Recurrent pneumococcal invasive disease in the region of Madrid during a five-year period.

PURPOSE: The aim of this study was to describe the clinical and microbiological characteristics of recurrent invasive pneumococcal disease (RIPD) cases identified in the Region of Madrid between January 2007 and December 2011. METHODS: Streptococcus pneumoniae serotyping was performed by Pneumotest-Latex and Quellung reaction. Molecular typing was carried out by pulsed-field gel electrophoresis (PFGE). A relapse was defined as any case of RIPD caused by strains with similar PFGE profile. Re-infections were defined by detection of recurrent episodes caused by strains with different PFGE patterns. RESULTS: During the study period, 2,929 S. pneumoniae strains isolated from 2,858 patients with invasive pneumococcal disease (IPD) were studied. In 61 patients (2.1 %), 132 episodes of RIPD were detected (two episodes in 52 patients, three in 8 and four in 1). Twelve patients had relapses, 47 had re-infections and two had re-infections followed by relapses. Common risk factors to developing RIPD were HIV (42.6 %) and haematological malignancies (16.4 %). The most frequent serotypes were 8 (16 episodes) and 19A (15 episodes). Fourteen strains that were resistant to levofloxacin were also resistant to erythromycin. The proportion of strains co-resistant to erythromycin and levofloxacin was significantly higher in relapses (11/29) than in re-infections (3/103). CONCLUSIONS: The occurrence of repeated episodes of IPD in the same patient over the time is not an exceptional issue. Some underlying conditions that may favour these recurrences, mainly immunosuppression, need to be considered in patients having an episode of IPD.

Pseudomonas aeruginosa bloodstream infections in children and adolescents: risk factors associated with carbapenem resistance and mortality.

BACKGROUND: Pseudomonas aeruginosa bloodstream infections (PA-BSIs) are a serious disease and a therapeutic challenge due to increasing resistance to carbapenems. Our objectives were to describe the prevalence and risk factors associated with carbapenem resistance (CR) and mortality in children with PA-BSI. METHODS: A retrospective, multi-centre study was carried out, including patients aged <20 years with PA-BSI in four tertiary hospitals in Madrid (Spain) during 2010-2020. Risk factors for CR PA-BSIs and 30-day mortality were evaluated in a multi-variable logistic regression model. RESULTS: In total, 151 patients with PA-BSI were included, with a median age of 29 months (interquartile range: 3.5-87.1). Forty-five (29.8%) cases were CR, 9.9% multi-drug resistant and 6.6% extensively drug resistant. The prevalence of CR remained stable throughout the study period, with 26.7% (12/45) of CR mediated by VIM-type carbapenemase. Patients with BSIs produced by CR-PA were more likely to receive inappropriate empiric treatment (53.3% vs 5.7%, P<0.001) and to have been previously colonized by CR-PA (8.9% vs 0%, P=0.002) than BSIs caused by carbapenem-susceptible P. aeruginosa. CR was associated with carbapenem treatment in the previous month (adjusted odds ratio (aOR) 11.15) and solid organ transplantation (aOR 7.64). The 30-day mortality was 23.2%, which was associated with mechanical ventilation (aOR 4.24), sepsis (aOR 5.72), inappropriate empiric antibiotic therapy (aOR 5.86), and source control as a protective factor (aOR 0.16). CONCLUSION: This study shows a concerning prevalence of CR in children with PA-BSIs, leading to high mortality. Inappropriate empiric treatment and sepsis were associated with mortality. The high prevalence of CR with an increased risk of inappropriate empiric treatment should be closely monitored.

Novel mechanisms of resistance to ß-lactam antibiotics in Haemophilus parainfluenzae: ß-lactamase-negative ampicillin resistance and inhibitor-resistant TEM ß-lactamases.

