scholarly journals Model System To Evaluate the Effect of ampD Mutations on AmpC-Mediated β-Lactam Resistance

2006 ◽  
Vol 50 (6) ◽  
pp. 2030-2037 ◽  
Author(s):  
Amber J. Schmidtke ◽  
Nancy D. Hanson

ABSTRACT Mutations within the structural gene of ampD can lead to AmpC overproduction and increases in β-lactam MICs in organisms with an inducible ampC. However, identification of mutations alone cannot predict the impact that those mutations have on AmpD function. Therefore, a model system was designed to determine the effect of ampD mutations on ceftazidime MICs using an AmpD− mutant Escherichia coli strain which produced an inducible plasmid-encoded AmpC. ampD genes were amplified by PCR from strains of E. coli, Citrobacter freundii, and Pseudomonas aeruginosa. Also, carboxy-terminal truncations of C. freundii ampD genes were constructed representing deletions of 10, 21, or 25 codons. Amplified ampD products were cloned into pACYC184 containing inducible bla ACT-1-ampR. Plasmids were transformed into E. coli strains JRG582 (AmpD−) and K-12 259 (AmpD+). The strains were evaluated for a derepressed phenotype using ceftazidime MICs. Some mutated ampD genes, including the ampD gene of a derepressed C. freundii isolate, resulted in substantial decreases in ceftazidime MICs (from >256 μg/ml to 12 to 24 μg/ml) for the AmpD− strain, indicating no role for these mutations in derepressed phenotypes. However, ampD truncation products and ampD from a partially derepressed P. aeruginosa strain resulted in ceftazidime MICs of >256 μg/ml, indicating a role for these gene modifications in derepressed phenotypes. The use of this model system indicated that alternative mechanisms were involved in the derepressed phenotype observed in strains of C. freundii and P. aeruginosa. The alternative mechanism involved in the derepressed phenotype of the C. freundii isolate was downregulation of ampD transcription.

2006 ◽  
Vol 74 (8) ◽  
pp. 4685-4693 ◽  
Author(s):  
Haiqing Sheng ◽  
Ji Youn Lim ◽  
Hannah J. Knecht ◽  
Jie Li ◽  
Carolyn J. Hovde

ABSTRACT The human pathogen Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening sequelae and transiently colonizes healthy cattle at the terminal rectal mucosa. This study analyzed virulence factors important for the clinical manifestations of human E. coli O157:H7 infection for their contribution to the persistence of E. coli in cattle. The colonizing ability of E. coli O157:H7 was compared with those of nonpathogenic E. coli K-12 and isogenic deletion mutants missing Shiga toxin (Stx), the adhesin intimin, its receptor Tir, hemolysin, or the ∼92-kb pO157. Fully ruminant steers received a single rectal application of one E. coli strain so that effects of mucosal attachment and survival at the terminal rectum could be measured without the impact of bacterial passage through the entire gastrointestinal tract. Colonization was monitored by sensitive recto-anal junction mucosal swab culture. Nonpathogenic E. coli K-12 did not colonize as well as E. coli O157:H7 at the bovine terminal rectal mucosa. The E. coli O157:H7 best able to persist had intimin, Tir, and the pO157. Strains missing even one of these factors were recovered in lower numbers and were cleared faster than the wild type. In contrast, E. coli O157:H7 strains that were missing Stx or hemolysin colonized like the wild type. For these three strains, the number of bacteria increased between days 1 and 4 postapplication and then decreased slowly. In contrast, the numbers of noncolonizing strains (K-12, Δtir, and Δeae) decreased from the day of application. These patterns consistently predicted long-term colonization or clearance of the bacteria from the bovine terminal rectal mucosa.


2021 ◽  
Vol 10 (3) ◽  
pp. e43910313516
Author(s):  
Lucas Harassim ◽  
Olibio Lopes Fiebig da Silva ◽  
Luiz Felipe Soares Pinheiro ◽  
Elber José Assaiante dos Santos ◽  
Cláudio Daniel Cerdeira ◽  
...  

