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Drug susceptibility testing was carried out using 14 antibiotics in order to identify trends in the antibiotic tolerance of 142 strains of Enterobacteriaceae isolated from horsemeat commercially available for raw consumption (basashi). A comparison of the sensitivity to the 14 antibiotics using the 90% MIC (minimum inhibitory concentration) values (MIC90) showed the strongest tolerance to ampicillin (ABPC) at a concentration of > 128 μg/mL, followed by that to fosfomycin (FOM) at a concentration of 128 μg/mL. When the sensitivity to these antibiotics was examined for each individual genus of tested bacteria, Hafnia spp. exhibited relative tolerance to ceftazidime (CAZ) and ceftriaxone (CTRX) at a concentration of 4 μg/mL and 2 μg/mL, respectively, which was high in comparison to that observed for the other strains. Furthermore, Raoultella spp. and Serratia spp. were found to be highly resistant to tetracycline (TC) at a concentration of 128 μg/mL and 64 μg/mL, respectively. Of the 142 strains of test bacteria, 140 (98.6%) demonstrated resistance to ABPC, with the exception of Hafnia alvei and Klebsiella pneumonia. In addition, a total of eight strains (5.6%), seven Serratia marcescens strains and one Raoultella terrigena strain, were found to be resistant to TC. Furthermore, one strain of Citrobacter freundii exhibited resistance to nalidixic acid (NA), while another displayed resistance to ofloxacin (OFLX) (0.7% each), and one strain (0.7%) each of Enterobacter cloacae, Serratia marcescens and Citrobacter youngae demonstrated resistance to fosfomycin (FOM), streptomycin (SM) and kanamycin (KM), respectively. A single strain of C. freundii was found to be resistant to three antibiotics, ABPC, NA and OFLX. Resistance to two antibiotics was confirmed in 11 strains, including seven strains of S. marcescens and one strain of R. terrigena (a total of eight strains) resistant to ABPC and TC, and one strain each of C. youngae, S. marcescens and E. cloacae resistant to ABPC and KM, ABPC and SM, and ABPC and FOM, respectively. In addition, 128 strains were resistant to the single antibiotic of ABPC alone. Of the 140 strains demonstrating antibiotic resistance, 137 (97.9%) retained the conjugative R-plasmid transfer factor, excluding three strains of S. marcescens. All transfer factors were ABPC and retained by a high proportion of the bacterial groups, with one strain (100%) being resistant to three antibiotics, nine (81.8%) of the 11 strains being resistant to two antibiotics, and 127 (99.2%) of the 128 strains being resistant to a single antibiotic. In addition, we examined ESBL productivity in the 140 strains of bacteria demonstrating drug tolerance; however, no strains exhibited this characteristic. Therefore, further observation is required to ascertain trends in antibiotic-tolerant bacteria.

Raoultella ornitholytica, an environmental organism, has only recently been reported as a cause of infections in humans, and is associated mainly with bacteraemia in elderly patients. Carbapenem resistance is unusual in Raoultella spp. We describe the first paediatric case of blaNDM-1 Raoultella ornitholytica bacteraemia in a preterm neonate that developed three days after hospitalisation. This case report discusses a rare organism that had an unexpected susceptibility profile and caused bacteraemia in a neonate. Treatment options are explored.

Rationale: Contact lens storage cases are known to be contaminated by a significant number of bacteria. However, histamine-producing Raoultella species has not been reported to contaminate contact lens storage case. Patient concerns: A 27-year-old woman with keratoconjunctivitis that developed in the left eye owing to a cosmetic contact lens and poor hygiene was referred to our hospital. The corrected visual acuity was hand motion. Diagnoses: Corneal infection other than Acanthamoeba keratitis (AK) and corneal hypoxia were excluded. Interventions: We initiated empirical therapy for AK, although no cysts or trophozoites were detected in the cornea and in the lens care solution. Analysis of 16S rDNA sequences from the lens care solution yielded the highest homology with Raoultella species, which are histamine-producing bacteria. Histamine was estimated to be 492 ng/mL in the lens care solution. Outcomes: Her clinical course was distinct from that of usual AK cases. The corrected visual acuity increased up to (1.2) only 5 days after initiating empirical therapy. Lessons: To our knowledge, this is the first report to indicate an association between histamine-producing bacteria and keratoconjunctivitis. We should pay an attention to the microbial contamination of contact lens storage cases by histamine producing bacteria.

Biofilm formation in a dairy waste irrigation system can reduce treatment capacity and increase maintenance and cleaning. An extreme biofilm observed in a primary treated dairy wastewater system blocked the irrigation line requiring manual cleaning. Both next-generation genomic sequencing and the culturable fraction showed the presence of predominantly Gram-negative bacteria. Isolates identified from current samples and stored samples from the extreme biofilm included Pseudomonas, Citrobacter, Klebsiella and Enterobacter, one Gram-positive spore former (Bacillus cereus) and one unique isolate from the biofilm, a member of the Raoultella genus. Raoultella spp was only cultured from the extreme biofilm; however, next-generation sequencing analysis of a wastewater sample showed this bacterium to be present in the wastewater system. The dominance of Gram negative may be due to the wastewater from the washing equipment used to handle raw milk. Six bacteria from the fresh samples were found to be strong biofilm formers along with the Raoultella spp from the extreme biofilm. Tests using multiple isolates showed Raoultella spp to be important in biofilm formation. This is the first report of the microbial composition of a dairy wastewater biofilm giving insight into the population and growth of microorganisms in the sections of a dairy wastewater irrigation system.

