Towards endemicity: large-scale expansion of the NDM-1-producing Klebsiella pneumoniae ST11 lineage in Poland, 2015-16.

OBJECTIVES: In 2015 and 2016 Poland recorded rapid proliferation of New Delhi MBL (NDM)-producing Enterobacterales, with at least 470 and 1780 cases, respectively. We addressed the roles of the Klebsiella pneumoniae ST11 NDM-1 outbreak genotype, already spreading in 2012-14, and of newly imported organisms in this increase. METHODS: The study included 2136 NDM-positive isolates identified between April 2015 and December 2016, following transfer of patients with K. pneumoniae ST147 NDM-1 from Tunisia to Warsaw in March 2015. The isolates were screened by PCR mapping for variants of blaNDM-carrying Tn125-like elements. Selected isolates were typed by PFGE and MLST. NDM-encoding plasmids were analysed by nuclease S1/hybridization, transfer assays, PCR-based replicon typing and PCR mapping. RESULTS: The organisms were mainly K. pneumoniae containing the Tn125A variant of the ST11 epidemic lineage (n = 2094; ∼98%). Their representatives were of the outbreak pulsotype and ST11, and produced NDM-1, encoded by specific IncFII (pKPX-1/pB-3002cz)-like plasmids. The isolates were recovered in 145 healthcare centres in 13/16 administrative regions, predominantly the Warsaw area. The 'Tunisian' genotype K. pneumoniae ST147 NDM-1 Tn125F comprised 18 isolates (0.8%) from eight institutions. The remaining 24 isolates, mostly K. pneumoniae and Escherichia coli of diverse STs, produced NDM-1 or NDM-5 specified by various Tn125 derivatives and plasmids. CONCLUSIONS: The K. pneumoniae ST11 NDM-1 outbreak has dramatically expanded in Poland since 2012, which may bring about a countrywide endemic situation in the near future. In addition, the so-far limited K. pneumoniae ST147 NDM-1 outbreak plus multiple NDM imports from different countries were observed in 2015-16.

Enterobacteriaceae producing OXA-48-like carbapenemases in Poland, 2013-January 2017.

Objectives: To analyse OXA-48 (OXA-48/181)-type carbapenemase-producing Enterobacteriaceae reported in Poland from 2013 until January 2017. Methods: Bacterial isolates were typed by PFGE and MLST. Genes coding for OXA-48/181 types and other ß-lactamases were amplified and sequenced. Mobile elements with blaOXA-48/181-like genes were PCR mapped. blaOXA-48/181-carrying plasmids were characterized by nuclease S1-hybridization profiling, transfer assays and PCR-based replicon typing, while the chromosomal location of the genes was confirmed by the I-CeuI analysis. Results: Fifty-four isolates from 52 patients in 20 hospitals (14 cities) were included, in 14 cases having probable foreign origins indicated. The organisms were genetically diverse and represented numerous pandemic clones, including Klebsiella pneumoniae ST395 (n = 23), ST11, ST15 and ST101, Escherichia coli ST38, ST410 and ST648, and Enterobacter cloacae complex ST78. These produced OXA-48 (n = 49), OXA-181 (n = 4) or OXA-232 (n = 1). One of five K. pneumoniae ST395 pulsotypes caused a multicentre outbreak with 18 cases, which significantly contributed to the total number of patients. Depending on the variant, the blaOXA-48/181-like genes were parts of the Tn1999-, Tn2013- or Tn2016-like transposons, with blaOXA-48 found in an ISEcp1-associated module (Tn2016-like) for the first time. Three genotypes, including E. coli ST38, had chromosomal blaOXA-48 genes, while others carried transmissible IncL (∼60 kb; blaOXA-48; n = 30), IncM (∼80-95 kb; blaOXA-48; n = 4), IncX3 (∼50 kb; blaOXA-181; n = 4) or non-typeable (∼90-160 kb; blaOXA-48 or blaOXA-232) plasmids. Conclusions: Even though OXA-48/181 producers seem to occur infrequently in Poland, their epidemiology has been marked by various phenomena, namely multiple imports, several limited transmissions plus one larger clonal outbreak, and possible plasmid transfers.

Molecular epidemiology of MRSA in 13 ICUs from eight European countries.

OBJECTIVES: The European epidemiology of MRSA is changing with the emergence of community-associated MRSA (CA-MRSA) and livestock-associated MRSA (LA-MRSA). In this study, we investigated the molecular epidemiology of MRSA during 2 years in 13 ICUs in France, Greece, Italy, Latvia, Luxemburg, Portugal, Slovenia and Spain. METHODS: Surveillance cultures for MRSA from nose and wounds were obtained on admission and twice weekly from all patients admitted to an ICU for ≥3 days. The first MRSA isolate per patient was genotyped in a central laboratory by MLST, spa typing, agr typing and SCCmec (sub)typing. Risk factors for patients with an unknown history of MRSA colonization were identified. RESULTS: Overall, 14 390 ICU patients were screened, of whom 8519 stayed in an ICU for ≥3 days. Overall MRSA admission prevalence was 3.9% and ranged from 1.0% to 7.0% for individual ICUs. Overall MRSA acquisition rate was 2.5/1000 patient days at risk and ranged from 0.2 to 8/1000 patient days at risk per ICU. In total, 557 putative MRSA isolates were submitted to the central laboratory for typing, of which 511 (92%) were confirmed as MRSA. Each country had a distinct epidemiology, with ST8-IVc (UK-EMRSA-2/-6, USA500) being most prevalent, especially in France and Spain, and detected in ICUs in five of eight countries. Seventeen (3%) and three (<1%) isolates were categorized as CA-MRSA and LA-MRSA, respectively. Risk factors for MRSA carriage on ICU admission were age >70 years and hospitalization within 1 year prior to ICU admission. CONCLUSIONS: The molecular epidemiology of MRSA in 13 European ICUs in eight countries was homogeneous within, but heterogeneous between, countries. CA-MRSA and LA-MRSA genotypes and Panton-Valentine leucocidin-producing isolates were detected sporadically.

