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604500.9941Lacticaseicin 30 and Colistin as a Promising Antibiotic Formulation against Gram-Negative β-Lactamase-Producing Strains and Colistin-Resistant Strains. Antimicrobial resistance is a global health concern across the world and it is foreseen to swell if no actions are taken now. To help curbing this well announced crisis different strategies are announced, and these include the use of antimicrobial peptides (AMP), which are remarkable molecules known for their killing activities towards pathogenic bacteria. Bacteriocins are ribosomally synthesized AMP produced by almost all prokaryotic lineages. Bacteriocins, unlike antibiotics, offer a set of advantages in terms of cytotoxicity towards eukaryotic cells, their mode of action, cross-resistance and impact of microbiota content. Most known bacteriocins are produced by Gram-positive bacteria, and specifically by lactic acid bacteria (LAB). LAB-bacteriocins were steadily reported and characterized for their activity against genetically related Gram-positive bacteria, and seldom against Gram-negative bacteria. The aim of this study is to show that lacticaseicin 30, which is one of the bacteriocins produced by Lacticaseibacillus paracasei CNCM I-5369, is active against Gram-negative clinical strains (Salmonella enterica Enteritidis H10, S. enterica Typhimurium H97, Enterobacter cloacae H51, Escherichia coli H45, E. coli H51, E. coli H66, Klebsiella oxytoca H40, K. pneumoniae H71, K. variicola H77, K. pneumoniae H79, K. pneumoniae H79), whereas antibiotics failed. In addition, lacticaseicin 30 and colistin enabled synergistic interactions towards the aforementioned target Gram-negative clinical strains. Further, the combinations of lacticaseicin 30 and colistin prompted a drastic downregulation of mcr-1 and mcr-9 genes, which are associated with the colistin resistance phenotypes of these clinical strains. This report shows that lacticaseicin 30 is active against Gram-negative clinical strains carrying a rainbow of mcr genes, and the combination of these antimicrobials constitutes a promising therapeutic option that needs to be further exploited.202135052897
514410.9939Genomic analysis of the nomenclatural type strain of the nematode-associated entomopathogenic bacterium Providencia vermicola. BACKGROUND: Enterobacteria of the genus Providencia are mainly known as opportunistic human pathogens but have been isolated from highly diverse natural environments. The species Providencia vermicola comprises insect pathogenic bacteria carried by entomoparasitic nematodes and is investigated as a possible insect biocontrol agent. The recent publication of several genome sequences from bacteria assigned to this species has given rise to inconsistent preliminary results. RESULTS: The genome of the nematode-derived P. vermicola type strain DSM_17385 has been assembled into a 4.2 Mb sequence comprising 5 scaffolds and 13 contigs. A total of 3969 protein-encoding genes were identified. Multilocus sequence typing with different marker sets revealed that none of the previously published presumed P. vermicola genomes represents this taxonomic species. Comparative genomic analysis has confirmed a close phylogenetic relationship of P. vermicola to the P. rettgeri species complex. P. vermicola DSM_17385 carries a type III secretion system (T3SS-1) with probable function in host cell invasion or intracellular survival. Potentially antibiotic resistance-associated genes comprising numerous efflux pumps and point-mutated house-keeping genes, have been identified across the P. vermicola genome. A single small (3.7 kb) plasmid identified, pPVER1, structurally belongs to the qnrD-type family of fluoroquinolone resistance conferring plasmids that is prominent in Providencia and Proteus bacteria, but lacks the qnrD resistance gene. CONCLUSIONS: The sequence reported represents the first well-supported published genome for the taxonomic species P. vermicola to be used as reference in further comparative genomics studies on Providencia bacteria. Due to a striking difference in the type of injectisome encoded by the respective genomes, P. vermicola might operate a fundamentally different mechanism of entomopathogenicity when compared to insect-pathogenic Providencia sneebia or Providencia burhodogranariea. The complete absence of antibiotic resistance gene carrying plasmids or mobile genetic elements as those causing multi drug resistance phenomena in clinical Providencia strains, is consistent with the invertebrate pathogen P. vermicola being in its natural environment efficiently excluded from the propagation routes of multidrug resistance (MDR) carrying genetic elements operating between human pathogens. Susceptibility to MDR plasmid acquisition will likely become a major criterion in the evaluation of P. vermicola for potential applications in biological pest control.202134598677
