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BACKGROUND: Several bacterial species belonging to the Gammaproteobacteria possess intrinsic class A ß-lactamase genes that may represent the source of further dissemination and acquisition to other Gram-negative species. Her,e we characterized the KSA-1 class A ß-lactamase which gene was identified into the chromosome of an environmental Enterobacterales species, namely Kosakonia sacchari, recently identified also progenitor of an MCR-like colistin resistance determinant. METHODS: In-silico analysis using the GenBank database identified a class A ß-lactamase gene in the chromosome of Kosakonia sacchari SP1 (GenBank accession no. WP_065368351). The corresponding protein KSA-1 shared 63% amino-acid identity with the intrinsic CKO-1 from Citrobacter koseri and 53% with TEM-1. Using K. sacchari DSM 100203 reference strain as template, the blaKSA-1 was amplified, cloned into plasmid pUCp24 and expressed in E. coli TOP10. MICs and kinetic parameters were obtained from the purified enzyme. RESULTS: Strain K. sacchari SP1 conferred resistance to amino-, carboyx- and ureidopenicillins only. Once produced in E. coli, KSA-1 showed a typical clavulanic-acid inhibited extended-spectrum ß-lactamase (ESBL) associated to a peculiar temocillin resistance profile. Kinetic assays were performed using a purified extract of KSA-1, showed a high hydrolysis rate for benzylpenicillin and piperacillin and weakly extended-spectrum cephalosporins. Determination of inhibitory constants showed IC50 values of 2.2, 3 and 1.8 nM for clavulanic acid, tazobactam and avibactam, respectively. Analysis of sequences surrounding the blaKSA-1 gene did not reveal any mobile element that could have been involved in the acquisition of this ß-lactamase gene in that species. CONCLUSION: KSA-1 is a class A ESBL, distantly related to known ESBLs, with a high activity also temocillin. The blaKSA-1 gene can be considered as intrinsic in the species.
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Objectives: To analyse the susceptibility profile to cefepime, carbapenems and new ß-lactam/ß-lactamase inhibitor combinations in Enterobacter cloacae complex and Klebsiella aerogenes isolated from intra-abdominal, urinary, respiratory and bloodstream infections in the SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study in Spain. Methods: The susceptibilities of 759 isolates (473 E. cloacae complex and 286 K. aerogenes) collected in 11 Spanish hospitals from 2016 to 2022 were analysed following the EUCAST 2023 criteria. Molecular characterization looking for ß-lactamase genes was performed through PCR and DNA sequencing analysis. Results: E. cloacae complex showed resistance to third-generation cephalosporins in 25% of the cases, whereas K. aerogenes was resistant in 35%. Regarding cefepime, resistance in E. cloacae was higher (10%) than in K. aerogenes (2%). Carbapenems showed >85% activity in both microorganisms. Ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam had good activity against these microorganisms (>95%). In contrast, the activity of ceftolozane/tazobactam was lower (80%). A high proportion of the isolates resistant to new ß-lactam/ß-lactamase inhibitor combinations carried a carbapenemase, mainly OXA-48-like and VIM-1. Conclusions: Ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam show high activity against both E. cloacae complex and K. aerogenes isolates recovered in the SMART-Spain study. In contrast, differences have been found in the case of cefepime, showing more activity against K. aerogenes than E. cloacae complex. These results are useful for antimicrobial stewardship programmes and for the implementation of local and national guidelines.
