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Citrobacter braakii AN-PRR1 is a potential salt-tolerant, plant growth-promoting rice rhizobacterium isolated from Pakistani soil. The 4.9-Mb draft genome sequence contributes to its taxonomic classification and will reveal the genes putatively responsible for its osmoprotectant and plant growth-promoting activity.

Background: Globally, Salmonella enterica is one of the leading causes of foodborne illness in humans. Food of animal origin is obligatorily tested for the presence of this pathogen. Unfortunately, in meat and meat products, this is often hampered by the presence of background microbiota, which may present as false-positive Salmonella. Methods: For the identification of Salmonella spp. from meat samples of beef, pork, and poultry, the authorized detection method is PN-EN ISO 6579-1:2017-04 with the White–Kauffmann–Le Minor scheme, two biochemical tests: API 20E and VITEK II, and a real-time PCR-based technique. Results: Out of 42 presumptive strains of Salmonella, 83.3% Salmonella enterica spp. enterica, 14.3% Citrobacter braakii, and 12.4% Proteus mirabilis were detected from 180 meat samples. Conclusions: Presumptive strains of Salmonella should be identified based on genotypic properties such as DNA-based methods. The aim of this study was the isolation and identification of Salmonella spp. from miscellaneous meat sorts: beef, pork, and poultry.

The anti-nutritional effects of dietary inositol phosphates (IP6 through IP3) have been recognized in broiler chickens; however, inositol hexaphosphate (IP6) is more potent than the lower IP esters. The efficacies of 2 commercial phytases, a Buttiauxella sp. phytase (BSP) and a Citrobacter braakii phytase (CBP) at 500 and 1,000 FTU/kg, were studied on IP6–3 concentrations in the crop, proventriculus + gizzard, and distal ileum digesta, and ileal IP6 disappearance in broilers at day 22. Apparent ileal P and Ca digestibility, and bone quality at days 22 and 33 were also measured. Female Ross 308 broilers (n = 1,890; 30 birds × 7 diets × 9 replicates) were fed corn-soy-based crumbled diets. The 7 diets included a primary breeder recommendation-based positive control diet (PC); the PC marginally reduced in available P by 0.146% and Ca by 0.134% of the diet, (NC1) or moderately reduced by 0.174 and 0.159% of the diet, respectively (NC2). Other diets were the NC1 + BSP or CBP at 500 FTU/kg (NC1+500BSP and NC1+500CBP) and the NC2 + BSP or CBP at 1,000 FTU/kg (NC2+1,000BSP and NC2+1,000CBP). Each of the NC1 and NC2 had distal ileum IP6 disappearance similar to that of PC, but each had lower P digestibility and the majority of measured bone quality parameters than the PC. The ileal IP6 levels were decreased by 52.0 and 32.7% for NC1+500BSP and NC1+500CBP, respectively, relative to NC1 and by 73.6 and 50.9% for NC2+1,000BSP and NC2+1,000CBP, respectively, relative to NC2 (P < 0.001), with a similar effect for distal ileum IP6 disappearance. Overall, phytase in the NC diets increased P digestibility, and femur breaking strength and cortical bone mineral density at days 22 and 33. Overall, each of the phytases at each dose degraded IP6–3 across the gastrointestinal tract segments to increase P digestibility and the P and Ca utilization in bone. However, dietary BSP at 1,000 FTU/kg was most effective. Supplemental phytase degrades phytate to decrease the anti-nutritional effects in a dose- and phytase-dependent manner.

Ying Li,1,2,&ast; Min Tang,3,&ast; Xiaoyi Dai,4,5 Yingshun Zhou,4,5 Zhikun Zhang,4,5 Yichuan Qiu,4,5 Chengwen Li,1,2 Luhua Zhang4,5 1Department of Immunology, School of Basic Medical Sciences, Southwest Medical University, Luzhou, Sichuan, People’s Republic of China; 2Immunological Technology Platform, Southwest Medical University, Luzhou, Sichuan, People’s Republic of China; 3Department of Laboratory Medicine, the Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, People’s Republic of China; 4Department of Pathogenic Biology, School of Basic Medical Sciences, Southwest Medical University, Luzhou, Sichuan, People’s Republic of China; 5Pathogen Biology Platform, Southwest Medical University, Luzhou, Sichuan, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Luhua Zhang; Chengwen Li Xiang Lin Road, Longmatan District, Luzhou City, Sichuan Province, 646000, People’s Republic of ChinaTel/Fax +86-0830-3160073Email zhluhua@swmu.edu.cn; Lichw110119@163.comPurpose: Three NDM-5-producing Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae, and Citrobacter braakii, one each) were isolated during a screening study for the presence of carbapenemase-producing Enterobacteriaceae (CPE) strains in urban rivers in China. The aim of the present study was to characterize these NDM-5-producing isolates by using whole-genome analysis.Methods: In vitro susceptibility testing was performed using the broth microdilution method. Conjugation assay was carried out to investigate the transferability of blaNDM-5-harboring plasmids. Whole-genome sequencing was performed using an Illumina HiSeq combined with the PacBio RSII system. The genetic characteristics of the blaNDM-5-harboring plasmids were analyzed. Antimicrobial resistance genes and virulence genes were identified from the genome sequences. Phylogenetic analysis was performed based on core genome.Results: Antimicrobial susceptibility testing showed that all three isolates were resistant to carbapenems, cephalosporins, quinolones, and aminoglycosides, and susceptible to colistin. Whole-genome sequencing showed that each isolate carried multiple antibiotic resistance genes mediating multidrug resistance, and harbored numerous virulence genes, some of which were located on plasmids. In these isolates, blaNDM-5 was carried by an IncX3 plasmid in K. pneumoniae and C. braakii, and on an IncR/IncX1 plasmid in E. coli. Conjugation experiments showed that these blaNDM-5-haboring plasmids were successfully transferred to E. coli J53. Phylogenetic analysis revealed that E. coli SCLZR49 was present in a cluster with isolates of different origin, K. pneumoniae SCLZR50 was mainly clustered with clinical isolates, and C. braakii SCLZR53 had closely genetic relationship with environmental isolates.Conclusion: This study revealed contamination of the urban river ecosystems by clinically significant carbapenemase gene blaNDM-5, raising the possibility of plasmid transmission into the environmental from humans and highlighting the need for a constant surveillance of CPE in the environment under the “One-Health” perspective.Keywords: carbapenemase, antimicrobial resistance, aquatic environment, river water