A Infecção no Trato Urinário (ITU) acometendo pacientes em Unidades de Terapia Intensiva (UTIs) é uma realidade preocupante, agravada pelo uso irracional de antimicrobianos e a alarmante multirresistência em microrganismos. Nós avaliamos o nível de assertividade quanto ao uso de antimicrobianos durante à antibioticoterapia empírica (ATE), em pacientes diagnosticados com ITU, comparando tal tratamento farmacológico empírico e o realizado após o antibiograma (antibioticoterapia direcionada), além disto, estimamos a prevalência dos agentes etiológicos e analisamos os fatores de risco associados. Este é um estudo observacional e transversal, realizado em 2015, no qual foram avaliados pacientes de ambos os sexos e todas as idades apresentando ITU e submetidos à antibioticoterapia, internados em uma UTI de um hospital no sul de Minas Gerais, Brasil. Dos 49 pacientes avaliados (28 mulheres [M] e 21 homens [H]), a média de idade foi 55±19 anos (IC(95) 49-61) e a faixa etária ≥70 anos foi a predominante. Quatorze diferentes microrganismos foram causadores de ITUs, sendo que 28,3% (IC(95%) 16,2-40,4) dos isolados clínicos tiveram Escherichia coli como o agente etiológico (33,3% H e 28,6% M); 18,9% (IC(95%) 8,3-29,4) Acinetobacter baumannii (33,3% H e 10,7% M); 15,1% (IC(95%) 5,5-24,7) Klebsiella pneumoniae (19% H e 14,3% M); 11,3% Pseudomonas aeruginosa (9,5% H e 14,3% M); 5,7% Enterobacter aerogenes (14,3% H); 3,8% Klebsiella oxytoca; 3,8% Staphylococcus aureus (7,1% M); e 1,9% para cada um dos seguintes microrganismos: Enterococcus faecalis (4,8% H); Proteus mirabilis (3,6% M); Enterobacter cloacae (3,6% M); Providencia rettgeri (4,8% H); Citrobacter koseri (3,6% M); Citrobacter freundii (3,6% M); e Fungos leveduriformes (4,8% H). As prevalências de ITUs causadas por A. baumannii e P. aeruginosa foram influenciadas pelo sexo (χ² com p<0,001). No sexo masculino, houve correlações positivas “substanciais” entre o aumento da idade (em anos) e a prevalência de ITU causada por E. coli (r = 0,69) ou entre idades menos avançadas e a prevalência de ITU causada por A. baumannii (r = -0,7). No sexo feminino, houve uma correlação positiva “extremamente forte” entre o aumento da idade e a prevalência de ITU causada por E. coli (r = 0,94; IC(95) 0,66-0,99; p<0,0014). Os antibióticos mais utilizados de forma empírica (ATE) foram: Ciprofloxacina (14,3% IC(95%) 4,7-24,1), Cefepima (14,3%) e Vancomicina (10%), e após o antibiograma (antibioticoterapia direcionada): Ceftazidima (16,3% IC(95%) 6-26,7), Ciprofloxacina (14,3% IC(95%) 4,5-24,1), Polimixina B (10,2%), Imipenem (10,2%) e Ampicilina + Sulbac. (8,2%). Em 20% dos casos, as terapias empíricas (ATE) foram consideradas “inapropriadas/não acertadas”. Contudo, também devemos ter ciência da necessidade clínica e quanto ao imediatismo para o tratamento de uma ITU em UTI, uma vez que a doença pode ser fatal se uma terapia não for instituída, portanto, nós aconselhamos avaliações mais minuciosas, tanto da racionalidade do uso de antibióticos, quanto dos fatores de risco para o desenvolvimento de ITUs em UTIs.


2002 ◽  
Vol 184 (16) ◽  
pp. 4374-4383 ◽  
Author(s):  
Abel Ferrández ◽  
Andrew C. Hawkins ◽  
Douglas T. Summerfield ◽  
Caroline S. Harwood