503320.9936A novel gene linked to Imipenem resistance in E. coli isolate lacking known Imipenem-resistance genes. Imipenem-resistant Escherichia coli strains represent a growing public health concern, posing a threat due to their resistance to last-resort antibiotics. Here, we present the discovery of the Imipenem-Linked Resistance Gene VIN (ILR-VIN) within E. coli isolates from Vietnam, revealing its absence in non-resistant E. coli and local bacterial species. ILR-VIN constitutes a previously unrecognized genetic element potentially linked to Imipenem resistance, with notable prevalence in Vietnamese E. coli strains.We conducted an in-depth examination of the genetic basis of Carbapenem resistance in E. coli strains causing urinary tract infections. In a set of 47 UTI strains, we identified five displaying Imipenem resistance, with four of them carrying known resistance genes. Interestingly, ECV219, despite exhibiting Imipenem resistance, lacked known resistance genes, suggesting an unreported resistance mechanism. Comparative genetic analysis revealed distinct genes in ECV219, indicating a novel Imipenem resistance gene. To assess its function, we conducted transformation experiments in E. coli Rosetta™(DE3)pLysS and performed bioinformatics analyses using BLASTp, InterProScan, and Pfam to characterize the gene's structure and potential functions.Our study identifies ILR-VIN as a novel gene linked to Imipenem resistance in E. coli isolate lacking known Imipenem-resistance genes. Experimental evidence confirmed that ILR-VIN expression enhances bacterial survival under Imipenem stress, providing direct evidence of its role in resistance. This discovery highlights the importance of ongoing research into antibiotic resistance genes to develop effective treatment strategies against antibiotic-resistant bacterial infections.202540097570
153630.9936Complete Genetic Analysis of Plasmids Carried by Two Nonclonal bla(NDM-5)- and mcr-1-Bearing Escherichia coli Strains: Insight into Plasmid Transmission among Foodborne Bacteria. Our objective was to characterize the genetic features of plasmids harbored by two genetically related, MCR-1 and NDM-5-producing Escherichia coli strains recovered from a chicken meat sample. The genetic profiles of all plasmids harbored by the two test strains, namely, 1106 and 1107, were determined by whole-genome sequencing, S1-pulsed-field gel electrophoresis (PFGE), Southern hybridization, and bioinformatics analysis. The transferability of plasmids harbored by the two strains was assessed by filter mating assay. Strains 1106 and 1107 were resistant to almost all the antibiotics, including colistin and fosfomycin, but remained susceptible to amikacin and tigecycline. The plasmids of p1107-NDM-5 and p1106-NDM-5 both contain a class I integron which lacks the ISAba125 element. The backbone of p1106-IncFII exhibited a high degree of similarity with that of p1106-NDM-5 and p1107-NDM-5, implying that events of plasmid fusion and resolution were involved in the formation of the two plasmids. The plasmids p1106-IncHI2MCR and p1107-IncHI2MCR belong to an IncHI2 replicon type, with three copies of ISApl1 being observed in p1106-IncHI2MCR, implying that the mcr-1 gene was transferable among bacteria that reside in the same food matrix. In this study, p1106-IncFIB, p1107-99K, p1107-111K, and p1107-118K were all found to be phage-like plasmids, with p1106-IncFIB and p1107-118K containing several virulence genes, including iroBCDEN, iucABCD, sitABCD, hlyF, and iss. Surprisingly, resistance genes such as aph(3')-Ia, sul3, and aac(3')-IId could also be found in p1107-118K, but resistance genes were not detected in other phage-like plasmids. In conclusion, enhanced surveillance is required to monitor and control the dissemination of various resistance determinants among foodborne pathogens. IMPORTANCE Carbapenem and colistin are last-resort antibiotics used to treat serious clinical infections caused by multidrug-resistant (MDR) bacterial pathogens. Plasmids encoding resistance to carbapenems and colistin have been reported in clinical pathogens in recent years, and yet few studies reported cocarriage of mcr and bla(NDM) genes in Escherichia coli strains of food origin. How plasmids encoding these two important resistance determinants are being evolved and transmitted in bacterial pathogens is not well understood. In this study, we investigated the genetic features of plasmids harbored by two nonclonal, mcr-1- and bla(NDM-5)-bearing E. coli strains (1106 and 1107) recovered from a fresh chicken meat sample to understand and provide evidence of the level and dynamics of MDR plasmid transmission. Our data confirmed that active plasmid fusion and resolution events were involved in the formation of plasmids that harbor multiple resistance genes, which provide insights into the further control of plasmid evolution in bacterial pathogens.202134468190
