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BackgroundColistin is used against multi-drug resistant pathogens, yet resistance emerges through dissemination of plasmid-mediated genes (mcr) or chromosomal mutation of genes involved in lipopolysaccharide synthesis (i.e.mgrB, phoPQ, pmrCAB).Phenotypic susceptibility testing is challenging due to poor diffusion of colistin in agar media, leading to an underestimation of resistance. Performance of five phenotypic approaches was compared in the context of different molecular mechanisms of resistance. We evaluated Vitek 2® (bioMérieux, AST N242), Colistin MIC Test Strip (Liofilchem Diagnostici), UMIC (Biocentric), and Rapid Polymyxin™ NP test (ELITechGroup) against the standard broth microdilution (BMD) method. We used whole genome sequencing (WGS) to infer molecular resistance mechanisms. We analysed 97Enterobacteralesand non-fermenting bacterial isolates, largely clinical isolates collected up to 2018. Data was analysed by comparing susceptibility categories (susceptible or resistant) and minimal inhibitory concentrations (MIC). Susceptibility category concordance is the percentage of test results sharing the same category to BMD. MIC concordance was calculated similarly but considering ±1 MIC titre error range. We determined genomic diversity by core genome multi locus sequencing typing (cgMLST) and identified putative antimicrobial resistance genes using NCBI and CARD databases, and manual annotation.ResultsOf 97 isolates, 54 (56%) were resistant with standard BMD. Highest susceptibility category concordance was achieved by Rapid Polymyxin™ NP (98.8%) followed by UMIC (97.9%), Colistin E-test MIC strip (96.9%) and Vitek 2® (95.6%). Highest MIC concordance was achieved by UMIC (80.4%), followed by Vitek 2® (72.5%) and Colistin E-test MIC strip (62.9%). Among resistant isolates, 23/54 (43%) were intrinsically resistant to colistin, whereas 31/54 (57%) isolates had acquired colistin resistance. Of these,mcr-1was detected in four isolates andmcr-2in one isolate. Non-synonymous mutations inmgrB, phoQ, pmrA, pmrB, andpmrCgenes were encountered inKlebsiella pneumoniae, Escherichia coli,andAcinetobacter bereziniaeresistant isolates. Mutations found inmgrBandpmrBwere only identified in isolates exhibiting MICs of ≥16 mg/L.ConclusionsThe Rapid Polymyxin™ NP test showed highest categorical concordance and the UMIC test provided MIC values with high concordance to BMD. We found colistin resistance in diverse species occurred predominantly through spontaneous chromosomal mutation rather than plasmid-mediated resistance.

Acinetobacter bereziniae, formerly Acinetobacter genomospecies 10, is an opportunistic pathogen possessing resistance to multiple antibiotics, and it has been reported to be responsible for hospital-associated infections in immunocompromised individuals. We report the draft genome sequences of four Acinetobacter bereziniae strains that were isolated from a single human milk sample collected with a personal breast pump and a hand-washed milk collection kit.

Made available in DSpace on 2020-12-11T15:32:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-03-09 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Acinetobacter baumannii is the main species of the Acinetobacter genus; however, non-baumannii Acinetobacter (NBA) species causing infections have been described for the past years, as well as antimicrobial resistance. In this study, we describe the occurrence of two multidrug-resistant (MDR) IMP-1-producing Acinetobacter bereziniae isolates recovered from bloodstream infections in different patients but in the same intensive care unit among 134 carbapenem-resistant Acinetobacter screened. Antimicrobial susceptibility testing revealed resistance to carbapenems, extended spectrum, and antipseudomonad cephalosporins, amikacin, and trimethoprim-sulfamethoxazole. Both A. bereziniae isolates shared the same ApaI-pulsed-field gel electrophoresis (PFGE) pattern. Whole-genome sequencing of both isolates revealed that bla(IMP-1) was embedded into an In86 Class I integron carrying also sul1, aac(6 ')-31, and aadA genes. A new sequence type (ST1309 Pasteur) was deposited. The virulence genes lpxC and ompA, seen in A. baumannii, were detected in the A. bereziniae strains. Recognition of A. bereziniae causing invasive MDR infection underscores the role of NBA species as human pathogens especially in at-risk patients. Adolfo Lutz Inst, Ctr Bacteriol, Ave Dr Arnaldo 355,9 Andar, BR-01246902 Sao Paulo, Brazil Adolfo Lutz Inst, Lab Estrateg, Sao Paulo, Brazil Univ Sao Paulo, Fac Med, Sao Paulo, Brazil Univ Sao Paulo, Fac Med Vet & Zootecnia, Sao Paulo, Brazil Univ Estadual Paulista, Fac Med Botucatu, Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biociencias Botucatu, Botucatu, SP, Brazil Univ Estadual Paulista, Fac Med Botucatu, Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biociencias Botucatu, Botucatu, SP, Brazil FAPESP: 2015/13179-4 FAPESP: 2016/20727-0 FAPESP: 2017/16988-6

