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1.
Zhonghua Shao Shang Za Zhi ; 36(1): 14-23, 2020 Jan 20.
Artigo em Chinês | MEDLINE | ID: mdl-32023713

RESUMO

Objective: To isolate a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient, and to study its biological characteristics, genomic information, and effects on bacterial biofilm. Methods: (1) In 2018, pan-drug resistant Klebsiella pneumoniae UA168 (hereinafter referred to as the host bacteria) solution isolated from the blood of a burn patient in Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (hereinafter referred to as Ruijin Hospital) was used to isolate and purify the bacteriophage against pan-drug resistant Klebsiella pneumoniae from the sewage of Ruijin Hospital with sewage co-culture method, drip plate method, and double-agar plate method. The bacteriophage was named as phage KP168 and the plaque morphology was observed. (2) The phage KP168 solution was taken for cesium chloride density gradient centrifugation and dialysis, and then the morphology of phage KP168 was observed through transmission electron microscope after phosphotungstic acid negative staining. (3) The phage KP168 solution was taken to determine the lytic ability of the phage KP168 against 20 strains of pan-drug resistant Klebsiella pneumoniae isolated from the burned patients' blood in Ruijin Hospital by the drip plate method, and then the lysis rate was calculated. (4) The phage KP168 solution at a initial titer of 9.3×10(11) plaque-forming unit (PFU)/mL (400 µL per tube) and the host bacteria solution at a concentration of 1×10(9) colony-forming unit (CFU)/mL (4 mL per tube) were conventionally shaking cultured together for 4 hours at multiplicity of infection (MOI) of 10.000, 1.000, 0.100, 0.010, or 0.001, respectively (1 tube per MOI). The titer of phage KP168 was measured by the double-agar plate method (the measurement method was the same below) to select the optimal MOI. The experiment was repeated three times. (5) The host bacteria solution at a concentration of 1×10(9) CFU/mL (4 mL per tube) and the phage KP168 solution at an adjusted titer of 5×10(7) PFU/mL (400 µL per tube) were mixed at the MOI of 0.005. The plaques were counted 0 (immediately), 1, 2, 3, 4, 5, 15, and 30 minutes (1 tube at each time point) after mixing by the double-agar plate method (the counting method was the same below), and the percentage of adsorbed phages was calculated to screen for the optimal adsorption time. The experiment was repeated three times. (6) The host bacteria solution at a concentration of 1×10(9) CFU/mL (300 µL per tube) and the phage KP168 solution at a titer of 5×10(8) PFU/mL (60 µL per tube) were mixed at MOI of 0.005 and conventionally shaking cultured after standing for the optimal adsorption time. The phage KP168 titer was measured 0 (immediately), 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 minutes after culture, and a one-step growth curve was drawn. The experiment was repeated three times. (7) The phage KP168 solution at a titer of 2.5×10(10) PFU/mL was left to stand for 1 hour at 37, 40, 50, 60, or 70 ℃ (3 tubes at each time point, 1 mL per tube) for counting the plaques, and then the thermal stability curve was drawn. SM buffer at a pH values of 5.0, 6.0, 7.0, 7.4, 8.0, 9.0, or 10.0 were added to the phage KP168 solution at a titer of 3.0×10(10) PFU/mL, respectively. The mixed solution was left to stand for 1 hour at 37 ℃ (3 tubes of each pH, each tube containing 100 µL phage KP168 solution and 900 µL SM buffer), and then the plaques were counted, and an acid-base stability curve was drawn. (8) The phage KP168 solution was taken for DNA extraction and sequencing after dialysis as in experiment (2). The whole genome was annotated with Prokka to obtain the coding sequence of phage KP168. Nucleotide's BLAST function was used to proceed nucleic acid sequence alignment for finding a known phage with the highest similarity to the phage KP168 nucleic acid sequence, and Blastx function was used to translate the coding sequence into protein for its function prediction. The comparison with Antibiotic Resistance Genes Database and Virulence Factors Database was proceeded. (9) In a 96-well plate, at a MOI of 1.000, 0.100, 0.010 or 0.001 (3 wells per MOI), 20 µL phage KP168 solution at a initial titer of 5.8×10(10) PFU/mL was added to 200 µL host bacteria solution at a concentration of 1.5×10(8) CFU/mL (the same concentration below) for co-cultivation for 48 hours. After 200 µL host bacteria solution was left to stand for 48 hours, 20 µL phage KP168 solution at a titer of 1×10(6,) 1×10(7,) 1×10(8,) 1×10(9,) or 1×10(10) PFU/mL (3 wells per titer) was added respectively for action for 4 hours. In both experiments, 200 µL host bacteria solution added with 20 µL SM buffer (3 wells) acted as a negative control, and 220 µL LB culture medium (3 wells) acted as a blank control. Absorbance values were measured by a microplate reader, and inhibition/destruction rates of biofilm were calculated. The experiments were both repeated three times. Results: (1) The plaques of phage KP168 successfully isolated and purified were transparent and round, and its diameter was approximately 1.5 mm. (2) The phage KP168 has a regular polyhedron structure with a diameter of about 50 nm and without a tail. (3) The phage KP168 could lyse 13 of 20 strains of Klebsiella pneumoniae from burned patients, with a lysis rate of 65.0%. (4) When MOI was 1.000, the titer was the highest after co-culturing the phage KP168 with the host bacteria for 4 hours, which was the optimal MOI. (5) After the mixing of the phage KP168 with the host bacteria for 4 minutes, the percentage of the adsorbed phage reached the highest, which was the optimal adsorption time. (6) The one-step growth curve showed that during the lysis of the host bacteria by phage KP168, the incubation period was about 10 minutes, and the lysis period was about 40 minutes. (7) With the condition of 40 ℃ or pH 7.4, the number of plaques and the activity of phage KP168 reached the highest. (8) The genome of phage KP168 was a linear double-stranded DNA with a length of 40 114 bp. There were 48 possible coding sequences. It had the highest similarity to Klebsiella phage_vB_Kp1. The most similar known proteins corresponding to the translated proteins of coding sequences contained 23 hypothetical proteins and 25 proteins with known functions. No resistance genes or virulence factor genes were found. The GeneBank accession number was KT367885. (9) After 48 hours of co-cultivation of the phage KP168 and the host bacteria at each MOI, the inhibition rates of biofilm were similar, with an average of about 45%. After the phage KP168 with a titer of 1×10(9) PFU/mL acted on the biofilm formed by the host bacteria for 4 h, the destruction rate of biofilm was the highest, reaching an average of 42%. Conclusions: In this study, a bacteriophage against pan-drug resistant Klebsiella pneumoniae from a burn patient, phage KP168, is isolated from sewage, which belongs to the tailless phage. It has a wide host spectrum, short adsorption time, and short incubation period, with certain thermal and acid-base stability. Its genomic information is clear, and it does not contain resistance genes or virulence factor genes. It also has an inhibitory effect on the formation of bacterial biofilm and a destructive effect on the formed bacterial biofilm.