Enzymes can aid in optimal feed stock utilization when used as feed additives. A range of toxicological studies were performed to evaluate the safety profile of a novel phytase (phytase HM) from Citrobacterbraakii produced in Aspergillus oryzae. Phytase HM was found to be non-mutagenic and non-clastogenic in in vitro tests. Further, the phytase HM preparation did not exhibit irritative potential to the eye and skin when applied in in vitro models. A 13-week subchronic toxicity study with oral administration of phytase HM to rats did not show any adverse effects. Efficacy studies showed that the dietary supplementation of this phytase significantly improved growth performance and bone mineralization in broiler chickens and piglets fed P-deficient diets, and increased retention of phosphorus (P) and calcium (Ca), and phytate-P degradation in excreta of broiler chickens in a dose-dependent manner. In conclusion, there are no safety concerns using phytase HM as a feed additive and the phytase is well tolerated by broiler chickens and pigs. Further, phytase HM improves with high efficacy the growth performance in both broiler chickens and pigs., Phytase; Aspergillus oryzae; Toxicology; Safety; Feed additive; Broiler chicken, Pig.

Background To investigate the antimicrobial profiles and genomic characteristics of MDR-Citrobacter spp. strains isolated from Fennec fox imported from Sudan to China. Methods Four Citrobacter spp. strains were isolated from stool samples. Individual fresh stool samples were collected and subsequently diluted in phosphate buffered saline as described previously. The diluted fecal samples were plated on MacConkey agar supplemented with 1 mg/l cefotaxime and incubated for 20 h at 37 °C. Matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI–TOF–MS) was used for identification. Antimicrobial susceptibility testing was performed using the broth microdilution method. Whole-genome sequencing was performed on an Illumina Novaseq-6000 platform. Acquired antimicrobial resistance genes and plasmid replicons were detected using ResFinder 4.1 and PlasmidFinder 1.3, respectively. Comparative genomic analysis of 277 Citrobacter genomes was also performed. Results Isolate FF141 was identified as Citrobacter cronae while isolate FF371, isolate FF414, and isolate FF423 were identified as Citrobacter braakii. Of these, three C. braakii isolates were further confirmed to be extended-spectrum β-lactamases (ESBL)-producer. All isolates are all multidrug resistance (MDR) with resistance to multiple antimicrobials. Plasmid of pKPC-CAV1321 belong to incompatibility (Inc) group. Comparative genomics analysis of Citrobacter isolates generated a large core-genome. Genetic diversity was observed in our bacterial collection, which clustered into five main clades. Human, environmental and animal Citrobacter isolates were distributed into five clusters. Conclusions To our knowledge, this is the first investigation of MDR-Citrobacter from Fennec Fox. Our phenotypic and genomic data further underscore the threat of increased ESBL prevalence in wildlife and emphasize that increased effort should be committed to monitoring the potentially rapid dissemination of ESBL-producers with one health perspective.

A 34-year-old woman presented due to progressive painful swelling around the nail of the right index finger. Onychectomy and drainage of the abscess of the affected finger were performed as the inflammation was progressive despite the previous antibiotic therapy. The microbiological culture revealed a ciprofloxacin-susceptible Citrobacter braakii.

Here, we report a draft genome sequence of a blaKPC-2-carrying Citrobacter braakii isolate from pediatric hospital effluent. Genome CF248 represents a multidrug-resistant C. braakii isolate derived from a clinical environment in Peru. Copyright © 2021 Salvatierra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. This study was funded by the Innóvate Perú grant number 289-2017 and FONDECYT grant number 088-2018. G.S. was supported by the CONCYTEC-FONDECYT-World Bank Group grant number E033-01-08-2018-FONDECYT/Banco Mundial-Programas de Doctorado en Áreas Estratégicas y Generales.

The phyCg gene encoding a new phytase from Citrobacter gillenii was optimized, synthesized, cloned and expressed in Pichia pastoris. Analysis of the amino acid sequence of the enzyme showed that it belongs to the histidine acid phosphatase family. The amino acid sequence of the PhyCg phytase has the highest homology (73.49%) with a phytase sequence from Citrobacter braakii. The main characteristics for the purified recombinant phytase were established. The optimum pH and temperature were 4.5 and 50°C, respectively. The specific activity of the enzyme was 1577 U/mg. The Michaelis constant (Km) and the maximum reaction rate (Vmax) for sodium phytate were 0.185 mM and 2185 U/mg, respectively. The enzyme showed the pH and trypsin stability and had a high activity over a wide pH range.