A taxonomic study was carried out on strain LW15T, which was isolated from the external lesions of diseased farmed Murray cod (Maccullochella peelii peelii) from an intensive culture pond. Cells of strain LW15T were Gram-negative, facultative-anaerobic, non-motile, and both coccobacillus- and bacillus-shaped. Growth was observed at NaCl concentrations of 0–2 % (w/v) (optimum, 0 %), 4–32 °C (optimum, 25–28 °C) and pH 5.0–9.0 (optimum, 7.0). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain LW15T was affiliated to the genus Acinetobacter , showing the highest similarity to Acinetobacter guillouiae CIP 63.46T (97.7 %) and other Acinetobacter species with validly published names (93.5–97.6 %). Whole-genome sequencing and phylogeny reconstruction based on a core set of 1061 Acinetobacter genes indicated that strain LW15T was most closely related to the clade formed by A. guillouiae CIP 63.46T and Acinetobacter bereziniae CIP 70.12T and distantly related to any of the described species of genus Acinetobacter . Furthermore, strain LW15T could be distinguished from all known Acinetobacter species by its ability to assimilate β-alanine and l-arginine, but not d-glucose. The principal fatty acids were C18 : 1ω9c, C16 : 0 and C16 : 1ω7c/C16 : 1ω6c. The major respiratory quinone was Q-9. Polar lipids of strain LW15T comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, four phospholipids, aminolipid and two unknown lipids. Based on its phenotypic and genotypic data, strain LW15T represents a novel species of the genus Acinetobacter , for which the name Acinetobacter piscicola sp. nov. is proposed. The type strain is LW15T (=MCCC 1K03337T=CICC 24241T=KCTC 62134T=JCM 32101T).

The aim of this study was to characterize the presence of carbapenemase-encoding genes in distinct species of Acinetobacter spp. isolated from Brazilian hospitals. Five carbapenem-resistant Acinetobacter spp. isolates (two Acinetobacter pittii, two Acinetobacter bereziniae and one Acinetobacter junii) recovered from two distinct hospitals between 2000 and 2016 were included in this study. All of the isolates harboured bla IMP-1, which was inserted into In86, a class 1 integron. Pulsed field gel eletrophoresis analysis showed that both A. pittii were identical, while the two A. berezinae isolates were considered to be clonally related. In this study, we demonstrated that mobile elements carrying carbapenemase-encoding genes such as In86 may persist for a long period, allowing their mobilization from A. baumannii to other Acinetobacter spp. that are usually susceptible to multiple antimicrobials.

Acinetobacter bereziniaeis an environmental microorganism with increasing clinical incidence, and may thus provide a model for a bacterial species bridging the gap between the environment and the clinical setting.A. bereziniaeplasmids have been poorly studied, and their characterization could offer clues on the causes underlying the leap between these two radically different habitats. Here we characterized the whole plasmid content ofA. bereziniaeHPC229, a clinical strain previously reported to harbor a 44-kbp plasmid, pNDM229, conferring carbapenem and aminoglycoside resistance. We identified five extra plasmids in HPC229 ranging from 114 to 1.3 kbp, including pAbe229-114 (114 kbp) encoding a MOBP111relaxase and carrying heavy metal resistance, a bacteriophage defense BREX system and four different toxin-antitoxin (TA) systems. Two other replicons, pAbe229-15 (15.4 kbp) and pAbe229-9 (9.1 kbp), both encoding MOBQ1relaxases and also carrying TA systems, were found. The three latter plasmids containedAcinetobacterRep_3 superfamily replication initiator protein genes. HPC229 also harbors two smaller plasmids, pAbe229-4 (4.4 kbp) and pAbe229-1 (1.3 kbp), the former bearing a ColE1-type replicon and a TA system, and the latter lacking known replication functions. Comparative sequence analyses against depositedAcinetobactergenomes indicated that the above five HPC229 plasmids were unique, although some regions were also present in other of these genomes. The transfer, replication, and adaptive modules in pAbe229-15, and the stability module in pAbe229-9, were bordered by sites potentially recognized by XerC/XerD site-specific tyrosine recombinases, thus suggesting a potential mechanism for their acquisition. The presence of Rep_3 and ColE1-based replication modules, differentmobgenes, distinct adaptive functions including resistance to heavy metal and other environmental stressors, as well as antimicrobial resistance genes, and a high content of XerC/XerD sites among HPC229 plasmids provide evidence of substantial links with bacterial species derived from both environmental and clinical habitats.

