Felipe Fernández-Cuenca

Biofilm formation in Acinetobacter baumannii : associated features and clinical implications

Biofilm formation in 92 unrelated strains of Acinetobacter baumannii isolated in a multicentre cohort study was investigated using a microtitre plate assay. Fifty-six (63%) isolates formed biofilm. These isolates were less frequently resistant to imipenem or ciprofloxacin than were non-biofilm-forming isolates (25% vs. 47%, p 0.04; and 66% vs. 94%, p 0.004, respectively). All catheter-related urinary or bloodstream infections and the sole case of shunt-related meningitis were caused by biofilm-forming strains. Multivariate analysis revealed that treatment in an intensive care unit, ciprofloxacin resistance and isolation from a respiratory sample were associated with non-biofilm-forming isolates, while previous aminoglycoside use was associated with biofilm-forming isolates.

CLINICAL FEATURES AND EPIDEMIOLOGY OF ACINETOBACTER BAUMANNII COLONIZATION AND INFECTION IN SPANISH HOSPITALS

OBJECTIVE To investigate the clinical features and the epidemiology of Acinetobacter baumannii in Spanish hospitals. DESIGN Prospective multicenter cohort study. SETTING Twenty-seven general hospitals and one paraplegic center in Spain. METHODS All cases of A. baumannii colonization or infection detected by clinical samples during November 2000 were included. Isolates were identified using phenotypic and genotypic methods. The molecular relatedness of the isolates was assessed by pulsed-field gel electrophoresis. RESULTS Twenty-five (89%) of the hospitals had 221 cases (pooled rate in general hospitals, 0.39 case per 1,000 patient-days; range, 0 to 1.17). The rate was highest in intensive care units (ICUs). Only 3 cases were pediatric. The mean age of the patients in the general hospitals was 63 years; 69% had a chronic underlying disease and 80% had previously received antimicrobial treatment. Fifty-three percent of the patients had an infection (respiratory tract, 51%; surgical site, 16%; and urinary tract, 11%). Crude mortality was higher in infected than in colonized patients (27% vs 10%; relative risk, 1.56; 95% confidence interval, 1.2 to 2.0; P = .003). Molecular analysis disclosed 79 different clones. In most hospitals, a predominant epidemic clone coexisted with other sporadic clones. Imipenem resistance was present in 39% of the hospitals. CONCLUSIONS A. baumannii was present in most participating Spanish hospitals (particularly in ICUs) with different rates among them. The organisms mainly affected predisposed patients; half of them were only colonized. Epidemic and sporadic clones coexisted in many centers.

Contribution of Efflux Pumps, Porins, and β-Lactamases to Multidrug Resistance in Clinical Isolates of Acinetobacter baumannii

ABSTRACT We investigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGE-ROC-1 (53 strains producing the OXA-58 β-lactamase enzyme and 18 strains with the OXA-24 β-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems). We used real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomal β-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-Arg β-naphthylamide dihydrochloride) and the TetA(39) system.

Diversidad clonal y sensibilidad a los antimicrobianos de Acinetobacter baumannii aislados en hospitales españoles. Estudio multicéntrico nacional: proyecto GEIH-Ab 2000