OBJECTIVES: To determine the mechanisms of resistance to ß-lactam antibiotics in clinical isolates of Haemophilus parainfluenzae. METHODS: Twenty clinical isolates of H. parainfluenzae with decreased susceptibility to aminopenicillins were examined and compared with a control group of 20 fully susceptible isolates. In this collection, the presence of amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3), ß-lactamase production and the surrounding genetic regions of blaTEM genes in selected isolates were analysed. RESULTS: Of the 20 non-susceptible isolates, 8 produced TEM ß-lactamase (gBLPAR), 7 had mutations in the transpeptidase domain of the ftsI gene related to decreased susceptibility to ß-lactams (gBLNAR) and 5 had both resistance mechanisms (gBLPACR). No resistance mechanisms were identified in the susceptible control group (gBLNAS). gBLNAR isolates had MIC90 values 4- to 16-fold higher than gBLNAS isolates for ampicillin, amoxicillin/clavulanic acid, cefuroxime, cefotaxime and cefixime, and the most common PBP3 mutation was Asn526Ser. The additional Ser385Thr substitution (III-like group) may confer decreased susceptibility to cefotaxime, cefixime and aztreonam, as in Haemophilus influenzae. In two ß-lactamase-positive isolates without PBP3 mutations, the inhibitor-resistant TEM (IRT) ß-lactamases TEM-34 and the novel TEM-182 were detected and carried by a TnA transposon of the Tn2 type; both isolates had an amoxicillin/clavulanic acid MIC of ≥8 mg/L. The TnA transposons of two ß-lactamase-positive isolates (TEM-1 and TEM-182) were inserted between the tfc20 and tfc21 genes, typically associated with integrative and conjugative elements in Haemophilus spp.; the TEM-34 IRT ß-lactamase was harboured in a ∼5.5 kb plasmid. CONCLUSIONS: Clinical isolates of H. parainfluenzae express a variety of aminopenicillin resistance mechanisms, either alone or in combination, including PBP3 modifications, blaTEM-1 and IRT ß-lactamase production.

What are the most relevant publications in Clinical Microbiology in the last two years?

This minireview describes some of the articles published in the last two years related to innovative technologies including CRISPR-Cas, surface-enhanced Raman spectroscopy, microfluidics, flow cytometry, Fourier transform infrared spectroscopy, and artificial intelligence and their application to microbiological diagnosis, molecular typing and antimicrobial susceptibility testing. In addition, some articles related to resistance to new antimicrobials (ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and cefiderocol) are also described.

Antibiotic resistance in bloodstream isolates from high-complexity paediatric units in Madrid, Spain: 2013-2021.

BACKGROUND: Antimicrobial resistance (AMR) has become a significant challenge in high-complexity healthcare settings. AIM: To evaluate the prevalence of AMR in bloodstream isolates from high-complexity paediatric units in Spain over a nine-year period. METHODS: A retrospective observational multicentre study was conducted in three tertiary hospitals, analysing bloodstream isolates from patients aged <18 years admitted to the paediatric intensive care, neonatology, and oncology-haematology units between 2013 and 2021. Demographics, antimicrobial susceptibility, and resistance mechanisms were analysed in two periods (2013-2017 and 2017-2021). FINDINGS: In all, 1255 isolates were included. AMR was more prevalent in older patients and those admitted to the oncology-haematology unit. Multidrug resistance was observed in 9.9% of Gram-negative bacteria (GNB); 20.0% of P. aeruginosa vs 8.6% of Entero-bacterales (P < 0.001), with an increase in Enterobacterales from 6.2% to 11.0% between the first and the second period (P = 0.021). Difficult-to-treat resistance was observed in 2.7% of GNB; 7.4% of P. aeruginosa vs 1.6% of Enterobacterales (P < 0.001), with an increasing trend in Enterobacterales from 0.8% to 2.5% (P = 0.076). Carbapenem resistance among Enterobacterales increased from 3.5% to 7.2% (P = 0.029), with 3.3% producing carbapenemases (67.9% VIM). Meticillin resistance was observed in 11.0% of S. aureus and vancomycin resistance in 1.4% of Enterococcus spp., with both rates remaining stable throughout the study period. CONCLUSION: This study reveals a high prevalence of AMR in high-complexity paediatric units. Enterobacterales showed a concerning increasing trend in resistant strains, with higher rates among older patients and those admitted to oncology-haematology units.

Activity of imipenem/relebactam against Enterobacterales and Pseudomonas aeruginosa in Spain. SMART 2016-2020.

