the presence of Sm strains, and its correlation with modified antifungal susceptibility is encouraged in patients with chronic respiratory infections by these 2 microorganisms

Are animals a source of Stenotrophomonas maltophilia in human infections? Contributions of a nationwide molecular study

Stenotrophomonas maltophilia (Sm) is an archetypal environmental opportunistic bacterium responsible for health care-associated infections. The role of animals in human Sm infections is unknown. This study aims to reveal the genetic and phylogenetic relationships between pathogenic strains of Sm, both animal and human, and identify a putative role for animals as a reservoir in human infection. We phenotypically and genotypically characterized 61 Sm strains responsible for animal infections (mainly respiratory tract infections in horses) from a French nationwide veterinary laboratory network. We tested antimicrobial susceptibility and performed MLST and genogrouping using the concatenation of the seven housekeeping genes from the original MLST scheme. Excluding the eight untypeable strains owing to the lack of gene amplification, only 10 out of the 53 strains yielded a known ST (ST5, ST39, ST162, ST8, ST27, ST126, ST131). The genogroup distribution highlighted not only genogroups (genogroups 5 and 9) comprised exclusively of animal strains but also genogroups shared by human and animal strains. Interestingly, these shared genogroups were primarily groups 2 and 6, which have previously been identified as the two most frequent genogroups among human-pathogenic Sm strains, especially among respiratory pathogens. The antimicrobial susceptibility testing underlined the presence of acquired resistance: 18.8 and 7.5% of the tested isolates were resistant to the sulfonamide-trimethoprim combination and ciprofloxacin, respectively. Animal strains of Sm shared phylogenetic traits with some of the most successful human strains. The exact relationships between the human and animal strains, and the genetic support of these common traits, need to be determined

Colistin resistance in Parisian inpatient faecal Escherichia coli as the result of two distinct evolutionary pathways

Beyond plasmid-encoded resistance (mcr genes) prevalence in strain collections, large epidemiological studies to estimate the human burden of colistin-resistant Escherichia coli gut carriage are lacking.Objectives: To evaluate the prevalence of colistin-resistant E. coli carriage in inpatients and decipher the molecular support of resistance and the genetic background of the strains.Methods: During a 3 month period in 2017, we prospectively screened patients in six Parisian hospitals for rectal carriage of colistin-resistant E. coli using a selective medium, a biochemical confirmatory test and MIC determination. WGS of the resistant strains and their corresponding plasmids was performed.Results: Among the 1217 screened patients, 153 colistin-resistant E. coli strains were isolated from 152 patients (12.5%). The mcr- 1 gene was identified in only seven isolates (4.6%) on different plasmid scaffolds. The genetic background of these MCR-1 producers argued for an animal origin. Conversely, the remaining 146 colistin-resistant E. coli exhibited a phylogenetic distribution corresponding to human gut commensal/clinical population structure (B2 and D phylogroup predominance); 72.6% of those isolates harboured convergent mutations in the PmrA and PmrB proteins, constituting a two-component system shown to be associated with colistin resistance.Conclusions: We showed that the occurrence at a high rate of colistin resistance in human faecal E. coli is the result of two distinct evolutionary pathways, i.e. the occurrence of chromosomal mutations in an endogenous E. coli population and the rare acquisition of exogenous mcr-1- bearing strains probably of animal origin. The involved selective pressures need to be identified in order to develop preventative strategies

mcr-9, An inducible gene encoding an acquired phosphoethanolamine transferase in Escherichia coli, and its origin

The plasmid-located mcr-9 gene, encoding a putative phosphoethanolamine transferase, was identified in a colistin-resistant human fecal Escherichia coli strain belonging to a very rare phylogroup, the D-ST69-O15:H6 clone. This MCR-9 protein shares 33% to 65% identity with the other plasmid-encoded MCR-type enzymes identified (MCR-1 to -8) that have been found as sources of acquired resistance to polymyxins in Enterobacteriaceae. Analysis of the lipopolysaccharide of the MCR-9- producing isolate revealed a function similar to that of MCR-1 by adding a phosphoethanolamine group to lipid A and subsequently modifying the structure of the lipopolysaccharide. However, a minor impact on susceptibility to polymyxins was noticed once the mcr-9 gene was cloned and produced in an E. coli K-12-derived strain. Nevertheless, we showed here that subinhibitory concentrations of colistin induced the expression of the mcr-9 gene, leading to increased MIC levels. This inducible expression was mediated by a two-component regulatory system encoded by the qseC and qseB genes located downstream of mcr-9. Genetic analysis showed that the mcr-9 gene was carried by an IncHI2 plasmid. In silico analysis revealed that the plasmid-encoded MCR-9 shared significant amino acid identity (ca. 80%) with the chromosomally encoded MCR-like proteins from Buttiauxella spp. In particular, Buttiauxella gaviniae was found to harbor a gene encoding MCR-BG, sharing 84% identity with MCR-9. That gene was neither expressed nor inducible in its original host, which was fully susceptible to polymyxins. This work showed that mcr genes may circulate silently and remain undetected unless induced by colistin