Genome sequence of OXA-23 producing Acinetobacter baumannii IHIT7853, a carbapenem-resistant strain from a cat belonging to international clone IC1

Background: Multidrug resistance in Acinetobacter baumannii has dramatically increased in recent years worldwide. Thus, last-line antibiotics like carbapenems are increasingly being used which in turn further augments selection pressure for resistant strains. Resistance to carbapenems in A. baumannii is frequently mediated by carbapenemases, particularly OXA-23 and OXA-58. Carbapenemase-producing bacteria are mainly described in human patients and the intestinal tract represents a common source for such pathogens. In this study, we sequenced and analyzed the genome of A. baumannii IHIT7853, a carbapenem-resistant, OXA-23 producing strain isolated from cystitis in a cat in 2000 in Germany. Results: Phylogenetic analysis revealed that IHIT7853 belonged to the globally distributed international clone IC1 and MLST type ST1/ST231 (Pasteur/Oxford MLST scheme). A phylogenetic tree based on the maximum common genome of 18 A. baumannii isolates placed IHIT7853 close to human clinical isolates, such as the multidrug-resistant (MDR) outbreak strain AYE that was isolated from a patient with pneumonia and cystitis in 2001 in France. The OXA-23 plasmid sequence could be determined as 53,995 bp in size, possessing resistance genes strA and strB in addition to bla OXA-23. Conclusions: The analysis of the genome of IHIT7853 reveals that companion animals carry MDR A. baumannii that resemble relevant clonal lineages involved in severe infections in humans. As urinary tract infections are often caused by bacteria that reside in the intestinal tract, future studies should unveil, if the animal gut serves as a source for MDR A. baumannii

Seasonal Occurrence and Carbapenem Susceptibility of Bovine Acinetobacter baumannii in Germany

Acinetobacter baumannii is one of the leading causes of nosocomial infections in humans. To investigate its prevalence, distribution of sequence types (STs), and antimicrobial resistance in cattle, we sampled 422 cattle, including 280 dairy cows, 59 beef cattle, and 83 calves over a 14-month period. Metadata, such as the previous use of antimicrobial agents and feeding, were collected to identify putative determining factors. Bacterial isolates were identified via MALDI-TOF/MS and PCR, antimicrobial susceptibility was evaluated via VITEK2 and antibiotic gradient tests, resistance genes were identified by PCR. Overall, 15.6% of the cattle harbored A. baumannii, predominantly in the nose (60.3% of the A. baumannii isolates). It was more frequent in dairy cows (21.1%) than in beef cattle (6.8%) and calves (2.4%). A seasonal occurrence was shown with a peak between May and August. The rate of occurrence of A. baumannii was correlated with a history of use of 3rd generation cephalosporins in the last 6 months prior to sampling Multilocus sequence typing (Pasteur scheme) revealed 83 STs among 126 unique isolates. Nine of the bovine STs have previously been implicated in human infections. Besides known intrinsic resistance of the species, the isolates did not show additional resistance to the antimicrobial substances tested, including carbapenems. Our data suggest that cattle are not a reservoir for nosocomial A. baumannii but carry a highly diverse population of this species. Nevertheless, some STs seem to be able to colonize both cattle and humans

Gastrointestinal Parasites and Bacteria in Free-Living South American Sea Lions (Otaria flavescens) in Chilean Comau Fjord and New Host Record of a Diphyllobothrium scoticum-Like Cestode

Present study aimed to characterize gastrointestinal parasites and culturable bacteria from free-living South American sea lions (Otaria flavescens) inhabiting waters of Comau Fjord, Patagonia, Chile. Therefore, a total of 28 individual fecal samples were collected from sea lions within their natural marine habitat during several diving expeditions. Using classical parasitological techniques, study revealed infections with five different gastrointestinal parasite genera. In addition, bacterial cultures showed presence of at least 28 different bacterial genera. Referring to parasites, protozoan, and metazoan species were found with some of them bearing anthropozoonotic potential and/or pathogenic impact for these marine mammals. As such, four of identified parasite genera harbored zoonotic potential (i.e., Entamoeba, Balantidium, Diphyllobothrium, Anisakis) and one genus (Parafilaroides) represented a specific lungworm of marine pinnipeds. Proglottids from fecal samples showed high morphological homology to “Diphyllobothrium” scoticum (Rennie and Reid, 1912; Meggitt, 1924), which was found in Antarctic sea leopards (Hydrurga leptonyx; Phocidae), but contained eggs of smaller size. Molecular characterization revealed 97–100% identity to a new “Diphyllobothrium” species which was recently isolated from a Californian sea lion (Zalophus californianus; Otariidae) in San Francisco. As such, O. flavescens represents a new host record for this parasite species. Furthermore, potential zoonotic bacteria (i.e., Clostridium, Escherichia, Vibrio, Yersinia, Salmonella) were identified amongst others in O. flavescens indicating a reservoir role for these pinnipeds in marine ecosystem. Current data should be considered as a baseline study for future monitoring surveys on anthropozoonotic pathogens circulating in wild free-living sea lions and their possible impact on public health issues and marine wildlife

Carbapenem-resistance and pathogenicity of bovine Acinetobacter indicus-like isolates.