OBJECTIVE: To determine susceptibility to the novel ß-lactam/ß-lactamase inhibitor combination imipenem/relebactam in clinical isolates recovered from intra-abdominal (IAI), urinary (UTI), respiratory (RTI) and bloodstream (BSI) infections in the SMART (Study for Monitoring Antimicrobial Resistance Trends) study in SPAIN during 2016 - 2020. METHODS: Broth microdilution MICs for imipenem/relebactam and comparators were determined by a central laboratory against isolates of Enterobacterales and Pseudomonas aeruginosa. MICs were interpreted using EUCAST-2021 breakpoints. RESULTS: In total, 5,210 Enterobacterales and 1,418 P. aeruginosa clinical isolates were analyzed. Imipenem/relebactam inhibited 98.8% of Enterobacterales. Distinguishing by source of infection susceptibility was 99.1% in BSI, 99.2% in IAI, 97.9% in RTI, and 99.2% in UTI. Of intensive care unit isolates (ICU) 97.4% were susceptible and of non-ICU isolates 99.2% were susceptible. In Enterobacterales, activity against Class A, Class B and Class D carbapenemases was 96.2%, 15.4% and 73.2%, respectively. In P. aeruginosa, imipenem/relebactam was active in 92.2% of isolates. By source of infection it was 94.8% in BSI, 92.9% in IAI, 91.7% in RTI, and 93.1% in UTI. An 88.7% of ICU isolates and 93.6% of non-ICU isolates were susceptible to imipenem/relebactam. Imipenem/relebactam remained active against P. aeruginosa ceftazidime-resistant (76.3%), cefepime-resistant (73.6%), imipenem-resistant (71.5%) and piperacillin-resistant (78.7%) isolates. Of all multidrug-resistant or difficult-to-treat resistance P. aeruginosa isolates, 75.1% and 46.2%, respectively, were susceptible to imipenem/relebactam. CONCLUSIONS: Imipenem/relebactam showed high rates of susceptibility in Enterobacterales and P. aeruginosa isolates from different sources of infection as well as depending on patients' location (ICU or non-ICU scenarios).

Role of universal 16S rRNA gene PCR and sequencing in diagnosis of prosthetic joint infection.

The etiological diagnosis of prosthetic joint infection (PJI) requires the isolation of microorganisms from periprosthetic samples. Microbiological cultures often yield false-positive and false-negative results. 16S rRNA gene PCR combined with sequencing (16SPCR) has proven useful for diagnosing various infections. We performed a prospective study to compare the utility of this approach with that of culture to diagnose PJI using intraoperative periprosthetic samples. We analyzed 176 samples from 40 patients with PJI and 321 samples from 82 noninfected patients using conventional culture and 16SPCR. Three statistical studies were undertaken following a previously validated mathematical model: sample-to-sample analysis, calculation of the number of samples to be studied, and calculation of the number of positive samples necessary to diagnose PJI. When only the number of positive samples is taken into consideration, a 16SPCR-positive result in one sample has good specificity and positive predictive value for PJI (specificity, 96.3%; positive predictive value, 91.7%; and likelihood ratio [LR], 22), while 3 positive cultures with the same microorganism are necessary to achieve similar specificity. The best combination of results for 16SPCR was observed when 5 samples were studied and the same microorganism was detected in 2 of them (sensitivity, 94%; specificity, 100%; and LR, 69.62). The results for 5 samples with 2 positive cultures were 96% and 82%, respectively, and the likelihood ratio was 1.06. 16SPCR is more specific and has a better positive predictive value than culture for diagnosis of PJI. A positive 16SPCR result is largely suggestive of PJI, even when few samples are analyzed; however, culture is generally more sensitive.

Rapid techniques for therapeutic optimization. Diagnostic stewardship.

Rapid microbiologic methods provide clinicians with information regarding the causative organisms of infections and their resistance to antimicrobials to optimize patient outcomes and antimicrobial use. Diagnostic stewardship requires that appropriate tests are requested and information is translated into appropriate management. The implementation of rapid techniques also provides collaborative opportunities between antimicrobial stewardship and diagnostic stewardship programs contributing to limiting the spread of antimicrobial resistance, and decreasing mortality, hospital length of stay, and healthcare costs.

What happened to microbiological diagnosis in 2020 beyond COVID-19?

