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This study aims to analyze the contamination and biofilm formation of foodborne and opportunistic pathogens in yellow-feathered chicken carcasses sampled in different seasons and to prove the relationship between biofilm-forming ability and bacterial extracellular polysaccharide (EPS) production. A total of 78 strains were isolated from chicken samples. The strains consisted of 30.8% Escherichia coli, 14.1% Pseudomonas aeruginosa, 12.8% Salmonella enteritidis, 12.8% Klebsiella pnenmoniae, 10.2% Enterobacter cloacae, 8.9% Proteus mirabilis, 5.1% Klebsiella oxytoca, 1.3% Staphylococcus aureus, and 1.3% Citrobacter braakii. Crystal violet staining assay revealed six strains with strong biofilm-forming ability, namely, E. coli S7, K. oxytoca B12, K. pnenmoniae B6, S. enteritidis H4, P. aeruginosa M5, and S. aureus G1, which showed had high abilities of cell motility and EPS production. Confocal laser scanning microscopy and scanning electron microscopy showed that all six strains can form mature biofilm architectures after 5 d of cultivation. This study may serve as a reference to control the contamination of foodborne pathogens in yellow-feathered chicken and enhance the quality and shelf life of these chicken products.

Introduction Citrobacter bacteria are gram-negative anaerobic bacilli commonly found in water, soil, food, and the intestinal tracts of animals and humans. Patients at highest risk of these nosocomial infections include neonates and adults who are ≥65 years of age, debilitated, or immunocompromised. Citrobacter braakii ( C. braakii), specifically, has been reported to cause sepsis in immunocompromised patients. Herein, we describe a case of an allogeneic stem cell transplant (SCT) adult patient with C. braakii bloodstream infection. Case Report We report our experience managing a central line-associated bloodstream infection (CLABSI) due to C. braakii in an allogeneic SCT patient. Management and Outcomes: Our patient was initially managed with cefepime. The central venous catheter (CVC) was removed. Blood cultures cleared 24 hours after antibiotic initiation. Therapy was transitioned to oral levofloxacin once susceptibilities resulted. Discussion The course of this patient highlights the important relationship between an unusual pathogen, C. braakii, in an immunocompromised allogeneic SCT patient. In our case, the source of the bacteremia was most likely a CLABSI given the positive catheter tip cultures. Although this report describes the successful utilization of cefepime and levofloxacin in the treatment of C. braakii infection, caution should be exercised when choosing empiric antimicrobial therapy as AmpC resistance. This clinical scenario can aid health care providers in making informed treatment decisions when faced with patients diagnosed with this relatively uncommon pathogen. Further reports should be published to determine C. braakii bacteremia management in hematopoietic stem cell transplant patients.

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.

Ying Li,1,2,* Min Tang,3,* 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*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

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.

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 plasmid-mediated colistin resistance gene mcr-1 has been found worldwide, but the diversity of organisms harbouring this gene is unknown. In this study, 12 colistin-resistant Citrobacter spp. isolates were obtained from diseased or dead chickens in China, and PCR analysis indicated that five were positive for mcr-1. One Citrobacter braakii strain (SCC4) with a multidrug-resistant phenotype was chosen for further analysis. SCC4 was resistant or intermediate-resistant to ten of the tested antibiotics, and the colistin minimum inhibitory concentration (MIC) was >4 µg/mL. A conjugation assay demonstrated successful transfer of colistin resistance to Escherichia coli strain J53 at a frequency of 10–7 cells per recipient cell. Whole-genome sequencing revealed that SCC4 contained 13 antibiotic resistance genes in its genome, and the mcr-1 gene resided on a 44-kb self-transmissible IncP-type plasmid of a recently discovered IncP-1 clade. In addition, the mcr-1 gene was part of an insertion element (ISApl1–mcr-1–orf–ISApl1) that was excised from the plasmid as a circular intermediate form. This is the first report of mcr-1-posiitve C. braakii of animal origin and these findings highlight the fact that the mcr-1 gene can be found in normal enteric flora as part of broad-host-range plasmids.

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).

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.

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.

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.

