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1.
Antimicrob Agents Chemother ; : e0013324, 2024 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-38624228

RESUMEN

A 2-year national genomic screening in the Czech Republic identified a notable prevalence of the New Delhi metallo-ß-lactamase 5 (NDM-5)-producing Escherichia coli sequence type 38 (ST38) in the city of Brno. Forty-two ST38 E. coli isolates harbored the blaNDM-5 gene on the chromosome. Virulence factors confirmed the persistence of these isolates through biofilm formation. Single Nucleotide Polymorphisms (SNPs)-based phylogeny and CRISPR assay typing showed minimal genomic variations, implying a clonally driven outbreak. Results suggest that this high-risk clone may impose a nationwide problem.

2.
Artículo en Inglés | MEDLINE | ID: mdl-38555080

RESUMEN

OBJECTIVE: Here we describe a novel IncFIA plasmid harboring mcr-10 gene in a clinical Enterobacter ludwigii strain isolated at the University Hospital in Pilsen in the Czech Republic. METHODS: The strain was subjected to antibiotic susceptibility testing. Whole genome sequencing was performed using Illumina for short read sequencing and Oxford Nanopore Technologies for long-read sequencing followed by hybrid assembly. The resulting genome was used to detect species using average nucleotide identity, resistance genes, plasmid replicon and MLST (using center for genomic epidemiology databases; ResFinder, PlasmidFinder and MLST repsectively) and virulence genes using VFDB. RESULTS: Τhe strain showed susceptibility against tetracycline, cefuroxime and chloramphenicol, and was susceptible to the 2nd and 3rd generation of cephalosporins, carbapenems and colistin. Genome analysis identified the strain as E. ludwigii sequence type ST20 and located the mcr-10 gene on an IncFIA (HI1)/IncFII (Yp) plasmid (pI9455333_MCR10; 129,863 bp). Upon blasting the nucleotide sequence of pI9455333_MCR10 against NCBI database; no similar plasmid sequence was detected implying a novel plasmid structure. Nevertheless, it showed a partial similarity with: pRHBSTW-00123_3 and FDAARGOS 1432 which were detected in Enterobacter cloacae complex (ECC) strains in wastewater samples in 2017 in UK and in 2021 in the USA respectively, and pEC81-mcr which was detected in a clinical Escherichia coli strain in 2020 in China. Moreover, I9455333cz genome carried virulence genes coding for: curli fibers, fimbrial adherence determinants, siderophore aerobactin, iron uptake proteins, and regulators of sigma factor. CONCLUSION: In conclusion, we identified a novel IncF plasmid harboring mcr-10 gene in a clinical Enterobacter ludwigii strain. To our knowledge, this is the first clinical report of mcr-10 in the Czech Republic.

3.
J Chemother ; 36(1): 24-30, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38156440

RESUMEN

The aim of the present study was to examine the impact of macrolides on the expression of virulence factors and QS-associated genes in clinical P. aeruginosa isolates. Among 60 clinical P. aeruginosa, pyocyanin production was detected in 27 (45%) isolates, which belonged to various STs. Erythromycin inhibited the production of pigments in 12 out of 27 isolates. Other antibiotic categories didn't have an impact on production of pigments. Additionally, results showed that erythromycin sub-MIC inhibited the growth-rate in 17 isolates. Of note, in six isolates, the inhibition of growth-rate was greater when using both erythromycin and meropenem than using each antibiotic individually. Finally, addition of erythromycin down-regulated the expression of QS-associated genes (65.5%-81.3%) and almost all virulence-associated genes. In conclusion, our results confirmed that macrolides could be used in combination with last-line antibiotics, such as carbapenems, to treat infections caused by multidrug-resistant Gram-negative bacteria.