587940.9935Isolation and phenotypic and genomic characterization of Tetragenococcus spp. from two Spanish traditional blue-veined cheeses made of raw milk. High throughput sequencing has recently revealed the presence of Tetragenococcus-related DNA sequences in dairy environments such as brine and cheeses. In the present work, a selective medium was developed to isolate Tetragenococcus spp. from two ripened, traditional, Spanish, blue-veined cheese varieties made from raw milk. The strains recovered belonged to either Tetragenococcus koreensis or Tetragenococcus halophilus species. Twenty of these isolates (15 of T. koreensis and 5 of T. halophilus) were then subjected to a battery of phenotypic and genetic tests, and six strains (4 T. koreensis and 2 T. halophilus) to genome sequencing. Wide genetic and phenotypic diversity was noted. All strains grew poorly in milk, producing small quantities of lactic and acetic acids. Most strains used lactose as a carbon source and ferment milk citrate. In agreement, genome analysis detected in the genome of the six strains analyzed gene clusters harboring several lactose/galactose-related genes and genes encoding citrate metabolic enzymes (permease, citrate lyase, and oxaloacetate decarboxylase). Most of the tested strains were resistant to erythromycin and clindamycin, and a few to other antimicrobial agents, but neither known mutations nor acquired genes conferring resistance to antibiotics were identified in their genomes. Neither were genes coding for pathogenicity or virulence factors detected. Decarboxylase-encoding genes involved in biogenic amine production were not identified, in keeping with the strains' negative biogenic amine-producer phenotype. Genome comparison revealed vast arrays of genes (similar in number to those described in other lactic acid bacteria) coding for components of proteolytic and lipolytic systems. Tetragenococcus strains showing desirable traits plus the absence of detrimental features might be exploitable in the form of secondary, adjunct or ripening cultures to ensure the typical bouquet of traditional blue-veined cheeses is obtained, or to diversify the final flavor in other varieties.202235427955
246150.9935In Vitro Activity of Cefiderocol on Multiresistant Bacterial Strains and Genomic Analysis of Two Cefiderocol Resistant Strains. Cefiderocol is a new siderophore cephalosporin that is effective against multidrug-resistant Gram-negative bacteria, including carbapenem-resistant strains. The aim of this study was to evaluate the activity of this new antimicrobial agent against a collection of pathogens using broth microdilution assays and to analyze the possible mechanism of cefiderocol resistance in two resistant Klebsiella pneumoniae isolates. One hundred and ten isolates were tested, comprising 67 Enterobacterales, two Acinetobacter baumannii, one Achromobacter xylosoxidans, 33 Pseudomonas aeruginosa and seven Stenotrophomonas maltophilia. Cefiderocol showed good in vitro activity, with an MIC < 2 μg/mL, and was able to inhibit 94% of the tested isolates. We observed a resistance rate of 6%. The resistant isolates consisted of six Klebsiella pneumoniae and one Escherichia coli, leading to a resistance rate of 10.4% among the Enterobacterales. Whole-genome sequencing analysis was performed on two cefiderocol-resistant Klebsiella pneumoniae isolates to investigate the possible mutations responsible for the observed resistance. Both strains belonged to ST383 and harbored different resistant and virulence genes. The analysis of genes involved in iron uptake and transport showed the presence of different mutations located in fhuA, fepA, iutA, cirA, sitC, apbC, fepG, fepC, fetB, yicI, yicJ, and yicL. Furthermore, for the first time, to the best of our knowledge, we described two Klebsiella pneumoniae isolates that synthesize a truncated fecA protein due to the transition from G to A, leading to a premature stop codon in the amino acid position 569, and a TonB protein carrying a 4-amino acid insertion (PKPK) after Lysine 103. In conclusion, our data show that cefiderocol is an effective drug against multidrug-resistant Gram-negative bacteria. However, the higher resistance rate observed in Enterobacterales underlines the need for active surveillance to limit the spread of these pathogens and to avoid the risks associated with the emergence of resistance to new drugs.202337107147