The global food chain may significantly promote the dissemination of bacteria resistant to antibiotics around the world. This study was aimed at determining the prevalence and genetic characteristics of Enterobacteriaceae with mcr-mediated colistin (CT) resistance in retail meat of different origins. Bacteria of the Enterobacteriaceae family carrying the mcr-1 gene were detected in 21% (18/86) of the examined samples, especially in turkey meat and liver originating from EU and non-EU countries (19%) and in rabbit meat imported from China (2%). The examined samples of the meat and liver of chicken and other poultry and of pork and beef were negative for the presence of bacteria carrying the mcr-1 to mcr-5 genes. A huge number of isolates belonging to Escherchia coli (n = 54), Klebsiella pneumoniae (n = 6), and Citrobacter braakii (n = 1) carrying the mcr-1 gene were obtained. Despite the high heterogeneity of the tested isolates, the mcr-1 gene was localized on only three types of plasmids (IncX4, IncHI2, and IncI2). The most frequent type of plasmid was IncX4, which carried the mcr-1 gene in 77% of E. coli and K. pneumoniae isolates from turkey meat and liver samples from the Czechia, Germany, Poland, and Brazil. Our findings indicate highly probable interspecies transfer of IncX4 and IncI2 plasmids within one meat sample. The co-resistance of plasmid-mediated CT resistance encoded by the mcr-1 and ESBL genes was detected in 18% of the isolates. Another noteworthy finding was the fosA3 gene coding for fosfomycin resistance in a multidrug-resistant isolate of E. coli from rabbit meat imported from China. The observed high level of Enterobacteriaceae with plasmids carrying the mcr-1 gene in retail meat reflects the need for Europe-wide monitoring of mcr-mediated CT resistance throughout the whole food chain.

The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) was attempted by engineering a strong methanol-inducible DAS1 promoter using Citrobacter braakii phytase production as a model case. Constitutive expression of KpTRM1, formerly PRM1—a positive transcription regulator for methanol-utilization (MUT) genes of K. phaffii,was demonstrated to produce phytase without addition of methanol, especially when a DAS1 promoter was used but not an AOX1 promoter. Another positive regulator, Mxr1p, did not have the same effect on the DAS1 promoter, while it was more effective than KpTrmp1 on the AOX1 promoter. Removing a potential upstream repression sequence (URS) and multiplying UAS1DAS1 in the DAS1 promoter significantly enhanced the yield of C. braakii phytase with methanol-feeding, which surpassed the native AOX1 promoter by 80%. However, multiplying UAS1DAS1 did not affect the yield of methanol-free expression by constitutive KpTrm1p. Another important region to enhance the effect of KpTrm1p under a methanol-free condition was identified in the DAS1 promoter, and was termed ESPDAS1. Nevertheless, methanol-free phytase production using an engineered DAS1 promoter outperformed phytase production with the GAP promoter by 25%. Difference in regulation by known transcription factors on the AOX1 promoter and the DAS1 promoter was also illustrated., Engineering the methanol-inducible DAS1 promoter to develop an efficient methanol-free expression system in K. phaffii (P. pastoris).

Citrobacter braakii and Citrobacter freundii are Gram-negative opportunistic pathogens associated with many infectious diseases, including septicemia, in humans and animals. Here, we report the draft genome sequences of seven C. braakii strains and one C. freundii strain isolated from Canadian wastewater treatment facilities.

[Objectives] This study aimed to isolate and characterise extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) isolates from animals and wastewater in Tunisia., [Methods] ESBL-E from wastewater (n = 123 samples), faeces of healthy animals (poultry, sheep, goats and calves) (n = 140) and raw milk from healthy cows (n = 42) and goats (n = 20) were investigated. Antimicrobial susceptibility was determined according to CLSI recommendations. The blaTEM, blaSHV, blaCTX-M and blaOXA-48 genes were analysed by PCR and sequencing. Phylogenetic groups were determined by PCR for Escherichia coli isolates. The clonality of E. coli and Klebsiella pneumoniae isolates was determined by XbaI-PFGE and MLST., [Results] A total of 81 E. coli, 20 K. pneumoniae, 4 Enterobacter cloacae, 1 Citrobacter freundii and 1 Citrobacter braakii were isolated. The blaCTX-M-1 and blaCTX-M-15 genes were predominant in E. coli and K. pneumoniae isolates. E. cloacae and C. braakii isolates harboured the blaSHV-12 gene. The C. freundii isolated from wastewater carried blaCTX-M-15, blaTEM-1 and blaOXA-204. E. coli isolates belonged to phylogroups A (37), B1 (25), B2 (7) and D (12). Seventy-eight E. coli isolates were typeable by PFGE and were classified into 34 pulsotypes. The K. pneumoniae isolates belonged to 11 pulsotypes. The E. coli isolates belonged to sequence types ST131, ST224, ST162, ST845, ST5204, ST69, ST141 and ST10. The K. pneumoniae isolates belonged to ST405, ST147, ST564, ST307, ST152, ST45, ST661 and ST1564., [Conclusion] This is the first report of O25b-B23-CTX-M-27-ST131 E. coli isolates and of C. freundii carrying blaCTX-M-15, blaTEM-1 and blaOXA-204 in Tunisia., This study was supported by the Tunisian Ministry of Higher Education and Scientific Research (Tunisia) and the University of Seville (Seville, Spain).

