Biosafety assessment of Acinetobacter strains isolated from the Three Gorges Reservoir region in nematode Caenorhabditis elegans.

Acinetobacter has been frequently detected in backwater areas of the Three Gorges Reservoir (TGR) region. We here employed Caenorhabditis elegans to perform biosafety assessment of Acinetobacter strains isolated from backwater area in the TGR region. Among 21 isolates and 5 reference strains of Acinetobacter, exposure to Acinetobacter strains of AC1, AC15, AC18, AC21, A. baumannii ATCC 19606T, A. junii NH88-14, and A. lwoffii DSM 2403T resulted in significant decrease in locomotion behavior and reduction in lifespan of Caenorhabditis elegans. In nematodes, exposure to Acinetobacter strains of AC1, AC15, AC18, AC21, A. baumannii, A. junii and A. lwoffii also resulted in significant reactive oxygen species (ROS) production. Moreover, exposure to Acinetobacter isolates of AC1, AC15, AC18, and AC21 led to significant increase in expressions of both SOD-3::GFP and some antimicrobial genes (lys-1, spp-12, lys-7, dod-6, spp-1, dod-22, lys-8, and/or F55G11.4) in nematodes. The Acinetobacter isolates of AC1, AC15, AC18, and AC21 had different morphological, biochemical, phylogenetical, and virulence gene properties. Our results suggested that exposure risk of some Acinetobacter strains isolated from the TGR region exists for environmental organisms and human health. In addition, C. elegans is useful to assess biosafety of Acinetobacter isolates from the environment.

Acinetobacter geminorum sp. nov., isolated from human throat swabs.

Two isolates of a non-fermenting, Gram-negative bacterial strain were cultured from two throat swabs that were taken from a pair of twins during routine microbiological surveillance screening. As these isolates could not be unambiguously identified using routine diagnostic methods, whole genome sequencing was performed followed by phylogenetic analysis based on the rpoB gene sequence and by whole genome datasets. The two strains compose a separate branch within the clade formed by the Acinetobacter calcoaceticus-baumannii (ACB) complex with Acinetobacter pittii CIP 70.29T as the most closely related species. The average nucleotide identity compared to all other species of the ACB complex was below 94.2% and digital DNA-DNA hybridization values were less than 60%. Biochemical characteristics confirm affiliation to the ACB complex with some specific phenotypic differences. As a result of the described data, a new Acinetobacter species is introduced, for which the name Acinetobacter geminorum sp. nov. is proposed. The type strain is J00019T with a G+C DNA content of 38.8 mol% and it is deposited in the DSMZ Germany (DSM 111094T) and CCUG Sweden (CCUG 74625T).

Rapid typing of carbapenem-resistant Acinetobacter baumannii and Acinetobacter nosocomialis by multiplex Pan- and OXA-PCR assays.

Introduction. Outbreaks of carbapenem-resistant A. baumannii and A. nosocomialis have occurred worldwide in healthcare settings. Rapid and reliable molecular typing of bacterial isolates is vital for the effective surveillance of institutional outbreaks. The Pan-PCR and OXA-PCR assays are two multiplex PCR-based assays for the molecular typing of Acinetobacter species.Gap statement. However, few studies have investigated the discriminatory power of two multiplex PCR assays in in the genotyping of Acinetobacter species.Aim. We aimed to evaluate the efficacies of the Pan-PCR and OXA-PCR assays for molecular typing of A. baumannii and A. nosocomialis.Methodology. A total of 105 carbapenem-resistant A. baumannii isolates (CRABs) and 93 carbapenem-resistant A. nosocomialis isolates (CRANs) obtained from blood cultures were used for molecular typing by the Pan-PCR and OXA-PCR assays and two multilocus sequence typing (MLST) schemes.Results. The isolates were individually divided into 12 and 21 different sequence types via the Pasteur and Oxford MLST schemes, respectively. Additionally, these isolates were distinguished into 18 different types by the Pan-PCR and OXA-PCR assays. The results of the Pan-PCR and OXA-PCR assays distinguished CRABs and CRANs with a sensitivity of 98.13 % and a specificity of 100 %.Conclusion. The Pan-PCR and OXA-PCR assays are promising alternative methods for rapid molecular typing of CRABs and CRANs in a routine laboratory setting.