Domestic ruminants are regarded as the major reservoir of Shiga toxin-producing Escherichia coli (STEC) closely related to human infection. A total of 363 ovine carcasses were swabbed in an Algiers city slaughterhouse for research on STEC. First of all, screening of the STECs was carried out by a multiplex PCR searching for the genes coding for the virulence factors stx1 , stx2 and eae. This step was followed by STEC isolation and serotyping. The presence of stx+ /stx+ eae+ genes was shown in 116 sheep carcasses (31·95%). From the 116 positive samples, 20 bacterial strains (17·24%) were isolated. Nineteen strains belonged to the species E. coli (STEC), and 1 belonged to Citrobacter braakii (eae+ stx1 + ). During this study, the presence of potentially pathogenic STEC for humans on the surface of sheep carcasses was confirmed. Corrective measures should be considered at the slaughterhouse level to avoid outbreaks of STEC in Algeria. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR screening revealed the significant presence of the genetic markers of Shiga toxin-producing Escherichia coli (STEC) (stx+ /stx+ eae+ ) on the surfaces of sheep carcasses. Citrobacter braakii (stx1 + eae+ ) was isolated for the first time in this study. The risk of foodborne diseases due to STEC must be taken into account in Algeria. To prevent the emergence of epidemic outbreaks among children and older by people, preventive measures should be taken.

Fresh vegetables are a frequent cause of foodborne disease outbreaks because they often do not undergo heat treatment prior to consumption. Pathogenic Escherichia coli strains are the major causative agents of produce-related outbreaks. In this study, we investigated the efficiency of conventional E. coli selective medium, MacConkey (MAC) agar, for the screening of fresh vegetable samples and evaluated the discrimination ability of Levine's eosin methylene blue (L-EMB) agar for E. coli-like colonies obtained from MAC agar. A total of 120 samples of lettuces (n = 60) and radish sprouts (n = 60) were analyzed. Among 17 (14.2%) MAC agar plates containing putative E. coli colonies, only one plate was confirmed E. coli-positive (positive predictive value = 5.9%). All 16 false-positive isolates were identified as Citrobacter braakii and formed E. coli-like colonies on L-EMB agar. These results indicate that in order to reduce false-positive results in screening fresh vegetable samples, both MAC and L-EMB selective agars should be modified with the aim of providing reliable differentiation between E. coli and C. braakii. Practical Applications Although MacConkey (MAC) and Levine's eosin methylene blue (L-EMB) agars are not specific for the detection of pathogenic Escherichia coli, the media are recommended for the isolation of pathogenic E. coli (except E. coli O157:H7) in the Food and Drug Administration Bacteriological Analytical Manual. This is due to the fact that it is difficult to develop each pathogenic strains-specific selective media as they were not classified by their biochemical characteristics. However, the unexpected high prevalence of Citrobacter braakii in fresh vegetables observed in this study indicates that the screening potential of these E. coli selective media needs to be improved.

Among the Citrobacter genus, the most commonly isolated bacteria from human specimens are Citrobacter freundii and Citrobacter koseri, and previous cases of infection due to Citrobacter braakii have been rarely reported. We present a case of bacteremia due to C. braakii in a 38-year-old woman with cervical cancer. She was admitted to our hospital with complaints of a fever, chills, and nausea. Blood culture results showed gram-negative bacilli identified as C. braakii via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, although biochemical testing findings were suggestive of C. freundii. Since a rare pathogen was detected in the present case and the results of additional biochemical studies were suggestive of both C. braakii and Citrobacter farmeri, genetic analysis was conducted. Finally, the gram-negative bacilli were confirmed as C. braakii, a member of the C. freundii complex since 1993, by 16S ribosomal RNA gene sequencing analysis. The gastrointestinal tract was considered the portal of entry, because the patient had a rectal fistula and other cultures such as urine and vaginal discharge incubated species other than C. braakii. The patient recovered after receiving treatment with ciprofloxacin for 14 days. The epidemiology and clinical characteristics of C. braakii infection are still unknown because of the limitations in accurate identification by using currently available commercial biochemical testing and previously, only 6 cases of C. braakii infection have been reported. Physicians should focus on this species, because it causes community-acquired infections, although further studies are needed to clarify the clinical characteristics of C. braakii infections.