Introduccion El objetivo del estudio fue determinar la relacion clonal y la sensibilidad a antimicrobianos de uso clinico de aislados de Acinetobacter baumannii obtenidos en Espana. Metodos Se incluyen 221 aislados consecutivos de A. baumannii obtenidos de muestras clinicas durante noviembre de 2000 en 25 hospitales espanoles (proyecto GEIH-Ab 2000). La identificacion se realizo mediante pruebas bioquimicas convencionales y genotipicas (analisis del patron de restriccion del acido desoxirribonucleico ribosomal [ADNr] 16S). La relacion clonal entre los aislados se determino mediante electroforesis en campo pulsante. Las concentraciones inhibitorias minimas (CIM) de ampicilina (AP), amikacina (AK), cefalotina (CF), cefepima (FP), cefoxitina (FX), ceftazidima (CZ), ciprofloxacino (CP), cotrimoxazol (T/S), doxiciclina (DX), gemifloxacino (GX), gentamicina (GN), imipenem (IP), minociclina (MI), meropenem (MP), piperacilina (PP), polimixina B (PB), rifampicina (RI), sulbactam (SB), tetraciclina (TT) y tobramicina (TO) se determinaron mediante microdilucion (normas del National Committee for Clinical Laboratory Standards [NCCLS], 2000). Resultados Se diferenciaron 79 clones de a. baumannii. las cim50/cim90 para los 221 a. baumannii fueron > 512/> 512 (pp), > 256/> 256 (ap, cf, fx), > 128/> 128 (tt, gn), 128/> 256 (cz), > 64/> 64 (cp), 64/256 (fp), 32/256 (ak), 32/64 (dx), > 16/> 16 (gx), 16/128 (to), 16/64 (sb, t/s), 8/> 128 (mp), 4/128 (ip), 4/8 (rf), 2/16 (mi) y 1/2 pb). los porcentajes de aislados sensibles fueron 100 (pb), 65,8 (mi), 52,5 (ip), 49,3 (rf), 46,7 (sb), 43,1 (mp), 34,7 (ak), 32,0 (dx), 21,3 (to) y Conclusiones Existe una gran diversidad de clones de A. baumannii aislados en Espana. Los antimicrobianos mas activos frente a A. baumannii fueron polimixina B, minociclina, imipenem, rifampicina, sulbactam, meropenem, amikacina y doxiciclina.

Contribution of OqxAB efflux pumps to quinolone resistance in extended-spectrum-β-lactamase-producing Klebsiella pneumoniae

OBJECTIVES The aims of this study were to analyse the presence of oqxA and oqxB genes in a collection of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae strains, to determine their chromosomal and/or plasmidic locations and to analyse expression levels in relation to susceptibility or resistance to quinolones. METHODS A collection of 114 non-repetitive isolates of ESBL-producing K. pneumoniae was used. K. pneumoniae ATCC 27799 and K. pneumoniae ATCC 700603 were also included. Detection of oqxA and oqxB genes was performed by PCR. Testing for chromosomal and/or plasmidic location was carried out using plasmid DNA and subsequent hybridization. oqxA gene expression was analysed using real-time RT-PCR. Transfer of the plasmid-encoded OqxAB was evaluated. RESULTS The prevalence of both oqxA and oqxB detected in K. pneumoniae was high: 76% and 75%, respectively. Hybridization assays showed that oqxA (16%) and oqxB (13%) were simultaneously present in locations on the chromosome and on large plasmids. The plasmids were transferable by transformation into K. pneumoniae. RT-PCR assays showed higher expression (4-fold) in strains with reduced susceptibility to quinolones than in susceptible strains. Interestingly, K. pneumoniae ATCC 700603 showed an 18-fold higher expression than K. pneumoniae ATCC 27799. These differences were in accordance with quinolone susceptibility. CONCLUSIONS The prevalence of the OqxAB efflux pump (both chromosomal and plasmid encoded) in ESBL-producing K. pneumoniae is high in Spain and represents a potential reservoir for the spread of these genes. High expression of this pump contributes to reduced susceptibility to quinolones in clinical isolates of ESBL-producing K. pneumoniae.

Colistin Resistance in a Clinical Acinetobacter baumannii Strain Appearing after Colistin Treatment: Effect on Virulence and Bacterial Fitness

ABSTRACT The fitness and virulence costs associated with the clinical acquisition of colistin resistance by Acinetobacter baumannii were evaluated. The growth of strain CR17 (colistin resistant) was less than that of strain CS01 (colistin susceptible) when the strains were grown in competition (72-h competition index, 0.008). In a murine sepsis model, CS01 and CR17 reached spleen concentrations when coinfecting of 9.31 and 6.97 log10 CFU/g, respectively, with an in vivo competition index of 0.016. Moreover, CS01 was more virulent than CR17 with respect to mortality and time to death.