We report the identification of citrocin, a 19-amino acid-long antimicrobial lasso peptide from the bacteria Citrobacter pasteurii and Citrobacter braakii. We refactored the citrocin gene cluster and heterologously expressed it in Escherichia coli. We determined citrocin's NMR structure in water and found that is reminiscent of that of microcin J25 (MccJ25), an RNA polymerase-inhibiting lasso peptide that hijacks the TonB-dependent transporter FhuA to gain entry into cells. Citrocin has moderate antimicrobial activity against E. coli and Citrobacter strains. We then performed an in vitro RNA polymerase (RNAP) inhibition assay using citrocin and microcin J25 against E. coli RNAP. Citrocin has a higher minimal inhibition concentration than microcin J25 does against E. coli but surprisingly is ∼100-fold more potent as an RNAP inhibitor. This suggests that citrocin uptake by E. coli is limited. We found that unlike MccJ25, citrocin's activity against E. coli relied on neither of the two proton motive force-linked systems, Ton and Tol–Pal, for transport across the outer membrane. The structure of citrocin contains a patch of positive charge consisting of Lys-5 and Arg-17. We performed mutagenesis on these residues and found that the R17Y construct was matured into a lasso peptide but no longer had activity, showing the importance of this side chain for antimicrobial activity. In summary, we heterologously expressed and structurally and biochemically characterized an antimicrobial lasso peptide, citrocin. Despite being similar to MccJ25 in sequence, citrocin has an altered activity profile and does not use the same outer-membrane transporter to enter susceptible cells.

Polymyxins are important lipopeptide antibiotics that serve as the last-line defense against multidrug-resistant (MDR) Gram-negative bacterial infections. Worryingly, the clinical utility of polymyxins is currently facing a serious threat with the global dissemination of mcr, plasmid-mediated polymyxin resistance. The first plasmid-mediated polymyxin resistance gene, termed as mcr-1 was identified in China in November 2015. Following its discovery, isolates carrying mcr, mainly mcr-1 and less commonly mcr-2 to -7, have been reported across Asia, Africa, Europe, North America, South America and Oceania. This review covers the epidemiological, microbiological and genomics aspects of this emerging threat to global human health. The mcr has been identified in various species of Gram-negative bacteria including Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella enterica, Cronobacter sakazakii, Kluyvera ascorbata, Shigella sonnei, Citrobacter freundii, Citrobacter braakii, Raoultella ornithinolytica, Proteus mirabilis, Aeromonas, Moraxella and Enterobacter species from animal, meat, food product, environment and human sources. More alarmingly is the detection of mcr in extended-spectrum-β-lactamases- and carbapenemases-producing bacteria. The mcr can be carried by different plasmids, demonstrating the high diversity of mcr plasmid reservoirs. Our review analyses the current knowledge on the emergence of mcr-mediated polymyxin resistance.

The emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) represent a major clinical problem and raise serious health concerns. The present study aimed to investigate and ascertain the occurrence of CRE among hospitalized patients of Mohamed VI University Hospital, Marrakech, Morocco. Biological samples were collected over a one-year period (2018). The bacterial isolates were identified by MALDI-TOF-MS. Antibiotic susceptibility testing was performed using disc diffusion and Etest. The modified Hodge test and combined disc diffusion test were used for phenotypic detection. CRE hydrolyzing enzyme encoding genes: blaOXA-48, blaKPC, blaIMP, blaVIM, and blaNDM were characterized by PCR and DNA sequencing. In total, 131 non-duplicate CRE clinical strains resistant to Ertapenem were isolated out of 1603 initial Enterobacteriaceae. Klebsiella pneumoniae was the most common species (59%), followed by Enterobacter cloacae (24%), E. coli (10%), Citrobacter freundii (3%), Klebsiellaoxycota (2%), Serratia marcescens (1%), and Citrobacter braakii (1%). Of these, 56.49%, 21.37%, 15.27%, 3.38%, and 3.05% were collected from blood, urine, pus, catheters and respiratory samples, respectively. Approximately 85.5% (112/131) of the isolates were carbapenemase producers (40 blaOXA-48, 27 blaNDM, 38 blaOXA-48 + blaNDM and 7 blaVIM). All metallo-β-lactamases isolates were NDM-1 and VIM-1 producers. This is the first documentation of blaOXA-48 genes from C. freundii and C. braakii in Morocco.

The aim of this work was to identify the main microbiota in raw cow milk from dairy farm of Slovakia and to describe the selected microorganisms responsible for thermostable protease and lipase production which can affected the quality of dairy products. The main bacterial classes identifying by MALDI-TOF MS were Gammaproteobacteria (62 %), Actinobacteria (19 %) and Bacilli (12 %). The dominant microbial genus of raw cow milk was Pseudomonas. From milk bacteria, the strain Lactococcus lactis and from the family Enterobacteriaceae, namely Enterococcus faecalis, Hafnia alvei, Citrobacter braakii and Raoultella ornithinolytica were observed in raw milk. The spoilage of milk products is caused by thermostable enzymes with lipolytic and proteolytic activity. Qualitative proteolytic and lipolytic activities were performed on skin milk agar and olive oil, respectively. From 16 identified microorganisms, only 8 strains (P. fragii, P. gessardii, P. lundesis, H. alvei, C. braakii, R. ornithinolytica, Kocuria rhizophila and Candida inconspicua) showed protease activity. Quantitative protease and lipase activities were determined by casein and olive oil, respectively. The highest both activities were measured for the genus Pseudomonas. While lipases produced by all isolated microbial species lose enzymatic activity at 77 °C for 30 – 40 min, almost proteases showed comparable activities during whole pasteurization experiment at selected experimental conditions (70 °C, 40 min).