Autochthonous bioaugmentation for metal phytoremediation is still little explored, particularly its application to estuarine salt marshes, but results obtained so far are promising. Nevertheless, understanding the behaviour of the microbial communities in the process of bioaugmentation and their role in improving metal phytoremediation is very important to fully validate the application of this biological technology. This study aimed to characterize the bacterial community dynamic associated with the application of autochthonous bioaugmentation in an experimentation which showed that Phragmites australis rhizosphere microorganisms could increase this salt marsh plant potential to phytoremediate Cu contaminated sediments. Bacterial communities present in the autochthonous microbial consortium resistant to Cu added to the medium and in the sediment at the beginning and at the end of the experiment were characterized by ARISA. Complementarily, the consortium and the sediment used for its production were characterized by next generation sequencing using the pyrosequencing platform 454. The microbial consortium resistant to Cu obtained from non-vegetated sediment was dominated by the genus Lactococcus (46%), Raoultella (25%), Bacillus (12%) and Acinetobacter (11%), whereas the one obtained form rhizosediment was dominated by the genus Gluconacetobacter (77%), Bacillus (17%) and Dyella (3%). Results clearly showed that, after two months of experiment, Cu caused a shift in the bacterial community structure of sediments, an effect that was observed either with or without addition of the metal resistant microbial consortium. Therefore, bioaugmentation application improved the process of phytoremediation (metal translocation by the plant was increased) without inducing long term changes in the bacterial community structure of the sediments. So, phytoremediation combined with autochthonous bioaugmentation can be a suitable technology for the recovery of estuarine areas, contributing for an efficient risk management strategy of these coastal zones. © 2017 To Rayra Santiago, Tatiana Necrasov and Catarina Magalhães for their help in the experiments assembling and dismantling of the vessels and to Paula Rodrigues for helping on samples preparation and metal analysis. This research was partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT – Foundation for Science and Technology and European Regional Development Fund (ERDF) , in the framework of the programme PT2020 and by the structured Program of R&D&I INNOVMAR - Innovation and Sustainability in the Management and Exploitation of Marine Resources , reference NORTE-01-0145-FEDER-000035 , namely within the Research Line ECOSERVICES (Assessing the environmental quality, vulnerability and risks for the sustainable management of the NW coast natural resources and ecosystem services in a changing world) within the R&D Institution CIIMAR (Interdisciplinary Centre of Marine and Environmental Research), supported by the Northern Regional Operational Programme ( NORTE2020 ), through the European Regional Development Fund (ERDF) .

The bacterial community of fermented horse milk (koumiss) from Mongolia was studied using three methods: cultivation, direct identification by 16S rRNA clone library and denaturing gradient gel electrophoresis (DGGE). Ninety-eight strains were isolated by traditional cultivation and 61 of those were randomly selected for further identification by 16S rRNA gene sequencing. The strains were dominated by lactic acid bacteria (LAB; six different lactobacilli), Acinetobacter, Bacillus and Psychrobacter. Construction of the clone library analysis revealed that 16S sequences of 220 clones, genus Lactobacillus was dominant, but Streptococcus thermophilus, Acetobacter pasteurianus and uncultured clones were also detected. Ten unique bands were sequenced from the DGGE and revealed: Lactococcus lactis, Lactococcus lactis subsp. lactis, Clostridium acidurici, Acinetobacter johnsonii, Dickeya sp., Enterobacter sp., Pseudomonas sp., Raoultella sp., and Ruminococcus sp.. In vitro growth inhibition of three human pathoge...

The aim of this study was to determine diversity in members of the family Enterobacteriaceae including the beta-lactamase producing isolates associated with the Spanish slug (Arion vulgaris). 81 isolates, each representing a distinct colonial morphotype, were obtained from fecal samples of Spanish slugs and analyzed. Genetically heterogeneous or similar groups were assessed among the isolates by enterobacterial repetitive intergenic consensus (ERIC)-PCR and further characterized by 16S rDNA sequence analysis. Representative strains were allocated to the genera Citrobacter, Raoultella, Escherichia, Klebsiella, Kluyvera, and Buttiauxella. Fifteen isolates, most closely related to Citrobacter freundii and Escherichia coli, displayed an AmpC phenotype, one E. coli isolate showed an ESBL phenotype. These isolates were phenotypically and genotypically characterized by their antimicrobial resistance pattern. Phylogenetic background and multi-locus sequence typing (MLST) of the E. coli isolates were determined. The multi-drug resistant TEM-1 E. coli positive isolate belongs to the phylogenetic group A and ST10. The CMY-2 positive E. coli isolate belongs to the phylogenetic group D and ST117. Our results show that the common garden slug represents an important potential vector of β-lactamase producing Enterobacteriaceae.

Restaurants used to prepare tenderloin beef medallions using different frying parameters in order to satisfy their customers. However, some of those cooking methods are not in accordance with food safety recommendations, because they do not reach internal temperatures recognized as save, in countries such as US, Canada and Brazil. This study aimed to assess the microbial inactivation in tenderloin beef medallions fried to five pre-determined degrees of doneness. Two different frying techniques were investigated, allowing beef pieces to reach the following degrees of doneness: rare, medium-rare, medium, medium-well and well-done. Before thermal processing, tenderloin beef medallions were artificially contaminated with approximately 106 CFU/g of Escherichia coli and the survival of this microorganism and mesophilic total counts were measured using VRBA + MUG and BHI plates, respectively. Bacterial colonies grew on BHI plates were also analyzed by Matrix-Assisted Laser and Time-Of-Flight desorption/ionization mass spectrometry (MALDI-TOF/MS) in order to identify survivors. Results indicated that complete E. coli inactivation was only observed on tenderloin beefs subjected to well-done degree established by both techniques, given that Technique 1 recommended a final heating inside oven after frying. Without this final oven processing, which is not always done in restaurants, more than 2 log CFU/g of viable E. coli were detected. Modeling of the kinetic parameters by GInaFIT revealed a D value (time needed to reduce 90% of the bacterial population) of 1.67 min for Technique 1 and D = 3.81 min for Technique 2 and this difference was associated to the difference of temperatures of olive oil. MALDI-TOF analyses demonstrated high diversity in natural microbiota of tenderloin beef pieces and among the main identified survivor genera was Citrobacter, Enterobacter, Hafnia, Serratia, Raoultella, Bacillus, Lactobacillus, Micrococcus, Pseudomonas, Serratia and Staphylococcus. Pathogens such as Salmonella and Listeria monocytogenes were not found. Based on the results, we recommend the use of high microbiological quality beef for the preparation of tenderloin beef medallions and the validation of frying process considering specific conditions of restaurants, because different degrees of fried may result in different counts and types of bacterial survivors.