Cloning, expression, and in silico structural modeling of cholesterol oxidase of Acinetobacter sp. strain RAMD in E. coli.

Cholesterol oxidases (CHOXs) are flavin-adenine dinucleotide-dependent oxidoreductases with a range of biotechnological applications. There remains an urgent need to identify novel CHOX family members to meet the demands of enzyme markets worldwide. Here, we report the cloning, heterologous expression, and structural modeling of the cholesterol oxidase of Acinetobacter sp. strain RAMD. The cholesterol oxidase gene was cloned and expressed in pGEM®-T and pET-28a(+) vectors, respectively, using a gene-specific primer based on the putative cholesterol oxidase ORF of Acinetobacter baumannii strain AB030 (GenBank [gb] locus tag: IX87_05230). The obtained nucleotide sequence (1671 bp, gb: MK575469.2), translated to a protein designated choxAB (556 amino acids), was overexpressed as inclusion bodies (IBs) (MW ˜ 62 kDa) in 1 mm IPTG-induced Escherichia coli BL21 (DE3) Rosetta cells. The optimized expression conditions (1 mm IPTG with 2% [v/v] glycerol and at room temperature) yielded soluble active choxAB of 0.45 U·mL-1 , with 56.25-fold enhancement. The recombinant choxAB was purified to homogeneity using Ni2+ -affinity agarose column with specific activity (0.054 U·mg-1 ), yield (8.1%), and fold purification (11.69). Capillary isoelectric-focusing indicated pI of 8.77 for choxAB. LC-MS/MS confirmed the IBs (62 kDa), with 82.6% of the covered sequence being exclusive to A. baumannii cholesterol oxidase (UniProtKB: A0A0E1FG24). The 3D structure of choxAB was predicted using the LOMETS webtool with the cholesterol oxidase template of Streptomyces sp. SA-COO (PDB: 2GEW). The predicted secondary structure included 18 α-helices and 12 ß-strands, a predicted catalytic triad (E220 , H380 , and N514 ), and a conserved FAD-binding sequence (GSGFGGSVSACRLTEKG). Future studies should consider fusion to solubilization tags and switching to the expression host Pichia pastoris to reduce IB formation.

Description of Acinetobacter kanungonis sp. nov., based on phylogenomic analysis.

A novel strain of a member of the genus Acinetobacter, strain PS-1T, was isolated from the skin of fresh water pufferfish (Tetraodon cutcutia) collected from Mahanadi River, India. Cells were Gram-stain-negative, aerobic, coccoid and non-motile. The predominant polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and phospholipid (PL) and the cell wall sugars were glucose, galactose and ribose. The major cellular fatty acids of PS-1T were C18 : 1ω9c (30.67 %), C16 : 1ω7c (19.54 %), C16 : 0 (15.87 %), C12 : 0 (7.35 %) and C12 : 0 3-OH (6.77 %). The genome size was 3.5 Mbp and the DNA G+C content was 41.97 %. Gene ontology study revealed that the major fraction of genes were associated with biological processes (53.99 %) followed by molecular function (30.42 %) and cellular components (15.58 %). Comparisons of 16S rRNA gene sequences revealed 97.94-97.05 % sequence similarity with the closely related type strains of species of the genus Acinetobacter. The average nucleotide identity (ANI) and average amino acid identity (AAI) of PS-1T with reference strains of species of the genus Acinetobacter with validly published names were bellow 95-96 and the corresponding in-silico DNA-DNA hybridization (DDH) values were below 70 %. A phylogenomic tree based on core genome analysis supported these results. Genotypic and phenotypic characteristics of PS-1T indicate that the strain represents a novel species of the genus Acinetobacter and the name Acinetobacter kanungonis sp. nov. is proposed. The type strain is PS-1T (=JCM 34131T=NCIMB 15260T).

Delineation of a novel environmental phylogroup of the genus Acinetobacter encompassing Acinetobacter terrae sp. nov., Acinetobacter terrestris sp. nov. and three other tentative species.