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Infective endocarditis (IE) caused by Enterococcus spp. represents the third most common cause of IE, with high rates of relapse compared with other bacteria. Interestingly, late relapses (>6 months) have only been described in Enterococcus faecalis, but here we describe the first reported IE relapse with Enterococcus faecium more than a year (17 months) after the initial endocarditis episode. Firstly, by multi locus sequence typing (MLST), we demonstrated that both isolates (EF646 and EF641) belong to the same sequence type (ST117). Considering that EF641 was able to overcome starvation and antibiotic treatment conditions surviving for a long period of time, we performed bioinformatic analysis in identifying potential genes involved in virulence and stringent response. Our results showed a 13-nucleotide duplication (positions 1638-1650) in the gene relA, resulting in a premature stop codon, with a loss of 167 amino acids from the C-terminal domains of the RelA enzyme. RelA mediates the stringent response in bacteria, modulating levels of the alarmone guanosine tetraphosphate (ppGpp). The relA mutant (EF641) was associated with lower growth capacity, the presence of small colony variants, and higher capacity to produce biofilms (compared with the strain EF646), but without differences in antimicrobial susceptibility patterns according to standard procedures during planktonic growth. Instead, EF641 demonstrated tolerance to high doses of teicoplanin when growing in a biofilm. We conclude that all these events would be closely related to the long-term survival of the E. faecium and the late relapse of the IE. These data represent the first clinical evidence of mutations in the stringent response (relA gene) related with E. faecium IE relapse.
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Endocarditis Bacteriana , Endocarditis , Enterococcus faecium , Infecciones por Bacterias Grampositivas , Humanos , Enterococcus faecium/genética , Enterococcus faecium/metabolismo , Tipificación de Secuencias Multilocus , Endocarditis Bacteriana/tratamiento farmacológico , Endocarditis Bacteriana/microbiología , Endocarditis/tratamiento farmacológico , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antibacterianos/metabolismo , Guanosina Tetrafosfato/metabolismo , Enterococcus faecalis/metabolismo , Recurrencia , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Infecciones por Bacterias Grampositivas/microbiologíaRESUMEN
OBJECTIVES: To evaluate the clinical and epidemiological impact of a new molecular surveillance strategy based on qPCR to control an outbreak by Serratia marcescens in a Neonatal Intensive Care Unit (NICU). METHODS: We design a specific qPCR for the detection of S. marcescens in rectal swabs of patients admitted to a NICU. We divided the surveillance study into two periods: (a) the pre-PCR, from the outbreak declaration to the qPCR introduction, and (b) the PCR period, from the introduction of the qPCR until the outbreak was solved. In all cases, S. marcescens isolates were recovered and their clonal relationship was analysed by PFGE. Control measures were implemented during the outbreak. Finally, the number of bloodstream infections (BSI) was investigated in order to evaluate the clinical impact of this molecular strategy. RESULTS: Nineteen patients colonized/infected by S. marcescens were detected in the pre-PCR period (October 2020-April 2021). On the contrary, after the PCR implementation, 16 new patients were detected. The PFGE revealed 24 different pulsotypes belonging to 7 different clonal groups, that were not overlapping at the same time. Regarding the clinical impact, 18 months after the qPCR implementation, no more outbreaks by S. marcescens have been declared in the NICU of our hospital, and only 1 episode of BSI has occurred, compared with 11 BSI episodes declared previously to the outbreak control. CONCLUSIONS: The implementation of this qPCR strategy has proved to be a useful tool to control the nosocomial spread of S. marcescens in the NICU.
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Infección Hospitalaria , Sepsis , Infecciones por Serratia , Recién Nacido , Humanos , Infección Hospitalaria/epidemiología , Infección Hospitalaria/prevención & control , Infección Hospitalaria/diagnóstico , Unidades de Cuidado Intensivo Neonatal , Serratia marcescens/genética , Infecciones por Serratia/epidemiología , Infecciones por Serratia/prevención & control , Infecciones por Serratia/diagnóstico , Reacción en Cadena de la Polimerasa , Sepsis/epidemiología , Brotes de EnfermedadesRESUMEN
OBJECTIVES: The BIChromET selective medium for detecting piperacillin-tazobactam (TZP) and cefepime (FEP) resistant Pseudomonas aeruginosa was developed. METHODS: The performance of this medium was first evaluated using a collection of 100 P. aeruginosa clinical strains (70 TZP-susceptible, 30 TZP-resistant, 58 FEP-susceptible, and 42 FEP-resistant). Then, we performed clinical validation by testing 173 respiratory clinical samples. RESULTS: The BIChromET medium showed excellent sensitivity (TZP (avg. 96.7%); FEP (avg. 92.7%)) and specificity (TZP (avg. 98.9%); FEP (avg. 98%)) in distinguishing the detection limit ranging from 104 to 108 CFU/mL. Then, testing the bronchoalveolar lavage (BAL) and tracheobronchial aspirate (TBA) clinical specimens (N = 173) revealed the excellent performance of the medium with P. aeruginosa, showing 100% and 92.6% of categorical agreements with the results obtained via the broth microdilution methods (BMD) for TZP and FEP, respectively. CONCLUSION: This medium allows for easy and accurate detection of TZP/FEP-resistant isolates regardless of their resistance mechanisms.