Species of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex are important human pathogens which can be recovered from animals and food, potential sources for their dissemination. The aim of the present study was to characterise the Acinetobacter isolates recovered from market meat samples in Peru. From July through August 2012, 138 meat samples from six traditional markets in Lima were cultured in Lysogeny and Selenite broths followed by screening of Gram-negative bacteria in selective media. Bacterial isolates were identified by MALDI-TOF MS and DNA-based methods and assessed for their clonal relatedness and antimicrobial susceptibility. Twelve Acinetobacter isolates were recovered from calf samples. All but one strain were identified as members of the clinically-relevant Acinetobacter calcoaceticus-Acinetobacter baumannii complex: 9 strains as Acinetobacter pittii, 1 strain as A. baumannii, and 1 strain as the recently described novel species A. dijkshoorniae. The remaining strain could not be identified at the species level unambiguously but all studies suggested close relatedness to A. bereziniae. All isolates were well susceptible to antibiotics. Based on macrorestriction analysis, six isolates were further selected and some of them were associated with novel MLST profiles. The presence of pathogenic Acinetobacter species in human consumption meat might pose a risk to public health as potential reservoirs for their further spread into the human population. Nevertheless, the Acinetobacter isolates from meat found in this study were not multidrug resistant and their prevalence was low. To our knowledge, this is also the first time that the A. dijkshoorniae species is reported in Peru.

Made available in DSpace on 2020-03-05T18:12:56Z (GMT). No. of bitstreams: 1 DetectionofOXA58producingAcinetobacterbereziniaeinBrazil.pdf: 213309 bytes, checksum: 726cd02e0f50ef408cdbc695c334f835 (MD5) Previous issue date: 2019

A novel strain, Acinetobacter bereziniae strain GWF(KM062029), was isolated from anaerobic activated sludge. The complete 16S rDNA sequence showed that strain GWF has closest homology (100%) with A. bereziniae strain BENAB7 (HE651923.1) and A. bereziniae strain ATCC 17924 (HE651922.1). Strain GWF is a Gram-negative, non-fermentative facultative anaerobe. The optimum growth conditions were shown to be 30 °C, pH 7.5 and acetate-to-perchlorate ratio of 10, respectively. In this study, anthraquinone-1-sulfonate (α-AQS) was the best redox mediator in the perchlorate reduction, and the optimum concentration range was 1.067 mmol/L. The perchlorate reduction rate was enhanced 1.33-fold using 1.067 mmol/L α-AQS. Meanwhile, strain GWF could not withstand the effect of concomitant anions (nitrate, chlorate, chlorite). The concomitant anions constants were 30 mg/L and 1.0 mmol/L for nitrate and chlorate, respectively. Chlorite completely inhibited the perchlorate reduction, and the inhibition did not depend on the concentration of chlorite.

The aim of this study was to investigate the phenotypic and genotypic antimicrobial resistance, integrons, and transferability of resistance markers in 243 aerobic bacteria recovered from pork at slaughter in the People's Republic of China. The organisms belonged to 22 genera of gram-negative bacteria (92.2%) and gram-positive bacteria (7.8%). High levels of resistance were detected to tetracycline, trimethoprim-sulfamethoxazole, and ampicillin (36.2 to 54.3%), and lower levels were detected to nitrofurantoin, cefotaxime, gentamicin, ciprofloxacin, and chloramphenicol (7.8 to 29.2%). Across species, genes conferring antimicrobial resistance were observed with the following frequencies: blaTEM, 40.7%; blaCMY-2, 15.2%; blaCTX-M, 11.5%; sul2, 27.2%; sul1, 14.4%; tet(A), 5.4%; tet(L), 5.4%; tet(M), 5.0%; tet(E), 3.7%; tet(C), 3.3%; tet(S), 2.5%; and tet(K), 0.8%. Various antimicrobial resistance genes were found in new carriers: blaTEM in Lactococcus garvieae, Myroides odoratimimus, Aeromonas hydrophila, Staphylococcus sciuri, Raoultella terrigena, Macrococcus caseolyticus, Acinetobacter ursingii, Sphingobacterium sp., and Oceanobacillus sp.; blaCMY-2 in Lactococcus lactis, Klebsiella oxytoca, Serratia marcescens, Acinetobacter baumannii, and Myroides phaeus; tet(L) in M. caseolyticus; sul1 in Vibrio cincinnatiensis; sul2 in Acinetobacter bereziniae, Acinetobacter johnsonii, and V. cincinnatiensis; and the class 1 integron and gene cassette aadA2 in V. cincinnatiensis. Approximately 6.6% of isolates contained class 1 integrons, and one isolate harbored class 2 integrons. Plasmid associated intI1 and androgen receptor- encoding genes were transferred into Escherichia coli J53 and E. coli DH5α by conjugation and transformation experiments, respectively. Our study highlights the importance of aerobic bacteria from pork as reservoirs for antimicrobial resistance genes and mobile genetic elements that can readily be transferred intra- and interspecies.

