MALDI-TOF/MS identification of species from the Acinetobacter baumannii (Ab) group revisited: inclusion of the novel A. seifertii and A. dijkshoorniae species.

OBJECTIVES: Rapid identification of Acinetobacter species is critical as members of the A. baumannii (Ab) group differ in antibiotic susceptibility and clinical outcomes. A. baumannii, A. pittii, and A. nosocomialis can be identified by MALDI-TOF/MS, while the novel species A. seifertii and A. dijkshoorniae cannot. Low identification rates for A. nosocomialis also have been reported. We evaluated the use of MALDI-TOF/MS to identify isolates of A. seifertii and A. dijkshoorniae and revisited the identification of A. nosocomialis to update the Bruker taxonomy database. METHODS: Species characterization was performed by rpoB-clustering and MLSA. MALDI-TOF/MS spectra were recovered from formic acid/acetonitrile bacterial extracts overlaid with α-cyano-4-hydroxy-cinnamic acid matrix on a MicroflexLT in linear positive mode and 2000-20 000 m/z range mass. Spectra were examined with the ClinProTools v2.2 software. Mean spectra (MSP) were created with the BioTyper software. RESULTS: Seventy-eight Acinetobacter isolates representative of the Ab group were used to calculate the average spectra/species and generate pattern recognition models. Species-specific peaks were identified for all species, and MSPs derived from three A. seifertii, two A. dijkshoorniae, and two A. nosocomialis strains were added to the Bruker taxonomy database, allowing successful identification of all isolates using spectra from either bacterial extracts or direct colonies, resulting in a positive predictive value (PPV) of 99.6% (777/780) and 96.8% (302/312), respectively. CONCLUSIONS: The use of post-processing data software identified statistically significant species-specific peaks to generate reference signatures for rapid accurate identification of species within the Ab group, providing relevant information for the clinical management of Acinetobacter infections.

Characterization of a novel international clonal complex (CC32) of Acinetobacter baumannii with epidemic potential.

Twelve non-replicate Acinetobacter baumannii isolates from five European hospitals, Kuwait, and the US military healthcare system collected between 1980 and 2005 revealed a new clone, CC32. These included representative isolates of outbreaks/cross-infections. Antimicrobial susceptibility and carbapenem-resistant genetic traits varied. The widespread occurrence, the association with an outbreak and the carbapenem resistance indicate that CC32 has epidemic potential.

Acinetobacter calcoaceticus-Acinetobacter baumannii complex species in clinical specimens in Singapore.

This study was performed to determine the prevalence, distribution of specimen sources, and antimicrobial susceptibility of the Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) species complex in Singapore. One hundred and ninety-three non-replicate Acb species complex clinical isolates were collected from six hospitals over a 1-month period in 2006. Of these, 152 (78·7%) were identified as A. baumannii, 18 (9·3%) as 'Acinetobacter pittii' [genomic species (gen. sp.) 3], and 23 (11·9%) as 'Acinetobacter nosocomialis' (gen. sp. 13TU). Carbapenem resistance was highest in A. baumannii (72·4%), followed by A. pittii (38·9%), and A. nosocomialis (34·8%). Most carbapenem-resistant A. baumannii and A. nosocomialis possessed the bla(OXA-23-like) gene whereas carbapenem-resistant A. pittii possessed the bla(OXA-58-like) gene. Two imipenem-resistant strains (A. baumannii and A. pittii) had the bla(IMP-like) gene. Representatives of carbapenem-resistant A. baumannii were related to European clones I and II.

Rapid and accurate identification of genomic species from the Acinetobacter baumannii (Ab) group by MALDI-TOF MS.