The objective of this study was to characterize blaOXA-23 harbouring Acinetobacter indicus-like strains from cattle including genomic and phylogenetic analyses, antimicrobial susceptibility testing and evaluation of pathogenicity in vitro and in vivo. Nasal and rectal swabs (n = 45) from cattle in Germany were screened for carbapenem-non-susceptible Acinetobacter spp. Thereby, two carbapenem resistant Acinetobacter spp. from the nasal cavities of two calves could be isolated. MALDI-TOF mass spectrometry and 16S rDNA sequencing identified these isolates as A. indicus-like. A phylogenetic tree based on partial rpoB sequences indicated closest relation of the two bovine isolates to the A. indicus type strain A648T and human clinical A. indicus isolates, while whole genome comparison revealed considerable intraspecies diversity. High mimimum inhibitory concentrations were observed for carbapenems and other antibiotics including fluoroquinolones and gentamicin. Whole genome sequencing and PCR mapping revealed that both isolates harboured blaOXA-23 localized on the chromosome and surrounded by interrupted Tn2008 transposon structures. Since the pathogenic potential of A. indicus is unknown, pathogenicity was assessed employing the Galleria (G.) mellonella infection model and an in vitro cytotoxicity assay using A549 human lung epithelial cells. Pathogenicity in vivo (G. mellonella killing assay) and in vitro (cytotoxicity assay) of the two A. indicus-like isolates was lower compared to A. baumannii ATCC 17978 and similar to A. lwoffii ATCC 15309. The reduced pathogenicity of A. indicus compared to A. baumannii correlated with the absence of important virulence genes encoding like phospholipase C1+C2, acinetobactin outer membrane protein BauA, RND-type efflux system proteins AdeRS and AdeAB or the trimeric autotransporter adhesin Ata. The emergence of carbapenem-resistant A. indicus-like strains from cattle carrying blaOXA-23 on transposable elements and revealing genetic relatedness to isolates from human clinical sources requires further investigations regarding the pathogenic potential, genomic characteristics, zoonotic risk and putative additional sources of this new Acinetobacter species

Seasonal Occurrence and Carbapenem Susceptibility of Bovine Acinetobacter baumannii in Germany

Acinetobacter baumannii is one of the leading causes of nosocomial infections in humans. To investigate its prevalence, distribution of sequence types (STs), and antimicrobial resistance in cattle, we sampled 422 cattle, including 280 dairy cows, 59 beef cattle, and 83 calves over a 14-month period. Metadata, such as the previous use of antimicrobial agents and feeding, were collected to identify putative determining factors. Bacterial isolates were identified via MALDI-TOF/MS and PCR, antimicrobial susceptibility was evaluated via VITEK2 and antibiotic gradient tests, resistance genes were identified by PCR. Overall, 15.6% of the cattle harbored A. baumannii, predominantly in the nose (60.3% of the A. baumannii isolates). It was more frequent in dairy cows (21.1%) than in beef cattle (6.8%) and calves (2.4%). A seasonal occurrence was shown with a peak between May and August. The rate of occurrence of A. baumannii was correlated with a history of use of 3rd generation cephalosporins in the last 6 months prior to sampling Multilocus sequence typing (Pasteur scheme) revealed 83 STs among 126 unique isolates. Nine of the bovine STs have previously been implicated in human infections. Besides known intrinsic resistance of the species, the isolates did not show additional resistance to the antimicrobial substances tested, including carbapenems. Our data suggest that cattle are not a reservoir for nosocomial A. baumannii but carry a highly diverse population of this species. Nevertheless, some STs seem to be able to colonize both cattle and humans

<i>Acinetobacter</i> spp. mediated cytotoxicity towards A549 human lung epithelial cells.

<p>Confluent A549 monolayers were infected for 20 h with an MOI of 100 with the indicated bacteria. Supernatants were harvested and LDH activities determined. Mean value for Triton X-100 (positive control) was 924.0 ± 93.7 U/L. Medium (DMEM) was used as a negative control. Shown are mean values ± standard deviations from n = 3–4 experiments. Asterisks indicate a significant difference between the control group (medium) and strains, respectively (* p<0.05; **p<0.001).</p

Neighbour-joining phylogenetic tree based on partial nucleotide sequences of the <i>rpoB</i> (823 bp) genes of <i>Acinetobacter indicus</i>-like strains IHIT27630 and IHIT27599 and nine published <i>A</i>. <i>indicus</i> strains, including relevant information about original host, geographical background, year of isolation and possession of carbapenemases.

<p>The tree was constructed using the maximum likelihood method. Bootstrap values (> 50%) after 1,000 simulations are shown at branch nodes. GenBank accession nos. are given in parentheses. Bar, 0.006 nucleotide substitutions per site. Carb-neg., carbapenemase negative.</p

Presence and absence of virulence gene/protein orthologs in four <i>Acintebacter</i> species strains used for virulence assays.

<p>Presence and absence of virulence gene/protein orthologs in four <i>Acintebacter</i> species strains used for virulence assays.</p

Schematic maps of the genetic environment of <i>bla</i>_OXA-23 in bovine <i>A</i>. <i>indicus</i>-like strains.

<p>The interrupted Tn<i>2008</i> is indicated with a solid line. Target site duplications are likely missing due to other insertion events, such as IS<i>Acsp2</i> at the left border and IS<i>Acra1</i> at the right border of Tn<i>2008</i>. IS, insertion site; IRL, inverted repeat left; IRR, inverted repeat right; <i>ATPase</i> (truncated), gene encoding the putative AAA ATPase; <i>merR</i>, gene encoding the transcriptional regulator MerR; <i>cesD</i>, gene encoding the cobalt-zinc-cadmium resistance protein CesD; <i>cro</i>, gene encoding the Cro-like protein; <i>cds</i>, encoding a hypothetical protein. The figure is not to scale. GenBank accession nos. KU833218 (IHIT27599) and KU833219 (IHIT27630).</p

Dose-dependent lethality of <i>Galleria mellonella</i> infected with <i>Acinetobacter</i> spp. strains.

<p>Larvae were injected with different cfu (5x10² to 5x10⁶) and survival was monitored over 72 h after infection. Mean values from at least four experiments are shown. Error bars show standard error of the mean.</p