Colistin- and carbapenem-resistant Klebsiella oxytoca harboring blaVIM-2 and an insertion in the mgrB gene isolated from blood culture.

A carbapenemase-producing colistin-resistant Klebsiella oxytoca isolate was recovered from a blood culture of a female patient without previous report of risk factors to obtain multidrug-resistant Gram-negative bacilli. A combination of biochemical and molecular methods was used to identify the resistance mechanism of this isolate. Carbapenemase production was mediated by Verona integron-encoded metallo-ß-lactamase (VIM)-2. Colistin resistance was not due to plasmid- borne mcr-1 gene, but we found an integration of IS5-like sequence in the mgrB gene of K. oxytoca. This gene is known to be an important regulator of the PhoPQ two-component system, and the disruption of this gene is most likely the cause of lipid A modification resulting in colistin resistance of our isolate. To the best of our knowledge this constitutes the first report of a carbapenemase-producing K. oxytoca with colistin resistance, a case that demonstrates the limited treatment options for infections with multidrug-resistant organisms.

Probing of microbial biofilm communities for coadhesion partners.

Investigations of interbacterial adhesion in dental plaque development are currently limited by the lack of a convenient assay to screen the multitude of species present in oral biofilms. To overcome this limitation, we developed a solid-phase fluorescence-based screening method to detect and identify coadhesive partner organisms in mixed-species biofilms. The applicability of this method was demonstrated using coaggregating strains of type 2 fimbrial adhesin-bearing actinomyces and receptor polysaccharide (RPS)-bearing streptococci. Specific adhesin/receptor-mediated coadhesion was detected by overlaying bacterial strains immobilized to a nitrocellulose membrane with a suspended, fluorescein-labeled bacterial partner strain. Coadhesion was comparable regardless of which cell type was labeled and which was immobilized. Formaldehyde treatment of bacteria, either in suspension or immobilized on nitrocellulose, abolished actinomyces type 2 fimbrial adhesin but not streptococcal RPS function, thereby providing a simple method for assigning complementary adhesins and glycan receptors to members of a coadhering pair. The method's broader applicability was shown by overlaying colony lifts of dental plaque biofilm cultures with fluorescein-labeled strains of type 2 fimbriated Actinomyces naeslundii or RPS-bearing Streptococcus oralis. Prominent coadhesion partners included not only streptococci and actinomyces, as expected, but also other bacteria not identified in previous coaggregation studies, such as adhesin- or receptor-bearing strains of Neisseria pharyngitis, Rothia dentocariosa, and Kingella oralis. The ability to comprehensively screen complex microbial communities for coadhesion partners of specific microorganisms opens a new approach in studies of dental plaque and other mixed-species biofilms.

Legionella contamination in warm water systems: A species-level survey.

Legionellae constitute a frequent contamination of warm water systems and can lead to serious infections. Therefore, in many countries it is mandatory to monitor warm water systems for their presence. The method of examination in Germany is regulated by guideline ISO 11731 and DIN EN ISO 11731-2, and the results are reported as concentration of Legionella spp. Only limited information is available on the presence of individual species of Legionellae in the examined systems, since most investigations and research focus solely on Legionella pneumophila as the most important human pathogen. In this study 76,220 samples obtained from 13,397 warm water systems originating from 24 different zip code districts covering an area of more than 71,000km2 in southern Germany were examined. This resulted in the identification of 47,924 Legionella isolates to the species level using a MALDI-TOF mass spectrometry-based method. Legionella species distribution was analyzed with respect to warm water system type, geographic region (defined as zip code district) and temperature during sample taking. Overall, 20.7% of the samples were found positive for Legionella species and 14 different species of Legionella were recovered. These were not equally present throughout the geographic area investigated, but instead an individual regional diversity of Legionella species was observed for the examined zip code districts. Although Legionella pneumophila represented 84% of all contaminations found, depending on the geographical region its proportion varied substantially between 57.5% and 91.2%. The occurrence of other species was also of importance since they accounted for up to 42% of contaminations regionally, with Legionella londiniensis being most prominent representing up to 38.8% of recovered colonies. In addition, the influence of temperature on the individual species was disparate, but the temperature range between 50°C and 59°C was identified as the optimal condition for facilitating emergence of the majority of recovered Legionella species. The identification of Legionella to the species level by MALDI-TOF allowed for a more concise depiction of the regional distribution and the ecology of this genus, and may be of additional value when counter measures need to be initiated.

Rapid and reliable identification of waterborne Legionella species by MALDI-TOF mass spectrometry.

Detection and enumeration of Legionella bacteria in drinking water is regulated in Germany by ISO 11731-2. The mandatory method for species identification employs parallel subculturing of suspicious colonies on selective media requiring the handling of a large number of cultivation plates. After changes to the drinking water quality regulation in Germany in 2012 the demand for Legionella contamination testing increased drastically. A more reliable, faster and less laborious method for species identification is therefore desirable. Matrix-assisted laser desorption ionization followed by time of flight detection mass spectrometry (MALDI-TOF MS) promises an accelerated identification of bacteria with high reliability and reduced expenditure. Our study shows that MS-based species identification results are in full concordance with cultural and biochemical detection and differentiation and that valuable additional information can be gained, even though the ISO regulation demands an extended incubation period for primary bacterial cultures that is actually in contrast to the prerequisites of the MALDI Biotyper system. In addition, the established identification algorithm is very economical and improves time-to-result. Based on our findings, the amendment of MALID-TOF MS identification to ISO11731-2 as an alternative identification method should be taken into consideration.

Mycobacterium monacense sp. nov.

Four bacterial strains were isolated from independent clinical specimens in different countries and their genotypic and phenotypic characters support their classification in a novel species within the genus Mycobacterium. One strain was clearly responsible for a severe, post-traumatic wound infection in a healthy boy. The novel species, for which the name Mycobacterium monacense sp. nov. is proposed, is yellow-pigmented, non-photochromogenic and grows in less than a week on solid medium. Based on phenotypic investigations alone, distinction of these four strains from known scotochromogenic rapidly growing strains is problematic. However, the novel strains differ from any other mycobacterium in each of the molecular species markers investigated: the 16S rRNA gene, the 16S-23S rRNA gene internal transcribed spacer and the hsp65 gene. Of the strains investigated, two different sequevars were detected for the hsp65 region. Phylogenetic analysis revealed that these four strains were most closely related to Mycobacterium doricum. The type strain of Mycobacterium monacense sp. nov. is B9-21-178T (=DSM 44395T=CIP 109237T).