Assuntos
Bacteriófagos , Queimaduras , Biofilmes , China , Genômica , Humanos , Klebsiella pneumoniae
2.
Euro Surveill ; 25(3)2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31992391

RESUMO

From September to October 2019, seven patients colonised or infected with a ceftazidime-avibactam (CZA)-resistant Klebsiella pneumoniae carbapenemase (KPC)-2-producing K. pneumoniae were detected in two intensive care units of a Greek general hospital. The outbreak strain was sequence type (ST)147 and co-produced KPC-2 and the novel plasmid-borne Vietnamese extended-spectrum ß-lactamase (VEB)-25 harbouring a K234R substitution associated with CZA resistance. Epidemiological investigations revealed that the resistance was probably acquired by horizontal transmission independently from previous CZA exposure.


Assuntos
Antibacterianos/farmacologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo , Idoso , Idoso de 80 Anos ou mais , Compostos Azabicíclicos , Ceftazidima , Surtos de Doenças , Combinação de Medicamentos , Farmacorresistência Bacteriana , Feminino , Genoma Bacteriano , Grécia , Humanos , Unidades de Terapia Intensiva , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação/genética , Sequenciamento Completo do Genoma
3.
J Med Microbiol ; 69(1): 82-86, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31904319

RESUMO

In recent years, Serratia marcescens has emerged as an important agent of hospital-acquired infections, such as pneumonia, urinary tract infection, septicaemia and meningitis, particularly in vulnerable patients. Compared to Klebsiella pneumoniae and Escherichia coli, S. marcescens is less commonly associated with bla KPC genes, yet few cases of plasmid transmission at the gastrointestinal level from K. pneumoniae carbapenemase (KPC)-producing Enterobacterales to S. marcescens have been described. Here we report a case of in vivo acquisition, during a 3-month period of hospitalization in the intensive care unit, of a bla KPC-3 gene carried by a pKpQIL-IT plasmid, and its probable transmission at the bronchial level among different species of Enterobacterales, including K. pneumoniae and S. marcescens. By using whole genome sequence analyses we were able provide insight into the dynamics of carbapenem-resistance determinants acquisition in the lower respiratory tract, a novel anatomical region for such plasmid transmission events, that usually involve the gastrointestinal tract. The co-presence at the same time of both wild-type and resistant Enterobacterales could have been the critical factor leading to the spread of plasmids harbouring carbapenem-resistance genes, of particular importance during surveillance screenings. The possibility of such an event may have significant consequences in terms of antimicrobial treatment, with a potential limitation of therapeutic options, thereby further complicating the clinical management of high-risk critically ill patients.


