Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems.

Automated antimicrobial susceptibility testing devices are widely implemented in clinical microbiology laboratories in Spain, mainly using EUCAST (European Committee on Antimicrobial Susceptibility Testing) breakpoints. In 2007, a group of experts published recommendations for including antimicrobial agents and selecting concentrations in these systems. Under the patronage of the Spanish Antibiogram Committee (Comité Español del Antibiograma, COESANT) and the Study Group on Mechanisms of Action and Resistance to Antimicrobial Agents (GEMARA) from the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), and aligned with the Spanish National Plan against Antimicrobial Resistance (PRAN), a group of experts have updated this document. The main modifications from the previous version comprise the inclusion of new antimicrobial agents, adaptation of the ranges of concentrations to cover the EUCAST breakpoints and epidemiological cut-off values (ECOFFs), and the inference of new resistance mechanisms. This proposal should be considered by different manufacturers and users when designing new panels or cards. In addition, recommendations for selective reporting are also included. With this approach, the implementation of EUCAST breakpoints will be easier, increasing the quality of antimicrobial susceptibility testing data and their microbiological interpretation. It will also benefit epidemiological surveillance studies as well as the clinical use of antimicrobials aligned with antimicrobial stewardship programs.

Molecular characterization of OXA-48 carbapenemase-producing Klebsiella pneumoniae strains after a carbapenem resistance increase in Catalonia.

INTRODUCTION: To characterize OXA-48 carbapenemase-producing Klebsiella pneumoniae strains isolated after an increase in carbapenem resistance in Catalonia. METHODOLOGY: K. pneumoniae identification, antimicrobial susceptibility studies, the Modified Hodge Test method, amplification of antimicrobial resistance genes (against ß-lactamases, quinolones and aminoglycosides), molecular typing (by PFGE and MLST), conjugation assays, plasmid characterization (PBRT-PCR and Southern blot), a description of mobile genetic elements and statistical analysis were done. RESULTS: OXA-48 was the only carbapenemase detected, with a prevalence of 1.9%. The blaOXA-48 gene was located in an IncL conjugative plasmid of 62kb and integrated into the transposons Tn1999.2 (91.7%) or Tn1999.1. Five PFGE profiles (A to E) were found, which exactly matched the MLST: ST101, ST17, ST1233, ST14 and ST405, respectively. ST1233 is described here for the first time. K. pneumoniae OXA-48-producing strains were also CTX-M-15 carriers, some producing OXA-1 and TEM-1 penicillinases. The acquired qnrB66 and qnrB1 and aac(3')-IIa, aac(6')-Ib genes were also identified. CONCLUSION: The K. pneumoniae ST405 clone has played an important role in the growing prevalence of OXA-48 in Catalonia. All clones described preserved the blaOXA-48 genetic environment and mobile genetic elements (Tn1999). Notably, the three strains with minor sequence types in this study are not multiresistant strains. These strains are expanding in elderly patients (average age of 76 years) with serious underlying diseases, mainly women (61.2%).

Prevalence of colistin resistance in clinical isolates of Enterobacteriaceae: A four-year cross-sectional study.

OBJECTIVES: The objectives were to determine the prevalence of colistin resistance in clinical isolates of Enterobacteriaceae, and to gain knowledge on the epidemiological and clinical features of the patients. METHODS: All colistin-resistant Enterobacteriaceae consecutively isolated from clinical samples in our institution from 2012 to 2015, were included in this cross-sectional study. Intrinsic-resistant species were excluded. Minimum inhibitory concentration was performed by gradient diffusion. Detection of plasmid-encoded colistin resistance genes mcr-1 and mcr-2 was performed by amplification. Epidemiological and clinical features were reviewed. RESULTS: Of 13579 Enterobacteriaceae isolates, 91 were colistin-resistant. The overall prevalence of colistin resistance was 0.67%. The rates were higher in Enterobacter cloacae (4.2%) than Escherichia coli (0.5%) and Klebsiella pneumoniae (0.4%). One third of the isolates were multi-drug resistant (MDR). While mcr-2 was not detected, mcr-1 was detected only in E. coli. Regarding these infections, 23% were community-acquired. 89% of the patients had not received colistin previously. There were no significant differences between infections caused by mcr-1 and non-mcr-1-carrying isolates. CONCLUSIONS: Colistin resistance was not restricted to MDR isolates and to clinical settings. Most patients had no record of previous administration of colistin.