This study aimed to define the taxonomic position and structure of a novel, taxonomically unique group of 26 Acinetobacter strains, provisionally designated Taxon 24 (T24). The strains were recovered from soil and freshwater ecosystems (n = 21) or animals (n = 5) in Czechia, Scotland, Germany, the Netherlands and Turkey between 1993 and 2015. They were non-glucose-acidifying, nonhemolytic, nonproteolytic, growing at 32 °C and on acetate and ethanol as single carbon sources, but not on 4-hydroxybenzoate and mostly not at 37 °C. Their whole-genome sequences were 3.0-3.7 Mb in size, with GC contents of 39.8-41.3%. Based on core genome phylogenetic analysis, the 26 strains formed a distinct clade within the genus Acinetobacter, with strongly supported subclades termed T24A (n = 11), T24B (n = 8), T24C (n = 2), T24D (n = 3) and T24E (n = 2). The internal genomic ANIb values for these subclades were >94.8%, while the ANIb values between them were <92.5%. The results of MALDI-TOF MS-based analyses agreed with this classification. The five subclades differed from each other in the results of one to six carbon source assimilation tests. Given the genomic and phenotypic distinctness, internal coherence, numbers of available strains and geographically diverse origin of T24A and T24B, we propose the names Acinetobacter terrae sp. nov. and Acinetobacter terrestris sp. nov. for these two taxa, respectively. The type strains are ANC 4282v (= CCM 8986T = CCUG 73811T = CNCTC 8082T) and ANC 4471T (= CCM 8985T = CCUG 73812T = CNCTC 8093T), respectively. We conclude that these two species together with the other T24 strains represent a widely dispersed Acinetobacter clade primarily associated with terrestrial ecosystems.

Acinetobacter pollinis sp. nov., Acinetobacter baretiae sp. nov. and Acinetobacter rathckeae sp. nov., isolated from floral nectar and honey bees.

A detailed evaluation of eight bacterial isolates from floral nectar and animal visitors to flowers shows evidence that they represent three novel species in the genus Acinetobacter. Phylogenomic analysis shows the closest relatives of these new isolates are Acinetobacter apis, Acinetobacter boissieri and Acinetobacter nectaris, previously described species associated with floral nectar and bees, but high genome-wide sequence divergence defines these isolates as novel species. Pairwise comparisons of the average nucleotide identity of the new isolates compared to known species is extremely low (<83 %), thus confirming that these samples are representative of three novel Acinetobacter species, for which the names Acinetobacter pollinis sp. nov., Acinetobacter baretiae sp. nov. and Acinetobacter rathckeae sp. nov. are proposed. The respective type strains are SCC477T (=TSD-214T=LMG 31655T), B10AT (=TSD-213T=LMG 31702T) and EC24T (=TSD-215T=LMG 31703T=DSM 111781T).

Comparative phylo-pangenomics reveals generalist lifestyles in representative Acinetobacter species and proposes candidate gene markers for species identification.

Acinetobacter species have the potential to invade and colonize immunocompromised patients, therefore being well-known as opportunistic pathogens. Among these bacteria, the species of the Acinetobacter calcoaceticus-Acinetobacter baumannii "complex" (Acb members) emerge as the main often isolated bacteria in clinical specimens. The unequivocal taxonomy is crucial to correctly identify these species and associated with comparative genomic analyses aids to understand their life-styles as well. In this study, all publicly available Acinetobacter species at the date of this study preparation were analyzed. The results revealed that the Acb members are in fact a complex when phenotypic methods are confronted, while for comparative and phylogenomics analyses this term is misleading, since they composed a monophyletic group instead. Nine best gene markers (response regulator, recJ, recG, phosphomannomutase, pepSY, monovalent cation/H + antiporter subunit D, mnmE, glnE, and bamA) were selected for identification of Acinetobacter species. Moreover, representative strains of each species were split according their isolation sources in the categories: environmental, human, insect and non-human vertebrate. Neither niche-specific genome signature nor niche-associated functional and pathogenic potential were associated with their isolation source, meaning it is not the main force acting on Acinetobacter adaptation in a given niche and corroborating that their ubiquitous distribution is a reflex of their generalist life-styles.

Multidrug resistance pattern of Acinetobacter species isolated from clinical specimens referred to the Ethiopian Public Health Institute: 2014 to 2018 trend anaylsis.