The closely related members of the Acinetobacter baumannii (Ab) group (A. baumannii, A. pittii and A. nosocomialis) are difficult to identify with phenotypic tests in diagnostic laboratories. Genotypic identification methods require special skills and most do not provide rapid results. The aim of this study was to investigate the ability of MALDI-TOF MS to identify members of the Ab group. Sixty epidemiologically unrelated Acinetobacter spp. isolates were investigated by MALDI-TOF MS: 18 A. baumannii, 17 A. pittii, 18 A. nosocomialis and seven additional isolates representing other Acinetobacter spp. All strains were verified by ARDRA, rRNA intergenic spacer (ITS), recA sequencing and bla(OXA-51) . MALDI-TOF MS correctly identified all the genomic strains but erroneously identified A. nosocomialis as A. baumannii because there was no reference strain within the Bruker database. Peak analysis of individual spectra from representative strains of each member of A. baumannii, A. pittii and A. nosocomialis suggested enough differences between their protein signatures to allow accurate identification using MALDI-TOF MS. Inclusion of specific signature profiles for A. nosocomialis within the Bruker database allowed the correct identification of this genomic species. MALDI-TOF MS spectra can be used as a fast, simple and reliable method to identify members of the Ab group. The rapid and accurate identification of clinically significant Acinetobacter strains will improve insight into their epidemiology and allow for targeted therapeutic and infection control measures against clinically important strains.

Widespread carbapenem resistant Acinetobacter baumannii clones in Italian hospitals revealed by a multicenter study.

Population diversity, susceptibility to antibiotics including carbapenems of 277 Acinetobacter baumannii strains collected in 17 Italian hospitals over a 6-months' period was assessed. Semi-automated rep-PCR was used for screening strains for genotypic relatedness. AFLP analysis and MLST were used as definitive methods for strain, species and/or clone identification. Among the 277 strains, 49 rep-PCR types were distinguished with four types (1-4) predominant, indicating both intra- and interhospital spread. AFLP analysis allowed to distinguish 51 types and largely confirmed rep-typing results. Isolates with predominant rep-types 1 and 2 (in 3 and 9 hospitals) were allocated to EU clones I and II, respectively. Rep-type 3 (8 hospitals) belonged to a new clone ("Italian clone"). Rep-type 4 was found in 2 neighbouring hospitals. Two isolates from 2 locations belonged to EU clone III. Twenty-five isolates were identified by AFLP-analysis to A. pittii, emphasizing misidentification by phenotypic methods. MLST confirmed clone identification by AFLP; demonstrating also that the "Italian clone" was ST78, recently detected in different Mediterranean countries. Multidrug resistance, defined as resistance to 9 out of the 11 drugs tested, was common in 10 out of 17 hospitals. The high prevalence of carbapenem resistance was associated with OXA-58 found in 9 out of the 10 hospitals. A high percentage of noted very major errors in susceptibility testing, especially for amikacin and meropenem, was probably due to heteroresistant strains. The occurrence of carbapenem and multidrug resistance in A. baumannii was mainly confined to a limited number of clonal lineages of A. baumannii.

Endemic and epidemic acinetobacter species in a university hospital: an 8-year survey.

The prevalence of the currently known Acinetobacter species and related trends of antimicrobial resistance in a Dutch university hospital were studied. Between 1999 and 2006, Acinetobacter isolates from clinical samples were collected prospectively. Isolates were analyzed by amplified fragment length polymorphism fingerprinting. For species identification, a profile similarity cutoff level of 50% was used, and for strain identification, a cutoff level of 90% was used. Susceptibility for antimicrobial agents was tested by disk diffusion by following the CLSI guideline. The incidences of Acinetobacter isolates ranged from 1.7 to 3.7 per 10,000 patients per year, without a trend of increase, during the study years. Twenty different species were distinguished. Acinetobacter baumannii (27%) and Acinetobacter genomic species (gen. sp.) 3 (26%) were the most prevalent. Other species seen relatively frequently were Acinetobacter lwoffii (11%), Acinetobacter ursingii (4%), Acinetobacter johnsonii (4%), and Acinetobacter junii (3%). One large cluster of A. baumannii, involving 31 patients, and 16 smaller clusters of various species, involving in total 39 patients, with at most 5 patients in 1 cluster, occurred. Overall, 37% of the A. baumannii isolates were fully susceptible to the tested antibiotics. There was a borderline significant (P = 0.059) trend of decreasing susceptibility. A. baumannii was the Acinetobacter species causing the largest burden of multiple-antibiotic resistance and transmissions in the hospital.