Assuntos
Proteínas de Bactérias/genética , Transferência Genética Horizontal , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Plasmídeos , Serratia marcescens/enzimologia , Serratia marcescens/genética , beta-Lactamases/genética , Adulto , Infecção Hospitalar/microbiologia , Humanos , Unidades de Terapia Intensiva , Infecções por Klebsiella/microbiologia , Masculino , Infecções Respiratórias/microbiologia , Infecções por Serratia/microbiologia , Sequenciamento Completo do Genoma
4.
Arch Virol ; 165(1): 97-104, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31734749

RESUMO

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has spread globally and emerged as an urgent public health threat. Bacteriophages are considered an effective weapon against multidrug-resistant pathogens. In this study, we report a novel lytic phage, kpssk3, which is able to lyse CRKP and degrade exopolysaccharide (EPS). The morphological characteristics of kpssk3 observed by transmission electron microscopy, including a polyhedral head and a short tail, indicate that it belongs to the family Podoviridae. A one-step growth curve revealed that kpssk3 has a latent period of 10 min and a burst size of 200 plaque-forming units (pfu) per cell. kpssk3 was able to lyse 25 out of 27 (92.59%) clinically isolated CRKP strains, and it also exhibited high stability to changes in temperature and pH. kpssk3 has a linear dsDNA genome of 40,539 bp with 52.80% G+C content and 42 putative open reading frames (ORFs). No antibiotic resistance genes, virulence factors, or integrases were identified in the genome. Based on bioinformatic analysis, the tail fiber protein of phage kpssk3 was speculated to possess depolymerase activity towards EPS. By comparative genomics and phylogenetic analysis, it was determined that kpssk3 is a new T7-like virus and belongs to the subfamily Autographivirinae. The characterization and genomic analysis of kpssk3 will promote our understanding of phage biology and diversity and provide a potential strategy for controlling CRKP infection.


Assuntos
Farmacorresistência Bacteriana , Klebsiella pneumoniae/virologia , Podoviridae/classificação , Sequenciamento Completo do Genoma/métodos , Composição de Bases , Carbapenêmicos , Genoma Viral , Concentração de Íons de Hidrogênio , Lisogenia , Microscopia Eletrônica de Transmissão , Filogenia , Podoviridae/genética , Podoviridae/fisiologia , Termodinâmica , Proteínas da Cauda Viral/genética
6.
APMIS ; 128(1): 48-60, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31693234

RESUMO

Klebsiella pneumoniae is an important opportunistic pathogen with significant potential for virulence and multidrug resistance. Treatment failure often occurs because the pathogen may couple virulence and drug resistance with the stringent response. This study assessed the role of the spoT gene in environmental and nutritional stress tolerance, exopolysaccharide capsule production and biofilm formation. spoT mutants were constructed using the lambda red recombinase technique, and mutant and wild-type (WT) strains were exposed to limiting concentrations of carbon (glucose), phosphate and aminoacid, and environmental stresses of ethanol, salt and heat. Cell viability, capsule production and cell length were assessed as well as the ability to grow biofilm under antibiotic pressure using gentamicin and ceftazidime. spoT mutants were more susceptible to stresses versus WT; the reverse was true for survival during biofilm susceptibility assay (p < 0.05), especially when carbon and phosphate were present. spoT mutants were elongated and lacked a capsule versus WT and non-starved strains. The inability to produce capsule in mutants before and after starvation was likely a general effect of spoT mutation. These data suggest that the spoT-mediated stringent response is important for K. pneumoniae in conditions of nutrient limitation, environmental stress and antimicrobial pressure.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/fisiologia , Estresse Fisiológico , Aminoácidos/química , Farmacorresistência Bacteriana , Etanol/farmacologia , Regulação Bacteriana da Expressão Gênica , Glucose/química , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Fosfatos/química , Cloreto de Sódio/farmacologia , Virulência/genética , Fatores de Virulência
7.
Artigo em Inglês | MEDLINE | ID: mdl-31859846

RESUMO

Nosocomial bacterial infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) is associated with high mortality in neurosurgical patients. There are few reports in the literature on meningitis caused by CRKP. We report two cases of CRKP meningitis after neurosurgery. The K. pneumoniae identification and antimicrobial susceptibility testing were performed using the Vitek Compact System. Minimum inhibitory concentrations of polymyxin B were determined using the broth microdilution method. Molecular typing of K. pneumoniae isolates was investigated using multilocus sequence typing. Antimicrobial susceptibility testing showed that the K. pneumoniae isolates were multidrug resistant and co-produced extended-spectrum ß-lactamases and KPC enzymes. The patients were treated with intrathecal polymyxin. Genetic polymorphism analyses revealed two different K. pneumoniae clones (ST1298 and ST2687), which were observed for the first time in CRKP infections. We recommend intravenous administration of intrathecal polymyxin for treating meningitis caused by multidrug-resistant K. pneumoniae .