Type 1 Integrons in Epidemiologically Unrelated Acinetobacter baumannii Isolates Collected at Spanish Hospitals

Acinetobacter baumannii is an opportunistic nosocomial pathogen, which is an important cause of pneumonia and bacteremia in patients in intensive care units (1). Increased resistance to all commercial antimicrobial agents, including colistin, in clinical isolates of A. baumannii has been reported (7, 12). An important factor for the development of multiresistance is the acquisition of genetic elements, such as integrons (6). Different reports have been published, identifying integrons as responsible for the presence and acquisition of antibiotic resistance in members of the genus Acinetobacter (2, 3, 5, 8, 9, 10, 13). The aim of this study was to investigate the role of type 1 integrons in mediating antibiotic resistance in Spanish clinical isolates of A. baumannii. Moreover, the epidemiological relationship between Spanish isolates containing type 1 integrons and seven isolates from Italian hospitals containing the same integrons was determined. For this purpose, 69 epidemiologically unrelated A. baumannii isolates were collected during November 2000 from 28 Spanish hospitals. All isolates were identified by amplified ribosomal DNA restriction analysis (11), and their epidemiological relationship was determined by pulsed-field gel electrophoresis (PFGE), following the method of Gautom (4). PCR amplification of type 1 integrons was done using the set of primers described by Ploy et al. (8) following conditions and procedures that will be published elsewhere (9). DNA sequencing of the inserted gene cassettes was performed with the dRhodamine terminator cycle sequencing kit and was analyzed in an automatic DNA sequencer (ABI Prism 377; Perkin-Elmer, Emeryville, Calif.) Of a total of 69 A. baumannii isolates, 19 (27.53%) possessed type 1 integrons. Fifteen of these 19 (78.94%) isolates showed the presence of a 700-bp band containing a single aadB allele (Table ​(Table1).1). One of the 19 isolates (5.26%) yielded an amplification product of approximately 2,400 bp (Table ​(Table1)1) with three gene cassettes, an aacA4 allele, an open reading frame (ORF) coding for a yet undetermined product named OrfO, and the blaOXA-20 gene (5, 8). Two of the 19 isolates (10.52%) gave an amplification product of approximately 800 bp (Table ​(Table1).1). Direct sequencing of this amplicon revealed the presence of a single gene cassette that contained an aacA4 gene, which was identical to that found in the integron mentioned above. Of the two isolates containing this integron, one was resistant to both tobramycin and amikacin, while the other isolate was resistant to tobramycin but was susceptible to amikacin. These results agreed with those found by Ploy et al. (8) who found two isolates with the same integron but susceptible to amikacin. Only one isolate (5.26%) showed an amplicon of approximately 2,800 bp containing four gene cassettes (Table ​(Table1),1), an aacC1 determinant, followed by two ORFs that code for unknown products and that are carried on two cassettes (5), and an aadA1a gene. To our knowledge, this type of integron carrying four gene cassettes has been described only once and is found in Italian isolates (5). TABLE 1. Integron gene composition related to the phenotype of resistance found in A. baumannii clinical isolates The integrons of 800, 2,400, and 2,800 bp, were found in Italian A. baumannii isolates. In order to elucidate whether Italian isolates with the same type of integrons (5) possessed the same clonal origin as the Spanish clinical isolates of A. baumannii, a PFGE was performed. The results showed that all the isolates were not epidemiologically related. In conclusion, our results reflect the potential risk of antimicrobial resistance dissemination, both within and between unrelated species. Moreover, we demonstrate that nonrelated isolates from different geographic areas are able to acquire common integrons, leading to the question of whether A. baumannii has a clear affinity for a specific type of integron.

[In vitro activity of 18 antimicrobial agents against clinical isolates of Acinetobacter spp.: multicenter national study GEIH-REIPI-Ab 2010].