Quorum sensing signal profile of Acinetobacter strains from nosocomial and environmental sources.

A set of 43 strains corresponding to 20 classified and unclassified genomic Acinetobacter species was analyzed for the production of typical N-acyl homoserine lactone quorum sensing molecules in culture broths. A large percentage of the strains (74%) displayed quorum sensing signals that could be separated into three statistically significantly different chromatographic groups (p < 0.001) based on their retention factor in TLC, i.e., Rf1 (0.22 +/- 0.02); Rf2 (0.40 +/- 0.02) and Rf3 (0.54 +/- 0.02). Noteworthy, 63% of the strains tested produced more than one quorum signal. The frequency of signal appearance was Rf3 > Rf2 > Rf1. None of the three signals could be specifically assigned to a particular species in the genus; furthermore, no distinction could be made between the quorum sensing signals secreted by typical opportunistic strains of the A. calcoaceticus-A. baumannii complex, isolated from patients, with respect to the other species of the genus, except for the Rf1 signal which was present in all the QS positive strains belonging to this complex and DNA group 13 TU. In conclusion, quorum sensors in Acinetobacter are not homogenously distributed among species and one of them is present in most of the A. calcoaceticus-baumannii complex.

Quorum sensing signal profile of Acinetobacter strains from nosocomial and environmental sources / Perfil de sensores de quórum en cepas nosocomiales y ambientales de Acinetobacter

A set of 43 strains corresponding to 20 classified and unclassified genomic Acinetobacter species was analyzed for the production of typical N-acyl homoserine lactone quorum sensing molecules in culture broths. A large percentage of the strains (74%) displayed quorum sensing signals that could be separated into three statistically significantly different chromatographic groups (p < 0.001) based on their retention factor in TLC, i.e. Rf1 (0.22 ± 0.02); Rf2 (0.40 ± 0.02) and Rf3 (0.54 ± 0.02). Noteworthy, 63% of the strains tested produced more than one quorum signal. The frequency of signal appearance was Rf3 > Rf2 > Rf1. None of the three signals could be specifically assigned to a particular species in the genus; furthermore, no distinction could be made between the quorum sensing signals secreted by typical opportunistic strains of the A. calcoaceticus-A. baumannii complex, isolated from patients, with respect to the other species of the genus, except for the Rf1 signal which was present in all the QS positive strains belonging to this complex and DNA group 13 TU. In conclusion, quorum sensors in Acinetobacter are not homogenously distributed among species and one of them is present in most of the A. calcoaceticus-baumannii complex.

Se analizó la producción de moléculas típicas de N-acil homoserina lactona con actividad de quorum sensing en cultivos líquidos de un grupo de 43 cepas correspondientes a 20 especies genómicas clasificadas y no clasificadas de Acinetobacter. Un porcentaje alto de las cepas (74%) mostraron señales de quorum sensing que pudieron ser separadas en tres grupos cromatográficos significativamente diferentes entre sí (p < 0,001) sobre la base de sus factores de retención en TLC, a saber: Rf1 (0.22 ± 0.02); Rf2 (0.40 ± 0.02) y Rf3 (0.54 ± 0.02). Es de notar que 63% de las cepas ensayadas produjeron más de una señal de quorum. La frecuencia de aparición de las señales fue Rf3 > Rf2 > Rf1. Ninguna de las tres señales pudo ser asignada a una especie en particular dentro del género; es más, no se encontró diferencia entre las señales producidas por las cepas típicamente oportunistas (complejo A. calcoaceticus-A. baumannii) aisladas de pacientes respecto de las producidas por otras cepas del mismo género, excepto para el caso de Rf1, que se encontró presente en todos los aislamientos quorum sensing positivos del mencionado complejo y en las cepas del grupo de DNA 13TU. En conclusión, los sensores de quórum en Acinetobacter no están homogéneamente distribuidos entre especies y uno de ellos (Rf1) está presente en la mayoría de los miembros del complejo calcoaceticus-baumannii.

