Resistance to antibiotics in Pseudomonas aeruginosa

Rezistencija Pseudomonas aeruginosa na antibiotike rezultat je smanjenja propusnosti stanične stijenke, pojačanog efluksa antibiotika iz bakterijske stanice, aktivnosti inaktivirajućih enzima te rjeđe promjene ciljnog mjesta. Urođena otpornost pseudomonasa na niz β-laktamskih antibiotika posljedica je aktivnosti kromosomske inducibilne ampC β-laktamaze (klasa C po Ambleru) čija derepresija dovodi do otpornosti na ureidopeniciline, cefalosporine 3. (i 4.) generacije, a u kombinaciji s gubitkom porina OprD i na karbapeneme. Stečene β-laktamaze u pseudomonasa mogu biti serinske, iz klase A i D te metalo-β-laktamaze (MBL) iz klase B koje posreduju rezistenciju na sve β-laktame uključujući karbapeneme. Rezistencija na aminoglikozide najčešće je posljedica aktivnosti modificirajućih enzima i efluksa. Rezistencija na fluorirane kinolone posljedica je uglavnom promjene ciljnog mjesta tj. mutacije DNA giraze ili topoizomeraze i pojačanog rada efluksnih proteina. Postotak rezistentnih izolata pseudomonasa nije se značajno promijenio u posljednjih 10 godina u Hrvatskoj. Među aminoglikozidima je 2008. godine bilo najmanje otpornih sojeva na amikacin, a najviše na gentamicin. Rezistencija na ciprofloksacin bila je 24%, na ceftazidim 8%, dok je karbapenem-rezistentnih izolata bilo oko 10%.Resistance of Pseudomonas aeruginosa to antibiotics is a result of cell wall impermeability, enhanced efflux of antibiotics from bacterial cells, inactivating enzymes and alterations in target molecules. Natural resistance of P. aeruginosa to a number of β-lactam antibiotics is a consequence of inducible ampC β-lactamase (Ambler class C) whose derepression leads to resistance to ureidopenicillines, 3rd (and 4th) generation cephalosporins, and in combination with the loss of porin OprD, to carbapenems. Acquired β-lactamases in pseudomonas can be of serine type from class Aand D, and zinc metallo-hydrolases, so called metallo-β-lactamases (MBLs) from class B. MBLs confer resistance to all β--lactams, including carbapenems. Resistance to aminoglycosides is usually a result of activities of modifying enzymes and efflux. Fluoroquinolone resistance in P. aeruginosa is mediated through alterations in DNA gyrase or topoisomerase, as well as through active efflux. Percentage of resistant pseudomonas isolates in Croatia was not significantly changed in the last 10 years. Among aminoglycosides the resistance rate in 2008 was lowest to amikacin and highest to gentamicin. Resistance to ciprofloxacin was 24%, to ceftazidime 8%, and to carbapenems around 10%

Carbapenemases

Carbapenems are usually regarded as the last treatment option for serious infections caused by Gram-negative and Gram-positive microorganisms. Although they are stable to hydrolysis by most β-lactamases, their usage as the last resort antbiotics was seriously compromised by the appearance of carbapenem-hydrolyzing enzymes called carbapenemases. These enzymes are produced mostly by Enterobacteriaceae and Gram-negative nonfermentative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii. True carbapenemases belonging to Ambler molecular classes A, B, and D are often encoded by genes embedded in mobile genetic elements like plasmids, integrons, and transposons, which often harbor multiple resistance determinants limiting further the treatment options. At present, large number of nosocomial and community-acquired infections caused by worldwide spread of carbapenem-resistant Gram-negative bacteria have become a major public health problem. Although polymyxins remain active, in vitro reports of benefits of combination schemes favor this strategy against carbapenemase-producing Gram-negative bacilli

In vitro effect of subinhibitory concentrations of ceftazidime and meropenem on the serum sensitivity of Pseudomonas aeruginosa strains

