Activity of polymyxin B combinations against genetically well-characterised Klebsiella pneumoniae producing NDM-1 and OXA-48-like carbapenemases.

BACKGROUND: Combination therapy can enhance the activity of available antibiotics against multidrug-resistant Gram-negative bacteria. This study assessed the effects of polymyxin B combinations against carbapenemase-producing Klebsiella pneumoniae (K. pneumoniae). METHODS: Twenty clinical K. pneumoniae strains producing NDM-1 (n = 8), OXA-48-like (n = 10), or both NDM-1 and OXA-48-like (n = 2) carbapenemases were used. Whole-genome sequencing was applied to detect resistance genes (e.g. encoding antibiotic-degrading enzymes) and sequence alterations influencing permeability or efflux. The activity of polymyxin B in combination with aztreonam, fosfomycin, meropenem, minocycline, or rifampicin was investigated in 24-hour time-lapse microscopy experiments. Endpoint samples were spotted on plates with and without polymyxin B at 4 x MIC to assess resistance development. Finally, associations between synergy and bacterial genetic traits were explored. RESULTS: Synergistic and bactericidal effects were observed with polymyxin B in combination with all other antibiotics: aztreonam (11 of 20 strains), fosfomycin (16 of 20), meropenem (10 of 20), minocycline (18 of 20), and rifampicin (15 of 20). Synergy was found with polymyxin B in combination with fosfomycin, minocycline, or rifampicin against all nine polymyxin-resistant strains. Wildtype mgrB was associated with polymyxin B and aztreonam synergy (P = 0.0499). An absence of arr-2 and arr-3 was associated with synergy of polymyxin B and rifampicin (P = 0.0260). Emergence of populations with reduced polymyxin B susceptibility was most frequently observed with aztreonam and meropenem. CONCLUSION: Combinations of polymyxin B and minocycline or rifampicin were most active against the tested NDM-1 and OXA-48-like-producing K. pneumoniae. Biologically plausible genotype-phenotype associations were found. Such information might accelerate the search for promising combinations and guide individualised treatment.

Interactions of Polymyxin B in Combination with Aztreonam, Minocycline, Meropenem, and Rifampin against Escherichia coli Producing NDM and OXA-48-Group Carbapenemases.

Carbapenemase-producing Enterobacterales pose an increasing medical threat. Combination therapy is often used for severe infections; however, there is little evidence supporting the optimal selection of drugs. This study aimed to determine the in vitro effects of polymyxin B combinations against carbapenemase-producing Escherichia coli. The interactions of polymyxin B in combination with aztreonam, meropenem, minocycline or rifampin against 20 clinical isolates of NDM and OXA-48-group-producing E. coli were evaluated using time-lapse microscopy; 24-h samples were spotted on plates with and without 4× MIC polymyxin B for viable counts. Whole-genome sequencing was applied to identify resistance genes and mutations. Finally, potential associations between combination effects and bacterial genotypes were assessed using Fisher's exact test. Synergistic and bactericidal effects were observed with polymyxin B and minocycline against 11/20 strains and with polymyxin B and rifampin against 9/20 strains. The combinations of polymyxin B and aztreonam or meropenem showed synergy against 2/20 strains. Negligible resistance development against polymyxin B was detected. Synergy with polymyxin B and minocycline was associated with genes involved in efflux (presence of tet[B], wild-type soxR, and the marB mutation H44Q) and lipopolysaccharide synthesis (eptA C27Y, lpxB mutations, and lpxK L323S). Synergy with polymyxin B and rifampin was associated with sequence variations in arnT, which plays a role in lipid A modification. Polymyxin B in combination with minocycline or rifampin frequently showed positive interactions against NDM- and OXA-48-group-producing E. coli. Synergy was associated with genes encoding efflux and components of the bacterial outer membrane.

High-risk clones of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae isolated from the University Hospital Establishment of Oran, Algeria (2011-2012).