Excessive nitrite in food is potentially harmful to human health because of carcinogenic effects caused by its nitroso-derivatives. Douchi, which widely distributed throughout the country, is a traditional solid fermented soybean food with low nitrite content. RESULTS In this study, bacteria which can degrade nitrite were isolated from Douchi and identified from their 16S rDNA sequences. Acinetobacter guillouiae, Acinetobacter bereziniae, Bacillus subtilis, Bacillus tequilensis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus aryabhattai and Bacillus methylotrophicus were selected. It was shown that all strains were able to degrade nitrite to some extent, including Bacillus subtilis NDS1, which was able to degrade 99.41% of nitrite. The enzyme activities of these strains were determined at 24 and 48?h and were shown to correspond with their nitrite degradation rates. The strains were used to inoculate Jiangshui, a kind of traditional fermented vegetable from northwest China that often has a high nitrite content. Of the strains tested, Bacillus subtilis NDS1, Bacillus tequilensis NDS3, Acinetobacter bereziniae NDS4, Bacillus subtilis NDS6, and Bacillus subtilis NDS12 were able to degrade nitrite in Jiangshui more rapidly, with Acinetobacter bereziniae NDS4 degrading almost all nitrite in 48?h compared with 180?h of control. CONCLUSION These results indicate that the selected strains have potential to be used as nitrite-degrading agents in food. ? 2018 Society of Chemical Industry

Acinetobacter bereziniae clinical relevance is starting to be recognized; however, very few descriptions of its carbapenem resistance currently exist. Here we characterize two carbapenem-resistant A. bereziniae isolates. Materials & methods: Isolates were obtained from environmental and clinical samples. Carbapenemases were searched by phenotypic, biochemical and PCR assays. Clonality was studied by ApaI-PFGE and genetic location for carbapenemase genes were assessed by I-CeuI and S1 hybridizations. Results: Isolates were not clonally related but both produced the exclusively Portuguese IMP-5, with the clinical isolate also producing an OXA-58. The carbapenemase genes were plasmid located. Conclusion: Our results emphasize the role of non-baumannii Acinetobacter species as important reservoirs of clinically relevant resistance genes that could also contribute to their emergence as nosocomial pathogens, This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors – COMPETE and by National Funds through FCT – Foundation for Science and Technology under the project Pest-C/EQB/LA0006/2013. L Silva and F Grosso were supported by grants from FCT (SFRH/BD/88028/2012 and SFRH/BPD/95556/2013, respectively). C Sousa was funded by an FCT contract CIENCIA2008. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

The highly variable nature of the internal transcribed spacer region (ITS) has been claimed to represent an ideal target for designing species-specific probes/primers capable of differentiating between closely related Acinetobacter species. However, several Acinetobacter species contain multiple ITS copies of variable lengths, and these include Acinetobacter bereziniae, Acinetobacter guillouiae and Acinetobacter baylyi. This study shows these length variations result from inter-genomic insertion/deletion events (indels) involving horizontal transfer of ITS fragments of other Acinetobacter species and possibly unrelated bacteria, as shown previously by us. In some instances, indel incorporation results in the loss of probe target sites in the recipient cell ITS. In other cases, some indel sequences contain target sites for probes designed from a single ITS sequence to target other Acinetobacter species. Hence, these can generate false positives. The largest of the indels that remove probe sites is 683 bp (labelled bay/i1-0), and it derives from the horizontal transfer of a complete ITS between A. bereziniae BCRC15423(T) and A. baylyi strain ADP1. As a consequence, ITS sequencing or fingerprinting cannot be used to distinguish between the 683 bp ITS in these two strains.

A Gram-stain-negative, non-spore-forming, non-motile and aerobic coccobacilli-shaped strain, designated BRTC-1T, was isolated from the gut of Omphisa fuscidentalis Hampson, which is a larva of a moth and was collected from Xishuangbanna Dai Autonomous Prefecture in China. The isolate was found to grow at NaCl concentrations of 0–5 % (w/v) (optimum: 0 %), 10–45 °C (optimum: 30–35 °C) and pH 5.0–9.0 (optimum: pH 6.0) on tryptic soy agar. Analysis of the 16S rRNA gene sequence indicated that the isolate belonged to the genus Acinetobacter and was most closely related to Acinetobacter rudis LMG 26107T, Acinetobacter guillouiae LMG 988T and Acinetobacter bereziniae LMG 1003T with 96.4, 96.3 and 96.3 % sequence similarity, respectively. The comparative sequence analyses of the rpo B and gyr B genes showed that BRTC-1T is distant from the species of the genus Acinetobacter with validly published names (≤84.0 and ≤82.0 % similarity, respectively). The average nucleotide identity and digital DNA–DNA hybridization values (≤77.0 and ≤22.8 %, respectively) between the whole-genome sequence of BRTC-1T and those of the known taxa were far below the thresholds used to discriminate bacterial species. The major fatty acids were determined to be C16 : 0, C18 : 1ω9c and C16 : 1ω7c/iso-C15 : 0 2-OH. The respiratory quinone was Q-9. The polar lipids were found to be diphosphatidyglycerol, phosphatidylglycerol, phosphatidylethanolamine, five phospholipids and one phosphatidylcholine. Based on its phenotypic and chemotaxonomic characteristics from this study, the isolate is concluded to represent a novel species of the genus Acinetobacter , for which the name Acinetobacter larvae sp. nov. is proposed. The type strain is BRTC-1T (=ACCC 19936T=JCM 31367T).

