A bacteriophage against Citrobacter braakii and its synergistic effect with antibiotics.

A bacteriophage BD49 specific for Citrobacter braakii was screened out and purified by double-layer plate method. It consists of a polyhedral head of 93.1 ± 1.2 nm long and 72.9 ± 4.2 nm wide, tail fibers, collar, sheath and baseplate. The bacteriophage was identified by morphology observed with transmission electron microscope (TEM), whole genome sequencing carried out by Illumina next generation sequencing (NGS) technique, and gene annotation based on Clusters of Orthologous Groups of proteins (COG) database. It was identified primarily as a member of Caudovirales by morphology and further determined as Caudovirales, Myoviridae, and Citrobacter bacteriophage by alignment of its whole genome sequence with the NCBI database and establishment of phylogenetic tree. The bacteriophage showed good environmental suitability with optimal multiplicity of infection (MOI) of 0.01, proliferation time of 80 min, optimum living temperature of 30-40 °C, and living pH of 5-10. In addition, it exhibited synergistic effect with ciprofloxacin against C. braakii in antibacterial tests.

Development of aptamers for rapid airborne bacteria detection.

Airborne microbes can rapidly spread and cause various infectious diseases worldwide. This necessitates the determination of a fast and highly sensitive detection method. There have been no studies on receptors targeting Citrobacter braakii (C. braakii), a pathogenic bacterium which can exist in the air. In this study, we rapidly isolate an aptamer, a nucleic acid molecule that can specifically bind to C. braakii by centrifugation-based partitioning method (CBPM) reported previously by our groups as omitting the repeated rounds of binding incubation, separation, and amplification that are indispensable for SELEX. The binding affinity and specificity of isolated aptamers are checked using bacteria in liquid culture and recollection solution from aerosolized bacteria. Recollection solutions of the recovered bacteria are obtained by nebulizing, drying, and recapturing with a biosampler. The CB-5 aptamer shows high affinity and specificity for C. braakii (Kd: 16.42 in liquid culture and 26.91 nM in recollection from aerosolized sample). Our results indicate the current protocol can be employed for the rapid development of reliable diagnostic receptors targeting airborne bacteria.

Citrobacter braakii CLABSI in a hematopoietic stem cell transplant patient.

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.

[Septic shock due to Citrobacter braakii following high-dose cytosine arabinoside therapy in a patient with acute myeloid leukemia].

Here, we report a case of a 67-year-old man who had septic shock due to Citrobacter braakii infection during the course of chemotherapy with high-dose cytosine arabinoside for acute myeloid leukemia. Treatment with cefepime rapidly improved his condition. The number of reported cases of sepsis due to Citrobacter braakii is limited. Further accumulation of cases is necessary to obtain accurate data such as the risk factors for Citrobacter braakii infections.

Citrobacter braakii bacteremia-induced septic shock after colonoscopy preparation with polyethylene glycol in a critically ill patient: a case report.

BACKGROUND: Polyethylene glycol (PEG) is widely used for bowel cleaning in preparation for colonoscopy because of its safety. Septic shock after PEG preparation is an extremely rare complication. Herein, we describe a case of septic shock that occurred immediately after colonoscopy preparation with PEG. CASE PRESENTATION: A 75-year-old Japanese male who had previously developed diabetes after total pancreatectomy received PEG in preparation for colonoscopy. He had been admitted to the emergency intensive care unit 4 days earlier due to hematochezia presenting with shock. He ingested PEG to prepare for a colonoscopy examination, which was performed to identify the source of his bleeding over a 5-h period, but suddenly exhibited septic shock and markedly elevated procalcitonin levels. A blood culture subsequently revealed Citrobacter braakii. Immediate resuscitation and intensive care with appropriate antibiotics improved his condition. CONCLUSIONS: Clinicians should be aware of the possibility of deteriorating conditions after bowel preparation with PEG among severely ill patients with recent episodes of hemorrhagic shock.

Bacteremia due to Citrobacter braakii: A case report and literature review.

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.

Characterization of cytotoxic Citrobacter braakii isolated from human stomach.

Citrobacter braakii (C. braakii) is an anaerobic, gram-negative bacterium that has been isolated from the environment, food, and humans. Infection by C. braakii has been associated with acute mucosal inflammation in the intestine, respiratory tract, and urinary tract. However, the pathogenesis of C. braakii in the gastric mucosa has not yet been clarified. In this study, the bacterium was detected in 35.5% (61/172) of patients with chronic gastritis (CG) and was closely associated with the severity of mucosal inflammation. Citrobacter braakii P1 isolated from a patient with CG exhibited urease activity and acid resistance. It contained multiple secretion systems, including a complete type I secretion system (T1SS), T5aSS and T6SS. We then predicted the potential pilus-related adhesins. Citrobacter braakii P1 diffusely adhered to AGS cells and significantly increased lactate dehydrogenase (LDH) release; the adhesion rate and LDH release were much lower in HEp-2 cells. Strain P1 also induced markedly increased mRNA and protein expression of IL-8 and TNF-α in AGS cells, and the fold increase was much higher than that in HEp-2 cells. Our results demonstrate proinflammatory and cytotoxic role of C. braakii in gastric epithelial cells, indicating the bacterium is potentially involved in inducing gastric mucosa inflammation.