International audience; The spread of carbapenemase-producing Enterobacteriaceae (CPE) is a significant problem for healthcare worldwide. The prevalence of carbapenem-resistant Enterobacteriaceae (CPE) in water environments in Algeria are unknown. The aim of this study was to screen for the presence of CPE isolates in the Soummam River in Bejaia, Algeria. Isolates of Enterobacteriaceae recovered from twelve samples of river water and showing reduced susceptibility to carbapenems were included in this study. The isolates were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Isolates were subjected to antimicrobial susceptibility testing and the modified Carba NP test. Carbapenemase and extendedspectrum b-lactamase (ESBL) determinants were studied by PCR amplification and sequencing. The clonal relatedness between isolates was studied by Multilocus Sequence Typing (MLST) method. A total of 20 carbapenem-resistant Enterobacteriaceae strains were included in this study, identified as Escherichia coli (n = 12), Klebsiella pneumoniae (n = 3), Raoultella ornithinolytica (n = 3), Citrobacter freundii (n = 1) and Citrobacter braakii (n = 1). Carbapenemase genes identified in this study included blaOXA-48, observed in 17 isolates (9 E. coli, 3 K. pneumoniae, 3 R. ornithinolytica, 1 C. freundii and 1 C. braakii), and blaOXA-244, a variant of blaOXA-48, was found in three E. coli isolates. MLST showed that 12 E. coli strains belonged to six different sequence types (ST559, ST38, ST212, ST3541, 1972 and ST2142), and we identified three different STs in K. pneumoniae isolates, including ST133, ST2055, and a new sequence type: ST2192. This study showed the presence of OXA-48-like-producing Enterobacteriaceae in water environments and highlighted the potential role of aquatic environments as reservoirs of clinically relevant antimicrobial-resistant bacteria, with the potential to spread throughout the community. (C) 2017 Elsevier Ltd. All rights reserved.

New Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae have become a threat to public health. Hospital sewage is generally unexplored, having the potential to harbour bacteria causing healthcare-associated infections. Hence, this study was initiated to monitor NDM-producing Enterobacteriaceae in hospital sewage water. A total of 32 isolates with blaNDM variants were detected in hospital sewage water, including 17 Escherichia coli, 8 Citrobacter freundii, 4 Shigella boydii, 2 Citrobacter braakii and 1 Citrobacter farmeri, showing resistance to all antibiotics except colistin. All 32 isolates carried blaNDM (9 blaNDM-1, 11 blaNDM-4, 10 blaNDM-5 and 2 blaNDM-7), 24 isolates carried blaCMY variants (1 blaCMY-2, 3 blaCMY-4, 5 blaCMY-6, 11 blaCMY-42, 2 blaCMY-86 and 2 blaCMY-139), 20 isolates carried blaOXA-type (17 blaOXA-1 and 3 blaOXA-48), 19 isolates carried blaCTX-M and 9 isolates carried ampC on conjugative plasmids of IncFIA, IncFIB, IncFIC, IncP, IncY, IncHI1 and IncI1 types. In E. coli, coexistence of blaNDM-1 with blaCMY-6 and blaCMY-139, of blaNDM-4 with blaCMY-6, blaCMY-42 and blaCMY-86, of blaNDM-5 with blaCMY-6 and blaCMY-42, and of blaNDM-7 with blaCMY-6 was observed. NDM-5-producing S. boydii and NDM-7-producing C. freundii were identified as well as detection of an association of blaNDM-4 and blaOXA-48 in C. braakii and C. farmeri. A class 1 integron was also found on a plasmid. ISAba125 and bleomycin genes were found surrounding all blaNDM variants. The emergence and dissemination of blaNDM variants in hospital sewage water is a matter of concern, creating an endemic scenario leading to the level of an outbreak.

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.