153960.9935WGS of a lytic phage targeting biofilm-forming carbapenem-resistant Klebsiella pneumoniae prevalent in a tertiary healthcare setup. Carbapenem-resistant Enterobacteriaceae (CRE) are listed as a priority-one critical pathogen category by the WHO because of their abysmal treatment outcomes owing to antibiotic inefficiency. Among CRE, Klebsiella pneumoniae is prevalent in acquiring resistance genes and withstanding the last-resort drugs. Additionally, its ability to form robust biofilms further exacerbates the treatment challenges. The escalating resistance and recalcitrance of biofilm-residing bacteria against standard antibiotic treatments demand an alternative to antibiotics. Phages, being nature-tailored, are a never-ending arsenal against the bacteria because of their capacity to lyse bacteria rapidly and co-evolve with bacteria. In our study, we isolated K. pneumoniae from patients at Madras Medical Mission Hospital (MMMH), India, and assessed their antibiogram profiles, presence of carbapenemase genes, and biofilm-forming abilities. 100 % of the strains were extended-spectrum beta-lactamase producing, multidrug-resistant (ESBL-MDR), with 95 % harbouring carbapenemase genes. Among the isolates, 65 % were strong biofilm formers, and the rest were moderate. Further, we isolated a bacteriophage, SAKp11, from the hospital sewage, which was able to lyse 62 out of 167 clinical isolates and successfully reduced 99.99 % viable bacterial cells of the 24-h-old biofilm of strong biofilm forming MDR K. pneumoniae strains. Whole genome analysis revealed that SAKp11, with a genome size of 59,338bp, belonged to the Casjensviridae family, one of the less explored bacteriophage families. Comprehensive characterization of SAKp11 indicated its suitability for therapeutic use. Our study highlights the severity of drug-resistant K. pneumoniae in Indian healthcare and the inadequacy of current antibiotics, underscoring the potential of phages as an alternative therapeutic option.202540348211
478170.9935Isolation and Characterization of New Bacteriophages against Staphylococcal Clinical Isolates from Diabetic Foot Ulcers. Staphylococcus sp. is the most common bacterial genus in infections related to diabetic foot ulcers (DFUs). The emergence of multidrug-resistant bacteria places a serious burden on public health systems. Phage therapy is an alternative treatment to antibiotics, overcoming the issue of antibiotic resistance. In this study, six phages (SAVM01 to SAVM06) were isolated from effluents and were used against a panel of staphylococcal clinical samples isolated from DFUs. A genomic analysis revealed that the phages belonged to the Herelleviridae family, with sequences similar to those of the Kayvirus genus. No lysogeny-associated genes, known virulence or drug resistance genes were identified in the phage genomes. The phages displayed a strong lytic and antibiofilm activity against DFU clinical isolates, as well as against opportunistic pathogenic coagulase-negative staphylococci. The results presented here suggest that these phages could be effective biocontrol agents against staphylococcal clinical isolates from DFUs.202338140529
166080.9934Emergence of Plasmid-Mediated Fosfomycin-Resistance Genes among Escherichia coli Isolates, France. FosA, a glutathione S-transferase that inactivates fosfomycin, has been reported as the cause of enzymatic resistance to fosfomycin. We show that multiple lineages of FosA-producing extended spectrum β-lactamase Escherichia coli have circulated in France since 2012, potentially reducing the efficacy of fosfomycin in treating infections with antimicrobial drug-resistant gram-negative bacilli.201728820368
494690.9934Escherichia coli B-Strains Are Intrinsically Resistant to Colistin and Not Suitable for Characterization and Identification of mcr Genes. Antimicrobial resistance is an increasing threat to human and animal health. Due to the rise of multi-, extensive, and pandrug resistance, last resort antibiotics, such as colistin, are extremely important in human medicine. While the distribution of colistin resistance genes can be tracked through sequencing methods, phenotypic characterization of putative antimicrobial resistance (AMR) genes is still important to confirm the phenotype conferred by different genes. While heterologous expression of AMR genes (e.g., in Escherichia coli) is a common approach, so far, no standard methods for heterologous expression and characterization of mcr genes exist. E. coli B-strains, designed for optimum protein expression, are frequently utilized. Here, we report that four E. coli B-strains are intrinsically resistant to colistin (MIC 8-16 μg/mL). The three tested B-strains that encode T7 RNA polymerase show growth defects when transformed with empty or mcr-expressing pET17b plasmids and grown in the presence of IPTG; K-12 or B-strains without T7 RNA polymerase do not show these growth defects. E. coli SHuffle T7 express carrying empty pET17b also skips wells in colistin MIC assays in the presence of IPTG. These phenotypes could explain why B-strains were erroneously reported as colistin susceptible. Analysis of existing genome data identified one nonsynonymous change in each pmrA and pmrB in all four E. coli B-strains; the E121K change in PmrB has previously been linked to intrinsic colistin resistance. We conclude that E. coli B-strains are not appropriate heterologous expression hosts for identification and characterization of mcr genes. IMPORTANCE Given the rise in multidrug, extensive drug, and pandrug resistance in bacteria and the increasing use of colistin to treat human infections, occurrence of mcr genes threatens human health, and