BACKGROUND: Acinetobacter species have been a leading cause of nosocomial infections, causing significant morbidity and mortality over the entire world including Ethiopia. The most important features of A. baumannii are its ability to persist in the hospital environment and rapidly develop resistance to a wide variety of antibiotics. This study aimed to determine trend of antimicrobial resistance in Acinetobacter species over a five years period. METHOD: A retrospective data regarding occurrence and antimicrobial resistance of Acinetobacter species recovered from clinical specimens referred to the national reference laboratory was extracted from microbiology laboratory data source covering a time range from 2014 to 2018. Socio-demographic characteristics and laboratory record data was analyzed using SPSS 20. RESULTS: A total of 102 strains of Acinetobacter species were analyzed from various clinical specimens. Majority of them were from pus (33.3%) followed by blood (23.5%), urine (15.6%) and body fluid (11.7%). Significant ascending trends of antimicrobial resistance was shown for meropenem (12.5% to 60.7%), ceftazidime (82.1% to 100%), ciprofloxacin (59.4% to 74.4%), ceftriaxone (87.1% to 98.6%), cefepime (80.0% to 93.3%) and pipracillin- tazobactam (67.8% to 96.3%). However, there was descending trend of antimicrobial resistance for tobramycin (56.5% to 42.8%), amikacin (42.1% to 31.4%) and trimethoprim-sulfamethoxazole (79.0 to 68.2%). The overall rate of carbapenem non-susceptible and multidrug resistance rates in Acinetobacter species were 56.7% and 71.6%.respectively. CONCLUSION: A five year antimicrobial resistance trend analysis of Acinetobacter species showed increasing MDR and resistance to high potent antimicrobial agents posing therapeutic challenge in our Hospitals and health care settings. Continuous surveillance and appropriate infection prevention and control strategies need to be strengthened to circumvent the spread of multidrug resistant pathogens in health care facilities.

Characterization of the microbiota and chemical properties of pork loins during dry aging.

Dry aging (DA) allows for the storage of meat without packaging at 0 to 3°C for several weeks. It enhances the production of pleasant flavors, tenderness, and juiciness in meat. Due to the long storage period and roles of indigenous microbiota in the maturation of several meat products, the microbiota of DA meat is of interest in terms of microbial contributions and food hygiene but has not yet been characterized in detail. This study identified the microbiota of pork loins during DA using culturing and culture-independent meta-16S rRNA gene sequencing and elucidated its characteristics. The amounts of free amino acids and profiles of aroma-active compounds were also monitored by high-performance liquid chromatography and gas chromatography, respectively. The meta-16S rRNA gene sequencing revealed that Pseudomonas spp. generally dominated the microbiota throughout DA; however, the culturing analysis showed marked changes in the species composition during DA. Acinetobacter spp. were the second most dominant bacteria before DA in the culture-independent analysis but became a minor population during DA. The cell numbers of yeasts showed an increased tendency during DA, and Debaryomyces hansenii was the only microorganism isolated from all meat samples throughout DA. Well-known foodborne pathogens were not observed in two microbiota analyses. The amounts of free amino acids were increased by DA, and the number of aroma-active compounds and their flavor dilution values markedly changed during DA. Most microbial isolates showed positive reactions with proteolytic and lipolytic activities, suggesting their contribution to tenderness and aroma production in DA meats.

Bacterial contaminants and their antibiotic susceptibility patterns in ready-to-eat foods vended in Ogun state, Nigeria.

Contamination of ready-to-eat (RTE) foods by pathogenic bacteria may predispose consumers to foodborne diseases. This study investigated the presence of bacterial contaminants and their antibiotic susceptibility patterns in three locally processed RTE foods (eko, fufu and zobo) vended in urban markets in Ogun state, Nigeria. Bacteria isolated from a total of 120 RTE food samples were identified by 16S rRNA gene phylogeny while susceptibility patterns to eight classes of antibiotics were determined by the disc diffusion method. Species belonging to the genera Acinetobacter and Enterobacter were recovered from all RTE food types investigated, Klebsiella and Staphylococcus were recovered from eko and fufu samples, while those of Shigella were recovered from eko samples. Enterobacter hormaechei was the most prevalent species in all three RTE food types. Precisely 99% of 149 isolates were multidrug-resistant, suggesting a high risk for RTE food handlers and consumers. Co-resistance to ampicillin and cephalothin was the most frequently observed resistance phenotype. Results demonstrate that improved hygiene practices by food processors and vendors are urgently required during RTE processing and retail. Also, adequate food safety guidelines, regulation and enforcement by relevant government agencies are needed to improve the safety of RTE foods and ensure the protection of consumer health.