Raoultella spp. representatives are Gram-negative capsulated, nonmotile rods. These bacteria are found in the natural environment: plants, water, soil and insects. R. ornithinolytica is one of the three species of Raoultella. R. ornithinolytica is the only species within the genus which has the ability to produce ornithine decarboxylase. Human infections related to R. ornithinolytica are exceedingly rare. The present case report describes catheter-related blood stream infection caused by R. ornithinolytica and successfully treated with antibiotic therapy.

Allovahlkampfia spelaea was identified for the first time in 2009. As a free living amoeba, it has been suggested to be a protective host for some bacterial pathogens against harsh environmental conditions and can transmit them to vulnerable hosts. We aimed in this study to test the interactions between Allovahlkampfia spelaea and some waterborne and foodborne bacteria and unravel if the tested bacteria can survive and multiply inside amoeba. We used a keratitis isolate of Allovahlkampfia spelaea grown in PYG medium containing proteose peptone, yeast extracts, and glucose. We examined amoeba interactions with Methicillin resistant Staphylococcus aureus, Escherichia coli 1, Klebsiella pneumoniae, Enterobacter aerogenes, Citrobacter cloaca, Proteus mirabilis, Raoultella terrigena, Raoultella ornitholytica, Aeromonas hydrophila and Pseudomonas aeruginosa using the co-culture assays. Amoebal survival rate with different bacterial strains were determined. With the exception of Proteus mirabilis that showed decreased survival rates inside amoebal cells, other bacterial isolates could survive and multiply inside Allovahlkampfia spelaea that was associated with decreased survival rates of the amoeba. Particularly, Pseudomonas aeruginosa, Aeromonas hydrophila and MRSA exhibited significantly increased multiplication rates inside amoeba. Our study demonstrated that Allovahlkampfia spelaea may act as a replicative host for pathogenic bacteria with environmental and clinical implications.

Background Raoultella ornitholytica (R. ornitholytica) is a waterborne Gram-negative bacilli increasingly found in hospitals. Multi-drug resistance has been reported, including to carbapenems. Our objective was to identify demographics of R. ornitholytica at Danville Regional Medical Center (DRMC) to determine predisposing factors to infection and potential antibiotic resistance. Methods Cultures positive for R. ornitholytica were identified through DRMC’s electronic medical records (EMR) from 1/2010 to 3/2017. Site of infection, concurrent infections, isolate susceptibilities, prior antibiotic exposure, and appropriateness of treatment were extracted from the EMR. Healthcare associated was defined as occurring in the hospital, nursing home, long-term acute care, or inpatient rehabilitation facility within the past 90 days. Those with diabetes, cancer, and end stage renal disease (ESRD) were qualified as immunosuppressed. Results Thirty-two cases were isolated, of which 20 had associated clinical data. One urine isolate was consistent with colonization. Of the 19 infections, the majority (n = 15) were urinary tract infections (UTIs) and one case each from bronchial washing, heel wound, blood culture, and vulvar lesion. Clinical demographics are shown in Figure 1. Thirteen (65%) had concurrent infections, of which 5 (26%) were co-infected with Enterococcus faecalis, one which was vancomycin resistant. Three had chronic Foley catheters, constituting 20% of the UTIs. Susceptibilities are reported in Figure 2. Prior antibiotic use is shown in Figure 3. Conclusion Most of the isolates from our institution were relatively sensitive, with most resistance to ampicillin. Two isolates were pansensitive, however one case was sensitive only to nitrofurantoin and ertapenem. All isolates which were resistant to cefazolin and ceftriaxone had prior exposure. The elderly and diabetics had the greatest association with infection. A majority of patients had a concurrent infection, which may suggest this as an opportunistic organism. Our findings warrant further studies to better characterize clinical associations and development of resistance in response to prior antibiotic exposure. Disclosures All authors: No reported disclosures.

The genus Raoultella has been separated from the genus Klebsiella in 2001. Two main species are responsible for human infections: R. ornithinolytica and R. planticola. The most frequent infections due to R. planticola include cystitis, pneumonia and bacteremia (mostly in immunocompromised hosts). To date, no joint or bone infection has been reported. We describe the first case of septic arthritis due to R. planticola following an arthroscopy with intra-articular injection of corticosteroids. Evolution was favorable after arthroscopic lavage and antibiotic therapy with quinolones. Raoultella planticola has been described rarely in human infection. It is mainly deemed responsible for cystitis, pneumonia and bacteremia (mostly in immunocompromised hosts) [1, 2, 3]. To our knowledge no case of bone or joint infection has been reported. We described here the first case of infective arthritis due to R. planticola involving a native knee joint following synovectomy and intra-articular injection of corticosteroids during arthroscopy.

Raultella planticola usually invaded immune compromised patients and has been considered a relatively harmless Gram-negative bacteria. We reported a case of a women with a new pancreatic cancer diagnosis that abruptly develop severe sepsis. A Raoultella planticola was yielded in blood cultures. This case suggest Raoultella as pathogen causing life threatening community acquired infection.

A 58-year-old woman presented to eye emergency with a chronic conjunctivitis which was diagnosed by laboratory microbiological testing to be due to the environmental pathogen Raoultella planticola. The organism was sensitive to Chloramphenicol and the patient made a rapid recovery on these drops. This is the first report of this organism infecting the eye.

Item does not contain fulltext A case series of 14 patients with Raoultella bacteremia was compared with 28 Klebsiella oxytoca and 28 Klebsiella pneumoniae bacteremia cases. Forty-three percent of Raoultella bacteremia cases were associated with biliary tract disease, compared to 32% and 22% of patients with K. oxytoca and K. pneumoniae bacteremia, respectively.