The global pandemic of COVID-19 has had negative repercussions on the activities and research in clinical microbiology laboratories other than those related to SARS-CoV-2. Nonetheless, the research activity has also continued in other fields. In this brief review, some of the recent publications related to new diagnostic tests, methods for rapid antimicrobial susceptibility testing and for the detection of resistance genes, new diagnostic technologies, and some aspects related to old and emergent pathogens (Candida auris, Elizabethkingia spp. Streptococcus pyogenes) are summarized.

Antimicrobial activity of ceftolozane-tazobactam against Enterobacterales and Pseudomonas aeruginosa recovered during the Study for Monitoring Antimicrobial Resistance Trends (SMART) program in Spain (2016-2018).

OBJECTIVE: To analyse the susceptibility to ceftolozane-tazobactam and comparators in Enterobacterales and Pseudomonas aeruginosa isolates recovered from intraabdominal (IAI), urinary (UTI), respiratory (RTI) and bloodstream infection (BSI) in the SMART (Study for Monitoring Antimicrobial Resistance Trends) study. METHODS: The susceptibility of 5,351 isolates collected in 11 Spanish hospitals (2016-2018) were analysed (EUCAST-2020 criteria) by broth microdilution and were phenotypically studied for the presence of extended-spectrum beta-lactamases (ESBL). Ceftolozane-tazobactam and/or carbapenem resistant isolates were genetically characterized for ESBL and carbapenemases. RESULTS: Escherichia coli was the most frequent pathogen (49.3% IAI, 54.9% UTI, 16.7% RTI and 50% BSI), followed by Klebsiella pneumoniae (11.9%, 19.1%, 13.1% and 15.4%, respectively). P. aeruginosa was isolated in 9.3%, 5.6%, 32% and 9%, respectively. The frequency of isolates with ESBLs (2016-2017) was: 30.5% K. pneumoniae, 8.6% E. coli, 2.3% Klebsiella oxytoca and 0.7% Proteus mirabilis. Ceftolozane-tazobactam was very active against non-ESBL-(99.3% susceptible) and ESBL-(95.2%) producing E. coli being less active against K. pneumoniae (98% and 43.1%, respectively) isolates. CTX-M-15 was the most prevalent ESBL in E. coli (27.5%) and K. pneumoniae (51.9%) frequently associated with OXA-48-like carbapenemase. Overall, 93% of P. aeruginosa isolates were susceptible to ceftolozane-tazobactam, preserving this activity (>75%) in isolates resistant to other beta-lactams except in those resistant to meropenen or ceftazidime-avibactam. GES-5, PER-1, VIM-1/2 were the most prevalent enzymes in isolates resistant to ceftolozane-tazobactam. CONCLUSIONS: Ceftolozane-tazobactam showed high activity rates against isolates recovered in the SMART study although it was affected in K. pneumoniae and P. aeruginosa isolates with ESBL and/or carbapenemases.

Ventilator-associated pneumonia in long-term ventilator-assisted individuals.

BACKGROUND: Information on the characteristics of pneumonia in long-term ventilator-assisted individuals is scarce. We evaluate the incidence, risk factors and outcome of ventilator-associated pneumonia (VAP) in a large series of chronically ventilated patients. METHODS: All patients assisted in a chronic ventilator-dependent unit were prospectively followed up for the development of VAP. Patients with a new and persistent lung infiltrate and a purulent tracheal aspirate were suspected to have VAP. Pneumonia was considered microbiologically confirmed in the presence of (1) a positive blood culture and/or (2) ≥105 CFU ml⁻¹ in quantitative bacterial culture of tracheal aspirates or ≥10³ CFU ml⁻¹ in quantitative mini-bronchoalveolar lavage cultures. RESULTS: In total, 100 consecutive long-term ventilated individuals with spinal cord injury (mean age 49 years) were prospectively followed up. The length of mechanical ventilation before admission in the unit was 54±37 days, and the follow-up after admission was 119±127 days. There were 32 episodes of VAP in 27 patients (1.74 episodes per 1000 days of mechanical ventilation). By logistic regression analysis, hypoalbuminaemia (P=0.03), administration of antacids (P=0.002) and length of mechanical ventilation (P=0.05) were independent risk factors for VAP. The most frequently isolated organisms were Pseudomonas aeruginosa (62%), methicillin-resistant Staphylococcus aureus (25%) and Acinetobacter baumannii (15%); 9 (28%) episodes were polymicrobial. Antimicrobial treatment, including monotherapy in 66%, was successful in most patients. Only three patients (11%) died in relation to VAP. CONCLUSIONS: Patients on long-term ventilation are at significant risk for the development of VAP, but the mortality is low.