Fermented milk containing Lactobacillus paracasei subsp. paracasei CNCM I-1518 reduces bacterial translocation in rats treated with carbon tetrachloride.

Probiotics can prevent pathological bacterial translocation by modulating intestinal microbiota and improving the gut barrier. The aim was to evaluate the effect of a fermented milk containing Lactobacillus paracasei subsp. paracasei CNCM I-1518 on bacterial translocation in rats with carbon tetrachloride (CCl4)-induced cirrhosis. Sprague-Dawley rats treated with CCl4 were randomized into a probiotic group that received fermented milk containing Lactobacillus paracasei subsp. paracasei CNCM I-1518 in drinking water or a water group that received water only. Laparotomy was performed one week after ascites development. We evaluated bacterial translocation, intestinal microbiota, the intestinal barrier and cytokines in mesenteric lymph nodes and serum. Bacterial translocation decreased and gut dysbiosis improved in the probiotic group compared to the water group. The ileal ß-defensin-1 concentration was higher and ileal malondialdehyde levels were lower in the probiotic group than in water group. There were no differences between groups in serum cytokines but TNF-α levels in mesenteric lymph nodes were lower in the probiotic group than in the water group. Fermented milk containing Lactobacillus paracasei subsp. paracasei CNCM I-1518 decreases bacterial translocation, gut dysbiosis and ileal oxidative damage and increases ileal ß-defensin-1 expression in rats treated with CCl4, suggesting an improvement in the intestinal barrier integrity.

CTX-M-15-H30Rx-ST131 subclone is one of the main causes of healthcare-associated ESBL-producing Escherichia coli bacteraemia of urinary origin in Spain.

OBJECTIVES: The objective of this study was to assess the prevalence and molecular epidemiology of ESBL-producing Escherichia coli causing healthcare-associated (HCA) and community-associated (CA) bacteraemia of urinary origin (BUO) in Spain. METHODS: An observational cohort study was conducted at eight hospitals from different Spanish geographical areas (2010-11). BUO episodes (n = 425) were classified as HCA (n = 215) and CA (n = 210), and one blood isolate per episode was collected. Susceptibility testing was performed, ESBLs were screened by double-disc diffusion test and ESBL and OXA-1 genes were characterized (PCR and sequencing). Population structure (phylogenetic groups, XbaI-PFGE and MLST) and ST131 subtyping (PCR) were determined. Virulence genes were detected by PCR and virulence score, profiles and extraintestinal pathogenic E. coli (ExPEC) status calculated. RESULTS: ESBL-producing E. coli prevalence was 9.2% (39/425). ESBL-producing E. coli episodes were significantly associated with HCA-BUO episodes [14% (30/215) versus 4.3% (9/210); P = 0.001]. The highest non-susceptibility proportions corresponded to ciprofloxacin (97.4%), amoxicillin/clavulanate (74.4%), co-trimoxazole (69.2%) and tobramycin (61.5%). Of the 39 ESBL-producing E. coli isolates, 34 produced CTX-M enzymes (21 CTX-M-15, 11 CTX-M-14 and 2 CTX-M-1). Fifteen STs were identified, the B2-ST131 clone being the most prevalent (54%; 21/39). All ST131 isolates were ExPEC and had the highest virulence scores, but they showed less diversity in virulence profiles than other STs. The H30Rx subclone accounted for most ST131 isolates (20/21), co-produced CTX-M-15 (20/20) and OXA-1 (19/20) enzymes and was associated with HCA episodes (16/20). CONCLUSIONS: The CTX-M-15-ST131-H30Rx subclone is a relevant MDR pathogen causing BUO, mainly HCA episodes. The dominance of this subclone with comparatively less diversity of virulence profiles reflects the spread of a successful and MDR ESBL ST131 lineage in Spain.

Detection of mcr-1 colistin resistance gene in polyclonal Escherichia coli isolates in Barcelona, Spain, 2012 to 2015.