Asunto(s)
Infecciones por Pseudomonas , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/genética , Macrólidos/metabolismo , Macrólidos/farmacología , Macrólidos/uso terapéutico , Proteínas Bacterianas/genética , Factores de Virulencia/metabolismo , Antibacterianos/uso terapéutico , Eritromicina/uso terapéutico , Infecciones por Pseudomonas/tratamiento farmacológico , Biopelículas
4.
Front Microbiol ; 14: 1147846, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37180238

RESUMEN

The occurrence of colistin resistance has increased rapidly among Enterobacterales around the world. We performed a national survey of plasmid-mediated colistin resistance in human clinical isolates through a retrospective analysis of samples from 2009 to 2017 and a prospective sampling in 2018-2020. The aim of this study was to identify and characterize isolates with mcr genes from various regions of the Czech Republic using whole genome sequencing (WGS). Of all 1932 colistin-resistant isolates analyzed, 73 (3.8%) were positive for mcr genes. Most isolates carried mcr-1 (48/73) and were identified as Escherichia coli (n = 44) and Klebsiella pneumoniae (n = 4) of various sequence types (ST). Twenty-five isolates, including Enterobacter spp. (n = 24) and Citrobacter freundii (n = 1) carrying the mcr-9 gene were detected; three of them (Enterobacter kobei ST54) co-harbored the mcr-4 and mcr-9 genes. Multi-drug resistance phenotype was a common feature of mcr isolates and 14% (10/73) isolates also co-harbored clinically important beta-lactamases, including two isolates with carbapenemases KPC-2 and OXA-48. Phylogenetic analysis of E. coli ST744, the dominant genotype in this study, with the global collection showed Czech isolates belonged to two major clades, one containing isolates from Europe, while the second composed of isolates from diverse geographical areas. The mcr-1 gene was carried by IncX4 (34/73, 47%), IncHI2/ST4 (6/73, 8%) and IncI2 (8/73, 11%) plasmid groups. Small plasmids belonging to the ColE10 group were associated with mcr-4 in three isolates, while mcr-9 was carried by IncHI2/ST1 plasmids (4/73, 5%) or the chromosome (18/73, 25%). We showed an overall low level of occurrence of mcr genes in colistin-resistant bacteria from human clinical samples in the Czech Republic.

5.
Mol Genet Genomics ; 297(6): 1767-1776, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-36197590

RESUMEN

The objective of this study was to investigate the prevalence of CRISPR/Cas systems in P. aeruginosa, isolated from a Greek hospital. Additionally, we aimed to determine the origin of the sequenced spacers. A collection of 100 nonrepetitive P. aeruginosa was analyzed. Isolates were typed by MLST. The presence of CRISPR/Cas systems, as well as amplification of CRISPR arrays, was examined by PCR using specific primers. CRISPR/Cas systems were detected in 36 isolates, of which 27 isolates exhibited resistance to carbapenems, with 10 of the later isolates producing a VIM-type MßL. The majority (n = 19) of CRISPR/Cas-positive isolates harbored a type I-F system, while I-C and I-E systems were found in 9 and 8 isolates, respectively. Based on MLST, isolates carrying I-E and I-F systems belonged to different STs and included CRISPR arrays with diverse number of spacers. Isolates with I-C systems belonged to clonal complex 235 and exhibited identical CRISPR arrays. Among 425 unique spacers, identified during this study, BLASTn search showed that they matched with P. aeruginosa chromosomal sequences (47.0%), phages (31.9%), plasmids, PAGIs, and an ICE. 16.3% of the spacers exhibited no significant similarity with sequences submitted to GenBank database. In conclusion, we observed the presence of type I-C, I-E and I-F CRISPR/Cas systems in P. aeruginosa of clinical origin. CRISPR/Cas were also observed among isolates carrying the carbapenemase-encoding blaVIM gene, which is usually associated with integrons, questioning the defense role against mobile elements. Therefore, further experimental characterization is needed to clarify their functional role.


