Whole-genome sequence-informed MALDI-TOF MS diagnostics reveal importance of Klebsiella oxytoca group in invasive infections: a retrospective clinical study.

BACKGROUND: Klebsiella spp. are opportunistic pathogens which can cause severe infections, are often multi-drug resistant and are a common cause of hospital-acquired infections. Multiple new Klebsiella species have recently been described, yet their clinical impact and antibiotic resistance profiles are largely unknown. We aimed to explore Klebsiella group- and species-specific clinical impact, antimicrobial resistance (AMR) and virulence. METHODS: We analysed whole-genome sequence data of a diverse selection of Klebsiella spp. isolates and identified resistance and virulence factors. Using the genomes of 3594 Klebsiella isolates, we predicted the masses of 56 ribosomal subunit proteins and identified species-specific marker masses. We then re-analysed over 22,000 Matrix-Assisted Laser Desorption Ionization - Time Of Flight (MALDI-TOF) mass spectra routinely acquired at eight healthcare institutions in four countries looking for these species-specific markers. Analyses of clinical and microbiological endpoints from a subset of 957 patients with infections from Klebsiella species were performed using generalized linear mixed-effects models. RESULTS: Our comparative genomic analysis shows group- and species-specific trends in accessory genome composition. With the identified species-specific marker masses, eight Klebsiella species can be distinguished using MALDI-TOF MS. We identified K. pneumoniae (71.2%; n = 12,523), K. quasipneumoniae (3.3%; n = 575), K. variicola (9.8%; n = 1717), "K. quasivariicola" (0.3%; n = 52), K. oxytoca (8.2%; n = 1445), K. michiganensis (4.8%; n = 836), K. grimontii (2.4%; n = 425) and K. huaxensis (0.1%; n = 12). Isolates belonging to the K. oxytoca group, which includes the species K. oxytoca, K. michiganensis and K. grimontii, were less often resistant to 4th-generation cephalosporins than isolates of the K. pneumoniae group, which includes the species K. pneumoniae, K. quasipneumoniae, K. variicola and "K. quasivariicola" (odds ratio = 0.17, p < 0.001, 95% confidence interval [0.09,0.28]). Within the K. pneumoniae group, isolates identified as K. pneumoniae were more often resistant to 4th-generation cephalosporins than K. variicola isolates (odds ratio = 2.61, p = 0.003, 95% confidence interval [1.38,5.06]). K. oxytoca group isolates were found to be more likely associated with invasive infection to primary sterile sites than K. pneumoniae group isolates (odds ratio = 2.39, p = 0.0044, 95% confidence interval [1.05,5.53]). CONCLUSIONS: Currently misdiagnosed Klebsiella spp. can be distinguished using a ribosomal marker-based approach for MALDI-TOF MS. Klebsiella groups and species differed in AMR profiles, and in their association with invasive infection, highlighting the importance for species identification to enable effective treatment options.

Stenotrophomonas maltophilia ventilator-associated pneumonia. A retrospective matched case-control study.

BACKGROUND: Stenotrophomonas maltophilia is increasingly identified in critically ill patients, but it is considered a pathogen with limited pathogenicity and it is therefore infrequently targeted. This study explores whether S. maltophilia may cause ventilator-associated pneumonia (VAP) and whether it affects intensive care unit (ICU) mortality and 28-day mortality when compared to VAP caused by other Gram-negative bacilli. METHODS: Retrospective analysis of a 19-year prospectively collected database. Stenotrophomonas maltophilia as a cause was considered in VAP-suspected cases when S. maltophilia growth of ≥10(4) cfu/ml was detected in bronchoalveolar lavage fluid analysis. Cases were matched on hospital, gender, age and acute physiology and chronic health evaluation II score in a 1:3 ratio with controls from the same database suffering from VAP caused by other Gram-negative bacilli. RESULTS: Eight cases met the inclusion criteria, of which three were labelled as 'probable' SM-VAP and three as 'possible' SM-VAP. These six patients constitute 1.8% of all VAPs in the studied period. No significant differences in baseline characteristics and duration of mechanical ventilation (p = 0.68), length of stay in the ICU (p = 0.55) and hospital (p = 0.84) between cases and controls were identified between cases and controls. Intensive care unit mortality odds ratio was 1.7 (p = 0.55; 95% CI 0.3-10.5) and 28-day mortality odds ratio was 1.4 (p = 0.70; 95% CI 0.2-9.1). CONCLUSIONS: Stenotrophomonas maltophilia is a possible, yet infrequent cause of VAP. No outcome differences were found when compared to matched VAP caused by other Gram-negative bacilli.

Ventilator-associated Pneumonia caused by commensal oropharyngeal Flora; [corrected] a retrospective Analysis of a prospectively collected Database.

BACKGROUND: The significance of commensal oropharyngeal flora (COF) as a potential cause of ventilator-associated pneumonia (VAP) is scarcely investigated and consequently unknown. Therefore, the aim of this study was to explore whether COF may cause VAP. METHODS: Retrospective clinical, microbiological and radiographic analysis of all prospectively collected suspected VAP cases in which bronchoalveolar lavage fluid exclusively yielded ≥ 10(4) cfu/ml COF during a 9.5-year period. Characteristics of 899 recent intensive care unit (ICU) admissions were used as a reference population. RESULTS: Out of the prospectively collected database containing 159 VAP cases, 23 patients were included. In these patients, VAP developed after a median of 8 days of mechanical ventilation. The patients faced a prolonged total ICU length of stay (35 days [P < .001]), hospital length of stay (45 days [P = .001]), and a trend to higher mortality (39 % vs. 26 %, [P = .158]; standardized mortality ratio 1.26 vs. 0.77, [P = .137]) compared to the reference population. After clinical, microbiological and radiographic analysis, COF was the most likely cause of respiratory deterioration in 15 patients (9.4 % of all VAP cases) and a possible cause in 2 patients. CONCLUSION: Commensal oropharyngeal flora appears to be a potential cause of VAP in limited numbers of ICU patients as is probably associated with an increased length of stay in both ICU and hospital. As COF-VAP develops late in the course of ICU admission, it is possibly associated with the immunocompromised status of ICU patients.