Acinetobacter bereziniae is an environmental microorganism with increasing clinical incidence, and may thus provide a model for a bacterial species bridging the gap between the environment and the clinical setting. A. bereziniae plasmids have been poorly studied, and their characterization could offer clues on the causes underlying the leap between these two different habitats. Here we characterized the whole plasmid content of A. bereziniae HPC229, a clinical strain previously reported to harbor a 44-kbp plasmid, pNDM229, conferring carbapenem and aminoglycoside resistance. We identified five extra plasmids in HPC229 ranging from 114 to 1.3 kbp, including pAbe229-114 (114 kbp) encoding a MOBP111 relaxase and carrying heavy metal resistance, a bacteriophage defense BREX system and four different toxin-antitoxin (TA) systems. Two other replicons, pAbe229-15 (15.4 kbp) and pAbe229-9 (9.1 kbp), both encoding MOBQ1 relaxases and also carrying TA systems, were found. The three latter plasmids contained Acinetobacter Rep_3 superfamily replication initiator protein genes, and functional analysis of their transfer regions revealed the mobilizable nature of them. HPC229 also harbors two smaller plasmids, pAbe229-4 (4.4 kbp) and pAbe229-1 (1.3 kbp), the former bearing a ColE1-type replicon and a TA system, and the latter lacking known replication functions. Comparative sequence analyses against deposited Acinetobacter genomes indicated that the above five HPC229 plasmids were unique, although some regions were also present in other of these genomes. The transfer, replication, and adaptive modules in pAbe229-15, and the stability module in pAbe229-9, were bordered by sites potentially recognized by XerC/XerD site-specific tyrosine recombinases, thus suggesting a potential mechanism for their acquisition. The presence of Rep_3 and ColE1-based replication modules, different mob genes, distinct adaptive functions including resistance to heavy metal and other environmental stressors, as well as antimicrobial resistance genes, and a high content of XerC/XerD sites among HPC229 plasmids provide evidence of substantial links with bacterial species derived from both environmental and clinical habitats.

The New Delhi metallo-β-lactamase (NDM-1) was initially identified in clinical isolates of Escherichia coli and Klebsiella pneumoniae in Sweden from a patient previously hospitalized in India (1). Since then, blaNDM-1 has frequently been reported in Enterobacteriaceae and Acinetobacter spp., with a fast dissemination in the Indian subcontinent, the Balkan countries, China, and the Middle East (2). NDM-1 producers generally associated with Enterobacteriaceae species have also been reported, albeit with much lower frequency, in Latin American countries, including Guatemala, Mexico, Colombia, and Brazil (3). Moreover, production of NDM-1 in this geographic region has also been noted in Acinetobacter baumannii in Honduras and Brazil (3, 4) and in Acinetobacter pittii in Paraguay and Brazil (5, 6). Here, we report the first case of an NDM-1-producing Acinetobacter species in Argentina, an Acinetobacter bereziniae clinical isolate. We describe also the complete sequence of a blaNDM-1-containing plasmid in this strain. Fil: Brovedan, Marco Alcides. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Marchiaro, Patricia M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Moran Barrio, Jorgelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Cameranesi, María Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Cera, Gabriela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Rinaudo, Mariángel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Viale, Alejandro Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Limansky, Adriana Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

Bacteria of the genus Acinetobacter are increasingly being isolated in hospitals and are recognized as emerging nosocomial pathogens. Species identification is difficult and there is a need for simple molecular methods to differentiate between the species. Naturally occurring oxacillinase genes (blaOXA) have been identified in several Acinetobacter species and their detection by PCR can aid in species identification. The aim of this study was to develop a multiplex PCR to identify intrinsic blaOXA genes (i.e. bla(OXA-134-like), bla(OXA-211-like), bla(OXA-213-like), bla(OXA-214-like) and bla(OXA-228-like)) from Acinetobacter spp. for use as a tool for rapid species identification.Primers were designed to selectively amplify internal fragments of intrinsic blaOXA from Acinetobacter lwoffii/Acinetobacter schindleri (bla(OXA-134-like)), Acinetobacter johnsonii (bla(OXA-211-like)), Acinetobacter calcoaceticus (bla(OXA-213-like)), Acinetobacter haemolyticus (bla(OXA-214-like)) and Acinetobacter bereziniae (bla(OXA-228-like)). Multiplex PCR was performed in a total of 100 Acinetobacter isolates. Flanking primers were designed for each blaOXA subgroup and products were sequenced.All A. lwoffii, A. schindleri, A. johnsonii, A. calcoaceticus, A. haemolyticus and A. bereziniae isolates were positive for their species-specific amplicons while other Acinetobacter species were negative. Thirty blaOXA novel variants were identified; the majority of these (21/30) were from A. calcoaceticus. ISAba11 was found upstream of bla(OXA-214) in four A. haemolyticus isolates, but was not associated with carbapenem resistance.This multiplex PCR specifically detected each of the five different blaOXA subgroups. Therefore, this method has the potential to aid in the identification of these species and monitor the spread of these genes into other Acinetobacter species.