Asunto(s)
Sistemas CRISPR-Cas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Grecia/epidemiología , Sistemas CRISPR-Cas/genética , Tipificación de Secuencias Multilocus , Prevalencia , Carbapenémicos/farmacología
6.
mSphere ; 7(4): e0023822, 2022 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-35862807

RESUMEN

Escherichia coli sequence type 963 (ST963) is a neglected lineage closely related to ST38, a globally widespread extraintestinal pathogenic ST causing urinary tract infections (UTI) as well as sepsis in humans. Our current study aimed to improve the knowledge of this understudied ST by carrying out a comprehensive comparative analysis of whole-genome sequencing data consisting of 31 isolates from silver gulls in Australia along with another 80 genomes from public resources originating from geographically scattered regions. ST963 was notable for carriage of cephalosporinase gene blaCMY-2, which was identified in 99 isolates and was generally chromosomally encoded. ST963 isolates showed otherwise low carriage of antibiotic resistance genes, in contrast with the closely related E. coli ST38. We found considerable phylogenetic variability among international ST963 isolates (up to 11,273 single nucleotide polymorphisms [SNPs]), forming three separate clades. A major clade that often differed by 20 SNPs or less consisted of Australian isolates of both human and animal origin, providing evidence of zoonotic or zooanthropogenic transmission. There was a high prevalence of virulence F29:A-:B10 pUTI89-like plasmids within E. coli ST963 (n = 88), carried especially by less variable isolates exhibiting ≤1,154 SNPs. We characterized a novel 115,443-bp pUTI89-like plasmid, pCE2050_A, that carried a traS:IS5 insertion absent from pUTI89. Since IS5 was also present in a transposition unit bearing blaCMY-2 on chromosomes of ST963 strains, IS5 insertion into pUTI89 may enable mobilization of the blaCMY-2 gene from the chromosome/transposition unit to pUTI89 via homologous recombination. IMPORTANCE We have provided the first comprehensive genomic study of E. coli ST963 by analyzing various genomic and phenotypic data sets of isolates from Australian silver gulls and comparison with genomes from geographically dispersed regions of human and animal origin. Our study suggests the emergence of a specific blaCMY-2-carrying E. coli ST963 clone in Australia that is widely spread across the continent by humans and birds. Genomic analysis has revealed that ST963 is a globally dispersed lineage with a remarkable set of virulence genes and virulence plasmids described in uropathogenic E. coli. While ST963 separated into three clusters, a unique specific clade of Australian ST963 isolates harboring a chromosomal copy of AmpC ß-lactamase encoding the gene blaCMY-2 and originating from both humans and wild birds was identified. This phylogenetically close cluster comprised isolates of both animal and human origin, thus providing evidence of interspecies zoonotic transmission. The analysis of the genetic environment of the AmpC ß-lactamase-encoding gene highlighted ongoing evolutionary events that shape the carriage of this gene in ST963.


Asunto(s)
Charadriiformes , Infecciones por Escherichia coli , Escherichia coli , Animales , Australia , Charadriiformes/microbiología , Escherichia coli/genética , Infecciones por Escherichia coli/transmisión , Infecciones por Escherichia coli/veterinaria , Humanos , Filogenia
7.
J Glob Antimicrob Resist ; 29: 116-119, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35231657

RESUMEN

OBJECTIVES: The aim of the study is to characterise the genomic features of three GES-producing Enterobacterales isolates from Czech hospitals. METHODS: In 2020, during a routine screening of the hospital's surfaces in Prague General Hospital, two strains (CZ862 and CZ863) that belonged to the Enterobacter cloacae complex were found to be blaGES positive. Another blaGES positive strain identified as Klebsiella oxytoca was recovered from a patient hospitalised in Pilsen. Antibiotic susceptibility profiling was done with broth microdilution assay. Conjugation/transformation experiments were performed on all three strains. Genomic DNA of the three isolates was subjected to whole genome sequencing using PacBio platform. RESULTS: Multilocus sequence types typing of CZ862 and CZ863 identified the strains as ST837 and a novel ST (ST1622). Both blaGES harbouring plasmids showed high sequence similarity and complete query coverage (100% and 99.98%) with pEcl-35771cz. Both plasmids had two copies of blaGES instead of one copy as found in pEcl-35771cz. The clinical isolate CZ598 belonged to ST180. The plasmid harboured blaGES-7 gene, cat and aac(6')-lb and the novel variant blaOXA-1011. No similar sequences were observed, suggesting a novel plasmid. CONCLUSION: The detection of the two blaGES-positive plasmids in the same hospital environment, the first report after 3 years, suggests a hidden source. This highlights the importance of the hidden sources and evolution of such plasmids on the route of spreading into clinical settings. Also, the detection of the new blaOXA-1011, which is thought in this case to be associated with carbapenem resistance, imposes a health risk if disseminated, limiting therapeutic options.