We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8-cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His 6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1 S )-2α-hydroxy-1,8-cineole or (1 R )-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida . Compared to P450 cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii , these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications. IMPORTANCE CYP101J2, CYP101J3, and CYP101J4 are cytochrome P450 monooxygenases from S. yanoikuyae B2 that hydroxylate the monoterpenoid 1,8-cineole. These enzymes not only play an important role in microbial degradation of this plant-based chemical but also provide an interesting route to synthesize oxygenated 1,8-cineole derivatives for applications as natural flavor and fragrance precursors or incorporation into polymers. The P450 cytochromes also provide an interesting basis from which to compare other enzymes with a similar function and expand the CYP101 family. This could eventually provide enough bacterial parental enzymes with similar amino acid sequences to enable in vitro evolution via DNA shuffling.

Sodium lauryl ether sulfate (SLES) is an anionic surfactant used in the formulation of several detergent products. The high concentration of organic materials and SLES present in the industrial wastewater poses a real problem. In the previous study, Dhouib et al. (Dhouib et al., 2003) showed that Citrobacter braakii has a large capacity to degrade anionic surfactants in the wastewater treatment of a cosmetics industry. The scale-down has made possible to isolate, from the aerobic membrane bioreactor (MBR) inoculated with Citrobacter braakii since after one year, four strains A4, A10, A13 and A14 which were identified as Pseudomonas aeruginosa , Pseudomonas stutzeri , Bacillus safensis and Staphylococcus arlettae , respectively. These isolates are able to degrade 100% of SLES at a concentration of 1000 mg l −1 in the BSM medium. Physical-chemical parameters have been followed during 104 days in the influent, bioreactor and effluent. Biomass concentration increased slightly in the reactor and reached 13 g VSS l −1 . The COD and surfactants average removals were about 92% and 99.6%, respectively.

The main objective of this study is to demonstrate the potential of microbial induced calcite precipitation (MICP) as rapid and effective process in the synthesis of fluorescent cadmium sulfide nanoparticles. A urease-producing bacterial isolate AX5, identified as Citrobacter braakii, from calcite rich shells of abalone that could tolerate cadmium at the concentration of 250 mg/l was used. In order to investigate the role of MICP in enhancing the synthesis of CdS nanoparticles, the biomimetic synthesis was carried out by C. braakii in modified nutrient broth without amending any additional organic or toxic surfactants back into the environment. The large amount of monodispersed CdS nanoparticles was harvested and found to bind with biocalcite. XRD analysis confirmed the presence of CdS nanoparticles bound with biocalcite minerals in the precipitation. HRTEM and SEM micrographs showed spherical CdS nanoparticles of particle size in the range of 50–100 nm. The other characteristics of harvested CdS nanoparticles were studied with UV–vis, PL spectra, FTIR, and fluorescence microscopy. The findings of present study have important insights into the application of MICP as a promising mechanism for the biosynthesis of CdS nanoparticles. Keywords: Biocalcite, Nanoparticles, Citrobacter braakii, Urease, CdS

Presence of extended spectrum beta-lactamases (ESBL) in bacteria is a growing health concern of global significance. The local, regional, national, and international epidemiological studies for extended spectrum beta-lactamases-producing Enterobacteriaceae and their encoding genes in foods are still incomplete. The objective of this study was to determine the occurrence of extended spectrum beta-lactamases-producing Enterobacteriaceae and the characteristics of their encoding genes from a total of 250 samples of various foods of animal-origin (100 raw chicken meat, 100 raw cow milk, and 50 raw cow milk cheese) sold in Turkey. Overall, 55 isolates were positive as extended spectrum beta-lactamases-producing Enterobacteriaceae. The most prevalent extended spectrum beta-lactamases-producing strain were identified as Escherichia coli (80%), followed by Enterobacter cloacae (9.1%), Citrobacter braakii (5.5%), Klebsiella pneumoniae (3.6%), and Citrobacter werkmanii (1.8%) by Vitek® MS. The simultaneous production of extended spectrum beta-lactamases and AmpC was detected in five isolates (9.1%) in E. coli (80%) and E. cloacae (20%). The frequency rates of blaTEM, blaCTX-M, and blaSHV were 96.4%, 53.7%, and 34.5%, respectively. The co-existence of bla -genes was observed in 82% of extended spectrum beta-lactamases producers with a distribution of blaTEM & blaCTX-M (52.7%), blaTEM & blaSHV (20%), blaTEM & blaCTX-M & blaSHV (12.7%), and blaSHV & blaCTX-M (1.8%). The most prevalent variant of blaCTX-M clusters was defined as blaCTX-M-1 (97.2%), followed by blaCTX-M-8 (2.8%). In summary, the analysed foods were found to be posing a health risk for Turkish consumers due to contamination by Enterobacteriaceae with a diversity of extended spectrum beta-lactamases encoding genes.