We report here the draft genome sequence of an NDM-1-producing Acinetobacter bereziniae clinical strain, HPC229. This strain harbors both plasmid and chromosomal resistance determinants toward different β-lactams and aminoglycosides as well as several types of multidrug efflux pumps, most likely representing an adaptation strategy for survival under different environments.

The genus Acinetobacter is amongst the most common causes of nosocomial bacterial infections. Even though Acinetobacter baumannii is the most frequent identified species causing a wide range of infections in humans, other species such as Acinetobacter pittii, Acinetobacter nosocomialis, Acinetobacter haemolyticus, Acinetobacter johnsonii, Acinetobacter lwoffii and Acinetobacter ursingii are sporadically observed as nosocomial pathogens. The four species that show similar phenotypes were grouped together as “Acinetobacter calcoaceticus – A. baumannii (ACB) complex”, i. e. A. baumannii, A. nosocomialis, A. pittii and less clinically important A. calcoaceticus. Owing to the high complexity within Acinetobacter genus, it is difficult to differentiate Acinetobacter into species level and these organisms are frequently misidentified. Acinetobacter spp. exhibit a great propensity to acquire antimicrobial resistance determinants and rapidly develop multidrug-resistant (MDR) phenotypes especially resistance to carbapenems, the last resort of antimicrobials to treat Acinetobacter infections. A high prevalence and endemic situations of carbapenem-resistant A. baumannii (CRAB) have been observed in multiple geographical areas such as Asia-Pacific and South America where predominant A. baumannii clonal lineages were noted. The international clone (IC) 2 is recognised as the most successful clone of A. baumannii causing outbreaks and persisting in hospital environments worldwide, particularly in Asia. Despite the increased amount of research on Acinetobacter epidemiology, potential virulence and evolution, little is known about the factors that may have contributed to the success of this well-known clone, IC2. The broad objectives of my PhD are to 1) develop a rapid method to assist in species identification of Acinetobacter non-baumannii, 2) determine the molecular epidemiology of A. baumannii, primarily from Thailand and 3) describe the genome of A. baumannii IC2 isolates from Thailand and compare these genome data with A. baumannii IC2 isolates from Japan, Malaysia and Singapore. Firstly, a multiplex PCR was developed to detect intrinsic oxacillinases encoding genes (blaOXAs), which assisted in rapid identification of multiple Acinetobacter species including A. lwoffii/Acinetobacter schindleri, A. johnsonii, A. calcoaceticus, A. haemolyticus and Acinetobacter bereziniae. Additionally, 30 novel blaOXAs variants were identified in this study. The investigation of Acinetobacter spp. collected worldwide revealed that acquired-type blaOXAs disseminated globally in Acinetobacter spp. as opposed to A. baumannii causing carbapenem resistance particularly in A. pittii. Carbapenem resistance was also observed in A. pittii from Australia and Thailand. Through the genomes of the two A. pittii strains ST119 and a novel ST655, several classes of antimicrobial resistance genes including a novel blaOXA-421, blaOXA-23, blaOXA-96, blaOXA-10, blaIMP-4, blaVEB-7, blaCRAB-2, floR, cmlA1, aar-2 and dfrA10 were identified. This emphasises the importance of A. pittii as an impending multidrug resistance pathogen in this region. Secondly, to explore an endemic situation of multidrug-resistant Acinetobacter spp., 300 non-repetitive ACB complex isolates, mainly A. baumannii from the largest tertiary hospital in Thailand were characterised for their molecular epidemiology and antimicrobial resistance mechanisms. Of these, 270 isolates were carbapenem-resistant and 92.2% resistant to amikacin. A. baumannii IC2 was the dominant clone of A. baumannii (80%) and blaOXA-23-like was detected in most of CRAB isolates. The genomes of 13 representative isolates of A. baumannii (n=11), A. nosocomialis (n=1) and A. pittii (n=1) were analysed via whole genome sequencing. Antimicrobial resistance island, AbaR4-type containing Tn2006, was found in all CRAB isolates. armA was the only 16S rRNA methylase gene found that caused resistance to amikacin (located within Tn1548) and in close proximity to macrolide resistance genes (mphE and msrE). csu locus, bap, bfmRS and pga locus associated with biofilm formation were found in all IC2 isolates in addition to the typical set of antimicrobial resistance genes; blaOXA-23, blaOXA-66, blaADC, strA, strB and tetB in this clone. Therefore, the presence of arrays of antimicrobial resistance genes and biofilm-related loci may contribute to the spread and persistence of A. baumannii IC2 in this hospital. Lastly, comparative genome analysis was performed on 21 representative Acinetobacter spp. isolates from Thailand and other countries, i.e. Japan, Malaysia and Singapore. The range of the CRAB genome size was 3.9 - 4.1 Mb with GC content approximately 39%. Interestingly, the susceptible A. baumannii genome size was 3.7-3.8 Mb. The size difference was due to the absence of resistance genes and regions for bacterial competition and biofilm formation in susceptible isolates. The genetic contexts of key antimicrobial resistance genes and resistance islands were investigated. Our study confirmed that the antimicrobial resistance genes and other genomic features of IC2 isolates were homogenous. The diversity was mainly found in the composition of the antimicrobial resistance genes of IC2 isolates and locus for capsule synthesis (K locus). The genome and composition of antimicrobial resistance genes between IC2 isolates from Thailand and all IC2 from other countries were closely related. Two IC2 carbapenem-susceptible isolates from Japan harboured less antimicrobial resistance genes (blaOXA-66, strA, strB, sul2 and tetB). The locus for the outer core of lipid A-core moiety (OC locus) was generally conserved - OCL1 was a common type within IC2. Such variations observed of the K locus may impact in the difficulties to generate human immune response to A. baumannii. Lastly, one key feature of IC2 was the integrity of all chromosomal regions for type VI secretion system (T6SS) and biofilm formation (csu). In contrast, these regions in non-IC2 isolates were disrupted. This may indicate the incapability of non-IC2 isolates to persist in the hospital environment. The study of various aspects of carbapenem-resistant Acinetobacter spp. in our region has revealed specific insights into this pathogen locally and globally. These include the species identification, molecular epidemiology and the genome of Acinetobacter spp. Genomic analysis has described all necessary attributes of the superiority of IC2 to spread further under antimicrobial pressure and harsh environments. Further research is required to understand greater detail of these many unique findings.