The purpose of the study was to characterize the resistome, virulome, mobilome and Clustered Regularly Interspaced Short Palindromic Repeats-associated (CRISPR-Cas) system of extended-spectrum ß-lactamase producing Klebsiella pneumoniae (ESBL-KP) clinical isolates and to determine their phylogenetic relatedness. The isolates were from Algeria, isolated at the University Hospital Establishment of Oran, between 2011 and 2012. ESBL-KP isolates (n = 193) were screened for several antibiotic resistance genes (ARGs) using qPCR followed by Pulsed-Field Gel Electrophoresis (PFGE). Representative isolates were selected from PFGE clusters and subjected to whole-genome sequencing (WGS). Genomic characterization of the WGS data by studying prophages, CRISPR-Cas systems, Multi-Locus Sequence Typing (MLST), serotype, ARGs, virulence genes, plasmid replicons, and their pMLST. Phylogenetic and comparative genomic were done using core genome MLST and SNP-Based analysis. Generally, the ESBL-KP isolates were polyclonal. The whole genome sequences of nineteen isolates were taken of main PFGE clusters. Sixteen sequence types (ST) were found including high-risk clones ST14, ST23, ST37, and ST147. Serotypes K1 (n = 1), K2 (n = 2), K3 (n = 1), K31 (n = 1), K62 (n = 1), and K151 (n = 1) are associated with hyper-virulence. CRISPR-Cas system was found in 47.4%, typed I-E and I-E*. About ARGs, from 193 ESBL-KP, the majority of strains were multidrug-resistant, the CTX-M-1 enzyme was predominant (99%) and the prevalence of plasmid-mediated quinolone resistance (PMQR) genes was high with aac(6')-lb-cr (72.5%) and qnr's (65.8%). From 19 sequenced isolates we identified ESBL, AmpC, and carbapenemase genes: blaCTX-M-15 (n = 19), blaOXA-48 (n = 1), blaCMY-2 (n = 2), and blaCMY-16 (n = 2), as well as non-ESBL genes: qnrB1 (n = 12), qnrS1 (n = 1) and armA (n = 2). We found IncF, IncN, IncL/M, IncA/C2, and Col replicon types, at least once per isolate. This study is the first to report qnrS in ESBL-KP in Algeria. Our analysis shows the concerning co-existence of virulence and resistance genes and would support that genomic surveillance should be a high priority in the hospital environment.

Clinical and molecular characteristics of carbapenem non-susceptible Escherichia coli: A nationwide survey from Oman.

The prevalence of carbapenem-resistant Enterobacterales (CRE) in the Arabian Peninsula is predicted to be high, as suggested from published case reports. Of particular concern, is carbapenem-resistant E. coli (CR-EC), due to the importance of this species as a community pathogen. Herein, we conducted a comprehensive molecular characterization of putative CR-EC strains from Oman. We aim to establish a baseline for future molecular monitoring. We performed whole-genome sequencing (WGS) for 35 putative CR-EC. Isolates were obtained from patients at multiple centers in 2015. Genetic relatedness was investigated using several typing approaches such as MLST, SNP calling, phylogroup and CRISPR typing. Maxiuium likelihood SNP-tree was performed by RAxML after variant calling and removal of recombination regions with Snippy and Gubbins, respectively. Resistance genes, plasmid replicon types, virulence genes, and prophage were also characterised. The online databases CGE, CRISPRcasFinder, Phaster and EnteroBase were used for the in silico analyses. Screening for mutations in genes regulating the expression of porins and efflux pump as well as mutations lead to fluoroquinolones resistance were performed with CLC Genomics Workbench. The genetic diversity suggests a polyclonal population structure with 21 sequence types (ST), of which ST38 being the most prevalent (11%). SNPs analysis revealed possible transmission episodes. Whereas, CRISPR typing helped to spot outlier strains belonged to phylogroups other than B2 which was CRISPR-free. The virulent phylogroups B2 and D were detected in 4 and 9 isolates, respectively. In some strains bacteriophages acted as vectors for virulence genes. Regarding resistance to ß-lactam, 22 were carbapenemase producers, 3 carbapenem non-susceptible but carbapenemase-negative, 9 resistant to expanded-spectrum cephalosporins, and one isolate with susceptibility to cephalosporins and carbapenems. Thirteen out of the 22 (59%) carbapenemase-producing isolates were NDM and 7 (23%) were OXA-48-like which mirrors the situation in Indian subcontinent. Two isolates co-produced NDM and OXA-48-like enzymes. In total, 80% (28/35) were CTX-M-15 producers and 23% (8/35) featured AmpC. The high-risk subclones ST131-H30Rx/C2, ST410-H24RxC and ST1193-H64RxC were detected, the latter associated with NDM. To our knowledge, this is the first report of ST1193-H64Rx subclone with NDM. In conclusion, strains showed polyclonal population structure with OXA-48 and NDM as the only carbapenemases in CR-EC from Oman. We detected the high-risk subclone ST131-H30Rx/C2, ST410-H24RxC and ST1193-H64RxC. The latter was reported with carbapenemase gene for the first time here.

Effects of the Antimicrobial Peptide LL-37 and Innate Effector Mechanisms in Colistin-Resistant Klebsiella pneumoniae With mgrB Insertions.