PGPB enhance plant growth through nitrogen fixation, IAA production, phosphorus and pottasium solubilization. PGPB as biofertilizer provide an alternative for chemical fertilizer to reduce environmental damage. PGPB in consortium could be more effective instead of single strain inoculant. The aim of this study is to investigate compatibility, design soil bacteria consortium isolated from rubber-Canna intercropping plantation and evaluate the consortium’s potential as biofertilizer. Functional roles of PGPB was tested such as nitrogen fixation, potassium and phosphate solubilization, and IAA production. Detection of pathogenic bacteria was tested by Blood Agar method. Compatibility test was performed by cross streak method. Bacteria were identified using sequencing of 16srRNA gene and verified using BLAST to database of NCBI. The results showed that among 19 bacterial isolates, all showed the nitrogen-fixing activity, G5 had the highest phosphorus and potassium solubilization index, whereas Citrobacter braakii strain 167 produced the highest IAA concentration. Compatibility analysis showed that Citrobacter freundii strain LMG 3246, Citrobacter braakii strain DSM 17596 and G5 are compatible as a bacteria consortium and may developed as biofertilizer. This study found a possible new and beneficial biofertilizer formulation to enhance plant growth and to reduce the application of chemical fertilizer.

Non-Human Primates are one of the most displayed exhibits in zoological facilities in Nigeria. They share phylogenetic similarities with humans and a high probability of pathogen transfer exists between humans and these species during zoo visit. The resident microbial flora plays a vital part in animal health. A total of 36 swab (18 oral and 18 rectal) samples were collected from anaesthetised apparently healthy NHPs of the family Cercopithecidae (sub family Cercopithecinae) from two urban wildlife facilities. Standard bacteriological procedures were used in the isolation and identification of the bacteria. Antimicrobial susceptibility testing was carried out using disc diffusion method according to Bauer-Kirby technique. All the samples yielded at least one bacterial organism. The oral swabs had 69.7% Gram positive and 30.3% Gram negative organisms while the rectal samples had 38.8% and 61.2% of Gram positive and negative organism respectively. The bacteria included; Bacillus spp., Staphylococcus saprophyticus, Micrococcus luteus, Proteus spp, Enterococcus faecalis, Escherichia coli, Klebsiella oxytoca, Citrobacter braakii, Serratia odorifera and Aeromonas hydrophila. . Antimicrobial susceptibility testing revealed high susceptibility of the isolates to ciprofloxacin (73.2%), sparfloxacin (70.7%), ofloxacin and pefloxacin (67.1%) and gentamycin (53.7%) while a high resistance was recorded against augmentin, amoxicillin, streptomycin and septrin. Some of the bacteria showed multidrug resistance to many of the antimicrobials. Isolation of cultivable bacteria with multidrug resistance from the oral and rectal orifices of NHPs strengthens the need for holistic sanitary and management regulations in zoological facilities housing these animals bearing in mind the probable routes of transmission of identified organisms.

The intestinal tract is a huge reservoir of Enterobacteriaceae, some of which are opportunist pathogens. Several genera of these bacteria harbour intrinsic antibiotic resistance genes, such as ampC genes in species of Citrobacter, Enterobacter or Escherichia genera. In this work, beta-lactamases and other resistance mechanisms have been characterized in Enterobacteriaceae isolates recovered from healthy human faecal samples, focusing on the ampC beta-lactamase genes. Fifty human faecal samples were obtained, and 70 Enterobacteriaceae bacteria were isolated: 44 Escherichia coli, 4 Citrobacter braakii, 9 Citrobacter freundii, 8 Enterobacter cloacae, 1 Proteus mirabilis, 1 Proteus vulgaris, 1 Klebsiella oxytoca, 1 Serratia sp. and 1 Cronobacter sp. A high percentage of resistance to ampicillin was detected (57%), observing the AmpC phenotype in 22 isolates (31%) and the ESBL phenotype in 3 isolates. AmpC molecular characterization showed high diversity into bla CMY and bla ACT genes from Citrobacter and Enterobacter species, respectively, and the pulsed-field gel electrophoresis (PFGE) analysis demonstrated low clonality among them. The prevalence of people colonized by strains carrying plasmid-mediated ampC genes obtained in this study was 2%. The unique plasmid-mediated bla AmpC identified in this study was the bla CMY-2 gene, detected in an E. coli isolate ascribed to the sequence type ST405 which belonged to phylogenetic group D. The hybridization and conjugation experiments demonstrated that the ISEcp1-bla CMY-2-blc structure was carried by a ~78-kb self-transferable IncK plasmid. This study shows a high polymorphism among beta-lactamase genes in Enterobacteriaceae from healthy people microbiota. Extensive AmpC-carrier studies would provide important information and could allow the anticipation of future global health problems.

Phosphorus (P) in most diets for sows is poorly digested because pigs do not produce salivary or gastric phytase to hydrolyze P from phytate (inositol hexaphosphate), thus leading to a relatively high amount of P in the manure. An experiment was carried out to study the efficacy of a novel bacterial 6-phytase produced by a strain of Aspergillus oryzae expressing two synthetic genes, both mimicking a phytase gene from a Citrobacter braakii strain. The apparent total tract digestibility coefficient (CATTD) of P, calcium (Ca), crude protein (CP), and gross energy, concentrations of plasma variables, and sow performance were used as response variables. In total, 45 crossbred gestating sows (Large white×Landrace) were individually housed with their litter. Three diets were prepared: (1) positive control (PC; 5.2 g available P/kg), regular sow diet containing inorganic P; (2) negative control (NC; 2.0 g available P/kg) without inorganic P; and (3) NC + 500 FTU/kg diet (NC + PHY). Each diet was fed to 15 sows for 21 days (from day 108 of gestation to day 15 of lactation). To the diets, 3.0 g titanium dioxide/kg was added as a marker to determine CATTD of nutrients. Blood samples were collected from the anterior vena cava at day 1 and 15 of lactation to determine plasma minerals (P, Ca, magnesium, copper, iron, and zinc) and plasma urea, glucose, total protein, albumin, and globulin. At day 15 of lactation, CATTD of P did not differ between PC and NC. Phytase supplementation to NC increased (P