characterization of these resistance genes becomes more important. We show that three commonly used heterologous expression strains are intrinsically resistant to colistin. This is important because these strains have previously been used to characterize and identify new mobile colistin resistance (mcr) genes. We also show that expression plasmids (i.e., pET17b) without inserts cause cell viability defects when carried by B-strains with T7 RNA polymerase and grown in the presence of IPTG. Our findings are important as they will facilitate improved selection of heterologous strains and plasmid combinations for characterizing AMR genes, which will be particularly important with a shift to Culture-independent diagnostic tests where bacterial isolates become increasingly less available for characterization.202337199645
5201100.9934Complete genome of Enterobacter sichuanensis strain SGAir0282 isolated from air in Singapore. BACKGROUND: Enterobacter cloacae complex (ECC) bacteria, such as E. cloacae, E. sichuanensis, E. kobei, and E. roggenkampii, have been emerging as nosocomial pathogens. Many strains isolated from medical clinics were found to be resistant to antibiotics, and in the worst cases, acquired multidrug resistance. We present the whole genome sequence of SGAir0282, isolated from the outdoor air in Singapore, and its relevance to other ECC bacteria by in silico genomic analysis. RESULTS: Complete genome assembly of E. sichuanensis strain SGAir0282 was generated using PacBio RSII and Illumina MiSeq platforms, and the datasets were used for de novo assembly using Hierarchical Genome Assembly Process (HGAP) and error corrected with Pilon. The genome assembly consisted of a single contig of 4.71 Mb and with a G+C content of 55.5%. No plasmid was detected in the assembly. The genome contained 4371 coding genes, 83 tRNA and 25 rRNA genes, as predicted by NCBI's Prokaryotic Genome Annotation Pipeline (PGAP). Among the genes, the antibiotic resistance related genes were included: Streptothricin acetdyltransferase (SatA), fosfomycin resistance protein (FosA) and metal-dependent hydrolases of the beta-lactamase superfamily I (BLI). CONCLUSION: Based on whole genome alignment and phylogenetic analysis, the strain SGAir0282 was identified to be Enterobacter sichuanensis. The strain possesses gene clusters for virulence, disease and defence, that can also be found in other multidrug resistant ECC type strains.202032127921
2471110.9934New sequence type of an Enterobacter cloacae complex strain with the potential to become a high-risk clone. OBJECTIVES: Enterobacter cloacae complex (ECC) has awakened interest recently because of its increasing resistance to carbapenems codified by several genes all over the globe. Even though there are some sequence types (STs) which represent high-risk clones, there is substantial clonal diversity in the ECC. This work aimed to perform whole-genome sequencing (WGS), genomic analysis, and phylogenetic studies of a Klebsiella pneumoniae carbapenemase (KPC) -producing multidrug-resistant (MDR) ECC isolate from Argentina. METHODS: We analysed the genome of an MDR KPC-producing ECC strain isolated from a urine sample from a patient in a hospital in Argentina. The WGS was done by Illumina MiSeq-I (Illumina, San Diego, CA). The genome was assembled with SPAdes 3.9.0, and annotated with PROKKA, RAST, and Blast. Plasmids were identified with PlasmidFinder. Antibiotic resistance genes were detected using RESfinder, CARD, and Blastn. STs were identified with pubMLST. RESULTS: The strain was identified as Enterobacter hormaechei, an important emerging human pathogen. No ST could be assigned; six of seven alleles of multilocus sequence typing (MLST) were the same as for E. hormaechei ST66, which is a high-risk clone. We found multiple acquired antibiotic resistance genes, including bla(KPC-2) in an IncM1 plasmid, and a secretion system VI, which can favour the prevalence of ECC strains while competing with other bacteria. CONCLUSION: Because of its MLST profile being so close to that of E. hormaechei ST66, the acquisition of multiple resistance genes, and the presence of the secretion systems, the potential of this strain for becoming a new high-risk clone cannot be discarded.202236049730