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

Bacterial growth and histamine formation in mackerel during storage at 0, 4, 15 and 25C were monitored. Histamine production and bacterial multiplication were accelerated with increase of the storage temperature. The highest histamine level of 2,080 mg/kg was detected after 2 days of storage at 25C. Selective media were used for isolation of histamine-producing bacteria (HPB) and the histamine production was confirmed by high-performance liquid chromatography analysis. A total number of 305 strains were isolated from spoiled mackerel and 32 of them were identified as HPB. Most of the HPB were isolated from violet red bile glucose agar. Morganella morganii, Citrobacter braakii, Hafnia alvei and Raoultella planticola were confirmed as high histamine-producing strains, whereas other microorganisms were found to possess weak histamine-producing ability. Practical Applications In order to effectively inhibit the activity of HPB, it is necessary to have a good understanding of bacterial species contributing to histamine production in mackerel during storage. Therefore, we studied the variation of histamine content and the growth of bacteria related to histamine production in mackerel during storage. Results of this study could be useful to assure the safety of mackerel products by controlling histamine content.

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.

Continuous European Union programmes with specified methods for enumeration of Escherichia coli in bivalves for human consumption are currently running. The objective of this research was to examine the species accuracy of the five times three tube Most Probable Number (MPN) EU reference method used for detection of E. coli in marine bivalves. Among 549 samples of bivalves harvested from Norwegian localities during 2014 and 2015, a total number of 200 bacterial isolates were prepared from randomly selected culture-positive bivalves. These presumptive E. coli isolates were characterized biochemically by the Analytical Profile Index (API) 20E, as well as by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). The majority of isolates (90%) were identified as E. coli, by both API 20E and MALDI-TOF MS. Ten isolates (5%) were identified as Klebsiella pneumoniae, while one isolate was identified as K. oxytoca by both methods, whereas three isolates were identified as Acinetobacter baumannii, Citrobacter braakii, and Enterobacter cloacae, respectively. The identification of the remaining six isolates were not in compliance between the two methods.

Bacteremia is frequently caused by gram-positive organisms such as Staphylococcus aureus or β hemolytic streptococci. While there is adequate information for the diagnosis and management of these common bacteria, less information is available to address bacteremia that is caused by uncommon bacteria such as Citrobacter. Citrobacter species are gram-negative bacilli that have been noted to cause infections in immune compromised patients. The re-speciation of Citrobacter by its varied genetic composition has produced 11 distinct species. While C. braakii is part of the genome species 6 of the previous Citrobacter freundii complex, there is a lack of definitive research on the subject of Citrobacter bacteremia caused by lesser- known species. We report an unusual case of Citrobacter braakii bacteremia in a patient with multiple comorbidities that presented to the hospital with cellulitis and pleural effusion. Blood cultures grew Citrobacter braakii. Despite good response to antibiotic treatment, the patient's infection proved to be persistent and he succumbed to the cirrhosis of the liver and subsequent renal failure. Multiple and fatal underlying disease processes seem to worsen the likelihood of recovery from unusual infections. We believe our case report can add to the limited data available for C. braakii.

The aim of this study was to isolate, identify and determine the ability of potential pathogenic bacteria hemolytic mite Disco ( Symphysodon discus ) and Neon Rainbow ( Melanotaenia praecox ), and define which of the antibiotic Trimethoprim, Florfenicol, Chloramphenicol, Norfloxacin, Erythromycin, Bacitracin, Enrofloxacin, Tetracycline, and Furazolidone Clindamycin has better inhibitory effect in vitro, and determine the minimum inhibitory concentration (MIC) for Chloramphenicol, Norfloxacin, Erythromycin and Enrofloxacin. In an outbreak of ornamental fish deaths were isolated nine strains of Gram negative, hemolytic where 3 were identified, two as Vibrio cholerae and one as Citrobacter braakii. With these strains were performed antibiogram and was determined the minimum inhibitory concentration at two different temperatures (22 and 30 ° C). Antibiotics Enrofloxacin, Norfloxacin, florfenicol, chloramphenicol and trimethoprim showed the best results in vitro inhibition against the Vibrio cholerae, and Citrobacter braakii. The MIC increased in 30 ° C for erythromycin and enrofloxacin against pathogens, while for Florfenicol and norfloxacin the MIC was not influenced by temperature.