The performance of a sulfidogenic bioreactor and the response of bacterial populations to influent alkalinity changes were investigated. The bioreactor reached 40% of sulfate removal efficiency (SRE) with 0 mg l −1 of alkalinity, and single-stranded conformation polymorphism profiles showed that some members of Bacteroides , Dysgonomonas , Sporobacter , Quinella , and Citrobacter became dominant populations. 16S rRNA gene library analysis indicated that the Actinobacteria group increased from 0% in seed to 23% in sludge. An increase in alkalinity to 1300 mg l −1 led to a rapid increase of SRE to 65% and changes in the bacterial community. Sequences representing Dysgonomonas , Raoultella , Kluyvera, and Phascolarctobacterium were now found. When alkalinity was deceased to 0 mg l −1 , SRE dropped and the bands representing Raoultella , Kluyvera, and Phascolarctobacterium disappeared, while bands representing Clostridium appeared. A second cycle of low/high alkalinity did not result in obvious changes to the bacterial community. These results indicate that the sulfidogenic bioreactor favored higher influent alkalinity and that the different functional microbial populations responded well to the alkalinity changes.

Raoultella planticola is a gram-negative bacterium of the Enterobacteriaceae family that is usually found in soil, plant and aquatic environments. It is an uncommon human pathogen and has been associated with cases of bacteremia, pneumonia, urinary tract infections, among others. Here, we present the case of an 85-year-old female that developed nosocomial pneumonia and bacteremia caused by Raoultella planticola. Pertinent microbiological studies detected carbapenemase production codified by the blaKPC gene. The patient was successfully treated with ceftazidime/avibactam and polymyxin. Our case illustrates the pathogenic potential of this organism and highlights the importance of phenotypic and genotypic assays for the appropriate identification of carbapenemase production.

Raoultella ornithinolyitca is a Gram-negative, non-motile bacillus belonging to the family Enterobacteriaceae and has been associated with hospital-acquired clinical cases. The pathogenic potential of R. ornithinolytica isolates in human disease has become increasingly important.

Raoultella planticola is a gram-negative, aerobic, nonmotile mostly found in environments with high prevalence in soil and water. This organism is a very rare human pathogen as only 29 cases of Raoultella planticola-related infections have been reported until 2017, with only 7 cases in the United States. Only 3 cases of urinary tract infection secondary to R planticola have been reported, 1 in a pediatric patient and 2 in adults. In this article, we present a case of R planticola urinary tract infection in a 65-year-old male with immunoglobulin A nephropathy. On investigation, the patient was found to be septic and empirical antibiotic was started for gram-negative coverage. The patient showed remarkable improvement and discharged on oral antibiotic for 7 days. R planticola rarely cause infection in humans, with overall good prognosis.

Raoultella terrigena ATCC 33257, recently reclassified from the genus Klebsiella, is a drinking water isolate and belongs to a large group of non-typeable Klebsiella and Raoultella strains. Using an O-antiserum against a capsule-deficient mutant of this strain, we could show a high prevalence (10.5%) of the R. terrigena O-serotype among non-typeable, clinical Klebsiella and Raoultella isolates. We observed a strong serological cross-reaction with the K. pneumoniae O12 reference strain, indicating that a large percentage of these non-typeable strains may belong to the O12 serotype, although these are currently not detectable by the K. pneumoniae O12 reference antiserum in use. Therefore, we analyzed the O-polysaccharide (O-PS) structure and genetic organization of the wb gene cluster of R. terrigena ATCC 33257, and both confirmed a close relation of R. terrigena and K. pneumoniae O12. The two strains possess an identical O-PS, lipopolysaccharide core structure, and genetic organization of the wb gene cluster. Heterologous expression of the R. terrigena wb gene cluster in Escherichia coli K-12 resulted in the WecA-dependent synthesis of an O-PS reactive with the K. pneumoniae O12 antiserum. The serological data presented here suggest a higher prevalence of the O12-serotype among Klebsiella and Raoultella isolates than generally assumed.

Although Raoultella planticola and Raoultella ornithinolytica were described more than 20 years ago, identifying them remains difficult. The reliability of the chromosomal bla gene for this identification was evaluated in comparison with that of the 16S rDNA and rpoB genes in 35 Raoultella strains from different origins. Of the 26 strains previously identified as R. planticola by biochemical tests alone or in association with molecular methods, 21 harboured a bla gene with 99.8% identity with the bla gene of two reference R. ornithinolytica strains (blaORN gene) and 5 harboured a bla gene with 99.2% identity with the bla gene of two reference R. planticola strains (blaPLA gene). The 9 isolates previously identified as R. ornithinolytica harboured a blaORN gene. The bla gene-based identification was confirmed by 16S rDNA and rpoB sequencing. The 21 isolates newly identified as R. ornithinolytica had a test negative for ornithine decarboxylase (ODC). Molecular experiments suggested one copy of ODC-encoding gene in both ODC-negative R. ornithinolytica and R. planticola strains and two copies in ODC-positive R. orninthinolytica strains. Analysis of the 35 bla genes allowed us (i) to confirm an identity of only 94% between the bla genes of the two Raoultella species while this identity was > 98% for rpoB and > 99% for 16S rDNA genes and (ii) to develop and successfully apply a bla PCR RFLP assay for Raoultella spp. identification. Overall, this study allowed us to discover ODC-negative R. ornithinolytica and to provide a reliable Raoultella identification method widely available as not requiring sequencing equipment.

Introduction : La taxonomie du genre Klebsiella est en perpétuel renouvèlement. Sur la base du séquençage des gènes de l’ARN 16S et rpoB, il a été proposé une modification de combinaison avec un reclassement en deux genres disctincts du genre Klebsiella avec la description du nouveau genre Raoultella. Raoultella ornithinolytica est positive pour l’ODC. Cependant les espèces du genre Raoultella restent difficiles à identifier tant par des systèmes d’identification phénotypiques que plus récemment par la spectrométrie de masse MALDI-ToF. Matériels et méthodes : trente isolats cliniques initialement identifiés comme R. ornithinolytica par MALDI-Biotyper ont été soumis à une identification par galerie API 20E et par séquençage des gènes rpoB, gyrB, speF. Résultats : Seuls deux isolats étaient ODC positifs. Aucun isolat clinique de R. planticola n’est d’identification certaine par MALDI-Biotyper. R. ornithinolytica et K. oxytoca sont difficilement discriminées. Le séquençage bigénique rpoB et gyrB permet une identification robuste de ces deux espèces et la sudivision de Klebsiella oxytoca en plusieurs phylo-groupes. L’analyse du gène speF confirme l’hétérogénicité des isolats de R. ornithinolytica constaté sur l’ODC variable. Discussion : L’ODC n’est pas un caractère fiable pour la confirmation de l’identification de R. ornithinolytica en routine. Le gène rpoB seul n’est pas suffisamment discriminant pour identifier le genre Raoultella. Il doit être complété par le gène gyrB. La construction d’une base utilisateur à partir des souches correctement caractérisées devrait permettre une identification fiable en routine.