1567120.9934Chromosomal Amplification of the blaOXA-58 Carbapenemase Gene in a Proteus mirabilis Clinical Isolate. Horizontal gene transfer may occur between distantly related bacteria, thus leading to genetic plasticity and in some cases to acquisition of novel resistance traits. Proteus mirabilis is an enterobacterial species responsible for human infections that may express various acquired β-lactam resistance genes, including different classes of carbapenemase genes. Here we report a Proteus mirabilis clinical isolate (strain 1091) displaying resistance to penicillin, including temocillin, together with reduced susceptibility to carbapenems and susceptibility to expanded-spectrum cephalosporins. Using biochemical tests, significant carbapenem hydrolysis was detected in P. mirabilis 1091. Since PCR failed to detect acquired carbapenemase genes commonly found in Enterobacteriaceae, we used a whole-genome sequencing approach that revealed the presence of bla(OXA-58) class D carbapenemase gene, so far identified only in Acinetobacter species. This gene was located on a 3.1-kb element coharboring a bla(AmpC)-like gene. Remarkably, these two genes were bracketed by putative XerC-XerD binding sites and inserted at a XerC-XerD site located between the terminase-like small- and large-subunit genes of a bacteriophage. Increased expression of the two bla genes resulted from a 6-time tandem amplification of the element as revealed by Southern blotting. This is the first isolation of a clinical P. mirabilis strain producing OXA-58, a class D carbapenemase, and the first description of a XerC-XerD-dependent insertion of antibiotic resistance genes within a bacteriophage. This study revealed a new role for the XerC-XerD recombinase in bacteriophage biology.201727855079
5208130.9934Complete genome sequence of Acinetobacter baumannii XH386 (ST208), a multi-drug resistant bacteria isolated from pediatric hospital in China. Acinetobacter baumannii is an important bacterium that emerged as a significant nosocomial pathogen worldwide. The rise of A. baumannii was due to its multi-drug resistance (MDR), while it was difficult to treat multi-drug resistant A. baumannii with antibiotics, especially in pediatric patients for the therapeutic options with antibiotics were quite limited in pediatric patients. A. baumannii ST208 was identified as predominant sequence type of carbapenem resistant A. baumannii in the United States and China. As we knew, there was no complete genome sequence reproted for A. baumannii ST208, although several whole genome shotgun sequences had been reported. Here, we sequenced the 4087-kilobase (kb) chromosome and 112-kb plasmid of A. baumannii XH386 (ST208), which was isolated from a pediatric hospital in China. The genome of A. baumannii XH386 contained 3968 protein-coding genes and 94 RNA-only encoding genes. Genomic analysis and Minimum inhibitory concentration assay showed that A. baumannii XH386 was multi-drug resistant strain, which showed resistance to most of antibiotics, except for tigecycline. The data may be accessed via the GenBank accession number CP010779 and CP010780.201626981403
1583140.9934Identification of Novel Mobilized Colistin Resistance Gene mcr-9 in a Multidrug-Resistant, Colistin-Susceptible Salmonella enterica Serotype Typhimurium Isolate. Mobilized colistin resistance (mcr) genes are plasmid-borne genes that confer resistance to colistin, an antibiotic used to treat severe bacterial infections. To date, eight known mcr homologues have been described (mcr-1 to -8). Here, we describe mcr-9, a novel mcr homologue detected during routine in silico screening of sequenced Salmonella genomes for antimicrobial resistance genes. The amino acid sequence of mcr-9, detected in a multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium (S Typhimurium) strain isolated from a human patient in Washington State in 2010, most closely resembled mcr-3, aligning with 64.5% amino acid identity and 99.5% coverage using Translated Nucleotide BLAST (tblastn). The S. Typhimurium strain was tested for phenotypic resistance to colistin and was found to be sensitive at the 2-mg/liter European Committee on Antimicrobial Susceptibility Testing breakpoint under the tested conditions. mcr-9 was cloned in colistin-susceptible Escherichia coli NEB5α under an IPTG (isopropyl-β-d-thiogalactopyranoside)-induced promoter to determine whether it was capable of conferring resistance to colistin when expressed in a heterologous host. Expression of mcr-9 conferred resistance to colistin in E. coli NEB5α at 1, 2, and 2.5 mg/liter colistin, albeit at a lower level than mcr-3 Pairwise comparisons of the predicted protein structures associated with all nine mcr homologues (Mcr-1 to -9) revealed that Mcr-9, Mcr-3, Mcr-4, and Mcr-7 share a high degree of similarity at the structural level. Our results indicate that mcr-9 is capable of conferring phenotypic resistance to colistin in Enterobacteriaceae and should be immediately considered when monitoring plasmid-mediated colistin resistance.IMPORTANCE Colistin is a last-resort antibiotic that is used to treat severe infections caused by MDR and extensively drug-resistant (XDR) bacteria. The World Health Organization (WHO) has designated colistin as a "highest priority critically important antimicrobial for human medicine" (WHO, Critically Important Antimicrobials for Human Medicine, 5th revision, 2017, https://www.who.int/foodsafety/publications/antimicrobials-fifth/en/), as it is often one of the only therapies available for treating serious bacterial infections in critically ill patients. Plasmid-borne mcr genes that confer resistance to colistin pose a threat to public health at an international scale, as they can be transmitted via horizontal gene transfer and have the potential to spread globally. Therefore, the establishment of a complete reference of mcr genes that can be used to screen for plasmid-mediated colistin resistance is essential for developing effective control strategies.201931064835