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Infecção Hospitalar/microbiologia , Klebsiella pneumoniae/isolamento & purificação , Meningites Bacterianas/microbiologia , Procedimentos Neurocirúrgicos/efeitos adversos , Complicações Pós-Operatórias/microbiologia , Adolescente , Antibacterianos/farmacologia , Infecção Hospitalar/diagnóstico , Infecção Hospitalar/tratamento farmacológico , Feminino , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Masculino , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/tratamento farmacológico , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade
8.
Bratisl Lek Listy ; 120(12): 935-940, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31855054

RESUMO

OBJECTIVES: We focused on detecting the most frequent resistance mechanisms in selected multidrug-resistant (MDR) strains and determining their antimicrobial resistance. BACKGROUND: MDR pathogens pose urgent public health threat due to limited treatment options, rigorous control measures and significant mortality. METHODS: We confirmed extended-spectrum ß-lactamase (ESBL) and carbapenemase producing Enterobacteriaceae through guidelines, as well following ß-lactamases: AmpC by cloxacillin, class A carbapenemase with phenylboronic acid, class B metallo-ß-lactamase with ethylenediaminetetraacetic acid. Multilocus sequence typing was used to investigate 20 Escherichia coli strains. RESULTS: Overall 205 mostly ESBL Escherichia coli demonstrated resistance against amikacin (4.7 %), tigecycline (1.2 %), and no resistance to ceftazidime/avibactam, meropenem, nitrofurantoin and fosfomycin. Out of 41 Klebsiella species (spp.), 37 (90.2 %) showed carbapenemase activity, 13 (35.1 %) of class A and 24 (64.9 %) of class B. Resistance was following: meropenem 66.7 %, tigecyclin 10.2 % and colistin 0 %. From Enterobacter spp. 21 strains, 14 (66.7 %) were ESBL, 5 produced ESBL and/or AmpC and 2 were MDR. We ascertained 14 (70 %) E. coli sequence type - ST131. CONCLUSIONS: The study revealed various resistance mechanisms in concert with different agents and association of specific ST131 within E. coli. These characteristics considerably contribute to emergence of antimicrobial resistance (Tab. 4, Ref. 30).


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Klebsiella pneumoniae/efeitos dos fármacos , beta-Lactamases/metabolismo , Adulto , Idoso , Proteínas de Bactérias/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/enzimologia , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Farmacorresistência Bacteriana , Enterobacteriaceae/classificação , Infecções por Enterobacteriaceae/microbiologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Humanos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , beta-Lactamases/genética
9.
Zhonghua Shao Shang Za Zhi ; 35(11): 798-803, 2019 Nov 20.
Artigo em Chinês | MEDLINE | ID: mdl-31775468

RESUMO

Objective: To explore the resistance mechanism and gene type of carbapenems-resistant Klebsiella pneumoniae (CRKP) in burn care unit. Methods: A total of 27 CRKP strains were primarily isolated from 22 patients [20 males, 2 females, aged (42±16) years] admitted to burn care unit of Institute of Burn Research of the First Affiliated Hospital of Army Medical University (the Third Military Medical University, hereinafter referred to as our department) from January to December 2017. After identification of bacteria, the months of detection and distribution of sample source were analyzed. Drug resistance tests of 15 antibiotics were conducted. Polymerase chain reaction was used to detect the drug resistant genes. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were used to analyze the gene type of strains. Results: (1) During the whole year of 2017, CRKP strains were mostly detected in August (8 strains), September (6 strains), and October (5 strains), with no CRKP in January, March, June, November, and December. Five strains from bed units were detected in August (2 strains), September (1 strain), and October (2 strains). (2) Twenty-seven CRKP strains were derived from blood samples (40.7%, 11/27), wound exudate samples (18.5%, 5/27), deep vein catheter samples (11.1%, 3/27), sputum samples (7.4%, 2/27), urine samples (3.7%, 1/27), and bed unit samples (18.5%, 5/27). (3) The 27 CRKP strains were detected with drug-resistance rates of 100.0% to 7 antibiotics including cefoperazone/sulbactam, piperacillin/tazobactam, cefazolin, ceftriaxone, cefepime, ertapenem, and compound sulfamethoxazole, no drug-resistance to tigecycline, with drug-resistance rates higher than 81.0% to the rest 7 antibiotics. (4) Detection rates for resistance gene bla(CTX-M-10), bla(SHV), bla(TEM), bla(CTX-M-14), bla(ACT), and bla(KPC) were all above 92.5%. (5) According to PFGE, the 27 CRKP strains had 6 types (A, A(1), A(2), B, C, and D). Strains of type A were mainly detected in February, May, and September, with detection rate of 37.0% (10/27). Strains of type C were mainly detected in July, August, and October, with detection rate of 48.1% (13/27). Strains of types A(1), A(2), B, and D were scatteredly detected, with detection rate of 3.7% (1/27) respectively. According to MLST, the 27 CRKP strains had 6 STs. ST11 was the most frequent type, accounting for 74.1% (20/27), which was detected in August to October. The detection rate of ST395, ST2230, ST215, ST260, and STnew ranged from 3.7%(1/27) to 7.4%(2/27), and the strains were scatteredly detected. Conclusions: The main source of CRKP from burn care unit of our department was bloodstream. All the CRKP strains showed high drug-resistance rate and complicated resistance mechanism. There were small scale outbreaks caused by CRKP of type A, type C, and ST11, which should be paid more attention to in clinical treatment and infection control.