Colistin resistance was detected in 53 of 10,011 Escherichia coli (0.5%) by prospective phenotypic testing of consecutive clinical isolates in a single hospital in Barcelona, Spain (2012-15). The mcr-1 gene was retrospectively identified by PCR and sequencing in 15 of 50 available isolates. Each isolate had a unique PFGE pattern except for two. This clonal diversity supports the hypothesis of horizontal dissemination of the mcr-1 gene in the local study population.

Validation of a colistin plasma concentration breakpoint as a predictor of nephrotoxicity in patients treated with colistin methanesulfonate.

Nephrotoxicity limits the effective use of colistin for the treatment of multidrug-resistant Gram-negative bacteria (MDR-GNB) infections. We previously defined a steady-state colistin plasma concentration (Css) of 2.42 mg/L that predicted nephrotoxicity at end of treatment (EOT). The objective of this study was to validate this breakpoint in a prospective cohort. This was a multicentre, prospective, observational study conducted at three hospitals with a cohort of patients treated for MDR-GNB infection with colistin methanesulfonate from September 2011 until January 2015. Nephrotoxicity was evaluated at Day 7 and at EOT using the RIFLE criteria. Css values were measured and analysed using HPLC. Taking the previously defined breakpoint for colistin concentration as a criterion, patients were divided into two groups (Css, ≤2.42 mg/L vs. >2.42 mg/L). Sixty-four patients were included. Seven patients (10.9%) had a Css > 2.42 mg/L and were compared with the remaining patients. Bivariate analysis showed that patients with a Css > 2.42 mg/L were older and had a significantly higher incidence of nephrotoxicity at Day 7 and EOT. Although not statistically significant, nephrotoxicity occurred earlier in these patients (6.2 days vs. 9.2 days in patients with lower Css; P = 0.091). Multivariate analysis of nephrotoxicity showed that Css > 2.42 mg/L was the only predictive factor. Nephrotoxicity was more frequent and occurred earlier in patients with colistin plasma concentrations higher than the previously defined breakpoint (2.42 mg/L). Colistin therapeutic drug monitoring should be routinely considered to avoid reaching this toxicity threshold and potential clinical consequences.

Molecular characterisation of acquired and overproduced chromosomal blaAmpC in Escherichia coli clinical isolates.

Escherichia coli recovered from three hospitals in Barcelona (Spain) were studied to determine the prevalence of isolates with acquired AmpC (ac-AmpC) and/or overproduced chromosomal AmpC (c-AmpC). Mechanisms involved in blac-AmpC overexpression, blaac-AmpC and the plasmids associated with their distribution as well as the prevalence of plasmid-mediated quinolone resistance (PMQR) in AmpC-producing isolates were also determined. Isolates were selected according to their resistance phenotype. blaac-AmpC, alterations in the blac-AmpC promoter/attenuator, and PMQR genes [qnrA, qnrB, qnrS, aac(6')-Ib-cr and qepA] were characterised by PCR and sequencing. blac-AmpC expression was determined by qRT-PCR. Population structure analysis was performed using PFGE, MLST and phylogenetic group PCR. Plasmids carrying blaac-AmpC were characterised by PCR-based replicon typing and S1-PFGE. IncI1 and IncF plasmids were also analysed by plasmid MLST and replicon sequence typing, respectively. Among 21563 E. coli isolates, 240 (1.1%) overproduced AmpC ß-lactamases, including 180 (75.0%) harbouring ac-AmpC (132 CMY-2 variants and 48 DHA-1) and 60 (25.0%) c-AmpC enzymes. Three mutation profiles in the blac-AmpC promoter/attenuator were associated with a 72.5-, 19.9- and 5.8-fold increased expression, respectively. Moreover, 63.3% of ac-AmpC and 43.3% of c-AmpC isolates belonged to B2, D, E or F phylogenetic groups. PMQR was found in 31% of ac-AmpC isolates [38 qnrB4, 8 aac(6')-Ib-cr, 6 qnrS1 and 3 qnrB19] and in 10% of c-AmpC isolates [5 aac(6')-Ib-cr and 1 qnrS1]. IncI1-ST12 and IncF were associated with blaCMY-2 and blaDHA-1, respectively. These results suggest that ac-AmpC ß-lactamases were the main mechanism of AmpC production. Isolates and plasmids both showed high genetic diversity.