Histidine, lysine, ornithine and tyrosine decarboxylase activities were tested in 79 strains of Enterobacteriaceae (41 of Hafnia alvei, 17 of Serratia liquefaciens, 5 of Enterobacter cloacae, 4 of Citrobacter braakii, 2 of Proteus vulgaris, 2 of Proteus mirabilis, 2 of Providencia stuartii, 2 of Klebsiella terrigena, 1 of Rahnella aquatilis, 1 of Salmonella arizonae, 1 of Citrobacter youngae and 1 of Escherichia coli) isolated from Botillo, a Spanish traditional sausage. In general, the strains were positive for all four activities, with the exception of two strains of H. alvei and the E. coli strain, which did not display histidine decarboxylase activity. The strains of P. mirabilis and P. stuartii did not exhibit any of the four activities tested. Accumulation of putrescine and cadaverine was studied throughout growth of the 75 strains that displayed ornithine and lysine decarboxylase activities. Biogenic amines were produced particularly in the exponential phase, with maximum accumulation occurring after between 12 to 72 h, depending on the biogenic amine and microbial species considered. Maximum accumulation of putrescine varied greatly between species and within the same species, and ranged from 18 mg/l in the R. aquatilis strain to 7325 mg/l in a H. alvei strain. Maximum accumulation of cadaverine varied less than that of putrescine, and ranged from 30 mg/l in the R. aquatilis strain to 1935 mg/l in a S. liquefaciens strain.

Rhizosphere microorganisms possessing phytase activity are considered important for rendering phytate-phosphorus (P) available to plants. In the present study, the Citrobacter braakii phytase gene (appA) was overexpressed in rhizobacteria possessing plant growth promoting (PGP) traits, for increasing their potential as bioinoculants. AppA was cloned under the lac promoter in the broadhost-range expression vector pBBR1MCS-2. Transformation of the recombinant construct pCBappA resulted in high constitutive phytase activity in all of the eight rhizobacterial strains belonging to genera Pantoea, Citrobacter, Enterobacter, Pseudomonas (two strains), Rhizobium (two strains), and Ensifer that were studied. Transgenic rhizobacterial strains were found to display varying levels of phytase activity, ranging from 10-folds to 538-folds higher than the corresponding control strains. The transgenic derivative of Pseudomonas fluorescens CHA0, a well-characterized plant growth promoting rhizobacterium, showed the highest expression of phytase (~8 U/mg) activity in crude extracts. Although all transformants showed high phytase activity, rhizobacteria having the ability to secrete organic acid showed significantly higher release of P from Caphytate in buffered minimal media. AppA overexpressing rhizobacteria showed increased P content, and dry weight (shoot) or shoot/ root ratio of mung bean (Vigna radiata) plants, to different extents, when grown in semisolid agar (SSA) medium containing Na-phytate or Ca-phytate as the P sources. This is the first report of the overexpression of phytase in rhizobacterial strains and its exploitation for plant growth enhancement.

Aims: To determine whether American alligators (Alligator mississippiensis) are an unrecognized poikilothermic source of faecal coliform and/or potential pathogenic bacteria in South Carolina’s coastal waters. Methods and Results: Bacteria from the cloaca of American alligators, as well as bacteria from surface water samples from their aquatic habitat, were isolated and identified. The predominant enteric bacteria identified from alligator samples using biochemical tests included Aeromonas hydrophila, Citrobacter braakii, Edwardsiella tarda, Escherichia coli, Enterobacter cloacae, Plesiomonas shigelloides and putative Salmonella, and these were similar to bacteria isolated from the surface waters in which the alligators inhabited. Based on most-probable-number enumeration estimates from captive alligator faeces, faecal coliform bacteria numbered 8·0 × 109 g−1 (wet weight) of alligator faecal material, a much higher concentration than many other documented endothermic animal sources. Conclusions: A prevalence of enteric bacteria, both faecal coliforms and potential pathogens, was observed in American alligators. The high faecal coliform bacterial density of alligator faeces may suggest that alligators are a potential source of bacterial contamination in South Carolina coastal waters. Significance and Impact of the Study: These findings help to increase our understanding of faecal coliform and potential pathogenic bacteria from poikilothermic reptilian sources, as there is the potential for these sources to raise bacterial water quality levels above regulatory thresholds.