Assuntos
Queimaduras/microbiologia , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/efeitos dos fármacos , Adulto , Antibacterianos/farmacologia , Unidades de Queimados , Feminino , Humanos , Klebsiella pneumoniae/classificação , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , beta-Lactamases/genética
10.
BMC Infect Dis ; 19(1): 928, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31684890

RESUMO

BACKGROUND: Endemic presence of Klebsiella pneumoniae resistant to carbapenem in Italy has been due principally to the clonal expansion of CC258 isolates; however, recent studies suggest an ongoing epidemiological change in this geographical area. METHODS: 50 K. pneumoniae strains, 25 carbapenem-resistant (CR-Kp) and 25 susceptible (CS-Kp), collected from march 2014 to march 2016 at the Laboratory of Bacteriology of the Paolo Giaccone Polyclinic University hospital of Palermo, Italy, were characterized for antibiotic susceptibility and fully sequenced by next generation sequencing (NGS) for the in silico analysis of resistome, virulome, multi-locus sequence typing (MLST) and core single nucleotide polymorphism (SNP) genotypes RESULTS: MLST in silico analysis of CR-Kp showed that 52% of isolates belonged to CC258, followed by ST395 (12%), ST307 (12%), ST392 (8%), ST348 (8%), ST405 (4%) and ST101 (4%). In the CS-Kp group, the most represented isolate was ST405 (20%), followed by ST392 and ST15 (12%), ST395, ST307 and ST1727 (8%). The in silico ß-lactamase analysis of the CR-Kp group showed that the most detected gene was blaSHV (100%), followed by blaTEM (92%), blaKPC (88%), blaOXA (88%) and blaCTX-M (32%). The virulome analysis detected mrk operon in all studied isolates, and wzi-2 was found in three CR-Kp isolates (12%). Furthermore, the distribution of virulence genes encoding for the yersiniabactin system, its receptor fyuA and the aerobactin system did not show significant distribution differences between CR-Kp and CS-Kp, whereas the Klebsiella ferrous iron uptake system (kfuA, kfuB and kfuC genes), the two-component system kvgAS and the microcin E495 were significantly (p < 0.05) prevalent in the CS-Kp group compared to the CR-Kp group. Core SNP genotyping, correlating with the MLST data, allowed greater strain tracking and discrimination than MLST analysis. CONCLUSIONS: Our data support the idea that an epidemiological change is ongoing in the Palermo area (Sicily, Italy). In addition, our analysis revealed the co-existence of antibiotic resistance and virulence factors in CR-Kp isolates; this characteristic should be considered for future genomic surveillance studies.


Assuntos
Farmacorresistência Bacteriana/genética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Fatores de Virulência/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Genótipo , Humanos , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/classificação , Klebsiella pneumoniae/isolamento & purificação , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Filogenia , Polimorfismo de Nucleotídeo Único , Sicília , beta-Lactamases/genética
11.
Arch Esp Urol ; 72(9): 939-947, 2019 Nov.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-31697255

RESUMO

During their journey through the female reproductive tract to reach the oocyte in the ampulla of the fallopian tube, spermatozoa interact with substances and microorganisms that affect sperm quality, thus altering their fertilizing capacity. OBJECTIVES: To determine in vitro the effect of Streptococcus agalactiae, Klebsiella pneumoniae and their soluble factors on sperm parameters, and to evaluate the ability of human sperm to interact with and transport these bacteria. METHODS: The effects of S. agalactiae, K. pneumoniae and their soluble factors on the viability, sperm motility and functional sperm parameters were quantified. In addition, motile spermatozoa were incubated with decreasing concentrations of bacteria for one hour, washed and post-infection treatments were performed with trypsin and transport capacity was assessed by quantitative cultures. RESULTS: Incubation of spermatozoa with K. pneumoniae decreased progressive motility. The soluble factors of K. pneumoniae increased the number of necrotic spermatozoa and the soluble factors of S. agalactiae increased lipid peroxidation of the sperm membrane (p<0.05). A strong interaction between sperm and bacteria was observed in the transport assays even in washed trypsin-treated samples. CONCLUSION: Human spermatozoa act as vectors for infections, generating strong interactions with K. pneumoniae and S. agalactiae favoring their diffusion through the female reproductive tract. This interaction affects male fertility by altering progressive motility, increasing the number of necrotic cells and inducing apoptosis.