Emerging non-13-valent pneumococcal conjugate vaccine (PCV13) serotypes causing adult invasive pneumococcal disease in the late-PCV13 period in Spain.

OBJECTIVE: An early reduction of adult invasive pneumococcal disease (IPD) was observed after the 13-valent pneumococcal conjugate vaccine (PCV13) introduction for children in Spain. We analysed the epidemiology of adult IPD in the late-PCV13 period. METHODS: This was a prospective multicentre study of adult IPD involving six hospitals. Strains were serotyped, genotyped and studied for antimicrobial susceptibility. The late-PCV13 period was compared with the pre- and early-PCV13 periods. RESULTS: A total of 2197 episodes were collected-949 in 2008-2009, 609 in 2012-2013 and 639 in 2015-2016. The initial decrease of IPD observed (from 12.3/100 000 to 8.1/100 000; 2008-2009 versus 2012-2013) plateaued in 2015-2016 (8.3/100 000). IPD due to PCV13 serotypes decreased (from 7.7 to 3.5 to 2.3/100 000; p < 0.05), whereas IPD caused by non-PCV13 serotypes increased (from 4.5 to 4.6 to 6.0/100 000; p < 0.05). The most frequent serotypes in the late-PCV13 period were: 8 (15.1%), 3 (10.5%), 12F (7.9%) and 9N (5.4%). These serotypes were related to major genotypes: CC53 (59.8%) and CC404 (30.4%) for serotype 8, CC180 (64.1%) and CC260 (28.1%) for serotype 3, CC989 (91.7%) for serotype 12F and CC67 (84.8%) for serotype 9N. Penicillin-non-susceptibility (21.2%) was associated with serotypes 11A (CC156), 14 (CC156) and 19A (CC320), and macrolide-resistance was related to serotypes 24F and 19A. Rates of pneumococcal meningitis remained stable throughout the periods (ranges 0.9, 0.8 and 1.0/100 000). CONCLUSIONS: The initial decrease of adult IPD observed after PCV13 introduction for children has been balanced by the rise of non-PCV13 serotypes. The spread of antibiotic-resistant lineages related to non-PCV13 serotypes (11A and 24F) could be a threat for the treatment of serious pneumococcal diseases.

Monitoring the antimicrobial susceptibility of Gram-negative organisms involved in intraabdominal and urinary tract infections recovered during the SMART study (Spain, 2016 and 2017).

OBJECTIVE: Continuous antimicrobial resistance surveillance is recommended by Public Health authorities. We up-dated data from the SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study in Spain. METHODS: The antimicrobial susceptibility data and extended-spectrum beta-lactamase (ESBL) production in isolates recovered from intra-abdominal (IAI) (n=1,429) and urinary tract (UTI) (n=937) infections during the 2016- 2017 SMART study in 10 Spanish hospitals were analysed. RESULTS: Escherichia coli was the most frequently microorganism isolated (48.3% and 53.7%) followed by Klebsiella spp. (11.5% and 21.9%) in IAIs and UTIs, respectively. Figures for Pseudomonas aeruginosa were 9.0% and 6.1%, being more frequently recovered from patients with nosocomial infections. Overall, 9.9% (IAI) and 14.0% (UTI) of E. coli, Klebsiella spp. and Proteus mirabilis isolates were ESBL-producers, being Klebsiella pneumoniae (34.5%) from UTI of nosocomial origin the most frequent. ESBL-producers were higher in patients >60 years in both IAIs and UTIs. As in previous years, amikacin (96.3%-100% susceptibility), ertapenem (84.2%-100%) and imipenem (70.3%- 100%) were the most active antimicrobials tested among Enterobacterales species. The activity of amoxicillin-clavulanic, piperacillin-tazobactam, and ciprofloxacin susceptibility was lower, particularly among ESBL-producers. Ertapenem susceptibility (88.9%-100%) was retained in ESBL-E. coli isolates that were resistant to these antimicrobials but decreased (28.6%-100%) in similar isolates of K. pneumoniae. CONCLUSIONS: Continuous antimicrobial resistance surveillance from the SMART study reveals overall maintenance of ESBL-producers in Spain, although with higher presence in isolates from UTIs than from IAIs. Moreover, ertapenem activity was high in E. coli irrespective of ESBL production but decreased in K. pneumoniae, particularly among ESBL-producers.