The efficacy of the agents was evaluated on the basis of minimum inhibitory concentration (MIC) and time-kill curve analysis in Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, Mycobacterium smegmatis, and Citrobacter braakii; MIC was found to be 0.5, 4.0, 0.015625, 0.0078125, and 0.0625 mg/L in FDC of ceftazidime + sulbactam, respectively, which is lower than ceftazidime and sulbactam individually. Time-kill curve analysis demonstrated maximum killing of bacteria after 4 h. A fixed dose combination of ceftazidime + sulbactam was found to have stronger antibacterial properties than ceftazidime and sulbactam alone at in vitro analysis.

Citrobacter braakii produced an intracellular acid glucose phosphatase (AgpC) which was purified 986 fold to homogeneity with the specific activity of 286 units/mg. AgpC hydrolyzed a wide variety of phosphorylated compounds with high activity for glucose-1-phosphate and glucose-6-phosphate. The optimum pH and temperature for the enzyme activity was pH 5.0 and 45 degrees C, respectively. The Km value for glucose-1-phosphate was 5.12 mM with a Vmax 27.8 U mg(-1). Its molecular weight was 46 kDa by SDS-PAGE gel and the sequence of N-terminal amino acid residues identified was Gln-Thr-Ala-Pro-Glu-Gly-Tyr-Gln-Leu-Gln. The glucose-1-phosphatase gene (agpC) was cloned from the C. braakii genomic library. This gene comprised 1,242 nucleotides and encoded a polypeptide of 413 amino acids. The result of its BLAST search showed a significant similarity with glucose-1-phosphatase from enterobacteria such as E. coli, Enterobacter, Shigella, and Salmonella.

In this study, we investigated the differences in histamine accumulation between blue scad and chub mackerel and methods of inhibiting histamine-forming bacteria and controlling histamine accumulation in fish. The free histidine contents in blue scad and chub mackerel were 1.45 and 2.75 mg/g, respectively. The histamine-forming bacteria isolated from them were identified as Citrobacter freundii, Citrobacter braakii, and Enterobacter aerogenes using 16S rDNA sequence analysis, the VITEK 2 Compact system, and MALDI-TOF MS. The histamine-producing capacities of C. freundii, C. braakii, and E. aerogenes were 470, 1,057, and 4,213 mg/liter, respectively, after culture at 37°C for 48 h. Among the different antimicrobials and preservatives tested, potassium sorbate and sodium diacetate effectively inhibited the histamine-forming bacteria and their histamine production. After chub mackerel was dipped into 0.5% potassium sorbate or sodium diacetate, its histamine content increased more slowly at room temperature. Therefore, a potassium sorbate or sodium diacetate dipping treatment could effectively control histamine accumulation in fish.

Citrobacter braakii is a Gram-negative bacterium belonging to the Enterobacteriaceae family. Here, we report 5.2- and 5.0-Mb genome assemblies for C. braakii strains GTA-CB01 and GTA-CB04, respectively.

In this study, we investigated the effects of enzyme-producing probiotic bacteria isolated from the gastrointestinal tract of rainbow trout on the growth performance, feed conversion ratio, and digestive enzyme activity, of fry (Oncorhynchus mykiss). Three isolates (G8/2013, T7/2013 and U5/2013) of candidate bacteria elicited the highest protease, lipase, and amylase activities, respectively. Isolates were identified as Aeromonas sp., Bacillus sp. and Citrobacter braakii by morphological, physiological, biochemical characterizations as well as 16S rRNA gene sequence analysis. The fry basal diet was supplemented with probiotics at varying concentrations; G8 group, Aeromonas sp. 1.72 x 108 CFU/g; U5 group, Bacillus sp. 3.01 x 108 CFU/g; T7 group, C. braakii 2.96 x 108 CFU/g and a mixed group (same bacterial concentrations), and control group (no bacteria). The rainbow trout fry were fed ad libitum in triplicate treatments with supplemented and non-supplemented probiotic diets for 70-days. The total bacterial count in the intestine was significantly higher in the mixed group (30th and 50th days) and U5 group (50th and 70th days) compared with the control group. However, there was no significant difference in weight gain, specific growth rate (SGR), feed conversion ratio (FCR), nutrient digestibility, or digestive enzyme activity among the groups.a