The bla NDM-1 gene is associated with extensive drug resistance in Gram-negative bacteria. This probably spread to Enterobacteriaceae from Acinetobacter spp., and we characterized plasmids associated with bla NDM-1 in Acinetobacter spp. to gain insight into their role in this dissemination. Four clinical NDM-1-producing Acinetobacter species strains from India and Pakistan were investigated. A plasmid harboring bla NDM-1 , pNDM-40-1, was characterized by whole-genome sequencing of Acinetobacter bereziniae CHI-40-1 and comparison with related plasmids. The presence of similar plasmids in strains from Pakistan was sought by PCR and sequencing of amplicons. Conjugation frequency was tested and stability of pNDM-40-1 investigated by real-time PCR of isolates passaged with and without antimicrobial selection pressure. A. bereziniae and Acinetobacter haemolyticus strains contained plasmids similar to the pNDM-BJ01-like plasmids identified in Acinetobacter spp. in China. The backbone of pNDM-40-1 was almost identical to that of pNDM-BJ01-like plasmids, but the transposon harboring bla NDM-1 , Tn 125 , contained two short deletions. Escherichia coli and Acinetobacter pittii transconjugants were readily obtained. Transconjugants retained pNDM-40-1 after a 14-day passage experiment, although stability was greater with meropenem selection. Fragments of pNDM-BJ01-like plasmid backbones are found near bla NDM-1 in some genetic contexts from Enterobacteriaceae , suggesting that cross-genus transfer has occurred. pNDM-BJ01-like plasmids have been described in isolates originating from a wide geographical region in southern Asia. In vitro data on plasmid transfer and stability suggest that these plasmids could have contributed to the spread of bla NDM-1 into Enterobacteriaceae .

This work was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Fundacao Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Oswaldo Cruz Institute (PAPES-FIOCRUZ).

Acinetobacter bereziniae (formerly Acinetobacter genomospecies 10) isolate Nec was recovered from a skin sample of a patient hospitalized in Paris, France. It was resistant to penicillins, penicillin-inhibitor combinations, and carbapenems. Cloning and expression in Escherichia coli identified the carbapenem-hydrolyzing class D β-lactamase OXA-229, which is weakly related to other oxacillinases (66% amino acid identity with the closest oxacillinase, OXA-58). It hydrolyzed penicillins, oxacillin, and imipenem but not expanded-spectrum cephalosporins. Sequencing of the genetic context of the bla OXA-229 gene did not identify an insertion sequence but did identify mutations in the promoter sequences in comparison to the fully susceptible A. bereziniae reference strain. The overexpression of bla OXA-229 in A. bereziniae Nec as a source of carbapenem resistance was identified by quantitative real-time PCR.