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Rapid determination of susceptibility to piperacillin-tazobactam (TZP) is very important since the development of antibiotic resistance and inadequate treatment could increase the risk of clinical failure in infected patients, especially if such resistance is unknown to the clinician. Therefore, based on color change from orange to yellow of phenol red due to glucose metabolism (bacterial growth) in the presence of an adequate concentration of TZP (10 mg/L piperacillin and 5 mg/L tazobactam), the RapidTZP test has been developed to detect TZP resistance in Escherichia coli and Klebsiella pneumoniae isolates in a maximum of 3 h. A total of 140 isolates, 43 of E. coli and 97 of K. pneumoniae, were used to evaluate the performance of the test, 60 being resistant to TZP. The sensitivity and specificity of the test were 98.24% and 100%, respectively. Additionally, the RapidTZP test was validated by a pellet obtained directly from blood culture bottles. A total of 37 positive blood cultures for E. coli and 43 for K. pneumoniae were used for validation, 8 of them resistant to TZP. The sensitivity and specificity shown in the evaluation were 100% for both parameters. This new test is easy, fast, and accurate, providing results in 3 h. IMPORTANCE TZP is an antibiotic widely used for the empirical treatment of severe infections such as bloodstream infections. However, resistance to TZP in K. pneumoniae and E. coli has been increasing in the last few years. Thus, rapid detection of TZP resistance is critical to optimize the empirical treatment of patients with severe infections. In this study, we developed and evaluated a rapid test (RapidTZP) for the detection of TZP resistance in K. pneumoniae and E. coli directly from positive hemocultures in just 3 h. This rapid test has been validated on 138 K. pneumoniae and E. coli clinical isolates directly from agar plates and 80 K. pneumoniae and E. coli isolates causing bloodstream infections. The results demonstrate that the RapidTZP test has great clinical potential to optimize the empirical treatment of patients with bloodstream infections.
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Background: Seven CTX-M-27-producing Shigella sonnei strains were isolated at the University Hospital Virgen del Rocío (Seville, Spain) microbiology service from October to November 2021. Objectives: To offer extensive information on the microbiological and molecular epidemiology results of the seven S. sonnei isolates and compare them with other previously documented CTX-M-27-producing S. sonnei associated with MSM transmission. Methods: S. sonnei isolated from stool samples of patients with acute diarrhoea were identified through biochemical and serological typing. Whole characterization of the seven isolates was performed by sequencing with MinION Mk1C followed by genomic and molecular analysis. Results: All the isolates were resistant to penicillins, cephalosporins, fluoroquinolones, cotrimoxazole and azithromycin. Sequencing showed the presence of several resistance determinants, outstanding bla CTX-M-27, azithromycin resistance genes [ermB and mph(A)], qnrB19 and mutations in the QRDRs. All isolates belonged to the same hierarchical clustering of cgMLST (HierCC) with five allele distance (HC5) scheme v1 from EnteroBase. However, they presented differences in plasmid composition, with all seven isolates harbouring IncFII, IncB/O/K/Z and ColE1-like while SH2, SH6 and SH7 had IncFIB only. Our isolates were closely related to others from Spain (HC5; 98748), Australia (HC5; 98748) and the UK (HC5; 98748), which were also associated with MSM transmission. Nevertheless, the structure of the non-chromosomal genetic elements and the genetic context of bla CTX-M-27 presented a certain variability compared with isolates from other countries and among them. Conclusions: This study confirms the emergence of CTX-M-27-producing S. sonnei (ST152) associated with MSM transmission in Spain, adding it to the Europe outbreak list and reinforcing the necessity of active surveillance and control of this high-risk clone.