Efficacy of the FilmArray blood culture identification panel for direct molecular diagnosis of infectious diseases from samples other than blood.

Molecular-based techniques reduce the delay in diagnosing infectious diseases and therefore contribute to better patient outcomes. We assessed the FilmArray blood culture identification (BCID) panel (Biofire Diagnostics/bioMérieux) directly on clinical specimens other than blood: cerebrospinal, joint, pleural and ascitic fluids, bronchoscopy samples and abscesses. We compared the results from 88 samples obtained by culture-based techniques. The percentage of agreement between the two methods was 75 % with a Cohen κ value of 0.51. Global sensitivity and specificity using the FilmArray BCID panel were 71 and 97 %, respectively. Sensitivity was poorer in samples with a low bacterial load, such as ascitic and pleural fluids (25 %), whereas the sensitivity for abscess samples was high (89 %). These findings suggest that the FilmArray BCID panel could be useful to perform microbiological diagnosis directly from samples other than positive blood cultures, as it offers acceptable sensitivity and moderate agreement with conventional microbiological methods. Nevertheless, cost-benefit studies should be performed before introducing this method into algorithms for microbiological diagnostics.

Effect of long-term acid gastric inhibition on bacterial translocation in cirrhotic rats.

BACKGROUND: Bacterial translocation (BT) related to intestinal bacterial overgrowth (IBO) plays an important role in the pathogenesis of bacterial infections in cirrhosis. Inhibition of acid gastric secretion promotes IBO and might favor BT. We evaluated the effect of long-term inhibition of acid gastric secretion on BT in cirrhotic rats. METHODS: Cirrhotic rats with and without ascites induced by oral CCl4 and controls were randomized to treatment with a daily subcutaneous injection of placebo, ranitidine (50 mg/kg), or pantoprazole (8 mg/kg) during 2 weeks. Continuous pH-metry was performed for 2 h before and at the end of treatment; thereafter, a laparotomy to obtain samples of blood, mesenteric lymph nodes, ascites, spleen, liver, and cecal stools was performed. RESULTS: Ranitidine and pantoprazole increased gastric pH as compared with placebo (P<0.001). However, antisecretory drugs increased the incidence of BT only in ascitic rats treated with ranitidine (P<0.05) or pantoprazole (P=0.07) when compared with placebo-treated ascitic rats or cirrhotic rats without ascites treated with the same drug. Cirrhotic ascitic rats treated with pantoprazole showed a trend toward an increased incidence of IBO (P=0.08), a higher ileal malondialdehyde level (P<0.01), and an increased production of tumor necrosis factor-α (P<0.05). CONCLUSION: Although inhibition of acid gastric secretion increased gastric pH in all animals, the incidence of BT increased only in ascitic rats, and it was associated with a trend toward an increase in IBO incidence, a higher ileal malondialdehyde level, and an increased production of serum tumor necrosis factor-α. Therefore, antisecretory drugs should be carefully administered to cirrhotic ascitic patients.

VSL#3 probiotic treatment decreases bacterial translocation in rats with carbon tetrachloride-induced cirrhosis.

BACKGROUND & AIMS: Probiotics can prevent pathological bacterial translocation in cirrhosis by modulating intestinal microbiota and improving gut barrier and immune disturbances. To evaluate the effect of probiotic VSL#3 on bacterial translocation, intestinal microbiota, gut barrier and inflammatory response in rats with experimental cirrhosis. METHODS: Forty-six Sprague-Dawley rats with CCl4 -induced cirrhosis were randomized into two groups: VSL#3 group (n = 22) that received VSL#3 in drinking water, and water group (n = 24) that received water only. Treatment began at week 6 of cirrhosis induction and continued until laparotomy, performed 1 week after development of ascites or at week 20. A control group included 11 healthy rats. At this study end, we evaluated bacterial translocation, intestinal flora, intestinal barrier (ileal claudin-2 and 4, ß-defensin-1, occludin and malondialdehyde as index of oxidative damage) and serum cytokines. RESULTS: Mortality during this study was similar in the VSL#3 group (10/22, 45%) and the water group (10/24, 42%) (P = 1). The incidence of bacterial translocation was 1/12 (8%) in the VSL#3 group, 7/14 (50%) in the water group (P = 0.03 vs. VSL#3 group) and 0/11 in the control group (P = 0.008 vs. water group). The concentration of ileal and caecal enterobacteria and enterococci was similar in the two groups of cirrhotic rats. The ileal occludin concentration was higher and ileal malondialdehyde and serum levels of TNF-α were lower in the VSL#3 group than in the water group (P < 0.05). CONCLUSIONS: VSL#3 decreases bacterial translocation, the pro-inflammatory state and ileal oxidative damage and increases ileal occludin expression in rats with experimental cirrhosis.