Quality changes, potential spoilage organisms, and shelf-life prediction of brackish river prawn (Macrobrachium macrobrachion) at different storage temperatures.

The brackish river prawn (Macrobrachium macrobrachion) is a species of commercial importance in West Africa. However, like other fishery products, it is prone to deterioration due mainly to microbial activities. The present study aimed at evaluating the spoilage characteristics of M. macrobrachion and predicting the growth of the main spoilage bacteria as well as the shelf-life of the product as a function of storage temperature. Freshly caught brackish river prawn samples from Lake Aheme were aerobically stored at 0, 7, 15, and 28 °C and, at pre-determined times during storage, they were taken for microbiological, chemical, and sensory analysis. At sensory rejection times, the spoilage potential of 185 isolates from specific groups of organisms enumerated was assessed in prawn of which the endogenous microbiota was heat inactivated. Isolates capable of producing strong off-odor were identified using 16S rRNA sequencing. Models predicting the maximum growth rate of Pseudomonas spp. and H2S-producing bacteria in the brackish river prawn as well as the shelf-life of the product were developed. These models were validated using an independent experiment during which prawn was stored at 0, 4, 10, and 25 °C. Results showed that Pseudomonas spp. at 0 °C, Pseudomonas spp. and H2S-producing bacteria at 7 °C, and H2S-producing bacteria at 15 °C and 28 °C were the dominant groups of microorganisms during storage. As expected, total volatile basic nitrogen, trimethylamine, and pH with initial values of 21.2 ± 3.0 mg-N/100 g, 4.1 ± 0.8 mg-N/100 g, and 7.46 ± 0.15 increased during storage reaching approximately 35 mg-N/100 g, 10 mg/ 100 g and 8, respectively at sensory rejection times which were 7 h at 28 °C, 1.2 d at 15 °C, 4.6 d at 7 °C, and 11.7 d at 0 °C. The main spoilage organisms were Citrobacter braakii at 28 °C, Citrobacter braakii, Pseudomonas kurunegalensis, and Shewanella bicestrii at 15 °C, Shewanella putrefaciens, Shewanella baltica, and Pseudomonas bubulae at 7 °C, and Pseudomonas versuta at 0 °C. The validation of the developed models showed an adequate agreement between the predicted and observed values. This study highlights the specific spoilage characteristics of the brackish river prawn and reveals that Gram-negative rod bacteria are the main spoilage organisms even at high storage temperatures, contrary to many earlier reports on the spoilage of tropical fishery products.

The Bacteriophage vB_CbrM_HP1 Protects Crucian Carp Against Citrobacter braakii Infection.

Citrobacter braakii is an opportunistic pathogen that induces aquatic infections in fish and turtles. In this study, a bacteriophage that infects C. braakii, named vB_CbrM_HP1, was isolated from sewage. This phage belongs to Myoviridae family, Ounavirinae subfamily, Mooglevirus genus. We also used the phage to treat crucian carp infection caused by C. braakii for the first time. vB_CbrM_HP1 was relatively stable at temperatures ranging from 4 to 60°C and pH values ranging from 3 to 11 but float slightly. When the multiplicities of infection (MOI) was 0.0001, the titer reached a maximum of 4.20 × 1010 PFU/ml. As revealed from the results of whole genomic sequence analysis, the total length of vB_CbrM_HP1 was 89335 bp, encoding 135 ORFs, 9 of which were <75% similar to the known sequences in NCBI. The phage vB_CbrM_HP1 showed a highly efficient bactericidal effect against C. braakii both in vitro and in vivo. In vitro, vB_CbrM_HP1 was capable of effectively killing bacteria (the colony count decreased by 4.7 log units at 5 h). In vivo, administration of vB_CbrM_HP1 (1 × 109 PFU) effectively protected crucian carp against fatal infection caused by C. braakii. Phage treatment reduced the levels of inflammatory factors. All these results demonstrated the potential of vB_CbrM_HP1 as an alternative treatment strategy for infections caused by C. braakii.

Co-Production of NDM-1 and OXA-10 ß-Lactamase in Citrobacter braakii Strain Causing Urinary Tract Infection.

In this study, we describe, for the first time, the co-existence of bla NDM-1and bla OXA-10 in a carbapenem-resistant Citrobacter braakii strain DY2019 isolated from a patient with urinary tract infection in China. We aimed to investigate the genomic context of two ß-lactamase-producing plasmids and characterize the transmission mechanism of the carbapenemase-encoding gene. Whole-genome sequencing of strain DY2019 was performed with Nanopore and Illumina platforms, which revealed a chromosome sequence with the length of 4,830,928 bp, an IncC group plasmid pDY2019-OXA (size of 178,134 bp), and a novel IncHI2 group plasmid pDY2019-NDM (length 348,495 bp). A total of 16 antimicrobial resistance genes (ARGs) that confer resistance to nine different antibiotic groups were identified in strain DY2019, and 11 of them were carried by plasmid pDY2019-OXA. These data and analyses suggest that the carbapenem-resistant C. braakii strains may serve as potential reservoir of carbapenemase and highlight the need for further close surveillance of this species in clinical settings.