OBJECTIVES To determine the prevalence of resistance to antimicrobials in Acinetobacter baumannii (A. baumannii) from Spain and to compare it with those obtained in the first national study (GEIH-Ab project 2000). METHODS A total of 446 isolates of A. baumannii obtained from 43 Spanish hospitals during February-March 2010 were studied. Identification of A. baumannii was confirmed by ARDRA and MALDI-TOF. Susceptibility to 18 antimicrobial agents was determined by microdilution (Clinical and Laboratory Standards Institute, CLSI). The CLSI break-points were used, except for doripenem, rifampin, sulbactam (Societé Française de Microbiologie [SFM] break-points) and tigecycline (European Committee on Antimicrobial Susceptibility Testing [EUCAST] break-points for Enterobacteriaceae). RESULTS The percentage of resistant isolates (intermediate susceptible plus resistant) was: > 94% (ceftazidime, piperacillin and ciprofloxacin), 82-86% (carbapenems, tetracycline), 60-70% (tobramycin, sulbactam, gentamicin, doxycycline), 49% (amikacin), 30% (minocycline, rifampin), 24% (tigecycline), and 3% (colistin). These isolates were, in comparison with those of the first study, more resistant (P < .01) to ceftazidime (99% vs 83%), carbapenems (82-86% vs 43-48%), sulbactam (65% vs 53%) and colistin (3% vs 0%), but more susceptible to aminoglycosides (particularly gentamicin: 70% vs 96% of resistant isolates), tetracycline (83% vs 91%) and rifampicin (30% vs 51%). CONCLUSION There is a high prevalence of A. baumannii resistant to antimicrobials, particularly to carbapenems. The resistance to carbapenems, ceftazidime and sulbactam was significantly higher than that observed for isolates from the GEIH-Ab project 2000. The resistance to aminoglycosides, tetracycline and rifampin, however, was significantly decreased.

Characterization of the Carbapenem-Hydrolyzing Oxacillinase Oxa-58 in an Acinetobacter Genospecies 3 Clinical Isolate

ABSTRACT Based on imipenem resistance in an Acinetobacter genospecies 3 clinical isolate, we were able to identify, for the first time in this genomic species, a plasmid-encoded blaOXA-58 gene that was 100% homologous to the same gene in Acinetobacter baumannii.

Epidemiologic and clinical impact of Acinetobacter baumannii colonization and infection: a reappraisal.

AbstractAcinetobacter baumannii is one of the most important antibiotic-resistant nosocomial bacteria. We investigated changes in the clinical and molecular epidemiology of A. baumannii over a 10-year period. We compared the data from 2 prospective multicenter cohort studies in Spain, one performed in 2000 (183 patients) and one in 2010 (246 patients), which included consecutive patients infected or colonized by A. baumannii. Molecular typing was performed by repetitive extragenic palindromic polymerase chain reaction (REP-PCR), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST).The incidence density of A. baumannii colonization or infection increased significantly from 0.14 in 2000 to 0.52 in 2010 in medical services (p < 0.001). The number of non-nosocomial health care-associated cases increased from 1.2% to 14.2%, respectively (p < 0.001). Previous exposure to carbapenems increased in 2010 (16.9% in 2000 vs 27.3% in 2010, p = 0.03). The drugs most frequently used for definitive treatment of patients with infections were carbapenems in 2000 (45%) and colistin in 2010 (50.3%). There was molecular-typing evidence of an increase in the frequency of A. baumannii acquisition in non-intensive care unit wards in 2010 (7.6% in 2000 vs 19.2% in 2010, p = 0.01). By MSLT, the ST2 clonal group predominated and increased in 2010. This epidemic clonal group was more frequently resistant to imipenem and was associated with an increased risk of sepsis, although not with severe sepsis or mortality.Some significant changes were noted in the epidemiology of A. baumannii, which is increasingly affecting patients admitted to conventional wards and is also the cause of non-nosocomial health care-associated infections. Epidemic clones seem to combine antimicrobial resistance and the ability to spread, while maintaining their clinical virulence.