[Importance of quality control for the detection of ß-lactam antibiotic resistance in Enterobacteriaceae]. / Importancia de los controles de calidad para la detección de la resistencia a antibióticos ß-lactámicos en enterobacterias.

ß-lactam antimicrobial agents are frequently used to treat infections caused by Enterobacteriaceae. The main mechanism of resistance to these antibiotics is the production of certain enzymes, collectively named ß-lactamases. Due to their substrate profile and their epidemiological implications, the most clinically important ß-lactamases are extended-spectrum ß-lactamases, class C ß-lactamases and carbapenemases. Phenotypic detection of these enzymes may be complicated and is based on the use of specific inhibitors of each ß-lactamase and on the loss of activity on some ß-lactam indicators. Various international committees postulate that it is no longer necessary to interpret the susceptibility results or determine the mechanism of resistance. Several critics disagree, however, and consider that susceptibility results should be interpreted until more data are available on the clinical efficacy of treatment with ß-lactams. Given these methodological difficulties and constant changes in the interpretation criteria, we consider that training and external quality controls are essential to keep updated in this field. For learning purposes, these external quality controls should always be accompanied by a review of the results and methodology used, and the analysis of errors. In this paper we review and contextualize all the aspects related to the detection and interpretation of these ß-lactamases.

Etiology of surgical site infections after primary total joint arthroplasties.

We sought to characterize the causative pathogens of surgical site infections (SSIs) following primary total joint arthroplasties and to evaluate trends in the microbial etiology. We analyzed the etiology of SSIs following 2,632 total hip arthroplasty and knee arthroplasty procedures performed at our institution from 2004 through 2010. We calculated the annual proportion of SSIs accounted for each of the most common organisms and evaluated trends using the χ(2) test for trend. SSIs were identified in 111 procedures (4.2%). The annual incidence of SSIs did not change significantly during the study period. Staphylococci were the most common cause of infection (59.6%) and most of infections were monomicrobial (82.8%). From 2004 to 2010, the annual proportion of infections due to gram-negative bacilli (GNB) increased from 21.4% to 66.7% (p = 0.085 for trend). This increase was accompanied by a decline in the proportion of SSIs from coagulase-negative staphylococci (p = 0.003). Additionally, we found an increase in the percentage of polymicrobial infections (from 7.1% in 2004 to 41.7% in 2010, p = 0.014). Multivariate analysis corroborated these trends. Our study reports an emergence of GNB as a cause of SSIs after primary total joint arthroplaties and an increase of polymicrobial infections.

Molecular diagnosis of bloodstream infections with a new dual-priming oligonucleotide-based multiplex PCR assay.

Mortality from bloodstream infections (BSIs) correlates with diagnostic delay and the use of inappropriate empirical treatment. Early PCR-based diagnosis could decrease inappropriate treatment, improving patient outcome. The aim of the present study was to assess the clinical utility of this molecular technology to diagnose BSIs. We assessed a new dual-priming oligonucleotide-based multiplex PCR assay, the Magicplex Sepsis Test (MST) (Seegene), along with blood culture (BC). A total of 267 patients from the intensive care unit and haematology and emergency departments were enrolled. Clinical data were also used by physicians to determine the likelihood of infection. Ninety-eight (37 %) specimens were positive: 29 (11 %) by both the MST and BC, 29 (11 %) by the MST only, and 40 (15 %) by BC only. The proportion of agreement between the two methods was 73 % (Cohen's κ: 0.45; 0.28-0.6; indicating fair to moderate agreement). According to clinical assessment, 63 (64 %) positive specimens were considered BSIs: 23 (36 %) were positive by both the MST and BC, 22 (35 %) were positive only by BC, and 18 (29 %) were positive only by the MST. Thirty-eight (14 %) positive specimens by the MST and/or BC were considered as contaminants. Of 101 specimens collected from patients receiving antibiotics, 20 (20 %) were positive by the MST and 32 (32 %) by BC. Sensitivity and specificity were 65 % and 92 %, respectively, for the MST and 71 % and 88 %, respectively for BC. We concluded that the MST shows a high specificity but changes in design are needed to increase bacteraemia detection. For viability in clinical laboratories, technical improvements are also required to further automate the process.