Endotracheal aspirate and bronchoalveolar lavage fluid analysis: interchangeable diagnostic modalities in suspected ventilator-associated pneumonia?

Authoritative guidelines state that the diagnosis of ventilator-associated pneumonia (VAP) can be established using either endotracheal aspirate (ETA) or bronchoalveolar lavage fluid (BALF) analysis, thereby suggesting that their results are considered to be in accordance. Therefore, the results of ETA Gram staining and semiquantitative cultures were compared to the results from a paired ETA-BALF analysis. Different thresholds for the positivity of ETAs were assessed. This was a prospective study of all patients who underwent bronchoalveolar lavage for suspected VAP in a 27-bed university intensive care unit during an 8-year period. VAP was diagnosed when ≥ 2% of the BALF cells contained intracellular organisms and/or when BALF quantitative culture revealed ≥ 10(4) CFU/ml of potentially pathogenic microorganisms. ETA Gram staining and semiquantitative cultures were compared to the results from paired BALF analysis by Cohen's kappa coefficients. VAP was suspected in 311 patients and diagnosed in 122 (39%) patients. In 288 (93%) patients, the results from the ETA analysis were available for comparison. Depending on the threshold used and the diagnostic modality, VAP incidences varied from 15% to 68%. For the diagnosis of VAP, the most accurate threshold for positivity of ETA semiquantitative cultures was moderate or heavy growth, whereas the optimal threshold for BALF Gram staining was ≥ 1 microorganisms per high power field. The Cohen's kappa coefficients were 0.22, 0.31, and 0.60 for ETA and paired BALF Gram stains, cultures, and BALF Gram stains, respectively. Since the ETA and BALF Gram stains and cultures agreed only fairly, they are probably not interchangeable for diagnosing VAP.

Surveillance cultures in intensive care units: a nationwide survey on current practice providing future perspectives.

PURPOSE: To explore the extent of surveillance culture (SC) implementation underlying motives for obtaining SC and decision making based on the results. MATERIALS AND METHODS: A questionnaire was distributed to Heads of Department (HODs) and microbiologists within all intensive care departments in the Netherlands. RESULTS: Response was provided by 75 (79%) of 95 HODs and 38 (64%) of 59 laboratories allied to an intensive care unit (ICU). Surveillance cultures were routinely obtained according to 55 (73%) of 75 HODs and 33 (87%) of 38 microbiologists. Surveillance cultures were obtained in more than 80% of higher-level ICUs and in 58% of lower-level ICUs (P < .05). Surveillance cultures were obtained twice weekly (88%) and sampled from trachea (87%), pharynx (74%), and rectum (68%). Thirty (58%) of 52 HODs obtained SC to optimize individual patient treatment. On suspicion of infection from an unknown source, microorganisms identified by SC were targeted according to 87%. One third of HODs targeted microorganisms identified by SC in the case of an infection not at the location where the SC was obtained. This was significantly more often than microbiologists in case of no infection (P = .02) or infection of unknown origin (P < .05). CONCLUSIONS: Surveillance culture implementation is common in Dutch ICUs to optimize individual patients' treatment. Consensus is lacking on how to deal with SC results when the focus of infection is not at the sampled site.

Molecular characterization of Staphylococcus aureus bloodstream isolates collected in a Dutch University Hospital between 1999 and 2006.

We observed that, between 1999 and 2006, up to 50% of the methicillin-susceptible Staphylococcus aureus (MSSA) bloodstream isolates in our hospital had a genetic background common to endemic methicillin-resistant S. aureus clones (clonal complex 5 [CC5], CC8, CC22, CC30, and CC45). Furthermore, several successful MSSA lineages, such as CC7 and CC15, were observed.

Identification of a new geographically widespread multiresistant Acinetobacter baumannii clone from European hospitals.

The aim of the study was to investigate the genetic diversity of Acinetobacter baumannii clinical strains that had previously been allocated to three major groups based on automated ribotyping. Forty-seven isolates from European hospitals and one isolate from a South African hospital, geographically representative of the three ribogroups (ribogroups 1, 2 and 3 with 10, 23 and 15 isolates, respectively), were analysed using the highly discriminatory fingerprinting methods AFLP and pulsed-field gel electrophoresis (PFGE). Based on AFLP data, the isolates clustered into three main groups, each corresponding to one ribogroup. Inclusion of reference strains of the previously described clones I and II, responsible for outbreaks in northwestern European hospitals, showed that ribogroups 1 and 2 correspond to clones I and II, respectively, whereas ribogroup 3 apparently represents a new clone. This clone III was found in France, The Netherlands, Italy and Spain. Clones I and II were not limited to northwestern European countries, as they were also recovered from Spain, South Africa, Poland and Italy (clone I) and from Spain, Portugal, South Africa, France, Greece and Turkey (clone II). Combined AFLP and PFGE data showed intraclonal diversity and led to the distinction of 23 different genotypes. Three genotypes, two of them belonging to clone II and one to clone III, were found in different hospitals and may correspond to subsets of isolates with a more recent clonal relationship, which emphasizes the epidemic potential of these organisms.