Metronidazole resistance in Clostridium difficile is heterogeneous.

At our institution, the prevalence of clinical isolates of Clostridium difficile with resistance to metronidazole is 6.3%. We observed that initial metronidazole MICs of 16 to 64 mg/liter against toxigenic, primary fresh C. difficile isolates, as determined by agar dilution, decreased to 0.125 mg/liter after the isolates were thawed. In this study, we examined the possibility of heterogeneous or inducible resistance. Totals of 14 metronidazole-resistant and 10 metronidazole-susceptible clinical isolates of toxigenic C. difficile were studied. The isolates were investigated for the presence of nim genes by PCR. After the isolates were thawed, susceptibility testing was done by agar dilution, by disc diffusion using a 5-mug metronidazole disc, and by the Etest method. An experiment for determining the effect of prolonged exposure to metronidazole was applied to all resistant isolates and to susceptible control strains. None of the isolates presented the nim genes. All initially metronidazole-resistant C. difficile isolates became susceptible after thawing; however, they presented slow-growing subpopulations within the inhibition zones of both the disk and the Etest strip. All metronidazole-susceptible isolates remained homogeneously susceptible by both methods. After prolonged exposure in vitro to metronidazole, no zone of inhibition was found around the 5-microg disk in any of the metronidazole-resistant isolates, and the MICs as determined by the Etest method ranged from 0.125 to >256 mg/liter, with colonies growing inside the inhibition zone. Our results indicate that (i) resistance to metronidazole was not due to the presence of nim genes, (ii) resistance to metronidazole in toxigenic C. difficile isolates is heterogeneous, and (iii) prolonged exposure to metronidazole can select for in vitro resistance. We recommend routine performance of the disk diffusion method (5-microg metronidazole disk) with primary fresh C. difficile isolates in order to ensure that metronidazole-heteroresistant populations do not go undetected.

Evolution of antimicrobial susceptibility patterns of aerobic and facultative gram-negative bacilli causing intra-abdominal infections: results from the SMART studies 2003-2007.

Study for Monitoring Antimicrobial Resistance Trends (SMART) is an ongoing global antimicrobial surveillance program focused on clinical isolates from intra-abdominal infections (IAI). The objective of this subanalysis was to assess the evolution of the antimicrobial susceptibility patterns among aerobic and facultative gram-negative bacilli (GNB) recovered over a 5-year period at our institution. We tested the in vitro activity of the antimicrobials, commonly used to treat IAI, against consecutive unique isolates from IAI using microdilution techniques according to the CLSI guidelines for MIC testing. All isolates were screened phenotypically for extended-spectrum beta-lactamase (ESBL) production. Isolates recovered within 48 h of hospitalization were considered community-acquired (CA). Over the study period a total of 572 aerobic and facultative gram-negative bacilli were recovered from 510 patients, of which 258 (45%) were CA. Enterobacteriaceae composed 91% of the total isolates. Escherichia coli was the most common isolated species (52%). Susceptibility rates of Enterobacteriaceae ranged from 96.5 %-100 % to ertapenem, 96.5 %-100 % to imipenem, 87.7%-94.3% to piperacillin-tazobactam, 85.1%-94.3% to cefotaxime, 89.5%-100% to cefepime, 76.3%-84.8% to ciprofloxacin, and 93.8%-100% to amikacin. ESBL were detected in 6.3% of E. coli, 5.7% of Klebsiella spp. and 2.7% of Enterobacter spp. ESBL producers generally had a more antibiotic- resistant profile than non-ESBL producers and 16% of them were CA. Susceptibility rates to ertapenem, imipenem, piperacillin-tazobactam, ceftazidime, cefepime, ciprofloxacin and amikacin were, respectively, for P. aeruginosa: 28.2 %, 58.9%, 82%, 84.6 %, 76.9 %, 71.8% and 82%; for Acinetobacter baumannii: 33.3 %, 100 %, 66.6 %, 66.6 %, 66.6%, 66.6% y 66.6%, and for Stenotrophomonas maltophilia: 0%, 0%, 0%, 28.6%, 0%, 42.9% and 14.3%. Over the 5 year-study period we have not observed significant increases in resistance of aerobic and facultative GNB causing IAI to commonly used beta-lactam antimicrobial drugs. A minority of ESBL-producing Enterobacteriaceae were CA. Carbapenems, including group I agents like ertapenem, were the most reliably active drugs in vitro against isolates producing IAI.

