M. Angeles Domínguez

Genetic Markers of Widespread Extensively Drug-Resistant Pseudomonas aeruginosa High-Risk Clones

ABSTRACT Recent reports have revealed the existence of widespread extensively drug-resistant (XDR) P. aeruginosa high-risk clones in health care settings, but there is still scarce information on their specific chromosomal (mutational) and acquired resistance mechanisms. Up to 20 (10.5%) of 190 bloodstream isolates collected from 10 Spanish hospitals met the XDR criteria. A representative number (15 per group) of isolates classified as multidrug-resistant (MDR) (22.6%), resistant to 1 to 2 classes (moderately resistant [modR]) (23.7%), or susceptible to all antibiotics (multiS) (43.2%) were investigated in parallel. Multilocus sequence typing (MLST) analysis revealed that all XDR isolates belonged to sequence type 175 (ST175) (n = 19) or ST111 (n = 1), both recognized as international high-risk clones. Clonal diversity was higher among the 15 MDR isolates (4 ST175, 2 ST111, and 8 additional STs) and especially high among the 15 modR (13 different STs) and multiS (14 STs) isolates. The XDR/MDR pattern in ST111 isolates correlated with the production of VIM-2, but none of the ST175 isolates produced acquired β-lactamases. In contrast, the analysis of resistance markers in 12 representative isolates (from 7 hospitals) of ST175 revealed that the XDR pattern was driven by the combination of AmpC hyperproduction, OprD inactivation (Q142X), 3 mutations conferring high-level fluoroquinolone resistance (GyrA T83I and D87N and ParC S87W), a G195E mutation in MexZ (involved in MexXY-OprM overexpression), and the production of a class 1 integron harboring the aadB gene (gentamicin and tobramycin resistance). Of particular interest, in nearly all the ST175 isolates, AmpC hyperproduction was driven by a novel AmpR-activating mutation (G154R), as demonstrated by complementation studies using an ampR mutant of PAO1. This work is the first to describe the specific resistance markers of widespread P. aeruginosa XDR high-risk clones producing invasive infections.

Biological Markers of Pseudomonas aeruginosa Epidemic High-Risk Clones

ABSTRACT A limited number of Pseudomonas aeruginosa genotypes (mainly ST-111, ST-175, and ST-235), known as high-risk clones, are responsible for epidemics of nosocomial infections by multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains worldwide. We explored the potential biological parameters that may explain the success of these clones. A total of 20 isolates from each of 4 resistance groups (XDR, MDR, ModR [resistant to 1 or 2 classes], and MultiS [susceptible to all antipseudomonals]), recovered from a multicenter study of P. aeruginosa bloodstream infections performed in 10 Spanish hospitals, were analyzed. A further set of 20 XDR isolates belonging to epidemic high-risk clones (ST-175 [n = 6], ST-111 [n = 7], and ST-235 [n = 7]) recovered from different geographical locations was also studied. When unknown, genotypes were documented through multilocus sequence typing. The biological parameters evaluated included twitching, swimming, and swarming motility, biofilm formation, production of pyoverdine and pyocyanin, spontaneous mutant frequencies, and the in vitro competition index (CI) obtained with a flow cytometry assay. All 20 (100%) XDR, 8 (40%) MDR, and 1 (5%) ModR bloodstream isolate from the multicenter study belonged to high-risk clones. No significant differences were observed between clonally diverse ModR and MultiS isolates for any of the parameters. In contrast, MDR/XDR high-risk clones showed significantly increased biofilm formation and mutant frequencies but significantly reduced motility (twitching, swimming, and swarming), production of pyoverdine and pyocyanin, and fitness. The defined biological markers of high-risk clones, which resemble those resulting from adaptation to chronic infections, could be useful for the design of specific treatment and infection control strategies.