Asunto(s)
Klebsiella oxytoca , beta-Lactamasas , República Checa , Genómica , Humanos , Klebsiella oxytoca/genética , Plásmidos/genética , beta-Lactamasas/genética
8.
Front Microbiol ; 13: 993240, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36687644

RESUMEN

Background: VIM metallo-ß-lactamases are enzymes characterized by the ability to hydrolyze all ß-lactams. Usually, bla VIM-like genes are carried by class 1 integrons. In the Czech Republic, only sporadic cases of VIM-producing Enterobacterales have been reported in which those isolates carried the VIM-1 carbapenemase-encoding integron In110. However, during 2019-2020, an increased number was reported. Therefore, the aim of the current study was to characterize the genetic elements involved in the increased spread of bla VIM genes. Materials and methods: 32 VIM-producing Enterobacterales collected between 2019 and 2020 were subjected to: antimicrobial susceptibility testing, integron analysis, and short reads sequencing. Based on the results, 19 isolates were selected as representative and sequenced using Sequel I platform. Results: The 32 VIM-producing isolates exhibited variations in the MICs of carbapenems. Based on short-read data, 26 of the 32 sequenced isolates harbored the bla VIM-1 allele while six isolates carried the bla VIM-4 gene. The most prevalent was the In110 integron (n = 24) and two isolates carried the In4873 class 1 integron. The bla VIM-4 allele was identified in class 1 integrons In1174 (n = 3), In416 (n = 1), In2143 (n = 1) and In2150. Long reads sequencing revealed that the bla VIM was carried by: pKPC-CAV1193-like (n = 6), HI1 (pNDM-CIT; n = 4), HI2 (n = 3), FIB (pECLA; n = 2) and N (n = 1) incompatibility groups. Two bla VIM-carrying plasmids could not be typed by the database, while another one was integrated into the chromosome. Conclusion: We observed the spread of VIM-encoding integrons, mainly of In110, among Enterobacterales isolated from Czech hospitals, but also an increased number of novel elements underlining the ongoing evolution.

9.
Sci Rep ; 11(1): 15732, 2021 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-34344951

RESUMEN

The aim of the present study is to describe the ongoing spread of the KPC-producing strains, which is evolving to an epidemic in Czech hospitals. During the period of 2018-2019, a total of 108 KPC-producing Enterobacterales were recovered from 20 hospitals. Analysis of long-read sequencing data revealed the presence of several types of blaKPC-carrying plasmids; 19 out of 25 blaKPC-carrying plasmids could be assigned to R (n = 12), N (n = 5), C (n = 1) and P6 (n = 1) incompatibility (Inc) groups. Five of the remaining blaKPC-carrying plasmids were multireplicon, while one plasmid couldn't be typed. Additionally, phylogenetic analysis confirmed the spread of blaKPC-carrying plasmids among different clones of diverse Enterobacterales species. Our findings demonstrated that the increased prevalence of KPC-producing isolates was due to plasmids spreading among different species. In some districts, the local dissemination of IncR and IncN plasmids was observed. Additionally, the ongoing evolution of blaKPC-carrying plasmids, through genetic rearrangements, favours the preservation and further dissemination of these mobile genetic elements. Therefore, the situation should be monitored, and immediate infection control should be implemented in hospitals reporting KPC-producing strains.