Identification of Nocardia species from clinical isolates using MALDI-TOF mass spectrometry.

The identification of Nocardia isolates still represents a challenge for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) despite its acceptance for most bacterial and fungal isolates. In this study we evaluate the identification of Nocardia isolates using direct spotting and an updated database. Overall, 82 Nocardia isolates belonging to 13 species were identified by DNA sequence analysis of the 16S rRNA and secA1 genes. Nine of these well-characterized isolates from 6 Nocardia species were used to create an in-house library. The remaining 73 isolates were directly spotted on the target plate and on-plate protein extraction was performed. The protein spectra obtained were analyzed by MALDI-TOF MS using the BDAL database (Bruker Daltonics) updated with 6,903 MSPs or the combination of this commercial database and our in-house library. As a result, the use of the commercial database alone and in combination with the in-house library yielded 94.5% and 95.9% of correct species-level identifications, respectively, No isolate was misidentified at the genus level with either database. Besides, the use of the in-house library allowed the species-level identification of a N. otitidiscaviarum isolate that could only be identified at the genus-level with a score value <1.6 using the commercial database. In conclusion, the implementation of the direct spotting method and the in-house database provided a high rate of correct species assignment of Nocardia isolates despite the low number of isolates added. Further addition of well-characterized Nocardia isolates may ensure the rapid, accurate and inexpensive identification of most isolates encountered in the routine of the microbiology laboratory.

Antimicrobial susceptibility trends and evolution of isolates with extended spectrum ß-lactamases among Gram-negative organisms recovered during the SMART study in Spain (2011-2015).

OBJECTIVE: The SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study monitors antimicrobial susceptibility and extended spectrum ß-lactamases (ESBLs) in Gram-negative bacilli recovered from intra-abdominal infections (IAI). METHODS: Antimicrobial susceptibility of 5,343 isolates from IAI recovered in 11 centres during the 2011-2015 SMART-Spain program was analysed by standard microdilution (EUCAST criteria) and compared with that from 2002-2010. ESBLs were phenotypically detected. RESULTS: Escherichia coli, the most common isolate, significantly decreased in community acquired IAI (60.9% 2002-2010 vs. 56.1% 2011-2015, P=0.0003). It was followed in prevalence by Klebsiella pneumoniae that increased both in the community (8.9% vs. 10.8%, P=0.016) and nosocomial (9.2% vs. 10.8%, P=0.029) IAI and P. aeruginosa, which significantly increased in community acquired IAI (5.6% vs. 8.0%, P=0.0003). ESBLs were more prevalent in K. pneumoniae (16.3%) than in E. coli (9.5%) of nosocomial origin and were more frequently isolated from elderly patients (>60 years). Considering all Enterobacteriaceae, ertapenem (92.3-100%) and amikacin (95.5%-100%) were the most active antimicrobials. Ertapenem activity, unlike amoxicillin-clavulanate or piperacillin-tazobactam, remained virtually unchanged in ESBL (100%) and non-ESBL (98.8%) E. coli producers. Its activity decreased in ESBL-K. pneumoniae (74.7%) but was higher than that of amoxicillin-clavulanate (14.0%) and piperacillin-tazobactam (24.0%). Interestingly, ertapenem susceptibility was maintained in >60% of ESBL isolates that were resistant to amoxicillin-clavulanate, piperacillin-tazobactam or fluoroquinolones. CONCLUSIONS: SMART-Spain results support current guidelines which include ertapenem as empiric treatment in mild-moderate community-acquired IAI, particularly with ESBL producers. These recommendations will need to be updated with the recently introduction of new antimicrobials.

Management of multidrug resistant Gram-negative bacilli infections in solid organ transplant recipients: SET/GESITRA-SEIMC/REIPI recommendations.