A large sustained endemic outbreak of multiresistant Pseudomonas aeruginosa: a new epidemiological scenario for nosocomial acquisition

BackgroundStudies of recent hospital outbreaks caused by multiresistant P.aeruginosa (MRPA) have often failed to identify a specific environmental reservoir. We describe an outbreak due to a single clone of multiresistant (MR) Pseudomonas aeruginosa (PA) and evaluate the effectiveness of the surveillance procedures and control measures applied.MethodsPatients with MRPA isolates were prospectively identified (January 2006-May 2008). A combined surveillance procedure (environmental survey, and active surveillance program in intensive care units [ICUs]) and an infection control strategy (closure of ICU and urology wards for decontamination, strict compliance with cross-transmission prevention protocols, and a program restricting the use of carbapenems in the ICUs) was designed and implemented.ResultsThree hundred and ninety patients were identified. ICU patients were the most numerous group (22%) followed by urology patients (18%). Environmental surveillance found that 3/19 (16%) non-ICU environmental samples and 4/63 (6%) ICU samples were positive for the MRPA clonal strain. In addition, active surveillance found that 19% of patients were fecal carriers of MRPA. Significant changes in the trends of incidence rates were noted after intervention 1 (reinforcement of cleaning procedures): -1.16 cases/1,000 patient-days (95%CI -1.86 to -0.46; p = 0.003) and intervention 2 (extensive decontamination): -1.36 cases/1,000 patient-days (95%CI -1.88 to -0.84; p < 0.001) in urology wards. In addition, restricted use of carbapenems was initiated in ICUs (January 2007), and their administration decreased from 190-170 DDD/1,000 patient-days (October-December 2006) to 40-60 DDD/1,000 patient-days (January-April 2007), with a reduction from 3.1 cases/1,000 patient-days in December 2006 to 2.0 cases/1,000 patient-days in May 2007. The level of initial carbapenem use rose again during 2008, and the incidence of MRPA increased progressively once more.ConclusionsIn the setting of sustained MRPA outbreaks, epidemiological findings suggest that patients may be a reservoir for further environmental contamination and cross-transmission. Although our control program was not successful in ending the outbreak, we think that our experience provides useful guidance for future approaches to this problem.

Impact of inappropriate empirical therapy for sepsis due to health care-associated methicillin-resistant Staphylococcus aureus

OBJECTIVES We investigated the influence of empirical therapy on the mortality of patients with health care-associated (HCA) sepsis caused by methicillin-resistant Staphylococcus aureus (MRSA) infections in a multicenter cohort, and the variables associated with inappropriate empirical therapy. METHODS All new cases of infection caused by HCA-MRSA presenting with sepsis syndrome in 59 Spanish hospitals during June 2003 were prospectively followed. The main outcome variable was mortality at day 30. Predictors of mortality and of inappropriate empirical therapy were studied using multivariate logistic regression. RESULTS We included 209 cases. Crude mortality was 23%. After controlling for severity of the underlying condition, ICU stay, presentation with severe sepsis or shock, and site of infection, inappropriate empirical therapy was associated with an increased odds of mortality (OR=3.0; 95% CI: 1.01-9.0; p=0.04). Only 21.1% of the patients received appropriate empirical therapy. Variables independently associated with appropriate therapy were recent surgery, central venous catheter and certain sites of infection (primary bacteraemia, intraabdominal infections, and respiratory tract infections). Cancer patients were at an increased risk of receiving inappropriate therapy. CONCLUSIONS Inappropriate empirical therapy was independently associated with increased mortality in this multicenter cohort. Clinicians should be aware of the need to consider coverage against MRSA more frequently.

Risk Factors for Colonization and Infection in a Hospital Outbreak Caused by a Strain of Klebsiella pneumoniae with Reduced Susceptibility to Expanded-Spectrum Cephalosporins

ABSTRACT Between February 2001 and January 2002, an increase in the number of Klebsiella pneumoniae isolates with reduced susceptibility to expanded-spectrum cephalosporins (RSKp) was detected in the neonatal unit of the Juan Canalejo Hospital, and 21 patients were either colonized or infected by the bacterial isolates. The current “gold standard” method for typing K. pneumoniae isolates is pulsed-field gel electrophoresis. However, this technique is expensive and time-consuming. In a search for faster and accurate alternatives to this method, we investigated PCR-based fingerprinting techniques (enterobacterial repetitive intergenic consensus sequence PCR [ERIC-PCR], repetitive extragenic palindromic sequence-based PCR [REP-PCR], and RAPD [randomly amplified polymorphic DNA]) for their ability to characterize K. pneumoniae isolates. The causal agent of the nosocomial outbreak was characterized by these techniques and was found to be a single epidemic strain (RSKp). A multiple regression logistic model was developed to identify potential independent factors associated with colonization and/or infection by RSKp. Logistic regression analysis was applied to all significant variables (P < 0.05) in the univariate analysis, and it was revealed that intubation (odds ratio [OR], 27.0; 95% confidence interval [95%CI], 5.39 to 135.14) and prematurity (OR, 4.4; 95%CI, 0.89 to 21.89) were such independent factors. Moreover, oxime cephalosporins did not appear to be statistically significant. Overall, the results showed that PCR-based techniques are expeditious and useful methods for typing K. pneumoniae isolates. Of the techniques studied, ERIC-PCR showed the highest discriminatory index (D = 0.828), followed by RAPD (D = 0.826) and REP-PCR (D = 0.773)