ABSTRACT Pseudomonas aeruginosa, a γ-proteobacterium, is motile by means of a single polar flagellum and is chemotactic to a variety of organic compounds and phosphate. P. aeruginosa has multiple homologues of Escherichia coli chemotaxis genes that are organized into five gene clusters. Previously, it was demonstrated that genes in cluster I and cluster V are essential for chemotaxis. A third cluster (cluster II) contains a complete set of che genes, as well as two genes, mcpA and mcpB, encoding methyl-accepting chemotaxis proteins. Mutations were constructed in several of the cluster II che genes and in the mcp genes to examine their possible contributions to P. aeruginosa chemotaxis. A cheB2 mutant was partially impaired in chemotaxis in soft-agar swarm plate assays. Providing cheB2 in trans complemented this defect. Further, overexpression of CheB2 restored chemotaxis to a completely nonchemotactic, cluster I, cheB-deficient strain to near wild-type levels. An mcpA mutant was defective in chemotaxis in media that were low in magnesium. The defect could be relieved by the addition of magnesium to the swarm plate medium. An mcpB mutant was defective in chemotaxis when assayed in dilute rich soft-agar swarm medium or in minimal-medium swarm plates containing any 1 of 60 chemoattractants. The mutant phenotype could be complemented by the addition of mcpB in trans. Overexpression of either McpA or McpB in P. aeruginosa or Escherichia coli resulted in impairment of chemotaxis, and these cells had smooth-swimming phenotypes when observed under the microscope. Expression of P. aeruginosa cheA2, cheB2, or cheW2 in E. coli K-12 completely disrupted wild-type chemotaxis, while expression of cheY2 had no effect. These results indicate that che cluster II genes are expressed in P. aeruginosa and are required for an optimal chemotactic response.


2016 ◽  
Author(s):  
Kristina Doxsee ◽  
Ryan Berthelot ◽  
Suresh Neethirajan

Electrotaxis or galvanotaxis refers to the migration pattern of cells induced in response to electrical potential. Although it has been extensively studied in mammalian cells, electrotaxis has not been explored in detail in bacterial cells; information regarding the impact of current on pathogenic bacteria is severely lacking. Therefore, we designed a series of single and multi-cue experiments to assess the impact of varying currents on bacterial motility dynamics in pathogenic multi-drug resistant (MDR) strains of Pseudomonas aeruginosa and Escherichia coli using a microfluidic platform. Motility plays key roles in bacterial migration and the colonization of surfaces during the formation of biofilms, which are inherently recalcitrant to removal and resistant to traditional disinfection strategies (e.g. antibiotics). Use of the microfluidic platform allows for exposure to current, which can be supplied at a range that is biocidal to bacteria, yet physiologically safe in humans (single cue). This system also allows for multi-cue experiments where acetic acid, a relatively safe compound with anti-fouling/antimicrobial properties, can be combined with current to enhance disinfection. These strategies may offer substantial therapeutic benefits, specifically for the treatment of biofilm infections, such as those found in the wound environment. Furthermore, microfluidic systems have been successfully used to model the unique microfluidic dynamics present in the wound environment, suggesting that these investigations could be extended to more complex biological systems. Our results showed that the application of current in combination with acetic acid has profound inhibitory effects on MDR strains of P. aeruginosa and E. coli, even with brief applications. Specifically, E. coli motility dynamics and cell survival were significantly impaired starting at a concentration of 125 μA DC and 0.31% acetic acid, while P. aeruginosa was impaired at 70 μA and 0.31% acetic acid. As these strains are relevant wound pathogens, it is likely that this strategy would be effective against similar strains in vivo and could represent a new approach to hasten wound healing.


1989 ◽  
Vol 102 (3) ◽  
pp. 447-458 ◽  
Author(s):  
G. R. Smith ◽  
D. Till ◽  
L. M. Wallace ◽  
D. E. Noakes

SUMMARYNecrobacillosis is caused byFusobacterium necrophorum(FN), but other organisms are often present in the lesions. Their possible role was studied in experiments made with a virulent FN strain which, by itself, produced fatal necrobacillosis in mice provided that large doses ( > 106organisms, subcutaneously) were given. Mice were inoculated subcutaneously with FN suspended in sub-lethal doses (0·1 ml) of undiluted or diluted broth cultures of other bacteria. Undiluted culture of a strain ofEscherichia colireduced the infective dose of FX to < 10 organisms: in the necrobacillosis lesions that developed, fusobacteria greatly outnumberedE. coli. A heat-killed preparation or sterile filtrate ofE. coliculture had little if any effect on FN.Citrobacter freundiiand comparativelv small numbers ofCorynebacterium (Actinomyces) pyogenesproduced effects similar to that ofE. coli. An α-haemolvtic streptococcus.Pseudomonas aeruginosa.Bacteroides fragilisandFusobacterium nudeatumalso enhanced the infectivity of FN. though less strikingly thanE. coli. FN increased the persistence invivoof the α-haemolytic streptococcus andB. fragilis, and enabled the latter to multiply profusely.