Phenotypical and genotypical characterization of Shewanella putrefaciens strains isolated from diseased freshwater fish.

Between 2007 and 2012, a variety of disease outbreaks most often characterized by skin disorders were observed among different species of freshwater fish in Poland. In most cases, the clinical signs included focally necrotized gills, necrotic skin lesions or ulcers. Internally, haemorrhages, oedematous kidney and abnormal spleen enlargement were generally noted. The disorders were accompanied by increased mortality. Most of the problems concerned cultured common carp Cyprinus carpio L. and rainbow trout Oncorhynchus mykiss (Walbaum). Fish have been examined from a number of these farms, and additionally, the wild and ornamental fish with similar clinical signs of diseases were also tested. Bacteria were isolated consistently from lesions and internal organs. They had characteristic orange-pigmented colonies which grew in pure culture or constituted 55-95% of total bacterial flora. One hundred and eighteen isolates were collected and biochemically identified as Shewanella putrefaciens group, and this was confirmed by sequencing. Challenge tests confirmed the pathogenicity of these bacteria. This is the first report characterizing and describing S. putrefaciens as a pathogen of different species of freshwater fish in Europe.

Dominant pathogenic species of mesophilic aeromonads isolated from diseased and healthy fish cultured in Poland.

Aeromonas isolates were collected from cultured fish, characterized phenotypically and identified to species using 16S rDNA. The pathogenicity of all isolates was assayed on the basis of haemolytic and proteolytic activity and challenge tests were performed for isolates from healthy fish. A total of 131 Aeromonas isolates were obtained and identified as follows: A. hydrophila (13), A. bestiarum (23), A. salmonicida (motile biogroup) (19), A. caviae (2), A. sobria (18), A. veronii bt. sobria (42), A. jandaei (1), A. encheleia (11) and A. allosaccharophila (2). All isolates of A. hydrophila and A. bestiarum and most isolates of A. salmonicida and A. veronii were classified as pathogenic. Aeromonas hydrophila was isolated only from diseased trout except for one isolate obtained from carp fry. The other potentially pathogenic Aeromonas species were present in diseased as well as healthy fish. The pathogenicity of isolates from healthy fish was correlated with their enzymatic activity and was also tested by challenge experiments. The dominant pathogenic species were A. veronii bt. sobria, A. bestiarum and A. salmonicida in common carp and A. hydrophila in rainbow trout.

The effect of various Aeromonas bestiarum vaccines on non-specific immune parameters and protection of carp (Cyprinus carpio L.).

Aeromonas bestiarum is one of the causal agents of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) in fish. Infections of the bacterium is an increasing problem in commercial carp (Cyprinus carpio L.) farmed in Poland. Non-specific immune response of the fish, vaccinated with oil-emulsified experimental vaccines containing formalin killed whole cells (WCs), formalin killed whole culture (WCt) or crude LPS (50 or 1250 microg per fish) were studied on days 7 and 30 after vaccination. Fish vaccinated for 30 days were challenged with the pathogen and mortalities recorded over 14 days. The cumulative mortalities were 10%, 0%, 20% and 20% in WCs, WCt, LPS-1250 and LPS-50 groups, respectively, whereas 70% fish died in the control group. Vaccinated fish showed significant increase of phagocytic activity (PA) and phagocytic index (PI). The total serum Ig (TSIg) level was significantly higher in most vaccinated fish groups than in control. Moreover, WCs and WCt induced significant increase of mucus lysozyme level in vaccinated fish.

Influence of temperature on the immune response of carp to lipopolysaccharide and whole cell Aeromonas bestiarum vaccines.

Immunisation of carp, Cyprinus carpio, with four different experimental vaccines for protection against A. bestiarum was tested. Carp immunized with different antigens of A. bestiarum P1s, and kept at 18 degrees C responded immunologically by relatively high titers of antibodies. Antibody production was inhibited at 12 degrees C.

Phenotypic characteristics and pathogenicity of Aeromonas genomospecies isolated from common carp (Cyprinus carpio L.).

AIMS: To evaluate the relationship between the genomospecies, phenotypic profile and pathogenicity for carp of 37 motile Aeromonas strains. METHODS AND RESULTS: Aeromonas strains were identified to genomospecies level by the 16S rDNA restriction fragment length polymorphism (RFLP) method and characterized phenotypically by the API 20E and API Zym systems and by conventional tube or plate methods. 16S rDNA RFLP analysis showed that the strains belonged to five species, Aeromonas bestiarum (5), Aerom. salmonicida (13), Aerom. veronii (11), Aerom. sobria (6) and Aerom. encheleia (2). Most strains of Aerom. bestiarum (80%) and Aerom. salmonicida (85%) could be separated by growth at 4 and 42 degrees C, autoagglutination after boiling, reaction for lipase (C14) and naphthol-AS-BI-phosphohydrolase. All strains of Aerom. veronii corresponded to Aerom. veronii biotype sobria and could be separated from Aerom. sobria by citrate utilization, growth at 37 and 42 degrees C, amygdalin and cellobiose fermentation. All strains of Aerom. bestiarum and most strains of Aerom. salmonicida (76.9%) and Aerom. veronii (63.6%) were pathogenic for carp. CONCLUSIONS: The biochemical identification of carp Aeromonas strains is not entirely clear. Some association between Aeromonas species, phenotypic profile and specific disease signs was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will be useful for ichthyopathology laboratories in the diagnosis of motile aeromonad septicaemia in carp.