The human gut microbiota contributes to host metabolic processes. Diverse microbial metabolic enzymes can affect therapeutic agents, resulting in chemical modifications that alter drug efficacy and toxicology. These interactions may result in ineffective treatments and dose-limiting side effects, as shown by bacterial modifications of the cardiac drug digoxin and chemotherapy drug irinotecan, respectively. Yet, few drug-microbiome interactions have been characterized. Here, a platform is developed to screen for drug-microbiome interactions, validated by the isolation of a gut bacterium capable of inactivating the antineoplastic drug doxorubicin. Two hundred gut strains isolated from a healthy patient fecal sample were cultured in the presence of antibiotic and antineoplastic drugs to enrich for resistance and possible inactivation. Raoultella planticola was identified for its ability to inactivate doxorubicin anaerobically through whole cell and crude lysate assays. This activity was also observed in other Enterobacteriaceae and resulted in doxorubicin inactivation by the removal of its daunosamine sugar, likely mediated by a molybdopterin-dependent enzyme. Other potential drug-microbiome interactions were identified in this screen and can be analyzed further. This platform enables the identification of drug-microbiome interactions that can be used to study drug pharmacology, improve the efficacy of therapeutic treatments, and advance personalized medicine. Thesis Bachelor of Science (BSc) The collection of microbes in the human intestinal tract, referred to as the gut microbiome, can modify therapeutic agents and change the efficacy of drug treatments. Identifying these interactions between drugs and the microbiome will help the study of drug metabolism, provide explanations for treatment failure, and enable more personalized health care. For this project, a platform was developed to isolate gut bacteria from human fecal samples and characterize bacteria that are capable of inactivating various antibiotics and anticancer drugs. Through this platform, the gut bacterium Raoultella planticola was found to inactivate doxorubicin, a commonly used anticancer drug. These results suggest that doxorubicin may be inactivated in the gut and demonstrates how this platform can be used to identify drug-microbiome interactions.

We developed a two-step PCR-based strategy to detect genes encoding OqxAB, allowing a specific assignment of Tn 6010 -associated oqxAB in Enterobacteriaceae . Chromosomal location in this setup was confirmed by hybridization with I- Ceu I-restricted genomes. This approach led us to find that Klebsiella sp. and Raoultella sp. reference strains chromosomally carried oqxAB .

Raoultella terrigena R1Gly is a diazotrophic endophyte isolated from surface-sterilized roots of Nicotiana tabacum . The whole-genome sequence was obtained to investigate the endophytic characteristics of this organism at the genetic level, as well as to compare this strain with its close relatives. To our knowledge, this is the first genome obtained from the Raoultella terrigena species and only the third genome from the Raoultella genus, after Raoultella ornitholytic and Raoultella planticola . This genome will provide a foundation for further comparative genomic, metagenomic, and functional studies of this genus.

Background Liquid antimicrobial soaps are commonly used in the dental health care setting for hand washing to minimize the potential spread of infectious agents to health care workers and patients. The purpose of the current study was to evaluate possible bacterial contamination of antimicrobial liquid soap dispensers located in 2 institutional comprehensive dental care clinics. Methods Fourteen soap dispensers and 16 original stock containers were sampled. A 1-milliliter aliquot was diluted in 10 mL of phosphate buffer (Tween-80; Acros). Serial dilutions were plated in duplicate on neutralizing agar and incubated for 7 days. Molecular identification was performed using 500 base pair comparisons of 16S ribosomal ribonucleic acid sequencing. Taq polymerase chain reaction was performed with sequence-specific primers for Raoultella species. Results Bacterial growth was observed at 18 hours for 57% (8 of 14) of soap dispenser samples. Bacterial densities ranged from 4 × 10 2 to 6 × 10 9 colony-forming units per milliliter. Original commercial containers exhibited no growth. Isolates were identified as Raoultella ( Klebsiella ) planticola. Conclusions This is the first study to the authors' knowledge indicating recovery of R planticola from antimicrobial liquid soap dispensers. R planticola is a recognized environmental opportunistic pathogen that potentially poses a health concern. Practical Implications These findings indicate compliance problems with infection prevention recommendations and support the US Centers for Disease Control and Prevention’s recommendation that dispensers should not be topped off. High bacterial loads of R planticola are inconsistent with infection control practices and are a concern because transmission and possible infection to the health care worker or the patient may occur.

Mallard ducks have demonstrated to be a likely reservoir for zoonotic E. coli strains; thus, it is possible that these ducks could also act as a reservoir for other Enterobacteriaceae members. The present study was initiated to evaluate the species distribution of Enterobacteriaceae other than E. coli in 175 fresh faecal samples collected from a population of mallard ducks. Sixty-four samples displayed detectable colonies of Enterobacteriaceae (excluding E. coli), which resulted in 75 pulsed-field gel electrophoresis (PFGE) types. Seventy-five single representatives of each PFGE type were subjected to identification with API 32NE and MALDI TOF MS systems due to the practical difficulties in species differentiation of Enterobacteriaceae. Those isolated were found to be from nine genera: Buttiauxella (15 %), Citrobacter (5 %), Enterobacter (32 %), Hafnia (1 %), Leclercia (1 %), Pantoea (7 %), Raoultella (21 %), Rahnella (7 %) and Serratia (11 %). Evaluation of antimicrobial resistance phenotypes using the disc method and detection of resistance genes using the microarray method revealed that these microbes possess resistance to β-lactams, aminoglycosides, macrolides, quinolones, rifamycine, sulphonamides, streptogramins and diaminopyrimidines. In conclusion, mallard ducks harbour a variety of non-pathogenic and pathogenic Enterobacteriaceae species like Enterobacter cloacae and Enterobacter amnigenus in their intestine and could act as a reservoir of resistant Enterobacteriaceae.