The aim of this study was to determine the effect of subminimal inhibitory concentrations (subMICs) of ceftazidime, meropenem and gentamicin on the in vitro serum sensitivity of Pseudomonas aeruginosa strains isolated from a variety of isolation sites at two medical wards and an intensive care unit in a government university hospital in Croatia. A total of 20 serum-resistant P aeruginosa strains isolated from different clinical specimens were selected. Bacteria were exposed to 1/2, 1/4, 1/8, 1/16, and 1/32 x MIC of each antibiotic tested. Sensitivity of P. aeruginosa strains to bactericidal activity of normal human serum before and after bacterial exposure to subMICs was determined. Significant difference in serum sensitivity of the strains was observed after the bacteria were exposed to subMICs of ceftazidime and meropenem (p < 0.01), while the exposure to subMICs of gentamicin did not affect significantly the resistance of tested strains to the serum bactericidal activity. Comparing the number of serum-resistant strains before and after exposure to subMICs of antibiotics, statistically significant differences were determined (p < 0.01) after exposure of the strains to 1/2, 1/4, 1/8 and 1/16 x MIC of meropenem, and after exposure to 1/2, 1/4 and 1/8 x MIC of ceftazidime. SubMICs of ceftazidime and meropenem affected not only the resistance to serum bactericidal activity of bacteria, but also their morphology. The alterations in bacterial morphology caused by subMICs of ceftazidime and meropenem could be connected with consecutive bacterial serum sensitivity

Detection of beta-lactamases

Postoje različite metode za detekciju ß-laktamaza. Metoda koja će biti korištena u određenom slučaju, ovisi o materijalu, tipu enzima i hitnosti rezultata. Većina ß-laktamaza koje proizvode gram-pozitivni organizmi su inducibilne i pojavljuju se u značajnim količinama jedino u prisustvu induktora. Mnogo je jednostavnije vršiti detekciju ß laktamaza gram-pozitivnih bakterija, jer one proizvode navedene enzime u većim količinama i izlučuju ih u okolni medij. Beta-laktamaze gram-negativnih bakterija su smještene intracelularno u periplazmatskom prostoru, pa ih je u većini slučajeva potrebno osloboditi iz stanice sonifikacijom ili osmotskim šokom. Iznimke su N. gonorrhoeae, H. influenzae, B. fragilis. Kod tih vrsta nema barijere između enzima ili supstrata, ili je ona vrlo slabo izražena. Metode koje koristimo za detekciju ß-laktamaza se dijele na acidimetriske, jodometrijske, mikrobiološke, kromogene i fluorescentne.There are different methods for detection of ß-lactamases. The method that will be used depends on the material, type of enzyme and urgency of results. Most ß-lactamases produced by gram-positive organisms are inducible enzymes that appear in significant amounts only in the presence of an inducer. It is easier to detect ß-lactamase production in gram-positive organisms because they produce these enzymes in higher amounts and they are secreted extracellulary. Gram-negative organisms produce ß-lactamases that are located intracellulary in the periplasmic space and it is therefore much more dificult to detect them. In most cases the enzymes must be released from the cells by sonication or osmotic shock. The exceptions are N. gonorrhoeae, H. influenze, B fragilis. In these strains there is little or no barrier separating enzyme and substrate. The methods most frequently used for the detection of ß-lactamase are acidimetric, iodometric, microbiological, chromogenic and fluorescent methods

Antibiotska osjetljivost i proizvodnja ß-laktamaza kod izolata Moraxella catarrhalis iz Zagreba, Hrvatska