Asunto(s)
Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana , Hospitales/estadística & datos numéricos , Infecciones por Klebsiella/epidemiología , Klebsiella pneumoniae/aislamiento & purificación , beta-Lactamasas/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , República Checa/epidemiología , Epidemias , Humanos , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/metabolismo
10.
Front Microbiol ; 12: 641415, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33633720

RESUMEN

The aim of this study was to characterize four Enterobacterales co-producing NDM- and OXA-48-like carbapenemases from Czech patients with travel history or/and previous hospitalization abroad. Klebsiella pneumoniae isolates belonged to "high risk" clones ST147, ST11, and ST15, while the Escherichia coli isolate was assigned to ST167. All isolates expressed resistance against most ß-lactams, including carbapenems, while retaining susceptibility to colistin. Furthermore, analysis of WGS data showed that all four isolates co-produced OXA-48- and NDM-type carbapenemases, in different combinations (Kpn47733: bla NDM- 5 + bla OXA- 181; Kpn50595: bla NDM- 1 + bla OXA- 181; Kpn51015: bla NDM- 1 + bla OXA- 244; Eco52418: bla NDM- 5 + bla OXA- 244). In Kpn51015, the bla OXA- 244 was found on plasmid p51015_OXA-244, while the respective gene was localized in the chromosomal contig of E. coli Eco52418. On the other hand, bla OXA- 181 was identified on a ColKP3 plasmid in isolate Kpn47733, while a bla OXA- 181-carrying plasmid being an IncX3-ColKP3 fusion was identified in Kpn50595. The bla NDM- 1 gene was found on two different plasmids, p51015_NDM-1 belonging to a novel IncH plasmid group and p51015_NDM-1 being an IncF K 1-FIB replicon. Furthermore, the bla NDM- 5 was found in two IncFII plasmids exhibiting limited nucleotide similarity to each other. In both plasmids, the genetic environment of bla NDM- 5 was identical. Finally, in all four carbapenemase-producing isolates, a diverse number of additional replicons, some of these associated with important resistance determinants, like bla CTX-M- 15, arr-2 and ermB, were identified. In conclusion, this study reports the first description of OXA-244-producing Enterobacterales isolated from Czech hospitals. Additionally, our findings indicated the genetic plurality involved in the acquisition and dissemination of determinants encoding OXA/NDM carbapenemases.

12.
Microorganisms ; 8(12)2020 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-33260774

RESUMEN

The objective of this study was to analyze the characteristics that contribute to the successful dissemination of VIM-producing Pseudomonas aeruginosa (P. aeruginosa), belonging to ST111 and ST235, in a Greek hospital. A total of 120 non-repetitive P. aeruginosa, which had meropenem minimal inhibitory concentrations (MICs) greater than 2 mg/L, were studied. VIM-encoding genes were amplified and sequenced within their integrons. Isolates were typed by multilocus sequence typing (MLST). Six VIM-producers, representative of different integron structures and sequence types (STs), were completely sequenced using Illumina platform. Sixty-one P. aeruginosa were confirmed to produce VIM-type carbapenemases. ST111 dominated (n = 34) among VIM-producers, while 15 VIM-producers belonged to ST235. The blaVIM-like genes were located in three integron types, including In59, In595 and In1760, which were integrated into P. aeruginosa chromosomes. Whole genome sequencing (WGS) data demonstrated that ST111 and ST235 MBL producers carried several resistance and virulence genes. Additionally, the presence of type I-C and type I-E clustered regularly interspaced short palindromic repeats (CRISPR)/Cas locus was observed in ST235 and ST395 isolates, respectively. In conclusion, our findings confirmed the clonal spread of ST111 P. aeruginosa, carrying the VIM-2-encoding integron In59, in the University Hospital of Larissa (UHL). In addition, they highlighted the important role of high-risk clones, ST111 and ST235, in the successful dissemination and establishment into hospital settings of clinically important pathogens carrying resistance determinants.