6183150.9934Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella Typhi. Multidrug resistance mediated by efflux pumps is a well-known phenomenon in infectious bacteria. Although much work has been carried out to characterize multidrug efflux pumps in Gram-negative and Gram-positive bacteria, such information is still lacking for many deadly pathogens. The aim of this study was to gain insight into the substrate specificity of previously uncharacterized transporters of Salmonella Typhi to identify their role in the development of multidrug resistance. S. Typhi genes encoding putative members of the major facilitator superfamily were cloned and expressed in the drug-hypersensitive Escherichia coli strain KAM42, and tested for transport of 25 antibacterial compounds, including representative antibiotics of various classes, antiseptics, dyes and detergents. Of the 15 tested putative transporters, STY0901, STY2458 and STY4874 exhibited a drug-resistance phenotype. Among these, STY4874 conferred resistance to at least ten of the tested antimicrobials: ciprofloxacin, norfloxacin, levofloxacin, kanamycin, streptomycin, gentamycin, nalidixic acid, chloramphenicol, ethidium bromide, and acriflavine, including fluoroquinolone antibiotics, which were drugs of choice to treat S. Typhi infections. Cell-based functional studies using ethidium bromide and acriflavine showed that STY4874 functions as a H(+)-dependent exporter. These results suggest that STY4874 may be an important drug target, which can now be tested by studying the susceptibility of a STY4874-deficient S. Typhi strain to antimicrobials.201525724589
5058160.9934Widespread Fosfomycin Resistance in Gram-Negative Bacteria Attributable to the Chromosomal fosA Gene. Fosfomycin is a decades-old antibiotic which is being revisited because of its perceived activity against many extensively drug-resistant Gram-negative pathogens. FosA proteins are Mn(2+) and K(+)-dependent glutathione S-transferases which confer fosfomycin resistance in Gram-negative bacteria by conjugation of glutathione to the antibiotic. Plasmid-borne fosA variants have been reported in fosfomycin-resistant Escherichia coli strains. However, the prevalence and distribution of fosA in other Gram-negative bacteria are not known. We systematically surveyed the presence of fosA in Gram-negative bacteria in over 18,000 published genomes from 18 Gram-negative species and investigated their contribution to fosfomycin resistance. We show that FosA homologues are present in the majority of genomes in some species (e.g., Klebsiella spp., Enterobacter spp., Serratia marcescens, and Pseudomonas aeruginosa), whereas they are largely absent in others (e.g., E. coli, Acinetobacter baumannii, and Burkholderia cepacia). FosA proteins in different bacterial pathogens are highly divergent, but key amino acid residues in the active site are conserved. Chromosomal fosA genes conferred high-level fosfomycin resistance when expressed in E. coli, and deletion of chromosomal fosA in S. marcescens eliminated fosfomycin resistance. Our results indicate that FosA is encoded by clinically relevant Gram-negative species and contributes to intrinsic fosfomycin resistance.IMPORTANCE There is a critical need to identify alternate approaches to treat infections caused by extensively drug-resistant (XDR) Gram-negative bacteria. Fosfomycin is an old antibiotic which is routinely used for the treatment of urinary tract infections, although there is substantial interest in expanding its use to systemic infections caused by XDR Gram-negative bacteria. In this study, we show that fosA genes, which encode dimeric Mn(2+)- and K(+)-dependent glutathione S-transferase, are widely distributed in the genomes of Gram-negative bacteria-particularly those belonging to the family Enterobacteriaceae-and confer fosfomycin resistance. This finding suggests that chromosomally located fosA genes represent a vast reservoir of fosfomycin resistance determinants that may be transferred to E. coli Furthermore, they suggest that inhibition of FosA activity may provide a viable strategy to potentiate the activity of fosfomycin against XDR Gram-negative bacteria.201728851843