Solid organ transplant (SOT) recipients are especially at risk of developing infections by multidrug resistant (MDR) Gram-negative bacilli (GNB), as they are frequently exposed to antibiotics and the healthcare setting, and are regulary subject to invasive procedures. Nevertheless, no recommendations concerning prevention and treatment are available. A panel of experts revised the available evidence; this document summarizes their recommendations: (1) it is important to characterize the isolate's phenotypic and genotypic resistance profile; (2) overall, donor colonization should not constitute a contraindication to transplantation, although active infected kidney and lung grafts should be avoided; (3) recipient colonization is associated with an increased risk of infection, but is not a contraindication to transplantation; (4) different surgical prophylaxis regimens are not recommended for patients colonized with carbapenem-resistant GNB; (5) timely detection of carriers, contact isolation precautions, hand hygiene compliance and antibiotic control policies are important preventive measures; (6) there is not sufficient data to recommend intestinal decolonization; (7) colonized lung transplant recipients could benefit from prophylactic inhaled antibiotics, specially for Pseudomonas aeruginosa; (8) colonized SOT recipients should receive an empirical treatment which includes active antibiotics, and directed therapy should be adjusted according to susceptibility study results and the severity of the infection.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Spain: a multicentre prevalence study (2002).

A point-prevalence study, performed in 2002 in 143 Spanish hospitals, collected 439 isolates of Staphylococcus aureus. Of these, 134 (30.5%) were resistant to methicillin (i.e., MRSA). Susceptibility testing was performed by a microdilution method, and mecA was detected by PCR. The isolates were characterised by phage typing, pulsed-field gel electrophoresis (PFGE) after SmaI digestion, and SCCmec typing. The 134 MRSA isolates showed resistance to ciprofloxacin (93.3%), tobramycin (88.8%), erythromycin (67.9%), clindamycin (59.7%), gentamicin (42.5%), mupirocin (17.9%), rifampicin (5.2%) and trimethoprim-sulphamethoxazole (5.2%). All of the isolates were susceptible to glycopeptides. Twenty-five resistance patterns were found, of which four accounted for 66% of the isolates. Phage group III was the most frequent (41.1%). PFGE revealed 31 different patterns, with ten major clones (including two predominant clones with variable antibiotypes that accounted for 43.3% of the MRSA isolates) and 21 sporadic patterns. Two isolates belonged to two variants of the Iberian clone (ST247-MRSA-I), one to the Brazilian clone (ST239-MRSA-III), and seven to the EMRSA-16 clone (ST36-MRSA-II). SCCmecIV accounted for 70.2% of the isolates (73.9% were type IVA), while SCCmecI, SCCmecII and SCCmecIII accounted for 22.1%, 6.9% and 0.8% of isolates, respectively, with three non-typeable isolates. Isolates of SCCmecIV and SCCmecIVA were predominantly nosocomial (95.8% and 97.1%, respectively). None of the isolates produced Panton-Valentine leukocidin. Thus, two clones carrying SCCmecIV and SCCmecIVA, respectively, were predominant among nosocomial MRSA isolates throughout Spain.

High rates of antimicrobial co-resistance among Enterobacteriaceae: comparative analysis between clinical isolates resistant and susceptible to third-generation cephalosporins.

We compared the antimicrobial co-resistance of 3,402 clinical isolates of Enterobacteriaceae resistant to third-generation cephalosporins (2,569 ESBL-producing and 833 AmpC overproducing) with that of 16,220 susceptible isolates, in order to determine the impact of resistance to third-generation cephalosporins on the likelihood of resistance to other antimicrobial classes. Enterobacteriaceae resistant to third-generation cephalosporins, independently of their mechanism of resistance, were significantly more resistant to other classes of antimicrobials than susceptible isolates (p <0.001). Percentages of co-resistance to ciprofloxacin, gentamicin, tobramycin and trimethoprim-sulfamethoxazole of resistant isolates were: 51%, 39%, 53% and 51%, respectively. However, among the susceptible isolates, percentages were 17%, 7%, 6% and 19%, respectively. Fosfomycin exhibited excellent in vitro activity against urinary isolates (92%), mainly against ESBL-producing organisms (90%), and is a good alternative treatment of infections caused by multidrug resistant Enterobacteriaceae. Amikacin and imipenem were the most active antimicrobials against all species tested.