Can Escherichia coli be used as an indicator organism for transmission events in hospitals?

Can Escherichia coli be used as an indicator organism for transmission events in hospitals? Perineal and pharyngeal swabs were obtained from patients admitted to a medical or surgical intensive care unit within 24 h of admission and then twice per week. Escherichia coli isolates were typed by random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) typing. Based on the typing results, transmission rates for RAPD and AFLP typing were 8.5 and 6.6 per 100 patient-days. Requiring in addition to similarity in genotype parity in time and place for a transmission event, the incidence dropped to 3.8 (RAPD) and 1.7 (AFLP) per 100 patient-days. The two typing methods not only differed with respect to numbers of transmissions identified, but also to individuals involved in transmissions. This study identified a number of problems regarding the use of Escherichia coli as indicator organism for transmission events. The use of Escherichia coli for this purpose cannot be recommended at the moment.

Multifactorial origin of high incidence of Serratia marcescens in a cardio-thoracic ICU: analysis of risk factors and epidemiological characteristics.

OBJECTIVE: A four-fold increase in the incidence of Serratia marcescens occurred in a cardio-thoracic ICU within a 13-month period. Clinical, epidemiological and molecular characteristics were analysed to elucidate the outbreak's origin. METHODS: Epidemiological data were analysed by mapping clustered cases; isolates were genotyped by AFLP analysis. A case-control study was performed to identify risk factors for the acquisition of S. marcescens. Data were obtained from files and electronic databases of the ICU and Department of Medical Microbiology. The adherence to hygiene protocols on the ICU was reviewed by a medical audit. RESULTS: Genotyping showed 16 distinct S. marcescens strains. Twenty-one cases and 39 controls were enrolled in the case-control study. Significant differences found by univariate analysis included the duration of surgery, APACHE-II-score on ICU admission, length of ICU stay, duration of mechanical ventilation, tube feeding and the sum of the number of days per invasive device. In a multivariate logistic regression model, the length of ICU stay and tube feeding were independent risk factors. Outbreak strains were not more frequently resistant to gentamicin, ciprofloxacin, meropenem or trimethoprim-sulfamethoxazole as compared to a reference group. Hygiene protocols, including hand washing, were insufficiently practiced by the ICU's medical staff. CONCLUSIONS: The heterogeneity of the strains points to transmission from various sources. This outbreak of S. marcescens was most probably caused by reduced hand washing and other breaks in infection prevention protocols in combination with the presence of the identified risk factors, which act by affecting the number and intensity of potential transmission events.

Dissemination of Bacillus cereus in a paediatric intensive care unit traced to insufficient disinfection of reusable ventilator air-flow sensors.

Over a two-week period in November 2006, vancomycin-resistant Bacillus cereus was isolated from respiratory samples from six ventilated paediatric intensive care unit (PICU) patients. To investigate the possibility of a common source and extent of the dissemination, all procedures related to mechanical ventilation were monitored and surveillance cultures performed. B. cereus was isolated from reusable air-flow sensors, before and after on-site disinfection with 70% alcohol. The organism was also isolated from respiratory samples from three other ventilated patients and from two ventilation grids in the ceiling of PICU, as well as from the alcohol solution itself. Using amplified fragment length polymorphism (AFLP) typing, B. cereus strains from the six PICU patients together with isolates recovered from the air-flow sensors and the alcohol solution were shown to be closely related. Isolates from the ventilation grids demonstrated different AFLP patterns to the outbreak strain. Intervening measures, including disinfection by autoclaving all reusable air-flow-guiding parts and the use of disposable non-autoclavable parts, resulted in rapid termination of the outbreak. B. cereus infections can cause significant morbidity, particularly in intensive care patients. Disinfection of all air-flow-guiding reusable parts for mechanical ventilation should be addressed with great care and should include effective autoclaving in order to eradicate spores.