Enterobacterial strains of Raoultella spp. display a penicillinase-related β-lactam resistance pattern suggesting the presence of a chromosomal bla gene. From whole-cell DNA of Raoultella planticola strain ATCC 33531 T and Raoultella ornithinolytica strain ATCC 31898 T , bla genes were cloned and expressed into Escherichia coli . Each gene encoded an Ambler class A β-lactamase, named PLA-1 and ORN-1 for R. planticola and R. ornithinolytica , respectively. These β-lactamases (291 amino acids), with the same pI value of 7.8, had a shared amino acid identity of 94%, 37 to 47% identity with the majority of the chromosome-encoded class A β-lactamases previously described for Enterobacteriaceae , and 66 to 69% identity with the two β-lactamases LEN-1 and SHV-1 from Klebsiella pneumoniae . However, the highest identity percentage (69 to 71%) was found with the plasmid-mediated β-lactamase TEM-1. PLA-1, which displayed very strong hydrolytic activity against penicillins, also displayed significant hydrolytic activity against cefepime and, to a lesser extent, against cefotaxime and aztreonam, but there was no hydrolytic activity against ceftazidime. Such a substrate profile suggests that the Raoultella β-lactamases PLA-1 and ORN-1 should be classified into the group 2be of the β-lactamase classification of K. Bush, G. A. Jacoby, and A. A. Medeiros (Antimicrob. Agents Chemother. 39: 1211-1233, 1995). The highly homologous regions upstream of the bla PLA-1A and bla ORN-1A genes comprised a nucleotide sequence identical to the −35 region and another one very close to the −10 region of the bla LEN-1 gene. From now on, as the bla gene sequences of the most frequent Raoultella and Klebsiella species are available, the bla gene amplification method can be used to differentiate these species from each other, which the biochemical tests currently carried out in the clinical laboratory are unable to do.

We report a case of an elderly Caucasian female who developed bacteremia secondary to Raoultella planticola following ingestion of undercooked seafood. There have been less than 25 cases reported worldwide. Clin Infect Immun. 2016;1(2):50-51 doi: https://doi.org/10.14740/cii17w

Histamine fish poisoning is caused by histamine-producing bacteria (HPB). Klebsiella pneumoniae and Klebsiella oxytoca are the best-known HPB in fish. However, 22 strains of HPB from fish first identified as K. pneumoniae or K. oxytoca by commercialized systems were later correctly identified as Raoultella planticola (formerly Klebsiella planticola ) by additional tests. Similarly, five strains of Raoultella ornithinolytica (formerly Klebsiella ornithinolytica ) were isolated from fish as new HPB. R. planticola and R. ornithinolytica strains were equal in their histamine-producing capabilities and were determined to possess the hdc genes, encoding histidine decarboxylase. On the other hand, a collection of 61 strains of K. pneumoniae and 18 strains of K. oxytoca produced no histamine.

In this study, we designed and evaluated a microalgal pretreatment method using cellulolytic bacteria that naturally degrades microalgae in their native habitat. Bacterial strains were isolated from each of two mollusk species in a medium containing 1% carboxymethyl cellulose agar. We selected nine bacterial strains that had endoglucanase activity: five strains from Mytilus chilensis , a Chilean mussel, and four strains from Mesodesma donacium , a clam found in the Southern Pacific. These strains were identified phylogenetically as belonging to the genera Aeromonas , Pseudomonas , Chryseobacterium , and Raoultella . The cellulase-producing capacities of these strains were characterized, and the degradation of cell walls in Botryococcus braunii and Nannochloropsis gaditana was tested with “whole-cell” cellulolytic experiments. Aeromonas bivalvium MA2, Raoultella ornithinolytica MA5, and Aeromonas salmonicida MC25 degraded B. braunii , and R. ornithinolytica MC3 and MA5 degraded N. gaditana . In addition, N. gaditana was pretreated with R. ornithinolytica strains MC3 and MA5 and was then subjected to an anaerobic digestion process, which increased the yield of methane by 140.32% and 158.68%, respectively, over that from nonpretreated microalgae. Therefore, a “whole-cell” cellulolytic pretreatment can increase the performance and efficiency of biogas production.

To develop a targeted metagenomics approach for the analysis of novel multispecies microbial consortia involved in the bioconversion of lignocellulose and furanic compounds, we applied replicated sequential batch aerobic enrichment cultures with either pretreated or untreated wheat straw as the sources of carbon and energy. After each transfer, exponential growth of bacteria was detected using microscopic cell counts, indicating that the substrate was being utilized. In batch, the final bacterial abundances increased from an estimated 5 to 8.7-9.5 log 16S rRNA gene copy numbers/ml. The abundances of fungal propagules showed greater variation, i.e., between 5.4 and 8.0 log ITS1 copies/ml. Denaturing gradient gel electrophoresis analyses showed that the bacterial consortia in both treatments reached approximate structural stability after six transfers. Moreover, the structures of the fungal communities were strongly influenced by substrate treatment. A total of 124 bacterial strains were isolated from the two types of enrichment cultures. The most abundant strains were affiliated with the genera Raoultella/Klebsiella, Kluyvera, Citrobacter, Enterobacter, Pseudomonas, Acinetobacter, Flavobacterium and Arthrobacter. Totals of 43 and 11 strains obtained from the untreated and pretreated substrates, respectively, showed (hemi)cellulolytic activity (CMC-ase and xylanase), whereas 96 strains were capable of growth in 7.5 mM 5-hydroxymethylfurfural. About 50 % of the latter showed extracellular oxidoreductase activity as detected by a novel iodide oxidation method. Also, (hemi)cellulolytic fungal strains related to Coniochaeta, Plectosphaerella and Penicillium were isolated. One Trichosporon strain was isolated from pretreated wheat straw. The two novel bacterial-fungal consortia are starting points for lignocellulose degradation applications.