13.
mSphere ; 5(6)2020 12 09.
Artículo en Inglés | MEDLINE | ID: mdl-33298573

RESUMEN

The aim of this study was to report the characterization of the first mcr-positive Enterobacterales isolated from Czech hospitals. In 2019, one Citrobacter freundii and four Enterobacter isolates were recovered from Czech hospitals. The production of carbapenemases was examined by a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) imipenem hydrolysis assay. Additionally, bacteria were screened for the presence of carbapenemase-encoding genes and plasmid-mediated colistin resistance genes by PCR. To define the genetic units carrying mcr genes, the genomic DNAs of mcr-carrying clinical isolates were sequenced on the PacBio Sequel I platform. Results showed that all isolates carried blaVIM- and mcr-like genes. Analysis of whole-genome sequencing (WGS) data revealed that all isolates carried mcr-9-like alleles. Furthermore, the three sequence type 106 (ST106) Enterobacter hormaechei isolates harbored the blaVIM-1 gene, while the ST764 E. hormaechei and ST95 C. freundii included blaVIM-4 Analysis of plasmid sequences showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. Additionally, at least one multidrug resistance (MDR) region was identified in each mcr-9-carrying IncHI2 plasmid. The blaVIM-4 gene was found in the MDR regions of p48880_MCR_VIM and p51929_MCR_VIM. In the three remaining isolates, blaVIM-1 was localized on plasmids (∼55 kb) exhibiting repA-like sequences 99% identical to the respective gene of pKPC-CAV1193. In conclusion, to the best of our knowledge, these 5 isolates were the first mcr-9-positive bacteria of clinical origin identified in the Czech Republic. Additionally, the carriage of the blaVIM-1 on pKPC-CAV1193-like plasmids is described for the first time. Thus, our findings underline the ongoing evolution of mobile elements implicated in the dissemination of clinically important resistance determinants.IMPORTANCE Infections caused by carbapenemase-producing bacteria have led to the revival of polymyxins as the "last-resort" antibiotic. Since 2016, several reports describing the presence of plasmid-mediated colistin resistance genes, mcr, in different host species and geographic areas were published. Here, we report the first detection of Enterobacterales carrying mcr-9-like alleles isolated from Czech hospitals in 2019. Furthermore, the three ST106 Enterobacter hormaechei isolates harbored blaVIM-1, while the ST764 E. hormaechei and ST95 Citrobacter freundii isolates included blaVIM-4 Analysis of WGS data showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. blaVIM-4 was found in the MDR regions of IncHI2 plasmids, while blaVIM-1 was localized on pKPC-CAV1193-like plasmids, described here for the first time. These findings underline the ongoing evolution of mobile elements implicated in dissemination of clinically important resistance determinants. Thus, WGS characterization of MDR bacteria is crucial to unravel the mechanisms involved in dissemination of resistance mechanisms.


Asunto(s)
Enterobacter/genética , beta-Lactamasas/genética , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Proteínas Bacterianas/genética , República Checa , Enterobacter/aislamiento & purificación , Hospitales , Humanos , Plásmidos/genética
14.
Artículo en Inglés | MEDLINE | ID: mdl-33139274

RESUMEN

Wild corvids were examined for the presence of carbapenemase-producing Gram-negative bacteria in the United States. A total of 13 isolates were detected among 590 fecal samples of American crow; 11 Providencia rettgeri isolates harboring blaIMP-27 on the chromosome as a class 2 integron gene cassette within the Tn7 transposon, 1 Klebsiella pneumoniae ST258 isolate carrying blaKPC-2 on a pKpQIL-like plasmid as a part of Tn4401a, and 1 Enterobacter bugandensis isolate with blaIMI-1 located within EcloIMEX-2.