Transmission of Pseudomonas aeruginosa in children with cystic fibrosis attending summer camps in The Netherlands.

BACKGROUND: Cross-infection of Pseudomonas aeruginosa has been reported to occur at holiday camps for children with Cystic Fibrosis (CF) with varying frequency. The study aimed to establish the degree of transmission resulting in subsequent infection of P. aeruginosa among CF children (n=80) attending holiday camps in The Netherlands. METHODS: The study was performed in the summer of 2001 in four camps organised simultaneously at different locations. Sputum was collected on day 1 of the holiday, and three and six months later. Different morphotypes of P. aeruginosa from sputum were genotyped by AFLP analysis. Criteria were defined for the degree of evidence of transmission. RESULTS: There were 18 cases possible, 2 cases of probable transmission and 1 case of highly probable transmission. Two predominant types of P. aeruginosa were found (types 18 and 23). Type 18 was already prevalent on day 1 mostly in younger children and was involved in eleven cases of transmission; type 23 was involved in six cases of transmission among older children. CONCLUSIONS: There was a considerable risk of transmission of P. aeruginosa during holiday camps for CF children in The Netherlands. Two genotypes of P. aeruginosa appeared to be easily transmissible, one of which seemed common in the Dutch CF population.

Guidelines for the validation and application of typing methods for use in bacterial epidemiology.

For bacterial typing to be useful, the development, validation and appropriate application of typing methods must follow unified criteria. Over a decade ago, ESGEM, the ESCMID (Europen Society for Clinical Microbiology and Infectious Diseases) Study Group on Epidemiological Markers, produced guidelines for optimal use and quality assessment of the then most frequently used typing procedures. We present here an update of these guidelines, taking into account the spectacular increase in the number and quality of typing methods made available over the past decade. Newer and older, phenotypic and genotypic methods for typing of all clinically relevant bacterial species are described according to their principles, advantages and disadvantages. Criteria for their evaluation and application and the interpretation of their results are proposed. Finally, the issues of reporting, standardisation, quality assessment and international networks are discussed. It must be emphasised that typing results can never stand alone and need to be interpreted in the context of all available epidemiological, clinical and demographical data relating to the infectious disease under investigation. A strategic effort on the part of all workers in the field is thus mandatory to combat emerging infectious diseases, as is financial support from national and international granting bodies and health authorities.

Identification of widespread, closely related Acinetobacter baumannii isolates in Portugal as a subgroup of European clone II.

The aims of this study were to determine whether a multidrug-resistant Acinetobacter baumannii clone, known to be endemic in three tertiary-care Portuguese hospitals, had disseminated throughout Portugal, and whether this clone was related to one of the European clones I-III described previously. The isolates were first screened by pulsed-field gel electrophoresis and/or M13 random amplified polymorphic DNA fingerprint analysis. Ten representative isolates were compared by amplified fragment length polymorphism (AFLP) analysis, and were also compared with isolates contained in the AFLP library of the Leiden University Medical Centre. All of the Portuguese isolates clustered in European clone II (clone delineation level >80%). Following AFLP analysis, seven isolates clustered at >96%, indicating a striking degree of genetic relatedness and suggesting recent spread of a (sub)clone. Three isolates were slightly more separated from this main group, but all isolates clustered at 87.4%. Thus, the Portuguese multidrug-resistant isolates formed a sub-cluster of European clone II, suggesting that they belong to a recent lineage within clone II.

Genotypic diversity and antibiotic susceptibility of Acinetobacter baumannii isolates in a Bulgarian hospital.