Citrobacter braakii (C. braakii) is widespread in nature, mainly found in human urinary tract. The current study was attempted to investigate the effect of Mr. Trivedi’s biofield treatment on C. braakii in lyophilized as well as revived state for antimicrobial susceptibility pattern, biochemical characteristics, and biotype number. Lyophilized vial of ATCC strain of C. braakii was divided into two parts, Group (Gr.) I: control and Gr. II: treated. Gr. II was further subdivided into two parts, Gr. IIA and Gr. IIB. Gr. IIA was analysed on day 10 while Gr. IIB was stored and analysed on day 159 (Study I). After retreatment on day 159, the sample (Study II) was divided into three separate tubes. First, second and third tube was analysed on day 5, 10 and 15, respectively. All experimental parameters were studied using automated MicroScan Walk-Away® system. The 16S rDNA sequencing of lyophilized treated sample was carried out to correlate the phylogenetic relationship of C. braakii with other bacterial species. The antimicrobial susceptibility and minimum inhibitory concentration showed 39.29% and 15.63% alteration respectively in treated cells of C. braakii as compared to control. Tetracycline showed improved sensitivity pattern, i.e., from resistant to susceptible after biofield treatment, with support of decreased MIC value (>8 to ≤ 4 µg/mL) by two-fold in all the treated samples as compared to the control. Biochemical reactions also showed significant (42.42%) alteration in the treated samples with respect to the control. Biotype numbers with species were substantially changed in Gr. IIA (53131052, Citrobacter freundii complex) on day 10 and in Gr. IIB, Study I (53111052; Citrobacter amalonaticus) on day 159 as compared to the control (77365776; Citrobacter braakii). Moreover, biotype numbers with species were substantially changed in Gr. IIB, Study II after retreatment on day 5 (53111042, Citrobacter amalonaticus) and (53131052; Citrobacter freundii complex) on day 10 and 15 as compared to the control. 16S rDNA analysis showed that the identified microbe as Citrobacter freundii (GenBank Accession Number: DQ517285) with 95% identity. The nearest homolog genus-species of C. braakii was found to be Citrobacter werkmanii (Accession No. AF025373). The results suggested that biofield treatment has a significant impact on C. braakii in lyophilized as well as revived state. https://www.trivedieffect.com/science/phenotyping-and-16s-rdna-analysis-after-biofield-treatment-on-citrobacter-braakii-a-urinary-pathogen https://www.omicsonline.org/open-access/phenotyping-and-16s-rdna-analysis-after-biofield-treatment-on-citrobacter-braakii-a-urinary-pathogen-2332-0672-1000130.php?aid=60954, Trivedi MK, Branton A, Trivedi D, Nayak G, Charan S, et al. (2015) Phenotyping and 16S rDNA Analysis after Biofield Treatment on Citrobacter braakii: A Urinary Pathogen. J Clin Med Genom 3: 129. doi: 10.4172/2472-128X.1000129

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.

Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL) and narrow spectrum (NSBL) beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter baumannii. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out of 13), blaGIM (2/2), blaKPC (27/27), blaNDM (5/5), blaIMP-2/4/7/8/13/14/15/16/31 (10/10), blaOXA-23 (12/13), blaOXA-40-group (7/7), blaOXA-48-group (32/33), blaOXA-51 (1/1) and blaOXA-58 (1/1). Furthermore, the test correctly identified additional beta-lactamases [blaOXA-1 (16/16), blaOXA-2 (4/4), blaOXA-9 (33/33), OXA-10 (3/3), blaOXA-51 (25/25), blaOXA-58 (2/2), CTX-M1/M15 (17/17) and blaVIM (1/1)]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4%) isolates, including Acinetobacter baumannii (28/28), Enterobacter spec. (5/5), Escherichia coli (4/4), Klebsiella pneumoniae (62/63), Klebsiella oxytoca (0/2), Pseudomonas aeruginosa (12/12), Citrobacter freundii (1/1) and Citrobacter braakii (2/2), were correctly identified by a panel of species specific probes. This assay might be easily extended, adapted and transferred to point of care platforms enabling fast surveillance, rapid detection and appropriate early treatment of infections caused by multiresistant Gram-negative bacteria.