Clinical and molecular epidemiology of community-acquired, healthcare-associated and nosocomial methicillin-resistant Staphylococcus aureus in Spain

A prospective cohort study including all new cases of methicillin-resistant Staphylococcus aureus (MRSA) colonization or infection in 64 Spanish hospitals during June 2003 was performed to investigate the epidemiology of MRSA in Spain. Only patients who yielded clinical MRSA-positive samples were included. Epidemiological and clinical data for a total of 370 cases were collected. Genotyping was performed using pulsed-field gel electrophoresis and multilocus sequence typing. Panton-Valentine leukocidin genes and the staphylococcal chromosomal cassette mec (SCCmec) were identified in representative isolates. MRSA was considered to be nosocomially acquired in 202 cases (55%), healthcare-associated (HCA) in 139 cases (38%), community-acquired (CA) in three cases, and of uncertain mode of acquisition in 26 (7%) cases. The pooled population-based rate was 2.31 cases/100,000 population/month, and the pooled nosocomial rate was 0.21 cases/1000 hospital stays (20.2% of S. aureus). Peripheral vascular disease, respiratory tract infections, catheter infections, bloodstream infections and crude mortality were more frequent among HCA cases, whereas neoplasia and urinary tract infections were more frequent among nosocomially acquired cases. Two clones related to the paediatric clone ST5-IV accounted for 71% of the isolates; EMRSA-16 has emerged in two different geographical areas. Only one isolate belonged to the formerly predominant Iberian clone. The three CA isolates were related to the USA300 clone. SCCmec type IV was the most frequent type in nosocomial and HCA isolates. The epidemiology of MRSA has changed in Spain; outpatients with previous healthcare contact represent a very important reservoir of MRSA, and community isolates are emerging.

Antibiotic Pressure is a Major Risk Factor for Rectal Colonization by Multidrug-resistant Pseudomonas aeruginosa in Critically ill Patients.

ABSTRACT The intestinal reservoir is central to the epidemiology of Pseudomonas aeruginosa, but the dynamics of intestinal colonization by different phenotypes have been poorly described. To determine the impact of antimicrobial exposure on intestinal colonization by multidrug-resistant (MDR) and extensively drug-resistant (XDR) P. aeruginosa, we screened intensive care unit (ICU) patients for rectal colonization on admission and at weekly intervals. During an 18-month study period, 414 ICU patients were enrolled, of whom 179 (43%) were colonized; 112 (63%) of these were identified at ICU admission and 67 (37%) during their ICU stay. At 10 days after ICU admission, the probabilities of carriage were 44%, 24%, and 24% for non-MDR, MDR-non-XDR, and XDR P. aeruginosa strains, respectively (log rank, 0.02). Pulsed-field gel electrophoresis showed 10 pairs of non-MDR P. aeruginosa and subsequent MDR-non-XDR strains isolated from the same patients to be clonally identical and another 13 pairs (8 MDR-non-XDR and 5 XDR) to be unrelated. There was one specific clone between the 8 MDR-non-XDR strains and an identical genotype in the 5 XDR isolates. The Cox regression analysis identified MDR P. aeruginosa acquisition as associated with the underlying disease severity (adjusted hazard ratio [aHR], 1.97; 95% confidence interval [CI], 1.22 to 3.18; P = 0.006) and prior use of fluoroquinolones (aHR, 1.02; 95% CI, 1.00 to 1.04; P = 0.039), group 2 carbapenems (aHR, 1.03; 95% CI, 1.00 to 1.07; P = 0.041), and ertapenem (aHR, 1.08; 95% CI, 1.02 to 1.14; P = 0.004). The epidemiology of MDR P. aeruginosa is complex, and different clusters may coexist. Interestingly, ertapenem was found to be associated with the emergence of MDR isolates.