Acinetobacter lanii sp. nov., Acinetobacter shaoyimingii sp. nov. and Acinetobacter wanghuae sp. nov., isolated from faeces of Equus kiang.

Six aerobic, non-motile, non-haemolytic, Gram-stain-negative, oxidase-negative strains (185T, 187, 323-1T, 194, dk386T and dk771) were recovered from different faecal samples of Equus kiang on the Qinghai-Tibet Plateau. In the 16S rRNA gene sequences, one strain pair, 185T/187, shared highest similarity to Acinetobacter equi 114T (97.9 %), and the other two (323-1T/194 and dk771T/dk386) to Acinetobacter harbinensis CGMCC 1.12528T (98.6 and 97.0 %, respectively). Phylogenomic tree analysis showed that these six strains formed three separate clades in the genus Acinetobacter. Digital DNA-DNA hybridization values of each pair of the isolates with all members of the genus Acinetobacter were far below 70 %. The main cellular fatty acids of all six strains were C18 : 1 ω9c, C16 : 0 and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). Q-9 was the predominant respiratory quinone for strains 185T, 323-1T and dk386T. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Based on the genotypic, phenotypic and biochemical analyses, these six strains represent three novel species of the genus Acinetobacter, for which the names Acinetobacter lanii sp. nov., Acinetobacter shaoyimingii sp. nov. and Acinetobacter wanghuae sp. nov. are proposed. The type strains are 185T (=CGMCC 1.13636T=JCM 33607T), 323-1T (=CGMCC 1.13940T=JCM 33608T) and dk386T (=CGMCC 1.16589T=JCM 33592T), respectively.

The status of the genus Prolinoborus (Pot et al. 1992) and the species Prolinoborus fasciculus (Pot et al. 1992).

On the basis of two other publications (Yarza et al. 2013; Nemec et al. 2019) and on the basis of resequencing of the 16S rRNA gene of Prolinoborus fasciculus CIP 103579T it is concluded that Prolinoborus fasciculus CIP 103579T, which is the only available strain of the species from culture collections, does not conform to the original description given by Pot et al. (1992). The strain investigated is a member of the genus Acinetobacter within the Moraxellaceae, a family of the Gammaproteobacteria and not a member of the Betaproteobacteria as originally proposed. Prolinoborus fasciculus CIP 103579T shared 99.8 % 16S rRNA gene sequence similarity with Acinetobacter lwoffii DSM 2403T. The two strains clustered together by rpoB- and core genome-based phylogenetic analyses and shared an average nucleotide identity of 96.47% (reciprocal, 96.56 %) and a digital genome distance calculation (GGDC) value of 66.9 %. Furthermore, the two strains shared matrix-assisted laser desorption/ionization time of flight MS profiles to a high extent and showed highly similar cellular fatty acid profiles and physiological substrate utilization patterns. It is proposed that the Judicial commission consider (1) that the strain currently deposited as CIP 103579 be recognized as a member of Acinetobacter lwoffii; (2) placing Prolinoborus fasciculus (Pot et al. 1992) on the list of rejected names if a suitable replacement strain, or a neotype strain cannot be found within 2 years of publication of this request; and (3) place the genus name Prolinoborus (Pot et al. 1992) on the list of rejected names [Recommendation 20D (3) of the Code].

Acinetobacter portensis sp. nov. and Acinetobacter guerrae sp. nov., isolated from raw meat.

The taxonomic status of six strains of Acinetobacter obtained from meat samples, collected from supermarkets in Porto, Portugal, was investigated using polyphasic analysis. Partial rpoB sequence similarities lower than 95 % to other Acinetobacter species with validly published names led to the hypothesis that these strains represented novel species. This was confirmed based on comparative multilocus sequence analysis, which included the gyrB, recA and 16S rRNA genes, revealing that these strains represented two coherent lineages that were distinct from each other and from all known species. The names Acinetobacter portensis sp. nov. (comprising four strains) and Acinetobacter guerrae sp. nov. (comprising two strains) are proposed for these novel species. The species status of these two groups was confirmed by low (below 95 %) whole-genome sequence average nucleotide identity values and low (below 70 %) digital DNA-DNA hybridization similarities between the whole-genome sequences of the proposed type strains of each novel species and the representatives of the known Acinetobacter species. Phylogenomic treeing from core genome analysis supported these results. The coherence of each new species lineage was supported by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry differentiation of the species at the protein level, by cellular fatty acid profiles, and by unique and differential combinations of metabolic and physiological properties shared by each novel species. The type strain of A. portensis sp. nov. is AC 877T (=CCUG 68672T=CCM 8789T) and the type strain of A. guerrae sp. nov. is AC 1271T (=CCUG 68674T=CCM 8791T).