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The NG-Test CTX-M MULTI immunochromatographic assay has been developed to identify CTX-M-type ß-lactamases in Enterobacterales, being the most widespread extended-spectrum ß-lactamases. We showed here that the chromosomally-encoded ß-lactamases from Citrobacter farmeri and Citrobacter amalonaticus generated false-positive NG-Test CTX-M MULTI results, compromising the specificity of the test.
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Citrobacter , beta-Lactamasas , Citrobacter/efectos de los fármacos , Citrobacter/genética , Pruebas de Sensibilidad Microbiana , beta-Lactamasas/genéticaRESUMEN
A multidrug-resistant (carbapenems, aztreonam + avibactam, and cefiderocol) ST167 Escherichia coli clinical isolate recovered from a patient hospitalized in Switzerland produced NDM-35 showing ca. 10-fold increased hydrolytic activity toward cefiderocol compared to NDM-1. The isolate co-produced a CMY-type ß-lactamase, exhibited a four amino-acid insertion in PBP3, and possessed a truncated iron transporter CirA protein. Our study identified an association of unrelated resistance mechanisms leading to resistance to virtually all ß-lactams in a high-risk E. coli clone.
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Infecciones por Escherichia coli , Escherichia coli , Antibacterianos/farmacología , Cefalosporinas , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Infecciones por Escherichia coli/tratamiento farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , beta-Lactamas/farmacología , CefiderocolRESUMEN
OBJECTIVES: To perform the first prospective surveillance evaluating the occurrence of genes encoding colistin resistance, fosfomycin resistance, carbapenemase, or extended-spectrum ß-lactamases (ESBLs) among Enterobacterial isolates recovered from the gut flora of pigs from Egypt. METHODS: Between February and April 2020, 81 rectal swabs were collected from pigs in a slaughterhouse, Cairo, Egypt. Samples were screened for different resistance mechanisms using SuperPolymyxin, ChromID ESBL, SuperFOS, and SuperCarba selective agar plates. Antimicrobial susceptibility testing was performed for all isolates using disk diffusion and broth microdilution techniques. PCR screening was performed for ESBLs, carbapenemases, mcr, and fosA genes. Mating-out assays, multilocus sequence typing analysis, and plasmid typing were also performed. RESULTS: A high prevalence of ESBLs, carbapenemases, fosfomycin, and colistin resistance genes was evidenced among those isolates. The predominant ESBL identified was blaCTX-M-15, followed by blaCTX-M-9. We also identified blaNDM-5 and blaOXA-244. fosA3, fosA4, and fosA6 were identified in E. coli isolates. In addition, 11 MCR-1 producers were recovered. Notably, co-occurrence of ESBL genes and mcr or fosA genes was observed. MLST analysis revealed a high clonal diversity, ruling out the dissemination of one major clone. IncFIB-type was predominantly present among ESBL and FosA producers. The blaNDM-5 gene was carried on an IncX4-type, although the blaOXA-244 gene was chromosomally located. The mcr-1 gene was carried on a diversity of plasmids (IncI2, IncX4, and IncHI2). CONCLUSION: These results raise serious public health concerns as Egyptian pig meat could serve as a reservoir for antimicrobial resistance genes (ARGs), leading to worldwide dissemination.