Clinical impact of imipenem-resistant Pseudomonas aeruginosa bloodstream infections

OBJECTIVES To describe the incidence and clinical characteristics of imipenem-resistant (IR) Pseudomonas aeruginosa bacteraemia. METHODS We performed a retrospective study including all episodes of IR P. aeruginosa bacteraemia seen from January 2003 to December 2005 in a tertiary teaching hospital. RESULTS There were 108 episodes of IR P. aeruginosa bacteraemia, which represented an incidence of 0.14 episodes per 1000 patient-days in 2003 and 0.11 episodes per 1000 patient-days in 2005. 83 of the episodes (77%) were nosocomially acquired. Most of patients had at least one underlying disease and had previously received antimicrobial treatment. The most frequent source was the urinary tract (31%), followed by unknown origin (22%). A total of 23 (21%) episodes were polymicrobial and 51 (47%) were caused by multidrug-resistant strains. The independent risk factors for mortality from IR P. aeruginosa bloodstream infection were a high-risk source of the bacteraemia (OR: 4.6; 95% CI 1.7-12.4; p=0.01), and presentation with severe sepsis (OR: 2.8; 95% CI 1-7.8; p=0.05). CONCLUSIONS Our study shows that the rates of IR P. aeruginosa bacteraemia remained stable throughout the study period. The source of bacteraemia and the clinical presentation with severe sepsis were the main determinants of the prognosis.

Cultivos de vigilancia epidemiológica de bacterias resistentes a los antimicrobianos de interés nosocomial

Los cultivos de vigilancia epidemiologica y la tipificacion molecular han sido importantes aportaciones de la Microbiologia Clinica al control de la infeccion nosocomial. En este documento se ofrece informacion sobre recogida, transporte, conservacion y procesamiento de muestras para cultivos de vigilancia, criterios para la interpretacion de los resultados y la emision de los mismos en relacion con las bacterias de mayor interes en infeccion nosocomial. Se incluyen Staphylococcus aureus resistente a meticilina (SARM), Enterococcus spp. resistentes a glucopeptidos, enterobacterias productoras de betalactamasas de espectro extendido (BLEE), Acinetobacter baumannii multirresistente y Pseudomonas aeruginosa resistente a carbapenems. Esta informacion pretende aportar una aproximacion general al problema, a partir de la cual el laboratorio desarrolle directrices propias, en funcion de las necesidades acordadas con el equipo multidisciplinar de control de infeccion nosocomial.

Molecular characterization of resistance to Rifampicin in an emerging hospital-associated Methicillin-resistant Staphylococcus aureus clone ST228, Spain

BackgroundMethicillin-resistant S. aureus (MRSA) has been endemic in Hospital Universitari de Bellvitge, Barcelona, since 1990. During the 1990-95 period the Iberian clone (ST-247; SCCmec-I) was dominant. Isolates of clonal complex 5 (ST-125; SCCmec-IV) gradually replaced the Iberian clone from 1996 to 2003. A new multiresistant MRSA phenotype showing rifampicin resistance emerged in 2004 and rapidly increased from 25% in 2004 to 45% in 2006. The aims of this study were i) the molecular characterisation of rifampicin resistant MRSA isolates, ii) the study of the rifampicin resistance expression by disk diffusion, microdilution and E-test, and iii) the analysis of the rpoB gene mutations involved in rifampicin resistance.ResultsA sample of representative 108 rifampicin-resistant MRSA isolates belonged to a single PFGE genotype, ST-228, SCCmec type I and spa type t041. Of 108 isolates, 104 (96%) had a low-level rifampicin resistance (MICs, 2 to 4 mg/L) and 4 a high-level rifampicin resistance (MICs, 128 - ≥ 256 mg/L). Disk diffusion and E-test methods failed to identify a low-level rifampicin resistance in 20 and 12 isolates, respectively. A low-level rifampicin resistance was associated with amino acid substitution 481His/Asn in the beta-subunit of RNA polymerase. Isolates with a high-level rifampicin resistance carried additional mutations in the rpoB gene.ConclusionsThe emergence of MRSA clone ST228-SCCmec I, related to the Southern Germany clone, involved a therapeutical challenge for treating serious MRSA infections. Decreased susceptibility to rifampicin in MRSA strains of ST228-SCCmecI was associated with one or two specific mutations in the rpoB gene. One fifth of isolates with low-level rifampicin-resistance were missed by the diffusion methods.