Assuntos
Espermatozoides , Infecções Estreptocócicas , Streptococcus agalactiae , Feminino , Humanos , Klebsiella pneumoniae/isolamento & purificação , Masculino , Motilidade Espermática , Espermatozoides/microbiologia , Infecções Estreptocócicas/transmissão , Streptococcus agalactiae/isolamento & purificação
12.
Medicine (Baltimore) ; 98(45): e17878, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31702658

RESUMO

RATIONALE: Donor-derived bacterial infection is a rare cause of morbidity after solid organ transplantation (SOT) but associated with significant morbidity and mortality, deaths caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) infection account for a considerable proportion of postoperation mortality rate in liver and kidney recipients. The arterial rupture as a result of fungal arteritis is occasionally described, while the rupture of graft vascular anastomosis after SOT due to donor-derived CRKP infection is rarely reported. PATIENTS CONCERNS: We reported 1 patient with donor-derived CRKP infection following liver transplantation and 2 patients following renal transplantation (1 liver and 2 kidneys were from the same donor), who experienced sudden abdominal pain and abdominal hemorrhage almost at the same time after organ transplantation. DIAGNOSIS: The patients were diagnosed as graft arteries rupture due to corrosion caused by CRKP infection based on computed tomography scan, blood culture, laparotomy, and pulse-field gel electrophoresis. INTERVENTIONS: Anti-shock treatment, exploratory laparotomy, broad-spectrum antibiotics, and abdominal puncture and drainage were given. OUTCOMES: The liver recipient survived as well as the liver graft, still under treatment of multiple abdominal infections. The 2 renal recipients were alive after resection of the renal grafts and underwent hemodialysis. LESSONS: Rupture of graft artery should be foreseen when donor-derived CRKP infection was confirmed and broad-spectrum antibiotics and other interventions need to be considered.


Assuntos
Transplante de Rim/efeitos adversos , Infecções por Klebsiella/etiologia , Transplante de Fígado/efeitos adversos , Adulto , Antibacterianos , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Feminino , Artéria Hepática/diagnóstico por imagem , Humanos , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Artéria Renal/diagnóstico por imagem , Ruptura/etiologia , Doadores de Tecidos
13.
J Basic Microbiol ; 59(12): 1195-1207, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31617952

RESUMO

Klebsiella pneumoniae can naturally synthesize 3-hydroxypropionic acid (3-HP), 1,3-propanediol (1,3-PD), and 2,3-butanediol (2,3-BD) from glycerol. However, biosynthesis of these industrially important chemicals is constrained by troublesome byproducts. To clarify the influences of byproducts on 3-HP production, in this study, a total of eight byproduct-producing enzyme genes including pmd, poxB, frdB, fumC, dhaT, ilvH, adhP, and pflB were individually deleted from the K. pneumoniae genome. The resultant eight mutants presented different levels of metabolites. In 24-h shake-flask cultivation, the adhP- and pflB-deletion mutants produced 0.41 and 0.44 g/L 3-HP, respectively. Notably, the adhP and pflB double deletion mutant K. pneumoniaeΔadhPΔpflB produced 1.58 g/L 3-HP in 24-h shake-flask cultivation. When K. pneumoniaeΔadhPΔpflB was harnessed as a host strain to overexpress PuuC, a native aldehyde dehydrogenase (ALDH) catalyzing 3-hydroxypropionaldehyde (3-HPA) to 3-HP, the resulting recombinant strain K. pneumoniaeΔadhPΔpflB(pTAC-puuC) (pTAC-puuC is PuuC expression vector) generated 66.91 g/L 3-HP with a cumulative yield of 70.84% on glycerol in 60-h bioreactor cultivation. Additionally, this strain showed 2.3-, 5.1-, and 0.67-fold decrease in the concentrations of 1,3-PD, 2,3-BD, and acetic acid compared with the reference strain K. pneumoniae(pTAC-puuC). These results indicated that the byproducts exerted differential impacts on the production of 3-HP, 1,3-PD, and 2,3-BD. Although combinatorial elimination of byproduct pathways could reprogram glycerol flux, the enzyme 1,3-propanediol oxidoreductase (DhaT) that catalyzes 3-HPA to 1,3-PD and the enzymes ALDHs, especially, PuuC are most pivotal for 3-HP production. This study provides a deep understanding of how byproducts affect the production of 3-HP, 1,3-PD, and 2,3-BD in K. pneumoniae.