Asunto(s)
Cuervos , Infecciones por Klebsiella , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Enterobacter , Klebsiella pneumoniae/genética , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Providencia , Estados Unidos , beta-Lactamasas/genética
15.
Folia Microbiol (Praha) ; 65(5): 895-900, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-32613406

RESUMEN

Rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is essential for proper initial antibiotic therapy and timely set up of hygienic measures. Recently, detection of MRSA using MALDI-TOF mass spectrometer mediated by the peptide-phenol-soluble modulin (PSM-mec)-linked to the class A mec gene complex present in SCCmec cassettes types II, III, and VIII of MRSA strains, has been commercially available. We present here a multicentre study on MALDI-TOF MS detection of MRSA evincing a poor repeatability and reproducibility of the assay. The sensitivity of the assay varies between 50 and 90% in strains carrying psmMEC and psmδ genes encoding for PSM-mec and δ-toxin (a member of the PSM peptide family), respectively. No false positive results were found. The very major error calculation was 30% and the major error achieved 0%. Interlaboratory repeatability varies between 0 and 100%. No significant difference was observed with the use of different cultivation media. Our data showed a poor sensitivity of the method excluding it from the use in routine laboratory testing.


Asunto(s)
Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Técnicas de Diagnóstico Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Infecciones Estafilocócicas/diagnóstico , Toxinas Bacterianas/genética , Errores Diagnósticos , Pruebas Diagnósticas de Rutina , Humanos , Staphylococcus aureus Resistente a Meticilina/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Infecciones Estafilocócicas/microbiología
16.
BMC Microbiol ; 20(1): 224, 2020 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-32711478

RESUMEN

BACKGROUND: The resistance of Streptococcus pneumoniae to macrolides is becoming an increasingly important issue and thus it is important to understand the genetics related to adaptation of this species to the widespread use of antibiotics in Europe. The 58 isolates of S. pneumoniae belonging to sequence type (ST) 416 and serotype 19A and to several different phenotypes originated from Italy, Portugal and Czech Republic were thus sequenced on Illumina MiSeq. The aim of the study was to describe genetical origine of isolates, investigate their macrolide resistance and suggest reasons for spread of ST416 in the Czech Republic. RESULTS: Investigation of genes associated with serotype determined serotype switch between 15B and 19A serotypes and core genome multilocus sequence typing (cgMLST) confirmed the origine of concerned isolates in Netherlands15B-37 clone. Inspected genomes proved variability of genes associated with the macrolide resistance even within closely genetically relative isolates. CONCLUSIONS: Participation of 19A/ST416 on the spread of Netherlands15B-37 is accompanied by serotype switch between 19A and 15B serotypes and with acquisition of genes involved in macrolide resistance to the clone that was originally macrolide susceptible. There is evident tendency to interchanging and modifications of these and surrounding genes, that could lead to accelerate spreading of this sequence type in regions with high macrolide consumption.


Asunto(s)
Farmacorresistencia Bacteriana , Macrólidos/farmacología , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/clasificación , Secuenciación Completa del Genoma/métodos , República Checa , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Italia , Tipificación de Secuencias Multilocus , Países Bajos , Filogenia , Filogeografía , Polimorfismo de Nucleótido Simple , Portugal , Serogrupo , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificación
18.
J Glob Antimicrob Resist ; 22: 275-282, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32247078