2013 ◽  
Vol 26 (5) ◽  
pp. 495-502 ◽  
Author(s):  
Vincent Walker ◽  
Maxime Bruto ◽  
Floriant Bellvert ◽  
René Bally ◽  
Daniel Muller ◽  
...  

Plant-beneficial effects of bacteria are often underestimated, especially for well-studied strains associated with pathogenicity or originating from other environments. We assessed the impact of seed inoculation with the emblematic bacterial models Agrobacterium tumefaciens C58 (plasmid-cured) or Escherichia coli K-12 on maize seedlings in nonsterile soil. Compared with the noninoculated control, root biomass (with A. tumefaciens or E. coli) and shoot biomass (with A. tumefaciens) were enhanced at 10 days for ‘PR37Y15’ but not ‘DK315’, as found with the phytostimulator Azospirillum brasilense UAP-154 (positive control). In roots as well as in shoots, Agrobacterium tumefaciens and E. coli triggered similar (in PR37Y15) or different (in DK315) changes in the high-performance liquid chromatography profiles of secondary metabolites (especially benzoxazinoids), distinct from those of Azospirillum brasilense UAP-154. Genome sequence analysis revealed homologs of nitrite reductase genes nirK and nirBD and siderophore synthesis genes for Agrobacterium tumefaciens, as well as homologs of nitrite reductase genes nirBD and phosphatase genes phoA and appA in E. coli, whose contribution to phytostimulation will require experimental assessment. In conclusion, the two emblematic bacterial models had a systemic impact on maize secondary metabolism and resulted in unexpected phytostimulation of seedlings in the Azospirillum sp.-responsive cultivar.


2012 ◽  
Vol 78 (10) ◽  
pp. 3580-3591 ◽  
Author(s):  
Monique Rothe ◽  
Carl Alpert ◽  
Wolfram Engst ◽  
Stephanie Musiol ◽  
Gunnar Loh ◽  
...  

ABSTRACTTo study the impact of nutritional factors on protein expression of intestinal bacteria, gnotobiotic mice monoassociated withEscherichia coliK-12 were fed three different diets: a diet rich in starch, a diet rich in nondigestible lactose, and a diet rich in casein. Two-dimensional gel electrophoresis and electrospray-tandem mass spectrometry were used to identify differentially expressed proteins of bacteria recovered from small intestine and cecum. Oxidative stress response proteins such as AhpF, Dps, and Fur, all of which belong to the oxyR regulon, were upregulated inE. coliisolates from mice fed the lactose-rich diet. Luciferase reporter gene assays demonstrated that osmotic stress caused by carbohydrates led to the expression ofahpCFanddps, which was not observed in anE. coliΔoxyRmutant. Growth ofahpCFandoxyRdeletion mutants was strongly impaired when nondigestible sucrose was present in the medium. The wild-type phenotype could be restored by complementation of the deletions with plasmids containing the corresponding genes and promoters. The results indicate that some OxyR-dependent proteins play a major role in the adaptation ofE. colito osmotic stress. We conclude that there is an overlap of osmotic and oxidative stress responses. Mice fed the lactose-rich diet possibly had a higher intestinal osmolality, leading to the upregulation of OxyR-dependent proteins, which enable intestinalE. colito better cope with diet-induced osmotic stress.


Microbiology ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 1143-1153 ◽  
Author(s):  
Nicola J. Holden ◽  
Makrina Totsika ◽  
Eva Mahler ◽  
Andrew J. Roe ◽  
Kirsteen Catherwood ◽  
...  

The majority of Escherichia coli strains isolated from urinary tract infections have the potential to express multiple fimbriae. Two of the most common fimbrial adhesins are type 1 fimbriae and pyelonephritis-associated pili (Pap). Previous research has shown that induced, plasmid-based expression of a Pap regulator, papB, and its close homologues can prevent inversion of the fim switch controlling the expression of type 1 fimbriae. The aim of the present study was to determine if this cross-regulation occurs when PapB is expressed from its native promoter in the chromosome of E. coli K-12 and clinical isolates. The regulation was examined in three ways: (1) mutated alleles of the pap regulatory region, including papB and papI, that maintain the pap promoter in either the off or the on phase were exchanged into the chromosome of both E. coli K-12 and the clinical isolate E. coli CFT073, and the effect on type 1 fimbrial expression was measured; (2) type 1 fimbrial expression was determined using a novel fimS : : gfp + reporter system in mutants of the clinical isolate E. coli 536 in which combinations of complete fimbrial clusters had been deleted; (3) type 1 fimbrial expression was determined in a range of clinical isolates and compared with both the number of P clusters and their expression. All three approaches demonstrated that P expression represses type 1 fimbrial expression. Using a number of novel genetic approaches, this work extends the initial finding that PapB inhibits FimB recombination to the impact of this regulation in clinical isolates.