Assuntos
Vias Biossintéticas/fisiologia , Glicerol/metabolismo , Klebsiella pneumoniae/metabolismo , Ácido Láctico/análogos & derivados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Reatores Biológicos , Vias Biossintéticas/genética , Butileno Glicóis/metabolismo , Expressão Gênica , Técnicas de Inativação de Genes , Klebsiella pneumoniae/genética , Ácido Láctico/metabolismo , Engenharia Metabólica , Propilenoglicóis/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
15.
Anal Bioanal Chem ; 411(27): 7315-7325, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31637462

RESUMO

Biofilms are communities of bacteria living embedded in a highly hydrated matrix composed of polysaccharides, proteins, and extracellular DNA. This life style confers numerous advantages to bacteria including protection against external threats. However, they also contribute to increase bacterial resistance against antimicrobials, an issue particularly relevant in dangerous infections. Due to the complexity of the matrix, few information is present in the literature on details of its architecture including the spatial distribution of the macromolecular components which might give hints on the way the biofilm scaffold is built up by bacteria. In this study, we investigated the possibility to combine well-established microbiological procedures with advanced microscopies to get information on composition and distribution of the macromolecular components of biofilm matrices. To this, confocal microscopy, diffraction-limited infrared (IR) spectral imaging, and atomic force microscopy (AFM) were used to explore biofilm produced by a clinical strain of Klebsiella pneumoniae. IR imaging permitted to have clues on how the biofilm grows and spreads on surfaces, and the local distribution of the components within it. Through the analysis of the pure component spectra, it was possible to assess the chemical and structural composition of the saccaridic matrix, confirming the data obtained by NMR. It was also possible to follow the time course of biofilm from 6 up to 48 h when the biofilm grew into a 3-dimensional multi-layered structure, characteristic of colonies of bacteria linked together by a complex matrix. In addition, nanoFTIR and AFM investigations allowed the estimation of biofilm growth in the vertical direction and the morphological analysis of bacterial colonies at different time points and the evaluation of the chemical composition at the nanoscale.


Assuntos
Biofilmes/crescimento & desenvolvimento , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/fisiologia , Matriz Extracelular de Substâncias Poliméricas/química , Matriz Extracelular de Substâncias Poliméricas/ultraestrutura , Humanos , Klebsiella pneumoniae/química , Klebsiella pneumoniae/ultraestrutura , Microscopia de Força Atômica , Microscopia Confocal , Espectrofotometria Infravermelho
16.
J Med Microbiol ; 68(12): 1787-1792, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31661052

RESUMO

Introduction. PFGE is the 'gold standard' method for bacterial subtyping. However, many strains are non-typable by this approach because of DNA degradation by nucleases action.Aim. To evaluate a modified PFGE protocol for typing nosocomial isolates of Klebsiella pneumoniae.Methods. Twenty- five K. pneumoniae isolates previously exposed to DNA degradation were used to optimize an extraction method for elimination of DNases activity before applying Xba1 enzyme. Introducing of sodium dodecyl sulfate (SDS) in different concentrations to the extraction buffer was evaluated for protecting genomic DNA molecule from degradation by nucleases.Results. Addition of 3 % SDS in combination with 3 % N-lauryl sarcosine to the extraction buffer was found to reduce the previously experienced nuclease activity. Pre-examination of plug quality prior to the digestion phase could efficiently reduce the expense of the wasted enzyme.Conclusion. We have successfully devised a PFGE protocol that enhanced the typeability of nosocomial K. pneumoniae.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Infecção Hospitalar/microbiologia , DNA Bacteriano/metabolismo , Eletroforese em Gel de Campo Pulsado/métodos , Klebsiella pneumoniae/classificação , Klebsiella pneumoniae/genética
18.
New Microbiol ; 42(4): 197-204, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31609454

RESUMO

Klebsiella pneumoniae due to the presence of multiple antibiotic resistance mechanisms is one of the most threatening human pathogens nowadays. The aim of the study was to characterize antimicrobial susceptibility, presence of resistance mechanisms and the prevalence of selected genes encoding ESBLs in 170 K. pneumoniae isolates recovered from children and adults hospitalized in two Polish medical centers from 2008 to 2015. The phenotypic identification of strains was confirmed by amplification of mdh gene. ESBLs, metallo-beta- lactamases, Klebsiella pneumoniae carbapenemases and OXA-48 were detected using phenotypic tests. The blaCTX-M-1, blaTEM and blaSHV ESBL genes were amplified by PCR. Pediatric K. pneumoniae isolates displayed significantly higher resistance to piperacillin/tazobactam, cefoxitin, imipenem, amikacin and ciprofloxacin than strains obtained from adults (P<0.05). The presence of ESBLs, OXA-48, KPC and MBL was confirmed in 80.6%, 21.8%, 8.2% and 2.4%, respectively, of the tested strains. The CTX-M-1 enzymes were predominant (91.2%), followed by TEM (63.5%) and SHV (11.8%). The blaTEM was significantly more common in adults than in children (P<0.05). Dual or triple bla genes were observed in 55.9% and 8.2% of K. pneumoniae isolates. Further local epidemiological studies are required to monitor the dissemination of multidrug-resistant K. pneumoniae strains.