Modulation of inflammatory response in a cirrhotic rat model with induced bacterial peritonitis.

Bacterial peritonitis is a severe complication in patients with cirrhosis and ascites and despite antibiotic treatment, the inflammatory response to infection may induce renal dysfunction leading to death. This investigation evaluated the effect of TNF-α blockade on the inflammatory response and mortality in cirrhotic rats with induced bacterial peritonitis treated or not with antibiotics. Sprague-Dawley rats with carbon-tetrachloride-induced cirrhosis were treated with an intraperitoneal injection of 10(9) CFU of Escherichia coli diluted in 20 mL of sterile water to induce bacterial peritonitis and randomized to receive subcutaneously-administered placebo, ceftriaxone, anti-TNF-α mAb and ceftriaxone, or anti-TNF-α mAb alone. No differences were observed between groups at baseline in respect to renal function, liver hepatic tests, serum levels of nitrite/nitrate and TNF-α. Treatment with ceftriaxone reduced mortality (73.3%) but differences did not reach statistical significance as compared to placebo. Mortality in rats treated with ceftriaxone and anti-TNF-α mAb was significantly lower than in animals receiving placebo (53% vs. 100%, p<0.01). Serum TNF-α decreased significantly in surviving rats treated with ceftriaxone plus anti-TNF-α mAb but not in treated with antibiotics alone. Additional studies including more animals are required to assess if the association of antibiotic therapy and TNF-α blockade might be a possible approach to reduce mortality in cirrhotic patients with bacterial peritonitis.

Epidemiology of Clostridium difficile infection and risk factors for unfavorable clinical outcomes: results of a hospital-based study in Barcelona, Spain.

Prospective hospital-based surveillance for Clostridium difficile-associated disease (CDAD) was conducted in Barcelona (Spain) to describe the epidemiology of this condition and investigate the risk factors for an unfavorable outcome. All patients diagnosed with CDAD during 2009 were included. Using logistic regression modeling, we analyzed the potential risk factors associated with recurrent and complicated CDAD, defined as a need for colectomy or death within 30 days. There were 365 episodes of CDAD, yielding an incidence of 22.5 cases/10(5) person-years, 1.22 cases/10(3) hospital discharges, and 1.93 cases/10(4) patient-days. The main PCR ribotypes identified were 241 (26%), 126 (18%), 078 (7%), and 020 (5%). PCR ribotype 027 was not detected. Among the 348 cases analyzed, 232 (67%) patients were cured, 63 (18%) had a recurrence of CDAD, and 53 (15%) developed complicated CDAD. Predictors of complicated CDAD were continued use of antibiotics following CDAD diagnosis (odds ratio [OR], 2.009; 95% confidence interval [CI], 1.012 to 3.988; P = 0.046), Charlson comorbidity index score (OR, 1.265; 95% CI, 1.105 to 1.449; P = 0.001), and age (OR, 1.028; 95% CI, 1.005 to 1.053; P = 0.019). A leukocyte count of >15 × 10(3) cells/ml (OR, 2.277; 95% CI, 1.189 to 4.362; P = 0.013), continuation of proton pump inhibitor (PPI) use after CDAD diagnosis (OR, 2.168; 95% CI, 1.081 to 4.347; P = 0.029), and age (OR, 1.021; 95% CI, 1.001 to 1.041; P = 0.036) were independently associated with higher odds of recurrence. The incidence of CDAD in Barcelona during 2009 was on the lower end of the previously described range for all of Europe. Our analysis suggests that the continuation of non-C. difficile antibiotics and use of PPIs in patients diagnosed with CDAD are associated with unfavorable clinical outcomes.