2017 ◽  
Vol 1 (2) ◽  
pp. 48-60
Author(s):  
A.G. Salmanov ◽  
A.V. Rudenko

Мета роботи — вивчити резистентність до антибіотиків бактеріальних збудників інфекцій сечових шляхів (ІСШ), виділених у пацієнтів урологічного стаціонару в м. Києві. Матеріали і методи. Досліджено 1612 штамів бактерій, виділених із сечі хворих з ІСШ (цистит, уретрит, пієлонефрит), госпіталізованих в урологічне відділення ДУ «Інститут урології НАМН України» у м. Києві протягом 2016 р. Серед пацієнтів переважали жінки — 1201 (74,5 %). Вік хворих становив від 17 до 74 років. Для збору даних використано медичну документацію лікарні. Мікробіологічні дослідження виконано у лабораторії мікробіології ДУ «Інститут урології НАМН України». Аналізували результати культурального дослідження зразків сечі, зібраних за наявності клінічних ознак ІСШ. Дослідження клінічного матеріалу та інтерпретацію отриманих результатів проводили загальноприйнятими методами. Вивчено чутливість уропатогенів до 31 антибіотика дискодифузійним методом відповідно до рекомендацій Інституту клінічних та лабораторних стандартів США (Clinical and Laboratory Standards Institute (CLSI)). Результати та обговорення. Аналіз мікробного спектра сечі виявив домінування серед уропатогенів штамів Escherichia coli (32,0 %), Enterococcus faecalis (19,5 %), Klebsiella pneumoniae (10,9 %), Staphylococcus epidermidis (8,9 %), S. haemolyticus (6,5 %) та Pseudomonas aeruginosa (6,4 %). Частка Enterococcus faecium, Enterobacter aerogenes і Streptococcus viridans становила відповідно 2,5, 2,2 і 1,6 %, Enterobacter cloacae, Klebsiella oxytoca, Acinetobacter baumannii, Proteus vulgaris та Providencia rettgeri — менше 1,0 %. У більшості випадків (69,7 %) мікроорганізми виділено у монокультурі, у решті випадків — у мікробних асоціа- ціях. Високу резистентність до тестованих антибіотиків виявили штами E. aerogenes (45,1 %), E. cloacae (45,7 %), E. faecium (40,9 %), E. faecalis (40,7 %), E. coli (39,9 %), P. aeruginosa (34,0 %), K. pneumoniae (28,6 %). Найбільш активними до уропатогенів були іміпенем (E. coli — 87,6 %, P. aeruginosa — 75,7 %, E. cloacae — 67,3 %, E. aerogenes — 72,6 %, K. pneumoniae — 93,2 %), меропенем (E. coli — 89,1 %, P. aeruginosa — 76,7 %, K. pneumoniae — 82,6 %), лефлоцин (E. coli — 74,5 %, ентерококи — 78,7 %, P. aeruginosa — 76,7 %, E. cloacae — 73,9 %, E. aerogenes — 80,4 %, K. pneumoniae — 83,5 %), амоксицилін/клавуланат (ентерококи — 84,6 %), фурагін (ентерококи — 82,6 %), цефоперазон (K. pneumoniae — 89,2 %, P. aeruginosa — 73,8 %), цефтріаксон (K. pneumoniae — 80,1 %). Висновки. Антибіотикорезистентність збудників ІСШ — важлива терапевтична проблема. Найбільшою активністю до уропатогенів характеризуються іміпенем, меропенем, лефлоцин, амоксицилін/ клавуланат, фурагін, цефоперазон, цефтріаксон, які можна розглядати як препарат вибору для призначення стартової терапії ІСШ. Необхідно здійснювати постійний моніторинг за резистентністю до дії антибіотиків. Політику використання антибіотиків у кожному стаціонарі слід визначати залежно від локальних даних щодо резистентності до протимікробних препаратів.


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