Self-fermentation of cellulosic substrates to produce biohydrogen without inoculum addition nor pretreatments was investigated. Dark fermentation of two different substrates made of leaf-shaped vegetable refuses (V) and leaf-shaped vegetable refuses plus potato peels (VP), was taken in consideration. Batch experiments were carried out, under two mesophilic anaerobic conditions (28 and 37 °C), in order to isolate and to identify potential H2-producing bacterial strains contained in the vegetable extracts. The effect of initial glucose concentration (at 1, 5 and 10 g/L) on fermentative H2 production by the isolates was also evaluated. H2 production from self-fermentation of both biomasses was found to be feasible, without methane evolution, showing the highest yield for V biomass at 28 °C (24 L/kg VS). The pH control of the culture medium proved to be a critical parameter. The isolates had sequence similarities ≥98% with already known strains, belonging to the family Enterobacteriaceae (γ-proteobacteria) and Streptococcaceae (Firmicutes). Four genera found in the samples, namely Pectobacterium, Raoultella, Rahnella and Lactococcus have not been previously described for H2 production from glucose. The isolates showed higher yield (1.6–2.2 mol H2/mol glucoseadded) at low glucose concentration (1 g/L), while the maximum H2 production ranged from 410 to 1016 mL/L and was obtained at a substrate concentration of 10 g/L. The results suggested that vegetable waste can be effectively used as both, substrate and source of suitable microflora for bio-hydrogen production.

Four isolates of Gram-negative facultatively anaerobic bacteria, three of them producing NDM-1 carbapenemase, were isolated from hospitalized patients and outpatients attending two military hospitals in Rawalpindi, Pakistan, and studied for their taxonomic position. Initially the strains were phenotypically identified as Citrobacter species. Comparative analysis of 16S rRNA gene sequences then showed that the four strains shared >97%, but in no case >98.3%, 16S rRNA gene sequence similarities to members of the genera Citrobacter, Kluyvera, Pantoea, Enterobacter and Raoultella, but always formed a separate cluster in respective phylogenetic trees. Based on multilocus sequence analysis (MLSA) including partial recN, rpoA, thdF and rpoB gene sequence and respective amino acid sequence analysis it turned out that the strains also here always formed separate clusters. Based on further comparative analyses including DNA-DNA hybridizations, genomic fingerprint analysis using rep- and RAPD-PCRs and physiological tests, it is proposed to classify these four strains into the novel genus Pseudocitrobacter gen. nov. with a new species Pseudocitrobacter faecalis sp. nov. with strain 25 CIT(T) (=CCM 8479(T)=LMG 27751(T)) and Pseudocitrobacter anthropi sp. nov. with strain C138(T) (=CCM 8478(T)=LMG 27750(T)), as the type strains, respectively.

The genus Raoultella is composed of Gram-negative, aerobic, oxidase-negative, catalase-positive, nonmotile, capsulated rods belonging to the Enterobacteriaceae family ([1][1]). Raoultella ornithinolytica is found in aquatic and hospital environments ([2][2]). Occurrence of Raoultella in human

The wood-boring beetleAnoplophora chinensisForster, native to China, has recently spread to North America and Europe causing serious damage to ornamental and forest trees. The gut microbial community associated with these xylophagous beetles is of interest for potential biotechnological applications in lignocellulose degradation and development of pest-control measures. In this study the gut bacterial community of larvae and adults ofA. chinensis, collected from different host trees in North Italy, was investigated by both culture and culture-independent methods. Larvae and adults harboured a moderately diverse bacterial community, dominated by Proteobacteria, Actinobacteria, and Firmicutes. The gammaproteobacterial family Enterobacteriaceae (generaGibbsiella, Enterobacter, Raoultella, andKlebsiella) was the best represented. The abundance of such bacteria in the insect gut is likely due to the various metabolic abilities ofEnterobacteriaceae, including fermentation of carbohydrates derived from lignocellulose degradation and contribution to nitrogen intake by nitrogen-fixing activity. In addition, bacteria previously shown to have some lignocellulose-degrading activity were detected at a relatively low level in the gut. These bacteria possibly act synergistically with endogenous and fungal enzymes in lignocellulose breakdown. The detection of actinobacterial symbionts could be explained by a possible role in the detoxification of secondary plant metabolites and/or protection against pathogens.

Item does not contain fulltext Ninety-nine clinical isolates previously identified as Klebsiella oxytoca were evaluated using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Eight isolates were identified as Raoultella spp., being 5 Raoultella spp. and 3 K. oxytoca, by 16S rRNA sequencing. These isolates were correctly identified by applying the 10% differential rule for the MALDI-TOF MS score values. This approach might be useful to discriminate Raoultella species from K. oxytoca. 01 april 2013

This study was conducted with the aim of isolating and identifying endophytic bacteria associated with bananas in Kenya and assessing their functional potentiality as biological fertilizers. Banana material was collected from two different banana cultivars in five different geographical regions and bacteria were isolated using five different isolation media. Whole-cell matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) analysis was used for microorganism profiling. Protein from the living cells were extracted using the ethanol/formic acid extraction procedure and intact molecular weights of the ionized proteins directly measured and the pattern of the protein molecular weights used as fingerprints. Forty three isolates were selected for partial 16S rRNA gene sequencing. Isolates were characterized on the basis of their in-vitro plant growth-promoting activities that included abilities to fix free nitrogen, solubilize phosphates and produce siderophores. The isolates were identified as Serratia, Pseudomonas, Rahnella, Enterobacter, Raoultella, Yokenella, Bacillus, Klebsiella, Yersinia and Ewingella species. Siderophore production activity was detected with all the Pseudomonas isolates as determined on blue Chrome Azurol S (CAS) agar plates. Twenty seven isolates were observed to solubilize phosphates, with Rahnella isolates showing the highest potential as determined on NBRIP growth medium. All the isolates grew on solid nitrogen-source free medium, suggesting their ability to fix nitrogen. In conclusion, endophytic bacteria of bananas in Kenya were isolated and identified, and Rahnella and Pseudomonas isolates proposed as potential microbial biofertilizers for sustainable banana production in Kenya.