Characterization of Acinetobacter chengduensis sp. nov., isolated from hospital sewage and capable of acquisition of carbapenem resistance genes.

Two strains of the genus Acinetobacter, WCHAc060005T and WCHAc060007, were isolated from hospital sewage in China. The two strains showed different patterns of resistance to clinically important antibiotics and their taxonomic positions were investigated. Cells are Gram-negative, obligate aerobic, non-motile, catalase-positive and oxidase-negative coccobacilli. A preliminary analysis based on the 16S rRNA gene sequences indicated that the two strains had the highest similarity to Acinetobacter cumulans WCHAc060092T (99.02%). Whole-genome sequencing of the two strains and genus-wide phylogeny reconstruction based on a set of 107 Acinetobacter core genes indicated that they formed a separate and internally cohesive clade within the genus. The average nucleotide identity based on BLAST and in silico DNA-DNA hybridization values between the two new genomes were 99.77% and 98.7% respectively, whereas those between the two genomes and the known Acinetobacter species were <88.93% and <34.0%, respectively. A total of 7 different genes were found in the two genome sequences which encode resistance to five classes of antimicrobial agents, including clinically important carbapenems, oxyimino-cephalosporins, and quinolones. In addition, the combination of their ability to assimilate gentisate, but not l-glutamate and d,l-lactate could distinguish the two strains from all known Acinetobacter species. Based on these combined data, we concluded that the two strains represent a novel species of the genus Acinetobacter, for which the name Acinetobacter chengduensis sp. nov. is proposed. The type strain is WCHAc060005T (CCTCC AB 2019139=GDMCC 1.1622=JCM 33509).

The epidemiology of carbapenem-non-susceptible Acinetobacter species in Europe: analysis of EARS-Net data from 2013 to 2017.

BACKGROUND: Due to limited therapeutic options and their association with high mortality and morbidity, carbapenem-non-susceptible Acinetobacter spp. (CNA) are of significant public health importance. This study aimed to describe current epidemiological trends of CNA proportions in Europe and to identify factors that are associated with carbapenem non-susceptibility of isolates from patients with invasive Acinetobacter spp. infections. METHODS: Data from routine carbapenem susceptibility testing of 18,412 invasive clinical Acinetobacter spp. isolates from 30 European countries in 2013-2017 were analysed using descriptive statistical analyses and uni- and multivariable regression analyses. These data were obtained from the European Antimicrobial Resistance Surveillance Network (EARS-Net). RESULTS: The population-weighted mean proportion of carbapenem-non-susceptible Acinetobacter spp. in Europe is 35.6% (95% confidence interval [CI] 29.7-42.0%). With CNA proportions of 75.5% (95% CI 71.2-79.4%) and 71.5% (95% CI 66.7-75.9%) the burden of CNA is particularly high in Southern and Eastern European regions. In contrast, Northern and Western European regions recorded CNA proportions of 2.8% (95% CI 1.2-6.0%) and 6.3% (95% CI 4.5-8.9%), respectively. Population-weighted mean CNA proportions are especially high in Acinetobacter spp. isolates from intensive care units (54.0% [95% CI 47.6-60.3%]). Male gender, age above 20 years and ICU admission were identified as independent factors associated with an increased likelihood of CNA. CONCLUSION: The burden of carbapenem-non-susceptible Acinetobacter spp. is particularly high in Southern and Eastern Europe. There is a risk that resistance could spread to other parts of Europe. Therefore, increased efforts in infection control and antibiotic stewardship, particularly in Intensive Care Units, are necessary to combat the spread of CNA in Europe.

Acinetobacter pullorum sp. nov., Isolated from Chicken Meat.