A 14-d experiment was conducted to study the effects of 2 dietary variables on efficacy of a 6-phytase from Citrobacter braakii on broiler growth performance and tibia mineralization. Diets were formulated with or without nutrient matrix values for phytase as negative or positive control (NC or PC, respectively) and with 2 Ca:total P (tP; 2:1 or 2.5:1). The diets were supplemented with 0, 1,000, or 2,000 phytase units (FYT)/kg, thus producing a 2 × 2 × 3 factorial arrangement. Birds and feed were weighed on d 7 and 21, and tibia bones were collected from all the birds on d 21. The main effects of nutrient matrix, Ca:tP, and phytase supplementation were significant (P0.05) for all the growth performance responses (except for G:F for which there was no effect of matrix). The Ca:tP × phytase and matrix × phytase interactions were significant (P0.05) for weight gain. In the PC diets, phytase increased weight gain (P0.05) relative to the control only in diets with 2,000 FYT/kg, whereas in NC diets weight gain increased (P0.01) only from 0 to 1,000 FYT/kg levels. Broilers consuming diets with 2.5:1 Ca:tP had lower (P0.05) tibia ash, whereas phytase increased (P0.01) tibia ash, Ca, P, and Zn but decreased (P0.01) tibia K. Phytase supplementation of diets with 2:1 Ca:tP increased (P0.05) tibia P in birds receiving 1,000 FYT/kg relative to the control with no further increase at 2,000 FYT/kg, whereas each level of phytase supplementation increased (P0.05) tibia P in the diets with 2.5:1 Ca:tP. It was concluded that the best response to lower phytase supplementation (1,000 FYT/kg) was in NC diets with narrow Ca:tP, whereas the best response to higher level of phytase supplementation (2,000 FYT/kg) was achieved in diets in PC diets with wide Ca:tP.

Glycerol dehydrogenase (GlyDH) catalyzes the regioselective oxidation of glycerol to yield 1,3-dihydroxyacetone (DHA); an important building block in chemical industry. Three recombinant GlyDHs from Geobacillus stearothermophilus, from Citrobacter braakii and from Cellulomonas sp. were stabilized by covalent immobilization. The highest activity recoveries (40–50%) of the insoluble preparations were obtained by immobilizing these enzymes in presence of polyethylene glycol (PEG). Noteworthy, these immobilized preparations were more stable and less inhibited by DHA than their soluble counterparts. In particular, GlyDH from G. stearothermophilus immobilized on agarose activated with both amine and glyoxyl groups and crosslinked with dextran aldehyde was 3.7-fold less inhibited by DHA than its soluble form and retained 100% of its initial activity after 18 h of incubation at 65 °C and pH 7. This is one of the few examples where the same immobilization protocol has minimized enzyme product inhibition and maximized thermal stability., This work has been supported by the Spanish Ministry of Economy and Innovation (project CTQ2009-07568). We would like to thank IKERBASQUE, Basque foundation for Science for the funding to PhD Fernando López Gallego. We also thank to Comunidad de Madrid gouverment for funding the PhD fellowship for J. Rocha-Martín.

Under European Economic Area Agreement, Romania and Norway have developed a project for monitoring the quality of raw and potable water (from production technology until distribution) supplied by two Romanian water companies. In the present study was investigated the microbiological quality of water, which was compared with organic matter loads. Resulting free chlorine and trihalomethane compounds after disinfection processes were also analysed. The raw water collected from Danube hydrographical basin showed significant pollution with potential pathogenic microorganisms with faecal origin such as Salmonella sp., Escherichia coli, Enterobacter sp., Klebsiella pneumoniae, Proteus mirabilis, Citrobacter braakii, Providencia stuartii, and Enterococcus sp. Also other bacteria such as Pseudomonas aeruginosa, Acinetobacter, total number of bacteria at 22 and 37 °C were detected, being related to high water oxidability. The microbial load was reduced considerably after the disinfection process. However, the total number of bacteria at 22 or 37 °C was constantly higher than the imposed limit and Clostridium perfringens was identified occasionally. The tap water presented a sporadic microbial loading with Enterococcus sp., Pseudomonas aeruginosa and E. coli and constantly with total number of bacteria due to an improper water distribution network.

Among five American Type Culture Collection (ATCC) Citrobacter strains, qnrB60 in Citrobacter freundii ATCC 6879, an isolate from the preantibiotic era, and qnrB61 in Citrobacter braakii ATCC 51113 T , a type strain belonging to the C. freundii complex, were identified. Meanwhile, a truncated qnrB -like pseudogene was identified in C. freundii ATCC 8090 T and ATCC 43864. No qnrB -like sequence was found in Citrobacter koseri ATCC 27028 T . These findings underscore the close relationship between this species and qnrB .