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Proteínas Bacterianas , Colistina , Farmacorresistencia Bacteriana , Proteínas de Escherichia coli , Escherichia coli , Fosfomicina , Klebsiella pneumoniae , Polimixinas , Porcinos , beta-Lactamasas , Animales , Proteínas Bacterianas/genética , Colistina/farmacología , Farmacorresistencia Bacteriana/genética , Egipto , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/genética , Fosfomicina/farmacología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , Carne/microbiología , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Polimixinas/farmacología , Estudios Prospectivos , Porcinos/microbiología , beta-Lactamasas/genéticaRESUMEN
The ability of broad-spectrum ß-lactamases to reduce the susceptibility to ceftazidime-avibactam (CZA), ceftolozane-tazobactam (C/T), imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam (AZA), and cefiderocol (FDC) was evaluated both in Pseudomonas aeruginosa and in Escherichia coli using isogenic backgrounds. Although metallo-ß-lactamases conferred resistance in most cases, except for AZA, several clavulanic-acid-inhibited extended-spectrum ß-lactamases (PER, BEL, SHV) had a significant impact on the susceptibility to CZA, C/T, and FDC.
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Infecciones por Escherichia coli , Inhibidores de beta-Lactamasas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Compuestos de Azabiciclo/farmacología , Aztreonam/farmacología , Ceftazidima/farmacología , Cefalosporinas , Combinación de Medicamentos , Escherichia coli , Infecciones por Escherichia coli/tratamiento farmacológico , Humanos , Lactamas , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/genética , CefiderocolRESUMEN
Chlorhexidine is a widely used antiseptic in hospital and community health care. Decreased susceptibility to this compound has been recently described in Klebsiella pneumoniae and Pseudomonas aeruginosa, together with cross-resistance to colistin. Surprisingly, few data are available for Escherichia coli, the main species responsible for community and health care-associated infections. In order to decipher chlorhexidine resistance mechanisms in E. coli, we studied both in vitro derived and clinical isolates through whole-genome sequence analysis. Comparison of strains grown in vitro under chlorhexidine pressure identified mutations in the gene mlaA coding for a phospholipid transport system. Phenotypic analyses of single-gene mutants from the Keio collection confirmed the role of this mutation in the decreased susceptibility to chlorhexidine. However, mutations in mlaA were not found in isolates from large clinical collections. In contrast, genome wide association studies (GWAS) showed that, in clinical strains, chlorhexidine reduced susceptibility was associated with the presence of tetA genes of class B coding for efflux pumps and located in a Tn10 transposon. Construction of recombinant strains in E. coli K-12 confirmed the role of tetA determinant in acquired resistance to both chlorhexidine and tetracycline. Our results reveal that two different evolutionary paths lead to chlorhexidine decreased susceptibility: one restricted to in vitro evolution conditions and involving a retrograde phospholipid transport system; the other observed in clinical isolates associated with efflux pump TetA. None of these mechanisms provide cross-resistance to colistin. This work demonstrates the GWAS power to identify new resistance mechanisms in bacterial species.
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Escherichia coli , Resistencia a la Tetraciclina , Antibacterianos/farmacología , Clorhexidina/farmacología , Escherichia coli/genética , Estudio de Asociación del Genoma Completo , Pruebas de Sensibilidad Microbiana , Tetraciclina/farmacología , Resistencia a la Tetraciclina/genéticaRESUMEN
In-silico analysis and cloning experiments identified a fosC2-like fosfomycin resistance gene in the chromosome of Aliidiomarina shirensis, with our data suggesting that this bacterium might be added to the list of species identified as reservoirs of fos-like genes that were subsequently acquired by other Gram-negative species. Indeed, the fosC2 gene was identified as acquired in Providencia huaxinensis and Aeromonas hydrophila isolates, with this gene being located in class 1 integron structures in the latter cases. Biochemical characterization and site-directed mutagenesis showed a higher catalytic efficiency for the intrinsic FosC2AS (from A. shirensis) than for the acquired FosC2 (from P. huaxinensis) enzyme due to a single substitution in the amino acid sequence (Gly43Glu). Notably, this study constitutes the first identification of the likely natural reservoir of a complete gene cassette (including its attC site).