RESUMEN

INTRODUCTION: Tuberculosis is considered one of the most fatal diseases worldwide, with an estimation of 10.1 million cases. In this study, whole-genome sequencing was used to determine the genomic characterisation of 40 Mycobacterium tuberculosis isolates from patients with different nationalities hospitalised in the Czech Republic. MATERIALS AND METHODS: Susceptibility testing for first-line drugs was performed. DNA was sequenced using the Illumina MiSeq platform. Spoligotype single-nucleotide polymorphisms and mutations in antibiotic-resistant genes were detected, and phylogenetic analysis was performed. RESULTS: Samples showing phenotypic resistance to at least one drug were 12 to streptomycin, 11 to isoniazid, 7 to rifampicin, 6 to ethambutol and 5 to pyrazinamide. Phenotypic and genotypic profiles did not match in all cases, suggesting the presence of a novel mutation in some cases and a low expression of resistant genes in others. The presented phylogeny enables the correct assignation of M. tuberculosis lineages and sublineages. Our results suggest that the most dominant lineage in our samples was lineage 4 (33/40). CONCLUSION: To our knowledge, this is the first study using this approach to be done in the Czech Republic. Lineage 4 was the predominant lineage identified among our samples. Nevertheless, the dominance of Lineage 4 along with other lineages suggests that infections can originate from different sources.


Asunto(s)
Antituberculosos , Farmacorresistencia Bacteriana Múltiple , Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Antituberculosos/farmacología , República Checa , Farmacorresistencia Bacteriana Múltiple/genética , Humanos , Mutación , Mycobacterium tuberculosis/genética , Filogenia
19.
Front Microbiol ; 11: 604067, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33519748

RESUMEN

The aim of our study was to determine complete nucleotide sequence of mcr-1-carrying plasmids from Enterobacterales isolates recovered from domestic and imported raw retailed meat and compare them with plasmids available at the GenBank sequence database. A set of 16 plasmids originating from Escherichia coli (n = 13), Klebsiella pneumoniae (n = 2), and Citrobacter braakii (n = 1) were analyzed. In our previous study, data from whole genome sequencing showed that mcr-1 gene was located on plasmids of different incompatibility groups (IncHI2, IncI2, and IncX4). The IncI2 (n = 3) and IncX4 (n = 8) plasmids harbored mcr-1.1 gene only, whereas IncHI2 sequence type 4 plasmids (n = 5) carried large multidrug resistance (MDR) regions. MDR regions of IncHI2 plasmids included additional antimicrobial resistance genes conferring resistance to ß-lactams (bla TEM-1), aminoglycosides [aadA1, aadA2, and aph(6)-Id], macrolides [mef (B)], tetracycline (tetA, tetR), and sulphonamides (sul1, sul2, and sul3). Likewise, IncHI2 plasmids carried several insertion sequences including IS1, IS3, IS26, IS1326, and ISApl1. In conclusion, our findings confirmed the involvement of IncX4, IncI2, and IncHI2 plasmids in the dissemination of mcr-1.1 gene in several environmental niches, as in samples of retail meat originating from different geographical regions. In contrast to IncX4 and IncI2, IncHI2 plasmids were more diverse and carried additional genes for resistance to heavy metals and multiple antimicrobials.

20.
Folia Microbiol (Praha) ; 65(2): 233-243, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-31713118

RESUMEN

In the last two decades, microbiology laboratories have radically changed by the introduction of novel technologies, like Next-Generation Sequencing (NGS) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Nevertheless, emergence of antibiotic-resistant microorganisms represents a global threat of current medicine, being responsible for increasing mortality and health-care direct and indirect costs. In addition, the identification of antibiotic-resistant microorganisms, like OXA-48 carbapenemase-producing Enterobacteriaceae, has been changeling for clinical microbiology laboratories. Even the cost of NGS technology and MALDI-TOF MS equipment is relatively high, both technologies are increasingly used in diagnostic and research protocols. Therefore, the aim of this review is to present applications of these technologies used in clinical microbiology, especially in detection of antibiotic resistance and its surveillance, and to propose a combinatory approach of MALDI-TOF MS and NGS for the investigation of microbial associated infections.


Asunto(s)
Bacterias/aislamiento & purificación , Infecciones Bacterianas/microbiología , Farmacorresistencia Bacteriana , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Espectrometría de Masas/métodos , Antibacterianos/farmacología , Bacterias/clasificación , Bacterias/efectos de los fármacos , Bacterias/genética , Infecciones Bacterianas/diagnóstico , Humanos , Laboratorios de Hospital
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