The kinetics of hexavalent chromium bio-reduction in draw-fill suspended and attached growth reactors was examined using sugar as substrate and indigenous microorganisms from the industrial sludge of the Hellenic Aerospace Industry. Initially, experiments in suspended growth batch reactors for Cr (VI) concentrations of 1.4–110 mg/l were carried out, to extensively study the behaviour of a mixed culture. The maximum Cr(VI) reduction rate of 2 mg/l h was achieved for initial concentration 12.85 mg/l with biomass production rate 4.1 mg biomass/l h. Analysis of the microbial structure in the batch reactor culture indicated that the dominant bacterial communities were constituted by bacterial members of Raoultella sp., Citrobacter sp., Klebsiella sp., Salmonella sp., Achromobacter sp. and Kerstersia sp. while the dominant fungal strain was that of Pichia jadinii . Experiments using the same mixed culture were also carried out in packed-bed reactors with plastic support media. High removal rates were achieved (2.0 mg/l h) even in high initial concentrations (109 mg/l). A combination of the model of Tsao and Hanson for growth enhancement and that of Aiba and Shoda for growth inhibition was used in order to describe and predict the process of Cr(VI) bio-reduction in suspended growth and packed-bed reactors. Kinetic constants of the equation obtained from both batch (or draw-fill) culture experiments. In the draw-fill experiments at the packed-bed reactor, hexavalent chromium inhibitory effects were minimized increasing the inhibitory constant value K i ′ at 148.5 mg/l, compared to suspended growth experiments which was K i = 8.219 mg/l. The model adequately predicts hexavalent chromium reduction in both batch reactors for all initial concentrations tested.

Raoultella spp. have recently been separated from the genus Klebsiella based on their molecular characteristics. It was discovered that Raoultella ornithinolytica can be misidentified as Klebsiella oxytoca by commonly used phenotypic identification systems. Therefore, this study evaluated the ability of three phenotypic systems to identify R. ornithinolytica compared with the genotypic methods sequence-specific primer PCR (SSP-PCR), 16S rRNA gene sequence analysis using the MicroSeq 500 system16S rDNA bacterial identification system or comparison with GenBank sequences using blast. The phenotypic systems examined in this study were the VITEK 2 GN ID card, the MicroScan Neg Combo 32 panel and API 20E. The SSP-PCR panel was able to distinguish the R. ornithinolytica reference strain from other Raoultella spp. and K. oxytoca. Of the 27 isolates identified as R. ornithinolytica by SSP-PCR, VITEK 2 identified all of them as R. ornithinolytica. MicroScan and API identified 25 isolates (92.6 %) and 24 isolates (88.9 %) as K. oxytoca, respectively. These isolates were ornithine decarboxylase (ODC) negative in all three phenotypic systems. MicroSeq 500 identified 24 isolates (88.9 %) as R. ornithinolytica, whereas GenBank identification was heterogeneous. Of the 68 isolates identified as K. oxytoca by SSP-PCR, 66 isolates (97.1 %) were identified as K. oxytoca by VITEK 2, MicroScan and API. MicroScan and API require additional biochemical tests to differentiate between ODC-negative R. ornithinolytica and K. oxytoca.

Fruit flies (Diptera: Tephritidae) are a highly successful, widespread group of insects that cause economic damage in agriculture. Data available so far on the composition of the bacterial community associated with their digestive tract indi- cate that members of Enterobacteriaceae are the species most often isolated. Bacteria naturally occurring in insect guts may be engineered and used to study the spatial and functional interactions of microbes within the insect system and offer one route to meet the demand for novel insect pest management strategies. With this aim we introduced by conjugation the gfp gene carried by the suicide plasmid pTn5gfpmut1 into Klebsiella oxytoca and Raoultella (formerly Klebsiella) spp. strains isolated from the oesophageal bulb of the fruit flies Ceratitis capitata (Wiedemann) and Rhagoletis completa Cresson, re- spectively. The GFP-encoding gene was stably maintained in two tested transgenic strains, both originally isolated from R. completa. In one case, GFP-labeled bacterial cells were used to feed larvae and adults of the original host. Genetically modified bacteria were able to colonize the gut of larvae and persisted through all larval instars to pupal stage. Resume : Les mouches a fruits (Dipteres : Tephritidae) forment un groupe d'insectes tres efficaces et repandus qui causent des dommages economiques a l'agriculture. Les donnees portant sur la composition de la communaute bacterienne associee a leur systeme digestif et disponibles jusqu'a maintenant indiquent que les membres des Enterobacteriaceae sont les especes le plus souvent isolees. Les bacteries naturellement presentes dans l'intestin des insectes peuvent etre modifiees par genie genetique et utilisees pour etudier les interactions spatiales et fonctionnelles des microbes a l'interieur du systeme des in- sectes, offrant une avenue pour repondre a la demande de developpement de nouvelles strategies de controle des insectes nuisibles. A cet effet, nous avons introduit par conjugaison le gene gfp contenu dans le plasmide suicide pTn5gfpmut1 dans les souches Klebsiella oxytoca et Raoultella (auparavant Klebsiella) spp., isolees du bulbe œsophagien des mouches a fruits Ceratitis capitata (Wiedemann) et Rhagoletis completa Cresson, respectivement. Le gene codant la GFP etait maintenu de facon stable dans deux souches transgeniques, toutes deux isolees a l'origine de R. completa. Dans un cas, les cellules bacte- riennes marquees a la GFP ont ete utilisees pour nourrir les larves et les adultes de l'hote original. Les bacteries modifiees genetiquement pouvaient coloniser l'intestin des larves et persistaient dans tous les instars larvaires jusqu'au stade pupal. Mots-cles : proteine fluorescente verte, conjugaison, Enterobacteriaceae, Tephritidae. (Traduit par la Redaction)