The growth of Listeria monocytogenes during the pathogen specific enrichment of food samples can be limited by the presence of additional microorganisms that are resistant to the selective conditions being applied. If growth is severely limited and minimum post-enrichment threshold levels are not met then the presence of L. monocytogenes may go undetected. Several food products were screened for non-pathogenic commensal or spoilage microorganisms that are capable of growth under the conditions commonly used by regulatory testing laboratories to select for Listeria species. The effect of these potential competitor microorganisms on the ability to detect L. monocytogenes by several common molecular screening assays was then determined. Eight species of bacteria were isolated from foods that demonstrated the ability to grow in buffered Listeria enrichment broth under selective conditions. Growth of these competitor microorganisms during the enrichment incubation resulted in a decrease ranging from 1 to 4 logs in the 48 h population of L. monocytogenes. Three strains of L. monocytogenes representing serotypes 1/2a, 1/2b, and 4b were included in this study but no one serotype appeared to be most or least sensitive to the presence of competitor microorganisms. One additional strain of L. monocytogenes was identified as displaying minimal growth during the enrichment period in the presence of the Citrobacter braakii with the final population only reaching approximately 2.6 log CFU/ml after 48 h which was a 2 log increase over the initial population. This particular strain was subsequently shown to be difficult to detect following enrichment by an automated immunofluorescence assay and an antibody-based lateral flow device assay. In some enrichments, this strain was also difficult to detect by real-time PCR.

The antigenic scheme for the Bethesda-Ballerup group of bacteria established by West and Edwards in 1954 has continued to be applied as a serotyping scheme for Citrobacter freundii. In 1993, however, the classification of the Citrobacter was drastically revised and the species C. freundii redefined by Brenner et al. Accordingly, to judge the propriety to continuously use a single antigenic scheme for the C. freundii complex, the 90 reference strains listed in the antigenic scheme for C. freundii by West and Edwards were characterized phenotypically and specified based on the revised classification. Of these 90 strains, two strains of Hafnia alvei and one of Escherichia coli were found. Among the remaining 87 reference strains, Citrobacter youngae was the predominant species (40 strains), followed by Citrobacter braakii (25 strains), Citrobacter werkmanii (13 strains), and the unnamed Citrobacter genospecies 10 of Brenner et al (six strains). Citrobacter freundii, as redefined, accounted for only three strains and ranked behind the other four species. No overlapping with most of the 42 O-groups and 82 H-antigens was recognized between species with few exceptions. O-groups 1-9 inclusive, which were estimated to represent more than 90% of the former C.freundii strains, occurred in strains of C. youngae and C. braakii; and all nine strains of O-group 29, formerly known as the Ballerup group, were identified as C. braakii. These findings suggest that further study of the serotyping system is needed for all H2S-producing Citrobacter species.

Phytases (EC 3.1.3) are widely used in animal feed to increase the availability of phosphorus and decrease the anti nutritive effect of myo-inositol hexakisphosphate (InsP₆). The aim of this work was to investigate the stereospecific degradation of InsP₆ in vitro and in vivo by a phytase from Citrobacter braakii (C. braakii), and to study gastric survival of the phytase as well as the site of action in the gastrointestinal tract. The in vitro results showed that the C. braakii phytase belongs to the group of 6-phytases (EC 3.1.3.26). However, in approximately one out of 10 instances the phytase initiated hydrolysis at the D-3 (L-1) position, demonstrating that phytase specificity is not unambiguous. Following the main degradation pathway, InsP₆ was degraded by stepwise removal of the phosphate groups on positions 6/1/5. The stereospecificity was found to be similar under in vitro and in vivo conditions. The phytase was found to be stable in the gastric environment and to be active in the stomach and possibly also in the proximal small intestine. While InsP₄ was accumulated under in vitro conditions this was not the case in vivo, where both InsP₅ and InsP₄ were seen to be hydrolysed in the small intestine, possibly as a combined action of the C. braakii phytase and endogenous phosphatases present in the mucosa. The ability of the C. braakii phytase to focus its activity on degrading InsP₆ to InsP₄ is believed to be a favourable complement to the endogenous phosphatases.