BACKGROUND: Colistin is a polypeptide antibiotic drug that targets lipopolysaccharides in the outer membrane of Gram-negative bacteria. Inactivation of the mgrB-gene is a common mechanism behind colistin-resistance in Klebsiella pneumoniae (Kpn). Since colistin is a cyclic polypeptide, it may exhibit cross-resistance with the antimicrobial peptide LL-37, and with other innate effector mechanisms, but previous results are inconclusive. OBJECTIVE: To study potential cross-resistance between colistin and LL-37, as well as with other innate effector mechanisms, and to compare virulence of colistin-resistant and susceptible Kpn strains. MATERIALS/METHODS: Carbapenemase-producing Kpn from Oman (n = 17) were subjected to antimicrobial susceptibility testing and whole genome sequencing. Susceptibility to colistin and LL-37 was studied. The surface charge was determined by zeta-potential measurements and the morphology of treated bacteria was analyzed with electron microscopy. Bacterial survival was assessed in human whole blood and serum, as well as in a zebrafish infection-model. RESULTS: Genome-analysis revealed insertion-sequences in the mgrB gene, as a cause of colistin resistance in 8/17 isolates. Colistin-resistant (Col-R) isolates were found to be more resistant to LL-37 compared to colistin-susceptible (Col-S) isolates, but only at concentrations ≥50 µg/ml. There was no significant difference in surface charge between the isolates. The morphological changes were similar in both Col-R and Col-S isolates after exposure to LL-37. Finally, no survival difference between the Col-R and Col-S isolates was observed in whole blood or serum, or in zebrafish embryos. CONCLUSION: Cross-resistance between colistin and LL-37 was observed at elevated concentrations of LL-37. However, Col-R and Col-S isolates exhibited similar survival in serum and whole blood, and in a zebrafish infection-model, suggesting that cross-resistance most likely play a limited role during physiological conditions. However, it cannot be ruled out that the observed cross-resistance could be relevant in conditions where LL-37 levels reach high concentrations, such as during infection or inflammation.

The prospect of malaria elimination in the Arabian Peninsula: a population genetic approach.

BACKGROUND: In the Arabian Peninsula malaria control is progressing steadily, backed by adequate logistic and political support. As a result, transmission has been interrupted throughout the region, with exception of limited sites in Yemen and Saudi Arabia. Here we examined Plasmodium falciparum parasites in these sites to assess if the above success has limited diversity and gene flow. METHODS: We examined 108 P. falciparum isolates in three sites in Yemen (Taiz, Dhamar and Hodeidah) and 91 isolates from Saudi Arabia (Jazan). Nine microsatellites were analyzed for allelic diversity, multi-locus haplotype and inter-population differentiation. RESULTS: Diversity at each locus (unbiased heterozygosity [H]) was relatively lower in Yemen; (Hodeidah, H=0.615, Taiz, H=0.66, Dhamar, H=0.481), compared to Saudi Arabia (Jazan, H=0.76). Microsatellites were distributed widely and private alleles, detected in a single population, were rare. Pairwise comparisons revealed that parasites population in Dhamar was relatively distanced (FST=0.19). However, Taiz (Yemen) (FST=0.065) and Hodeidah (FST=0.107) populations were closer to that in Jazan (Saudi Arabia). Nonetheless, parasites in the four sites can be considered as one population. CONCLUSION: Although malaria risk in Saudi Arabia has been cut considerably, the extent of diversity and parasite genetic structure are indicative of a large population size. Elimination strategy should target demographic factors that favor parasite dispersal and flow of imported malaria.

Genetic diversity of Plasmodium falciparum and distribution of drug resistance haplotypes in Yemen.