A set of 18 Acinetobacter baumannii isolates, collected prospectively in a Bulgarian hospital during episodes of increased A. baumannii occurrence during 2000-2002, was investigated for genotypic diversity and antibiotic susceptibility. Four genotypes were identified by amplified fragment length polymorphism genomic fingerprinting, one of which (type 1) accounted for 13 isolates, indicating that a specific strain was predominant. The single isolate allocated to type 2 was identified to European clone I. All isolates were resistant to multiple antibiotics, but most retained susceptibility to tobramycin and colistin, and all except one were susceptible to imipenem.

Epidemiology of multiple Acinetobacter outbreaks in The Netherlands during the period 1999-2001.

An increase in the number of outbreaks of Acinetobacter infection was notified in The Netherlands during 1999-2001. The present study compared the outbreaks at the species and strain levels, and analysed the epidemiology and control measures at the different locations. For each institute, three representative isolates from three patients were identified to the species and strain levels by genotyping methods. A questionnaire investigated the impact of the outbreak, the control measures that were taken, and the possible effects of the measures. Seven outbreaks were associated with Acinetobacter baumannii (three outbreaks with a strain designated strain A, two outbreaks with a strain designated strain B, and one outbreak each with strains designated C and D). An additional outbreak was caused by genomic species 13TU, which is related closely to A. baumannii. Strains B and D were identified as European clones III and II, respectively. Except for two hospitals with outbreaks caused by strain A, there was no known epidemiological link between the participating hospitals. In all hospitals the outbreak occurred on one or several intensive care units, and spread to other departments was noted in two hospitals. The number of patients affected ranged from six to 66 over a period of 2-22 months. In most outbreaks, patients were the likely reservoir from which spread occurred. In all hospitals, a large panel of measures was required to bring the outbreak to an end. Extensive environmental sampling yielded numerous positive samples in most but not all hospitals.

Prevalence of Acinetobacter baumannii and other Acinetobacter spp. in faecal samples from non-hospitalised individuals.

In total, 226 individuals from the community were investigated for faecal carriage of Acinetobacter spp. by broth enrichment culture, followed by growth on blood agar and/or Leeds Acinetobacter Medium (LAM). Acinetobacter baumannii was isolated on both LAM and blood agar from one of 100 specimens in the UK and one of 126 specimens in The Netherlands. The predominant species were Acinetobactor johnsonii and genomic sp. 11, which were cultured from 22 and five specimens, respectively. A. baumannii did not seem to be widespread in the faecal flora of individuals in the community.

High resolution DNA fingerprinting by AFLP to study the genetic variation among Oesophagostomum bifurcum (Nematoda) from human and non-human primates from Ghana.

An AFLP approach was established to investigate genetic diversity within Oesophagostomum bifurcum (order Strongylida) from human and non-human primates. Evaluation of different combinations of restriction enzymes (n = 8) and primers (n = 29) demonstrated that the use of HindIII/BglII digested templates and primers with the selective nucleotides + AG/ +AC, respectively, was the most effective for the analysis of O. bifurcum DNA. A total of 63 O. bifurcum adults from human, Patas monkey, Mona monkey and Olive baboon hosts from different geographical regions in Ghana were subjected to analysis using this method. Cluster analysis revealed 4 genetically distinct groups, namely O. bifurcum from the Patas monkey (I), from the Mona monkey (II), from humans (III) and from the Olive baboon (IV). These findings were concordant with those achieved previously using RAPD analysis and supports population genetic substructuring within O. bifurcum according to host species. The results demonstrated the effectiveness of the present AFLP method for establishing genetic variation within O. bifurcum, and indicates its applicability to other parasitic nematodes of human and/or veterinary health importance.

Persistent Acinetobacter baumannii? Look inside your medical equipment.

Two outbreaks of multidrug-resistant Acinetobacter baumannii occurred in our hospital. The outbreak strains were eventually isolated from respiratory ventilators, an apparatus used to cool or warm patients, and four continuous veno-venous hemofiltration machines. Removing dust from the machines and replacing all dust filters brought the outbreaks to an end.