Citrobacter braakii Yield False-Positive Identification as Salmonella, a Note of Caution.

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.

Green nail syndrome caused by Citrobacter braakii.

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.

Novel IncR/IncP6 Hybrid Plasmid pCRE3-KPC Recovered from a Clinical KPC-2-Producing Citrobacter braakii Isolate.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have become widespread in hospitals and the environment. Here, we describe a blaKPC-2-carrying plasmid called pCRE3-KPC, which was recovered from a clinical multidrug-resistant Citrobacter braakii CRE3 strain in China. The complete nucleotide sequence of pCRE3-KPC was determined by combining MiSeq and MinION sequencing and then compared with those of three related plasmids. Plasmid conjugal transfer and electroporation tests, modified carbapenem inactivation method, and bacterial antimicrobial susceptibility test were carried out. We compared this plasmid with three related plasmids to verify that the backbone of pCRE3-KPC was composed of the backbones of the IncR plasmid and IncP6 plasmid. Further bioinformatics analysis showed that pCRE3-KPC carried two resistance-related regions (the blaKPC-2 gene cluster and the aacC2-tmrB-related region). The aacC2-tmrB-related region included two novel insertion sequences (ISCfr28 and ISCfr16).IMPORTANCE Reports of human-pathogenic C. braakii strains, especially of strains showing resistance to carbapenems, are rare. To the best of our knowledge, our results represent the first detection of carbapenemase gene blaKPC-2 in C. braakii strains. In addition, we have studied detailed genetic characteristics of the novel IncR/IncP6 hybrid plasmid pCRE3-KPC, which was isolated from a clinical multidrug-resistant Citrobacter braakii CRE3 strain. Our results may provide further insight into the horizontal transfer of multidrug resistance genes in bacteria and into the genomic diversity and molecular evolution of plasmids.

Evaluation of a new generation phytase on phytate phosphorus release for egg production and tibia strength in hens fed a corn-soybean meal diet.

To test the effect of several inclusion levels of Citrobacter braakii phytase (CBP), on phytate P release, 420 50-wk-old-Bovans White hens were randomly allocated to 7 treatments with 5 replicates of 12 hens each. The experimental period lasted 12 weeks, first 8 for adaptation and last 4 for data collection. Feed and water were provided ad libitum. Treatments were: (1) a 0.12% basal corn-soybean meal diet deficient only in non-phytate P. Treatments 2 and 3 were added with constant increases of 0.11% inorganic P, to get a linear hen response to P addition. Treatments 4 to 7 were the addition of 300; 600; 1,200; and 1,800 phytase units (FYT)/kg to the basal diet. Variables analyzed were hen productive performance (HPP) and tibia resistance to fracture (TRF), and mineral content. Data were analyzed as a Complete Randomized Design (CRD). The results from treatments 1 to 3 were analyzed by a regression model to test for a significant linear response (P < 0.05). Then for every level of CBP added (treatments 4 to 7), the linear regression equation was solved to find out the equivalent value of released P. Based on hen health and welfare, the response variables that yielded realistic P equivalence values for the CBP levels used in the present trial were the tibia data. Following the significant (P < 0.001) linear response, the equations; TRF, kg (Y = 28.16X + 17.42 R2 = 0.84); Tibia Ca, % (Y = 11.6X + 14.2 R2 = 0.80); Tibia P, % (Y = 11.6X + 6.1 R2 = 0.81); and T ash, % (Y = 33.3X + 38.1 R2 = 0.80). Under the experimental conditions of this trial, the HPP variables were not a sensitive parameter to measure P release; whereas, tibia parameters showed the following average P release values per level of CBP inclusion in the corn- soybean meal diet; 300 FYT/kg = 0.099%, 600 FYT/kg = 0.141%, 1,200 FYT/kg = 0.182%, and 1,800 FYT/kg = 0.198%.

Isolation of an IncP-1 plasmid harbouring mcr-1 from a chicken isolate of Citrobacter braakii in China.

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.

A Case of Citrobacter braakii Sepsis Complicated with Hepatocellular Carcinoma / 감염과화학요법

Citrobacter species is a gram-negative bacilli that can cause opportunistic infections in immunocompromised hosts. Citrobacter braakii refers to the genomospecies 6 of the Citrobacter freundii complex. There are no detailed studies on infections caused by this newly identified specific genetic species in Korea. We herein report a case of a patient with hepatocellular carcinoma who, after undergoing transcatheter arterial chemoembolization, developed biloma which later progressed to C.braakii sepsis and did not respond to treatment. To our knowledge, this is the first reported case in Korea on C. braakii infection resulting in septic shock in a patient with malignancy in Korea.