Objectives: There is little information about carbapenemase-producing (CP) Citrobacter spp. We studied the molecular epidemiology and microbiological features of CP Citrobacter spp. isolates collected in Spain (2013-15). Methods: In total, 119 isolates suspected of being CP by the EUCAST screening cut-off values were analysed. Carbapenemases and ESBLs were characterized using PCR and sequencing. The genetic relationship among Citrobacter freundii isolates was studied by PFGE. Results: Of the 119 isolates, 63 (52.9%) produced carbapenemases, of which 37 (58.7%) produced VIM-1, 20 (31.7%) produced OXA-48, 12 (19%) produced KPC-2, 2 (3.2%) produced NDM-1 and 1 (1.6%) produced VIM-2; 9 C. freundii isolates co-produced VIM-1 plus OXA-48. Fourteen isolates (22.2%) also carried ESBLs: 8 CTX-M-9 plus SHV-12, 2 CTX-M-9, 2 SHV-12 and 2 CTX-M-15. Fifty-seven isolates (90.5%) were C. freundii, 4 (6.3%) were Citrobacter koseri, 1 (1.6%) was Citrobacter amalonaticus and 1 (1.6%) was Citrobacter braakii. By EUCAST breakpoints, eight (12.7%) of the CP isolates were susceptible to the four carbapenems tested. In the 53 CP C. freundii analysed by PFGE, a total of 44 different band patterns were observed. Four PFGE clusters were identified: cluster 1 included eight isolates co-producing VIM-1 and OXA-48; blaVIM-1 was carried in a class 1 integron (intI-blaVIM-1-aacA4-dfrB1-aadA1-catB2-qacEΔ1/sul1) and blaOXA-48 was carried in a Tn1999.2 transposon. Conclusions: We observed the clonal and polyclonal spread of CP Citrobacter spp. across several Spanish geographical areas. Four species of Citrobacter spp. produced up to five carbapenemase types, including co-production of VIM-1 plus OXA-48. Some CP Citrobacter spp. isolates were susceptible to the four carbapenems tested, a finding with potential clinical implications. This work was supported by Plan Nacional de I + D+i 2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16CIII/0004/0002) - co-financed by European Development Regional Fund (ERDF) ‘A way to achieve Europe’, Operative Program Intelligent Growth 2014–2020. This work was also supported by a grant from the Instituto de Salud Carlos III (grant number MPY 1135/16), by the Antibiotic Resistance Surveillance Program of the Centro Nacional de Microbiología (Instituto de Salud Carlos III, Ministerio de Economía y Competitividad) of Spain and by a grant from the Agencia Española de Medicamentos y Productos Sanitarios (grant number MEG 1281/14). Sí

Objective The objective of the present study was to analyze the presence of qnr genes among different clinical isolates and, subsequently, antibacterial activity of drugs was tested against all of the clinical isolates. Next, we examined the effect of different concentrations of ethylenediaminetetraacetic acid (EDTA) and half of minimum inhibitory concentration (MIC) of drugs on conjugation. Methods Analysis of the presence of qnr genes ( qnrA , qnrB and qnrS ) was done using previously reported primers by polymerase chain reaction. The antibacterial activity of all drugs was determined by MIC and antimicrobial susceptibility (AST) according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2009). Conjugation was carried out according to the broth mating method. Results The qnrA gene was detected in Acinetobacter baumannii and Citrobacter braakii , whereas qnrB gene was found in Escherichia coli , A. baumannii , C. braakii , Pseudomonas aeruginosa and Klebsiella pneumoniae . However, qnrS was not detected in any of the clinical isolates. The results revealed that Potentox has potential antimicrobial activity against all of the clinical isolates compared to other drugs. Conjugal transfer of qnrB gene from donor to recipient was inhibited at 10 mM EDTA. Similarly, when various drugs at half of MIC were evaluated on conjugation, interestingly, only Potentox could inhibit conjugal transfer of qnrB gene, whereas rest of the drugs failed to inhibit conjugation. Conclusion Potentox has enhanced in vitro antibacterial activity compared to other drugs against quinolone resistant clinical isolates. 10 mM EDTA effectively prevents the conjugal transfer of qnrB gene from donor to recipient. When the same concentration of EDTA was used as a solvent for Potentox, it has again inhibited the conjugal transfer of qnrB gene from donor to recipient. Therefore, inhibition of conjugation can be a novel antimicrobial approach to combat spreading of antibiotic resistance which can be achieved only with Potentox the novel antibiotic adjuvant entity (US patent - US8178501B2).

Microbial competition during selective enrichment negatively affects Listeria monocytogenes populations and may hinder the subsequent detection or recovery of this organism. Competition assays among 10 selected strains of Listeria and Citrobacter braakii were performed in buffered Listeria enrichment broth, 3-(N-morpholino)propanesulfonic acid-buffered Listeria enrichment broth, University of Vermont medium-modified Listeria enrichment broth, and Fraser broth. The individual contributions of each selective agent in these media were also assessed, as well as the contribution of incubation temperature. Acriflavine hydrochloride and sodium nalidixate were ineffective at preventing the overgrowth of C. braakii ; this resulted in substantially lower populations of Listeria than when the competitor was absent. At the higher levels, both of these selective agents were detrimental to Listeria populations. The highest enrichment populations of Listeria were observed when either NaCl or LiCl was present. In the absence of selective agents, the final populations of Listeria following competitive growth with C. braakii were not substantially affected by temperature; however, in the presence of selective agents, the Listeria populations were statistically higher at the higher incubation temperature. There are a limited number of selective agents available for use in Listeria -specific enrichment media, resulting in formulations that are only somewhat selective for this species. The optimization of current formulations may help researchers to improve Listeria recovery, particularly from products with a high microbial load. The understanding of the behavior and interactions between target and nontarget microorganisms in the presence of these available selective agents is a necessary step in the optimization of Listeria selective enrichment formulations.