Moraxella catarrhalis, a commensal of the nasopharynx, has been recognized with increasing frequency as a potential pathogen in respiratory tract infections. The ß-lactamase production in Moraxella catarrhalis, first described in 1977, has been ever more frequently reported in many countries of the world. However, there have no such reports from Croatia so far. The aim of this study was to investigate antibiotic susceptibilities and ß-lactamase production in Moraxella catarrhalis isolates from Croatia. Fifty Moraxella catarrhalis strains were collected from various clinical specimens at Zagreb University Children\u27s Hospital during the 1990-1992 period. Antibiotic susceptibilities to a wide range of antibiotics were determined by the broth microdilution method according to NCCLS. In all strains, ß-lactamases were detected by the disk chromogenic substrate (nitrocefin) test. The prevalence of ß-lactamase positive strains in the study period was 100%. No resistance to amoxicillin/clavulanate, cephalexin, ceftibuten, tetracycline, erythromycin, azithromycin and chloramphenicol was observed. In all strains, the activity of amoxicillin was strongly enhanced in the presence of clavulanic acid. Older cephalosporins were equally active, however, the thirdgeneration cephalosporin ceftibuten showed significantly lower minimal inhibitory concentrations compared with older compounds. Among non ß-lactam antibiotics, tetracycline and erithromycin showed similar activity. Azithromycin had a markedly stronger inhibitory activity in comparison with erythromycin and tetracycline. According to our results, amoxicillin combined with clavulanic acid should be the antibiotic of choice for the treatment of infections caused by ß-lactamase positive isolates of Moraxella catarrhalis. Oral cephalosporins, tetracycline, macrolides or azithromycin could be an option too.Moraxella catarrhalis se je ranije smatrala komenzalom u respiracijskom traktu, ali se danas zna da je ona značajan respiracijski patogen. Proizvodnja ß-laktamaze, prvi puta opisana 1977., javlja se sa sve većom učestalošću u mnogim zemljama svijeta. Dosadnije bilo izvještaja iz Hrvatske. Cilj ovoga istraživanja bio je ispitati osjetljivost na antibiotike i proizvodnju ß-laktamaze u kliničkim izolatima Moraxella catarrhalis iz Hrvatske. Pedeset izolata Moraxella catarrhalis prikupljeno je iz različitih kliničkih uzoraka u Klinici za dječje bolesti Zagreb od 1990. do 1992. godine. Osjetljivost na antibiotike je određivana mikrodilucijskom metodom prema NCCLS. Proizvodnja ß-laktamaza je utvrđivana metodom diska s kromogenim supstratom (nitrocefinski test). Supstratni profil je određivan biološkom metodom. ß-Laktamaze su dokazane pomoću nitrocefinskog testa u svim sojevima. Učestalost ß-laktamaza pozitivnih izolata Moraxella catarrhalis u ispitivanom razdoblju je iznosila 100%. Nije zapažena rezistencija na amoksicilin/klavulanat, cefaleksin, ceftibuten, tetraciklin, eritromicin, azitromicin i kloramfenikol. Aktivnost amoksicilina se je značajno pojačala u prisutnosti klavulanske kiseline u svim sojevima. Stariji cefalosporini su bili podjednako djelatni, dok je ceftibuten kao cefalosporin treće generacije imao značajno niže minimalne inhibicijske koncentracije u odnosu na starije generacije. Među ne ß-laktamskim antibioticima tetraciklini i eritromicin su imali slične minimalne inhibicijske koncentracije. Azitromicin je imao jače inhibicijsko djelovanje od tetraciklina i eritromicina. Prema rezultatima našega istraživanja amoksicilin u kombinaciji s klavulanskom kiselinom bi bio antibiotik izbora za liječenje infekcija uzrokovanih sojevima Moraxella catarrhalis pozitivnim na b-laktamazu. Oralni cefalosporini, eritromicin, tetraciklini i azitromicin bi također mogli predstavljati moguću terapiju

ß-lactamases of Moraxellae catarrhalis and susceptibility to antibiotics

M. catarrhalis dobiva sve veće značenje kao uzročnik infekcija respiratornog trakta. Visok postotak kliničkih izolata proizvodi ß laktamazu koja je odgovorna za pojavu rezistencije sojeva na ß laktamske antibiotike, prije svega na penicilin i ampicilin. M. catarrhalis stvara dva osnovna tipa ß-laktamaze. To su BRO-1 i BRO-2. Oba enzima su plazmidnog podrijetla, po supstratnom profilu su penicilinaze, a biosinteza im je konstitutivna. Vrlo su osjetljive na inhibiciju klavulanskom kiselinom. Sojevi koji proizvode ß-laktamazu su, u pravilu, rezistentni na peniciline. Kombinacija amoksicilina i klavulanske kiseline lijek je izbora u liječenju infekcija uzrokovanih M. catarrhalis, a mogu se koristiti i eritromicin i cefalosporini.M. catarrhalis is gaining increasing interest as respiratory tract pathogen. High percent of clinical isolats rpoduces ß- lactamase which is responsible for resistance to bea-lactam antibiotics, especially to ampicillin and penicillin. M. catarrhalis produces two main types of ß lastamase: BRO-1 and BRO-2. The both are plasmid mediated, hydrolize penicillin at much higher rate the cephalosporins and are biosinthetised constitutively. They are very sensitive to inhibition by clavulanic acid. The B-lactamase producing strains are in, general resistant, to penicillins. The combination of amoxycillin and clavuranic acid represents the first choice antibiotic in the therapy of infections caused by M. catarrhalis. Eritromycin and cephalosporins can be used too