Assuntos
Anti-Infecciosos , Infecções por Klebsiella , Klebsiella pneumoniae , beta-Lactamases , Adulto , Anti-Infecciosos/farmacologia , Criança , Hospitais/estatística & dados numéricos , Humanos , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Polônia/epidemiologia , Prevalência , beta-Lactamases/metabolismo
19.
BMC Infect Dis ; 19(1): 830, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31590648

RESUMO

BACKGROUND: Many gaps in the burden of resistant pathogens exist in endemic areas of low- and middle-income economies, especially those endemic for carbapenem resistance. The aim of this study is to evaluate risk factors for carbapenem-resistance, to estimate the association between carbapenem-resistance and all-cause 30-day mortality and to examine whether mortality is mediated by inappropriate therapy. METHODS: A case-control and a cohort study were conducted in one tertiary-care hospital in Medellín, Colombia from 2014 to 2015. Phenotypic and genotypic characterization of isolates was performed. In the case-control study, cases were defined as patients infected with carbapenem-resistant K. pneumoniae (CRKP) and controls as patients infected with carbapenem-susceptible K. pneumoniae (CSKP). A risk factor analysis was conducted using logistic regression models. In the cohort study, the exposed group was defined as patients infected with CRKP and the non-exposed group as patients infected with CSKP. A survival analysis using an accelerated failure time model with a lognormal distribution was performed to estimate the association between carbapenem resistance and all-cause 30-day-mortality and to examine whether mortality is mediated by inappropriate therapy. RESULTS: A total of 338 patients were enrolled; 49 were infected with CRKP and 289 with CSKP. Among CRKP isolates CG258 (n = 29), ST25 (n = 5) and ST307 (n = 4) were detected. Of importance, every day of meropenem (OR 1.18, 95%CI 1.10-1.28) and cefepime (OR 1.22, 95%CI 1.03-1.49) use increase the risk of carbapenem resistance. Additional risk factors were previous use of ciprofloxacin (OR 2.37, 95%CI 1.00-5.35) and urinary catheter (OR 2.60, 95%CI 1.25-5.37). Furthermore, a significant lower survival time was estimated for patients infected with CRKP compared to CSKP (Relative Times 0.44, 95%CI 0.24-0.82). The strength of association was reduced when appropriate therapy was included in the model (RT = 0.81 95%CI 0.48-1.37). CONCLUSION: Short antibiotic courses had the potential to reduce the selection and transmission of CRKP. A high burden in mortality occurred in patients infected with CRKP in a KPC endemic setting and CRKP leads to increased mortality via inappropriate antibiotic treatment. Furthermore, dissemination of recognized hypervirulent clones could add to the list of challenges for antibiotic resistance control.


Assuntos
Antibacterianos/uso terapêutico , Enterobacteriáceas Resistentes a Carbapenêmicos , Doenças Endêmicas , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/mortalidade , Klebsiella pneumoniae/genética , Meropeném/uso terapêutico , Idoso , Antibacterianos/efeitos adversos , Estudos de Casos e Controles , Cefepima/efeitos adversos , Cefepima/uso terapêutico , Ciprofloxacino/efeitos adversos , Ciprofloxacino/uso terapêutico , Colômbia , Farmacorresistência Bacteriana Múltipla , Feminino , Genótipo , Humanos , Estimativa de Kaplan-Meier , Klebsiella pneumoniae/isolamento & purificação , Modelos Logísticos , Masculino , Meropeném/efeitos adversos , Pessoa de Meia-Idade , Fenótipo , Estudos Prospectivos , Fatores de Risco , Análise de Sobrevida , Cateteres Urinários/efeitos adversos
20.
Medicine (Baltimore) ; 98(39): e17362, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31574882

RESUMO

RATIONALE: Klebsiella pneumoniae infection can induce multiple invasive abscesses, and the invasive infection is severe and life-threatening. PATIENT CONCERNS: A 69-year-old previously healthy Chinese male presented with fever, chill, backache, and ocular pain. DIAGNOSIS: The blood culture results indicated Klebsiella pneumoniae of the K1 serotype. Multiple invasive abscesses in liver, lung, eye, soft tissue, and central nervous system were identified by imaging examination. Subsequently, the patient experienced right ocular pain accompanied by visual disturbance. Tyndall sign was strongly positive, and lens opacity was observed by the ophthalmologist. INTERVENTIONS: Full-dose and long-term treatment with meropenem was performed. Intraventricular injection of glass and anterior chamber puncture with antibiotics were performed twice. The patient also underwent an evacuation of the brain abscess. OUTCOMES: The patient's headache and lumbar backache were relieved, his ophthalmodynia disappeared, and his vision recovered after nearly 3 months of treatment. LESSONS: Imaging examination is very important for severe Klebsiella pneumoniae infection. The choice of antibiotics is complex, and the antimicrobial regimen should be adjusted according to the assessment of illness and the therapeutic effect. Surgical intervention must be considered for patients with multiple invasive abscesses.


Assuntos
Abscesso/microbiologia , Antibacterianos/uso terapêutico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae , Meropeném/uso terapêutico , Idoso , Humanos , Masculino
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