Although many studies have been performed on carbapenem-resistant Acinetobacter baumannii, only a few studies have addressed carbapenem resistance in Acinetobacter spp. other than A. baumannii (non-baumannii Acinetobacter). Amongst 287 Acinetobacter spp. isolates from patients with bacteraemia in a South Korean hospital collected between 2003 and 2010, 160 (55.7%) were non-baumannii Acinetobacter spp. Antimicrobial susceptibility testing was performed and the effect of efflux pump inhibitors was examined. Antimicrobial resistance genes were identified and pulsed-field gel electrophoresis (PFGE) analysis was performed. OprD expression was also evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), and CarO disruption was investigated by PCR. Seventeen non-baumannii Acinetobacter isolates (10.6%) were resistant to imipenem or meropenem, comprising eight Acinetobacter pittii (or Acinetobacter genomospecies 3), four Acinetobacter nosocomialis (or Acinetobacter genomospecies 13TU), two Acinetobacter genomospecies 'close to 13TU', two Acinetobacter bereziniae (or Acinetobacter genomospecies 10) and one Acinetobacter genomospecies 16. bla(IMP-1) genes were detected in seven and two carbapenem-resistant A. pittii and A. bereziniae isolates, respectively. PFGE showed that A. pittii isolates carrying bla(IMP-1) belonged to the same clone. In addition, bla(SIM-1) and bla(PER-1) genes were simultaneously identified in two A. nosocomialis isolates. In four isolates (one each of A. pittii, A. nosocomialis, Acinetobacter genomospecies 'close to 13TU' and Acinetobacter genomospecies 16), efflux pumps were implicated in the increase in carbapenem minimum inhibitory concentrations. No decreased expression of OprD was identified in any carbapenem-resistant non-baumannii Acinetobacter isolates, and disruption of carO was also not detected. Clonal spread of carbapenem-resistant A. pittii carrying bla(IMP-1), which contributes to a high resistance rate in this species, was identified. The bla(IMP-1) and bla(SIM-1) genes were first identified in A. bereziniae and A. nosocomialis, respectively. Since no carbapenem resistance mechanisms could be identified, further efforts to find the resistance mechanism should be made.

Acinetobacter genospecies (genomic species) 10 and 11 were described by Bouvet and Grimont in 1986 on the basis of DNA–DNA reassociation studies and comprehensive phenotypic analysis. In the present study, the names Acinetobacter bereziniae sp. nov. and Acinetobacter guillouiae sp. nov., respectively, are proposed for these genomic species based on the congruence of results of polyphasic analysis of 33 strains (16 and 17 strains of genomic species 10 and 11, respectively). All strains were investigated by selective restriction fragment amplification (i.e. AFLP) analysis rpoB sequence analysis, amplified rDNA restriction analysis and tDNA intergenic length polymorphism analysis, and their nutritional and physiological properties were determined. Subsets of the strains were studied by 16S rRNA gene sequence analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS or had been classified previously by DNA–DNA reassociation. Results indicate that A. bereziniae and A. guillouiae represent two phenetically and phylogenetically distinct groups within the genus Acinetobacter. Based on the comparative analysis of housekeeping genes (16S rRNA and rpoB genes), these species together represent a monophyletic branch within the genus. Despite their overall phenotypic similarity, the ability to oxidize d-glucose and to grow at 38 °C can be used in the presumptive differentiation of these two species from each other: with the exception of three strains that were positive for only one test, A. bereziniae strains were positive for both tests, whereas A. guillouiae strains were negative in these tests. The strains of A. bereziniae originated mainly from human clinical specimens, whereas A. guillouiae strains were isolated from different environmental sources in addition to human specimens. The type strain of A. bereziniae sp. nov. is LMG 1003T (=CIP 70.12T =ATCC 17924T) and that of A. guillouiae sp. nov. is LMG 988T (=CIP 63.46T =ATCC 11171T =CCUG 2491T).

Metallo-β-lactamase (MBL) producers have been reported among the various Acinetobacter species worldwide. In this study, the epidemiology and molecular characteristics of carbapenemase-encoding genes and mobile elements were studied to analyse the regional dissemination of MBL genes in Acinetobacter species. From January 2001 to December 2006, 48 Acinetobacter isolates harbouring MBL genes identified from five hospitals in Kyoto and Shiga Prefecture, Japan were collected and analysed. The partial rpoB gene or the 16S-23S ribosomal RNA intergenic spacer region was sequenced to obtain a species-level identification. Molecular typing using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) was performed. Twenty-five Acinetobacter pittii isolates were divided into eight PFGE types and five sequence types (STs) using MLST. Nine Acinetobacter bereziniae isolates belonged to five PFGE types. Five Acinetobacter nosocomialis isolates were divided into two PFGE types and two STs. Three unclassified Acinetobacter species isolates were divided into two PFGE types. Eighteen of the 25 A. pittii isolates belonged to ST119 and were identified from four hospitals. The bla(IMP-19) gene was detected in 41 of 48 isolates, including all of the A. pittii ST119 isolates. The bla(IMP-1) and bla(IMP-11) genes were detected in four and three isolates, respectively. The MBL genes were all embedded within a class 1 integron as a gene cassette array: bla(IMP-19) -aac(6')-31-bla(OXA-21) -aadA1, catB8-like/aacA4-bla(IMP-1) and bla(IMP-11). This study is the first report demonstrating the regional dissemination of MBL-producing Acinetobacter species. A. pittii ST119 harbouring blaIMP-19 was widely spread throughout the Kyoto-Shiga region.