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Fosfomicina , Antibacterianos/farmacología , Fosfomicina/farmacología , Gammaproteobacteria , Integrones/genética , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Reacción en Cadena de la PolimerasaRESUMEN
A selective medium for screening fosfomycin (FOS)-resistant Enterobacterales was developed. Performances of this medium were first evaluated by using cultures of a collection of 84 enterobacterial clinical strains (42 FOS susceptible and 42 FOS resistant). The SuperFOS medium showed excellent sensitivity and specificity of detection (100%) in those conditions. Then, by testing spiked stool and spiked urine specimens, it revealed excellent performances, with lower limits of identification ranging from 101 to 102 CFU/ml. This screening medium allows easy and accurate detection of FOS-resistant isolates regardless of their resistance mechanisms.
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Fosfomicina , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Fosfomicina/farmacología , Humanos , Klebsiella pneumoniae , Pruebas de Sensibilidad Microbiana , beta-LactamasasRESUMEN
Escherichia coli is the most commonly identified human pathogen and a prominent microorganism of the gut microbiota. Acquired resistance to antibiotics in this species is driven mainly by horizontal gene transfer and plasmid acquisition. Currently, the main concern is the acquisition of extended-spectrum ß-lactamases of the CTX-M type in E. coli, a worldwide-observed phenomenon. Plasmids encoding CTX-M enzymes have different scaffolds and conjugate at different frequencies. Here, we show that the conjugation rates of several plasmid types encoding broad-spectrum ß-lactamases are increased when the E. coli donor strain is exposed to subinhibitory concentrations of diverse orally given antibiotics, including fluoroquinolones, such as ciprofloxacin and levofloxacin, but also trimethoprim and nitrofurantoin. This study provides insights into underlying mechanisms leading to increased plasmid conjugation frequency in relation to DNA synthesis inhibitor-type antibiotics, involving reactive oxygen species (ROS) production and probably increased expression of genes involved in the SOS response. Furthermore, we show that some antioxidant molecules currently approved for unrelated clinical uses, such as edaravone, p-coumaric acid, and N-acetylcysteine, may antagonize the ability of antibiotics to increase plasmid conjugation rates. These results suggest that several antioxidative molecules might be used in combination with these "inducer" antibiotics to mitigate the unwanted increased resistance plasmid dissemination.
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Antibacterianos , Infecciones por Escherichia coli , Antibacterianos/farmacología , Antioxidantes/farmacología , Farmacorresistencia Microbiana , Escherichia coli/genética , Transferencia de Gen Horizontal , Humanos , Plásmidos/genética , beta-Lactamasas/genéticaRESUMEN
Colistin is considered as a last resort agent for treatment of severe infections caused by carbapenem-resistant Enterobacterales (CRE). Recently, plasmid-mediated colistin resistance genes (mcr type) have been reported, mainly corresponding to mcr-1 producers. Those mcr-1-positive Enterobacterales have been identified not only from human isolates, but also from food samples, from animal specimens and from environmental samples in various parts of the world. Our study focused on the occurrence and characterization of mcr-1-positive Enterobacterales recovered from retail raw chicken in Egypt. From the 345 retail chicken carcasses collected, a total of 20 samples allowed to recover mcr-1-positive isolates (Escherichia coli, n = 19; Citrobacter freundii, n = 1). No mcr-2- to mcr-10-positive isolate was identified from those samples. The colistin resistance trait was confirmed for all those 20 isolates with a positivity of the Rapid Polymyxin NP (Nordmann-Poirel) test. Minimum inhibitory concentrations (MICs) of colistin for all MCR-1-producing isolates ranged between 4 and 16 µg/mL. Noticeably, 9 out of the 20 mcr-1-positive isolates produced an extended-spectrum ß-lactamase (ESBL), respectively producing CTX-M-9 (n = 2), CTX-M-14 (n = 4), CTX-M-15 (n = 2), and SHV-12 (n = 1). Noteworthy, the fosA4 gene encoding resistance to fosfomycin was found in a single mcr-1-positive E. coli isolate, in which both genes were located on different conjugative plasmids. The pulsed-field gel electrophoresis (PFGE) patterns were identified, corresponding to 10 different sequence types (STs), highlighting the genetic diversity of those different E. coli. Whole-genome sequencing revealed three major types of mcr-1-bearing plasmids, corresponding to IncI2, IncX4, and IncHI2 scaffolds. The occurrence of MCR-1-producing multidrug-resistant Enterobacterales in retail raw chicken is of great concern, considering the possibility of transmission to humans through the food chain.