1670170.9933KPC-2-producing Klebsiella pneumoniae isolated from a Czech patient previously hospitalized in Greece and in vivo selection of colistin resistance. Carbapenemase-producing Gram-negative bacteria peak clinical interest due to their ability to hydrolyze most β-lactams, including carbapenems; moreover, their genes spread through bacterial populations by horizontal transfer. Bacteria with acquired carbapenemase have sporadically been reported in the Czech Republic, so far only in Enterobacteriaceae and Pseudomonas aeruginosa. In this study, we described the first finding of a KPC-2-producing strain of Klebsiella pneumoniae, which was isolated from a surgical wound swab, decubitus ulcer, and urine of a patient previously hospitalized in Greece. The patient underwent various antibiotic therapies including a colistin treatment. However, after approximately 20 days of the colistin therapy, the strain developed a high-level resistance to this drug. All the isolates were indistinguishable by pulsed field gel electrophoretic analysis and belonged to the international clone ST258, which is typical of KPC-producing K. pneumoniae isolates. The bla (KPC-2) gene was located on a Tn4401a transposon variant. The OmpK35 and OmpK36 genes analysis performed due to the high resistance level of the strains to β-lactams exhibited no changes in their sequence or in their expression when compared with carbapenem-susceptible isolates.201121818609
6173180.9933Mutation in crrB encoding a sensor kinase increases expression of the RND-type multidrug efflux pump KexD in Klebsiella pneumoniae. BACKGROUND: RND-type multidrug efflux systems in Gram-negative bacteria protect them against antimicrobial agents. Gram-negative bacteria generally possess several genes which encode such efflux pumps, but these pumps sometimes fail to show expression. Generally, some multidrug efflux pumps are silent or expressed only at low levels. However, genome mutations often increase the expression of such genes, conferring the bacteria with multidrug-resistant phenotypes. We previously reported mutants with increased expression of the multidrug efflux pump KexD. We aimed to identify the cause of KexD overexpression in our isolates. Furthermore, we also examined the colistin resistant levels in our mutants. METHODS: A transposon (Tn) was inserted into the genome of Klebsiella pneumoniae Em16-1, a KexD-overexpressing mutant, to identify the gene(s) responsible for KexD overexpression. RESULTS: Thirty-two strains with decreased kexD expression after Tn insertion were isolated. In 12 of these 32 strains, Tn was identified in crrB, which encodes a sensor kinase of a two-component regulatory system. DNA sequencing of crrB in Em16-1 showed that the 452nd cytosine on crrB was replaced by thymine, and this mutation changed the 151st proline into leucine. The same mutation was found in all other KexD-overexpressing mutants. The expression of crrA increased in the mutant overexpressing kexD, and the strains in which crrA was complemented by a plasmid showed elevated expression of kexD and crrB from the genome. The complementation of the mutant-type crrB also increased the expression of kexD and crrA from the genome, but the complementation of the wild-type crrB did not. Deletion of crrB decreased antibiotic resistance levels and KexD expression. CrrB was reported as a factor of colistin resistance, and the colistin resistance of our strains was tested. However, our mutants and strains carrying kexD on a plasmid did not show increased colistin resistance. CONCLUSION: Mutation in crrB is important for KexD overexpression. Increased CrrA may also be associated with KexD overexpression.202337331490
1769190.9933DNA sequence and comparative genomics of pAPEC-O2-R, an avian pathogenic Escherichia coli transmissible R plasmid. In this study, a 101-kb IncF plasmid from an avian pathogenic Escherichia coli (APEC) strain (APEC O2) was sequenced and analyzed, providing the first completed APEC plasmid sequence. This plasmid, pAPEC-O2-R, has functional transfer and antimicrobial resistance-encoding regions. The resistance-encoding region encodes resistance to eight groups of antimicrobial agents, including silver and other heavy metals, quaternary ammonium compounds, tetracycline, sulfonamides, aminoglycosides, trimethoprim, and beta-lactam antimicrobial agents. This region of the plasmid is unique among previously described IncF plasmids in that it possesses a class 1 integron that harbors three gene cassettes and a heavy metal resistance operon. This region spans 33 kb and is flanked by the RepFII plasmid replicon and an assortment of plasmid maintenance genes. pAPEC-O2-R also contains a 32-kb transfer region that is nearly identical to that found in the E. coli F plasmid, rendering it transferable by conjugation to plasmid-less strains of bacteria, including an APEC strain, a fecal E. coli strain from an apparently healthy bird, a Salmonella enterica serovar Typhimurium strain, and a uropathogenic E. coli strain from humans. Differences in the G+C contents of individual open reading frames suggest that various regions of pAPEC-O2-R had dissimilar origins. The presence of pAPEC-O2-R-like plasmids that encode resistance to multiple antimicrobial agents and that are readily transmissible from APEC to other bacteria suggests the possibility that such plasmids may serve as a reservoir of resistance genes for other bacteria of animal and human health significance.200516251312