A bacterial strain, designated B301T and isolated from raw chicken meat obtained from a local market in Korea, was characterized and identified using a polyphasic taxonomic approach. Cells were gram-negative, non-motile, obligate-aerobic coccobacilli that were catalase-positive and oxidase-negative. The optimum growth conditions were 30°C, pH 7.0, and 0% NaCl in tryptic soy broth. Colonies were round, convex, smooth, and cream-colored on tryptic soy agar. Strain B301T has a genome size of 3,102,684 bp, with 2,840 protein-coding genes and 102 RNA genes. The 16S rRNA gene analysis revealed that strain B301T belongs to the genus Acinetobacter and shares highest sequence similarity (97.12%) with A. celticus ANC 4603T and A. sichuanensis WCHAc060041T. The average nucleotide identity and digital DNA-DNA hybridization values for closely related species were below the cutoff values for species delineation (95-96% and 70%, respectively). The DNA G+C content of strain B301T was 37.0%. The major respiratory quinone was Q-9, and the cellular fatty acids were primarily summed feature 3 (C16:1 ω6c/C16:1 ω7c), C16:0, and C18:1 ω9c. The major polar lipids were phosphatidylethanolamine, diphosphatidyl-glycerol, phosphatidylglycerol, and phosphatidyl-serine. The antimicrobial resistance profile of strain B301T revealed the absence of antibiotic-resistance genes. Susceptibility to a wide range of antimicrobials, including imipenem, minocycline, ampicillin, and tetracycline, was also observed. The results of the phenotypic, chemotaxonomic, and phylogenetic analyses indicate that strain B301T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter pullorum sp. nov. is proposed. The type strain is B301T (=KACC 21653T = JCM 33942T).

Response of particle-associated bacteria to long-term heavy metal contamination in a tropical estuary.

Estuaries being the connecting link between terrestrial and marine environment, experience spatial variations in the hydrographic variables as well as concentrations of pollutants. The present study reports a contrasting difference in the metal tolerance and enzyme activity of particle-associated bacteria (PAB) isolated from the upstream and downstream reaches of a tropical estuary [Cochin Estuary (CE) in the southwest coast of India], exposed to different levels of heavy metal contamination. The upstream of the estuary has been overloaded with heavy metals in the last few decades, while the downstream is less polluted. There were only 25% of culturable PAB phylogenetically common in both upstream and downstream. The PAB isolated from the upstream were dominated by γ-proteobacteria (48.1%) followed by α-proteobacteria (25.0%), while it was in the reverse order of α-proteobacteria (45.9%) and γ-proteobacteria (36.1%) in the downstream. More number of PAB from the upstream showed tolerance to higher concentrations of Zn and Cd. The Acinetobacter sp. MMRF1051 isolated from the upstream showed tolerance up to 250 mM Zn, 100 mM Cd, and 250 mM Ni. The enzyme expression profile of PAB from downstream was in the order of lipase > phosphatase > ß-glucosidase > aminopeptidase, while it was in the order of ß-glucosidase > lipase > aminopeptidase > phosphatase in the upstream of the estuary. The present study shows the selective pressure exerted by heavy metal pollution on the diversity of culturable bacteria associated with particulate matter in a tropical estuary. Also, the variation in their enzyme activities may impinge the remineralization of particulate organic matter (POM) in the system and may impart adverse impacts on ecosystem functioning.

Abundance of mobile genetic elements in an Acinetobacter lwoffii strain isolated from Transylvanian honey sample.

Based on phylogenetic analyses, strain M2a isolated from honey, an unexpected source of acinetobacters, was classified as Acinetobacter lwoffii. The genome of this strain is strikingly crowded with mobile genetic elements. It harbours more than 250 IS elements of 15 IS-families, several unit and compound transposons and 15 different plasmids. These IS elements, including 30 newly identified ones, could be classified into at least 53 IS species. Regarding the plasmids, 13 of the 15 belong to the Rep-3 superfamily and only one plasmid, belonging to the "Low-GC" family, possesses a seemingly complete conjugative system. The other plasmids, with one exception, have a mobilization region of common pattern, consisting of the divergent mobA/mobL-family and mobS-, mobC- or traD-like genes separated by an oriT-like sequence. Although two plasmids of M2a are almost identical to those of A. lwoffi strains isolated from gold mine or Pleistocene sediments, most of them have no close relatives. The presence of numerous plasmid-borne and chromosomal metal resistance determinants suggests that M2a previously has also evolved in a metal-polluted environment. The numerous, possibly transferable, plasmids and the outstanding number of transposable elements may reflect the high potential of M2a for rapid evolution.