BACKGROUND: Despite evident success of malaria control in many sites in the Arabian Peninsula, malaria remains endemic in a few spots, in Yemen and south-west of Saudi Arabia. In addition to local transmission, imported malaria sustains an extra source of parasites that can challenge the strengths of local control strategies. This study examined the genetic diversity of Plasmodium falciparum in Yemen and mutations of drug resistant genes, to elucidate parasite structure and distribution of drug resistance genotypes in the region. METHODS: Five polymorphic loci (MSP-2, Pfg377 and three microsatellites on chromosome 8) not involved in anti-malarial drug resistance, and four drug resistant genes (pfcrt, pfmdr1, dhfr and dhps) were genotyped in 108 P. falciparum isolates collected in three sites in Yemen: Dhamar, Hodeidah and Taiz. RESULTS: High diversity was seen in non-drug genes, pfg377 (He = 0.66), msp-2 (He = 0.80) and three microsatellites on chr 8, 7.7 kb (He = 0.88), 4.3 kb (He = 0.77) and 0.8 kb (He = 0.71). There was a high level of mixed-genotype infections (57%), with an average 1.8 genotypes per patient. No linkage disequilibrium was seen between drug resistant genes and the non-drug markers (p < 0.05). Genetic differentiation between populations was low (most pair-wise FST values <0.03), indicating extensive gene flow between the parasites in the three sites. CONCLUSION: The high diversity of P. falciparum in Yemen is indicative of a large parasite reservoir, which represents a challenge to control efforts. The presence of two distinct pfcrt genotype, CVIET and SVMNT, suggests that chloroquine resistance can possibly be related to a migratory path from Africa and Asia. The absence of the triple mutant dhfr genotype (IRN) and dhps mutations supports the use of artesunate + sulphadoxine-pyrimethamine as first-line therapy. However, the prevalent pfmdr1 genotype NFSND [21%] has previously been associated with tolerance/resistance response to artemisinin combination therapy (ACT). Regular surveys are, therefore, important to monitor spread of pfmdr1 and dhfr mutations and response to ACT.

Source of drug resistant Plasmodium falciparum in a potential malaria elimination site in Saudi Arabia.

A major challenge to the success of malaria control program in Saudi Arabia is the high influx of expatriates and holy visitors from malaria endemic countries. In the present study we examined whether drug resistant parasite genotypes reported in Jazan region, southwest of Saudi Arabia are imported or developed locally. We examined 178 Plasmodium falciparum isolates for alleles of dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr), associated with Sulfadoxine-Pyrimethamine (SP) resistance, and three microsatellites flanking each gene. In addition, we examined a neutral polymorphic gene (Pfg377). We compared the dhfr and dhps haplotypes in Jazan, using network analysis, to an existing similar data set of 94 P. falciparum isolates from eastern Sudan. In Jazan, double mutant dhfr allele (51I, 108N) occurred with a prevalence of 33%. The vast majority (99%) of dhps were wild-type alleles. The mean expected heterozygosity (H(e)) of microsatellites around mutant dhfr alleles (H(e)=0.312; n=60) was lower (P ≤ 0.05) than that around the wild-type allele (H(e)=0.834; n=116). Also, the mutant dhfr isolates showed high H(e) for dhps (H(e)=0.80) and the non-drug resistance locus Pfg377 (H(e)=0.63) indicative of selection for mutant dhfr only. The predominant double mutant dhfr haplotype in Jazan (73%), was prevalent among P. falciparum in east Africa. Network analysis suggests the mutant haplotype of dhfr gene was possibly introduced into Jazan from East Africa. The absence of mutations in dhps as well as triple mutant dhfr haplotype associated with SP failure support the current use of SP as a partner with artesunate as a first line therapy in Saudi Arabia. However, the close relationship between the major mutant dhfr haplotype in Sudan and Saudi Arabia, favour the hypothesis of recent migration as a source of the major resistant dhfr lineage. Thus, regular monitoring of the dhfr and dhps haplotypes is of high priority to guard possible importation of high level SP resistant lineages.

Distribution of drug resistance genotypes in Plasmodium falciparum in an area of limited parasite diversity in Saudi Arabia.

Two hundred and three Plasmodium falciparum isolates from Jazan area, southwest Saudi Arabia, were typed for Pfcrt, Pfmdr1, dhps, and dhfr mutations associated with resistance to chloroquine, mefloquine, halofantrine, artemisinin, sulfadoxine-pyrimethamine, and the neutral polymorphic gene Pfg377. A large proportion (33%) of isolates harbored double mutant dhfr genotype (51I,59C,108N). However, only one isolate contained mutation dhps-437G. For Pfcrt, almost all examined isolates (163; 99%) harbored the mutant genotype (72C,73V,74I,75E,76T), whereas only 49 (31%) contained the mutant Pfmdr1 genotype (86Y,184F,1034S,1042N), 109 (66%) harbored the single mutant genotype (86N,184F,1034S,1042N), and no mutations were seen in codons 1034, 1042, and 1246. Nonetheless, three new single-nucleotide polymorphisms were detected at codons 182, 192, and 102. No differences were seen in distribution of drug resistance genes among Saudis and expatriates. There was a limited multiplicity (5%), mean number of clones (1.05), and two dominant multilocus genotypes among infected individuals in Jazan. A pattern consistent with limited cross-mating and recombination among local parasite was apparent.