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Many transferable quinolone resistance mechanisms have been identified in Gram-negative bacteria. The plasmid-encoded 65-amino-acid-long ciprofloxacin-modifying enzyme CrpP was recently identified in Pseudomonas aeruginosa isolates. We analyzed a collection of 100 clonally unrelated and multidrug-resistant P. aeruginosa clinical isolates, among which 46 were positive for crpP-like genes, encoding five CrpP variants conferring variable levels of reduced susceptibility to fluoroquinolones. These crpP-like genes were chromosomally located as part of pathogenicity genomic islands.
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Fluoroquinolonas , Pseudomonas aeruginosa , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana , Europa (Continente) , Fluoroquinolonas/farmacología , Islas Genómicas/genética , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/genética , VirulenciaRESUMEN
OBJECTIVES: To decipher the genetics of acquisition of carbapenemase-encoding genes identified in two carbapenem-resistant Enterobacteriaceae recovered from a single patient in Portugal. METHODS: Carbapenemase genes were searched by PCR assays and mating-out assays were performed to further characterize the plasmid support of the carbapenemase genes. Genetic characterization of the plasmid supports was performed by whole-plasmid sequencing using the Illumina technology. RESULTS: We identified here two NDM-1-producing isolates, namely a Morganella morganii and a Proteus mirabilis, sharing the same blaNDM-1-positive plasmid. This 154 kb plasmid belonged to the IncA/C2 type, recently renamed IncC, and co-harboured two AmpC ß-lactamase genes, namely blaCMY-4 and blaDHA-1, in addition to the 16S rRNA methylase gene armA encoding high-level resistance to aminoglycosides. In addition, the M. morganii isolate produced the CTX-M-33 extended-spectrum ß-lactamase possessing weak carbapenemase activity, encoded by another plasmid. CONCLUSIONS: We showed here that, in addition to KPC-type and OXA-181 carbapenemases, which have been identified as widespread in this country, another concern is the emergence of NDM-1-producing enterobacterial isolates in Portugal. We demonstrated here the in vivo plasmid transfer of a blaNDM-1-positive plasmid leading to dissemination of this carbapenemase gene within different enterobacterial species in a single patient.
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Infecciones por Enterobacteriaceae , Morganella morganii , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Infecciones por Enterobacteriaceae/epidemiología , Humanos , Pruebas de Sensibilidad Microbiana , Morganella morganii/genética , Plásmidos/genética , Portugal , Proteus mirabilis/genética , ARN Ribosómico 16S , beta-Lactamasas/genéticaRESUMEN
Escherichia coli is the most frequent Gram-negative bacilli involved in intra-abdominal infections. However, despite high mortality rates associated with biliary tract infections due to E. coli, there is no study focusing on this pathogen. In this study, we have characterized a group of 15 E. coli isolates obtained from 12 patients with biliary tract infections. Demographic and clinical data of the patients were recovered. Phylogeny, resistome, and virulome analysis through whole genome sequencing and biofilm formation were investigated. Among the 15 E. coli isolates, no predominant sequence type (ST) was identified, although 3 of them belonged to unknown STs (20%). Resistance to ampicillin, amoxicillin/clavulanic acid, cotrimoxazole, and quinolones was more present in these isolates; whereas, third and fourth generation cephalosporins, carbapenems, amikacin, tigecycline, and colistin were highly active. Moreover, high diversity of virulence factors has been found, with sfa, fimH, and gad the most frequently detected genes. Interestingly, 26.6% of the E. coli isolates were high biofilm-producers. Altogether, our data characterized for the first time E. coli isolates associated with biliary tract infections in terms of genomic relationship, resistome, and virulome.